The Fungal Spore and Disease Initiation in Plants and Animals

The Fungal Spore and Disease Initiation in Plants and Anirnals

Edited by Garry T. Gofe The University of Texas at Austin Austin, Texas

and Harvey C. Hoch Corne/1 University New York State Agricultural Experiment Station Geneva, New York

Springer Science+Business Media, LLC

Llbrary of Congress Cata1oglng-ln-Pub11catlon 01t1

The Fungal spare and ~lsease tnlttatlon ln plant& and anl•a1s I edltad by Garry T. Cola and Harvey c. Hoch.

p. Cl. Includas blbllographlcal rafarences and Index. ISBN 978-1-4899-2637-1 ISBN 978-1-4899-2635-7 (eBook) DOI 10.1007/978-1-4899-2635-7 1. Mycoses--Pathogenasls. 2. Fungel dtsaases of plants­

-Pathogenesls. 3. Fungi--Spores. I. Cola, Garry T •• 1941-II. Hoch, Harvey C.

IDNLM: 1. Spores, Fungal--pathogantclty. QH 180 F9818l QR245.F854 1991 BS2 • .4--dc20 DNLMIDLC for Ltbrary of Congress 90-14316

ISBN 978-1-4899-2637-1

© 1991 Springer Science+Business Media New York Originally published by Plenum Press, New York in 1991 Softcover reprint of the hardcover 1st edition 1991

All rights reserved

CIP

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Dedicated to our students and colleagues;

their diligence and inspiration are deeply appreciated.

Gontributars

James R. Aist • Department of Plant Pathology, Cornell University, lthaca, New York 14853

Drion G. Boucias • Department of Entomology and Nematology, University of Florida, Gaines­ville, Florida 32611~0711

William R. Bushnell • USOA-ARS Cereal Rust Laboratory, University of Minnesota, St. Paul, Minnesota 55108

A. K. Charnley • School of Biological Sciences, University of Bath, Bath, Avon BA2 7AY, England

Garry T. Gote • Department of Botany, University of Texas, Austin, Texas 78713

Pierre J. G. M. De Wit • Department of Phytopathology, Wageningen Agricultural University, 6700 EE Wageningen, The Netherlands

R. J. Dillon • School ofBiological Sciences, University ofBath, Bath, Avon BA2 7AY, England

Dennis M. Dixon • Laboratories for Mycology, Wadsworth Center for Laboratories and Re­search, New York State Department of Health, Albany, New York 12201-0509

Lynn Epstein • Department of Plant Pathology, University of California, Berkeley, California 94720

/wao Furusawa • Labaratory ofPlant Pathology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

Robert C. Garher • Biotechnology Center and Department of Plant Pathology, Ohio State University, Columbus, Ohio 43210. Present address: Springer-Verlag, New York, New York 10010

Randall E. Gold • BASF AG, Agricultural Research Station, D-6703 Limburgerhof, Federal Republic of Germany

Tadayo Hashimoto • Department of Microbiology, Loyola University Stritch School of Medi­cine, Maywood, Illinois 60153

H. C. Hoch • Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456

Matthieu H. A. J. Joosten • Department of Phytopathology, Wageningen Agricultural Univer­sity, 6700 EE Wageningen, The Netherlands

vii

viii Contributors

Michael J. Kennedy • The Upjohn Company, Kalamazoo, Michigan 49001

Theo N. Kirkland • Veterans Administration Medical Center, San Diego, California 92161

Wo{fram Köller • Department of Plant Pathology, Cornell University, New York State Agri­cultural Experiment Station, Geneva, New York 14456

Yasuyuki Kubo • Labaratory ofPlant Pathology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

Joseph Kuc • Department of Plant Pat.hology, University of Kentucky, Lexington, Kentucky 40546-0091

J. P. Latge • Mycology Unit, Pasteur Institute, 75015 Paris, France

Nageswara Rao Madamanchi • Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0331

Kurt Mendgen • Department of Plant Pathology, University of Constance, D-7750 Constance,. Federal Republic of Germany

Ralph L. Nieholsan • Department of Botany and Plant Pathology, Purduc University, West Lafayette, lndiana 47906

Anton Novaclzy • Department of Plant Pathology, University of Missouri, Columbia, Missouri 65211

Frank C. Odds • Department of Bacteriology and Mycology, Janssen Research Foundation, B-2340 Beerse, Belgium

S. Paris • Mycology Unit, Pasteur Institute, 75015 Paris, France

Jacquelyn C. Pendland • Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32611-0711

Annemarie Polak • E Hoffmann-La Roche Ltd., CH 4002 Basel, Switzerland

Donald W. Roberts • Insect Pathology Resource Center, Boyce Thompson Institute, Cornell University, Ithaca, New York 14853

Maura D. Robertson • Institute of Occupational Medicine, Edinburgh EH8 9SU, Scotland

lngrid M. J. Scholtens-Toma • Department ofPhytopathology, Wageningen Agricultural Univer­sity, 6700 EE Wageningen, The Netherlands

M. G. Smart • Northern Regional Research Center, Agricultural Research Service, U.S. Depart­ment of Agriculture, Peoria, Illinois 61604. Present address: Kraft General Foods, Inc., Glen­view, Illinois 60025

