The Fifth Kingdom An Introduction To Mycology by Bryce Kendrick

The Fifth Kingdom

An Introduction to Mycology Fourth Edition

Bryce Kendrick

The Fifth Kingdom An Introduction to Mycology

Fourth Edition

The Fifth Kingdom An Introduction to Mycology

Fourth Edition

Bryce Kendrick

Focus

an imprint of Hackett Publishing Company, Inc. Indianapolis/Cambridge

I dedicate this book to my wife, Laurie A Focus book

Focus an imprint of

1234567

For further information, please address Hackett Publishing Company, Inc. P.O. Box 44937 Indianapolis, Indiana 46244-0937 www.hackettpublishing.com Cover design by Rick Todhunter Interior design by Elizabeth L. Wilson and Mireidys Garcia Composition by Integrated Composition Systems Library of Congress Cataloging-in-Publication Data Names: Kendrick, Bryce, author. Title: The fifth kingdom / Bryce Kendrick. Description: Fourth edition. | Indianapolis : Focus an imprint of Hackett Publishing Company, Inc., [2017] | Includes bibliographical references and index. Identifiers: LCCN 2017009567 | ISBN 9781585104598 (paper) Subjects: LCSH: Fungi. | Mycology. Classification: LCC QK603 .K46 2017 | DDC 579.5—dc23 LC record available at https://lccn.loc.gov/2017009567 Adobe PDF ebook ISBN: 978-1-58510-862-6

Contents

Preface Introduction 1. Kingdoms, Classification, Nomenclature, and Biodiversity

vii viii 1

2. A Mixed Bag: Protozoan ‘Pseudofungi’—Kingdom Protozoa (the So-Called Slime Moulds—Phyla Myxostelida, Dictyostelida, Labyrinthulida, Plasmodiophorida); Pseudofungi—Kingdom Chromista: Phyla Hyphochytriomycota and Oomycota; True Fungi—Kingdom Eumycota: Phylum 1 Chytridiomycota, Phylum 2 Blastocladiomycota, Phylum 3 Neocallimastigomycota

3. Eumycotan Fungi—the Mainstream and Others: Phylum 4 Zygomycota, Phylum 5 Glomeromycota, Phylum 6 Microsporidia

4. Kingdom Eumycota (True Fungi), Subkingdom Dikarya: Phylum 7 Ascomycota—the Ascomycetes

5. Kingdom Eumycota (True Fungi): Subkingdom Dikarya (Fungi with a Dikaryophase); Phylum 8 Basidiomycota: the Basidiomycetes; Subphyla: Agaricomycotina, Pucciniomycotina, Ustilaginomycotina

105

6. Yeasts—Compact Polyphyletic Extremophile Fungi

139

7. Lichens—Dual (or Even Triple) Extremophile Organisms

145

8. Spore Dispersal in Fungi—Airborne Spores and Allergy

9. Fungal Physiology and Metabolism

175

10. Fungal Genetics—Mendelian and Molecular

195

11. Fungal Ecology

227

12. Fungal Diseases of Crops and Trees

247

13. Fungicides —Several Generations—More Needed

265

14. Fungi as Agents of Biological Control

276

15. Fungi Exploiting Microscopic Animals

295

16. Mutualistic Symbioses between Animals and Fungi

308

17. Mycorrhizas—Mutualistic Plant-Fungus Symbioses

323

18. Fungi as Food—Mycophagy

345

19. Fungi in Food Processing

359

Contents 20. Food Spoilage by Fungi and How to Prevent It

365

21. Mycotoxins in Food and Feed

372

22. Poisonous and Hallucinogenic Mushrooms

392

23. Medical Mycology

409

24. Commercial Exploitation of Fungal Metabolites and Mycelia

420

25. But How Do You Actually Do Mycology? And How Can You Earn a Living Doing It?

429

Glossary Sources of Illustrations Index

455 489 491

Preface

Fungi probably rival flowering plants in their species diversity, and outweigh the animal kingdom. Whilst wielding great destructive power as agents of disease and decay, they drive the global carbon cycle, sustain our forests and grasslands via mycorrhizal associations, and clothe, as lichens, what would otherwise be bare parts of the planet. Their developmentally versatile body forms provide immense scope for industrial exploitation as well as experimentally accessible systems for studying fundamental biological issues. Yet most people’s appreciation of fungi stops at mushrooms, mouldy food and fairy tales. Challenged by such ignorance, mycologists need to overcome some deeply rooted prejudices. On the one hand, the variety, edibility and toxicity of fungal fruit bodies has always been a source of fascination which can be relied on to deliver new recruits to the cause of mycology, but if that fascination becomes an obsession, the cause is lost. On the other hand, mycologists working on disease control, taxonomy or some industrial process often find it difficult to communicate the wider interest of what they are doing. Because of the vicious cycle of neglect, their task is made harder by the need to use “technical” terms: plant scientists can assume that their audience knows what leaves, roots and stems are; mycologists always have to explain what hyphae and mycelium are. So there are two images of the mycologist: one of the eccentric amateur, the other of the remote professional working on esoteric problems. Both are damaging. So writes Professor Alan Rayner, one of mycology’s most articulate spokespersons, and it is impossible to disagree with him. Perhaps this book can do something to produce a more balanced understanding and appreciation of fungi among university students and intelligent laypersons. Interest is the best stimulant to learning, and at least some of the stories in this book will surely tickle even the most jaded palate, since the fungal lifestyle is so bizarre, the facts so strange. Science fiction writers, look no further. Plots lie within. So far, we have described about 100,000 fungi, yet we estimate these to represent less than one-tenth of the Earth’s mycota. Part of this book, then, is a celebration of biodiversity—just think, there are over 10,000 species of mushrooms alone. Tragically, the world is gradually losing its biological richness. As a result of human activities, species of living organisms, fungi among them, are being driven irretrievably into extinction every day. We need you, the readers of this book, to help stop those losses. There are many kinds of environmental action, and I urge you to become personally involved in some of them. Our grandchildren will thank us, but only if we succeed.

vii

Introduction

Imagine the picnic of your dreams. You and your loved one float across a flower-filled meadow, coming to rest under the shade of a giant white pine. There you spread out the magic ingredients—the champagne (or wine, or beer, if you prefer), the fresh crusty bread, the pâté au truffe, the creamy Camembert (or Brie or Roquefort) cheese. You may add other ingredients—many spring to mind—but up to this point, your tryst is a tribute to the beneficent influence of fungi on our diet and our scenery. The grass, the flowers in the meadow, and the pine tree have specialized nutrientgathering fungi growing in and around their roots in an obligately symbiotic relationship. Without these fungi, we believe that neither grass nor tree would exist. Champagne, wine, and beer are all direct products of fungal action on specific substrates, and the alcohol they contain is a fungal metabolite highly prized by those who need an occasional escape from reality. Even those who prefer to keep reality at arm’s length with psilocybin or LSD usually know that these psychotropic substances are also fungal metabolites. Bread owes its lightness and texture to the ‘raising’ activities of a fungus. The mouthwatering flavour of your pâté is enhanced by the presence of pieces of black truffle, a subterranean fungus from Europe. The cheeses are ripened and given their unique texture and taste by specific moulds. But fungi, like people, have a darker side. On closer inspection, the flowers in the meadow may be found to be suffering from a host of fungal diseases—leaf spots, wilts, mildews, blights, and more—and the pine tree may have problems with root rot, heart rot, blister rust, and needle cast, all caused by fungi. Some of the food in your picnic may have been insidiously infiltrated by fungi. Those ripe, juicy peaches you brought along for dessert may reveal rapidly spreading brown areas. You must trust that the bread wasn’t made from wheat containing vomitoxin or, if it’s rye bread, that no ergot, with its multitude of alkaloids and hallucinogens, was in the grain. Even your blue cheese and your peanut butter could possibly contain mycotoxins. Homeowners know this other face, too. Has your wooden fence or your deck become rotten and needed rebuilding? Has your prize elm tree died or your chestnut been defoliated by leaf blight? Are your roses besmirched by black spot, your lilacs by powdery mildew, your hollyhocks by rust? Do your tomatoes suffer from early blight, your potatoes from late blight, your grapes from downy mildew, your strawberries from grey mould? Are your peaches attacked by leaf curl or soft rot, your apples by scab? Does damping-off cause your seedlings to keel over? Does food go mouldy, turning green, pink, or brown, or growing whiskers, even in the refrigerator? And is there a creeping black stain around the door of that appliance? Have you ever had athlete’s foot, jock itch, or ringworm of the scalp? All of these, too, are the results of fungal activity. But there’s still another side to the fungi. The blister rust on your pine tree may itself be attacked and controlled by another fungus, as may the powdery mildew on the grass. Specialized fungi can control infestations of insects in your garden. A fungal metabolite is used worldwide to control many bacterial infections (including gonorrhea), while viii

Introduction another can cure some of the fungal diseases that afflict people from time to time. Organ transplants now have much-improved chances of success because of a fungal metabolite that safely prevents the body from rejecting the new organ, and this same substance seems to stop the development of some kinds of diabetes—the first actual cure ever discovered for this disease. These are a few of the reasons you should know something about fungi. I hope that by the end of this book, which I have tried to prepare as a tasty mycological smorgasbord, you will be able to add other reasons of your own. For additional images and online resources, visit www.mycolog.com.

Fig. 1.1

Patterson and Sogin diagram

Prokaryota

Agrobacterium

Mycoplasma

Eukaryota

Microsporidians

Euglenoids

Entamoebae Protozoa Kinetoplastids

Myxostelida

Alveolates (Rhodophyta)

Trichom*onads Diplomonads

Halobacteria

Fungi

Archaea

Sulfolobus Thermoplasma Methanobacteria

Enterobacteria

Plant mitochondria

Bacteria

Cyanobacteria

Plant chloroplasts

Animalia

Chromista Plantae

1 Kingdoms, Classification, Nomenclature, and Biodiversity Kingdoms—Now There Are Seven! As you read the text that follows, you may come across words that are new to you. Most of them will be in the glossary. If even the definitions given there leave you scratching your head, I suggest you find and read a first-year university biology textbook before going much further. You may not know it, but you are about to become a member of an elite group. Although many people are aware that there are millions of different types (species) of living organisms on Earth today (although our own species is doing its best to drive many of them into extinction), surprisingly few people are aware that these organisms are now divided up among no fewer than seven kingdoms. Before I can effectively develop the theme of this book, I must explain these major patterns and those of some of the almost 100 distinctive evolutionary pathways known as phyla (botanists sometimes call them divisions) which make up those kingdoms. The really basic division among life forms is between the simpler prokaryotes and the more complex eukaryotes. The diagram below (after Patterson and Sogin 1992) shows the way in which we think the kingdoms evolved. It is based on molecular evidence, consisting of base sequences from ribosomal RNA (rRNA). The earliest forms of life, which appeared about 3,500–3,800 million years ago, were prokaryotes. We tend to define them by their relative morphological simplicity (although they have enormous biochemical complexity) and by the absence of many features found in more modern cells. Although their modern descendants have a single circular chromosome, this is not found inside a nucleus, and their cytoplasm contains no mitochondria or plastids (cytoplasmic organelles). These organisms make up the baseline kingdoms Archaebacteria (or Archaea) and Eubacteria. The prokaryotes had the world to themselves for 1,500 million years (they did, however, invent photosynthesis during that time). Not until about 2 billion years ago did life take the next giant step, the evolution of the eukaryotic cell. Many biologists now believe that this arose as a result of the mutually beneficial symbiotic union of several different types of prokaryotes within another host prokaryote. At least two types of modern organelles have been found to retain some of their original independent DNA: (1) mitochondria, which specialize in the oxidation of 3-carbon organic acids (the Krebs cycle), providing an immediately available energy supply in the form of ATP; and (2) plastids, which may contain photosynthetic pigments and enzymes 1

Chapter 1 (chloroplasts) or may store food. Some biologists also think that flagella were once free-living prokaryotes, though they are very different from the motility organelles found in bacteria. Eukaryotic cells also have their DNA organized into a number of discrete chromosomes, which are found inside a nucleus which is surrounded by a membrane. Cell division in eukaryotes involves a complex process called mitosis. The nuclear membrane breaks down, a mitotic spindle of microtubules develops, and the chromosomes are duplicated. Then the daughter chromosomes separate and are pulled to opposite poles by the contracting spindle fibres. Each set of chromosomes then becomes enclosed by a new nuclear membrane, and the cell finally divides into two. Prokaryotic cells have only a single, usually circular, chromosome and do not undergo mitosis. They usually divide by a much simpler process called binary fission. Mitosis, with its very accurate duplication and sharing of the genetic material, seems to have been a crucial invention. Only eukaryotic cells, with their precisely regulated genetic mechanisms, apparently had the potential to evolve into more complex, multicellular organisms in which cells are organized into different tissues and organs. All prokaryotes are still microbes. Now look at the kingdom diagram once more. The Archaebacteria (or Archaea) and the Eubacteria are prokaryotes. The eukaryotes encompass the other five kingdoms, and it is in these other kingdoms that the dazzling evolutionary explosion of new taxa has occurred. The diagram shows five eukaryote kingdoms: Protozoa, Chromista (or Stramenopila), Plantae, Animalia, and Eumycota (or Fungi). New information on the fully sequenced genome of an amoeboid (protozoan) pseudofungus, Dictyostelium discoideum, has now thrown some light on the timing of the origin of the kingdoms. The burst of eukaryotic evolution was made possible by, among other things, a modified form of mitosis called meiosis or reduction division. In many organisms, this produces special sex cells called gametes. Each of these sex cells has a single set of chromosomes (we say that the gametes are haploid). When two gametes from compatible parent organisms fuse, the resulting cell (the zygote) has two sets of chromosomes (we call this condition diploid). In plants and animals, zygotes develop into diploid, multicellular organisms, but in true fungi, the vegetative phase is always haploid, and therefore, meiosis must take place in the zygote. Whether meiosis happens in the zygote or at the other end of the life cycle, during the formation of gametes, it is responsible for the reassortment of the genetic information built into the chromosomes by the process known as crossing-over. New features are constantly being added to the pool of genetic material by the process of mutation, but sexual reproduction is the mechanism by which this pool is recombined each generation in most eukaryotic organisms, producing an endless supply of variation upon which the processes of natural selection can work. This is one of the key secrets of eukaryote diversity. The radiation of the two unicellular kingdoms (Protozoa and Chromista) shows how the evolution of the eukaryotic cell expanded life’s horizons. But the full potential of the new teamwork—I call it that because several prokaryotes cooperate to make one functional eukaryotic cell—was not realized until cells, as well as cell components, began to cooperate. When organisms became multicellular, different cells could assume different, specialized functions. This division of labour eventually led to the

Kingdoms, Classification, Nomenclature, and Biodiversity development of tissues and organs and ultimately permitted the evolution of complex beings such as ourselves, beings with almost infinitely expanded capabilities (both wonderful and terrible). Three new multicellular kingdoms arose, exemplifying three different ways of life. Multicellular organisms which could photosynthesize—make their own food from simple inorganic precursors—were eaten by other multicellular organisms that lacked this talent, and both were recycled after death by a third group. These groups we call producers, consumers, and decomposers, which roughly correspond to the plants, the animals, and the fungi. We recognize about nine phyla of plants, about thirtytwo phyla of animals, and eleven phyla of fungi (two chromistan and [currently] nine eumycotan). The world being what it is, the picture is not as simple as we might like. Some of the divergent paths of evolution have come together again, almost as they did at the birth of the eukaryotes, and many organisms that seem unitary are, in fact, partnerships or even consortia. Lichens, for example, always incorporate both an alga (eukaryotic or prokaryotic) and a fungus. Other examples are reef-building corals and some medusae that co-exist with endosymbiotic dinoflagellates, many sponges and some molluscs and flatworms that co-exist with endosymbiotic green algae, plants that often co-exist with systemic endophytic fungi which may function in repelling herbivores, and almost all plants that also have mycorrhizal fungi intimately associated with their roots. How can fungi fit into two kingdoms? The answer lies in the way we define the term fungus (plural: fungi). Fungi are eukaryotic (their cells contain nuclei); heterotrophic (they can’t make their own food); osmotrophic (they absorb, don’t ingest, food); develop a rather diffuse, branched, tubular body (radiating hyphae making up mycelia or colonies); and reproduce by means of spores. This describes not a single phylogenetic line but, rather, a way of life shared by two kingdoms of different evolutionary backgrounds. We recognize chromistan fungi (or pseudofungi) as well as eumycotan (or true) fungi. If you find this strange, consider the algae for a moment—they include representatives of three kingdoms: eubacterial prokaryotes (the blue-green cyanobacteria), chromistans (brown algae, diatoms, etc.), and plantae (green algae). Both algae and fungi are in fact defined functionally or ecologically, rather than phylogenetically.

Recent Additions to the True Fungi Whittaker (1969) led to a general acceptance that true fungi constitute a separate kingdom of organisms (the fifth, but not the last, to be proposed). Yet the idea of a more or less universal hyphal module and microscopic spores as characterizing fungi led to their being mixed together with ‘outgroups’, such as the hypha-producing oomycetes, which are primarily related to brown algae (kingdom Chromista), the Hyphochytriomycetes, and, for no obvious reason that I can see, the Myxostelida (Mycetozoa; plasmodial slime moulds), Plasmodiophorida, and Labyrinthulida (all three placed in kingdom Protozoa). These will all be briefly discussed later. Once these outgroups had been expelled, the true fungi were considered to embrace five phyla—Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota, and

Chapter 1 Basidiomycota. All of these are heterotrophic, lack plastids, and have chitinous cell walls. But those characteristics alone are no longer adequate to define the fungi (or, since some fungi, as you will see, have lost major characteristics, this definition may be too demanding).

Blastocladiomycota—a sixth fungal phylum This group was considered to be an order within the Chytridiomycetes until 2006, when it was promoted to phylum rank based on molecular evidence.

Microsporidia—a seventh fungal phylum Molecular evidence has now added to the fungi a seventh, unlikely group of phylum rank, the Microsporidia (e.g., Nosema)—obligate intracellular parasites of animals, lacking walls in the somatic condition and long considered to be Protozoa. There are 1,400 species of Microsporidia in 144 genera and probably many more. Why is the naked, mitochondria-lacking, peroxisome-lacking Nosema a fungus? Because it has chitin-walled spores and appropriate DNA sequences. During the course of evolution, the Microsporidia have lost many fungal characteristics, but their DNA gives them away.

Neocallimastigomycota—an eighth fungal phylum The Neocallimastigomycota, recently recognized and similar to aberrant Chytridiomycetes living anaerobically in the rumen of herbivores, and with multiflagellate spores, have also lost mitochondria and peroxisomes but are nevertheless true fungi.

Cryptomycota—a ninth fungal phylum? These so-called hidden fungi turned up in a pond in England as unknown DNA. Once it was better characterized and visualized, this group was found to have cells 3–5 microns in diameter and to lack the chitinous cell walls so common in fungi. This meant that these organisms could feed by engulfing food particles, which is most un-funguslike, or live inside host cells. But then, rules in biology are often broken and we have to think again. DNA sequences typical of this new group have now been found in lakes in France, farms in the United States, and deep ocean sediment, and they may turn out to be very numerous, although they have not yet been grown in culture. It is also still possible that they are not really fungi. It is important to realize that the Oomycota are not hom*ologues of the true fungi, but highly convergent analogues. They have cellulosic walls and are somatically diploid, having arisen from a very different group of organisms, which includes the diatoms and the brown algae (kingdom Chromista). It is now thought that fungus-like organisms arose at least six times—walled somatic cells, no plastids, heterotrophic—but that only one of these became the true fungi, or Eumycota. Thus, the definition of a fungus is (sigh!) more complex than we might wish. It involves a mixture of ultrastructural and molecular characteristics. Cilia (flagella) of true fungi, when they are present (as in the Chytridiomycota and Neocallimastigomycota), never have flimmers (tripartite hairs) like those found on

Kingdoms, Classification, Nomenclature, and Biodiversity tinsel flagella (as in the Oomycota). The cristae of true fungal mitochondria are flattened (like those in animals), while those of Chromista are tubular (like those in Protozoa). Fungi synthesize lysine by the alpha amino adipic acid pathway, while Chromista (and plants) employ the diaminopimelic acid pathway. A minimal definition of fungi might now be as follows: Absorptive heterotrophs (lacking plastids and therefore not photosynthetic, and not phagotrophic) that usually produce spores with chitinous walls and often have multinucleate, walled hyphae. I will close this brief outline with a caution. We tend to accept molecular evidence without raising too many serious questions, yet the outline of fungal phylogeny is still rather vague, because rRNA, the only molecule extensively used in constructing fungal phylogenies, evolves at such different rates in different groups of fungi that the phylogenies suffer from ‘systematic biases as well as random noise’. At this point, it becomes clear that this book does not, as its title implies, deal exclusively with the fifth kingdom, Eumycota, but also discusses some elements of kingdom Chromista, and even the nonfungal slime moulds. But these are all relatively minor players in the biosphere when compared with the huge numbers and biomass of the true fungi.

Biological Classification The first part of this book deals with the classification of the fungi. You could certainly ignore chapters 2–7 and move quickly to the more accessible and, to many people, more interesting chapters later in the book which describe the many ways in which fungi impact human existence. However, I don’t think I am overstating the case if I say that unless you understand something about how the main groups of fungi differ in morphology and behaviour, you will not be able to make much sense of the more ‘relevant’ sections of the book. If you can develop a sort of ‘cognitive map’ of the main groups, recognize them on sight, and understand the unique abilities of each, you will find the study of fungi—mycology—infinitely more rewarding.

A New Phylogenetic Classification of True Fungi—2007 This new system has just been completed. It is based on all available information, but especially on molecular data. It is contained in a study that appeared in the journal Mycologia titled “A Higher-Level Phylogenetic Classification of the Fungi” by no fewer than sixty-six authors (Hibbett et al. 2007). Note that it does not deal with the Oomycetes nor with the various types of slime moulds, none of which are true fungi. I am adding it here as a preliminary to reorganizing the first several chapters of this book. Please consult these tables so that you can place the taxa discussed later in their ‘correct’ places. This first table deals with the six older and simpler groups of true fungi, including the Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Zygomycota, Glomeromycota, and Microsporidia.

Chapter 1 The second table covers the Basidiomycota, including rust and smut fungi. The third table covers the Ascomycota, including almost all lichens.

MICROSPORIDIA Kickxellales Dimargaritales Harpellales Asellariales Zoopagales Entomophthorales Blastocladiales Mucorales Endogonales Mortierellales

Kickxellomycotina Zoopagomycotina Entomophthoromycotina BLASTOCLADIOMYCOTA Blastocladiomycetes Mucoromycotina

NEOCALLIMASTIGOMYCOTA Neocallimastigomycetes Monoblepharidales Monoblepharidomycetes Chytridiales CHYTRIDIOMYCOTA Spizellomycetales Chytridiomycetes Rhizophydiales Neocallimastigales

Archaeosporales Diversisporales Glomerales Paraglomerales

GLOMEROMYCOTA Glomeromycetes

BASIDIOMYCOTA DIKARYA ASCOMYCOTA

Traditional Zygomycota Table 1.1

Traditional Chytridiomycota

Kingdoms, Classification, Nomenclature, and Biodiversity Wallemiales, Wallemiomycetes Entorrhizales, Entorrhizomycetes Classiculales, Classiculomycetes Cryptomycocolacales, Cryptomycocolacomycetes Mixiales, Mixiomycetes Atractiellales, Atractiellomycetes Spiculogloeales Agaricostilbomycetes Agaricostilbales Cystobasidiales Cystobasidiomycetes Erythrobasidiales Naohideales Helicobasidiales Platygloeales Septobasidiales Pucciniomycetes Pucciniales Pachnocybales Heterogastridiales Leucosporidiales Microbotryomycetes Microbotryales Sporidiobolales Urocystales Ustilaginomycetes Ustilaginales Malasseziales Doassansiales Entylomatales Exobasidiales Exobasidiomycetes Georgefischeriales Microstromatales Tilletiales Cystofilobasidiales Tremellomycetes Filobasidiales Tremellales Dacrymycetales, Dacrymycetes Auriculariales Sebacinales Agaricomycetes Cantharellales Trechisporales Geastrales Gomphales Phallomycetidae Hysterangiales Phallales Hymenochaetales Corticiales Gloeophyllales Polyporales Thelephorales Russulales Agaricales Atheliales Boletales

Table 1.2

Agaricomycetidae

Pucciniomycotina

Ustilaginomycotina

Agaricomycotina

Chapter 1 Neolectales, Neolectomycetes Pneumocystidales, Pneumocystidomycetes Taphrinomycotina Schizosaccharomycetales, Schizosaccharomycetes Taphrinales, Taphrinomycetes Saccharomycotina Saccharomycelales, Saccharomycetes Orbiliales, Orbiliomycetes Pezizales, Pezizomycetes Lahmiales Pezizomycotina Medeolariales Triblidiales Capnodiales Dothideomycetidae Dothideales Myrianglales Pleosporales, Pleosporomycetidae Dothideomycetes Botryosphaeriales Hysteriales Patellariales Jahnulales Arthoniales, Arthoniomycetes Chaetothyriales Chaetothyrlomycetidae Pyrenulales Verrucariales Mycocaliciales, Mycocaliciomycetidae Eurotiales Eurotiomycetidae Onygenales Coryneliales Laboulbeniales Pyxidiophorales Lichinales, Lichinomycetes Acarosporales, Acarosporomycetidae Candelariales Umbilicariales Lecanorales Peltigerales Lecanoromycetidae Teloschistales Agyriales Baeomycetales Ostropales Pertusariales

Laboulbeniomycetes

Lecanoromycetes

Ostropomycetidae

Cyttariales Erysiphales Helotiales Rhytismatales Thelebolales Calosphaeriales Lulworthiales Meliolales Phyllachorales Trichosphaeriales Xylariales, Xylariomycetidae Coronophorales Hypocreales Hypocreomycetidae Microascales Melanosporales Boliniales Chaetosphaeriales Coniochaetales Sordariomycetidae Diaporthales Ophiostomatales Sordariales

Table 1.3

Eurotiomycetes

Leotiomycetes

Sordariomycetes

Kingdoms, Classification, Nomenclature, and Biodiversity

Biological Nomenclature Every species of living organism is a collection of individuals which are very similar (genetically, if not always in appearance), and each species has a unique name made up of two words, which may actually be from the ancient Latin lexicon but are far more often new, pseudo-Latin words coined for the occasion. This two-epithet name is the binomial. You and I belong to the species hom*o sapiens. The supermarket mushroom belongs to the species Agaricus brunnescens. In each case, the first of these two Latin words is the generic name or epithet (this places the organism in a genus, a collection of similar and/or related species). The second Latin word is the epithet applied to one particular species of the genus. But notice that the name of the species always consists of both epithets together. This is because only the two-word combination is actually unique to that species. The generic epithet is shared by all other species in that genus. The same species epithet may also be applied to species in other genera (for example, many Canadian spring flowers, although belonging to different genera, have the same species epithet, canadensis, as does the national animal of Canada, the beaver). Therefore, remember that only the two epithets together—the binomial—properly specify a species. hom*o sapiens is the only extant species in the genus hom*o, but most genera contain more than one species, and some, for example, the mushroom genus Cortinarius, are made up of hundreds of species. For purposes of classification, which is actually a method of information storage and retrieval, related genera are grouped into families, families are grouped into orders, orders into classes, classes into phyla, and phyla into kingdoms. Here is a sample of how an organism is classified in this hierarchical (boxes within boxes) system. Note that the binomial is in italics, as it is in most scientific publications, while the names of higher-ranking taxa (families, orders, etc.) are not in italics. If you refer to a binomial in writing, it should be underlined to show that it would be printed in italics. Kingdom: EUMYCOTA or FUNGI Phylum: Basidiomycota Subphylum: Basidiomycotina Class: Agaricomycetes Order: Agaricales Family: Agaricaceae Genus: Agaricus Species: Agaricus brunnescens—the edible (supermarket) mushroom. Why do we use this binomial nomenclature, which is so unfamiliar to the man or woman in the street? Why not use common names wherever they exist? For three good reasons: (1) The common names of many organisms differ from country to country, and even from district to district. (2) The same common name is sometimes applied to different organisms— for example, the British, North American, and Australian ‘Robins’. (3) Common names can be downright misleading: Irish moss is a red alga, Spanish moss is a flowering plant, clubmoss is a fern ally, and reindeer moss is a lichen.

Chapter 1 Let’s face it, common names are too unreliable and confusing to be of any use to scientists, who rely heavily on international communication and cooperation. Please take the time to learn the proper scientific names of the more important organisms you encounter in these pages. If you ever want to know more about any of them, you’ll find that their binomials are the key to almost everything that has been written about them. Having said that, I must admit that I can occasionally remember the common name of an organism when I can’t pull the scientific name out of my memory banks (this is increasingly true with advancing years; as of November 2016, I am almost eighty-three). Google and Wikipedia are wonderful memory aids which I (and I am sure all of you) use often. Many identification manuals list common names, and, therefore, they can in times of forgetfulness be one way to get at the scientific name. But that’s as far as I am willing to go, unless you have to give a talk to nonscientists or small children, when common names are all they will be able or willing to accept. ‘But why Latin or pseudo-Latin binomials?’ I can hear you saying plaintively. That’s easy too: (1) Latin is officially a dead language, and although scientists do coin new words, the grammar, vocabulary, and usage will change much more slowly than those of all living languages. In a changing world, we need the relative stability of Latin for our scientific names. (2) The use of Latin for names and, until very recently, diagnoses of all new taxa also means that no one can be offended by being forced to use someone else’s language. Latin has again become a useful international standard. I do have one problem with the Latin terminology, and it is that the same generic names can be used in different kingdoms. For example, there is a hyphomycete genus called Graphium (an asexual phase of an ascomycete—see picture, below, left, and chapter 4). There is also a genus of butterflies called Graphium (below, right). I do not think that this is necessary or reasonable, and I personally would disallow it—especially since the affiliations of kingdoms may change: the fungi are now considered quite closely allied with the animals, rather than the plants.

Fig. 1.2

Two different examples of Graphium.

It is unlikely that anything will be done about this in the near future, but you should be aware of the problem.

Kingdoms, Classification, Nomenclature, and Biodiversity

Biodiversity Different strokes for different folks. For some people, place names are evocative, calling up vivid memories of past experiences. For me it is the names of organisms I have seen that bring back those experiences. Metrosideros excelsa conjures up Christmas in Auckland, New Zealand, where the red flowers of this tree adorn beaches in December. Protea cynaroides takes me back to the amazingly rich Fynbos plant community in South Africa, where this flamboyant shrub flourishes, and has been adopted as the national flower. Amphiprion places me on the Barrier Reef in Australia, where these agile little damselfish with blue-white stripes live unscathed among the tentacles of large sea anemones. Such organisms and the communities and ecosystems of which they are a part are the real reasons I leave home. I hope that some of you will also come to think of the biodiversity you encounter as a measure of your quality of life. Biodiversity was one of the buzzwords of the nineties and has continued to be a newsmaker in the new millennium. Biologists know from the earliest years of their training that the Earth is blessed with an amazing number of different living things. As you have just read, systematists have tried to catalogue and describe all these riches, but with widely varying degrees of success. We know practically all the birds (about 8,000 species), and almost all the mammals, that inhabit the Earth. But we know only a small fraction of the arthropods and fungi. How can I make those two statements so confidently? Because although there are many ornithologists and mammalogists scouring the globe for new taxa, they rarely find any. At the other end of the scale, entomologists and mycologists find new taxa every day. They have so far described approximately a million insects and about 100,000 fungi, but it is obvious to the professionals who work in these areas that huge numbers of both groups have not yet been described (and new sequencing techniques are revealing thousands of new taxa in many habitats). I began my mycological career examining the sequence of fungi involved in the slow decomposition of Scots pine (Pinus sylvestris) needles. Almost immediately, I found several microscopic fungi which turned out to be new to science, and I had the privilege of describing these fungi (you can see some of them in chapter 11 of www.mycolog.com). Since I moved to Vancouver Island, off the west coast of Canada, in 1994, I have seen many mushrooms that could not be identified using the existing literature. I am convinced that many of these fungi are actually undescribed. How many fungi are waiting to be discovered? David Hawksworth came up with an ingenious answer a few years ago. Noting that Britain is among the most intensively investigated areas on Earth for plants and for fungi, he pointed out that almost all the flowering plants in Britain are known and that there are about 2,000 species. Although the fungi of Britain are definitely not as completely known, as new ones are still being described, about 12,000 species have been recorded there. This gives a ratio of about six fungi to each plant species. Extrapolating (perhaps rather ambitiously) from Britain to the entire globe, Hawksworth suggested that because there appears to be about 250,000 species of flowering plants in the world, there are probably six times as many fungi—about 1,500,000 fungi, in fact. Even if this figure is an overestimate, and there are only half a million fungi, we have still described only 20% of the total, and a huge task lies before us.

Chapter 1 If we accept this figure as a working approximation (and no one has yet come up with a different formula), it brings us to a realization that about two centuries of mycology have so far succeeded in describing only about 6%–7% of the world’s mycota—a pretty shocking state of affairs. Over the years, I have been involved in describing probably 300 fungal taxa and have now basically run out of steam in this area of mycology. But even if all the mycologists alive today were to publish 100 species apiece, they would still manage to describe only about 300,000 taxa. Thus, it is a multigenerational task. The preceding discussion assumes a steady state in which no species are added, and none are subtracted, from the global total. However, we know that this is not the case. New species are constantly arising, albeit at an unknown rate, as a result of the combined effects of selection pressure and genetic recombination. Our own species, by sticking its fingers into every existing niche and ecosystem, as well as creating new ones, is undoubtedly providing the fungi with new challenges at every turn, and they are surely responding to those challenges by spawning new taxa. Now we come to the tragic bit. Human activities are undoubtedly driving some fungi into extinction. We don’t know which or how many are being lost, and it is absurd for anyone to suggest that we are losing two species each week or twenty each day. Information on extinctions is extremely hard to obtain. How can you tell when a particular microscopic fungus, which can be detected only by culturing the soil, or a macroscopic fungus that may fruit only once in twenty years, has finally succumbed? Nevertheless, we have good reason to suspect that these things are happening. For example, the huge fruit bodies of Bridgeoporus (Oxyporus) nobilissimus, a long-lived bracket fungus that grows only in old-growth forests on the west coast of North America (see chapter 5), are seen less and less frequently, and, along with the forests in which it lives, this species may certainly be considered endangered. One of our problems in North America is that we do not have extensive records of the mycota from the past—a baseline with which present-day comparisons can be made. Fortunately, some European countries, with centuries of data collection to draw upon, have been able to document the decline in numbers of many fungi and have published what they call Red Lists. These lists highlight the increasing rarity of many fungi and the apparent disappearance of some. What does a red-bellied toad from North Vietnam have to do with preserving fungal diversity? Read on. Dr. Bob Murphy, then director of the Royal Ontario Museum’s Centre for Biodiversity and Conservation Biology, reported in the Globe and Mail for July 12, 1999, that he had just returned from collecting herpetiles (amphibians and reptiles) in the North Vietnam rainforest. His comments about his animals are worth repeating here: ‘Because they’re beautiful, aesthetically appealing, wonderful animals, you can get them protected. If you can get them protected, you protect the forest. If you protect the forest, you protect everything that’s there, especially the fungi [my italics], which are producing the majority of new pharmaceuticals that are coming out’. You will find an echo of that statement in chapter 24, in the discussion of antibiotics, immunosuppressants, and other fungal metabolites. Therefore, our efforts to collect and describe the world’s mycota need to be redoubled. As you will learn from many of the other chapters in this book, and perhaps most accessibly from chapter 24, many fungi confer enormous benefits to mankind. I am

Kingdoms, Classification, Nomenclature, and Biodiversity not referring just to the producers of antibiotics such as penicillin and immunosuppressants such as cyclosporine but also to the myriad species which recycle organic matter, especially plant debris, and to the others that establish obligate mutualistic symbioses with many of our most important plants. This awareness of our ignorance has led to proposals to compile what are called alltaxa biodiversity inventories, or ATBI for short. A meeting to discuss such an ATBI for a forested area in Costa Rica came up with a figure of $20 million for the fungi alone. Although the Costa Rican venture didn’t fly, it spawned a marginally less ambitious project for the Great Smoky Mountains National Park in Tennessee. Setting other organisms aside, it has been estimated that there are 20,000 fungi in the park, of which only 2,250 have so far been described. (Hey, that’s more than 10%, so we are already ahead of the game.) A two-year pilot project aimed at refining sampling methods and data protocols was begun in March 1999, and mycologists everywhere will be watching with interest (or being co-opted) as this unfolds. To learn more about this project, visit the website http://www.discoverlife.org/ATBI_brochure.html.

Less Than 10% of the Park’s Estimated 100,000 Species Are Currently Known

Known Species 9,800

Fig. 1.3 Estimated Species >100,000

Vertebrates

Estimated total in park: 475 Number currently known: 450

Fungi

Estimated total in park: 20,000 Number currently known: 2,250

Plants

Estimated total in park: 5,400 Number currently known: 2,816

Invertebrates

Estimated total in park: 76,000 Number currently known: 4,280

(includes protists)

Chapter 1

Further Reading on Classification and Biodiversity Ainsworth, G. C. 1976. Introduction to the History of Mycology. Cambridge, U.K.: Cambridge University Press. Carlile, M. J., S. C. Watkinson, and G. W. Gooday. 2001. The Fungi. 2nd ed. New York: Academic Press. Cavalier-Smith, T. 2001. “What Are Fungi?” In The Mycota, edited by D. J. McLaughlin, E. G. McLaughlin, and P. A. Lemke, 3–37, vol. 7A. Berlin: Springer. Crowson, R. A. 1970. Classification and Biology. London: Heinemann. Dawkins, R. 1995. River out of Eden: A Darwinian View of Life. New York: Harper-Collins [Paperback ed. 1996 W.W. Norton]. Hawksworth, D. L. 1991. “The Fungal Dimension of Biodiversity: Magnitude, Significance and Conservation.” Mycological Research 95:641–55. ———. 2004. “Fungal Diversity and Its Implications for Genetic Resource Collections.” Studies in Mycology 50:9–18. Heckman, D. S., D. M. Geiser, B. R. Eidell, R. L. Stauffer, N. L. Kardos, and S. B. Hedges. 2001. “Molecular Evidence for the Early Colonization of Land by Fungi and Plants.” Science 293:1129–33. Hibbett, David S., Manfred Binder, Joseph F. Bischoff, Meredith Blackwell, Paul F. Cannon, Ove E. Eriksson, Sabine Huhndorf, et al. 2007. “A Higher-Level Phylogenetic Classification of the Fungi.” Mycological Research 111:509–47. James, T. Y., P. M. Letcher, J. E. Longcore, S. E. Mozley-Standridge, D. Porter, M. J. Powell, G. W. Griffith, and R. Vilgalys. 2006. “A Molecular Phylogeny of the Flagellated Fungi (Chytridiomycota) and Description of a New Phylum (Blastocladiomycota).” Mycologia 98:860–71. doi: 10.3852. Jaques, H. E. 1946. Living Things: How to Know Them. Dubuque, IA: Wm. C. Brown. Jones, M. D. M., I. Forn, C. Gadelha, M. J. Egan, D. Bass, R. Massana, and T. A. Richards. 2011. “Discovery of Novel Intermediate Forms Redefines the Fungal Tree of Life.” Nature 474:200– 203. doi: 10.1038/nature09984 [sv. Phylum Cryptomycota]. Kendrick, B. 1981. “The History of Conidial Fungi.” In Biology of Conidial Fungi, edited by G. T. Cole and B. Kendrick, 3–18, vol. 1. New York: Academic Press. Kendrick, B., and T. R. Nag Raj. 1979. “Morphological Terminology in Fungi Imperfecti.” In The Whole Fungus, edited by B. Kendrick, 43–62, vol. 1. Ottawa, Canada: Ottawa National Museum of Natural Sciences. Kirk, P. M., P. F. Cannon, J. C. David, and J. A. Stalpers, eds. 2001. Ainsworth and Bisby’s Dictionary of the Fungi, 9th ed. Egham, UK: CABI Bioscience, UK Centre. Lucking, R., S. Huhndorf, D. H. Pfister, E. R. Plata, and H. T. Lumbsch. 2009. “Fungi Evolved Right on Track.” Mycologia 101:810–22. Margulis, L., and R. Guerrero. 1991. “Kingdoms in Turmoil.” New Scientist, March 23, 46–50. Margulis, L., and K. V. Schwartz. 1982. Five Kingdoms. San Francisco: Freeman. Miller, O. K. Jr., T. Henkel, T. Y. James, and S. L. Miller. 2001. “Pseudotulostoma, a Remarkable New Volvate Genus in the Elaphomycetaceae from Guyana.” Mycological Research 105:1268–72. Mueller, G., G. Bills, and M. Foster, eds. 2004. Biodiversity of Fungi. Inventory and Monitoring Methods. Amsterdam: Elsevier/Academic Press.

Kingdoms, Classification, Nomenclature, and Biodiversity Orpin, C. G. 1975. “Studies on the Rumen Flagellate Neocallimastix frontalis J. Gen.” Microbiology 91:249–62. Patterson, D. J., and M. L. Sogin. 1992. “Eukaryote Origins and Protistan Diversity.” In The Origin and Evolution of Prokaryotic and Eukaryotic Cells, edited by H. Hartman and K. Matsuno. 13–46 New Jersey: World Scientific Pub. Co. Rogers, D. P. 1978. A Brief History of Mycology in North America. In “Proceedings of the Second International Mycological Congress,” edited by E. G. Simmons, 253–65. Tampa: University of South Florida. Ross, H. H. 1974. Biological Systematics. Reading, PA: Addison Wesley. Taylor, T. N., M. Krings, and H. Kerp. 2006. “Hassiella monospora gen. et sp. nov., a Microfungus from the 400 Million Year Old Rhynie Chert.” Mycological Research 110:628–32. Whittaker, R. H. 1969. “New Concepts of Kingdoms of Organisms.” Science 163:150–60. Woese, C. R., O. Kandler, and M. L. Wheelis. 1990. “Towards a Natural System of Organisms: Proposal for the Domains Archaea, Bacteria, and Eucarya.” Proceedings of the National Academy of Science (PNAS) 87:4576–79.

2 A Mixed Bag Protozoan ‘Pseudofungi’—Kingdom Protozoa (the So-Called Slime Moulds—Phyla Myxostelida, Dictyostelida, Labyrinthulida, Plasmodiophorida) Pseudofungi—Kingdom Chromista: Phyla Hyphochytriomycota and Oomycota True Fungi—Kingdom Eumycota: Phylum 1 Chytridiomycota, Phylum 2 Blastocladiomycota, Phylum 3 Neocallimastigomycota First, let me elaborate a little on the definition of fungi given earlier. Fungi (whether chromistan or eumycotan) are heterotrophic (nonphotosynthesizing) eukaryotes that absorb their food (osmotrophic), typically at the many growing points of their rather diffuse, indefinite ‘body’ (often called a thallus or mycelium), which is made up of fine branching tubes called hyphae. The wall of the tubes is mainly composed of chitin (Eumycota) or cellulose (Oomycota), which offer protection from the outside world, and within this wall, the cytoplasm and nuclei live and move and are able to explore small areas of it (the humongous fungus [see chapter 11] manages fairly large areas) inside their apically extending, microscopic, hyphal tunnels and much larger areas by means of their detachable reproductive units, called spores. Before I discuss real fungi, four ‘outsider’ phyla must be mentioned. These are the so-called slime moulds, the inappropriately named Myxomycota, Acrasiomycota, Labyrinthulomycota, and Plasmodiophoromycota. They’re slimy, but they’re not mouldy! Although they used to be thought of as fungi, and still appear in some current mycological literature, this is at least phylogenetically incorrect. Here’s why. The assimilative or somatic phases of three of these four groups are basically amoeboid, where none of the four ever produces hyphae (a diagnostic feature of most true fungi), and the assimilative plasmodia have no cell walls. The names currently applied to some of these groups are misleading, in that they imply a fungal nature, so in three cases, I have supplied new names reflecting their nonfungal affiliations. I hope you will agree with me after you have read the thumbnail sketches below and compared them with my later descriptions of the wall-possessing hyphal fungi. These phyla are included in some mycology courses because some of them (particularly Myxostelida) tend to 16

A Mixed Bag turn up when we look for fungi. If mycologists, who have historically looked after them, abandon them, what other group of organismic biologists will agree to add these organisms to their already crowded course schedules?

Phylum Myxostelida (Formerly Myxomycota or Mycetozoa) This is the only one of these four nonfungal phyla you are likely to find if you go out looking for fungi in autumn. The macroscopic, slimy, amoeboid plasmodium (the somatic or assimilative phase of the organism) is populated by diploid nuclei and oozes around in the soil or in decaying wood or other organic matter, eating (yes, actually engulfing or ingesting) bacteria and other tiny food particles. You can keep the plasmodia of some myxostelids as pets, crawling around in petri dishes and eating oldfashioned rolled oats. Eventually, the slowly pulsating plasmodium will dramatically metamorphose into a cluster of stalked, dry sporangia full of powdery resting spores. Spore formation involves reduction division (meiosis), so when each haploid spore germinates to release a cell that may be either amoeboid or biflagellate, this cell can act as a gamete. Fusion of two compatible gametes produces a diploid zygote which can then fuse or aggregate with other zygotes, begin to feed, and eventually grow into a plasmodium once more. A typical myxostelid life cycle is shown in Fig. 2.1.

Fig. 2.1

Myxostelida: life cycle of Stemonitis fusca.

Chapter 2

Phylum Dictyostelida (Formerly Acrasiomycota) In the assimilative phase, Dictyostelium discoideum consists of independent, individual amoebae which feed by ingesting bacteria. The reproductive phase begins when the amoebae begin to secrete cyclic adenosine monophosphate (cAMP), a type of pheromone which causes the amoebae to be mutually attracted. They stream together and form aggregations called pseudoplasmodia, or ‘slugs’, and these differ from true plasmodia in that each amoeba retains its cell membrane. Each slug crawls around until dry conditions prompt it to undergo differentiation, heaping itself up and eventually forming a structure called a sorocarp, which has a slim cellulosic stalk and an expanded head containing spores. Dictyostelium has been used as an experimental organism by many scientists because it provides a simple system for studying differentiation (Fig. 2.2).

Fig. 2.2 Dictyostelida: life cycle of Dictyostelium discoideum.

A Mixed Bag

Phylum Labyrinthulida (Formerly Labyrinthulomycota) The colourless colonies of Labyrinthula cause a wasting disease of eel grass (Zostera, one of the few marine flowering plants). The spindle-shaped, naked cells of the colony live and move entirely within a network of narrow, tubular, polysaccharide sheaths which they themselves secrete. They release biflagellate gametes, and the zygote divides mitotically to generate a new colony, whose cells are presumably diploid. Most other members of this group are also marine-parasitizing algae (Fig. 2.3).

Fig. 2.3 Labyrinthulida: Labyrinthula.

Phylum Plasmodiophorida (Formerly Plasmodiophoromycota) All members of this group are obligate parasites. Plasmodiophora brassicae produces uninucleate, biflagellate primary zoospores which penetrate a root hair of its host, cabbage (Brassica oleracea). Inside, they grow into multinucleate but still microscopic primary plasmodia. These eventually develop a wall and divide internally into uninucleate secondary sporangia. These germinate, releasing four secondary, biflagellate zoospores which leave the host. These may also act as gametes, fusing in pairs, but soon infect a root again, developing within host cells into multinucleate secondary plasmodia. At maturity, these can cleave into uninucleate cysts, each containing a single spore, which can persist in the soil for many years. This parasite stimulates the cabbage roots to become grossly swollen, a serious disease condition known as ‘club root’ (Fig. 2.4).

Chapter 2

Fig. 2.4 Plasmodiophorida: life cycle of Plasmodiophora brassicae.

Kingdom Chromista Phylum 1: Hyphochytriomycota This group is like the Chytridiomycota in many ways: they live in fresh water or soil; they can be parasites or saprobes; they may be holocarpic or eucarpic, the latter kind having assimilative rhizoids; and the sporangia release uniflagellate zoospores. So why aren’t they Chytridiomycetes? A single characteristic visible under the light microscope gave the original clue and was soon supplemented by several ultrastructural features accessible only to the electron microscope (Fig. 2.5). The ‘visible’ feature concerns the behaviour of the flagellum on the zoospore. In Chytridiomycetes, this propels the spore from the rear, but in hyphochytriomycetes, the flagellum is seen to be attached at the front of the cell, where it works like a swimmer’s arm or an oar. Other

A Mixed Bag differences are revealed by the transmission electron microscope. In Chytridiomycota, the axis of the flagellum is smooth (a whiplash flagellum), while in the Hyphochytriomycota, the axis of the flagellum bears many fine lateral filaments called mastigonemes or flimmers that give it the name tinsel flagellum. These may appear to be insignificant differences, but biologists consider the numbers and kinds of flagella on zoospores to be an extremely conservative characteristic—one likely to remain unchanged over vast stretches of time, perhaps even hundreds of millions of years. This makes it an important indicator, which is amply confirmed by the ultrastructural differences illustrated above, and justifies the recognition of phylum Hyphochytriomycota. There are few well-documented species in this phylum, but Hyphochytrium catenoides is common in

Fig. 2.5 Ultrastructure of fungal zoospores: er, endoplasmic reticulum; F, flagellum; G, Golgi apparatus; K, kinetosome; L, lipid; M, mitochondrion; m, microbody; mn, mastigoneme; mt, microtubules; N, nucleus; NC, nuclear cap (ribosomes); nfc, nonfunctional centrioles; Nu, nucleolus; R, ribosomes; Ro, rootlet; Ru, rumposome; SD, striated disc.

Chapter 2 soil and is often (like the chytrids Spizellomyces and Chytridium) found in pollen when this is added to soil as ‘bait’ in the laboratory. This species may be of some importance in the natural biological control of plant-pathogenic Oomycota (see below), by parasitizing their oospores. No hyphochytrid has yet been seen to sexually reproduce.

Fig 2.6

Asexual reproduction in Oomycota. A: Saprolegniales; B–D: Peronosporales.

A Mixed Bag

Kingdom Chromista Phylum 2: Oomycota Having just read about the minute but basic differences between the two previous groups, you won’t be surprised to learn that similar inconspicuous features distinguish the Oomycota, our last and most important group of chromistan fungi. Basic features: (1) Oomycetous zoospores have two flagella: one tinsel, one whiplash (Fig. 2.6A), and these arise from the side of the cell rather than at the front or the rear, as in other groups. The zoospores swim with their tinsel flagellum pointing forward, while the whiplash flagellum is directed backward. (2) Unlike the nuclei of all true fungi, those in the assimilative hyphae of oomycetes are diploid. (3) The walls of the hyphae usually contain a cellulose-like material (a poorly crystallized hexose polymer), although this makes up only a fraction of the cell wall, and chitin has also been found in some oomycetes. All of these characteristics separate them from the eumycotan fungi. But the feature that gives the group its name is its oogamous sexual reproduction and the fact that each zygote develops into a thickwalled, persistent oospore (Fig. 2.7A, B). Note, however, that fungal oogamy is not unique to the Oomycota—some Chytridiomycota such as Monoblepharis do it, too.

A Fig 2.7

B Sexual reproduction in Oomycota. A: Saprolegniales; B: Peronosporales.

Chytridiomycota and Hyphochytriomycota are often extremely inconspicuous, as befits organisms that in some cases devote an (albeit brief) lifetime to exploiting a single pollen grain. Oomycota, although they also include some holocarpic and eucarpic unicells, often produce extensive hyphal networks (mycelia). These fungi, covering a dead fish like a whitish fur coat (as I saw on the dead salmon in the Adams River in 2010), or devastating crops such as grapes, hops, lettuce, cabbage, radishes, potatoes, and tobacco, have spawned a number of common names—water moulds and white rusts, downy mildews, and damping-off.

Chapter 2

Fig 2.8

Saprolegniales: life cycle of Saprolegnia.

Order Saprolegniales. The water mould, Saprolegnia parasitica, attacks fish and their eggs. After establishing itself, the fungus reproduces asexually. The tips of the normally nonseptate hyphae become modified into long mitosporangia (Fig. 2.8) delimited by a cross-wall at the base. These asexual sporangia release biflagellate zoospores, often called swarm spores, which actively swim for a while and then encyst;

A Mixed Bag this means that they stop swimming and develop a thick wall. Later, they germinate again as secondary zoospores which, if they are lucky, will find a new substrate and develop into new assimilative thalli. This process of encystment, followed by a repetition of germination, is a strategy that gives the spores a second chance at finding food if they aren’t so lucky when they are first released. Many Saprolegnia and Achlya species form compatible antheridia and oogonia on the same mycelium, which means that they are hom*othallic. Since the assimilative thallus is diploid, meiosis must take place inside the gametangia. Each globose oogonium contains several eggs (Fig. 2.7A). A number of antheridia may grow toward and touch a single oogonium, penetrating its wall at preformed thin spots and sending in fertilization tubes which deliver the male nuclei to the eggs. Fertilization is more reliable because neither gamete is exposed to the vagaries of a free-swimming existence. The whole double life cycle is illustrated in Fig. 2.8. The zygotes develop thick, resistant walls and obviously function as survival spores that can live through such catastrophes as the drying up of a pond or stream. hom*othallic species have lost the enhanced variation provided by outbreeding, but they still benefit from the thick-walled resting oospores produced by the sexual cycle. Order Peronosporales. Many members of this order are obligately parasitic on higher plants. In some cases, they cause epidemics that devastate important crops. The buildup of these epidemics is made possible (1) by our need to grow dense stands of single plant species (monoculture crops), and (2) by aerial transmission of the fungi, which have evolved airborne mitosporangia (Fig. 2.6B, C). Note that these are often wrongly called conidia, as they are analogous but not hom*ologous to those spores (which are discussed under the subkingdom Dikarya). Oogonia are also formed, each containing a single egg (Fig. 2.7B). Sexual reproduction is usually hom*othallic (the antheridium arises from the same thallus as the oogonium). We will examine representatives of five genera from three families, Pythiaceae (Pythium, Phytophthora), Peronosporaceae (Peronospora, Plasmopara), and Albuginaceae (Albugo).

Damping-off disease of seedlings (Pythium—Pythiaceae) This is a soilborne disease, so its causal agents, species of Pythium, have no need for airborne sporangia, since they persist saprobically in most soils and spread by zoospores during wet conditions. When these motile cells find young plants, they cause infections which release toxins and also produce a pectinase enzyme which dissolves the middle lamella, which glues plant cells together. Seedlings of many plants collapse rapidly when this disease strikes at the base of their delicate shoots. Damping-off is, unhappily, familiar to gardeners who try to get a head start on the growing season by germinating seeds indoors. The disease can be controlled by using heat-sterilized soil, by dusting seeds with Benomyl (a very safe fungicide, although now delisted; see chapter 13), or by watering seedlings with other fungicides such as zineb or No Damp Off.

Chapter 2

Fig 2.9

Peronosporales: life cycle of Pythium.

A Mixed Bag

Late blight of potato (Phytophthora—Pythiaceae) In the mid-nineteenth century, a North American oomycete caused havoc in Europe. Unintentionally imported to Ireland and reintroduced to a susceptible host from which it had been separated 250 years earlier, Phytophthora infestans wiped out the Irish potato crop in the damp, cool summers of the years 1845–1847, causing widespread famine in that poor, one-crop economy. At first, the disease was not attributed to the fungus but to an excess of water in the plants, or even to the effects of the newly introduced steam locomotives. However, the Reverend M.J. Berkeley, England’s leading mycologist, drew sporangiophores emerging from the potato leaves and correctly concluded that the fungus caused the disease. His contemporaries eventually admitted that he was right, and this episode led to the founding of the discipline now known as ‘plant pathology’. The ravages of potato blight contributed to a million deaths and drove millions more to emigrate from Ireland. Ten years after the first epidemic, the population of Ireland had crashed from 8 million to 4 million. Contemporary pictures from the London Illustrated News convey some of the misery caused by the recurring epidemics. Ragged and starving peasant girls gleaned desperately in the fields for anything edible, and whole families were forced to leave their homes forever. Some died. Many sailed for North America, but 187 doubly unfortunate souls were shipwrecked and drowned off Forillon National Park near the tip of the Gaspé Peninsula, Quebec—in sight of the promised land. There are websites that explore the potato famine, easily found on Google. Until 1976, this heterothallic pathogen (except in its homeland, Mexico) was asexual, representing only one of the two mating types. In that dry year, many crop failures led to importation of potatoes that carried the other mating type with them. It has also been suggested that new genotypes were spread in seed tubers, in tomato tissues, and even in tropical storm systems. However it came about, new sexual populations have certainly supplanted the older asexual strain and have led to a resurgence of the disease, which now costs the United States alone about $3 billion per year in attempts at control by spraying fungicides and by trying to breed resistant varieties of potato. The airborne sporangium of the potato blight fungus is clearly an efficient shortrange dispersal mechanism, although humans were just as clearly the long-range vectors of this disease. Potato blight is still a threat, although it can be controlled by (1) spraying with fungicide at times carefully chosen by plant pathologists (nowadays with the aid of special blight-forecasting computer programs) (2) destroying infected foliage before harvest, and (3) planting disease-resistant seed potatoes. In addition to its most infamous species, the genus Phytophthora contains sixty other species. Some of them are also, as one might suspect, serious plant pathogens. Phytophthora sojae plagues soybean farmers in North America. Phytophthora megakarya attacks cacao trees in West Africa. A hybrid between P. cambivora and P. fragariae has killed about 10% of the Alder trees (Alnus spp.) in Britain and is now spreading to France, the Netherlands, Sweden, Germany, and Austria. Phytophthora cinnamomi is destroying the Jarrah (Eucalyptus) forest and other natural ecosystems in western

Chapter 2 Australia and cork oak forests in Spain, and it is a serious problem for growers of avocados, pineapples, and ericaceous shrubs elsewhere in the world. No wonder this genus provides work for hundreds of plant pathologists and mycologists worldwide.

Downy mildew diseases (Plasmopara, Peronospora—Peronosporaceae) The downy mildews include blue mould of tobacco and downy mildew of grape. Since these diseases have historical or economic importance, I’ll tell you something about them. In this group, the mitosporangia are no longer unspecialized hyphal tips, but are borne on highly differentiated, branched, aerial sporangiophores. The sporangia don’t only release zoospores but are themselves set free and blown or splashed away. The sporangia of Peronospora germinate by producing a hypha, although those of most other members of the group still release motile zoospores.

Downy mildew of grape (Plasmopara—Peronosporaceae) Plasmopara viticola, an oomycete native to America, causes downy mildew of grapes. It can be found attacking wild grapes every summer. But because it evolved along with its North American host, a biological balance has been struck, and the wild Vitis species aren’t seriously damaged. When this fungus was accidentally introduced to Europe in the 1870s, it was a different story. The French grape vines (Vitis vinifera) had no resistance to the new pathogen and were quickly devastated. Fortunately for the oenophiles (wine lovers) of the world, the concoction of Bordeaux Mixture, one of the world’s first practical fungicides, by a university professor (yes, we profs occasionally have good ideas!) saved the day. The rather strange story behind this invention is told in chapter 13. The life cycle of Plasmopara is illustrated in Fig. 2.10. Look at this set of diagrams carefully and decide at which stage you think it would be most vulnerable to chemical attack (here’s a clue: the answer begins with z).

Blue mould of tobacco (Peronospora—Peronosporaceae) My last example of a downy mildew fungus is Peronospora tabacina, which causes blue mould of tobacco. This disease was first recorded in Ontario in 1938, was epidemic in 1945–1947, and had not been seen since 1966. But in 1979, seedlings infected with the pathogen were imported to Canada from the United States, escaping detection at the border. The weather that year favoured the development and spread of the fungus, and a large-scale epidemic ensued. About 30% of the Ontario crop, worth $100 million, was lost. Blue mould helped to put the Ontario tobacco industry on a slippery slope, and the decline in tobacco acreage is still continuing, although it is now driven by changing societal attitudes toward smoking. Peronospora once again became a serious problem (a coffin nail?) for Ontario tobacco growers in 1997.

White rust disease of crucifers (Albugo—Albuginaceae) This disease attacks all members of the family Brassicaceae such as cabbage and radish and is caused by Albugo candida, which produces extensive white blisters on leaves and stems (see www.mycolog.com). These unique blisters contain innumerable unicellular

A Mixed Bag

Fig 2.10

Peronosporales: life cycle of Plasmopara viticola.

mitosporangia developing in chains from the tips of short, tightly packed sporangiophores (Fig. 2.6D). When the host epidermis bursts, the sporangia are dispersed by wind or rain splashes to other host plants, where each can germinate to release eight biflagellate zoospores. Oogonia develop later, inside the host stem or leaves, and sexual reproduction is usually heterothallic, or outbreeding.

Chapter 2

True Fungi—Kingdom Eumycota As I mentioned in chapter 1, what we call ‘fungi’ share many morphological and behavioural similarities in their assimilative phase, but they do not have a uniform genetic background. It now seems obvious that they have evolved from at least two ancestral lineages. Some fungi can produce cells that swim by means of one or two very fine whip-like extensions called flagella (hom*ologous to the tails of sperms). Five phyla—Hyphochytriomycota, Oomycota, Chytridiomycota, Blastocladiomycota, and Neocallimastigomycota—fall into this category. But molecular evidence tells us that they are members of two different kingdoms, the first two being chromistan in origin, and related to the brown algae, and the last three (despite their flagellate phase), eumycotan. The other six generally recognized true fungal phyla never have motile cells, and they—the Microsporidia, Zygomycota, Cryptomycota, Glomeromycota, Ascomycota, and Basidiomycota—make up the rest of kingdom Eumycota, although only the Ascomycota and Basidiomycota produce highly differentiated, multicellular reproductive structures, as you will see. Before I introduce you to the three flagellate eumycotan phyla, a few words about flagella (singular: flagellum). These are very long, narrow organelles, essentially contractile extensions of a cell, which have the ability to beat or make whip-like motions that confer motility to the cell. Flagella move the cell about in water, giving it the ability to swim up chemical gradients (that is, to move from a lower concentration of a substance toward a higher concentration), such as those which lead toward a sexual partner or a suitable host organism. The amazing thing about flagella is that wherever we find them among eukaryotic organisms, they have essentially the same fine structure, consisting of nine pairs of peripheral microtubules that run along the flagellum shaft and two central microtubules—the 9 + 2 pattern. Each microtubule is built from a protein called tubulin, which has subunits that are arranged in thirteen vertical stacks (count them in Fig. 2.11) around a hollow centre.

Fig 2.11 Transverse section of a typical 9 + 2 microtubule flagellum (diameter about 170 nm).

A Mixed Bag This image of a transverse section of a flagellum (which is about one-sixth [0.16] of a micron thick; 100,000 of them laid side by side would be less than an inch thick) would look more or less the same whether the flagellum came from one of the simple fungi discussed here or from a protozoan such as Paramoecium, which has lots of short flagella called cilia all over the outside of the cell, or from a unicellular or colonial green alga such as Chlamydomonas or Volvox, or a sea gooseberry (a member of phylum Ctenophora), which has plates of cilia that beat in rhythm and move the whole macroscopic organism, or from the sperm (male gamete) of a brown alga, a sea urchin, a moss, a fern, or a human. You see, all eukaryotes really are related! By the way, the flagella of bacteria are completely different, but that’s another story.

Kingdom Eumycota Phylum 1: Chytridiomycota The somatic phases of the microscopic Chytridiomycetes vary widely in appearance, but when they undergo asexual reproduction, most produce zoospores with a single, backwardly directed whiplash flagellum. I will discuss members of three orders: the Chytridiales, Spizellomycetales, and Monoblepharidales. Although these orders were formerly separated by the morphology of their microscopic thalli, we now know that this is too variable to be reliable. Emphasis has switched to ultrastructural features of the zoospore, some of which are illustrated in Fig. 2.5. Unfortunately, although conservative, and therefore taxonomically valuable, these characteristics can be seen only in the transmission electron microscope after elaborate preparative techniques. Fortunately, you can see enough of an Allomyces zoospore under the light microscope (using phase contrast optics) to identify the phylum (although some members of this phylum recently discovered in the rumen of large herbivorous mammals have many flagella on each cell). Orders Chytridiales and Spizellomycetales. These orders look very similar under the light microscope, and it takes an expert to tell them apart. However, most Chytridiales are aquatic, while most Spizellomycetales live in soil. Until important differences were found in the ultrastructure of their zoospores (Fig. 2.5), the two orders were considered to be one. These simple fungi do not produce hyphae. They are often parasitic, and their assimilative thallus often consists of a single cell. This cell is either (1) entirely converted into a reproductive sporangium (the holocarpic mode, Fig. 2.12B), as in Olpidium brassicae, or (2) differentiated into assimilative rhizoids and a sporangium (the eucarpic mode, Fig. 2.12A), as in Chytridium lagenaria or Spizellomyces punctatus. Other chytrids have a more extensive system of rhizoids, called a rhizomycelium, which may nourish several sporangia, as in Cladochytrium (Fig. 2.12C). We describe this multisporangial condition as polycentric to differentiate it from the monocentric forms just mentioned, which produce only a single sporangium. The difference between rhizoids and rhizomycelium is that rhizoids generally have no nuclei in them and are usually less than a millimetre long, while rhizomycelia contain nuclei and can be much more extensive.

Chapter 2 You might be interested in the activities of some of the members of these two orders, including the fungi just named. The eucarpic Spizellomyces punctatus and Chytridium lagenaria parasitize pollen grains. The polycentric Cladochytrium is saprobic, growing on decaying aquatic vegetation. At least one chytrid, Synchytrium endobioticum, is a serious problem for farmers because it causes wart disease of potato. This fungus produces dark brown, cauliflower-like growths on the tubers and a catastrophic reduction in yield. Fortunately, although the pathogen is widespread in Europe and has spread to Newfoundland, resistant varieties of potato help to keep the disease under control. Other microscopic chytrids parasitize algae (see the website) and can be so numerous as to cause epidemics which significantly, if temporarily, reduce primary production in lakes.

The Frog Problem Most recently, a eucarpic chytrid has been found attacking amphibia in many regions of the world. It has been associated with significant die-offs of frogs in Australia, in Central America, and at the National Zoo in Washington, D.C. The condition has been quite reasonably called chytridiomycosis since no other organisms are yet known to be involved. The fungus lives inside epidermal cells and causes thickening of the skin, which may interfere with normal respiration, as frogs breathe partly through their skins. Its zoospores have ultrastructural characteristics (the kind that can be seen only under a transmission electron microscope) that put it into a new genus, Batrachochytrium. Joyce Longcore, the chytrid taxonomist who determined this, published a short article in Inoculum, the newsletter of the Mycological Society of America, for October 1998 and gave illustrations of both the eucarpic thallus and the zoospores. The study describing the new genus appeared in Mycologia in April 1999, and the full reference is given at the end of this chapter. No one knows why this fungus would suddenly begin killing frogs in places as diverse as Australia and Panama. The fungus may have been transported to these places only recently, perhaps even on the boots or equipment of researchers studying the disappearance of frogs. It may be the case that the fungus was present for a long time, but frogs are now succumbing because their immune systems have been impaired by recent environmental changes. One obvious change is increased ultraviolet (UV) light, which is known to damage the immune systems of animals. Recently, chlorinated chemicals released by humans have attacked the ozone layer in the upper atmosphere, allowing 10% more UV light to reach the Earth’s surface. Industrial chemicals may also be damaging the frogs’ immune systems. Retinoids are under suspicion because they cause birth defects in many animals, including frogs and humans. Accutane, used to treat acne, is a retinoid known to cause birth defects in humans. If you are interested in pursuing this topic, I suggest you search for more information on the internet— check out the archives of Rachel, an internet environmental magazine. Sexual reproduction in chytrids and Spizellomycetales should be reexamined, as it used to be assumed that any zoospore with two flagella and every resting spore resulted from nuclear fusion. Now we know that some biflagellate zoospores originate by

A Mixed Bag incomplete differentiation of the cytoplasm during zoospore formation and that many resting spores are just thick-walled asexual sporangia which can survive dry periods. Order Monoblepharidales. Monoblepharis polymorpha (Figs. 2.5, 2.12D), which is found on twigs of birch, ash, elm, or oak submerged in slightly alkaline freshwater pools, is the first fungus we have met that has gone all the way to complete sexual differentiation of gametes. The male gamete is motile (a sperm), but the female (an egg) is

Fig 2.12 Types of thalli and reproductive structures among the Chytridiomycota. A: eucarpic thallus of Spizellomyces punctatus (Spizellomycetales) in pine pollen; B: holocarpic thallus of Olpidium brassicae (Chytridiales) in a cell of cabbage root; C: polycentric thallus of Cladochytrium (Chytridiales); D: stages of oogamous reproduction in Monoblepharis polymorpha (Monoblepharidales).

Chapter 2 not. This style of sexuality is called oogamy. Sperms form in gametangia called antheridia, while eggs develop in oogonia, which are found on the same hypha just below the antheridia. Sperm is often released before the adjacent oogonium is ripe. This may be a mechanism for avoiding self-fertilization and thus ensuring outbreeding (called heterothallism in fungi). After the egg has been fertilized, the resulting zygote becomes amoeboid, moves out onto the top of the oogonium, and encysts, developing a thick wall. Meiosis probably occurs when this resting spore germinates, producing a germ tube (another name for a first hypha). Although the Chytridiomycota vary in many things, such as in the morphology of their assimilative phase, in their patterns of sexuality, and in their adoption of parasitic or saprobic lifestyles, most of them have motile spores (zoospores or gametes), with one flagellum at the back (posteriorly uniflagellate—the cell swims like a sperm). In addition, their cell walls, like those of the other, more complex, eumycotan fungi, are largely made of chitin, a polysaccharide very similar to the stuff of which insect exoskeletons are made. They synthesize lysine by the same pathway (see chapter 8), and the more advanced members of the group produce true hyphae. The Chytridiomycota apparently represent modern survival of the ancestral line that evolved into the eumycotan fungi. Perhaps surprisingly, two presumed members of the Chytridiomycota, the genera Olpidium and Rozella, have been discovered to be completely separate clades. Indeed, Rozella was found to be the earliest-diverging lineage in the fungi and perhaps a member of yet another new phylum, the Cryptomycota. The parasitic Olpidium appears closely related to the Zygomycota.

Kingdom Eumycota Phylum 2: Blastocladiomycota This phylum was long considered no more than an order within the Chytridiomycota. But in 2006, the order Blastocladiales was promoted to the rank of phylum by James et al. from molecular evidence. Here, the thallus has both broad true hyphae and narrow rhizoids. Allomyces arbusculus, whose lifecycle is illustrated in Fig. 2.13, exhibits what we call a rotation between haploid and diploid thalli. Haploid thalli produce gametes in specialized gametangia, while diploid thalli produce flagellate zoospores and resting sporangia. In Allomyces, the gametes come in two sizes, which is a condition called anisogamy. The general principle underlying anisogamy is division of labour, whereby the smaller, more mobile gamete (which we can now think of as male) actively seeks out the larger, less mobile (female) gamete, which has sacrificed some speed in order to carry enough food to give the next generation a good start. In Allomyces arbusculus, both kinds of gamete are formed on the same haploid thallus. The colourless female gametangia are borne at the tips of hyphal branches, with the orange male gametangia immediately below. Zygotes develop into diploid thalli which bear two kinds of sporangia, thin walled and thick walled. The nuclei of thin-walled sporangia undergo repeated mitosis and

A Mixed Bag

Fig 2.13. Blastocladiales: life cycle of Allomyces arbusculus.

produce mitospores, which in this case are diploid, uniflagellate zoospores that can establish new diploid thalli. The other kind of reproductive structures, resistant sporangia, shown on the right side of the diagram, are thick walled, brown, and can survive for up to thirty years. Eventually, some environmental stimulus triggers reduction division (meiosis) in these sporangia, and the resultant haploid meiospores are liberated and develop into sexual thalli. Coelomomyces is another genus of the Blastocladiomycota in which some species are obligate parasites of mosquito larvae, and attempts are being made to use them in biological control of these insects (see chapter 14).

Chapter 2

Kingdom Eumycota Phylum 3: Neocallimastigomycota Just to spoil our picture of the Chytridiomycota even further, in 1975, Orpin discovered some new and very different chytridiomycetous fungi living in the rumens of large herbivorous mammals. These fungi, mostly species of Neocallimastix, were obligately anaerobic. They resembled their aerobic relatives in many ways but had no mitochondria and often had multiflagellate zoospores. Fifteen species of anaerobic chytrids had been described by 1994. They produce rhizomycelia which efficiently penetrate plant material and have enzymes that more effectively break down cellulose than the cellulases of the mould Trichoderma (see chapter 14 part 3 and chapter 24). They are now classified as the order Callimastigales in phylum Neocallimastigomycota.

Further Reading Barr, D. J. S. 1990. “Phylum Chytridiomycota.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 454–66. Boston: Jones and Bartlett. Barron, G. L. 1991. “Protoplasm in Motion.” Seasons 31, no. 2:20–25. Bigelow, D. M., M. W. Olsen, R. L. Gilbertson. 2005. “Labyrinthula terrestris sp. nov., a New Pathogen of Turf Grass.” Mycologia 97:185–90. Bonner, J. T. 1967. The Cellular Slime Molds. 2nd ed. Princeton, NJ: Princeton University Press. ———. 1993. Life Cycles: Reflections of an Evolutionary Biologist. Princeton, NJ: Princeton University Press. Buczacki, S. T., ed. 1983. Zoosporic Plant Pathogens, a Modern Perspective. New York: Academic Press. Cavender, J. C. 1990. “Phylum Dictyostelida.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 88–101. Boston: Jones and Bartlett. Dick, M. W. 1990. “Phylum Oomycota.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 661–85. Boston: Jones and Bartlett. Dylewski, D. P. 1990. “Phylum Plasmodiophoromycota.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 399–416. Boston: Jones and Bartlett. Eichinger, L., et al. 2005. “The Genome of the Social Amoeba Dictyostelium discoideum.” Nature 435:43–57. Erwin, D. C., and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. St. Paul, MN: APS Press. Frederick, L. 1990. “Phylum Plasmodial Slime Molds: Class Myxomycota.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 467–83. Boston: Jones and Bartlett. Fuller, M. S. 1990. “Phylum Hyphochytriomycota.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 380–87. Boston: Jones and Bartlett. Fuller, M. S., A. Jaworski, eds. 1987. Zoosporic Fungi in Teaching and Research. Athens, GA: Southeastern Publishing Co.

A Mixed Bag Gray, W. D., and C. J. Alexopoulos. 1968. Biology of the Myxomycetes. New York: Ronald. Hagiwara, H. 1989. “The Taxonomic Study of Japanese Dictyostelid Slime Molds.” Tokyo: National Science Museum. James, T. Y., P. M. Letcher, J. E. Longcore, S. E. Mozley-Standridge, D. Porter, M. J. Powell, G. W. Griffith, and R. Vilgalys. 2006. “A Molecular Phylogeny of the Flagellated Fungi (Chytridiomycota) and Description of a New Phylum (Blastocladiomycota).” Mycologia 98:860–71. doi:10.3852. Karling, J. S. 1977. Chytridiomycetarum Iconographia. Vaduz, Liechtenstein: Cramer. Large, E. C. 1962. The Advance of the Fungi. New York: Dover. Longcore, J. E., A. P. Pessier, D. K. Nichols. 1999. “Batrachochytrium dendrobatidis gen. et sp. nov., a Chytrid Pathogenic to Amphibians.” Mycologia 91:219–27. Margulis, L., J. O. Corliss, M. Melkonian, D. J. Chapman, eds. 1990. Handbook of Protoctista. Boston: Jones and Bartlett. Moore, R. T. 2000. “Mycological Dispatches [about Phytophthora].” Mycologist 14:93. Muehlstein, L. K., D. Porter, F. T. Short. 1991. “Labyrinthula zosterae sp. nov., the Causative Agent of Wasting Disease of Eelgrass, Zostera marina.” Mycologia 83:180–91. Olive, L. S. 1975. The Mycetozoans. New York: Academic Press. Orpin, C. G. 1975. “Studies on the Rumen Flagellate Neocallimastix frontalis.” Microbiology 91, no. 2: 249–62. Porter, D. 1990. “Phylum Labyrinthulomycota.” In Handbook of Protoctista, edited by L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, 388–98. Boston: Jones and Bartlett. Quammen, D. 2000. The Boilerplate Rhino: Nature in the Eye of the Beholder. New York: Scribner. Sparrow, F. K. 1960. Aquatic Phycomycetes. 2nd ed. Ann Arbor: University of Michigan Press. Spencer, D. M., ed. 1981. The Downy Mildews. New York: Academic Press. Webster, J. 1980. Introduction to Fungi. 2nd ed. Cambridge, UK: Cambridge University Press.

3 Eumycotan Fungi—the Mainstream and Others Phylum 4 Zygomycota Phylum 5 Glomeromycota Phylum 6 Microsporidia First, a line-item display of the classification to show what goes where.

Kingdom Eumycota Phylum 4 Zygomycota Subphylum Mucoromycotina Order Mucorales Order Endogonales Order Mortierellales Groups currently of uncertain position Subphylum Entomophthoromycotina Order Entomophthorales Subphylum Zoopagomycotina Order Zoopagales Subphylum Kickxellomycotina Order Kickxellales Order Dimargaritales Order Harpellales Order Asellariales

Kingdom Eumycota Phylum 5 Glomeromycota The arbuscular mycorrhizal or endomycorrhizal fungi Class Glomeromycetes (formerly placed in the Zygomycota)

Eumycotan Fungi—the Mainstream and Others

Kingdom Eumycota Phylum 6 Microsporidia e.g., Nosema, intracellular parasites, mainly of insects (formerly considered protozoa but now regarded as extremely reduced fungi—see Corradi and Keeling 2009). Rapidly evolving and possibly derived from the Zygomycota.

Introduction Kingdom Eumycota was for many years assumed to be made up of four phyla— Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. These, and particularly the last two, far outnumber the chromistan pseudofungi in species diversity. We already know about 100,000 eumycotan fungi, and it is obvious to those of us who work with them that these are just the tip of the iceberg. We estimate that there are well over a million species waiting to be found and described. Hundreds of new fungal taxa are described every year. For example, in 1990, in a single publication, Rafael Castañeda and I described fourteen new genera and forty new species of microscopic moulds from dead leaves of Cuban rainforest plants. And we have become aware of several new phyla in recent years, as the outline of the phylogenetic system shows. This wealth of species is a measure of fungal success in evolutionary terms, just as the existence of millions of species of insects tells us that they, too, are winners (although their total biomass is far less than that of the fungi). Before we look at the eumycotan fungi in detail, it is worth enquiring into the reasons for their success. Earlier, I introduced the idea that the number, kind, and arrangement of motility organelles (flagella) found in the chromistan pseudofungi (Oomycota, Hyphochytriomycota) and some eumycotan fungi (Chytridiomycota, Neocallimastigomycota, Blastocladiomycota) are very basic, highly conserved features. As a corollary of this, the absence of motile cells from the life cycle of most eumycotan fungi must also be considered important. This seems to reflect a radical shift in evolutionary direction. It shows very clearly that most true fungi are basically terrestrial, and must have been so for a long time (even in geological terms). Many more ecological niches and substrates are available on land than in the water, and the challenges of survival and dispersal are very different. Those true fungi that live in fresh or salt water are secondarily adapted to those habitats (as are, for example, all aquatic mammals). Fungi are heterotrophic, which means that they depend on energy-rich carbon compounds manufactured by other organisms. But this doesn’t seem to have been a serious disadvantage. Fungi have evolved enzymes that can digest some extremely tough substrates. Chitin (arthropod exoskeletons), keratin (mammalian and avian skin, hair, horn, and feathers), cellulose (most plant debris—the largest reservoir of biological material), and lignin (a major constituent of wood) nourish many fungi, although we must keep in mind that cellulose and lignin remain completely unavailable to almost all animals (except with the collaboration of microbial symbionts). The unusual ability of some saprobic fungi to exploit cellulose and lignin gives them almost exclusive access to the massive quantities of plant debris produced every year and may well make them the world’s number one recyclers. Only human-made plastics are, perhaps

Chapter 3 unfortunately, mostly immune to their attacks. This means that we, not the fungi, must take responsibility for recycling these substances. I should add that we are now beginning to develop and use biodegradable plastics, which should somewhat mitigate the problem. There are two major reasons for the incredible success the fungi enjoy. The first is the fungal spore, the second the fungal hypha. Spores permit rapid dispersal and a kind of scattershot saturation of the biosphere—fungal spores are (almost) everywhere. Hyphae permit the thorough and intimate exploration and exploitation of newly available substrates. SPORES. The nonmotile microscopic spores of eumycotan fungi, which come in a dazzling array of forms (Fig. 3.1) to fit specific functions, are often produced very

Fig. 3.1

Diversity of spores among eumycotan fungi.

Eumycotan Fungi—the Mainstream and Others quickly (in a matter of days or even hours after the initial colonization of the substrate) and in enormous numbers. They are dispersed by wind, by water, or by animal vectors, and they can often survive long periods, sometimes even years, of unfavourable conditions such as freezing, starvation, or desiccation (which means drying out and is spelled with one ‘s’ and two ‘c’s). Like bacteria, fungal spores are everywhere, especially in the soil (in astronomic numbers) and in the air we breathe (sometimes 10,000 or more in a cubic metre—occasionally many more!). If you are curious about the ways in which we describe and name these spores, zip off to chapter 4 and find out. HYPHAE. These are the vegetative, assimilative organs of most fungi. When a spore germinates, what emerges is a hypha (sometimes more than one hypha), which grows at its tip and explores the microscopic world in which it landed. The picture below shows hyphae emerging from spores and looking for food. In order to explore their immediate surroundings properly, hyphae must branch repeatedly as they spread out. This is shown in the drawing below. It shows a young fungal colony (Fig. 3.2) arising from a single spore (the black dot in the middle). Its strong, waterproof, chitinous hyphae; its richly branched growth pattern; the digestive enzymes it secretes at its growing tips; and the hydrostatic pressures it can bring to bear—all these make it ideally suited for actively penetrating, exploring, and exploiting solid substrates in a manner that the bacteria, chief competitors of the fungi in the recycling business, cannot match. (How many hyphal tips do you think there are in this illustration of a very young colony? 176? 294? 388? 502? The answer is 388, and this number will double every hour or so.) If a fungus is growing in liquid culture or in a solid substrate and producing a spherical colony, the rate of increase in the number of hyphal tips is much faster, and the final number in a mature colony, astronomical. The transmission electron micrograph (Fig. 3.3) (from Cole and Samson 1979) shows how vesicles (v) containing new wall material or enzymes concentrate at the

Fig. 3.2 Young colony of Phycomyces arising from a mitospore. Note the large number of hyphal tips and the absence of septa that is typical of Mucorales.

Chapter 3

Fig. 3.3 Transmission electron micrograph from Cole and Samson 1979.

hyphal tip, while the energy engines of the cell, the mitochondria (m), sit a little farther back. The many little round black dots in clusters are ribosomes, the sites of protein synthesis. It is at the hyphal tip that the fungus interacts with the world outside, and where all growth in length takes place. Fungi have learned to cope with environmental extremes. They can grow at temperatures as low as –5°Celsius and as high as 60°Celsius. They include the most xerotolerant organisms known: some moulds will grow at the amazingly low water activity of 0.65 (most plants wilt permanently at a water activity of 0.98). Other moulds grow in oxygen concentrations as low as 0.2% (air now contains 20% oxygen). Certain fungi can grow under extremely acid conditions (pH 1); others can tolerate alkalinity up to pH 9. These topics are covered in more detail in chapter 9 (Fungal Physiology and Metabolism) and chapter 20 (Food Spoilage by Fungi and How to Prevent It). As I have already noted, the saprobic fungi are recyclers par excellence, but they are also among the world’s greatest opportunists and don’t restrict their attentions to naturally occurring dead wood and leaves. Wherever there is a trace of moisture, their omnipresent spores will germinate, and the hyphae arising from them will attack food and fabric, paper and paint, or almost any other kind of organic matter. Some of their metabolites (mycotoxins) are extremely dangerous—even carcinogenic—if they contaminate food (chapter 21—Mycotoxins in Food and Feed). And parasitic fungi cause the majority of serious plant diseases (chapter 12—Fungal Diseases of Crops and Trees) as well as some of animals and people (chapter 23—Medical Mycology). Fortunately, there is a brighter side to fungal intervention in human affairs: we have harnessed the biochemical virtuosity of the saprobic fungi in the production of beer, wine, bread, some gourmet cheeses, soy sauce, some antibiotics and immunosuppressants, organic acids, and many other useful chemicals. Fungi are even being used to convert plant waste into high-protein animal feed and into packing materials. We ourselves eat a number of the large, spore-producing structures developed by fungi: mushrooms, chanterelles, morels, and truffles are all familiar to devotees of French cuisine,

Eumycotan Fungi—the Mainstream and Others who prize them for their unique flavours. Some of the parasitic forms are now being recruited to attack insects, weeds, and other fungi which threaten our welfare (chapter 14). And fungi in intimate, obligatory association with the roots of almost all higher plants (forming mycorrhizas), silently, invisibly, and influentially perpetuate one of the world’s oldest and most successful forms of mutualistic symbiosis (chapter 17). Now for a few more details.

Phylum 4 Zygomycota This phylum is the least well defined at present, since several of the groups traditionally placed in it are now of uncertain disposition, and it is not regarded as monophyletic. Although the subphylum Mucoromycotina contains only about 1% of the known species of fungi, its members are distinctive, and some of them are common, successful saprobes, fast-growing, primary colonizers of substrates containing accessible carbon sources like sugar or starch. Zygosporangia. The name of the phylum is derived from the way in which its members reproduce sexually by the physical blending—fusion or conjugation—of morphologically similar gametangia to form a zygosporangium (the teleomorphic phase). ‘Zygos’ is Greek for a yoke or joining. The gametangia arise from hyphae of a single mycelium in hom*othallic species or from different but sexually compatible mycelia in heterothallic species. Zygosporangia usually develop thick walls and act as resting spores. The four diagrams in Fig. 3.4 show how a zygosporangium develops. When compatible mycelia of Phycomyces blakesleeanus meet, individual hyphae establish intimate contact, developing finger-like outgrowths and giving the appearance of grappling with one another. This lets them exchange chemical signals which establish that they are indeed sexually compatible. Then the two hyphae grow apart again, only to loop back, swelling as they approach each other, and finally meeting head on. They have become gametangia, which fuse when their tips touch. Note that there isn’t any sexual differentiation in size or shape here: since we can’t call them male and female, we simply label the mycelia ‘+’ and ‘–’. After the walls between the two gametangial tips have broken down and their multinucleate contents have mixed, the mixture is quickly isolated by two septa, one at each side, and the paired-off nuclei fuse. The structure is now called a zygosporangium, and it develops a thick and often ornamented wall, even while still supported on either side by the former gametangia, which are now called suspensors. Although the two suspensors are now just empty appendages, they make it easy to recognize a zygosporangium when you see one. Anamorphs. You won’t often see zygosporangia in field collections, although I sometimes find a hom*othallic species of Syzygites producing them profusely as it parasitizes over-mature wild mushrooms. But asexual or anamorphic phases of zygomycetes are easy to find on mouldy bread or peaches or on horse dung and dog poop. A number of examples (including a couple of teleomorphs) are illustrated in Figs. 3.5 and 3.6.

Chapter 3

Fig. 3.4 Development of zygosporangium (teleomorph) in Phycomyces blakesleeanus (Mucorales).

Eumycotan Fungi—the Mainstream and Others

Fig. 3.5 Anamorphs in the Zygomycota. A–D: Mucorales; E, F: Kickxellales.

Chapter 3

Fig. 3.6 Pilobolus (Mucorales). A: habit of sporangiophores (anamorph) on dung: beaded appearance is caused by condensation droplets; B, C: action of subsporangial vesicle and retinal area in phototropism; D: zygosporangium (teleomorph). E, F: Entomophthora (Entomophthorales). E: section through the anamorph sporulating on a fly; F: zygosporangium (teleomorph) arising by fusion of hyphal bodies.

Collect some fresh horse dung (from a horse eating naturally in a field), keep it in a damp chamber, and look at it through a dissecting microscope, or even a hand lens, every day. You should be able to follow a sequence of specialized coprophilous fungi—and the first to develop will probably be the spectacular anamorph of Pilobolus (Fig. 3.6A–C), which is discussed below and in chapter 11. The nonmotile asexual mitospores are usually formed inside mitosporangia borne at the tips of specialized sporangiophores. Zygomycetous cell walls are mainly of chitin, and the nuclei in their somatic hyphae are haploid. Now for a brief taxonomic survey of the phylum.

Subphylum 1 Mucoromycotina I will introduce you to three orders: the Mucorales, Mortierellales, and Endogonales. (1) Order Mucorales. This order of about 300 species, most of which grow readily and rapidly in culture, includes all the common saprobic zygomycetes. Here belong the ubiquitous bread mould, Rhizopus stolonifer (Fig. 3.5A), and the almost equally common

Eumycotan Fungi—the Mainstream and Others genus Mucor. Each globose mitosporangium of these fungi contains hundreds of nonmotile, asexual spores, and these sporangia are produced at the ends of tall, stout, simple, or branched hyphae called sporangiophores. The trademark of this group is a swollen extension of the sporangiophore called a columella (Fig. 3.5A, D), which protrudes into the sporangium and often persists after the delicate outer skin or peridium of the sporangium has disappeared and the sporangiospores have been dispersed. Other groups often have fewer spores per sporangium, and their sporangia have no columella. Thamnidium elegans (Fig. 3.5D) seems to compromise. Its tall sporangiophores have one large, terminal, columellate sporangium, but lower on the stalk there are branches which fork repeatedly in a dichotomous manner, the final branchlets ending in tiny mitosporangia (sporangioles) which contain only a few spores. The reductionist tendency is also evident in Helicostylum, which has ten to twenty spores per sporangium, and in Blakeslea trispora (Fig. 3.5B), which has only three spores per sporangium. This trend reaches its logical conclusion in Cunninghamella (Fig. 3.5C), which has only one spore per mitosporangium, and in which the walls of spore and sporangium appear to have fused. Now the whole mitosporangium becomes detached and acts as a dispersal unit. Although Mucorales can go through cycle after cycle—spore, mycelium, sporangium, spore—producing only the anamorph, they sometimes form sexual zygosporangia (the teleomorph), perhaps as a survival mechanism, perhaps for the benefits of genetic recombination, or perhaps just because compatible strains have met. The photo (Fig. 3.7) shows both teleomorph and anamorph of Phycomyces.

Fig. 3.7 Teleomorph and anamorph of Phycomyces.

Chapter 3 The anamorph-teleomorph alternation is diagrammed in Fig. 3.8 for one of the most common and successful members of the Mucorales, Rhizopus stolonifer. Note that when the zygosporangium germinates, it produces a mitosporangium rather than a germ tube.

Fig. 3.8.

Mucorales: life cycle of Rhizopus stolonifer.

Eumycotan Fungi—the Mainstream and Others Zygosporangia vary in minor ways from one genus to another and among families and orders, but they are generally rather similar (Figs. 3.4; 3.6D, F; 3.7; 3.8): if they are present, they are the easiest way to tell if a fungus is a zygomycete. By contrast, the anamorphs of zygomycetes—mitosporangia and the structures on which they are borne—have evolved some amazing and bizarre adaptations. This contrast between teleomorphic constancy and anamorphic diversity is presumably the result of differing evolutionary pressures. Long-term survival, one of the main objectives of the teleomorph, is presumably best ensured by structures with minimal surface area and thick, protective walls. Dispersal, the main purpose of anamorphs, can be achieved in many ways. Let’s look at three of the more specialized zygomycetous anamorphs. Pilobolus crystallinus (Fig. 3.6A–C) is an atypical but fascinating coprophilous (dung-inhabiting) member of the order Mucorales. It grows very rapidly and is one of the first fungi to fruit in the extended succession that occurs on dung (see chapter 11 for a detailed exposé of this succession). Its unbranched sporangiophores are 2–4 centimetres tall and have a unique explosive dispersal mechanism. The whole sporangiophore is a single large cell. Beneath the black apical mitosporangium is a lens-like subsporangial vesicle, with a light-sensitive ‘retina’ at its base that controls the growth of the sporangiophore very precisely (Fig. 3.6B–C), aiming it accurately toward any light source. In a word, it is phototropic. The generation of osmotically active compounds inside this giant cell causes hydrostatic pressure in the sporangiophore and the subsporangial vesicle to build up until it is over 100 pounds per square inch (7 kg per square cm). This eventually causes the vesicle to explode, hurling the black sporangium away to a distance of up to 2 metres, directly toward the light. The mucilaginous contents of the subsporangial vesicle go with the sporangium and glue it to whatever it lands on. Can you explain why Pilobolus needs such a specialized mechanism for spore dispersal: such a powerful cannon, so carefully aimed? You can find the answer in chapter 11. Note that the originality of Pilobolus extends only to the behaviour of its anamorph— the teleomorph (the zygosporangium, shown in Fig. 3.6D) is fairly conventional. (2) Order Mortierellales, almost 100 species in 6 genera, but 90 of those species in Mortierella. The zygosporangia of these fungi resemble those of the Mucorales, but the asexual sporangia do not have a columella. (3) Order Endogonales, four genera. These fungi are among the relatively few that establish mycorrhizal symbioses with plants. Some produce pea-sized sporocarps that contain numerous zygosporangia (see www.mycolog.com).

Subphylum 2 Kickxellomycotina This contains the orders Kickxellales, Dimargaritales, Harpellales, and Asellariales. This seems rather strange because members of the first order are largely coprophilous saprobes, the second order is parasitic, while the last two are biotrophic commensals inside the guts of living insects and have been traditionally separated as the class Trichomycetes (but see note below defining this group in an ecological rather than phylogenetic way).

Chapter 3 (1) Order Kickxellales (named after a mycologist called Kickx). Members of this order are atypical of the Zygomycetes in that they often have regularly septate hyphae. Their teleomorphs are unremarkable, but they develop some of the most complex anamorphs known (Fig. 3.5E, F). I have found Coemansia (Fig. 3.5E) on bat dung from a cave. Its tall sporangiophore bears many fertile side branches called sporocladia. Each of these produces a row of lateral cells called pseudophialides (true phialides are discussed in chapter 4). Finally, from the apex of each pseudophialide arises an elongate, one-spored mitosporangium (often called a sporangiole). That’s complex enough, but it looks simple beside Spirodactylon (Fig. 3.5F). This, surely the most elaborate of all zygomycetous anamorphs, produces a tall, branched sporangiophore that is repeatedly thrown into tight coils. Within these coils arise the sporocladia, which bear pseudophialides, and these in turn bear one-spored sporangioles. It is hard to imagine why this strange configuration might have evolved, until one learns that the fungus grows on mouse and rat dung. Coprophilous fungi have various highly evolved strategies for getting back inside the gut of the animals that produce their preferred substrate. This isn’t too difficult for genera like Pilobolus, which grow on herbivore dung, since all they have to do is get their spores onto the animal’s food, which is all around. But rats and mice are omnivores, not herbivores, and it is essentially impossible for the fungus to ensure that its spores will be present on their food. The only alternative (as I see it) is to attach spores to the animal itself in the hope that they will be ingested during grooming activities. Rats and mice are creatures of habit, using well-trodden paths each day. Along these trails they deposit dung, and there, later, the coils of Spirodactylon become entangled in their hair. Only the zygosporangia of the Kickxellales convince us that these strange fungi are indeed zygomycetes. (2) Orders Harpellales and Asellariales. These eccentric groups of fungi live almost exclusively attached to the lining of the guts of living arthropods, which is why you won’t run into them very often (if at all). But they are examples of the opportunism displayed by fungi and the determination shown by mycologists in winkling out fungi wherever they are to be found. Bob Lichtwardt’s 1986 book gives a fine account of these offbeat groups. A plate (Fig. 3.9) of phase-contrast photomicrographs by Richard Benjamin (1979) shows the characteristic structures of some ‘Trichomycetes’. Top left (A) are developing trichospores of Smittium. Top right (B) are trichospores of Stachylina showing the hair-like appendages that give them their name (these are not to be confused with flagella). Bottom left (C) is a trichospore of Smittium. Bottom centre (D) is a developing zygospore of Trichozygospora, and bottom right (E) a released, mature zygosporangium with a collar and a bunch of hair-like appendages below it. This last structure (look at the conical ‘suspensors’, top and bottom) places these ‘Trichomycetes’ in phylum Zygomycota. I must mention here that the ‘Trichomycetes’ are now regarded as a polyphyletic group defined in ecological terms. In addition to the two orders mentioned above, there are two groups often called ‘Trichomycetes’ that are actually Protozoans—the orders Amoebidiales and the Eccrinales—which occur, just as the Harpellales and Asellariales do, as commensals attached to the gut walls of living invertebrates.

Eumycotan Fungi—the Mainstream and Others

Fig. 3.9 Phase-contrast photomicrographs of Trichomycetes.

Chapter 3

Subphylum 3 Entomophthoromycotina (1) Order Entomophthorales. As the name implies, these fungi often attack insects. Entomophthora muscae infects, and eventually kills, houseflies. Dying flies, their bodies riddled by the fungus, usually crawl into exposed situations—I find them on windows and on the growing tips of shrubs in my wife’s garden—where the fungal infection bursts through the exoskeleton and produces tightly packed masses of sporangiophores (Fig. 3.6E). Each sporangiophore bears one unicellular, sticky mitosporangium that is shot away at maturity. When the fly dies on a window, this barrage produces a whitish halo of mitosporangia on the glass. These sporangia can infect other unsuspecting flies that come to pay their last respects. As you may already have guessed, species of Entomophthora are being investigated for their potential use in biological control of insect pests (see chapter 14). Note again that the zygosporangia of Entomophthora, although developing in an unusual way, by the fusion of hyphal bodies inside the fly, are still recognizable as zygosporangia (Fig. 3.6F). Basidiobolus and Conidiobolus cause serious animal, including human, diseases.

Subphylum 4 Zoopagomycotina Order Zoopagales. Most of the almost 200 species of this inconspicuous order are parasites or predators of microscopic organisms, such as amoebae.

Phylum 5 Glomeromycota Endomycorrhizal or Arbuscular Mycorrhizal Fungi Five orders, 29 genera, about 230 species These fungi are dealt with in some detail in chapter 17, because they are involved in more mycorrhizal relationships (with plants) than any other group. The soil-inhabiting Glomeromycota were placed in the Zygomycota only tentatively, since the only genus that develops zygosporangia is now known to belong elsewhere. Nevertheless, they are extremely important fungi, because their hyphae enter the living root cells of perhaps 90% of all higher plants and establish with them obligate mutualistic symbioses called arbuscular mycorrhizas (AM) or endomycorrhizas. These are illustrated and discussed in chapter 17, my main treatment of mycorrhizas. AM fungi won’t grow in axenic culture: they must be associated with a living plant root. Their generally very large (up to 600 microns) and thick-walled resting spores are common in most soils and are stimulated to germinate by the proximity of plant roots (almost any plant will do, because these fungi have such wide host ranges). Their usually nonseptate hyphae spread through the soil and enter living roots, where they develop diagnostic structures—intracellular, finely branched, tree-like arbuscules (Fig. 17.3), which are the interface across which the fungus exchanges mineral nutrients, especially phosphorus, for photosynthates (sugars, etc.) provided

Eumycotan Fungi—the Mainstream and Others by the plant. Many of the Glomeromycota produce both arbuscules and lipid-filled structures, called vesicles or intramatrical spores, inside plant roots. The soil-inhabiting mycelium is very efficient at mobilizing insoluble phosphorus and translocating (moving) it and other mineral nutrients to the plant. Since phosphorus is often the limiting nutrient for plant growth, AM fungi help plants to thrive in poor soils. These fungi are therefore vital in many natural habitats and of great potential value in agriculture. Again, for details, consult chapter 17. Molecular biology strongly suggests that the Glomeromycota nest with the Ascomycota and Basidiomycota.

Phylum 6 Microsporidia These organisms (about 1,500 known species) are all intracellular parasites of animals, mainly insects but also fish, crustaceans, and even humans. They have no mitochondria or flagella and reproduce mostly by forming extremely resistant spores. Nosema is the best-known example, and parasitizes many different insects. It is highly unlikely that you will ever see members of this group.

Further Reading Benjamin, R. K. 1959. “The Merosporangiferous Mucorales.” Aliso 4:321–453. ———. 1979. “Zygomycetes and Their Spores.” In The Whole Fungus, edited by B. Kendrick, 573–621. Vol. 2. Ottawa: National Museums of Canada. Bentivenga, S. P. 1998. “Ecology and Evolution of Arbuscular Mycorrhizal Fungi.” McIlvainea 13:30–39. Brundrett, M., L. Melville, and L. Peterson, eds. 1994. “Practical Methods in Mycorrhiza Research.” Mycologue Publications, 8727 Lochside Dr., Sidney, BC V8L 1M8, Canada [CD-ROM]. Cerda-Olmedo, E., and E. D. Lepson, eds. 1987. Phycomyces. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Cole, G. T., and R. A. Samson. 1979. Patterns of Development in Conidial Fungi. London: Pitman Publishing. Corradi, N., and P. J. Keeling. 2009. “Microsporidia: A Journey through Radical Taxonomic Revisions.” Fungal Biology Reviews 23:1–8. Fuller, M. S., ed. 1978. Lower Fungi in the Laboratory. Athens: University of Georgia Press. Ingold, C. T. 1978. The Biology of Mucor and Its Allies. London: Edward Arnold. Kendrick, B., and S. M. Berch. 1985. “Mycorrhizae: Applications in Agriculture and Forestry.” In Comprehensive Biotechnology, edited by C. Robinson, 109–15. Vol. 3. Oxford: Pergamon. Lichtwardt, R. W. 1986. The Trichomycetes. New York: Springer-Verlag. Morton, J. B., and G. L. Benny. 1990. “Revised Classification of Arbuscular Mycorrhizal Fungi (Zygomycetes): A New Order, Glomales, Two New Suborders, Glomineae and Gigasporineae,

Chapter 3 and Two New Families, Acaulosporaceae and Gigasporaceae, with an Emendation of Glomaceae.” Mycotaxon 37:471–91. O’Donnell, K. L. 1979. Zygomycetes in Culture. Athens: University of Georgia Press. Zycha, H., R. Siepmann, and G. Linnemann. 1969. Mucorales. Eine Beschreibung aller Gattungen und Arten dieser Pilzgruppe. Lehre, Germany: Cramer.

4 Kingdom Eumycota (True Fungi), Subkingdom Dikarya Phylum 7 Ascomycota—the Ascomycetes Introduction Zygomycetes are terrestrial fungi: there’s no doubt about that. But they usually thrive and sporulate only in damp places where the atmosphere is more or less saturated with moisture. For example, Rhizopus stolonifer will colonize the moist interior of a loaf of bread but won’t produce its characteristic sporangiophores and mitosporangia on the outside of the bread unless the surrounding atmosphere is humid. If we persuade the fungus to sporulate by keeping the loaf in a damp chamber (a plastic bag containing a few drops of water will do) and then take it out of the bag, the sporangiophores will quickly collapse. Most of the fungi covered in this chapter and the next are much less delicate and thrive in or on a much wider range of substrates.

Subkingdom Dikarya Phylum 7 Ascomycota Introduction (Also comparing Phylum 8 Basidiomycota; see chapter 5) Hyphae of most zygomycetes are wide, thin walled, and coenocytic—continuous tubes with no cross-walls, as shown in Fig. 3.2. Hyphae of subkingdom Dikarya (phylum Ascomycota plus phylum Basidiomycota) are usually narrower. Hyphae average about 5 microns in width, but in aggregate they are very long (sometimes kilometres per gram of soil). They are also septate—they have cross-walls called septa at regular intervals (Fig. 5.1). These miniature bulkheads give the hyphae some physical rigidity and limit loss of cytoplasm if the outer hyphal wall is ruptured. As a result, we find that the Dikarya can grow in a wide range of conditions; for example, they can often spread and fruit in drier situations than zygomycetes could tolerate. Some dikaryan anamorphs (especially coelomycetes—those with closed fructifications) grow in dead leaves and stems of desert plants, and some moulds are the most drought tolerant of all

Chapter 4 organisms, able to grow at water activities below 0.70 (for example, on jams, salt fish, and other substrates of extremely high osmotic pressure; see chapter 20). While many zygomycetes can assimilate only ‘accessible’ substrates like sugars and starch, ascomycetes can often exploit cellulose, and many basidiomycetes can digest both cellulose and lignin, carbon sources that are available to remarkably few other organisms. Although fungi cannot fix atmospheric nitrogen (this talent is restricted to some prokaryotes), dikaryan fungi can use many different forms of combined nitrogen; some ascomycetes even specialize in metabolizing the protein keratin, which is the main component of hair, skin, and nails. In case you were wondering if those fungi might constitute a health hazard—some of them do. Some other orders of ascomycetes are obligate parasites of plants. Remember the ‘downy mildews’ caused by Oomycota? There are also plant diseases called ‘powdery mildews’ that are caused by Ascomycota. The similarity of ‘common names’ is unfortunate, but try to remember the difference, because although the groups of fungi involved are both obligately biotrophic, the diseases are different in many important ways, such as host ranges and methods of control. This is just one example of how taxonomy has practical implications (see chapter 12). Thousands of basidiomycetes, and quite a few ascomycetes, establish intimate mutualistic symbioses (mycorrhizas) with the roots of trees, especially conifers (see chapter 17). Nearly 20,000 ascomycetes, and a few basidiomycetes, have ‘domesticated’ algae and/or cyanobacteria, thus becoming lichens, which can live in some of the world’s harshest climates and colonize the barest and most inhospitable substrates (see chapter 7). Some dikaryan fungi have even re-entered the water and, lacking motile cells, have evolved other mechanisms, such as long appendages, to aid spore dispersal. Dikaryan fungi range from unspecialized, almost omnivorous saprobes to fungi so specialized and ecologically demanding that they are found only on one particular leg of one species of insect. Some dikaryan fruit bodies are microscopic (as in most ascomycetes), but often (especially among the basidiomycetes) they are large and complex, and most of the common names applied to fungi refer to the visible teleomorphs of basidiomycetes (mushrooms, puffballs, bracket fungi) and, in a few cases, ascomycetes (morels, cup fungi). You may already be acquainted with some of these; I will introduce you to many more in the pages ahead.

Characteristics of Teleomorphs Most dikaryan fungi share a number of important features: (1) chitinous cell walls; (2) hyphae with regular cross-walls called septa (centrally perforated to allow movement of cytoplasm, and sometimes nuclei, between compartments); (3) the ability of somatic, assimilative hyphae to fuse with one another (anastomosis) and to exchange nuclei; and (4) the occurrence in their life cycles (or at least in those which produce a teleomorph) of a unique nuclear phenomenon called the dikaryon. After sexually compatible nuclei from different mycelia have been brought together by anastomosis, they pair off but do not fuse immediately to form a diploid zygote. Instead, they go on dividing synchronously to populate the cells of what are called dikaryotic hyphae, in which each cell or compartment has two sexually compatible haploid nuclei. Oh yes, they do fuse eventually, but not before some remarkable developments have taken place. (In

Kingdom Eumycota (True Fungi), Subkingdom Dikarya basidiomycetes, that ultimate sexual fusion may be delayed for years, and the number of fusions multiplied enormously.) If ascomycetes and basidiomycetes share all these things, how do they differ? Actually, in many ways, and with experience it’s usually easy to tell their sexual fructifications apart with the naked eye. But their microscopic, unicellular meiosporangia are most diagnostic of all.

Fig. 4.1 Comparison of ascus and basidium development.

The meiosporangia of ascomycetes are asci (singular, ascus). They are cylindrical or sack-like and at maturity usually contain eight haploid spores (ascospores) which are in most cases expelled into the air through the top of the ascus. The meiosporangia of basidiomycetes are basidia (singular, basidium). They usually have four tiny projections called sterigmata, each bearing a haploid spore (basidiospore) which is shot away individually at maturity. The formation of asci or basidia marks the end of the dikaryophase. The paired nuclei have fused, and the resulting zygote has undergone meiosis (and a subsequent mitosis in ascomycetes) to produce eight haploid ascospores or four haploid basidiospores. Compare the two sets of diagrams in Fig. 4.1, and note how similar the developmental processes are until the final stages.

Chapter 4

Teleomorph Stage of Ascomycota Now let’s examine the sexual (teleomorphic) part of the ascomycete life cycle from the beginning. When an ascospore germinates, it establishes a haploid mycelium. In heterothallic ascomycetes, this can’t undergo sexual reproduction until it meets another compatible haploid mycelium. When this relatively rare event takes place, the fungus cleverly maximizes the ensuing potential for genetic recombination in a unique way. One would expect a single sexual fusion, resulting in a single zygote. But most ascomycetes interpolate a dikaryophase, during which the number of pairs of compatible nuclei is multiplied, often enormously, as dikaryotic hyphae (often called ascogenous hyphae, as in Fig. 4.2) grow and branch within a mass of monokaryotic (haploid) tissue, which is the framework of the fruit body (the ascoma). Eventually, the ultimate branches of the dikaryotic (ascogenous) hyphae, of which there will be millions in larger cup fungi, reach their ordained positions in the future hymenium and the long-delayed sexual fusions take place in cells called asci. The genome is reshuffled during the ensuing meiosis in each ascus (this genetic recombination is due to crossing-over, which is explained in chapter 10). Each meiosis, having different patterns of crossing-over, will produce a somewhat different arrangement of the genome. In this way the products of a single hyphal encounter are first multiplied; then, on top of that, the reshuffling of genes inherent in meiosis generates a lot of genetic diversity. Not only is the dikaryon itself an unusual phenomenon, but during the dikaryophase an effectively diploid mycelium is growing within, and drawing nourishment from, the haploid ascoma tissue. This phenomenon has interesting parallels in the red algae, although molecular evidence doesn’t support a close relationship between the two groups (both lack motile gametes and appear to have simply hit upon the same solution to the problem posed by the rarity of sexual encounters). Ascospores are not motile, in the sense of self-propelling, but most ascomycetes nevertheless send their ascospores off with a burst of kinetic energy. Most asci are tiny spore guns, which work by building up internal pressure then releasing it through the tip. The job of most asci is to get their ascospores into the turbulent airflow above the ascoma. Mature asci of the dung-inhabiting Ascobolus (Fig. 4.2) project above the hymenium and point toward the light before discharging their spores. In this way they ensure that the spores will not run into any obstacles on their upward flight (see chapter 8).

Four Kinds of Ascoma The multicellular structures (ascomata) that produce the asci, and act as the platforms from which the spores are launched, come in four main designs, sectional views of which are drawn in Fig. 4.3 and photomicrographs given in Fig. 4.4 (1) Apothecial Ascomata allow many asci to discharge simultaneously because the entire fertile layer or hymenium is exposed. (2) Perithecial Ascomata have a narrow opening which permits discharge of the contents of only one ascus, or a few asci, at a time, as do (3) Pseudothecial Ascomata (similar type of discharge, but different development, different [bitunicate] asci).

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.2 Teleomorphic cycle of an apothecial ascomycete, Ascobolus (Pezizales) (see text for full explanation).

(4) Cleistothecial Ascomata lack an opening entirely. This usually indicates that the asci are spherical, as in these illustrations, and no longer shoot their spores; the fungus has evolved another dispersal strategy. That may have happened because the fungus fruits in a confined space (for example, under bark, or below the surface of the ground) where airborne dispersal cannot operate. We often find that the spores of such fungi are dispersed by animals (usually because the fungus has evolved chemical attractants).

Chapter 4

Fig. 4.3 Teleomorphs: asci, ascophore, and sectional views of ascomata.

Four Kinds of Ascus Before we go on to explore the many orders of Ascomycota, we must take a closer look at the ascus itself. All asci are not the same. There are four flavours (Figs. 4.3, 4.5): (1) Unitunicate-Operculate Asci, which have a single functional wall with a builtin lid or operculum at the tip—at maturity this pops open so that the spores can be ejected. Unitunicate-operculate asci are found only in apothecial ascomata. (2) Unitunicate-Inoperculate Asci, which have no operculum but have a special elastic ring mechanism built into their tip. This is a pre-set pressure release valve, or sphincter, and the ring eventually stretches momentarily, or turns inside out, to let the

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.4. A: Apothecial ascomata; B: Perithecial ascoma; C: Pseudothecial ascoma in vertical section; D: Cleistothecial ascoma.

spores shoot through. Such inoperculate asci are found in perithecial and some apothecial ascomata. (3) Prototunicate Asci, which have no active spore-shooting mechanism. These asci are usually more or less spherical and are most often found in cleistothecial (closed) and hypogeous (underground) ascomata. Sometimes the wall of this kind of ascus dissolves at maturity and releases the ascospores, which can then ooze, rather than be shot, out of the ascoma; or they may wait inside until it decays or is ruptured. These asci are often called prototunicate. Yet perhaps because they are found in several otherwise rather different orders, it seems likely that they represent a secondary condition

Chapter 4

Fig. 4.5 Five photomicrographs. A: Operculate asci of Peziza, with the opercula stained blue; B: Asci and ascospores of Caloscypha; C: Bitunicate ascus with pigmented dictyoseptate ascospores; D: Transmission electron micrograph of inoperculate ascus showing vertical section of sphincter; E: Prototunicate asci of Tuber californicum.

