Environmental and Microbial Relationships

The MycotaEdited byK. Esser

The MycotaIIIIIIIVVVIVIIGrowth, Differentiation and Sexuality1st edition ed. by J.G.H. Wessels and F. Meinhardt2ndeditioned.byU.KüesandR.FischerGenetics and BiotechnologyEd.byU.KückBiochemistry and Molecular BiologyEd. by R. Brambl and G. MarzlufEnvironmentalandMicrobialRelationships1steditioned.byD.WicklowandB.Söderström2nd edition ed. by C.P. Kubicek and I.S. DruzhininaPlant Relationships1st edition ed. by G. Carroll and P. Tudzynski2ndeditioned.byH.B.DeisingHuman and Animal Relationships1st edition ed. by D.H. Howard and J.D. Miller2nd edition ed. by A. Brakhage and P. ZipfelSystematics and EvolutionEd. by D.J. McLaughlin, E.G. McLaughlin, and P.A. Lemke †VIII Biology of the Fungal CellEd. by R.J. Howard and N.A.R. GowIXXXIXIIFungal AssociationsEd. by B. HockIndustrial ApplicationsEd.byH.D.OsiewaczAgricultural ApplicationsEd. by F. KempkenHuman Fungal PathogensEd. by J.E. Domer and G.S. KobayashiXIII Fungal GenomicsEd. by A.J.P. Brown

The MycotaA Comprehensive Treatiseon Fungi as Experimental Systemsfor Basic and Applied ResearchEdited by K. EsserIVEnvironmentaland Microbial Relationships2nd EditionVolume Editors:C.P. Kubicek · I.S. DruzhininaWith 55 Figures and 16 Tables123

Series EditorProfessor Dr. Dr. h.c. mult. Karl EsserAllgemeine BotanikRuhr-Universität44780 Bochum, GermanyTel.: +49 (234)32-22211Fax.: +49 (234)32-14211e-mail: Karl.Esser@rub.deVolume EditorsProfessor Dr. Christian P. KubicekTel.: + 43 1 58801 17250Fax.: + 43 1 58801 17299e-mail: ckubicek@mail.zserv.tuwien.ac.atDr. Irina S. DruzhininaTel.: + 43 1 58801 17202Fax.: + 43 1 58801 17299e-mail: druzhini@mail.zserv.tuwien.ac.atTU WienInstitut für VerfahrenstechnikUmwelttechnik und Technische BiowissenschaftenGetreidemarkt 91060 WienAustriaLibrary of Congress Control Number: 2007927885ISBN 978-3-540-71839-0 Springer Berlin Heidelberg New YorkISBN 3-540-58005-0 1st ed. Springer Berlin Heidelberg New YorkThis work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically therights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way,andstorageindatabanks.DuplicationofthispublicationorpartsthereofispermittedonlyundertheprovisionsoftheGermanCopyright Law of September 9, 1965, in its current version, and permissions for use must always be obtained from Springer-Verlag.Violations are liable for prosecution under the German Copyright Law.Springer is a part of Springer Science+Business Mediaspringer.com© Springer-Verlag Berlin Heidelberg 1997, 2007The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence ofa specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for generaluse.Editor: Dr. Dieter Czeschlik, Heidelberg, GermanyDesk editor: Dr. Andrea Schlitzberger, Heidelberg, GermanyCover design: Erich Kirchner and WMXDesign GmbH, Heidelberg, GermanyProduction and typesetting: LE-TEXJelonek,Schmidt&VöcklerGbR,Leipzig,GermanyPrinted on acid-free paper SPIN 10987223 31/3180 543210