David R. Soll• Department of Biology, University of Iowa, Iowa City, Iowa 52242

R. J. St. Leger • Boyce Thompson Institute, Cornell University, Ithaca, New York 14853

R. C. Staples • Boyce Thompson Institute, Cornell University, lthaca, New York 14853

Paul J. Szaniszlo • Department of Microbiology, University of Texas, Austin, Texas 78713

Alayn R. Waldorf• Department of Biological Sciences, California State University, Hayward, California 94542

Preface

This treatise is focused on early aspects of fungal pathogenesis in plant and animal hosts. Our aim in choosing the topics and contributors was to demonstrate common approaches to studies of fungal-plant and fungal-animal interactions, particularly at the biochemical and molecular Ievels. For example, the initial events of adh«sion of fungal spores to the exposed surface tissues of the host are essential for subsequent invasion of the plant or animal and establishment of pathogenesis. A point of consensus among investigators who have directed their attention to such events in plants, insects, and vertebrates isthat spore adhesion to the host cuticle or epithelium is more than a simple binding event. lt is a complex and potentially pivotal process in fungal-plant interactions which "may involve the secretion of ftuids that prepare the infection court for the development of morphological stages of the germling" and subsequent invasion of the host (Nicholson and Epstein, Chapter 1). The attachment of the fungal propagule to the arthropod cuticle is also "mediated by the chemical components present on the outer layer of the spore wall and the epicuticle .... Initial attachment may be reinforced further by either the active secretion of adhesive materials or the modification of spore wall materiallocated at the [fungal spore­arthropod] cuticle interface (Boucias and Pendland, Chapter 5). The parallel nature of the research approaches and comparability of experimental methods used for examinations of spore attachment to and invasion of both plant and animal hosts is illustrated in the first eight chapters of this volumc.

The plant and insect cuticle, and epithelial surfaces of vertebrales, represent the principal passive barriers to fungal invasion of respective hosts. lt is intuitive that many fungi actively breach thesepassive barriers. Evidence has been presented in Chapters lO, 12, and 13 that specific hydrolytic enzymes produced by certain fungal pathogens of plants and animals are associated with cuticular/epithelial penetration. Common goals ·are underscored by the authors of these chapters, namely the isolation and characterization of these key enzymes (virulence factors) and identification of compounds that selectively inhibit the enzyme activity in vivo. lt is also evident from these and other chapters in this book that address the topic of fungal spore products and pathogenesis that future research must focus on the molecular basis of synthesis and release of substances derived from the pathogen and host which are involved in fungal penetration. Results of these exciting new approaches to old problems will contribute significantly to our understand­ing of the early mechanisms of host invasion (Odds, Chapter 13).

Although there are clear differences in the nature of host response to early fungal invasion of plants and animals, an understanding of host "defenses and how the fungus avoids or overcomes them is rudimentary" (Aist and Bushnell, Chapter 15). Parallels of oxygen free radical production

ix

X Preface

by both plant and animal defense systcms are reported in Chapters 17 and 20, respectively. Discussions of active and passive mechanisms by which fungal spores or highly specialized infection structures modüy, suppress, or resist the battery of plant and animal host defenses are presented in Chapters 15, 19, and 21.

The development of recombinant DNA technology has introduced new and potentially rewarding possibilities for investigators of fungal pathogenesis of plants and animals. Under the topic "Molecular Aspects of Disease Initiation" discusssions of fungal genes responsible for putative pathogenic factors are presentcd (Chaptcrs 22 and 23). The application of molecular probes to assess strain relatedness and explore basic questions of fungal epidemiology and pathogenesis is also examined in Chapter 23. These chapters again emphasize the similarities of experimental design which plant pathologists and medical mycologists have devised for investi­gations of molecular aspects of fungal-host interactions. At least three, not mutually exclusive, approaches for future research on fungal pathogenicity genes emerge from these discussions. "The first, and most developed to date, is to study the regulation of a genein the organism from which it was isolated. . . . A second approach is to transfer genes into nonpathogens or into pathogens with different hosts .... A third and extremely underexploited route to the problern of isolating fungal pathogenicity genes is through mutant analysis" (Garber, Chapter 22).

We are most grateful for the contributions of the authors as weil as the support of many other individuals who provided assistance during the preparation of this treatise.

Austin, Texas Geneva, New York

Garry T. Cole Harvey C. Hoch

INTRODUCTION

Funga/-Host lnteractions Opportunities for lnterdisciplinary Research

Donald W. Roberts

This book provides, for the first time, the opportunity for researchers and educators interested in fungal diseases of animals (vertebrales and invertebrates) and plants to examine in one volume the salient features of disease initiation in both types of hosts. Are there sufficient commonalities between fungal infections of the two host groups to make the information from fungal-plant interactions enlightening to specialists of animal mycoses, and vice versa? The answer, in my opinion, is an unqualified "yes." lt is probably safe to suppose that the great majority of readers of the following pages will be specialists actively involved with either plants or animals, but not both. As such, the natural tendency will be to view the book as a comprehensive, current review of their specialty which happens to be duttered with intervening, peripheral treatises on other host systems. This view is diametrically opposed tothat of the Editors (see Preface), and to me (see Roberts and Aist, 1984). With few exceptions, there is little contact between students of animal and plant mycoses. This book was conceived as one tool to facilitate comparing basic knowledge, current problems, and methods of research on fungal infective units and early disease events between the two host groups.