Kingdom Eumycota (True Fungi), Subkingdom Dikarya and have evolved several times from unitunicate asci (as they clearly did in the truffles; see Tuberaceae later in this chapter). (4) Bitunicate Asci, which have a double wall. A thin, inextensible outer wall covers a thick, elastic inner wall. At maturity the thin outer wall splits, and the thick inner wall absorbs water and expands upward, carrying the ascospores with it. This ‘jack-inthe-box’ design allows the ascus to stretch up into the neck of the ascoma to expel its spores. The bitunicate ascus, although still a spore-shooting device, is so different from the unitunicate ascus that they clearly diverged a long time ago. In many unitunicate ascomycetes, the perithecial ascoma develops only after the sexual stimulus, so that the asci can grow into an actively enlarging cavity. In many bitunicate ascomycetes, fertilization doesn’t happen until after a solid primordium or stroma has developed, so room has to be made for the asci by dissolving away existing tissue. In some cases the asci themselves do the job, but in others it is carried out by special sterile hyphae (pseudoparaphyses) growing down from the upper layer of the stroma; the asci then grow up between them. Remember that ascomata which develop in this way (pseudothecial ascomata) always produce bitunicate asci. Now for the other half of the story . . .

Anamorph Stages of Ascomycota Here is a mantra to begin with—say it until you know it: Holomorph = Anamorph + (the whole fungus) = (asexual reproduction) +

Teleomorph (sexual reproduction)

In any modern consideration of the ascomycetes, we cannot ignore their asexual reproductive phases, many of which are called moulds (hyphomycetes and coelomycetes). You already know that Zygomycota have diverse asexual phases (often also called moulds). So you won’t be surprised to discover that many ascomycetes have comparable asexual (anamorphic) phases during which they reproduce rapidly, and often relatively cheaply, by means of mitospores called conidia, which are often just modified bits of hyphae. The asexually reproducing phase of the ascomycete life cycle was more or less ignored for many years in favour of teleomorph studies. But when we consider that the anamorph is an important (and sometimes the only) phenotypic expression of many ascomycotan genotypes, we realize that it has much to tell us. Besides, one can get DNA and RNA from anamorphs just as easily as from teleomorphs, so we are beginning to understand the relationships and taxonomic dispositions of anamorphs much better, even in many cases where no teleomorph is known. Although they play essentially the same role in the life cycle, the anamorphs of ascomycetes differ from those of zygomycetes in two very important respects: (1) While zygomycetous mitospores commonly originate by free cell formation inside a sporangium, many spores cleaving from a single mass of cytoplasm, the mitospores (conidia) of ascomycetes are basically just modified bits of hyphae, either budded out as a new structure or converted from a whole existing cell. (2) In Zygomycota, anamorph and teleomorph sometimes occur together (especially in hom*othallic species) and always share the same binomial. In Ascomycota,

Chapter 4 anamorph and teleomorph often develop at different times and on different substrates. Each phase has often been collected in total ignorance of the existence of the other, and because of this, the International Code of Botanical Nomenclature has (until very recently) maintained that it is legal to give them separate binomials—a useful option but one giving rise to a great deal of confusion in some students’ minds. (How can one organism have two names?) Recently, this statute has been overturned, but we now have to decide which name takes precedence, and the idea of calling a well-known sexual fungus by the name of its asexual phase (just because the latter was described first) perturbs many experienced mycologists. It will be a while before things settle down. Several thousand anamorph-teleomorph connections have now been established in the ascomycetes. Known connections represent only a small proportion of the total number of taxa, either of anamorphs or teleomorphs (we know about 30,000 of each). Because anamorphs so often occur alone, as mentioned above it is still common practice to use separate names for them, as you will see, although the latest Code of Nomenclature specifies that there can be only one binomial per species—a decision with many as yet undetermined consequences, since sometimes the anamorph was described first, while in other cases the teleomorph was seen first. I have always found it completely unacceptable to talk about conidial fungi, like many other texts do, as if they constitute a separate major high-level taxon called the ‘Deuteromycotina’. This ignores both the evidence that they are all expressions of dikaryan genomes and the thousands of connections that have already been established with teleomorphs—a number which grows every year. The word Deuteromycotina should be excised from the mycological lexicon! I advocated this in a paper many years ago (Kendrick 1989—see references). In addition, we no longer call the anamorphic fungi ‘Fungi imperfecti’. Of about 30,000 known ascomycetes, about 5,000 have so far been connected to their anamorphs. What about the many thousands of conidial (anamorphic) fungi that are still ‘orphans’? I think there is good reason to believe that many of them have given up sex altogether, and become ‘anamorphic-holomorphs’, although they seem to have retained some genetic flexibility by (a) having more than one kind of nucleus in their mycelia (heterokaryosis) as a result of occasional hyphal fusions (anastomoses); and (b) sometimes undergoing a complex parasexual process involving rare somatic diploidization, mitotic crossing-over, and finally a return to the haploid condition (this process is more fully explained in chapter 10). Molecular biology now allows us to place anamorphs in teleomorphic families and orders, so the confusion is decreasing. It turns out that the conidial fungi are a mixed bag. Although most of them are, or were, part of ascomycete life cycles, others are, or were, connected with basidiomycetes. Despite this mixed ancestry, we have had to set up a single classification for all of them, because it is often impossible to tell, just by looking at them, in which phylum the connection lies. Unfortunately, our scheme for classifying the anamorphs was formerly unable to make little reference to teleomorphs, for several reasons: (1) teleomorphs are known for only 10%–15% of anamorphic species; (2) members of what seems to be a single morphologically coherent anamorph genus may have teleomorphs in many different holomorph genera (even from different orders)—this must be due to convergent evolution among anamorphs; and (3) anamorphs belonging to several different anamorph genera can have sexual phases in a single holomorph genus. This

Kingdom Eumycota (True Fungi), Subkingdom Dikarya must be due to radiative evolution among anamorphs. In addition, quite a few teleomorphs have more than one anamorph (we call such anamorphs synanamorphs). So our attempts to classify anamorphs have concentrated on (a) their mitospores (conidia); (b) the diverse structures (conidiogenous cells, conidiophores, and conidiomata) which bear them; (c) the ways in which the mitospores develop (conidiogenesis); (d) the way in which they are liberated (dehiscence); and, more recently, (e) molecular techniques, which are now helping us to elucidate their relationships (Seifert et al. 2011).

Morphology of Anamorphs There are at least 2,000 described genera of conidial fungi and almost 30,000 described species, and these numbers are increasing rapidly. The first really useful classification of dikaryan anamorphs, established in the late nineteenth century, was based on mature morphology. The principal characteristics used were (1) colour, septation (cross-walls), and shape of conidia; (2) conidiophore aggregation or lack of it; and (3) the production of conidia in enclosed structures or the absence of such enclosure. These characteristics are illustrated in Fig.4.6. The division of anamorphs into two large groups (hyphomycetes and coelomycetes) is informal, is based on characteristic (3) above, and is largely for convenience, since the easiest decision to make is usually (although not always) whether a conidial fungus is a hyphomycete or a coelomycete. You can recognize a hyphomycete because its conidiophores can be single or aggregated in various ways but are never enclosed within a covered conidioma. Coelomycetes form their conidia in initially enclosed conidiomata, which usually develop just beneath the surface of their plant substrate. I am not going to discuss coelomycetes in detail (although there are thousands of them, and they cause many important diseases of crop plants), but I must mention a few basic facts.

Coelomycetes = covered or ostiolate conidiomata A covered conidioma is called an acervular conidioma or acervulus (Fig. 4.6, bottom row). This may develop at various depths within the host: it can be subcuticular (covered only by the host cuticle); intraepidermal (arising within the cells of the epidermis, as in the diagram); subepidermal; or developing beneath several layers of host cells. Under this roof of host material, fungal hyphae aggregate and form a flat fertile layer, or hymenium, of short conidiophores that produce many conidia (shown in a vertical section). The pressure of accumulating conidia, and often of accessory mucilage, eventually splits the host epidermis and allows the conidia to escape. At the other end of the spectrum is the flask-shaped, ostiolate pycnidial conidioma, or pycnidium, seen in vertical section, in which the fungus itself provides the enclosing wall and conidia eventually ooze out through a narrow apical pore or ostiole. Between these extremes lies a range of sometimes hard-to-categorize fructifications that are discussed succinctly by Kendrick and Nag Raj (1979). Browsing the superbly illustrated books by Sutton (1980) and Nag Raj (1993) will give you an appreciation for this amazing plasticity.

Chapter 4

Fig. 4.6

Anamorphs: conidia, conidiophores, and conidiomata.

Hyphomycetes = exposed conidiophores or conidiomata Among the hyphomycetes, conidiophores are usually solitary, although they sometimes form columnar aggregations, called synnematal conidiomata, or cushionshaped masses, called sporodochial conidiomata (all shown in Fig. 4.6).

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Conidium Morphology Presence or absence of pigment is important, as are shape and septation. There are seven shape and septation categories. (Fig. 4.6 top row). (A) Commonest of all are single-celled amerospores. (B) The addition of one cross-wall makes them didymospores. (C) Conidia curved through more than a half-circle, or coiled in two or three dimensions, are called helicospores. (D) Those with several conspicuous, radiating arms or other projections are called staurospores. (E) Septa running two ways, like the meshes of a net or like the mortar layers of a brick wall, identify dictyospores. (F) Two or more transverse septa, arranged like the rungs of a ladder, characterize phragmospores. (G) Finally, those which are long and thin (more than fifteen times as long as they are wide) are called scolecospores (which means ‘worm-like’).

Conidiogenesis (Mitospore Development) More recently, it was discovered that conidial fungi use a number of different techniques to produce their spores. Since these often represent genuine ‘embryological’ differences, they have become important characteristics in our classification. Spores which look alike can develop in different ways. We begin by checking an anamorph to see which of two basic patterns of development—blastic or thallic—it exhibits (Fig. 4.7A, B):

Fig. 4.7 Basic modes of conidium development and release.

Chapter 4

Fig. 4.8

Representative anamorphs with blastic conidiogenesis.

In blastic conidiogenesis (Fig. 4.7A), the young conidium is recognizable before it is cut off by a cross-wall (this is an extension of the idea of cells ‘budding’). In thallic conidiogenesis (Fig. 4.7B), the cross-wall is laid down before differentiation of the conidium begins. Ripe conidia may also be liberated in two different ways, described as schizolytic and rhexolytic dehiscence.

Kingdom Eumycota (True Fungi), Subkingdom Dikarya In schizolytic secession (Fig. 4.7C), the halves of a double septum at the base of the conidium split apart by the breakdown of a kind of middle lamella. In rhexolytic secession (Fig. 4.7D), the outer wall of a cell beneath or between conidia breaks down or ruptures. We will examine ten different kinds of conidium development: seven blastic (Fig.4.8) and three thallic (Fig. 4.10).

Type I: blastic-acropetal conidiogenesis The Monilia anamorph of Monilinia fructicola, the soft brown rot fungus of peach and other stone fruits (see chapter 12), and the Cladosporium anamorph of Mycosphaerella tassiana, an extremely common mould on decaying organic matter (and in the air), both produce conidia in chains by apical budding. The youngest conidium is at the tip of the chain. The chain branches when two buds, rather than one, develop on the conidium at the tip of the chain (this rather diagnostic conidium is then called a ramoconidium). This is clearly just a modified form of hyphal growth (Fig. 4.8).

Type II: blastic-synchronous conidiogenesis The hyphomycetes Botrytis and Gonatobotryum produce many conidia synchronously on a swollen cell: Gonatobotryum goes on to form acropetal chains of secondary conidia (by apical budding), while Botrytis does not. Despite this ‘deficiency’, Botrytis is one of the most successful moulds (Fig. 4.8). Other blastic-synchronous moulds are Chromelosporium and Botryosporium.

Type III: blastic-sympodial conidiogenesis In species of Beauveria, insect pathogens which are now being used in biological control of potato beetle, the narrow apex of the conidiogenous cell extends sympodially; this means that each new apex becomes converted into a blastic conidium and then the next apex grows out from behind and to one side of it. The more conidia are produced, the longer the conidiogenous cell becomes. Although Leptographium anamorphs of Ophiostoma have single conidiophores, these have complex heads with several tiers of supporting cells (metulae), the ultimate ones in some species bearing many sympodially extending conidiogenous cells, and innumerable conidia accumulate in a slimy head. These spores are insect dispersed. Basifimbria (teleomorph unknown), which is common on horse dung, has simple conidiophores that elongate sympodially during conidiation (Fig. 4.8).

Type IV: blastic-annellidic or blastic-percurrent conidiogenesis In the Spilocaea anamorph of Venturia inaequalis, the apple scab fungus, each seceding conidium leaves a ring-like scar, an annellation, around the conidiogenous cell, which then grows through the scar (‘percurrently’) to produce the next conidium (Fig. 4.8). Conidiogenous cells that have produced x spores bear x annular scars— hence the name annellidic. Scopulariopsis (Fig. 4.8) has several annellidic conidiogenous cells on each branched conidiophore. Leptographium conidiophores may have many such cells at their apex (Fig. 4.8).

Chapter 4

Fig. 4.9

Analysis of three modes of blastic conidiogenesis.

Note that Leptographium has been mentioned under both sympodial and percurrent headings. It has recently been confirmed that some individual anamorphs can be both percurrent and sympodial. I’ll give you two examples of how this knowledge may change our classification. When it was thought, not many years ago, that conidiogenesis in the synnematal anamorphs of Ophiostoma species was either exclusively sympodial or exclusively percurrent, they were segregated into two anamorph genera (Pesotum and Graphium). Now that it has been shown that both kinds of conidiogenesis can occur on the same conidiophore, they are being united again in the older genus, Graphium. In exactly the same way, some complex anamorphs of Ophiostoma species were segregated into the ‘exclusively sympodial’ Verticicladiella (which I monographed) and the ‘exclusively percurrent’ Leptographium but are now united under the older name, Leptographium.

Type V: blastic-phialidic conidiogenesis Many common moulds produce conidia in rapid succession from the open end of special conidiogenous cells called phialides. Common genera such as Penicillium, Aspergillus, Stachybotrys, and Chalara are all phialidic (Fig. 4.8). Many plant-pathogenic hyphomycetes, such as Fusarium and Verticillium, both causing serious wilt diseases of crop plants, also produce phialides. Penicillium and Aspergillus are dry spored;

Kingdom Eumycota (True Fungi), Subkingdom Dikarya Fusarium, Verticillium, and Stachybotrys have slimy spores. Phialidic ontogeny is basically rather similar to type IV—percurrent (Fig. 4.9A). Most phialides don’t change in length while producing many successive conidia, although many wall layers build up inside the open end of the cell (Fig. 4.9B). This accumulation of wall layers may eventually plug the opening, and in such phialides there is a tendency to produce sympodial extensions that develop new fertile apertures. Such phialides are called polyphialides since they have more than one conidiogenous locus.

Type VI: blastic-retrogressive conidiogenesis In Basipetospora (a thermotolerant fungus used in Indonesia in the preparation of a red food colouring), a conidium blows out at the tip of the initially undifferentiated conidiogenous hypha and is delimited by a basal cross-wall; then a short zone of the hypha just below the conidium balloons out to produce the second conidium. After this has developed its basal septum, the next segment of the hypha just below plasticizes and blows out. This process can be repeated many times. As the chain of conidia elongates, the conidiogenous hypha becomes shorter. Similar development occurs in Trichothecium and Cladobotryum, but this is a very unusual kind of conidiogenesis— in fact, prior to our time-lapse analysis of Basipetospora, it had been interpreted as being phialidic (Fig. 4.9C).

Type VII: basauxic conidiogenesis In the Pseudoidium anamorph of Erysiphe graminis, whitish or transparent bead-like chains of conidia (the ‘powdery mildew’) cover the host leaves. Each chain consists of a graded series of gradually maturing conidia, the oldest at the tip, the youngest barely differentiated from the hyphal cell just below it. New material is added at the base of the chain in a form of intercalary growth, arising from a sometimes swollen mother cell which appears to be a highly modified phialide (Fig. 4.21). Although powdery mildew anamorphs are almost ubiquitous, basauxic conidiogenesis is not common among the anamorphic fungi.

Type VIII: thallic-arthric conidiogenesis In the Geotrichum anamorphs of Dipodascus spp., an assimilative hypha stops growing and then becomes divided up into short lengths by irregularly arising septa. These are double septa which split apart schizolytically to give a ‘chain’ of short cylindrical ‘fission arthroconidia’ that disarticulates and appears jointed (hence ‘arthric’). In Oidiodendron (Fig. 4.10A), a common soil mould, the branches of a tree-like conidiophore disarticulate into conidia, ultimately leaving only the denuded ‘trunk’ (the stipe). Many basidiomycetes also produce thallic-arthric conidia.

Type IX: thallic alternate-arthric conidia In Coremiella (Fig. 4.10B) some hyphal cells degenerate to release the intervening cells as rhexolytically ‘alternate arthroconidia’. This arrangement is also seen in Sporendonema, Amblyosporium, and Geomyces but is not very common.

Chapter 4

Type X: thallic-solitary conidiogenesis The Microsporum anamorphs of Nannizzia (Fig. 4.10C), which can digest keratin and cause skin diseases in humans (see chapter 23), develop single, large, thallic phragmospores at the ends of hyphae. These conidia are liberated rhexolytically, as the final diagram shows.

Fig. 4.10 Developmental analyses of thallic conidiogenesis.

Kingdom Eumycota (True Fungi), Subkingdom Dikarya One of the earliest kinds of ‘developmental’ information was the observation that some moulds produced conidia in chains, while others did not. But looking back over the various kinds of conidiogenesis I have just described, we find no fewer than seven ways in which look-alike ‘chains’ of conidia can develop. These are seven good reasons we no longer rely on mature morphology alone to establish our classification of conidial fungi.

Importance of Conidial Anamorphs Why is it important to be able to identify conidial fungi? Some of them literally grow on you. The most prevalent fungal diseases of humans (mycoses) are caused by conidial fungi. Various superficial mycoses, ranging from ringworm of the scalp through jock itch to athlete’s foot, are caused by keratin-attacking species of Microsporum, Epidermophyton, and Trichophyton (see chapter 23). When I compiled the fungi causing important plant diseases, I found that 62 were conidial fungi (hyphomycetes and coelomycetes), as compared to 111 from all other fungal groups combined. One of the most serious outbreaks of plant disease in the last half-century was the southern corn blight, caused by Drechslera (Helminthosporium) maydis, the anamorph of Cochliobolus heterostrophus, which devastated the U.S. corn crop in 1970. Unknown to farmers, the special ‘Texas male sterile’ strain of corn that was becoming widely used for seed at that time was highly susceptible to the fungus, which produces a toxin that disrupts membranes, especially those of the mitochondria, reducing the production of ATP. The toxin also reduces photosynthesis: it inhibits uptake of potassium by the guard cells of the stomates, causing the stomates to close and thereby reducing the intake of carbon dioxide. Southern corn blight was brought under control by changing the strain of seed corn used by growers. Plant diseases are covered in more detail in chapter 12. Cellulolytic hyphomycetes cause blue stain and soft rot of wood and discolouration and loss of strength of cotton materials (the phialidic Stachybotrys is particularly troublesome in the tropics) and can cause moulding of almost any damp organic substrate. Stachybotrys chartarum is a common fungus growing on paper (such as that covering gypsum wallboard) in damp buildings and is now regarded in some quarters as a threat to human health, although there is little evidence to support this hypothesis (see chapter 8). Many moulds spoil food in storage (even in your refrigerator). Food spoilage is the subject of chapter 20 and undoubtedly causes huge losses. These may be hard to quantify, since nobody writes it down when they throw out mouldy food. Worse yet, Aspergillus flavus grows on and in peanuts and many other substrates, producing a mycotoxin called aflatoxin, which contaminates food and causes liver damage even at very low concentrations. It is perhaps the most potent carcinogenic substance known. Fusarium graminearum, growing on feed corn, produces another mycotoxin, zearalenone, which is a steroid and causes oestrogenic syndrome—vagin*l and rectal prolapse—in young female pigs. Many other mycotoxins have been discovered in recent years. They are potential threats to human and animal health of which we are only now becoming fully aware, and they have necessitated the development of new

Chapter 4 techniques for toxin monitoring and new programs for plant protection and food storage. The subject of mycotoxins is explored more fully in chapter 21. On the positive side, hyphomycetous and coelomycetous anamorphs are among the prime colonizers and decomposers of plant debris, playing vital roles in the carbon and nitrogen cycles. Hyphomycetes are important components of the saprobic soil fungi in many forests. The energy regime of many streams is based on the dead leaves of landbased plants. These are colonized by aquatic hyphomycetes, which usually form tetraradiate (four-armed) conidia, and are tolerant of low temperatures so can grow during the winter and even under ice. These fungi make the dead leaves much more palatable and nutritious for the various detritivorous invertebrates which eat them, and thus the fungi act as vital intermediaries in energy flow in northern stream systems. The terrestrial and aquatic ecology of conidial fungi is explored in chapter 11. Some soil-inhabiting hyphomycetous anamorphs have evolved specialized traps with which they catch small animals—nematodes, rotifers, tardigrades, amoebae, and even springtails. These truly predatory fungi can be visited in chapter 15. In the same chapter you can read about fungi that make spores designed to adhere to, or be swallowed by, and infect tiny animals. Conidial fungi are not just out there doing their own thing. We have also learned to exploit some of them for our own purposes. The enzymes of Penicillium camembertii produce the soft, smooth texture of Camembert and Brie cheeses. Penicillium roquefortii puts that inimitable zippy flavour in blue cheeses such as Roquefort, Danish Blue, Stilton, and Gorgonzola. Now there’s even Cambozola, which blends the buttery texture of Camembert with the assertive flavour of Gorgonzola (see www.mycolog.com). Aspergillus oryzae is used in the Far East to turn soya protein into such delicacies as soy sauce (or its sweet Indonesian variant, ket-jap, the word which became ketchup in English; see chapter 19). Other anamorphs are also exploited in Asia to prepare a variety of ‘fermented foods’, and at least one, a species of Fusarium, is now mass cultured to produce food for people (chapter 19). Finally, despite the insidious threat of mycotoxins, secondary metabolites of moulds have come to play important roles on the human stage; for example, a substance which gave Penicillium chrysogenum an edge over competing bacteria in the natural habitat became one of our most potent weapons against bacterial disease—penicillin. Cyclosporine, a secondary metabolite isolated from the mould Tolypocladium niveum, is one of the most effective and least toxic immunosuppressants yet discovered. It has enormously improved the success rate of organ transplant operations by preventing recipients’ immune systems from rejecting the implant, but not leaving them totally defenceless against infection. This substance or its derivatives also hold out some hope for treatment of severe autoimmune diseases such as juvenile diabetes, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, aplastic anaemia, Addison’s disease, Hashimoto’s thyroiditis, and systemic lupus erythematosus (SLE). See chapter 24 for a more detailed discussion of the cyclosporine phenomenon. In addition, conidial anamorphs can now be genetically transformed to act as hosts for vectors carrying multiple copies of genes from other eukaryotic organisms (including humans) and have already been persuaded to express and secrete a number of eukaryotic gene products, including insulin, human growth factor, human tissue plasminogen activator (used to dissolve blood clots), bovine chymosin (an enzyme used in

Kingdom Eumycota (True Fungi), Subkingdom Dikarya cheese making), and amylase and cellulase enzymes (see chapters 10, 24). Obviously, the biotechnological potential of the moulds is tremendous.

A Survey of Ascomycetous Holomorphs Now to put anamorph and teleomorph together and talk about the whole fungus (the holomorph). I will briefly survey the more important orders of ascomycetes, linking the different life forms together in as many cases as possible. Although mycologists now recognize sixty orders of Ascomycota (quite a few of them almost entirely lichenized), you will be (marginally) relieved to learn that I will mention only twenty-two (mainly nonlichenized ones—some of the larger lichenized orders are listed in chapter 7). That is still a large number to deal with, and you will become familiar with them only if you see them and work with them. Phylum Ascomycota is now divided into three subphyla (remember, you can Google any of these names, and Wikipedia will help you out), Taphrinomycotina, Saccharomycotina, and Pezizomycotina, the last being by far the largest, within which the orders fit as follows. Taphrinomycotina: (1) Taphrinales, (2) Pneumocystidiales, (3) Schizosaccharomycetales Saccharomycotina: (4) Saccharomycetales Pezizomycotina: (5) Pezizales, (6) Dothideales, (7) Pleosporales, (8) Capnodiales, (9) Elaphomycetales, (10) Onygenales, (11) Eurotiales, (12) Laboulbeniales, (13) Helotiales, (14) Rhytismatales, (15) Erysiphales, (16) Xylariales, (17) Meliolales, (18) Microascales, (19) Hypocreales, (20) Sordariales, (21) Diaporthales, (22) Ophiostomatales Each order has its own visual and ecological flavour, which you can learn only by experiencing them. Many of them are easy to recognize after you have seen them once.

Subphylum 1 Taphrinomycotina Order 1—Taphrinales: 9 genera, 120 species. This is an outlying group which causes serious diseases of some plants in the Rosaceae (e.g., Taphrina deformans causing peach leaf curl) and the Salicaceae (e.g., Taphrina populina on poplar). Fig. 4.11 shows Taphrina deformans attacking a peach leaf. Leaves become thickened, distorted, and often yellow or reddish in colour. This fungus has four unique or unusual features: (1) The assimilative mycelium is dikaryotic—this would immediately distinguish it from most other ascomycetes. (2) It produces an exposed layer of asci on the surface of the host leaf (Fig. 4.11). Since there is no surrounding or supporting fungal tissue, there is nothing we could call an ascoma. (3) The ascospores often bud in a rather yeast-like manner, even while still inside the ascus. (4) When the asci open to release their spores, they tend to split across the tip, rather than around it (Fig. 4.11), so they are not like the rest of the operculate group; compare them with the asci of the Pezizales.

Chapter 4

Fig. 4.11 Taphrinales: Taphrina deformans.

Order 2—Pneumocystidiales: These parasites are single celled and amoeboid and closely associated with host lung cells. Thick-walled spherical cysts containing eight spores eventually develop. The spores are released by rupture of the cyst wall. Meiosis may occur in the cysts, but we do not yet know the nuclear status of the different cell types, at least partly because none of the species will grow in pure culture. These primitive fungi have become notorious because one species, Pneumocystis jirovecii, causes a serious form of pneumonia very commonly found in AIDS patients and sometimes in other immunosuppressed or immunodeficient people such as those having had bone marrow transplants. Order 3—Schizosaccharomycetales: Schizosaccharomyces pombe, also called ‘fission yeast’, has rod-shaped cells measuring 3–4×7–14 microns. Cells grow at opposite ends and divide across the middle after laying down a transverse septum (in other words, they don’t bud, but they do appear to be thallic and to have schizolytic secession—you can see how anamorphic terminology fits in). This species was originally isolated from millet beer in Africa (pombe is the Swahili word for beer), and its method of division has since made it a good experimental organism for yeast genetics and for studies of the cell cycle (Nurse, Hartwell, and Hunt won the 2001 Nobel Prize in Physiology or Medicine for their work on these aspects of the fungus). The base sequence of its genome was published in 2002. It was only the sixth eukaryotic organism to have been entirely sequenced. Its genome has about 14 million base pairs, and it is estimated to contain just under 5,000 protein-coding genes. It has become an important model for studying cellular responses to DNA damage and the process of DNA replication.

Kingdom Eumycota (True Fungi), Subkingdom Dikarya As you can see from their position in the new phylogenetic classification, the Schizosaccharomycetales are not very closely related to most other yeasts (see chapter 6).

Subphylum 2 Saccharomycotina Order 4—Saccharomycetales: The main group of yeasts. These, being rather different in appearance and behaviour from most other fungi, are dealt with separately in chapter 6 (check it out). For now I will content myself by saying that yeasts are frequently unicellular (although some produce hyphae) and are often involved in fermentative processes, such as the production of alcoholic beverages, bread, and some Asian fermented foods. Some yeasts are also active human pathogens. Yeasts usually exist in the anamorphic condition (see chapter 6).

Subphylum 3 Pezizomycotina This huge assemblage embraces ten major classes. I am going to list six of these, but only to establish baselines for the twenty-two orders I will briefly characterize. I will mention and illustrate examples of many of the families in the group because they are common, conspicuous, well known, or particularly interesting. Most are illustrated at www.mycolog.com.

Class 1 Pezizomycetes Order 5—Pezizales (have unitunicate-operculate asci):

150 genera, 900 species; the ‘oper-

culate discomycetes’. (a) Family Pezizaceae. Classic ‘cup fungi’ producing apothecial ascomata that are usually shaped more like saucers or goblets, usually without stalks, and found growing on wood, dung, or soil. They vary so much in size, colour, texture, and ornamentation that most discomycete specialists split the Pezizaceae into several tribes or even families. Their asci have a diagnostic pop-open lid or operculum (Figs. 4.3, 4.5A), and the tips of the asci are amyloid (sometimes expressed as I+ or J+; this means giving a blue, starchlike reaction in an iodine solution known as Melzer’s reagent). A small species of Peziza often crops up on soil in greenhouses, frequently preceded or accompanied by its blasticsynchronous Chromelosporium anamorph (Fig. 4.12A). Larger species of Peziza, producing thin, rather brittle apothecial ascomata several centimetres across, with light brown or orange hymenia, can be found on the ground in spring and fall. The exposed hymenium of each of these ascomata contains many millions of asci, and if you find a ripe specimen and blow on it, you can sometimes provoke a massive synchronized spore-shooting, triggered by the change in humidity, in which the thousands of simultaneously expelled ascospores look like a puff of smoke. (b) Family Sarcosomataceae. Wood-inhabiting fungi with apothecia that are often stalked, relatively tough, and brightly coloured. The asci are suboperculate and nonamyloid. The scarlet cups of Sarcoscypha coccinea, growing from buried hemlock branches, brighten up the early spring in eastern Canadian woodlands. The brightly coloured, stalked apothecia of Cookeina are a common sight in the Neotropics and may provide a camouflaged perch for the tiny but equally colourful poison arrow frog, as one National Geographic cover showed.

Chapter 4

Fig. 4.12 Representative Pezizales (Unitunicatae-Operculatae) and truffles.

(c) Family Otideaceae. Scutellinia scutellata, its orange apothecia rimmed with dark hairs (which give it the name ‘eyelash fungus’; Fig. 4.12D) and producing nonamyloid asci, is one of the commonest cup fungi, growing on rotting wood. Anamorphs don’t seem to be produced in this family. Some genera, such as Genea (Fig. 4.13), produce closed but hollow ascomata. The asci are cylindrical or clavate and are arranged in a flat hymenium lining the ascoma, but they do not shoot their spores. These apparently contradictory features show that members of this family are becoming sequestrate (meaning that their fruit bodies do not liberate spores at maturity) and hypogeous (which means that they produce

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.13 Suggested evolutionary sequence from epigeous to hypogeous Pezizales.

their ascomata underground). Another member of this family, Geopora (Fig. 4.13), is also hypogeous, but now the air space inside the fruit body is much less than that in Genea—another step on the way to becoming a truffle (see Fig. 4.12 and family Tuberaceae). This evolutionary process is diagrammatically illustrated in Fig. 4.13. The fourth and final step sees the elimination of air spaces altogether and is a solid truffle of the genus Tuber, family Tuberaceae. Evolution toward the sequestrate and hypogeous condition is not restricted to the Otideaceae but can also be seen operating in several other families of the operculate discomycetes. (d) Family Ascobolaceae. Students who have followed the succession of fungal fructifications appearing on horse dung (and that should be all students of mycology) will be familiar with the two most important genera of this largely coprophilous family— Ascobolus and Saccobolus. Both produce minute, translucent apothecia (seen under the dissecting microscope, Figs. 4.2, 4.4A, 4.12C). The dark dots are mature asci, which are broad and project from the hymenium when mature, so that their tips may orient themselves to point toward the light. The ascospores have a purple or brown outer wall layer. Ascobolus (Fig. 4.2), like most other ascomycetes, shoots ascospores individually. Saccobolus atypically sticks all eight ascospores together in a bundle, and they are

Chapter 4 expelled as a single projectile, which gives them extra range (see the website). I haven’t personally seen any anamorphs in this family, although a few are known. (e) Family Helvellaceae. These mostly spring-fruiting fungi have large and unusually configured apothecial ascomata. All are stalked, with beige to brown, hymeniumcovered caps. Many Helvella species (Fig. 4.12E) have a drooping flap on either side and for that reason are called saddle fungi. The ascomata of Gyromitra species are among the largest ascomycete fructifications (one species has the rather extravagant species epithet gigas), and some species contain the toxin gyromitrin, a precursor of the deadly monomethylhydrazine. By causing some fatal poisonings despite its name, the springfruiting Gyromitra esculenta (Fig. 22.4) has earned its place in chapter 22 on poisonous mushrooms. It is vital for morel hunters to be able to distinguish the convoluted head of Gyromitra, the false morel, from the ridged and pitted head of the delicious true morel (Fig. 18.2; see below). (f) Family Morchellaceae. While Gyromitra, above, is one of the few lethally toxic fungi, its cousin Morchella, the true morel (Fig. 18.2), is one of the finest of all edible fungi. Species of Morchella have a broad, hollow stalk and a pitted and ridged, spongelike, more or less conical head. Since the hymenium doesn’t cover the ridges, it seems likely that a morel is a compound ascoma, each pit representing an individual apothecium. The anamorph of the morel is a blastic-sympodial hyphomycete, Costantinella, which I often found growing on soil by trails in Algonquin Park, Ontario, in the fall (note that the teleomorph fruits in spring). Morels have a broad geographic range but are common in relatively few areas, of which Michigan is perhaps the best known. People throng to the woods in May to hunt this elusive delicacy, and Boyne City hosts a popular annual morel-hunting championship. When Dutch elm disease was killing millions of elm trees, morels sometimes fruited profusely around recently dead trees. In recent years they have also been collected in large numbers on burned-over areas of western forests. Morels are discussed as a delicacy in chapter 18. Just to confuse the issue a little, a second genus of Morchellaceae, Verpa, also fruits in May. Species of Verpa aren’t toxic, but neither are they good to eat. The wrinkled thimble-cap, Verpa bohemica, looks like a morel, but it is easy to tell the difference by bisecting the fruit bodies. While the cap and stalk of the true morels are firmly united, the cap of Verpa is attached only at the apex. In addition, the stipe of Verpa is ‘stuffed’ with cottony mycelium, while that of Morchella species is completely hollow. (g) Family Tuberaceae—the truffles. Here, the evolutionary process still active in the Otideaceae has run its course. The ascomata are sequestrate, hypogeous, and solid (no air spaces anymore—as you can see in the bisected specimen of Tuber aestivum in Fig. 4.12F, which a truffle dog brought to me at Scheggino in Italy). The asci of truffles, produced in a highly convoluted hymenium, are rounded and thin walled (Fig. 4.12G), with no trace of an operculum or other shooting mechanism, and usually contain only one to three spores. The ascospores of truffles have complex, highly ornamented walls. They come in two basic patterns—spiny and lacunose (Fig. 4.12G). Only by examining a series of microscopic characteristics—and by considering some intermediate forms that trace the probable course of evolution in the group (Fig. 4.13) or by comparing DNA sequences—can we tell that these fungi are related to the ‘operculate discomycetes’.

Kingdom Eumycota (True Fungi), Subkingdom Dikarya Although it doesn’t make taxonomy any easier, we must now logically place these hypogeous (underground) families with their epigeous (aboveground) forebears in the order Pezizales. The hypogeous habit has necessitated the evolution of new methods for passive spore dispersal, in which some agency other than the fungus supplies the energy for dispersal. Members of the Tuberaceae, especially species of the genus Tuber (the true truffles), have achieved this by developing what can only be called fascinating smells. These odours are released when the ascospores are mature and lead many mammals unerringly to the ascomata, which they unearth and consume, subsequently depositing the still-viable spores elsewhere. Tuber melanosporum, the black diamond, Queen, or Perigord truffle of French gastronomy, is dependent not only on mammalian vectors but also on the roots of oak and hazelnut trees, with which it establishes a symbiotic ectomycorrhizal relationship (see chapter 17). Tuber melanosporum and Tuber magnatum are, respectively, the black and white truffles of French and Italian haute cuisine, perhaps the most highly esteemed (and certainly the most expensive) of all edible fungi and so are discussed in detail in chapter 18.

Class 2 Dothideomycetes (produce bitunicate asci) Fifty families, 650 genera, 6,300 species. Phragmoseptate or dictyoseptate ascospores are common in this class; in fact, if a fungus has ascospores of this kind, the odds are about nine to one that it is a member of the Dothideomycetes. Coelomycetous anamorphs are common in this group.

Order 6—Dothideales

(a) Family Venturiaceae. Venturia inaequalis causes apple scab, an economically important disease. You’ll find the Spilocaea pomi hyphomycetous anamorph causing large brownish spots on the leaves and disfiguring blackish scabs on the fruit. It produces its blastic-annellidic (percurrent) conidiogenous cells (in Fig. 4.14A you can see the rings clearly) and obclavate conidia on those spots and scabs. But you won’t find the teleomorph during the growing season. Its pseudothecial ascomata (seen in section in Fig. 4.14A) develop slowly in the dead apple leaves over the winter, and the ascospores are shot in spring when the susceptible young leaves appear. Apiosporina morbosa causes the extremely common and disfiguring black knot disease of some rosaceous trees, especially wild cherry and damson plum, its pseudothecial ascomata developing on conspicuous black fungal stromata; you shouldn’t have too much trouble spotting these on wild cherry trees. This fungus damaged and disfigured my own damson plum tree despite my best efforts at controlling it by pruning.

Order 7—Pleosporales

Pleospora occurs on dead herbaceous stems and has anamorphs in the common hyphomycete genus Stemphylium (Fig. 4.14B) and the coelomycete genus Phoma. The even more common mould Alternaria is the anamorph of the less frequently found Lewia.

Order 8—Capnodiales

Commonly known as ‘sooty moulds’, these fungi grow on the sugary excreta of various insects such as mealy bugs and scale insects. I have found blankets of their thick black mycelia covering the trunks and leaves of southern beech trees (Nothofa*gus) in the forests of South Island, New Zealand (Fig. 4.15A, B). The black, feathery branches of the numerous synanamorphs are easily seen by the naked eye, but soots frequently grow so intermingled that it is very difficult to sort out

Chapter 4

Fig. 4.14 Representative Dothideales (Bitunicatae).

the various allegiances. Sooty moulds occur in many warmer and wetter parts of the world.

Class 3 Eurotiomycetes (many have prototunicate asci) Order 9—Elaphomycetales: One genus, twenty species.

At first sight, the hypogeous ascomata of Elaphomyces look just like truffles; and they’re even called ‘deer truffles’. But they have no discrete hymenium—the basically spherical,

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.15 A: Epiphytic sooty moulds growing on a twig; B: Mixed colonies of sooty moulds covering the normally light-coloured bark of Southern Beech trees (Nothofa*gus) in New Zealand.

Chapter 4 nonshooting asci are produced randomly throughout the interior of the ascoma. Elaphomyces no longer offers much in the way of visual clues about its possible epigeous ancestors, so only molecular techniques can help us decide its relationships. Order 10—Onygenales: 40 genera, 120 species. Here belong some unusual fungi which cause skin diseases in people and can digest hair, horn (see the website), and feather—all because some of them have the unusual ability to metabolize the resistant protein, keratin. Family Arthrodermataceae contains the infamous dermatophytes which cause superficial mycoses ranging from the inappropriately named ‘ringworm’ of the scalp to another misnomer, ‘athlete’s foot’ (you certainly don’t need to be athletic to catch it); see chapter 23. Other members of the Onygenales can degrade cellulose, and yet others are coprophilous (dung inhabiting). They all produce ascomata, but although these are theoretically cleistothecial, their walls are often very loosely woven, and in some the ascospores can simply fall out through the gaps. The asci are always more or less spherical, never shoot their spores, and tend to break down at maturity. Because of my earlier conclusion that asci evolved as spore-shooting devices, I assume that the ascoma and asci in the Onygenales are ‘reduced’ forms, simplified during evolution from an earlier spore-shooting design. The ascomata often bear highly characteristic coiled or branched appendages that can make identification easy—if the teleomorph is present. The ascospores, appendages, ascomata, and anamorphs of various genera are illustrated in Fig. 4.16. If you isolate dermatophytes in pure culture, they may or may not produce teleomorphs. But they will develop characteristic thallic conidial anamorphs (Fig. 4.16). Sometimes these produce small, thallic-arthric conidia (Chrysosporium or Malbranchea); sometimes large, spindle-shaped, transversely septate, solitary thallic conidia (Trichophyton or Microsporum); and sometimes the same culture will produce both kinds of conidia. When a fungus has two or more different anamorphs, these are called synanamorphs. The three most important anamorph genera of dermatophytes are Epidermophyton, Microsporum, and Trichophyton. Of these, Epidermophyton has no known teleomorph, nine species of Microsporum have teleomorphs in Nannizzia, and seven species of Trichophyton have teleomorphs in Arthroderma. Order 11—Eurotiales: 50 genera, 140 species. This largely cleistothecial order contains the teleomorphs of some of the most successful of all anamorphs—the common green and blue moulds of the hyphomycete genera Penicillium and Aspergillus. Aspergillus conidiophores have characteristic apical vesicles (Figs. 4.8 and 4.17A); the apices of Penicillium conidiophores are brush-like (Fig. 4.8). These ubiquitous and almost omnivorous anamorphs are blastic-phialidic and produce masses of dry, wind-dispersed conidia. These moulds aren’t just extremely successful, they are of considerable importance to us because they produce antibiotics and mycotoxins and cause huge amounts of food spoilage. Species of Aspergillus have teleomorphs in Eurotium (sections through cleistothecial ascomata are shown in Figs. 4.4D and 4.17A) or Emericella, while many penicillia have teleomorphs in the rather similar Talaromyces or Eupenicillium. The cleistothecial ascomata of the teleomorphs have impermeable walls one or more cells thick. The asci (Fig. 4.17A) are scattered throughout the cavity of the ascoma (i.e., never in a hymenium); they are spherical

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Families of the Onygenales Family

Ascospores

Peridia and Appendages

Anamorphs

Substrate* and Habitats

carnivore dung

ONYGENACEAE

soil enriched with keratin or dung

pitted; spherical, oblate, aliantoid

CHRYSOSPORIUM MALBRANCHEA SPORENDONEMA

ARTHRODERMATACEAE

KERATIN*

decaying hoof, horn, feathers, hair, and skin

smooth; oblate to oblate-discold or oblate-convex

CHRYSOSPORIUM MICROSPORUM TRICHOPHYTON

some are parastic on animals

processed or decaying plant materials

MYXOTRICHACEAE

CELLULOSE*

smooth or striate; fusiform, ellipsoid

GEOMYCES MALBRANCHEA OIDIODENDRON

paper, straw, soil around the roots of plants decaying vegetation soil rich in organic matter

GYMNOASCACEAE

VARIABLE* smooth or slightly irregular (’lumpy’); often with polar and/or equatorial thickenings

Fig. 4.16

Families of the Onygenales.

absent or of un-named arthroconidia

various types of dung

Chapter 4 and thin walled and break down when the spores mature. The ascospores often have a pulley wheel shape. Again, it is thought that these fungi are ‘reduced’ derivatives of spore-shooting ancestors.

Fig. 4.17 Prototunicatae. A: Eurotiales; B: Ophiostomatales.

Class 4 Laboulbeniomycetes Order 12—Laboulbeniales: 75 genera, 1,700 species (Fig. 4.18).

All are invariably found attached to the exoskeleton of insects or occasionally millipedes and mites. The development of Stigmatomyces baerii, which is found on houseflies, is followed in Fig. 4.18C. An ascospore becomes attached to the animal, germinates, and sends a foot into the exoskeleton to absorb nutrients. Although haustoria may penetrate as far as the epidermal cells, puzzlingly there is never any real invasion of host tissues. The ascospore develops a median septum, and the upper cell becomes differentiated into a male organ, with several phialide-like cells that produce spermatia. The lower cell then develops an ascogonium with a trichogyne, which is fertilized by the spermatia. Several asci then develop from the ascogonium and eventually deliquesce. The mature ascoma is spine-like, projecting from the exterior of the host, and can be seen with a hand lens. Other genera exhibit the same basic features (Corethromyces, Fig. 4.18A; Hesperomyces, Fig. 4.18B). The Laboulbeniales apparently don’t produce anamorphs, so are presumably spread from animal to animal by adhesive ascospores during mating of the hosts or when insects form dense swarms. This goes some way toward explaining the almost incredible site specificity of many Laboulbeniales. Various species are restricted to one part of the insect, for example, one side of a particular left limb, or even to one sex of their host, although most species are not quite so limited.

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.18 Representative Laboulbeniales: A: Corethromyces; B: Hesperomyces; C: Stigmatomyces.

Now for the Classical Unitunicate-Inoperculates Although none have lids (opercula), the asci of this group are not as uniform in appearance or structure as we might like (Fig. 4.19). Most have thickened walls at their tips pierced by a fine pore. Inside the apices, many have diagnostic sphincter-like rings, which control the expulsion of the spores. Some of those rings are amyloid, or I+ (they stain blue in iodine); others don’t react with iodine and are called chitinoid; some don’t have rings at all.

Chapter 4

Fig. 4.19 Unitunicate-inoperculate asci. A: Nectria (Hypocreales); B: Sordaria (Sordariales); C: Melanconis (Diaporthales); D: Claviceps (Clavicipitales); E: Microglossum (amyloid ring, Leotiales); F: Rosellinia (amyloid ring, Sphaeriales); G: Lecanora (Lecanorales).

Class 5 Leotiomycetes (have unitunicate-inoperculate asci) Order 13—Helotiales: 13 families, 400 genera, 2,000 species.

These are the classical ‘inoperculate discomycetes’. The apothecial ascomata are superficially similar to those of the Pezizales but are often stalked; the asci are inoperculate and usually have amyloid apical rings. This suggests to me that the two major kinds of apothecial ascomata are examples of parallel or convergent evolution. Several of the families in this order are common and well known, so five of them are dealt with below. (a) Family Sclerotiniaceae. As the name implies, these frequently parasitic fungi often form sclerotia, which may be solid masses of fungal tissue or may be of mixed origin—fungal hyphae riddling a mummified host such as a peach, plum, or cherry or a tree catkin. Having overwintered in this guise, they germinate in spring and use the stored energy to produce stalked apothecial ascomata (Fig. 4.20A). Ascospores, the primary inoculum, are shot when the host is in flower and gain entrance through the stigma. The anamorphs are generally responsible for secondary dispersal during the growing season, and some cause serious plant diseases. For example, the soft brown rot of peaches (Fig. 4.20A) and cherries is produced by a Monilia anamorph of Monilinia. The beige or greyish powder on the surface of the peach and the cherry are made up of branched chains of conidia. The longer I left the ripe cherries on my cherry tree, the more of them succumbed to the Monilinia soft brown rot, as the conidia being produced on one cherry infected others (see chapter 12). Another Monilinia produces spur blight of wild cherry, killing back young shoots and forming new conidia on the leaves. Many members of this family have distinctive anamorphs, while the teleomorphs are relatively uniform. So some of the genera erected for the teleomorphs have rather atypically been distinguished by characteristics of their anamorphs—and even named after them. So we have Sclerotinia with Sclerotium (sclerotial) anamorphs, Monilinia with Monilia anamorphs (blastic-acropetal), Botryotinia with Botrytis anamorphs (blasticsynchronous), and Streptotinia with Streptobotrys anamorphs (blastic-sympodial). Sclerotium, Monilia, and Botrytis cause serious plant diseases—grey mould of strawberry is caused by Botrytis cinerea (chapter 12), but when Botrytis grows on overripe grapes in certain areas of Canada, France, Germany, Hungary, and South Africa it is

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.20 Representative ‘inoperculate discomycetes’. A: Monilinia and its Monilia anamorph; B: Geoglossum; C: Leotia; D: Phacidium and its Ceuthospora coelomycetous anamorph.

called the ‘noble rot’ (pourriture noble, Edelfäule) because the small quantities of sweet dessert wine that can be made from such shriveled grapes have intense and exquisite flavour and can be sold for very high prices. Find out what a bottle of Chateau d’Yquem sauternes from France (or a Trockenbeerenauslese from Germany) costs at your local wine store: be prepared for a shock. The full story can be found in chapter 19.

Chapter 4 (b) Family Phacidiaceae. Some Phacidium spp. cause snow blight diseases of conifers. If we look more closely, we will see that this family is not typical of discomycetous fungi in general, since the ascomata develop inside host tissue and are at first covered by a roof of dark fungal tissue (Fig. 4.20D). But at maturity the roof splits open and exposes the hymenium. The apical ring in the asci is amyloid (I+). Compare this family with order 14, Rhytismatales, below. How do the orders differ? Phacidium has coelomycetous anamorphs: those of pathogenic species such as P. coniferarum belong to Apostrasseria, while those of saprobic species like P. betulinum belong to Ceuthospora (Fig. 4.20D). (c) Family Geoglossaceae—literally ‘earth tongues’—produce unusual stalked, somewhat flattened and tongue-like ascomata which emerge from the ground (Fig. 4.20B). The hymenium doesn’t line a cup or saucer but covers the convex upper surface of the ascoma, which is fleshy and yellow in Spathularia, tough and black in Microglossum, Geoglossum, and Trichoglossum. If you squash a tiny piece of the hymenium of a mature Geoglossum ascoma, you will see the asci, each of which contains a bundle of eight long, parallel, phragmoseptate brown ascospores (Fig. 4.20B). (d) Family Leotiaceae contains more typical ‘discomycetes’, such as Bisporella, which produces those tiny bright yellow discoid apothecia so common on fallen, decorticated tree trunks, while Chlorociboria, also fairly common, stains wood green and forms small green apothecia. Less typical are the spectacular ascomata of Leotia (Fig. 4.20C); these are much larger, are stalked and jelly-like, and have convex fertile heads, a beautiful velvety green in Leotia viscosa, that contrasts with its vivid yellow stipe. Another rather spectacular member of this order is Bulgaria inquinans, found on wood of deciduous trees. The clustered apothecial ascomata have a rubbery texture, and the hymenium is jet black. All of the above are illustrated on the website. (e) Family Dermateaceae includes Diplocarpon rosae (which, with its Marssonina coelomycetous anamorph, causes the dreaded black spot of roses) and another common fungus, Trochila ilicicola, which fruits on the upper surface of dead leaves of holly (Ilex) in our garden. Its ascomata have a hinged lid which opens when the leaf is kept in a damp chamber. Order 14—Rhytismatales: 70 genera, 400 species. The ascomata, like those of the Phacidiaceae, develop immersed in host tissue or a fungal stroma, which ultimately ruptures to expose the hymenium. The asci often have apical rings, but these are small and chitinoid (do not stain blue in iodine). The ascospores are usually long and thin and have a slimy sheath (absent in the Phacidiaceae). The genus Lophodermium is sometimes endophytic and asymptomatic in pine needles for much of its life but eventually fruits after the needles die (see chapter 11). Rhytisma acerinum causes ‘tar spot’ of red maple leaves in eastern North America. Rhytisma punctatum produces a similar syndrome on big-leaf maple in western North America, but the small, individual ascomata do not fuse (see the website). Order 15—Erysiphales: 28 genera, 100 species. All members of this order are obligate parasites on leaves and fruits of higher plants, causing diseases called powdery mildews. These fungi have a superficial mycelium which extracts nourishment from the host plant through specialized hyphae that penetrate the epidermal cells of the host and develop absorbing organs called haustoria (Fig. 4.21). You should have no difficulty spotting a few powdery mildews in summer because their whitish colonies growing on living leaves are unlike anything else. In dry

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.21 Representative Erysiphales.

summers they are particularly common on grass in shady parts of lawns, on squash plants (cucurbits), on perennial Phlox, on Alnus rugosa, and on many other angiosperms. Basauxic chains of conidia of the Oidium-like anamorph (Fig. 4.21), whose powdery, whitish appearance gives these diseases their name, arise from the mycelium in early summer. Airborne conidia spread the disease from plant to plant and are later succeeded by dark ascomata, which mature slowly in fall and release ascospores the following spring. This order parasitizes well over 1,000 higher plant species, and the powdery mildews of grapes, hops, gooseberries, and cereals are economically important diseases.

Chapter 4 The generic concepts in the teleomorphs of this order are unusually straightforward and easy to apply, since they depend on two major features of the ascoma: (1) the number of asci within it, and (2) the kind of appendage growing out from it (Fig. 4.21). In one way, the Erysiphales are the antithesis of the Sclerotiniaceae. There, the anamorphs were far more distinctive and diverse than the teleomorphs; here, the reverse is true. Most anamorphs of the Erysiphales belong to the hyphomycete genus Oidium or other very similar genera. Although the order Erysiphales is very easy to characterize and recognize, its systematic position is controversial. Some mycologists insist that its asci are bitunicate, which would place it alongside the Dothideales (see above), but many mycologists do not accept this and place the order among the unitunicate ascomycetes. The asci are sometimes rather thick walled, but one of the world experts on the group, Dr. Zheng Ru-yong of Beijing, told me that she had seen distinctive inner and outer wall layers only in an undescribed taxon from Tibet and had never seen the ‘jack-inthe-box’ mechanism so typical of bitunicate asci. The asci seem to have neither an operculum nor an apical ring apparatus. This information, plus their strange arrangements for dispersal and dehiscence (see chapter 8), their unique basauxic anamorphs, and their obligately parasitic yet strangely superficial lifestyle, make them a rather atypical (although important and interesting) group. Alder (Alnus) leaves often bear extensive colonies of Phyllactinia. Under the dissecting microscope you can see ascomata with unique appendages (Fig. 4.21). The basal bulbs of the appendages develop first, then the needle-like extensions grow out. At maturity, these appendages all bend downward in unison and lever the ascoma off the leaf surface, breaking its connections with the mycelium. It is then free to be blown or splashed away, becoming attached to a new substrate by an apical blob of mucilage secreted by specialized hyphae. Paradoxically, this leaves the asci, which are designed to shoot their spores, facing downward. The final chapter is written when the ascoma splits around the equator at a built-in line of weakness and hinges open so that the spores can finally be shot away (Fig. 8.3).

Class 6 Sordariomycetes (have unitunicate-inoperculate asci) 225 genera, 1,300 species. Many members of this order produce dark, brittle, globose to pear-shaped individual perithecial ascomata with prominent ostioles (narrow apical openings; Fig. 4.22A). Others have many perithecial cavities immersed in a single stroma to form a compound fructification (Fig. 4.22C, D). The asci often have an apical ring or sphincter, which is usually, although not always, amyloid (stains blue in iodine). Thread-like, sterile elements called paraphyses are present between the asci in the hymenium of some members but absent from others. Ascospores can be light or dark, simple or septate, with or without germ pore or slit, sometimes with gelatinous sheaths or appendages. The compound fructification of Xylaria, a common wood-inhabiting genus (Fig. 4.22C), has hundreds of individual perithecial cavities just below the surface. Each of these contains many asci. The asci are inoperculate, with an amyloid apical ring, and contain eight darkly pigmented, asymmetrical ascospores. These will eventually be shot out through the narrow ostiole.

Order 16—Xylariales:

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.22

Representative perithecial ascomycetes.

This order also includes such pathogens as Hypoxylon mammatum, which causes poplar canker, a disease that kills millions of trees every year. The extensive, more or less elliptical cankers develop groups of perithecial ascomata after the tree cambium has been killed. Family Diatrypaceae, with its extensive stromatic multiascomatal fructifications spreading out under bark of trees, and its tiny sausage-shaped ascospores (Fig. 4.22D), has now been folded into the Xylariales

Chapter 4 Order 17—Meliolales

These are in some ways tropical, darkly pigmented analogues of the Erysiphales (powdery mildews). They are obligate biotrophs that live superficially on their hosts, anchored by numerous conspicuous hyphopodia and deriving food from haustoria that penetrate only the epidermis. Although their spreading black colonies look ominous, they do not usually cause serious damage to their hosts (see the website).

Order 18—Microascales

Ceratocystis fa*gacearum and its Chalara quercina phialidic anamorph are the cause of another widespread and serious tree disease, oak wilt. The teleomorphs of Ophiostoma (Ophiostomatales, below) and Ceratocystis are very similar, but the genera are easily distinguished by their anamorphs: the Chalara anamorphs of Ceratocystis have solitary phialides with long, tubular collarettes and form long, cylindrical conidia (Fig. 4.8). Order 19—Hypocreales: 80 genera, 550 species. This order is recognized by its brightly coloured, simple or compound, perithecial ascomata—usually yellow, orange, or red—which are fleshy or waxy in texture and usually borne on a supporting layer of mycelium (a subiculum) or in stromata. Four genera are especially well known, and the family Clavicipitaceae is among the most fascinating of all fungi, as you will read. (a) Nectria (Fig. 4.23) often has bright red, superficial perithecia containing twocelled (didymosporous) ascospores. Some species cause cankers and diebacks of trees. Nectria sensu lato has a variety of conidial anamorphs (Fig. 4.24), but all of them are phialidic. The erumpent sporodochia of one commonly encountered anamorph, Tubercularia (Fig. 4.23), cause a condition known as coral spot. As you can see, the Tubercularia anamorph often grows beside the dark red Nectria perithecial ascomata. It is interesting and a little unusual to see both phenotypic expressions of the genome being produced simultaneously. However, the most economically important of the nectriaceous anamorphs are certain Fusarium species (Figs. 4.24, 14.2, 21.1B), many of which cause destructive wilt diseases of higher plants or produce dangerous mycotoxins. (b) Gibberella also has Fusarium anamorphs. A member of this genus causes a disease of rice called ‘foolish seedling’ in which seedlings grow too rapidly and consequently fall over. The active principle, a plant growth hormone called gibberellic acid, has been extracted and is now widely used to study and stimulate plant growth. (c) Hypomyces lactifluorum is an orange-red fungus which parasitizes the agaric (mushroom) genera Lactarius and Russula, producing a layer of tissue that completely covers the gills and suppresses their development and then develops thousands of bright orange-red perithecial ascomata all over the surface of the host. The Hypomyces completely envelops the aborted mushroom, and its colour gives the host-parasite combination the name ‘lobster fungus’. Strangely enough, this monstrosity is edible, although I regret to have to tell you that it does not taste like the divine crustacean. Hypomyces has extremely characteristic spindle-shaped, two-celled, colourless ascospores. The anamorphs of Hypomyces species belong to the hyphomycete genus Cladobotryum, which has an unusual blastic-retrogressive method of forming conidia (type VI above). (d) Hypocrea forms fleshy stromata on wood. The dark spots on these stromata are the ostioles of the embedded perithecial cavities. The teleomorph of Hypocrea,

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.23

Hypocreales: Nectria cinnabarina and its Tubercularia anamorph.

although relatively common, is recorded far less often than its green-spored, phialidic anamorph, Trichoderma (Fig. 14.3), which, because it is a broad-spectrum mycoparasite and produces cellulases and antibiotics, is one of the most important moulds in forest soils. It is now being exploited in biological control of pathogenic fungi (see chapter 14) and in the production of enzymes which can convert cellulose to glucose (chapter 24).

Chapter 4

Fig. 4.24 Hypocreales: anamorphs of Nectria sensu lato. Arrows indicate possible lines of derivation.

Family Clavicipitaceae: 27 genera, 270 species. This order comprises a group of highly evolved and sophisticated, all obligately parasitic fungi with (a) frequently stalked, all-fungus stromata (Fig. 4.25B–E); (b) very long asci without apical rings but with thickened tips (Fig. 4.25F); and (c) extremely long, thread-like ascospores that in some taxa fragment at or following release (Fig. 4.25F). Three bizarre and spectacular genera, Claviceps, Cordyceps, and Epichloë, will give us a snapshot of this fascinating family. (a) Claviceps purpurea (Fig. 4.25A–C) discharges its ascospores when its main host, rye, is in flower, and infection takes place through the stigma. As the infection progresses, the fungus takes over the food being channeled into seed production by the host. The ovarian tissues are replaced by a mycelial mat that produces masses of conidia of the Sphacelia anamorph in a sweet-smelling nectar. Insects are attracted to the nectar and spread the conidia to other host plants. The mycelial mat hardens and becomes a purplish sclerotium—the ergot—which replaces the grain (Figs. 4.25A, 4.26). I have found very large ergots on Elymus mollis, a large grass that grows along the shore in the Pacific Northwest. The largest ergot was 4 centimetres long and almost 5 millimetres wide. These sclerotia fall to the ground in autumn, overwinter, and germinate the following spring, each producing several stalked stromata (Fig. 4.25B, C). Each stroma has a spherical head within which many perithecia develop just below the surface. Each perithecial cavity contains many asci, each ascus having eight extremely long ascospores that are forcibly ejected at maturity. Because this fungus has a small target, the stigma of the grass flower, which is available only during a narrow time window, and because spores reach it only by chance, the fungus must disperse a large number of ascospores in a short time. A rough

Kingdom Eumycota (True Fungi), Subkingdom Dikarya

Fig. 4.25 Representative Clavicipitaceae. A–C: Claviceps; D–F: Cordyceps.

calculation suggests that a single ergot can give rise to 5 stromata, and each of those may contain 100 perithecial cavities, each cavity with 50 asci, and each ascus producing 8 ascospores: a total of 5 × 100 × 50 × 8 = 200,000 propagules per ergot. If the sclerotia are accidentally consumed by cattle, or if rye bread made from ergoty rye is eaten by humans, a large number of alkaloids found in the ergot cause a form of poisoning known as ergotism, or, more picturesquely, St. Anthony’s Fire. Human victims frequently hallucinate and feel that they are burning (see chapter 21 for a fuller account of this mycotoxicosis). The alkaloids ergotamine and ergotaline

Chapter 4

Fig. 4.26 Ergots on a grass.

cause contractions of the smooth muscles, and the ensuing restriction of the peripheral blood supply can lead to gangrene and even death. St. Anthony’s Fire was fairly common in the Middle Ages, and sporadic outbreaks occurred until recently. Ergot, the only fungal structure in the British Pharmaceutical Codex, has been used in obstetrics both to induce childbirth and to control postpartum bleeding. Another species of Claviceps brought the genus renewed fame, or perhaps I should say notoriety, as the prime source of LSD (lysergic acid diethylamide), one of the most powerful psychedelic drugs (it is a hundred times more potent than psilocybin, the active ingredient of ‘magic’ mushrooms). (b) Cordyceps species (Fig. 4.25D–F) are bizarre: they generally parasitize insects or spiders or hypogeous (underground) fungi, and their large stromata spring up directly from their victims. These perithecial stromata arising from an insect larva or pupa are known as ‘vegetable caterpillars’, in recognition of the fact that they always incorporate elements from more than one kingdom. These strange ‘two-kingdom’ structures (see the website) are used in traditional Chinese medicine as a treatment for many conditions: general debility after illness, weakness, spitting of blood caused by TB, chronic coughing and asthma, night sweating, anaemia, and even malignant tumours—you name it. As mentioned, a few species of Cordyceps don’t pick on arthropods but cannibalistically attack another fungus. Actually, it’s even another ascomycete—the underground

Kingdom Eumycota (True Fungi), Subkingdom Dikarya deer truffle (Elaphomyces). The large, stalked stroma of the Cordyceps can be seen emerging from the host truffle in Fig. 4.25E. Every September for many years, during a mycology field course I taught, we found Cordyceps parasitizing Elaphomyces along one of the hiking trails in Algonquin Park, Ontario. Once one of the students had spotted the club-shaped stroma of the parasite, excitement ensued as we dug down, following the yellow rhizomorphs of the fungus, until we finally excavated the host. This find was often dubbed—and with good reason—‘fungus of the day’, although perhaps the first word of that title should have been pluralized. Cordyceps, which must infect target organisms that are clearly far scarcer than rye flowers, goes a big step further than Claviceps in the multiplication of spores. Each of the eight long ascospores breaks up into about 100 part-spores, often while still in the ascus (Fig. 4.25F). I estimate that the usually single large stroma produced by this genus from its fungal or insect host may bear as many as 800 perithecial ascomata, each containing at least 100 asci, each ascus containing 8 spores, and each of them fragmenting into 100 part-spores, for a total of 800 × 100 × 8 × 100 = 64,000,000 propagules: 64 million airborne spores from a single stroma. In 1996 Cordyceps subsessilis was discovered to be the teleomorph of Tolypocladium niveum. So what? you might say, until you realized that Tolypocladium inflatum is the fungus that produces the medically important, selective immunosuppressant cyclosporine, which has made the organ transplant revolution possible. For the story of that amazing pharmaceutical, see chapter 24. (c) Epichloë causes ‘choke’ disease of grasses. A grass called Glyceria normally produces open, nodding inflorescences. When Epichloë attacks, the energy for the inflorescences is stolen by the fungus and used to produce creamy yellow perithecial stromata, each incorporating many perithecia, which surround the stalk of the now-sterile grass (see the website). In a recently discovered twist to this story, this apparently damaging parasitic fungus has been found to have a mutualistic symbiosis with the grass. Epichloë’s simple, phialidic anamorph, Neotyphodium (which looks very like Acremonium), grows systemically throughout the grass plant without producing any disease symptoms and actually protects the grass from herbivores by producing a virulent neurotoxin. A more detailed discussion of this relationship is given in chapter 21. Order 20—Sordariales: 5 families, 75 genera, 600 species. This is a generally saprobic group producing solitary perithecial ascomata and found on dung or decaying plant remains. Their asci sometimes have nonamyloid apical sphincters and sometimes lack any apical apparatus. Several members of this order are important tools in fungal genetics and biochemistry. First and foremost is Neurospora, which has justifiably been called the ‘Drosophila of the fungus world’. It was on Neurospora crassa that the science of haploid genetics was founded. The uses of Neurospora and Sordaria mutants are explored in chapter 10. Many species of Sordaria and Podospora (Fig. 4.22A) fruit on herbivore dung and shoot their ascospores from perithecial ascomata whose necks are phototropic (point toward the light). Different members of the genus Podospora, which has over 100 species, have 4, 8, 16, 32, 64, 128, 256, 512, 1,024, or 2,048 ascospores per ascus (see the website). The various combinations of tubular and gelatinous ascospore appendages in Podospora not only help in species identification but also stick the spores to grass after they have been shot away from the dung on which the ascomata develop. Some species of Podospora have Phialophora

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Chapter 4 anamorphs (Fig. 4.22A). Chaetomium (Fig. 4.22B) is an important cellulolytic genus that damages fabrics and paper, especially in the tropics. It is unlike most other Sordariales in that its asci, although cylindrical, deliquesce or autolyze at maturity. Since they don’t shoot their spores, they have no apical ring mechanism, and the mucilaginous, lemon-shaped, or (American) football-shaped ascospores ooze out of the ascoma into a characteristic mass of coiled or dichotomously branched hairs that develop on the top of the ascoma. Dispersal must be by rain or arthropods. Chaetomium has Botryotrichum anamorphs (Fig. 4.22B). Neurospora has Chrysonilia anamorphs (which look very like Monilia, although that genus belongs in order 13, the Helotiales of class Leotiomycetes). Order 21—Diaporthales: 90 genera, 500 species. Here several beaked, perithecioid ascomata are usually immersed in a single stroma (as in Diaporthe impulsa; Fig. 4.27). The asci, which have an amyloid apical ring, become free inside the ascoma and then autolyze. This rather paradoxical situation suggests that evolution is in active progress here. Two important genera stand out. Cryphonectria (Endothia) parasitica causes chestnut blight, which almost extinguished an important species of North American tree in about fifty years; you can read the full story in chapter 12. Because of this near extinction, you will probably not be able to find specimens of Cryphonectria, but another member of this order, Gaeumannomyces graminis, which causes ‘whiteheads’ or ‘take-all’ of wheat, is common. It rots the roots of afflicted plants and so causes premature drying out of the plant, sometimes reducing yields to zero.

Fig. 4.27 Diaporthales: Diaporthe impulsa. Order 22—Ophiostomatales: 15 genera, 130 species.

The ascomata of this order generally have long, tubular necks, with the ostiole at the tip (Fig. 4.17B). The asci are not arranged in a hymenium and autolyze early. The spores ooze out of the ostiole and form a slimy droplet that is supported by a radiating ring of

Kingdom Eumycota (True Fungi), Subkingdom Dikarya specialized, hair-like hyphae at the top of the neck. These fungi often fruit in bark beetle tunnels, and the elevated spore drop has evolved to ensure that the beetle carries spores with it when it flies off in search of another tree. The most important genus in this order is Ophiostoma. Ophiostoma ulmi (Fig. 4.17B) causes Dutch elm disease, which was introduced into the United States in 1930 and into Canada in 1944, has since spread right across the continent, and has much more than decimated the American elm (to decimate means to take out one in ten). This beetle-transmitted fungus has a Graphium anamorph that produces many tall, synnematal conidiomata (Fig. 4.17B), each bearing a slimy droplet of conidia at its tip. In producing this stalked spore drop, the anamorph is completely analogous to the teleomorph; both have evolved to ensure that beetles don’t leave home without them on their way to the next tree. It has proved very difficult to breed resistant strains of the American elm (Ulmus americana), but some naturally resistant trees have been found, and scientists are now multiplying these in cell culture with a view to generating millions of resistant trees that can replace the magnificent vase-shaped elms that not long ago shaded many city streets in North America (see the website).

(Mostly) Dichotomous Key to Some Groups of Ascomycetes 1 No ascoma produced, asci solitary, in chains or in layer on host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 1 Ascoma produced (open or closed, epigeous [above ground] or hypogeous [below ground]) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 2 Hyphae often absent; ascus-like meiosporangia free or produced on individual hyphae or absent . . . . . . . . . . . . . . . . . . . . .(yeasts: chapter 6—Saccharomycetes, Schizosaccharomycetes) 2 Hyphae always present; asci borne in a naked layer on the surface of the host plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Taphrinales 3 Thallus containing algal cells or filaments . . . . . . . . . . . . . . . . . . . (lichens: chapter 7—Lecanoromycetes, Chaetothyriomycetidae) 3 Thallus without algae or cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 4 Ascus wall thick, with two functionally different layers (bitunicate) . . . . . Dothideomycetes (Dothideales, Pleosporales, Botryosphaeriales, Capnodiales) 4 Ascus wall thin, functionally single layered, lysing at or before maturity in some orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 5 Ascus wall lysing before spore maturity; ascospores not discharged (prototunicate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

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Chapter 4 5 Ascus wall persistent; ascospores discharged except in hypogeous forms (unitunicate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 6 Assimilative hyphae absent; ascomata on exterior of insects, spine-like outgrowths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboulbeniomycetes (Laboulbeniales) 6 Assimilative hyphae well developed, immersed or superficial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 7 Ascomata usually with ostiole; asci lysing early . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ophiostomatales 7 Ascomata closed (cleistothecial), occasionally ostiolate; asci spherical, randomly arranged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 8 Ascomatal peridium complete; ascus wall lysing before maturity; anamorphs usually blastic-phialidic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eurotiales 8 Peridium often loosely woven, with appendages; anamorphs thallic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Onygenales 9 Asci operculate, or with thin apex; ascomata apothecial or hypogeous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 9 Asci opening by apical pore or canal, with apical sphincter or thick apex (inoperculate); ascomata perithecial or apothecial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 9 Asci without lid or sphincter; ascomata closed; obligate plant parasites with superficial assimilative hyphae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erysiphales 10 Ascomata apothecial or hypogeous; asci in hymenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pezizales 10 Ascomata closed, hypogeous; asci random . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elaphomycetales 11 Mature ascomata have an exposed hymenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 11 Mature ascomata perithecial (closed but with ostiole) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 12 Asci with apical sphincter blueing in iodine (amyloid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leotiales 12 Asci with nonamyloid sphincter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Rhytismatales 13 Asci with apical sphincter amyloid (blueing in iodine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Kingdom Eumycota (True Fungi), Subkingdom Dikarya 13 Asci with nonamyloid ring or ring absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 14 Ascomata compound, perithecia embedded in a black stroma; ascospores small, sausage shaped (allantoid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diatrypales 14 Ascomata single, or compound in a stroma; ascospores not sausage shaped . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Xylariales 15 Ascomata single . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sordariales 15 Ascomata grouped in a stroma or on a subicular layer . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 16 Stroma often stalked; asci long, narrow, lacking sphincter, but apex thick, with pore; ascospores long and thread-like, often fragmenting at maturity; all obligately parasitic (on plants, arthropods, or fungi) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clavicipitaceae 16 Stroma never stalked; asci and ascospores not as above . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 17 Ascomata compound, perithecia immersed, with long neck or beak; asci with apical ring but lysing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Diaporthales 17 Perithecia not beaked, often brightly coloured, embedded in a stroma or superficial on a subiculum; asci not lysing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypocreales

Further Reading on Ascomycetes and Their Anamorphs Arx, J. A. von. 1981. The Genera of Fungi Sporulating in Pure Culture. Vaduz, Germany: Cramer. Arx, J. A. von, and E. Müller. 1975. A Reevaluation of the Bitunicate Ascomycetes with Keys to Families and Genera. Studies in Mycology 9. Baarn, Netherlands: Centraalbureau voor Schimmelcultures. Barron, G. L. 1968. The Genera of Hyphomycetes from Soil. Baltimore, MD: Williams and Wilkins. Breitenbach, J., and F. Kränzlin. 1984. Fungi of Switzerland. Vol. 1: Ascomycetes. Lucerne, Switzerland: Verlag Mykologia [the gold standard for books on ascomycetes]. Carmichael, J. W., B. Kendrick, I. L. Conners, and L. Sigler. 1980. Genera of Hyphomycetes. Edmonton, Canada: University of Alberta Press. Cole, G. T., and B. Kendrick, eds. 1981. Biology of Conidial Fungi. 2 vols. New York: Academic Press. Cole, G. T., and R. A. Samson. 1979. Patterns of Development in Conidial Fungi. London: Pitman. Dennis, R. W. G. 1968. British Ascomycetes. 2nd ed. Lehre, Germany: Cramer. Domsch, K. H., W. Gams, and T.-H. Anderson. 1980. Compendium of Soil Fungi. Vols. 1 and 2. London: Academic Press.

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Chapter 4 Ellis, M. B. 1971. Dematiaceous Hyphomycetes. Kew, U.K.: Commonwealth Mycological Institute. ———. 1976. More Dematiaceous Hyphomycetes. Kew, U.K.: Commonwealth Mycological Institute. Ellis, M. B., and J. P. Ellis. 1985. Microfungi on Land Plants: An Identification Handbook. London: Croom Helm. ———. 1988. Microfungi on Miscellaneous Substrates: An Identification Handbook. London: Croom Helm. Hughes, S. J. 1976. “Sooty Moulds.” Mycologia 68:693–820. Ken, J., R. Summerbell, L. Sigler, S. Krajden, and G. Land. 1997. Laboratory Handbook of Dermatophytes. Belmont, CA: Star Publications. Kendrick, B. ed. 1979. The Whole Fungus. Vols. 1 and 2. Ottawa: National Museums of Canada. ———. 1989. “Subdivision Deuteromycotina—a Fungal Chimera.” Sydowia 41:6–14. Kendrick, B., and T. R. Nag Raj. 1979. “Morphological Terms in Fungi Imperfecti.” The Whole Fungus 1:43–61 Kirk, P. M., P. F. Cannon, J. C. David, and J. A. Stalpers. 2001. Dictionary of the Fungi. 9th ed. Wallingford, UK: CAB International. Nag Raj, T. R. 1993. Coelomycetous Anamorphs with Appendage-Bearing Conidia. Sidney, Canada: Mycologue Publications [www.mycolog.com]. Nag Raj, T. R., and G. Morgan-Jones et al. 1972–1982. Icones generum coelomycetum. Fascicles I–XIII. Waterloo, Canada: Department of Biology, University of Waterloo. Seaver, F. J. 1978a. North American Cup-Fungi—Inoperculates. Reprint. Monticello, NY: Lubrecht and Cramer. ———1978b. North American Cup-Fungi—Operculates. Reprint. Monticello, NY: Lubrecht and Cramer. Seifert K., G. Morgan-Jones, W. Gams, and B. Kendrick. 2011. The Genera of Hyphomycetes. CBS Biodiversity Series 9. Utrecht, Netherlands: CBS-KNAW Fungal Biodiversity Centre. Sivanesan, A. 1984. The Bitunicate Ascomycetes and their Anamorphs. Vaduz, Germany: Cramer. Sutton, B. C. 1980. The Coelomycetes. Kew, U.K.: Commonwealth Mycological Institute. Wingfield, M. J., K. A. Seifert, and J. F. Webber, eds. 1993. Ceratocystis and Ophiostoma, Taxonomy, Ecology and Pathogenicity. St. Paul, MN: APS Press. Zheng, Ru-yong. 1985. “Genera of the Erysiphaceae.” Mycotaxon 22:209–63. A few relevant websites http://www.mycolog.com/CHAP4a.htm. http://www.mycolog.com/CHAP4b.htm Rogers, A. “Unraveling the Mystery of the Canadian Whiskey Fungus.” http://www.wired.com /2011/05/ff_angelsshare/ —the fascinating story of Baudoinia, a conspicuous mould with a taste for alcohol, that was named only in 2007.

5 Kingdom Eumycota (True Fungi) Subkingdom Dikarya (Fungi with a Dikaryophase) Phylum 8: Basidiomycota: the Basidiomycetes

Subphyla: Agaricomycotina, Pucciniomycotina, Ustilaginomycotina

Introduction With the ascomycetes under your belt, you should now find it (slightly) easier to cope with the other half of subkingdom Dikarya. Phylum Basidiomycota has many important features in common with the Ascomycota: (1) haploid nuclei in somatic hyphae; (2) chitinous hyphal walls; (3) regularly septate hyphae; (4) the presence of central pores piercing the septa (albeit with different ultrastructure); (5) the potential for somatic, assimilative hyphae to anastomose (fuse and merge contents); (6) the production of complex and often macroscopic sexual fruit bodies (basidiomata vs. ascomata); (7) the presence of a dikaryophase in the life cycle (except in anamorphic holomorphs); (8) a specialized mechanism for launching the meiospores into the air (basidium vs. ascus); (9) production of a conidial anamorph by many species. Make no mistake, ascomycetes and basidiomycetes evolved from a common stock, but molecular biology tells us that this happened hundreds of millions of years ago, so they are usually relatively easy to tell apart, macroscopically, microscopically, and ultrastructurally. Here are some of the differences. (a) Walls. The walls of ascomycete hyphae are basically two layered, those of basidiomycete hyphae are multilayered. Don’t worry about this, because it can be determined only with the transmission electron microscope. (b) Septa (cross-walls). Dikaryan hyphae are regularly septate, but the structure of the septal pore in different classes of the two phyla differs, as you can see in Fig. 5.1. The differences are important, but again can usually be seen only with the electron microscope. Ascomycete septa (Fig. 5.1A) are pierced by a simple, central pore, with a round Woronin body hovering on each side, ready to plug the pore if the hypha is damaged.

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Fig. 5.1

Types of septa typical of various fungal groups.

Septa of class Saccharomycetes (many yeasts and related fungi that form ascus-like meiosporangia; see chapter 6), are often perforated by many micropores (Fig. 5.1B). In many basidiomycetes the septa have a central barrel-shaped structure called a dolipore covered on both sides by a cap of membrane called a parenthesome (Fig. 5.1C). The septal pore of the rust fungi (Pucciniomycotina), is simpler, but is often blocked by a pulley wheel occlusion (Fig. 5.1D). Both basidiomycotan pore mechanisms seem to almost entirely block the migration of nuclei from cell to cell: the importance of this will soon become clear. (c) Dikaryophase. In ascomycetes, anastomosis of somatic hyphae may establish a heterokaryon (a hypha containing more than one kind of nucleus), but doesn’t usually initiate a strict dikaryophase. This is restricted to the special system of ascogenous hyphae arising from the ascogonium within the ascoma (sexual fruit body). But when monokaryotic basidiomycete hyphae anastomose, they may, if they are of compatible mating types, be establishing a strict dikaryophase, which can then grow for months or years in a dikaryotic condition before indulging in any overtly sexual behaviour. To

Kingdom Eumycota (True Fungi) put it in a nutshell: ascomycetous teleomorphs have a restricted dikaryophase (usually within the ascoma only), basidiomycetes often have an extended dikaryophase (in the somatic-feeding mycelia, long before the basidiomata develop), and even their asexual anamorphs can be dikaryotic, a phenomenon not found among the ascomycetes. We can tell that because some of those basidiomycetous anamorphs have clamp connections (see below), a diagnostic feature of the dikaryotic condition. (d) Clamp connections. In both groups the dikaryophase climaxes and ends in the hymenium of the teleomorph. In many ascomycetes, at this point, the tip of each ascogenous hypha bends back to form a crozier, which allows the two nuclei of the dikaryon to divide simultaneously, one in the hypha, one in the hook of the crozier, in such a way that the ascus mother cell contains only a compatible pair of nuclei (Fig. 5.2A). In ascomycetes, this phenomenon is generally restricted to the hymenium, but in many basidiomycetes, similar nuclear bypasses are found, not just at the base of the basidium, but at every septum in the dikaryophase (no matter how long that lasts). In basidiomycetes, they are called clamp connections (boucles in French), and their development is shown in Fig. 5.2B. If a septate, somatic hypha has regular clamp connections, it must be dikaryotic. If clamps are absent, the hyphae could still be those of a dikaryotic basidiomycete (many of the boletes [fleshy mushrooms with tubes] have no clamps on their hyphae), but they could equally be those of a monokaryotic basidiomycete, or of an ascomycete, or even those of a zygomycete, since members of the order Kickxellales have regularly septate hyphae. (e) Basidia. These constitute perhaps the most basic difference between the phyla, and one of the easiest to see under the microscope. While the meiospores of ascomycetes

Fig. 5.2

Comparison of ascus and basidium development (see text).

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Chapter 5 are developed inside tubular or globose meiosporangia called asci (Fig. 5.2A, Fig 4.3), those of basidiomycetes are formed outside specialized meiosporangia called basidia (Fig. 5.2B). Nuclear fusion and meiosis happen inside the cell, but the spores blow out like tiny balloons at the ends of (usually) four tiny tapered outgrowths called sterigmata. Most basidiomycetes shoot their basidiospores actively from hymenia that are exposed at maturity (puffballs, earthstars, basidiotruffles, and stinkhorns don’t). Basidiospores which are to be forcibly discharged (ballistospores) blow out at an angle to the fine sterigma that bears them: in other words, they are asymmetrically mounted, or offset, as can be seen in Fig. 5.3.

Fig. 5.3 SEM of shooting basidiospore, asymmetrically placed on sterigma.

Then they are actively expelled from their perches by a mechanism explained in Fig. 5.4. Just before discharge, a droplet of fluid, enclosed within a membrane, appears at one side of the spore base, and within seconds the spore is shot away. A minute quantity of mannitol and hexose sugars is secreted from a small area at the base of the spore, forming a hygroscopic spot on which water condenses from the saturated air surrounding the basidium. When it becomes large enough, the droplet coalesces instantaneously with a film of water on the surface of the spore, as in Fig. 5.4, causing a rapid displacement of the spore’s centre of gravity. This redistribution of mass is opposed by the sterigma. The sterigma is under high turgor pressure, and immediately breaks, so the spore shoots away with very high initial acceleration, although it doesn’t go very far. The mechanism has been described as a surface tension catapult. It is fascinating that Fig. 5.4 Current explanation of basidiospore essentially the same mechanism is found discharge mechanism. From N. P. Money 1998. in the basidia of mushrooms, bracket fungi,

Kingdom Eumycota (True Fungi) crust fungi, jelly fungi, rust fungi, and some yeasts. It is a strong argument for the monophyly of the basidiomycetes. If you want to follow the extended trail of experiment and observation that led to the current explanation, I recommend that you read Money (1998). There is also new evidence that some mushrooms chill their fruit bodies by evaporative cooling. This enhances condensation on their spores, which need a layer of free water if the shooting mechanism just described is to work (see Husher et al. 1999). Remember that, just as in the ascomycetes, there is a significant minority of basidiomycetes which develop basidia, but have lost the spore-shooting mechanism (Fig.5.5). These we call sequestrate, because the mature basidiospores are kept inside the basidioma (which may simply remain closed, or may develop below ground). Spores are released later in a variety of ways, some involving animal vectors, some wind, some rain, as we shall see.

Fig. 5.5

Sequestrate basidiomycetes which lack active spore discharge.

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Chapter 5 You may be relieved to learn that you don’t have to search for any of these features in order to recognize a basidiomycete when you see one. The reason for that is that many diagnostic features are visible to the naked eye. What else do you know that looks like a mushroom? Or a puffball? Or an earthstar (Fig. 5.5)? They are unique. The same is also true of most other basidiomycete fructifications. You can also recognize certain basidiomycete mycelia on sight, because they tend to form delicate but visible fan-like arrangements on decaying wood (Fig. 5.6).

Fig. 5.6

Mycelial fan typical of Basidiomycetes.

Three subphyla are recognized within the phylum Basidiomycota. These are the Agaricomycotina, the Pucciniomycotina, and the Ustilaginomycotina. They are morphologically dissimilar, but are linked by their meiosporangia, which are microscopic basidia with the unique spore-shooting mechanism discussed above—often hard to find in the last two subphyla, since they arise from individual spores, and even harder to see in action. However, you won’t find it hard to spot members of all three subphyla.

Subphylum (1) Agaricomycotina Classes Agaricomycetes, Dacrymycetes, Tremellomycetes This huge group incorporates all mushrooms, bracket fungi, puffballs, earthstars, bird’s-nest fungi, stinkhorns, crust fungi, and the various jelly fungi. As their common names suggest, there are visible features that enable us to recognize 99% of all basidiomycetes as fungi belonging to this group. If you don’t have these features at your fingertips already, time spent looking at the various illustrations in this text, the even more numerous pictures on the website (www.mycolog.com), and one or more of the beautifully illustrated field guides listed at the end of this chapter, or a variety of mushroom websites, will pay off handsomely when you go outdoors to look for these fungi in their natural habitats. Believe it or not, it’s quite exciting to suddenly recognize a mushroom in the woods when you’ve only seen its picture.

Kingdom Eumycota (True Fungi)

Fig. 5.7 Morphological diversity in Boletales. a. Bondarzevomyces taxi; b. B. taxi, pores; c. Coniophora puteana; d. Leucogyrophana mollusca; e. Hygrophoropsis aurantiaca; f. Suillus granulatus; g. Chroogomphus vinicolor; h. Boletinellus merulioides, hymenophore; i. Calostoma cinnabarinum; j. Scleroderma septentrionale; k. Meiorganum neocaledonicum, young hymenophore; l. ‘Tylopilus’ chromapes; m. Phylloporus centroamericanus; n. Xerocomus sp. From Mycologia 2006 (credit from original: Pictures a and b courtesy Y.-C. Dai; m courtesy M.-A. Neves.)

1—Class Agaricomycetes Most members of the major class Agaricomycetes develop characteristic fleshy, corky, or woody basidiomata, albeit of an amazing range of sizes and shapes as noted above: those of the minor classes Dacrymycetes and Tremellomycetes are gelatinous or rubbery (some yeast-like Tremellomycetes don’t make fruit bodies at all). Class 1 Agaricomycetes is by far the largest and most diverse, with 17 orders, 100 families, 1,147 genera, and 21,000 species. I must begin with a warning. The huge influx of molecular data over the past decade has more or less put the old taxonomic system through the blender. Just as an example, the boletes (a subgroup of the Agaricomycetes), are no longer simply those chunky mushrooms with fleshy tubes instead of

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Chapter 5 gills, that all mushroom gatherers know so well. They now come in a very wide range of morphologies (Fig. 5.7). Many still, of course, have tubes, but some have gills like regular mushrooms; some have wrinkled hymenia, some are crustose or spread out (resupinate), some look like bracket fungi, some are stalked puffballs, some are earthballs. What this means to the identifier, to be blunt, is that you may still be able to run things down to the species level with the existing literature—BUT you may not know to which of the higher groups (family, order, class) it belongs (a hint—once you have the genus, use Wikipedia to find out!). The class Agaricomycetes is partly subdivided into two subclasses, but many of the orders have not yet been fitted into a systematic superstructure. I will not consider the orders given in square brackets below—it would make life too complicated for you. But since many well-known genera are now so widely scattered, I had to include some orders for their sake.

Subclass 1—Agaricomycetidae Order Agaricales

puffballs

(32 families, 410+ genera) mushrooms, club fungi, puffballs, stalked

(16 families, 95+ genera) boletes and hom*ologues (morphology extremely diverse, as the colour plate Fig. 5.7, from Binder and Hibbett 2006, shows). Mostly ectomycorrhizal. No white rots, some brown rots of conifers. [Order Atheliales (1 family, 22 genera) crust fungi] Order Boletales

Subclass 2—Phallomycetidae Order Geastrales (1 family, 8 genera) earthstars (Fig. 5.5I) Order Gomphales (3 families, 18 genera) club fungi and chanterelle-like fungi Order Hysterangiales (5 families, 18 genera) hypogeous fungi, basidiotruffles Order Phallales (2 families, 26 genera) stinkhorns (morphology very diverse) (Fig. 5.9)

Group 3—Incertae sedis (position uncertain) The orders below are of uncertain position and are not yet allocated to a subclass. Order Auriculariales (6–7 families, 30+ genera) ear fungi, wood ears (see www.mycolog.com) Order Cantharellales (7 families, 38 genera) chanterelles and relatives (see the website) Order Corticiales (3 families, 30+ genera) crust fungi (see the website) Order Gloeophyllales (1 family, 4 genera) bracket fungi causing brown rot Order Hymenochaetales (3 families, 50+ genera) mostly crusts or bracket fungi Order Polyporales (9 families, ~200 genera) saprobic bracket fungi and relatives (see the

website)

Order Russulales (12 families, 80+ genera) brittle mushrooms and milky caps Order Thelephorales (2 families, 18 genera) crusts, tooth fungi, bracket fungi, club fungi:

all ectomycorrhizal (see the website) Order Trechisporales (1 family, 15 genera) crusts Compared to the leathery, corky, or woody polypores, agarics are relatively ephemeral, the basidiomata persisting for anything from a few hours to a few weeks, depending

Kingdom Eumycota (True Fungi) on the species. They occur seasonally, fruiting mainly in late summer or fall, although on the west coast of North America they can be found during most months of the year. The Mycological Society of San Francisco holds its annual Mushroom Fair in December, when agarics are only memories in Minnesota and Manitoba. Los Angeles celebrates in January. Agaric fruit bodies arise from an extensive, usually perennial mycelium which ramifies, invisible to the eye, around roots, through soil, plant debris, or wood, gathering energy for that once-a-year (or once every several to many years) splurge. Some fairy rings (which are enormous, radially extending fungal colonies) are estimated to be over four hundred years old, and have presumably produced flushes of basidiomata in many of those years. A single colony (genet) of a species of honey fungus, Armillaria ostoyae (the humongous fungus) growing in the forests of western North America, has been found to cover 600 hectares, and to have biomass exceeding that of a blue whale. Such colonies are probably more than 1,000 years old. Most agarics are either saprobic, exploiting dead organic matter, or ectomycorrhizal, establishing mutualistic symbioses with the roots of woody plants, especially conifers of the economically important family Pinaceae. This explains immediately why woodlands are often such excellent places to look for agarics. A detailed discussion of the ectomycorrhizal relationship is given in chapter 17. Only a few genera are parasitic, and even Armillaria (see Forest Pathology in chapter 12) is sometimes saprobic. A few genera such as Squamanita and Asterophora are parasitic on other mushrooms (these are illustrated on the website). Most agarics share the same somewhat umbrella-like basic design (Fig. 5.8A–D). There is a central, vertical stalk or stipe, with a horizontally spreading cap or pileus at the top. The underside of the cap usually bears delicate, radially arranged, vertical plates called gills or lamellae, although some have vertical fleshy tubes instead. The hymenium covers both sides of each gill, or lines each tube. Basidiospores are gently launched from the basidia, drop through the space between adjacent gills, and enter

Fig. 5.8

Agarics.

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MACROSCOPIC CHARACTERISTICS

Margin (choose one or more):

Kingdom Eumycota (True Fungi)

MICROSCOPIC CHARACTERISTICS

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Chapter 5 the more turbulent outside air which carries them away. In some genera, for example Amanita, the developing basidioma is totally enclosed within a membranous universal veil, the remains of which can be seen on the mature, expanded agaric in the form of a sheath or volva around the base of the stipe, and spots, warts or patches on the cap. There may also be a partial veil connecting the stipe and the edge of the cap in young specimens, enclosing and protecting the developing gills. This, too, may remain on the stipe of the mature, expanded agaric as a membranous ring or annulus (as in Amanita and Agaricus), or as a filamentous, cobwebby veil or cortina (as in Cortinarius). A few agarics have both ring and volva; some others have only one of these features, and the majority have neither. Agarics are complex, rather variable structures, and have many other taxonomically valuable features. The checklist on page 114 includes many (although not nearly all) of them, and shows the kinds of information we need to collect in order to classify the thousands of different agarics. If you are going to make a serious stab at it, you could do worse than photocopy the chart and fill in as many of the blanks as you can before going to the books, or to a mushroom identification software program such as Matchmaker (Google it). If you try to find all of those characteristics in several agarics (admittedly a counsel of perfection), you will learn a tremendous amount about them, which will help you in all future encounters with mushrooms. In fact, you can usually identify them to genus with a small fraction of those characteristics, although getting them to species will probably call for much more information (depending on the number of species in the genus). Read what follows, in which I introduce you to representatives of many families, and you will see which of the characteristics mentioned above are the most important in separating them. I am convinced that the basidium originally evolved as a spore-shooting mechanism, but that for various ecological reasons, which we will explore, it has on many separate occasions lost that function. Puffballs, basidiotruffles, earthstars and stinkhorns have often been called ‘Gasteromycetes’ (stomach fungi), because even if they have hymenia, they don’t expose them when the spores are mature, so the spores stay on or in the fruit body, and because the spores are symmetrically placed on the sterigmata, and are never actively shot away. But since these features have evolved from many different groups by convergent evolution, the ‘Gasteromycete’ label is now falling into disuse, and we tend to call them all ‘sequestrate’ fungi.

Order Agaricales This order has 33 families, 413 genera and over 13,000 described species. A rather informal classification involving six clades: Agaricoid, Tricholomatoid, Marasmioid, Hygrophoroid, Pluteoid, and Plicaturopsidoid is currently being used for the Agaricales. You will now be presented with many generic names. To see representative pictures of these, look in one of the manuals mentioned below, or on www.mycolog.com, or in Google and Wikipedia.

Kingdom Eumycota (True Fungi)

1—Agaricoid Clade—Thirteen Families Family 1 Agaricaceae. The genus Agaricus (Fig. 5.8B), to which the supermarket mushroom

belongs, has a ring on the stem, lacks a volva (that is, it has a partial veil but no universal veil), and has gills that are not attached to the stipe (they are described as free); its spore print is dark. Other members of the family such as Leucoagaricus may have spore prints of different colours, but they are never rusty-brown or cinnamon. Leucoagaricus naucinus, which is common on lawns, is an all-white or cream-coloured agaric with a ring, but no volva. It has an uncomfortable resemblance to the deadly poisonous Amanita virosa, so, although it is not dangerous (edible to some, a gastric irritant to others), I always advise people against making a meal of it. Lepiota clypeolaria has a scaly cap and a ring, both typical of the genus. Macrolepiota rachodes is a much larger, edible species. Again, note the large cap scales and the conspicuous ring (there are pictures on the website). This species was placed in Lepiota until recently. So was what is now called Chlorophyllum molybdites, a green-spored, poisonous Lepiota lookalike. The genus Coprinus, a common mushroom which produces dark spores and whose gills autolyze (liquefy) at maturity, formerly had about 100 species, and formed the basis for a family, Coprinaceae, but some of them, including the type species, C. comatus (the shaggy mane; Fig. 5.8C), have now been moved into the Agaricaceae, and the rest into the Psathyrellaceae. Perhaps not surprisingly, Podaxis, the desert shaggy mane, a similar-looking but sequestrate relative of Coprinus comatus, which I have seen fruiting on three continents, has also been put here. It has black spores, but no recognizable gills, and its sporiferous tissue doesn’t autolyze. The spore mass (gleba) is dry, and disperses on the wind only when the fruit body disintegrates or is disrupted (there is a short video of this on the website). Now we come to some entirely un-mushroom-like members of the Agaricaceae. The puffball genera Lycoperdon (Fig. 5.5H), Bovista, and Calvatia—no cap, no stipe, no gills; and Tulostoma, a puffball on a stalk (Fig. 5.5G). You can find illustrations of all these on the website or in Google images (note that the Google algorithm for images is far from perfect and that many of the images lower on the page are misleading). Family 2 Strophariaceae. A saprobic family that contains many ‘magic’ mushrooms. The spores are purple-black or brown, are smooth walled, and have a germ pore; the pileipellis (surface layer of the cap) is filamentous, and the gills are attached to the stipe and often bear accessory cells called chrysocystidia, which have contents that stain yellow in alkali. Some species of Psilocybe (e.g., P. cubense) and Stropharia contain the hallucinogen psilocybin, and the flesh of such species often turns blue when bruised. It is easy to grow Psilocybe cubense in culture, but for some peculiar paranoid societal reason, possession of psilocybin-containing species is still illegal. This topic is explored further in chapter 22. The very common nonhallucinogenic genera Pholiota and Hypholoma usually fruit on wood. Sequestrate derivatives of the Strophariaceae found in New Zealand and Australia are placed in the brightly coloured genus Weraroa (see the website). Family 3 Hymenogastraceae (formerly part of the Cortinariaceae). Genera Alnicola, Galerina, Hebeloma, Hymenogaster, Phaeocollybia. (Remember that all the names mentioned in this chapter can be looked up on Google, where you will find both descriptions and illustrations.) Galerina is a genus of small mushrooms, some of which are extremely

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Chapter 5 poisonous. Hebeloma has some species that are important mycorrhizal partners of young conifers. Family 4 Inocybaceae. Inocybe, Phaeomarasmius, Tubaria—gilled genera with brown spores. Inocybe (fibre head) is a very large genus with hundreds of species. Many are toxic; a few rare species are hallucinogenic. Many species have conical caps with radial fibres and cannot be identified without recourse to microscopic characteristics. Some species have unique nodulose basidiospores (see the website). Family 5 Crepidotaceae. Crepidotus, a brown-spored, gilled genus generally has small, delicate, one-sided, stipeless (sessile) basidiomata, often on wood. Family 6 Panaeoleae. Panaeolus. This genus is black spored and saprobic, growing mostly in fields or on dung. The gills tend to be variegated or spotted. Family 7 Gymnopileae. Gymnopilus is a gilled genus of about 200 species, 14 of which are hallucinogenic (but bitter). Basidiomata tend to be reddish-brown to rusty-orange to yellow and rather large, emerging in clusters from wood (there is a fine picture on the website). Family 8 Cortinariaceae. Cortinarius, a gilled mycorrhizal genus with warty, rusty-brown spores and a delicate web-like partial veil (cortina), has 2,100 species(!), which are mostly nearly impossible to identify. The deadly toxin orellanine occurs in at least thirty-four Cortinariaceae. Amanitin has also been found in at least seven species. But at least one member of the Cortinariaceae, Cortinarius (Rozites) caperata, is a wellknown and highly regarded edible (see chapter 18). Most other genera formerly placed in this family have been relocated to other families. Some members of the Cortinariaceae have become sequestrate. The genus Thaxterogaster closely resembles Cortinarius in many ways, but its cap never expands, and its gills have become so convoluted that even if they were to be exposed, they could not successfully drop many spores into the air. While on sabbatical in New Zealand, I often found a beautiful purple species of Thaxterogaster in the Southern beech (Nothofa*gus) forests (see the website). A brown species of Thaxterogaster that I also found in New Zealand had even lost its external stipe and looked rather like a puffball, but a vertical section of the fruit body revealed a central column of stipe tissue—the transformation to truffle still isn’t complete. The stalked, browncapped Thaxterogaster pingue is relatively common in western North America. Family 9 Bolbitiaceae. Seventeen genera, including Bolbitius and Conocybe. Bolbitius has numerous fragile species, of which B. vitellinus (the fried egg mushroom) is the best known. The surface layer of the cap (the pileipellis) is epithelial (the cells are swollen and don’t appear filamentous); the spore print is ochraceous to rustybrown; and the spores have a germ pore. Conocybe has over 200 species of delicate, usually conical mushrooms, sometimes resembling Galerina but with a cellular cap cuticle. Conocybe filaris contains deadly amatoxins (see chapter 22). In addition to normal agaricoid species, the Bolbitiaceae has sequestrate members with basidia that don’t shoot their spores. The genus Gastrocybe still looks like an agaric, but its spores are symmetrically mounted on the sterigmata, its cap does not open, and it has a habit of always falling over as soon as it comes up (see the website). These features suggest that it is even now actively evolving and that its spores are dispersed by invertebrates rather than wind. An alternative theory suggests that its regular collapse is due to a bacterial infection, but that does not explain the nonshooting basidia.

Kingdom Eumycota (True Fungi) Family 10 Psathyrellaceae. Psathyrella, with 400 species, produces delicate, dark-spored

agarics. Three genera recently segregated from Coprinus by molecular data have also been placed here—Coprinellus, Coprinopsis, and Parasola. The basidiomata of the first two tend to deliquesce or self-digest. Family 11 Hydnangiaceae. This family seems very strange, because it contains two seemingly very different genera: Hydnangium and Laccaria (plus a few others you need not concern yourselves with). Hydnangium is a hypogeous fungus (a basidiotruffle), while Laccaria is a common and important epigeous agaric genus. Both are ectomycorrhizal with conifers. You may never see a Hydnangium, but you are almost certain to come across Laccaria. Its species have thick, widely spaced, purple to peach, adnate to slightly decurrent gills and produce colourless, globose, or ellipsoidal, spiny, amyloid basidiospores. Family 12 Nidulariaceae. The bird’s-nest fungi, with four common genera—Crucibulum, Cyathus (Fig. 5.9A), Nidula, and Nidularia. These are not in the least like agarics; they have tiny basidiomata that look like bird’s nests filled with eggs (which contain basidiospores and are called peridioles) that are dispersed by rain splash. Family 13 Cystodermateae. Cystoderma is a fairly common mushroom with a rather granulose cap, a ring, and colourless spores. It is sometimes classified in the Agaricaceae.

2—Tricholomatoid Clade—Four Families Family 1 Lyophyllaceae. Eight genera and almost 160 species. We note Asterophora, Lyo-

phyllum, and Termitomyces (all are illustrated on the website). Asterophora is a very unusual agaric which parasitizes other mushrooms and in which much of the cap becomes converted into distinctive (asexual) chlamydospores. Lyophyllum is best represented by L. decastes, which grows in large fleshy clumps and is widely eaten. Termitomyces is another unusual agaric and has a symbiotic association with moundbuilding termites in Africa and Australasia (you won’t find it in North America or Europe). Since the termites knowingly cultivate and feed the fungus, some species can produce very large (and edible) basidiomata. This relationship is discussed in chapter 16 and is illustrated on the website. Family 2 Entolomataceae. This mostly ground-fruiting family has pinkish spore prints and unique angular basidiospores shown on the website. The best-known genera are the mycorrhizal Entoloma, with about 1,000 species, and the saprobic Nolanea, Leptonia, and Rhodocybe. cl*topilus has longitudinally ridged spores. The Entolomataceae has given rise to a sequestrate offshoot, Richoniella, whose basidiospores are angular, exactly like those of Entoloma, and unerringly reveal its relationship. Family 3 Tricholomataceae. This used to be the largest family of agarics, because everything that apparently could not be fit elsewhere was dumped here. Now several segregate families have been erected and reduced the congestion (if not the confusion). The family now has a mere 80 genera and 1,000 species. Some of the better-known genera are Tricholoma, Arrhenia, Catathelasma, cl*tocybe, and Collybia (a once numerous genus now reduced to three small species that arise from sclerotia [see the website], most other species having been transferred to the Omphalotaceae). As a mould specialist, I note especially Dendrocollybia racemosa, a unique species which develops many conspicuous synnemata of its Tilachlidiopsis anamorph along the stipe (there’s a

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Chapter 5 beautiful photo of this on the website). Other genera are Lepista, Leucopaxillus, Melanoleuca, and Phyllotopsis; the rare Squamanita (a parasite of other mushrooms) and Tricholomopsis (Tricholoma) rutilans also belong here. Family 4 Mycenaceae. Mycena, a very common genus of small, delicate mushrooms usually with translucent cap flesh, has about 500 species. All are saprobic, and 30 are bioluminescent. Other common genera are Panellus (P. stipticus is also bioluminescent) and Xeromphalina, a small mushroom commonly found in large clusters on rotting wood.

3—Marasmioid Clade—Four Families Family 1 Omphalotaceae. Gymnopus and Rhodocollybia have been segregated from Collybia

and relocated here. The other two important genera are Lentinula (home of the prized edible shiitake mushroom, L. edodes) and Omphalotus. Family 2 Marasmiaceae. Although there are about 50 genera in this family, Marasmius (often with thin, tough stipes) is the most common and well known, with about 500 species. Family 3 Schizophyllaceae. This family contains two rather dissimilar wood-inhabiting genera, Schizophyllum, the sessile ‘split-gill’ (see the website), and Fistulina, a fleshy bracket fungus. Family 4 Physalacriaceae. This wood-inhabiting family contains one really well-known and important genus, Armillaria (formerly in the Tricholomataceae). Armillaria mellea, the so-called honey mushroom (colour not flavour), is a dangerous tree pathogen, although what used to be thought of as a single species is now known to comprise several distinct taxa—for example, the common Armillaria of the west coast of North America is Armillaria ostoyae, one genet (a single colony) of which extends over 600 hectares (the humongous fungus; see chapter 11). Armillaria mellea produces characteristic blackish mycelial strands called rhizomorphs under the bark of affected trees, and its mycelia in wood are often bioluminescent. The other well-known genus in this family is Flammulina, known as the velvet-stalk, which, when grown in culture, becomes the well-known edible enokitake, which has extremely elongated stalks and tiny caps (see chapter 18).

4—Hygrophoroid Clade—Two Families Family 1 Hygrophoraceae. These are the waxy caps. The best-known genera are the often

brightly coloured Hygrocybe, the duller Hygrophorus, and the very common and successful lichenized genus Lichenomphalia. Family 2 Typhulaceae. Typhula is an unusual club fungus which is generally saprobic, although some species cause diseases of turf and some crops.

5—Pluteoid Clade—Four Families Family 1 Pleurotaceae. The edible oyster mushroom (Pleurotus) and relatives are saprobic

on wood and in many cases also prey on nematodes.

Family 2 Amanitaceae. One of the most famous families, because it contains the genus

Amanita (Fig. 5.8A), known for a few extremely toxic species (dealt with in detail in chapter 22) and for striking appearance. Species of Amanita are extremely common and

Kingdom Eumycota (True Fungi) are mycorrhizal. Many species have both a ring (partial veil) and a volva (universal veil), and their gills are generally ‘free’ (not directly connected with the stem). Because some Amanita species are deadly poisonous (see chapter 22), the genus has even made the cover of Scientific American, and mushroom hunters (especially those planning to eat what they collect) should always make sure they get to the base of the stipe of any agaric they pick, so they can see whether or not there’s a volva. The ‘destroying angel’, Amanita virosa, is pure white, with ring, conspicuous volva, and even a white spore print. But this species, like A. fulva, does not have spots (patches of universal veil) on the cap. Like most other members of the family, this lethal species is ectomycorrhizal, and so fruits only near tree species with which it is symbiotic (see chapter 17). Family 3 Pluteaceae. This saprobic family has free gills and pink spore print (but its spores are rounded, unlike the angular ones of the also pink-spored Entolomataceae). The genus Pluteus is the most common, but Volvariella is widely cultivated in the tropics for food. Volvariella volvacea, the straw mushroom, although not native to North America, is the best-known member of the genus, since it is widely cultivated in Asia (see chapter 18). Next time you eat mushrooms at a Chinese restaurant, see if they belong to this species—you can easily spot the persistent volva almost enclosing the whole basidioma. When in Java, I got some living specimens of this fungus from a mushroom grower and watched them open and make a profuse pink spore print. Family 4 Limnoperdaceae. This contains one genus with a single species. We will meet Limnoperdon, a weird little aquatic sequestrate puffball, in the chapter on fungal ecology (chapter 11).

6—Plicaturopsidoid Clade Mostly upward-growing coral or club fungi: Clavaria, Clavulinopsis, Ramariopsis. But basidiomata of Mucronella have individual downward-growing teeth (see www.mycolog.com).

Family 1 Clavariaceae.

Boletales—Six Clades The Boletales is a more or less monophyletic order, but there are six major clades, and the morphology of the basidiomata is extremely variable, as the images in Fig. 5.7 exemplify. 1 Boletineae. Boletus (Fig. 5.8D), Strobilomyces, Xerocomus, Boletellus, and Phylloporus— the first four with fleshy tubes, the last with gills. And now comes Spongiforma, a recently described epigeous sequestrate bolete (its second species, described in 2011, was named S. squarepantsii). 2 Paxillineae. Paxillus, Gyrodon, and Melanogaster—the first gilled, the second with fleshy tubes, and the last sequestrate and hypogeous (a basidiotruffle). This group, like the Boletales, is a perfect example of the difficulties implicit in the new classification. 3 Sclerodermatineae. Another really mixed bag, morphologically, with Scleroderma (earthballs), Gyroporus (typical bolete), Pisolithus (dye maker’s puffball, dead man’s foot, an important sequestrate mycorrhizal genus in dry, poor, or toxic soils), Calostoma (a brightly coloured stalked puffball), and Boletinellus (the Ash bolete, symbiotic with root aphids and secondarily parasitic on the ash tree; see chapter 16).

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Chapter 5 4 Suillineae. Yet another mixed bunch. The genera Suillus (very common typical poroid

boletes, usually with viscid caps and rings on the stipe), Gomphidius, and Chroogomphus (gilled boletes). Truncocolumella, a basidiotruffle which has a vestigial stipe, and Rhizopogon, which does not, are both sequestrate, hypogeous offshoots of Suillus. Like the parent genus, they are important ectomycorrhizal partners of conifers in western North America. Rhizopogon parksii, a very common western species, has a spongy, lacunose basidioma. The spores, however, are just like those of a Suillus, and DNA studies have established that Rhizopogon is very closely related to Suillus. 5 Coniophorineae. Coniophora (wood-rotting crust fungi). 6 Tapinellineae. Tapinella (formerly Paxillus) atrotomentosa—a gilled bolete.

Subclass Phallomycetidae—Four Orders Order 1—Geastrales Earthstars: genera Geastrum (Fig. 5.5I) and Myriostoma. These genera both contain somewhat specialized puffballs with thick external peridium layers that split radially and bend backward to raise the inner spore mass (gleba) in its thin peridium above the litter; Myriostoma has not one but many ostioles (examples are shown on the website).

Family Geastraceae.

Order 2—Gomphales Family Clavariadelphaceae. Clavariadelphus (yellow club fungi). Family Gomphaceae. A mixed bag: Gomphus—substantial chanterelle-like mushrooms,

Gautieria—hypogeous basidiotruffles, Ramaria—highly branched coral fungi.

Order 3—Hysterangiales Five families of hypogeous basidiotruffles.

Order 4—Phallales Collectively known as the stinkhorns. Despite their amazingly variable shapes, one unusual similarity runs through the family: all produce their spores in an evil-smelling slime that attracts flying insects, which act as vectors. The genera are Phallus, Mutinus, Colus, Pseudocolus, Clathrus (Fig. 5.9F), Anthurus (Fig. 5.9C), Aseroë (Fig. 5.9E), Dictyophora, and Lysurus; pictures of all these are on the website as well. Phallus (Fig. 5.9D) and Mutinus (Fig. 5.9B) have the simplest morphology: the stipe becomes sponge-like and elongates rapidly, carrying their respectively thimble-shaped and conical heads (often called receptacles) into the air, where they can expose their gleba, release their effluvium, and attract flies more easily. Europeans have sufficient knowledge of these fungi to enjoy cartoons of them. North Americans, to whom they are less familiar, look at such artwork askance (see the website). The genus Dictyophora (whose name means ‘net bearer’) does indeed have a visually striking lacy skirt hanging below the receptacle. My guess is that this is a landing platform for flies queuing up for a sample of spores. In Anthurus (Fig. 5.9C), the gleba at

Family Phallaceae.

Kingdom Eumycota (True Fungi)

Fig. 5.9

Gasteromycetes. A: Nidulariales; B–F: Phallales.

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Chapter 5 first is central in the egg. Later the spore mass covers the inner side of several octopuslike arms. In Clathrus (Fig. 5.9F) the arms remain fused, and in some species form an open lattice, again with the gleba on the inside. Aseroë (Fig. 5.9E) is surely one of the most flamboyant members of a truly spectacular order. Bright orange-red extensions of its receptacle radiate out like the petals of a flower and also look rather like meat, providing a diverse range of visual as well as olfactory clues to would-be vectors, which can be drawn from among the meat eaters (e.g., wasps), the nectar eaters (e.g., butterflies), and the visitors to excreta (e.g., dipteran flies).

Orders not yet allocated to a subclass 1—Order Auriculariales Ear fungi, wood ears, jelly tooth—jelly fungi growing on wood. Family Auriculariaceae. Auricularia (see www.mycolog.com) and Exidia are sessile with smooth hymenia, but the extremely common Pseudohydnum is toothed, is one sided, and has a stalk.

2—Order Cantharellales Four Families—chanterelles and relatives Family Cantharellaceae. Chanterelles, Cantharellus (see the website), Craterellus Family Clavulinaceae. Club, coral, and crust fungi Family Hydnaceae. Hydnum, Hydnellum (see the website), Sistotrema—mostly tooth fungi Family Tulasnellaceae. Corticioid with distinctive basidia (Fig. 5.11B).