Karl Esser(born 1924) is retired Professor of General Botany and Directorof the Botanical Garden at the Ruhr-Universität Bochum (Germany).His scientific work focused on basic research in classicaland molecular genetics in relation to practical application. Hisstudies were carried out mostly on fungi. Together with his collaboratorshe was the first to detect plasmids in higher fungi. Thishas led to the integration of fungal genetics in biotechnology. Hisscientific work was distinguished by many national and internationalhonors, especially three honorary doctoral degrees.Christian P. Kubicek(born 1951) studied chemistry with specialization in Biotechnologyand Food Chemistry. In 1977, he graduated at the Universityof Technology of Vienna with a thesis on citric acid productionby Aspergillus niger. He later on habilitated at the Technical Universityin Microbial Biochemistry in 1983, at the Biocenter ofthe University of Vienna in Applied Microbiology in 1988, andreceived the honorary title of Professor for Microbial Biochemistryat the Vienna University of Technology in 1991. In 2003, hewas appointed full Professor for Biotechnology and Microbiologyat the same university. He is the author of about 250 papersin peer-reviewed journals, more than 30 book articles, severalpatents and three books, an editorial board member for severalmicrobiological journals, and has been editor of Applied andEnvironmental Microbiology for 10 years. His scientific interestsare various aspects of the biochemistry, molecular genetics,evolution and genomics of mitosporic fungi, particularly Trichoderma,Aspergillus and PenicilliumIrina S. Druzhinina(born 1974) graduated at the Department of Mycology and Algologyof Lomonosov’s Moscow State University (Russia). HerMaster’s thesis was dedicated to the species concept in fungiand population genetics of Pleurotus ostreatus (Fr.) Kumm. In1998, she moved to Austria and switched her attention to radioecologyand environmental risk assessment, by studying thephysiology of radionuclide accumulation in fruit bodies of ediblemushrooms. In 2001, she completed her thesis “Radioactive contaminationof wild mushrooms: mycological approach and riskperception” at the University of Vienna (Austria), and receivedher doctoral degree with specialization in Botany. Soon thereafter,she was appointed Assistant Professor in the Departmentof Gene Technology and Applied Biochemistry at the Institute ofChemical Engineering of the Vienna University of Technology(Austria). Here, she returned to her main area of scientific interestand established a research group on Fungal Evolution andSpeciation. Her work focuses on the phylogeny of Hypocrealenfungi, and on the development of bioinformatics tools for themolecular identification of fungi. In a second line of research,she is interested in applying phenotype microarray techniquesin investigating the metabolomics of filamentous fungi.

Series PrefaceMycology, the study of fungi, originated as a subdiscipline of botany and was a descriptivediscipline, largely neglected as an experimental science until the early years of thiscentury. A seminal paper by Blakeslee in 1904 provided evidence for selfincompatibility,termed “heterothallism”, and stimulated interest in studies related to the controlof sexual reproduction in fungi by mating-type specificities. Soon to follow was thedemonstration that sexually reproducing fungi exhibit Mendelian inheritance and thatit was possible to conduct formal genetic analysis with fungi. The names Burgeff, Kniepand Lindegren are all associated with this early period of fungal genetics research.These studies and the discovery of penicillin by Fleming, who shared a Nobel Prizein 1945, provided further impetus for experimental research with fungi. Thus began aperiod of interest in mutation induction and analysis of mutants for biochemical traits.Such fundamental research, conducted largely with Neurospora crassa, ledtotheonegene: one enzyme hypothesis and to a second Nobel Prize for fungal research awarded toBeadle and Tatum in 1958. Fundamental research in biochemical genetics was extendedto other fungi, especially to Saccharomyces cerevisiae, and by the mid-1960s fungalsystems were much favored for studies in eukaryotic molecular biology and were soonable to compete with bacterial systems in the molecular arena.The experimental achievements in research on the genetics and molecular biology offungi have benefited more generally studies in the related fields of fungal biochemistry,plant pathology, medical mycology, and systematics. Today, there is much interest in thegenetic manipulation of fungi for applied research. This current interest in biotechnicalgenetics has been augmented by the development of DNA-mediated transformationsystems in fungi and by an understanding of gene expression and regulation at themolecular level. Applied research initiatives involving fungi extend broadly to areas ofinterest not only to industry but to agricultural and environmental sciences as well.It is this burgeoning interest in fungi as experimental systems for applied as well asbasic research that has prompted publication of this series of books under the title TheMycota. This title knowingly relegates fungi into a separate realm, distinct from that ofeither plants, animals, or protozoa. For consistency throughout this Series of Volumesthe names adopted for major groups of fungi (representative genera in parentheses) areas follows:PseudomycotaDivision:Division:EumycotaDivision:Division:Division:Subdivision:Oomycota (Achlya, Phytophthora, Pythium)HyphochytriomycotaChytridiomycota (Allomyces)Zygomycota (Mucor, Phycomyces, Blakeslea)DikaryomycotaAscomycotina