I recommend that readers first read the chapters on the host group foreign to them. It is very likely that this will provide new insights and methods for research in the area of primary interest. Of equal importance, each treatise specifies a nurober of unresolved but important questions pertaining tothat chapter's topic-and readers working with other systems may have approaches based on their own experience to resolve some of these questions. If the book succeeds in its goals, it will stimulate cross-disciplinary collaboration and incite significant research progress in mycoses of both host groups.

The study of fungi is justified on many grounds. For example, fungi are eukaryotic microorganisms which usually are haploid most of their life cycle, making them powerful tools for basic genetic and biochemical studies. They tend to secrete, rather than sequester, metabo-

Donafd W. Roberts • Insect Pathology Resource Center, Boyce Thompson Institute, Cornell University, Ithaca, New York 14853.

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xii lntroduction

Iites, and this enhances their value as industrial fermentation agents. Fungi produce pharmaco­logically important and edible metabolites and, in some cases, the fungal biomass is itself a foodstuff. Although bacteria were the first tools of the modern molecular geneticists, methods to utilize yeasts in molecular biology were soon developed and pioneering research has been done with these microorganisms. More recently, filamentous fungi have yielded to transformation attempts, and, as described in the next-to-last chapter oftbis book, they have been added to the Iist of organisms available as tools for molecular genetics. From the viewpoint of man, the above are all beneficial characteristics which can and are being exploited. Fungi, however, can be detrimen­tal to man. Some infest improperly stored grain and nuts and produce mycotoxins, and there has been considemble mycological research directed at first understanding, and then reducing, the hazard of these compounds to man and domesticated animals.

Despite the importance of the above (and other) activities of fungi which have stimulated research on them, the single activity which probably is used more than any other to justify mycological and mycologically related research is that of pathogenicity of some fungi to other organisms. Their hosts include other fungi, vascular plants, invertebmtes, and both warm- and cold-blooded vertebrates. Most plant diseases are caused by fungi (Aist, 1984 ). All classes of the Eumycotina include at least a few plant pathogens (Alexopoulos and Mims, 1979; Agrios, 1988; Strobel and Mathre, 1970). At least 90 genera and more than 700 species of fungi have been identified as closely associated with invertebmtes. Virtually every major fungal taxonomic group except the higher basidiomycetes and dematiaceous Hyphomycetes has members pathogenic to invertebrates, primarily insects (Roberts and Humber, 1981). The numbers of fungal species which are primarily pathogenic, as opposed to adventitiously pathogenic, to warm-bloaded animals are mther small. There are only about 20 fungi which routinely cause systemic mycoses in these animals, and about a dozen associated with subcutaneous disease. In addition, there are approximately 20 dermatophytes which are restricted to the outer (keratin-containing) layers of the skin (Rippon, 1988). The latter are not generally considered a threat to life, whereas the former are very much so. The recent dramatic increase in numbers of immunosuppressed medical patients, either from leukemia, from intentional immunosuppression with chemieals for such medical events as organ transplants, or from the immunosuppression associated with AIDS cases, has been followed by a sharply escalated number of fungal infections (Campbell and White, 1989). In fact, esophageal candidosis and cryptococcosis of the central nervous system are valuable indicators of AIDS. Additional fungal diseases frequently found in HIV-positive patients include histoplasmosis and candidosis of the bronchi or lungs. The range of mycoses in AIDS patients, however, is much wider than the four Iisted here. Almost all of the well­recognized fungal pathogens of man have been described from these patients, in addition to a number of very rare types. The sharp increase in human mycoses has engendered a new sense of urgency to understanding and Controlling these diseases.

In addition to protecting man and bis domestic animals, the pathogenic processes of fungi have been studied to devise methods of protection in other hosts upon which man depends, such as food plants, and beneficial invertebmtes (primarily honeybee, silkworm, and shrimp). The scientific specialties of plant pathology and medical mycology basically originated around the protection concept. In addition, the potentially important roJe of fungi in control of pest organisms has stimulated concerted efforts to discover and develop virulent strains of fungi pathogenic to pest insects and pest plants (weeds). This pest-control concept has been an important force in the development of insect pathology, and recently it has played a significant roJe in plant pathology where fungi are being selected and/or engineered for weed and plant pathogen (primarily fungal) control.

Regardless of the motivation for study, the type of host, or the fungal pathogen, there is one common event underlying the understanding of all fungal diseases, viz. the initiation of the infection event. As pointed out by the Editors in the Preface, the early events of infection can be

lntroduction xiii

divided inlo lhree major areas: (l) spore attachmenl and invasion, (2) fungal spore products and palhogenesis, and (3) host response to early fungal invasion. They delineate one more area to emphasize its ernerging power and utility in understanding disease initiation: (4) molecular aspects of disease initiation. The reader is referred to the Preface for a hrief discussion of these topics. ll is Understandahle that all topics are not examined at the same depth for each host group. For example, the Iack of a good animal model for dermatophytes has prevented clear-cut, ultrastruclure-level elucidalion of lhe events associated with host invasion. In fact, according lo Hashimoto (Chapter 8), the first convincing evidence that lhe presume~ infective units of dermalophytes (i.e., arthroconidia) are indeed infectious was not puhlished unlil 1987.

ll is not my inlent in lhis inlroductory chapter to provide reviews or summaries of the individual treatises that follow, hut rather to provide some hrief comments on a few of many possihle examples of similarities and differences in findings with the various host groups.