3—Order Corticiales Wood-rotting crust fungi.

4—Order Gloeophyllales Bracket fungi causing brown rot—for example, Gloeophyllum and Lentinus.

5—Order Hymenochaetales Many genera of wood-rotting fungi, mostly crusts or brackets. This order includes the species Fomitiporia ellipsoidea, which in Hainan, China has, as of 2010, developed the largest basidioma ever recorded. This specimen when measured was about twenty years old and 10.85 metres long × 0.82–0.88 metres wide and 4.6–5.5 centimetres thick, with a volume calculated as 409,000—525,000 cubic centimetres and a fresh weight estimated at 400–500 kilograms. With some 452 million pores, it might be producing around one trillion spores per day.

6—Order Polyporales Most saprobic bracket fungi belong here—there are several families and hundreds of species. Common (mostly poroid) genera are Cryptoporus, Daedalea, Fomes (see the

Kingdom Eumycota (True Fungi) website), Fomitopsis, Ganoderma, Laetiporus, Lenzites, Merulius (hymenium wrinkled), Neolentinus, Oligoporus, Phaeolus, Piptoporus, Polyporus, Poria, Pycnoporus, Sparassis (cauliflower fungus), Trametes, Trichaptum, Tyromyces, and many more. Many produce perennial basidiomata, adding a layer of tubes each year; others are smaller and annual. Anamorphs of this group of fungi may be thallic-arthric conidia produced when clamped hyphae disarticulate at the septa, as in the Osteomorpha anamorph of Trechispora, or more specialized conidiophores producing blastic-sympodial conidia, as in the unnamed anamorph of Sistotrema (see Kendrick and Watling 1979). Bridgeoporus (Oxyporus) nobilissimus is a rare and threatened species found only in old-growth forests of the Pacific Northwest. U.S. law at present mandates that 240 hectares (600 acres) of forest must remain undisturbed around each known site of this fungus. Progress! A basidioma of this species was formerly noted in the Guinness Book of Records as the world’s largest fungal fruit body, but it was supplanted by a specimen of Rigidoporus ulmarius that is still growing actively at Kew, England, and has now been displaced by the Fomitiporia described above under Hymenochaetales (see the website). Polyporus squamosus, the dryad’s saddle, is easily recognized by the conspicuous scales on the upper surface of its fruit body and its relatively soft texture. Fomes fomentarius has very tough, hoof-shaped basidiomata. A few polypores, such as Laetiporus sulphureus, which produces spectacular orange and yellow fruitings on fallen trees, are soft enough when young to be eaten (common name: chicken-of-the-woods), although they should be avoided if the substrate is Eucalyptus. Albatrellus ovinus is another unusual polypore which looks very like an agaric, grows on the ground, and has soft flesh. The pores are very narrow and shallow. Heterobasidion annosum is highly pathogenic to many conifers and causes serious root rots. I have seen a forest clearing in California produced entirely by this fungus: it had killed representatives of fourteen different conifers, many of them introduced species. Piptoporus betulinus, on the other hand, kills only birch trees. Trametes versicolor (often called the turkey tail) is one of the smaller and most common saprobic polypores and has annual, rather than perennial, basidiomata. Like many other fungi, polypores often have anamorphs (see Kendrick and Watling 1979), although these may be very inconspicuous. Heterobasidion annosum has a hyphomycetous anamorph in the genus Spiniger, which forms many conidia synchronously on an apical vesicle. The Ptychogaster anamorph of Trametes forms alternatearthric conidia (to review conidium development, return to chapter 4 or check the website). Many polypores, e.g., Poria, Polyporus, and Ganoderma, may not kill trees, but they cause serious decays of both standing and structural timber. These rots cost us many millions of dollars every year. The general division here is into brown rots, where only the cellulose is digested, and white rots, where both cellulose and lignin are metabolized. Coltricia cinnamomea, a centrally stalked, ground-fruiting polypore, is atypical in being ectomycorrhizal (see chapter 17). To the uninitiated, most polypores tend to appear rather similar—a bracket-shaped excrescence protruding from a branch or trunk of a tree. Most of the bracket fungi were at one time put into the genus Polyporus. No more! Mycologists now recognize

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Chapter 5 almost 100 genera of bracket fungi because new microscopic and biochemical characteristics have been recognized. And all this happened even before the molecular biologists got really busy. These characteristics are as follows. (1) The kind of hyphal system. All fungal fruit bodies are built up of hyphae, but those of polypores can have as many as three different major kinds of interwoven hyphae and are called monomitic, dimitic, or trimitic, according to whether they have one, two, or three major hyphal systems. Monomitic fruit bodies are made up of what we call generative hyphae, which are septate, can be thick or thin walled, and may or may not have clamps. Most such species are relatively soft in texture (e.g., the white cheese polypore, Tyromyces chioneus). Dimitic basidiomata have two hyphal systems, the generative being supplemented by either thick-walled, nonseptate skeletal hyphae which give basidiomata a hard, tough texture (e.g., the artist’s conk, Ganoderma applanatum) or by thin-walled, highly branched binding hyphae (e.g., the sulphur shelf, Laetiporus sulphureus). Trimitic basidiomata are composed of generative hyphae, plus skeletal hyphae, plus binding hyphae (e.g., the turkey tail, Trametes versicolor). (2) The kinds of digestive or degradative enzymes produced by the fungus. Brown rot fungi digest cellulose but not lignin. White rot fungi digest lignin but tend to leave some cellulose. Mycorrhizal fungi may not degrade wood at all. (3) The septation of the generative hyphae. In some species they are simple septate, while in others they are regularly clamped. (4) The kinds of cystidia (sterile cells in the hymenium) produced, and their origin. (5) The reaction of basidiospores with Melzer’s reagent (they are amyloid [stain blue] in Bondarzewia, dextrinoid [stain brown] in Perenniporia). (6) The size, shape, ornamentation, and walls of basidiospores (spores have a conspicuously truncate apex and a double wall in Ganoderma and are minutely spiny in Heterobasidion). Perhaps the best keys available are to be found in the two-volume North American Polypores by Gilbertson and Ryvarden, (1986–1987) and in the very recent publication by Ginns (2017) but many mushroom field guides also contain relatively good coverage of polypores, and if you include these persistent fungi among your collections, you Fig. 5.10 Diagram of trimitic will come home with something interesting at any time hyphae. After H. J. Hudson, of year—even in the depths of winter or the driest month of Fungal Biology, 1986. summer.

7—Order Russulales The two best-known genera of this order are gilled mushrooms with numerous species: Russula, common and conspicuous mycorrhizal mushrooms with brittle flesh, and Lactarius (milky caps), also brittle because the flesh is partly made up of inflated,

Kingdom Eumycota (True Fungi) thin-walled cells called sphaerocysts, and in addition, it exudes latex when broken. The basidiospores are unique in having elaborate ornamentation of ridges and warts. This ornamentation, but not the rest of the spore wall, stains darkly (usually blueblack) in Melzer’s reagent (a concoction including iodine). This is known as the amyloid (or starch-like) reaction. The spore print of the Russulaceae is white, cream, or yellow. A section through a species of the sequestrate genus Macowanites reveals a reduced stipe and shows that the gills are distorted and clearly not the vertical plates of tissue seen in true Russulas. Nevertheless, the spores of Macowanites have amyloid ornamentation and are clearly russulalean. Russula has apparently given rise to two separate sequestrate lines, Macowanites➝ Gymnomyces, and Elasmomyces➝ Martellia. Both involve an agaricoid and a hypogeous form, and both retain microanatomical characteristics, like sphaerocysts and amyloid spore ornamentation that give surprising proof of their origin in Russula. Two sequestrate genera have also evolved from Lactarius: Arcangeliella (still mushroom-like, but with a cap that encloses the gills and nonshooting basidia) and Zelleromyces (which has become hypogeous and truffle-like). All three genera produce latex. The order also contains poroid, crustose, and sequestrate relatives: a dozen families, which contain an odd-looking assortment of taxa that on initial inspection would not seem to be related to Russula. These include Albatrellus, fleshy poroid mushrooms; Auriscalpium, tiny toothed mushrooms; Echinodontium, woody, toothed tree fungi; Hericium, fleshy, sessile tree fungi with long teeth; Peniophora, common crustose fungi; and Stereum, small sessile annual tree fungi with smooth hymenia. All these are illustrated on the website.

8—Order Thelephorales The genera Thelephora (earth fans, see the website) and Polyozellus (blue chanterelles). Also Hydnellum, Sarcodon, and Phellodon, all toothed mushrooms, all are mycorrhizal. And that brings us to the end of the class Agaricomycetes.

2—Class Dacrymycetes These are all ‘jelly fungi’ that grow on rotting wood. Their gelatinous, yellow basidiomata are common and conspicuous in wet weather but shrivel up and almost disappear in dry periods. The basidiomata of Dacrymyces are irregular to the point of shapelessness and could easily be confused with the Tremellomycetes (below). But a quick look at the basidia will settle the issue. Tremelloid basidia are vertically and cruciately septate (see the website), but dacrymycetoid basidia are not. They have a unique appearance: we call them tuning fork basidia because they develop two long arms that grow up to the surface of the jelly, where their basidiospores are produced and shot away (see the website). The basidiospores are also very unusual in becoming phragmoseptate after liberation. The gelatinous fruit body of Dacrymyces plays a double role in that it often produces an unnamed thallic-arthric conidial anamorph (see the website) before the basidia develop. Guepiniopsis (Heterotextus) is bell shaped and a translucent gold in colour. On the northwest coast of North America it is common on rotting branches in fall. A third common genus is Calocera, which looks a bit like a slender, gelatinous Clavaria. There are several pictures on the website.

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3—Class Tremellomycetes Three orders, 11 families, 50 genera, and 380 species. All teleomorphs in the order Tremellales, despite their innocent appearance, are vicious parasites of other wood-inhabiting fungi. Basidiomata are gelatinous and frequently rather shapeless. Most anamorphs are yeasts. The order currently comprises 8 families with 300 species. Important genera include Tremella (see the website), with two commercially grown edible species. The anamorphic yeast genus Cryptococcus (teleomorph Filobasidiella) contains human pathogens (see chapter 23). If you want to know more about the strange and fascinating sequestrate agarics— taxa derived from no fewer than fourteen families of agarics—which no longer shoot their spores, you can read two papers dealing with this issue (Kendrick 1994a, 1994b). In 1989 Canada joined the many countries that have issued postage stamps depicting macrofungi, producing handsome (if slightly stylized) stamps of Clavulinopsis fusiformis, Boletus mirabilis, Cantharellus cinnabarinus, and Morchella esculenta; these are shown on the website. (I have been trying to get the Canadian post office to bring out some stamps on moulds, thus far without success, despite the important roles these fungi play as producers of penicillin, griseofulvin, cyclosporine, aflatoxin, etc.) The foregoing is no more than a gesture sketch of the world of the agarics. If you want to learn more about what many people consider the most fascinating of all fungi, you must buy or borrow one of the field guides listed under ‘Further Reading’ at the end of this chapter. The large tome by René Pomerleau has all the minutiae a Northeasterner needs (although the colour illustrations are poor), but the pocket-sized Audubon guide by Lincoff, although less detailed, covers the whole continent and can go anywhere with you. The larger format Mushrooms of North America by Roger Phillips, published in 1991, has over 1,000 colour photographs and includes many more species of, for example, Cortinarius (93 species), Amanita (41), Lactarius (64), and Russula (81), than other guides. Mushrooms Demystified by Arora, although oriented toward western North America, is a mine of useful and often amusing information for all mushroom fanciers; it also covers a wide range of taxa (even dealing with many sequestrate forms). The New Savory Wild Mushroom has excellent colour photographs but is mainly useful to those in the Pacific Northwest. The latest additions to these field guides are Fungi of Eastern Canada and the Northeastern United States by George Barron and Mushrooms of the Pacific Northwest by Steve Trudell and Joe Ammirati. A more regional but very recent guide is The Outer Spores—Mushrooms of Haida Gwaii by Paul Kroeger, Bryce Kendrick, Oluna Ceska, and Christine Roberts. The most recent book about west coast fungi is Mushrooms of the Redwood Coast, by Noah Siegel and Christian Schwarz. Its nomenclature is particularly up-to-date and it has excellent pictures. In addition to agarics, these books cover the more conspicuous sequestrate fungi (often called Gasteromycetes), as well as ascomycetes and polypores. Some of the larger and more difficult genera call for separate keys. ‘Matchmaker’ is a new, profusely illustrated synoptic key to thousands of western mushrooms (Google it).

Kingdom Eumycota (True Fungi)

Fig. 5.11 A: Septobasidium; B: basidia of Tulasnella; C: basidia of Puccinia; D: basidia of Ustilago; E: basidia of Tilletia.

Subphylum Pucciniomycotina Class Pucciniomycetes The new taxonomic system, based on molecular studies, divides the Pucciniomycotina up into eight classes, of which seven are rather obscure. The names can be found in Wikipedia, but the fungi are not important or conspicuous elements of the mycota. We will explore the eighth class, the Pucciniomycetes, in some detail because this subphylum contains economically important fungi that cause destructive rust diseases of plants.

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Chapter 5 Even within the chosen class, there are five orders, four of which are relatively obscure and will receive only brief notice here. Order Septobasidiales. Septobasidium (Fig. 5.11A) basidiomata are not gelatinous, and it parasitizes scale insects, which do not die but are rendered sterile. They become buried in a weft of fungal hyphae that produces basidia on its surface and provides shelter for other healthy scales. Order Helicobasidiales. Soil fungi. One member causes violet root rot of carrots. Order Platygloeales. Eocronartium, with a gelatinous basidioma, parasitizes mosses. Order Pachnocybales. Pachnocybe ferruginea, often found on rotting wood in houses, was long interpreted as a synnematal hyphomycete but was eventually recognized as a basidiomycetous teleomorph. See why on the website. Order Pucciniales. All members of this large and important order are obligate parasites, and although they do not produce what could be called a basidioma (fruit body), they tend to have complicated life cycles involving several different kinds of spores with different purposes, and they have great economic significance, because human beings are ultimately dependent on plants for subsistence. Members of this group have simple septal pores with pulley wheel occlusions (Fig. 5.1D) rather than the dolipores characteristic of most other basidiomycetes. The rust fungi are all obligately biotrophic on vascular plants and often have very narrow host ranges, being restricted to a single family, a single genus, or even a single species. Although they have obviously co-evolved with their hosts for millions of years and don’t usually kill them, rust fungi can severely reduce yields of our domesticated plants, particularly the cereal grasses on which we are so dependent. The rust fungi produce basidia from overwintering spores (teliospores), so they don’t form basidiomata. But they do produce no fewer than five different kinds of spore, each specialized for a particular step or phase in the life cycle. And they often alternate between two hosts, which tend to be from taxonomically distant groups. This is important information because, as you will see in chapter 12, our efforts to control many diseases of our food crops depend on our knowledge of the life history of the pathogens. In any case, these most complex of all fungal cycles are intrinsically fascinating. Puccinia graminis subspecies tritici, the fungus causing black stem rust of wheat, can exemplify macrocyclic, heteroecious rusts (those producing all five spore forms and moving back and forth between two different hosts). The different stages of the life cycle are shown in Fig. 5.12. Basidiospores, which are of + and – mating types, land on a young leaf of barberry Berberis) in spring and initiate localized monokaryotic infections. The hyphae are intercellular, but they send haustoria into host cells to absorb food. Soon, these monokaryotic mycelia develop tiny flask-shaped spermagonia (stage 0) in the upper layers of the leaf. They produce only small brown spots and don’t do any significant damage to the barberry. Each spermagonium forms innumerable tiny spermatia (nonmotile male gametes), which ooze out in a sweet-smelling nectar. A tuft of receptive hyphae also grows out from the neck of each spermagonium. Insects are attracted by the nectar and walk or fly from one spermagonium to another, unwittingly transferring spermatia of each mating type to receptive hyphae of the other type. This process, which is somewhat analogous to pollination, initiates the dikaryophase. The dikaryotization

Kingdom Eumycota (True Fungi)

Fig. 5.12 Pucciniales: life cycle of Puccinia graminis.

spreads to the lower surface of the barberry leaf, where the fungus has already produced the primordia of cup-like structures called aecia (stage I)—two are shown in this section. The flower-like aecia burst through the host epidermis and liberate dikaryotic aeciospores—but these spores can’t infect the barberry. Only if they land on a wheat plant (Triticum) can they establish new dikaryotic infections. That is why I call them ‘transfer spores’. The dikaryotic mycelia in the wheat plant soon produce uredinia— pustules of reddish-brown, dikaryotic urediniospores (summer spores—stage II)— which again burst through the host epidermis and are wind dispersed to other wheat plants.

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Chapter 5 Note that the urediniospores are unicellular and rather thick walled but have distinct equatorial germ pores. The many new infections generated by these spores soon produce further inoculum, and waves of urediniospores, borne on the prevailing winds, cause the massive epidemics of wheat rust that periodically sweep North America. Toward the end of summer, these same pustules switch over to producing another kind of spore, the dark, two-celled, thick-walled teliospores (winter spores—stage III). Each cell of the teliospore is binucleate at first, but karyogamy soon occurs and the spores overwinter in the genuinely diploid or zygotic condition. In spring, each cell germinates and gives rise to a short hypha which becomes a transversely septate basidium (Fig. 5.11C, rather like those of the Auriculariales and Septobasidiales). Each cell develops a short sterigma which in turn bears a basidiospore (stage IV). Basidiospores are borne asymmetrically and are shot away in typical basidiomycete manner. They must land on a barberry leaf if the cycle is to continue. Some rust fungi don’t produce all five spore forms and are described as microcyclic. Some complete their cycle on a single host and are called autoecious. Puccinia poaenemoralis, a normally heteroecious rust fungus, persists in the Canadian arctic through the ability of its urediniospores to overwinter. It never forms teliospores and so needs no other host. Some tropical rust fungi don’t form teliospores either, but in this case it is because there is no need for an overwintering spore. Stage I (aecia) and stage II (uredinia) may be regarded as the two asexually reproductive stages (anamorphs) of a rust fungus. Stage III, the teliospore, is regarded as the sexual state or teleomorph, so the host on which these spores develop is called the primary host. In the case of Puccinia graminis subspecies tritici, wheat (Triticum, Poaceae, monocots) is the primary host, and barberry (Berberis, Berberidaceae, dicots) the alternate host. Because of the threat they pose to our food supplies, the cereal rusts have been intensively studied, and they have repaid that scrutiny with a rich harvest of taxonomic and genetic information. The species Puccinia graminis attacks many different grasses. Several subspecies have been recognized by their apparent restriction to individual grass genera, for example, P. graminis ssp. avenae on oats (Avena); P. graminis ssp. hordei on barley (Hordeum); and, of course, P. graminis ssp. tritici on wheat (Triticum). Each of these subspecies is subdivided into many physiological races which differ in their ability to attack specified commercial varieties of the host genus. Puccinia graminis ssp. tritici has over 200 such races, and new ones are discovered every year. Wheat breeders have to work hard to stay one jump ahead of the pathogen. Breeding of resistant plants is discussed in chapter 12. Some heteroecious rusts move between angiosperm and gymnosperm hosts, and sometimes it is the alternate host, rather than the primary host, that is economically important. Gymnosporangium is an interesting heteroecious rust. One of its hosts is a rosaceous plant like pear. It produces spermagonia on the upper side of the leaves. These liberate both nectar, to attract arthropods, and spermatia (nonmotile, but effectively male gametes), which the visiting animals transfer to spermagonia of the opposite mating type, whereupon dikaryotization happens. After dikaryotization, the fungus goes on to produce its aecia on the same lesion, but on the other side of the leaf. On another rosaceous host, serviceberry (Amelanchier), it produces aecia on the

Kingdom Eumycota (True Fungi) hypertrophied fruit. The aeciospores can infect only the other host, a conifer such as juniper (Juniperus), on which the spectacular gelatinous telial horns are produced (these are obvious only during wet weather; see the website). A similar alternation happens in the genus Cronartium. Cronartium ribicola (blister rust of white pine) produces its spermatia and aeciospores on five-needled white pines (e.g., eastern white pine, Pinus strobus, and western white pine, Pinus monticola), and its urediniospores and teliospores on wild currant (Ribes). The aecial hosts of Cronartium comandrae (Comandra blister rust), are two- and three-needled pines, including Pinus ponderosa (ponderosa pine) and Pinus contorta var. latifolia (lodgepole pine). The telial hosts are the herbs California comandra (Comandra umbellata var. californica) and bastard toadflax (Comandra livida). The name of these diseases, ‘blister rust’, refers to the conspicuous aecia, and it is the perennial aecial cankers on the pines that gradually spread and often eventually girdle and kill the tree. Cronartium fusiforme, another blister rust, alternates between various southern pines (on which it develops its aecia) and oaks (Quercus spp). Chrysomyxa arctostaphyli moves between black spruce (Picea mariana), on which it produces its aecia, and Arctostaphylos, an ericaceous shrub on which it produces its telia. The genus Phragmidium commonly occurs on members of the Rosaceae. I found a telial specimen in the garden on one of my wife’s precious roses. She was not impressed. However, I was nostalgic about it, because the first microscopic fungus I ever collected, way back in the 1950s, was a Phragmidium producing clusters of its dark, stalked, phragmosporous teliospores on blackberry leaves. When I mounted the spores I immediately assumed it was a dark-spored hyphomycete (see phragmospores in chapter 4). I can’t remember who disabused me of this notion, but if you get the class and order right, this is one of the easiest rusts to identify. The teliospores from our rose leaf had a distinct, although colourless, stalk, six darkly pigmented cells, and an apical spine. These characteristics and its host preference identified it as Phragmidium mucronatum, which is the commonest of the nine species that occur on roses. These teliospores are big, as fungal spores go: the body of the spore is about 77 microns long (Fig. 5.13).

5.13. Two pictures of Phragmidium. A: infected rose leaves, B: phragmosporous teliospores (note germ pore in each cell) (photos by Bryce Kendrick).

Some rust fungi are systemic, spreading throughout their host plants before sporulating. This means that the sudden eruption of uredinia all over the leaves can be rather spectacular. In Java I found coffee leaves infected with the infamous coffee rust, Hemilaea vastatrix, which causes defoliation of the coffee plants (see the website). It was the

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Chapter 5 invasion of this rust fungus that caused Sri Lanka (formerly called Ceylon) to abandon coffee as its prime crop and make the radical shift to growing—and exporting—tea. The plant Euphorbia cyparissias is commonly infected by the rust fungus Uromyces pisi. Infected E. cyparissias apparently cannot flower, but the fungus induces it to form pseudoflowers—rosettes of yellow leaves that look rather like flowers (see the website). On these, the fungus produces spermagonia which ooze gametes (spermatia) in a sweet-smelling fungal nectar. Since this fungus is heterothallic, it depends on insects to cross-fertilize it. Experiments in which insects were excluded showed this to be true.

Subphylum Ustilaginomycotina This subphylum contains two major classes, the Ustilaginomycetes and the Exobasidiomycetes.

Class 1—Ustilaginomycetes Order Ustilaginales. Fifty genera and 850 species (300 in Ustilago). Like the rust fungi, the

smut fungi are all parasites of vascular plants and produce basidiospores on transversely septate basidia arising from overwintering teliospores. But the two groups differ in many respects, as Table 5.1 shows.

Table 5.1. Differences between Rust and Smut Fungi Uredinales

Ustilaginales

1) Teliospores terminal

Telispores intercalary

2) Basidiospores 4, shot from sterigmata

Basidiospore number variable, not on sterigmata, not discharged

3) Spermagonia produced (sex organs)

No sex organs, any two compatible cells can fuse

4) Clamp connections absent

Clamp connections common

5) Often require 2 hosts

Never require 2 hosts

6) Obligately biotrophic

Facultatively biotrophic, yeast-like in culture

7) Infections usually localized

Infections usually systemic

8) Teliospores in telial sori, location unspecific

Teliospores replace host organ, e.g., ovary, anther

9) Attack ferns, gymnosperms, and angiosperms

Attack only angiosperms

Kingdom Eumycota (True Fungi) In this group the teliospore is karyologically equivalent to that of a rust fungus, so the short hypha arising from a germinating teliospore of Ustilago (Fig.5.11D) becomes three-septate and buds off a yeast-like basidiospore from each compartment. Compatible elements soon fuse to restore the dikaryon. In hom*othallic species this can rather conveniently involve basidiospores from the same basidium, or a basidiospore can fuse with a cell of the basidium, or two cells of the same basidium may fuse, or teliospores may germinate and form a mycelium between whose hyphae fusions can occur. Many smut fungi, however, are heterothallic, so fusions must be between cells of different and compatible parents. The teliospores of Ustilago violacea are present on the seeds of its host, Silene (Caryophyllaceae), and germinate when the seeds do. After the dikaryotization process described above, the newly dikaryotic mycelium infects the seedling. Although the mycelium becomes systemic, spreading throughout the host, it incites no pathological symptoms until the flowers develop. Then the pollen is suddenly replaced by a mass of dikaryotic mycelium, which eventually disarticulates into teliospores. The disease is called anther smut (see www.mycolog.com). Many other smuts are also organ specific: in corn smut, caused by Ustilago maydis, some or all kernels are replaced by grossly swollen masses of black teliospores. In onion smut, caused by Urocystis cepulae (our only example of the order Urocystales), the teliospores develop in the bulb. Note that the organ attacked (and often replaced) by smut fungi is always one into which the plant directs high-energy resources—anthers, seeds, bulbs. Since humans are often interested in the host storage organs as sources of food, it may not be too surprising that frustration at the apparent loss of that food led people to sample the fungus instead. As you can read in chapter 18, at least two smuts are widely eaten: the black spore masses of corn smut (Ustilago maydis) are regarded as a delicacy in Mexico (huitlacoche), and Ustilago esculenta, which causes hypertrophy in the stems of wild rice, is widely eaten in China. (All of these examples can be seen on the website).

Family Ustilaginaceae.

Class 2—Exobasidiomycetes Order Exobasidiales. Exobasidium doesn’t produce a fruit body (a basidioma)—just a

whitish layer of basidia on the surface of the host plant. The host in this case is a member of the Ericaceae. Other Exobasidiales occur on members of the family Commelinaceae. Exobasidium produces symptoms like those caused by the Taphrinales—excessive growth of the leaf tissues and disturbances in photosynthesis that often cause the leaves to turn red (check the website). In the Family Tilletiaceae, which attack grasses, karyogamy, meiosis, and mitosis all happen inside the teliospore. When this germinates, the resulting basidium produces a cluster of slender, parallel basidiospores from its apex (see the website). These soon copulate in pairs to restore the dikaryon. Tilletia caries, the cause of ‘bunt’ or stinking smut of wheat, is just as important an economic problem as stem rust, because it has so far proved impossible to breed strains of wheat resistant to this fungus.

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Further Reading on Basidiomycetes Aime, M. C., P. B. Matheny, D. A. Henk, E. M. Frieders, R. H. Nilsson, M. Piepenbring, D. J. McLaughlin, et al. 2006. “An Overview of the Higher Level Classification of Pucciniomycotina Based on Combined Analyses of Nuclear Large and Small Subunit rDNA Sequences.” Mycologia 98:896–905. Arora, D. 1986. Mushrooms Demystified. 2nd ed. Berkeley, CA: Ten Speed Press. Bandoni, R. J. 1987. “Taxonomic Overview of the Tremellales.” In The Expanding Realm of Yeastlike Fungi, edited by G. S. de Hoog, M. T. Smith, and A. C. M. Weijman, 87–110. Baarn, Netherlands: Centraalbureau voor Schimmelcultures. Barron, George. L. 1999. Fungi of Eastern Canada and the Northeastern United States. Edmonton, Canada: Lone Pine. Begerow, D., M. Stol, and R. Bauer. 2006. “A Phylogenetic Hypothesis of Ustilaginomycotina Based on Multiple Gene Analyses and Morphological Data.” Mycologia 98:906–16. Binder, M., and D. S. Hibbett. 2006. “Molecular Systematics and Biological Diversification of Boletales.” Mycologia 98:971–81. Breitenbach, J., and F. Kränzlin. 1986. Fungi of Switzerland. Vol. 2: Non-gilled Basidiomycetes. Lucerne, Switzerland: Verlag Mykologia. ———. 1991. Fungi of Switzerland. Vol. 3: Boletes and Agarics Part 1. Lucerne, Switzerland: Verlag Mykologia. ———. 1995. Fungi of Switzerland. Vol. 4: Agarics Part 2. Lucerne, Switzerland: Verlag Mykologia. co*ker, W. C., and J. N. Couch. 1928. Gasteromycetes of the Eastern United States and Canada. Chapel Hill: University of North Carolina Press. Corner, E. J. H. 1950. A Monograph of Clavaria and Allied Genera. Annals of Botany Memoirs, Commonwealth Agricultural Bureau, Kew 1. ———. 1968. A Monograph of Cantharelloid Fungi. London: Oxford University Press. Couch, J. N. 1938. The Genus Septobasidium. Chapel Hill: University of North Carolina Press. Cummins, G. B., and Y. Hiratsuka. 1983. Illustrated Genera of Rust Fungi. Revised ed. Minneapolis, MN: American Phytopathological Society. Dai, Y.-C., and B.-K. Cui. 2011. “Fomitiporia ellipsoidea Has the Largest Fruiting Body among the Fungi.” Fungal Biology 115:813–14. Desjardin, D. E., K. G. Peay, and T. D. Bruns. 2011. “Spongiforma squarepantsii, a New Species of Gasteroid Bolete from Borneo.” Mycologia 103:1119–23. Eriksson, J., K. Hjortstam, and L. Ryvarden. 1973–1981. The Corticiaceae of North Europe. Vols. 1– 6. Oslo: Fungiflora. Fischer, G. W. 1953. Manual of the North American Smut Fungi. New York: Ronald. Gibbett, D. S. 2006. “A Phylogenetic Overview of the Agaricomycotina.” Mycologia 98:917–25. Gilbertson, R. L., and Leif Ryvarden. 1986–1987. North American Polypores. 2 vols. Oslo: Fungiflora. Ginns, J. 2017. “Polypores of British Columbia (Fungi: Basidiomycota).” FLNRO Technical Report 104. Hosaka, K., S. T. Bates, R. E. Beever, M. A. Castellano, W. Colgan, L. S. Dominguez, E. R. Nouhra, et al. 2006. “Molecular Phylogenetics of the Gomphoid-Phalloid Fungi with an Establishment of the New Subclass Phallomycetidae and Two New Orders.” Mycologia 98:949–59.

Kingdom Eumycota (True Fungi) Husher, J., S. Cesarov, C. M. Davis, T. S. Fletcher, K. Mbuthia, L. Richey, R. Sparks, L. A. Turpin, and N. P. Money. 1999. “Evaporative Cooling of Mushrooms.” Mycologia 91:351–52. Jahn, H. 1979. Pilze die an Holz wachsen. Herford, Germany: Busse. Kendrick, B. 1994a. “Evolution in Action: From Mushrooms to Truffles: Part 1.” McIlvainea 11:34–38. ———. 1994b. “Evolution in Action: From Mushrooms to Truffles: Part 2.” McIlvainea 11:39–47. ———. 2017. “All About Fungi.” Accessed April 19, 2017. http://www.mycolog.com. Kendrick, B., and R. Watling. 1979. “Mitospores in Basidiomycetes.” In The Whole Fungus, 473– 545. Vol. 2. Ottawa: National Museum of Canada. Available only from Mycologue Publications, Sidney, Canada. Kibby, G., and R. Fatto. 1990. Keys to the Species of Russula in Northeastern North America. 3rd ed. Somerville, NJ: Kibby Fatto Enterprises. Kroeger, P., B. Kendrick, O. Ceska, and C. Roberts. 2012. The Outer Spores—Mushrooms of Haida Gwaii. Sidney, Canada: Mycologue Publications and Haida Gwaii, Canada: Haida Gwaii Museum. Lincoff, G. H. 1981. The Audubon Society Field Guide to North American Mushrooms. New York: Knopf. Matheny, B., J. M. Curtis, V. Hofstetter, M. C. Aime, J.-M. Moncalvo, Z.-W. Ge, Z.-L. Yang, et al. 2006. “Major Clades of Agaricales: A Multilocus Phylogenetic Overview.” Mycologia 98:982–95. McKenny, M., D. E. Stuntz, and J. Ammirati. 1987. The New Savory Wild Mushroom. 3rd ed. Seattle: University of Washington Press. Miller, S. L., E. Larsson, K.-H. Larsson, A. Verbeken, and J. Nuytinck. 2006. “Perspectives in the New Russulales.” Mycologia 98:960–70. Moncalvo, J.-M., R. H. Nilsson, B. Koster, S. M. Dunham, T. Bernauer, P. B. Matheny, T. M. Porter, et al. 2006. “The Cantharelloid Clade: Dealing with Incongruent Gene Trees and Phylogenetic Reconstruction Methods.” Mycologia 98:937–48. Money, N. P. 1998. “More g’s Than the Space Shuttle: Ballistospore Discharge.” Mycologia 90:547–58. Moser, M. 1983. Keys to Agarics and Boleti (Polyporales, Boletales, Agaricales, Russulales). London: Roger Phillips. Oberwinkler, F. 1982. “The Significance of the Morphology of the Basidium in the Phylogeny of Basidiomycetes.” In Basidium and Basidiocarp, edited by K. Wells and E. K. Wells, 9–35. New York: Springer-Verlag. Pfunder, M., and B. A. Roy. 2000. “Pollinator-Mediated Interactions between a Pathogenic Fungus, Uromyces pisi (Pucciniaceae), and Its Host Plant, Euphorbia cyparissias (Euphorbiaceae).” American Journal of Botany 87:48–55. Phillips, R. 1991. Mushrooms of North America. Boston: Phillips, Little, Brown & Co. Pomerleau, R. 1980. Flore des Champignons au Québec. Ottawa, Canada: Les Editions La Presse. Ramsbottom, J. 1953. Mushrooms and Toadstools. London: Collins. Reijnders, A. F. M. 1963. Les Problèmes du Developpement des Carpophores des Agaricales et de Quelques Voisins. The Hague, Netherlands: Junk. Siegel, N., and C. Schwarz. 2016. Mushrooms of the Redwood Coast. Berkeley, California: Ten Speed Press.

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Chapter 5 Singer, R. 1975. The Agaricales in Modern Taxonomy. 2nd ed. Weinheim, Germany: Cramer. Smith, A. H., and H. D. Thiers. 1971. The Boletes of Michigan. Ann Arbor: University of Michigan Press. Trudell, S., and J. Ammirati. 2009. Mushrooms of the Pacific Northwest. Portland: Timber Press. Ziller, W. G. 1974. The Tree Rusts of Western Canada. Ottawa: Information Canada.

6 Yeasts—Compact Polyphyletic Extremophile Fungi Everyone knows the word ‘yeast’, but very few people have much idea what yeasts really are (since they are invariably microscopic), and fewer still are aware that the name is applied to organisms of very different origins. You are about to join that elite group. The word ‘yeast’ has been widely interpreted in terms of morphology alone, but as you will see, that is an oversimplified view, and yeast-like forms have evolved in many different groups of fungi, from Zygomycota to Basidiomycota. Currently, about 1,500 species of yeasts are known, distributed among about 100 genera. Yeasts are mostly (but not all) unicellular organisms, some of which are useful to us because they make bread rise; put the alcohol in beer, wine, and spirits; and are a highprotein food supplement as well as a rich source of B vitamins. But there is much more to them than that, and they have a darker side: some are implicated in food spoilage, and a few yeasts, such as Candida albicans, cause potentially serious diseases of humans. Although yeasts are still often characterized as single-celled fungi that do not produce hyphae, Candida and quite a few other yeasts clearly do produce hyphae, as well as what we call ‘yeast cells’. The yeast cells of Candida are basically conidia and develop in what we would call ‘blastic-acropetal’ branched chains (see chapter 4). Because of their economic and medical importance, there was a need to identify microscopically similar but physiologically different yeasts. So zymologists (yeast experts) developed a taxonomic scheme based on physiological tests such as the ability of yeasts to ferment or assimilate a variety of sugars, their nitrogen and vitamin requirements, antibiotic resistance, and so on. More recently, sophisticated techniques such as magnetic resonance analysis of cell wall components, electrophoretic enzyme analysis, cytochrome spectrophotometric analysis, serological tests, DNA reassociation, and DNA base composition have all been pressed into service in the search for useful taxonomic characteristics in yeasts, and the genomes of some yeasts have been sequenced. One assumption underlying much of this activity was that yeasts had relatively few morphological characteristics to work with. It was thought that yeasts reproduced by one of two processes, which were simplistically called ‘budding’ or ‘fission’. But yeasts exhibit morphological and developmental features whose significance has only fairly recently been appreciated. These characteristics even offer clues to the underlying phylogenetic diversity of the group. We now think of assimilative yeast cells as essentially conidia and have identified several different kinds of conidiogenesis among them, as Fig 6.1 shows. Many yeasts (about 600 species in 22 genera) never develop a teleomorph and are essentially anamorphic conidial fungi. Here is a quick overview of the different groups in which yeasts or yeast-like forms occur.

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Fig 6.1

Conidiogenesis in yeasts (compare with Figs. 4.5–4.8).

Phylum Zygomycota—Mucor rouxii Phylum Ascomycota Incertae sedis—Blastomyces Subphylum Saccharomycotina (true yeasts)

Yeasts—Compact Polyphyletic Extremophile Fungi Class Saccharomycetes—(Families Ascoideaceae, Candidaceae, Cephaloascaceae, Dipodascaceae, Endomycetaceae, Eremotheciaceae, Lipomycetaceae, Metschnikowiaceae, Pichiaceae, Saccharomycetaceae, Saccharomycodaceae, Saccharomycopsidaceae) genera Saccharomyces, Saccharomycodes, Dipodascus (anamorph = Geotrichum) Saccharomycopsis (anamorph = Candida) Hanseniaspora (anamorph = Kloeckera) Subphylum Taphrinomycotina Order Taphrinales—Taphrina (germinating ascospores) Order Pneumocystidiales—Pneumocystis Class Schizosaccharomycetes—Schizosaccharomyces Subphylum Pezizomycotina Order Onygenales—Histoplasma (in human tissue) Phylum Basidiomycota Subphylum Agaricomycotina Class Tremellomycetes—(germinating basidiospores) Filobasidium and Filobasidiella (anamorph = Cryptococcus) Subphylum Pucciniomycotina Class Microbotryomycetes—Leucosporidium Rhodosporidium (anamorph = Rhodotorula) Sporidiobolus (anamorph = Sporobolomyces) Subphylum Ustilaginomycotina (have saprobic yeast phases) Multilateral budding yeasts bud from many different points on the cell, producing only one daughter cell (conidium) from each site and leaving many scars (Saccharomyces; Fig 6.1A). What have commonly been called apiculate, bipolar budding yeasts have long cells that bud repeatedly from each end, extending percurrently in the process (e.g., Saccharomycodes; Fig. 6.1C, lower diagram). Cells of what have been inaccurately termed ‘fission’ yeasts also extend percurrently, but on a much broader base (e.g., Schizosaccharomyces; Fig. 6.1C, upper diagram). Some hyphal yeasts produce thallic-arthric conidia (Geotrichum; Fig. 6.1E). Basidiomycetous yeasts may be blastic-sympodial (Cryptococcus; Fig. 6.1B) or blastic-phialidic (Rhodotorula and Sporobolomyces; Fig. 6.1D). Some of these unicellular anamorphs can switch into the teleomorphic mode and produce structures that would appear to place them among the Dikarya (Ascomycetes or Basidiomycetes), although since sex involves fusion of individual cells to form a zygote, there is no dikaryophase. Yeast phases of smut fungi (Ustilaginomycotina) can, however, have a dikaryophase. Some yeasts form endogenous meiospores inside meiosporangia that are karyologically exactly comparable with asci (several such meiosporangia, most containing four spores, are shown in Fig. 6.2A), although the wall chemistry (a good indicator of phylogeny) is somewhat different from that of mainline ascomycetes, and they are never produced on or in a fruit body (ascoma). The ability of some yeasts to produce hyphae is emphasized in Fig. 6.2. Saccharomycopsis (Fig. 6.2A) produces Candida anamorphs. Dipodascus (Fig. 6.2B) produces a Geotrichum anamorph, shown here beside the meiosporangium. Other yeasts are members of the Basidiomycota. Some of these (e.g., Sporobolomyces; Fig. 6.2F) even produce exogenous spores borne asymmetrically on pointed

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Fig 6.2

Some unusual yeasts. A–C: Saccharomycetes; D, E, F: Basidiomycetes.

outgrowths of the cell. These spores are forcibly discharged, and the mechanism involved is obviously that of the basidium. Some, which produce hyphae, even make clamp connections (Fig. 6.2D). Others, such as Cryptococcus, produce blastic-phialidic (Fig.6.1D, centre) or blastic-sympodial (Fig. 6.1B) conidia. To outline the full taxonomic diversity of yeasts, I must also add that when members of the Ustilaginales (Ustilaginomycetes) or Taphrinales (Ascomycetes) are grown in axenic culture, they become yeast-like. Basidiospores of Tremellales germinate to produce a haploid yeast phase. Several fungal pathogens of humans, while filamentous in culture, are yeast-like when growing inside us (e.g., Histoplasma capsulatum, Blastomyces dermatitidis; see chapter 23). Finally, a few fungi such as Mucor rouxii (Zygomycetes) can be changed from a hyphal to a yeast-like morphology, or vice versa, by varying levels of carbon dioxide or of various nutrients. So yeast morphology is sometimes a response to environmental factors such as osmotic stress, a response that has evolved many times in different groups (just as the lichenization process [chapter 7] and the change from agaric to sequestrate derivative [chapter 5] have occurred many times). As a final twist to this tale, mycologists have discovered that some fungi which consistently produce hyphae (e.g., Arthroascus,

Yeasts—Compact Polyphyletic Extremophile Fungi Ashbya, Candida, Crebrothecium, Dipodascus, Eremothecium, Guilliermondiella, Saccharomycopsis) are closely related to the unicellular yeasts. This conclusion is based on five kinds of evidence: (1) even in hyphal forms, there is never a dikaryophase; (2) they produce ascus-like meiosporangia in isolation, or singly, in clusters, or in chains, on individual somatic hyphae, but never in any kind of ascoma; (3) their cell walls contain less chitin and more mannan than those of regular ascomycetes; (4) some of them produce yeast-like anamorphs; and (5) some of them have many extremely narrow micropores (Fig. 5.1B) piercing each septum, rather than a single central pore. These features, among others, argue for the recognition of the ascus-forming yeasts as a group distinct from the ascomycetes proper. Class Saccharomycetes, Order Saccharomycetales. Representative genera are Dipodascus with Geotrichum anamorphs, Hanseniaspora with Kloeckera anamorphs, and Saccharomycopsis with Candida anamorphs. Candida albicans, which produces aerial hyphae (Fig. 6.2C), and whose teleomorph (if any exists) is unknown, causes candidiasis, a disease which affects mucous membranes in various parts of the body or may even become systemic. This is more fully discussed in chapter 23. The Geotrichum anamorph of Dipodascus produces hyphae which break up into thallic-arthric conidia, but since this is a yeast, there are subtle differences between this and, for example, the many basidiomycetous anamorphs that also produce thallicarthric conidia. Wall chemistry is different and the septa have many micropores rather than the single, central septal pore of most other hyphal fungi. Class Tremellomycetes, Order Tremellales (in part). A second, very different group of yeasts have chitin-mannan walls which also contain some xylose or fucose (both absent from the Saccharomycetes). The anamorphs in this group also have two modes of conidiogenesis. Most are blastic-sympodial (e.g., the Cryptococcus anamorphs of Filobasidiella). Others are blastic-phialidic (e.g., Cryptococcus anamorphs of Filobasidium). The teleomorphs, where these are known, produce clamp connections and basidium-like structures. The holomorphs are placed in the family Filobasidiaceae, and Cryptococcus neoformans, the anamorph of Filobasidiella neoformans, causes a potentially serious lung disease, cryptococcosis, which is further discussed in chapter 23. Some other genera, such as Phaffia and Bullera, are known only as anamorphs. And although the teleomorph of Trichosporon, if one exists, is unknown, this anamorphic yeast probably belongs here, because its hyphae have dolipore septa (Fig. 6.2E). As the diagram shows, this genus forms conidia sympodially, and the hyphae also tend to break up into thallicarthric conidia.

Subphylum Pucciniomycotina. Although 99% of this group are the rust fungi, the other 1% is extremely variable. Class Microbotryomycetes, Order Sporidiobolales (in part). The third group are called the red yeasts, because they contain carotenoids (although some species of Cryptococcus and Phaffia also produce these compounds). Rhodotorula, which produces pinkish or reddish colonies, forms blastic-phialidic conidia from the attenuated ends of the yeast cells. Sporobolomyces cells (Fig. 6.2F) develop sterigmata from which asymmetrically borne spores are forcibly ejected. A series of such ballistospores is formed by sympodial extension of the sterigma. Note that although the spore-shooting technique being employed here is that of the basidium, the spores being formed are asexual mitospores (conidia). Yeasts of this group sometimes produce a teleomorph: a chlamydospore-like

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Chapter 6 teliospore which germinates to form an outgrowth from whose tip meiosporic ballistospores are formed and discharged (e.g., Aessosporon and Sporidiobolus with Sporobolomyces anamorphs; Rhodosporidium with Rhodotorula anamorphs). It is now possible to differentiate basidiomycetous yeasts from Saccharomycetes by staining with buffered diazonium blue B. But in order to identify yeasts to genus and species, one now has to check such features as (1) the minimum, optimum, and maximum temperatures for growth and sporulation; (2) growth in the presence of some toxic compounds; (3) osmotolerance (growth in high sugar or salt concentrations); (4) cell morphology and method of conidiogenesis; and (5) DNA sequences. Yeasts have always been important to us, primarily as the producers of bread and alcohol, which is still, despite competition from other fungal metabolites, and despite its manifest dangers, our most widely used and accepted social drug. Accounts of the involvement of yeasts in human affairs can be found in chapters 10 (Fungal Genetics), 18 (Fungi as Food), 19 (Fungi in Food Processing), and 23 (Medical Mycology).

Further Reading on Yeasts Barnett, J. A. 2011. Yeast Research: A Historical Overview. Washington, DC: ASM Press. de Hoog, G. S., M. Smith, and A. C. M. Weijman, eds. 1987. The Expanding Realm of Yeast-like Fungi. Studies in Mycology 30. Baarn: Centraalbureau voor Schimmelcultures. Feldmann, H. 2010. Yeast: Molecular and Cell Biology. Hoboken: Wiley-Blackwell. Kurtzman, C. P., and J. W. Fell, eds. 1997. The Yeasts: A Taxonomic Study. 4th ed. New York: Elsevier. [A comprehensive treatment by thirty-eight authors] Kurtzman, C. P., and J. W. Fell. 2006. “Yeast Systematics and Phylogeny—Implications of Molecular Identification Methods for Studies in Ecology.” In Biodiversity and Ecophysiology of Yeasts, edited by Carlos A. Rosa and Peter Gabor, 11–30. Berlin: Springer. Kutty, S. N., and R. Philip. 2008. “Marine Yeasts—a Review.” Yeast 25, no. 7: 465–83. Samson, R. A., M. Smith, and E. S. van Reenen-Hoekstra. 1988. Introduction to Food-Borne Fungi 3rd. ed. Centraalbureau voor Schimmelcultures, Institute of the Royal Netherlands Academy of Arts and Sciences, Baarn. Skinner, F. A., S. M. Passmore, and R. R. Davenport, eds. 1980. Biology and Activities of Yeasts. New York: Academic Press. Von Arx, J. A. 1979. “Propagation in the Yeasts and Yeast-Like Fungi.” In The Whole Fungus, edited by B. Kendrick, 555–71. Vol. 2. Ottawa: National Museums of Canada. ———. 1980. “A Mycologist’s View of Yeasts.” In Biology and Activities of Yeasts, edited by F. A. Skinner, M. Passmore, and R. R. Davenport, 53–61. London: Academic Press. ———. 1981. “Systematics of Conidial Yeasts.” In Biology of Conidial Fungi, edited by G. T. Cole and B. Kendrick, 85–96. Vol. 1. New York: Academic Press. Von Arx, J. A., L. Rodrigues de Miranda, M. T. Smith, and D. Yarrow. 1977. The Genera of Yeasts and the Yeast-Like Fungi. Studies in Mycology 14. Baarn: Centraalbureau voor Schimmelcultures. Wood, V., R. Gwilliam, M. A. Rajandream, M. Lyne, R. Lyne, A. Stewart, J. Sgouros, et al. 2002. “The Genome Sequence of Schizosaccharomyces pombe.” Nature 415, no. 6874: 871–80.

7 Lichens—Dual (or Even Triple) Extremophile Organisms Habitats Lichens are among the toughest macroscopic organisms: what other group can grow on bare rock; in exposed situations subjected to extremes of temperature, radiation, and desiccation; from hot deserts to the sub-zero arctic; and from the seashore to the highest mountains (lichens are found at over 7,000 metres on Mount Everest)? As you can see at www.mycolog.com, the rocky cliffs along the north shore of Lake Superior are covered with lichens. So is the ground in the forest near Schefferville in northern Quebec. So is the bark of a tree at Dingo Beach in Queensland, Australia. The intertidal zone is another tough neighbourhood, and the rocks just above high tide are exposed to drying out, salt spray, rain, sun, and frost, often in rapid succession. Yet many rocks along the east and west coasts of North America are coated with black lichens of the genera Hydropunctaria and Wahlenbergiella. Simple lichen associations also grow beneath the surfaces of rocks in the Namib desert of southwestern Africa. They will grow wherever the air is clean.

Anatomy: Lichen = Fungus + Alga(e) What is their secret? Most of them are dual organisms. Each lichen combines the talents and strengths of a fungus (the mycobiont) with those of at least one type of alga and/or cyanobacteria (the photobiont). The fungus obtains water and minerals, builds a complex thallus (body), and produces sexual and asexual reproductive structures. The alga lives and photosynthesizes inside the fungal thallus, and although algae mostly constitute only 5%–10% of the total biomass of lichens, usually concentrated in a zone just below the upper surface of the thallus, they supply energy-rich carbon compounds to the entire organism. Only very few of the 15,000–20,000 fully lichenized fungi (almost a fifth of all known fungi and 40% of all ascomycetes) are found in nature without their domesticated alga, although the algae involved can be free-living (and many of the fungi have been grown in axenic culture). The degree to which the association has led to physical, as opposed to physiological, integration varies. In the simplest case, that of the amazing cryptoendolithic associations of fungi and algae recently discovered beneath the surface of sandstone in the deserts of Antarctica and of Namibia, there are no special 145

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Fig 7.1 Lichens. A, B: Crustose thalli; C, D: foliose thalli; E: squamulose thallus; F–H; fruticose thalli, note squamules at base of podetium in H; I: thallus releasing soredia; J: coralloid isidia; K: cylindrical isidia; L: lichen synthesis, Trebouxia being enveloped by its mycobiont; M: v.s. discolichen through apothecial ascoma.

Lichens—Dual (or Even Triple) Extremophile Organisms dual structures. There are a few ‘filamentous’ lichens, in which the algal filaments determine the form of the association. But almost all lichens are at least 95% fungus, and so the fungus determines the shape of the entire organism. Fig. 7.1M shows a section through part of a lichen thallus (or body). Most of the thallus is clearly made up of fungal hyphae. Those composing the upper and lower surfaces are densely aggregated, forming protective cortical layers. Inside the thallus there is more room between the hyphae, and the round algal cells usually sit just below the upper cortex, surrounded by hyphae. In fact, the fungus has effectively ‘captured’ or ‘domesticated’ the alga, and the relationship is one of exploitation or balanced parasitism rather than of mutualistic symbiosis, since about 50% of the food synthesized by the alga is pirated by the fungal hyphae, which form tight little cages around the algal cells (Fig. 7.1L). What does the fungus give in return? It provides mineral elements and protection from rapid drying out, which doesn’t sound like much of a bargain, but the combination of kingdoms is incredibly tough and successful, and all lichens are now regarded as essentially fungi.

The Photobionts Although there are over 500 genera of lichens and up to 28,000 species, these are all associated with only about 25 genera of eukaryotic green algae (Chlorophyta) and 15 genera of prokaryotic blue-green algae (Cyanobacteria). About 80% of lichens contain unicellular green algae (most of them contain the unicellular Trebouxia), about 10% contain filamentous green algae, and about 10% contain cyanobacteria. The photobionts of more than 90% of all lichens are drawn from only three genera: two green algae, the unicellular Trebouxia and the filamentous Trentepohlia; and the filamentous cyanobacterium Nostoc. So lichen taxonomy has very little to do with algae—it is essentially fungal taxonomy, and the names given to lichens are always those of the fungal component, or mycobiont.

Fig 7.2

Lichen photobionts—Trebouxia and Nostoc.

About 500 lichens with green phytobionts also have areas on or in their thalli which contain blue-green algae (cyanobacteria). These anomalous, often wart-like, areas are called cephalodia. Three-part lichens are found in such common genera as Lobaria, Peltigera, Pseudocyphellaria, and Sticta. In lichens containing green algae, the carbohydrates (photosynthates) that move from alga to fungus are sugar alcohols; in lichens containing cyanobacteria, glucose migrates to the fungus. We do not yet know how the fungus controls this transfer. Cyanobacteria have the incredibly useful and very rare

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Chapter 7 ability to fix atmospheric nitrogen, and some of this fixed nitrogen is also passed on to the fungus. In three-part lichens, the cyanobacteria have numerous heterocysts, cells responsible for nitrogen fixation, and hence it is likely that their main function in the symbiosis is the supply of nitrogen.

The Mycobionts We know between 15,000 and 20,000 lichenized fungi. About 10,000 produce only teleomorphic (sexual) fructifications, while the rest produce conidial anamorphs as well, and there are some genera of lichens which produce only anamorphs or are sterile. Over 98% of all lichenized fungi are ascomycetes, but there are a few lichenized basidiomycetes, one of which is shown on the cover of this book.

Morphology—Thallus Types: Crustose, Foliose, Fruticose, Squamulose, Leprose, Gelatinous Most lichens are what we call discolichens, because their fungal fructifications are apothecial ascomata of the kind found in discomycetes. Icmadophila ericetorum is a bit of a mouthful, but it is the name of an easily recognized lichen which grows as a layer on the surface of rotten wood. Its thallus is blue-green, while the scattered apothecial ascomata are pink. Lecanora xylophila produces elongated whitish thalli embedded in the surface layer of many of the larger decorticated logs that have washed up along the beach below my house. Its apothecial ascomata are a deep red with a whitish margin. Caloplaca produces brilliant orange apothecial ascomata on finely lobed orange thalli that grow over rock surfaces. Parmelia produces grey to green lobed thalli that often form large circular colonies on rocks. Its apothecial ascomata are brownish cups concentrated toward the centre of the colony. Letharia columbiana is an apothecial lichen that grows on dead branches of trees in western North America. It has a delicately branched, bright yellow, lacy thallus bearing brown apothecial ascomata. The fine, branched thalli of Usnea and Alectoria hang down in strands from tree branches and may bear small apothecia. We need to categorize these very different thalli, at least for the sake of communication and convenience. All these lichens are illustrated on the website. Of the lichens just mentioned, Icmadophila, Caloplaca, and Lecanora are closely pressed to, or even embedded in, their substrate, and are termed crustose (Fig. 7.1A, B). Parmelia is attached to the substrate by thread-like rhizines produced from its lower cortex and is (at least in theory) separable from the substrate. Its lobed appearance gives such thalli the name foliose (Fig. 7.1C, D). Letharia and Usnea grow away from their substrates and often branch repeatedly: they are called fruticose (which means bush-like and has nothing to do with fruit; Fig. 7.1F–H). A fourth type of thallus is exemplified by many species of Cladonia, which are basically made up of many small upturned scales, sometimes derived from the cracking and fragmentation of a crustose thallus. This type of thallus, often found on soil, is called squamulose (Fig. 7.1E). Species of Cladonia are unusual in that they often have dimorphic thalli, with squamules on the substrate, and upright, sometimes branched structures called podetia, which frequently bear reproductive organs—apothecial ascomata, pycnidial conidiomata, and

Lichens—Dual (or Even Triple) Extremophile Organisms even soredia (see below). The fifth type of thallus is the leprose, in which the entire thallus is made up of loose, powdery material. All of these, and the rather unusual sixth type of thallus found in the gelatinous lichens, are illustrated on the website.

Sexual Reproduction Although most lichens, as discussed above, are apothecial, some, such as Verrucaria, produce flask-shaped perithecia (see chapter 4), and some, such as Roccella, Pyrenula, and Arthonia, have pseudothecia containing bitunicate asci (chapter 4). Some, like Lepraria, never reproduce sexually. About twenty lichens involve basidiomycetes. Perhaps the most common of those is Lichenomphalia (Omphalina) ericetorum, shown on the cover of this book, in which the mycobiont is (as the name implies) a small agaric.

Lichen Asci and Ascospores

Fig. 7.3 Lichen asci and ascospores. From Brodo, Sharnoff, and Sharnoff, Lichens of North America (Yale University Press, 2001).

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Chapter 7 Many lichen asci tend to mature slowly. At any given time, relatively few of the asci in a lichen hymenium will be ripe, and the type of ‘puffing’ so often seen in freeliving apothecial fungi such as Peziza never happens in lichens. Some lichen asci are unitunicate-inoperculate, some are prototunicate, and others are bitunicate. Some are of the ‘jack-in-the-box’ type we have already seen in the Dothideales (chapter 4), but others, especially in the very large order Lecanorales (which has nearly 6,000 species), have a different type of asci that split at the tip. Although they have two wall layers, they are functionally unitunicate. Lichen ascospores are diverse. In Pertusaria the ascospores are relatively large and multinuclear, often with a reduced number (1–4) per ascus. In Acarospora there may be up to 100 spores per ascus. Ascospores may be amerosporous (single celled), didymosporous (two celled), or dictyosporous (many celled). As mentioned, they often ripen slowly, only a few being mature at any given time. But there are also pioneers, such as Gregorella (or foliicolous lichens) that develop asci within a year and are then outcompeted by faster-developing species.

Asexual and Somatic Reproduction Although the obvious fruiting structures of most lichens are the fungal teleomorphs, many lichens also produce pycnidial anamorphs, which can be seen, for example, around the edges of Cladonia pyxidata podetia as small dark dots. Many lichens also produce specialized ‘somatic propagules’. In some, the upper surface of the thallus ruptures, exposing a powdery mass of propagules called soredia, which are small groups of algal cells entangled in fungal hyphae. Sometimes the entire thallus is basically powdery (leprose). Another type of asexual reproduction involves small, fingerlike or branched structures called isidia, which grow up from the thallus, then break off. Some representatives of certain genera produce only isidia, others only soredia. Lichens that reproduce asexually are less likely to form ascomata. Perhaps the most common accessory structures are the podetia of such squamulose genera as Cladonia. These are large, upright, often branched structures which generally have one or more apothecium-like cup at the top. These may in fact become single red apothecia, as in Cladonia coccifera, or they may bear smaller, variously coloured apothecia around the rim, or they may have tiny anamorphic, flask-shaped pycnidial conidiomata around the edge, as in Cladonia pyxidata. The surface of the podetium may also be covered with powdery soredia and not produce any asci. In such cases, what began as a fungal reproductive structure has evolved into a photosynthetic organ.

Taxonomic Groupings The proportions of the various classes of Ascomycetes and Basidiomycetes that are lichenized are still only partially known because the phylogenetic placement of some lichenized groups is not yet known. Thus the numbers given below are only rough estimates. The most important things to note here are (1) the process of lichenization appears to have occurred independently in many different groups of fungi, although a

Lichens—Dual (or Even Triple) Extremophile Organisms single origin of the lichen lifestyle in Ascomycetes cannot be entirely rejected with the data available at the moment; and (2) about 40% of all Ascomycetes are lichenized, although very few Basidiomycetes are. About 98% of all lichens have an ascomycetous fungal component. Percentage of fungal groups lichenized Ascomycota Arthoniomycetes—circa 95% (Arthoniales 95%) Dothideomycetes—circa 5% (Pleosporales s. lat. 5%, Trypetheliales 100%) Eurotiomycetes—circa 40% (Pyrenulales 80%, Verrucariales 90%) Lichinomycetes—100% (Lichinales 100%) Lecanoromycetes—circa 95% (Acarosporales 98%, Baeomycetales 100%, Candelariales 100%, Rhizocarpales 100%, Lecideales 100%, Peltigerales 100%, Lecanorales 95%, Teloschistales 100%, Pertusariales 98%, Baeomycetales 100%, Ostropales 75%, Umbilicariales 100%) Basidiomycota Agaricomycetes—about 1% Although eighteen largely or entirely lichenized orders of Ascomycetes are listed above, and several more have some or many lichenized members, I am going to mention features of only seven of the larger or more common orders. (1) Order Arthoniales: (95% lichenized) 45 genera, 1,500 species, with green algae as photobionts. Thalli mostly crustose or sometimes fruticose, with apothecioid or lirellate (long and narrow) ascomata, producing bitunicate asci. (2) Order Ostropales: circa 50 genera, 2,500 species, with green photobionts. Thalli crustose, with apothecioid or lirellate ascomata, containing unitunicate-inoperculate asci with a thickened apex. The highest diversity is in the tropics, and the number of species is probably much higher than currently assumed. (3) Order Lecanorales: (95% lichenized) 300 genera, 5,700 species, with green photobionts. Crustose, squamulose, foliose, or fruticose thalli, with apothecioid ascomata producing lecanoralean asci. This huge order is home to many of our commonest lichen genera— Cladonia (including ‘reindeer moss’ [C. rangiferina], and ‘British soldier’ [C. cristatella]), Hypogymnia, Letharia, Parmelia, and Umbilicaria (‘rock tripe’). (4) Order Peltigerales: (100% lichenized) 18 genera, 600 species, with blue-green or green photobionts. Some species have both a green and blue-green photobiont and the cyanobacteria are often concentrated in cephalodia. Mostly foliose or fruticose thalli with apothecioid ascomata producing lecanoralean asci. (5) Order Pyrenulales: (80% lichenized) 35 genera, 1,150 species, with green photobionts. Mainly crustose, with pseudothecial ascomata with ascohymenial development, containing bitunicate asci. (6) Order Teloschistales: (100% lichenized) 11 genera, 600 species, with green photobionts. The thalli are of all four main types, bearing apothecioid ascomata with lecanoralean asci, and also producing pycnidial anamorphs. (7) Order Verrucariales: (90% lichenized) 25 genera, 700 species, with green photobionts. Usually crustose, rock-inhabiting lichens with pseudothecial ascomata, ascohymenial development, and bitunicate asci.

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Chapter 7 The discipline of lichenology, until fairly recently, has been conducted outside the mainstream of mycology, because the dual organisms were considered so radically different from nonlichenized fungi in nutrition, ecology, and lifespan. But it is being increasingly realized that the life processes of lichens, including their biotrophic nutrition, are not really alien to those of many other fungi, and we can anticipate increased integration of this large minority group, as specialists in lichenized and nonlichenized fungi exchange information and ideas. At least one important reference work, the Dictionary of the Fungi, now covers both groups.

Identification of Lichens Lichens produce about 300 unique compounds called ‘lichen substances’. These are mainly weak phenolic acids, derivatives of orcinol or β-orcinol. They include depsides, depsidones, and dibenzonfuran derivatives, among other substances, such as usnic acid, which has antibiotic properties. The indicator, litmus, is obtained from depside-containing lichens. Some of these unique lichen substances are routinely used to identify the genera and species that produce them. Keys to lichens often call for chemical tests with 10% aqueous potassium hydroxide (KOH), chlorine bleach (Cl), and 5% alcoholic paraphenylenediamine (PPD). These, when applied in various sequences, combine with secondary compounds to give characteristic yellow, orange, or red colour reactions. Professional lichenologists can’t stop at this level: accurate identification of many lichens calls for more refined techniques. (1) Recrystallization of lichen substances: these are first leached out of the thalli by acetone, then redissolved in a glycerine/alcohol mix with some water, orthotoluidine, aniline, or quinoline added. Heating causes recrystallization, generating characteristic shapes and colours observed under UV. More precise identification can be attained by resorting to (2) thin layer chromatography, (3) high performance liquid chromatography, or (4) DNA analysis. (These procedures are necessitated by the existence of as many as six ‘chemical strains’ within some lichen species. These strains may look exactly alike, but their chemistry differs, and although they often have different distributions, they frequently overlap.)

Lichen Synthesis The very existence of the slowly developing lichen ascospores is something of a puzzle, because when they are eventually released, in many but not all cases, no algal cells go with them. This means that if the ascospores are to establish a new generation of lichens, they must encounter an appropriate photosynthetic partner, and this in turn implies that lichens must be constantly resynthesized in nature. The only problem with this was that for many years all our best efforts to synthesize lichens from their component fungi and algae failed. The trick was finally mastered by having each of the prospective partners in a thoroughly debilitated condition. Only then, it seems, will the fungus literally embrace the alga, and only then will the alga permit itself to be coopted without making the ultimate protest.

Lichens—Dual (or Even Triple) Extremophile Organisms In a successful synthesis, the fungal hyphae grow around each algal cell and produce appressoria on its surface. It appears that, once the alga is in this situation, its physiology is subtly altered. While it metabolizes more or less normally, it becomes very leaky, losing large quantities of soluble carbohydrates. Trebouxia leaks ribitol, Trentepohlia leaks erythritol, and Nostoc leaks glucose. All of these are quickly absorbed by the fungus and converted into typical fungal carbohydrates such as trehalose. This is interesting in view of research suggesting that high levels of this sugar are one of the secrets of surviving extreme desiccation.

Growth Rate and Longevity Since the sustaining alga usually makes up no more than 5%–10% of the thallus, and since lichens in exposed situations will be dried out during much of the year, it is apparent that we can’t expect lichens to grow very fast. And in fact, they don’t. A respectable growth rate for many lichens is 1–4 mm per year, although some grow faster and some much slower than that. Their tough, resistant thalli and their ability to sit out dry or cold conditions, resuming metabolism quickly when wetted, seem to have conferred on them great longevity; some lichen colonies are reputed to be 4,500 years old, giving the ancient bristlecone pines (Pinus aristata) of the White Mountains in California some inconspicuous competition for the title ‘World’s oldest living thing’. This represents an amazing change from the Discomycetes (which provided most of the fungal partners), with their ephemeral fruiting bodies.

Lichenometry Lichenologists calculate past growth rates based on measurements of colonies found on dated gravestones. This enables them, for example, to help glaciologists determine how long it has been since particular rock faces emerged from under the ice of retreating glaciers.

Lichens and Air Pollution Lichens can tell even urbanites something important about their habitat. Since lichens have no roots or other specialized absorptive organs, and since they often live in soilless habitats, they are dependent on the rain to bring them mineral nutrients. As we know, the rain over much of eastern North America contains dissolved pollutants— especially sulphur dioxide as sulphurous acid. Lichens are extremely susceptible to the deleterious effects of acid rain, and many cities are essentially lichen deserts. Fish, trees, lichens: all are like the canaries that miners used to take down the pit—ultrasensitive indicators of dangers to ourselves and to the entire biosphere. As a footnote to this chapter, I feel I must mention the case of the bitunicate Ascomycete Mycosphaerella ascophylli and its host, the brown marine alga Ascophyllum nodosum (an inhabitant of the Atlantic Ocean). Ascomata of Mycosphaerella are invariably found embedded in the thallus of Ascophyllum. Since the fungus is always

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Chapter 7 present, this may indicate a type of reversed lichenization, with the alga providing the thallus, and the fungus some type of growth substances (or perhaps it is just a universally distributed parasite—no one has yet done the research necessary to establish the facts).

Lichen Phylogeny and Evolution Unlike many other groups, lichens have no common ancestor—only a widely shared symbiotic process that has (probably) arisen time and time again as a result of natural affinity, opportunity, or need. The polyphyletic nature of lichens has been conclusively demonstrated with molecular techniques. The lichens are really a nutritional, rather than a taxonomic, group. Since the basidioma of Lichenomphalia (Omphalina) ericetorum does not contain algal cells, and since other related species are not lichenized, we think that this one may still be evolving into the lichen condition. Other fungi may be evolving away from the lichen state. These could include nonlichenized members of the Ostropales and Arthoniales. It has been suggested that if life were to be found on another planet, it might well be lichen-like. I consider this to be highly unlikely, because the existence of lichens depends on the previous existence of the two founding races—fungi and algae—neither of which is nearly as tough as the lichen association. If conditions on other planets are as nasty as we suspect, we should look in the Archaea for the type of really extremophile life forms that might be found.

Further Reading about Lichens Ahmadjian, V., and M. E. Hale, eds. 1973. The Lichens. New York: Academic Press. Ahmadjian, V., and J. B. Jacobs. 1981. “Relationship between Fungus and Alga in the Lichen Cladonia cristatella.” Nature 289:169–71. Brodo, I. M. 1981. “Lichens of the Ottawa Region.” Syllogeus 29. Ottawa: National Museums of Canada. Brodo, I. M., S. D. Sharnoff, and S. Sharnoff. 2001. Lichens of North America. New Haven, CT: Yale University Press. Ferry, B. W., M. S. Baddeley, and D. L. Hawksworth. 1973. Air Pollution and Lichens. London: Athlone Press, University of London. Friedmann, E. I. 1982. “Endolithic Microorganisms in the Antarctic Cold Desert.” Science 215:1045–53. Hale, M. E. 1979. How to Know the Lichens. 2nd ed. Dubuque, IA: Wm. Brown. Hale, M. E. 1983. The Biology of Lichens. 3rd ed. London: Edward Arnold. Hawksworth, D. L. 1988a. “Coevolution of Fungi with Algae and Cyanobacteria in Lichen Symbioses.” In Coevolution of Fungi with Plants and Animals, edited by K. A. Pirozynski and D. L. Hawksworth, 125–48. London: Academic Press.

Lichens—Dual (or Even Triple) Extremophile Organisms ———. 1988b. “The Variety of Fungal-Algal Symbioses, Their Evolutionary Significance, and the Nature of Lichens.” Botanical Journal of the Linnean Society 96:3–20. Hawksworth, D. L., and D. J. Hill. 1984. The Lichen-Forming Fungi. Glasgow: Blackie. Hawksworth, D. L., and F. Rose. 1976. Lichens as Pollution Monitors. London: Edward Arnold. Henssen, A., and H. M. Jahns. 1974. Lichenes. Eine Einführung in die Flechtenkunde. Stuttgart: Georg Thieme Verlag. Kendrick, B. 1991. “Fungal Symbiosis and Evolutionary Innovations.” In Symbiosis as a Source of Evolutionary Innovation, edited by L. Margulis and R. Fester, 249–61. Cambridge, MA: MIT Press. “Lichens of North America Information.” Accessed April 22, 2017. www.lichen.com. [a comprehensive and beautifully illustrated website which explores many aspects of lichens and their ecology] Lücking, R. 2008. Foliicolous Lichenized Fungi. Bronx, NY: Published for the Organization for Flora Neotropica by The New York Botanical Garden Press. Lumbsch, H. T., and H. M. Huhndorf. 2010. “Myconet Volume 14. Part One. Outline of Ascomycota—2009.” Fieldiana (Life and Earth Sciences) 1:1–42. Nash, T. H. 2008. Lichen Biology. 2nd ed. Cambridge, UK: Cambridge University Press. Richardson, D. H. S. 1975. The Vanishing Lichens. Their History, Biology and Importance. Newton Abbot, UK: David and Charles. ———. 1992. Pollution Monitoring with Lichens. Slough, UK: Richmond. Seaward, M. R. D. 1977. Lichen Ecology. London: Academic Press. Smith, D. C. 1973. The Lichen Symbiosis. Oxford: Oxford University Press. ———. 1978. “What Can Lichens Tell Us about Real Fungi?” Mycologia 70:915–34. Vitt, D. H., J. E. Marsh, and R. B. Bovey. 1988. Mosses, Lichens and Ferns of Northwest North America. Edmonton, Canada: Lone Pine. Vobis, G., and D. L. Hawksworth. 1981. “Conidial Lichen-Forming Fungi.” In Biology of Conidial Fungi, edited by G. T. Cole and B. Kendrick, 245–73, Vol. 1. New York: Academic Press.

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8 Spore Dispersal in Fungi— Airborne Spores and Allergy Introduction Fungi cannot walk or run, but some can swim, most can soar, a few can jump, and some must be carried. From your reading of the taxonomic survey in this book, you can probably put a few names in each of the categories I have just mentioned. At the beginning of the book, when I was defining the word ‘fungus’, I concentrated on the unusual somatic morphology and the heterotrophic, osmotrophic nutrition shared by most fungi. But perhaps I did not place enough emphasis on one of the main reasons for the success of the fungi, which is their ability to produce and disperse vast numbers of tiny but often highly characteristic and specialized spores. By sheer fecundity the fungi make sure that, whenever and wherever a new food substrate becomes available, they will be on hand to exploit it. Many fungi are cosmopolitan: you can find them almost anywhere in the world. The air we breathe sometimes contains more than 10,000 spores per cubic metre. The soil contains astronomical numbers of spores, waiting for food. Why are there so many? How did they get there? What significance do these numbers hold for us? This chapter will try to answer those questions.

Chemotaxis—Swim for It! The taxonomic survey recognizes nine phyla of fungi. Each of these has solved the problems of dispersal in its own way, although some methods of dispersal have been invented more than once, and sometimes the parallelisms are striking, as you will see. The phyla Chytridiomycota, Hyphochytriomycota, and Oomycota are basically aquatic, so their spores are often equipped with flagella. In the Chytridiomycota, each zoospore has one, backwardly directed whiplash flagellum, and it swims like a sperm. In the Hyphochytriomycota, the single tinsel flagellum is forwardly directed. In the Oomycota, each zoospore has two flagella, one whiplash, and one tinsel. These zoospores, once liberated from their mitosporangia, may embark on a random search for a new substrate, in which case their chances of survival aren’t good: more fortunate spores are given a chance to use a special talent they possess for ascending a chemical gradient toward a food substrate. For example, the zoospores of Pythium and Phytophthora, many species of which parasitize the roots of plants, find their hosts by tracing the source of the sugars and other chemicals that 156

Spore Dispersal in Fungi—Airborne Spores and Allergy leak out of root cells. Some chytrids that attack nematodes detect and swim toward substances emanating from the bodily orifices of the worms. This process is called chemotaxis (see also chapter 15).

Airborne Sporangia and Where They Got To Some oomycetes have become parasites of the aerial parts of plants (remember the downy mildews in chapter 2). Hopping from one plant to another is no job for a spore that’s designed to swim. In response to this selection pressure, two completely new structures, the aerial sporangiophore and the detachable, wind-dispersed mitosporangium, evolved, apparently in tandem. And they have changed the biological history of the planet. The microscopic sporangia of downy mildews develop at the tips of sporangiophore branches, have a very narrow attachment to the cell that produces them, and are easily dislodged by wind or rain. Landing by chance on another leaf, those of most species revert to their ancestral behaviour and require the presence of a film of free water so that they can release motile, biflagellate zoospores which swim off to infect the plant, usually entering through the stomates. In a few of the most highly evolved oomycetes, members of the family Peronosporaceae, the airborne sporangia produce a germ tube when they land on a new host. These fungi appear to be cutting their last link with the aquatic life of their ancestors. As I mentioned in chapter 2, the human species is probably the most important vector for many fungi. One important example of this is the oomycete Phytophthora infestans, which causes late blight of potato. Human transoceanic commerce inadvertently carried this Central American fungus to northeastern North America in 1843 and to Europe in 1845. I say ‘inadvertently’ because at that time no one even knew the fungus existed or what it was capable of. The maps reproduced here (Fig. 8.1) show how, after these introductions, its natural spreading proceeded. Airborne sporangia were obviously a successful evolutionary invention. The structures associated with sexual reproduction in zygomycetes are very conservative. Zygosporangia are basically tiny, lookalike, thick-walled, resistant capsules designed to survive hard times. But in a few cases they may also have some adaptations for dispersal. The antler-like outgrowths of the suspensors in Phycomyces blakesleeanus make the whole structure a ‘microburr’ that can be unknowingly picked up and carried away by a passing arthropod.

Nonmotile Sporangiospores—Zygomycetous Anamorphs When we look at the anamorphs of zygomycetes, we find a bewildering diversity of form and function. We can distinguish four main types of dispersal mechanism and several subcategories. (1) Large, spherical, columellate mitosporangia each containing hundreds or thousands of spores (Fig. 3.5A, D). But the generally similar form of these sporangia is not reflected in their dispersal techniques.

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Fig 8.1 Spread of Phytophthora infestans. A: In eastern North America 1843–1845; B: in Europe during 1845.

Spore Dispersal in Fungi—Airborne Spores and Allergy (a) In some examples the spores are produced in a slimy matrix. This may be surrounded by (i) a thin but persistent membrane (peridium), as in Phycomyces nitens, or (ii) an equally thin membrane that dissolves and exposes the spore drop. In many Mucor species, the exposed mucilage imbibes water and swells to several times its original size, often supported by a collar-like remnant of the peridium. This is a stalked spore drop and is often an adaptation for dispersal by small animal vectors. (b) In other cases the spores are dry, so that when the peridium ruptures, they can blow away on the wind, e.g., Rhizopus stolonifer. (c) In the third group, the spore mass is violently discharged. This technique has been evolved by only one genus of the Zygomycota, Pilobolus (Fig. 3.6A–C), a specialized inhabitant of the dung of herbivorous mammals. In order to survive, this fungus must get its spores away from the dung and onto the prospective diet of the animal concerned. The subsporangial vesicle of this fungus ruptures when internal pressure reaches about 7 atmospheres and expels the spore mass plus glue to a distance of up to 2 metres, far enough to get it away from even an elephant’s huge deposit. (2) Small, few-spored sporangia (sometimes called ‘sporangioles’). In such putatively intermediate genera as Thamnidium (Fig. 3.5D), large and small sporangia coexist. The small sporangia often break off and are wind dispersed, while the large sporangia remain in place and act as slimy spore drops. Thamnidium is unusual in this two-pronged allocation of reproductive resources. Helicostylum produces multiple sporangia with reduced spore numbers. Other genera like Blakeslea produce sporangia with very few spores (Fig. 3.5B). (3) Specialized merosporangia. These are unusual small mitosporangia which often contain a row of spores, as in Syncephalis (Fig. 8.2A) and Piptocephalis (Fig. 8.2B). At maturity the sporangial wall breaks down and the spores are set free. Multiple merosporangia are usually formed on a special head cell, which may break off and carry the spores away with it. Merosporangia may be (a) dry and their spores wind dispersed, or (b) slimy and sticky. (4) The logical endpoint of this reductive process is a single-spored mitosporangium, which is in fact found in many genera. These are often virtually indistinguishable from the conidia of dikaryan anamorphs under the light microscope, but they have an outer sporangial wall and a complete inner spore wall which often also fills and occludes (blocks off) the stalk (Fig. 8.2C), so release is often by rhexolytic rupture of the stalk. Dikaryan conidia (the mitospores liberated by moulds), probably the most numerous and important fungal propagules of all, don’t have separate inner and outer walls and retain cytoplasmic continuity with their parent cell until release, which usually occurs by schizolytic separation of the components of the basal double septum (Fig. 8.2D). The one-spored sporangia of Cunninghamella are dry and wind dispersed. The umbellate sporocladia of Kickxella produce their sporangioles in a drop of slime. The sporangiophores of some dung-inhabiting genera are very tall and elaborately branched or coiled, as in Spirodactylon (Fig. 3.5F). I think these structures play an important part in spore dispersal, becoming tangled in the hair of the sedentary rodents on whose dung they grow and being ingested during grooming activities. In the Entomophthorales, one-spored mitosporangia are essentially ballistospores, actively shot away by three different mechanisms: (a) In Entomophthora muscae (Fig. 3.6E), the apex of the sporangiophore ruptures to expel the sporangium.

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Fig 8.2

Merosporangia, sporangioles, and conidia.

Cytoplasm from the sporangiophore goes with the propagule and may help it to stick to the substrate when it lands. (b) Species of Basidiobolus have a line of weakness around the sporangiophore just below the apex. At maturity, the wall splits there, and the spore flies away with part of the sporangiophore attached. As in a two-stage rocket, the sporangiophore fragment falls away during flight. (c) In some species of Entomophthora, and in Conidiobolus, the mitosporangium is projected by the release of pressure built up between the sporangium and a tiny, intrusive columella.

Spore Dispersal in Fungi—Airborne Spores and Allergy

Ascospores—Shot or Not In the taxonomic section of this book it is made clear that Ascomycetes and Basidiomycetes, although they may look very different, appear to have arisen from a common ancestral group. Many of the superficial differences between their teleomorphic fructifications can be traced back to the different types of meiosporangia they produce: asci and basidia. It is worth comparing the mechanisms of these vital cells and the ways in which they have probably affected the evolution of their respective teleomorphs. The ascus (Fig. 4.3) seems originally to have evolved as a tubular spore gun—an elongated cell within which, once the spores have matured, turgor pressure builds up until the tip of the cell bursts and the ascospores fly out. You will remember the two basic lines of asci. First, the unitunicate ascus with either (a) an operculum or lid, opening around a built-in line of weakness at the moment of discharge, or (b) an elastic apical ring which either stretches enough to let the spores pop out through an apical pore, or turns inside out as the spores leave. Second, the bitunicate ascus, in which the inner wall expands upward after the thin outer wall splits, then shoots the spores out of an apical canal. These are different ways of achieving the same end. In each case, the basic function of the ascus is more or less the same. And most asci still conform to this norm. At least, those which have some access to the outside world do. Some are borne on apothecial ascomata in exposed layers (hymenia). They can ‘shoot at will’, whenever they are mature. Anyone who spends much time looking for fungi will experience the phenomenon of ‘puffing’ in apothecial ascomata. This happens when atmospheric humidity changes suddenly and thousands of mature asci expel their ascospores simultaneously, producing a smoke-like cloud of ascospores. The theory of puffing is that the many simultaneous jets of ascospores generate a general movement of the air above the hymenium, which carries the spores much farther than if the asci fired individually. Cookeina sulcipes (Ascomycetes, Pezizales), a colourful tropical cup-fungus, produces 215,000 asci per square centimetre, which collectively liberate 1,720,000 ascospores. Cookeina asci are straight, but their lids develop on the side toward the light, so the spores shoot in the right direction. The asci in some apothecia have evolved light-sensitive mechanisms in their tips, so that they can aim their spores toward the light (e.g., Ascobolus). Other asci develop inside ascomata that open to the outside world only by a narrow pore (an ostiole). These asci are more protected during development, but they can’t all fire at once; they have to take turns. Each ascus, as it ripens, must stretch right up the neck of the ascoma to the ostiole before it can shoot its spores. Some ostiolate (perithecial or pseudothecial) ascomata have light-sensitive necks, making sure that their asci shoot toward the light. This is especially important to fungi growing on the dung of herbivores (e.g., Sordaria and Podospora), which must get their spores away from the dung and onto the plants their host animal will eat. The size of a projectile has a considerable influence on its range. In the dung-inhabiting Saccobolus, the eight ripe ascospores are glued together and expelled as a single projectile (see www.mycolog.com). Since Saccobolus is one of the commonest fungi found on dung, there must be some advantage to this strategy. I think the advantage is that the larger propagule made up of eight spores has more mass and momentum, so it

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Chapter 8 carries farther. Podospora fimicola, whose ascospores are extremely large anyway (averaging 54 × 37 μm), shoots all eight at once and so achieves the phenomenal range (for an ascus) of 50 centimetres. Most asci, however, expel their spores separately, either in a single burst or at short intervals. Cordyceps militaris (Fig. 4.25D, F) has very long, narrow asci, with a very fine pore in their thickened tip. The ascospores are 400–500 microns long, 2 microns thick, and arranged in a parallel bundle. A ripe ascus suddenly protrudes from the ostiole, the first ascospore flashes out after a second or two, followed at one-second intervals by the others. After the eighth spore has been shot, the tip of the ascus disappears and is soon replaced by that of another ripe one. No one knows how this precise sequence is executed. Some asci don’t shoot their spores. Mycologists believe that the shooting mechanism has been lost only in situations where it has become useless or inadaptive. This happens if the fungus adopts a cryptic habitat: if it fruits under bark or underground, for example. The known teleomorphs of the common moulds Penicillium (e.g., Talaromyces) and Aspergillus (e.g., Eurotium, Fig. 4.17A) produce closed (cleistothecial) ascomata, and their asci are spherical and have no shooting mechanism. Truffles fruit underground, so their ascomata are closed, and their asci are spherical (Fig. 4.12G). Their spores are dispersed by mammalian vectors, which can find and dig up the buried ascomata only because these emit uniquely attractive aromas. For many years, the French hunted truffles with the aid of female pigs, because these pigs had such good noses for truffles and were so enthusiastic. We did not know until recently that the sows were literally ‘turned on’ by a chemical they normally encounter only when it emanates from rutting boars. In some strange way, truffles have evolved a spore-dispersal mechanism that involves a mammalian pheromone. It is easy to understand why asci that develop in closed ascomata don’t shoot their spores. But quite a few ascomycetes with ostiolate ascomata have adopted the same habit. In the cellulolytic genus Chaetomium (Fig. 4.22B), the walls of the asci break down as the spores mature, liberating them into the cavity of the ascoma in a mucilaginous matrix. As the mucilage imbibes water and expands, it oozes out through the ostiole and forms a long tendril or a gooey mass. Chaetomium species typically have a mass of coiled or branched hairs growing from the upper part of the ascoma; these hairs act as a natural holder for the spore drop. It seems likely that these spores are set up for arthropod or rain dispersal, rather than the original airborne route. The genus Ophiostoma (Fig. 4.17B) follows a very similar pattern, but here the ascoma usually has a long, tubular neck. When the mucilage expands, it can escape only by moving up the neck, carrying the ascospores with it, and forming a spore drop at the top which sits on a fringe of specialized supporting hyphae. These fungi often fruit in the tunnels of bark beetles, which pick up the ascospores (or the conidia of the anamorph, another stalked spore drop) as they crawl along the tunnels, then fly off to other trees. This is the devastating secret of Dutch elm disease. Again, human vectors brought the fungus to North America, but flying beetle vectors have spread it very effectively within this continent. First found in Ohio in 1930, it reached Tennessee by 1946, California by 1975. In Canada it was first detected in Quebec in 1946. In fifteen years it had killed more than 600,000 trees in an area of 25,000 square miles. It has since spread to the maritime provinces, to Ontario, and as far west as Manitoba, although the discontinuous distribution of the elm in Canada suggests that the fungus reached Manitoba by human agency, or from the south.

Spore Dispersal in Fungi—Airborne Spores and Allergy The ascomata of the powdery mildew fungi (Erysiphales, Fig. 4.21) are closed and might easily be described as cleistothecial. But their asci are elongate, often grow in a radially oriented cluster, and can shoot their spores (molecular techniques have recently shown them to be related to the Discomycetes). So we must assume, despite the absence of an ostiole, that the ascomata open at some point. How this happens in the genus Phyllactinia is a strange story with some interesting twists. The ascomata of this fungus have two unusual features: (1) a ring of radiating, tapered, needle-like appendages, and (2) a tuft of secretory hairs on top of the ascoma, that produce a blob of mucilage (Fig. 8.3A). When the ascoma is mature, the appendages all bend downward and lever it upward, breaking its hyphal connections with the leaf (Fig. 8.3B). It is now free to be dispersed by wind or water. When the ascoma lands on a new substrate, the blob of mucilage will hold it there, in the upside-down position, so that the business end of each ascus now points futilely at the substrate (Fig. 8.3C). Fortunately, there is a line of weakness around the equator of the ascoma. This now splits open, and the lower half of the ascoma swings over through 180 degrees (Fig. 8.3D). Lo and behold, the asci now point outward, and the spores can finally be shot away into the air.

Fig. 8.3

Adventures of Phyllactinia (Erysiphales).

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Fig. 8.4 Spread of powdery mildew of grapes in Europe, 1845–1852.

The dramatic spread of some fungi which cause powdery mildews has been well documented, since they affect economically important crops. Fig. 8.4 shows the way in which the powdery mildew of grapes, caused by Uncinula necator, spread in Europe after its introduction to England (from America or Japan) in 1845. By 1851–1852 its airborne conidia had carried it throughout the wine-producing countries of the Mediterranean.

Basidiospores—A Gentle Rain (But Not from Heaven) The distances to which ascospores are projected ranges from less than a millimetre to 50 centimetres. Basidiospores are much more uniform in size and ballistic mechanism, and are projected for much smaller distances: 0.005–0.1 centimetres. Let us explore the reasons for that. Everyone is familiar with the appearance of a mushroom but probably much less aware of how it works. As a basidioma develops, its primordium is at first negatively geotropic, as the stipe grows upward; then as the cap (pileus) spreads out sideways, it is diageotropic; finally, as the gills grow downward, it is positively geotropic. Once the structure is mature, we can see some of its potential and some of its limitations. The gills represent a huge area of hymenium, capable of producing millions of basidiospores. But gills are usually very closely packed. If a basidiospore was shot farther than the distance between adjacent gills, it would simply hit the next gill, and probably lodge there. To take advantage of their enormous fertile area, agaric evolution has fine-tuned their spore-shooting mechanism. The spores must be launched delicately from their basidia and then allowed to free-fall straight down between the gills until they reach the open air below the cap, when natural turbulence will carry them away.

Spore Dispersal in Fungi—Airborne Spores and Allergy The latest ideas about the shooting mechanism of the basidium have already been presented in chapter 5, and there is no need to describe them exhaustively here. In short, a large droplet secreted at the base of the asymmetrically borne spore just before dehiscence merges suddenly with a film of water already present around the body of the spore. This provides more than adequate impetus for disjunction. There are more than 10,000 species of agaric, and all, within fairly broad limits, share a similar design: they tend to look like open umbrellas. This is because they are in fact biological umbrellas. If the hymenia of most basidiomycetes get wet, they stop shooting spores, since the droplet-film interaction is ‘drowned’. The umbrella shape is so effective that it has evolved over and over again. Basidiomata that look rather like umbrellas (or, in some cases, half-umbrellas) are found among the Cantharellaceae, stipitate Hydnaceae, and Polyporaceae; the Tremellales (Pseudohydnum, Phlogiotis); and the Auriculariales, as well as the ubiquitous agarics and boletes. The annual weeds are among the most successful and most recently evolved of the flowering plants. They have an equivalent among the agarics: the genus Coprinus. Members of this genus and its close relatives have many unique and sophisticated features that earn them a place of honour in this chapter. Coprinus comatus (the shaggy mane: Fig. 8.5) is a large and common agaric that fruits on disturbed ground in late fall, the first frosts triggering formation of basidiomata before it is too late. Almost everything about this mushroom increases its efficiency as a producer of spores. The stipe, although tall, is hollow, economizing on material. The cap does not spread out like those of most other agarics, but it is very deep and almost cylindrical. Most of the basidioma is made up of a tightly packed array of extremely thin gills. These gills are so close together that each has to produce a sprinkling of large, specialized cells called cystidia to keep its neighbours from touching it. In addition, the gills are not rigorously kept vertical, as they are in most agarics. There is no way that spores could be fired from the surface of these gills and reach the world outside. No way, that is, except one. In most agarics, any area of a gill will have basidia at various stages of development. But in Coprinus the process is highly regimented. Only the basidia at the bottom edge of a gill are permitted to mature. Their exposed position allows them to shoot their spores. But what about all the other basidia higher up? In order to expose them, the tissue at the lower edge of the gill undergoes self-digestion (autolysis). The process resolves into a beautifully orchestrated sequence. A perfectly timed wave of spore maturation and spore shooting, followed immediately by a wave of autolysis, sweeps up the gills. The cystidia near the gill edge autolyze first, so that they will not be in the way of the spores. As spore-shooting proceeds, the entire cylinder of gills gradually melts away, as the top three diagrams in Fig. 8.5 show. The basidia of agarics may differ in size and shape, and even in the number of spores they produce, but they are remarkably uniform in the way they carry their spores. These always develop at the ends of tapering outgrowths of the basidium. And in functional (which is to say, spore-discharging) agarics, they always sit asymmetrically (Fig. 5.3). This asymmetry seems to be absolutely diagnostic of basidiospores that are actively shot away. If you find a basidium with spores symmetrically mounted on their sterigmata (Fig. 5.5A–E), you can say definitely that those spores will not be actively propelled from their perches. Why should that matter? Well, it is an indicator of many

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Fig. 8.5 Agaricales: Coprinus comatus showing gill spacing and autolysis.

other things that have happened to the fungus during the course of evolution. It marks a radical change in the biology of the organism. As we saw in chapter 5, most families of agarics have produced what we call sequestrate offshoots which have lost their ability to shoot basidiospores, a loss that has gone hand in hand with changes in the development and morphology of the mature basidioma. In general, gills become crumpled, or even indistinguishable, as the hymeniumbearing tissues assume a loculate or spongy aspect, and in many cases the edge of the pileus, or the partial veil, encloses the spore-bearing tissues even at maturity. Stipes may be lost, and basidiomata may adopt a hypogeous habit.

Spore Dispersal in Fungi—Airborne Spores and Allergy We believe that these forms represent some of the most recent evolutionary developments in the fungi. Sequestrate forms seem to have arisen independently in no fewer than fourteen families of agarics, probably as a response to exceptionally dry conditions which would damage the exposed hymenia of normal agarics. Spore dispersal must now be delayed until the basidioma breaks up, or it may be carried out by insects or mammals. In fact, the California red-backed vole Clethrionomys californicus lives almost exclusively on basidiomycetous truffles such as Rhizopogon, which is a sequestrate, hypogeous derivative of Suillus (Boletaceae), and the vole may be an important distributor of their spores. In Australia, the Gilbert’s potoroo, a threatened marsupial the size of a rabbit, also exists almost entirely on truffles. Some of the orders of Gasteromycetes have colourful common names (puffballs, earthstars, bird’s-nest fungi, earthballs, stinkhorns) that suggest their specialized methods of spore dispersal. Puffballs (Lycoperdales) produce a mass of dry basidiospores (the gleba) inside a papery shell with a hole in the top. Raindrops cause this thin outer peridium to dimple, forcing a small puff of air mixed with spores out through the opening. Wind blowing across the opening can also suck out spores. Earthstars are just puffballs with an outer, fleshy peridium that develops several splits running radially outward from the centre. The segments thus formed open by bending backward, and as they reflex farther and farther, they lift the gleba above the surrounding leaf litter, exposing it to the rain and wind. Myriostoma has several evenly spaced ostioles. In the bird’s-nest fungi (Nidulariales), the basidiospores of most genera form inside several small seed-like packets called peridioles or, more colloquially, eggs. These sit in a deeply funnel-shaped splash-cup which focuses and reflects the kinetic energy of falling raindrops. Some of that energy is transferred to the peridioles, which are thrown for some distance. In the earthballs (Sclerodermatales), a group otherwise extremely passive in its spore dispersal, there is one aberrant family, the Sphaerobolaceae, which has rather surprisingly evolved a new type of active spore dispersal. The positively phototropic basidioma of Sphaerobolus stellatus is only 2 millimetres in diameter but can catapult its gleba (1 mm in diameter) up to 7 metres. The peridium in this fungus has several different layers. At maturity, the top splits and reflexes to expose the spherical gleba. The lower part of the peridium separates into two nesting cups which touch each other only at the rims. Glycogen in the cells of the inner cup is converted to glucose, and turgor pressure builds up until the inner cup abruptly turns inside out and flings the gleba into space. Sphaerobolus often occurs on old dung, and the evolutionary rationale for its explosive spore dispersal is clearly similar to those for the very different mechanisms we examined earlier in Pilobolus, Saccobolus, and Podospora. The stinkhorns (Phallales) are perhaps the most bizarre members of this strange menagerie. The young fruit body is called an egg. In Phallus (Fig. 5.9D), the soft shell splits in the morning, as a dense mass of specialized tissue inside takes up water from the mucilage that surrounds the embryonic basidioma, and elongates and expands quickly to produce a tall, spongy stalk. At the top is a receptacle, covered with a sugary but evil-smelling greenish slime in which the basidiospores are embedded. The smell attracts a procession of flying insects, particularly dipteran flies, which gorge excitedly on the slime and carry spores away on their feet. By evening, the green slime is gone, its mission accomplished. The most highly evolved phalloids seem to be those which,

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Chapter 8 like Aseroë, have basidiomata with long, bright red, radiating rays that can only be intended to supplement their olfactory message with a visual one. As a passing vector (probably a flying arthropod) might say: ‘It’s a flower. No, it’s rotting meat. No, it’s faeces!’ Strange fungi indeed, that in the name of dispersal combine the qualities of flowers and excrement.

Airborne Spores and Health Spores are microscopic, sealed units, typically formed and released in enormous numbers, and usually passively dispersed through the air, so that they are present almost everywhere. They contain one or more nuclei, some cytoplasm, and a minute food reserve. Like tiny seeds, they will germinate in damp conditions to produce hyphae which, if they are lucky enough to find food and moisture, can extend and multiply to become a mycelium, accumulate energy surpluses, and eventually make more spores. The problem with spores is the same as that with pollen. Both lack any motive power or navigational equipment. To make sure that at least a few spores land in places where they will find food (especially if they are picky), fungi must liberate them in astronomical numbers. Some fungi make and release spores from early spring until late fall. Some will even release them in winter whenever the temperature rises above freezing. Mycologists have described nearly 100,000 fungi, and there is no doubt that hundreds of thousands more remain to be discovered (the total number of fungi in existence has been estimated at 1,500,000). Let’s see just how many spores a few individual fungi contribute to the total. One specimen of the common bracket fungus Ganoderma applanatum (see chapter 5) can discharge 30,000,000,000 spores a day, every day from the beginning of May to the end of September (4,500,000,000,000 spores over the season). One fructification of another wood-inhabiting ascomycete, Daldinia concentrica, can liberate more than 100,000,000 spores a day for many days. A single wheat grain infected with stinking smut (Tilletia caries) can contain 12,000,000 spores. One 2.5-centimetre-diameter colony of the green mould Penicillium can produce 400,000,000 spores. Of course, even these huge numbers become greatly attenuated when the spores are dispersed in the vastness of the atmosphere, but the total spore load of the outside air is always significant and can on occasion be a real threat to health.

Fungal Allergies The prime suspects in respiratory allergies provoked by airborne particles were originally the pollen grains of plants, and ragweed (Ambrosia spp., Asteraceae) became the villain of the piece (even though most people have no idea what the plants look like), causing what is widely and inaccurately known as ‘hay fever’. But people tended to forget that allergenic pollen is actually only a summer problem, while many respiratory allergies persist in fall and winter. So scientists had to look elsewhere for other, less seasonal causative agents and found them in the form of fungal spores. Skin tests proved that such spores can indeed be allergenic. About 20% of the population is atopic and easily sensitized by normal spore concentrations. These people may react by

Spore Dispersal in Fungi—Airborne Spores and Allergy developing ‘hay fever’ or asthma and may become sensitized to a number of common allergens. The other 80% of the population do not develop allergies so easily. They would require exposure to higher spore concentrations such as occur only during events such as haymaking, harvesting, or grain handling. These concentrations may then produce allergic alveolitis (hypersensitivity pneumonitis), resulting in breathlessness. Such sensitivity is usually restricted to a single allergen, and the condition is usually related to the person’s occupation (farmer, grain handler). Many common fungi are now known to be allergenic, and more allergens are being recognized as time goes on. So all fungal spores should be regarded as potentially allergenic. Sufferers from allergies induced by fungal spores could gain some relief by moving to hot or cold deserts, or to the mountains, or by taking an ocean cruise. Very high local concentrations of spores can be encountered during epidemics of fungal plant diseases such as wheat rust, and the spore concentrations to which farm workers handling mouldy hay are exposed can eventually cause a serious and sometimes fatal allergic disease called ‘farmer’s lung’ (Rippon 1974). Repeated exposure to high concentrations of spores from a number of different allergenic fungi (often species of Penicillium and Aspergillus) can lead to sensitization and produce acute or chronic symptoms. The acute stage is usually found in harvesters and threshers, who are briefly exposed to overwhelming spore loads. They experience chills and fever and generally feel unwell, but they recover. The chronic stage is found among silo and mill workers who have low-level but constant exposure to the allergens. This stage is much more serious, because it causes degenerative changes in the respiratory tract which lead to obstruction of the airway. Patients become breathless after exertion, cough constantly, and feel weak. The chronic stage may be a cause of emphysema, and it may eventually be fatal. This disease was first described in Canada (Cadham 1924) and is commonest in temperate regions where high rainfall encourages moulding of hay. A similar complaint has been seen in some office workers when hidden air conditioning systems have supported massive growth of similar moulds. Bronchial asthma is also frequently provoked by airborne fungal spores, usually belonging to the mould genera Alternaria, Aspergillus, Drechslera (‘Helminthosporium’), and Penicillium. These spores reach their highest numbers in fall, with another lower peak in spring.

A Cautionary Tale: Bleeding Lung Syndrome and Stachybotrys chartarum In 1993, ten cases of bleeding lung syndrome in infants in Cleveland were tentatively linked by the Centers for Disease Control and Prevention (CDC) to Stachybotrys chartarum (also known as Stachybotrys atra), a microscopic black mould that grows on damp paper, especially the paper covering wallboard. Recently, the Tottenville Branch Library on Staten Island, New York, which had a damp basem*nt, was closed after a staff member had trouble breathing, and Stachybotrys was found. Because of recurring Stachybotrys contamination, there is a real possibility that the entire Gorge Hospital in Victoria, British Columbia, may be pulled down and rebuilt, at a cost of well over $30 million. The CDC has recorded at least 100 similar instances in which a health problem has been associated with the presence of Stachybotrys, although (as the CDC has now admitted) there is still no solid proof that the health problems were caused by the mould. There is, as scientists well know, a considerable distance between correlation and causation. Are the administrative decisions reported above reasonable? Some

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Chapter 8 scientists believe that Stachybotrys has been made a scapegoat for a variety of symptoms which it may not be causing. Unfortunately, Stachybotrys does have a history. There is no doubt that it produces some nasty mycotoxins (see chapter 21), but—and this is a big but—these have caused problems only in animals (usually horses), and even then only if those animals ate hay on which the fungus was growing; in other words, the horses actually consumed measurable amounts of the fungus. But the Stachybotrys in buildings usually grows inside wall cavities or crawl spaces. Certainly, no humans are eating the fungus (even little kids don’t normally lick the wall). So how can it get inside them and cause sickness? As far as I can see, only by the inhalation of spores. How likely is that? Not so much. Stachybotrys chartarum produces its spores in tiny, slimy droplets, so the spores stay where they are and are not released into the air. The only way in which they can become airborne is after the colony has stopped growing and has dried out, and if the spores are somehow disturbed by physical means. Interestingly enough, this physical disturbance is likely to happen only when active attempts are made to remove the fungus. As of May 2017 I am inclined to believe that the Stachybotrys scare is a form of hysteria based on little more than the co-occurrence of the fungus and the symptoms, and that we should not do drastic or expensive things to remove or avoid the mould until we are absolutely sure it is guilty as charged. The best remediation is to prevent the entrance of water and treat existing mould colonies with bleach, which kills the fungus. In severe cases, the wallboard should probably be replaced (although if the new board becomes damp, the mould will probably develop all over again). It might be a good idea to encapsulate the mould with some type of sealant spray before removing the affected areas. Proper masks and protective clothing should be worn by those doing the work, and the mould should not be allowed to come in contact with skin. Areas such as wall cavities or basem*nts that are subject to continuous or sporadic dampness should probably be treated with a persistent fungicide which has low toxicity to humans. But, in the words of the Hitchhiker’s Guide to the Galaxy, DON’T PANIC!

Spore Sampling and Counting Once we know that airborne fungal spores cause plant diseases, and that they can also cause allergies and even some lung infections (Aspergillosis, Histoplasmosis, Coccidioidomycosis), it is apparent that we need to quantify and identify them. In theory, this could be very difficult, because (1) there are almost 100,000 known species of fungi, most of which make spores; (2) there is no comprehensive publication dealing with the identification of fungal spores; (3) the spores of many fungi, and especially those that are unicellular, are not particularly distinctive; with the corollary that (4) many of them (especially the unicellular ones) cannot be easily identified in the absence of the structures which produced them. Fortunately, the situation is not quite as bad as it looks. All 100,000 fungi do not contribute equally to the continuous rain of spores. In fact, the vast majority of spores found in the air are produced by very few fungi. Originally an allergy sufferer himself, Grant Smith retaliated by taking a large number of photomicrographs of spores trapped by a new spore sampler which he had developed. It is still impossible to identify every photomicrograph with the kind of precision we would like, but the pictures

Spore Dispersal in Fungi—Airborne Spores and Allergy presented in Smith’s 1990 book Sampling and Identifying Allergenic Pollens and Molds are more representative and more accurately identified than any previously assembled for the purpose of identifying airborne spores. The spores in the air can be counted and identified in two ways: by ‘viable’ techniques which depend on the germination, growth, and subsequent sporulation of spores trapped or sedimented onto a nutritive agar medium, or by ‘nonviable’ techniques, which trap the spores by impaction onto a transparent slide and so allow them to be directly observed under the microscope. Each technique has its advantages and disadvantages. Viable (culturing) methods allow us to identify such common spores as those of Aspergillus and Penicillium to species, while nonviable (observational) methods will permit only the recognition of a general ‘Aspergillus-Penicillium’ component, since the spores of these two genera are very small and very similar. But, more importantly, viable methods will not detect many types of spores at all, including slime mould spores, lichen spores, spores of obligate plant parasitic fungi such as powdery mildews, and spores of many basidiomycetes (agarics, polypores, gasteromycetes, rust fungi, smut fungi), since these do not germinate on standard culture media. In addition, ‘viable’ techniques take no account of dead spores, which may make up half of the sample. Fortunately, the so-called nonviable methods enable us to record and identify most of these. If a general or introductory survey is called for, I would recommend the nonviable approach, since it detects the widest range of taxa, whereas if a more detailed breakdown of some of the common fungi such as Aspergillus and Penicillium were needed, I would suggest that a viable technique be added. My students and I have usually used a Samplair impaction trap in which a small fan draws air at a constant and calibrated rate (e.g., 15 l/min) through a narrow slit, just below which sits a standard microscope slide, its upper side covered with a thin layer of transparent silicone grease or agar. When the air jet entering through the slit impinges upon and is deflected by the slide, the spores (and any other particles) tend to strike the grease/agar layer and stick to it. After a ten-minute collection period, the fan turns off and the slide is automatically moved horizontally a few millimetres to expose a new area for the next sampling period. Thus a single slide can collect a series of samples at one location at chosen intervals or at a succession of localities. A meta-analysis of 200 reports from around the world showed that Cladosporium (mostly Cladosporium herbarum) represented an average of 33% (highest value 65%) of spore counts in air samples; basidiomycetes (agarics, polypores, gasteromycetes, rusts, smuts) 22% (43%); ascospores 14% (highest count 45%); Alternaria 4.5% (13%); and Aspergillus/Penicillium 3.5% (18%). Twenty-one other individual taxa or groups were also present in significant numbers. The total number of taxa actually recorded is of course much higher (I gave illustrations of over 200 taxa in the chapter I wrote for Smith’s book), and the ‘top forty’ are described, keyed out, and illustrated on www.my colog.com. The spores of mushrooms (mostly Basidiomycetes: Agaricales) are numerous in fall when the fruiting of these fungi climaxes. Since many of them live in forests, the spore concentrations recorded there will be much higher than those taken in cities. Rust aeciospores will be most numerous in spring, urediniospores will be recorded mostly during summer, and teliospores in fall. Superimposed on these seasonal variations are daily fluctuations, some of which are due to the timing of spore release in certain fungi.

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Chapter 8 Airborne Cladosporium spores are most numerous around midday, probably as a function of the wind velocity, which is usually highest then, whereas Sporobolomyces spore counts peak during the night. A European study showed that at an altitude of 2,000 metres there are only one-quarter as many airborne spores as at altitudes below 1,000 metres. Since most fungi grow on plant material, the highest counts are recorded from the countryside, those in cities being appreciably lower. Where viability has been tested, it ranged from 20% to 90%, with the average sample showing over 50% viable spores. Wind increases spore counts. Rain has three distinct effects. The impact of the first drops tends to release spores. Soon after rain begins, ascospore discharge increases. But if the rain is heavy or prolonged, it removes most spores from the air. The air inside buildings tends to reflect the condition of the outside air, although spore counts are usually lower indoors because there is no wind effect. However, if rooms are damp, or if there is soil or plant material present, these may represent new spore sources and counts may increase and become more diverse. Bathrooms are damp, and moulds are often found sporulating around windows. Kitchens have refrigerators and garbage containers, both of which may be spore sources. Living rooms often contain houseplants, and both soil and plants may be spore sources. Air conditioning systems, which involve condensation of moisture, may become major sources of fungal spores or bacteria. Activities such as house cleaning (especially vacuuming, except in the case of a central vacuum system exhausted to the exterior) and food preparation are known to increase airborne spore counts. The effects of such changes on the development of allergic symptoms are being researched, and although there are as yet few proven connections between individual fungal taxa and the onset of respiratory allergies, properly designed epidemiological studies will, in my opinion, lead to the confirmation of current suspicions and the unmasking of many fungal culprits.

Identification of Airborne Spores One can acquire a true impression of the almost infinite variety of spores only by personally exploring them over a period of years. Some idea of that diversity can be gained, however, from the illustrations in books such as those by Domsch et al. (1980), Ellis (1971, 1976), Ellis and Ellis (1985, 1988), Carmichael et al. (1980), Nag Raj (1993), Seaver (1942, 1951), Dennis (1978), Sivanesan (1984), and a series of volumes by Breitenbach and Kränzlin (1984 et seq.). More recently, Smith (1990), collaborating with a number of mycologists (including the author of this book), compiled a book of colour photomicrographs of pollen and spores trapped by the Samplair quantitative impaction spore trap which he had devised. The widest coverage of mould spores is given in a 2011 book, The Genera of Hyphomycetes, by K. Seifert et al.

Further Reading about Spore Dispersal and Allergies Adams, K. F., H. A. Hyde, and D. A. Williams. 1968. “Woodlands as a Source of Allergens, with Special Reference to Basidiospores.” Acta Allergologica 23:265–81.

Spore Dispersal in Fungi—Airborne Spores and Allergy Breitenbach, J., and F. Kränzlin, eds. 1984–2005. Fungi of Switzerland. Vols. 1–6 Lucerne, Switzerland: Verlag Mykologia. Buller, A. H. R. 1909, 1922, 1924, 1931. Researches on Fungi. Vols. I–IV. London: Longmans, Green and Co. Burnett, J. H. 1976. Fundamentals of Mycology. 2nd ed. London: Edward Arnold. Cadham, F. T. 1924. “Asthma due to Grain Rusts.” Journal of the American Medical Association 83, no. 1: 27. Carmichael, J. W., B. Kendrick, I. L. Conners, and L. Sigler. 1980. Genera of Hyphomycetes. Edmonton, Canada: University of Alberta Press. Dennis, R. W. G. 1978. British Ascomycetes. Vaduz, Liechtenstein: Cramer. Domsch, K. H., and W. Gams. 1972. Fungi in Agricultural Soils. Translated by P. S. Hudson. New York: Wiley. Domsch, K. H., W. Gams, and T. H. Anderson. 1980. Compendium of Soil Fungi. Vols. 1 and 2. New York: Academic Press. Ebner, M. R., K. Haselwandter, and A. Frank. 1989. “Seasonal Fluctuations of Airborne Fungal Allergens.” Mycological Research 92:170–76. Ellis, M. B., 1971. Dematiaceous Hyphomycetes. Kew, UK: Commonwealth Mycological Institute. ———. 1976. More Dematiaceous Hyphomycetes. Kew, UK: Commonwealth Mycological Institute. Ellis, M. B., and J. P. Ellis. 1985. Microfungi on Land Plants. London: Croom Helm. ———. 1988. Microfungi on Miscellaneous Substrates. London: Croom Helm. Gregory, P. H. 1973. Microbiology of the Atmosphere. 2nd ed. Aylesbury, UK: Leonard Hill. Gregory, P. H., and T. Sreeramulu. 1958. “Air Spora of an Estuary.” Transactions of the British Mycological Society 41:145–56. Hirst, J. M. 1953. “Changes in Atmospheric Spore Content: Diurnal Periodicity and the Effects of Weather.” Transactions of the British Mycological Society 36:375–93. Ingold, C. T. 1971. Fungal Spores: Their Liberation and Dispersal. Oxford: Clarendon Press. Kendrick, B. 1990. “Fungal Allergens.” In Sampling and Identifying Allergenic Pollens and Molds, edited by E. G. Smith, 41–50, 133–65. San Antonio, TX: Blewstone Press. Kendrick, B., and F. DiCosmo. 1979. “Anamorph-Teleomorph Connections in Ascomycetes.” In The Whole Fungus, edited by B. Kendrick, 283–410. Vol. 1. Ottawa: National Museum of Natural Sciences. Kendrick, B., and T. R. Nag Raj. 1979. “Morphological Terminology in Fungi Imperfecti.” In The Whole Fungus, edited by B. Kendrick, 43–62. Vol. 1. Ottawa: National Museum of Natural Sciences. Kendrick, B., and R. Watling. 1979. “Mitospores in Basidiomycetes.” In The Whole Fungus, edited by B. Kendrick, 473–545. Vol. 2. Ottawa: National Museum of Natural Sciences. Lacey, J. 1981. “The Aerobiology of Conidial Fungi.” In Biology of Conidial Fungi, edited by G. T. Cole and B. Kendrick, 373–416. Vol. 1. New York: Academic Press. Nag Raj, T. R. 1993. Coelomycetous Anamorphs with Appendage-Bearing Conidia. Waterloo, Canada: Mycologue Publications. Rippon, J. W. 1974. Medical Mycology. Philadelphia: Saunders. Seaver, F. J. (1942) 1978. The North American Cup Fungi (Operculates). New York: Seaver. Reprint, Monticello, NY: Lubrecht and Cramer.