VIIIClass:Class:Subdivision:Class:Class:Series PrefaceSaccharomycetes (Saccharomyces, Schizosaccharomyces)Ascomycetes (Neurospora, Podospora, Aspergillus)BasidiomycotinaHeterobasidiomycetes (Ustilago, Tremella)Homobasidiomycetes (Schizophyllum, Coprinus)We have made the decision to exclude from The Mycota the slime molds which, althoughthey have traditional and strong ties to mycology, truly represent nonfungal formsinsofar as they ingest nutrients by phagocytosis, lack a cell wall during the assimilativephase, and clearly show affinities with certain protozoan taxa.The Series throughout will address three basic questions: what are the fungi, what dothey do, and what is their relevance to human affairs? Such a focused and comprehensivetreatment of the fungi is long overdue in the opinion of the editors.A volume devoted to systematics would ordinarily have been the first to appearin this Series. However, the scope of such a volume, coupled with the need to giveserious and sustained consideration to any reclassification of major fungal groups, hasdelayed early publication. We wish, however, to provide a preamble on the nature offungi, to acquaint readers who are unfamiliar with fungi with certain characteristicsthat are representative of these organisms and which make them attractive subjects forexperimentation.The fungi represent a heterogeneous assemblage of eukaryotic microorganisms.Fungal metabolism is characteristically heterotrophic or assimilative for organic carbonand some nonelemental source of nitrogen. Fungal cells characteristically imbibe or absorb,ratherthaningest,nutrientsandthey have rigidcell walls. The vastmajority offungiare haploid organisms reproducing either sexually or asexually through spores. Thespore forms and details on their method of production have been used to delineate mostfungal taxa. Although there is a multitude of spore forms, fungal spores are basically onlyof two types: (i) asexual spores are formed following mitosis (mitospores) and culminatevegetative growth, and (ii) sexual spores are formed following meiosis (meiospores) andare borne in or upon specialized generative structures, the latter frequently clustered ina fruit body. The vegetative forms of fungi are either unicellular, yeasts are an example,or hyphal; the latter may be branched to form an extensive mycelium.Regardless of these details, it is the accessibility of spores, especially the directrecovery of meiospores coupled with extended vegetative haploidy, that have madefungi especially attractive as objects for experimental research.Theabilityoffungi,especiallythesaprobicfungi,toabsorbandgrowonrathersimple and defined substrates and to convert these substances, not only into essentialmetabolites but into important secondary metabolites, is also noteworthy. The metaboliccapacities of fungi have attracted much interest in natural products chemistry and inthe production of antibiotics and other bioactive compounds. Fungi, especially yeasts,are important in fermentation processes. Other fungi are important in the production ofenzymes, citric acid and other organic compounds as well as in the fermentation of foods.Fungi have invaded every conceivable ecological niche. Saprobic forms abound,especially in the decay of organic debris. Pathogenic forms exist with both plant andanimal hosts. Fungi even grow on other fungi. They are found in aquatic as well assoil environments, and their spores may pollute the air. Some are edible; others arepoisonous. Many are variously associated with plants as copartners in the formation oflichens and mycorrhizae, as symbiotic endophytes or as overt pathogens. Associationwith animal systems varies; examples include the predaceous fungi that trap nematodes,the microfungi that grow in the anaerobic environment of the rumen, the many insectassociatedfungiandthemedicallyimportantpathogensafflictinghumans.Yes,fungiare ubiquitous and important.

Series PrefaceIXThere are many fungi, conservative estimates are in the order of 100,000 species,and there are many ways to study them, from descriptive accounts of organisms foundin nature to laboratory experimentation at the cellular and molecular level. All suchstudies expand our knowledge of fungi and of fungal processes and improve our abilityto utilize and to control fungi for the benefit of humankind.We have invited leading research specialists in the field of mycology to contributeto this Series. We are especially indebted and grateful for the initiative and leadershipshownbytheVolumeEditorsinselectingtopicsandassemblingtheexperts.Wehaveallbeen a bit ambitious in producing these Volumes on a timely basis and therein lies thepossibility of mistakes and oversights in this first edition. We encourage the readershipto draw our attention to any error, omission or inconsistency in this Series in order thatimprovements can be made in any subsequent edition.Finally, we wish to acknowledge the willingness of Springer-Verlag to host thisproject, which is envisioned to require more than 5 years of effort and the publicationof at least nine Volumes.Bochum, GermanyAuburn, AL, USAApril 1994Karl EsserPaul A. LemkeSeries Editors

Addendum to the Series PrefaceIn early 1989, encouraged by Dieter Czeschlik, Springer-Verlag, Paul A. Lemke andI began to plan The Mycota. The first volume was released in 1994, 12 volumes followedin the subsequent years. Unfortunately, after a long and serious illness, Paul A. Lemkedied in November 1995. Thus, it was my responsibility to proceed with the continuationof this series, which was supported by Joan W. Bennett for Volumes X–XII.The series was evidently accepted by the scientific community, because severalvolumes are out of print. Therefore, Springer-Verlag has decided to publish completelyrevised and updated new editions of Volumes I, II, III, IV, V, VI, and VIII. I am glad thatmost of the volume editors and authors have agreed to join our project again. I would liketo take this opportunity to thank Dieter Czeschlik, his colleague, Andrea Schlitzberger,and Springer-Verlag for their help in realizing this enterprise and for their excellentcooperation for many years.Bochum, GermanyFebruary 2007Karl Esser