It is apparent that almost all of the contrihutors have little firstband knowledge of fungal diseases of organisms outside their primary area of study. The Iiterature cited seldom includes references to infections of other host groups. This is almost completely the case with plant pathologists and medical mycologists. Invertehrate pathologists, on the other hand, do cite some plant pathology studies-prohahly because of the hasic similarities of the fungi involved and common characteristics of the hosts' outer surfaces (hard, wax-covered), which is the normal invasion site. Also, mycological plant pathologisls prohahly outnumher mycological invertehrate pathologisls hy more lhan ten to one, and this is reftected in the numhers of publications on invasion processes in these two host types. Invertebrale pathologists have undouhledly gained from their horrowing of ideas and methods from other areas. The clear message is that lhe same potential for cross-fertilization of ideas exisls for those working with plants and vertebrales. An example is the discussion by Kennedy (Chapter 7) on the adhesion of Candida hlastospores to the gaslroinlestinal tract of vertebrales. For plant or invertebrate pathologists, comments on this yeastlike fungus in a vertebrate-specific organ would he assumed of little relevance to their host systems-although the plant-pathologist authors, Nicholson and Epstein (Chapter 1), do cite studies describing Ieetin-mediated binding of Candida to human epithelium. Nevertheless, in trying to explain the events leading to human gut infections, Kennedy presents concepts of importance to underslanding adhesion of any small, charged, chemically active body to another structure. He invokes the classic lyophohic colloid theory (Derjagiun and Landau, 1941; Verwey and Overbeek, 1948) (DLVO theory) and its derivations (e.g., Jones and Isaacson, 1983) to explain adhesive interactions between the surfaces of hlastospores and the gut mucosa, which theorelically should repel one another since they are hoth negatively charged. He suggests that the DLVO theory describes long-range (> 10 nm) adhesive interactions, whereas close-range interactions ( < l nm) are regu1ated by adhesion-receptor binding.

A large numher of research opportunities are mentioned in this book which probably are amenable lo successful resolution hy experts of other host/palhogen syslems. As menlioned previously, virtually all the authors point out lacunae in knowledge concerning their research areas. Sludents, professors, and researcti scientists alike will be well advised to carefully search these pages for research opportunities with mycoses of both plants and animals. This may Iead lo collaboralions wilh specialists working with hosls new to them. I can attesl from personal experience that such collaborative research can have synergistic, favorable effects on both research teams (for recent results of work on an insect pathogen in collahoration -with a plant pathologist, Richard C. Staples, see Goettel et al., 1989, 1990; St. Leger et al., 1989a-1990).

Characterizing and modifying nucleic acids of palhogenic fungi, without douhl, will provide new insighls inlo processes of disease initiation and development. Molecular approaches currently available to sludents of palhogenic fungi are described hy Garher for phylopathogens (Chapter 22) and by Soll for Candida infecling man (Chapter 23). Cloning by lransformation is a parlicularly powerful tool for working with components of disease, e.g., specificity and viru-

xiv lntroduction

lence, where the biochemical bases of the traits are not known. The ability afforded by this approach to isolate genes in the absence ofknowledge about gene products is, as stated by Garber, "invaluable."

A clearly expressed plea to students of pathogenesis to utilize the powerful modern molecular biology tools in their investigations, but to be certain that these studies are based on sound biological information, constitutes the final paragraph of Soll's contribution (Chapter 23). This, in my opinion, should be read, and reread periodically, to encourage relevance to the "real" world of virulence in molecular genetics experiments.

Dimorphism (the production of yeast and filamentaus growth forms by a fungus) has been noted for several entomopathogenic fungi. The yeast phase is almost exclusively produced in the hemocoel of the arthropod host; but one species, Nomuraea rileyi, readily produces the yeast phase on agar media-switching after a few days to filamentaus growth. Neither the importance of this phenomenon to virulence nor the underlying mechanisms involved in determining which type of growth will occur have been subjected to close scrutiny with entomopathogenic fungi. Dimorphism and phenotypic switching of Candida are important, however, to Candida strains pathogenic to humans; and these topics have been, and continue tobe, very active research areas with this organism (Soll, Chapter 23). There are obvious, but to date underexploited, oppor­tunities for research on "blastospore "-producing entomopathogenic fungi based on Candida findings and methods.

Metabolites produced by pathogenic fungi during their growth are suspected to have important roles in disease development. Even though these metabolites are numerous and some are fully described chemically, their biological effects on the host usually are still unknown. As mentioned by Garber (Chapter 22), some toxic peptides produced by plant pathogenic fungi are assembled by specific synthetase enzymes rather than by the normal ribosomal system. The very existence of these specific synthetase enzymes suggests strongly that the fungi find the resulting peptides of significant survival value. Their role in pathogenesis is best documented for certain plant pathogens (Deol et al., 1978; Macko, 1983; Macko, et al., 1985; Wolpert et al., 1985; Springer, et al., 1984). Beauvericin, bassianolide, and destruxins, insect-toxic depsipeptides produced by the entomopathogenic fungi Verticillium lecanii, Beauveria bassiana, and Meta­rhizium anisopliae, respective1y, also probably are synthesized by the enzymatic method (Grove and Pople, 1980; Roberts, 1981). The destruxins are particularly relevant to this book in that they are toxic to both insects and plants (Bains and Thwari, 1987; Gupta et al., 1990). Although the methods are technically demanding, it is probable that the examination of the synthesis of other active peptides produced by pathogenic fungi will reveal synthetase enzymes. The next burdie will be to modify these enzymes by molecular genetics methodologies. This will be of great utility in defining the roJe of the toxic peptides in disease and, perhaps, in producing fungal strains with increased or reduced virulence-according to their intended use.