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Chapter 8 ———. (1951) 1978. The North American Cup Fungi (Inoperculates). New York: Seaver. Reprint, Monticello, NY: Lubrecht and Cramer. Seifert, K., G. Morgan-Jones, W. Gams, and B. Kendrick. 2011. The Genera of Hyphomycetes. Utrecht: CBS-KNAW Fungal Biodiversity Centre. Sivanesan, A. 1984. The Bitunicate Ascomycetes and Their Anamorphs. Vaduz, Liechtenstein: Cramer. Smith, E. G., ed. 1990. Sampling and Identifying Allergenic Pollens and Molds. San Antonio, TX: Blewstone Press/Allergenco. Von Arx, J. A. 1979. “Propagation in the Yeasts and Yeast-Like Fungi.” In The Whole Fungus, edited by B. Kendrick, 555–71. Ottawa: National Museums of Canada [Now available only from Mycologue Publications, 8727 Lochside Drive, Sidney, BC, V8L 1M8, Canada].

9 Fungal Physiology and Metabolism Fungi, being eukaryotic organisms, have many physiological processes in common with other eukaryotes. But just as they have unique sets of morphological and behavioural features, so some aspects of their cellular chemistry differ from those of other organisms. If you already know all about basic cell chemistry, you can skip the next section and go directly to the rest of the chapter. But why not read through it anyway, just to refresh your memory?

Cell Components Proteins are large, complex molecules, made up of various mixtures and configurations of twenty different amino acids, held together by peptide bonds. Because of the essentially infinite number of structural possibilities that the building of a protein molecule presents, most organisms make many unique proteins. Fungal proteins are unique, yet they function just like those of other organisms. Some are enzymes and structural components, others are associated with nucleic acids to form nucleoproteins, and a third group is conjugated with carbohydrates to form glycoproteins, which are found in membranes and the cell wall, as well as being secreted as extracellular enzymes. The precise folding of proteins after they have been assembled is essential for the functioning of enzymes, since it produces docking stations for the substrates on which they act. Nucleic acids are of two kinds, commonly known as DNA and RNA. DNA is the central repository of genetic information. DNA incorporates the genetic code, in which sequences of three bases (codons) code for individual amino acids and thus specify the order in which these will be joined together to form the various proteins. DNA replicates itself and also transcribes encoded information into RNA. Some RNA is associated with proteins in ribosomes, some occurs as messenger RNA, and some as transfer RNA. Ribosomes move along messenger RNA strands, reading the succession of 3-base codons and stringing together amino acids brought in by transfer RNA. In this way, proteins are assembled. Both DNA and RNA have a sugar-phosphate spine, with purines and pyrimidines attached to the sugars. The sugar in DNA is 2-deoxyribose, which, in RNA, is ribose. One of the pyrimidines of DNA, thymine, is replaced by uracil in RNA. DNA molecules are usually in pairs, helically intertwined: RNA is single stranded. DNA is concentrated in the nuclei of eukaryotic cells, although some is also associated with the

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Chapter 9 mitochondria (this is because these organelles were originally independent prokaryotes). One way of categorizing DNA is by its base ratio (percent guanine + cytosine). In the eumycotan fungi, reported values range from 38% to 63%. The DNA content of fungi has been found to be very low, 0.15%–0.30%. Their RNA content is much higher, 1%–10% dry mass. Of this, the greater part is accounted for by ribosomal RNA, with a much smaller amount of transfer RNA and even less messenger RNA. Carbohydrates are sugars, sugar alcohols, and polysaccharides (polymers of sugars), all with the empirical formula (CH2O)n. Most fungal carbohydrates are polysaccharides, such as chitin, chitosan, mannan, glucan, starch, glycogen, and, in the pseudofungal oomycetes, something resembling cellulose. Chitin, a principal wall component in the Dikarya, is a polymer of β-1,4 N-acetylglucosamine. Cellulose is a polymer of β-1,4 glucose. The main storage carbohydrate in true fungi is glycogen, but the disaccharide, trehalose, and sugar alcohols like mannitol, are also used. Lipids all have an aliphatic hydrocarbon chain as part of their makeup. Their structure may be complicated by substitution with hydroxyl and carboxyl groups, they can be saturated or unsaturated, they may have aromatic moieties, and they can be combined with carbohydrates and amino acids. All are soluble in nonpolar solvents. They include the fatty acids (saturated and unsaturated), fats and oils (fatty acids combined with glycerol), phospholipids, and sphingolipids. The main fatty acids found in fungi are palmitic (C16:0), oleic (C18:1), and linoleic (C18:2). The first number in parens indicates the number of carbon atoms in the molecule, and the second number indicates the number of double (unsaturated) bonds. Most fatty acids are combined with glycerol to form oils and fats, widely used as storage compounds. Phospholipids and sphingolipids are components of membranes, where they are often complexed with proteins. Isoprenoid lipids are based on isoprene, a 5-carbon branched chain molecule. Terpenes contain two isoprenes, sesquiterpenes three, diterpenes four, and triterpenes six. Carotenoids are diterpenes, and sterols are triterpenes. Although ergosterol is the main fungal steroid, many other sterols are also present.

Metabolism Metabolism may be defined as the sum total of all chemical reactions that support life. These may be divided into anabolic and catabolic functions. Anabolic metabolism converts food substrates into fungal biomass, catabolic metabolism extracts energy from various substrates, producing adenosine triphosphate (ATP), reduced nicotinamideadenine dinucleotide (NADH), and NADPH, as well as intermediates used in various anabolic processes. All important reactions in biological systems are initiated and controlled by enzymes. In the absence of enzymes, most reactions would go on too slowly to sustain life (if they proceeded at all). Enzymes increase rates of reaction dramatically, by factors up to 107. An enzyme consists of a protein, often with a coenzyme such as a vitamin, and an activator such as Mg ions. Enzymes often work in sequence, each catalyzing a particular step in a metabolic pathway. Many fungi can produce enzymes (e.g., ligninases and cellulases) that are rarely found in other organisms.

Fungal Physiology and Metabolism Glycolysis. Of the three pathways by which glucose can be converted to pyruvate before it is oxidized in the citric acid cycle, most fungi use two: the Embden-Meyerhof (EM) and the hexose monophosphate (HM). The EM pathway yields ATP and pyruvate. The HM pathway yields NADPH, the main reducing agent in the biosynthesis of fatty acids and sugar alcohols, and ribose, used to make RNA, DNA, and other nucleotides. Most fungi respire aerobically, regenerating NAD by transfer of electrons from NADH to an external acceptor, oxygen. Fungal fermentation involves the regeneration of NAD by transfer of electrons to pyruvate, which is produced while the substrate is being metabolized. This kind of fermentation can produce alcohol or lactic acid. Everyone knows about the fermentation of pyruvate to alcohol and CO2 by yeasts such as Saccharomyces cerevisiae, but species of Aspergillus, Fusarium, and Mucor can do it too. Oomycetes and zygomycetes carry out lactic acid fermentations. Respiration involves three processes: the citric acid cycle, electron transport, and oxidative phosphorylation, all associated with the mitochondria, as they are in all eukaryotic organisms. The citric acid cycle accepts acetyl units, builds them into citrate, then oxidizes them to CO2, reducing NAD as it does so. The electron transport mechanism transfers electrons to oxygen to make water, and along the way involves cytochromes in the phosphorylation of ADP to make ATP. Respiration can be inhibited in two ways: by uncoupling phosphorylation from electron transport, or by blocking electron transport. Many fungicides are uncouplers or blockers. Biosynthesis of amino acids. The average protein contains twenty or so different amino acids, but two of these amino acids, lysine and tryptophan, are of particular interest to students of the fungi. Eumycotan fungi and chytridiomycetes make lysine by a pathway that is different from that used by all other organisms, except the euglenids (unicellular, wall-less, flagellated algae). Eumycotan fungi, chytridiomycetes, and euglenids synthesize lysine via eight steps, one of which involves a unique intermediate, α-aminoadipic acid (AAA). All other eukaryotic organisms that can make their own lysine (oomycetes, green algae, and vascular plants) do so by a pathway involving seven steps and a unique intermediate called diaminopimelic acid (DAP). The two pathways have nothing in common: every step and every intermediate is different. (Animals are lysine auxotrophs—they can’t make it at all, and they must get it in their diet.) Apart from anything else, the two radically different synthetic pathways suggest that the eumycotan fungi, chytridiomycetes, and euglenids may have evolved from a common ancestral group, a group different from that which gave rise to the oomycetes, green algae, and higher plants, and different from that which produced the animals. Only one synthetic pathway is known for tryptophan, but genetic analysis of the loci controlling the sequence of enzymes has shown that, while in two mycelial fungi (Neurospora and Aspergillus) four genes are involved, the yeast Saccharomyces requires five genes. The sedimentation of the various enzymes on sucrose gradients shows four patterns of enzyme aggregation, which are taxonomically distributed as follows: (1) chytridiomycetes, ascomycetes, most basidiomycetes; (2) yeasts; (3) zygomycetes, pucciniomycetes; (4) oomycetes. Although we can’t draw firm phylogenetic conclusions from such data, we can suggest that groups with different enzyme aggregation patterns are unlikely to be closely related. Polysaccharide synthesis. I need hardly emphasize the importance of the hyphal wall to most fungi: it is simply the main skeletal component of the mycelium, imparting

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Chapter 9 mechanical strength to the hyphae and also protecting the living contents from deleterious outside influences just as the cellulose wall of the average plant cell does. The principal components of most fungal walls are polysaccharides, such as chitin (in the eumycotan fungi and chytridiomycetes) and a cellulose-like compound (in the pseudofungal oomycetes). The sugar monomers from which these polysaccharides are built (N-acetylglucosamine and glucose, respectively) are linked with a nucleotide, then enzymically added to the end of a growing chain. The spores of many fungi are embedded in slimy mucopolysaccharides, which may be important in dispersal. Little need be said about the metabolism of specific elements, except to mention that fungi are carbon and nitrogen heterotrophs, so they must be supplied with both in some combined form. A further restriction in the case of carbon is that the carbon source must be an energy-rich substance previously synthesized by another organism. Common carbon sources are sugars, hemicellulose, cellulose, and lignin: the last two are relatively recalcitrant polymers that few other organisms can metabolize, presumably because they lack the necessary enzymes. Nitrogen can often be assimilated in the form of nitrate or ammonia, but amino acids, polypeptides, and proteins can also be digested by many fungi. Again, some rather resistant structural proteins such as keratin can be attacked by certain groups of fungi (see chapter 23). Secondary metabolism is a strange phrase. Surely the one word, metabolism, describes the total spectrum of chemical activities going on inside a living organism? But on close inspection, it becomes apparent that some organisms produce quantities of certain substances that do not seem to be part of the ordinary, ongoing business of existence (called ‘primary metabolism’). These substances are now called ‘secondary metabolites’, and may be defined as natural products that are not necessary for growth, are often produced only by specific groups of organisms, during only part of their life cycle, and are derived from a few precursors formed during primary metabolism. Why then are we so conscious of these substances? Although they occur only sporadically, secondary metabolites tend to accumulate, since organisms usually produce them steadily but do not degrade them. In addition, they are often biologically active. Penicillin, griseofulvin, cyclosporine, aflatoxin, ergot alkaloids, and psilocybin all are secondary metabolites of fungi, and all are famous for their effects on other organisms, which are described elsewhere in this book. Although secondary metabolites are rare in animals, they are common in plants, bacteria, and fungi. Many are formed only after the requirements of cell growth have been satisfied. When growth stops, it seems that some biochemical pathways are not shut off, and things like fatty acids and amino acids accumulate, while the tricarboxylic acid cycle keeps on cycling. The organism now uses these raw materials, and a few others, to manufacture new end products. For example, fatty acids give rise to polyacetylenes, amino acids to ergot alkaloids and penicillin. We now have a large catalogue of secondary metabolites, all derived from a relatively small number of precursors. Secondary metabolites can be placed in five groups, according to the area of primary metabolism from which they are derived: (1) glucose-derived substances like polysaccharides, peptidopolysaccharides, and sugar alcohols; (2) condensation products of acetate derived from the acetate-malonate pathway of fatty acid synthesis (e.g., polyketides and phenolics); (3) condensation products of acetate derived from the mevalonic acid pathway (e.g., terpenes); (4) phenolics derived from the shikimic

Fungal Physiology and Metabolism acid pathway of aromatic amino acid synthesis; and (5) derivatives of other amino acid syntheses. Acetate is the raw material from which polyacetylenes, polyketides, steroids, and terpenes are synthesized. Polyacetylenes are straight-chain compounds containing conjugated acetylenic systems. These compounds fluoresce brightly in UV and so are easily detected. Of the more than 400 polyacetylenes known, about 80 are fungal and found exclusively in basidiomycetes. Polyketides are produced by many ascomycetes and conidial anamorphs by condensation of acetyl units with malonyl units, with simultaneous decarboxylation. Examples are the antifungal antibiotic griseofulvin (see chapter 23), and the mycotoxins ochratoxin and aflatoxin (see chapter 21). Terpenes and steroids are biosynthesized from isopentenyl pyrophosphate, which is itself an acetate derivative. The trichothecene mycotoxins are sesquiterpenoids. Gibberellic acid is another fungal terpene that has also been found to be an important plant growth regulator. The mycotoxin zearalenone is a steroid. Although it produces profound sexual dysfunctions in young pigs, it is now widely used in the form of implants to promote growth of beef cattle. Amino acids are the building blocks of proteins, but they are also the raw materials for many fungal secondary metabolites. Four groups stand out: (1) cyclic oligopeptides, which may be death-dealing toxins like the amanitins (eight amino acids), or lifesaving immunosuppressants like cyclosporine (eleven amino acids); (2) indole alkaloids, such as the hallucinogen psilocybin and the dangerous ergot alkaloids; (3) the β-lactam antibiotics penicillin and cephalosporin; and (4) plant growth regulators such as auxin, cytokinin, and ethylene, which are formed by many fungi. So far, I have taken the usual anthropocentric attitude in this brief survey of fungal secondary metabolites, concerning myself only with those that are of some direct importance to humans. In the interests of the impartial intellectual approach, it might be a good idea to now consider briefly the possible roles of these substances in the lives of the organisms that produce them. What value can a highly poisonous and extremely carcinogenic substance like aflatoxin have for Aspergillus flavus? What good, if any, is penicillin to Penicillium chrysogenum, or zearalenone to Fusarium graminearum? It seems highly probable that these substances are not just waste products. Aflatoxin may well give A. flavus an advantage when it is competing with animals for food. Small mammals may learn to avoid mouldy nuts or seeds; if they don’t, they may be poisoned. Either way, the fungus wins. Penicillin, with its powerful bacteriostatic activity, may be presumed to give P. chrysogenum an edge over competing bacteria. Zearalenone, a mycotoxin that acts like a steroid sex hormone in pigs, is now believed to play a role in the development of the Gibberella zeae teleomorph of Fusarium graminearum. Regulatory mechanisms. On reflection, it must be obvious that all of the thousands of reactions and cycles that make up the metabolic activity of an organism must be under some kind of control. Genes are turned on and off by environmental and inherent developmental factors, but many processes have to go on all the time, or at least be constantly ready for action. Obviously, there isn’t an overseer in the nucleus turning all these switches on and off as needs are perceived and fulfilled. Most processes investigated have built-in controls, which are often simple but elegant feedback mechanisms, which affect the enzymes that drive most reactions. For example, a different enzyme catalyzes each step in the production of leucine. If the raw material for this synthesis is

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Chapter 9 in good supply, the pathway could churn out far more leucine than is needed. So, in a breathtakingly simple solution, the synthesis of the first enzyme in the series is inhibited by increased concentrations of the end product, leucine. The pathway effectively turns itself off when it isn’t needed.

Growth Growth is often defined as irreversible increase in volume, but it usually implies some other kinds of change as well: changes in components, metabolism, shape, and function. A mycelial fungus will extend in all directions as its hyphae grow at their tips. The hyphae become longer, they often branch repeatedly, a lot of wall material is laid down, and the amount of protoplasm and the number of nuclei in the colony increase. If the fungus is lucky, it will find more food than it uses up in the search, so it can both grow and accumulate reserves that will enable it to sporulate. Fungal growth is usually measured as increase in fresh weight (unreliable because of variations in water content), or in dry mass (which for obvious reasons can be measured only once for any particular colony), or by increase in the diameter or radius of the colony (which can be measured repeatedly). In unicellular yeasts, growth is measured by counting cells, or by measuring the increase in turbidity of the culture medium. If we were trying to produce conidial inoculum for use in a program of biological control (see chapter 14), we might express the success of the organism in terms of the numbers of propagules it formed in a certain time, at a certain temperature, or on a particular substrate. A mushroom grower would be interested only in the mass of basidiomata produced. Beginning from the spore, growth proceeds in stages, which can be categorized as germination, assimilative growth, and sporulation. Each stage may require conditions very different from the others. I will examine them in turn. Much of our information about the early stages of growth has been obtained by plant pathologists, who are very interested in how pathogens get into their host plants and in how they can be killed at their most vulnerable point, which is often the germ tube. Other groups of organisms produce spores, but none produce them with such singleminded dedication, in such prodigal abundance, or in such an exuberant variety of forms, as the fungi (Fig. 3.1). The spore, almost as much as the hypha, is a fungal trademark. Spores may be single cells, or they may be divided up in various ways into tens or even hundreds of compartments. Some live for a few hours; others live for years. But all have two characteristics in common: they ensure the survival of the fungus through time, space, or both; and sooner or later they germinate. A dormancy phase usually precedes germination. Spores are often formed when conditions are deteriorating for the fungus. Temperatures may be falling, water drying up, and food running out. If the spores germinated immediately, they would face the very problems they were produced to circumvent. Many spores have a built-in timer, an endogenous constraint that will not allow them to germinate until a certain time has elapsed. Many require prolonged cold treatment. Spores of many coprophilous fungi won’t germinate until they have been exposed to elevated temperatures or to a specific chemical treatment: things that mimic what happens to them inside the gut of an animal. The signals they receive while passing through the gut prepare them to

Fungal Physiology and Metabolism germinate in the dung as soon as it has been deposited, and so to have first access to the abundant nutrients it contains. Spores of some rust fungi won’t germinate if they land too close to the parent mycelium or to sibling spores—this phenomenon is called selfinhibition. However, spores of less specialized fungi often have no such built-in inhibitions and will germinate as soon as appropriate conditions arise. In these cases, any dormancy must be regarded as exogenous, that is, imposed from outside by lack of environmental encouragement. Germination of powdery mildew conidia requires only oxygen, and the spores of some other obligately parasitic fungi need only oxygen and water. Most saprobic fungi need other factors, ranging from inorganic salts to various organic carbon sources. If a spore is to produce an extension of itself in the form of a germ tube (the first hypha), it must increase in volume. The only way to do this in the absence of an external food supply is to imbibe water or to produce it metabolically. Of course, the food reserves of the spore permit some synthesis of cytoplasm and wall material, but this is a limited resource and is soon exhausted. If the young hypha is to survive, it must find food. The walls of resting spores are often chemically different from those of hyphae and are relatively impermeable. When they are ready to germinate, however, enzymes render the walls permeable, so that the spore can receive chemical stimuli from outside. When germination begins, enzyme action intensifies, softening the wall, often at a preformed thin area called a germ pore or germ slit. A germ tube emerges, the constituents of its cytoplasm, nuclei, and wall material supplied by a renascent metabolism. Once germination has happened, growth and differentiation are the next phases. Growth involves elongation of the young hypha at its tip, often with concurrent migration of cytoplasm and nuclei in the direction of growth. Growth implies increase in volume and increase in dry weight. These increases may be achieved by absorption of soluble nutrients already available in the environment. More usually, exoenzymes have to be secreted from the hyphal tip: they degrade the substrate into smaller, water-soluble molecules which can be absorbed and metabolized. However, if a hypha simply grew in a straight line, it could not effectively explore or exploit the substrate, and no appreciable biomass could ever be accumulated. This is where differentiation comes in. Soon after a germ tube appears, it branches. Then each of the two resulting hyphae branch, and so on, and so on (see Fig. 3.2). Soon, hyphae are growing in all directions possible, minutely exploring the substrate and forming the typical fungal colony, which will be spherical if the fungus can grow in three dimensions (as it does in a liquid medium) or circular if it is growing mainly in two dimensions (as it does when it spreads across the thin film of nutrient agar in a petri dish culture). Growth rate. Since more and more fungi are being used for industrial or biotechnological purposes, it is important that fermentations be carried out under the best possible conditions of temperature, pH, and nutrition. Fungal physiologists have laid the groundwork for such applications. If we want to find out the best temperature at which to grow our fungus, we set up a series of experiments in which conditions are the same in all replicates, except for the temperature. The two common measures of growth rate are (1) increase in radius of colony over time and (2) increase in dry mass of colony over time. The first has the advantage that sequential records can be obtained from each colony. The second is a more absolute measurement but can be performed only once for each colony. If we are growing the fungus on a solid substrate like cellulose, it

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Chapter 9 is difficult to separate the mycelium from the substrate at the end of the experiment, so we have to measure growth in another way: (3) by determining protein content. Such studies show that growth can be divided into several phases: (a) the lag phase, when growth begins slowly, then gradually accelerates; (b) the exponential phase, when growth continues at a high and steady rate; (c) the decline phase, as growth slows down and finally stops; and (d) death, often accompanied by various degrees of autolysis. This is the kind of picture we get when we study fungi in what is sometimes called batch culture. The fungus is grown in a flask or a fermenter, with a limited amount of food and space. When growth slows down, it might be because the fungus is running out of food. But the decline often comes long before the food is exhausted. This deterioration is often called staling and is attributed to the accumulation of waste products that inhibit metabolism. If a fungus is inoculated at one end of a very long horizontal tube with a bed of nutrient medium along its length, it can remain in the exponential growth phase for as long as there is fresh medium to explore. Needless to say, this cannot happen often in nature, so staling must be accepted as the norm. It may even be the switch that shifts some fungi into the reproductive mode. Different fungi may grow at widely differing rates during their exponential phase, as may the same fungus grown under different conditions or on different substrates. One measure of growth rate is the time it takes a fungus to double its dry mass. A more easily determined measure is the specific growth rate, which is derived from measurements of a colony’s mass at one-hour intervals. A fungus which increases in mass by 20% in an hour is said to have a specific growth rate of 0.2. Fungi have a wide range of specific growth rates. Chaetomium virescens has given a specific growth rate of 0.4 on glucose, and a value of 0.6 has been recorded for Neurospora crassa (or rather, for its Chrysonilia anamorph). These examples have much higher specific growth rates than most fungi. Yet some other fungi, including some common saprobic zygomycetes (e.g., Rhizopus oligosporus) and some yeasts, also have growth rates high enough to lead to their commercial exploitation, or to their denunciation as ‘weeds’. Clearly, if we could understand and perhaps circumvent the mechanisms that limit the growth rate of many potentially useful fungi, biotechnological applications of these organisms would multiply. Localization of growth. The walls of assimilative hyphae are not impermeable: they have pores similar in size to those in higher plant cell walls. Hyphal walls reduce but do not prevent the outward movement of water in dry conditions (desiccation), and the inward movement of deleterious substances from the environment. They do prevent the passage of most enzymes (large, proteinaceous molecules), and the exoenzymes on which fungi depend are in fact secreted almost entirely at or near the hyphal tip. This suggests that most hyphal wall material must be laid down just behind the hyphal tip. And experiments indicate that this is actually what happens. What kind of experiments? Early observations showed that the distances between septa, and between the origins of successive hyphal branches, did not change with time. Exposing growing hyphae to osmotic shock produced abnormalities only at their tips. Fluorescent antibodies have been used to distinguish between old and new wall material, and the resulting pattern of fluorescence showed that new material was introduced only at the hyphal tip. Tritiated N-acetylglucosamine was fed to growing hyphae and its incorporation

Fungal Physiology and Metabolism pinpointed by autoradiography: again, incorporation was largely restricted to the apical micron. These are very significant observations. We can now see why a fungus needs so many hyphal tips, and how every fungal colony is essentially similar to the ‘fairy ring’, driven to ever-expanding radial growth by its need for food and by the exhaustion or staling of the substrate left behind. Since branching is so vital to the success of the fungus in exploring its substrate, the mechanisms that control branching are of great interest. The phenomenon of apical dominance is well known, for example, in coniferous trees. It appears that a similar dominance can be detected in the hyphal tips of many fungi. Fungi growing in culture often have a characteristic distance from the hyphal tip to the first branch. Different metabolic inhibitors affect this relationship in different ways. Mitomycin C inhibits branch formation but not hyphal elongation; sodium fluoride has the opposite effect. (Mitomycin is known to inhibit DNA synthesis, NaF to inhibit metal-activated enzymes, but this knowledge hardly helps us to explain their differential effects on hyphal growth.) In most fungi, the leader and the first one or two branches grow faster than the others: if the faster growing tips are cut off, the subordinate ones are ‘released’, and grow faster. If inhibited branches are severed at their base, they proceed to grow faster, showing that the inhibition originated within the fungal thallus. The amount and kind of some nutrients, such as sulphur and nitrogen, available to the fungus also influence apical dominance. The complexity of the growth regulation system is indicated by the mapping of no fewer than ninety genes that control some aspect of colony morphology in Neurospora. Septa (cross-walls) are a diagnostic feature of the hyphae of most true fungi, especially the Dikarya. A septum is not usually laid down as a thin membrane, but rather begins as a ring of material around the inside of the hypha. The ring grows inward, eventually becoming an almost complete bulkhead which physically reinforces the intrinsically strong tubular configuration of the hypha. If we could watch a septum form, the process would resemble the closing of an iris diaphragm in a camera. In some fungal hyphae there is a clear relationship between septum formation and distribution of nuclei; for example, in dikaryotic hyphae there is a septum for every two nuclei. But in many fungi there is no such relationship, nuclei pass through the septal pore between compartments with relative ease, and the mechanism that decides where septa will be laid down is obscure. Of course, many fungi are not restricted to the formation of individual hyphae, even during the assimilative phase. Various stimuli trigger the development of mycelial strands, rhizomorphs, and sclerotia. But the true capacity for differentiation that is innate in most fungi is expressed only in the reproductive phase. Media. In nature, fungi grow on just about any organic substrate. When we grow them in pure culture in the laboratory, we usually provide them with a special nutritive medium. This may be liquid (a broth) or solid (a gel). Most identification is done on solid media, but most fermentations and many physiological experiments are carried out in liquid media. Liquid media are based on water and may contain a variety of nutrients, buffers, and so on. The fungus is usually grown submerged, and its oxygen requirements are met by shaking (small flasks) or stirring and aerating (large fermenters). In solid media, the water and other components are held in a gel by agar. This complex polysaccharide, derived from a red alga, melts at 100°C but does not solidify until it cools to 45°C and is not metabolized by most fungi. As little as 1%–2% agar

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Chapter 9 solidifies most media. Agar media are usually used to form thin layers covering the bottom of petri plates or to fill the bottom third or so of test tubes (called ‘slants’ because they are placed at an angle while the agar is setting). Plates are used to grow cultures for identification. Typically, a small inoculum will be placed in the middle of the plate, then incubated. The colony which develops, growing partly above the surface of the medium and partly below it, will often show diagnostic features (colour, texture, sporulation, etc.). Living cultures are often stored in slants, or they may be lyophilized (freeze-dried and sealed in a high vacuum) for long-term storage. If the culture is to be axenic (or as we say, uncontaminated), the medium, which is attractive and accessible to many kinds of microorganisms, must first be sterilized. This has usually involved pressure cooking the medium in an autoclave at a steam pressure of 15 pounds/square inch (2 atm) for 15 minutes. The temperature reaches 120°C, effectively killing all microorganisms. But it also tends to caramelize sugars and to destroy thermolabile substances like thiamin (a vitamin) and some antibiotics. Autoclaving is acceptable for routine work, but for critical physiological studies, it is better to filter the medium through membrane filters that effectively remove bacteria and fungal spores.

Nutritional Requirements Carbon nutrition. One of the principal distinguishing features of most fungi is their inability to fix inorganic carbon. The simplest compound most fungi can use as a source of energy is the monosaccharide glucose. Unlike most other carbon sources, this doesn’t need to be enzymically broken down to anything simpler before it can be absorbed. Virtually all fungi are ready to metabolize glucose at a moment’s notice: they already have all the necessary enzymes, which are thus described as constitutive. Fructose, mannose, and galactose are also readily used, but there is often a delay before assimilation begins. This is because the enzymes involved in processing these sugars aren’t necessarily ready and waiting. The fungus takes a little while to recognize the nature of the substrate and to synthesize the proper enzymes. This process, called induction, produces adaptive enzymes. If a lot of glucose is present, it may suppress the production of the enzymes that deal with other substrates: the fungus takes the easy route. A little glucose, on the other hand, may fuel the induction process and shorten the lag phase on many substrates. Although many experiments have been done to compare the ability of fungi to use different single-carbon sources, these may not tell the whole story. In nature, fungi usually have to deal with mixtures, and their behaviour in this situation can’t always be forecast from single-substrate tests. We’ve already seen that the presence of glucose can suppress the utilization of other substrates. Perhaps the most important example of the mixed substrate situation involves lignin. Although the ability to degrade lignin to carbon dioxide is one of the things for which many basidiomycetes (the white rot fungi) are most notorious, they can’t use lignin as the sole carbon source and will break it down only in the presence of another accessible carbon source, such as cellulose, cellobiose, or glucose. Fungi may break down lignin only to gain better access to the cellulose or in order to release available nitrogen.

Fungal Physiology and Metabolism Culture media must also contain a source of nitrogen. No fungus (in fact, no eukaryote) can fix atmospheric nitrogen. Many fungi can use nitrate, although ammonium nitrogen is even more universally metabolized. Urea, amino acids, and various polypeptides and proteins are accessible to some, but not all, fungi. A good nitrogen source for many fungi is hydrolyzed casein, a mixture of amino acids. Sulphur requirements can almost always be met by incorporating sulphate in the medium, although some chytridiomycetes require sulphur-containing amino acids such as methionine. Vitamins are coenzymes that are required in minute amounts. Although some fungi can make many of their own vitamins, many are deficient for thiamin (vitamin B1, involved in carboxylation), biotin (B7, carboxylation), riboflavin (B2, dehydrogenation), pyridoxine (B6, transamination), nicotinic acid (B3, dehydrogenation), and others. Vitamin deficiency is sometimes absolute, in which case the fungus can be described as auxotrophic in this respect, and won’t grow unless one or more vitamins are supplied. In other cases it is only partial, so that additions of vitamins may merely increase growth rather than making it possible. Vitamin deficiency may be temporary: Myrothecium needs biotin for spore germination but not for mycelial growth. While many fungi require vitamins, others can synthesize them from precursors. Thiamin, for example, consists of a pyrimidine ring and a thiazole ring, linked by methylene. Some fungi, if provided with the two rings, can link them and complete the molecule; other fungi need only the pyrimidine ring; yet others need only the thiazole ring. Fungi also need a range of elements, which can be divided into two groups according to the amounts required for normal growth. The macronutrients include potassium (K), which is used in carbohydrate metabolism, enzyme activity, and to maintain ionic balance; phosphorus (P), an essential component of nucleic acids and of energy transfer mechanisms; magnesium (Mg), an enzyme activator required in ATP metabolism; sulphur (S), a component of some amino acids, vitamins, and other sulfhydryl compounds; and calcium (Ca), an enzyme activator that is also often found in membranes. Micronutrients, sometimes called trace elements, include iron (Fe), found in cytochromes, haem apoenzymes, and pigments; copper (Cu), an enzyme activator also involved in pigments; and manganese (Mn), zinc (Zn), and molybdenum (Mo), all enzyme activators. Fungi get along without boron, chlorine, fluorine, iodine, and silicon, although these elements are apparently essential to many other organisms. Incorporating iron in a growth medium can be a problem, since ferric iron is extremely insoluble at pH values above 4, and ferrous iron is quickly oxidized to ferric by the free oxygen most fungi need. Fortunately, a chelating agent such as EDTA (ethylenediamine tetraacetic acid), which acts as a metal ion buffer, will increase the biological availability of iron. Although culture media must contain some available water, some conidial fungi and yeasts are the most xerotolerant organisms known, able to grow at water activities (aw) as low as 0.65. If we consider that most animals grow only above aw 0.99, most green plants wilt irreversibly at aw 0.97, and most bacteria will grow only at aw 0.95 or higher, this must be recognized as a truly remarkable talent, although for us it is an expensive nuisance, as you will read in chapter 20. Fungi that grow at low external water activities have comparably low internal aw as well. Yeasts control their internal osmotic pressure by interconverting sugars and polyhydric alcohols such as glycerol and mannitol, and it seems probable that mycelial fungi may well do this too, although

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Chapter 9 that has not yet been established. Most enzymes normally operate in an aqueous medium, and if a fungus is to function at low internal aw, some enzyme-compatible water substitute must be present. It has been found that glycerol can play this role. Using the information given above, you should be able to concoct a culture medium on which many fungi would grow well. By combining many individual chemicals, you can make specific provision for their basic metabolic needs. The medium you produce will be a ‘defined’ or even possibly a ‘minimal’ medium. But many fungi grow even better on very complex substrates: things such as extracts of malt, or of potatoes, or of yeast. These rich mixtures, although ‘undefined’, appear to be nutritionally optimal, and it is much easier to use one of them than to painstakingly measure out increasingly minute amounts of a long list of trace elements and expensive purified growth factors. Unless you are doing critical physiological experiments, you will probably grow most fungi on potato dextrose agar (PDA), malt extract agar (MEA), or some other undefined medium. Recipes of media suitable for a wide range of applications can be found in Methods in Microbiology, vol. 4 (Booth 1971) and The Mycology Guidebook (Stevens 1974). Also, remember that your best efforts to culture many groups of fungi are doomed to frustration and failure. The physiology of many obligately parasitic fungi is intimately linked with that of their hosts, so no ordinary medium will support growth of members of the Pucciniomycetes, Peronosporaceae, Erysiphales, Laboulbeniomycetes, Glomeromycota, and others. The simplest system in which most of these organisms can be studied is a ‘dual’ culture: fungus + host. Transport. Fungi can absorb food only in the form of relatively small molecules like glucose. Water moves into hyphal tips by passive diffusion, driven by osmotic differentials. Although the cell wall is more or less permeable in either direction to the kind of molecules the cell seeks to accumulate, it limits the inflow of water by offering a physical resistance (wall pressure) to expansion. The plasmalemma is semipermeable and controls the movements of solutes. The membrane itself is largely lipid—actually a double layer of phospholipid molecules—which tends to keep water-soluble compounds out. Transport can be passive or active. In passive transport, the substrate moves along a concentration gradient or an electropotential gradient. Active transport requires an investment of energy, usually ATP, by the organism. Desirable substances may be carried in, or unwanted material expelled. In active transport, the substance being moved is believed to be pumped through special channels lined with proteins called permeases, or to become bound to a specific carrier protein, which is responsible for transporting it across the membrane. Carrier proteins also aid in passive ‘facilitated diffusion’. Cations such as potassium, ammonium, magnesium, calcium, manganese, and iron are all accumulated against high concentration gradients, showing that active, carriermediated transport mechanisms are involved. If a fungus is loaded with sodium, then supplied with potassium, sodium will be expelled as potassium is taken up. This kind of behaviour is called countertransport. The divalent cations, Mg++, Ca++, and Mn++, will be taken up only if phosphate is also available, and Fe+++ is chelated with siderochromes before being transported. The transport of ions such as phosphate and sulphate is also carrier mediated. Once phosphate is inside the cell, it is converted to polyphosphate, and internal concentrations of orthophosphate don’t change. The study

Fungal Physiology and Metabolism of nitrate uptake has been hindered by the lack of a really sensitive measuring technique, so it isn’t certain that carriers are involved. Glucose and other sugars move across the plasmalemma of fungi by facilitated diffusion, or by active transport, or by a combination of the two. A single fungus may have several different mechanisms. Some of the active mechanisms are constitutive (always present and ready), while some are inducible. Amino acids are also transported actively. Neurospora crassa has been shown to have at least five different amino acid transport systems: one carries only methionine; another, only acidic amino acids; a third, only basic amino acids; the fourth, aromatic and aliphatic amino acids; the fifth, aromatic, aliphatic, and basic amino acids. Amino acid transport systems differ from those for sugars and ions in that no countertransport has been detected. Cellulose and Lignin Decomposition. Fungi produce an extraordinary spectrum of enzymes and can degrade just about any organic substrate. Perhaps the most important of these substrates are cellulose and lignin. Billions of tonnes of cellulose are produced by the higher plants every year. It forms the greater part of their cell walls, and, being apparently unable to recycle it themselves, they discard it in vast quantities every year. Autumn-shed leaves, the entire biomass of annual plants, and eventually the corpses of the much longer-lived trees: all are bequeathed to the fungi, because no other organisms can initially unlock the energy they contain. The key to this wealth is the complex of fungal cellulases (some bacteria produce cellulases but generally operate under wetter, more alkaline conditions). Trichoderma viride is a well-known cellulolytic fungus that is commonly isolated from some forest soils and from decaying plant material. It produces three distinct cellulolytic enzymes: cellulase, which hydrolyzes all kinds of cellulose; glucan cellobiohydrolase, which degrades crystalline cellulose to cellobiose; and the glucanases, which hydrolyze amorphous cellulose. There are two kinds of glucanase: the endoglucanases, which produce cellulose oligomers, and the exoglucanases, which attack those oligomers, cleaving off one glucose unit at a time. Exoglucanases and cellobiases digest cellobiose, releasing glucose. The enzymes of the cellulase complex are all glycoproteins and are resistant to thermal denaturation. Plants also produce vast quantities of lignin. This recalcitrant polymer strengthens the walls of many plant tissues, especially those involved in secondary thickening. Their deposition in wood tends to mask the cellulose content of the cell walls. Some fungi (the white rot basidiomycetes) are the only organisms we know that can unequivocally degrade lignin, and even they, as already mentioned, cannot use it as their sole source of carbon. Ligninases are oxidative rather than hydrolytic enzymes. Many white rot organisms produce an extracellular polyphenol oxidase called laccase, which must play a critical role in lignin breakdown, since organisms that don’t have it can’t attack lignin. Other as yet undiscovered enzymes are also almost certain to be involved. Protein digestion. Although some proteins are water soluble, they cannot cross the plasmalemma into the fungal cell unless they are broken down into small oligopeptide fragments containing no more than three to five amino acid molecules. Hence the need for extracellular proteases. Fungi like Saccharomyces cerevisiae, which produce only intracellular proteases, cannot assimilate proteins. At the other end of the scale are organisms like Trichophyton and other dermatophytes, which can attack keratin, a

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Chapter 9 tough structural protein. There are two main kinds of protease: exohydrolases, which nibble individual amino acids from the ends of peptide chains; and endohydrolases, which will cleave a chain into two large fragments, doubling the number of ends on which the exohydrolases can operate.

Environmental Effects Physical parameters like temperature, light, and gravity have profound effects on many fungi, but generalizations are dangerous. Some psychrophilic fungi grow at temperatures below 0°C; some thermophiles can function at temperatures above 50°C. Some fungi need light in order to fruit; others seem indifferent to illumination. Many macrofungi are extremely sensitive to gravity (especially when producing fruit bodies); many microfungi are totally oblivious to it. It is helpful to know the cardinal temperatures of any fungus we want to work with. These are its minimum, optimum, and maximum temperatures for growth. Most researchers find it convenient to grow a fungus at its optimum temperature, but this ignores the fluctuating and often extreme temperatures the organism must face in much of North America. I have already pointed out that falling temperatures in autumn may induce fruiting in some fungi (such as Coprinus comatus), dormancy in others; that resting stages of many fungi (such as Monilinia) must be chilled before they will germinate; and that heat treatment produces the same effect in others. From study of their assimilative growth, fungi can generally be categorized as psychrophilic, mesophilic, or thermophilic. Psychrophiles have minimum growth temperatures below 0°C, maxima below 20°C, and optima in the range 0°C–17°C. Mesophiles (the great majority of fungi) have minima above 0°C, maxima below 50°C, and optima between 15°C and 40°C. Thermophiles have minima above 20°C, maxima above 50°C, and optima between 35°C and 50°C. Establishing true optima may not be simple, as Fig. 9.1 shows. If a complete growth curve is not plotted at each of the chosen temperatures, incorrect conclusions could be drawn. Compare the answer you would get if you harvested the experiment detailed in Fig. 9.1 at day 7 with that you would get at day 14. Also note that the quickest start-up does not necessarily eventually produce the most dry mass. Some of the most spectacular effects of light on fungi are documented in chapter 8 (dispersal) and chapter 11 (ecology). Pilobolus aims its explosive sporangial mechanism at the light; Podospora points the neck of its perithecial ascoma toward the light; the individual asci of Ascobolus point toward the light. Each of these mechanisms involves positive phototropism. Phototropism implies the existence of a photoreceptor. Most phototropic fungi respond best to blue light, and this is strongly absorbed by β-carotene, which is usually present in the photosensitive organs. Circadian rhythms. Some fungi in culture display daily rhythms of growth, pigment production, or sporulation, which seem to be responses to the alternation of light and darkness. Although Pilobolus sphaerosporus (Mucorales) didn’t need light in order to produce sporangia, establishment of a regular twelve-hour light/twelve-hour dark regime increased the number of sporangia produced and led to a peak of discharge six hours after the lights were turned on. Continuous light destroyed this synchrony, but in continuous darkness the cyclical discharge continued for several days, although with

Fungal Physiology and Metabolism

Fig 9.1. Effect of time and temperature on growth of Phycomyces in a defined medium. From Robbins and Kavanagh (1944).

gradually decreasing intensity. Raising or lowering the temperature did not change this 24-hour rhythm. Many other such circadian rhythms have been recorded, including some ‘clock’ mutants of Neurospora, but the underlying mechanisms of these cycles are not yet understood.

Reproduction: The Formation of Propagules The foregoing paragraph leads me to a consideration of reproductive physiology in fungi. If hyphae are the secret of the remarkable success of fungi in exploiting their myriad substrates, spores are the secret of their ubiquity. Spores are omnipresent,

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Chapter 9 ensuring that whenever a new substrate becomes available, fungi will always be there to colonize it. We can express the strategy of many fungi quite simply: in the assimilative mode, fungi produce hyphae; as long as there is food to be had, the fungus concentrates on accumulating reserves of energy, some to be invested in producing more hyphae, some to be stored. When food runs out, or staling factors build up, or reserves reach an appropriate level, or specific environmental signals are received, the fungus switches into the reproductive mode and produces spores. Some fungi produce spores directly on the assimilative hyphae; others form specialized but simple, one- or few-celled spore-bearing structures. In these cases, the onset of reproduction can be very rapid. In hyphomycetes like Penicillium, while assimilative hyphae at the margin of the colony are still advancing, the older hyphae are producing simple conidiophores and conidia. This situation can be recognized at a glance: the margin is white, while the rest of the colony is covered by a mass of green conidia. In other fungi, such as the agarics, the spore-bearing structure is large and complex. It takes longer for these fungi to prepare for the actual production of spores, but, once again, they are eventually liberated in astronomical numbers. Careful physiological work on the hyphomycete Aspergillus niger has established that several stages lead up to sporulation. These can be recognized by their differing nutritional requirements. (1) Low levels of nitrogen, with adequate glucose and aeration, permitted the development of foot cells and the subsequent elongation of conidiophores. Addition of ammonium ion would prevent this. (2) Addition of ammonium nitrogen and a tricarboxylic acid (TCA) cycle acid permitted development of the apical vesicle and the phialidic conidiogenous cells. (3) Glucose and nitrate were required for the formation of conidia. We cannot assume that exactly the same process operates in other fungi. I have already mentioned the period of endogenous dormancy that is apparently built into spores. The hyphae arising from spores also seem to have a minimum growth period before they will sporulate. Hyphae which have not emerged from this phase will not sporulate, even in conditions that normally induce fruiting. Toward the end of this refractory period, the fungus becomes less able to take up glucose and other nutrients. This suggests some kind of membrane control as part of the induction process. The range of temperature which permits sporulation is narrower than that over which assimilative growth can occur. For example, mycelia of Penicillium species grow at temperatures ranging from 2°C to 43°C; conidia are formed between 3°C and 40°C. Gnomonia vulgaris grows between 5°C and 30°C but produces ascomata only between 10°C and 25°C. Since fungi grow in so many different habitats and have such varied ecological requirements, it isn’t surprising that we can’t generalize about the link between light and sporulation. Light may inhibit, it may stimulate, or it may have opposite effects at different points in development. It stimulates the production of conidia in the Aspergillus anamorph of a Eurotium species, while inhibiting development of ascomata of the teleomorph. The effects of light have been investigated from two angles: which wavelengths are active (the action spectrum) and how much light is needed (the dosage response). I noted earlier that blue light and near-UV stimulated phototropic responses in Phycomyces (Mucorales), and the same wavelengths (420–485 and 350–390 nm)

Fungal Physiology and Metabolism induce formation of perithecial ascomata in Gelasinospora. Some ascomycetes and conidial fungi respond to UV but not to visible light. Although many fungi fruit only after exposure to light, the actual amount of light energy needed can be very small. Initiation of Coprinopsis lagopus basidiomata is triggered by only 8 joules (J) per square metre (5 seconds at 0.1 foot candle). To induce pseudothecial ascomata of Leptosphaerulina requires even less light (0.64 J m–2). Most fungal responses need only 0.5–20 J m–2—remarkably little, considering the magnitude of the induced effect. The development of reproductive structures obviously necessitates changes in morphology and development, but the nature of the physiological and biochemical changes involved is not immediately apparent. Detailed comparisons of the mycelia and conidia of the Chrysonilia anamorph of Neurospora crassa show that some substances such as trehalose, glutamic acid, glutathione, carotenoids, and phospholipid, which are present at low levels in mycelium, are found at much higher levels in conidia. Others, such as arginine, ornithine, and adenine nucleotides, are more plentiful in mycelium.

The Physiology of Sex Reproduction in fungi frequently involves sex, although their sexual behaviour is sometimes obscure, and one mode of sexuality evolved by fungi is unique and extremely prolonged. Diffusible chemical substances that trigger sexual activity are found in many organisms. A differentiation has usually been made between hormones, which act on the organism that produces them, and pheromones, which act on other sexually compatible organisms. This differentiation is harder to make in the fungi. Closely related taxa may be hom*othallic and heterothallic, respectively, so a shared sexually active substance could be referred to as a hormone in the first case and as a pheromone in the second. In the fungi it is simpler to call them all hormones. The blastocladiomycete Allomyces has a water-diffusible sex hormone called sirenin. This is released into the water by the female gametes, and the smaller, more motile male gametes swim toward them by detecting the concentration gradient. In vitro experiments with Allomyces showed that response decreased at hormone concentrations above 10–6M. Presumably at this concentration the receptor sites on the male gametes were saturated, and they could no longer find their way up the concentration gradient. Male gametes normally maintain their sensitivity by breaking down the sirenin they intercept. The oomycete Achlya ambisexualis produces sex hormones in a ping-pong sequence, to coordinate the development of the male and female sex organs. A potentially female mycelium secretes hormone A, which causes any nearby potentially male mycelium to develop antheridial branches. The male strain then releases hormone B, which triggers the development of oogonia on the female mycelium. The developing oogonia then release hormone C, which attracts the antheridial initials. These initials produce hormone D, which causes the oogonial initials to mature. The antheridia mature when they touch the oogonia, but hormone E might also be hypothesized. Two of these hormones have been isolated and characterized. Hormone A is called antheridiol, and

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Chapter 9 hormone B, oogoniol. Pure antheridiol will also induce chemotropism and maturation of antheridia, so it may also represent hormones C and E. The zygomycetes provide classic laboratory demonstrations of fungal sexuality. We plant a ‘+’ strain of Phycomyces or Mucor on one side of an agar plate and the corresponding ‘–’ strain on the other. When the two meet, gametangia are formed, then zygosporangia. Because these events seem to happen when the mycelia touch, we don’t necessarily think of diffusible hormones. Yet there is chemistry here, too. Sixty years ago, it was demonstrated that compatible strains of Mucor would form gametangia, even when separated by a semipermeable membrane. Much later, it was found that both mycelia produce a sex hormone called trisporic acid when grown close together. This induces the formation of gametangia. The final rendezvous of the gametangia is guided by volatile, mating-type specific substances which, although demonstrably present, have not yet been characterized. The conjugation of yeast cells is governed by diffusible hormones and by agglutination factors that are bound to the cell walls. Each mating type of Saccharomyces cerevisiae has its own hormone. One consists of oligopeptides of twelve and thirteen amino acids. The other has a molecular weight of about 600,000 and contains protein and polysaccharide. Although they are so different, these substances have similar effects on the appropriate mating type: they inhibit the initiation of DNA synthesis, effectively locking the cell into interphase. Budding stops, and cells of opposite mating type become mutually adhesive. Since isolated protoplasts won’t stick together unless they manage to regenerate walls, the agglutination factor must be wall bound. Cells of opposite mating type have distinct but complementary peptidopolysaccharide agglutination factors. Conjugation follows agglutination. Sometimes the zygote multiplies to form a generation of diploid cells, and sometimes it develops into an ascus-like meiosporangium. Among the ascomycetes proper, sex hormones have been partially purified for Neurospora (Sordariales), and there is evidence for the existence of comparable hormones in Ascobolus (Pezizales) and Bombardia (Sordariales). The well-known mycotoxin zearalenone, produced by the hyphomycete Fusarium graminearum, apparently stimulates the development of perithecial ascomata of its teleomorph, Gibberella zeae (Hypocreales). Among the basidiomycetes, it has been shown that opposite mating types of Tremella (Basidiomycota, Tremellomycetes) have individual, constitutive sex hormones. One of them has been partially characterized: tremerogen, as it is called, is a twelveamino acid lipopeptide with an isoprenoid conjugated to the sulphur of the cysteine at one end. When the yeast-like basidiospores are exposed to this, they stop budding and produce a conjugation tube. The red yeast Rhodotorula has similar hormones. One of these, named rhodotorucine, inhibits budding and induces formation of conjugation tubes in the opposite mating type. The resultant teleomorph is Rhodosporidium (Ustilaginomycetes). The situation in many basidiomycetes is complicated by the fact that although the first prerequisite for sexual reproduction—the bringing together of compatible nuclei— happens at the moment of dikaryotization, the ultimate sexual fusion of nuclei may be long delayed and happens only to distant descendants of the original nuclear pair. Although sex hormones may facilitate the meeting of monokaryotic mycelia, other

Fungal Physiology and Metabolism factors, nutritional and environmental, probably determine the timing of nuclear fusion and meiosis. Although very few fungi have been investigated for the presence of sex hormones, it seems likely that their secretion is the norm rather than the exception. If ascomycete and basidiomycete sex hormones are shown to have some uniformity of structure and action, it would be fascinating to apply them to the vast number of dikaryan anamorphs for which no teleomorph is known, to see if sexual development could be initiated, and many longstanding mysteries solved (although molecular techniques can now give us answers).

Antifungal Compounds The chemical industry synthesizes thousands of new compounds every year. Many are routinely screened for various possible uses. Two questions commonly asked are (1) Are they antibiotic? (2) Are they fungicidal? So, by empirical testing, new fungicides are found. Until recently it was only after a compound had been discovered to be fungicidal that its mode of action was investigated, although enough is now known about fungicidal action that new fungicides can be designed at the molecular level, with appropriate prosthetic groups. Here are some groups of fungicides and their sites of action. (1) Copper, mercury, dithiocarbamates, phthalimides, and quinones tend to be nonspecific enzyme poisons that bind to functional groups that normally maintain the secondary structure of proteins. (2) The antifungal polyene antibiotics, nystatin, and amphotericin B form complexes with sterols and thus disrupt membrane formation. Oomycetes and bacteria, which have no sterols in their membranes, are unaffected. (3) The sterol inhibitors, such as bitertanol, triadimefon, and triforine, prevent the biosynthesis of ergosterol, the major sterol in many fungi, and so presumably interfere with membrane synthesis. (4) Polyoxins interfere with chitin synthesis in vitro by competing with chitin synthetase for its monomer substrate but have a disappointingly limited range of activity in vivo. (5) Cycloheximide is a pyrimidine analogue and blocks protein synthesis by binding to ribosomes. (6) Benzimidazoles (e.g., Benomyl) bind to the tubulin that normally forms the mitotic spindle and so disrupt nuclear division. Once again, oomycetes are not sensitive to Benomyl, although their division is inhibited in a similar way by colchicine. (7) Carboxins interfere with the metabolism of mitochondria in many dikaryan fungi, causing succinate accumulation. As we learn more about the physiology and biochemistry of fungi, we should be able to design molecules that will interfere in aspects of metabolism that are specific to fungi, leaving nontarget organisms unaffected. We will also find new uses for many fungal metabolites.

Further Reading Aronson, J. M. 1981. “Cell Wall Chemistry, Ultrastructure and Metabolism.” In Biology of Conidial Fungi, edited by G. T. Cole and B. Kendrick, 459–507. Vol. 2. New York: Academic Press.

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Chapter 9 Bartnicki-Garcia, S. 1966. “Cell Wall Chemistry, Morphogenesis, and Taxonomy of Fungi.” Annual Review of Microbiology 22:87–108. Berry, D. R. 1975. “The Environmental Control of the Physiology of Filamentous Fungi.” In The Filamentous Fungi, edited by J. E. Smith and D. R. Berry, 16–32. Vol. 1. London: Arnold. Booth, C. 1971. Methods in Microbiology. Vol. 4: 1–795. The Netherlands: Elsevier. Burnett, J. H. 1976. Fundamentals of Mycology. London: Arnold. Carlile, M. J. 1970. “The Photoresponses of Fungi.” In Photobiology of Microorganisms, edited by P. Halldal, 309–44. New York: Wiley. Griffin, D. H. 1996. Fungal Physiology. 2nd ed. New York: Wiley. Hall, R. 1981. “Physiology of Conidial Fungi.” In Biology of Conidial Fungi, edited by G. T. Cole and B. Kendrick, 417–57. Vol. 2. New York: Academic Press. Hawker, L. E. 1957. The Physiology of Reproduction in Fungi. Cambridge, UK: Cambridge University Press. Jennings, D. H. 2007. The Physiology of Fungal Nutrition. Cambridge, UK: Cambridge University Press. Lowe, D. A., and R. P. Elander. 1983. “Contribution of Mycology to the Antibiotic Industry.” Mycologia 75:361–73. Mueller, E. 1971. “Imperfect-Perfect Connections in Ascomycetes.” In Taxonomy of Fungi Imperfecti, edited by B. Kendrick, 184–201. Toronto: University of Toronto Press. Robinson, P. M. 1978. Practical Fungal Physiology. New York: Wiley. Smith, J. E., and D. R. Berry, eds. 1975, 1976, 1978. The Filamentous Fungi. Vols. 1–3. London: Arnold. Smith, J. E., D. R. Berry, and B. Kristiansen, eds. 1983. The Filamentous Fungi. Vol. 4. London: Arnold. Stevens, R. B. 1974. Mycology Guidebook. Seattle, WA: University of Washington Press. Turian, G. 1966. “Morphogenesis in Ascomycetes.” In The Fungi, edited by G. C. Ainsworth and A. S. Sussman, 339–85. Vol. 2. New York: Academic Press. ———. 1969. Differenciation Fongique. Paris: Masson. Wheeler, M. N., and B. R. Johnston. 2014. Fungicides: Classification, Role in Disease Management and Toxicity Effects. Hauppauge: Nova Science Publishers.

10 Fungal Genetics—Mendelian and Molecular

Introduction Genetics is the discipline that seeks to understand the ways in which the information needed to reproduce an organism is stored within it and how that information may change and be presorted before it is passed on to the next generation. In recent years, we have also become concerned with how this information can be changed in a directed way by human intervention. This chapter attempts to show how fungi are useful tools in some areas of both Mendelian and molecular genetics. If your background in these areas is sparse, you will find some useful introductory information in chapters 1 and 9. If you still have trouble with what follows, I recommend that you consult an elementary genetics text before trying again. In the simplest terms, genetic information (called, in its entirety, the genome) is maintained in the cell as long, linear sequences of nucleotide base pairs (bps) which make up DNA molecules. The order in which these bases occur constitutes the genetic code, and triplets of the code specify the sequences of amino acids required to build all the proteins necessary for the construction and operation of the living organism. DNA molecules can be very long, incorporating millions of bps, and are called chromosomes. The genome of prokaryotes is contained in a single, usually circular chromosome found in the cytoplasm. The genome of eukaryotes is contained in two or more (often many more, even up to several hundred) chromosomes, which are contained in a nucleus, a special command module separated from the cytoplasm by two membranes. The eukaryotic plants and animals differ from each other in many ways, but both are basically diploid. This means that their nuclei contain two matched sets of chromosomes (usually one set originally derived from a male gamete and one set from a female gamete). So each chromosome has a ‘double’. Most genes on each chromosome have a counterpart, called an allele, on the ‘double’. This allele affects the same characteristics, although not necessarily in the same way. For example, one allele of a particular gene makes pea plants tall, while the other allele makes them dwarf. If a tall plant is crossed with a dwarf plant, there will be more tall offspring than dwarf offspring. Plants will be dwarf only if both alleles are of the dwarfing kind. This shows that one allele can mask another: we say that the ‘tall’ allele is dominant, the ‘dwarf ’ allele recessive. This makes genetic analysis difficult, and also makes it hard to breed pure lines of many diploid organisms, because it is almost impossible to eradicate recessive genes, 195

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Chapter 10 since you can’t tell whether they are present or not (although it is easy to pure breed for recessive colour genes, such as those expressed in white rats and mice). The vast majority of true fungi are somatically haploid, which means that their nuclei contain only a single set of chromosomes. This gives them certain advantages over diploid organisms for genetic studies, since there are no competing alleles, and every gene is potentially capable of being expressed in the phenotype (the physical manifestation or incarnation of the organism). This absence of masking makes genetic analysis much easier. The advantages of using fungi in genetic studies are as follows: (1) The mycelia of almost all fungi are populated with haploid nuclei (but oomycetes, being members of kingdom Chromista rather than kingdom Eumycota, are somatically diploid), and many fungi form large numbers of uninucleate, haploid spores. These can be used to study naturally occurring or induced mutations. (2) The hyphae of compatible conspecific eumycotan fungi can fuse (anastomose) with one another locally during normal assimilative growth, exchanging nuclei and thereby producing heterokaryons (mycelia containing genetically different nuclei). The heterokaryotic condition confers great flexibility on many fungi, helping them to cope with different substrates and conditions. Heterokaryons can be investigated under controlled conditions by isolating spores or hyphal fragments and are used by geneticists in the complementation test (see Parasexuality below). The production of heterokaryons may also be an essential step toward a long-delayed sexual reproduction, as when basidiomycetes initiate dikaryotization by anastomosis between sexually compatible mycelia. (3) Hyphal fusions also lead to exchange of cytoplasm, producing heteroplasmons. These make it possible to study extranuclear genetic phenomena, and fungi are particularly valuable for the investigation of cytoplasmic inheritance. (4) The phenomenon of crossing-over, a vital part of the process of genetic recombination, can be most elegantly studied in ascomycetes like Neurospora or Sordaria. These fungi have very short life cycles and conveniently arrange the eight nuclei resulting from meiosis and the subsequent mitosis in a linear sequence within the ascus. One nucleus goes into each ascospore, and the ascospores are arranged in single file within the narrowly cylindrical ascus. The ascospores in this ‘ordered tetrad’ can be individually cultured and tested in various ways. Using appropriate marker genes, (a) first-division segregation can be distinguished from second-division segregation, (b) reciprocal and nonreciprocal chromosomal exchanges can be detected, (c) chromosomes can be mapped, and (d) interference can be studied. (All the terms just mentioned are discussed in more detail in Investigating Crossing-Over below.) (5) The phenomenon of somatic crossing-over was first seen in the fruit fly Drosophila, but it can be much more easily studied in fungi. Somatic nuclear fusions occur, with low but predictable frequency in fungal heterokaryons. The resulting diploid nuclei occasionally undergo mitotic crossover. Some of the somatic diploid nuclei which have undergone mitotic crossover can revert to the haploid condition through irregular forms of mitosis. These haploid nuclei have thus undergone genetic recombination without benefit of sex. The process is called parasexuality. Thanks to their production of large numbers of uninucleate spores expressing specific genetic markers (e.g., colour, or nutritional deficiencies), conidial fungi such as Aspergillus nidulans are especially well suited for investigations of this phenomenon.

Fungal Genetics—Mendelian and Molecular (6) Fungi can be handled rather like bacteria—many pure cultures can be stored in a small space, and the generation time is short—yet fungi are eukaryotic, so results are much more applicable to the other major kingdoms, both animals and plants. Fungal genetics is not without its difficulties. Fungal nuclei are often very small, and we cannot do the kind of analysis of chromosomal arrangement at the metaphase stage of nuclear division that is possible in many plants and animals. The drawings in Fig. 10.1 follow a fungal nucleus through a normal mitosis, which takes about five minutes. It is immediately apparent that fungal division is not like that in other organisms. The spindle develops inside the nuclear envelope. There is no metaphase plate. The chromosomes are very small and not very clearly visualized. Their disjunction is not synchronous. Most of the division happens inside an intact nuclear envelope, which eventually elongates, constricts, and finally gives rise to two daughter nuclei.

Fig. 10.1

Nuclear division in fungi. Courtesy of Dr. J. Aist.

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Chapter 10 Because fungal chromosomes are so tiny, it is not possible to count them as easily as in many other organisms (pulsed gel electrophoresis takes seven to ten days and produces fuzzy bands on the gel), so we have to rely on features discussed below, such as spore colour and assimilative abilities, to investigate genetic traits. The small size of conidia, ascospores, and basidiospores makes them difficult to handle individually, and the necessity for sterile technique to avoid contamination doesn’t make things any easier. But with practice, all these handling difficulties can be overcome. Fungi have been widely used to study recombination and gene action, but they have been little exploited in studies of population genetics. This may be partly because it is often hard to decide what a fungal individual is: it has such a diffuse ‘body’, and through anastomosis the mycelia of eumycotan fungi often become heterokaryotic, containing nuclear material from several different genomes. Nevertheless, the potential for such studies remains and is beginning to be exploited in studies of biological species complexes such as that represented by the classical concept ‘Armillaria mellea’.

Investigating Crossing-Over in a Fungus Using Marker Genes Crossing-over is a normal part of the major process called meiosis or reduction division. As meiosis begins, the diploid cell has two sets of chromosomes. Each chromosome has already replicated itself, and so is composed of two parallel strands or chromatids. Each chromosome comes to lie parallel to the same (hom*ologous) chromosome from the other set: in Fig. 10.2 the two ‘white’ chromatids represent one hom*ologous chromosome, and the two ‘black’ ones represent the other. If we assume that the ‘black’ chromatids carry a gene for dark-coloured ascospores and the ‘white’ chromatids carry a different allele of the same gene, one that will produce light-coloured ascospores, then Fig. 10.2A shows what happens in the absence of crossing-over, and Fig. 10.2B shows what transpires when a crossover occurs. In the simplest crossover, shown in Fig. 10.2B, a break occurs at the same place in one of the ‘white’ chromatids and one of the ‘black’ chromatids. The ends rejoin, but in a new arrangement: the part of the ‘black’ chromatid carrying the dark ascospore gene is now joined to part of a ‘white’ chromatid and vice versa. When the four chromatids separate, they will represent new combinations of genes. This happens in the real world with ascospore colour in the dung-inhabiting fungus Sordaria, as you can see in Fig. 10.3, when a dark-spored ‘wild-type’ strain is crossed with a pale-spored mutant. To complicate matters, as you can see in Fig. 10.4, more than one crossover can happen between two hom*ologous chromosomes. This strange but vitally important process of genetic recombination accounts for the unpredictable mixes of parental genes that occur in the offspring of sexual eukaryotes. Crossing-over ensures that sexually reproducing organisms vary in many ways and so remain physiologically flexible. Crossing-over is one of the main mechanisms involved in providing the pool of variability on which natural selection acts. If we have appropriate marker genes, like the ascospore colour gene just mentioned, we can use the incidence of crossing-over to find out roughly where these genes are in

Fungal Genetics—Mendelian and Molecular

Fig. 10.2 A: No crossing-over = first division segregation pattern. B: crossing-over between ascospore colour gene and centromere = second division segregation pattern (see text).

relation to the centromere (the point at which the chromatids are functionally joined, and the last thing to separate at mitosis). How can we do this? We begin by assuming that a chromosome is equally likely to break anywhere along its length. If this is true, then the farther away from the centromere a marker gene is, the more likely it is to be involved in a crossover. Also, if we have two linked marker genes, the farther apart they are on a chromosome, the more likely they are to be separated by a crossover. This kind of information allows us to make chromosome maps showing the relative (although not the absolute) locations of our marker genes. Our map-making rests on the assumption that we can keep track of the products of meiosis. In most organisms we simply cannot recover and analyze all the nuclei arising from one meiosis. But amazingly enough, we can do it in some ascomycetes, because their meiosis takes place in a long, narrow tube called an ascus. Fig. 10.3 shows how the products of the divisions lie in a straight line, so that their exact origin can be traced. The example I gave above involving light- and dark-coloured ascospores is in fact a real one. In Sordaria fimicola, ascospore colour is determined by a single gene. Wild-type ascospores are dark, but there is a mutant strain with pale spores. Since Sordaria fimicola is heterothallic (outbreeding), the mating of a normal dark-spored strain with a mutant pale-spored strain can be used to demonstrate some features of crossing-over. In this particular mating, if no crossover involving the ascospore colour gene has happened, there will be four dark ascospores at one end of the ascus, four light ones at the other end, as in Fig. 10.2A. But if the segment of chromosome bearing the colour gene has been crossed over, then each half of the ascus will contain a pair of light spores and a pair of dark ones, as shown in Fig. 10.2B. These pairs can appear in several different sequences, depending on which of the chromatids undergo crossing-over. Crossovers can take place between any two of the hom*ologous chromatids, so there are four possibilities for single crossovers: 1–3, 1–4, 2–3, 2–4.

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Chapter 10 In fact, crossing-over can be even more complex than I have just described, because it can happen twice between a particular pair of chromatids; or one chromatid can exchange segments with both of its hom*ologues. Some of these possibilities are shown in Fig. 10.4. Of course, we can’t watch these events, but we can explain the ascospore arrangements resulting from crosses between strains with two marker genes by diagrams such as those in Fig. 10.4. Not all genes express themselves so immediately and unequivocally as that determining ascospore colour, but the process of segregation works just the same for any gene. In order to analyze other kinds of markers which don’t express themselves visibly in the ascospore, we have to physically pick out the ascospores (this calls for great dexterity and lots of practice) and grow them individually in culture. The sequence of the spores inside the ascus is recorded, and this helps in the interpretation of the subsequent genetic analysis.

Fig. 10.3

Perithecial squash.

As we have already seen, if no crossing-over happens between a particular gene and the centromere, the four ascospores at one end of the ascus will all be of one genotype, and the four at the other end will all be of the other genotype. This arrangement is called the ‘first-division segregation pattern’ because the two versions of the gene separate at first-division meiosis (see Fig. 10.2A). But if crossing-over has happened between the gene and the centromere, the two different versions of the gene are not separated until the second division of meiosis. This arrangement is called a ‘seconddivision segregation pattern’, and there can be four such patterns, which occur with about equal frequency. Any particular gene will show a definite frequency of crossingover, which naturally increases as its distance from the centromere increases. The recombination frequency for any gene will equal half of its frequency of crossing-over. This is because only two of the four chromatids are involved in any particular crossover. If, in observing a squashed perithecium, we find that eight of twenty asci show

Fungal Genetics—Mendelian and Molecular evidence of crossing-over in the ascospore colour gene, we can say that the frequency of crossing-over for our marker gene is 40% and the recombination frequency is 20%. Those figures are also a useful way of placing the marker gene on a chromosome map. One map unit is arbitrarily defined as the distance between linked genes (genes on the same chromatid) that will give 1% recombination. The gene mentioned above is 20 map units from the centromere. If a second marker gene has a recombination frequency of 30%, this means that it is 10 map units farther from the centromere than the first marker. It could be only 10 map units from that first marker, but it could also be 50 map units away, on the other side of the centromere.

Fig. 10.4 Consequences of various double crossovers.

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Chapter 10 With patience and dexterity, a two-factor cross can be done with the ascomycete Neurospora crassa (Sordariales), using two linked marker genes (with alleles A and a, B and b). Three main ascospore patterns will emerge. (1) The parental ditype, AB AB AB AB ab ab ab ab: if there is no crossing-over between the two marker genes, the two tetrads of ascospores will reflect the characteristics of the respective parents. (2) The tetratype pattern, e.g., AB AB Ab Ab aB aB ab ab: when a single crossover happens somewhere between the two marker genes, four kinds of ascospore result, two parental types and two recombinants. (3) The nonparental ditype, Ab Ab Ab Ab aB aB aB aB: if two crossovers occur between the marker genes, all the products will be reciprocal recombinants, arranged in two tetrads. None of the products have the same combination of genes as either of the parents. The relative frequencies of these three patterns can be used to calculate the linkage distance between the two marker genes and to deduce their positions relative to each other and the centromere. It can also be used to discover which of the two markers is closer to the centromere and whether the markers are on the same or opposite sides of the centromere. For example, we analyze the ascospore arrangements resulting from a two-factor cross and find that there are fifty-six parental ditype asci, forty-four tetratype asci, and zero nonparental ditype asci. What can we deduce from these data? If the marker genes were unlinked (i.e., not on the same chromosome), the frequency of parental ditype and nonparental ditype asci would be expected to be the same. Since no nonparental ditype asci are recorded, we can assume that the two markers are linked (i.e., on the same chromosome). In order to be able to place the markers in their correct relationship to each other and the centromere, we need to analyze the forty-four tetratype asci further. We note that there are three arrangements: (i) Twenty-four are AB AB Ab Ab aB aB ab ab (ii) Nineteen are AB AB ab ab AB AB ab ab (iii) One is AB AB aB aB Ab Ab ab ab The marker genes could theoretically be arranged in one of three ways with respect to the centromere: (/) Centromere—Aa—Bb (//) Centromere—Bb—Aa (///) Aa—Centromere—Bb Ascospore pattern (i) above is a result of first-division segregation of the Aa marker, and second-division segregation of the Bb marker (the B and b alleles have been exchanged, whereas the A and a alleles haven’t). The crossover that produced this arrangement must have happened between the Bb gene and the centromere, but not between the Aa gene and the centromere. If the two markers are on the same side of the centromere, Aa must be closer to the centromere than is Bb (gene arrangement /). But ascospore pattern (i) could also be explained by gene arrangement (///), in which the marker genes are on opposite sides of the centromere. So far, only gene arrangement (//) can be ruled out. However, if we now look at ascospore pattern (ii), it is clear that both Aa and Bb segregated at second division. If we assume there was only a single crossover, this means that it must have taken place nearer to the centromere than either Aa or Bb, and

Fungal Genetics—Mendelian and Molecular between the centromere and both markers. So both marker genes must be on the same side of the centromere, and gene arrangement (///) can be excluded. So, by a process of elimination, we have shown that only one of the three possible gene arrangements, (i) Centromere—Aa—Bb, fits all the observed facts. Even the ‘oddball’ ascospore pattern (iii) can be explained by a two-chromatid double crossover, between Aa and the centromere, and between Aa and Bb (you can easily work this out on paper; it is not one of the examples shown in Fig. 10.4, but it can be visualized if the uppermost example is revamped with the crossover nearest the centromere happening between Aa and the centromere, rather than between Aa and Bb). We can now calculate the ‘map distances’ of the marker genes from each other and from the centromere. Of the 100 asci examined, 25 (patterns [i] + [iii]) had a crossover between Aa and Bb, while 20 (patterns [ii] + [iii]) had a crossover between the centromere and Aa. Applying the appropriate formula (half the number of recombinants, divided by the total asci observed, multiplied by 100), we find that the distance between Aa and Bb is 12.5 map units, and the distance between the centromere and Aa is 10 map units. Interference occurs when crossing-over at one point reduces the chance of another crossover in nearby regions of the chromosome. This phenomenon is detected by studying crossovers of three or more linked genes. Since the centromere itself acts as a marker, we have essentially a three-gene system in tetrad analysis, which is therefore a good way of studying interference. The events discussed above involved truly reciprocal crossovers, in which exactly equivalent segments of chromatids are exchanged. But sometimes the exchange is not exactly equal. This is called nonreciprocal recombination, or gene conversion, and if very closely linked marker genes are studied, it is found that crossovers are actually more often nonreciprocal than reciprocal. This phenomenon is explained by the breakdown or excision of short lengths of DNA during recombination, and their replacement by replication from another chromatid. Once again, fungi like Neurospora have been very useful in elucidating gene conversion. Mutant genes can act as markers enabling us to investigate the genetics of fungi. The kinds of mutant genes available affect such features as morphology, colour, mating type, and nutritional requirements. In some morphological mutants, the growth rate or branching pattern of hyphae is altered, with various effects on colony morphology. Neurospora crassa has ‘button’ and ‘ropy’ mutants. Other morphological mutations affect reproductive structures: Aspergillus nidulans has stunted conidiophore and ‘bristle’, in which the conidiophore has no conidium-producing apparatus at its apex. Colour mutants usually affect spore colour: Aspergillus niger, normally very dark brown to black, has white, fawn, and olive mutants. Biochemical mutants are perhaps the most useful markers. Biochemical mutants usually require some nutrient that is not needed by the wild type. Such mutants are called auxotrophs. A minimal medium is concocted for the wild type (for Neurospora crassa, this contains only inorganic salts, including a nitrogen source, sucrose, biotin, and agar). Samples of the fungus are exposed to a mutagenic agent such as UV light, then plated out on the minimal medium and also on a complete medium, which contains malt extract and yeast extract in addition to the ingredients listed for the minimal medium. If a strain is found that will grow on complete medium but not on

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Chapter 10 minimal medium, some biochemical deficiency is suspected. Now a little detective work is called for. This strain must be systematically tested to see what it needs. This can be done by attempting to grow it on minimal medium with additions of mixed vitamins, or mixed amino acids, or nucleic acids. If the minimal medium plus mixed vitamins keep it alive, then it is grown on minimal medium supplemented with individual vitamins. In this way the specific requirement of the auxotroph can be pinpointed. Fermentation mutants arise spontaneously in yeasts, resulting in inability to ferment a particular sugar. Resistance mutants also arise spontaneously in wild populations, but their frequency of occurrence increases if the organisms are exposed to antibiotics, antimetabolites, or other deleterious influences: such mutants are actively sought in the laboratory. The fungus is grown in a concentration of the deleterious substance high enough to inhibit normal growth: resistant mutants are the only ones to survive. Suppressor mutants overcome or compensate for any deficiency induced by an earlier mutation and cause an apparent reversion to the wild type. Physiological mutants apparently change the biochemistry of the fungus subtly, altering its reactions to some environmental influence, such as temperature or light. One mutant of the zygomycete Phycomyces blakesleeanus has normal morphology, but its sporangiophores no longer grow toward the light. Another group of biochemical mutants are those which produce greater than normal amounts of particular substances. Although this kind of mutant hasn’t been subjected to very much genetic analysis, it is sometimes economically important. The commercially exploited strains of Penicillium chrysogenum that produce such large amounts of penicillin are mutants of this kind.

One Sex, Two Sexes, Many Sexes Sexual reproduction will introduce more genetic variation to a population if the genomes which meet, and are then reassorted during meiosis, come from different individuals. That statement may sound strange and even superfluous to you, since you belong to a species in which such behaviour is not only natural but obligatory. But in many cases, an individual fungal mycelium can and does keep its sexuality to itself— its hyphae can produce sex organs of both kinds, which go through the processes of sexual fusion and produce a viable zygote. This condition is called hom*othallism. hom*othallic taxa are very useful if we simply want to demonstrate sexual behaviour in fungi, since we don’t have to worry about providing a suitable mate. The advantage of this system in nature is probably twofold: (1) to permit sexual reproduction when no appropriate compatible mycelium can be found (the lonely spore hypothesis) and (2) to perpetuate particularly successful genotypes, which would tend to be reassorted, and therefore diluted, by outbreeding. Many fungi, however, have evolved some form of reproductive differentiation of individual mycelia: we call this phenomenon heterothallism, and it enforces outbreeding. One approach is sexual dimorphism, that is, the production of two kinds of sexual structure which look and act differently and are often developed on different mycelia. In some fungi, both kinds of sex organ can be formed by a single mycelium, but only

Fungal Genetics—Mendelian and Molecular gametes originating from different mycelia can fuse. This implies genetic control of sexual reproduction through the development of mating types that incorporate incompatibility genes. These make sex impossible between strains of the same mating type. In many fungi, mycelia may be morphologically indistinguishable, yet invisible incompatibility factors can prevent their mating. Incompatibility can prevent anastomosis or prevent karyogamy. In fungi like the ascomycetes, where fusion of assimilative hyphae does not initiate the sexual process, vegetative incompatibility is not a barrier to sexual reproduction and is often determined by entirely separate genes, so that a single species may be divided up into a number of vegetative compatibility groups (VCGs). Such ascomycetous taxa as Cryphonectria parasitica (Diaporthales), Neurospora crassa (Sordariales), and Fusarium moniliforme (anamorphic Hypocreales) contain many VCGs. In the basidiomycetes, where fusion of ordinary, undifferentiated assimilative hyphae is a prerequisite to the establishment of the dikaryophase, and dikaryotization a prerequisite of karyogamy (sexual fusion of nuclei), vegetative incompatibility can effectively prevent sexual reproduction. Basically, heterothallism implies that a haploid nucleus can complete the life cycle only if it mates with another haploid nucleus carrying a different mating-type factor. Heterothallism is the fungal equivalent of the separate sexes found in many plants and animals. As we shall see, the fungi, despite their restricted genome and relatively consistent organization, have evolved many and complex variations on this sexual theme. The simplest kind of genetic system that can ensure outbreeding consists of two different alleles, which we can label ‘A’ and ‘a’, at the same locus. Pairs of mycelia carrying the same allele will be incompatible (A with A, or a with a), while pairs of mycelia with different alleles (A and a) will be compatible. This system effectively divides a population into two categories and has the same effect as division into two sexes. This twoallele system is found in all groups of fungi other than the most highly evolved basidiomycetes. Examples are the zygomycetous Rhizopus and Phycomyces; species of the ascomycetous genera Neurospora, Ascobolus, and Sclerotinia; and species of the pucciniomycete genera Puccinia and Ustilago.

Bipolar and Tetrapolar Mating Systems In many basidiomycetes, compatibility is determined by one or two genes, but each of these may have many different alleles. Only two or four alleles are present in any given dikaryon, at a single locus or at two loci. If all compatibility alleles occur interchangeably at one locus, the mating system of the fungus is called ‘bipolar’; if they are found at two loci, the mating system is called tetrapolar. If the alleles occur at a single locus, the offspring of a single basidioma will be of two different mating types. If the alleles are at two loci, offspring of a single basidioma will be of four mating types. Although the products of meiosis in the basidiomycetes are not an ordered tetrad, as they are in the cylindrical asci of some ascomycetes, it is still possible to culture the four basidiospores arising from an individual meiosis and use them in compatibility trials. In bipolar fungi (most smuts, some gasteromycetes, Coprinus comatus), the single locus at which all compatibility alleles occur can be called A. Now we can label the alleles in a given dikaryon A1 A2 (they must be different or the dikaryon won’t form in

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Chapter 10 the first place). Other dikaryons will probably have different alleles, which we can call A3 A4, A5A6, and so forth. Random matings in populations with such diverse matingtype alleles can be almost 100% successful. Remember that random matings in populations of two-allele organisms can be only 50% successful. Now we can see why a multiple-allele system may be more desirable than a two-allele system. In tetrapolar fungi (most polypores, Agaricales, and gasteromycetes), we can label the two loci A and B. For a dikaryon to be fertile, the alleles at each of the loci must be different—we can label them A1 B1 A2 B2. The haploid (monokaryotic) mycelia derived from this dikaryon will be of four kinds: A1 B1, A2 B2, A1 B2, and A2 B1. You can easily work out that only 25% of random matings among these siblings will be successful. Of course, matings of nonsibling monokaryons will again work much better: if we match up the four genotypes just listed with monokaryons derived from a dikaryon that is A3 B3 A4 B4, success should be complete. But what if the allele at one of the loci is the same as in our original strain, so that its alleles can be listed as A1 B3 A4 B4? What should be the percentage success of matings between this and the products of this and the original strain (A1 B1 A2 B2)? Work it out on paper. Your answer (which should be 50%) represents the number of fertile dikaryons that will result. But if you do this experiment, you will probably find that you finish up with many more dikaryons than you expected. This is because dikaryons can form between partially incompatible monokaryons, although such dikaryons will not be able to produce fruit bodies. In the example I just gave, there could be as many as 87.5% dikaryons (37.5% of them sterile) and only 12.5% total incompatibility. It has been found that the genes at the two loci often control different parts of the dikaryotization process. In the basidiomycetes Coprinopsis lagopus and Schizophyllum commune, clamp connections develop only if the dikaryon is heterozygous (has different alleles) for the A locus. For example, A1 B1 A2 B1 would have hyphae with clamps, A1 B1 A1 B2 would not. Nuclear migration is controlled by the B locus and would fail in A1 B1 A2 B1. Of 230 species of polypores, Agaricales, and gasteromycetes examined, 10%–15% were hom*othallic, about 35% were bipolar heterothallic, and about 55% tetrapolar heterothallic. It has been estimated that Schizophyllum commune probably has about 460 different A alleles, and 76 different B alleles. Estimates in some other basidiomycetes are of the order of 100 different alleles for each locus, although the bird’s-nest fungi, Cyathus striatus and Crucibulum vulgare, are believed to have only about 10 alleles for each locus. Secondary hom*othallism can occur in heterothallic fungi. If an ascus contains only four spores, as in Neurospora tetrasperma, instead of eight, there can be a compatible pair of nuclei in each spore. Similarly, if a basidium bears only two spores, as in Agaricus brunnescens, each of these may also contain two compatible nuclei. hom*othallism is possible, even in species with four-spored basidia. If an extra mitosis happens in the basidia, two compatible nuclei may find their way into some of the basidiospores. hom*othallism can also be introduced in what would otherwise be a heterothallic fungus by mating-type switching. In addition to the functional mating-type allele at the active locus, Saccharomyces cerevisiae has ‘silent’ copies of mating-type alleles at two other loci. A site-specific endonuclease cuts the double-stranded DNA at the active locus. The resulting gap is then repaired by splicing in DNA from one of the loci at

Fungal Genetics—Mendelian and Molecular which the silent copies reside. This often means that one allele is replaced by the other, so the mating type of the organism is switched. Similar switching occurs in another yeast, Schizosaccharomyces pombe, and in the filamentous ascomycetes Sclerotinia trifoliorum, Chromocrea spinulosa, and Glomerella cingulata, although the mechanism is still obscure in those fungi. The switching in Chromocrea and Sclerotinia happens in only one direction. If the mechanisms involved are like the mechanism found in Saccharomyces, it is likely that only one of the mating-type alleles is present in a silent form. We do not yet know how much fungal hom*othallism can be accounted for by mating-type switching. In some fungi, both self-sterile spores with a single nucleus and self-fertile spores with two nuclei are developed in the same fruit body. This kind of mating behaviour is called amphithallism. Recognizing the existence of compatibility genes is one thing, understanding how they work is another. The best-documented compatibility system is that of the yeast, Saccharomyces cerevisiae. Here there is a single locus with two alleles. Each mating type secretes a constitutive polypeptide pheromone which causes cells of the opposite mating type to become arrested in the G1 stage of the cell cycle. Such arrested cells agglutinate and undergo plasmogamy and karyogamy. If the resultant diploid cells are starved, they will undergo meiosis and produce haploid meiospores. Each stage of this process is apparently under the control of mating-type genes. These genes are regulated by DNA-binding proteins encoded by the mating-type alleles. One of the alleles contains a unique sequence of 747 bps and encodes two regulatory polypeptides. The other allele has a unique sequence of 642 bps and encodes two polypeptides, of which only one is known to be regulatory. The mating-type genes of other fungi are currently being isolated and characterized, and we should soon know how representative S. cerevisiae really is. It will be a tremendous challenge to explain how the hundreds of separate alleles we know to exist in some individual basidiomycete taxa differ and are regulated.

Intersterility Compatible mating types are not always enough to ensure successful sex. Sometimes, mating fails despite apparent compatibility. There is therefore another genetic system, which we can call an intersterility system, that can override the usual incompatibility system. Unfortunately, we don’t know nearly as much about the basis of this system as we do about incompatibility. The kinds of barriers involved are either prezygotic, preventing fertilization, or postzygotic, resulting in hybrids of reduced fertility or meiotic offspring less fit than the parents. Prezygotic barriers exist between closely related populations of many well-known basidiomycetes, including Armillaria, Collybia, Coprinus, Laccaria, Paxillus, Pleurotus, Ganoderma, and Heterobasidion. Since intersterility is usually complete, particularly in sympatric populations, the intersterile groups are equivalent to biological species. In some of these fungi, DNA reassociation or DNA restriction fragment patterns have shown that the intersterile groups are also genomically divergent. But sometimes two entirely intersterile sympatric populations are partly interfertile with a third population from another area. This happens in Cladosporium and is an example of syntopy,

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Chapter 10 a concept more widely used in animal studies. In Ustilago cynodontis, intersterile populations and partly interfertile ‘bridging’ strains co-exist within what appears to be a single complex species. Postzygotic barriers are present when mating occurs, but most of the resulting spores are not viable. In closely related heterothallic Neurospora species, the reproductive barriers appear to be mostly postzygotic. Intraspecific crosses yield viable ascospores, but interspecific crosses produce largely nonviable ascospores.

Parasexuality Ascomycetes and basidiomycetes can be easily distinguished when they reproduce sexually. In this phase (the teleomorph), they form characteristic fruiting bodies (ascomata and basidiomata) bearing unique meiosporangia (asci and basidia) from which, as we have seen, the products of a single meiotic event can be isolated and analyzed. Many of these fungi reproduce asexually as well, producing what are called anamorphs, which form mitospores called conidia, and often occur well separated in time and space from the teleomorph. In fact, we know thousands of anamorphs which have not yet been persuaded to metamorphose into a teleomorph. Many of these go on, generation after generation, in the asexual condition, and it now appears highly probable that many of them have entirely lost the ability to produce a teleomorph, thus becoming anamorphic holomorphs (although their DNA will reveal their affinity to either ascomycetes or basidiomycetes). We know that one of the most vital functions performed by the teleomorph is genetic recombination. This reassortment of the gene pool during meiosis broadens the ability of the population to cope with the stresses imposed by changing environments. Conidial fungi, which are often highly opportunistic and grow on a wide range of substrates, might seem to be especially in need of the flexibility conferred by genetic recombination. One of their responses to this perceived need for genetic diversification is to become heterokaryotic: to acquire more than one kind of nucleus as a result of one or more anastomoses. But we now know that they have also evolved a special mechanism for generating some genetic recombination without sex. We call this process parasexuality. The parasexual cycle has four stages: (1) fusion (anastomosis) of adjacent somatic hyphae, and exchange of nuclei, establishing a heterokaryon; (2) fusion of different nuclei in the vegetative hyphae, to form somatic diploids; (3) somatic recombination (mitotic crossing-over); and (4) nonmeiotic reduction of the altered nuclei via aneuploidy (loss of individual chromosomes) to the haploid condition. This sequence of events is rare, happening in fewer than one conidium in a million, but the number of conidia produced by most conidial anamorphs is astronomical, so parasexuality is a practical means for producing genetic variation. We don’t yet know how widespread this phenomenon is among the conidial fungi, but it has been detected in species of Aspergillus, Acremonium, Fusarium, and Verticillium, and is probably common. It is worthwhile to compare sexuality and parasexuality. (1) Sexual reproduction is a highly organized, often precisely timed process, which is genetically programmed. Parasexuality involves a rare sequence of uncommon events which seems to operate by

Fungal Genetics—Mendelian and Molecular chance, rather than by design. (2) In sexual reproduction, nuclear fusion is often mediated by genetic factors, expressed as ‘mating types’; happens in highly specific structures; and often involves many pairs of compatible nuclei. In the parasexual cycle, nuclear fusion is an isolated event, not mediated by mating-type factors, not found in specialized structures, and involving only individual nuclei. (3) During meiosis, crossingover probably takes place in every hom*ologous pair of chromosomes, and multiple crossovers are common. During somatic recombination, crossing-over commonly involves only one or a few chromosomes and never happens as often as during meiosis. (4) In meiosis, segregation happens in a highly organized way during two specialized nuclear divisions. Somatic haploidization probably occurs as a result of successive chromosome losses from an aneuploid nucleus (2n – 1) over several mitotic divisions until the stable haploid is reached. The factors that initiate sexual reproduction vary enormously from one fungus to the next, presumably because of their diverse ecological adaptations, so it is very difficult to make generalizations, although special media have been concocted to persuade such important genetic tools as Neurospora to undergo sexual reproduction on demand. The parasexual cycle can be encouraged in various ways. Camphor vapour selects for somatic diploids in some fungi. In species with uninucleate conidia, the best approach is to produce a heterokaryon between two auxotrophic mutants (each of which has a different biochemical deficiency), then grow its conidia on minimal medium. Neither of the original auxotrophs will be able to grow, but diploid conidia will grow, since the chromosomes from one parent compensate for the deficiency in the other set, and vice versa (this is called complementation, and the diploid is described as being prototrophic). This technique has been used in Verticillium albo-atrum, Aspergillus niger, and Aspergillus nidulans, and yields about one diploid conidium in 106–107 conidia. The subsequent frequency of mitotic recombination can be increased by X-rays, UV, mitomycin, and nitrous acid. Finally, to complete the cycle, low concentrations of p-fluorophenylalanine or Benomyl (Benlate) stimulate haploidization. We can see the potential advantages of the parasexual cycle to an asexual fungus, but is it of any use to the geneticist? As it happens, it can be used to determine linkage groups, the order of genes, and the position of the centromere. The genetic recombination achieved is on a much smaller scale than in meiosis: only one or two chromosomes are involved, and the possibility of multiple crossovers is so low that it can be ignored. This means that linkage analysis is much easier. The original diploids are heterozygous for the various marker genes. Those in which crossing-over subsequently occurs will become hom*ozygous for any marker genes that are distal to the point of crossover. The relative frequencies with which such markers become hom*ozygous is an indication of their relative distances from the centromere.

Extranuclear Inheritance Some genetic phenomena can’t be explained by reference to nuclear or chromosomal events. The logical corollary of this is that the determinants may be transmitted in cytoplasm rather than in nuclei. In some heterothallic fungi, the volume of cytoplasm that accompanies one of the nuclei during a sexual fusion may be much greater than

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Chapter 10 that associated with the other nucleus. Alternatively, if one side of the fusion involves a microconidium or a spermatium, this must inevitably bring much less cytoplasm to the union than does the receiving partner. This sometimes results in the offspring resembling the parent that contributed more cytoplasm and implies the existence of cytoplasmic genes. It has been shown that in Aspergillus glaucus, attributes such as spore germination, growth rate, pigmentation, and density of perithecia are under cytoplasmic control. A well-known example of cytoplasmic control is the ‘poky’ mutant of Neurospora crassa. This grows more slowly than the wild-type strain and cannot be speeded up by dietary supplements. If ‘poky’ is crossed with the wild type, the ‘poky’ condition is transmitted only when the ‘poky’ strain forms the perithecium initial, which means that it is essentially the maternal parent. Another well-known example of extranuclear inheritance is the ‘petite’ strain of Saccharomyces cerevisiae, which arises with a frequency of about 1 cell in 500. Such cells give rise to smaller than normal colonies, which can respire only anaerobically, even when oxygen is present. This deficiency is due to the absence of important respiratory enzymes such as cytochrome oxidase and succinic dehydrogenase. The mitochondria are defective. Whole colonies can be converted to ‘petite’ cells by growing them on a medium containing 3 parts per million of acriflavine. Diploid ‘petite’ cells don’t reproduce sexually, but diploid hybrids derived from ‘petite’ and normal haploid cells respire aerobically and can form ascus-like meiosporangia. If the meiospores are cultured, all are normal, and ‘petite’ cells arise among their offspring only in the ordinary 1:500 ratio. Normal yeast cells contain cytoplasmic genes (in mitochondria) controlling the synthesis of respiratory enzymes. ‘Petites’ arise by mutations in the mitochondrial DNA. When a cell containing this cytoplasmic mutant fuses with a normal haploid cell, the extranuclear genes from the normal cell render the resulting diploid normal again, and normal mitochondria find their way into any resultant haploid cells, which will all therefore be normal.

Genetics and Plant Pathology Plant breeders try to produce not only higher-yielding varieties of crop and garden plants but also new disease-resistant strains. This is done by finding natural defence mechanisms that are present in wild relatives of the economically important host plant. Painstakingly, the plant breeders introduce the resistance genes to the crop plants. Although such new cultivars may be immune to a particular fungal disease for a few years, eventually a new race of the fungal pathogen appears which can overcome the resistance of the plant. Analysis of this endlessly repetitive cycle of resistance and susceptibility led to the theory of the gene-for-gene relationship between host and pathogen. This theory suggests that the evolutionary paths of host and pathogen have been so closely linked for so long that for every gene in the host that can mutate to give resistance, there is a corresponding gene in the pathogen which can mutate to overcome that resistance. Cladosporium (Fulvia) fulvum, a hyphomycete, causes leaf mould of tomato. Three genes that confer resistance to this fungus are known, and tomato varieties exist which carry none, one, two, or all three of these genes. With the aid of these host varieties,

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Fungal Genetics—Mendelian and Molecular eight races of Cladosporium fulvum can be discriminated. The most efficient way to differentiate these races is with three tomato varieties which have, respectively, resistance genes 1, 2, and 3, as can be seen from Table 10.1. If you examine the eight columns which give the responses of the three tomato varieties to the different fungal races, you will see that each column differs from all the others. This means that any of the eight races can be identified by testing it against only three tomato varieties.

Table 10.1. Interactions of races of Cladosporium fulvum with three tomato varieties. Cladosporium races

Tomato with resistance gene

1+2

1+3

2+3

1+2 +3

That is how prevalence and spread of many important plant pathogenic fungi are monitored. It is also the mechanism by which the existence of new physiologic races of pathogens are discovered. The more genes for resistance we recognize, the more pathogenic races can be distinguished. Almost 200 races of the flax rust fungus Melampsora lini have been identified by their reactions with eighteen host varieties. Puccinia graminis subsp. tritici, which causes wheat rust, has well over 200 races, and the number is growing steadily in response to the efforts of the plant breeders. The genetics of resistance have also been explored in Venturia inaequalis, the apple scab fungus, which is a bitunicate ascomycete. It was found that the genes controlling virulence exist in virulent and avirulent alleles, which segregate regularly in the ascus. Seven of these genes were discovered, and seven apple varieties were found that would enable their presence to be recognized. For example, the avirulent allele of gene 1 didn’t affect McIntosh apples (which might simply mean that McIntosh carried a corresponding gene for resistance to that allele). Yellow Transparent apple was resistant not only to avirulent 1, but also to the avirulent alleles of genes 3 and 4. Each of the seven apple varieties had a different resistance gene, or genes, which could be identified by exposing the host to various races of V. inaequalis. The natural testing ground for resistance of potatoes to the late blight pseudofungus Phytophthora infestans (Oomycota) is central Mexico, where both mating types of the pathogen are present and new physiologic races can arise more readily than elsewhere. Working in this environment, potato breeders have found it more useful to aim for ‘field resistance’, which is mediated by many genes with small individual effects, rather than concentrating on a few major resistance genes with all-or-none effects. The war goes on.

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Recombinant DNA and Gene Cloning in Fungi Since fungi have not been among the most important contributors to our knowledge of DNA and how it works, I will not burden you with the usual spiel on DNA, its functions, and its replication: you can get that from any first-year biology textbook. In addition, for an overview of recombinant DNA technology, you should refer to a recent text on gene cloning. However, although fungal DNA is essentially the same as that of animals and plants, it is present in relatively much smaller quantities: the fungal genome is only about six to ten times larger than that of the bacterium Escherichia coli, having about 2.6–4.3 × 104 kilobases (kb). Repetitive DNA makes up less than 20% of the nuclear genome. Most fungal DNA is found in normal eukaryotic chromosomes, but there is also a circular mitochondrial chromosome of about 30–200 kb, mitochondrial plasmids, and often some small, supernumerary chromosomes in the nucleus, which do not appear to be essential for survival. The techniques of molecular biology have not only given us a great deal of detailed information about the genetic material, and even the actual sequence of bps which make up parts of the genomic DNA, but also permit the movement of genetic material from one organism to another and the expression of certain genes from one organism in another. I will set the scene by outlining the processes involved in moving genes from one organism to another. Recombinant DNA technology usually involves the following steps: (1) Cells of a host (often the bacterium Escherichia coli) are broken, and their DNA extracted. (2) This DNA includes plasmids, small closed rings of extrachromosomal DNA, which may be used as vectors for the introduction of foreign DNA. The vectors are separated from the other DNA by ultracentrifugation. (3) Special enzymes called restriction endonucleases cleave the plasmid vectors and leave them as linear sequences of DNA with ‘sticky’ ends (unpaired bases). (4) DNA from the donor organism (the source of the desired gene) is isolated and then treated with the same restriction endonucleases, producing additional linear sequences with sticky ends that match those of the cleaved plasmids. (5) Vector DNA and donor DNA are mixed: sticky ends rejoin, by complementary base pairing, in various configurations—vector ends rejoin, vector joins donor, donor joins donor. In a few cases the desired joinings happen, producing a closed loop, which is part vector DNA, part donor DNA. (6) The sugar-phosphate backbone of the DNA is then properly repaired by an enzyme called a DNA ligase. (7) The modified vectors are mixed with E. coli made permeable by treatment with a calcium salt. This allows some of the bacteria to pick up modified vectors and so be transformed. (8) The transformed cells that bear the desired donor gene can now be isolated, with the help of selectable marker genes previously incorporated in the vector, and can subsequently be propagated on a large scale. Why are yeasts and filamentous fungi now being used in gene cloning, if bacteria are such suitable hosts? Fungi are valuable because of the following reasons: (1) Many of the donor genes we want to clone are eukaryotic. Bacteria aren’t ideal hosts for this job, because their mechanisms of gene transcription and translation are so different from those in eukaryotes. In fact, even if suitably modified plasmids are successfully introduced to Escherichia coli, relatively few eukaryotic genes will be expressed by this prokaryote. This is obviously not a problem in the fungi, which are all eukaryotes. The

Fungal Genetics—Mendelian and Molecular yeast Saccharomyces cerevisiae bypasses some of the roadblocks encountered when E. coli was the principal host available. S. cerevisiae, for example, can glycosylate proteins, fold them, or carry out other posttranslational modifications which must be made if some eukaryotic proteins are to become functional. (2) Saccharomyces cerevisiae and such filamentous fungi as Aspergillus nidulans are genetically well explored, and useful mutations are available in many of their biochemical pathways. (3) Yeasts can be grown and handled in very much the same way as the bacterium Escherichia coli: simplicity itself compared to the tissue culturing of animal cells. Two techniques are commonly used to transform yeast cells; one requires the removal of the cell walls, the other uses entire cells. The first technique is carried out as follows: (1) The yeast cells are treated with β-mercaptoethanol, a reducing agent, which facilitates subsequent digestion of the cell wall. (2) The wall is digested by mixtures of glucanases derived from snails or bacteria. (3) The resulting protoplasts (often called spheroplasts) are washed and suspended in a stabilizing solution (0.6M KCl or 0.8M sorbitol) to which is added the foreign DNA (vector incorporating the desired sequences). (4) Uptake of the plasmid DNA during protoplast fusion is promoted by adding polyethylene glycol. (5) The transformed protoplasts are then allowed to regenerate a wall and are grown on a selective medium (one containing a specific antibiotic, or with a particular food substrate, depending on which marker genes were used) which will allow only appropriately transformed cells to grow (because only they carry the appropriate marker gene, which came with the vector and confers resistance to that antibiotic or the ability to metabolize that particular substrate). Alternatively, the alkali salt method permits transformation of intact cells. Cells are incubated in lithium acetate to make them competent, that is, receptive to exogenous DNA. The DNA is then incorporated in the presence of polyethylene glycol 4000. Although transformation is less efficient than with protoplasts, the procedure is simple and quick, cells can be stored for weeks without loss of competence, and the problem of diploid formation during protoplast fusion is avoided. The first demonstration that yeast could be transformed with exogenous DNA was made in 1978, using a recombinant bacterial plasmid carrying the Saccharomyces cerevisiae gene for an enzyme needed in the synthesis of leucine (LEU 2). This gene had earlier been recognized in E. coli because it complemented a mutation in the bacterium that had caused the loss of the same enzyme. Several other yeast genes have now been cloned in E. coli by complementation of other bacterial mutants. These are useful markers which can be incorporated in the exogenous DNA along with the desired gene: their uptake and subsequent expression in yeast cells allow recognition and selection of yeast cells which have been appropriately transformed, that is, which now carry the desired donor gene. Most strains of Saccharomyces cerevisiae contain up to a hundred 2µm plasmids per cell. Each plasmid has about 6,300 bps. Hybrid plasmids made up of the entire 2µm sequence, plus the LEU 2 yeast gene, plus a bacterial vector sequence, efficiently transform yeast cells that lack the LEU 2 gene. (As a consequence of the bacterial vector sequence DNA having been replicated in a bacterium, the 2 µm plasmid also works in E. coli, so it can serve as a ‘shuttle vector’.) The complementation of the LEU 2 gene means that those cells which have been properly transformed can be selectively isolated on leucine-free medium and subsequently multiplied. It has also been

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Chapter 10 demonstrated that the hybrid plasmid replicates in transformed cells. However, it appears that smaller plasmids containing only a fragment of 2µm DNA are more versatile, giving higher frequencies of transformation and more copies of the plasmid in each transformed cell (up to 300). All stages of gene cloning can be carried out in yeast, but it is usually more efficient to amplify recombinant plasmids in E. coli. The most important aim of the cloning exercise may be to obtain gene products, but cloning also lets us produce a lot of hom*ogeneous DNA, which can then be used in the sequencing of genes.

Expression of Exogenous Genes in Yeast Yeast genes generally have the following components: (1) upstream promoter elements which include constitutive or regulated promoters (positive or negative); (2) 20–400 bp downstream, a TATA promoter element (so named because it incorporates the base sequence, thymine-adenine-thymine-adenine); (3) 30–90 bp downstream, a transcription initiating site which initiates production of mRNA; (4) protein-coding sequences; (5) transcription termination signals (see Fig. 10.5). Transcription of the inserted DNA depends on the presence of a promoter sequence that is recognized by the host RNA polymerase. Highly expressed yeast genes such as alcohol dehydrogenase 1 (ADH1) or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) usually have very high mRNA levels, so most methods of expressing exogenous genes in yeast have concentrated on the production of high mRNA levels. This involves using multiple copy plasmids to boost the number of gene sequences per cell, and fusing coding sequences to efficient yeast promoters to increase transcription. Yeast genes may contain both constitutive and regulated promoters, which serve to initiate transcription. Where both are present, the constitutive sequences are active at all times during cell growth and may produce a base level of gene expression which can be modified by other upstream sequences. Regulated promoters need to be activated before they will initiate transcription but will, in some cases, produce much higher levels of gene expression. Researchers initially tended to use constitutive promoters with high mRNA levels, especially those from the genes mentioned above (ADH1 and G3PDH), but found that while these gave high yields of hom*ologous proteins, they produced much lower amounts of heterologous (donor) proteins. If large amounts of the desired protein are deleterious to the host cell, it is better to use regulated promoters, which are not derepressed until required. Good examples are those involved in galactose metabolism, such as GAL1, GAL7, and GAL10, which are repressed by glucose and derepressed by the addition of galactose to the medium. If cultures are grown with glucose or glycerol as the carbon source, these promoters will remain repressed until almost all the glucose has been metabolized. If glucose is absent, and galactose is added to the medium, these promoters can be induced about 1,000-fold. Galactose induction is controlled by the GAL4 and GAL80 proteins. The GAL4 protein is a positive promoter that binds to specific DNA sequences upstream of the coding sequences of genes regulated by galactose. The GAL80 protein is a negative regulator which binds to the GAL4 protein, preventing it from activating transcription. If galactose is added,

Fungal Genetics—Mendelian and Molecular it binds to the GAL80 protein, stopping this from interfering with the GAL4 protein, which can then go about its business of promoting transcription. Hybrid promoters have been developed, combining strong constitutive promoters with upstream sequences of regulated genes. One of these has been used in the controlled expression of human interferon. Brewer’s yeast must be provided with sugars if it is to produce alcohol. If yeast could be transformed so that it possessed an amylase (a starch-degrading enzyme), production of ethanol would be simpler and cheaper. Various amylase genes from bacteria, yeasts, and filamentous fungi have now been cloned and expressed in Saccharomyces cerevisiae: commercial exploitation of these should soon be possible. The cellulose and hemicellulose in wood represent almost limitless potential substrates for the fermentation industry. Complete degradation of cellulose to glucose requires the activities of three successive enzymes: endoglucanase, exocellobiohydrolase, and β-glucosidase. Trichoderma reesei secretes all three cellulolytic enzymes, but normal cultures of the fungus aren’t used to produce these enzymes commercially because all three enzymes are inhibited by their end products. The strategy has been to isolate mutant, highly cellulolytic strains of Trichoderma reesei, then isolate the genes for the three enzymes and place them in vectors with control sequences appropriate for their expression in a suitable host. In 1987 the T. reesei gene for endoglucanase was cloned, characterized, and expressed in S. cerevisiae. Endoglucanase and exoglucanase from the bacterium Cellulomonas fimi have been cloned, expressed, and secreted in yeast in a regulated manner by attaching their coding sequences to the melibiase promoter and signal sequences from Saccharomyces carlsbergensis. Although yields are still too low for commercial exploitation, there is optimism that higher-yielding strains will be developed and that cellulolytic brewer’s yeast will be able to clarify beer and provide cheaper fuel alcohol. It appears that recipient yeast strains can take up and maintain exogenous DNA even without the mediation of vectors. The brewing industry has achieved this in two ways. Beer normally contains dextrins that are not degraded by brewing yeasts. These dextrins give a beer greater body and a higher caloric content. The light beers which are so popular today (for reasons that escape me) have had these dextrins removed by an exogenous enzyme, glucoamylase. This enzyme is produced naturally by some nonbrewing yeasts (e.g., Saccharomyces diastaticus). Brewers have therefore tried to get the ability to make this enzyme into their brewing strains, so that these could produce a light beer without assistance. One approach has been to incubate the protoplasts of the brewing yeast with partially purified high molecular weight DNA from the donor yeast. A second approach has been to fuse entire protoplasts of the two yeasts. Unfortunately for this second method, the S. diastaticus brought with it not only the glucoamylase but also 4-vinyl guaiacol, which ruined the flavour of the beer. Classical hybridization techniques were then used to segregate the glucoamylase gene from the 4-vinyl guaiacol gene. The flavour of Brazilian wines is often spoiled by an excess of 1-malic acid. Fusion of the wine yeast protoplasts with those of Schizosaccharomyces pombe, which metabolizes 1-malic acid, produced a hybrid that successfully reduced 1-malic acid levels in the wine. Protoplast fusion has some potential, because some characteristics important in baking, brewing, and distilling are polygenic (controlled by many different

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Chapter 10 genes) or are not well understood genetically. Such characteristics aren’t suitable for enhancement by gene cloning or transformation. In addition, protoplast fusion combines whole genomes, and it is known that increases in ploidy may increase productivity. Intergeneric fusions are often unstable, but if one ‘petite’ parent (which has nonfunctional mitochondria) is used, more stable hybrids are produced, probably because the hybrid contains functional mitochondria from only one parent.

Expression of Eukaryotic Genes in Filamentous Fungi Although for several years yeasts were the hosts favoured by gene cloners seeking to express heterologous eukaryotic genes, they cannot secrete enzymes in the quantities produced by bacteria. But mycelial conidial fungi such as Aspergillus niger can secrete enzymes more efficiently than either yeasts or bacteria and are therefore becoming the hosts of choice for expression and secretion of many enzymes, antibiotics, and even mammalian pharmaceutical proteins. Transformation in filamentous fungi was first reported for Neurospora crassa in 1979, and transformation systems have since been developed for many other filamentous fungi. Genes in filamentous fungi are composed of a promoter, a translation initiation region, DNA encoding a secretory signal peptide (where necessary), DNA encoding for the gene product, and DNA sequences for terminating transcription and for polyadenylation (Fig. 10.5). Translation initiation signals DNA encoding desired gene product

Translation control region (promoter) DNA encoding signal peptide for secretion Fig 10.5

Polyadenylation and transcription termination signals

Components of a fungal gene.

When a gene is to be moved to a new host, it is first assembled in a vector plasmid which can replicate in E. coli. A selectable marker may also be incorporated in the plasmid. As mentioned earlier, markers may be selectable because they compensate for some auxotrophic deficiency in the host, or (preferably, since in many cases we are not dealing with auxotrophic hosts) because they confer upon the host resistance to an antibiotic, or the ability to metabolize a particular food substrate. The hyphae of the host fungus are enzymically stripped of their walls, and the transformation of the resultant protoplasts proceeds much as it does in yeasts. Following transformation, the protoplasts convalesce on a regenerative medium which lets them reconstitute their

Fungal Genetics—Mendelian and Molecular hyphal walls. In fungal transformations, the vector DNA usually becomes integrated into the host genome. Bovine chymosin, a mammalian protease used in cheese making, has been successfully expressed and secreted in Aspergillus nidulans. The cDNA sequence encoding the chymosin was attached to transcriptional, translational, and secretory control elements of the glucoamylase gene from Aspergillus niger. All four units were incorporated into a suitable vector, which was used to transform Aspergillus nidulans. Active human interferon α2s and a bacterial endoglucanase have also been cloned in A. nidulans, this time using promoters from the A. niger glucoamylase gene and the A. nidulans alcohol dehydrogenase gene. Human tissue plasminogen activator (tPA, a protease used to dissolve blood clots) has been expressed and secreted in A. nidulans. One of the vectors constructed for this purpose is illustrated in Fig.10.6.

Fig 10.6

A plasmid vector.

Molecular Taxonomy and Population Genetics Fungal identification is often difficult, even with good, mature specimens of the usual reproductive structures in hand. If all we have are sterile mycelia, or fungus-inhabited substrates, identification has been virtually impossible. But now, with the advent of a variety of molecular techniques, such identification has become possible (see A Proposed DNA Barcode for Organisms, below).

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Chapter 10 We can detect the activity of specific enzymes. We can use probes to identify particular base sequences in the DNA. We can do immunoassays, using antibodies raised in a mammal against unique components of the organism. Or we can use sodium dodecylsulphate polyacrylamide gel electrophoresis (more succinctly known as SDS-PAGE). This technique separates the proteins in any mixture by their molecular weight. An electrical field is used to draw the protein molecules through a porous gel. Smaller molecules move more quickly and so travel farther in a given time. Eventually, the concentrations of the various protein molecules are made visible by staining with dyes or silver-based reagents, and the resulting spatial and intensity pattern of bands compared, often by computer, with those derived from known organisms. References describing these techniques are given at the end of the chapter. It has become possible to compare parts of the DNA or RNA sequences in the genome of different organisms with a view to establishing their degrees of biological relatedness. Rhizopogon is a ‘false’ or basidiotruffle, a hypogeous, mycorrhizal basidiomycete with a closed basidioma, a convoluted hymenium, and nonshooting basidia. For anatomical reasons, this fungus has been thought to be related (although nobody knew how closely) to ‘normal’ epigeous boletes, which produce basidiomata with a stipe, a cap, hymenium-lined tubes, and spore-shooting basidia. In recent molecular studies of these fungi, a number of fragments of Suillus mitochondrial DNA (mtDNA) were cloned and hybridized with mtDNA from other boletes. This showed that fifteen different regions of the mitochondrial genome of Rhizopogon subcaerulescens are virtually identical to those of fourteen species of the ‘normal’ bolete genus, Suillus. This is surprising because not only does the order of these fifteen regions differ among species of Suillus, but Rhizopogon and Suillus have traditionally been placed in different families or even different orders. Their molecular similarity, at least as far as this has been explored, is in striking contrast to their morphological divergence. This kind of revelation has happened in many areas of the fungi.

PCR Using the polymerase chain reaction (PCR) technique, researchers can now routinely replicate very small samples of DNA thousands of times and ultimately produce enough DNA to permit sequential analysis of its bps. Base sequences from the mitochondrial large subunit of the ribosomal RNA gene show that Rhizopogon and Suillus are very closely related and that both genera have diverged sharply from other boletes tested. In defence of classical taxonomy, I must point out that many mycologists have long believed that Rhizopogon is a secondarily reduced or sequestrate (nonspore-shooting) derivative of the genus Suillus. It is encouraging that this relationship has now been dramatically affirmed. This work also demonstrates either that major morphological changes may not be reflected by corresponding changes in the genome or that we have not been looking in the right places to find the genetic reflection of those differences. It also emphasizes that our concepts of fungal relationships must be based on as many kinds of information as possible (not just morphological and not just molecular).

Fungal Genetics—Mendelian and Molecular Armillaria ostoyae (sometimes called Armillaria solidipes) is known to be one of the largest living organisms. It is estimated that a single genet found in Malheur National Forest in Oregon is 2,400 years old, covering 3.4 square miles (8.4 km2). When hom*okaryons anastomose, nuclei migrate but mitochondria do not, so the resulting mycelium is uniform in its nuclear component but has at least two sectors with different mitochondrial genotypes. Therefore, an examination of mitochondrial DNA polymorphisms can now help us discover the history of those clones. The uses of molecular techniques in mycology are multiplying. I must begin this discussion by warning my readers that it is almost impossible for any publication to keep pace with the latest developments in this field. However, the attempt must be made! DNA sequencing, usually after amplification by PCR, is now being used to identify important fungi, such as commercially grown species or serious plant pathogens, or to find the appropriate taxonomic niche for fungi whose taxonomic position is problematic. A small region of the genome of several individuals in one species is compared to the DNA sequence of the equivalent region in other taxa. Currently the region being sequenced and compared most commonly is the rRNA (ribosomal RNA) operon. This operon consists of three genes under the control of a single promoter. The genes are (1) the small subunit gene (SSU), (2) the 5.8S gene, and (3) the large subunit gene (LSU). Fortunately, in most fungi these genes are arranged in that order, and so they can be located and compared. The first gene transcribed is the SSU. Then comes an internal transcribed spacer region (ITS 1), the 5.8S gene, and a second ITS region (ITS 2). At the other end is the LSU. The ITS 1—5.8S—ITS 2 is collectively generally called the ITS. The rRNA operon is part of a multigene family consisting of repeated arrays of operons. Different genes and regions in the rRNA operon have different degrees of sequence conservation (the likelihood that changes will enter the sequence of bps over time). The varying pressure for sequence conservation is due to the differing selection pressure on the different sequences: areas that are less important in the function of the genes tend to vary more than areas that are crucial. This is because any changes in those crucial areas might wreck the gene’s ability to make its product. The rather mysterious ITS regions, since they do not themselves code for a gene product, are far more mutable than the genes. The ITS regions have proved most suitable for comparisons between related species (see A Proposed DNA Barcode for Organisms, below). The 5.8S gene is small and highly conserved, and, because it is so short (about 150 bp), it is of limited value for phylogenetic comparison. The SSU is also conserved, but since it is larger than the 5.8S gene, more variation has crept in, and it has been used for comparisons among genera and higher taxa. The LSU is the least conserved of these three genes and has allowed comparisons within and between species. Nonribosomal genes are now routinely used to identify strains of economically important fungi such as Fusarium, Aspergillus, or Penicillium. Sequences of genes such as beta-tubulin, calmodulin, or actin, and myriad other genes are often used, but they are not standardized across different genera. Species concepts are often studied by comparing the results of analyses of different genera, a process usually described as genealogical concordance or multigene sequencing typing. DNA sequencing after PCR amplification has been used to identify species of the mushroom genus Armillaria (and

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Chapter 10 many other fungi). But these techniques are not restricted to identification of known species. They can also be used to connect or compare previously unidentifiable mycelial (nonsporulating) cultures with fungi that have been identified using their sporulating structures (whether sexual [teleomorphic] or asexual [anamorphic]). The DNA profiles of nonfruiting cultures have been compared with the profiles from DNA isolated from fruit bodies of Armillaria, and with this evidence in hand, new species of Armillaria have been described. The specificity of the PCR reaction also offers possibilities for identification. DNAbased primers can be identified which have a sequence that is unique—found only in a particular species. Thus, when PCR amplification using these primers works, we can say that the fungal isolate being examined belongs to a given species or group of fungi. Such techniques also lend themselves to fluorescent quantification. Commercial techniques using fluorescent quantification are now available for some fungi and are being used for a number of fungal and bacterial pathogens of humans. In the years ahead we can look forward to molecular help with some of our more difficult taxonomic problems, although I do not foresee a day when our taxonomic concepts are based entirely on DNA sequencing. It is important to stress that a fungus is much more than the sum of its bps! It looks as if there are few genes that actually ‘code’ for morphological features. Morphology (or shape, anyway) seems very much to be an emergent phenomenon, in other words, a consequence of the interaction of various developmental or regulatory genes in pathways or cascades. Higher expression of one gene leads to longer cells and ellipsoid rather than globose cells. How would you like to be told that two groups of organisms that can be distinguished only by their DNA are being described as different species? Looking at DNA sequence data derived from species we know to be distinct, we are getting an idea, at least in some groups, of how different fungi must be before they can be considered as distinct taxa. It might be suggested that if two groups have occupied the same kind of niche, this may have caused them to retain morphological similarities, but they may also have been genetically isolated from each other for so long that the base sequence of their DNA has changed. However, we have to remember that ultimately the whole purpose of taxonomy is to help us to identify organisms, and if it becomes too difficult to do this using morphology, can we justify using the more expensive techniques? The answer may be that we need ‘functional classification’ and phylogenetic classification. The Botanical Code of Nomenclature requires a diagnosis of new taxa which states how they differ from existing taxa. It is not unusual for such diagnoses to be largely focused on DNA sequences. Many mycologists think that molecular data are important and exciting but should be correlated with morphological comparisons and with comprehensive population and mating-type studies. Nowadays, new DNA sequencing techniques, originally developed for genomic studies are revolutionizing molecular ecology. So-called environmental metagenomics, using techniques such as 454 pyrosequencing, generate literally millions of DNA sequences (often ITS barcodes), allowing thousands of species of fungi to be detected and tentatively identified from complex substrates such as soil. Although the computer analyses involved in these experiments are complex, these powerful techniques are rapidly replacing traditional studies involving collection of fruiting bodies, or culturing and identification of cultures by morphology. The reliability of the results depends

Fungal Genetics—Mendelian and Molecular on the reliability of the reference databases used to identify the sequences, making the development of DNA barcode databases particularly critical.

Genome Projects Most people are aware that a large number of laboratories collaborated in the sequencing of the entire human genome. The entire genomes of many other organisms have also been sequenced: these include many viruses, bacteria, and a current project aimed for no fewer than 1,000 fungi. See: http://genome.jgi.doe.gov/programs/fungi/index. jsf. I have learned from those involved in the project that this target was reached in early 2017. The project has given rise to a new assessment of the evolution of the major fungal groups, shown in Fig. 10.7. Compare that with the tables in chapter 1, which date from 2007. The genome of Saccharomyces (the first fungus to be fully sequenced) was found to contain about 12 million bps with about 6,000 recognizable genes, divided among 16 chromosomes. Pucciniomycotina Ustilaginomycotina Agaricomycotina Pezizomycetes

Basidiomycota

Eurotiomycetes

Pezizomycotina Dikarya Ascomycota

Dothideomycetes Lecanoromycetes Leotiomycetes Sordariomycetes Xylonomycetes Saccharomycotina Taphrinomycotina Glomeromycota Mucoromycotina Zoopagomycotina Entomophthoromycotina

Fungi

Kickxellomycotina Blastocladiomycota Chytridiomycota Neocallimastigomycota Microsporidia Cryptomycota

Fig. 10.7 Evolutionary diagram of major fungal groups as of 2017.

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Proteomics It is apparent that the very large number of proteins required for the complex activities of a cell, and the extremely complex ways they are folded and configured in order to do what is needed, are not simply coded into the DNA as a series of sequences, since the functional DNA is not enough to do all this without further manipulations, and different proteins may be required at different times. In fact proteins are constructed by the carefully timed operations of small parts of the genome, controlled and coordinated by regulatory elements, turned on and off as required. So now it is not enough to know the base sequence of the DNA, but we also need to know much more about the control mechanisms and their transcription factors. Wikipedia puts it succinctly: ‘The proteome is the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses, that a cell or organism undergoes.’ The term ‘proteomics’ was coined in 1997 and is now a growing part of genetic investigations.

Epigenetics Just when geneticists were all set to concentrate on sequencing genomes and barcodes as the ‘standard model’ of genetics, it became apparent that, although the base sequences in the DNA were not changed in a new generation, the ways and extent to which the DNA instructions were carried out could indeed be altered by the introduction of elements that could turn genes on and off. This was a game-changing discovery! Now we need to be aware of not only sequences but also how they can be ‘methylated’, which slows down or limits their expression. Other external features can increase the expression of a gene. What surprised most people is that such epigenetic changes can be passed from one generation to the next. This is a rather recent discovery, and its implications are just beginning to be worked out. You will have to refer to your prof, or to the very recent literature, to find out the latest information in this area, since no textbook can keep up with everything that is going on.

High-Throughput Sequencing Analysis in Molecular Fungal Ecology High-throughput sequencing analysis methods allow us to make detailed analysis of fungal communities, using large sets of samples, and provide ecological information that is far beyond anything previously possible. As more and more named fungal sequences become available in repositories such as GenBank and UNITE (the latter purely fungal), more sequences recovered can be linked to known species. The procedures require careful attention to protocols, and it is vital that users be aware of potential problems. Several world leaders in fungal community molecular ecology have come together to produce a valuable and pragmatic ‘user’s guide’ (Lindahl et al. 2013).

Fungal Genetics—Mendelian and Molecular Researchers visited twenty-six pine forests across North America and collected a total of six hundred 10-centimetre-deep soil cores. The samples were quickly preserved, and the fungal DNA in them was extracted and isolated. The researchers then sequenced unique stretches of the DNA that allowed them to identify all of the fungal species present in each sample (Talbot et al. 2014). This revealed more than 10,000 species of fungi in the 600 samples, which the researchers then analyzed to determine biodiversity, distribution, and function by geographical location and soil depth. Interestingly, there was relatively little overlap in the fungal species from different regions—many East Coast fungi were not found on the West Coast or in the Midwest, and vice versa. But although ‘regional endemism’ was strong, similar suites of enzymes showed that they performed the same saprobic and symbiotic roles in all locations. This paper by Talbot et al. is a very fine example of the way in which molecular techniques can throw light on major ecological problems. It has excellent illustrations, and I strongly recommend that you read it.

A Proposed DNA Barcode for Organisms DNA barcoding is a taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to a particular species. This is not an attempt to identify unknown taxa but simply to compare a short, known segment of bases with equally short segments in other known organisms, which is believed to establish conspecificity. It is obviously much easier and quicker to use such small and precisely chosen sequences than to compare whole genomes or large segments of them. Currently, the standard genes used for each kingdom are as follows: (1) For animals and many other eukaryotes, the mitochondrial CO1 gene: a 648bp segment of mitochondrial cytochrome c oxidase 1 (CO1) is the standard barcode region. As of 2009, databases of CO1 sequences included at least 620,000 specimens from over 58,000 species of animals. (2) For land plants, it is the concatenation of the rbcL and matK chloroplast genes. (3) For fungi, cytochrome c oxidase 1 (CO1), although not yet widely tested, was found to be suitable in fungi, including many Penicillium spp. (Ascomycota). Because introns were present in some taxa, the barcode region potentially ranges from 642 bp in Hypocrea jecorina to 12.3 kb in Podospora anserina (both Ascomycota). In 2012, agreement was reached (see Schoch et al. [more than 150 collaborators]) on the barcode to be used for Fungi. An evaluation of several DNA regions—the mitochondrial cytochrome c oxidase subunit 1, three regions from the nuclear ribosomal RNA cistron, regions of three representative protein-coding genes (RPB1, RPB2, and MCM7), nuclear ribosomal small subunit (SSU), and nuclear ribosomal large subunit (LSU)—concluded that the ITS region was the most appropriate, having the highest probability of successful identification of the regions within the ribosomal cistron across the broadest range of fungi. This region most often provided a barcode gap between the levels of within-species and between-species sequence variation, so it was formally adopted, although supplementary barcodes may be needed for some taxonomic groups.

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Mycoviruses It is now understood that a considerable fraction of the human genome is made up of viral sequences, which have somehow become incorporated over geological time. The story of how they got there and what, if anything, they do, are still shrouded in mystery. There is a suggestion that during their evolutionary history, mammals have been assailed by many viral plagues, which may have started off by being highly lethal. As usual with such things, there were survivors, and as usual with viruses, they may have insinuated themselves in the genome and stayed there long after their initial deadliness had waned. Whether any of this applies to viruses that affect fungi I do not know, but we do now know that fungal viruses exist, something that was not known fifty years ago. Hypovirulence or reduced virulence associated with the presence of dsRNA has become a well-recognized phenomenon in some fungal pathogens. The first of these mycoviruses to be well characterized was the ‘hypovirus’, so named because it causes hypovirulence (reduced pathogenicity) in the chestnut blight fungus Cryphonectria parasitica. This virus apparently has no coat protein and can’t exist outside the fungal cell. It is transmitted from one strain of the fungus to the next during anastomosis (the fusion of somatic hyphae). Fungal mycelia are capable of anastomosis only when they belong to the same VCG. Diverse fungal populations with many different VCGs will tend to inhibit the spread of the virus. So, while mycoviruses are potentially potent biocontrol agents for fungal pathogens, they are likely to be more effective in pathogen populations with low genetic diversity. A growing number of mycoviruses have been found in fungi. Some, like the Cryphonectria parasitica hypovirus, are associated with symptoms such as reduced virulence of the pathogen (similar viruses have been found in other ascomycetes such as Ophiostoma ulmi and Diaporthe ambigua). Other mycoviruses are cryptic. The best known of the more cryptic mycoviruses are found in the well-studied yeast Saccharomyces cerevisiae. These viruses are associated with the killer yeast phenomenon, where one of the viral genomes codes only for a protein toxin. The other genomes code only for a coat protein and an RNA-dependent RNA polymerase. Neither of these last two genomes have any apparent negative effect on their host. Most mycoviruses do not appear to be closely related, and a number of them are based on DNA sequences rather similar to those of some plant viruses.

Bioinformatics One final note to end this complex chapter: the new subject of bioinformatics, which is basically the involvement of computers in accepting, digesting, collating, analyzing, coordinating, and ultimately allowing us to make sense of the huge amounts of information being made available by molecular techniques, has already entered the biological curriculum. Just remember that every organism has millions of bps and has to be compared, pair by pair, with innumerable others. So we must be grateful for fast computers and to those who write the software that makes these dizzying manipulations possible.

Fungal Genetics—Mendelian and Molecular

CRISPR-Cas9 CRISPR-Cas9 is a clumsy acronym for the 2015 discovery of what may be the most important development in genetics during the past century. It is defined in the Glossary, and is essentially a prokaryotic immune system evolved by bacteria to fend off viruses. Science has now learned to use it to edit genomes with incredible precision, allowing the removal of specific sequences (and also insert their replacement if desired). This enables us to modify genomes without inserting alien sequences, and has almost limitless possibilities for precise editing of genomes. In closing this chapter, I must express my gratitude to Erin Feldman of UBC for her careful analysis of the text, and her invaluable suggestions.

Further Reading Bruns, T. D., R. Fogel, T. J. White, and J. D. Palmer. 1989. “Accelerated Evolution of a False-Truffle from a Mushroom Ancestor.” Nature 339:140–42. Bruns, T. D., and J. D. Palmer. 1989. “Evolution of Mushroom Mitochondrial DNA: Suillus and Related Genera.” Journal of Molecular Evolution 28:349–62. Bruns, T. D., T. J. White, and J. W. Taylor. 1991. “Fungal Molecular Systematics.” Annual Review of Ecology and Systematics 22:525–64. Burnett, J. H. 1975. Mycogenetics. London: Wiley. Burnie, J. P., R. C. Matthews, I. Clark, and L. J. R. Milne. 1989. “Immunoblot Fingerprinting of Aspergillus fumigatus.” Journal of Immunological Methods 118:179–86. Glass, N. L., and G. Donaldson. 1995. “Development of Primer Sets Designed for Use with PCR to Amplify Conserved Genes from Filamentous Ascomycetes.” Applied and Environmental Microbiology 61:1323–30. Harrington, T. C., and D. M. Rizzo. 1999. “Defining Species in the Fungi.” In Structure and Dynamics of Fungal Populations, edited by J. J. Worrall, 43–71. Dordrecht: Kluwer Academic Press. Harrington, T. C., and B. D. Wingfield. 1995. “A PCR-Based Identification Method for Species of Armillaria.” Mycologia 87:280–88. Hibbett, D. S., Y. f*ckumasa-Nakai, A. Tsuneda, and M. J. Donoghue. 1995. “Phylogenetic Diversity in Shiitake Inferred from Nuclear Ribosomal DNA Sequences.” Mycologia 87:618–38. Jahnke, K.-D., G. Bahnweg, and J. J. Worrall. 1987. “Species Delimitation in the Armillaria mellea Complex by Analysis of Nuclear and Mitochondrial DNAs.” Transactions of the British Mycological Society 88:572–75. Lindahl, B. D., R. H. Nilsson, L. Tedersoo, K. Abarenkov, T. Carlsen, R. Kjøller, U. Kõljalg, et al. 2013. “Fungal Community Analysis by High-Throughput Sequencing of Amplified Markers— A User’s Guide.” New Phytologist 199:288–99. Martin, C. E., and S. Scheinbach. 1989. “Expression of Proteins Encoded by Foreign Genes in Saccharomyces cerevisiae.” Biotech Advances 7:155–85. Murat, C., and F. Martin. 2016. “Truffle Genomics: Investigating an Early Diverging Lineage of Pezizomycotina.” In True Truffle (Tuber spp.) in the World, edited by A. Zambonelli, M. Iotti, and C. Murat, 137–49. Switzerland: Springer.

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Chapter 10 O’Donnell, K., E. Cigelnik, and H. Nirenberg. 1998. “Molecular Systematics and Phylogeography of the Gibberella fujikuroi Species Complex.” Mycologia 90:465–93. Scheinbach, S. 1983. “Protoplast Fusion as a Means of Producing New Industrial Yeast Strains.” Biotechnology Advances 1:289–300. Schoch, C. L., K. A. Seifert, S. Huhndorf, V. Robert, J. L. Spouge, C. A. Levesque, W. Chen, et al. 2012. “Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi.” Proceedings of the National Academy of Sciences USA 109:6241–46. Seifert, K. A., R. A. Samson, J. R. deWaard, J. Houbraken, C. A. Lévesque, J.-M. Moncalvo, G. Louis-Seize, and P. D. N. Hebert. 2007. “Prospects for Fungus Identification Using CO1 DNA Barcodes, with Penicillium as a Test Case.” Proceedings of the National Academy of Sciences USA 104:3901–906. Talbot, J. M., T. D. Bruns, J. W. Taylor, D. P. Smith, S. Branco, S. I. Glassman, S. Erlandson, et al. 2014. “Endemism and Functional Convergence across the North American Soil Mycobiome.” Proceedings of the National Academy of Sciences USA 111:6341–46. Taylor, J. W. 1986. “Fungal Evolutionary Biology and the Mitochondrial DNA.” Experimental Mycology 10:259–69. Van Brunt, J. 1986. “Fungi: The Perfect Hosts?” Biotechnology 4:1057–62. Volk, T. J. 2002. “The Humongous Fungus—Ten Years Later.” Inoculum 53(2): 4–8. Volk, T. J., H. H. Burdsall, and M. T. Banik. 1996. “Armillaria nabsnona, a New Species from Western North America.” Mycologia 88:484–91.

11 Fungal Ecology Ecology is the study of organisms as they relate to each other and their environment. It must be apparent that even in the taxonomic chapters I gave a lot of ecological information. Think of the effects that fungi have had on people: the potato famine, the downy mildew of the French grapevines, the blue mould of Canadian tobacco, the way chestnut blight removed an important species from the forests of eastern North America, and the more recent loss of the beautiful American elm trees to Dutch elm disease. Fungi may alter the ecology of our gardens, as their depredations persuade some people to give up growing roses (because of the prevalence of black spot disease, powdery mildew, and rust) or phlox (because of its susceptibility to powdery mildew). The early drop inflicted on horse chestnut trees by Guignardia blight (at least in eastern North America) may persuade us to plant other shade trees. But in this chapter I want to explore some other areas of fungal ecology: some of the ways in which fungi influence the course of events in a variety of natural, as opposed to human-made, habitats. I will explore their roles in four natural habitats I and my undergraduate or graduate students have personally examined in some detail, and then I will give a few more general comments. (For images of the fungi involved in the dung succession, visit http://mycolog.com /chapter11a.htm.)

The Succession of Coprophilous Fungi The first habitat is dung. We may turn up our noses, but to some other organisms, dung is a considerable resource, which is constantly being produced in large quantities by billions of animals all over the world. You may think that because it has passed through an animal’s digestive tract, every bit of nutritional value will have been extracted from it. Not true! There may not be a lot of high-quality protein left, but there is a great deal of microbial biomass, as well as many food components, for example, cellulose, that neither the animal nor its gut flora managed to digest. There are also excretory products which, although they are of no further value to the animal, are high in nitrogen: herbivore dung may contain 4% nitrogen—more, in fact, than the plant material originally eaten by the animal. So, at frequent intervals throughout its life, every mammal evacuates from its gut a mass of first-class fungal substrate, simply asking to be exploited. Are there, then, fungi which specialize in exploiting dung? And if there are, how do they gain access to this substrate when it becomes available? The answers may surprise you. About 175 genera of ascomycetes are largely or exclusively found on dung. The 227

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Chapter 11 extremely advanced and successful agaric genus Coprinus and its close relatives have many species that occur exclusively on dung. There are also many specialized dunginhabiting zygomycetes, among which Pilobolus and some of the elaborate anamorphs in the order Kickxellales are perhaps the most spectacular. So there is indeed a numerous and specialized mycota of dung-inhabiting (coprophilous) fungi. But how do they compete successfully for this substrate? The answer here may be a little unexpected, but it is nevertheless perfectly logical. These fungi contrive to be first to exploit the dung by the simple expedient of being in it when it is deposited. And what is the only way to achieve that? To be eaten by the animal. Coprophilous fungi manage this trick in several ingenious ways. These processes must take into account some immutable logic. (1) The fungi are growing in the dung and will therefore have to fruit on it. (2) Animals do not, in general, eat their own dung (although rabbits do, raising interesting questions about the coprophilous fungi associated with them). (3) Therefore, the spores must be somehow distanced from the dung in such a way as to increase their likelihood of being eaten by herbivorous mammals. You have already read in earlier chapters about how several fungi of herbivore dung achieve this trick: How the zygomycete Pilobolus aims and shoots its sporangia up to 2 metres toward the light. How the ascus tips of the apothecial ascomycete Ascobolus protrude from the hymenium and bend toward the light before shooting their spores. How the necks of the perithecial ascomata of Podospora and Sordaria bend toward the light before their ascospores are expelled. Each of these independently evolved phototropic mechanisms is obviously evolved to direct the spores away from any other adjacent dung and to get the spores deposited on nearby vegetation that has a good chance of being eaten by the animal. Many other dung-inhabiting fungi are less specialized than those I have just mentioned or have specializations so subtle that we have not yet detected them. Nevertheless, the fact remains that with patient and repeated examination, we can find a large number of fungi representing most of the major fungal groups on the dung of many herbivorous mammals. Repeated observations have shown that the various fungi tend to sporulate in a reasonably definite sequence. First the Zygomycetes will appear: Pilobolus, then the dichotomously branched sporangiophores of Piptocephalis, which attacks other zygomycetes; the tall sporangiophores of Syncephalis with their swollen apices and linear merosporangia; the graceful multiple recurved sporangia of Circinella minor; Rhopalomyces elegans, which parasitizes nematode eggs; Cunninghamella, with its apical vesicle and unispored sporangia. (Pictures of these are easily accessible on http://mycolog.com/chapter11a.htm.) Then the Ascomycetes: apothecial fungi like Ascobolus, Saccobolus, Thecotheus, and the perithecial Podospora and Sordaria, accompanied by a variety of conidial anamorphs (Hyphomycetes) such as the blastic-sympodial Basifimbria, the nematodetrapping Arthrobotrys with its clustered didymosporous (two-celled) conidia and its various ways of snaring nematodes, including three-dimensional nets; the synnematal, slimy-spored (arthropod-dispersed) Graphium; the synnematal, dry-spored Cephalotrichum; and Trichurus, another synnematal hyphomycete with twisted, hair-like setae arising all over the fertile head. You can also find pictures of all these on http://myco log.com/chapter11a.htm.

Fungal Ecology And finally the Basidiomycetes, mainly small (but profuse) species of the agaric genus Coprinus and its allies, Coprinopsis and Coprinellus, with tiny caps, black spores, and autolysing gills. It has been suggested that this is a true ecological succession, albeit a miniature and condensed one. Initially it was postulated that the sequence was a nutritional one. Zygomycetes can generally assimilate only fairly accessible carbon sources, such as sugars. Their fast growth was assumed to give them an advantage in finding these, and their early disappearance was thought to be due to the exhaustion of this substrate. The ascomycetes and conidial anamorphs that appeared next were assumed to be able to assimilate more complex carbon sources such as hemicellulose and cellulose; while the basidiomycetes, appearing last and persisting longest, were able to exploit both cellulose and lignin. But when this hypothesis was scrutinized more carefully and tested by experiment and further observation, it did not hold up. The growth rates of the various fungi were found to be relatively similar, and the various carbon sources were not exhausted as quickly as had been assumed. So a second hypothesis was advanced. This one was based on the time it took for each kind of fungus to accumulate enough food reserves to permit it to fruit. It was argued that the simple sporangiophores of the zygomycetes could be developed after only a short period, while the more elaborate fruit bodies of the ascomycetes would require a longer buildup, and the even larger basidiomata of the coprini would need the longest preparation of all. This is a more reasonable hypothesis, because if we grow some of the dung fungi on laboratory media, we find that it takes Mucor hiemalis two to three days to sporulate, while Sordaria fimicola needs nine to ten days, and Coprinellus heptemerus seven to thirteen days. Some of the Kickxellales, advanced zygomycetes often found on the dung of sedentary mammals (such as rats, which have a defined home base, a small territory, and habitually used paths), produce extremely complex and convoluted anamorphs. Spirodactylon, possibly the most complex of all, produces tall, branched sporangiophores that bear tiny coils within which innumerable one-spored sporangia develop (Fig.3.5F). The whole structure must be designed to catch on the hairs of the rat or mouse as it passes by. This is made possible by the habits of the animal, which, although it doesn’t eat its own dung, at least deposits it somewhere along one of the trails it follows every day in its journeys to and from its den or burrow. The final step, the ingestion of the spores, is presumably taken when the animal grooms itself, as mammals (other than human children) habitually do. Some coprophilous hyphomycetes (e.g., Graphium) produce slimy droplets of conidia at the top of tall conidiophores or synnematal conidiomata. These spores are presumably dispersed by arthropods which may themselves specialize in seeking out dung and may thus act as specific, and very efficient, vectors for the slimy-spored fungi. So we can assume that an assortment of spores of coprophilous fungi will be present in dung when it is deposited and that these will all have been triggered to germinate during passage through the mammalian gut. While Pilobolus is producing its miniature artillery extravaganza, the other fungi are growing and assimilating steadily within the dung, preparing for their own appearance at the surface. The new hypothesis had neglected only one important factor: antagonism. After a few weeks, almost the only fungi still sporulating on the dung will be species of Coprinellus and

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Chapter 11 Coprinopsis. These can go on producing a sequence of ephemeral basidiomata for months. We now know that the various components of the substrate are far from exhausted after the initial flushes of growth and sporulation. What has really happened is that Coprinopsis or Coprinellus has seized control by suppressing most of the other fungi. Hyphae of these genera are actually extremely antagonistic to those of many other coprophilous fungi. If one of their hyphae touches one belonging to Ascobolus, the Ascobolus hypha collapses within minutes. We don’t understand exactly how this trick is done, but it is extremely effective and turns out to be a fairly common stratagem among the fungi, whose main competitors for many substrates are other fungi. One interesting gambit used by Coprinopsis involves repeated anastomoses. Spores are more or less evenly dispersed throughout the dung when it is deposited (Fig. 11.1A), and they all germinate more or less simultaneously, producing small mycelia within the dung (Fig. 11.1B, C). When compatible mycelia meet, they will anastomose, and soon the entire dung deposit is permeated by what is now essentially a single mycelium (Fig. 11.1D), which can then pool its resources and produce more and larger basidiomata. Cooperation pays off for Coprinopsis. And don’t forget the interesting subplots that run concurrently with the main story. Several of the zygomycetes that usually appear (e.g., Piptocephalis) are actually parasitic on other zygomycetes. One common zygomycete, Rhopalomyces elegans, parasitizes nematode eggs. Nematode-trapping fungi such as Arthrobotrys develop their characteristic ring and net traps (see chapter 15). Keratinolytic hyphomycetes such as Microsporum may appear on hair that the animal has accidentally eaten during grooming.

Fig. 11.1

Behaviour of Coprinopsis in dung.

Fungal Ecology Occasionally, an undescribed species of fungus may be seen. For many years my third-year mycology class at Waterloo followed the dung succession as a laboratory exercise. These undergraduates saw the zygomycete Stylopage anomala on horse dung several years before it was formally described in 1983. They also found an undescribed species of Podospora (Ascomycetes), which is perhaps the 102nd species of this genus. They also found the rare zygomycete Helicocephalum, which I had never seen before. (Who says your students can’t teach you anything?) Horse dung is easy to obtain in most areas, comes in discrete units, and can be handled and observed without creating much personal distress. As many as forty species of fungi representing most major groups of eumycotan fungi are commonly recorded from a single collection of horse dung. Most of them can be identified fairly easily to genus with the help of the specialized taxonomic literature that is now readily available (and by looking at the pictures at www.mycolog.com), although I admit that some of the zygomycetes are not easily recognized as such by beginners. Many of the fungi can be isolated in pure culture without too much difficulty, and with a little imagination, interesting experiments can be devised to investigate various aspects of their behaviour. Perhaps now you can understand why I and many other teaching mycologists ask our classes to put their culturally determined attitudes on hold, adopt an objective scientific approach, and study the succession of fungi on horse dung, then think about the biological mechanisms and manoeuvring that lie behind the visible manifestations. It’s a truly thought-provoking mycological experience. Now for another, smaller terrestrial system, the pine needle.

The Pine Needle Microsere When I began my own Ph.D. studies back in 1955 (Could it be that long ago?), I was presented with a problem which, briefly stated, was as follows: ‘When we isolate fungi from the soil, the majority of cultures will be of light-coloured fungi, while a majority of the hyphae seen in the soil are darkly pigmented. Figure out what’s going on.’ I tried innumerable times to grow the dark hyphae, picking them out with a micromanipulator and giving them a variety of delicious media. But they refused to grow, so I eventually decided that most or all of them must be dead and that they must have grown at some other time and in some other place. I looked in the organic horizon above the mineral soil and found there a thriving community of litter-decomposing fungi, which I proceeded to investigate (I did not realize it at the time, but this is fairly typical of Ph.D. projects, which are often changed in midcourse by unforeseen events). The pine needles making up the litter underwent a gradual transition as they gradually sank through several recognizable layers from L (litter) through F1 and F2 (fermentation) to H (humus) layers of the organic horizon of the soil. I decided to examine as many needles from each layer and sublayer as I could process each month (the number turned out to be 300). Living needles from the tree represented stage one in fungal colonization (some fungi grew on the surface, some inside the needle as endophytes). Recently dead needles constituted the L layer (pale brown), below which lay the upper F1 (needles much darker, but still tough), then the lower F1 (needles blackish and softer), and finally the F2 (needles greyish and fragmenting). By the time litter material

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Chapter 11 entered the H layer, it was no longer recognizable as entire individual needles. Needles were treated in various ways. (1) Some were washed repeatedly to remove loose surface spores and plated out in segments to isolate fungi growing on and in the needles. (2) Some were surface sterilized before plating out, to select for internal colonizers. (3) Some were wax embedded and sectioned to permit spatial analysis. (4) Some were observed directly as they grew and sporulated over a period of incubation in damp chambers.

Table 11.1. Provisional biomass estimates and annual litter production for Meathhop Wood soil (after Satchell 1970) Group

Dry matter biomass (kg/ha)

Bacteria Actinomycetes Fungi Protozoa Nematodes Earthworms Enchytraeidae Molluscs Acari Collembola Diptera Other arthropods

7.3 0.2 454.0 1.0 2.0 12.0 4.0 5.0 1.0 2.0 3.0 6.0

Total biomass

497.5

Annual litter production

7640.0

One of the first dramatic changes is the development of numerous ascomata of Lophodermium pinastri, which apparently often colonizes the interior of living needles without producing overt symptoms—in fact, it appears to be an endophyte involved in deterring herbivores from eating the living needles. The death and fall of the needle stimulates this fungus to fruit. The large number of lenticular black ascomata of Lophodermium that can occur in a single needle indicates that it is a dominant colonizer. Other fungi fruit in other needles—individual needles may thus travel along several distinct decompositional paths, although the average time it takes a needle to become humified is about nine years. One pathway begins with Lophodermium, another with pycnidial conidiomata of a coelomycetous anamorph, Fusicoccum (its teleomorph probably Botryosphaeria). The interior of the needle begins to break down under the attacks of the fungi.

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Fungal Ecology Meanwhile, on the surface of the needle, networks of dark hyphae develop. But what fungi do they represent? Mycelium without sporulating structures is not very helpful unless one has access to molecular techniques (which hadn’t been discovered when I was doing this work). Fortunately, several of these fungi fruited either in nature or in damp chambers. The first of these was Slimacomyces monospora (which I mistakenly described in 1958 as a new species of Helicoma). A second major surface colonizer was Sympodiella acicola, which I described as the type species of a new genus (it still stands). Again, the conidiophores often form an almost pure stand. Its unique characteristics are that while its conidiophore extends sympodially, the conidia are thallic-arthric (for those of you who are fans of conidium development—otherwise look back to chapter 4). Another fungus that produced pure stands of tall external conidiophores was Verticicladium trifidum (an anamorph connected to an apothecial teleomorphic fungus, Desmazierella acicola, which for some reason I never saw). Now I began to find needles divided up between neighbouring fungi. Sections of partitioned needles showed a black, melanized barrier between species. New participants enter the picture. Once the needles have been softened up by the fungi, arthropods can eat the needle material. Oribatid mites, miniature armoured tanks, eat both fungi and needles. In the lower F1, the interior of the needle has collapsed or been eaten, and the upper surface is coated with a deposit of arthropod frass, which contains many fragments of fungal hyphae and spores. Fig 11.2 plots the overall picture, following the needles through nine years of mainly fungal decay. The width of each bar represents the relative importance of the fungus at each stage. Darker bands show fruiting periods. At far left the fungi are those that grow on or in living needles. As we move to the right, the fungi involved in later stages of decay are traced. Most of these fungi are illustrated on the website. Coniosporium

Pullularia

Lophodermium

Desmazierella Fusicoccum

Sympodiella

Penicillium

Helicoma

Trichoderma

Fig. 11.2

Generalized scheme of the fungal succession.

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Chapter 11 Read the numbers in Table 11.1 carefully—they will amaze you, and they show just how important fungi really are in the forest ecosystem. Not merely important, but producing greater biomass than any group other than the plants. The huge numbers of fungi involved (both saprobic and symbiotic) have just been worked out, using highthroughput DNA sequencing, by Talbot et al. (2014). Other special substrates have evoked specialized fungi: keratin is attacked by some of the Onygenales and their anamorphs; wood by many Aphyllophorales. Extreme physical conditions have selected specialist fungi which, by evolving the ability to cope with high or low temperatures or low water activity, have essentially escaped from competition and gained access to untapped food supplies. Some fungi are the most osmotolerant organisms known (see chapter 20). The cycling of anamorph and teleomorph, which I mention many times in connection with plant disease fungi in chapters 4 and 12, is often largely a matter of their response to specific ecological conditions, which turn on and off large segments of the genome. The fungal ecology of sewage; compost; mushroom beds; agricultural and forest soils; naturally decomposing plant remains; some cheeses, bread, wine, and beer; crops in the field and after harvest; the air; the space between your toes; and the tissues of immune-deficient or immune-suppressed people: all can be the subjects of worthwhile, and even important, studies of fungal ecology. Many of the food webs illustrated in ecology textbooks miss out on more than half of the organisms involved in the transfer of energy and nutrients. They often stress macroscopic organisms, while omitting microscopic organisms such as the saprobic and mycorrhizal fungi. This neglect is unfortunate, especially since we now appreciate that microorganisms, being at the base of food webs, provide nutrients and mutualistic symbionts for almost all plants and animals. The basic links in terrestrial food webs lie in the soil, which is, of course, where a huge number of fungi and bacteria still live. Every attempt to understand terrestrial trophic systems must start and finish with soil organisms. And surely the fungi are among the most important of those.

Amphibious Fungi in Streams The second area of fungal ecology I want to examine is a stream flowing through a woodland, somewhere in the temperate zone. We already know that tiny chytrids and oomycetes live here, but we might not expect to find many of the typically terrestrial dikaryomycotan fungi. However, if you collect some stream foam and examine it under the microscope, you will see that the bubbles have trapped a rather unusual kind of multiarmed spore (this is simply a physical phenomenon—a surface tension effect— and there is no other relationship between the bubbles and the spores). In fact, the water contains what we call tetraradiate spores of many sizes. Pass a litre of stream water through a filter, then stain the filter in cotton blue and examine it through the microscope, and you will see more of these often large and strikingly shaped tetraradiate fungal spores (Fig. 11.3). Other spores will be unbranched, long, thin, and arc shaped, sinuate or sigmoid (s-shaped). All are produced by conidial anamorphs that are specially adapted for living in streams. Where do these spores come from, and how do the fungi that produce them make a living?

Fungal Ecology

Fig. 11.3

Tetraradiate conidia of stream-inhabiting fungi.

The first clue came when limnologists (biologists specializing in freshwater systems) began to examine the energy budgets of streams. Because some streams flow through forests, they are heavily shaded during the growing season. This means that few green plants (primary producers) can grow in them. It was found that more than half, and sometimes nearly all, of the energy-supporting organisms that live in streams comes from autumn-shed leaves of trees that grow over the streams. This source of energy is described as ‘allochthonous’ (a fancy Greek derivative which just means ‘coming from somewhere else’). When they first fall into the water, these leaves are extremely unpalatable to stream invertebrates, but as they are colonized and ‘conditioned’ by microorganisms, they

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Chapter 11 apparently become tastier. Experiments in which batches of leaves were treated with either antifungal or antibacterial antibiotics showed that the fungi were chiefly instrumental in making leaves palatable to animals such as Gammarus pseudolimnaeus, a numerous amphipod crustacean living in the stream (another amphipod lives on the beach below my house in millions, eating decaying tidal jetsam, mostly seaweeds and, no doubt, the fungi growing on and in them). In a feeding experiment, Gammarus chose to eat fungal mycelium rather than unconditioned leaf discs. Later experiments with leaves conditioned by individual stream fungi showed that not only were some of the fungi that produce tetraradiate or sigmoid conidia most active in conditioning leaves, but their mycelia and sporulating structures were also highly nutritious food for detritivorous stream animals such as Gammarus. An important ecological role had been established for these fungi. So aquatic hyphomycetes make a living by breaking down plant polysaccharides and other recalcitrant compounds. They use the liberated subunits, primarily sugars, for growth and reproduction. Fungal biomass accumulating on leaves attracts and, at least in part, feeds various leaf-shredding invertebrates (Canhoto and Graça 2008), whereas released spores might be captured by filter feeders (Baerlocher and Brendelberger 2004). Fungal exoenzymes release small molecules and cause softening or maceration of leaf tissues, which contributes to the production of fine particulate organic carbon in streams (Suberkropp and Klug 1980; Chamier and Dixon 1982). But many questions remained. Were those fungi with tetraradiate spores related to one another? Did they have teleomorphs? (This would help to answer the first question.) Since streams always flow the same way and have a natural tendency to carry small things like spores downstream, where did the inoculum for the upper reaches come from? What were the advantages of the tetraradiate and sigmoid spore shapes? The information we needed was gradually accumulated over several years of experiments, until eventually we were in a position to give some answers. Many of the tetraradiate (four-armed) spores, although similar in configuration at maturity, developed in rather different ways. I will describe just two of these. In some, three arms grew upward and outward from the top of the first-formed arm (e.g., Tetrachaetum in Fig. 11.3). In others, one arm grew upward, the other three or four outward and downward at the same time from a central cell (e.g., Lemonniera in Fig. 11.3). Some of these conidia were thallic, some blastic. A few had clamp connections; most didn’t. This impression of diversity was confirmed when some of the teleomorphs were discovered. Some were unitunicate ascomycetes, both operculate and inoperculate, producing apothecial and perithecial ascomata. Some were bitunicate ascomycetes. Some were basidiomycetes. It became clear that the morphologically similar anamorphs were actually a mixed bunch: fungi of very different origins that had undergone convergent evolution, molded by selection pressure into similar shapes. The teleomorphs also provided one answer to the question of how these fungi got upstream: ascomata and basidiomata, unlike the anamorphs, were not submerged in streams, and they liberated airborne ascospores or basidiospores. The group has been christened the amphibious fungi because of its immersed anamorphs and emergent teleomorphs. But why did so many of these taxonomically diverse amphibious fungi evolve conidia with similar shapes? It was found that as they were carried along by the water,

Fungal Ecology tetraradiate spores sometimes entered the layer of still water just above the surface of submerged leaves and then made three-point landings on these leaves. We know that a tripod is the most stable configuration, able to stand firm on irregular surfaces. The spores formed microscopic tripods that gave them a foothold on the dead leaves for long enough to germinate from the ends of the three arms and attach themselves to the substrate before being swept away. The reason for the sigmoid shape has not yet been fully established. After colonizing the leaves, the amphibious fungi sporulate again, and it was found that they would do this only in highly oxygenated conditions and with the physical stimulus provided by flowing water. If the spore numbers are charted over the entire year, it will be seen that their numbers peak in fall and spring. In the first place, the massive new input of autumn-shed leaves provides the necessary substrate. In the second case, spring runoff will also carry plant debris into the stream. The entire process is diagrammatically summarized below, showing that the fungi are vital intermediaries of energy flow in streams, providing a link between dead leaves and trout (Fig. 11.4). There are about 300 described species of aquatic hyphomycetes, mostly specialized to live in streams. This ecological group is clearly polyphyletic. The number of species observed in any twelve-month period is relatively constant, but only 40% consistently occurred in five successive periods (Baerlocher 2000). In a recent study, estimated Operational Taxonomic Units varied between 69 and 174, whereas aquatic hyphomycete species based on conidium identifications varied between 12 and 41.

Fig. 11.4 Energy flow in streams.

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Chapter 11 New methods of estimating fungal biomass (equating it to ergosterol, a fungusspecific indicator molecule; reviewed by Gessner et al. 2003) and rate of growth (incorporation of 14C-acetate into ergosterol; Newell et al. 1988) have established that aquatic hyphomycetes make significant contributions to stream communities. Studies of leaf decomposition estimate fungal biomass by measuring ergosterol content of the decomposing substrate. This has permitted an estimate of total production of leaf-decaying fungi (Suberkropp and Gessner 2005). Over an annual cycle in a small, nutrient-poor woodland stream, up to 17% of decaying leaf biomass has been attributed to fungal biomass (Gessner 1997; Gessner et al. 2003), and fungal productivity per unit stream bed area is close to or exceeds that of bacteria or invertebrates (Suberkropp 1997; Carter and Suberkropp 2004). Barcoding relies on a short DNA sequence from a specified region of the genome that can be extracted and amplified reliably and has low-intraspecific and highinterspecific variability (Hebert et al. 2003). The consensus recommendation of the first meeting of the All Fungi Barcode Initiative (AFBI) was to use the entire internal transcribed spacer (ITS) sequence as a first step for species identification, with a second sequence added for more precise identification and phylogenetic placement. Compared with traditional techniques (identification of newly released conidia), molecular techniques have revealed higher species richness in the first seven to fourteen days after the leaves enter the stream. In temperate streams, alder leaves are among the best baits for aquatic hyphomycetes, while conifer needles are among the worst (Baerlocher 1992).

Aero-Aquatic Fungi in Ponds One good aquatic habitat deserves another, so after sorting out the role of fungi in streams, we switched our attention to woodland ponds. The pond in question lay in the heart of the woods behind my house in Waterloo. Again, primary production within the pond was limited by the forest canopy. Again, there was a specialized group of fungi living in the pond, although no one knew if these fungi played an important role in the ecology of the pond. In this case the fungal propagules commonly found were hollow and floated. Again, this end was achieved in several different ways, of which I will describe only two: The pond gradually dries out in summer, and the colonized leaves come to lie just below the surface of the water. (1) A conidiophore emerges from a dead leaf, emerges into the air, and branches like a tree. Eventually, the ends of the fine branches all swell up and fuse with their neighbours to form an air-filled, watertight structure. This is the propagule of Beverwykella (Fig. 11.5). (2) Another conidiophore grows up from a dead leaf, it emerges through the water surface, and its tip begins to grow in circles. Coiling repeatedly on itself in wider and wider, then narrower and narrower gyres, it eventually builds a barrel-shaped, airfilled, watertight structure. This is the propagule of Helicoon (Fig. 11.6). Another apparently rare pond fungus is the tiny floating gasteromycete Limnoperdon. It has been recorded only from our pond in Ontario and somewhere near Seattle, Washington, although it surely occurs at many places between those widely separated localities—people just haven’t looked carefully. The fruit bodies are hollow and are lined

Fungal Ecology

Fig. 11.5 Development of the floating propagule of Beverwykella.

with nonshooting basidia; the spores are symmetrically mounted and the sterigma is not pointed (see discussion in chapter 5). Young basidia have a clamp connection at their base. Because these fungi live and grow under water, but produce their spores only above the surface, they are called the aero-aquatic fungi. It’s obvious that the structures of the two kinds of conidia described above, although functionally equivalent, are not closely

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Fig. 11.6 Development of the floating propagule of Helicoon.

related. Again, convergent evolution has been at work, the selection pressure applied by some ecological imperative. We finally discovered what this was. It was the need to be first on the scene when new substrate appears. When a dead leaf falls into a pond, it does not sink immediately. It may actually fall on top of some of the floating propagules, or the propagules may be drawn

Fungal Ecology to the floating leaves by surface tension. In either case, these fungi will be the first pond-adapted species to enter this new substrate. The leaves soon sink to the bottom of the pond, carrying their new colonizers— hyphomycete or gasteromycete—with them. These fungi also have the ability to grow at low oxygen levels and to survive the virtually anaerobic conditions that prevail at the bottom of a pond for extended periods during the winter. Sporulation will happen again when the pond begins to dry out during the following summer, and the water level subsides until the colonized leaves are once more just below the surface. We found that these aero-aquatic hyphomycetes play an ecological role parallel to that of the amphibious fungi in streams: conditioning the dead leaves and making them palatable to the detritus-eating invertebrates such as snails and vertebrates such as frogs, whose tadpoles live in the pond and skeletonize leaves after the fungi have ‘conditioned’ Fig. 11.7 Melanized boundaries between them, eventually metamorphosing into tree frogs, which represent the apex of the pyra‘territories’. mid of life in the pond.

Other Habitats The biosphere has myriad other habitats, each unique in various ways and each making special demands of the organisms that live in it. The roots of plants create special conditions around themselves and have established especially intimate relations with hundreds of endotrophic and thousands of ectotrophic mycorrhizal fungi (which have chapter 17 to themselves). Other rather less specialized saprobic and parasitic fungi also abound on and near roots. The surface of living leaves is inhabited by a specialized mycota, while dead and decaying leaves are substrates for a succession of other species. The soil, into which most leaf remains are incorporated, is itself a mass of microhabitats and is the richest reservoir of fungal diversity. And of course the leaves of different plants, and the various soil types, will have different subsets of the total mycota. Not all fungi can be parceled out neatly into successive steps of a succession. Often, fungi compete for access to a substrate. Sometimes a natural phenomenon will give us an unexpected insight into this struggle. Wood is often colonized by many different mycelia. The boundaries between the ‘territories’ of different mycelia can often be clearly seen as black lines or zones, and the wood is described as ‘spalted’ (Fig. 11.7). The black material is melanin-like, oxidized, and polymerized phenolics deposited by wood-rotting fungi, and although the biological function of the zones isn’t entirely clear,

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Chapter 11 melanins are the precursors of the humic acids, which are long-lived and important determinants of soil fertility.

Fire Fungi Forest fires, slash burns, camp fires, and even volcanic eruptions will all trigger the fruiting of a rather specialized group of macrofungi. We are all becoming familiar with the massive fruitings of morels, particularly the black morels, Morchella elata and allies, which appear in the spring on areas burned the previous year. Foresters may be familiar with the ascomycete Rhizina undulata, which resembles, quite literally, a pile of dung, because this species attacks conifer seedlings on burn sites. Less familiar are the many other species in the genera Pholiota, Myxomphalia, Omphalina, Tephrocybe, Psathyrella, Coprinus, and the cup fungi Pyronema, Lamprospora, Octospora, and Peziza that appear in similar situations. While some of the cup fungi and pholiotas are brightly coloured and conspicuous, others are black, dark grey, or brown and therefore are hard to see among charred wood and soil. If you want to find these fungi, squat down and slowly scan small areas, particularly those showing some regeneration of mosses. Many of the ‘fire fungi’ are in fact bryophilous species associated with the mosses and liverworts that also characterize old burns. Fire fungi may be induced almost anywhere by controlled burning (and a little patience) or in the laboratory by various heat shock treatments of soil samples.

Macrofungal Ecology—Help Wanted! Most of the situations I have described in this chapter are small or localized. If we consider the macroscopic fungi, and their roles in such extensive ecosystems as forests, we find that the state of fungal ecology is relatively primitive, meaning that we simply don’t know very much about how those fungi act and interact under natural conditions. If you doubt this, you could explore the mycological literature for information on where to find morels (in my opinion, the best of all edible fungi). You will be led a merry dance, from old apple orchards and dead elms to recently burned forests. Until relatively recently, no one even seemed to know whether morels were mycorrhizal or not (my understanding is that they are opportunistic saprobes, exploiting new substrates, then fading away, only to appear somewhere else when new food sources present themselves). As for the ubiquitous agarics, which are undoubtedly the most widely collected and studied of all fungi, I have to report that things aren’t much better. Only Europe holds out a candle in the darkness. Since Europeans have been collecting and recording macrofungi for centuries, they have the kind of database that allows the present generation of mycologists to draw comparisons with the past. This is why several European countries have ‘red lists’: compilations of macrofungi which seem to have undergone serious declines in recent years or even to have disappeared altogether. It is impossible to produce such red lists for anywhere in North America because records do not go back far enough and are, in any case, still fragmentary. Although we may suspect that certain

Fungal Ecology species are declining or disappearing, we have no well-documented historical reason for saying so. You can find out more about red lists by going to Google and entering ‘red list endangered fungi’. We understand that about half of the known agarics are mycorrhizal—they have an intimate, mutually beneficial relationship with many of our forest trees. Ecological research has recently begun to focus on the effects on such fungi of various forest practices, and especially the clear-cutting of old-growth forests, which still (regrettably) goes on in many jurisdictions, and most blatantly in British Columbia where I live. One of my own graduate students has recently established that many of the fungi associated with old-growth forests do not recolonize clear-cut habitats for forty to fifty years. And his suspicion is that the recolonization happens by means of airborne basidiospores which originate in nearby old growth. What if there is no longer any nearby old growth to provide this inoculum? But international logging companies carry on in blissful ignorance of any such concerns. Just when we think we have established a few principles based on the occurrence of fruiting bodies of the mycorrhizal fungi, it is demonstrated by molecular techniques that in many cases the fungi producing the fungal sheaths around the roots of the trees are not those whose basidiomata are appearing above ground. Are we back to square one? No one seems sure at present. But I mention this to demonstrate how little we actually know about macrofungal ecology. A fascinating study by Tofts and Orton (1998) points out that although they collected agarics regularly in a particular woodland in Scotland for twenty-one years and recorded 502 species in that time, in each subsequent year they could still find species they had never seen before. Over twenty years of collecting, and they still could not say that they had a proper handle on agaric biodiversity in that woodland. They suggested that at least twenty-five to thirty years of collecting, and possibly more, would be necessary before that goal could be attained. This is not intended to put you off, to deter you from getting involved in fungal ecology. Rather the reverse. It is clear that the need for research in this area is critical. We need good ecological studies just as much as we need molecular research on fungi. Some groundwork has been done in Britain, where the macrofungal assemblages characteristic of many habitats have been broadly outlined. But this is still far from an understanding of the full role played by those fungi in the habitats being considered. The need for seminal research has never been greater. I describe one such project at the end of chapter 10, which gives an entirely new view of the spectrum and roles of soil fungi across North America (Talbot et al. 2014).

What’s an ATBI? Of course, you can’t do fungal ecology unless you know what fungi are present. There is almost certainly no habitat in the world whose fungi have been fully enumerated. A group of twenty-two mycologists gathered in Costa Rica in 1995 and came up with a strategy for isolating and identifying all of the fungi (an estimated 50,000) in a particular habitat (the Guanacaste Conservation Area)—an All-Taxa Biodiversity Inventory for fungi (Rossman et al. 1998). This ambitious plan called for a staff of a hundred,

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Chapter 11 $1 million worth of agar media, and 1.8 million slants to isolate the endophytic fungi alone. Unfortunately all this would have cost about US$25 million, so it hasn’t been done. But the need remains, and the general lack of knowledge about fungi means that they are not usually considered when conservation issues are raised. Perhaps you can help to change all that. A less ambitious ATBI is now under way in Great Smoky Mountains National Park, but it is a long-term endeavour. And having outlined that currently unsatisfactory state of affairs, we must turn the page to another, completely different aspect of mycology which came to prominence in the middle of the nineteenth century and has remained front and centre ever since.

Further Reading on Fungal Ecology Baerlocher, F., ed. 1992. The Ecology of Aquatic Hyphomycetes. Ecological Studies 94. Berlin: Springer. ———. 2000. “Water-Borne Conidia of Aquatic Hyphomycetes: Seasonal and Yearly Patterns in Catamaran Brook, New Brunswick, Canada.” Canadian Journal of Botany 78:157–67. ———. 2009. “Reproduction and Dispersal in Aquatic Hyphomycetes.” Mycoscience 50:3–8. Baerlocher, F., and H. Brendelberger. 2004. “Filtration of Aquatic Hyphomycete Spores by a Benthic Suspension Feeder.” Limnology Oceanography 49:2292–96. Baerlocher, F., and B. Kendrick. 1974. “Dynamics of the Fungal Population on Leaves in a Stream.” Journal of Ecology 62:761–91. ———. 1981. “The Role of Aquatic Hyphomycetes in the Trophic Structure of Streams.” In The Fungal Community: Its Organization and Role in the Ecosystem, edited by E. T. Wicklow and G. C. Carroll, 743–60. New York: Marcel Dekker. Bell, A. 1983. Dung Fungi: An Illustrated Guide to Coprophilous Fungi in New Zealand. Wellington, NZ: Victoria University Press. Belliveau, M., and F. Baerlocher. 2005. “Molecular Evidence Confirms Multiple Origin of Aquatic Hyphomycetes.” Mycological Research 109:1407–17. Canhoto, C., and M. H. S. Graça. 2008. “Interactions between Fungi and Stream Invertebrates: Back to the Future.” In Novel Techniques and Ideas in Mycology, edited by K. R. Sridha, F. Baerlocher, and K. D. Hyde, 305–25. Fungal Diversity Research Series 20. Kunming: People’s Republic of China. Cannon, P. F. 1995. “An ATBI—How to Find One and What to Do with It.” Inoculum 46:1–4. Carter, M. D., and K. Suberkropp. 2004. “Respiration and Annual Fungal Production Associated with Decomposing Leaf Litter in Two Streams.” Freshwater Biology 49:1112–22. Chamier, A.-C., and P. A. Dixon. 1982. “Pectinases in Leaf Degradation by Aquatic Hyphomycetes: The Enzymes and Leaf Maceration.” Journal of General Microbiology 128:2469–83. Frankland, J. C. 1998. “Fungal Succession—Unravelling the Unpredictable.” Mycological Research 102:1–15. Gessner, M. O. 1997. “Fungal Biomass, Production and Sporulation Associated with Particulate Organic Matter in Streams.” Limnetica 13:33–44. Gessner, M. O., F. Baerlocher, and E. Chauvet. 2003. “Qualitative and Quantitative Analyses of Aquatic Hyphomycetes in Streams.” In Freshwater Mycology: A Practical Approach, edited

Fungal Ecology by C. K. M. Tsui, K. D. Hyde, and W. H. Ho, 127–57. Hong Kong: University of Hong Kong Press. Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. DeWaard. 2003. “Biological Identifications through DNA Barcodes.” Proceedings of the Royal Society of London B 270:313–21. Hudson, H. J. 1980. Fungal Saprophytism. 2nd ed. London: Arnold. Kendrick, B. 1958a. “Microfungi in Pine Litter.” Nature 181:432. ———. 1958b. “Sympodiella, a New Hyphomycete Genus.” Transactions of the British Mycological Society 41:519–21. ———. 1959. “The Time Factor in the Decomposition of Conifer[ous] Leaf Litter.” Canadian Journal of Botany 37:907–12. ———. 1961. “Hyphomycetes of Conifer Leaf Litter. Thysanophora gen. nov.” Canadian Journal of Botany 39:817–32. Kendrick, B., and A. Burges. 1962. “Biological Aspects of the Decay of Pinus sylvestris Leaf Litter.” Nova Hedwigia 4:313–42. Michaelides, J., and B. Kendrick. 1982. “The Bubble-Trap Propagules of Beverwykella, Helicoon and Other Aero-Aquatic Fungi.” Mycotaxon 14:247–60. Newell, S. Y., T. L. Arsuffi, and R. D. Fallon. 1988. “Fundamental Procedures for Determining Ergosterol Content of Decaying Plant Material by Liquid Chromatography.” Applied and Environmental Microbiology 54:1876–79. Newell, S.Y., and R. D. Fallon. 1991. “Toward a Method for Measuring Instantaneous Fungal Growth Rates in Field Samples.” Ecology 72:1547–59. Nikolcheva, L. G., and F. Baerlocher. 2004. “Taxon-Specific Primers Reveal Unexpectedly High Diversity during Leaf Decomposition in a Stream.” Mycol Prog. 3:41–49. Nikolcheva, L. G., A. M. co*ckshutt, and F. Baerlocher. 2003. “Diversity of Freshwater Fungi on Decaying Leaves: Comparing Traditional and Molecular Approaches.” Applied Environmental Microbiology 69:2548–54. Price, P. W. 1988. “An Overview of Organismal Interactions in Ecosystems in Evolutionary and Ecological Time.” Agriculture, Ecosystems and Environment 24:369–77. Richardson, M. J., and R. Watling. 1974. Keys to Fungi on Dung. Cambridge, UK: British Mycological Society. Rossman, A. Y., R. E. Tulloss, T. O’Dell, and R. G. Thorn, eds. 1998. Protocols for an All Taxa Biodiversity Inventory in a Costa Rican Conservation Area. Boone, NC: Parkway Publishers. Seena, S., N. Wynberg, and F. Baerlocher. 2008. “Fungal Diversity during Leaf Decomposition in a Stream Assessed through Clone Libraries.” Fungal Diversity 30:1–14. Seifert, K. A., B. Kendrick, and G. Murase. 1983. A Key to Hyphomycetes on Dung. University of Waterloo Biology Series 27. Waterloo, Canada: Department of Biology, University of Waterloo. Seifert, K. A., R. A. Samson, J. R. DeWaard, J. Houbraken, C. A. Levesque, J. M. Moncalvo, G. Louis-Seize, and P. D. N. Hebert. 2007. “Prospects for Fungus Identification Using CO1 DNA Barcodes, with Penicillium as a Test Case.” Proceedings of the National Acadeny of Sciences USA 104:3901–906. Shearer, C. A., and L. C. Lane. 1983. “Comparison of Three Techniques for the Study of Aquatic Hyphomycete Communities.” Mycologia 75:498–508. Suberkropp, K. 1997. “Annual Production of Leaf-Decaying Fungi in a Woodland Stream.” Freshwater Biology 38:169–78.

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Chapter 11 Suberkropp, K., and M. O. Gesssner. 2005. “Acetate Incorporation into Ergosterol to Determine Fungal Growth Rates and Production.” In Methods to Study Litter Decomposition, edited by M.A.S. Graça, F. Baerlocher, and M. O. Gessner, 197–202. Dordrecht: Springer. Suberkropp, K., and M. J. Klug. 1980. “The Maceration of Deciduous Leaf Litter by Aquatic Hyphomycetes.” Canadian Journal of Botany 58:1025–31. Talbot, J. M., T. D. Bruns, J. W. Taylor, D. P. Smith, S. Branco, S. I. Glassman, S. Erlandson, et al. 2014. “Endemism and Functional Convergence across the North American Soil Mycobiome.” Proceedings of the National Academy of Sciences USA 111:6341–46. Tofts, R. J., and P. D. Orton. 1998. “The Species Accumulation Curve for Agarics and Boleti from a Caledonian Pinewood.” Mycologist 12:98–102. Webster, J. 1970. “Coprophilous Fungi.” Transactions of the British Mycological Society 54:161–80. Webster, J., and E. Descals. 1981. “Morphology, Distribution, and Ecology of Conidial Fungi in Freshwater Habitats.” In Biology of Conidial Fungi, vol. 1, edited by G. T. Cole and B. Kendrick, 295–355. New York: Academic Press.

12 Fungal Diseases of Crops and Trees Introduction We are utterly dependent on plants. Directly or indirectly they supply all our food. So it is an extremely serious matter if something prevents our domesticated plants from living up to their genetic potential in terms of growth and yield. Outside influences that do this are said to cause disease and are dealt with by a broad collection of disciplines grouped under the heading of plant pathology. At many universities, whole academic departments are devoted to it; entire government laboratories do nothing else. This is because our crops, in field or forest, are threatened by thousands of diseases. Plant pathology concerns itself both with noninfectious or physiological diseases caused by factors such as mineral deficiencies, climate, or pollutants and with infectious diseases caused by a horde of different organisms: nematodes, bacteria, mycoplasmas, viruses—and fungi. This chapter, as you might expect, will be concerned only with diseases caused by fungi. Although insects are our chief competitors for food, fungi are a good second. Crops in Ohio are attacked by about 50 bacterial diseases, 100 viral diseases, and 1,000 fungal diseases. About 60% of all plant disease literature concerns fungal diseases. You have already read about some of these in the taxonomic chapters earlier in this book: rusts, smuts, blights, downy mildews, powdery mildews, etc. I am not going to repeat myself; you should refresh your memory of the organisms involved by looking back into that section. You could even glance through it now, before you go on with the rest of this chapter. (That’s the great thing about a book; it’s a very flexible teaching machine.) But I will mention some additional diseases here, just to broaden your perspective. Ever since people became farmers, they have had problems with fungal diseases of plants. These diseases weren’t visited upon them by the gods, as the ancient Romans thought, but were a natural consequence of growing plants in extensive pure stands, or monocultures. Whether the fungi grow, swim, float, ride, or blow from one host plant to the next, they will find a new home much more readily in a monoculture than in most natural plant communities. This is because most plant disease fungi have a limited host range, and the very diversity of the community means that individuals of a particular host species are often well separated so may escape infection emanating from their relatives. Although fungal diseases have been recognized for thousands of years, they were not connected with the organisms that caused them until the mid-nineteenth century. Fortunately, the scientific revolution was in full swing when the potato famine caused by Phytophthora infestans (Oomycota) struck Europe (Fig. 12.1), and it was not many years 247

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Fig. 12.1

Progressive attack of Phytophthora infestans on potato plants.

before the relationship between fungus and disease was firmly established. This knowledge has not prevented many subsequent disastrous episodes when particular fungi have ravaged field or forest. A few examples may help to establish this: the downy mildew of grape, caused by Plasmopara viticola (Oomycota), which almost destroyed the French wine industry; the chestnut blight caused by Cryphonectria (Endothia) parasitica (ascomycetes), which has killed almost all mature sweet chestnut trees in North America; the wheat rust epidemics of the ‘dirty thirties’, caused by Puccinia graminis (pucciniomycetes), which made the depression an all the more bitter experience for prairie farmers; the southern corn blight epidemic of 1970, caused by Drechslera maydis (ascomycetous anamorph), which destroyed up to 70% of the crop in several corn-growing states; the Dutch elm disease, caused by Ophiostoma ulmi (ascomycetes),

Fungal Diseases of Crops and Trees which continues to decimate our beautiful native American elm over much of the continent; blue mould of tobacco, caused by Peronospora tabacina (Oomycota), which destroyed $100 million worth of Ontario tobacco in 1979; the ‘bayoud’ fungus, Fusarium oxysporum (ascomycetous anamorph), which has killed 12 million date palms in Morocco and 3 million in Algeria, speeding up desertification; and the whimsically named but incurable ‘brewer’s droop’ of hops, caused by a Verticillium (ascomycetous anamorph), which is now spreading through the hop-growing areas of Britain. And there will be more surprises, more defeats, and many ongoing battles against the army of fungi that encroach on our chosen plants. Why are fungi such a threat? Why can’t we breed totally resistant plants or synthesize ultimate fungicides? The answer lies in the remarkable genetic flexibility of the fungi, some aspects of which are discussed in chapter 10. Why, to ask an even more basic question, do the fungi attack living plants in the first place? The answer may well be hidden in the distant past, but it seems to me that once there is a division between autotrophic and heterotrophic organisms, it will only be a matter of time before some of the heterotrophs seize the advantage by attacking the autotrophs before they die, rather than respectfully waiting until afterward. This calls for the development of new talents: ways of overcoming the natural defence mechanisms of the target organisms, of penetrating their cell walls, of ensuring dispersal from one host to the next. The fungi have responded nobly to this challenge. I’ve already pointed out that for thousands of years we didn’t know that fungi caused plant diseases; but even now, pinning the blame on the pathogen isn’t always easy. Finding a fungus fruiting on a diseased plant isn’t enough to allow us to blame that fungus for the disease. Preexisting disease, and the necrotic tissue that often results, may open the way for exploitation of that tissue by secondary colonizers, which may or may not be parasites themselves. We can’t hope to deal with a disease until we know exactly which organism is causing it. Fungi can be unequivocally labelled as pathogens only after a number of conditions, known as Koch’s postulates, have been satisfied: (1) The fungus must be consistently associated with the disease. (2) The fungus must be isolated in pure, axenic culture (a culture on a susceptible host has to suffice for obligate biotrophs). (3) When the fungus is reinoculated onto healthy host plants, it must produce the original disease. (4) The fungus must then be reisolated from the diseased plant. If you have been through all those steps, there won’t be much doubt in your mind about the pathogenicity of the fungus in question. Actually, even Koch relaxed these postulates a little because they were so difficult to meet in their entirety.

Classification of Plant Diseases There are many possible approaches to an understanding of plant disease and its control. We need to know the precise identity of the organism causing the disease. But before we can hope to control the disease, we need to know much more than that. We have to establish the nature of the relationship between the fungus and its host; how and where and when it gets into the plant; the kind of symptoms it causes; the parts of the plant it attacks; and, above all, its life history, which may reveal a weak point at which it may be attacked. This kind of information can be treated systematically in several different ways, each of which can materially assist us in our planning.

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Chapter 12 To kill one’s host is not the ideal strategy. Just as the parasite is settling in comfortably to enjoy the amenities of its new home, the host collapses and dies. It is almost axiomatic that a host and a parasite that have been associated for a long time have co-evolved to produce a balanced relationship in which the host doesn’t expel the parasite, and the parasite doesn’t kill the host. There is a finely tuned genetic equilibrium between pathogenicity and resistance (or tolerance). When a pathogen is introduced to a new host as it migrates around the globe, generally one related to its usual host but lacking the accumulated genes for resistance, the results are likely to be catastrophic. See if you can pick out these fateful meetings in the examples discussed below. The adoption of a parasitic existence necessitated many specialized adaptations. Some fungi evolved new enzymes: cellulases that could dissolve the substance of plant cell walls and pectinases to dissolve the cement that holds plant cells together. At one extreme, some fungi evolved diffusible toxins that killed host cells at long range and circumvented the problems inherent in exploiting living cells. At the other extreme, some fungi became so intimate with their hosts that they ultimately became dependent upon the living host cytoplasm for many things—not just food but also a variety of vital enzymes, or even whole biochemical pathways. Some fungi produced plant growth regulators that either increased or decreased the ability of host cells to grow and divide. In this way, three different kinds of pathogen evolved. (1) Some are facultative parasites. These versatile organisms can live either saprobically or parasitically. Many of them are pathogens of annual herbaceous crop plants and must survive between growing seasons as members of the normal soil mycota. This ability makes them particularly difficult to control and virtually impossible to eradicate; the hyphomycete Fusarium oxysporum var. cubense, which causes Panama disease of bananas, can survive in the soil for at least forty years in the absence of the host. (2) Other fungi are necrotrophs, basically saprobic but producing toxins specific to susceptible host cells. The hyphomycetous Monilia anamorphs of Monilinia species (ascomycetes), causing brown rot of peaches and other stone fruits, belong to this category. (3) The last group are called obligate parasites or obligate biotrophs, because they have long since lost their independence and cannot grow at all except on or in a suitable host. In fact, the dependence is often so complete that only one host species, or a few cultivars of that species, will support a particular race of such a pathogen. The rust fungi, for example the genus Puccinia, are good examples of this third category. Life cycle studies. Usually we can’t decide on the best way to tackle a fungal disease until we know a great deal about the life cycle of the fungus. For example, many of the obligately biotrophic rust fungi require two hosts (such rust fungi are called heteroecious), with an obligatory annual migration from one to the other. We may be able to control the fungus by eradicating one of the hosts (assuming that it is neither of economic importance nor rare, of course) wherever it grows too close to the other. Perhaps the bestknown example of this practice is the widespread eradication of barberry (Berberis spp.) in North America to break the life cycle of the wheat rust fungus Puccinia graminis (pucciniomycetes, Pucciniales), or at least reduce its opportunities for genetic recombination, since dikaryotization happens on the barberry. Host-pathogen relationships.

Fungal Diseases of Crops and Trees The Spilocaea hyphomycetous anamorph of Venturia inaequalis (ascomycetes) is a virulent parasite which attacks the leaves and fruit of apple trees and causes the unsightly scab disease. But it can also live saprobically, because the ascomata of the teleomorph develop in the dead apple leaves over the winter, releasing ascospores (the primary inoculum) in spring. To control the hyphomycetous anamorph requires repeated spraying throughout the growing season and, as chapter 13 on fungicides clearly shows, the fungus quickly develops resistance to each new fungicide. Removal of dead leaves from the orchard floor, and spraying with disinfectants while the trees are dormant, are valuable ways of reducing the amount of ascospore inoculum released in spring. Phytophthora infestans (Oomycota, Peronosporales) produces easily detached and subsequently airborne mitosporangia; but when these land on a new potato plant, they still release swimming spores. These are delicate, are short lived, and can function only when free water is present on the potato leaf. This stage might be described as the Achilles’ heel of the fungus, since minute quantities of fungicide in the water will kill the zoospores. But once the fungus is inside the host plant, control becomes much more difficult (Fig. 12.1). It is clear that we must have detailed knowledge of the life cycles of pathogenic fungi if we want to develop optimal disease control strategies. Stage of host development affected. Diseases can strike a crop at any point in its development. Some important diseases, such as loose smut of wheat (Ustilago tritici: Ustilaginomycetes) are seed borne. Others, such as damping-off (Pythium spp.: Oomycota) devastate tender seedlings. Yet others attack the growing or mature plant (the hyphomycete Alternaria solani causes early blight of potato; the oomycete Phytophthora infestans causes late blight). Finally, some diseases cause serious losses after harvest: the hyphomycetes Monilia and Botrytis cause soft rot of peaches and grey mould of strawberries, respectively. And many root disease organisms simply sit in the soil and wait for an appropriate host to appear. Their propagules are so long lived that it’s not critical which year the host returns. Some infections, notably those caused by smut fungi, some rust fungi, and some members of the Clavicipitales, spread throughout the plant and are described as systemic. Others, such as vascular wilts and heart rots, are restricted to a single tissue. Yet others attack a single organ, as in fruit or seed diseases or anther smut. Finally, individual infections of some, like the wheat rust just mentioned, are extremely localized and may form only a tiny leaf spot. As a systemic infection spreads, or as the number of small, individual infections increases, the host will usually develop physical symptoms, such as reduction in growth, necrotic or discoloured areas, hypertrophied tissues, etc. Each disease has its own trademark, producing a particular set of symptoms, although some diseases (such as smuts) can remain asymptomatic for long periods, and in others, such as the heart rots of trees, the symptoms are cryptic. But sooner or later, the fungus will reproduce, making it much easier to name the culprit. It will be sooner in wheat rust, in which the anamorphic uredinia produce spores that can infect only wheat and pass through several eight-day generations as an epidemic grows. It will be later in the case of smuts or wood-rotting fungi. But always the spores are produced in astronomical numbers, because the odds are so heavily against their individual survival.

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Chapter 12 It is interesting to consider just how diseases damage their hosts. Damping-off (Pythium spp.: Oomycota) causes breakdown of seedling tissue by enzymically dissolving the pectic middle lamella between cells and also produces toxins. This fungus can obviously derive its food from dead cells. The vascular wilts caused by species of Fusarium and Verticillium (hyphomycetes) and Ophiostoma (ascomycetes) drastically reduce the upward movement of water in the xylem. The vessels become blocked with hyphae and spores, fungal polysaccharides, or tyloses (outgrowths developed by the plant into the lumen of the vessels in an attempt to stop the spread of the fungus). The transpiration stream is reduced to 2%–4% of normal, and wilting and death inevitably follow. The damping-off and wilt fungi don’t strike me as particularly well adapted to their hosts. Rust fungi (pucciniomycetes) are obligately biotrophic, so they don’t kill the cells of their hosts; and the combined biomass of many tiny rust infections is probably not large enough to cause a serious drain on the plant. So why are yields often so drastically reduced by these fungi? The answer becomes clear when they rupture the host epidermis to release their urediniospores. The plant’s waterproof skin is broken in so many places that in a dry prairie summer it can no longer maintain turgor pressure, and its physiology is disrupted. Some other highly adapted pathogens don’t kill, or even seriously damage, the vegetative organs of the plant. But they do subvert the energy the plant normally accumulates for reproductive purposes. Smut fungi (ustilaginomycetes) enter the plant when the seed germinates or may already be present as mycelium in the grain. The ergot fungus (Claviceps: ascomycetes) gains access through the stigma of the flower. But both fungi eventually home in on the developing ovary and ultimately replace it with their own reproductive structures. If we grew corn and rye as we do lettuce, just for the leaves, these diseases wouldn’t be so serious. And if carrot leaf blight didn’t reduce the efficiency of the harvesting machinery (the leaves are weakened and break off, leaving the carrot in the ground), it might not be taken nearly so seriously by the growers, since it doesn’t drastically reduce the actual carrot crop. Host organs attacked. Diseases can be described as root rots, vascular wilts, leaf spots, etc. When a disease is first noticed, the fungus causing it will not usually be producing diagnostic structures. Symptoms may well develop in parts of the plant that aren’t being directly attacked: symptoms of root disease will often manifest themselves in the shoot system. Consequently, many plant disease manuals concentrate on describing and illustrating sets of symptoms by which diseases can be diagnosed early. Although positive identification of the fungus may not be possible until it eventually fruits, or is isolated and identified in pure culture, treatment must begin as early as possible to prevent the buildup of an epidemic. It is easy to talk about such diseases as Alternaria blight and Cercospora blight of carrots, as if these were easily recognizable entities like mushrooms or mice, but the truth is that the early symptoms are often very inconspicuous, that they change continuously as the condition develops, and that it takes a very practised eye to make an early diagnosis of most diseases. Plant diseases can be classified according to the symptoms they elicit as follows. (1) Necrosis, generalized cell death, is the most extreme reaction. It can affect the base of the shoot, as in damping-off (caused by Pythium species: Oomycota); or the leaves, as in late blight of potato (Phytophthora infestans: Oomycota); or storage tissues, as in soft rot of peaches. Necrosis goes by many names: anthracnose, blight, canker, scab, leaf spot, shot hole.

Fungal Diseases of Crops and Trees (2) Permanent wilting, caused by blockage of the xylem by hyphae or as a reaction to a fungal toxin, as in wilt of tomato (caused by Verticillium: hyphomycetes, ascomycetous anamorphs), Panama disease of banana (Fusarium oxysporum f. sp. cubense: hyphomycetes, ascomycetous anamorphs), and Dutch elm disease (Ophiostoma ulmi: ascomycetes). (3) Hypertrophy or hyperplasia, caused by growth hormones (auxins) liberated by the pathogen, as in white rust of crucifers (caused by Albugo candida: Oomycota), corn smut (Ustilago maydis: ustilaginomycetes), and peach leaf curl (Taphrina deformans: ascomycetes). (4) Leaf abscission, caused by hormones produced or stimulated by the pathogen, as in powdery mildew of gooseberry (Sphaerotheca: ascomycetes) and coffee rust (Hemileia vastatrix: pucciniomycetes). (5) Etiolation, excessive extension growth, caused by a growth hormone (gibberellic acid) produced by the pathogen, as in ‘foolish seedling’ disease of rice (caused by Gibberella fujikuroi: ascomycetes). (6) Prevention of reproduction, caused in various ways: choke of grasses (Epichloë typhina: ascomycetes, Clavicipitales) prevents flowering; ergot of grasses (Claviceps purpurea: ascomycetes) replaces the grain with a fungal sclerotium; and anther smut (Ustilago violacea: ustilaginomycetes) replaces the pollen with fungal spores. Irrespective of how we classify and diagnose fungal plant diseases, the prime objective of plant pathology is to thwart the game plan of the pathogens, and many disciplines now contribute to this end. The meteorologist provides data which will allow the plant pathologist to forecast outbreaks of certain diseases and prescribe appropriate preventive measures: this technique is particularly valuable in dealing with late blight of potato. Some plant pathologists have delved into micrometeorology and aerodynamics to probe the way conditions within the canopy of a forest or of a field crop affect spore dispersal and germination. The chemist synthesizes new and ever more sophisticated fungicides. The plant breeder produces cultivars with built-in resistance to specific diseases. The plant pathologist is dealing not simply with an isolated interaction between a fungus and a plant but with the overriding effects of climate and microclimate on how that interaction develops through time. Most imponderable of all is the fungus itself, which, with its endless genetic flexibility, is never more than one jump behind the plant breeders and the fungicide formulators. Sometimes there are other complicating factors: the mysterious wanderings of animal vectors such as the bark beetle that carries Dutch elm disease from tree to tree; and since Dutch elm disease was brought, albeit accidentally, by people from Europe to North America, the control of all plant imports assumes tremendous importance. Although some diseases are almost ubiquitous, many are still relatively localized, and governments try, with mixed success, to exclude exotic pathogens by quarantine regulations, employing plant pathologists to inspect incoming shipments of plant products. Much of what we know about long-range dispersal of fungi (see chapter 8) concerns the spread of crop diseases, which will make themselves felt, and hence be documented, wherever they appear. It must be clear by now that there is no simple formula for dealing with plant diseases. Each is a special case; and each outbreak will differ from all others in various ways. Plant pathologists will never put themselves out of a job.

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Establishment of Disease When pathogen meets plant, a number of factors determine whether disease will develop. The plant may be entirely resistant, extremely susceptible, or somewhere between those extremes. The fungus may be extremely virulent, almost avirulent, or somewhere between the two. The stage of development of the host may be important: damping-off attacks only young seedlings; ergot ascospores can infect only grasses in flower. The weather may be critical: many downy mildews have an absolute requirement for free water. Perhaps the most crucial phase in the development of any disease is the initial penetration of the host. A microscopic spore (one in a hundred? one in a million?) lands on a leaf. If the spore is initiating the first infection of the growing season, it is called a primary inoculum and causes a primary infection. Spores produced by primary infections are called secondary inocula. So an ascospore of Venturia inaequalis, liberated from the overwintered dead leaves, is a primary inoculum, while the conidia of the Spilocaea anamorph, produced on the new season’s growth, are secondary inocula. In smuts, there is only primary inoculum, since the fungus doesn’t form any further spores until the end of the season. In either case, the more primary inoculum there is, the more serious the disease is likely to be. But all this assumes that the spore successfully infects the host, and we mustn’t take that for granted. Many things can go wrong. Does the leaf belong to a susceptible host? Is the temperature suitable for spore germination? Is free water available, or is the relative humidity high enough? And will it stay that way long enough for the spore to germinate and penetrate the plant? If our spore is typical, it will die, before or after germination. But one in a hundred, or one in a million, go on to establish themselves. Some germ tubes, and the zoospores of some downy mildews, find their way to a stomate, but other fungi make a frontal assault (Fig. 12.2). The germ tube establishes an appressorium, a small swelling of the hypha, that adheres very tightly to the plant surface. This gives the fungus the physical leverage it needs in order to go ahead with the actual penetration. Now a very narrow hypha partly digests, partly Fig. 12.2 TEMicrograph. forces its way through the cuticle and then the cell wall. Some pathogens always enter a cell; others just as regularly grow between host cells. Once inside, the hypha broadens out, extends, branches, and establishes an infection if the plant is susceptible. Plant resistance may take a number of forms: the cuticle

Fungal Diseases of Crops and Trees and/or the epidermal cell wall may be thick and tough enough to resist penetration. In hypersensitive plants, the penetrated cell dies almost at once. This is enough to discourage many parasites. Obligately biotrophic fungi, deprived of the kind of nutrients living cells provide, will certainly die (of course, if the fungus is a necrotroph, the hypersensitive reaction won’t faze it). The host may lay down a sheath of material around the invader, and this encapsulation sometimes starves the fungus out. Specially produced cork layers or abscission layers can effectively isolate the pathogen at a later stage: in shot hole disease of leaves, the infected part simply drops out. Many defences are biochemical. Most pathogens, although their spores obviously land on an enormous number of different plants, can infect relatively few of them (sometimes only one). All the others must in some way deter or resist the fungus; this is what we call nonhost resistance. Some plants contain substances like phenolics, which inhibit the development of many fungi. Others produce special antifungal compounds called phytoalexins, also often phenolic, only when attacked. The genetic aspects of disease resistance are discussed in chapter 10.

Epidemiology Epidemics of different diseases develop in different ways. The incidence of smuts is predetermined by the level of infection or spore contamination of the seed when the crop is planted. But the severity of most other diseases depends on their success not only in producing primary infection but in multiplying their secondary inoculum during the growing season. This success, of course, is a product of the interaction of many factors: virulence in the fungus, susceptibility in the host, favourable weather, or lack of action by laissez-faire farmers. Conditions for the development of major epidemics of many diseases do not happen every year, but some crops are always threatened. For example, if apple growers did not spray 8–20 times per season to control apple scab (the hyphomycetous Spilocaea pomi anamorph of Venturia inaequalis: ascomycetes), between 70% and 100% of their crop would be unsaleable. If peanut growers didn’t spray 8–10 times a season for foliar diseases, they would lose 20%–75% of their crop. Peaches need foliar and postharvest treatments if 80% of the crop is not to be lost. Strawberry growers stand to lose 70% of their crop if it is not sprayed several times. Grains, on the other hand, can often be protected against their worst diseases by a single seed treatment, which prevents losses of up to 35%. Let’s see how some of those diseases develop. Apple scab begins the season with ascospores, primary inoculum shot into the air from ascomata developed in last year’s dead leaves. But it immediately switches into anamorph mode: each infection derived from a single ascospore soon produces many conidia on the current season’s leaves. These spread the infection to new leaves, and the new infections soon produce even more conidia. The number of conidia increases in a geometric progression, and unless this cycle of infection and conidium production is interrupted, the outlook is bad. You will find a detailed discussion of the difficulties involved in controlling apple scab in chapter 13. It is an interesting story, with no end in sight. Other crops are threatened by more than one serious disease. Potatoes, for example, suffer from an early blight caused by Alternaria (hyphomycetes) then from late blight

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Chapter 12 caused by Phytophthora (oomycetes). No single fungicide will effectively control both fungi. Mancozeb is commonly used for the Alternaria, and Ridomil for the Phytophthora, and a close watch is kept on the weather to determine when it is necessary to spray for late blight. Experienced plant pathologists perform an invaluable service when they go out into a field and diagnose a disease, but they cannot keep track of every disease on every crop. And if the development of epidemics, and the results of control measures, are to be documented, we need some objective ways of deciding how much disease is present and how serious it is. Sometimes these take the form of descriptions, as in the following excerpts from a key for assessing potato blight: ‘Up to 10 spots per plant = 1% of crop diseased’. ‘Every plant affected and about one-half of leaf area destroyed by blight; field looks green flecked with brown = 50% of crop diseased’. In other cases, visual aids are provided, which allow farmers to assess the severity of attack for themselves. Figure 12.1 shows a chart for assessing damage caused to potato plants by late blight. Fig.12.3A shows a chart used with Septoria glume blotch of wheat, and Fig. 12.3B one used with powdery mildew of cereals.

Fig. 12.3 L Diagrams used to assess severity of disease. A: Septoria glume blotch of wheat; B: Erysiphe powdery mildew of cereals.

Some diseases begin very inconspicuously. The ergot fungus, Claviceps purpurea, can gain entry to its host only through the stigma of the open flower. This indicates considerable coevolution of plant and fungus. If the fungus did not shoot its ascospores during the relatively brief time that the grass flowers are receptive, it would miss the boat. The very precision of this timing may be turned against the fungus if we can develop rye cultivars that flower earlier or later than normal. The disease remains virtually asymptomatic until the fruit begins to develop, when it soon becomes apparent that the resources allocated for that purpose have been misappropriated. At the other extreme are diseases which manifest themselves as soon as infection occurs. The soft

Fungal Diseases of Crops and Trees rot of peach becomes visible as a spot of necrotic tissue within hours of fungal penetration. This is caused by a specific phytotoxin produced by the hyphomycetous Monilia anamorph of Monilinia fructicola (ascomycetes). Some fungal diseases kill plants—Phytophthora infestans can turn a field of thriving potato plants into a mass of rotting vegetation (cf. the sequence shown in Fig. 12.1); repeated defoliation by the coffee rust fungus Hemileia vastatrix (pucciniomycetes) can kill a coffee tree; Cryphonectria parasitica and Ophiostoma ulmi (both ascomycetes) have killed millions of chestnut and elm trees, respectively. But many diseases do no more than significantly reduce the photosynthetic activity of the plant. So why are they taken so seriously? The answer lies in the economics of the situation. Does the disease attack one of our crop plants? Does it significantly reduce crop yield? If the answer to both questions is yes, the disease automatically enters the province of the plant pathologist. But even after a disease has been recognized as a problem, it is by no means certain that anything will be done about it. Economic considerations are again paramount in matters of disease control. Some diseases are easy and cheap to control. It would be possible to suppress many others with an appropriate regime of sanitation and prophylaxis, but the return in increased yield would not cover the cost of the program. This is particularly true of many forest diseases. At the other end of the scale, greenhouse crops are often so valuable that expensive control programs (from soil fumigation to repeated applications of fungicide) are routine.

Control of Plant Diseases Control measures range from hot water treatment of seeds to kill hitchhiking spores of Ustilago tritici to chilling of fruit to retard spoilage by Monilia. We try to control fungal plant diseases in four ways: by exclusion, by eradication, by protection, and by immunization. I’ll discuss these in turn. (1) We can exclude pathogens from susceptible hosts in a variety of ways. (a) By keeping them out of the country. This involves quarantine regulations and careful inspection of all incoming plant material and soil. The price of freedom from introduced diseases is ceaseless vigilance. The potato blight fungus was carried across the Atlantic to Europe before anyone knew that fungi caused plant disease. But even now, fully aware as we are of the threat, pathogens are still carried inadvertently from continent to continent as an unfortunate side effect of international trade. Ophiostoma ulmi entered the United States from Europe in 1930 on elm burl logs imported for veneer. Regulations to prevent the import of elm products to Canada were introduced in 1928 and 1934, but the disease was seen in Quebec in 1944, having probably been introduced on crates made of diseased elm wood. The contents of the crates not having anything to do with elm trees, the crates themselves probably escaped scrutiny. This shows how difficult it is for bureaucracies to cope with microscopic fungi. Other ways of excluding pathogens include (b) growing susceptible plants in conditions that are inimical to the development of the fungus, and (c) in the case of some obligate parasites which cannot survive without the host, using disease-free seed or stock. (2) It is worth trying to control some pathogens by eradicating them. (a) By rigorously destroying all diseased plants. Most of the magnificent old elms in Fredericton,

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Chapter 12 New Brunswick, and along the Niagara Parkway in southern Ontario have been preserved by the consistent and immediate removal of any tree found to be infected by Dutch elm disease. (b) By pruning out affected branches (this wouldn’t work for Dutch elm disease, because the pathogen spreads through the vascular system of the tree rather rapidly by means of microconidia). (c) By applying fungicides. Systemic fungicides will kill the fungus both outside and inside the host plant, so eradication is sometimes possible. (3) In some cases, it is worth protecting healthy plants from the predictable attacks of pathogens by dusting or spraying them with protectant fungicides. Through much of the growing season the leaves of French grape vines look blue because they are covered with a residue of a copper-containing spray which is repeatedly applied to discourage the ingress of the downy mildew fungus, Plasmopara viticola. (4) The use of cultivars which are immune or resistant to specific pathogens is now very common, but actual immunization, the induction of antibodies to particular disease organisms by challenging the host organism with a dead or attenuated pathogen, which is so important in dealing with many diseases of animals and people, is impractical in plants: they simply don’t have the sort of immune system found in animals. Nevertheless, the hypersensitive reaction, in which the host cell dies as soon as it is penetrated by a pathogen, can deny some parasites, especially biotrophic ones, a foothold. This reaction and the production of specifically antifungal substances, called phytoalexins, by many plants may be regarded as analogous responses. To give you some idea of what practising plant pathologists are expected to know, I will list the fungal diseases to which two crops are susceptible. Onions are attacked by (1) purple blotch (Alternaria porri: hyphomycetes); (2) neck rot and grey mould rot (Botrytis allii, Botrytis squamosa, and Botrytis cinerea: hyphomycetes); (3) leaf blight (Botrytis squamosa); (4) smudge or anthracnose (Colletotrichum circinans: coelomycetes); (5) Fusarium bulb rot (Fusarium oxysporum f. sp. cepae: hyphomycetes); (6) downy mildew (Peronospora destructor: Oomycota); (7) pink root (Pyrenochaeta terrestris: coelomycetes); (8) white rot (Sclerotium cepivorum: sterile anamorphs); (9) smut (Urocystis magica: ustilaginomycetes). Of course, a given crop will not develop all of these diseases at once, but the control strategies used by farmers are sufficiently diverse to be worth outlining. Four main strategies are adopted: (1) Crop sanitation. Since many pathogens overwinter and complete their life cycle on the decaying remains of their host plant, it makes sense to destroy crop debris by burning it or ploughing it under. (2) Crop rotation. Another way of reducing the populations of pathogenic organisms is to alternate susceptible with nonsusceptible crops. The length of rotation depends on how long the pathogen can survive without the host. Sometimes a two- or three-year rotation will do, but since Sclerotium, Colletotrichum, Pyrenochaeta, and Urocystis can survive in the soil for a long time, rotations of at least five years would be necessary to reduce their inoculum potential to a reasonable level. Such long rotations are often impractical. (3) Fungicide treatments. Seeds are treated with fungicides to prevent damping-off and smut, because the inoculum for these diseases is seed borne. Leaf diseases are difficult to treat once they have appeared and require repeated, and therefore expensive, sprayings. So a protectant spray is often applied before any disease symptoms appear.

Fungal Diseases of Crops and Trees Preventive spraying may be repeated if weather conditions or disease forecasts call for it. (4) Resistant cultivars are under constant development by plant breeders. When available, they are the most effective and the cheapest way of avoiding losses. More specifically, in onion fields, purple blotch, neck rot, and leaf blight are controlled during the growing season by fungicides, and their inoculum potential is reduced after harvest by removing and destroying plant residues. Neck rot is basically a storage rot and can be minimized by harvesting the onions in dry weather, air-drying them before storing, then keeping them at 0°C and a relative humidity (R.H.) below 70%. Smudge occurs at harvest time and in storage, so drying onions properly, and keeping them at 0°C and 70% R.H., is recommended. Fusarium bulb rot is difficult to eliminate, since Fusarium species commonly survive in the soil as chlamydospores or grow saprobically on crop residues. A three-year rotation is required to keep this disease in check, and use of resistant cultivars is recommended. To become established, the downy mildew fungus Peronospora needs cool temperatures and rain or dew on the leaves (just like potato blight, and for the same reasons). A few hours of dry, sunny weather will slow Peronospora down considerably. The farmer can help by avoiding mildew-contaminated sets, practising a two-year rotation, spraying regularly with fungicide, and destroying infected crop debris. If pink root becomes a problem, a long rotation will be necessary. White rot also necessitates a long rotation, although soil treatment with fungicide may deal with small problem areas. Control of smut is achieved by coating the onion seed with a systemic fungicide, practising rotation, and using onion transplants if the soil is already contaminated; established plants will not be affected by the disease. It is instructive to compare the fungal diseases attacking onions with those found on carrots. Carrots are prone to Alternaria blight (Alternaria dauci: hyphomycetes); Cercospora blight (Cercospora carotae: hyphomycetes); rusty root (Pythium spp.: Oomycota); violet root rot (Rhizoctonia crocorum anamorph of Helicobasidium purpureum: basidiomycetes); Rhizoctonia crown rot (Rhizoctonia solani anamorph of Thanatephorus cucumeris: basidiomycetes); Sclerotinia rot (sclerotial anamorph of Sclerotinia sclerotiorum: ascomycetes); and black rot (Stemphylium radicinum: hyphomycetes). Are any genera common to both lists? Only Alternaria. This gives you some hint of the diversity of pathogenic fungi and the difficulties faced by the plant pathologist. Which group of fungi is most prominent in both lists? Dikaryan anamorphs, mainly those we call hyphomycetes. This is a generalization that holds across the entire spectrum of plant disease fungi. Of course, the diseases vary in distribution and in their economic impact. Cercospora blight is favoured by hot, humid weather and develops in high summer, while Alternaria blight and Sclerotinia rot like it cooler and develop later. Rusty root is most severe in wet soils. Rhizoctonia crown rot in organic soils after repeated carrot crops. Sclerotinia rot probably causes the greatest losses. Since the range of control measures available to us is much more restricted than the variety of fungi involved, I don’t need to elaborate on this except to say that the measures used will reflect such features of the fungus as its longevity in the soil, the part of the plant it attacks, and economics. In the case of the two Rhizoctonia diseases, no effective control is possible at present, so the only possible recourse is to grow carrots in uninfected soil. It is hoped that resistant varieties will eventually be developed.

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Plant Disease Forecasting Plant disease forecasting is not, of course, designed simply to tell the farmer when to expect an outbreak of a particular disease; it is meant to give him a chance to apply preventive measures that will effectively stop the development of the epidemic. But how does it work? In some areas, potato growers can now telephone computerized systems, supply specific weather data, and receive advice on the necessity or otherwise for crop spraying to prevent late blight. Simpler programs call for the grower to use a hygrothermograph and a plastic rain gauge to keep track of the daily maximum, minimum, and mean temperature and daily rainfall on a standardized record sheet. A blight-favourable day is recorded as a ‘+’, and the first spray is dictated by ten successive ‘+’ days. Every seven days after this, the need for spraying is reassessed, using humidity, temperature, and rainfall data. A weather-timed spray program has been devised to keep Alternaria and Cercospora leaf blights of carrot from killing more than 15% of the leaf area. If this level is exceeded, harvesting machinery tends to tear off the weakened leaves, leaving the carrot in the ground. No fungicide is applied until the blight covers 1%–2% of the leaf area. Then fungicide is applied before the next forecast rain or before the next night with a forecast minimum temperature of 16°C or higher. At least seven to ten days are allowed between subsequent sprays, which are applied in conditions similar to those for the first spraying. Sprays are not needed before forecast rains when the night temperature will be below 9°C, because the fungal spores infect the leaves only when these are both warm and wet for an extended period. Although wetness is vital for the successful establishment of most leaf diseases, the xerotolerant powdery mildew fungi are less affected by moisture and more by the availability of inoculum: levels of airborne conidia. A third factor is the increasing susceptibility of some crops as they age. Programs have now been worked out to determine the critical date for a single application of fungicide to forestall powdery mildew of barley, the date of the first spray to control powdery mildew of rubber, and the timing of successive sprays against powdery mildew of apple. The value of plant disease forecasting is, as one might expect, economic. In the carrot blight situation, an average of two to five sprayings were saved by following the program. The saving resulted from delaying the first spraying, and making subsequent spraying contingent upon the existence of conditions favourable to infection, rather than ritually spraying every so many days. Each program must be designed or modified to take into account conditions prevailing in the area where it will be applied.

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Table 12.1: Some Important Fungal Diseases of Plants Pathogen

Host(s)

Disease

Control

Alternaria solani (hyphomycetes)

potato, tomato

early blight

Mancozeb

Armillaria mellea (Agaricales)

forest trees

butt rot

Botrytis cinerea (hyphomycetes)

lettuce, tomato, strawberry, etc.

grey mould

Captan, Benomyl

Bremia lactucae (oomycetes)

lettuce

downy mildew

Dithiocarb

Ceratocystis fimbriata (ascomycetes)

sweet potato

black rot

Cercosporella herpotrichoides (hyphomycetes)

wheat, barley

eyespot

cultivar

Claviceps purpurea (ascomycetes)

rye, other grasses

ergot

clean seed

Colletotrichum lindemuthianum (coelomycetes)

beans

anthracnose

clean seed

Diplocarpon rosae (ascomycetes)

roses

black spot

Captan

Erysiphe graminis + Blumeria (ascomycetes)

cereals (grasses)

powdery mildew

cultivar, Tridemorph

Heterobasidion annosum (basidiomycetes)

conifers

root rot, butt rot

shorten rotation

Forest Pathology The American sweet chestnut, Castanea dentata, once grew from Maine to Alabama. It was a fine tree that thrived even in poor soil and on steep hillsides. Some specimens in the Great Smoky Mountains were 4 metres in diameter and 40 metres high. Chestnut wood was extensively used for fencing and roofing, to make furniture and to build barns. During the burgeoning industrial revolution of the nineteenth century it was used for lining mine shafts and in mine roof supports, as railroad ties, as telegraph

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Chapter 12 poles, and as fuel. Chestnut trees shared pride of place with elms as street and shade trees. Appalachian farmers fattened their hogs on chestnuts, which were also roasted and used in meat stuffings. The chestnut was the most economically important tree in the eastern hardwood forests. Near the end of the nineteenth century, chestnut seedlings imported from Asia to New York brought with them the fungus Cryphonectria (Endothia) parasitica (ascomycetes, Dothiorales). This introduction found a defenceless host in the American chestnut. The first diseased trees were noticed in 1904 in the New York Zoo, and from then on, the epidemic mounted and spread remorselessly. The early issues of Mycologia, the journal of the Mycological Society of America, contain repeated rather plaintive references to the advancing plague. Despite their agonizing concern, mycologists and foresters could only watch helplessly as millions of chestnut trees died, and the face of the forests in eastern North America changed. By the 1940s or 1950s practically all the mature American chestnut trees had died, although living roots are still sending up sprouts that reach a fair size before being killed by the fungus, and some large trees still survive, because they are apparently infected by a hypovirulent strain of the pathogen (apparently the result of its becoming infected by a virus). All that is history now, and it’s too late to do much about it, except to slowly reintroduce scions of surviving trees to areas in which the species used to grow. What of current concerns in forestry? The lumber industry is a mainstay of the economy in many areas of North America, but until recent years forests were ‘mined’ with little thought to replacement, since the resource was assumed to be virtually infinite, or at least entirely self-renewing. In Canada, a combination of depletion of first-growth forests by clear-cutting, heavy tree mortality due to insects and diseases (which together cause an annual loss of almost 130 million cubic metres of wood), and extensive forest fires combined to produce a potential wood shortage. Even if only 20 million cubic metres could be saved, this would provide 39,000 jobs, $800 million in wages and salaries, and forest products worth $2.9 billion. All this makes fungal diseases important, because they are one of the main factors contributing to the losses. Tree diseases are often distinctly unspectacular in appearance, and their effects are insidious rather than dramatic. Trunk decays and root rots progress steadily, year in, year out. Once established in a tree, they cannot be eradicated. In Canada alone, the various rots cause a combined loss estimated at 30 million cubic metres of wood each year. Root rot caused by Phellinus (Poria) weirii (basidiomycetes, Hymenochaetales) is widespread in West Coast forests and is especially destructive in Douglas fir (Pseudotsuga menziesii). Despite extensive studies, we still have no cost-effective way of preventing or eliminating this problem. Nevertheless, it appears that losses could be reduced by earlier cutting of infected stands, selective cutting to favour the establishment of less susceptible tree species, and the use of red alder in rotation to reduce the amount of Phellinus inoculum in the soil. Other silvicultural practices that would be helpful include stump removal, fumigation, prescribed burning, fertilization, interplanting, sanitation, biological control, and host tolerance or resistance. Heart rot caused by Fomes pini (basidiomycetes) is also a severe problem in western forests. Trees with signs of internal decay should be cut because Fomes pini, unlike many other rot fungi, doesn’t cause further decay after harvest. Root rot caused by

Fungal Diseases of Crops and Trees Heterobasidion annosum (basidiomycetes), an aggressive parasite that infects cut stumps, spreads from root system to root system, and kills many different conifers, was the subject of nearly 600 publications between 1960 and 1970. Recommended management procedures include wide spacing in new plantations; preventing stump infection by applying inoculum of Peniophora gigantea (basidiomycetes; biological control by a saprobic competitor); decreasing the number of thinnings per rotation; removing as much of the stump and taproot as possible during logging; regenerating by seeding instead of planting; and using resistant species. At present, the single best way to reduce losses caused by decay fungi appears to be to shorten the rotation time; in the Southern United States, heart rot losses in pines have been reduced from 30% to below 1% simply by decreasing the age at which trees are harvested. A survey of forest pathologists across Canada showed that root rot caused by Armillaria mellea (Agaricales) was the only disease placed in the ten most important diseases for all six forest regions and was among the top three for all except the Quebec region. White pine blister rust, caused by Cronartium ribicola (Pucciniomycetes), was among the top ten diseases in all regions of Canada but one. This disease produces spreading cankers on branches or the main trunk that may eventually girdle and kill the tree, meanwhile producing the aecial stage of the fungus. This fungus, like many others causing rust diseases, has two hosts, so eradication of the Ribes spp. has been widely practised. Cronartium comandrae, cause of the similar comandra rust on lodgepole pine and other two- and three-needled pines, is not susceptible to this form of control, because its alternate host is a common and inconspicuous herbaceous plant. In the Southern United States another stem rust, called fusiform rust, caused by Cronartium fusiforme, is responsible for losses of more than $30 million a year. This disease is on the increase as a result of (1) the use of infected nursery stock; (2) widespread monoculture of susceptible tree species; and (3) an increase in the alternate host, red oak, following improved fire prevention. The only practical control measures are the use of fungicides in nurseries and the breeding of resistant tree cultivars. Gremmeniella abietina (ascomycetes) causes a serious canker of conifers, especially pines, in Northeastern North America. The fungus has two races. The American race is very widespread in Ontario north of latitude 45 and kills many young trees in their first decade. Once more than two metres tall, trees seem able to survive the depredations of this race. But since 1975 the European race has been found killing mature pines in New York State and in a few locations in Quebec, New Brunswick, and Newfoundland. All infected material discovered has been destroyed, and the situation is being closely monitored.

Integrated Pest Management Integrated pest management (IPM) is now a concept to be reckoned with. Although the idea hasn’t yet been fully incorporated into agricultural and forestry practice, it is the next logical step for plant pathology. This approach considers all the pests and pathogens which attack a particular crop and develops an overall plan to control them. The crop is considered as an ecosystem, and all factors influencing that system are taken

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Chapter 12 into account. Instead of simply applying chemical sprays at regular intervals, all possible control measures are considered: sanitation, crop rotation, cultivation practices, sowing date, plant spacing, use of resistant cultivars, disease forecasting, and biological control as well as chemical control. Sprays may be fewer but more complex, with components aimed at widely differing organisms, such as fungi and insects. Obviously, IPM calls for a lot of preliminary analysis and detailed but flexible planning—processes that are facilitated by computers. We can expect to see a lot more IPM in future, because its sophistication will result in less expensive pest control and will reduce our use of, and dependence on, chemical pesticides.

Websites and Further Reading on Plant Pathology The University of Kansas Department of Plant Pathology has a series of extension Service web pages which give information on many plant diseases and their management. The lead-in page is at http://www.ksu.edu/plantpath/extension. The University of Florida has a plant disease management guide on the web at http:// edis.ifas.ufl.edu/topic_book_plant_disease_management_guide. Agrios, G. N. 2005. Plant Pathology. 5th ed. Burlington: Elsevier. American Phytopathological Society. 1977–1988. Disease Compendium Series. St. Paul. [alfalfa, barley, beet, citrus, corn, cotton, elm, grape, ornamental foliage plants, pea, peanut, potato, rhododendron, azalea, rose, sorghum, soybean, strawberry, sweet potato, turfgrass, and wheat] Carefoot, G. L., and E. R. Sprott. 1967. Famine on the Wind. New York: Rand McNally. Gurr, S. J., M. J. McPherson, and D. J. Bowles. 1992. Molecular Plant Pathology: A Practical Approach. Vol. 1. Oxford: Oxford University Press. Holliday, P. 2001. A Dictionary of Plant Pathology. Cambridge, UK: Cambridge University Press. James, C. 1971. A Manual of Assessment Keys for Plant Diseases. Canada Department of Agriculture Publication No. 1458. St. Paul: American Phytopathological Society. Johnston, A., and C. Booth, eds. 1983. Plant Pathologists’ Pocketbook. 2nd ed. Kew: Commonwealth Mycological Institute. Large, E. C. 1962. The Advance of the Fungi. New York: Dover. [A timeless standard and a good read] Roane, M. K., G. J. Griffin, and J. R. Elkins. 1986. Chestnut Blight, Other Endothia Diseases, and the Genus Endothia. St. Paul: APS Press. Roberts, D. A., and C. W. Boothroyd. 1972. Fundamentals of Plant Pathology. San Francisco: Freeman. Schmidt, O. 2006. Wood and Tree Fungi. Biology, Damage, Protection, and Use. Berlin: Springer.

13 Fungicides—Several Generations—More Needed Necessary but Toxic? Protectant or Systemic? Evolution of Resistance in Pathogens Introduction Agriculture was the single invention that allowed the human population to increase exponentially. Without it, most of us would not be here today. There could never have been very many hunter-gatherers—that way of life did not permit population explosions. But with agriculture came many headaches, not the least of which were plant diseases and pests. Fungi, insects, bacteria, and viruses have ravaged our crops ever since we domesticated plants. As soon as we started to grow many of the same kind of plant close together (a monoculture), any other organisms that made a living from that kind of plant suddenly found life much easier, since the next meal (called a host plant) was sitting right beside the previous one. This chapter deals only with problems caused by true fungi (Eumycota) and pseudofungi (Oomycota). Until about 150 years ago, humans had no idea what caused most plant diseases, and until we learned that pathogenic fungi were actually extraneous, spore-dispersed living organisms, rather than ‘humours’ or ‘effluvia of the earth, or of thunder, or of snakes’, we couldn’t do anything about it. So, for example, the destruction of the Irish potato crop by Phytophthora infestans (Oomycota) during the 1840s went completely unchecked, for all its terrible effects on the human population. The first practical fungicide wasn’t devised until forty years later, by a university professor in France. Even today, over a third of all crop losses are due to fungal diseases. They cost American farmers billions of dollars each year in lost produce. Some pathogenic fungi (e.g., Puccinia graminis: Pucciniomycotina) can be controlled by breeding resistant plant varieties. But many commercial apple varieties are still susceptible to apple scab (caused by the Spilocaea pomi conidial anamorph of the ascomycete Venturia inaequalis). The conditions conducive to infection are precisely known and occur up to twenty times each growing season. Unprotected orchards may produce no saleable fruit at all, so fungicide must be applied six to fifteen times each year. 265

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Chapter 13 Billions of dollars are now spent, worldwide, on fungicides of all kinds. This chapter is an exploration of our increasingly sophisticated efforts to combat pathogenic fungi with chemicals—we started out by poisoning them and then progressed to less toxic agents that interfered with various parts of the growth or reproduction of the fungi, and from external treatments to those that travel throughout the plant.

The First Generation: Inorganic Fungicides In the 1880s, the famous vineyards of Bordeaux in France were being devastated by a recent accidental introduction from America, Plasmopara viticola (Oomycota), which causes downy mildew of grape. Strolling through a vineyard at St. Julien in the Medoc, Professor Millardet was surprised to see that the vines bordering the path looked much healthier than those farther away. When he asked the vigneron how these plants had been treated, he was told that it was the custom to spatter the vines near the path with some conspicuous, poisonous-looking substance such as verdigris, to deter passersby from eating the grapes. Millardet, who knew a lot about the fungus and its habits (much had been learned since the terrible potato blight epidemics), went away and concocted a variety of witches’ brews, optimistic that he could poison the fungus when it was at its most vulnerable, just after the wind-dispersed mitosporangia had released their delicate zoospores on the wet surface of the plant. He finally settled on a blend of copper sulphate and calcium hydroxide (quicklime). This soon became famous as Bordeaux mixture (in French, ‘Bouillie Bordelaise’). Other copper sulphate–based fungicides followed: Burgundy mixture, in which the lime was replaced by sodium carbonate, and Cheshunt mixture, in which it was replaced by ammonium carbonate. Although Bordeaux mixture is an efficient, wide-spectrum fungicide, it has now largely been replaced by formulations of copper oxide, copper hydroxide, and copper oxychloride (Fig. 13.1).

Fig. 13.1 Cupro-Euparene label.

Fungicides—Several Generations—More Needed Bordeaux mixture, and almost all other fungicides developed before 1960, are called protectants: they are toxic to pathogenic fungi, but only if they intercept the fungi outside the host plant. If the plant’s exterior is not coated with fungicide, the fungus can slip through the defence. Once inside their host, many pathogens can’t be reached by the chemical and have only the plant’s internal defences to deal with. Inorganic fungicides also have the potential to damage the plant itself, especially if they get inside it (they are phytotoxic as well as fungitoxic), and they can be washed off by rain. This loss of fungicide plus new plant growth necessitates repeated spraying during the growing season and ultimately, after many years of use, can lead to a buildup of toxic substances in the soil. Long-term use of Bordeaux mixture on grape vines has produced concentrations of up to 130 ppm copper in the soil. One early alternative to Bordeaux mixture was sulphur, applied as elemental sulphur or as lime sulphur. It is not toxic to animals and is still occasionally used to control powdery mildews, apple scab, and peach leaf curl, but it may ‘scorch’ leaves, causing them to drop, and can have a dwarfing effect on plants. Mercurous chloride was also found to be an excellent broad-spectrum fungicide (heavy metals denature a wide range of enzymes), but its residues can cause both acute and chronic toxicity in animals. Its LD50 to rats—the amount that will kill half of the animals exposed to it—is 1–5 mg/kg, and long-term exposure to even very low levels of mercury eventually causes severe brain damage (check out Minamata disease).