Many, and probably most, toxins produced by pathogenic fungi are not peptides. For example, cytochalasins C and D, two members of a biologically active class of compounds, were first isolated from cultures of an entomopathogenic fungus, M. anisopliae. Their function in insect disease has not been elucidated. Accordingly, the comment by Robertson (Chapter 21) that Aspergillus fumigatus conidia release a diffusible substance into their hosts' lungs which, although not chemically defined, is similar to cytochalasins in biological activity-viz., anti­phagocytic-immediately suggests an appropriate approach to defining the role of cytochalasins produced by M. anisopliae in insects. It should be mentioned that depsipeptides (destruxins) produced by M. anisopliae have been reported to inhibit insect phagocytes (Huxham et al., 1989).

The theme of this book is the similarities of fungal infections, regardless of host. lt is patently clear in the following chapters that such similarities abound in all four Subdivisions ofthe infection process around which the book is organized. Also, there is extensive commonality of

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concepts and methods, and therefore the potential for truly meaningful dialogue between pathologists working with very different hosts certainly exists. As mentioned previously, such recognition of common ground will facilitate development of all areas of the understanding of mycoses. In addition, there are, without doubt, some important differences between the hosts, pathogenic fungi, and fungus/host interactions. For example, although many of the fungi which infect plants and invertebrates fall within the same taxonomic groups, there is very little overlap at the fungal genus Ievel, and virtually none at the species Ievel. It follows that the characteristics needed by fungi to successfully establish disease in plants must be fundamentally different in some ways from those needed to infect animals. These differences, when clearly delineated, will indicate probable key virulence characters for pathogens of the two host groups. One approach to identifying specialized pathogenicity traits will be to compare available knowledge on all pathogenic fungi. Another function of the information in this book, therefore, could be its use by students engaged in the study of evolution of pathogenicity in fungi.

REFERENCES

Agrios, G. N., 1988, Plant Pathology, 3rd ed., Academic Press, New York. Aist, J. R., 1984, A survey of plant diseases caused by fungi that attack cuticularized plant surfaces, in: lnfection

Processes of Fungi (D. W. Roberts and J. R. Aist, eds. ), The Rockefeiler Foundation, New York, pp. 13-15. Alexopoulos, C. J., and Mims, C. W., 1979, Introductory Mycology, 3rd ed., Wiley, New York. Bains, P. S., and Thwari, J. P., 1987, Purification, cbemical characterization and host-specificity of the toxin

produced by Alternaria brassicae, Physiol. Mol. Pinnt Pathol. 30:259-271. Campbell, C. K., and White, G. C., 1989, Fungal infection in 'AIDS' patients, Mycologist 3:7-9. Deol, B. S., Ridley, D. D., and Singh, P., 1978, Isolation of cyclodepsipeptides from plant pathogenic fungi, Aust. J.

Chem. 31:1397-1399. Derjagiun, B. V., and Landau, L., 1941, Theory of the stability of strongly charged lyophobic sols and of tbe

adhesion of strongly charged particles in solutions of electrolytes, Acta Physicochim. URSS 14:633-662. Goettel, M. S., St. Leger, R. J., Rizzo, N. W., Staples, R. C., and Roberts, D. W.,1989, Ultrastructurallocalization

of a cuticle-degrading prolease produced by the entornopathogenic fungus Metarhizium anisopliae during penetration of host cuticle, J. Gen. Microbiol. 135:2233-2239.

Goettel, M. S., St. Leger, R. J., Bhairi, S., Jung, M. K., Oakley, B. R., Roberts, D. W., and Staples, R. C., 1990, Pathogenicity and growth of Metarhizium anisopliae stably transformed to benomyl resistance, Curr. Genet. 17:129-138.

Grove, J. F., and Pople, M., 1980, The insecticidal activity of beauvericin and the enniatin complex, Mycopatho­logia 70:103-105.

Gupta, S., Roberts, D. W., and Renwick, J. A. A., 1989, Insecticidal cyclodepsipeptides from Metarhizium anisopliae, J. Chem. Soc. Perkin Trans. 1:2347-2357.

Huxham, I. M., Lackie, A. M., and McCorkindale, N. J., 1989, Inhibitory effects of cyclodepsipeptides, destruxins, from the fungus Metarhizium anisopliae, on cellular immunity in insects, J. Insect Physiol. 35:97-105.

Jones, G. W., and Isaacson, R. E., 1983, Proteinaceous bacterial adhesins and their receptors, CRC Crit. Rev. Microbiol. 10:229-260.

Macko, V., 1983, Structural aspects of toxins, in: Toxinsand Plant Pathogenesis (J. M. Daly and B. J. Deverall, eds.), Academic Press, New York, pp. 41-80.

Macko, V., Wolpert, T. J., Acklin, W., Jaun, B., Seibl, J., Meili, J., and Arigoni, D., 1985, Characterization of victorin C, the major host-selective toxin from Cochliobolus victoriae: Structure of degradation products, Experientia 41:1366-1370.

Peeters, H., Zocher, R. and Kleinkauf, H., 1988, Synthesis ofbeauvericin by a multifunctional enzyme, J. Antibiot. 41:352-259.