The Second Generation: Organic Fungicides and Seed Dressings The organomercurials were the first of a new generation of fungicides developed in response to the problems with mercurous chloride. They retained the persistent fungitoxicity of mercury, but in compounds that were less poisonous to animals (their LD50 [rat] ranges from 30 to 200 mg/kg). The general formula for many organomercurials is RHgX, where R=aryl or alkyl and X=chloride, acetate, etc. For example, the protectant organomercurial Ceresan is 2-methoxyethyl mercuric chloride. It was obviously unwise to spray or broadcast such toxic compounds, and fungicides containing mercury were mostly used as seed dressings (although phenylmercuric acetate was used in orchards for twenty years until it was officially banned in 1971). Organomercurials successfully controlled many seed-borne and soilborne diseases such as rots, seedling blights, and damping-off, but they have now been replaced by less toxic chemicals (see Thiram, Captan, Carboxin). Organotin fungicides were similar in principle to the organomercurials. They were often relatively phytotoxic, but one of them, triphenyltin hydroxide (Du-ter), was widely used to control potato blight. Its LD50 (rat) is 108 mg/kg, and it is believed to act by uncoupling oxidative phosphorylation. An organocopper, copper naphthenate (Cuprinol), cannot be used as a plant spray or a seed treatment: it is a broad-spectrum biocide (kills everything) and is still used as a wood preservative. The phenols, another group of organic fungicides, are like copper naphthenate in that they are disinfectants (general biocides) rather than protectants. Pentachlorophenol is widely used as a wood preservative and in the control of slime development

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Chapter 13 during papermaking, although environmental concerns are now inhibiting some of its applications. Another phenol, 4,6-dinitro-o-cresol (DNOC), is used as a disinfectant spray for orchard floors. It is aimed at the saprobic overwintering stages of such pathogenic fungi as Venturia inaequalis, whose teleomorph develops in dead apple leaves, and provides the ascospore inoculum that reinfects the host in spring. DNOC is also used in ‘dormant sprays’ (sprays which would damage living leaves so are applied to kill fungi and other pests while fruit trees are dormant). Phenols apparently work by uncoupling oxidative phosphorylation. The LD50 (rat) of pentachlorophenol is 210 mg/kg, and that of DNOC is 25–40 mg/kg. The 1930s saw the introduction of the dithiocarbamates, an important family of organic, protectant fungicides with very low phytotoxicity. There are dimethyl-dithiocarbamates and ethylene-bis-dithiocarbamates. The dimethyl-dithiocarbamates include Thiram, Ferbam, and Ziram. Thiram is used as a seed treatment to control damping-off diseases. It has an LD50 (rat) of 400–900 mg/kg. Ferbam and Ziram are used to combat leaf pathogens and have LD50s (rat) of 17,000 and 1,400 mg/kg, respectively. The ethylene-bis-dithiocarbamates (EBDCs) include Nabam, Maneb, Mancozeb, and Zineb. The LD50s (rat) of these four fungicides are 400, 7,000, over 8,000, and 5,200 mg/kg, respectively. As you can see, the last three seem to be particularly nonthreatening to animals. But the EBDC fungicides are not really safe: when they break down, ethylene thiourea, a carcinogen, is formed. This happens when EBDC-contaminated plant parts are cooked. Ethylene thiourea causes teratological effects (malformed offspring) in rats at a dosage of only 10 mg/kg. Despite this drawback, the dithiocarbamates are still important organic, protectant fungicides. This is because when copper compounds were replaced by dithiocarbamates, potato yields rose dramatically because the dithiocarbamates caused so much less damage to the plants. As you might expect, special precautions are now taken to avoid contamination of food with dithiocarbamate residues. What do you think the most important of these precautions might be? Another important group of protectant, organic fungicides are the phthalimides. The best-known of these is Captan, although Captafol (Difolatan) and Folpet (Phaltan) were also widely used. Captan was registered in Canada in 1951 as a foliar treatment and a seed dressing, often in mixes with other fungicides. It has a very short half-life in soil or water, and has little toxicity to mammals: its LD50 (rat) is 9,000 mg/kg. Like heavy metals, it acts on many sites in the target fungi, so resistance is unlikely to develop. Captan is widely used on fruit crops, especially apples, peaches, and strawberries, to control many pathogens. It has been estimated that without this fungicide, 25% of the fruit crop would be lost. It is also used to protect conifer seedlings against grey mould (Botrytis) and powdery mildew. Millions of kilograms of Captan are used each year. It is the centre of some controversy, since it has been alleged to have some carcinogenic effects, and has been partially banned in Sweden. Recent North American studies find the accusations unproven, point out that no fully effective substitutes are available, and suggest that fruit growers go on using it, conservatively, and with strict precautions. Some quinones are used as organic, protectant fungicides. Dichlone (Phygon) is one of the most effective treatments currently approved for use against apple scab. Chloranil and Dichlorane control downy mildews (Oomycota); Chloranil is so

Fungicides—Several Generations—More Needed

Fig. 13.2 Roadside billboard advertising Difolatan, an organochloride, for use on peanuts. It is no longer used because it has been identified as a carcinogen.

effective that $775,000 invested in it brought a return of $19,000,000 to the pea industry in a single year.

The Third Generation: Systemic Fungicides A new generation of fungicides commenced with the appearance of the benzimidazoles in the 1960s. Benomyl (Benlate) (Fig. 13.2) was the first systemic or eradicant fungicide—the first to get inside the plant and kill the fungus where it had previously been safe from attack. Benomyl is apoplastic—it accumulates mainly between, rather than in, living cells and travels upward in the transpiration stream flowing through the nonliving xylem vessels. Since it is not retained in living cells, it does not move downward in the phloem. A benzimidazole fungicide applied near the ground may travel up to the growing tips of a plant but not down into the roots. Benomyl was stable and nontoxic—its LD50 (rat) is 10,000 mg/kg—and it was effective against many ascomycetes and their conidial anamorphs at extremely low doses. Whereas Maneb was applied at 5.4 kg/ha (kilograms of active ingredient per hectare) to control apple scab, a mere 0.3 kg/ha of Benomyl also did the job. Better still, because Benomyl was systemic, fewer sprays were needed to achieve control. Even Benomyl’s mode of action was new—it is absorbed by the spindle fibres of dividing ascomycetous nuclei, disrupting microtubule assembly and so aborting the division process. But even Benomyl was not a panacea. It became widely available for use on apples in 1973 and was immediately widely adopted by apple farmers because it controlled all

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Chapter 13 major fungal diseases of this crop. By 1975—only two years later—some apple pathogens had begun to develop resistance to the new fungicide. Before very long, both Venturia inaequalis (the cause of apple scab) and Penicillium expansum (which causes a storage rot) had become completely resistant to Benomyl. The registration of Benomyl was subsequently allowed to lapse (registration must normally be renewed every few years). Some of the new ambimobile systemic fungicides can move into new plant growth—tissue that was not directly treated with the fungicide. The amount of mobility varies: some can move up in the phloem and down in the xylem, but many cannot move out of a treated leaf (DeMethylation Inhibitors, or DMIs) and some cannot move more than just through the thickness of the leaf (some Quinone Outside Inhibitors, or QoIs). But even this low mobility can be valuable, because it allows the fungicide to reach the underside, where many plant pathogens concentrate their activities and their sporulation. Some are also selective: they kill certain groups of fungi and not others. This should enable us to treat certain diseases with low potential for disrupting the soil mycota or discouraging the vital mycorrhizal fungi. Some of the newer phosphorous acid fungicides, for example Fosetyl-Al (Aliette, aluminum ethyl phosphite) and the Phenyl amidesmefenoxam (Ridomil, Subdue, Apron), act selectively on members of phylum Oomycota: genera such as Phytophthora, Pythium, Plasmopara, and Peronospora, which cause root rots, Fig. 13.3 Benomyl label. This damping-off, and downy mildews. Mefenoxam, like metawas the first systemic fungicide, laxyl, the related active ingredient commercialized before but it is no longer used. it, controls many of these fungi, including Pythium and Phytophthora on soybeans, Phytophthora on strawberries, and Plasmopara on grapes. After the very destructive epidemic of blue mould (Peronospora tabacina), which rocked the tobacco farmers of Ontario in 1979, Metalaxyl was quickly registered for soil application to control this disease. Aliette is registered for use against many diseases. As a bonus, it has been shown to stimulate the growth of certain endomycorrhizal fungi. Another newer family of systemic but nonselective fungicides are the sterol inhibitors. These act by preventing the biosynthesis of ergosterol, the major sterol in many fungi. Since ergosterol is a basic component of fungal membranes, any shortage will severely curtail fungal growth. Examples of this new group are Bitertanol (Baycor), Fenarimol (Rubigan, Bloc), Triforine (Funginox, Saprol), Etaconazole (Vangard), Triarimol (Trimidal), Prochloraz (Sportak), and Fendapanil. On apple, sterol inhibitors give good control of scab (Spilocaea anamorph of Venturia inaequalis: ascomycetes), powdery mildew (Oidium anamorph of Podosphaera leucotricha: ascomycetes), and cedar-apple rust (Gymnosporangium juniperi-virginianae:

Fungicides—Several Generations—More Needed Pucciniomycotina) if applied weekly—they lack long-term residual activity. On stone fruits, sterol inhibitors control brown rot (Monilia anamorphs of Monilinia spp.: ascomycetes), leaf curl (Taphrina spp.: ascomycetes), shot hole (Coryneum sp.: coelomycetes), and cherry leaf spot (Coccomyces hiemalis: ascomycetes). In other words, they inhibit the development of a wide range of unitunicate and bitunicate ascomycetes, ascomycetous anamorphs, and some basidiomycetes as well. Earlier, I mentioned that the dithiocarbamate protectant Maneb, used to control apple scab at 5.4 kg/ha, could be replaced by the benzimidazole systemic Benomyl at 0.28 kg/ha. Now that fungicide has been delisted, but the sterol inhibitor systemic Fenarimol will provide control at an even lower dosage: only 0.065 kg/ha. But as you probably suspect, sterol inhibitors aren’t perfect, either. On stone fruits they don’t work well against peach scab (Cladosporium carpophilum: hyphomycetes), Rhizopus (Zygomycota) fruit rot, or Alternaria (hyphomycetes) fruit rot. And some resistance has already developed in certain pathogens, such as powdery mildews (Erysiphales: ascomycetes).

An Unexpected Fourth Generation—the Strobilurins In the early 1980s, it was found that a small agaric, Strobilurus tenacellus, found fruiting on pine cones, produced a fungicidal substance, soon called Strobilurin A (β-methoxyacrylic acid). In the laboratory, it was effective against almost all fungi tested. However, the strobilurin molecule was sensitive to light and oxygen, and it did not work well in tests involving plants infected by fungi. In 1983, BASF chemists set about modifying the molecule in such a way that it would no longer be unstable. By herculean efforts, over Fig. 13.4 A small agaric, Strobilurus trullisatus. This thirteen years they synthesized more than 15,000 variants of the genus is the source of some widely used fungicides. natural ingredient, which were then tested for suitability as fungicides. Eventually they were successful, and the first commercial fungicides of this new class went on the market in 1996. These fungicides rapidly assumed great importance, because they have protectant, systemic, and eradicant activities. All strobilurin, or QoI fungicides, bind at the Qo site on cytochrome b, inhibiting mitochondrial respiration. Strobilurin fungicides are unique in that they are the first synthetic, site-specific compounds to provide significant control of plant diseases caused by pathogens from both major groups of true fungi, Ascomycota and Basidiomycota, as well as from the Oomycota (chromistan pseudofungi). Azoxystrobin,

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Chapter 13 under the trade names Quadris and Abound, was the first strobilurin registered by the U.S. Environmental Protection Agency (EPA). Trifloxystrobin (Flint, Stratego, and Compass) was next. Pyraclostrobin (Cabrio and Headline) followed. Strobilurins are Qo inhibitors (QoI), or quinone outside inhibitors—chemical compounds which act at the quinol outer binding site of the cytochrome bc1 complex. QoIs include three fungicide families, the strobilurins and two new families, represented by fenamidone and famoxadone. I quote from an article by Margaret Tuttle McGrath: Quadris [Azoxystrobin] is now registered for use on almost all vegetable crops... for managing Cladosporium leaf blotch, purple blotch, rust, downy mildew, and Rhizoctonia damping-off in bulb crops (onion, garlic, etc.); early blight, late blight, and Rhizoctonia damping-off in carrot and in celery; rust, gray leaf spot, Northern corn leaf blight, Northern corn leaf spot, and Rhizoctonia root and stalk rot in corn; anthracnose, belly rot, downy mildew, gummy stem blight/black rot, Alternaria and Cercospora leaf spots, Myrothecium canker, powdery mildew and Rhizoctonia root rot in cucurbits; Alternaria, Cercospora, and Septoria leaf spots, anthracnose, downy mildew, powdery mildew, white rust, and Rhizoctonia diseases (e.g. bottom rot) in leafy vegetables; early blight, late blight, black dot, powdery mildew, black scurf and silver scurf in potato; anthracnose, early blight, late blight, black mold, powdery mildew, buckeye rot, Septoria leaf spot, and target spot in tomato; and Alternaria, Cercospora, and Ascochyta leaf spots, powdery mildew, rust, white rust, Aphanomyces root rot, southern blight, Pythium root rot, and Rhizoctonia crown rot in the root and tuber subgroup (beets, carrots, ginseng, radishes, etc.) and in the tuberous and corm subgroup (artichokes, sweet potatoes, etc.). Strobilurin fungicides accounted for over 20% of the global fungicide market within ten years of their commercial release. Azoxystrobin is one of the world’s most heavily used fungicides and is applied extensively to control a wide range of important fungal pathogens of vegetables. All fungicides in each chemical group act in the same way and must be managed carefully to avoid the appearance of fungicide resistance, since some resistance has already been observed in many crops. It is worth noting that strobilurins are also being marketed for improving yield—an effect achieved through their growth regulatory activity—and, because of their mode of action, have also been used as experimental antibiotics and anticancer agents (see chapter 23). The last fungicide I will mention really belongs in a chapter on biological control (although not the one which follows this chapter, since that deals only with uses of fungi in biocontrol). Serenade Garden is a formulation of Bacillus subtilis strain QST 713 that attacks and controls powdery mildew fungi on squash, cucumber, melons, and pumpkins—in fact the family Cucurbitaceae—as well as anthracnose, Botrytis blights (grey mould), downy mildew, head and leaf drop, leaf spot, pink rot, and Sclerotinia stem rot. It is EPA approved and can be used in organic gardening. It is apparently nontoxic to plants and beneficial insects, and crops treated with it can be harvested the same day if necessary. Sounds almost too good to be true....

Fungicides—Several Generations—More Needed

Resistance to Fungicides As you read through the earlier sections of this chapter, you probably noticed that the phenomenon of target resistance to fungicides became troublesome only after the introduction of the systemic fungicides. It transpired that the resistance was developing, not because these fungicides were systemic but because they acted on very specific sites within the fungus. A broad-spectrum fungicide like mercury poisons so many enzyme systems—it is a multisite fungicide—that only an absolutely inconceivable number of simultaneous genetic alterations could confer resistance on a pathogen. But Benomyl operated by interfering specifically with microtubule assembly in ascomycetes, and it became apparent that some target organisms, when repeatedly exposed to Benomyl, rapidly evolved strains that were resistant to this and other MBC (methyl benzimidazole carbonate) fungicides. This story has been repeated with each new family of systemics. Resistance also has been reported to phenylamides, carboxamides, strobilurins, sterol inhibitors, and succinate dehydrogenase inhibitors. The first documented case of resistance was to Dodine, a protectant/eradicant (LD50 [rat]= 1,000 mg/kg) used against apple scab, rather than to a systemic. Resistance was reported in 1969, after this fungicide had been used exclusively in some orchards for ten years. Resistance to Benomyl was noted in 1975, after that fungicide had been used exclusively and repeatedly for only two years. Orchardists who used Benomyl+ Dodine began to experience resistance to the combination in 1978. We now realize that it is often best to use mixtures of unrelated fungicides or to apply a sequence of different fungicides, as part of an integrated pest management scheme. Had this been done initially with Benomyl, we might have experienced fewer resistance problems with this fungicide. A species of the mould genus Lauriomyces (named after my wife by Rafael Castañeda in 1990), has now been discovered by Japanese biochemists to produce unique compounds that are strongly antifungal. It remains to be seen whether these compounds will be developed for commercial use.

Choice, Formulation, and Application of Fungicides The many different diseases to which single crops are subject often call for a variety of fungicides. Mefenoxam, a systemic acylalanine, is very effective against an oomycete like Phytophthora infestans, which causes late blight of potato, but not against an ascomycetous anamorph like Alternaria solani, which causes early blight. So the farmer has to use something like Mancozeb, an ethylene-bis-dithiocarbamate (EBDC), as well, to control the Alternaria. Interestingly enough, the Phytophthora has also developed resistance to mefenoxam, and a mefenoxam/Mancozeb mix works better than either fungicide alone. Ridomil, a commercial formulation of mefenoxam, and some other phenylamides are now sold only mixed with protectant, residual fungicides (mancozeb, copper, or chlorothalonil). Stone fruit fungicide trials involved mixing or alternating sterol inhibitors with other fungicides such as Benomyl, Captafol, Captan, Chlorothalonil, Dichlone, Dichloran, Dodine, and Thiophanate-methyl.

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Chapter 13 Perhaps I can place the development of fungicides and our attitudes toward them in perspective by giving a case history. Hops (Humulus lupulus), an invaluable flavour ingredient in beer (adds the bitterness), suffer from a destructive downy mildew caused by Pseudoperonospora humuli (Oomycota). At the beginning of the twentieth century this was treated with Bordeaux mixture. More recently, Zineb, an EBDC, was the fungicide of choice. But this breaks down to the carcinogenic ethylene thiourea, so the brewing industry asked that it be abandoned. Mefenoxam is an excellent substitute, but signs of resistance have appeared, so this ambimobile systemic is now often mixed with a protectant such as copper oxychloride. Satisfactory control involves using (1) resistant hop cultivars, (2) sanitation—removing infected material, (3) timely application of fungicides. Many systemic fungicides are almost insoluble in water, and the plant cuticle keeps waterborne substances out as well as in. So if these fungicides are dispensed as wettable powders (WP), after the spray droplet has dried, most of the fungicide will still be outside the plant. More fungicide gets in if it is supplied as an emulsifiable concentrate (EC) and if surfactants (to lower the surface tension of the spray and make it spread out over the surface of the plant) and humectants (to slow the drying of the droplets) are incorporated. These measures allow lower dosages to be used. Sprayers and nozzles have been developed which increase the efficiency of application by dispersing the fungicide in finer droplets than ever before, or by placing an electrostatic charge on the droplets, so that they will be drawn directly to the plant. The most efficient use of fungicides for seed and root diseases is obviously as seed dressings. Corn is grown on over 40 million hectares in the United States, and over 90% of the seed is treated with fungicide. Without this treatment, it is estimated that yield would be reduced by 10%–12% in most years. Seed treatment results in some contamination of the soil, but since the newer fungicides are not very persistent, and the amounts applied per hectare are minute, this is not a serious problem. Seed and root dips are also sometimes employed. Many crops need more than seed dressings to adequately control their diseases, especially those occurring on aboveground parts. Cotton receives seed treatment, in-furrow treatments, and some foliar sprays for cotton rust. Without fungicides, it is estimated that 20% of the cotton crop would be lost. Peanut leaf spot (Cercospora: hyphomycetes) is potentially devastating. No resistant cultivars exist; crop rotation doesn’t help in controlling leaf diseases; and the conditions for infection (leaves wet for four to six hours or near 100% R.H. at temperatures above 22°C) exist almost every day in the Southeastern United States. Fungicides have to be applied every two weeks. Losses due to leaf spot diseases are now 2.5%–15.0%. Without fungicide, this figure would soar to 20%–75%, and peanuts would not be worth growing. I quote from a 2011 article by Wyenandt and Maxwell: To date, there are 43 numbered and three lettered FRAC (Fungicide Resistance Action Committee) codes for the approximately 86 chemical codes and 171 common names of fungicides listed (FRAC, 2009). Accordingly, fungicides listed within a given FRAC code may share a similar mode-of-action and therefore have similar risks for resistance development and similar use patterns on single or multiple crops, and they may also exhibit the potential for cross-resistance

Fungicides—Several Generations—More Needed development. Other industry-sponsored resistance management groups have been established to monitor resistance development in weeds (Herbicide Resistance Action Committee, HRAC) and insect pests (Insecticide Resistance Action Committee, IRAC). No discussion of fungicides would be complete without some mention of other techniques for controlling or eradicating fungi. (1) Soil sterilization may involve steam or dry heat treatment or chemicals such as formalin, chloropicrin, and methyl bromide (although this last is now widely banned). Some of the same chemicals are also used to control arthropods and fungi in stored food. The Canadian government has recently banned five chemical fumigants, including ethylene dibromide, which has been identified as a carcinogen, and most other available fumigants are under investigation. (2) Antimould compounds are often added to paints, fabrics, paper, cosmetics, soaps, etc. Many modern fungicides are good candidates for such uses, because they have low solubility in water, are nontoxic to mammals, are biodegradable, and are not very persistent. (3) Mould inhibitors—weak acids such as sorbic, benzoic, acetic, or propionic acid, or their esters, which are fungus inhibitors rather than fungicides—are added to some foods. Calcium propionate, for example, is added to bread to extend its shelf life—a segue into chapter 20.

Further Reading Bartlett, D. W., J. M. Clough, J. R. Godwin, A. A. Hall, M. Hamer, and R. Parr-Dobrzanski. 2002. “The Strobilurin Fungicides.” Pest Management Science 58, no. 7:649–62. doi:10.1002/ps.520. Canadian Journal of Plant Pathology—recent issues. Fungicide Resistance Action Committee. http://www.frac.info. Johnston, A., and C. Booth, eds. 1983. Plant Pathologist’s Pocketbook. 2nd ed. Kew, UK: Commonwealth Mycological Institute. Marsh, R. W., ed. 1977. Systemic Fungicides. 2nd ed. London: Longman. McGrath, M. T. 2009. “Efficacy of Various Biological and Microbial Fungicides—Does That Really Work?” Paper presented at the New England Vegetable and Fruit Conference, Manchester, NH, December 15–17. Naqvi, S. A. M. H. 2004. Diseases of Fruits and Vegetables: Diagnosis and Management. Vol. 2. New York: Springer. [704 pp.] Phytopathology—recent issues Wyenandt, A., and N. L. Maxwell. 2011. “Evaluating Fungicide Recommendations for Vegetable Crops in the United States: Should More Be Done to Limit the Risks of Fungicide Resistance Development?” Journal of Extension 49, no. 3. https://www.joe.org/joe/2011june/a8.php.

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14 Fungi as Agents of Biological Control Introduction In recent years we have begun to understand the consequences of the widespread and repeated use of chemical biocides to control the host of organisms, such as insects, weeds, and fungi, that threatens human interests. You probably know that while many pests became resistant to persistent pesticides like DDT (a chlorinated hydrocarbon), predatory birds such as the peregrine falcon suffered population crashes as a result of the biological accumulation of DDT residues. Since we, too, are at the top of many food webs, this and other examples could hardly be ignored. We soon phased out the more persistent pesticides, at least in North America, and intensified the search for replacements. Newer generations of pesticides are less persistent but are often very toxic to many nontarget organisms, including the natural enemies of the pests and, not too surprisingly, humans. The elimination of natural enemies may allow outbreaks of secondary pests, and rapid resurgence of the target species, once the pesticide loses its activity. To make things worse, some pests soon develop resistance to each new formulation. Nevertheless, many chemical pesticides give quick results and a high level of control, and no substitutes are yet available for most of them, so we will inevitably go on using them for many purposes; but it makes good sense to look for less dangerous alternatives. Biological control—often shortened to biocontrol—is one of these alternatives. How does biocontrol work? We begin by looking for a natural predator, parasite, or competitor of the organism to be controlled, then we try to shift the ecological equilibrium in favour of this biocontrol agent so that it can drastically reduce the population of the target organism. These are simple principles, but their practical application is often difficult. Natural enemies of pests and pathogens may be few, rare, or inconspicuous. They may be found only in restricted areas or at specific times of year; they may have complex life histories involving two hosts; and they may attack friend as well as foe. They may even have been left behind when the pest ‘migrated’ to a new area or continent. It often takes patient detective work to bring them to light and then years to test their host range, develop techniques to mass produce them, and learn the most effective ways and times to introduce them to the host population. It is encouraging to know that biocontrol has already had several spectacular successes—you may already be aware of the moth (Cactoblastis: Lepidoptera) which was introduced to Australia to control the prickly pear (Opuntia: Cactaceae) which was taking over vast areas of grazing land, and the myxomatosis virus which was 276

Fungi as Agents of Biological Control introduced to control the population explosion of rabbits. I will tell you about a few other famous victories (which naturally involve fungi) and about some promising or potential applications of fungi in this area. Most early attempts at biocontrol pitted one arthropod against another, for example, ladybugs (Coleoptera, Coccinellidae) against aphids (hom*optera, Aphididae). But the fungi are, potentially at least, better biocontrol agents than any arthropod because (1) Fungi have an extremely high reproductive capacity. (2) Fungi have a very short generation time. (3) Fungi are often highly specific in their action, attacking only the host with which they have co-evolved. (4) Fungi often produce resting stages or saprobic phases that can survive for a long time when no host organism is available. If you will also compare these four features with the characteristics of chemical pesticides, you will understand the advantages of using fungal biocontrol whenever possible. So why haven’t the fungi cornered the market? The problem was partly one of perception, partly one of practice. Under natural conditions, the population of a fungal parasite may build up to very high levels, but not quickly enough to control the target organism during the period when it causes the most damage. There’s not much advantage in a fungus killing off most of our houseflies in September when the nuisance has been around all summer and the frosts of October would have done the job anyway. So fungi have several potential shortcomings as biocontrol agents. (1) They may only damage, rather than kill, their host. (2) They may only reduce, rather than eliminate, the target population. (3) They may do both of these things relatively slowly. These outcomes are not entirely satisfactory to humans, who are used to the quick action and high kill rate of chemical biocides. But the nontoxic, target-specific, self-reproducing, self-perpetuating characteristics of fungi are persuasive incentives for considering them as alternatives, so we are making efforts to overcome their deficiencies. Several critical factors must be manipulated before we can count on success. (1) It must be established that the biocontrol fungus is not pathogenic to any economically valuable organisms that might be exposed to it. (2) A large amount of inoculum must be available. (3) This must be properly distributed early enough to saturate the target population well before that reaches its peak. (4) Climatic conditions must favour growth, sporulation, and dispersal of the fungus. Where can we make effective use of fungi in biocontrol? Principally in three areas: (1) control of arthropod or other invertebrate pests, (2) control of weeds, and (3) control of fungi causing plant diseases or biodeterioration. I’ll examine these areas in turn and give some case histories. Even where fungi alone cannot give effective biocontrol, they may often be usefully combined with other agents, biological and/or chemical, in an integrated pest management program. (Integrated pest management, or IPM, is discussed in chapter 12.)

(1) Control of Animal Pests by Entomogenous Fungi Arthropods, and particularly insects, are our greatest competitors. They damage or destroy our crops before and after harvest and transmit many fatal or debilitating diseases. In the developing countries, insect control is often a matter of life or death. A

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Chapter 14 number of fungi are lethal parasites of arthropods; in most cases the fungal spores are released in enormous numbers and can infect the host at any stage of its life cycle. The spores germinate on the host cuticle, the germ tube penetrates the chitinous exoskeleton, and branching hyphae riddle the viscera. Spore-bearing structures of the fungus eventually emerge from the corpse, liberating fresh inoculum. We are only now learning how to exploit entomogenous fungi in biocontrol. Several of these fungi cause spectacular epidemics in natural insect populations and are now being grown in large-scale artificial culture to produce inoculum with which we hope to induce epidemics on demand. I will mention examples from three fungal phyla; these range from established success stories to promising newcomers, with one case of tantalizing, but as yet unfulfilled, potential. Most of my examples are illustrated in Fig. 14.1. Several hyphomycetes have proved so successful that spray concentrates

Fig. 14.1

Genera of fungi used in biocontrol of arthropods.

Fungi as Agents of Biological Control containing their spores are now sold under trade names as mycoinsecticides—but don’t look for them yet at your local hardware store or nursery. (1) Beauveria bassiana (hyphomycetes) has a fascinating history. In the early 1800s the so-called muscardine disease was ravaging the silkworm industries of Europe. Silkworms died, their corpses hardened, and a white bloom appeared on them. The disease spread rapidly through silkworm colonies, but no one knew what caused it, although there was some notion that it was probably ‘environmental’ in origin. Bassi, an Italian scientist, subjected the larvae to the most barbarous treatments: ‘the poor creatures died by thousands and in a thousand ways’. Eventually he discovered that the disease was caused by an ‘infectious principle’, which he identified as the white powder on the mummified corpses. He even recognized that it was a parasitic fungus. So originated, in 1834, the germ theory of disease—a milestone in the history of biology. Conidia of the fungus around which the theory was conceived are now mass produced as a preparation called Boverin and have been used in Russia since 1965 to control the Colorado potato beetle (Leptinotarsa: Coleoptera). This pest, if left unchecked, will completely strip potato plants of their leaves. The Boverin, which contains 30 billion conidia/g, is sprayed onto the potato fields twice, at a rate of 1.0–1.5 kg/ha, with fifteen to twenty-one days between applications. Boverin also controls codling moth (Carpocapsa: Lepidoptera), whose larvae cause enormous losses by tunnelling into young apples. In damp seasons, naturally occurring B. bassiana helps control chinch bugs (Blissus leucopterus) in lawns. Beauveria bassiana strain GHA (trade names Mycotrol GH-OF and Mycotrol GH-ES) is registered to control grasshoppers, locusts, and Mormon crickets on rangeland, improved pastures, alfalfa, corn, cotton, potatoes, rapeseed, safflower, small grain crops, soybeans, sugar beets, and sunflowers. The host range of Beauveria includes such important pests as whiteflies, aphids, grasshoppers, termites, Colorado potato beetle, Mexican bean beetle, Japanese beetle, boll weevil, cereal leaf beetle, bark beetles, lygus bugs, chinch bug, fire ants, European corn borer, codling moth, and Douglas fir tussock moth. (2) Metarhizium anisopliae (hyphomycetes), available as a commercial mycoinsecticide under the name Metaquino, is widely used in Brazil to control spittlebugs (hom*optera, Cercopidae) on sugarcane and in pastures (the nymphs suck large quantities of sap from their host plant to make a protective foam; the adults are called froghoppers). The same hyphomycete has also been used with great success as part of an integrated pest management program in the South Pacific islands of Tongatapu and Western Samoa. The Rhinoceros beetle (Oryctes) arrived on these islands in about 1930. It subsequently killed all newly planted coconut palms by chewing up the young shoots and similarly prevented most of the existing palms from reproducing. The introduction of Metarhizium and an entomopathogenic virus in 1968 soon controlled the beetle. Now, young trees survive, and old trees once again bear fruit. Mosquito larvae of the genera Anopheles, Aedes, and Culex are also attacked by Metarhizium, as are spruce budworm larvae. Metarhizium is registered in the United States for control of household co*ckroaches. (3) Hirsutella thompsonii (hyphomycetes) causes spectacular epidemics each year among populations of citrus rust mite (Phyllocoptruta: Acarina) in Florida—but only after the fruit has been damaged by the pest. Mass produced as a mycoacaricide under

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Chapter 14 the trade name Mycar, it is now sprayed early in the season to prevent the buildup of mite populations. (4) Verticillium lecanii (hyphomycetes) causes natural epidemics in two groups of plant-sucking pests: aphids (hom*optera, Aphidoidea) which cause malformation and transmit viruses, and scale insects (hom*optera, Coccoidea) in the tropics and in greenhouses. Its conidia are now available commercially under the trade names Vertalec and Mycotal. Vertalec contains a strain which is highly pathogenic to aphids, while Mycotal incorporates another strain that is lethal to greenhouse whitefly. (5) Paecilomyces fumosoroseus Apopka strain 97 (hyphomycetes) has been approved for use on ornamentals, nonfood crops in greenhouses and interiorscapes to manage whiteflies, aphids, thrips, and spider mites. (6) Nomuraea rileyi (hyphomycetes) doesn’t yet have a trade name but is nevertheless an effective mycoinsecticide, causing high mortality in caterpillar pests (Lepidoptera, Noctuidae) on cabbage, clover, and soybean. It is being intensively studied for potential large-scale agricultural applications. (7) My last example of a fungus being mass produced for use in biocontrol of insects is Entomophthora (Entomophthoromycotina, Entomophthorales). This may well be the only entomogenous fungus that most people will ever see. It infects and kills houseflies, which for some reason usually crawl into an exposed location before dying. The cause of death can be ascertained from the masses of sporangiophores emerging through the insect’s cuticle, or from the halo of discharged sporangia around the victim. Roland Thaxter, who made an incredibly productive life’s work of exploring the fungi growing on insects, was drawn to this field when an epidemic of Entomophthora decimated the fly colony maintained at Harvard for experimental purposes. Although species of Entomophthora attack aphids, houseflies, caterpillars, and grasshoppers, their use in biocontrol has been hindered by the short lifespan of their sporangia and by the great dependence of the fungus on such factors as rainfall, temperature, and host density. The spotted alfalfa aphid (Therioaphis) was first detected in Australia in 1977. It had somehow contrived to arrive without the Entomophthora pathogens that often kill it in North America. Entomophthora was introduced into Australian populations of the aphid in 1979 and is apparently spreading. This theme, of a pest reaching a new country and leaving its parasites or predators behind, is a recurring one and often presents an opportunity for biocontrol. (8) Dutch elm disease is caused by an ascomycete, Ophiostoma ulmi, but is transmitted by bark beetles. In Britain it has recently been observed that a coelomycetous anamorph, Phom*opsis oblonga, occurring naturally in the bark of the elms, discourages or disrupts the breeding of the local vectors, Scolytus scolytus and Scolytus multistriatus (Coleoptera). This may help control the spread of the disease. (9) Coelomomyces (Blastocladiales) is an obligate parasite of mosquito larvae and sometimes causes heavy mortality in natural populations of such important diseasecarrying mosquitoes as Anopheles gambiae, a notorious vector of malaria. Although natural epidemics are fairly common occurrences, attempts to infect larval mosquito populations were unsuccessful. The reason for this became clear in the mid-1970s, when it was discovered that Coelomomyces requires a copepod or an ostracod as an obligate alternate host if it is to complete its life cycle. This problem may well prevent

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Fungi as Agents of Biological Control this fungus from being exploited in the biocontrol of mosquitoes. Yet since these insects are probably the most important pests in the world—there are 7 million cases of malaria each year in Africa and half a million deaths, almost all of them children— strenuous efforts are being made to overcome this impasse. If you have sympathy to spare for insects, save it for larval scale insects (hom*optera, Coccoidea) and whiteflies (hom*optera, Aleyrodidae)—they are susceptible to the widest range of entomogenous fungi. They can be attacked by members of the Chytridiomycetes, Zygomycetes, unitunicate and bitunicate ascomycetes, and conidial fungi (hyphomycetous and coelomycetous anamorphs of ascomycetes and basidiomycetes). Since scales and whiteflies are difficult to control by chemical means, I think we may eventually use mycoinsecticides routinely to keep them in check. Another potential application for biocontrol is in the suppression of arthropods that infest stored food, where it is impossible, for obvious reasons, to use regular pesticides. Table 14.1 lists some of the actual and potential uses of fungi in biocontrol of arthropods.

Table 14.1. Genera of fungi used in biocontrol of arthropods Genus

Trade Name

Phylum

Principal Target

Coelomomyces

Chytridiomycota

Mosquito larvae

Entomophthora

Zygomycota

Aphids

Conidiobolus

Zygomycota

Aphids

Beauveria

Boverin

Ascomycota (hyphomycetes)

Colorado beetle, codling moth

Hirsutella

Mycar

Ascomycota (hyphomycetes)

Citrus rust mite

Metarhizium

Metaquino

Ascomycota (hyphomycetes)

Spittlebug, mosquito larvae, rhinoceros beetle, lepidopteran larvae

Verticillium

Vertalec

Ascomycota (hyphomycetes)

Aphids

Verticillium

Mycotal

Ascomycota (hyphomycetes)

Whitefly

Nomuraea

Ascomycota (hyphomycetes)

Lepidopteran larvae

Aschersonia

Ascomycota (coelomycetes)

Whitefly, scale insects

Nematodes, rotifers, copepods, tardigrades, collembola, soil amoebae, and other microscopic animals are also parasitized or preyed upon by fungi. I use the latter phrase advisedly, because a number of microfungi (again from several major taxonomic

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Chapter 14 groups) are actually predators of these animals: they have evolved special trapping devices with which they physically catch their prey, thereupon sending in hyphae to exploit the newly acquired substrate. Other parasitic fungi have small spores of unusual shapes, which when eaten by the unsuspecting animal, catch in its gullet and colonize its viscera. But these stories, the pictures that will help you to visualize this strange microcosm, and the possible roles of such fungi in biocontrol can be found in chapter 15.

(2) Control of Weeds by Plant Pathogenic Fungi Now for a look at the second major area of fungal involvement in biocontrol. The target organisms here are higher plants: pioneer species of remarkable vigour which compete only too well with our domesticated plants. Weeds—they have even spawned a strange verb, ‘to weed’ (which actually means ‘to deweed’), and every gardener pays tribute on his or her knees to their success. Until recently, farmers could control weeds only with various forms of cultivation, but now they can call on broad-spectrum herbicides like Paraquat and selective weed killers such as 2,4-D and 2,4,5-T. These control dicotyledonous weeds in monocotyledonous crops (fortunately, many of our staple food plants are grasses—wheat, corn, rice, millet, sorghum, oats, barley, rye). And, of course, they used to help us to keep marginally ahead of the dandelions which grow so well in our lawns. But these weed killers, after being freely broadcast for years, were discovered to have ingredients that are toxic and teratogenic (causing developmental defects). And no herbicide is available to control annual grasses in small grains. Even at their best, chemical herbicides lack the finely tuned selectivity of many plant pathogenic fungi, which often restrict their attacks to a single host species. For a discussion of how fungi attack and damage or destroy plants, refer to chapter 12. Of the more than 300,000 plants in the world, a mere 200 species cause almost all of our weed problems. Two-thirds of the world’s worst weeds are present in North America, and crop losses caused by weeds in the United States are estimated to cost $14 billion a year. Many weeds are plants which have been accidentally introduced to a new area without their natural enemies. Of 117 common weeds in Canada, more than three-quarters were introduced from other continents. The classical biocontrol strategy is to search in the weed’s homeland for fungal pathogens that help to keep it in check there. This section details several examples of fungi (most of which are illustrated in Fig. 14.2) which are actual or potential biocontrol agents for weeds. Rust fungi (pucciniomycetes, Pucciniales) are often extremely host specific, and it is fitting that my first two examples of fungal herbicides should be members of this group. (1) When European blackberry (Rubus sp., Rosaceae) began to encroach on ranges and pastures in Chile, introduction of a European rust fungus, Phragmidium violaceum, successfully suppressed its spread. Unlike wheat rust, this species needs only one host to complete its life cycle—we say it is autoecious—so no other plants were threatened by its introduction. (2) A Mediterranean member of the Asteraceae, Chondrilla juncea (‘rush skeleton weed’), was accidentally introduced into Australia in the early 1900s, leaving its natural enemies behind. It spread rapidly and infested hundreds of thousands of hectares

Fungi as Agents of Biological Control

Fig. 14.2 Genera of fungi used in biocontrol of weeds.

of wheatland, competing with the wheat for water and nitrogen and clogging harvesting equipment. By the 1940s some farmers had given up growing wheat. Those who persevered were later able to spray with 2,4,5,-T to control it, although the cost of spraying vast areas, which in Australia give low yields anyway, was almost prohibitive. In 1966 a search for potential biocontrol agents was mounted in the Mediterranean region. By 1971 a rust fungus from Italy, Puccinia chondrillina, was being released in Australia. The introduction was so successful that almost half a million hectares no longer need to be sprayed. This program has already saved Australia 112 times its

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Chapter 14 cost, in one of the most spectacular successes ever achieved with biocontrol. There is a footnote to this story: on its home territory in the Mediterranean region, Puccinia chondrillina itself has a fungal hyperparasite (Eudarluca caricis: coelomycetes), and the material being introduced to Australia had to be carefully checked to make sure it was not contaminated by this hyperparasite, which might have reduced its effectiveness. (Unhappily, Chondrilla is also the generic name of a sponge.) In each of the examples just given, the fungus is obligately biotrophic, so inoculum cannot be mass produced in artificial culture. In these cases, small amounts of natural inoculum have been introduced to the area in which the host plant is growing, and further spread of the pathogen has been by natural spore dispersal. In other examples of fungal biocontrol of weeds, of which I will mention five, it has been necessary to mass produce the fungal propagules and apply them as a mycoherbicide spray. This massive inoculum swamps any host resistance, and if conditions are right initiates an epidemic. (3) Northern joint vetch, Aeschynomene virginica (Fabaceae), infests rice and soybeans in the United States. It is severely attacked and often killed by the coelomycete Colletotrichum gloeosporioides, but in many areas low levels of natural inoculum seem to preclude development of an epidemic. Accordingly, plants were sprayed with a suspension containing 2–6 million conidia/mL, and 95%–100% of the sprayed plants subsequently succumbed. This was the first practical mycoherbicide, which has now been patented and is being produced under the trade name Collego. The fungus over winters on and in joint vetch seeds, but this natural inoculum must be augmented each year. (4) Strangler vine, Morrenia odorata (Apocynaceae), is a weed of Florida citrus groves that can overgrow mature citrus trees. It is now controlled by a commercial mycoherbicide, Devine, which contains an oomycetous pathogen, Phytophthora palmivora. The pseudofungus causes a root and stem rot and can kill mature vines in three to four weeks. One pint of the liquid suspension contains nearly a million chlamydospores/mL and treats an acre when diluted in 50 gallons of water. The oomycete, once established in the soil, persists well from year to year. (5) A search has recently been made for potential biocontrol agents of water hyacinth (Eichhornia crassipes: Commelinales), a beautiful but prolific aquatic plant which has clogged waterways, lakes, and reservoirs in many parts of the tropics. In 1976 a previously unknown species of hyphomycete, Cercospora rodmanii, was found causing a local epidemic on Eichhornia in Florida. This fungus has now been patented as a mycoherbicide and is being produced commercially, following extensive research to ensure that it is not harmful to nontarget plants or animals. (6) Another hyphomycete, Acremonium zonatum, which is also pathogenic to water hyacinth, may yet be used along with the Cercospora and other agents in an integrated weed control program, but there is still some concern about its host range, since it is known to attack two valuable crop plants, figs (Ficus) and coffee (Coffea). This demonstrates the care that must be taken to ensure that biocontrol agents are without unwanted side effects. But since the potential for fungal control of many weeds exists, it is surely a matter of time before this is developed, especially as the more insidious effects of chemical herbicides come to light.

Fungi as Agents of Biological Control (7) A species of Colletotrichum (coelomycetes) is now used in some parts of China to control the parasitic flowering plant Cuscuta (‘dodder’), which can be a serious weed on certain crops. (8) Phom*opsis convolvulus is being tested in Canada as a potential biocontrol agent for field bindweed, Convolvulus arvensis. Other examples of actual or potential mycoherbicides are listed in Table 14.2.

Table 14.2. Actual (A) and potential (P) applications of fungi in weed control Fungal Biocontrol Agent

Weed Target

Crop Infested and Area

(A) Puccinia chondrillina (Uredinales)

Chondrilla juncea (rush skeleton weed)

wheat, Australia

(A) Phragmidium violaceum (Uredinales)

Rubus sp. (blackberry)

pastures, Chile

(A) Colletotrichum xanthii (coelomycetes)

Xanthium spinosum (Bathurst burr)

rangeland, Australia

(A) C. gloeosporioides (coelomycetes) Collego

Aeschynomene virginica (northern joint vetch)

rice, soybean, United States

(A) Phytophthora palmivorum (Oomycota) Devine

Morrenia odorata (strangler vine)

citrus, Florida

(P) Ascochyta pteridium (coelomycetes)

Pteridium aquilinum (bracken fern)

pastures, Britain

(P) Colletotrichum malvarum (coelomycetes)

Sida spinosa (prickly sida)

cotton, soybeans, United States

(P) Colletotrichum dematium (coelomycetes)

Cassia occidentalis (coffee senna)

pastures, United States

(P) C. dematium (coelomycetes)

Convolvulus arvensis (field bindweed)

sorghum, United States

(P) C. coccodes (coelomycetes)

Abutilon theophrasti (velvetleaf)

lima beans, United States

(P) C. gloeosporioides (coelomycetes)

Jussiaea decurrens (winged water primrose)

rice, United States

(P) Acremonium sp. (hyphomycetes)

Cassia surattensis (kolomana)

pastures, Hawaii

(A) Acremonium diospyri (hyphomycetes)

Diospyros virginiana (persimmon)

rangeland, United States

(P) Cercospora lantanae (hyphomycetes)

Lantana camara (lantana)

rangeland, Hawaii

(A) Cercospora eupatorii (hyphomycetes)

Eupatorium adenophorum (crofton weed)

Australia

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Table 14.2. (continued) Fungal Biocontrol Agent

Weed Target

Crop Infested and Area

(P) Alternaria cassiae (hyphomycetes)

Cassia obtusifolia (sicklepod)

cotton, soybean, United States

(P) Alternaria macrospora (hyphomycetes)

Anoda cristata (spurred anoda)

cotton, United States

(P) Fusarium lateritium (hyphomycetes)

Anoda cristata

cotton, United States

(P) Fusarium lateritium (hyphomycetes)

Sida spinosa (prickly sida)

cotton, soybean, United States

(P) Fusarium solani (hyphomycetes)

Cucurbita texana (Texas gourd)

(A) Cercospora rodmanii (hyphomycetes)

Eichornia crassipes (water hyacinth)

water, tropics

(P) Acremonium zonatum (hyphomycetes)

Eichornia crassipes (water hyacinth)

water, tropics

(P) Cercospora piaropi (hyphomycetes)

Eichornia crassipes (water hyacinth)

water, tropics

(P) Fusarium roseum (hyphomycetes)

Eichornia crassipes (water hyacinth)

water, tropics

(P) Sclerotinia sclerotiorum (ascomycetes)

Cirsium arvense (Canada thistle)

many crops, Canada, United States

(P) Phom*opsis convolvulus

Convolvulus arvensis (field bindweed)

many crops, many countries

(A) Chondrostereum purpureum (basidiomycetes) Chontrol and Ecoclear

(Alders, aspen and other hardwoods)

rights of way, Canada

(3) Fungi in Biocontrol of Other Fungi This third area in which fungi have biocontrol potential may initially seem strange, but I’m sure you will quickly appreciate the logic of setting a fungus to control a fungus. The main reasons can be stated very briefly: (a) Some fungi are parasitic on other fungi—I’ve already mentioned one example in discussing the biocontrol of Chondrilla in Australia. (b) Fungi often compete strenuously with one another for substrate. (c) Preinoculation of a host plant with avirulent strains of some normally pathogenic

Fungi as Agents of Biological Control fungi, or with close but nonpathogenic relatives of those fungi, can protect the plant from attack by virulent strains of the same fungi. I will discuss several examples of each approach (Table 14.3). (1) Sphaerellopsis filum (coelomycetes), often discussed in the literature under an older name, Darluca filum, is parasitic on many rust fungi (pucciniomycetes, Pucciniales). It is credited with keeping some rust diseases down to low levels in natural host populations, and it has been proposed as a potential biocontrol agent against the heteroecious rusts Cronartium strobilinum and Cronartium fusiforme (which cause serious blister rust diseases of pines), while they are growing on their other hosts, oak trees. The fungus can move from the oak to the pine only if teliospores are produced, so it is significant that in some natural populations of Cronartium strobilinum on oak, 93% of the rust sori were found to be infected with Sphaerellopsis, and only 0.8% formed teliospores. Researchers have concluded that the parasite was more likely to control C. strobilinum, which was growing actively in the oaks all summer, than C. fusiforme, whose few weeks of activity on the host did not give the Sphaerellopsis enough time to colonize the rust and control it. The future of Sphaerellopsis in rust control is still uncertain.

Table 14.3. Some actual and potential applications of fungi in the control of fungal plant diseases Target Fungus

Disease and Host(s)

Biocontrol Fungus

Rhizoctonia solani (anamorphic basidiomycetes)

damping-off, root rot, stem canker: many crops

Trichoderma viride (hyphomycetes)

Armillaria mellea (Agaricales)

root disease: trees

Trichoderma viride (hyphomycetes)

Stereum purpureum (basidiomycetes)

silverleaf: plum

Trichoderma viride (hyphomycetes)

Heterobasidion annosum (basidiomycetes)

root disease: trees

Trichoderma viride (hyphomycetes)

Heterobasidion annosum (basidiomycetes)

root disease: trees

Peniophora gigantea (basidiomycetes)

Pythium spp. (oomycetes)

damping-off: seedlings

Trichoderma hamatum (hyphomycetes)

Sclerotium rolfsii (sclerotial anamorph)

stem blight: peanuts

Trichoderma harzianum (hyphomycetes)

Verticillium fungicola (hyphomycetes)

dry bubble: mushrooms

Trichoderma sp. (hyphomycetes)

Fusarium roseum (hyphomycetes)

seedling blight: corn

Chaetomium globosum (ascomycetes)

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Table 14.3. (continued) Target Fungus

Disease and Host(s)

Biocontrol Fungus

Phytophthora cinnamomi (oomycetes)

root rot: trees and herbs (forty-eight families)

Leucopaxillus sp. (Agaricales)

Verticillium alboatrum (hyphomycetes)

wilt: cotton

Verticillium alboatrum (hyphomycetes)

Verticillium dahliae (hyphomycetes)

wilt: eggplant

Talaromyces flavus (ascomycetes)

Verticillium dahliae (hyphomycetes)

wilt: mint

Verticillium nigrescens (hyphomycetes)

Alternaria zinneae (hyphomycetes)

leaf spot: beans

Alternaria tenuissima (hyphomycetes)

Claviceps purpurea (ascomycetes)

ergot: grasses

Fusarium roseum (hyphomycetes)

Sphaerotheca fuliginea (ascomycetes)

powdery mildew: cucumbers

Cicinnobolus cesatii (coelomycetes)

Cronartium ribicola (basidiomycetes)

blister rust: pines

Tuberculina maxima (hyphomycetes)

Cronartium strobilinum (basidiomycetes)

blister rust: pines

Sphaerellopsis filum (coelomycetes)

Puccinia spp. (basidiomycetes)

rust: many crops

Sphaerellopsis filum (coelomycetes)

Sclerotinia sclerotiorum (ascomycetes)

watery rot: many crops

Coniothyrium minitans (coelomycetes)

Gaeumannomyces graminis (ascomycetes)

take-all: wheat

Phialophora radicicola (hyphomycetes)

Crinipellis perniciosa (basidiomycetes)

witches’ broom: cocoa

Cladobotryum amazonense (hyphomycetes)

Botrytis cinerea (hyphomycetes)

grey mould of strawberry

Gliocladium roseum (hyphomycetes)

(2) Tuberculina maxima (hyphomycetes) is another parasite of rust fungi that is active against Cronartium ribicola (pucciniomycetes), the cause of white pine blister rust, but although its biocontrol potential has been hinted at by various forest pathologists, it has not yet been exploited. (3) Cicinnobolus cesatii (coelomycetes) parasitizes powdery mildews (Ascomycota, Erysiphales) and is now being used as a spray to control Sphaerotheca on greenhouse cucumbers.

Fungi as Agents of Biological Control

Fig. 14.3 Fungal pathogens controlled by Trichoderma and Peniophora.

(4) Cladobotryum amazonense (hyphomycetes) gives control of Crinipellis perniciosa (Agaricales, Marasmiaceae), which causes a serious disease of cocoa, called witches’ broom. Competition between fungi is the area in which biocontrol of fungal pathogens has achieved its greatest successes. Species of Trichoderma (hyphomycetes), green moulds common in some forest soils, are powerful antagonists to many pathogens (Fig. 14.3). (5) Trichoderma viride both parasitizes the hyphae of many other fungi and produces an antibiotic. This double-barreled approach allows it to deal effectively with soil pathogens such as Rhizoctonia solani (a sterile basidiomycetous anamorph that causes a variety of diseases on many hosts) and Armillaria mellea (Agaricales, which kills many species of trees.

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Chapter 14 (6) Trichoderma harzianum, mass produced in culture and applied to soil, controls Sclerotium rolfsii (another sterile fungus which causes diseases of many hosts) on tomatoes and peanuts. A pharmaceutical company is developing T. harzianum as a commercial biocontrol for Sclerotium rolfsii. This could be indirectly frustrated by the fact that peanuts are very susceptible to attack by Cercospora (hyphomycetes), which causes a serious leaf spot disease. Repeated fungicidal sprays needed to control the Cercospora also reduce the population of Trichoderma and lead to an increase in stem blight caused by Sclerotium rolfsii. (7) Nevertheless, in France, Trichoderma spray concentrate was competitive in price with the first systemic fungicide, Benomyl (now deregistered) and is used to control Verticillium fungicola (hyphomycetes), a serious pathogen of the cultivated mushroom Agaricus brunnescens (Agaricales). (8) Some basidiomycetous pathogens of trees often gain entrance to their host through wounds. It has been found that application of Trichoderma spores to fresh wounds, such as those caused by pruning of plum trees, will prevent subsequent infection by Stereum purpureum (basidiomycetes, Russulales), which causes silverleaf disease. (9) Freshly cut tree stumps painted with a Trichoderma spore suspension will not be invaded by Heterobasidion annosum (basidiomycetes, Russulales, Bondarzewiaceae), a very serious root pathogen that spreads from tree to tree through root contact. (10) A commercial preparation of the saprobic Peniophora gigantea (Russulales, Peniophoraceae) is available for treating newly cut pine stumps to protect them from invasion by Heterobasidion. (11) Spraying apple leaves with spore suspensions of Chaetomium globosum (ascomycetes) reduces infection by the apple scab fungus, the Spilocaea anamorph of Venturia inaequalis. (12) Protection from some soilborne diseases can be obtained by treating seeds with biocontrol fungi. For example, spores of Chaetomium globosum will protect corn against seedling blight caused by Fusarium roseum (hyphomycetes). Spores of Penicillium spp. (hyphomycetes) will confer similar protection on peas. (13) Eggplant is started in pots before outplanting. Inoculation of the potting medium with spores of Talaromyces flavus (Ascomycetes, Eurotiales) has been found to reduce the incidence of wilt caused by Verticillium dahliae (hyphomycetes) by 67%– 76% and to increase yield by 18%–54%. Preinoculation, or, as it is sometimes called, cross-protection, is now receiving serious attention from plant pathologists (Fig. 14.4). (14) Application of weakly pathogenic strains of Verticillium alboatrum (hyphomycetes) protected cotton plants from more virulent strains of the same wilt disease fungus. This protection appears to accrue from a kind of immunization process. The weak pathogen, while doing little damage, stimulates the host plant to produce phytoalexins— specifically antifungal compounds—which are then ready to repel subsequent attacks by more pathogenic strains. Sometimes different species, rather than different strains of the same species, are used to induce cross-protection. (15) For example, the weakly pathogenic Verticillium nigrescens (hyphomycetes) induces resistance in mint plants to the more pathogenic, wilt-producing Verticillium dahliae.

Fungi as Agents of Biological Control

Fig. 14.4 Fungal biocontrol agents and their target pathogens (see text).

In 1980, Ontario growers harvested 33,000 tonnes of peaches worth over $14 million. But another 57,000 tonnes had to be imported. Ontario should be growing more peaches, yet peach production is gradually declining. Why is this? It is largely due to a fungal disease called peach canker, caused by the coelomycetous Cytospora anamorph of Leucostoma (ascomycetes). The Cytospora can’t attack healthy trees: it can gain access only through wounds, such as those regularly caused by pruning. Infections begin and spread during fall and spring dormancy. Each year the cankers spread and yield declines, and eventually the tree dies. Because of the deep-seated nature of the disease, only limited chemical control has been possible, even with the newest fungicides. Fortunately, there is now some prospect of biological control of peach canker by species of

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Fig. 14.5 More fungal biocontrol agents and their target pathogens (see text).

Trichoderma and Gliocladium (hyphomycetes), which are highly competitive saprobes and also actively parasitize many fungi. Grey mould of strawberries is caused by Botrytis cinerea (hyphomycetes) and has usually been combatted with the fungicide Captan. In recent years, the advisability of using Captan has been questioned. Fortunately, it has been established that the mycoparasite Gliocladium roseum (hyphomycetes) can also control Botrytis. We know that the crucial stage in the development of the problem is during the flowering of the strawberry, well before the fruit forms. An ingenious delivery system has been designed in

Fungi as Agents of Biological Control which honeybees leaving the hive are automatically dusted with about 50,000 Gliocladium conidia, which they deliver directly to the flowers. The years ahead should see many advances in biocontrol by fungi; certainly our increasing knowledge of the adverse effects of chemical biocides on the biosphere and on ourselves can only accelerate the search for alternatives.

Further Reading Anon. 1980. Proceedings of Workshop on Insect Pest Management with Microbial Agents. Ithaca, NY: Boyce Thompson Institute. Baker, K. F., and R. J. Cook. 1974. Biological Control of Plant Pathogens. San Francisco: Freeman. Baker, R., P. Hanchey, and S. D. Dottarar. 1978. “Protection of Carnation against Fusarium Stem Rot by Fungi.” Phytopathology 68:1495–501. Bastos, C. N., H. C. Evans, and R. A. Samson. 1981. “A New Hyperparasitic Fungus, Cladobotryum amazonense, with Potential for Control of Fungal Pathogens of Cocoa.” Transactions of the British Mycological Society 77:273–78. Burges, H. D., ed. 1981. Microbial Control of Pests and Plant Diseases 1970–1980. New York: Academic Press. Butt, T. M., C. W. Jackson, and N. Magano. 2001. Fungi as Biocontrol Agents: Progress, Problems and Potential. Wallingford, UK: CABI. Charudattan, R., and H. L. Walker. 1982. Biological Control of Weeds with Plant Pathogens. New York: Wiley. Cullen, D., F. M. Berbee, and J. H. Andrews. 1984. “Chaetomium globosum Antagonizes the Apple Scab Pathogen, Venturia inaequalis, under Field Conditions.” Canadian Journal of Botany 62:1814–18. Cullen, J. M., P. F. Kable, M. Catt. 1973. “Epidemic Spread of a Rust Imported for Biological Control.” Nature 244:462–64. Ferron, P. 1978. “Biological Control of Insect Pests by Entomogenous Fungi.” Annual Review of Entomology 23:409–42. Freeman, T. E. 1981. “Use of Conidial Fungi in Biological Control.” In Biology of Conidial Fungi. Vol. 2. Edited by G. T. Cole and B. Kendrick, 143–65. New York: Academic Press. Gutteridge, R. J., and D. B. Slope. 1978. “Effect of Inoculating Soils with Phialophora Radicicola var. Graminicola on Take-All Disease of Wheat.” Plant Pathology 27:131–35. Harman, G. E., I. Chet, and R. Baker. 1980. “Trichoderma hamatum Effects on Seed and Seedling Disease Induced in Radish and Pea by Pythium spp. or Rhizoctonia solani.” Phytopathology 70:1167–72. Hasan, S. 1981. “A New Strain of the Rust Fungus Puccinia chondrillina for Biological Control of Skeleton Weed in Australia.” Annals of Applied Biology 99:119–24. Julien, M. H., ed. 1987. Biological Control of Weeds: A World Catalogue of Agents and Their Target Weeds. 2nd edition. Wallingford, UK: CABI Publishing. Kelleher, J. S., and M. A. Hulme, eds. 1984. Biological Control Programmes against Insects and Weeds in Canada: 1969–1980. Farnham Royal, UK: Commonwealth Agricultural Bureaux. Lee, B. 1981. “Pests Control Pests: But at What Price?” New Scientist 89 (1236): 150–52.

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Chapter 14 Marois, J. J., S. A. Johnston, M. T. Dunn, and G. C. Papavizas. 1982. “Biological Control of Verticillium Wilt of Egg Plant in the Field.” Plant Disease 66:1166–68. Roberts, D. W., and R. A. Humber. 1981. “Entomogenous Fungi.” In Biology of Conidial Fungi. Vol. 2. Edited by G. T. Cole and B. Kendrick, 201–36. New York: Academic Press. Shah, P. H., and J. K. Pell. 2003. “Entomopathogenic Fungi as Biological Control Agents.” Applied Microbiology and Biotechnology 61:413–23. TeBeest, D. O., ed. 1991. Microbial Control of Weeds. New York: Chapman and Hall. TeBeest, D. O., and G. E. Templeton. 1985. “Mycoherbicides: Progress in the Biological Control of Weeds.” Plant Disease 69:6–10. Templeton, G. E., D. O. TeBeest, and R. J. Smith, Jr. 1979. “Biological Control of Weeds with Mycoherbicides.” Annual Review of Phytopathology 17:301–10. Trutmann, P., P. J. Keane, and P. R. Merriman. 1982. “Biological Control of Sclerotinia sclerotiorum on Aerial Parts of Plants by the Hyperparasite Coniothyrium minitans.” Transactions of the British Mycological Society 78:521–29. Verma, M., S. K. Brar, R. D. Tyagi, R. Y. Surampalli, and J. R. Valero. 2007. “Antagonistic Fungi: Trichoderma spp. Panoply of Biological Control.” Biochemical Engineering Journal 37:1–20. Webber, J. 1981. “A Natural Biological Control of Dutch Elm Disease.” Nature 292:449–51.

15 Fungi Exploiting Microscopic Animals Introduction As a tiny soil nematode wriggles along, its head passes through a tiny hoop. Its body follows, sliding smoothly through. Just as it is about to clear the hoop, the hoop suddenly inflates inward and grips the worm tightly. Thrash about as it will, the worm cannot escape. Soon its tail end triggers another ring trap. It has been captured by a fungus, and it is doomed to die (Fig. 15.1). Fungi are usually thought of as being slow and insidious in their lifestyle, insinuating themselves stealthily, silently penetrating and permeating the substrate with their hyphae and their enzymes. It comes as something of a shock to encounter fungi that set traps to catch animals or have spores that instantaneously inject their contents into their unsuspecting target. Yet these fungi, and others almost equally bizarre, exist in the microcosms of the soil, the compost pile, and the rotting log. Relatively few fungi attack large animals, and those which do are often specialists with a taste for keratin or opportunists able to grow at the body temperature of Fig. 15.1 A nematode caught by two constrict- birds and mammals or to attack injured fish. But as we descend the scale of size, we ing ring traps. eventually reach a point where any physical confrontation between fungus and animal becomes a much more even contest. The tiny animals that roam through the soil make excellent proteinaceous dietary supplements, so perhaps it is not surprising that the 150 or so fungi that have adopted this ‘carnivorous’ lifestyle are drawn from four major fungal phyla and one pseudofungal phylum (Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, and Oomycota). The mechanisms they have evolved in order to exploit this resource are diverse and ingenious. We will consider eleven such mechanisms. Bet you didn’t think there would be that many.

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Mechanisms for Infecting Nematodes and Other Small Animals (1) Motile spores. The Chytridiomycota and Oomycota have motile cells (cells with flagella), and in ‘carnivorous’ species these have taken on the responsibility of finding the prey. The uniflagellate spores of Catenaria (Chytridiomycota; Fig. 15.2A) swim to a nematode by chemotaxis and encyst on it near its mouth or anus before penetrating the cuticle and attacking its internal organs. The biflagellate spores of some species of Myzocytium (Oomycota; Fig. 15.2B) do the same thing, encysting on the surface of a new host and then penetrating its cuticle. The zoospores of other species of Myzocytium disperse actively for a short while, but if they don’t find a host soon, they conserve energy by encysting and developing a special adhesive bud, which can stick to a passing nematode. This makes these species a combination of categories 1 and 3 (see below).

Fig. 15.2 A: Catenaria (Chytridiomycota) infecting nematodes by motile zoospores; B: Myzocytium (Oomycota) releasing infective motile zoospores; C: Haptoglossa (Oomycota) releasing infective harpoon cells (see also Fig. 15.3); D: Meria (Hyphomycetes) producing infective sticky conidia.

Fungi Exploiting Microscopic Animals (2) Injected spores. The oomycetous genus Haptoglossa (Figs. 15.2C, 15.3) is unique among fungi. It produces spores which, although nonmotile, are sophisticated ‘harpoon cells’. A harpoon cell adheres to the substrate and sits with the ‘barrel’ pointing upward at a low angle (Fig. 15.3). It has a high turgor pressure and is triggered by contact with prey: a built-in line of weakness ruptures, and an internal tube with a harpoonlike tip is rapidly everted with sufficient force to penetrate the integument of the prey and inject sufficient genetic material into the animal to form a tiny infection unit. This is an extremely highly evolved mechanism: its considerable mechanical complexity can be clearly seen in Fig. 15.3. (The only comparable mechanisms I can think of are (a) that of the zoospore in Plasmodiophora, a colonial protozoan; and (b) that of the nematocysts of the animal phylum Cnidaria [corals, sea anemones, hydrozoans, and jellyfish, or medusae]. There is, of course, no suggestion of hom*ology here. These superficially similar mechanisms evolved entirely separately).

Fig. 15.3 Details of the harpoon cell of Haptoglossa.

After Haptoglossa gets inside the animal, it grows through the internal organs, absorbing nourishment from them. This is not healthy for the animal, which soon expires. Its corpse eventually houses several to many mitosporangia, which liberate large numbers of flagellate or injective propagules to begin the cycle again. Sexual reproduction can also occur, and another chromistan fungus, Myzocytium, sometimes fills its hosts with oogonia, each oogonium ultimately containing one thick-walled, resting zygote (oospore)—the whole looking rather like a pea pod. (3) Adhesive spores. Meristacrum is a common nematode-exploiting zygomycete. From a parasitized nematode arise tall sporangiophores with helically twisted apical regions. Sticky one-spored mitosporangia are produced on these and are forcibly shot away. If they don’t make contact with a nematode, they will germinate and form a small, adhesive-coated secondary spore at the top of a little stalk. These stick to nematodes; some cause infection, while others form secondary spores, and the host worm may thus spread the infection to other nematodes while it can still move about. Hyphomycetes are well represented among the nematode-exploiting fungi and have evolved the widest range of techniques for gaining access to the interiors of nematodes. Verticillium and Meria (Fig. 15.2D) use the sticky spore technique already mentioned.

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Chapter 15 Once penetrated by the germ tube arising from these conidia, the worm is doomed. After a few days, its body is riddled with assimilative hyphae. Then the fungus breaks out of this ‘capsule’ and produces characteristic conidiophores, bearing adhesive conidia. The conidia of Nematoctonus leiosporus (Fig. 15.4A), after becoming detached, develop a vertical extension that ends in a sticky, infective swelling. The assimilative hyphae inside the host have clamp connections, showing that this is a dikaryotic basidiomycetous anamorph. Several species of Nematoctonus have been shown to be anamorphs of species of Hohenbuehelia (basidiomycetes; see also method 8, below). (4) Ingested spores. Some hyphomycetes have evolved conidia that are designed to be eaten by their victims. The conidia of Harposporium anguillulae (Fig. 15.4B) are crescent shaped, with a sharp point at one end. These conidia literally stick in the craw

Fig. 15.4 A: Nematoctonus leiosporus (dikaryotic basidiomycetous anamorph) sticky conidia penetrating the host nematode and developing assimilative hyphae with clamp connections; B: Harposporium anguillulae (anamorph of Atricordyceps harposporifera: ascomycetes, Clavicipitales) producing chlamydospores and curved, pointed conidia that are ingested by the host worm.

Fungi Exploiting Microscopic Animals (actually the oesophagus) of the worm, and from this initial bridgehead their hyphae soon permeate the host. Eventually, new conidiophores arise from the defunct nematode. It has been discovered that the teleomorph of Harposporium anguillulae is Atricordyceps harposporifera (ascomycetes, Hypocreales), which attacks millipedes. This is the only case I know of in which the anamorph exploits one group of animals, the teleomorph another. Other species of Harposporium also have ‘edible’ conidia. Those of H. diceraeum (Fig. 15.5J) have a striking resemblance to a high-heeled shoe or clog: those of H. rhynchosporum (Fig. 15.5K) look like cartoons of small birds minus legs. In each case there is a subtle asymmetry and one or more sharp points, which undoubtedly combine to help the conidia lodge in the muscle of the worm’s buccal cavity or oesophagus. The longer conidia of H. helicoides (Fig. 15.5L) don’t pierce the gut wall mechanically but germinate in the intestine and infect the worm just as effectively from there.

Fig. 15.5 Conidia of nematode-exploiting fungi; the large spores (A–G) are of trapforming species, the small spores (H–O) either stick to nematodes or are eaten by them. A–G: Arthrobotrys spp.; H–L: Harposporium spp.; H: H. bysmatosporum; I: H. anguillulae; J: H. diceraeum; K: H. rhynchosporum; L: H. helicoides; M: Meria conospora; N, O: Nematoctonus spp.

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Chapter 15 Harposporium spirosporum has sinuate, twisted conidia which are very sharp at both ends. These conidia are eaten by rotifers and lodge in the gullet or mastax to initiate an infection. At least twelve species of the hyphomycete genus Pochonia parasitize rotifers after they ingest conidia. Although these conidia are not pointed, they still lodge in the mouth, gullet, or mastax and penetrate the body cavity of the animal in the usual way. Species of Pochonia are being developed as biocontrol agents for endoparasitic plant nematodes.

Mechanisms for Trapping Nematodes The remaining fungi that exploit nematodes do so by trapping or snaring them. The traps are of six different kinds but can initially be categorized as either adhesive or nonadhesive. Some fungi have evolved an efficient nematode glue (or glues) to which the cuticle of nematodes adheres instantly and strongly. Others lack this feature and have developed even more interesting alternatives: (5) Adhesive-assimilative hyphae. In some Zygomycota, the assimilative hyphae are covered with glue. Cystopage can be recognized by its thick-walled chlamydospores (it does not produce mitosporangia). Stylopage produces a few large spores on upright hyphae. (6) Adhesive side branches (Fig. 15.6D). A few species of Dactylella (hyphomycetes) have specialized adhesive-coated side branches on their otherwise nonsticky assimilative hyphae. These branches project from the substrate just far enough to ensure proper contact with passing nematodes. We presume that this simple form of trap gave rise to the more elaborate and sophisticated types reported below. Dactylella copepodii manages to capture copepods with adhesive branches (it also uses adhesive knobs; see method 7). (7) Adhesive knobs (Figs. 15.6A–C, 15.7A) are specialized, swollen cells, coated with nematode glue and are often situated at the ends of short side branches. They are found in nearly twenty species of Arthrobotrys, Dactylella (hyphomycetes) and Nematoctonus (anamorphic Basidiomycota). Sometimes the knobs are firmly enough attached to prevent a nematode from leaving the scene, particularly if the animal has stuck to several of them at once. Often the nematode tears a knob loose from its moorings and makes good its escape. But the ensuing freedom is short lived. The knob remains firmly attached to the worm’s cuticle and soon sends in an infective hypha. Game over. Some species of Nematoctonus which are anamorphs of the gilled fungi Hohenbuehelia and Resupinatus (Basidiomycota) produce unique adhesive knobs shaped like hourglasses enveloped in a drop of glue (Fig. 15.6C). These knobs do not break off, but hold nematodes firmly while infection proceeds. The clamped hyphae of Nematoctonus also bear conidia (Fig. 15.5N), but these are not infectious until they have germinated and formed a sticky knob at the end of the germ tube. (8) Adhesive nets (Figs. 15.6F, G) are probably the commonest trapping device, since they have been recorded in nearly forty species of fungi. They may originally have evolved by anastomosis of adjacent adhesive branches of assimilative hyphae (only eumycotan fungi can undergo anastomosis of assimilative hyphae), and some of them are

Fungi Exploiting Microscopic Animals

Fig. 15.6 Trapping devices of nematode-exploiting fungi. A–C: sticky knobs; A, B: Arthrobotrys candida (hyphomycetes); C: Nematoctonus sp. (anamorph of Hohenbuehelia: basidiomycetes, Agaricales); D: sticky branches of Dactylella cionopaga; E: nonconstricting rings of Arthrobotrys candida; F: net of Dactylella gephyropaga; G: net of Arthrobotrys oligospora; H: constricting rings of Arthrobotrys anchonia.

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Chapter 15 still simple hoops. Others are more complex, ranging from two-dimensional ladder-like arrangements to the contorted three-dimensional labyrinths of Arthrobotrys oligospora, the commonest nematode-trapping hyphomycete (Fig. 15.6G). These networks can arise only as a result of repeated anastomoses. The advantage of the more complex systems is not simply their greater extent but also the fact that larger nematodes are likely to become stuck at more than one point and are therefore less likely to escape. The adhesive works equally well on dry and wet nematodes (although not on other soil animals). The nematodes are not just blind, insensate victims: they often recoil violently when they touch an adhesive net—an aversive reaction that sometimes saves them from certain death. When the fungus is successful in establishing adhesion, an infective hypha soon penetrates the body of the prey, and the worm becomes comatose within an hour—so quickly, in fact, that the fungus is suspected of producing a toxin (see mechanism 11). After the prey has been riddled by absorptive hyphae, the energy in the converted biomass is translocated to the external hyphae, which use it, spider-like, to spin new nets and also to produce the distinctive tall conidiophores of Arthrobotrys oligospora, with their successive clusters of two-celled, colourless conidia (didymospores). (9) Nonconstricting rings (Figs. 15.6E, 15.7A), which could also be called detachable rings, are produced by four species of Arthrobotrys and Dactylella (hyphomycetes). A single hypha grows around in a perfect circle, finally anastomosing with a new bud waiting at the top of the stalk-to-be. The three-celled ring is stouter than the stalk. When a nematode crawls through the ring, this fits snugly around its body (a not too surprising evolutionary adaptation) and easily breaks away from the narrow stalk. The worm goes on its way wearing its newly acquired collar. As you will have deduced, infection and assimilation soon follow. All species with nonconstricting rings produce sticky knobs as well (Fig. 15.7A). In terms of the spread of the pathogen, it is interesting to note that fungi producing sticky spores, or detachable knobs or loops, may be carried some distance by the animal before it becomes incapacitated, so possible future inoculum may be more widely dispersed. (10) Constricting rings (Figs. 15.1, 15.6 H, 15.7 B–D, 15.8A, B) are the most sophisticated nematode traps of all. They are produced by twelve hyphomycetes, especially species of Arthrobotrys and Dactylella. At first sight, these traps seem very similar to nonconstricting rings: the ring is composed of three cells, borne on a stalk. But here the stalk is shorter and stronger: these traps are designed to stay put. Their true nature is revealed only when they are triggered. If a nematode passes through the loop and touches the inside of one or more of the cells, all three cells simultaneously inflate inward, in about one-tenth of a second, and the nematode is held in a vise-like grip (Figs. 15.1, 15.7D). The inflated cells soon squeeze the worm so tightly that it is virtually garroted. Rings can be triggered by mechanical stimulation or by heat, when no nematode is present, and in this case the three cells expand to three times their original volume—until they touch one another and the centre of the ring is completely occluded. Between the contact stimulus and the implosive response there is a delay of a few seconds. If a worm is lucky, it may retract during that period of grace, leaving a trap sprung but empty. How do these traps work? A variety of experimental and observational techniques have now provided us with a reasonable hypothesis. The three cells of each constricting ring trap have a high turgor pressure, generated by a high internal osmotic pressure.

Fungi Exploiting Microscopic Animals

Fig. 15.7 Arthrobotrys (hyphomycetes). A: A. candida with detachable sticky knobs and detachable nonconstricting rings; B–D: A. brochopaga; B: developing constricting ring trap; C: germinated conidium which has formed a constricting ring trap; D: conidium with a constricting ring trap which has caught a nematode.

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Chapter 15 The cell membranes are freely permeable to water, and the cytoplasm would take up more but is prevented from doing so by the presence of the outer cell walls, which are exerting an equal and opposite wall pressure. The trap waits for its victim in a state of hydrostatic tension. A clue to future events is given by the presence of two inconspicuous lines of weakness running around the inner faces of the ring cells. And if we look inside the cells, near the lines of weakness, we find folded reserves of wall material and membrane (Fig. 15.8B).

B Fig 15.8 A, B: Constricting ring traps. The first pair of images shows the same trap before and after it is triggered. The first of the second pair, a scanning electron micrograph, clearly shows the line of weakness in the outer cell wall that has split when the inner wall of the trap expanded. The second shows two triggered traps.

Fungi Exploiting Microscopic Animals When a nematode enters the ring and touches the cells, it triggers a rapid sequence of events. The outer walls rupture along the lines of weakness, and the ring cells take up water very quickly over their entire surface. The cells expand into the central gap from the line of weakness, deploying the reserve wall and membrane (Fig. 15.8A). The three inwardly expanding cells grip the nematode but do not crush it immediately, because their increase in volume has reduced their osmotic pressure to about a third of its former value. The osmotic pressure is quickly pumped up again, turgor pressure increases, and the worm is strangled. All that remains is for the fungus to send in assimilative hyphae, which will extract the vital nitrogen supplement from the animal. This is clearly an unusual kind of mechanism to find in a fungus. If we try to unravel the evolutionary steps that led to it, we begin with the ability of eumycotan hyphae to anastomose. Without that, no trap is possible. When hyphae anastomose repeatedly, they can produce a network; some nematode-trapping fungi have gone no further than this. But worms can wriggle out of passive networks, and there was obviously a selective advantage if the fungus did something extra to detain the moving meal. As is often the case, the various intermediate steps in the elaboration of the trap mechanism are nowhere to be seen. Like so many other missing links, they have inconveniently vanished in the mists of time. But we can draw some analogies with other fungal mechanisms that depend on a buildup of turgor pressure followed by its rapid release: the ascus, the basidium, the harpoon cell of Haptoglossa, the subsporangial vesicle of Pilobolus. These show how a basic physical phenomenon like turgor pressure, teamed with an appropriately placed line of wall weakness, can be used for purposes as diverse as shooting spores and catching food. Many of these fungi exploit nematodes only as a dietary nitrogen supplement, and they may often grow in places where an adequate supply of nematodes is not forthcoming. Here, the development of traps would be a pointless waste of energy. To avoid this possibility, the fungi will not produce traps unless they detect certain chemical trademarks that indicate the presence of nematodes. It has also been confirmed that the communication goes both ways. Some fungi secrete a chemical attractant which lures nematodes to their doom. This attractant may be ammonia or carbon dioxide. The conidia of some of the trap-forming fungi will sometimes produce a trap almost immediately after germinating (Fig. 15.7C, D). This suggests that nematodes are a very important part of the diet of these particular fungi. It is also significant that the conidia of trap formers are usually quite large, enabling them to carry enough reserves to build a trap all by themselves (Figs.15.5A–G, 15.7C, D). Spores of species that rely on adhesive, mobile, or ingested propagules are usually much smaller (Fig. 15.5H–O).

Chemical Warfare (11) Toxins. Mycelia of the widely eaten and cultivated ‘oyster mushroom’, Pleurotus ostreatus, and several other Pleurotus species secrete a substance that rapidly inactivates nematodes, allowing the fungus to colonize their inert bodies. Since Pleurotus species are often primary colonizers of dead wood, a substrate notoriously deficient in nitrogen, the nematodes may be an important component of the fungal diet, as they appeared to be for the agarics (Hohenbuehelia and Resupinatus) mentioned earlier.

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Chapter 15 A few fungi parasitize nematode eggs. Rhopalomyces elegans, a striking zygomycete commonly encountered on dung (see chapter 11), is one of these. Nematode eggs appear to release some kind of attractant which causes hyphae of Rhopalomyces to grow toward them. On arrival, the hyphae establish appressoria, then penetrate the egg and assimilate its contents. Rhopalomyces hyphae can also parasitize adult nematodes.

Exploitation of Other Animals and Protists Nematodes, as you will already have noticed, are not the only animals preyed upon by fungi. Amoebae, rotifers, tardigrades, copepods, and even collembola (springtails) are also exploited. The largest animal known to be captured by a predaceous fungus is a small springtail. Arthrobotrys entomopaga (hyphomycetes) produces a prostrate hyphal network from which arise clusters of two-celled traps, the upper cell of each bearing a large droplet of glue. In a confrontation like that of Gulliver and the Lilliputians, the collembolan, which is up to 130 µm long, sticks to several microscopic droplets at once and cannot escape their combined effect. Another hyphomycete, a species of Harposporium, has been found attacking tardigrades (water bears). Zoophagus (Oomycota) traps rotifers by means of ‘lethal lollipops’— sticky knobs which the animals unwisely try to eat (Fig. 15.9). Six hyphomycetes trap amoebae, usually testaceous rhizopods. These fungi are drawn from four genera: Dactylella, Pedilospora, Tridentaria, and Triposporina. The amoeba Geococcus vulgaris normally feeds on fungi by attaching itself to the wall of a spore or hypha and sucking out the cytoplasm. But when it encounters Dactylella passalopaga, the tables are turned. The fungus responds to the attack of the amoeba by gagging it with a rapidly swelling bulbous outgrowth that effectively prevents escape. Assimilative hyphae subsequently digest the amoeba. Most amoeba-trapping hyphomycetes rely on sticky knobs to catch their prey. The rarity of amoeba-trapping hyphomycetes can probably be explained by a difference of scale. A robust hyphomycete would need to exploit Fig. 15.9 A rotifer caught by one of the a large number of the tiny amoebae in order ‘lethal lollipops’ of Zoophagus (Oomycota). to accumulate enough energy to form conidiophores and conidia.

Fungi Exploiting Microscopic Animals Some years ago, when we first became aware of the ubiquity of the nematodeexploiting fungi, it was suggested that they might be useful in controlling the populations of plant-parasitic nematodes in the soil. A number of attempts were made to obtain biological control of eelworms by boosting natural populations of the fungi or by shifting ecological equilibria in their favour. Unfortunately, although small-scale experiments often gave promising results, field trials were generally less successful. A combination of green manuring and additions of nematode-destroying fungi gave the best results. Nematode-exploiting fungi are naturally present in all agricultural soils. If they are already actively exploiting nematode populations, the impact of adding more fungal inoculum might well be less than expected. Even though biocontrol of plant-pathogenic nematodes was disappointing, control of problematic nematodes in the guts of several species of domestic animals has been achieved by daily incorporation of numerous chlamydospores of Duddingtonia (Arthrobotrys) flagrans in their diets. Recent references can be found on Google by entering ‘Duddingtonia flagrans’.

Further Reading Barron, G. L. 1977. The Nematode-Destroying Fungi. Guelph: Canadian Biological Publications. ———. 1981. “Predators and Parasites of Microscopic Animals.” In Biology of Conidial Fungi. Vol. 2. Edited by G. T. Cole and B. Kendrick, 167–200. New York: Academic Press. ———. 1985. “Fungal Parasites of Bdelloid Rotifers: Diheterospora [Now Pochonia].” Canadian Journal of Botany 63:211–22. ———. 1986. “A New Harposporium Parasitic in Bdelloid Rotifers.” Canadian Journal of Botany 64:2379–82. ———. 1987. “The Gun Cell of Haptoglossa mirabilis.” Mycologia 79:877–83. ———. 1990. “A New Predatory Hyphomycete Capturing Copepods.” Canadian Journal of Botany 68:691–96. Duddington, C. L. 1962. “Predacious Fungi and the Control of Eelworms.” Viewpoints in Biology 1:151–200. Gray, N. F. 1987. “Nematophagous Fungi with Particular Reference to Their Ecology.” Biological Review 62:245–304. Nordbring-Hertz, B. 1988. “Ecology and Recognition in the Nematode/Nematophagous Fungus System.” Advances in Microbial Ecology 10:81–114. Samuels, G. J. 1983. “Ascomycetes of New Zealand 6. Atricordyceps harposporifera gen. et sp. nov. and Its Harposporium Anamorph.” New Zealand Journal of Botany 21:171–76. Thorn, R. G., and G. L. Barron. 1984. “Carnivorous Mushrooms.” Science 224:76–78. ———. 1986. “Nematoctonus and the Tribe Resupinatae in Ontario, Canada.” Mycotaxon 25:321– 453.

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16 Mutualistic Symbioses between Animals and Fungi Introduction At first sight, such relationships sound bizarre and improbable. What could a fungus do for an animal that would cause it to modify its lifestyle to accommodate such aliens? And what could be in it for the fungi, which usually compete with animals for food? The first and most important driving force is the inability of almost all animals to digest cellulose and lignin. Some animals, like the detritivores in streams and ponds, wait until amphibious and aero-aquatic hyphomycetes have exploited the plant remains and then seek out and eat the hyphae and conidiophores of these fungi. Many other animals, including the herbivorous mammals and some termites, have overcome this deficiency in a much more efficient and reliable way, by maintaining large populations of cellulolytic microorganisms in their gut. Then they can eat the cellulose and lignin directly, leaving their gut microbiota to digest these substrates for them. But some social insects—the mound-building termites of Africa and Asia and the leaf-cutting ants of Central and South America—have evolved a third strategy. They cultivate specific cellulolytic fungi in underground gardens. And I use the words ‘cultivate’ and ‘garden’ deliberately. The insects establish pure cultures of special co-evolved fungi, keep them constantly supplied with food and moisture, and weed out contaminants. The fungus, then, receives very special treatment, and there is no doubt that it benefits from the arrangement. How many other fungi have guardians that keep out the competition and bring endless supplies of food? But then the ants and termites have their turn. As you have no doubt guessed, they are exclusively mycophagous. The fungi have transformed the wood brought by the termites and the leaves supplied by the ants into a digestible and nutritious fungal biomass. The following extended quotation from E. O. Wilson’ fine book Biophilia expresses the leaf-cutter ant scenario beautifully, from the ants’ point of view.

An insect I most wanted to find made its appearance soon after my arrival at Fazenda Esteio, with no effort of my own and literally at my feet. It was the leafcutter ant (Atta cephalotes), one of the most abundant and visually striking animals of the New World tropics. The sauva, as it is called locally, is a prime consumer of fresh vegetation, rivaled only by man, and a leading agricultural pest in Brazil. I had devoted years of research to the species in the laboratory but never

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Mutualistic Symbioses between Animals and Fungi studied it in the field. At dusk on the first day in camp, as the light failed to the point where we found it difficult to make out small objects on the ground, the first worker ants came scurrying purposefully out of the surrounding forest. They were brick red in color, about a quarter inch [6 mm] in length, and bristling with short, sharp spines. Within minutes, several hundred had arrived and formed two irregular files that passed on either side of the hammock shelter. They ran in a nearly straight line across the clearing, their paired antennae scanning right and left, as though drawn by some directional beam from the other side. Within an hour, the trickle expanded to twin rivers of tens of thousands of ants running ten or more abreast. The columns could be traced easily with the aid of a flashlight. They came up from a huge earthen nest a hundred yards from the camp on a descending slope, crossed the clearing, and disappeared again into the forest. By climbing through tangled undergrowth we were able to locate one of their main targets, a tall tree bearing white flowers high in its crown. The ants streamed up the trunk, scissored out pieces of leaves and petals with their sharp-toothed mandibles, and headed home carrying the fragments over their heads like little parasols. Some floated the pieces to the ground, where most were picked up and carried away by newly arriving nestmates. At maximum activity, shortly before midnight, the trails were a tumult of ants bobbing and weaving past each other like miniature mechanical toys. For many visitors to the forest, even experienced naturalists, the foraging expeditions are the whole of the matter, and individual leafcutter ants seem to be inconsequential ruddy specks on a pointless mission. But a closer look transforms them into beings of another order. If we magnify the scene to human scale, so that an ant’s quarter-inch length grows into six feet, the forager runs along the trail for a distance of about ten miles at a velocity of 16 miles an hour. Each successive mile is covered in three minutes and forty-five seconds, about the current (human) world record. The forager picks up a burden of 750 pounds and speeds back toward the nest at 15 miles an hour—hence, four-minute miles. This marathon is repeated many times during the night and in many localities on through the day as well. From research conducted jointly by biologists and chemists, it is known that the ants are guided by a secretion paid onto the soil through the sting, in the manner of ink being drawn out of a pen. The crucial molecule is methyl-4methyl-pyrrole-2-carboxylate, which is composed of a tight ring of carbon and nitrogen atoms with short side chains made of carbon and oxygen. The pure substance has an innocuous odour, judged by various people to be faintly grassy, sulphurous, or fruitlike with a hint of naphtha (I’m not sure I can smell it at all). But whatever the impact on human beings, it is an ichor of extraordinary power for the ants. One milligram, a quantity that would just about cover the printed letters in this sentence, if dispensed with theoretical maximum efficiency, is enough to excite billions of workers into activity or to lead a short column of them three times around the world. The vast difference between us and them has

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Chapter 16 nothing to do with the trail substance itself, which is a biochemical material of unexceptional structure. It lies entirely in the unique sensitivity of the sensory organs and brains of the insects. One millimetre above the ground, where ants exist, things are radically different from what they seem to the gigantic creatures who peer down from a thousand times that distance. The ants do not follow the trail substance as a liquid trace on the soil, as we are prone to think. It comes up to them as a cloud of molecules diffusing through still air at the ground surface. The foragers move inside a long ellipsoidal space in which the gaseous material is dense enough to be detected. They sweep their paired antennae back and forth in advance of the head to catch the odorant molecules. The antennae are the primary sensory centres of the ant. Their surfaces are furred with thousands of nearly invisible hairs and pegs, among which are scattered diminutive plates and bottle-necked pits. Each of these sense organs is serviced by cells that carry electrical impulses into the central nerve of the antenna. Then relay cells take over and transmit the messages to the integrating regions of the brain. Some of the antennal organs react to touch, while others are sensitive to slight movements of air, so that the ant responds instantaneously whenever the nest is breached by intruders. But most of the sensors monitor the chemicals that swirl around the ant in combinations that change through each second of its life. Human beings live in a world of sight and sound, but social insects exist primarily by smell and taste. In a word, we are audiovisual where they are chemical. The oddness of the insect sensory world is illustrated by the swift sequence of events that occurs along the odour trail. When a forager takes a wrong turn to the left and starts to run away from the track, its left antenna breaks out of the odour space first and is no longer stimulated by the guiding substance. In a few thousandths of a second, the ant perceives the change and pulls back to the right. Twisting right and left in response to the vanishing molecules, it follows a tightly undulating course between the nest and tree. During the navigation it must also dodge moment by moment through a tumult of other runners. If you watch a foraging worker from a few inches away with the unaided eye, it seems to touch each passerby with its antennae, a kind of tactile probe. Slow-motion photography reveals that it is actually sweeping the tips of the antennae over parts of the other ant’s body to smell it. If the surface does not present exactly the right combination of chemicals—the colony’s unique odour signature—the ant attacks at once. It may simultaneously spray an alarm chemical from special glands located in the head capsule, causing others in the vicinity to rush to the site with their mandibles gaping. An ant colony is organized by no more than ten or twenty such signals, most of which are chemical secretions leaked or sprayed from glands. The workers move with swiftness and precision through a life that human beings have come to understand only with the aid of mathematical diagrams and molecular formulas. We can also simulate the behaviour. Computer technology has made it theoretically possible to create a mechanical ant that duplicates the observed activity.

Mutualistic Symbioses between Animals and Fungi But the machine, if for some reason we chose to build one, would be the size of a small automobile, and even then I doubt if it would tell us anything new about the ant’s inner nature. At the end of the trail the burdened foragers rush down the nest hole, into throngs of nestmates and along tortuous channels that end near the water table fifteen feet or more below. The ants drop the leaf sections onto the floor of a chamber, to be picked up by workers of a slightly smaller size who clip them into fragments about a millimetre across. Within minutes still smaller ants take over, crush and mold the fragments into moist pellets, and carefully insert them into a mass of similar material. This mass ranges in size between a clenched fist and a human head, is riddled with channels, and resembles a gray cleaning sponge. It is the garden of the ants: on its surface a symbiotic fungus grows which, along with the leaf sap, forms the ants’ sole nourishment. The fungus spreads like a white frost, sinking its hyphae into the leaf paste to digest the abundant cellulose and proteins held there in partial solution. The gardening cycle proceeds. Worker ants even smaller than those just described pluck loose strands of the fungus from places of dense growth and plant them onto the newly constructed surfaces. Finally, the very smallest—and most abundant—workers patrol the beds of fungal strands, delicately probing them with their antennae, licking their surfaces clean, and plucking out the spores and hyphae of alien species of mold. These colony dwarfs are able to travel through the narrowest channels deep within the garden masses. From time to time they pull tufts of fungus loose and carry them out to feed their larger nestmates. The leafcutter economy is organized around this division of labour based on size. The foraging workers, about as big as houseflies, can slice leaves but are too bulky to cultivate the almost microscopic fungal strands. The tiny gardener workers, somewhat smaller than this printed letter I, can grow the fungus but are too weak to cut the leaves. So the ants form an assembly line, each successive step being performed by correspondingly smaller workers, from the collection of pieces of leaves out of doors to the manufacture of leaf paste to the cultivation of dietary fungi deep within the nest. The defence of the colony is also organized according to size. Among the scurrying workers can be seen a few soldier ants, three hundred times heavier than the gardener workers. Their sharp mandibles are powered by massive adductor muscles that fill the swollen, quarter-inch-wide head capsules. Working like miniature wire clippers, they chop enemy insects into pieces and easily slice through human skin. These behemoths are especially adept at repelling large invaders. When entomologists digging into a nest grow careless, their hands become nicked all over as if pulled through a thorn bush. I have occasionally had to pause to stanch the flow of blood from a single bite, impressed by the fact that a creature one-millionth my size could stop me with nothing but its jaws. No other animals have evolved the ability to turn fresh vegetation into mushrooms. The evolutionary event occurred only once, millions of years ago,

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Chapter 16 somewhere in South America. It gave the ants an enormous advantage: they could now send out specialized workers to collect the vegetation while keeping the bulk of their populations safe in subterranean retreats. As a result, all of the different kinds of leafcutters together, comprising fourteen species in the genus Atta and twenty-three in Acromyrmex, dominate a large part of the American tropics. They consume more vegetation than any other group of animals, including the more abundant forms of caterpillars, grasshoppers, birds, and mammals. A single colony can strip an orange tree or bean patch overnight, and the combined populations inflict over a billion dollars’ worth of damage yearly. It was with good reason that the early Portuguese settlers called Brazil the Kingdom of the Ants. At full size, a colony contains three to four million workers and occupies three thousand or more underground chambers. The earth it excavates forms a pile twenty feet across and three to four feet high. Deep inside the nest sits the queen, a giant insect the size of a newborn mouse. She can live for at least ten years, and perhaps as long as twenty. No one has had the persistence to determine the true longevity. In my laboratory I have an individual collected in Guyana fourteen years ago. When she reaches eighteen, and breaks the proved longevity of the seventeen-year locusts, my students and I will open a bottle of champagne to celebrate. In her lifetime an individual can produce over twenty million offspring, which translates into the following: a mere three hundred ants, a small fraction of the number emerging from a single colony in a year, can give birth to more ants than there are human beings on earth. The queen is born as a tiny egg, among thousands laid daily by the old mother queen. The egg hatches as a grub-like larva which is fed and laved incessantly throughout its month-long existence by the adult worker nurses. Through some unknown treatment, perhaps a special diet controlled by the workers, the larva grows to a relatively huge size. She then transforms into a pupa, whose waxy casem*nt is shaped like an adult queen in fetal position, with legs, wings, and antennae folded tightly against the body. After several weeks the full complement of adult organs develops within this cuticle, and the new queen emerges. From the beginning she is fully adult and grows no more in size. She also possesses the same genes as her sisters, the colony workers. Their smaller size and pedestrian behavior is not due to heredity but rather to the different treatment they received as larvae. In bright sunshine following a heavy rain, the virgin queen comes to the surface of the nest and flies up into the air to join other queens and the darkly pigmented, big-eyed males. Four or five males seize and inseminate her in quick succession, while she is still flying through the air. Their sole function now completed, they die within hours, without returning to the home nest. The queen stores their sperm in her spermatheca, a tough muscular bag located just above and behind her ovaries. These reproductive cells live like independent microorganisms for years, passively waiting until they are released into the oviduct to meet an egg and create a new female ant. If the egg passes through the oviduct

Mutualistic Symbioses between Animals and Fungi and to the outside without receiving a sperm, it produces a male. The queen can control the sex of her offspring, as well as the number of new workers and queens she produces, by opening or shutting the passage leading from her sperm-storage organ to the oviduct. The newly inseminated queen descends to the ground. Raking her legs forward, she breaks off her wings, painlessly because they are composed of dead, membranous tissue. She wanders in a random pattern until she finds a patch of soft, bare soil, then commences to excavate a narrow tunnel straight down. Several hours later, when the shaft has been sunk to a depth of about ten inches, the queen widens its bottom into a small room. She is now set to start a garden and a colony of her own. But there is a problem in this life-cycle strategy. The queen has completely separated herself from the mother colony. Where can she obtain a culture of the vital symbiotic fungus to start the garden? Answer: she has been carrying it all along in her mouth. Just before leaving home, the young queen gathered a wad of fungal strands and inserted it into a pocket in the floor of her oral cavity, just back of the tongue. Now she passes the pellet out onto the floor of the nest and fertilizes it with droplets of feces. As the fungus proliferates in the form of a whitish mat, the queen lays eggs on and around its surface. When the young larvae hatch, they are fed with other eggs given to them by the queen. At the end of their development, six weeks later, they transform into small workers. These new adults quickly take over the ordinary tasks of the colony. When still only a few days old, they proceed to enlarge the nest, work the garden, and feed the queen and larvae with tufts of the increasingly abundant fungus. In a year the little band has expanded into a force of a thousand workers, and the queen has ceased almost all activity to become a passive eating and egg-laying machine. She retains that exclusive role for the rest of her life. The measure of her Darwinian success is whether some of her daughters born five or ten years down the line will grow into queens, leave on nuptial flights, and—rarest of all achievements—found new colonies of their own. In the world of the social insects, by the canons of biological organization, colonies beget colonies; individuals do not directly beget individuals. People often ask me whether I see any human qualities in an ant colony, any form of behaviour that even remotely mimics human thought and feeling. Insects and human beings are separated by more than 600 million years of evolution, but a common ancestor did exist in the form of one of the earliest multicellular organisms. Does some remnant of psychological continuity exist across that immense phylogenetic gulf? The answer is that I open an ant colony as I would the back of a Swiss watch. I am enchanted by the intricacy of its parts and the clean, thrumming precision. But I never see the colony as anything more than an organic machine. Let me qualify that metaphor. The leafcutter colony is a superorganism. The queen sits deep in the central chambers, the vibrant growing tip from which all the workers and new queens originate. But she is not in any sense the leader or the repository of an organizational blueprint. No command center directs the

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Chapter 16 colony. The social master plan is partitioned into the brains of the all-female workers, whose separate programs fit together to form a balanced whole. Each ant automatically performs certain tasks and avoids others according to its size and age. The superorganism’s brain is the entire society; the workers are the crude analogue of its nerve cells. Seen from above and at a distance, the leafcutter colony resembles a gigantic amoeba. Its foraging columns snake out like pseudopods to engulf and shred plants, while their stems pull the green pieces down holes into the fungus gardens. Through a unique step in evolution taken millions of years ago, the ants captured a fungus, incorporated it into the superorganism, and so gained the power to digest leaves. Or perhaps the relation is the other way around: perhaps the fungus captured the ants and employed them as a mobile extension to take leaves into the moist underground chambers. In either case, the two now own each other and will never pull apart. The antfungus combination is one of evolution’s master clockworks, tireless, repetitive, and precise, more complicated than any human invention and unimaginably old. To find a colony in the South American forest is like coming upon some device left in place ages ago by an extraterrestrial visitor for a still undisclosed purpose. Biologists have only begun to puzzle out its many parts. Because of modern science the frontier is no longer located along the retreating wall of the great rain forest. It is in the bodies and lives of the leafcutters and thousands of other species found for the most part on the other side of that tragic line.

Leaf-Cutting Ants, Leucoagaricus and Lepiota The gardening ants of the New World make up the tribe Attini. Although you have probably never heard of them before, people in South America are only too familiar with them. Searching for leaves to feed to their tame fungus, these ants will defoliate trees and growing crops. In the sixteenth century, the invading Spaniards may have conquered the native peoples of South America, but the native ants got the better of them—their failure to grow cassava and citrus fruits was attributed to attine ants, whose nests, at the base of the trees, were ‘white as snow’ (presumably with fungal mycelium). Leaf-cutting ants of the genera Atta and Acromyrmex have long been regarded as serious pests, and they still make farming difficult in some primitive areas. The native peoples eat the large females, but this, unfortunately, doesn’t seem an adequate population control for the ants. Nevertheless, in tropical rainforests, these insects and their fungi have an important ecological role to play. In these forests, the turnover of organic matter and mineral nutrients at the soil surface is very rapid, and few organisms, including the trees, penetrate far into the soil. Here, a large nest of Atta, with hundreds of fungus gardens, vastly increases the organic matter content of the soil and opens it up for subsequent colonization by many other organisms. In some areas of Trinidad, small species of attine ants achieve densities of one nest for every two square metres, and are a dominant feature in soil ecology and nutritional status.

Mutualistic Symbioses between Animals and Fungi Although the Attini comprise many genera, Atta sexdens is the most economically important species, and therefore the most intensively studied. A winged female, carrying inoculum of the all-important fungus in a special pocket at the back of her mouth, and with her spermatheca stocked with perhaps 300 million sperm, establishes the colony. First she expels the fungal pellet from her mouth and finds some plant material for it. As soon as the fungus starts growing, the queen lays eggs on it. Soon she is laying about fifty eggs a day, but she eats most of them herself until the worker population is established, which takes about three months. A second entrance to the nest is added after another year, and then entrances proliferate; there are about 75 by the end of the second year and about 1,000 by the end of the third year. From now on, new females emerge each year to establish colonies elsewhere. In case you are wondering why these animals haven’t taken over South America, it is worth pointing out that an estimated 99.7% of all new nests are destroyed in their first six months. When a four-year-old nest was excavated, it was found to contain 1,027 subterranean chambers, of which 390 had fungus gardens. Another Atta nest, more than six years old, had 1,920 chambers, of which 248 contained fungus gardens and 1,219 were empty. The gardens were usually 20–30 cm in diameter and weighed about 300 g. It was calculated that this colony had consumed nearly 6,000 kg of vegetation. Fig. 16.1B shows a section of a representative nest, with many entrances and many interconnected chambers. Note that most of the larger chambers contain fungus gardens (shown in white). In the early days of the colony, the queen and the first broods establish the first fungus garden, excavating a chamber, filling it with vegetation brought by workers, and

Fig. 16.1 Sectional views of A: termite mound; B: attine ant nest. Fungal gardens or combs are shown in white.

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Fig. 16.2 Mycophagous insects and the fungal structures they eat.

inoculating the substrate with the fungus. Leaf-cutting ants forage for leaves along well-marked trails which often extend up into the crowns of trees. The ants have no difficulty in scissoring out large pieces of leaf, petal, or twig with their formidable jaws, although they may have a little trouble manoeuvring on their way home. Back in the nest, they cut the material into smaller pieces, lick it all over, chew the edges, and often deposit an anal droplet on it. Then they wedge it into the garden and put tufts of mycelium on it. Gardens have a sponge-like construction containing many cavities. The

Mutualistic Symbioses between Animals and Fungi ants walk all over the garden, probing the fungus with their antennae, licking and sometimes eating hyphae, and depositing anal droplets, as in Fig. 16.2A. Some Attines are not leaf cutters, but they nevertheless grow perfectly functional fungal gardens on such substrates as plant debris and insect excreta. The gardens invariably contain only one species of fungus. This is surprising, because decaying organic substrates are usually competitively colonized by a wide range of different fungi. If a garden is removed from the colony, it soon becomes overgrown by extraneous fungi or bacteria. From this, we deduce that the ants must have some kind of chemical inhibitors that prevent the growth of unwanted microorganisms. It seems likely that these substances, and perhaps others that promote the growth of the proper fungus, are present in the ants’ saliva and anal fluid, with which they constantly anoint their tame fungus. In return, it flourishes and develops clusters of inflated hyphal tips (Fig. 16.2B), which the ants eat. I must emphasize that although the insects cut up and chew up the leaves they bring to the nest, leaves are never eaten. The ants are exclusively mycophagous. The fungi cultivated by the attine ants never seem to fruit in or near the gardens, so various attempts have been made to isolate them in pure culture, and mature ascomata and basidiomata have sometimes developed. These belonged to species of Leucoagaricus or Lepiota (basidiomycetes, Agaricales). Species of Xylaria (ascomycetes) and Auricularia (basidiomycetes) may also be involved. It has been suggested that the cultivation of fungi by ants evolved only once, about fifty million years ago, when ants domesticated an agaric belonging to the family Lepiotaceae. Not long ago, it was discovered that the fungal gardens of the Attines were contaminated by a parasitic hyphomycetous mould, Escovopsis. Now it has been found that the ants themselves carry colonies of an actinomycetous bacterium, Streptomyces, on specific areas of the cuticle of their exoskeleton and that this bacterium produces antibiotic substances which help to control the Escovopsis. So it looks as if the symbiosis actually involves three partners.

Termites and Termitomyces Across the South Atlantic ocean from the territory of the leaf-cutting ants begins the realm of the fungus-growing termites. The subfamily Macrotermitinae is found in the Old World, its twelve genera being variously found in sub-Saharan Africa, Madagascar, the Indian subcontinent, and much of Southeast Asia, including the Indonesian archipelago. Unlike many other termites, these have no cellulolytic protozoans in their gut, so, like the attine ants, but independently, they have established a mutualistic symbiosis with fungi. Each colony is founded by a winged male and female, which wall themselves up in an underground chamber. The queen lays eggs, and the resulting workers bring food to the sequestered couple, take eggs away for incubation, and build the nest. The fungus garden or comb surrounds the royal chamber. Above their nests, many fungus-growing termites construct mounds (termitaria), which can be an impressive 6 metres tall and

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Fig. 16.3

Termitomyces basidiomata arising from a subterranean fungal comb.

3 metres across at the base. The mounds are penetrated by air shafts leading to the nest below, particularly to the fungus garden, which may be a large, central structure, 50 cm in diameter and weighing as much as 25 kg, or a series of smaller combs (as in honeycomb: riddled with holes) in individual chambers (Fig. 16.1A). A large colony may contain a million termites, which forage for wood and other plant debris. Unlike the attine ants, the termites eat this material on the spot, so their fungus gardens are made entirely from faecal material. The gardens have a sponge-like or deeply convoluted appearance, and at many points on their surface spherical sporodochial conidiomata develop (Fig. 16.2C) with monilioid conidiophores bearing dikaryotic conidia (Fig. 16.2D). The workers eat these and nibble the garden itself, redepositing the resultant faecal material on the comb. The cellulases of the fungus remain active in the gut of the insect. The soldiers, nymphs, king, and queen don’t eat the fungus directly but live on salivary secretions provided by the workers. The taxonomy of the termite fungi is better understood than that of the ant fungi, for the simple reason that termite fungi fruit in nature (Fig. 16.3). When termites of the genus Pseudacanthotermes desert a comb, the fungus produces basidiomatal primordia on its surface. When the rainy season starts, rainfall of more than 2 cm/day stimulates the primordia to develop long stipes, which grow up to the soil surface and produce a large pileus. These mushrooms are identifiable as Termitomyces striatus

Mutualistic Symbioses between Animals and Fungi (Agaricales, Lyophyllaceae). Interestingly, the combs of fungus-growing termites are often inhabited by an additional fungus, a species of Xylaria, which may also produce stromata on the comb. Although about thirty species of Termitomyces have been described, only two species of Xylaria have been found associated with termites. Macrotermitinae are regarded as major pests of tropical agriculture, and they are destructive to wooden buildings. They take scarce organic matter underground, where its nutrients may remain locked up for years. However, there are a few minor compensations. Termites are food for many other animals, and many Termitomyces species are among the most highly prized, and the largest, edible tropical agarics; so much so that attempts have been made to domesticate them.

Beetles and Ambrosia Fungi Although social insects like ants and termites are the most visible, the most dramatic, and probably the most highly evolved examples of animal-fungus mutualism, they are not the only animals in such relationships. A similar bond exists between wood-boring beetles of the families Scolytidae, Platypodidae, and Lymexylidae and the ambrosia fungi they carry in special organs called mycangia as they travel from tree to tree and on which their larvae feed exclusively. The fungus is introduced to weakened or freshly logged trees when a female beetle burrows into them to lay its eggs. By the time the larvae hatch, the fungus has colonized the surrounding wood and is sporulating all over the walls of the beetle tunnels. Since they cannot digest wood, the larvae eat the fungal biomass, which is called ambrosia. Before an adult female beetle leaves the tree to seek fresh habitat, it will rock back and forth to make sure that its mycangium is stocked with the fungus (Fig. 16.2E, F). Many species of beetle have specific ambrosia fungi, although their larvae may feed on other fungi that are also found sporulating in the tunnels. The full spectrum of such fungi takes in some yeasts (Saccharomycetes: Ascoidea, Dipodascus, Endomyces, Endomycopsis, Hansenula, Saccharomyces), ascomycetous anamorphs (Acremonium, Ambrosiella, Diplodia, Scopulariopsis), and some basidiomycetes and their conidial anamorphs.

Scales and Septobasidiales There are almost 200 species of the Urediniomycete order Septobasidiales. All grow on the surfaces of plants and are associated with scale insects (hom*optera, Coccoidea). Some of the insects are parasitized by the fungus but do not die. And although the infection renders them dwarfed and sterile, they continue to feed from the plant, supplying the enclosing fungus with a reliable flow of nutrients. The tough mat of fungal hyphae (150–1,000 µm thick) that develops around parasitized insects protects many other healthy scales from predators and parasitoid hymenoptera (Fig. 5.11). The mutualism is not perfectly balanced, because the insects occasionally survive without the fungus.

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Midges and Macrophoma Some gall midges (Diptera, Cecidomyiidae) have mutualistic relationships with members of the coelomycetous anamorph Macrophoma (Teleomorph = Botryosphaeria [Ascomycota, Dothideomycetes]), which inhabit ‘ambrosia galls’. The dipteran larvae must eat the fungal mycelium, and the fungus needs the adult midge as its vector. The female gall midge carries the fungus away in a pair of specialized pouches called mycangia and lays some conidia with her eggs.

Woodwasps and Wood-Rotting Fungi Woodwasps of the genus Sirex (Hymenoptera, Siricidae) often invade dead or dying trees but may also be implicated in the death of healthy ones. They drill through the bark of the tree with a long ovipositor and lay eggs in the xylem. A pair of mycangia associated with the ovipositor are full of thallic-arthric conidia of a basidiomycete, some of which are deposited with the eggs. Female larvae also have mycangia in which the fungus is maintained in a dormant condition, embedded in wax plates. The role of the fungus is not fully understood, and different workers have suggested that (1) the fungus regulates moisture content and provides a suitable microclimate for egg incubation, (2) the fungus reduces the intensity of the tree’s response to attack, (3) the fungus is eaten by the larvae. Whatever the relationship is based on, it is apparently an obligatory one, since it has been experimentally established that fungus-free females cannot reproduce successfully. The fungi associated with woodwasps have been identified as species of Stereum and Amylostereum (Agaricomycetes, Russulales).

Anobiid Beetles and Endosymbiotic Yeasts Anobiid beetles (Coleoptera, Anobiidae) live in wood. These beetles have pouches called mycetomes at the beginning of their midgut. These are full of yeast-like fungi of the genus Symbiotaphrina (Ascomycota). Adult beetles transmit the symbionts to their offspring by smearing the eggs with fungal cells. The newly hatched larva eats some of the eggshell and becomes ‘infected’. The fungus apparently supplies vitamins and essential amino acids. Its role is mainly to recycle nitrogen in a rather nitrogen-deficient habitat. It has been demonstrated (by disinfecting the eggs) that beetles without endosymbionts cannot grow, even when given their normal diet.

Boletinellus and Root Aphids Some years ago, another apparently mutualistic relationship between fungi and insects was discovered in the mycology laboratory at Waterloo, this time between a bolete and a root aphid. Boletinellus merulioides (Agaricomycetes, Boletaceae) is commonly associated with ash trees (Fraxinus) and was once thought to be their ectomycorrhizal

Mutualistic Symbioses between Animals and Fungi partner. Mark Brundrett examined many ash roots closely and found that they were exclusively endomycorrhizal. The Boletinellus was subsequently found to produce hollow black sclerotia near the roots, and within these sclerotia lived individuals of a root aphid, Meliarhizophagus fraxinifolii (hom*optera, Aphidae). From within this safe haven, the aphids pierced the roots and sucked sap at their leisure. We suggested that in exchange for housing and protecting the aphid, the fungus obtained nutrients, especially sugars, excreted by the aphid in its honeydew.

Red-Backed Voles, Gilbert’s Potoroo, and Truffles My last examples are perhaps less clear-cut than those above, since there isn’t a true ‘living with’ involved. Nevertheless, the diet of the California red-backed vole (Clethrionomys californicus) consists almost exclusively of the hypogeous basidiomata of ectomycorrhizal fungi such as the genus Rhizopogon (Agaricomycetes, sequestrate Boletales). This establishes the dependence of the vole on the fungi, but although the fungal spores can survive passage through the vole gut, and are therefore spread by the animal, it is unlikely that the fungus depends entirely on this small mammal for dispersal. In West Australia, a highly endangered marsupial called the Gilbert’s potoroo also lives almost exclusively on hypogeous fungi. Its diet, as well as maintaining sufficient territory, must be at the centre of efforts to preserve the species. Although that more or less exhausts the known examples of animal-fungus mutualistic symbiosis, there are almost certainly others out there waiting to be recognized. Perhaps you will discover and describe one of them.

Further Reading Batra, L. R., ed. 1979. Insect-Fungus Symbiosis. Montclair: Allanheld, Osmun. Batra, L. R., and S. W. T. Batra. 1967. “The Fungus Gardens of Insects.” Scientific American 217: 112–20. Bissett, J., and A. Borkent. 1988. “Ambrosia Galls: The Significance of Fungal Nutrition in the Evolution of the Cecidomyiidae (Diptera).” In Coevolution of Fungi with Plants and Animals, edited by K. A. Pirozynski and D. L. Hawksworth, 203–25. New York: Academic Press. Brundrett, M. C., and B. Kendrick. 1987. “The Relationship between the Ash Bolete (Boletinellus merulioides) and an Aphid Parasitic on Ash Tree Roots.” Symbiosis 3:315–20. Buchner, P. 1965. Endosymbiosis of Animals with Plant Microorganisms. New York: Wiley. Couch, J. N. 1938. The Genus Septobasidium. Chapel Hill: University of North Carolina Press. Currie, C. R., J. A. Scott, R. C. Summerbell, and D. Malloch. 1999. “Fungus-Growing Ants Use Antibiotic-Producing Bacteria to Control Garden Parasites.” Nature 398:701–4. Fisher, P. J., D. J. Stradling, and D. N. Pegler. 1994. “Leaf Cutting Ants, Their Fungus Gardens and the Formation of Basidiomata of Leucoagaricus gongylophorus.” Mycologist 8:128–31. Morrison, L., and A. Collins. 2017. “Hunt for a Safe Haven for World’s Rarest Marsupial, Continues.” ABC Great Southern, February 21, 2017. http://www.abc.net.au/news/2017-02-22/hunt -for-a-home-for-gilberts-potoroo/8290948.

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Chapter 16 Pirozynski, K. A., and D. L. Hawksworth, eds. 1988. Coevolution of Fungi with Plants and Animals. New York: Academic Press. Weber, N. A. 1972. Gardening Ants: The Attines. Memoir 92. Philadelphia: American Philosophical Society. Wilson, E. O. 1984. Biophilia. Cambridge, MA: Harvard University Press. Excerpt from Biophilia by Edward O. Wilson, Copyright © 1984 by the president and fellows of Harvard College. Used by permission.