Rippon, J. W., 1988, Medical Mycology: The Pathogenic Fungi and the Pathogenic Actinomycetes, 3rd ed., Saunders, Philadelphia.

Roberts, D. W., 1981, Toxins of entomopathogenicfungi, in: Microbial Control of Pestsand Plant Diseases: 1970-1980 (H. D. Burges, ed.), Academic Press, New York, pp. 441-464.

Roberts, D. W., and Aist, J. R., eds., 1984,/nfection Processes of Fungi, The Rockefeiler Foundation, New York.

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Roberts, D. W., and Humber, R. A., 1981, Entomopathogeoic fungi, in: The Biology ofConidial Fungi, Vo1ume 2 (G. T. Cole and B. Kendrick, eds.), Academic Press, New York, pp. 201-236.

Springer, J. P., Cole, R. J., Dorner,l W., Cox, R. H., Richard, I. L., Barnes, C. L., and van der Helm, D., 1984, Sttucture and conformation of roseotoxin 8, l Am. Chem. Soc. 106:2388-2392.

St. Leger, R. J., Butt, T. M., Goettel, M. S., Staples, R. C., and Roberts, D. W., 1989a, Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae, Exp. Mycol. 13:274-288.

St. Leger, R. J. Butt, T. M., Staples, R. C., and Roberts, D. W. 1989b, Synthesis of proteins including a cuticle­degrading prolease durlog differentiation of the entomopathogenic fungus Metarhizium anisopliae, Exp. Mycol. 13:253-262.

St. Leger, R. J., Roberts, D. W., and Staples, R. C., 1989c, Calcium and calmodulin mediated protein synthesis and protein phosphorylation durlog germination, growth and prolease production by Metarhizium anisopliae ,l Gen. Microbiol. 135:2141-2154.

St. Leger, R. I., Roberts, D. W., and Staples, R. C., 1989d, Novel GTP-binding proteins in plasma membranes ofthe fungus Metarhizium anisopliae, Biochem. Biophys. Res. Commun. 164:562-566.

St. Leger, R. J., Laccetti, L. 8., Staples, R. C., and Roberts, D. W., 1990, Protein kinases in the entomopathogeoic fungus Metarhizium anisopliae,l Gen. Microbiol. 136:1401-1411.

Strobel, G. A., and Mathre, D. E., 1970, Outlines of Plant Pathology, Van Nosttand-Reinhold, Princeton, N.I. Verwey, E. J. W., and Overbeek, J. T. G., 1948, Theory ofthe Stability of Lyophobic Colloids, Elsevier, Amsterdam. Wolpen, T. J., Macko, M., Acklin, W., Iaun, 8., Seibl, I., Meili, I., and Arigoni, D.,1985, Structure ofvictorin C,

the major host-selective toxin from Cochliobolus victoriae, Experientia 41:1524-1529.

Contents

I. Spore Attachment and Invasion

1. Adhesion of Fungi to the Plant Surface: Prerequisite for Pathogenesis . . 3

Ralph L. Nieholsan and Lynn Epstein

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2. Initiation of the Infection Process, the Production of Extracellular Matrices and

Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3. Composition and Significance of Non-adhesive Compounds in the Extracellular

Matrix of Colletotrichum graminicola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4. Functional Significance of Adhesion and Extracellular Matrices . . . . . . . . . . . . . 9 5. Mechanisms of Fungal Adhesion to Plant Surfaces . . . . . . . . . . . . . . . . . . . . . . . 13 6. Methodology for the Study of Extracellular Matrices and Adhcsives . . . . . . . . . 16 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2. Signaling for lnfection Structure Formation in Fungi . . . . . . . . . . . . . . . . . . . . 25

H. C. Hoch and R. C. Staples

1. Introducdon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2. Signaling for Growth Odentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3. Signals for Infection Structure Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4. Differentiation Proteins-A Consequence of Signal Reception . . . . . . . . . . . . . . . 39 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3. The Plant Ce// Wall as a Barrier to Fungal Invasion . . . . . . . . . . . . . . . . . . . . . 47

M. G. Smart

l. lntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2. Structure of the Plant Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3. Resistance of Native Cell Walls to Fungi . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . 51

xvii

xviii Contents

4. Modification of Cell Walls in Resistance to Fungi . . . . . . . . . . . . . . . . . . . . . . . . 53 5. Cooperative Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 6. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 I

4. Rust Basidiospore Germlings and Disease Initiation . . . . . . . . . . . . . . . . . . . . 67

Randall E. Gold and Kurt Mendgen

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 2. Basidiospore Morphology and Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 3. Basidiospore Germlings in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 4. Basidiospore Germlings in Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

5. Attachment of Mycopathogens to Cuticle: The Initial Event of Mycoses in Arthropod Hosts .................................................. 101

Drion G. Boucias and Jacquelyn C. Pendland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 2. Structural Sturlies of the Outer Wall Layer of Fungal Propagules Infectious to

Arthropod Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 3. Chemical Properties of the Outer Layer of Fungal Propagules Infectious to

Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 4. Structure and Composition of the Epicuticle: The Thrget Substrate for Spore

Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 5. Relationship between Fungal Propagules and the Host Cuticle . . . . . . . . . . . . . . I 18 6. Summary ......................................................... 123 7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

6. The Fate of Fungal Spores in the lnsect Gut .......................... 129

R. J. Di/lon and A. K. Charnley

1. lntroduction ................................................. 0 o o. o. 129 2. The Structure of the Insect Gut and the Associated Microbiotia 0 o ••••• o o • • • • 130 3. Retention of Spores in the Gut ........................................ 132 4. lnftuence on Spore Germination of Passage through the Gut .......... o • • • • • 133 5. Conditions Imposed by the lnsect and Gut Microbiotia Which Influen~e Spore

Adhesion and Germination ...... o o •••••••••• o •••• o o ••• o • o o •••••• o • • • • 134 6. Gut Infections in lnsects ............................... o ••••••••••••• 141 7. Conclusions .. 0 ••••••••••••• o •••••••••••••••••• o o •••••••••••••••••• 149 8. References 0 •••••••••• o •••••••••••••••••••••• o o •••••• o ••••••••• o ••• 150

7. Candida Blastospore Adhesion, Association, and Invasion of the Gastrointestinal Tract of Vertabrates .................................. 157

MichaeiJ. Kennedy

1. Introduction ........ 0 •••••••••••••••••• o ••• o •• o o •• o ••• o ••••••• o • • • • 157

Contents xix

2. Sequential Steps in Mucosal Association and Invasion ..................... 158 3. Physicochemical Aspects of Mucosal Association ........................ 159 4. Mechanisms of Adhesion and Association with Gastrointestinal Mucosa ...... 161 5. Mechanisms of Mucosal Invasion ..................................... 167 6. Candida Cell Surface Composition, Ultrastructure, and Possible Adhesins .... 171 7. Factors Inftuencing Adhesion, Invasion, and Colonization .................. 174 8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

8. lnfectious Propagules of Dermatophytes .............................. 181

Tadayo Hashimoto

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 2. Infective Conidia Produced by the Dermatopbytes ........................ 183 3. Formation of Conidia ............................................... 185 4. Biological Characteristics ............................................ 193 5. Infectivity and Pathogenicity ......................................... 196 6. Mechanisms of Disease Initiation in Dermatophytosis ..................... 198 7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

II. Fungal Spore Products and Pathogenesis

9. Melanin Biosynthesis: Prerequisite for Successfullnvasion of the Plant Host by Appressoria of Colletotrichum and Pyricularia ................. 205 Yasuyuki Kubo and lwao Furusawa

1. Introduction ....................................................... 205 2. Mode of Appressorial Penetration ..................................... 206 3. Penetration Ability of Melanin-Deficient Mutants ........................ 206 4. Location and Form of Melanin ........................................ 208 5. Action Mechanism of Melanin-Inhibiting Fungieides ...................... 209 6. Regulation of Melanin Biosynthesis during Appressorium Formation ......... 215 7. Conclusion ........................................................ 215 8. References ........................................................ 215

10. The Plant Cuticle: A Barrier toBe Overcome by Fungal Plant Pathogens .......................................................... 219

Wolfram Köller

1. Introduction ....................................................... 219 2. The Plant Cuticle ................................................... 220 3. Cutinase from Fungal Pathogens ...................................... 222 4. Cutinase as a Factor in Pathogenicity .................................. 227 5. Modes of Penetration and Involvement of Cutinase ....................... 231 6. Cutinase as a Target for Plant Disease Control ........................... 237 7. Ecology of Cutinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 8. References ........................................................ 240

XX Contents

11. Appearance of Pathogen-Related Proteins in Plant Hosts: Relationships between Compatib/e and lncompatible Intersetions .................... 247

/ngrid M. J. Scho/tens-Toma, Matthieu H. A. J. Joosten, and Pierre J. G. M. Oe Wit

I. Plant-Pathogen Interactions .......................................... 247 2. The Compatible Interaction ........................................... 252 3. The Incompatible Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 4. Concluding Remarks ................................................ 261 5. References ........................................................ 261

12. The RoJe of Cuticle-Degrading Enzymes in Fungal Pathogenesis in lnsects ............................................................. 267

A. K. Charn/ey and R. J. St. Leger

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 2. The Invasion Process ................................................ 268 3. Cuticle as a Barrier to Fungal Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 4. Cuticle-Degrading Enzymes .......................................... 271 5. Proteolytic Enzymes ................................................ 273 6. Chitinolytic Enzymes ............................................... 278 7. Lipolytic and Esterolytic Enzymes ..................................... 279 8. Host Defense against Cuticle-Degrading Enzymes ........................ 281 9. Cuticle-Degrading Enzymes as Virulence Determinants ................... 282

10. References ........................................................ 283

13. Potential for Penetration of Passive Barriers to Funga/Invasion in Humans ............................................................ 287

Frank C. Odds

1. Fungi with Potential for Penetration of Epithelial Surfaces ................. 287 2. Proteolysis (and Keratinolysis) as a Mechanism for Epithelial Penetration ..... 288 3. Other Hydrolytic Enzymes as Agents for Epithelial Penetration ............. 292 4. Conclusions ....................................................... 292 5. References ........................................................ 292

14. Dihydroxynaphthalene (DHN) Melanin and lts Relationship with Virulence in the Early Stages of Phaeohyphomycosis .................. 297

Dennis M. Dixon, Pau/ J. Szanisz/o, and Annemarie Polak

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 2. Virulence of the Dematiaceous Fungi .................................. 298 3. DHN Melanin and Wangiella dermatitidis ............................... 300 4. Initial Studies of Virulence ........................................... 306 5. Studies of me/3 in Additional Mouse Strains . . . . . . . . .. . . . . . . . . . . . . . . . . . . 308 6. Studies with Additional Mutants in Outbred Mice ........................ 313 7. Preliminary Studies with Broth-Cultivated Inoculum ...................... 314 8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