17 Mycorrhizas: Mutualistic Plant-Fungus Symbioses Introduction When green plants first colonized the land more than 400 million years ago, the invasion may have succeeded only because they established an intimate alliance—a mutualistic symbiosis—with fungi. Early land plants could photosynthesize effectively but hadn’t yet developed extensive root systems and must have been hard-pressed to acquire water and mineral nutrients from what passed for soil. This ‘soil’ likely had little organic matter below the surface. The filamentous fungi, which had relatively recently emerged from the water, were perfectly adapted for exploring this medium and finding those very scarce mineral nutrients, but they themselves desperately needed energyrich carbon compounds of the kind produced by the plants. Traces of sugars and amino acids leak out of plants, and Devonian fungi were undoubtedly attracted by these. The relationships presumably developed in more than one direction: some fungi remained saprobic; others became destructive parasites, causing wilts and root rots; yet others evolved into a mutually beneficial symbiosis. Proof of this lies in the fact that fossils of some Devonian plants contain well-preserved arbuscular mycorrhizal (AM) fungal structures just like those we can find in the roots of more than 90% of healthy modern plant species. Over a century ago, several biologists noticed that some plant roots, although extensively invaded by fungi, were not diseased. The name mycorrhiza (fungus root) was coined in 1885. We now know that, especially in poor soils, mycorrhizal plants grow better than nonmycorrhizal plants. This is because the hyphae of the fungal symbionts permeate large volumes of soil and obtain scarce elements—especially water, nitrogen, and phosphorus (this last often being limiting for plant growth)—which they pass on to the plant in exchange for photosynthates. Interest in these symbioses has escalated dramatically in recent years because of their potential benefits to agriculture, forestry, and the revegetation of ecosystems damaged by human activities such as mining. Many plants cannot become established or grow normally without an appropriate fungal partner (often called the mycobiont). Even when plants can survive without mycorrhizas, those with ‘fungus roots’ need less fertilizer, withstand heavy metal and acid rain pollution better, and grow better on the infertile soils of marginal lands, on mine spoils, on other areas needing revegetation, and at high elevations. They also survive transplant shock better, are more resistant to soilborne diseases, and withstand higher soil temperatures, higher soil salinity, and wider extremes of soil pH. So mycorrhizal fungi are almost ubiquitous. Over 90% of all

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Chapter 17 higher plant species are normally mycorrhizal; the plants can be called photobionts, while the fungi involved are called mycobionts. Two main kinds of mycorrhizas are constantly found in association with our agricultural and forest crops. By far the commoner of the two is the arbuscular mycorrhiza (AM), in which specialized hyphae of the fungus enter the cells of the root cortex and set up finely branched, microscopic intracellular interfaces called arbuscules (Fig. 17.1, left side). Although about 300,000 plant species are believed to have AMs, only 230 species of fungi, all members of the phylum Glomeromycota, have so far been described from such relationships. These fungi will grow only in association with plant roots (i.e., they are obligate biotrophs), and only one of them has ever been seen to reproduce sexually.

Fig. 17.1

Diagrams of endo- and ectomycorrhizal structures.

Mycorrhizas: Mutualistic Plant-Fungus Symbioses The second kind of mycorrhiza is the ectomycorrhiza (EM), so called because the fungus grows around the root and between its cortical cells but never actually penetrates the cells (Fig. 17.1, right side). This kind of mycorrhiza is found in only about 2,000 species of plants, but these include some of the most important forest trees, including Pinaceae (pine, spruce, hemlock, Douglas fir, true firs, etc.), fa*gaceae (beech, oak, southern beech), and Myrtaceae (eucalypts). These plants have about 5,000 fungal partners. Although these fungi are usually found only in association with tree roots, many of them can be grown in pure culture, and almost all produce sexual fructifications in their natural habitat. They are nearly all members of the subkingdom Dikarya, mostly Basidiomycota, although a number are Ascomycota. The mycorrhizal symbiosis, whether ectomycorrhizal or arbuscular, must have three basic functioning components: (1) fungal mycelium exploring large volumes of soil and retrieving mineral nutrients; (2) a fungus-plant interface where the exchange of chemicals can go on; (3) plant tissues which produce and store carbohydrates.

Development and Morphology of Ectomycorrhizas (EM) Ectomycorrhizas (Figs. 17.1, 17.2A–C) normally develop one to three months after the tree seed germinates, forming on the ‘short’ or ‘feeder’ roots near the surface of the soil. Roots may be colonized by hyphae which grow through the soil from another mycorrhizal root or by germinating spores, which can subsist on root exudates until they reach the root. Colonization occurs only in the unsuberized zone behind the root tip. The process of colonization involves (1) penetration of hyphae between the cells of the root cortex to form a characteristic Hartig net, (2) establishment of a mantle of hyphae around the outside of the root, and (3) extension of hyphae from the mantle into the surrounding soil. The fungus produces plant growth hormones which cause the short roots of the plant to grow faster, to become thicker, and, often, to branch in characteristic ways (e.g., dichotomously). The Hartig net may be restricted to the outermost layer(s) of the root, or it may spread slowly through the cortex until it reaches the endodermis, which effectively bars any penetration of the stele. As the hyphae insinuate themselves between the cortical cells, these separate at the middle lamella, and an almost complete single layer of fungal hyphae eventually surrounds each cell, although plasmodesmata still connect many cortical cells. Far from being deleterious, the presence of the Hartig net actually seems to prolong the life of the cortical cells and of the root as a whole. The fungal mantle surrounding the root varies from a relatively loose weft of hyphae to a thick, pseudoparenchymatous layer which accounts for nearly half the biomass of the mycorrhiza. The formation of root hairs by the plant is suppressed, since they have been rendered redundant by mycelial strands and/or individual hyphae radiating from the mantle. Compared to nonmycorrhizal roots, EMs are (1) a different colour; (2) thicker; and (3) much more often branched—pinnately and racemosely in Abies, fa*gus, and Eucalyptus and dichotomously in Pinus (Fig. 17.2A). The truly diagnostic structure, however, is the Hartig net, the functional extracellular interface between the symbionts (Fig. 17.2B, C).

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Fig. 17.2 Ectomycorrhizas. A: dichotomous mycorrhizal short roots of Pinus; B: sectional view of part of Hartig net (note that the cortical cell is completely surrounded by hyphae); C: surface view of part of Hartig net, showing convoluted, interlocking hyphae.

Individual ectomycorrhizal roots remain active for up to three years. Roots and mantles often extend at the same rate, but the root sometimes breaks through and grows beyond the mantle. The root tip may then be colonized by other opportunistic ectomycorrhizal fungi, which may be better adapted than the original partner to the specific soil microhabitat being encountered. So a tree may have a number of different fungal partners on its root system at the same time. Many of the fungi responsible for EMs can be isolated in axenic culture without much difficulty, but most grow slowly, will not fruit in culture, and require vitamins like thiamine, some amino acids, and other normally root-derived substances, as well as simple carbohydrates. Most of them

Mycorrhizas: Mutualistic Plant-Fungus Symbioses are completely incapable of degrading cellulose or lignin, although these substances are the principal diet of many other saprobic basidiomycetes. If we add to this picture the information that, when not associated with a tree, these fungi cannot compete with fungi of the saprobic soil mycota and are adversely affected by toxins present in humus and leaf litter, we gain the impression that in nature these fungi are more or less obligate root symbionts. Sugars are translocated from the root to the fungal mantle, where they are converted into trehalose (a disaccharide), mannitol (a polyhydric alcohol), and glycogen—all typical fungal carbohydrates. The glycogen is insoluble and therefore unavailable for possible reabsorption by the plant. More surprisingly, although the mannitol and trehalose remain in solution in the fungus, the plant is incapable of reabsorbing them. Thus the fungal sheath acts as a sink where reserves of carbohydrates derived from the plant are stored. This has some interesting consequences: (1) As autumn approaches, many of the fungi mobilize their stored carbohydrates and produce flushes of large, aboveground mushrooms or underground fruit bodies near the tree. (2) Carbohydrates can be translocated through mycelial strands from established trees to seedlings of the same species. This must help in the maintenance of pure stands. (3) The tree can reclaim some of the stored energy if conditions become appropriate for a new surge of plant growth. How much does a tree invest in its mycorrhizal partners? If we add up the various organs of the fungus—the conspicuous fruit bodies, the extensive although inconspicuous mycelium ramifying through the soil, and the rootlet mantles—we find that trees often invest at least 10% of their total production of photosynthates in their mycobionts. The drain of photosynthates from the tree is clearly more than compensated for by the increased efficiency of mineral absorption and by the fact that the fungal mantle can also store mineral nutrients, e.g., chloride, ammonium, and especially phosphate, that aren’t immediately needed by the tree. These can subsequently be released to the plant during periods of deficiency or of active growth. Since the mycorrhizal rootlets are perennial, the mantle can repeatedly act as a storage organ when the root is not acting as a growing organ. This, and the ability of the fungus to fruit massively during a relatively short time window, indicate that ectomycorrhizal plants and fungi are both adapted to grow in climates where seasonal changes are often dramatic, causing wide fluctuations in growth rate and in the supply of nutrients. This goes some way toward explaining why ectomycorrhizal plants are common in cool temperate climates, in boreal forests, in montane regions, and in other habitats subjected to environmental extremes.

Systematics of Ectomycorrhizal Fungi and Their Hosts Most ectomycorrhizal fungi are basidiomycetes: members of at least seventy-three genera in nine orders. They are (1) agarics; (2) former agarics which have become sequestrate (closed, not shooting spores at maturity), sometimes fruiting aboveground, sometimes underground (basidiotruffles); and (3) some club fungi, chanterelles, tooth fungi, and resupinate basidiomycetes. There are also ectomycorrhizal fungi in sixteen

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Chapter 17 unitunicate ascomycete genera from two orders. All but one of these ascomycete genera fruit underground (they are described as being hypogeous). The principal EM fungal taxa are listed in Table 17.1, and many are illustrated in chapters 4, 5, and 22.

Table 17.1. Taxonomic distribution of ectomycorrhizal fungi Number of Genera

Subkingdom Dikarya Phylum Basidiomycota Subphylum: Agaricomycotina Class: Agaricomycetes Order: Agaricales Family: Amanitaceae

Hygrophoraceae

Tricholomataceae

Entolomataceae

Cortinariaceae

Paxillaceae

Gomphidiaceae

Boletaceae Strobilomycetaceae

13 3

Order Russulales

Order: Gautieriales

Order: Hymenogastrales

Order: Phallales

Order: Lycoperdales

Order: Melanogastrales

Order: Sclerodermatales

Order: Aphyllophorales

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Subkingdom Dikarya

Number of Genera

Subphylum: Ascomycotina Order: Pezizales Family: Pezizaceae

Balsamiaceae

Geneaceae

Helvellaceae

Pyronemataceae

Terfeziaceae

Tuberaceae

Order: Elaphomycetales Total genera

1 85

Most EMs (the actual fungus roots) are rather similar, so the 5,000 fungi involved must usually be identified from their DNA or their macroscopic fruit bodies (basidiomata or ascomata), which are produced during a relatively short season each year. Many EM fungi belong to cosmopolitan agaric genera like Russula, Lactarius, Cortinarius, Amanita, Tricholoma, Inocybe, and Laccaria. Cortinarius alone is estimated to have 2,000 species. Some families are almost entirely ectomycorrhizal—Boletaceae, Gomphidiaceae, Russulaceae, Strobilomycetaceae, and Cantharellaceae. So are all or most species of the genera Amanita, Cortinarius, Hebeloma, Laccaria, Pisolithus, Scleroderma, and Tricholoma, as well as almost all known hypogeous basidiomycetes (e.g., Rhizopogon, Truncocolumella, Hymenogaster). Among the ascomycetes, members of the hypogeous Tuberaceae (true truffles) are probably all mycorrhizal. One generalization may be in order: if a fungus produces sizeable underground fruit bodies, then it is extremely likely to be mycorrhizal, whether it is a basidiomycete or an ascomycete. Some EM fungi have a wide host range, for example, Amanita muscaria, Boletus edulis, Cantharellus cibarius, Cenococcum geophilum, Laccaria laccata, Pisolithus tinctorius, and Thelephora terrestris. Others seem to be more selective, and some are virtually host specific—Suillus grevillei associates only with Larix (larch or tamarack), Suillus lakei with Pseudotsuga menziesii (Douglas fir). As I have already pointed out, one tree may have several, or even many, different ectomycorrhizal partners on its roots at the same time, and these may be replaced by others as the tree ages. So a single tree species can have a large number of potential mycobionts. Douglas fir may be able to form EMs with as many as 2,000 different species of fungi. And different isolates of the same fungal species may relate differently to the same tree species.

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Table 17.2. Comparison of the photobionts of ecto- and endomycorrhizal fungi Ectomycorrhizal (EM) Fungi

Endomycorrhizal (AM) Fungi

2,000 spp. of plants: mainly trees

300,000+ spp. of plants: herbaceous, woody

Gymnosperms:

380 families:

ALL Pinaceae and some Cupressaceae Angiosperms: ALL fa*gaceae, Betulaceae, Salicaceae, Dipterocarpoideae; MOST Myrtaceae; MANY legumes; SOME Aceraceae, Euphorbiaceae, Rosaceae, Tiliaceae, Ulmaceae, and members of seven other families

(EXCLUDING: ALL Brassicaceae, Commelinaceae, Cyperaceae, Juncaceae, Proteaceae; SOME Amaranthaceae, Caryophyllaceae, Chenopodiaceae, Polygonaceae; members of three other families; and MOST ectomycorrhizal spp.)

The approximately 2,000 ectomycorrhizal plant species are almost all woody and perennial. Many of them grow in extensive pure stands. Many are indigenous to the Northern Hemisphere, and some are the main components of the circumglobal boreal forest. The Pinaceae are the single most important ectomycorrhizal plant family, since they cover vast areas of the world and are harvested and replanted in astronomical numbers each year. Selection of appropriate mycorrhizal partners for our forest trees, and inoculation of seedlings before outplanting or encouragement of ectomycorrhizal fungi indigenous to outplanting sites, could improve the survival and growth of tree seedlings and therefore offer important economic benefits.

Evaluation and Selection of Ectomycorrhizal Fungi The thousands of different species of ectomycorrhizal fungi probably evolved in response to the diverse needs of many hosts in many habitats. How many host-fungussoil-climate combinations are there? No one knows, but for example, one 250km transect running east from the coast of Oregon passes through seventeen major forest zones and hundreds of kinds of habitat and includes at least ten genera of economically important ectomycorrhizal trees. So how can we select the best possible mycobiont for each combination? Fortunately for the decision-making process, one or two characteristics are often of overriding importance. For example, if a fungus cannot be grown in pure culture for the large-scale production of mycelial inoculum, it is often effectively excluded, no matter how good a mycorrhizal partner it is—although in some cases, such as Rhizopogon, Scleroderma, and Pisolithus, fruit bodies can provide adequate inoculum. All potential host-fungus pairs should ideally be tested for all of the following characteristics. (1) Rate and extent of mycorrhization. EMs can be seen with the naked eye and can be readily quantified. Entire root systems of seedlings can be examined, but in older trees

Mycorrhizas: Mutualistic Plant-Fungus Symbioses only a sample obtained by soil coring or local excavation can be studied. The percentage of mycorrhizal short roots can be determined visually, and the weights of these structures determined. Results may be expressed as number and weight of ectomycorrhizal structures per unit area or per unit volume of soil. (2) Host response. The reaction of a plant to mycorrhizal colonization can be measured in various ways. An easy, nondestructive method is to follow seedling survival, expressed as percentages of the initial uninoculated and inoculated populations. Such data can be gathered at various ages, before and after outplanting. Other nondestructive measures are plant height, thickness of stalk at ground level, number of leaves, leaf length, and leaf area. More definitive measurements involve determining the dry weight of the whole plant or of separate root and shoot systems. Measurements of stem height and stem diameter at soil line are eventually replaced by diameter at breast height (1.4 m) in older trees. (3) Mineral nutrition. Nitrogen and phosphorus uptake, and levels achieved in the mantle and in the plant, have been determined using radiotracer techniques and are among the most important reflections of the effects of EM fungi on their hosts. Ectomycorrhizal plants also absorb many other minerals (e.g., calcium, potassium, copper, molybdenum, magnesium, and zinc) from the soil more efficiently than nonmycorrhizal plants can. The fungal mantle can store inorganic nutrients (e.g., chloride, ammonium, and phosphate) and release them to the plant during periods of deficiency or of active growth. Pisolithus tinctorius thrives in soils of extremely low fertility, such as mine spoils, while Paxillus involutus does well only on sites with relatively abundant available nitrogen. (4) Water relations. The fungus Cenococcum geophilum is especially tolerant of low water potential, which correlates well with its propensity for forming EMs in dry areas. In fact, because Cenococcum grows best at a water potential of –15 bars, it can be difficult to establish in irrigated nurseries, where it may be soon replaced by Thelephora terrestris. (5) Temperature. Pinus cembra destined for high-altitude outplanting is inoculated with a cold-adapted strain of Suillus plorans. Other fungi, especially Pisolithus tinctorius, have been found to be adapted to high temperatures. Cenococcum geophilum appears to tolerate both extremes relatively well. (6) pH. Pine seedlings with Pisolithus tinctorius EMs survive and grow better on acid coal mine spoils than do nonmycorrhizal seedlings. This fungus can tolerate a pH range of 2.6–8.4. Cenococcum geophilum forms mycorrhizas from pH 3.4–7.5. Other ectomycorrhizal fungi, especially truffle fungi spore-inoculated onto seedlings, improve the growth of trees in alkaline soil. (7) Toxicity. Studies on the spoils derived from nickel mining at Sudbury, Ontario, indicate that some EM fungi can tolerate fairly high levels of heavy metals in the substrate. (8) Persistence. The choice of an ectomycorrhizal partner for a tree should be based either on its short-term benefits or on the longer-term production of edible fruit bodies. The initial mycobiont may be supplanted or supplemented by other EM fungi after the seedling has been outplanted, but its presence in the early days may well make the difference between death and survival. (9) Disease resistance. The presence of EM fungi on the roots of trees gives them some protection against the attacks of root-pathogenic fungi. Boletus bovinus helps to protect

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Chapter 17 Picea abies from Heterobasidion annosum. Pisolithus tinctorius increases the survival rate of Pinus taeda seedlings exposed to Rhizoctonia solani. Mycorrhizas formed by Suillus granulatus seem to protect seedlings of Pinus excelsa from a root-rotting Rhizoctonia. Seedlings of Pinus clausa are protected against Phytophthora cinnamomi by mycorrhizas of Pisolithus tinctorius. These effects are not fully explained but may be due to competition between fungi for nutrients and for access to the root. (10) Mycelial strand formation. These aggregations of parallel hyphae serve as effective agents for the spread of the fungi through the soil and in the long-distance translocation of nutrients to the mycorrhizal roots. Other things being equal, it would seem reasonable to choose a strand-forming fungus, such as Rhizopogon, over one that does not produce them. (11) Ease of isolation. Pure cultures of ectomycorrhizal fungi are usually derived from fruit body tissue, although they can also be obtained from surface-sterilized mycorrhizal roots, sclerotia, rhizomorphs, or mycelial strands. It is difficult to germinate basidiospores, and this is rarely attempted. Isolation from fruit bodies allows precise identification of the fungus at the outset. Members of the following genera are often fairly easy to isolate: Amanita, Boletus, Cortinarius, Hebeloma, Hysterangium, Laccaria, Lactarius, Leccinum, Paxillus, Pisolithus, Rhizopogon, Scleroderma, Suillus, and Tricholoma. Happily, these include some of the better mycorrhizal partners with the broadest host ranges. But only a few species of Russula have yet been cultured. (12) Large-scale inoculum production. Since Pisolithus tinctorius has been shown to establish mycorrhizas with almost fifty different tree species and it thrives over a wide range of soil pH, tolerates high temperatures well, and can establish mycorrhizas in the poorest soils, it was touted as a panacea for all our ectomycorrhizal problems. It was also the first EM fungus to be made available in the form of commercially produced mycelial inoculum. But Pisolithus can’t be all things to all EM trees. It is probably at its best coping with heat and drought stress. It has been collected only a few times in Canada and is not the perfect partner for boreal conifers. Rhizopogon is replacing it in some areas. Some research suggests that when conifer seedlings with Pisolithus mycorrhizas are outplanted in northern Quebec, the Pisolithus is soon replaced by indigenous species. Inoculum of Cenococcum geophilum, Rhizopogon spp., Suillus spp., Thelephora terrestris, and others is now available (see http://www.mycorrhizae.com). (13) Edibility. If several potential partners seem more or less equivalent, the ultimate choice may be dictated by secondary factors such as the edibility or otherwise of the fruit bodies of the fungi being considered. For example, if a hypothetical choice lay between a species of Amanita known to be highly toxic and another agaric, such as Boletus edulis, that was edible and choice, the decision would be straightforward. Less obvious, but also important, is the caution that species known to have toxic fruit bodies should not be introduced to new areas as mycorrhizal partners, even if they might seem otherwise desirable. One of the most toxic of all agarics, Amanita phalloides, was inadvertently introduced into South America as a mycobiont of oak seedlings imported from Europe early in the last century. The cyclopeptide toxins (amatoxins) in this fungus have since caused many fatalities. The Australian government prevented another problem by refusing to allow the importation of cultures of Amanita pantherina, which is a good mycorrhizal partner but produces basidiomata containing high levels of ibotenic acid (see chapter 22). At the other end of the scale are the originally French experiments

Mycorrhizas: Mutualistic Plant-Fungus Symbioses with ‘trufficulture’: the deliberate use of Tuber melanosporum as a mycorrhizal partner, with an eye to the production of truffles, an extremely valuable crop, which is also now being cultivated in New Zealand, Australia, the United States, and Canada. The first steps toward the culture of other choice edible fungi have been made, again by the French. Using pure cultures of the famous ‘cep’ (Boletus edulis) and three other boletes as well as Lactarius deliciosus and Tricholoma flavovirens, EMs have been established on Pinus pinaster and Pinus radiata in test tubes and in greenhouse pots. In New Zealand, Lactarius deliciosus is fruiting, providing an interesting and perhaps valuable byproduct of afforestation.

Sources of Ectomycorrhizal Inoculum Ectomycorrhizas may be initiated by several different kinds of inoculum: (1) naturally dispersed spores; (2) colonized soil; (3) mycorrhizal seedlings; (4) ascomata, basidiomata, spores, or sclerotia specifically collected for the purpose; and (5) fungal mycelium produced in axenic culture. It is worth comparing the merits of these different kinds of inoculum. (1) Natural spore inoculum is, of course, one of the prime dispersal mechanisms for fungi, but it can’t always be relied on to colonize nursery or outplanted seedlings because (a) it is available only during a relatively short season, since most agarics fruit in late summer or early fall. (b) Even when spores are being released, they may not reach the seedlings in adequate numbers, especially if the seedlings are a long way from the nearest stand of ectomycorrhizal trees. (c) We have no control over the nature of the fungal partners being introduced. This is important because EM fungi vary widely in their efficiency. (d) If seedlings are being started at a low-elevation nursery for highelevation outplanting, they may acquire local mycobionts unsuited to conditions at the intended growth site. (2) In Western Australia, pine seeds planted at fourteen new nurseries germinated and grew relatively well for a few months and then began to decline and die. This was because most of them had not encountered appropriate ectomycorrhizal partner fungi. The few healthy seedlings were found to have developed EMs. When soil from around the healthy seedlings was used to inoculate other seedbeds, the seedlings there recovered. Soil from beneath established ectomycorrhizal trees is a fairly reliable source of inoculum if 10% by volume is added to a new nursery bed. Colonized soil has been used to establish exotic pines in many parts of the southern hemisphere, and soil transfer is a regular procedure in many developing countries. (3) Introduced mycorrhizal seedlings have also worked relatively well. This technique was first used on a large scale in Indonesia and is still used there. Mycorrhizal seedlings are planted in seedbeds at 1–2 m intervals. At outplanting time, some seedlings are left to inoculate the next crop. (4) The deliberate collection and introduction of spores, fruit bodies, or sclerotia would seem to be an obvious way of improving upon nature, but there are problems: (a) Naturally occurring fruit bodies of most EM fungi are available only during a small part of the year. (b) The amount of inoculum available will fluctuate from year to year. (c) Fruit bodies occur sporadically and scattered over large areas, so collection in the

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Chapter 17 amounts needed would be almost impossible. Only the particularly concentrated spore source represented by gasteromycetes such as Rhizopogon and Pisolithus could be obtained in the necessary quantity. (d) Storage of perishable basidiomata would be difficult. (e) Initiation of mycorrhizas by basidiospore inoculum takes three to four weeks longer than when mycelial inoculum is used. This gives pathogens longer to attack the roots, and the later-developing mycorrhizas also provide less growth stimulation during the crucial early stages of seedling development. One widespread mycobiont, Cenococcum geophilum, produces structures called sclerotia that are much less perishable than either spores or basidiomata, since they evolved as a long-term survival mechanism. These sclerotia occur naturally in the soil in huge numbers and could be harvested and used as inoculum. In another case, over 450 kg of Pisolithus tinctorius basidiomata were collected on coal mine spoils in Alabama in seventy-five person-days. Since less than 1 mg of spores is needed to inoculate a plant, this collection provided enough inoculum for hundreds of millions of pine seeds. The basidiospores were used in a seed-pelletizing mix, each seed being coated with 500,000 to 5 million spores which became dispersed around and below the seed after planting as a result of rain or irrigation and colonized the roots as these developed. (5) If mycelial inoculum derived from pure cultures of known mycobionts is used, the identity of the fungus will be known and pests and pathogens will be absent. However, this too has inherent problems: (a) it is expensive; (b) some EM fungi are difficult to isolate in pure culture; (c) cultures are expensive to maintain, grow slowly, and take a long time to produce enough biomass for large-scale applications; (d) we don’t know how well such inoculum survives in the soil in the face of predation and competition from indigenous organisms; (e) we have not yet defined the best possible fungus-host combinations for many soil-climate combinations; and (f) it is not worth going to the expense of mass producing mycelium of many species until we are sure that the results will be economically worthwhile. However, some EM fungi, often those with relatively small basidiomata (e.g., Thelephora, Laccaria), are early colonizers and are associated with young trees, while others, often with large fruit bodies (e.g., Boletus) are late colonizers and are often associated with larger, older trees. It would seem appropriate to concentrate on early colonizers. The production and field application of mycelial inoculum of EM fungi are still expensive. Yet many foresters believe that the inoculation of both bare root and containergrown seedlings with appropriate EM fungi will eventually become routine. However, in many parts of the world it is not afforestation, but reforestation, that is important. In this case, the soil of the site to be replanted will often already contain good mycorrhizal fungi.

Morphology and Development of Arbuscular Mycorrhizas (AM) The AM is a more subtle phenomenon than the EM. The presence of an AM fungus in a root is usually undetectable by the naked eye. There is no obvious morphological change, no mycelial mantle, no sudden flush of large fungal fruit bodies. Yet, as appropriate clearing and staining will show, roots are often extensively colonized (Fig. 17.1A). The life cycle of an AM fungus goes more or less as follows: Spores in the soil

Mycorrhizas: Mutualistic Plant-Fungus Symbioses germinate, usually in conditions appropriate for plant seed germination and root growth. If the fungus encounters a receptive root or root hair, an appressorium is formed, and penetration occurs (often through ‘short cells’ of the exodermis, if these are present, because they are not yet suberized) in the elongation zone of the root. Symbiosis is initiated in juvenile tissues. Hyphae grow through or between the cortical cells but never enter meristematic cells or endodermal cells. Specialized hyphal branches penetrate the walls of individual cortical cells and form finely branched, treelike structures called arbuscules (Figs. 17.1, 17.3), which are completely encapsulated by the host plasmalemma (so not really inside the cytoplasm), and these are the main sites of exchange between the fungus and the plant. The nucleus of the root cell is enlarged, and the volume of cytoplasm increases. We assume that nitrogen and phosphorus are being actively transferred to the plant throughout the life of each arbuscule. Polyphosphate granules, involved in P transport in the fungus, are present in hyphae but not in the finest branches of the arbuscules, which contain acid and alkaline phosphatases. After four to fifteen days, the arbuscule gradually breaks down, and the root cell returns to normal.

Fig. 17.3 Finely branched arbuscule of an endomycorrhizal fungus inside a root cell of the photobiont.

Many, although not all, AM fungi also form vesicles in the root. These are thinwalled, inflated structures without a basal septum and are often full of lipids. I have seen up to 500 vesicles/cm in older leek roots, the root cortex looking like an almost solid mass of vesicles. Despite this, the root remains functional, since the stele (the central vascular cylinder) is not colonized and can still translocate substances to and from the active root tips. Intramatrical vesicles are not formed by the genus Gigaspora. This is an important reason we now call this kind of mycorrhiza AM, not vesiculararbuscular mycorrhiza (VAM), which used to be the preferred name.

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Chapter 17 While the fungus is developing its intramatrical phase within the root, it is also developing an extramatrical hyphal network in the soil. Extramatrical hyphae extend 8 cm or more from the root. This means that a mycorrhizal plant can exploit many times the volume of soil available to a nonmycorrhizal plant. The extramatrical hyphae obtain nitrogen and phosphorus and translocate them to the plant, which reciprocates by supplying the fungus with photosynthates. These enable the latter to extend its hyphal network and to produce its large, asexual spores. Spores may form in the soil singly or in aggregations up to 2 cm in diameter called sporocarps. Individual spores are very large by fungal standards: 50–600 µm in diameter. They may have walls up to 30 µm thick, are often darkly pigmented, and are filled with storage lipids—all features that emphasize their role in long-term survival when host plants are absent or dormant. Vesicles are sometimes regarded as ‘intramatrical spores’. The spores will eventually germinate, producing hyphae which will once more grow through the soil and perhaps encounter another plant. The identity of the plant may not matter much, since AM fungi can usually relate successfully to a very large number of host species (ca. 230 fungal taxa with 300,000 plant taxa).

Systematics of Arbuscular Mycorrhizal Fungi and Their Hosts The taxonomy of the AM fungi is in a state of ferment. Thirty taxa were recognized in 1974, about 120 by 1985, and 130 by 1991. And as of 2012, mycologists recognize 3 classes, 5 orders, 14 families, 29 genera, and about 230 species. They are now classified as a separate phylum, Glomeromycota, which are now understood to be related to the subkingdom Dikarya. Until quite recently only five AM genera were recognized: Acaulospora, Entrophospora, Gigaspora, Glomus, and Scutellospora (Fig. 17.4). Since these fungi are present in most soils around the world, I will provide a dichotomous key to these five genera.

Dichotomous Key to Five Genera of AM Fungi (after Morton and Benny 1990) 1 Only arbuscules formed in mycorrhizal roots; ‘azygospores’ produced at apex of a fertile hypha; auxiliary cells formed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 1 Both arbuscules and vesicles formed in mycorrhizal roots; ‘chlamydospores’ produced terminally or laterally on or in fertile hyphae; auxiliary cells not produced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 2 Germ tubes produced directly through spore wall; inner flexible wall group absent; auxiliary cells ornamented (papillate or echinulate) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gigaspora 2 Germ tubes arise from germination shield; inner flexible wall group always present; auxiliary cells knobby, papillate, or smooth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Scutellospora

Mycorrhizas: Mutualistic Plant-Fungus Symbioses 3 Chlamydospores formed apically from fertile hyphae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glomus 3 Chlamydospores formed from or within the neck of a sporiferous vesicle ........................................................................... 4 4 Spores arise laterally from the neck of a sporiferous vesicle (saccule) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acaulospora 4 Spores formed in the neck of the sporiferous vesicle (saccule) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Entrophospora Glomus (Fig. 17.4), the commonest genus, now has ninety species (including former members of Sclerocystis, which was distinguished by its multispored sporocarps). Most species of Glomus form globose, ellipsoid, or irregular spores that are 20–400 µm in diameter and have walls up to 30 µm thick. They are colourless, yellow, red-brown, brown, or black. They are attached to a single hypha and are produced in the soil near plant roots, at the soil surface, or, occasionally, inside roots, and they may be solitary, in groups, or in large aggregates called sporocarps that are 1–20 mm across. The sporocarps of a few species are formed at the surface of the soil, those of other species buried in the soil or in leaf litter. Sporocarps are commonest in undisturbed forest communities with perennial plants and a thick organic horizon. The spores of Acaulospora species (Fig. 17.4) form on the side of thin-walled, terminal swellings that later collapse and vanish or leave only an inconspicuous remnant. The spores are globose or ellipsoid, are 100–400 µm in diameter, and have walls up to 12 µm thick. Spores are colourless to reddish-brown and never occur in sporocarps. Individual spores of Gigaspora species (Fig. 17.4) are 200–600 µm in diameter, with walls up to 20 µm thick, and develop singly at the end of a persistent, bulbous hypha, which may bear short lateral projections, the remains of collapsed hyphal branches. Spores range from colourless to black and may be ornamented. Gigaspora species also form unique ‘ornamented vesicles’, 20–50 µm in diameter, borne singly or in clusters of twelve or more, typically on spiral hyphae. But of course, largely as a result of molecular techniques, no fewer than twenty-nine genera are now recognized. I am not going to burden you with the other twenty-four, but you can find them in Oehl et al. (2011). AMs have actually been seen in only 1,000 or so genera of plants from about 200 families. This is a small but still highly significant sample of the 350,000 extant species of higher plants, and we believe that about 90% of vascular plants will normally have AM fungi in their roots, especially if they are growing in poor soil. I can’t mention all the families which we know or assume are AM. It is easier to list those few that are mostly non-AM. These are the Brassicaceae, Commelinaceae, Cyperaceae, Juncaceae, and Proteaceae; as well as some members of the Capparaceae, Polygonaceae, Resedaceae, Urticaceae, and herbaceous members of the Caryophyllales (Amaranthaceae, Caryophyllaceae, Chenopodiaceae, Portulacaceae); plus, of course, most of the 2,000 woody species that are ectomycorrhizal. Apart from this last group, most of the plants just mentioned are entirely nonmycorrhizal, and most are herbaceous (see Table 17.2 for a comparison between the hosts of AM and ectomycorrhizal fungi).

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Fig. 17.4

Diagnostic spores and sporocarps of endomycorrhizal fungi (Glomeromycota).

The only entirely nonmycorrhizal woody family is the Proteaceae, whose members have fine, brush-like roots and abundant root hairs. Many of the rest are weedy— vigorous pioneer herbaceous annuals with highly opportunistic lifestyles. They germinate quickly in poor soils, and some can flower and set seed in a few weeks (try chickweed and some mustards!). This means that they cannot wait for the local AM fungi to find and colonize their roots. They have evolved finely branched roots with many root hairs, which enable them to dispense with mycobionts.

Mycorrhizas: Mutualistic Plant-Fungus Symbioses The AM relationship is extremely old—at least 400 million years—and it is hardly surprising that some plants may now be evolving different lifestyles. This is especially true of the herbaceous annuals, the newest group of plants. As well as being weedy, members of the Brassicaceae (formerly Cruciferae) and related families have evolved chemical defences to repel herbivorous animals and thus may have inadvertently discouraged their now inessential mycorrhizal fungi. Since there are just under 400 families of higher plants, we estimate that all or most members of over 380 of these families associate with AM fungi. Many of the 50 or so families of nonflowering vascular plants (Gymnosperms, Pteridophytes, etc.) are also AM. This means that about 300,000 plant species are receptive hosts for the incomparably smaller number of AM fungi.

Sources of Arbuscular Mycorrhizal Inoculum AM fungi are obligately biotrophic and can be maintained or multiplied only in dual culture with a host plant. All experimental work with these fungi involves the establishment of such cultures. The roots of experimental plants can be inoculated with infected soil, infected roots, or spores, or a mixture of all three. But soil often contains more than one mycorrhizal fungus and may even contain pathogens. Root inoculum can be used if it has been grown under more or less aseptic conditions and if the original inoculum used to infect the roots was of a single, named species. The structures developing inside roots, although diagnostic of AM fungi as a group, are not usually identifiable to species. Most species can be identified only by their spores, which develop primarily on hyphae outside the roots. So spores may still be the best inoculum for laboratory experiments. Since AM fungi won’t grow in axenic culture, the only sources of spores are either the soil near the roots of colonized plants or in vitro culture on genetically modified roots. This latter technique is explained by Khaliq et al. (2010). Plant tissues genetically transformed by the Ri (root-inducing) plasmid of Agrobacterium rhizogenes exhibit a neoplastic disease syndrome known as ‘hairy root’: the transformed tissue grows rapidly in Atropa belladonna and Nicotiana tabacum (doubling in forty-eight hours). The Ri plasmid also carries a gene responsible for producing amino acid derivatives called ‘-opines’, such as agropine, mannopine, cuccumopine, and mikimopine, the detection of which indicate transformation. Hairy roots can be subcultured as excised roots on solid media, where they are repeatedly treated with antibiotics such as carbenicillin or cephalexin to render the roots free of any bacteria and thus essentially clonal. Hairy roots can be developed from suitably treated stem and leaf tissue. Choice of AM inoculum used in colonization of hairy roots is important. Researchers have variously used sporocarps of Glomus mosseae and spores of Gigaspora margarita. The isolation of such inocula requires wet sieving and decanting, followed by density gradient centrifugation to clean the spores. Spores must then be surface sterilized before use. Most researchers have used chloramine-T, gentamycin, and streptomycin sulphate, followed by rinsing in sterile water. So colonized roots can now be produced in large quantities. Mycologists in England are also inoculating tomato plants with AM fungi and then growing them with their

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Chapter 17 roots in a thin film of recirculating nutrient solution. The colonized plants will give an improved yield of tomatoes, and later their chopped roots can be used to infect the next crop. The Myke-Pro website, built by a company that can supply inoculum in commercial quantities, provides useful information on the storage and effects of mycorrhizal inoculum: http://www.usemykepro.com/mycorrhizae-benefits-application-and-research .aspx.

Evaluation of Arbuscular Mycorrhizal Fungal Partners If we want to follow the development of colonization during an experiment, or to assess the level of colonization in native plants collected in the field, we look at stained roots using the gridline intersect method. Roots are washed, cleared in KOH or hydrogen peroxide, and stained in acid fuchsin or trypan blue. Stained roots are spread out evenly over the bottom of a petri dish which is marked with a grid of lines delimiting 1/2-inch squares. The gridlines are scanned under a dissecting microscope, and presence or absence of colonization is recorded at each point at which a root intersects a line. If three sets of 100 intersects are recorded, and the mean value determined, the percentage of the root length colonized will be estimated fairly accurately. This technique also allows the total length of roots in the dish to be determined. If the distance between lines is 14/11 of the chosen measuring unit, the number of intersects equals the length of the roots, expressed in that unit. Conveniently, 14/11 cm = 1/2 inch. So with a 1/2-inch grid, the number of grid intersects equals the total length of roots in centimetres (see Brundrett et al. 1994, which explains many techniques for studying mycorrhizas). The responses of host plants to AM fungi can also be measured in several ways. An obvious parameter is dry weight production. Dry weight of the root and shoot systems should be recorded separately, since their responses may differ. Other, nondestructive measures are differences in plant height, stem diameter, shoot volume, and leaf number and area—all of which can be measured repeatedly on the same plants at intervals. Crop yield, transplant survival, and disease resistance are other valid criteria. Since the AM fungus is often essentially parasitic on its host until it becomes established, the experimental growth period must be long enough for the positive effects of mycorrhization to become apparent. Extramatrical hyphae have been shown to retrieve soluble and insoluble nitrogen and phosphorus nearly 3 cm from a root—minerals that were completely unavailable to nonmycorrhizal roots at similar distances. The zone of depletion around mycorrhizal roots can be twice that around nonmycorrhizal roots. But even the most efficient AM fungus will have little or no effect on a plant if available phosphorus and nitrogen are present at luxury levels. A relative mycorrhiza dependency factor can be calculated from the following formula to be calculated for any given level of P availability: 100 ×

dry wt. mycorrhizal plant – dry wt. nonmycorrhizal plant dry wt. of mycorrhizal plant

Mycorrhizas: Mutualistic Plant-Fungus Symbioses For example, at 100 μg/g available P, the relative mycorrhiza dependency of carrot is 99.2%, and that of wheat is 0%. This calculation also lets us compare the responses of any species of plant to different sources of P and to different mycorrhizal fungi. As yet we don’t know much about the contributions to plant growth made by indigenous AM fungi in field soils (although we extrapolate from the results of laboratory experiments). This is partly because any treatment that will kill all spores in the soil and allow comparisons to be made is sufficiently drastic that it will also change the chemical makeup of the soil and alter its nutrient status.

Effects of Arbuscular Mycorrhizas on Plant Disease The presence of AM fungi in the roots of plants tends to reduce the incidence and severity of soilborne diseases in those plants. I have found many reports of interactions between AM fungi and soilborne plant pathogenic fungi, plant parasitic nematodes, and viruses, involving a total of twenty-one different crop plants. Most experiments showed that mycorrhizal plants had less disease, although virus symptoms were more severe. Various explanations have been put forward to explain the reductions in fungal disease: (1) the mycorrhizal plants were healthier and more able to resist the attacks of pathogenic fungi; (2) the cells of mycorrhizal plants may partly digest the senescent arbuscules of the fungus, and the same chitinolytic enzymes might be used on other invading fungi; and (3) possible infection sites on the surface of the plant roots may be preferentially occupied by the mycorrhizal fungus, to which the plant may well be more susceptible.

Prospects It has been said that ‘most woody plants require mycorrhizas to survive, and most herbaceous plants need them to thrive’, so it seems only logical that both AM and EM fungi must be of vital concern to hom*o sapiens, a supposedly intelligent species that knows it is totally dependent on plants. Tragically, the endomycorrhizal rainforests are rapidly being destroyed by logging, agriculture, mining, road building, urbanization, and other human activities. Since these forests do not regenerate easily or quickly, they will often be replaced by plantations of fast-growing oil palms and exotic ectomycorrhizal conifers. Conifers are also being established in many treeless areas. Existing forests need to be replanted after harvesting. It looks to me as though any research and biotechnological investments society makes in ectomycorrhizal fungi will be well repaid by the ease and speed with which the trees become established and by the accelerated growth of the maturing forests. Tree nurseries and field crops are often heavily fertilized. This effectively discourages many mycorrhizal fungi, which are highly adapted to infertile soils. Commercial mycorrhizal inoculum may eventually wean forest nursery workers and farmers away from chemical fertilizers: increasing energy costs may provide the required impetus.

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Other Kinds of Mycorrhizas Although AM and EM relationships are the dominant expressions of this kind of plant-fungus relationship, several other patterns are known. The family of flowering plants with by far the largest number of species is the Orchidaceae, and they have their own unique kind of (possibly nonmutualistic) mycorrhiza. What if anything does the fungus get out of it? Is it tricked by the orchid, and if so how? Lots of fascinating questions.

Orchid Mycorrhizas Orchids produce astronomical numbers of seeds, which are so tiny that they can carry little or no food. For the first two to eleven years of their lives—until their first chlorophyll-bearing leaf develops—these plants depend on being colonized by fungi, commonly anamorphs of the genus Rhizoctonia (whose holomorphs are crust fungi belonging to basidiomycetous genera such as Thanatephorus, Corticium, and Ceratobasidium). Since these fungi are active saprobes in the soil, they are in a position to provide the orchid seedlings with nutrients. The fungus enters cells of the root cortex and develops coils, or pelotons, which eventually swell, degenerate, and are absorbed by the plant cell. If we observe the germination of many orchid seeds, it becomes clear that there are three common outcomes: (1) the seed becomes colonized by an appropriate fungus and thrives; (2) the fungal infection takes over and kills the seedling; (3) the fungal invasion fails, the fungus is eliminated, and the seedling stops growing. This does not appear to be a mutualistic symbiosis, but rather a delicate balancing act on the part of both partners, perhaps still in the process of evolving, although spectacularly successful when it works. Another widespread and important family, the Ericaceae, also have a special kind of fungus-plant interface, as do the Gentianaceae and the Monotropaceae. We’ll take a quick look at these offbeat symbioses (also check out www.mycolog.com). Note that none of these phenomena has been investigated anything like as thoroughly as have the AMs and EMs—much remains to be done before we will understand them properly.

Gentianaceous Mycorrhizas These look rather like orchid mycorrhizas, producing coils inside cortical root cells. However, in this case the plants can provide nutrients to the fungus. So the gentianaceous root cell environment seems to induce a morphological variant of normal endomycorrhizas.

Ericoid Mycorrhizas These, mostly involving Rhizoscyphus (formerly Hymenoscyphus), an inoperculate discomycete (Ascomycota, Leotiomycetes), and ericaceous plant genera such as Erica, Gaultheria, and Vaccinium, look intermediate between EMs and AMs (they usually have a mantle covering the root), and for that reason they used to be called ‘ectendomycorrhizas’. In roots of the ericaceous Arctostaphylos, the mantle is less obvious, but

Mycorrhizas: Mutualistic Plant-Fungus Symbioses the fungal coils clearly fill the cortical cells. Ericoid mycorrhizas have intracellular colonization but no superficial hyphae—no mantle on the root surface.

Arbutoid Mycorrhizas In Arbutus menziesii, a beautiful evergreen ericaceous tree that grows on the west coast of North America, the short roots have a sheathing fungal mantle and branch in a characteristic way, but instead of producing a Hartig net between the host cells, the fungus actually penetrates the cortical cells and fills them with densely coiled hyphae (not arbuscules). The fungi involved here are typical EM fungi behaving atypically.

Monotropoid Mycorrhizas The relationship between the colourless flowering plant Monotropa (‘Indian pipe’) and its fungus is strange. Monotropa is white and has no chlorophyll, so it is not in a position to provide carbohydrates to a mycorrhizal fungus. Yet the roots have a fungal mantle outside and something resembling a Hartig net inside. The distinguishing morphological feature is that the fungus sends a single haustorium-like peg into each root cell. Whatever the morphology suggests, the fact is that the achlorophyllous Monotropa is taking food from the fungus, and no one knows if it is giving anything at all in exchange. The key to the situation is that, using radiotracers, the food in question has been found to be coming from a neighbouring plant (a green one this time) with which the fungus in question has a normal ectomycorrhizal relationship. So we are looking at a tripartite relationship: In the case of Monotropa uniflora, a conifer makes sugars and passes them to Russula, its ectomycorrhizal partner. Russula translocates them through its mycelium and part is hijacked by the Monotropa, which thus seems to exploit the fungus directly, and the conifer at second hand. Monotropa hypopitys parasitizes Tricholoma. Sarcodes sanguinea and Pterospora andromeda parasitize Rhizopogon. Needless to say, both Tricholoma and Rhizopogon are ectomycorrhizal fungi, so the ultimate victim is always a tree. So, despite what you read in even the most up-to-date dictionary, Monotropa is not a ‘saprophyte’. Interestingly, mycologists have known this since 1960. It makes me wonder about lexicologists. You may have the impression that all these kinds of mycorrhiza are quite distinct phenomena. Yet it has been demonstrated that a number of fungi—Astraeus hygrometricus (an earthstar), Tricholoma flavovirens, Suillus albidipes, Suillus tomentosus, Lactarius paradoxus (all agarics), and Coltricia perennis (Hymenochaetales)—can all form normal EMs with Pinus banksiana (Pinaceae) and ectoendomycorrhizas with Arctostaphylos uva-ursi (Ericaceae). This has opened a whole new area of study.

Further Reading on Mycorrhizas Allen, M. F. 1992. Mycorrhizal Functioning: An Integrative Plant-Fungal Process. New York: Chapman & Hall. Brundrett, M. L., L. Melville, and L. Peterson, eds. 1994. Practical Methods in Mycorrhiza Research. Sidney, Canada: Mycologue Publications. www.mycolog.com.

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Chapter 17 Harley, J. L., and S. E. Smith. 1983. Mycorrhizal Symbiosis. London: Academic Press. Kendrick, B., and S. M. Berch. 1985. “Mycorrhizae: Applications in Agriculture and Forestry.” In Comprehensive Biotechnology. Vol. 3. Edited by C. W. Robinson, 109–52. Oxford: Pergamon. Khaliq, A., D. J. Bagyaraj, and M. Alam. 2010. “Advances in Mass Production Technology of Arbuscular Mycorrhiza.” In Mycorrhizal Biotechnology, edited by D. Thangadurai, C. A. Basso, and M. Hijri. Enfield, NH: Science Publishers. Morton, J. B., and G. L. Benny. 1990. “Revised Classification of Arbuscular Mycorrhizal Fungi (Zygomycetes): A New Order, Glomales, Two New Suborders, Glomineae and Gigasporineae, and Two New Families, Acaulosporaceae and Gigasporaceae, with an Emendation of Glomaceae.” Mycotaxon 37:471–91. Oehl, F., E. Sieverding, J. Palenzuela, K. Ineichen, and G. A. da Silva. 2011. “Advances in Glomeromycota Taxonomy and Classification.” IMA Fungus 2:191–99. Accessed May 14, 2017. https:// www.scienceopen.com/document/read?vid=4479da29-962d-4be5-8edf-a01baddffa15. Peterson, R. L., H. B. Massicotte, and L. H. Melville. 2004. Mycorrhizas: Anatomy and Cell Biology. Ottawa: NRC Press. Read, D. J., D. H. Lewis, A. H. Fitter, and I. J. Alexander. 1992. Mycorrhizas in Ecosystems. CAB. International. Smith, S. E., and D. J. Read. 1997. Mycorrhizal Symbiosis. 2nd ed. San Diego: Academic Press. Taylor, T. N., W. Remy, H. Hass, and H. Kerp. 1995. “Fossil Arbuscular Mycorrhizae from the Early Devonian.” Mycologia 87:560–73. van der Heijden, M. G. A., J. N. Klironomos, M. Ursic, P. Moutoglis, R. Streitwolf-Engel, T. Boller, A. Wiemken, and I. Sanders. 1998. “Mycorrhizal Fungal Diversity Determines Plant Biodiversity, Ecosystem Variability and Productivity.” Nature 396:69–72. Varma, A. 2008. Mycorrhiza: State of the Art, Genetics and Molecular Biology, Eco-Function, Biotechnology, Eco-Physiology, Structure and Systematics. Berlin: Springer. Varma, A., and A. C. Kharkwal, eds. 2009. Symbiotic Fungi. Berlin: Springer. There are numerous illustrations on http://mycorrhizas.info/. Mycorrhizal Associations: The Web Resource. [A fairly comprehensive website, built by Mark Brundrett, which gives very extremely background and focuses on Australian plants.] See also http://www.mycolog.com /Chapter17.htm.

18 Fungi as Food: Mycophagy Introduction In the English-speaking world, fungi are widely viewed with suspicion. Toadstools, perhaps because of their sudden appearance, strange shapes, bizarre colours, and reputedly poisonous nature, became associated in folklore with fairies, witches, or even the devil. These superstitions probably saved many lives over the centuries. People in forested areas of Central and Eastern Europe apparently directed their superstitious awe elsewhere (see Grimms’ Fairy Tales) and did not hesitate to eat mushrooms whenever they appeared. Information derived from these ‘experiments’ gradually accumulated and was passed on, first in the oral tradition, later in books. A few agarics gained a reputation for killing those who ate them. These are dealt with in chapter 22. At the other end of the scale, a very small number of fungi eventually entered the culinary hall of fame. They are the chief subjects of this chapter: Ravioli filled with fresh black truffles and celery in melted butter and parmesan cheese... sweetbreads with a soya sauce, cloud ears and julienne of many vegetables... lamb with kidney stuffing and potato crepe stuffed with spinach and mushrooms... warm sweetbread salad with girolles (chanterelles), oysters and leeks in truffle sauce... tiny veal kidneys with chanterelle mushrooms... cloudlike mousse of fattened livers of Bresse chickens with truffles raining over it. These quotations are taken from a series of articles written by the restaurant critic of the Toronto Globe and Mail, Joanne Kates, after a grand gourmet tour of France. The common denominator in this outpouring of haute cuisine: fungi as ingredients contributing flavour and texture. Can we make any generalizations about the edibility of large fungi? Let’s try a few. (1) Of more than 10,000 species of fleshy fungi, only a handful are lethal—deadly poisonous. (2) Unfortunately, some of those are relatively common. (3) Representatives of only about twenty genera are regarded as prime edible fungi: check the list below. (4) There are no simple ways of distinguishing between the edible and the poisonous; all folkloric tests such as, ‘If the cap peels, it’s edible’ and ‘If it doesn’t blacken a silver spoon, it’s OK,’ are misleading and dangerous fictions. (5) You should eat a mushroom only if you know its name with considerable precision (and by that, I mean its scientific name, its Latin binomial, a unique pair of epithets which specify its genus and species). Don’t assume that all is well if it looks like a picture in a book, or even if you can identify it to genus: genera which contain prized edible species may also have disagreeable or dangerous members and vice versa—this is true of both Agaricus and Amanita. (6) 345

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Chapter 18 In order to discover the proper name, you will probably have to refer to an expert: every handbook ever published is incomplete and fallible, and you will often need to examine microscopic features (such as basidiospores). (7) Do not accept the word of selfstyled ‘experts’ without checking their credentials: after all, it’s your life, not theirs. (8) The first few times you eat a mushroom that is new to you, don’t eat much of it, because some people develop severe allergic reactions, even to species generally considered safe. (9) Sort your collections very carefully: don’t mix species, and never eat old or shrivelled specimens. (10) If you are still determined to become a mycophagist, buy one or more of the well-illustrated manuals listed at the end of this chapter (the Lincoff, Phillips, and Arora books are the most comprehensive), and join your local natural history or mushroom society. If you become fascinated by the strange and mysterious world of the fungi, then you will be ready to join NAMA—the North American Mycological Association—and perhaps subscribe to Mushroom magazine. Good hunting. If you are prepared to wait until spring and fall each year before indulging in mushroom feasts, well and good. There is something special about seasonality that gives you something to look forward to (think also fresh-picked strawberries in June). But if you’d rather be able to eat mushrooms all year round, someone will have to grow them for you. That means bringing them into cultivation. We can’t do that with chanterelles or matsutake, no matter how much we would like to have them. That is because they are mycorrhizal, and will fruit only with their tree symbiont, and at their genetically determined season. So we must look among the saprobic mushrooms for suitable candidates. Most of those listed below will grow on composted manure, or sawdust, or logs, under the right conditions. Now for a quick tour of the better-known edible mushrooms. I will begin with seven that have been brought into large-scale cultivation and then move on to others, some grown on natural substrates and others available only in nature. Mushroom cultivation may have an extremely bright future. Consider the following: If we use a hectare of land to produce beef, the yield of protein is about 80 kg/ha. If we use the same area for fish farming, the yield may be as much as 660 kg/ha. But if we grow mushrooms, the protein yield is commonly 80,000 kg/ha; and fungi have the added advantage that they bioconvert cellulosic debris such as straw, sawdust, and animal manure, which are produced in large quantities as essentially worthless byproducts of other industries. Please note that all of the edible fungi reported in this chapter are illustrated at www .mycolog.com. (1) Agaricus brunnescens or A. bisporus (depending on whether you follow Malloch or Singer [Agaricales, Agaricaceae])—the mushroom of the Western world. To many people, all other agarics are ‘toadstools’ to be avoided. Sautéed in butter, the supermarket mushroom is an excellent accompaniment to steaks; dipped in batter and deep fried, it makes a truly greasy but tasty snack at many country pubs. This is the one edible mushroom that every Westerner knows about. It was domesticated in the seventeenth century by the French and has spawned a considerable industry in North America (sorry about the pun; it was originally unintentional). Annual world production of this species is estimated to be about 1,000,000 tonnes and growing. As an interesting footnote to the end of the Cold War, the nuclear missile silos at Császár in Hungary are now being used to grow mushrooms. The crop is exported to Germany, the very country at which the missiles were formerly aimed.

Fungi as Food: Mycophagy Canadian consumption of A. brunnescens increased sevenfold between 1963 and 1983. Before I move on to the many other edible fungi, I must insert a warning. Mushrooms should never be eaten raw (as in salads), because they contain significant amounts (0.6 ppm) of the carcinogenic (cancer causing) 4-(hydroxymethyl)benzenediazonium ion. Fortunately, this unstable ion is destroyed by cooking. (2) Pleurotus ostreatus (Agaricales, Pleurotaceae), the oyster mushroom, is another good find. It forms overlapping clusters of large, nonstipitate basidiomata on dead or dying trees. In fact, no fewer than seven species of Pleurotus have been domesticated and marketed, notably Pleurotus sajor-caju, with P. ostreatus a distant second, but some growers ran into an unexpected snag: the basidiospores are extremely allergenic and cause severe reactions in many of the workers. World production is more than 20,000 tonnes/year. (3) Lentinula edodes (Agaricales, Marasmiaceae), the shiitake (shee-tah-kay) of Japan, the xiang-gu of China, is widely used in East Asian cuisine. For 2,000 years the Japanese and Chinese have cultivated it by boring holes in specially stacked oak and chestnut logs and inoculating them with plugs of infected sawdust or wood. The fungus takes a year or more to fruit, although repeated crops (flushes) will arise from each log. A refined culture technique now grows the mycelium in a much cheaper hardwood sawdust medium supplemented with 8%–25% of a starch-protein mixture and some calcium sulphate. This medium is autoclaved in a heat-resistant plastic bag and then inoculated. Twenty days in darkness are followed by thirty to sixty days in the light, all at 24ºC–28ºC, and then ten more days in darkness at 13ºC–18ºC. Finally, fruiting is induced by opening the bags, exposing them to light, and deliberately varying the temperature between 10ºC and 18ºC. Fruiting may continue for ten months. The effort is worthwhile, because dried shiitake retail for up to $40/kg in gourmet shops. In southeast Asia, it is estimated that 200,000 growers produce 150,000 tonnes of shiitake per year. It is one of the best edibles. (4) Volvariella volvacea (Agaricales, Pluteaceae), the paddy straw mushroom, is widely used in the Far East as well as in Chinese cuisine in the West. It has been cultivated for centuries by preparing heaps of plant debris and watering them. It is now being grown on a more scientific basis, and pure mycelial inoculum (spawn) is available. When encountered in Chinese food, it can be easily recognized by the extremely well-developed volva or universal veil that virtually envelops the young basidioma. We usually see this fungus only in an immature condition, since it is harvested and canned before the cap expands. I was fortunate enough to visit a straw mushroom factory in Java in 1989. I took a few specimens back to my room and watched them first expand and then deposit a heavy pinkish spore print. World production is more than 60,000 tonnes/year. (5) Flammulina velutipes (Agaricales, Physalacriaceae), the winter or velvet stem mushroom, long cultivated in Japan, where it is called enokitake, is now grown on a sawdust-bran mixture in North America. It is too early to say whether it will catch on here, but it suits our climate, since it will grow and fruit at low temperatures. The cultivated form has little resemblance to the natural fruit bodies, since it consists of long, narrow stipes with tiny caps at the top. World production is about 40,000 tonnes/year. (6) Pholiota nameko (Agaricales, Strophariaceae) is another lignicolous mushroom that has been domesticated in Japan, where it is called nameko, and about 15,000 tonnes a year are produced.

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Chapter 18 (7) Auricularia polytricha (Auriculariales, Auriculariaceae), the tree ear or cloud ear or mu-er of Chinese cuisine, is a jelly fungus that grows on dead trees. Its flexible ear-like basidiomata are added to a variety of Chinese dishes; mostly, I suspect, for their slippery, crunchy texture. It has recently been suggested that something in these fungi reduces the clotting tendencies of human blood and may help to explain the low rate of heart disease among the Chinese. About 8,000 tonnes a year are consumed. (8) Tuber melanosporum (Pezizales, Tuberaceae), the black, queen, or Perigord truffle (Figs. 4.12F, G). This is what the French call the diamond of the kitchen. They also say: ‘Ta femme, tes truffes et ton jardin, garde-les bien de ton voisin’ (Your wife, your truffles, and your garden; guard them well from your neighbour). This old French saying will give you some idea of the high esteem in which truffles are held. The great French author, Molière, who wrote much better stuff than that, was obviously fascinated by truffles, since he gave the old French name for a truffle, ‘Tartuffe’, to the main character in his comedy of the same name and named his estate ‘Perigord’ after an area famous for its black truffles. My introductory quotations from Joanne Kates’ gastronomic tour show how heavily the best French cuisine leans on the subtle aroma emanating from truffles. Italian chefs place an equal premium on the extremely odoriferous white or Piedmont truffle (Tuber magnatum) that grows in northern Italy—Alba holds a great yearly truffle festival in its honour, which I hope some day to attend. In December of 1984 I visited the tiny mountain village of Scheggino in Umbria, another centre of truffle collecting, where both black and white truffles grow. As a guest of the Urbani family, who appear to have cornered much of the international trade in truffles, I overdosed on dishes laced with black and white truffles and came to the conclusion that the smell of the white truffle was too overpowering for my taste (reminiscent of the overripe sock) but that the black truffle added real cachet to a meal. Needless to say, I did not mention these treasonable thoughts to my Italian hosts, voicing them to my companions only after we had crossed the border into France. At the truffle market in Carpentras, France, I watched as buyers and sellers haggled quietly and skillfully over baskets of the clay-daubed tubers (yes, you pay for the dirt, too) and acted as a slightly bemused translator while one of my American companions paid $60 for a single fist-sized truffle. Such is the power of the legend. Unfortunately for most of humanity, these culinary gems are native only to Europe (although more plebeian edible species like Tuber gibbosum are common in western North America). The truffles of haute cuisine are also hard to find: they fruit underground, and their subterranean ripeness must be sniffed out by female pigs or trained dogs such as Kiki, who stars in Fig. 18.1. The secrets of ‘trufficulture’ are gradually being unravelled by French scientists, who now inoculate the roots of oak and hazelnut seedlings (the mycorrhizal partners of Perigord truffles; see chapter 17) with a suspension of truffle ascospores and obtain truffles as little as three years later. During my truffle tour, I visited two French truffle orchards, and in each case the trained dogs had little difficulty in sniffing out and unearthing a few of the black diamonds (Fig. 18.1). I also visited a new French company named Agritruffe, which markets mycorrhizal seedlings. Almost all of my American fellow travellers took home a batch of mycorrhizal seedlings, in the hope of establishing truffle orchards in Texas, Oregon, New York, and places between. One of their incentives was the unpredictable and apparently diminishing French harvest (2,000 tonnes in 1889, 200 tonnes in 1976), which

Fungi as Food: Mycophagy

Fig. 18.1 Kiki, the truffle dog, at work.

means that the North American price can be nearly a thousand dollars a kilogram. This isn’t as absolutely prohibitive as it may seem, because unlike Agaricus and most other edible fungi, truffles are usually added to food only in small quantities to enhance or embellish the flavours of the principal ingredients. According to some people, truffles are also an aphrodisiac. Although this was long considered apocryphal, and perhaps just a story invented to jack up their price even further, it has been discovered that one of the components of the odour of truffles is a

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Chapter 18 steroid called α-androstenol (5-androst-16-en-3-ol). α-androstenol is also found in the saliva, and hence on the breath, of rutting boars, where it serves as a pheromone, overcoming the sexual inhibitions of the young female pig. This explains the natural talent sows have for truffle hunting. The same substance is found in the underarm perspiration of men and in the urine of women. Although its sexual role in humans hasn’t been clearly established, men rating photographs of normally dressed women for sexual attractiveness gave higher marks while sniffing α-androstenol. Women’s reactions either haven’t been tested or we are being sheltered from the results; although if the advertising industry’s usual dire warnings about underarm perspiration are to be believed, androstenol is unlikely to fulfil male fantasies. However, it is already being added to certain cosmetics. (9) Species of Morchella (Pezizales, Morchellaceae), the spring-fruiting morels with their distinctive ridged and pitted heads (Fig. 18.2), are to many people (including me), the supreme edible fungi. As an accompaniment to steak and a good bottle of red wine, they are unbeatable. Although morels occur over a wide geographical range, in most areas it is hard to find them in quantity (people are very secretive about their morel patches), and to make things worse, in most places they fruit only in May (but later the further north you go). There is, however, at least one place where everyone can share the bounty: Michigan. In mid-May, half a million people head for the wooded hills to hunt, first for the black morel, Morchella angusticeps, then, a week or so later, for the white morel, Morchella esculenta. The rather similar Verpa bohemica (wrinkled thimblecap) is also picked, although it doesn’t taste as good as the true morels and can make you disoriented if you eat too many caps. Remember that poisonous false morels (Gyromitra species) also fruit at that time in the same places. Morels famously become profuse on burned areas the year after forest fires, but watch out for whatever nasty chemicals they may have used to put the fire out! Several morel festivals are held in Michigan, the most famous being that at Boyne City. This event celebrated its twenty-fifth year in 1984, when the winner of the National Mushroom Hunting Championship found over 500 morels in ninety minutes. But even that total pales into insignificance before the all-time record of over 900 morels collected by the champion in 1970. As part of the research involved in the preparation of this book, I attended the 1984, 1985, and 1991 festivals and can personally vouch that even a newcomer to the area can find enough morels for a good feed if he or she is willing to tramp the woods for a few hours and cultivate a morel spotter’s eye. In fact, in the 1985 championships, a visiting professional mycologist, Dr. Nancy Weber, outdid the local ladies by collecting 129 morels in the Fig. 18.2 Morels: Morchella esculenta. finals. It is worth noting that although morels

Fungi as Food: Mycophagy are easy to recognize, several other fleshy ascomycetes, including the dangerous false morel, Gyromitra esculenta, fruit at the same time (see chapter 22). In 1991 a quart basket of black morels went for $5, but the same quantity of white morels would set you back $20. Although the white morel is thicker fleshed and firmer than the black, I don’t think the price differential reflects any real superiority: both species have a strong and unforgettable flavour. Morel mycelium has been grown in pure culture on artificial media for many years, but only in 1982 was it finally persuaded to fruit. The dream of fresh morels available year round may soon be realized. U.S. patent number 4,594,809 was granted in 1986 for the method by which it can be induced to fruit in culture. Morels are now being produced at a rate of about 250 kg per week on a regular basis, and all those who have ever eaten them hope that the process can be scaled up to a commercial level before long. (10) Dictyophora duplicata (Phallales, Phallaceae), a large and beautiful stinkhorn which has a white lace-like skirt below its receptacle, has long been avidly collected in its natural habitat under bamboos in Guangdong Province, China, where it is called zhu-sun or ‘bamboo sprouts’. It is now cultured on a small scale on a medium composed of sawdust, bagasse (sugarcane debris), and bamboo. The dried product, which is reported to have a ‘pleasant sweetish smell’, sells for US$400–1,000/kg in Hong Kong. Because of its rarity it is served as a delicacy at state banquets, but it is widely sought because according to Chinese medicals, it not only cures high blood pressure, but also reduces cholesterol content of the blood, and through long use, it effectively reduces the belly fat. (11) Tremella fuciformis (Tremellales, Tremellaceae), a jelly fungus, is widely cultured on wood blocks and in bags of sawdust in China, where it is called ‘silver ear’ or yin-er and is used in soups and as a tonic. I saw great mounds of the spherical, frilly colonies for sale in the fascinating market at Guangzhou (formerly Canton). (12) Ustilago esculenta (Ustilaginomycetes, Ustilaginales), a smut fungus, is inoculated into wild rice, Zizania caduciflora, in China. It causes the stem to become very thick and fleshy, and at maturity the fungus produces small pockets of teliospores scattered throughout this tissue. During my first visit to China, I enjoyed the fungusriddled, hypertrophied stem many times as a vegetable. For the Chinese it has the added advantage that it is supposed to have curative powers against fevers, conjunctivitis, and kidney and bladder problems. Although ‘mushrooms’ of thirty-two species in sixteen genera are currently being cultivated commercially, only four or five are grown on a large scale. Many of them satisfy our demand for recycling and reuse, because they can be grown on various kinds of plant debris, a plentiful resource. Agaricus, Pleurotus, and Volvariella are grown on straw or similar substrates: byproducts of agriculture. Lentinula, Flammulina, Pholiota, Auricularia, and Ganoderma (this last is called reishi in Japan, and ling chi in China, where it is used for medicinal purposes rather than being eaten) are grown on wood, sawdust, or wood chips: byproducts of forestry. For those of you who would like to grow mushrooms in your basem*nt or garden, there is now an excellent handbook called The Mushroom Cultivator. This gives detailed instructions on all aspects of home mushroom culture and deals with many species, including several which are hallucinogenic. Those specifically interested in cultivating Lentinula edodes should consult the Shiitake Growers Handbook.

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Chapter 18 Now we move on to species that have never been commercially cultivated. These include some of the best of all edible fungi, so there is still room for research and lots of entrepreneurial spirit in bringing some of them to an eager public. (13) Boletus edulis (Boletales, Boletaceae) is the celebrated cep of France, the Steinpilz of Germany, the porcini of Italy. Their large, plump basidiomata bear fleshy tubes rather than gills (Fig. 5.8D). They are the basis for some European dried ‘mushroom’ soups and are also imported in see-through plastic packages. The aroma emanating from an unopened packet, even after it has been sitting in a cupboard for months or even years, is unbelievably appetizing. Most members of the family Boletaceae are edible, although species with reddish or orange pore mouths, and those whose flesh turns blue when bruised, should be avoided. Boletus edulis is mycorrhizal with conifers in western North America, where it fruits from June to November. I collected it in June 1984 near the edges of melting snowbanks near Bend, Oregon, and in November 1990 along the southern coast of Oregon. I have also found it in the east, although it is less common there. About twenty-five varieties of this species have been described. The importance of the boletes to European mushroom hunters can be gauged by the fact that of about 850 tonnes of mushrooms a year offered in the Munich market at the turn of the century, over 500 tonnes were accounted for by Boletus edulis and another bolete, Leccinum scabrum. These mushrooms are the filling for crêpes à la bordelaise. The tubes are removed, the caps are cut into slices 1 cm thick, seasoned with salt and pepper, and browned in olive oil or butter for 5 minutes. Then they are cooked for two minutes in egg yolks beaten with sour cream and finally rolled up inside thin pancakes and reheated in the oven. I also find Boletus edulis delightful on its own, simply sliced and fried in butter. A former colleague of mine, Dr. Maria Pantidou, was the first to persuade boletes to fruit in pure culture. However, the fruit bodies were tiny, and despite further efforts at refining the medium, no one has yet managed to grow a commercially viable product. (14) Tricholoma matsutake (Agaricales, Tricholomataceae), the highly prized matsutake or pine mushroom of Japan, is thought by some to be the best of all edible mushrooms, and it brings astronomical prices in Japan: up to $200/kg, fresh. It does not grow in North America, but the Japanese now import fresh Tricholoma magnivelare by air from western North America to supplement their own matsutake crop. Prices are volatile, depending on supply. Pickers in Oregon and Washington have been paid as much as $80/kg for grade 1 matsutake, as little as $1/kg for grade 7. The grading system works like this: grade 1—tight buttons more than 6 cm long, veil not evident; grade 2—larger button with partial veil apparent but unbroken; grade 3—as grade 2, but with breaks in partial veil; grade 4—partial veil completely ruptured, gills fully visible, cap not expanded, still inrolled at margin; grade 5—cap expanded, but undamaged; grade 6—cap expanded, with breaks, holes, or stains; grade 7—wormy but still firm. In early December 1990, during a sabbatical visit to Oregon, I was fortunate enough to find a few good specimens of about grade 3 on the mountains of the coast range. Their spicy aroma is unmistakable, although it reminds different people of different things: some liken it to cinnamon; I thought it smelled like fresh watercress. These few large, solid agarics added a delicious new dimension to our diet for several days. I have since learned that, because of the high prices being paid, competition between mushroom hunters has reached the point at which guns are being pointed, and occasionally

Fungi as Food: Mycophagy discharged, in disputes over collecting territory. The wild west has not totally vanished, it seems. (15) Cantharellus cibarius (Cantharellales, Cantharellaceae), the chanterelle or girolle of France, or Pfifferlinge of Germany, is widely collected, partly because its yellow, funnel-shaped basidiomata are easy to recognize. It is eaten fresh, often sautéed in butter with a little parsley, chopped garlic, and freshly cracked pepper served on generously buttered hot toast. Since in many places it is found only during a short period of the year, it is commonly dried; many European kitchens I have visited boast a big jar of dried chanterelles, which add their delicate but distinctive flavour to stews and other dishes year round. Fresh chanterelles have sold for $22/kg in Toronto. In Oregon, the white chanterelle, Cantharellus subalbidus, commonly accompanies C. cibarius and is also considered desirable. A closely related species, the black Craterellus cornucopioides, the horn of plenty, although less common and less substantial, is reputed to taste better than the chanterelle. As much as 2 million kg of chanterelles are picked in the Northwestern United States in a good year and shipped in barrels of brine to Germany. (16) Lactarius deliciosus (Russulales, Russulaceae), the saffron milk cap, was mentioned by the Roman author Pliny and is represented in frescoes excavated at Pompeii, the Roman city buried in 79 CE by an eruption of the volcano Vesuvius. Lactarius, which is mycorrhizal, fruits under conifers in autumn. I must admit that as far as I am concerned, this species has two drawbacks: it is often already riddled with the borings of dipteran larvae by the time I encounter it; and I don’t find the flavour particularly interesting. However, since I am not an early riser, and some of my acquaintances enjoy this species, I felt obliged to include it. (17) cl*tocybe nuda (Agaricales, Tricholomataceae), the blewit, is a rare exception to the widespread British mycophobia and is sold at markets in central and Northern England. I find this strange, since this species is entirely lilac to blue or purple: distinctly poisonous looking. But having been raised in Northern England, I can confirm that blewits make excellent eating. These species were formerly classified in Tricholoma or Lepista. (18) Marasmius oreades (Agaricales, Marasmiaceae), the fairy ring mushroom, is a small but numerous, summer-fruiting, pasture species that can be collected in fair quantity and is usually dried for use as a flavouring. Make sure the pasture in which you collect has not been treated with selective weed killers. (19) Coprinus comatus (Agaricales, Agaricaceae), the shaggy mane or shaggy ink cap (Figs. 5.5C, 8.5), is common on new lawns and disturbed ground in late autumn. Unfortunately, the ease with which we find it is in inverse proportion to its flavour. I find shaggy manes, which must be eaten before the gills begin to darken and deliquesce, rather watery, although they can make a good contribution to a soup. (20) Langermannia gigantea (Agaricales, Lycoperdaceae), the giant puffball (Fig. 18.3), which can be eaten when the fruit body is young and its interior the colour and texture of marshmallow, is sold in farmers’ markets in Ontario. It has an interesting texture and can be a vehicle for other flavours, but its own is too delicate to be of much interest. I suppose one of its attractions is that a single basidioma can feed a large number of people. (21) Armillaria mellea (Agaricales, Physalacriaceae), the honey mushroom (Fig. 14.3), actually comprises at least eleven genetically different strains in North America.

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Chapter 18 It has been involved in a few poisonings, but this problem should not arise if only fresh young specimens are eaten and if it is always well cooked. Many amateur mycologists swear by this species, which is very common in most woodlands, but it would seem prudent to eat only a few when trying them for the first time (as with any mushroom you have not eaten before). I have had some tasty dishes incorporating this fungus, but the common name refers to its colour, not its flavour. (22) Amanita caesarea (Agaricales, Amanitaceae) was the favourite mushroom of Roman emperors (who presumably had access to the best). Amanita rubescens (the blusher) is also reputed to be edible. Note that these species are congeneric with Amanita phalloides and Amanita virosa, perhaps the two deadliest mushrooms in the world. Although I have not eaten (nor even seen) A. caesarea, a close relative of that species, Amanita umbonata, grows in North America. Amanita caesarea starred in one of history’s dramatic moments. Agrippina, wife of the Roman emperor Claudius, coveted her husband’s job for her son Nero. She tried to kill Claudius with a dish of his favourite mushrooms, rendered lethal by judicious additions of Amanita phalloides juice. This plan went awry when Claudius threw up his dinner (emperors often did this bulimic trick to make room for another meal). But the resourceful Agrippina had a backup plan: the imperial physician was in her pay, and poison was resolutely and, as we know, successfully administered to the doomed Claudius by enema. (Nero was another matter altogether, as we know!) (23) Various species of Termitomyces (Agaricales, Lyophyllaceae), whose often very large basidiomata are found associated with termite mounds in Africa (Fig. 16.3), are collected and eaten. Perhaps the largest edible mushroom on record was a Termitomyces collected in Zambia which had a cap 66 cm across and weighed 2.5 kg. The size of these basidiomata is easier to understand when one knows that the mycelia from which these mushrooms arise are actually cultivated in special underground fungal gardens by the termites, which supply them with chewed-up wood and rigorously exclude other fungi (see chapter 16 for the full story). (24) Charles Darwin, circumnavigating South America in the Beagle, noted that the golf-ball-like compound ascomata of what was subsequently named Cyttaria darwinii (Cyttariales, Cyttariaceae), which were parasitic on the southern beech tree Nothofa*gus, were an important part of the diet of the natives of Tierra del Fuego. The Araucans of Chile take advantage of the fact that Cyttaria contains up to 15% of fermentable sugars and has on its surface the yeast Saccharomyces to prepare an alcoholic beverage Fig. 18.3 Man holding one of the world’s from ripe ascomata. largest edible fungi, Langermannia gigantea.

Fungi as Food: Mycophagy Various amateur mycophagists of my acquaintance have told me that they really enjoy one or more of the following macrofungi (for those I have tried myself, I add my own evaluation): (25) ‘hen-of-the-woods’, Grifola frondosa (Polyporales, Meripilaceae); (26) ‘umbrella polypore’, Polyporus umbellatus (Polyporales, Polyporaceae); (27) ‘chicken mushroom’ or ‘sulphur shelf’, Laetiporus sulphureus (Polyporales, Polyporaceae) [excellent, but only the edges of fruit bodies when young, and cooked thoroughly]; (28) ‘sweet tooth’, Dentinum repandum and D. umbilicatum (Cantharellales, Hydnaceae) [not exciting]; (29) ‘clustered coral’, Ramaria botrytis (Gomphales, Gomphaceae); (30) ‘cauliflower mushroom’, Sparassis crispa (Polyporales, Sparassidaceae) [acceptable]; and (31) the ‘beefsteak fungus’, Fistulina hepatica (Agaricales, Schizophyllaceae). Finally, (32) fried teliospore masses of corn smut, Ustilago maydis (Ustilaginales), are regarded as a treat by many Mexicans, under the name huitlacoche [I was not impressed...]. As a souvenir of my lecture tour of China in 1987, I have a beautifully illustrated book (all in Chinese except for Latin binomials) which a Chinese graduate student of mine assures me is titled Edible Mushrooms. This book contains paintings of representatives of seventy genera of fungi, at least some of which I would be extremely reluctant to eat (e.g., Paxillus involutus [Boletales, Paxillaceae], the ‘poison pax’). I can only assume that the Chinese are more adventurous than we are in these matters.

A Warning In Eastern Europe, other hazards accompany mycophagy. Since the Chernobyl disaster, wild mushrooms in Poland and Russia contain levels of radioactive cesium many times higher than those found in other food sources, and radio stations frequently warn people not to eat too many. But it is only fair to point out that, wherever they grow, wild mushrooms tend to accumulate cadmium and other toxic elements. By means of the Ames test for mutagenicity, it has also been established that some ‘edible’ mushrooms, including Agaricus brunnescens, shiitake (Lentinula edodes), and Boletus edulis contain base pair substitution mutagens, and B. edulis also contains frameshift mutagens. While the mutagenic activity of L. edodes was not reduced by boiling for twenty minutes, that of A. brunnescens and B. edulis declined, but only by 50%. Since the mutagens have not yet been isolated and characterized, we don’t know their implications for human health. They may or may not turn out to be significantly carcinogenic. So while eating mushrooms as an occasional treat may be fine, they should never be regarded as a dietary staple.

Other Edible Fungi Many filamentous fungi do not produce large fruit bodies but can grow on cheap substrates and produce large amounts of mycelia high in protein. In Britain, a strain of Fusarium graminearum, the conidial anamorph of Gibberella zeae (Hypocreales, Nectriaceae), is being grown in submerged culture in 1,300-litre fermentors on glucose syrup and ammonia. The dried mycelium has 45% protein of acceptable amino acid composition, 10%–15% fat, and 20%–25% fibre. The nucleic acid content starts out at a

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Chapter 18 gout-inducing 10% but is reduced to an acceptable 1% by heating the mycelium to 64ºC for twenty minutes. This inactivates proteolytic enzymes but allows the organism’s own ribonucleases to break down the nucleic acids to products that can be washed out of the cells. Successful feeding trials on animals and volunteer humans have led to commercial production of this high-protein food, which is flavoured and textured to resemble traditional foods and sold in various forms under the name Quorn. No discourse on fungi as food would be complete without some mention of yeasts. Species of Saccharomyces and so-called Torula (a misnomer) have been used as food supplements for many years, largely because they contain high levels of the B vitamins. But yeasts also commonly contain 40%–50% dry weight of protein, can be grown on substrates such as the effluents from food-processing plants, and have a short generation time. They would seem to be obvious sources of SCP (single-cell protein), yet they have not yet been fully exploited. Why is this? First, the presence of high levels of nucleic acids has made yeast proteins unacceptable: if humans eat more than 2 grams of nucleic acid per day, hyperuricaemia (elevated levels of uric acid in the blood) will result, possibly leading to kidney stones and gout. Second, yeast proteins are nutritionally inferior because they are low in the amino acids methionine and tryptophan. For yeast protein to be suitable as food, it must be separated from the indigestible chitinous wall material, must not be denatured, must have its nucleic acid level reduced, and should be supplemented with methionine. Apparently, if yeast cells are disrupted at pH 8.5 in the presence of succinic anhydride, 90% of the protein can be extracted without denaturation. If the pH is then lowered to 4.2–4.5, the protein is precipitated, leaving most of the nucleic acid (mainly RNA) in the supernatant fluid. The development of processes like this may bring acceptance of yeast SCP closer. Of course, no one could be expected to eat yeast protein as it comes; it would presumably be used (as the still cheaper soy protein is used) to supplement the protein concentration of other, tastier foods. Yeastburgers, anyone?

Animal Mycophagy I am sure none of you imagines that human beings are the only animals to appreciate the dietary value of fungi. One of the more dangerous myths used to establish edibility of mushrooms asserts that if they have been nibbled by larvae, slugs, snails, or squirrels, they must be edible. In fact, many animals, both vertebrates and invertebrates, seem to be unaffected by the toxins found in agarics poisonous to us (this is especially true of some Drosophila species, as you will learn in chapter 22). Elaphomyces, the deer truffle or hart’s truffle, is apparently eagerly sought and relished by the animals for which it is named. In the forests of California the red-backed vole, Clethrionomys californicus, lives almost exclusively on truffles (Tuber spp.) and false truffles (e.g., Rhizopogon); and many other small rodents feed on, and disperse, these hypogeous fungi. Wildlife biologists were surprised to find remains of flying squirrels, which live high in trees, in the faeces of bobcats and coyotes. Then mycologists spotted truffle ascospores and false truffle basidiospores in the guts of the flying squirrels: apparently the squirrels had been fatally lured onto the ground, away from the safety of the trees, by the

Fungi as Food: Mycophagy odour of ripe truffles. Birds migrating across the deserts of Kuwait, including nine species of lark, find and eat desert truffles of the genus Phaeangium. Flies of the genus Helomyza are also tuned in to truffles, since their larvae will eat nothing else. It is sometimes possible to find truffles by the swarms of egg-laden flies hovering over their hiding places. Many other insects are also extremely fond of mushrooms, as any collector of Lactarius deliciosus or boletes knows. Adult Mycetophilidae (fungus gnats) and members of many other groups (including some species of Drosophila) seek out fungal fructifications unerringly, so that their larval instars can fatten on the proper diet. Aphids, nematodes, and amoebae have been found selectively feeding on the mycorrhizal fungi growing around conifer roots. Some Amphipoda (freshwater crustaceans) graze preferentially on the conidiophores of amphibious hyphomycetes—fungi which colonize leaves that fall into streams (see chapter 11). Many Collembola (springtails) and oribatid mites (Acarina) have also been found to prefer a diet of fungal spores and mycelium. Perhaps the most interesting examples of animal mycophagy are found among groups of insects that cannot themselves digest cellulose or lignin but still manage to exploit these substrates through the mediation of specific fungi. The insects either carry the fungi around from tree to tree (ambrosia beetles) or actually cultivate them in special subterranean gardens (the attine ants of Central and South America and the mound-building termites of Africa and Asia). These special relationships are discussed in chapter 16.

Further Reading Arora, D. 1986. Mushrooms Demystified. 2nd ed. Berkeley: Ten Speed Press. [One of the most comprehensive field guides yet published; everyone should have it, but it does focus on Western taxa.] Chang, S. T., and W. A. Hayes. 1978. The Biology and Cultivation of Edible Mushrooms. New York: Academic Press. Ingratta, F. J., and T. J. Blom. 1980. Commercial Mushroom Growing. Ontario: Ministry of Agriculture and Food. Kroeger, P., B. Kendrick, O. Ceska, and C. Roberts. 2012. The Outer Spores—Mushrooms of Haida Gwaii. Sidney, Canada: Mycologue Publications. [189 pp.; good ecological information.] Lincoff, G. H. 1981. The Audubon Society Field Guide to North American Mushrooms. New York: Knopf. [Relatively inexpensive; lots of good colour photographs; one of the more comprehensive guides for those in Eastern North America.] Malloch, D. 1976. “Agaricus brunnescens, the Cultivated Mushroom.” Mycologia 68:910–19. Maser, Z., C. Maser, and J. M. Trappe. 1985. “Food Habits of the Northern Flying Squirrel (Glaucomys sabrinus) in Oregon.” Canadian Journal of Zoology 63:1084–88. McIlvainea: Journal of the North American Mycological Association. [This journal comes with your membership in NAMA: 336 Lenox Ave., Oakland, CA 94610.] McKenny, M., D. E. Stuntz, and J. F. Ammirati. 1987. The New Savory Wild Mushroom. Seattle: University of Washington Press. [Fine colour photographs; very suitable for beginners in the Pacific Northwest.]

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Chapter 18 Miller, O. K. 1979. Mushrooms of North America. New York: Dutton. [The first of the modern guides, now superseded by more comprehensive books, including that of the same title by Roger Phillips.] Mushroom: The Journal of Wild Mushrooming. [861 Harold St., Moscow, ID 83843; appears quarterly and will keep you up-to-date on all things agaric.] Nout, M. J. R., and K. E. Aidoo. 2011. “Asian Fungal Fermented Food.” In The Mycota X Industrial Applications. Edited by M. Hofrichter, 29–58. Berlin: Heidelberg Springer. Pacioni, G. 1981. Simon & Schuster’s Guide to Mushrooms (U.S. editor, G. Lincoff). New York: Simon & Schuster. [Inexpensive; lots of fine colour photographs.] Phillips, R. 1991. Mushrooms of North America. Boston: Phillips, Little, Brown & Co. [The most comprehensive of North American guides with over 1,000 colour photographs; best coverage of Amanita, Cortinarius, Hygrophorus, Lactarius, and Russula.] Pomerleau, R. 1980. Flore des Champignons au Québec. Montreal: Les Editions La Presse. [French text; excellent technical descriptions and line drawings but poor colour photographs; suitable for anywhere in Northeastern North America.] Przybylowicz, P., and J. Donoghue. 1988. Shiitake Growers Handbook. Dubuque, IA: Kendall/ Hunt. [If you really want to get serious.] Singer, R. 1984. “Agaricus brunnescens Peck and Agaricus bisporus (Lange) Imbach.” Mycotaxon 20:479–82. Smith-Weber, N. 1988. A Morel Hunter’s Companion. Lansing: Two Peninsula Press. [Probably the best book about morels and their relatives, although taxonomy is now much updated.] Stamets, P., and J. S. Chilton. 1983. The Mushroom Cultivator: A Practical Guide to Growing Mushrooms at Home. Olympia: Agarikon Press. [A good field guide.] Toth, B., K. Patil, and H.-S. Jae. 1981. “Carcinogenesis of 4(hydroxymethyl)-benzenediazonium Ion (Tetrafluoroborate) of Agaricus bisporus.” Cancer Research 41:2444–49. Trudell, S., and J. Ammirati. 2009. Mushrooms of the Pacific Northwest. Portland: Timber Press. [351 pp.] Ying, J. Z., J. D. Zhao, X. L. Mao, Q. M. Ma, L. W. Xu, and Y. C. Zeng. 1982. Edible Mushrooms. Beijing: Academic Press. [Beautiful, full-page paintings of many well-known, and some not-so-familiar, fungi.]

19 Fungi in Food Processing ‘A loaf of bread, a jug of wine, and thou’. Omar Khayyam had it right. Even if the right ‘thou’ happens to be away, good bread and wine offer some consolation. Eating fungi in recognizable form can be fun (or occasionally fatal), as you may read in chapters 18 and 22, but even those who make a point of avoiding such gastronomic adventures eat fungi (or fungal byproducts) without even being aware of it. The reason for this is that a number of the basic items in our diet, as well as some of the most interesting tidbits, are ‘processed’ by fungi. No one seems the slightest bit interested in the presence of the fungus itself, only in the changes it produces in the substrate. And what dramatic changes they are. Without fungi, French bread would be matzoh. Blue cheeses would be blah. Beer and wine might not exist. The wonderful texture of bread is created by the yeast Saccharomyces cerevisiae, which ferments small amounts of sugars and liberates bubbles of carbon dioxide that become trapped in the dough and leaven it. Bread without yeast would be like a day without sunshine, or steak without wine, or watching a football match without beer. Whatever your tipple, its alcoholic component is ultimately derived from the activities of yeasts, again of the genus Saccharomyces, which ferment the sugar in grapes or malted barley and liberate alcohol and carbon dioxide. The substrate and end products balance as follows: C6H12O6 → 2C2H5OH + 2CO2 The process is actually very complex, involving twenty-two enzymes, at least six coenzymes, and Mg and K ions. Bottled beer, crackling wines, and champagne all owe their fizz as well as their kick to yeast. So how come bread doesn’t have any alcohol? Perhaps fortunately, it evaporates during the baking process, so we aren’t all on the road to alcoholism with our first peanut butter sandwich (although we could be on our way to a different kind of intoxication, as you can read in chapter 21 on mycotoxins). The manufacture of beer begins with the malting of barley, during which the barley’s own amylase converts the starch in the grain to sugar, which is then fermented by the chosen yeast. Lager is produced by Saccharomyces carlsbergensis, ale by Saccharomyces cerevisiae. Although the making of beer appears to have been mechanized and standardized to a high degree, with tailor-made yeasts (see chapter 10) and precisely controlled conditions at every step, I have found many different brews during sabbatical trips around the world, most of them pleasant enough. Although I was born in Britain, my preference is for German beer, so I was delighted to find a reasonable facsimile in western Samoa—which used to be administered by the Germans. British beer 359

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Chapter 19 (‘best bitter’), drunk flat and almost warm, represents one end of the beer spectrum; some European lagers, rather carbonated and served cold, the other. Wine has been aptly described as a chemical symphony, although the kind of organoleptic (Google that word) impression left on the consumer can vary enormously. For thousands of years people have made wine by crushing grapes to produce ‘must’ and letting the wild yeasts always found on grape skins perform the alcoholic fermentation. Wine making has been brought to a high art in parts of France (Bordeaux and Burgundy). Even some inexpensive French wines can be excellent, and the better ones are delectable. Having reverently assisted at the consumption of a bottle of Chateau Lafite Rothschild, a Premier Grand Cru, I can attest to the almost magical complexity and perfection of the best. Some French wines (e.g., Chateau Margaux and a select few other Bordeaux) sell for astronomical sums (to whom, I do not know). The reasons for the continued, at least partial, supremacy of the best French wines are several. First, the grape species used. Make no mistake, the best wine is made only from varieties of Vitis vinifera. For many years the winegrowers of New York and Ontario used a native North American grape, Vitis labrusca, with disastrous results. This species, used because it is hardier and more pest and disease resistant than the European grape, has now been almost entirely replaced by V. vinifera hybrids, with considerable benefit to the resulting wines. The great wine-growing regions of France use different vinifera varieties. The great red Burgundies are made from the Pinot Noir grape, the red Bordeaux from Cabernet Sauvignon. Beaujolais is made from the Gamay grape. Excellent white wines are made from such varieties as Chardonnay, Sauvignon Blanc, and Chenin Blanc. Other wines are blends of two or more varieties. Châteauneufdu-Pape permits thirteen different varieties of grape, including some white grapes— although the blend is now almost exclusively red, and predominantly Grenache, with admixtures of Mourvèdre and Syrah. You can gather something about wine snobbery from the fact that since the influential wine critic Robert Parker began to push the excellence of Châteauneuf-du-Pape, its price has quadrupled. Second, the climate should provide a long growing season, to give a high sugar content, but should not be too hot, so that the sweetness of the grapes will be balanced by their acidity. These conditions are found in Bordeaux, the Rioja area of northern Spain, northern Italy, Yugoslavia, and Northern California. Less reliable summers in Germany, Austria, Switzerland, Oregon, Washington, New York, Ontario, and even Burgundy mean that the grapes do not always become sweet enough, and the wine may be unbalanced. This explains the concept of ‘vintage years’. Wine from warm summers may be much better than that from cooler, wetter years. Hot, sunny climates like those in Southern California, southern Spain, Algeria, Cyprus, South Africa, and Australia produce grapes with lots of sugar but relatively low acidity, which are easy drinking in any year. No need for ‘vintage’ years in those areas. Third, the soil (or terroir). Some of the finest Burgundies are grown in some of the worst-looking soils I have ever seen: basically just a collection of small limestone rocks. Clearly, the soil must be limestone based and extremely well drained, to put it mildly. Fourth, the yeast. If early winemakers were lucky, the yeast that got the lion’s share of the action was Saccharomyces cerevisiae var. ellipsoideus. But other wild yeasts often dominated, with less than desirable results, and sometimes Acetobacter ruined things

Fungi in Food Processing completely. It was that greatest of all Frenchmen, Louis Pasteur, who finally put the crucial fermentation stage of wine making on a scientific footing in 1866 with his classic paper, ‘Études sur le vin’. Most winemakers now suppress wild yeasts with sulphur dioxide and add pure cultures of highly selected wine yeasts. Needless to say, there are yeasts for every kind of wine: Riesling yeasts, Chablis yeasts, etc. The actual fermentation is now kept relatively cool to boost the production and retention of esters and other aromatic compounds which are largely responsible for the bouquet and aroma of the wine. The introduction of modern winemaking techniques to many parts of the world has given us a much wider selection of excellent wines than has ever been available before. Price is not the sole arbiter of quality. I have recently imbibed first-class wines from Chile, Argentina, Spain, and Australia, all costing much less than French wines of equivalent quality. Fifth, the age and method of storage. Although Beaujolais (made with the Gamay grape) should be drunk young, some other red wines, particularly those made with the Cabernet Sauvignon grape, contain an amazingly complex mixture of chemicals which sometimes needs years of ageing to blend and smooth out into an ideal flavour. Such wines are often stored for a while in oak casks, which impart more tannin (firmness) to the wine. Then the wine spends several more years in the bottle before arriving at its peak. Wines are of two main kinds. Table wines usually have 10%–14% alcohol. Aperitifs and dessert wines like port and sherry are fortified with brandy, which is itself distilled from wine, and have about 20% alcohol (40 proof). The brandy is sometimes added before the natural fermentation of the sugars is complete, leaving a sweet wine like port or muscatel. There is another way of making a sweet wine that depends on a hyphomycete, Botrytis cinerea. This causes the ‘noble rot’—pourriture noble in France, Edelfäule in Germany, aszú in Hungary. In good years, the grapes are left on the vines until they are overripe, and the Botrytis grows on them. The grapes crack open, the mould lives on the juice, and the sugar content increases. At just the right stage, they are ultimately individually picked, all mouldy and shrivelled, and made into wine. The very best Sauternes are made this way, as are the German Trockenbeerenauslesen and the Hungarian Tokay. I acquired some Botrytis wine in South Africa and found that its velvety texture, its concentration, and its great sweetness balanced by acidity made a really unusual combination, but one to be sipped only occasionally and in small quantity. Ontario vintners recently began to make Eiswein (ice wine), long a European specialty. Like Botrytis wine, it is made from grapes that have been left on the vine until well past the normal harvest. In this case the grapes do not rot but become frozen, in which state they are picked and pressed. As with the noble rot, a small amount of rich, sweet wine is produced. Ontario Eiswein, made from the Vidal grape, is now regarded as the best in the world and sells for more than $50 per half bottle. Many books have been written about wine. If your interest in the subject has been whetted, I suggest you proceed to reading and tasting. Never forget that most wine is at its best as an accompaniment to food. ‘Hard’ drinks such as the whiskies also originate from a yeast fermentation. Bourbon is made from fermented corn, Scotch from fermented barley, Rye from fermented rye. The fermented grain broth is distilled to concentrate the alcohol, usually to 40%

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Chapter 19 (80 proof). In the case of Scotch, smoke from the peat fire and subsequent ageing in wood supplies further character. The Araucans of Chile ferment the fleshy ascomata of Cyttaria harioti (Ascomycetes, Cyttariales), which contain as much as 15% of fermentable carbohydrates and, like grapes, bear natural populations of Saccharomyces. The natives dry the fungus, grind it up, and mix it with warm water. Natural fermentation produces a refreshing, mildly alcoholic drink called chicha del llau-llau. Cheese has probably been with us since the domestication of animals, since it is an effective way of storing milk proteins for long periods. There are more than 500 different cheeses, many of them characteristic of a particular area. Only a small handful are processed by fungi, but they are among the most interesting we have in terms of texture and flavour. They are of two kinds: the soft-ripened Camembert type and the blue Roquefort type. Camembert, Brie, Thenay, Troyes, and Vendome cheeses are all ripened by Penicillium camembertii or Penicillium caseiolum (Hyphomycetes). These moulds form a dense white mycelial mat on the outside of the cheese, and their extracellular proteases diffuse inward, digesting milk proteins and giving the cheese a wonderfully smooth, soft, almost buttery consistency. The interior of a really ripe Brie (my favourite) or Camembert will have a rather viscous, almost fluid texture and may ooze gently when cut. The flavour of these cheeses is mild and is probably generated largely by lactobacilli. It is unfortunate that these cheeses have a very high content of saturated fats. The blue cheeses—Roquefort, Stilton, Gorgonzola, Danish Blue, and Wensleydale— are ripened by Penicillium roquefortii. When the curd is ready, breadcrumbs with P. roquefortii growing on them are added and then the cheese is incubated until ripe. P. roquefortii can tolerate high carbon dioxide concentrations and low levels of oxygen, so it grows throughout the curd, assisted to some extent by holes punched in the substrate by the cheesemaker. The colour of the blue-green veins that develop is due to the presence of innumerable conidia. Not only the appearance but also the taste of the cheese is transformed by the mould. P. roquefortii oxidizes fatty acids to methyl ketones, particularly 2-heptanone, which are believed to give the cheese its penetrating smell and its unique, pungent flavour. Under some conditions, P. roquefortii produces a dangerous mycotoxin called PR toxin; happily this is not formed during the cheesemaking process. And with alcoholic beverages, bread, and a few cheeses, we have exhausted the repertoire of fungus-processed foods manufactured in the Western world. But if we look to the Far East, we find a whole range of fungus-fermented foods, some of which are now gradually becoming more familiar to Western palates. Shoyu, better known to us as soy sauce, is such a standard part of the everyday Japanese diet that annual consumption in Japan is nearly 15 litres per person. Shoyu is made from a mixture of wheat and soybeans or soybean meal. One version of the process is as follows. The soybean is cooked and mixed with crushed, roasted wheat and then inoculated with Aspergillus oryzae and incubated at 25°C for eighteen days. It is then stirred and incubated at 30°C–35°C for forty-eight hours and then stirred again with brine and inoculated with a yeast and a Lactobacillus. Now it is incubated for up to six months, and finally it is matured for anything up to two years. I’ll bet you had no idea soy sauce was so complicated to make.

Fungi in Food Processing Ket-jap is a simpler, Indonesian variant on the soy sauce theme. It is made from black soybeans, which are boiled and then inoculated with Aspergillus oryzae or, as in the cottage ket-jap factory I visited in Java, with whatever mould spores fall into it from the air. It is incubated for two to three days and then kept in brine for eight days. The result is filtered, cooked in several changes of water to extract all soluble components, mixed with sugar (the Javanese have a sweet tooth) and other flavouring ingredients, and, finally, concentrated by slow boiling to a thick, syrupy consistency. Miso is second in importance only to shoyu among fungus-fermented foods in Japan. It is a thick paste used as a spread and can be made from soybeans and rice, soybeans and barley, or soybeans alone. Rice is steamed and then inoculated with Aspergillus oryzae and incubated for two days at 28°C to produce a starter called koji. Meanwhile, soybeans are crushed, washed, and soaked for a few hours and then steamed. After cooling, the soybeans are inoculated with a yeast, Saccharomyces rouxii, and mixed with the koji and salt. The mixture ferments for a week at 28°C and then for two months at 35°C, and it is finally ripened for two weeks at room temperature. Miso soup is a standard item in the Japanese diet. Hamanatto is another Japanese soybean product. Soybeans are soaked for four hours, steamed for ten hours, cooled to 30°C, and then inoculated with Aspergillus oryzae. After incubation in trays for twenty hours, the beans become covered with A. oryzae (basically, green mould). They are then dried, put in baskets with ginger, soaked in brine, and aged for six months. A similar product is known as tu-su in China and tao-si in the Philippines. Tempeh, a kind of soybean cheese, is an important food in Indonesia. It is an attempt to make the notoriously indigestible soybean both edible and tasty by exploiting fungal enzymes. Soybeans are cooked and then inoculated with Rhizopus oligosporus (Zygomycetes, Mucorales). When I visited a cottage tempeh factory in Java, I found that the fungal inoculum is now bought as a whitish powder in small plastic bags. The mycelium of this fast-growing fungus permeates the soybean, and its extracellular proteases break down some of the bean protein within a day. When fried, the result is not unpleasant to Western taste buds. In Java, the basic tempeh is often made more tasty by adding sugar and sometimes hot peppers. Sufu is a Chinese version of soybean cheese, the fungus involved being Actinomucor elegans (Zygomycetes, Mucorales). Ont-jom is another Indonesian food, this time made from press cake, which is the residue left after oil has been expressed from peanuts. The press cake is washed, steamed, put in small containers, and sprinkled with pink conidia of the Chrysonilia anamorph of Neurospora sitophila (Ascomycetes, Sordariales) from the previous batch of ont-jom. The containers are incubated until the fast-growing fungus has thoroughly colonized the substrate, and then the result is cut up and cooked. Katsuobushi is made in Japan by fermenting cooked bonito fish with Aspergillus glaucus until it dries out. Shavings of the resulting hard, dark substance are used to flavour other foods. It is obvious that we have no more than begun to explore the possible uses of fungi in predigesting and flavouring many basically nutritious but indigestible or tasteless food substrates. A world that welcomes the kind of gustatory trivia presented by most fast foods will surely embrace whatever comes after beer and wine, Brie and Roquefort.

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Further Reading Gray, W. D. 1959. The Relation of Fungi to Human Affairs. New York: Holt. ———. 1981. “Food Technology and Industrial Mycology.” In Biology of Conidial Fungi. Vol. 2. Edited by G. T. Cole and B. Kendrick, 237–67. New York: Academic Press. Hesseltine, C. W. 1965. “A Millenium of Fungi, Food and Fermentation.” Mycologia 57:148–97. Hesseltine, C. W., and H. L. Wang. 1967. “Traditional Fermented Foods.” Biotechnology and Bioengineering 9:275–88. Wood, B. J. B. 1998. Microbiology of Fermented Foods. Vol. 1. 2nd ed. London: Blackie Academic & Professional. Wood, B. J. B., and F. M. Yong. 1975. “Oriental Food Fermentations.” In The Filamentous Fungi. Vol 1. Edited by J. E. Smith and D. R. Berry, 265–80. London: Arnold.

20 Food Spoilage by Fungi and How to Prevent It I have a great fondness for damson plum jam, with all the tangy skin of the fruit in it. You just can’t buy stuff like that at the supermarket. So when I found some damson plums at our local farmers’ market, I carried them home in triumph, intending to make jam—very soon. A week later I remembered my plan and retrieved the fruit, only to find that some of the plums had a mould sporulating on them. Can you suggest, from the nature of the substrate (a stone fruit), what that mould might have been? (Did you remember Monilia?) I quickly sorted out the mouldy fruit and put it aside for my undergraduate class. I then pitted the sound fruit and cooked it very briefly with what seemed like an enormous amount of sugar. I ladled the jam into sterilized jars and covered it with a thin layer of melted paraffin wax before sealing the jars tightly. That story exemplifies the problem of food spoilage and what we have to do to prevent it. What is food spoilage? You may think the answer is obvious. But, as often happens when we sit down and try to define a phenomenon, it’s not as clear-cut as it might seem. How about this definition: ‘Changes in the appearance, texture, smell, or taste of food that cause it to be discarded’? One important flaw in this definition is that the criteria for acceptability are strongly affected by cultural or economic conditioning. So the quantification of the various criteria may have to be reformulated for different countries or ethnic groups. Many kinds of food become visually unappealing only when fungi are seen to be fruiting on the surface. But you must be aware by now that the interior of the food will be riddled by assimilative hyphae and permeated by fungal metabolites before visible sporulation occurs (especially if the surrounding air is dry). Those fungal metabolites may include mycotoxins (see chapter 21). Even the most fastidious consumer can’t always tell when these are present. Many kinds of food are processed before they reach us, so the mouldiness that might be evident on raw peanuts will no longer be apparent after they have been turned into peanut butter. Since ideas of what constitutes spoilage vary so much, no definition can be completely objective and scientific. But science has now added an important new criterion. Food must be regarded as spoiled, no matter how appealing it may look, smell, or taste, if it contains potentially harmful levels of mycotoxins. (In bacterial terms, the same would obviously be true if it contained botulinus toxin.) Food is spoiled by fungi when they bring about unacceptable changes in its appearance, texture, smell, or taste or contaminate it with potentially harmful levels of one or more mycotoxins. Toxigenic fungi are identified by an asterisk (*) in the text that follows. Having defined the condition, our next problem is to establish how prevalent and important it is. Food may be living or dead, fresh or preserved, raw or processed. In the 365

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Chapter 20 case of fruits and vegetables, spoilage can occur in the field before harvest. But since North Americans no longer shop every day, insist on eating many seasonal foods all year round, and demand many exotic foods, most spoilage problems occur during transportation or storage. Food that is wet or has a high moisture content and an alkaline pH tends to be spoiled by bacteria. Drier or more acidic foods are usually spoiled by fungi. It has been estimated that about a quarter of all produce harvested spoils before it can be eaten. Fungal spoilage falls into several categories, depending on the strategy of the fungus. (1) Some losses are caused by the ongoing activities of plant pathogenic fungi already established in the substrate. (2) Some fungi that cause storage problems, such as Monilia fructigena (soft rot of peaches and other stone fruits), are actually necrotrophs. Necrotrophs are fungi which, although they grow on living hosts, are not really biotrophic. They liberate toxins that kill the plant cells before the fungal hyphae reach them, so the fungus is actually feeding saprobically. (3) Other storage fungi are strictly saprobic but have one or more of the following five unusual abilities: (a) coping with substrates of low moisture and/or high osmotic pressure (xerotolerance), (b) surviving high temperatures (thermotolerance), (c) growing at low temperatures (psychrotolerance), (d) growing in extremely acid media, or (e) growing at low levels of oxygen and/or high levels of carbon dioxide. The growth of any fungus presupposes (1) the presence or introduction of fungal propagules or inocula of some kind, (2) an appropriate source of energy-rich carbon compounds and other basic nutrients, (3) an appropriate level of moisture in the substrate, (4) a tolerable pH, (5) adequate oxygen, or (6) absence of inhibitory substances. I will examine each of these in turn and (fungal physiology revisited) establish the limits of fungal tolerance and thus show the types of criteria that our food processing and storage techniques must meet or exceed if we are to be able to eat more of what we grow. Since fungal spoilage of food can happen only if a fungus is present and active, there are two possible avenues of prevention. The first is to rigorously exclude fungi from the substrate; the second is to prevent them from growing even if they are present. Which do you think is the easier route? There isn’t much doubt in my mind: the environment is teeming with fungal spores, and it is extremely difficult to exclude them completely. Only in laminar flow benches and ‘clean’ rooms are fungal spores virtually absent. In most places, the air contains from hundreds to many thousands of spores per cubic metre. The surfaces of many substrates are colonized by fungi, and it is extremely difficult to remove them all. So we should accept fungal contamination as a fact of life. It is worth remembering, however, that the inner tissues of healthy plants and animals are usually uncontaminated by spoilage organisms (although symptomless fungal endophytes are common in many plants; see chapter 21) and that natural coverings such as the skin of fruits, the husks of grains, and the shells of nuts often protect these tissues from spoilage for extended periods. What practical ways are there of preventing fungi from exploiting our food? Fortunately we have devised several very effective techniques, which can be grouped into two categories: (1) those which kill or remove the microorganisms; and (2) those which inhibit their growth. I will list the methods in each category and then discuss them in turn: (1a) heat sterilization, (1b) irradiation, (1c) filtration; (2a) drying, (2b) refrigeration, (2c) addition of chemical inhibitors, (2d) exclusion of oxygen.