Contents xxi

III. Host Response to Early Fungal Invasion

15. Invasion of Plants by Powdery Mildew Fungi, and Cellular Mechanisms of Resistance ....................................................... 321

James R. Aist and William R. Bushne/1

1. Introduction ....................................................... 321 2. Primary Germ Thbes ................................................ 324 3. Cytoplasmic Aggregation ............................................ 325 4. Halo Formation .................................................... 326 5. Papilla Formation .................................................. 327 6. Haustoria ......................................................... 330 7. Hypersensitive Response ............................................. 333 8. Levels of Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 9. Concluding Statement ............................................... 339

10. References ........................................................ 339

16. lnduced Systemic Resistance in Plants ............................... 347

Nageswara Rao Madamanchi and Joseph Kuc I. Introduction ....................................................... 347 2. The Phenomenon ................................................... 348 3. Mechanisms Activated .............................................. 350 4. Gene Expression as it Relates to Induced Systemic Resistance-Applications

of Molecular Biology ............................................... 355 5. Concepts and Applications for the Future .. · ............................. 356 6. References ........................................................ 357

17. The Plant Membrane and lts Response to Disease .................... 363

Anton Novacky

l . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 2. Membrane Structure and Function ..................................... 364 3. Interaction with the Plasma Membrane H+-ATPase ....................... 364 4. Interaction with a Single Ion Channel .................................. 367 5. The Plasma Membrane as a Possible Toxin Target . . . . . . . . . . . . . . . . . . . . . . . . 368 6. Recognition of an Incompatible Pathogen ............................... 372 7. Conclusion ........................................................ 374 8. References ........................................................ 375

18. The Fungal Spore: Reservoir of Allergens ............................. 379

J. P. Latge and S. Paris

I. Introduction ....................................................... 379 2. Methodology Available to Study Fungal Spore Allergy .................... 380 3. Characterization of Spore Allergens .................................... 388 4. Other Fungi ........................................................ 396 5. Perspectives for Future Research ...................................... 397 6. References ........................................................ 398

xxii Contents

19. Conidia of Coccidioides immitis: Their Significance in Disaase Initiation 403 Garry r Co/e and Theo N. Kirkland

1. Introduction ....................................................... 403 2. Saprobie and Parasitic Cycles of Coccidioides immitis ..................... 404 3. Techniques of Isolation and Characterization of Conidial Wall Fractions ...... 408 4. Outer and Inner Conidial Wall Fractions ................................ 416 5. Water-Soluble Conidial Wall Fraction ................................... 428 6. Possible Biological Functions of Isolated Conidial Wall Components ......... 435 7. Summary ......................................................... 438 8. References ........................................................ 439

20. Ce/1-Mediated Host Response to Funga/Aggression ................... 445 Alayn R. Waldorf

1. Introduction ....................................................... 445 2. Fungicidal Mechanisms .............................................. 446 3. Rote of the Mononuclear Phagocyte .................................... 447 4. Rote of the Polymorphonuclear Leukocyte .............................. 452 5. Conclusion ........................................................ 456 6. References ........................................................ 456

21. Suppression of Phagocytic Ce/1 Responses by Conidia and Conidial Products of Aspergillus fumigatus .................................... 461

Maura 0. Robertson

1. Introduction ....................................................... 461 2. Pathogenicity of A. fumigatus ........................................ 462 3. Aspergillus fumigatus in the Lung ..................................... 462 4. Phagocytic Cells versus Aspergillus jumigatus ........................... 463 5. Effect of Conidial Products on Phagocytic Cell Function .................. 4 73 6. Summary ......................................................... 476 7. References ........................................................ 4 77

IV. Molecular Aspects of Disease Initiation

22. Molecu/ar Approaches to the Analysis of Pathogenicity Genes from Fungi Causing Plant Disaase ......................................... 483

Robert C. Garber

1. Introduction ....................................................... 483 2. Technology for Isolation of Fungal Genes ............................... 484 3. Isolation of Pathogenicity Genes with Known Products .................... 491 4. Isolation of Pathogenicity Genes with Unknown Products but Genetically

Established Roles in Disease ......................................... 495 5. Prospects for Progress ............................................... 497 6. References ........................................................ 498

Contents xxiii

23. Current Status of the Molecu/ar Basis of Candida Pathogenicity ........ 503

David R. Soll

1. lntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503 2. The Basic Biology of Candida Dimorphism ............................. 503 3. The Basic Biology of High-Frequency Switching in C. albicans ............ 507 4. Basic Biological Characteristics Other Than Dimorphism and Switching Which

May Play Roles in Pathogenesis ....................................... 514 5. The Molecular Genetics of C. albicans ................................. 514 6. The Molecular Biology of Dimorphism ................................. 517 7. The Molecular Biology of Switching ................................... 519 8. Applying Molecular Technologies to the BasicQuestions of Candida

Epidemiology and Disease ........................................... 521 9. Concluding Remarks ................................................ 534

10. References ........................................................ 535

Taxonomie Index ........................................................ 541

Subject Index ........................................................... 547

The Fungal Spore and Disease Initiation in Plants and Animals