Food Spoilage by Fungi and How to Prevent It (1a) Heat sterilization involves heating the food to a high enough temperature for long enough to kill all the fungal propagules present on or in it. This method often has to be teamed with other techniques for preventing subsequent recontamination or for inhibiting moulds that do find their way in. Food may, for example, be heat processed and then vacuum packed, canned, bottled, or frozen. Some moulds are thermotolerant and can grow at 55°C–60°C. These include species of Aspergillus*, Chrysosporium, Humicola, Malbranchea, Paecilomyces, and Penicillium*, all hyphomycetes (dikaryan anamorphs). Some species of Talaromyces and Byssochlamys*, ascomycetous teleomorphs of Penicillium and Paecilomyces, have ascospores that can survive heating to 80°C. Certain foods have delicate textures or flavours that would be ruined by heating to 100°C, so other methods of preservation are often used. A final note of caution: heat sterilization is not a very effective way of getting rid of mycotoxins—some of them are very heat stable. (1b) Irradiation is one of the newest and most promising forms of sterilization. Food is sealed in airtight containers to prevent later recontamination and treated with appropriate doses of gamma radiation. This kills all living things—bacteria, fungi, even insects—in the food and endows it with a very long shelf life. This method should be suitable for a wide range of foods, although it has not been very successful with fresh vegetables. Some misguided environmentalists have condemned this technique, ostensibly because it reduces vitamin levels and produces free radicals (but more probably because it is associated with the hated nuclear industry). The free radicals, although in some cases potentially carcinogenic, are unstable and break down naturally after a while, and I am personally convinced that hungry people would rather eat food with reduced vitamin levels than lose it entirely to bacteria, fungi, or insects. (1c) Filtration is obviously of very limited application; in fact it can probably be used only for clear fluids such as beer, wine, some fruit juices, and soft drinks. This is because the pores in the filter have to be small enough to filter out microbial propagules. If we include bacteria in this instance (which is logical because they tend to be the major spoilers of liquids), the pores must be less than 1 micron in diameter, and the filter will soon clog up and need replacing. (2a) Drying is almost certainly the oldest method of preserving food. It works quite simply by denying the microorganisms the water they need to grow. Dryness is relative—by that I mean that we never need to remove all the water from food in order to extend its shelf life. In fact we sometimes remove very little of the water, simply making it unavailable to fungi by manipulating the osmotic pressure of the system. This is commonly done by adding salt or sugar. These substances reduce the water activity of the food just as effectively as if we had physically removed the water. Water activity (aw) is an expression of the moistness of the food; to measure it, we keep a sample of the food in a small airtight enclosure until the water in the sample and in the air within the container have equilibrated. If the relative humidity of the air within the container is then 85%, the water activity of the food is said to be 0.85. Life can go on over a range of water activities from 1.0 down to about 0.6. Animals can function only when virtually saturated—at water activities from 0.99–1.00. Many plants wilt permanently at 0.98. Most microorganisms can grow only above 0.95. These figures make the drought tolerance of some fungi all the more remarkable. In fact, some conidial fungi and yeasts are the most xerotolerant organisms known. Wallemia

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Chapter 20 sebi, which grows on salt fish, can tolerate a water activity of 0.75. Chrysosporium fastidium can grow at 0.69 and causes spoilage of dried prunes in Australia. But Aspergillus echinulatus is the champion, able to grow at water activities as low as 0.62. Some osmotolerant yeasts do almost as well. The xerotolerance of many moulds means simply that cheddar cheese at its normal moisture content will go mouldy if exposed to a little air, and the fact that many conidial fungi are also psychrotolerant means that this will happen even in the refrigerator. If we did dry the cheese to the point that fungi would not grow, it would not be cheddar cheese anymore but would take on the arid texture of the parmesan cheese we grate onto spaghetti. Some moulds are so xerotolerant that not even jams and jellies, with their high sugar content, are immune to spoilage, although the high osmotic pressure of jam slows the fungi down considerably. So when preparing homemade jam we have to sterilize the jam jars, put a layer of wax on top of the jam, and seal the jars tightly before storing them. Dried fruit, grains, and powdered milk have an aw of less than 0.75 and are therefore susceptible to attack by only a few fungi (e.g., species of Aspergillus, Wallemia, and Chrysosporium). Only by reducing the water activity of foods to 0.65 or less can we virtually eliminate fungal deterioration. (2b) Refrigeration is commonly used to retard food spoilage, but everyone knows that if you leave food in the refrigerator long enough, it will spoil. Many moulds will grow, slowly but surely, at 4°C, and even at 0°C. Some psychrotolerant fungi will grow even at temperatures several degrees below freezing. Several Penicillium spp.* grow at –2°C. Cladosporium herbarum* grows at temperatures as low as –5°C, and Fusarium poae* as low as –7°C. So refrigerators are not foolproof insurance policies against spoilage: they merely extend the storage life of especially perishable foods like milk and meat. If we really want to use low temperature to prevent fungal spoilage of foods containing moderate amounts of moisture, we have to keep them at –18°C or below, the temperature range maintained by modern freezers. (2c) Addition of chemical inhibitors. If you read the list of ingredients on the packaging of a loaf of bread, you will often find calcium propionate among them. It slows down the germination and growth of moulds and extends the shelf life of bread by several days. Since it has little effect on yeasts, it can be added during the preparation of bread dough. Calcium propionate is also added to cheese spreads. Sodium benzoate is often added to jams, jellies, margarine, carbonated soft drinks, fruit salads, fruit juices, pickles, etc. to inhibit yeasts and moulds in acidic conditions (pH 2.5–4.0) that some of them could normally tolerate. Calcium, sodium, and potassium sorbate are used to inhibit moulds in the same range of foods, but at pH values above 4.0. Sulphur dioxide is used to disinfect winemaking equipment and to preserve fruit juices and dried fruit. Lactic acid is developed during the natural fermentation of sauerkraut, dill pickles, green olives, many cheeses, and some sausages, and propionic acid forms naturally during the ripening of swiss cheese; but these acids are usually supplemented by other preservatives. In case you were wondering, the substances mentioned above are ‘generally regarded as safe’ (GRAS) food additives. (2d) Exclusion of oxygen. Fungi are almost all aerobic (oxygen requiring), so such strategies as canning, bottling, sealing under paraffin wax, or wrapping food in impermeable membranes can effectively inhibit growth of most moulds—so can storage of food in inert atmospheres of carbon dioxide or nitrogen. A few species, such as

Food Spoilage by Fungi and How to Prevent It Penicillium roquefortii*, the mould that ripens blue cheeses, can grow at oxygen tensions only 10% of normal, but if most other cheeses are tightly sealed in aluminum foil or plastic wrap they will not become mouldy. Once the wrapping has been opened, conidia present on the cheese will soon germinate (even in the refrigerator), and visible mouldiness will not be far behind. Some common saprobic moulds such as Gliocladium roseum* and Trichoderma koningii* grow quite well in 1.0% oxygen, and the oxygen level has to be reduced to 0.2% before the growth of Fusarium oxysporum* is much curtailed. Keeping in mind the kind of techniques we have developed for preventing or retarding mould spoilage of foods, we can now make a quick survey of the main groups of foods and the kinds of moulds that cause spoilage problems in each. Foods from plants fall into four broad categories: (1) cereals and nuts, (2) high sugar, (3) vegetables, and (4) fruits. Those of animal origin provide a further four categories: (5) meat and poultry, (6) eggs, (7) fish and other seafood, and (8) milk and milk products. We will examine these eight in that order. (1) Cereals and nuts. If promptly harvested and stored under dry conditions, most of these naturally dry foods are relatively immune to fungal spoilage. But if the growing season is wet, grain can become mouldy while still in the field. Some field moulds are notorious mycotoxin producers—Fusarium spp.* produce zearalenone, T-2 toxin, and vomitoxin. Some storage moulds that attack grain and nuts are equally infamous—Aspergillus flavus* and A. parasiticus* produce aflatoxins. Rice, which is grown in warm, wet conditions, is often moulded by species of Aspergillus or Penicillium; and rice stained yellow by P. citrinum*, P. islandicum*, and P. citreoviride often contains mycotoxins. Barley is often contaminated with ochratoxin produced by P. verrucosum var. verrucosum*. Corn can be spoiled by Fusarium graminearum* or Aspergillus flavus*, which produce zearalenone and aflatoxins, respectively. Corn and winter wheat in the field are threatened by Fusarium graminearum*, the source of vomitoxin, and wheat in storage is sometimes spoiled by members of the Aspergillus glaucus series, A. candidus, A. flavus*, and A. ochraceus*, and by some penicillia*. If flour is stored at a moisture content of 13% or less, it won’t mould, but the addition of water during baking makes the finished product available to fungi. The commonest moulds on bread are the zygomycete Rhizopus stolonifer, with its tall sporangiophores and black pinhead sporangia; the green-spored hyphomycetes Penicillium expansum* and P. stoloniferum; the black-spored Aspergillus niger; and the pinkish Chrysonilia sitophila anamorph of Neurospora. Species of the zygomycete genus Mucor and the filamentous yeast Geotrichum may also be involved. Wheat bread is often moulded by Penicillium brevicompactum*, P. chrysogenum, and P. verrucosum var. cyclopium*; rye bread by the last species and P. roquefortii*; pastries by Aspergillus repens and P. verrucosum var. cyclopium*. (2) High sugar. Properly made, with enough sugar, jams and jellies have an aw of 0.7 or lower and are generally inaccessible to fungi. But because manufacturers are now trying to cut down on calories or cost, many jams have a water activity nearer to 0.8. This opens the door to such xerotolerant hyphomycetes as members of the Aspergillus glaucus series, Penicillium corylophilum, and Wallemia sebi and means that jam makers have had to resort to such supplementary measures as sterile filling of containers, refrigeration, and even chemical preservatives.

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Chapter 20 (3) Vegetables. Some vegetables, such as cabbages, potatoes, and turnips, are relatively resistant to fungal spoilage. Others, such as lettuce and ripe tomatoes, have a much shorter shelf life. The most serious fungal diseases of market vegetables are grey mould rot caused by Botrytis (Hyphomycetes), watery soft rot caused by the sclerotial anamorph of Sclerotinia (Ascomycetes, Leotiales), blue mould rot caused by Penicillium*, Fusarium* rot, Alternaria* rot, Cladosporium* rot, black mould rot caused by Aspergillus niger (the last five all hyphomycetes), and soft rot caused by Rhizopus (Zygomycetes, Mucorales). Cabbage can be preserved by fermenting it with lactic acid bacteria, turning it into sauerkraut. Few organisms can compete with the lactobacilli, so that other than reducing the pH to very low values, the lactobacilli do not spoil the cabbage. (4) Fruits. The combination of senescent cells and high sugar and acid content makes many ripe fruits particularly susceptible to fungal spoilage by several hyphomycetes. Botrytis cinerea causes the extremely destructive grey mould of strawberries. Penicillium expansum* and Gloeosporium spp. produce storage rots of apples. Penicillium digitatum and P. italicum destroy oranges and lemons. Monilia brown rot causes heavy losses of peaches, cherries, other stone fruits and pears. Rhizopus stolonifer (Zygomycetes, Mucorales) is also a common problem on ripe peaches. Tropical fruits are often attacked by species of Colletotrichum* (Coelomycetes). Fruits are often preserved by drying or canning (or by making into jam, as mentioned above), and fruit juices now support a whole industry. Apricots and peaches for drying are impregnated with sulphur dioxide to preserve their colour, and this also effectively prevents fungal spoilage. Unsulphured dried fruit can go mouldy. Prunes, especially, are sold in moist packs which have an aw of 0.80–0.85. This makes them accessible to most xerotolerant moulds, which can be inhibited by sorbic or benzoic acid. Canned goods are usually heated to a high enough temperature to destroy fungal spores, but fruits with delicate textures are pasteurized at lower temperatures. Heating to 80°C will kill all zygomycete sporangiospores and all hyphomycete conidia, but it won’t kill ascospores of Byssochlamys fulva* or B. nivea. These thermotolerant ascomycetes sometimes spoil canned strawberries, soft drinks, and fruit juices (prune, grape, pineapple) and home-bottled fruit. If these fungi are cultured, their Paecilomyces anamorphs usually develop. Fruit juices are naturally contaminated with yeasts, and the normal course of events would involve an alcoholic fermentation which would ultimately, as in the case of wine, effectively preserve the substrate. But if the juice is refrigerated, moulds, rather than yeasts, will be favoured. Some fruit juices (blackcurrant, grape) are preserved with sulphur dioxide. In conclusion, it is worth pointing out that we can’t blame all fruit losses on the fungi—bananas become brown and mushy through the action of their own enzymes. (5 and 6) Meat and eggs. Although most serious spoilage of foods of animal origin is caused by bacteria, species of Penicillium and Aspergillus are commonly recorded and may produce mycotoxins such as cyclopiazonic acid, penitrem A, ochratoxin, patulin, and aflatoxin. Fungi can be inhibited by refrigeration, vacuum packing, drying, or irradiation. (7) Fish, usually dried, is most frequently contaminated by Eurotium, Scopulariopsis, Penicillium species, and Wallemia. Mycotoxins produced are ochratoxin A and

Food Spoilage by Fungi and How to Prevent It citreoviridin. Reducing the water activity of the substrate is the best way of preventing fungal spoilage. (8) Raw milk is usually spoiled by bacteria before fungi can begin to affect it. Pasteurization and airtight, refrigerated storage are the best ways to prolong its shelf life. Conversion to dried milk or cheese will also extend its life, but cheese is commonly attacked by fungi, especially Penicillium commune, but also by other penicillia, Aspergillus versicolor, Scopulariopsis species, and Eurotium herbariorum. Spoilage is delayed by reducing water activity, by vacuum packing, and by refrigeration. Preserving 101

There are countless ways to preserve foods, and some methods actually enhance an ingredient’s flavour: a confit of duck, salted and packed in oil, takes on a concentrated, autumnal flavour; small cucumbers are more recognizable as pickles, bottled in salt and vinegar brine; simmering fruit with sugar heightens the flavour of a jam or jelly. (The Globe and Mail, November 5, 2000) That’s as may be—but none of those things would ever have been discovered were it not absolutely necessary to preserve food from attack by fungi and bacteria. If you’d like to see some time-lapse movies of food items being attacked by fungi, just go to Google and type in ‘mold time lapse’.

Further Reading Dijksterhuis, J., and R. A. Samson. 2007. Food Mycology: A Multifaceted Approach to Fungi and Food. Boca Raton, FL: CRC Press. [403 pp.] Frazier, W. C., and D. C. Westhoff. 1978. Food Microbiology. 3rd ed. New York: McGraw-Hill. Pitt, J. I. 1981. “Food Spoilage and Biodeterioration.” In Biology of Conidial Fungi. Vol. 2. Edited by G. T. Cole and B. Kendrick. New York: Academic Press. Pitt, J. I., and A. D. Hocking. 2009. Fungi and Food Spoilage. Berlin Heidelberg: Springer. [519pp.] Samson, R. A., and E. S. van Reenen-Hoekstra, eds. 1988. Introduction to Food-borne Fungi. 3rd ed. Baarn: Centraalbureau voor Schimmelcultures. [With contributions by twelve other authors.]

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21 Mycotoxins in Food and Feed Introduction Many foods (e.g., bread, meat, milk, cheese) are nonliving but natural organic substances, which are obvious targets for saprobic fungi and accordingly tend to go mouldy if kept too long. The people of Western countries, generally relatively wealthy and fastidious, will throw out most food that is obviously mouldy. But in the poorer nations food is often a precious commodity, not to be rejected because of a little surface discolouration. This attitude is reinforced in the Far East by the common use of moulds to prepare traditional fermented foods (see chapter 19). Some indigenous peoples in Africa and Asia actually prefer the spicy flavour foods acquire when they go mouldy (they aren’t alone—note the blue cheese eaten by Westerners). Animals will often accept mouldy feed. To compound the problem, the term mouldy refers only to food on which the contaminating fungi are already sporulating. But even food which looks perfectly edible may be riddled by the invisible assimilative hyphae of moulds and permeated by their questionable metabolites. Do these things matter? Is mouldy food dangerous? This chapter will give you some answers. Some fungi, including many common moulds (mostly hyphomycetous anamorphs of Ascomycetes), produce secondary metabolites—usually steroids, carotenoids, alkaloids, cyclopeptides, and coumarins. Many substances in the last three categories are toxic to animals and to other microorganisms at very low dosages and are also persistent and often heat stable. Such compounds are commonly produced in funguscontaminated foodstuffs; remain there even after processing, sterilization, or cooking; and are unsuspectingly eaten. They are called mycotoxins. Well over 200 such substances, produced by more than 150 different fungi, are now known, and more are being discovered every year.

Ergot Alkaloids and St. Anthony’s Fire We have become aware of most mycotoxins and their insidious effects only since 1960, but the condition called ergotism has been known for thousands of years. This mysterious and dreadful disease struck the Spartans in 430 BCE during their war with Athens. Epidemics of ergotism during the Middle Ages have left bizarre accounts in which the screams of the dying, the stench of rotting flesh, and limbs actually dropping off are recorded in grisly detail. Supplications were naturally made to the saints, and especially to one St. Anthony, the founder of monastic life, since sufferers who made 372

Mycotoxins in Food and Feed pilgrimages to his shrine often gained relief. Grateful survivors founded a hospital brotherhood in his name, and to this day the condition is known as St. Anthony’s fire. What was really happening to the victims? How can we explain those ‘miraculous’ cures? We now know that the victims had all eaten bread made from grain contaminated with sclerotia of the ergot fungus, Claviceps purpurea (Figs. 4.25A–C). These sclerotia contain a co*cktail of physiologically active substances: 10 different groups with over 100 compounds. The disease-producing substances are alkaloids of two main types: (1) clavine alkaloids, and (2) derivatives of lysergic acid—amides or peptides. What was the disease like? Two main patterns were seen. (1) Gangrenous ergotism. This began with fatigue and cold or prickling sensations in the limbs and then severe muscular pains. Limbs later became swollen and inflamed, and burning pains and sensations of heat alternated with those of icy coldness. Gradually the affected parts became numb. Feet and legs turned black and mummified. This dry gangrene spread upward, and the fingers and toes of victims, or even their hands and feet, literally sloughed off. (2) Convulsive ergotism. Here the central nervous system was most severely affected. Symptoms ranged from formication (check the spelling—it means feeling as if ants are crawling under your skin) to itching, numbness of hands and feet, twitching, and muscular cramps escalating into sustained convulsions. Fists became clenched, the hands in acute flexion—the body either rolled up in a ball or bent rigidly backward as in tetanus. Convulsions alternated with periods of drowsiness. Between convulsions, patients often ate voraciously and could not sleep. Some died within hours, others lingered for weeks. Severe nonfatal cases lasted up to two months, and brain damage was common. Mortality ranged from 11% to 60%. At least sixty-five epidemics were documented between 1581 and 1889. A Frenchman named Dodart exposed the cause of the disease as early as 1673, but ignorance and stubbornness prevented much reduction in the death rate for another century. Finally, when a famine in 1770 precipitated many terrible epidemics, effective control measures were introduced. But Claviceps also attacks the ovaries of many wild grasses, and grazing livestock still sometimes suffer from ergotism. The symptoms can be analyzed into two main components, caused by different groups of chemicals. (1) Effects on muscles and blood vessels: the alkaloids affect smooth muscle and cause vasoconstriction (which cuts off circulation and can cause gangrene). This effect has been put to medical use in two areas: (a) Ergotamine tartrate is useful in the management of migraine. It reduces the diameter of the cranial arterioles, thus reducing pulsation pressure and the attendant headache. (b) Ergometrine has been widely used to induce labour (but not to procure abortions, since the uterine muscles become really sensitive to the compound only at full term) and to control bleeding after childbirth. (2) Effects on the central nervous system: lysergic acid amides, particularly lysergic acid diethylamide (LSD), are dramatically potent hallucinogens. Since this was discovered, thousands of research papers have appeared on the subject, and many more unofficial experiments have been conducted. Although the medical profession drily records the effects of LSD as hyperthermia, hyperglycemia, mydriasis, piloerection, tachypnea, and hyperreflexia (I’ll leave your own curiosity to drive you to look those words up:

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Chapter 21 they aren’t in the glossary), the really important immediate effects appear to be extremely subjective and less easily described. However, I’m sure you have all read, or heard at firsthand, accounts of trips taken by devotees, with their dazzling visual hallucinations (Lucy in the Sky with Diamonds) and intense sexual experiences. Those who are tempted by such glowing accounts should be warned, not only of the temporary personality-dissolving effects but also of the less well-charted long-term sequelae. Perhaps the most promising use for this drug lies in the treatment of certain mental disorders—it is not recommended for most of us, who might describe ourselves as nearly normal neurotics. So, on the one hand, ergot is now produced deliberately by the pharmaceutical industry, spores of the fungus being mechanically inoculated into the flowers of rye; on the other hand, ergot is now rigorously excluded from grain that will be ground for flour. No longer do we need to take pilgrimages to St. Anthony’s shrine to give ourselves a break from an ergot-contaminated diet (yes, that seems to have been the secret of those ‘miraculous’ cures). Only animals and the occasional alkaloid overdose patient now suffer from St. Anthony’s fire. A few years ago in England, ergotamine tartrate was prescribed for a woman suffering from migraine. Unhappily, the amount prescribed was a massive overdose, she developed gangrene in her feet, and her toes had to be amputated. In Canada a hog farmer lost most of his pigs to ergot poisoning. He was awarded $100,000 in damages against the feed supplier, despite the fact that the grain had not exceeded the level of ergot contamination permitted by the Canadian Grain Commission. Claviceps purpurea has not lost its ancient powers, and its toxic effects can be avoided only by constant vigilance.

Aflatoxins—Toxins, Mutagens, Carcinogens The modern awakening of scientists to an awareness of mycotoxins began in 1960, when tens of thousands of turkey poults in England began to die from a mysterious disease—they sickened, they haemorrhaged subcutaneously, they died. Postmortems showed that their livers had undergone extensive necrosis, and their bile ducts had hypertrophied. This condition was called ‘Turkey X disease’, which is easy to remember but not very informative. Elsewhere in Britain, large losses of partridges, pheasants, and ducklings were reported, and, later, calves and pigs were affected. Some slick detective work showed that the only factor common to all these outbreaks was the incorporation of Brazilian peanut meal in the animals’ feed. A high incidence of hepatoma in hatchery-raised trout was also linked to peanut meal. It wasn’t long before the toxin was isolated and found to originate with a fungus growing on the peanuts, rather than with the peanuts themselves. The fungus responsible was found to be a very common mould, Aspergillus flavus (Fig. 21.1C), so the toxin was called aflatoxin. It soon transpired that there were four toxins, rather than one. When separated chromatographically and examined under UV light, two fluoresced blue, and two fluoresced green. The toxins were called aflatoxin B1 and B2 and aflatoxin G1 and G2. Under most conditions, aflatoxin B1 is the major toxin produced. The chemical name for this group of compounds is bisfuranocoumarins.

Mycotoxins in Food and Feed The factor that made aflatoxins big news was not simply that they are toxic but, as was soon discovered, that they are extremely potent carcinogens in rats (and therefore, by implication, in humans). To make matters worse, the aflatoxins were discovered at a time when UNICEF (the United Nations Children’s Fund), among others, was pushing peanut meal, with its high protein content, as a dietary supplement to prevent kwashiorkor (gross protein deficiency) among children in many tropical countries. Did the discovery of aflatoxins mean that these people were at risk? Were they liable to sustain liver damage or even develop liver cancer? It became very important to know just what conditions led to the formation of aflatoxins, especially since Aspergillus flavus occurs on many grains in storage and is even used in the Far East to prepare some types of vegetable cheese and soy sauce. Not only that—cows whose diet is contaminated with aflatoxin produce milk containing a derivative called aflatoxin M1, and peanut butter is the staple diet of most North American children. Although it was soon found that the range of conditions under which Aspergillus flavus could grow was much wider than that over which it formed aflatoxins, it was also discovered just how toxic aflatoxins are. Some typical LD50 figures (the dosage in mg/kg which will kill half of a population of experimental animals) are ducklings—0.335, rabbits—0.300, cats—0.550, pigs—0.620, sheep—1.000. That’s bad enough, but it was discovered that even if the diet didn’t contain sufficient toxin to cause acute poisoning, prolonged exposure to much lower levels (chronic poisoning) will often cause liver cancer. Many experiments have demonstrated this in rats. As little as 0.015 ppm (parts per million) in the diet over seventy weeks caused neoplasms in all rats tested. At 1 ppm this took only forty weeks. At 5 ppm it took nine weeks. Shasta trout are so sensitive that as little as 0.5 mg/kg (0.5 ppb) in their food over twenty months will produce similar results. If we examine the incidence of liver cancer in human populations, we find it is exceptionally common in some developing countries, especially those of sub-Saharan Africa and the Far East. The problem is well documented in Uganda, Swaziland, Kenya, and Thailand. All have elevated levels of liver cancer, and the diet in each country is significantly contaminated with aflatoxins. Although we cannot, of course, prove experimentally that aflatoxins cause liver cancer in humans, the suspicion is strong enough to be almost a certainty (as with the connection between smoking and lung cancer). And we do know that in some other mammals, aflatoxins are the most potent carcinogens yet discovered. What is being done to monitor and control the levels of aflatoxins in our diet? In Canada the allowable limit of contamination, originally 20 ppb in a finished product, has been reduced to 15 ppb. Germany allows only 10 ppb. I personally believe that no detectable aflatoxin should be permitted. If proper attention was paid to storage and selection of peanuts for human consumption, and to appropriate dilution of mildly contaminated nuts, this standard could be attained. Aspergillus flavus competes best in warm climates on substrates that have low water contents (low ‘water activities’; see chapter 20). It was originally classified as a storage mould, but some contamination has now been traced back to the field; the fungus can be an adventitious parasite, invading insect-damaged tissue. Serious aflatoxin contamination has been found in peanuts, brazil nuts, pistachios, almonds, walnuts, pecans, filberts, cottonseed, copra, corn, grain sorghum, millet, palm kernels, beans, wine, milk,

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Fig. 21.1 The principal genera of toxigenic anamorphs.

cheese, dried fish, garlic, pasta, bread, flour, and figs. Aflatoxicosis is mainly encountered in warmer climates, and it is interesting to note that peanuts now grown in Ontario (as a replacement for tobacco) seem to be generally free of aflatoxin—one advantage to living so far north. In Nigeria, corn farmers are now broadcasting in their fields a new strain of Aspergillus flavus that not only does not produce aflatoxin but outcompetes the strain that

Mycotoxins in Food and Feed does and thus reduces or prevents the contamination of their crop with aflatoxin. This is a good example of biological control.

Mycotoxins and Oesophageal Cancer—Fumonisins In at least two areas of the world, the incidence of oesophageal cancer is many times higher than might be expected. The reasons for this have only recently begun to be understood. In Lin Xian, China, not many years ago, oesophageal cancer killed 25% of the population, and the disease had tragically become an accepted part of existence. Folk wisdom held that if you had trouble swallowing it was because you were unhappy. Scientists, however, suspected that nitrosamines were responsible. The foods people ate did not contain elevated levels of nitrosamines, but it was established that these compounds could easily be produced in the stomach from nitrites and amines. Gradually, the pieces of the puzzle were fitted together. Although the local water was not initially very high in nitrite, people simmered their corn for hours, concentrating the nitrite, and then used the water to make soups. The soil was low in molybdenum, a deficiency which caused crop plants to accumulate nitrite in their leaves. So human levels of nitrite were found to be high, while vitamin C intake was low. Apparently, without adequate vitamin C, the body could not rid itself of the nitrites. The poorest workers made steamed bread from the corn. Nothing unusual in that, except that they made enough at one time to last for three weeks. Needless to say, the bread started to go mouldy after a few days. But this didn’t bother the people of Lin Xian: they liked the spicy flavour of the mouldy bread. (Remember my comments on cultural determination of what constitutes food spoilage?) Scientists found that two of the fungi involved in the moulding caused levels of amines in the bread to increase seventeen-fold. Here was another piece of the puzzle. Rats fed nitrites and mouldy bread manufactured nitrosamines and developed cancer. But it was also noted that some of the control rats, those fed only mouldy bread, also developed cancer. Some of the fungi must produce unknown carcinogens. Doctors also found that 90% of the cancers surgically removed from patients actually had these moulds growing inside them. This was another important observation, because many apparently healthy people already had precancerous thickenings of their oesophagus, 72% of which harboured living moulds. The etiology of the disease was now much clearer. Molybdenum-deficient soil led to nitrite accumulation in crops; vitamin C deficiencies permitted high body nitrite levels. Mouldy bread was high in amines and harboured fungi that produced carcinogens. Now it was time to institute preventive measures. Seeds are being treated with molybdenum. People are supplied with piped water free of nitrite. People are told to eat fresh vegetables and to avoid mouldy food at all costs. Unfortunately, it takes years for a cancer to develop, and it will be years before the results of the cancer prevention campaign can be assessed. But this is certainly one disease for which prevention, if possible, is a thousand times better than the attempt to cure, which involves drastic surgery and follow-up radiation treatments and has a poor prognosis. A similarly high incidence of oesophageal cancer among the Xhosa people in some parts of the Transkei, South Africa, has also been connected with the consumption of

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Chapter 21 mould metabolites in corn and in the native beer, which is made by preference from mouldy grain. This time the suspect fungus is Fusarium verticillioides, and the suspect metabolites are fumonisins, secondary metabolites that were characterized only after fifteen years of investigations. Again, preventive dietary changes would seem to be the answer. But as we all know, it is hard to get people to give up dangerous habits they enjoy, particularly those involving food or legal drugs such as alcohol and tobacco. Each year in South Africa about 200 people suffer from an unnamed haemorrhagic disease with additional tremorgenic (neurotoxic) effects, plus associated liver and kidney symptoms. This complex syndrome is believed to result from drinking homebrewed native sorghum beer, which is often contaminated with at least two mycotoxins: tenuazonic acid produced by Alternaria alternata and Phoma sorghina and cytochalasin produced by Aspergillus clavatus.

Equine Leucoencephalomalacia: ‘Hole in the Head’ Disease of Horses Both terrible names for a horrible disease! In horses, donkeys, and mules, the first signs of this condition are apathy, protruding tongue, unwillingness to move backward, and walking in circles. Eventually the animal becomes delirious and may run full tilt into fences. Finally it falls over, thrashes its legs in the air, and dies. Death may occur in seven hours or several days. A postmortem reveals areas of brain necrosis— large, irregular holes where the white matter has disintegrated. It was found that the disease condition could be reproduced by feeding the animals corn moulded by Fusarium verticillioides. Field outbreaks of this mycotoxicosis have occurred in Argentina, Brazil, China, Egypt, South Africa, and the United States, and the mycotoxins involved are now known to belong to the fumonisins.

Trichothecenes and Haemorrhagic Syndrome During and after World War II (1942–1947), a serious and widespread haemorrhagic syndrome called alimentary toxic aleukia (ATA) affected people in Siberia. Similar illnesses had been noted on earlier occasions but had never before reached such epidemic proportions—in some areas 10% of the population developed the disease, and most cases were fatal. ATA was characterized by nausea, vomiting, haemorrhages in many organs, bleeding from nose and throat, bloody diarrhea, low leucocyte (white blood cell) count, exhaustion of bone marrow, and throat sepsis and necrosis. About a third of the deaths were due to asphyxiation resulting from internal swelling of the throat. This disease occurred when much of the Russian population was starving, and manpower shortages had prevented fall harvesting of grain. When the grain was finally harvested in spring, it proved to be extremely toxic. Not until years afterward was the epidemic linked to mycotoxins produced by moulds growing on the overwintered grain. Symptoms of ATA appeared after about 2 kg of contaminated grain had been

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Mycotoxins in Food and Feed O

CH3

O CO CH3

O O

OCH3

Vomitoxin HO

H N

O H

CONH

CH3

N H

Slaframine

CH3 CH2OH

Aflatoxin B1

H2N

O HO

O N

Zearalenone

Ergometrine

N H

CH3 CI

CHO CH3

CH3OO

Patulin

Ochratoxin A

CH3

PR Toxin

H O

H3C

CH3

OH O

O O

Ergotamine

CH3

LSD

T2 Toxin

CH3 CI HO O

HO O

S S

COCH3

N H

C2H5

N H

CH3

Sporidesmin

Fig. 21.2 Structural formulae of some important mycotoxins.

Tenuazonic Acid

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Chapter 21 eaten. Consumption of 6 kg was fatal. A similar haemorrhagic syndrome in animals is called mouldy corn toxicosis. The two fungi found to be mainly responsible were Fusarium poae and Fusarium sporotrichioides (hyphomycetous anamorphs of Ascomycetes; cf. Fig. 21.1B), and the toxins they produced belonged to the group of tetracyclic sesquiterpenoids called trichothecenes, which are extremely poisonous, having an LD50 for many animals of less than 10 mg/kg. ATA is provoked by a trichothecene called T-2 toxin. The molecules of the more than forty naturally occurring trichothecenes all contain a ring system called trichothecane, an olefinic bond at C-9,10, and an epoxy group at C-12,13 (Fig. 21.2). T-2 toxin has an LD50 for mice of 5.2 mg/kg.

Trichothecenes and Stachybotryotoxicosis in Horses During the 1930s, horses in Ukraine began to die in large numbers. They suffered mouth ulceration and swelling, fever, severe inflammation of the skin and respiratory tract, and depletion of leucocytes and blood platelets leading to complete failure of the blood-clotting mechanism. Death could occur in less than a day or up to a month after the onset of symptoms. Scientists eventually connected the disease to fodder contaminated with macrocyclic trichothecenes produced by Stachybotrys chartarum (see Fig. 21.1F), a common cellulose-decomposing hyphomycete growing on hay used as food and bedding. No fewer than five stable and persistent trichothecenes are produced by this fungus: verrucarin J, roridin E, and satratoxins F, G, and H, although only satratoxins have been demonstrated in naturally contaminated straw associated with an outbreak among sheep in Hungary. Although typically reported in horses, this toxicosis can also affect cattle, pigs, poultry, sheep, and even humans (but only if they eat the fungus). Nikita Khrushchev, who later became the Russian head of state, owed some of his early career success to his recognition that horses, so vital to the Russian economy and the Red Army transport system of the day, needed clean, dry fodder. Note that animals had to eat mouldy hay before they became ill. Back in chapter 8, I noted that people became very excited when they detected the same mould in their houses—but none of them had eaten the mould, and breathing in a few spores is not going to hurt anyone, unless they are severely allergic to the mould. This was in most cases a hysterical reaction. Check back there for a full discussion.

Trichothecenes and Yellow Rain During the Vietnam War the U.S. government received many reports of chemical attacks launched by the invading Vietnamese in Laos and Kampuchea against troops and the civilian Hmong people. According to the reports, victims were sprayed with ‘yellow rain’, an aerosol containing substances with toxic effects that did not match those of any known agents of chemical warfare. Eventually it was recognized that the symptoms—prolonged vomiting, diarrhea, headaches and dizziness, respiratory problems, blisters, internal haemorrhages, sometimes culminating in death—were like

Mycotoxins in Food and Feed those produced by the trichothecenes. Samples of blood, urine, and body tissues from victims and leaves, water, and soil from sprayed areas were found to contain several trichothecenes and zearalenone, typical Fusarium toxins. Control samples, including cereal grains, from areas adjacent to the places where attacks had taken place contained no Fusarium toxins. However, this issue was clouded by the remoteness of the location and the unsatisfactory and inconclusive nature of the available evidence. Samples of the ‘yellow rain’ deposits were later shown to be largely made up of pollen, and it seems probable that these deposits were in fact nothing more than bee faeces, dropped during communal cleansing flights. The issue was carefully documented in an article titled ‘Political Science’ in The Atlantic Monthly, and I leave you to draw your own conclusions from that.

Trichothecenes: Vomitoxin, Refusal, and Emesis in Pigs Fusarium graminearum (Fig. 21.1B), a common mould on damp corn, produces a trichothecene (3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one) named deoxynivalenol. It has also been called vomitoxin because it was first discovered as a result of its powerful emetic effect on pigs, which will quickly learn to refuse food contaminated with it. Up to 40 ppm have been found in corn from Austria, Canada, France, Japan, South Africa, and the United States. It also contaminates barley and winter wheat. Since it affects pigs at concentrations as low as 0.7 ppm, many countries won’t buy grain contaminated with it. Current vomitoxin limits for wheat used in pastry flour and in bread or breakfast cereal are 0.3 and 0.1 ppm, respectively. After one recent wet season, Ontario farmers growing winter wheat lost $17 million on a harvest of 670,000 tonnes because of extensive vomitoxin contamination of the grain.

Zearalenone (F2 Toxin) and Oestrogenic Syndrome in Pigs Pig farmers sometimes find that their young female pigs (‘gilts’) develop swelling of the vulva, enlargement of mammary glands, enlargement of the uterus, and sometimes even rectal and vagin*l prolapse—the vagin* and associated structures swell and are often literally extruded. Internally, the ovaries atrophy. At the same time, the testes of young male pigs shrivel, and their mammary glands enlarge. Since all of these symptoms affect primary and secondary sexual characteristics, the involvement of some type of sex hormone might be suspected. Once characterized, the syndrome was quickly linked with the presence of mouldy corn in the feed, and the fungus concerned was found to be the hyphomycete Fusarium graminearum (Fig. 21.1B). The toxin was named zearalenone. It obviously has many of the properties of oestrogen, one of the principal female sex hormones (it actually seems to be involved in regulating the development of sexual fructifications of the fungus). The toxin doesn’t usually kill the animal, but complications following rectovagin*l prolapse sometimes cause the whole herd to be destroyed. Lower levels of exposure are also serious in pigs, because they can cause infertility, small litters, and stillbirths.

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Chapter 21 Fusarium graminearum, the anamorph of Gibberella zeae (Ascomycetes, Hypocreales), develops as a pinkish mycelium on corncobs stored wet over winter, and the toxin may reach levels of 50–100 ppm in the grain. Animals which aren’t allowed to eat such mouldy feed will not develop oestrogenic syndrome. Zearalenone is not inevitably detrimental: when added to feed in very low doses, its hormonal properties produce accelerated growth in cattle and sheep, and it has been patented as a feed supplement (but not for pigs). It has also been used to treat postmenopausal syndrome in humans and as an oral contraceptive.

Mouldy Sweet Potato Toxicosis Cattle fed sweet potatoes affected by Fusarium solani storage rot develop acute symptoms of respiratory distress, and they may die. The disease affects only the lungs, which become swollen and congested, with scattered haemorrhages. It has been found that four furanoterpenoid toxins are responsible for this condition: 4-ipomeanol, 1-ipomeanol, ipomeanine, and 1,4-ipomeadiol. These compounds are not simply fungal metabolites but are produced by a host-pathogen interaction. In response to stress, the sweet potato produces phytoalexins, such as 4-hydroxymyoporone, which are catabolized by F. solani to produce the toxic end products. Humans in New Guinea, for whom sweet potatoes are an important dietary item, often suffer from a chronic respiratory syndrome that is sometimes fatal. The causation of this disease is still obscure, but the compounds discussed above must be strong suspects.

Ochratoxin, Mycotoxic Nephropathy in Pigs, and Balkan Nephropathy in Humans In 1928 a new kidney disease of pigs was discovered in Denmark, but it was not until 1966 that the condition was proved to be a mycotoxicosis. It is caused by ochratoxin A, a metabolite of Aspergillus ochraceus (and six other species of Aspergillus; Fig. 21.1C), and Penicillium viridicatum (and five other species of Penicillium; Fig. 21.1A). These fungi grow on damp feed grain, usually barley or oats. Epidemiological studies also link ochratoxin with endemic Balkan nephropathy, a fatal kidney condition reported among people living near the Danube River and its tributaries in Yugoslavia, Bulgaria, and Romania. The LD50 of ochratoxin A for rats is 20 mg/kg. The structure of ochratoxin is shown in Fig. 21.2.

Sporidesmin and Facial Eczema of Sheep For many years, New Zealand sheep (one of that country’s principal industries) have been plagued by outbreaks of a condition known as facial eczema. They stop eating, develop diarrhea, and then develop inflamed swellings on their lips, face, eyelids, and vulva. They also become photophobic, trying to avoid bright light. Internally there is

Mycotoxins in Food and Feed hypertrophy of the bile ducts and extensive liver necrosis. Affected animals sometimes die. The disease usually occurs in late summer after warm rains. Eventually, in 1963, the cause of the disease was found to be a toxin called sporidesmin, which is produced by a saprobic hyphomycete, Pithomyces chartarum (Fig. 21.1E), growing on dead parts of forage grasses. The name of the disease is misleading, because the liver damage, rather than the skin problem, is life threatening. The photosensitivity causing the ‘facial eczema’ is a secondary symptom caused by a porphyrin, phylloerythrin. This is a product of chlorophyll digestion that builds up in the peripheral circulation because the damaged liver cannot excrete it. Since it would be very expensive to spray large areas of grassland with fungicide, attempts are made to avoid the disease by forecasting weather conducive to sporulation of the fungus and moving sheep to less susceptible grazing areas during such periods.

Lupinosis of Sheep Sheep or other animals grazing on lupin stubble in Australia, New Zealand, South Africa, and Europe, especially a week or so after heavy rains, may become anorexic, feverish, and listless and then jaundiced. Up to half of affected animals may die. The liver is clearly the main organ affected. It was first suggested as long ago as 1880 that the disease might be caused by toxins derived from fungi growing on the lupins, but final proof of this was not forthcoming until 1970. Phom*opsin A, the hepatotoxin responsible, is a cyclic hexapeptide produced by Phom*opsis leptostromiformis (the coelomycetous anamorph of Diaporthe woodii: Ascomycetes, Diaporthales). This fungus produces black pycnidial conidiomata on stems and pods of Lupinus, and the teleomorph has also been reported from this substrate. The fungus is a pathogen that continues growing saprobically after the death of the host, producing toxins in both phases. In Western Australia, where lupinosis is a serious problem, attempts are being made to breed lupins resistant to the Phom*opsis.

Slaframine: Slobber Factor In the U.S. Midwest, cattle which were seen to slobber or drool and then refuse to eat may well have been feeding on red clover (Trifolium) that was attacked by a sterile basidiomycetous anamorph, Rhizoctonia leguminicola (Fig. 21.1D), especially after cool, wet weather. The fungal metabolite that produced this reaction was named slaframine. It is an acetate ester of a bicyclic amine synthesized partly from lysine. This compound itself is physiologically inactive but is metabolically transformed to a quaternary amine similar in activity to acetylcholine and stimulates all cholinergic exocrine glands. Although we know what causes the disease, it has proved impossible either to breed red clover resistant to the fungus or to control the fungus with fungicides. Fortunately, there is a simple answer: farmers in the Midwestern United States have given up sowing red clover as a forage crop.

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Apple Juice and Patulin Patulin first attracted attention as a potential antibacterial antibiotic isolated in 1943 from Penicillium patulum and also from Penicillium expansum and Penicillium claviforme, as well as several other penicillia (Fig. 21.1A) and aspergilli (Fig. 21.1C). Interest in its antibacterial activity has now given way to concern about its toxic effects on plants and animals and its possible role as a carcinogen. Penicillium expansum causes a very common storage rot in apples, so contamination of apple juice is likely and should be monitored. The LD50 of patulin in mice is 8–10 mg/kg. Its structural formula is given in Fig. 21.2.

Tremorgens, the Shakes, and Staggers People in western Nigeria are sometimes afflicted by a condition known as Ijesha shakes. After eating, they become almost completely incapacitated by tremors of the legs. The condition may last for several days, but patients eventually recover completely. It is suspected that foods containing tremorgens—neurotoxins of fungal origin—are responsible. A disease of sheep and cattle known as grass staggers presents symptoms identical to those produced by the tremorgenic toxin penitrem A of Penicillium cyclopium. There is a strong possibility that the toxins responsible for this disease are produced by symptomless endophytic fungi, including the Neotyphodium anamorph of Epichloë typhina (Ascomycetes, Clavicipitales) or other members of this order.

PR Toxin and Blue Cheese You will have noted that many of the mycotoxins discussed in this chapter are produced by species of Penicillium or Aspergillus (Figs. 21.1A, C). It so happens that various species of both genera have been used for many generations in the preparation of traditional foods. Aspergilli are responsible for many fungally fermented Asian foods, while penicillia impart the unique and delicious flavours to some of our finest cheeses. The conjunction of these facts did not go unnoticed. Could both Westerners and Asians be insidiously poisoning themselves with these delicacies? Scientists who set out to answer that question were rather dismayed to find that Penicillium roquefortii, the fungus responsible for ripening all blue cheeses (Roquefort, Gorgonzola, Danish blue, Stilton), did in fact produce a toxin, which they called PR toxin. This substance was lethal to mice: when injected intraperitoneally it had an LD50 of 6 mg/kg. Fortunately for those of us who are addicted to blue cheese, no trace of this toxin has been found in the cheese itself, and it appears that conditions prevailing during the cheesemaking process prevent toxin formation. It has also been found that toxins are not normally produced during the preparation of soy sauce (shoyu), ket-jap, miso, hamanatto, or katsuobushi, all of which involve species of Aspergillus. It seems that some type of selection process has excluded toxigenic strains from most food-processing applications, or the conditions involved have inhibited toxin production. However,

Mycotoxins in Food and Feed some concerns remain. Penicillium roquefortii has also been found to produce two other toxins, roquefortine and patulin, and Penicillium camembertii, which ripens soft cheeses such as Brie and Camembert, produces cyclopiazonic acid. Whether these toxins are produced in dangerous quantities, or occur at all in cheeses, is not yet fully established.

Alternaria and Tenuazonic Acid Alternaria (Fig. 21.1H) is one of the commonest moulds on various crops, such as apples and tomatoes, and on deteriorating food, and it is now known to produce several mycotoxins, of which the most poisonous is tenuazonic acid. This has been detected in commercial tomato pastes at levels of 0.1–1.0 ppm and warrants further attention. In South Africa it has been shown that the growth of native cattle is considerably retarded by the presence of tenuazonic acid in their diet.

Cladosporium and Epicladosporic Acid Cladosporium (Fig. 21.1G) is another extremely common mould of deteriorating plant materials and, again, produces several mycotoxins. One of these, epicladosporic acid, may have been implicated in the outbreaks of alimentary toxic aleukia, since it was produced by one of the cladosporia isolated from samples of the grain consumed by ATA patients. It must be obvious by now that mycotoxins are a growing cause for concern. Obviously, their presence in certain foods, such as peanut butter, must be continuously monitored. Can we detoxify contaminated food or, better still, prevent moulds from growing on it in the first place? If the substrate can be kept dry and cool, moulds cannot grow and mycotoxins will not be produced. Mycotoxin-producing moulds are sometimes classified as either field moulds, which grow in substrates containing 22%– 25% moisture, or storage moulds, which need only 13%–18% moisture. This is only a rough division, since some fungi can exploit both ranges, but it gives us some idea of the type of conditions to aim for in food storage. Certain species of Aspergillus are the world’s most xerotolerant organisms and are capable of growing at extremely low water activities (below 0.7), so it is not easy to eliminate them completely. Temperature is another important factor. The three most important toxigenic mould genera are Penicillium, Fusarium, and Aspergillus (Fig. 21.1A–C). The optimum temperatures for the members of these genera are rather different. Many Fusarium species grow best in the range 8°C–15°C; the optimum for Penicillium species is usually 25°C–30°C; and that for Aspergillus species is often 30°C–40°C. This information gives us some idea of where to expect problems with each of these genera. A recent compilation found that representatives of forty-six genera of fungi are known to produce mycotoxins. When anamorph-teleomorph connections are taken into account, the number of holomorphic genera decreases to thirty-five. Given that there are thousands of fungal genera, the number reported as toxigenic seems very low.

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Chapter 21 But is that so surprising when we consider that we have detected most mycotoxins only after reacting to reports of toxicity, and we have not yet taken a proactive attitude toward testing a wide range of fungi for toxin production? If and when such a survey is carried out, I predict a dramatic increase in the ranks of the toxigenic fungi.

Detection of Mycotoxins The continuous monitoring of food for mycotoxin contamination requires regular sampling, efficient methods for extracting and purifying mycotoxins, and sensitive methods of detecting and quantifying them. Sampling can be a problem, because of the uneven distribution of mould growth. (How many mouldy peanuts are there in a sack?) Mycotoxins are extracted with an organic solvent: chloroform, dichloromethane, acetonitrile, ethyl acetate, acetone, or methanol. Repeated column chromatographic purification is often necessary, using such substances as silica gel, alumina, and Sephadex. Thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) help in the final separation of the mycotoxins from other compounds extracted with them. Aflatoxins can then be detected directly by their UV fluorescence. Other toxins do not autofluoresce, for example, sterigmatocystin fluoresces only after being sprayed with AlCl3 in ethanol. The possibility that nontoxins will behave like toxins and give false-positive results can be reduced by running toxin standards in tandem with samples, but this can be completely ruled out only by positive identification of the toxin. This is most reliably done by high resolution mass spectroscopy (MS), but in many cases, relatively simple chemical tests will suffice. For example, after one-dimensional TLC, sterigmatocystin can be reacted with trifluoroacetic acid (TFA) to form a product with an R F value lower than that of sterigmatocystin itself. This compound can then be detected by running the plate again in the second dimension, using the same solvent. Interfering substances that do not react with the TFA will finish up on the diagonal of the plate, since they will move the same distance each time. The sterigmatocystin-TFA derivative will move a smaller distance on the second run and will thus stand out. It can also be checked against a similarly treated sterigmatocystin standard.

Detoxification It seems that we will always have to deal with mycotoxin-contaminated food and feed. Are there ways of removing or destroying mycotoxins? Aflatoxin has been the subject of most detoxification research. Although this toxin is relatively heat stable, heating a contaminated substrate to 100°C for two hours can degrade 80% of the aflatoxin present. Dry roasting nuts has a similar effect. But heat treatment won’t eliminate aflatoxin. Chemical treatment can give more complete detoxification. Aflatoxins are degraded by aqueous solutions of strong acids and bases, so crude edible oils are now treated with NaOH solution to remove aflatoxin. Ammonia treatment will almost eliminate aflatoxin from peanut meal and grains but may reduce their food value. Oxidizing agents

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Mycotoxins in Food and Feed will also destroy aflatoxins, for example, NaOCl and hydrogen peroxide hold some promise. Bisulphite, already accepted as an antimicrobial additive in fruit juices and dried fruits, degrades aflatoxins and may become widely used for this purpose if current research shows that it doesn’t damage other food constituents. Although more mycotoxins are being discovered and characterized each year, it seems unlikely that we will ever again have to resort to pilgrimages to give us a vital respite from a mycotoxin-contaminated diet. We should remember, however, that many people in the underdeveloped countries are still at risk, as are wild and domesticated animals. Mycotoxins may play no direct role in the metabolism of the fungi that produce them, but in a situation in which a fungus and an animal are competing for a grain of corn, mycotoxins may have evolved as powerful weapons that help the fungi to deter or destroy the competition. On a fact sheet by Charmley and Trenholm (2015) it says, It has been estimated that at least 25% of the grain produced each year worldwide is contaminated with mycotoxins. In temperate climates such as Canada, the mycotoxins of major concern are the trichothecenes (including deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin), zearalenone (ZEN), the fumonisins (FB) predominantly fumonisin B1 (FB1), the ochratoxins, predominantly ochratoxin A (OA), and ergot alkaloids. However, aflatoxins (AF) are of concern in food and feedstuffs imported from warmer tropical and subtropical regions. Canada’s indigenous mycotoxins occur mainly in cereal grains and corn, although occasionally there have been reports of contamination of other crops such as alfalfa and oilseeds, and foods such as coffee, cocoa, rice, beer and wine. As analytical techniques evolve to become more sensitive and widely available, the documentation of widespread contamination in a variety of commodities and of new mycotoxins no doubt, will increase. Tables 21.1 and 21.2 show what is happening in the real world.

Table 21.1. Legislated maximum tolerated levels of aflatoxins and regulatory guidelines of other mycotoxins in some foodstuffs, feedstuffs, and dairy products Mycotoxins

Commodity

Canada

Commodity

U.S.

Deoxynivalenol (Vomitoxin) (mg/kg)

Uncleaned soft wheat for human consumption

Finished wheat products

Deoxynivalenol (Vomitoxin) (mg/kg)

Diets for cattle and poultry

Grains and grain byproducts destined for ruminating beef and feedlot cattle older than 4 months and chickens (not exceeding 50% of the cattle or chicken total diet)

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Table 21.1 (continued) Mycotoxins

Commodity

Canada

Commodity

U.S.

Deoxynivalenol (Vomitoxin) (mg/kg)

Diets for swine, young calves, and lactating dairy animals

Grains and grain byproducts (not exceeding 40% of the diet)

Deoxynivalenol (Vomitoxin) (mg/kg)

Grains and grain byproducts destined for swine (not exceeding 20% of the diet)

HT-2 toxin mg/ kg (ppm)

Diets for cattle and poultry

0.1

HT-2 toxin mg/ kg (ppm)

Diets for dairy animals

0.025

Aflatoxins µg/ kg (ppb)

Nut products for human consumption

All foods

Aflatoxins µg/ kg (ppb)

Animal feedstuffs

Dairy products (AFM1)

0.5

Aflatoxins µg/ kg (ppb)

Feedstuff ingredients

Aflatoxins µg/ kg (ppb)

Cottonseed meal intended for beef cattle, swine, or mature poultry (regardless of age or breeding status)

300

Aflatoxins µg/ kg (ppb)

Corn and peanut products intended for breeding beef cattle, swine, or mature poultry

100

Aflatoxins µg/ kg (ppb)

Corn and peanut products intended for finishing swine of 100 lbs or more

200

Aflatoxins µg/ kg (ppb)

Corn and peanut products intended for finishing beef cattle

300

Source. H. O. van Egmond and W. H. Dekker, Worldwide Regulations for Mycotoxins in 1995—A Compendium. FAO Food and Nutrition Paper 64 (Rome, Italy: FAO, 1997).

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Table 21.2. Recommended tolerance levels (mg/kg) of some mycotoxins in Canada and the United States Mycotoxin

Canada: Recommended Tolerance Levels

U.S. Guidelines

Diacetoxyscirpenol (DAS)

Swine feed < 2 Poultry feed < 1

T-2 toxin

Swine and poultry feed < 1

Zearalenone (ZEN)

Gilt diets < 1–3 Cow diets 10 (1.5 if other toxins present) Swine industry has voiced concern over levels of 0.25– 5.00 in diets for sheep and pigs

Ochratoxin A (OA)

Swine diets (kidney damage) 0.2 Swine diets (reduced weight gain) 2 Poultry diets 2

Ergot

Maximum alkaloid content in feed of Cattle, sheep, horses 2–3 Swine 4–6 Chicks 6–9

Fumonisin

Animal Feeds Total ration in feed for horses and rabbits, 1 Total ration for pigs, 10 Total ration for cattle, sheep, and goats more than 3 months old, 30 Total ration for ruminant and poultry breeding stock, 15 Total ration for poultry fed for slaughter, 50 Human Foods Degermed dry-milled corn products, 2 Dry-milled corn bran, 4 Cleaned corn, for masa, 4

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Further Reading Charmley, L. L., and D. B. Prelusky. 1994. “Decontamination of Fusarium Mycotoxins.” In Mycotoxins in Grain. Compounds Other Than Aflatoxin, edited by J. D. Miller and H. L. Trenholm, 421–35. St. Paul, MN: Eagan Press. Charmley, L. L., and H. L. Trenholm. 2015. A Review of Current Literature on Mycotoxins and Their Regulations. [Unpublished review for Canadian Food Inspection Agency, Government of Canada.] Charmley, L. L., H. L. Trenholm, and D. B. Prelusky. 1995. “Mycotoxins: Their Origin, Impact and Importance: Insights into Common Methods of Control and Elimination.” In Biotechnology in the Feed Industry: Proceedings of Alltech’s Eleventh Annual Symposium, edited by T. P. Lyons and K. A. Jacques, 41–63. Thrumpton: Nottingham University Press. Christensen, C. M. 1975. Molds, Mushrooms and Mycotoxins. Minneapolis: University of Minnesota Press. Clay, K. 1988. “Fungal Endophytes of Grasses: A Defensive Mutualism between Plants and Fungi.” Ecology 69:10–16. Egmond, H. P. van. 1988. “Mycotoxins, Sampling and Chemical Detection.” In Introduction to Food-borne Fungi, edited by R. A. Samson and E. S. van Reenen-Hoekstra, 250–61. 3rd ed. Baarn: Centraalbureau voor Schimmelcultures. Hocking, A. D., J. I. Pitt, R. A. Samson, and U. Thrane, eds. 2005. Advances in Food Mycology. Berlin: Heidelberg Springer. Howell, M. V. 1982. “The Detection and Determination of Mycotoxins in Food and Feedingstuffs.” Journal of the Science of Food and Agriculture 33:590–91. Keller, N. P., G. Turner, and J. W. Bennett. 2005. “Fungal Secondary Metabolism—from Biochemistry to Genomics.” Nature Reviews Microbiology 3, no. 12:937–47. Krogh, P., ed. 1988. Mycotoxins in Food. New York: Academic Press. Marasas, W. O., and P. E. Nelson. 1987. Mycotoxicology. University Park: Pennsylvania State University Press. Mirocha, C. J., R. A. Pawlosky, K. Chatterjee, S. Watson, and W. Hayes. 1983. “Analysis for Fusarium Toxins in Various Samples Implicated in Biological Warfare in Southeast Asia.” Journal of the Association of Official Analytical Chemists 66:1485–99. Pringle, P. 1985. “Political Science.” The Atlantic Monthly 256, no. 4:67–81. Purchase, I. F. H., ed. 1974. Mycotoxins. Amsterdam: Elsevier. Richard, J. L. 2007. “Some Major Mycotoxins and Their Mycotoxicoses—an Overview.” International Journal of Food Microbiology 119, nos. 1–2:3–10. Rodicks, J. V., C. W. Hesseltine, and M. A. Mehlman. 1977. Mycotoxins in Human and Animal Health. Park Forest South, IL: Pathotox Publishers. Samson, R. A., E. S. Hoekstra, and J. C. Frisvad. 2001. Introduction to Food-borne Fungi. 6th ed. Centraal Bureau voor Schimmelcultures, American Society of Microbiology. Scott, P. M., H. L. Trenholm, and M. D. Sutton, eds. 1985. Mycotoxins: A Canadian Perspective. Ottawa: National Research Council of Canada. Shephard, G. S. 2008. “Determination of Mycotoxins in Human Foods.” Chemical Society Reviews 37, no. 11:2468–77.

Mycotoxins in Food and Feed Stoloff, L., S. Nesheim, L. Yin, J. V. Rodricks, M. Stack, and A. D. Campbell. 1971. “A MultiMycotoxin Detection Method for Aflatoxins, Ochratoxins, Zearalenone, Sterigmatocystin and Patulin.” Journal of the Association of Official Analytical Chemists 54:91–97. Trenholm, H. L., D. W. Friend, R. M. G. Hamilton, and B. K. Thompson. 1982. Vomitoxin and Zearalenone in Animal Feeds. Ottawa: Agriculture Canada. [publication 1745E] Trenholm, H. L., D. B. Prelusky, J. C. Young, and J. D. Miller. 1988. Reducing Mycotoxins in Animal Feeds. Ottawa: Agriculture Canada. [Publication 1827E.] Tucker, T. B. 2001. “The Yellow Rain Controversy: Lessons for Arms Control Compliance.” The Nonproliferation Review. Spring 2001: 25–42. Wylie, T. D., and L. G. Morehouse, eds. 1977–1978. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. Vols. 1–3. New York: Marcel Dekker.

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22 Poisonous and Hallucinogenic Mushrooms Introduction A man is brought to the emergency department of a hospital suffering from diarrhea, abdominal cramps, nausea, and vomiting. His problem is diagnosed as gastroenteritis. He is given atropine, Donnatal (phenobarbital, hyoscyamine, atropine, and scopolamine), and intravenous fluids to combat dehydration and then sent home. The vomiting and diarrhea go on for another 24 hours. By now he is severely dehydrated and has to be admitted to the hospital. Over the next two days, his liver, kidney, and heart begin to fail. Despite treatment of his symptoms with a battery of antibiotics, corticosteroids, vitamins, stimulants, and intravenous fluid, he dies. This is a true story. The only thing I didn’t tell you was that the man had eaten a meal of wild mushrooms about twelve hours before the onset of his symptoms. By the time you have read this chapter, you should be able to diagnose his illness correctly and suggest treatments that might have saved his life. People can conveniently be divided into two groups: those who love to eat wild mushrooms and those who would never dream of doing such a thing. There doesn’t seem to be any middle ground on this issue: you are either a ‘picker’ or a ‘kicker’. This characteristic seems to be culturally determined. Most people of Anglo-Saxon origin are kickers, while those from Central and Eastern Europe are pickers. Picking is a pastime that occasionally gets them into trouble. There are more than 10,000 different species of fleshy fungi. The vast majority are perfectly innocuous. A relatively small number are hunted for their delicious flavour, and a cooperative few have been domesticated (see chapter 18). But another few are deadly poisonous, and many others can cause more or less serious discomfort if they are unwittingly eaten. During the course of human history, I would suspect that all 10,000+ species of agarics have been eaten. This chapter considers what we have learned from the trying, tragic, or transcendental experiences of those who made random or unconventional choices of mushroom for their free meal. The main problem is one of identification. There is no simple rule or test that will tell whether a mushroom is edible or deadly poisonous. Many people are blissfully unaware of this and rely on tests which are irrelevant and fallacious. They are playing Russian roulette. You should eat wild mushrooms only if you know, or can precisely determine, their scientific names. If you are sure, from observation (some of it through the microscope) or experience, that all of a particular collection of fungi belongs to, say, Cantharellus cibarius (the chanterelle) or Morchella esculenta (the morel), and that the fruit bodies are young and freshly picked, experience tells us that you can eat and 392

Poisonous and Hallucinogenic Mushrooms enjoy them, as mycophagists have done for thousands of years (although some people are allergic to mushrooms). Every year, many people take unnecessary chances by eating unfamiliar mushrooms or confuse poisonous species with edible ones, and every year some unfortunates are fatally poisoned. Since most North Americans are kickers, they tend not to become mushroom poisoning statistics. Europeans, however, are pickers and have suffered as many as 100 fatalities in two weeks. In 1975, a Swiss newspaper reported fifty-four local deaths during a short period in late summer. Which fungi killed these people? What are the toxins involved? We recognize twelve different types of mushroom poisoning, which are listed in Table 22.1. A quick look at this table will show that fatalities are usually caused only by groups I, II, and III. In fact, 50% of all serious mushroom poisonings and 95% of all fatalities are caused by a few members of a single genus, Amanita, which fruits in late summer and fall.

Table 22.1. Mushroom Toxins and Their Occurrence Toxins

Fungi

I. Amanitins (cyclopeptides)

Amanita bisporigera, A. ocreata, A. phalloides, A. verna, A. virosa, etc. Galerina autumnalis, G. marginata, etc. Lepiota spp. Conocybe filaris

II. Gyromitrin, monomethylhydrazine

Gyromitra brunnea, G. caroliniana, G. esculenta, G. fastigiata, G. infula (?) Helvella elastica, (?) H. lacunosa (?) Paxina spp. Sarcosphaera crassa

III. Orellanine

Cortinarius orellanus, C. orellanoides, C. rainierensis

IV. Coprine

Coprinopsis atramentaria, (?) Coprinus spp. cl*tocybe clavipes

V. Muscarine

cl*tocybe cerussata, C. dealbata, C. rivulosa, C. sudorifica Inocybe geophylla, I. lilacina, I. pudica

VI. Ibotenic acid, muscimol

Amanita co*keri, A. cothurnata, A. gemmata, A. muscaria, A. pantherina (?) Panaeolus campanulatus

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Table 22.1 (continued) Toxins

Fungi

VII. Psilocybin, psilocin

Conocybe cyanopus Gymnopilus spectabilis, (?) Gymnopilus spp. Panaeolus foenisecii, P. subbalteatus Psilocybe cubensis, P. cyanescens, P. semilanceata, P. silvatica

VIII. Gastrointestinal irritants

Agaricus hondensis, A. placomyces Amanita brunnescens Boletus luridus, other blueing Boletus spp. Chlorophyllum molybdites Entoloma grande, E. lividum, E. sinuatum, E. strictius Hebeloma crustuliniforme Lactarius piperatus, L. rufus, L. uvidus, L. vellereus Marasmius urens Naematoloma fasciculare Omphalotus olearius Pholiota squarrosa Russula emetica Scleroderma aurantium Tricholoma pardinum, etc.

IX. Cycloprop-2-ene carboxylic acid

Tricholoma equestre Russula subnigricans

X. Unidentified

Pleurocybella porrigens

XI. Unidentified

Paxillus involutus

XII. Unidentified

Amanita smithiana

Group I: Amanitin Poisoning As a result of many inadvertent, and often fatal, experiments made by hapless or foolhardy volunteers, we can say that the basidiomata of several species of the agaric genera Amanita (Fig. 22.1), Galerina (Fig. 22.2), and Conocybe (Fig. 22.3) contain toxins that are lethal to humans in extremely small doses. The same toxins have also been found in species of Lepiota, Omphalotus, and cl*tocybe. Deadly poisonous species include Amanita abrupta, A. arocheae, A. bisporigera (eastern North American destroying angel), Amanita exitialis (Guangzhou destroying angel), A. magnivelaris, A. ocreata (western North American destroying angel), A. phalloides

Poisonous and Hallucinogenic Mushrooms (death cap), A. subjunquillea (East Asian death cap), A. verna (fool’s mushroom), and A. virosa (European destroying angel)—notorious killers all. They contain such high levels of toxin that a single bite can be fatal to a debilitated individual. The same toxin has also been found in at least seven members of the Cortinariaceae. The toxin molecules are made up of amino acids in a double ring and so are called cyclic oligopeptides or cyclopeptides. They come in two varieties, Fig. 22.1 Amanita phalloides. known as amatoxins (amanitins), which contain eight amino acid molecules, and phallotoxins (phalloidins), which contain seven amino acid molecules. When injected into mice, the phallotoxins are ten times more lethal than cyanide: their LD50 is 2 mg/kg. But when taken by mouth, they have no effect. They may be neutralized or broken down by digestive juices or may not be absorbed by the gut. In contrast, the much more deadly amatoxins are actively toxic when eaten (LD50 = 0.1 mg/kg). Amatoxins rapidly damage intestine, kidney, and liver. Alpha-amanitin attacks the nucleus of cells, binding to RNA polymerase II, the enzyme that transcribes DNA and produces messenger RNA. The synthesis of RNA ceases, and so, as a direct consequence, does the synthesis of protein. This ultimately brings the machinery of the cell to a standstill, and it dies. Cells of the intestinal lining, liver, and kidney have a rapid turnover, so their loss and nonreplacement will soon have serious effects on the organism. This is bad news, but there is worse to come. We still have no specific antidote to these toxins. And worst of all, the outward symptoms of amatoxin poisoning do not begin until after a great deal of the cell damage has been done. This makes it one of the most difficult forms of poisoning to treat. The most poisonous mushroom in eastern North America is probably Amanita virosa. The large basidiomata of this innocent-looking species are pure white throughout and have both ring and volva. In the west, the situation is complicated by the presence of Amanita phalloides, with a greenish cap, which is probably just as toxic as A. virosa. Those who have eaten these species report that they have a mild flavour. Subsequent events may be divided into four stages. (1) A latent period of six to twenty-four hours, most commonly about twelve hours. This asymptomatic interlude is long enough that the patient frequently does not even connect the subsequent illness with mushrooms. During this hiatus, the amanitin is attacking the cells of the liver, kidney, and intestine. (2) Violent vomiting, diarrhea, and abdominal pain, which last for a day or so. (3) A brief, misleading remission of symptoms. (4) Collapse of kidney and liver function, with secondary effects on the heart and brain, leading to coma and death. What can be done for victims of amanitin poisoning? The biggest problem is the long delay in the appearance of overt symptoms. By the time the patient seeks medical aid, massive cell damage may already have been done. The first hurdle is to arrive at a

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Fig. 22.2 Galerina marginata.

correct diagnosis. Amanita poisoning is relatively rare in North America, and many doctors may not think of it unless the patient mentions mushrooms. Even then, most physicians (and this is no discredit to them) know virtually nothing about diagnosing mushrooms. No identifiable specimens may be available. With luck, the local mycologist will be called in and will recognize the danger inherent in the delayed symptoms. Even if the problem is correctly diagnosed, there is currently no antidote for amanitin. Treatment consists of attempts to (1) remove the toxin from the system; (2) increase the rate at which the patient excretes it; and (3) support the patient’s various systems. One important recent development is the availability of Legalon SIL (silybinin— extract of milk thistle, given intravenously). This new treatment was introduced by Madeus Inc. and is made available free of charge if physicians call a 24-hour hotline 866-520-4412 when confronted with an amatoxin poisoning. (1) Removal of toxin. If the condition is diagnosed within an hour or two, it is obviously appropriate to empty the stomach by emesis and gastric lavage (getting the patient to throw up and then washing out the stomach). If the usual latent period has elapsed, this approach would be pointless. Three blood-cleansing techniques have been applied to late-diagnosed amanitin poisoning. (a) Haemodialysis (circulating the blood through a semipermeable membrane bathed in an isotonic medium) is used in long-term treatment of kidney failure or until a transplant becomes available. Its use in Amanita poisoning is questionable, since it removes substances of molecular weight 300 or less. Amanitin itself has a molecular weight of 900, and it may often become complexed with much larger molecules, such as proteins. Nevertheless, in serious cases, where kidney failure threatens, haemodialysis may be useful under heading 3. (b) Haemoperfusion (circulating the blood over activated charcoal) is used to support the detoxifying function of ailing livers. It has been used experimentally to treat Amanita poisoning in recent years and has been shown to remove some toxin from amanitin-spiked blood. Unfortunately, the amounts of amanitin detected in the blood of poisoning victims are usually very low, especially if more than twelve hours have elapsed since the

Poisonous and Hallucinogenic Mushrooms mushrooms were eaten. Keeping in mind the possible unfavourable effect of haemoperfusion on a blood coagulatory pattern already unbalanced by the effects of the toxin, this technique should be applied with caution. It may, of course, be indicated if the effects of the toxin cause liver failure. (c) Apheresis (centrifuging the blood to segregate its major components—plasma, erythrocytes, leucocytes, platelets—then discarding and replacing the fraction containing the unwanted agent) is being increasingly used to treat many diseases of the immune system, and some types of poisoning. Again, the actual kinetics of amanitin in the body are not yet well enough understood for us to be sure which blood fraction, if any, should be replaced. This technique may prove to be of value when the movements of amanitin in the body are better understood. (2) Increase excretion of toxin. Although the body’s natural excretion of amanitin is obviously not efficient enough to prevent cell damage, a new and sensitive radioimmunoassay for amanitin in body fluids has shown that the toxin is present in the urine of patients at far higher levels than can be found in their blood. This suggests that attempts to increase urine production by giving intravenous fluids, and an appropriate diuretic, might be helpful. (3) Supportive measures involve careful monitoring of electrolyte and fluid balance and blood sugar, with appropriate replacement therapy as required. Liver and kidney functions must be closely followed. If kidney and/or liver failure occurs, haemodialysis and/or haemoperfusion may be necessary. In addition, various researchers have suggested several other strategies to support the damaged liver or kidneys: intravenous infusion of B vitamins, vitamin K, penicillin-G, corticosteroids, and thioctic acid (a coenzyme in the Krebs cycle). The therapeutic value of some of these agents has not been firmly established, but in a potentially fatal condition, the shotgun approach is worth trying. The Bastien Treatment. A French physician, Dr. P. Bastien, has developed a treatment for Amanita poisoning. It has three parts: (1) intravenous injections of 1 gram vitamin C twice a day; (2) two capsules of nifuroxazide three times a day; (3) two tablets of dihydrostreptomycin three times a day. The treatment is supplemented by measures to control fluid and electrolyte balance and by penicillin. Bastien successfully treated fifteen cases of A. phalloides poisoning between 1957 and 1969. In 1974 he ate 65 grams of A. phalloides and survived. In 1981 he ate 70 grams of A. phalloides and again successfully treated himself. It is reported that the Bastien treatment is now used throughout France, where it saves the lives of all those whose treatment has not been delayed until massive liver and kidney damage has occurred. This method should obviously be widely publicized and tested in North America. As a fascinating biological footnote, I must mention that several mycophagous species of Drosophila (the fruit fly genus) eat poisonous amanitas with impunity. It has been demonstrated that they can survive concentrations of amanitin hundreds of times greater than can Fig. 22.3 Conocybe filaris. their fruit-feeding relatives. They are, in fact,

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Chapter 22 the most amanitin-tolerant species known. Although we do not know exactly how they deal with the toxin, we now know two things which may have driven the evolution of this tolerance. First, although the agarics in which mycophagous drosophilas breed are also sought out by crane flies and wood gnats, much larger insects, those competitors cannot survive in mushrooms that contain amanitin. Second, Drosophila larvae in amanitin-containing mushrooms are never parasitized by the nematode Howardula. This is an important selective advantage, because parasitized adults, which can represent up to 35% of the population, are often sterile or have reduced breeding success. There is an old saying: ‘It’s an ill wind that blows nobody any good’. It certainly seems that the presence of amanitin in some agarics is a real boon to Drosophila, if not to people.

Group II: Monomethylhydrazine Poisoning Morels, various species of Morchella, are among the finest edible fungi. A meal of morels, steak, and good red wine is a truly memorable occasion. So the compound ascomata of this species are avidly hunted in spring. Fruiting at the same time, and looking similar to the untutored eye, is the false morel, Gyromitra esculenta (Fig. 22.4). This species is responsible for 2%–4% of all fatal mushroom poisonings. Over a tenyear period in Poland, 100 people were hospitalized and six died as a result of eating Gyromitra. The toxin precursor in G. esculenta is called gyromitrin. When this is hydrolyzed, it becomes monomethylhydrazine (MMH), which is used as a rocket fuel and is, as researchers for the space program realized, extremely toxic. All species of Gyromitra are poisonous. Reports that those who ate the mushrooms were unaffected while the cook became ill were often discounted. But there is a rational explanation of these seemingly bizarre stories. The monomethylhydrazine has a boiling point of 87.5°C, and its vapours are toxic. Certain cases in which some diners were unaffected while others became very ill were also difficult to explain until it was shown that there is a narrow margin between no effect and a lethal dose—an ‘all-or-nothing’ response. The same individual could eat Gyromitra several times without apparent effect and then on one occasion unwittingly exceed the limit and be poisoned. Symptoms of poisoning appear two to twelve hours (typically six to eight hours) after the meal. An Fig. 22.4 Gyromitra esculenta. initial bloated feeling is followed by nausea, vomiting, diarrhea, and abdominal cramps. Victims often experience faintness, loss of muscular control, and fever. In severe cases, jaundice and convulsions occur, and coma and death may ensue after two to seven days. The delay in the onset of symptoms gives a clue to the action of the toxin. As with the cyclopeptides, it is at the cellular level. It is haemolytic, toxic to the central nervous

Poisonous and Hallucinogenic Mushrooms system, irritates the gastrointestinal tract, and damages the liver. Methaemoglobin and free haemoglobin are present in the blood. Levels of bilirubin and liver enzymes rise, and blood sugar falls. Unless the toxic nature of the mushroom is diagnosed almost immediately after it has been eaten, there is little point in evacuating the gut. Pyridoxine hydrochloride should be administered as a specific physiological antagonist of MMH. The patient’s blood sugar, liver and kidney function, and free haemoglobin level should be monitored. Intravenous glucose, forced diuresis if free haemoglobin rises, haemodialysis in severe cases, and other supportive measures may be needed.

Group III: Orellanine Poisoning In 1957 a report emerged from Poland of three outbreaks of mushroom poisoning caused by eating Cortinarius orellanus (Fig. 22.5). Among 132 people poisoned, 19 died. Death was caused by kidney failure, and in most cases occurred two to three weeks after symptoms began (although some children died within a few days, and other deaths were delayed for months). The most unusual feature of this poisoning was the extremely delayed onset of symptoms. Even in severe and ultimately fatal cases, no symptoms were reported until three to four days after the mushrooms had been eaten. In milder cases, the latent period was lonFig. 22.5 Cortinarius orellanus. ger, extending to ten to seventeen days. Initial symptoms were an intense thirst, accompanied by burning and dryness of the mouth. Headache, chills, loin or abdominal pain, nausea, and vomiting followed. Although urination was initially stimulated, it was soon reduced and in some cases ceased altogether. In serious cases, the BUN (blood urea nitrogen) rose, as might be expected following kidney damage. Once this form of poisoning was recognized and described, it was reported from France, Germany, Switzerland, and Czechoslovakia. No confirmed cases have been reported in North America. The nephrotoxin involved is called orellanine and has been detected in another European species, Cortinarius orellanoides, as well as in the North American species Cortinarius rainierensis. Orellanine has an oral LD50 (cat) of 4.9 mg/kg. It is estimated that 100–200 g of fresh C. orellanus contains enough orellanine to cause complete kidney failure. Although there do not appear to be any reports of kidney transplants in the literature, that procedure would now seem to be an appropriate response to total renal shutdown. Orellanine has been found in at least thirtyfour species of Cortinariaceae.

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Group IV: Coprine (Antabuse-like) Poisoning This type of poisoning can occur at any time for up to five days after Coprinopsis atramentaria (Fig. 22.6) has been eaten. Strangely enough, the mushrooms by themselves are not toxic. Symptoms appear thirty to sixty minutes after the mushroom eater has a drink containing alcohol; they include hot flushes of the face and neck, a metallic taste in the mouth, tingling sensations in the limbs, numbness in the hands, palpitations, a throbbing headache, nausea, and vomiting. This is an unpleasant combination, but it isn’t fatal. The only treatment necessary may be to control arrhythmia (irregular heartbeat). The syndrome will persist as long as there is any alcohol in the system—usually two to four hours— after which recovery is spontaneous, and the victim may well swear off booze. The condition arises because Coprinopsis atramentaria contains coprine, a Fig. 22.6 Coprinopsis atramentaria. unique amino acid that blocks the metabolism of ethyl alcohol at the acetaldehyde stage. Coprine poisoning is really acetaldehyde poisoning. The rather excessive duration of this potential booby trap for drinkers is due to the persistence of coprine in the body. Antabuse (disulfiram), which is prescribed to help alcoholics stay on the wagon, has an action almost identical to that of coprine, although the two substances are chemically different.

Group V: Muscarine Poisoning—PSL Syndrome It is a good idea to avoid eating little white or brown mushrooms that grow in the grass, since some members of two common genera, cl*tocybe (Fig. 22.7) and Inocybe (Fig.22.8), contain significant amounts of muscarine. This is a toxin that, within thirty

Fig. 22.7

cl*tocybe dealbata.

Fig. 22.8

Inocybe geophylla.

Poisonous and Hallucinogenic Mushrooms minutes to two hours, stimulates the exocrine glands—the producers of sweat, saliva, and tears—in what is called the ‘PSL’ syndrome (perspiration, salivation, lachrymation [sweat, drool, and tears]). It also causes constriction of the pupils, blurred vision, muscle spasms, diarrhea, slow heartbeat, and a drop in blood pressure. The only real danger here is that the heart may actually stop, if enough toxin has been absorbed. This has happened only rarely and only in patients with existing cardiovascular disease. The appropriate treatment is carefully administered intravenous atropine.

Group VI: Ibotenic Acid-Muscimol Poisoning Earlier in this chapter, I described what happens when someone eats the deadly species of Amanita, those containing amatoxins. But other species of Amanita produce very different effects. They include Amanita gemmata, A. pantherina, and A. muscaria; this last one is the famous scarlet-capped, white-spotted mushroom so beloved of fairy tale illustrators, shown in Fig. 22.9. They induce muscle spasms, dizziness (and vomiting, if too many mushrooms have been eaten), and then a deep sleep full of fantastic dreams, lasting about two hours. On waking, the subject usually experiences a ‘good trip’: a feeling of elation that persists for several hours. People often become hyperactive, making compulsive and uncoordinated movements, perhaps talking nonstop, and having altered perceptions of reality. Occasionally the experience is a ‘downer’. Clearly, these amanitas contain a subFig. 22.9 Amanita muscaria. stance that specifically affects the central nervous system. Needless to say, this was discovered long ago and has been exploited by various peoples. The Soma hymns of the 3,000-year-old sacred Indian book, the Rig Veda, have been interpreted as a glorification of A.muscaria and its effects. Many tribes in Siberia used it for centuries as a religious or recreational intoxicant, and although it has now largely been replaced by vodka, some Siberians still prefer mushrooms. Historical accounts suggest that the active principle is not destroyed in the body but is excreted unaltered in the urine. Probably by watching their reindeer, which have a fondness for urine, the Siberians learned that the inebriant could be recycled. When mushrooms were in short supply, and only the richer tribesmen could afford them, the poor folk waited for the guests to relieve themselves and then drank the intoxicating liquid. Clearly, the motivation was very strong. Although fresh mushrooms contain ibotenic acid, which has some effect on the nervous system, dried mushrooms have been found to be much more potent. This is

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Chapter 22 because ibotenic acid degrades to muscimol on drying. Muscimol is five to ten times more psychoactive than ibotenic acid. Dried mushrooms retain their potency for five to ten years. Although very few deaths have been reported from this type of poisoning, ten or more mushrooms can constitute a fatal dose. In most cases, the best treatment is no treatment. Recovery is spontaneous and complete within twenty-four hours. If many mushrooms have been eaten, severe convulsions may have to be controlled, and the stomach should be emptied. On no account should atropine be given: it will exacerbate the symptoms.

Group VII: Psilocybin-Psilocin ‘Poisoning’ Teonanacatl—‘the flesh of the gods’. This is how the Aztecs of Mexico described the sacred mushrooms which have been used for thousands of years in Central America for religious rites of divining and curing. The practice was suppressed by the ‘Christian’ Spanish conquistadors, and the secret of teonanacatl was lost to the outside world until the twentieth century. The story of its rediscovery is a mycological classic. After penetrating the wall of silence with which the people of Oaxaca protected their shamanic ceremonies, two Americans were eventually allowed to participate and to partake of the sacred mushrooms. After eating the mushrooms, they waited with their hosts in the dark. The two visitors vomited. Again they waited. Nothing happened. Then one whispered to the other, ‘I’m seeing things’. ‘That’s all right’, said the other, ‘so am I’. One of them (R. Gordon Wasson) later described the visions as being in colour . . . kaleidoscopic... they were angular and they would go fast or slow, responding to my wishes... I had delightful feelings... euphoria, peaceful feelings. The effects lasted about 4 1/2 hours. Then imperceptibly we all... fell asleep on the ground. After many similar experiences, the same author wrote, The sacred mushrooms of Mexico seize hold of you with irresistible power. They lead to a temporary (state)... in which your body lies, heavy as lead, on the mat, and you take notes and compare experiences with your neighbour, while your soul flies off to the ends of the world and, indeed, to other planes of existence... some seem to experience only a divine euphoria, which may translate itself into uncontrollable laughter... I experienced hallucinations... visions of palaces, gardens, seascapes, and mountains.... With the speed of thought you are translated wherever you desire to be, and you are there, a disembodied eye, poised in space, seeing, not seen, invisible, incorporeal. I have placed stress on the visual hallucinations, but all the senses are equally affected, and the human organism as a whole is lifted to a plane of intense experience. [Everyday experiences are] transformed, leaving you breathless with wonder and delight. The emotions and intellect are similarly stepped up. Your whole being is aquiver with life. Experiences like these can be triggered by members of four agaric genera: Psilocybe (Fig. 22.10), Panaeolus, Conocybe, and Gymnopilus.

Poisonous and Hallucinogenic Mushrooms

Fig. 22.10

Psilocybe cyanescens.

The Mexican rites usually employ one of several Psilocybe species, particularly P. caerulescens, P. zapotecorum, and P. mexicana. In the United States and in coastal areas of British Columbia devotees of ‘magic mushrooms’ often collect Psilocybe cyanescens (Fig. 22.10) and hallucinogenic species of Panaeolus and Conocybe. Psilocybe species are often cultivated, since spore prints of, for example, Psilocybe cubensis can be ordered by mail, and people in the Pacific Northwest avidly hunt for Psilocybe semilanceata and P. pellicula, which are the fabled ‘liberty caps’. The psychoactive principles in these agarics are indole alkaloids called psilocybin and psilocin, which are hydroxyltryptamine derivatives related to the neurotransmitter serotonin. An average effective dose of psilocybin is 4–8 mg, the amount contained in about 2 g of dried mushrooms. If larger quantities of mushrooms are eaten, the hallucinogenic effects may be rather overwhelming, but serious poisoning is unlikely unless huge numbers of mushrooms are consumed. Adults on ‘bad trips’ may become extremely anxious or even paranoid and may need considerable reassurance or, more rarely, tranquilizers. Children who eat hallucinogenic mushrooms may develop a high fever or convulsions. They should not be given aspirin. Tepid baths or wet sheets should be used. Hallucinations may be suppressed by chlorpromazine, and convulsions by diazepam.

Psilocybin for Treatment of Anxiety and Depression in Cancer Patients This is the most recent nonreligious and nonrecreational use for psilocybin. Since 2013, clinical trials of psilocybin for reducing anxiety and depression in cancer patients at Johns Hopkins and New York State University have shown great promise, as described in the Journal of Psychopharmacology (Ross et al. 2016). The studies involved eighty

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Chapter 22 patients, many of whom were given a single dose of synthetic psilocybin. Results were long lasting, with minimal side effects: 80% of patients showed clinically significant reductions in both psychological disorders for at least seven months after the study. Three years later one participant reported that he was not anxious about cancer, or about dying, anymore. There is obviously huge potential to be explored here.

Group VIII: Gastrointestinal Irritants The seven types of poisoning already described are now fairly well understood. Most of the relatively few fungi involved are clearly identified as containing specific toxins which cause well-defined sets of symptoms. In contrast, the seventh type of poisoning is caused by a grab bag of fleshy fungi belonging to many different genera. They have only one thing in common: within thirty to ninety minutes of being eaten, all cause various degrees of digestive upset. The commonest symptoms are vomiting and diarrhea, with abdominal cramps. Fortunately, the similarity to amanitin poisoning ends there. Symptoms generally clear up spontaneously in three to four hours, and complete recovery takes only a day or so. Little or nothing is known about the toxins involved, although the diversity of fungi causing these symptoms suggests that a number of different substances may eventually be implicated. Digestive disturbances can be caused by various members of the following genera: Agaricus, Amanita, Boletus, Chlorophyllum, Entoloma, Hebeloma, Lactarius, Marasmius, Naematoloma, Omphalotus, Pholiota, Russula, Scleroderma, Tricholoma, and possibly many others. Since we don’t know what chemicals are causing the problem, treatment is restricted to emptying the stomach and, in elderly or debilitated patients, monitoring for dehydration, reduced blood pressure, or impaired kidney function. Occasional fatalities have been caused by almost all of these fungi.

Group IX: Rhabdomyolysis—Poisoning by Tricholoma equestre (= T. flavovirens) and Russula subnigricans Twelve cases of delayed rhabdomyolysis (breakdown of skeletal muscles), three of them fatal, have been reported from France among people who ate several consecutive meals of Tricholoma flavovirens. It caused fatigue, weakness, and myalgia, especially in the thighs, twenty-four to seventy-two hours after the last meal. This worsened over three to four days, with dark urine, facial redness, sweating, nausea, high temperature, myocarditis, arrhythmia, and, in three cases, cardiovascular collapse. Heart muscle lesions and kidney lesions were found on autopsy. Tricholoma equestre/T. flavovirens (Fig. 22.11) has been widely regarded as edible and choice, but this evaluation has obviously changed as of 2001. Even more recently, some fatal poisonings by Russula subnigricans have been reported in Japan. Again, rhabdomyolysis is a major symptom, the muscle breakdown releasing myoglobin into the bloodstream and causing kidney failure. The toxin involved here has been identified as cycloprop-2-ene carboxylic acid. Since most people do not

Poisonous and Hallucinogenic Mushrooms

Fig. 22.11. Tricholoma equestre.

eat Russula anyway, this type of poisoning is not expected to be a major problem, at least in the Western world.

Group X: Poisoning by Pleurocybella porrigens (Angel Wings) During 2004 at least seventeen people in Japan died of acute brain damage after eating this delicate and visually attractive species (which is locally called sugihiratake; Fig. 22.12). All those who died had serious preexisting kidney disease. Research using mice has subsequently shown that the fungus does contain one or more toxins, but their nature has not yet been determined.

Fig. 22.12 Pleurocybella porrigens.

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Group XI: Poisoning by Paxillus involutus (Poison Pax) This mushroom (Fig. 22.13) is not widely eaten in North America, but the Chinese regard it as edible, and it is the third most common cause of gastrointestinal symptoms in Eastern Europe. However, the most serious problem is acute haemolytic anaemia, arising in people who have eaten the fungus many times, apparently without problems; this may make the diagnosis difficult.

Fig. 22.13. Paxillus involutus.

Group XII: Poisoning by Amanita smithiana Amanita smithiana (Fig. 22.14) has caused many serious poisonings in the Pacific Northwest of North America, mainly because people confuse it with the desirable matsutake (Tricholoma magnivelare; see chapter 18). The nature of the toxin is unknown, although it does not seem to be an amanitin. Its effects are similar to those of orellanine, but the onset is much quicker (four to eleven hours as opposed to thirty-six hours to three weeks). It causes kidney and liver failure, so it can be very dangerous.

Fig. 22.14

Amanita smithiana.

Poisonous and Hallucinogenic Mushrooms If we look back on the various types of poisoning examined in this chapter and try to analyze the mechanisms involved, we can discern four basic patterns: 1. 2. 3. 4.

toxins that cause extensive cell destruction but that produce overt symptoms only after a significant, and potentially fatal, delay (amanitin, orellanine, monomethylhydrazine) toxins that act on the autonomic nervous system, causing symptoms either as soon as they have been absorbed or whenever the appropriate substrate enters the system (muscarine, coprine) toxins that act on the central nervous system, causing symptoms as soon as they have been absorbed (muscimol, psilocybin) undetermined toxins that act on the alimentary canal, causing symptoms as soon as they have been absorbed

Perhaps it is worthwhile, after that rather alarming litany of toxic effects, to suggest again that unless you are a mushroom expert, you should either refrain from eating wild mushrooms altogether or stick to a few species whose characteristics you have learned in great detail. In addition to checking your fungi in the Audubon Society Field Guide to North American Mushrooms by Lincoff, Mushrooms Demystified by Arora, or Mushrooms of North America by Phillips, I recommend Funghi Velenosi by Azzaretti et al., which has excellent colour pictures of most of the really dangerous species. In any case, if someone you know should be unfortunate enough to be poisoned by mushrooms, you will now be in a position to offer some practical advice, even to the medical profession.

Further Reading Arora, D. 1986. Mushrooms Demystified. 2nd ed. Berkeley, CA: Ten Speed Press. Azzaretti, G., R. Galli, A. Bernini, and F. Polani. 1983. Funghi Velenosi. Milan: Edizioni La Tipotecnica. Bastien, P. 1985. J’ai dû manger des Amanites mortelles. Paris: Flammarion. Bauchet, J. M. 1983. “Treatment of Amanita phalloides Poisoning—the Bastien Method.” Bulletin of the British Mycological Society 17, no. 2:110–11. Bedry, R., I. Baudrimont, G. Deffieux, E. E. Creppy, J. P. Pomies, J. M. Ragnaud, M. Dupon, et al. 2001. “Wild Mushroom Intoxication as a Cause of Rhabdomyolysis.” New England Journal of Medicine 345:798–802. Benjamin, D. R. 1995. Mushrooms: Poisons and Panaceas—a Handbook for Naturalists, Mycologists and Physicians. New York: W. H. Freeman and Company. Beug, M. W. 2004. “An Overview of Mushroom Poisonings in North America.” Mycophile 45, no. 2:4–5. Faulstich, H., B. Kommerell, and T. Wieland. 1980. Amanita Toxins and Poisoning. BadenBaden: Gerhard Witzstrock. Hall, I., S. S. Stephenson, P. K. Buchanan, W. Yun, and A. L. J. Cole. 2003. Edible and Poisonous Mushrooms of the World. Portland: Timber Press, Inc. [372 pp.] Heim, R. 1978. Les Champignons Toxiques et Hallucinogènes. Paris: Editions Boubée.

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Chapter 22 Jaenike, J. 1987. “Of Toxic Mushrooms, Flies, and Worms.” McIlvainea 8, no. 1:32–34. Lampe, K. F. 1991. “Human Poisoning by Mushrooms of the Genus Cortinarius.” In Toxicology of Plant and Fungal Compounds, edited by R. S. Keeler and A. T. Tu, 497–521. New York: Marcel Dekker. Lincoff, G. H. 1981. The Audubon Society Field Guide to North American Mushrooms. New York: Knopf. Lincoff, G., and D. H. Mitchel. 1977. Toxic and Hallucinogenic Mushroom Poisoning. New York: Van Nostrand Reinhold. Litten, W. 1975. “The Most Poisonous Mushrooms.” Scientific American 231, no. 3:90–101. Phillips, R. 1991. Mushrooms of North America. Boston: Phillips, Little, Brown & Co. Ross, S., A. Bossis, J. Guss, G. Agin-Liebes, T. Malone, B. Cohen, S. E. Mennenga, et al. 2016. “Rapid and Sustained Symptom Reduction Following Psilocybin Treatment for Anxiety and Depression in Patients with Life-Threatening Cancer: A Randomized Controlled Trial.” Journal of Psychopharmacology 30:1165–80.

23 Medical Mycology Introduction Since fungi can make use of so many different substrates, it’s hardly surprising that some of them can and do grow on and in people. Some are little more than a nuisance, but others can cause serious and even fatal diseases, about which many in the medical profession know little. Three rather different groups of fungi actually cause specific diseases. (1) A few obligately parasitic fungi (dermatophytes) have evolved specifically to attack the outer surface of human beings. (2) A few other fungi which cause disease in people are normally soil organisms but have also adapted to life in the unusual and rather hostile environment of the human body, often responding to this environment by developing a different morphology (thermal dimorphic saprobes). (3) A third group of opportunistic saprobes can attack us only when our defences are down—when our immune systems themselves are diseased or deficient or when we artificially suppress them, as we must to prevent the rejection of transplanted organs. We can divide fungal attacks on our persons into (1) cutaneous infections, which involve the outer layers of the skin and cause an allergic or inflammatory response; (2) subcutaneous infections, usually involving fungi of low inherent virulence which have been introduced to the tissues through a wound of some type and which remain localized or spread only by direct mycelial growth; and (3) systemic infections, which are caused either by true pathogenic fungi which can establish themselves in normal hosts or by opportunistic saprobic fungi which could not infect a healthy host but can attack individuals whose immune system is not working. Both types of fungi in systemic infections sometimes become widely disseminated through the body of the host.

Cutaneous Infections Most cutaneous mycoses are caused by a specialized group of keratinolytic fungi called the dermatophytes, of which you have already learned something. There are about forty species of dermatophytic hyphomycetes placed in three genera. Epidermophyton has two species, Microsporum (Fig. 4.10C) has seventeen species, and Trichophyton has twenty-four species and varieties. Eight species of Trichophyton have teleomorphs in Arthroderma, and nine species of Microsporum have teleomorphs in Nannizzia. These

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Chapter 23 teleomorphic genera are both members of the family Arthrodermataceae (Ascomycetes, Onygenales). About half of the dermatophytes are found only on people, causing diseases commonly called tinea or, more colloquially, ringworm. These have no reservoir of infection in the soil or on animals: they can grow only on humans, although their arthric conidia can survive in carpets and upholstery for up to two years. Many of the other half are usually isolated from other mammals. Microsporum canis, despite its name, has its reservoir in cats. It may move to dogs or humans but will die out after one or two person-to-person transfers. If it is to survive, it must return to a cat for rejuvenation. About five species are recorded from both man and animals. The irritation caused by the presence of the fungus stimulates the epithelial cells of the host to divide more often than usual. This increases the amount of keratin available to the fungus and also means that more flakes of skin (dander) containing infective mycelium will be shed. Epidermophyton floccosum causes transient infections and relies on this exfoliated material for quick spread to other hosts. Trichophyton rubrum tends to cause chronic infections of the foot and toenails, so the host produces infective material over a period of years. Almost everyone is susceptible to short-term infection by Epidermophyton floccosum, but a chronic Trichophyton rubrum infection of one spouse may never be transmitted to the other. Trichophyton concentricum causes a chronic ringworm of the body in Polynesians (tinea imbricata, Tokelau) but is never transmitted to whites or blacks living in the same communities. Trichophyton rubrum can attack any part of the skin, but Microsporum audouinii and Trichophyton tonsurans are found mainly on the head (tinea capitis), and Epidermophyton floccosum usually infects the feet (tinea pedis, athlete’s foot) or the groin (tinea cruris, jock itch, crotch rot). It must be emphasized that these fungi are not growing on living tissue. Their clinical effects are due to the various irritants they produce: enzymes such as proteases, peptidases, and elastases and other metabolites. The condition is really a form of toxic dermatitis. There is no social stigma to tinea—my daughter contracted athlete’s foot repeatedly when she was a young girl; she seemed to be highly susceptible to the fungus. Fortunately, she grew out of it.Here is the reaction of Timothy Patterson: I’m growing fungus on my feet. To tell the truth, it’s kinda neat. I grew it for my science class. It’s got so big, I’m bound to pass. But it’s not easy growing mould. You must keep it dark and from the cold. Put your socks on when they’re wet, And feed your fungus lots of sweat. It’s been a month since last I showered, And because of this, it’s truly flowered. It’s amazing just how fast it grows. You’ve never seen such fuzzy toes.

Medical Mycology It has the most delightful hue. It’s sorta green and sorta blue. But there are drawbacks to its fungal riches. You won’t believe how much it itches. And the smell is gross, I have to say. But it’s worth it all to get an ‘A’

Dandruff and Seborrhoeic Dermatitis—Malassezia globosa The most common cause of dandruff is a basidiomycetous yeast called Malassezia globosa (which also causes seborrhoeic dermatitis and tinea versicolor). This fungus has a very small genome (4,285 genes—about 1/300th the size of the human genome) and cannot make its own fatty acids so must rely on those produced by other organisms. Most of us carry this fungus, which is feeding on sebum, the fatty or oily secretion produced by the sebaceous glands in our skin (and especially on the scalp). We make sebum in order to protect and waterproof our hair and skin and prevent it from drying out. The fungus produces eight different lipases and three phospholipases to digest the sebum, in the process producing oleic acid, which penetrates the skin and causes dandruff in about half of the population. Any of the eleven fungal enzymes noted above would make a good target for future antidandruff preparations. The study which sequenced the entire Malassezia genome was by Xu et al. (twentyone authors; see Further Reading) and was published in Proceedings of the National Academy of Sciences on November 20, 2007. This paper may be viewed as a pdf file at http://www.pnas.org/cgi/reprint/104/47/18730

Candidiasis The yeast Candida albicans (Fig. 6.2C) is a normal component of the gut microbiota, but excessive wetness, or very tight clothing, can trigger rapid overgrowth of skin by this fungus. It can cause diaper rash or infections around fingernails, in armpits and crotch, and under breasts. Mucous membranes are particularly susceptible to inflammation by the toxins of this fungus: oral candidiasis (thrush) is common in the newborn, arising when the normal flora of lactobacilli doesn’t develop quickly enough. Pregnant women produce vagin*l secretions with altered levels of glycogen. This encourages the growth of Candida, and vagin*l candidiasis is common in pregnant women. Their husbands sometimes contract candida balanitis, a nasty infection of the penis, although this may also be a consequence of diabetes. If cutaneous candidiasis becomes chronic, it may be a sign of various abnormalities of the thymus, of the thyroid, of white blood cells (leucocytes), etc.

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Subcutaneous Infections This category includes such diseases as chromoblastomycosis, entomophthoromycosis, mycetoma, and sporotrichosis. These are caused by fungi that are normally saprobic but that, when introduced to wounds, can adapt to growth in humans, often changing their morphology or physiology in the process. Chromoblastomycosis (verrucous dermatitis) is common throughout the tropics among people who go barefoot. The disease-causing agent is one of three soil hyphomycetes (Phialophora verrucosa, Cladosporium carrioni, or Fonsecaea pedrosoi) which is ‘inoculated’ by a thorn or a sliver. When the fungus starts to grow, the host cells respond by dividing rapidly and produce unsightly, stalked, warty growths on the feet or legs. The fungus may spread through the lymphatic system. Entomophthoromycosis is a clumsy word but tells us that the disease is caused by a member of the order Entomophthorales (Zygomycota). Basidiobolus ranarum usually lives in rotting vegetation and in the guts of amphibia and reptiles. It may be introduced to the human body by insect bites and causes the formation of a subcutaneous tumour that grows steadily and may involve a whole limb or the chest or shoulder. Mycotic mycetoma is another disease of barefoot tropical peoples. Again, the fungal agent enters the body through a wound. The fungus attacks various tissues and stimulates the formation of a tumour, within which are many compact fungal colonies called grains. If the surface of the skin eventually ruptures, some of these colonies may be extruded. They have been found to belong to fungi such as Madurella mycetomatis (Hyphomycetes), Exophiala jeanselmei (Hyphomycetes), Pseudallescheria boydii (Ascomycetes), and Leptosphaeria senegalensis (Ascomycetes). Sporotrichosis is caused by Sporothrix schenckii, a cosmopolitan hyphomycete which may be an anamorph of Ophiostoma. The fungus enters the host through a wound made, for example, by a contaminated thorn. Once inside the host, the normally mycelial fungus becomes yeast-like (it is therefore dimorphic but not related to the other dimorphic fungi discussed elsewhere in this chapter). The initial, localized infection may ulcerate, drain, and heal. But all is not well. The infection spreads through the lymphatic system, and many secondary lesions may form. Eventually, the disease may become systemic, spreading first to the joints, then the bones, and finally the internal organs, through the bloodstream.

Systemic Mycoses These diseases are of two very different types: those produced by specialized pathogens and those caused by opportunistic saprobes. There are four true pathogens, all of which are dimorphic—this means they have one type of morphology outside the host, another inside the host. Three of these diseases are extremely common in North America, and the fourth in South America. In the first three mentioned below, the causative fungi are readily isolated from soil. Although these and other mycoses occasionally have horrifying effects on the human body, I am not going to gross you out with pictures. If you really must know how bad things can occasionally get, there are many pathetic photographs in the works listed in the Further Reading, and at www.mycolog.com.

Medical Mycology

Dimorphic Pathogens (1) Histoplasmosis, commonly abbreviated to ‘histo’, is caused by the Histoplasma capsulatum anamorph of Ajellomyces capsulatus (Ascomycetes). This anamorph grows well in high-nitrogen substrates like wild bird droppings, chicken manure, and bat guano. Anyone who disturbs such deposits, or spends much time around them, is likely to become infected. Conidia of the fungus are inhaled and cause primary infections in the lungs. About 95% of all cases produce no obvious clinical symptoms and heal spontaneously, leaving the subject with only a small calcified lesion in the lung and with resistance to reinfection. In the other 5%, various clinical symptoms develop. The inhaled conidia assume a yeast-like form and become parasitic within histiocytes (phagocytic host cells). At first flu-like, the disease may go on to produce a progressive lung disease that mimics tuberculosis. If untreated, it may even develop into a generalized, systemic infection which can attack all internal organs, ultimately with fatal results. Histoplasmosis is endemic in the Mississippi and Ohio valleys of the United States, where about forty million people have had the disease (most of them without knowing it). It attacks males more commonly than females. (2) Coccidioidomycosis is a nasty tongue twister of a name, often contracted to ‘coccy’ (pronounced ‘coxy’), for the disease caused by Coccidioides immitis. This fungus thrives in dry, saline soils and is endemic in desert areas of the Southwestern United States, where the disease is often called ‘valley fever’, and Mexico (although it is strangely absent from the deserts of Africa and Asia). The process of infection, progress of the disease, and clinical symptoms are very similar to those of histoplasmosis, although the fungus is not intracellular, and it forms spherical structures containing spores. In culture, the same fungus produces chains of alternate thallic-arthric conidia and has no known teleomorph. Millions of people in the Southwest United States have contracted the disease. Fortunately, as in histoplasmosis, most cases are benign, and healing is spontaneous. A few become systemic and are usually fatal if untreated or misdiagnosed. The disseminated form of this disease is commoner among males than females and among people with darkly pigmented skin. (3) Paracoccidioidomycosis is a disease exclusive to Central and South America. It is caused by Paracoccidioides brasiliensis, which seems to occur mainly in tropical mountain forests. Inhalation of conidia causes a primary infection in the lungs. However, as with the other two diseases just discussed, although infection is common in endemic areas, serious disease is rare. When secondary infections do occur, they tend to provoke ulceration of the mucosa of mouth and nose, often causing loss of teeth. Less commonly, the pulmonary infection progresses, mimicking tuberculosis, and sometimes eventually involves other internal organs. In the host, the fungus occurs as large, multipolar budding yeast cells (see. Fig. 6.1A), but in culture it is a mycelial, Chrysosporium-like anamorph (see. Fig. 4.16). The teleomorph, if one exists, has not been discovered. (4) Blastomycosis is caused by Blastomyces dermatitidis, a fungus rarely isolated in culture from soil or other natural substrates. However, the disease is endemic to several areas, including the Eastern United States and Canada. Infection often seems to be a result of disturbing plant debris. Again, the primary infection is in the lungs, where large granulomas that contain many tiny abscesses form. These lesions may heal, but

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Chapter 23 the organism then crops up in another area, frequently the exposed parts of the face and neck. Warty, thickened patches develop, which spread widely and cause extensive scarring and destruction of tissue. Internally, bones may become involved. Eventually other organs, especially the prostate and the brain, are attacked. Blastomyces is seen as a yeast cell in host tissue but forms a Chrysosporium-like anamorph in culture, converting to a yeast-like form at 37°C. Mating of compatible strains produces the teleomorph Ajellomyces dermatitidis (Ascomycetes).

Opportunistic Pathogens Opportunistic infections are caused by diverse fungi—a few species of Aspergillus, Candida, and Cryptococcus and some members of the Mucorales. All grow well at body temperature but do not otherwise seem particularly different from closely related nonpathogenic species. None of them can usually cause an infection in a normal, healthy individual. All rely on some breakdown in the mechanisms of resistance. This type of systemic fungal infection is often a complication of diabetes, AIDS (Acquired Immune Deficiency Syndrome), or advanced cancer or is a sequel of steroid or antibiotic therapy. Candidiasis (also called candidosis). The causal organism is Candida albicans. In children, oral candidiasis that becomes chronic and spreads down the oesophagus is probably a sign of genetic defects or multiple endocrine deficiencies. In adults, alimentary candidiasis may be associated with diabetes, AIDS, steroid or antibiotic therapy, cancer, blood disease, endocrine deficiencies, or other debilitating conditions. In leukaemic patients, candidiasis may become truly systemic or may produce a form of septicaemia. Either way, it can ultimately be fatal. This condition may also be produced by repeated entry of the fungus with injections self-administered by drug addicts or as a sequel of long-term antibiotic or steroid therapy or indwelling catheters. Candida septicaemia may also arise as a result of parenteral hyperalimentation (feeding by continuous direct injection of fluids, often undertaken during treatment of severe gastrointestinal disease). More than half of patients who remain on hyperalimentation for more than twenty days develop Candida septicaemia. Fortunately, in patients with intact immune responses, the infection often clears up if the needle is removed. Zygomycosis is caused by several opportunistic members of Mucorales (Zygomycota). Rhizopus arrhizus and Rhizopus oryzae are most commonly involved, but species of Mucor, Rhizomucor, and Absidia have also been reported. Four types of systemic disease occur: rhinocerebral, thoracic, gastrointestinal, and cutaneous. Rhinocerebral zygomycosis attacks acidotic diabetics (who have high blood sugar, high ketone levels, and usually some leucocyte dysfunction). The infection begins in the sinuses and then grows with dramatic rapidity outward to the eyes and inward to the brain. The eyes bulge and may become paralyzed, the eyelids droop, and there is often some degree of facial paralysis. The disease usually progresses with devastating rapidity and is often fatal within seven days. Thoracic zygomycosis strikes people already suffering from leukaemia or lymphoma and occasionally also diabetics, transplant patients undergoing steroid therapy,

Medical Mycology or patients on dialysis. The symptoms are those of bronchitis and pneumonia, with complications like thrombosis or infarction (blockage) when the fungus invades blood vessels. This disease is also fatal if untreated. Gastrointestinal zygomycosis occurs almost always in developing countries, attacking children who are already suffering from kwashiorkor (chronic protein deficiency). The causal agent, Absidia corymbifera (Zygomycetes), invades the walls of the stomach and intestine, blocking the arteries. The resulting necrosis and perforations are fatal. Cutaneous zygomycosis occurs when zygomycetous fungi colonize burns. In a severely burned, and therefore extremely debilitated, patient, the infection may spread rapidly and be quickly fatal. Cryptococcosis is caused by an encapsulated, budding basidiomycetous yeast, Cryptococcus neoformans, the anamorph of Filobasidiella neoformans. The anamorph commonly grows on pigeon droppings, so everyone is exposed to the propagules of the fungus. Many people contract subclinical or asymptomatic cryptococcosis, which resolves spontaneously. An unfortunate minority, often already suffering from leukaemia or lymphoma or on immunosuppressive therapy following organ transplants, develop lung disease, which may then become systemic. This phase involves bones or organs such as heart, testicl*, prostate, or eye and is often fatal. A second form of the disease is cryptococcal meningitis. Patients complain of increasingly severe headaches, which eventually escalate into meningitis. Untreated cryptococcal meningitis is always fatal. A second species, Cryptococcus gattii, has also been reported as growing on Eucalyptus trees in Australia, South America, or South Africa and in other tropical and subtropical areas of the world. This species has now been recorded on and near the east coast of Vancouver Island, British Columbia, Canada, where it has been isolated from Douglas fir, Grand fir, alder, cedar, Garry oak, air, and soil and has apparently caused 129 cases, resulting in four deaths (or approximately one per year) among residents or visitors to Vancouver Island from 1999 to 2005. A number of additional cases have now been reported on the island and now on the mainland as well, extending the distribution of the fungus and bringing the total to 216 cases (8 deaths) as of February 2008 (mostly in immunocompromised, elderly, or very young people). This is a very large cluster compared to the former rate of about two cases per year in the same area and leads to the suspicion that the introduction of C. gattii is recent and that this species may even have mutated to become more pathogenic. Symptoms appear from two to nine months after exposure and include prolonged cough, sharp chest pain, shortness of breath, weight loss, night sweats, and a severe headache. If untreated, the disease may eventually involve the brain, producing a fatal meningitis. Fortunately, infected people do not transmit the disease. More than thirty-five cases have also been recorded in animals on or near Vancouver Island, including dogs, cats, llamas, a ferret, and wild porpoises. Animal symptoms range from runny noses and lumps under the skin all the way to fatal infections of the lungs or nervous system. The medical authorities point out that the human infection rate is only 3 per 100,000 population per year, which does not merit panic. But this is still three times the incidence in Australia, where the fungus is more common. The fungus is turning up in increasing numbers of air, soil, and tree samples along the east coast of Vancouver Island, and it is now suspected that climate change may be responsible for the outbreak.

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Chapter 23 The yeast may have been carried from Hawaii on the so-called Pineapple Express weather system, but this does not explain why it has not affected many people on the west coast of the island. As of 2009 it has been determined that Cryptococcus has spread to the mainland of British Columbia and subsequently to Washington State, Oregon, and California. About fifty human cases have been diagnosed in these three states, and ten people have died. Check www.mycolog.com for maps showing the distribution of cases. Fortunately the disease, if diagnosed reasonably early, can be treated with antifungal antibiotics, although the treatment may need to be prolonged. The prognosis is good for otherwise healthy people but not so good for those with other underlying illnesses. Aspergillosis. Although species of Aspergillus (Hyphomycetes) cause other health problems, such as acute and chronic aflatoxin poisoning, we are concerned here only with diseases caused by the growth of the fungus itself somewhere in the body. (1) Bronchopulmonary aspergillosis is usually caused by Aspergillus fumigatus, which colonizes mucus within the bronchi, evoking a severe allergic reaction. (2) In aspergilloma, the fungus forms a mycelial ball in a lung cavity produced by an earlier attack of tuberculosis. The wall of the cavity may erode, causing the patient to spit blood and necessitating surgical intervention. (3) Invasive aspergillosis is found only in patients who are severely debilitated or are immunosuppressed, as in AIDS. The fungus grows outward from the lung, invading blood vessels and spreading to other organs through the bloodstream. This insidious disease is usually fatal and is often diagnosed only when an autopsy is performed. Since the rise in the number of immunocompromised patients, those suffering from cancer and AIDS as well as those with transplanted organs, new groups of opportunistic fungi have been observed. Phaeohyphomycosis is the general term for such diseases produced by about forty darkly pigmented conidial fungi. Hyalohyphomycosis is a general term for opportunistic infections caused by about a dozen nonpigmented conidial fungi. Fusarium is one of the commonest among these.

AIDS and Mycoses Since the searing advent of AIDS to our societal consciousness, medical mycologists have become aware that many AIDS patients suffer from a variety of mycoses. Patients who develop certain opportunistic mycoses in the absence of any obvious predisposing factors are automatically investigated to see whether they also have AIDS. The “diagnostic” mycoses include aspergillosis, candidiasis, cryptococcosis, and zygomycosis. Oesophageal candidiasis and cryptococcosis of the central nervous system are regarded as being particularly strong indicators of AIDS. Candidiasis of mucous membranes is seen in two-thirds of AIDS patients. Cryptococcosis is found in 6%–10% of AIDS patients in North America but in one-third of patients in Zaire. The dermatophytes Trichophyton rubrum and T. interdigitale also cause more protracted and more severe infections in AIDS patients.

Medical Mycology

Treatment of Mycoses Although potassium iodide (KI) has been used successfully in the treatment of sporotrichosis since 1903, until recently there were no really effective drugs to combat most other fungal diseases. Until fairly recently, several of these diseases, such as blastomycosis, mucormycosis, and disseminated forms of histoplasmosis, coccidioidomycosis, cryptococcosis, candidiasis, and aspergillosis, were almost always fatal. Antibacterial antibiotics usually had little or no effect on fungi and might actually make things worse by knocking out the competition. Fortunately, a number of effective antifungal antibiotics are now available (although they are not without side effects). Most important until very recently were two polyene antibiotics produced by Streptomyces spp. The first, Nystatin, introduced in 1950, is an effective treatment for superficial and oesophageal candidiasis. Drs. Rachel Brown and Elizabeth Hazen, the scientists who discovered this antibiotic, philanthropically gave their profits to establish a foundation that finances research in medical mycology. The second polyene antibiotic, Amphotericin B (Fungizone), which became available in 1957, represented a major breakthrough. It is effective against most of the potentially fatal deep-seated mycoses when administered intravenously. But it is a very toxic substance with many side effects: patients often need secondary treatment to cope with the attendant nausea, phlebitis (inflammation of major blood vessels), headaches, and impairment of kidney function. This drug should be given only to patients whose condition is potentially fatal. Although Amphotericin B usually works, some resistant strains of fungi, especially Aspergillus flavus, have been encountered, and one of my colleagues has had the truly terrible experience of watching a fourteen-year-old slowly die as his brain was gradually destroyed by this fungus. Miconazole (Monistat) is also used intravenously to treat several of the major systemic fungal infections, but it, too, has unpleasant side effects, especially nausea and phlebitis. Cryptococcosis is now treated with a combination of 5-fluorocytosine and Amphotericin B. Another antibiotic, Griseofulvin, derived from Penicillium griseofulvum (Hyphomycetes), is useful in oral doses of up to 1 g per day for treating dermatophyte infections. Newer, topical treatments for athlete’s foot and ringworm are Tolnaftate (Tinactin), Canesten (Clotrimazole), haloprogin, miconazole nitrate, iodochlorhydroxyquin, thiabendazole, or glutaraldehyde. Topical and systemic treatments are often used in parallel for stubborn cases. In 1981, a new antifungal antibiotic, Ketoconazole (Nizoral), became available. But although it can cure severe cases of some systemic mycoses, it should be used only in extreme cases, because it has severe side effects: adrenal suppression in both sexes and aspermia and impotence in males. I note the use of derivatives of a new generation of fungicides found in the small cone-inhabiting mushroom genus Strobilurus as antibiotics and anticancer agents (Anke et al.1977). Table 23.1 lists most of the antifungal agents in current use or to be introduced soon. Note the different aspects of the pathogen’s metabolism at which these are aimed.

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Chapter 23

Table 23.1. Antifungal Agents Allylamine ergosterol synthesis inhibitors:

Amorolfine (topical) Butenafine (topical) Naftifine (topical) Terbinafine (topical, oral)

Azole ergosterol biosynthesis inhibitors:

Clotrimazole (topical) Econazole (topical) Miconazole (topical) Oxiconazole (topical) Sulconazole (topical) Terconazole (topical) Tioconazole (topical)

Chitin synthase inhibitors:

Nikkomycin Z (being developed) Caspofungim (intravenous)

Glucan synthase inhibitors:

Micafungin (intravenous) Anidulafungin (intravenous) Amphotericin B (topical, intravenous)

Antimetabolites:

Flucytosine (oral) Fluconazole (oral, intravenous) Itraconazole (oral, intravenous) Ketoconazole (topical, oral) Posaconazole (oral) Ravuconazole (being developed) Voriconazole (oral, intravenous)

Polyenes:

Liposomal Amphotericin B (intravenous) Amphotericin B oral suspension (oral) Topical Nystatin (topical) Pimaricin (ophthalmic)

Other systemics:

Griseofulvin (oral) Ciclopiroxolamine (topical)

The Fifth Kingdom An Introduction To Mycology by Bryce Kendrick - PDFCOFFEE.COM (2024)

References