Volume Preface to the Second EditionIn the concept of The Mycota series, Karl Esser (Series Editor) felt a need for a volumewhich summarized existing knowledge on fungal communities, their interactions witheach other and with biotic and abiotic factors of the environment, and which emphasizedthe role of fungi in ecosystem processes such as symbiotic relations and nutrientcycling. This was consequently published as Volume IV, Environmental and MicrobialRelationships, in 1997. Now – 10 years later and after a revolution in molecular techniquesand tools in this domain – there appeared an urgency for updating the volume,and we were invited to organize a second edition. Consequently, some reconstructionof content became necessary, particularly because some new chapters were deemed appropriateto document the progress in recently emerged areas of research. The originalconcept that authors should concentrate largely on overviews of main phenomena andmost recent achievements was maintained.The volume is partitioned into fours sections: ‘Life History and Genetic Strategies’;‘Determinants of Fungal Communities’; ‘Fungal Interactions and Biological ControlStrategies’; and ‘Decomposition, Biomass and Industrial Applications’.In the first, J.H. Andrews and R.F. Harris attempt to relate the evolution of fungito their ecological and physiological properties, approaching this question by addingthe dimension of trophic property to the conventional phylogenetic tree, and exploringbiochemical aspects further by using bioenergetic electron flow diagrams. The chapterby K. Brunner, S. Zeilinger and R.L. Mach reviews various modern molecular biologicalmethods applied for the correct identification of environmental isolates and communities,which have been essential in the progress in unravelling fungal ecology this pastdecade.The second section deals with the role of main environmental factors and ecosystemdisturbances in determining the species and population structure of fungal communities.S.J.Morris,C.F.FrieseandM.F.Allenevaluatethediversityandbiomassofmicrobialcommunities as direct indicators of the extent of the functional role played by these organismsin the dynamics of different ecosystems. They conclude that, if anthropogenicchanges alter the structure and biodiversity of microbial communities, then criticalfunctional roles in ecosystem- and global-level nutrient cycling are also likely impacted.R.M. Miller and D.J. Lodge elaborate on the impacts of various management practicesassociated with agriculture and forestry in influencing fungal structure and function,and discuss the hierarchical nature of soils and how this affects system response todisturbance by fungi. G.M. Gadd emphasizes how newly developed approaches usingmolecular biology and biomarkers are enabling a better understanding of communitystructure and responses to environmental factors, and pollutants. He concludes that –becauseofthecomplexityofthefungalgrowthform,multiplicityofbiologicalresponsesand interactions with pollutants, coupled with the complexity of terrestrial (and other)environments – “a wealth of knowledge still awaits discovery”. In reviewing examples offungi living in extreme environments, N. Magan stresses a critical need for experimentswhich better simulate fluctuating abiotic parameters and their impacts on the ecophysiologyof these fungi. E.M.J. Arnolds illustrates how a knowledge of global, regional

XIVVolume Preface to the Second Editionand endemic patterns of fungal distribution can serve to enhance understanding ofevolutionary processes and biodiversity patterns.The use of fungi for the biological control of various agricultural pests has beena leading theme in applied mycology during the past 10 years. This is dealt with in thethird section, on fungal interactions and biological control strategies. Mycoparasitism isconsidered to have an important influence on competitive interactions involving fungi innature, and in the biological control of phytoparasitic fungi. A. Viterbo, J. Inbar, Y. Hadarand I. Chet use Trichoderma as a model system of a typical mycoparasite, and criticallyreview how research on this phenomenon can be used to improve plant resistance tofungal pathogens. In the following chapter, S. Casas-Flores and A. Herrera-Estrellaemphasize the application of fungi in the biocontrol of nematodes, highlighting thebiochemical and molecular information available to date on this process. A.K. Charnleyand S.A. Collins outline the current state of knowledge of insect fungal pathogens as itrelates to their present use and future potential as mycoinsecticides. W.G.D. Fernando,R. Ramarathnam and T. de Kievit illustrate the use of bacteria in the biocontrol ofSclerotinia stem rot and blackleg diseases in canola. T.P. McGonigle shows how grazingcan affect fungal communities through effects on species richness, community diversity,and replacement of some species by others. P. Bayman illustrates the recent progressmade in the detection and understanding of endophytic fungi. M. Girlanda, S. Perottoand P. Bonfante emphasize the position of mycorrhizal fungi as a“tie that binds thehost plant to the biotic and abiotic environment”. J.B. Gloer offers numerous examplesshowing how observations in fungal ecology have generated hypotheses about fungalantagonism and defence which, in turn, have led to the discovery of novel bioactivefungal metabolites.The fourth section highlights fungus-mediated decomposition and the nutrientmobilizingpotentials of fungi in both aquatic (marine and freshwater) and terrestrialecosystems. J. Dighton illustrates how traditional concepts of nutrient cycling, whichstill today rely mainly on an understanding of leaf litter and wood decomposition,must be expanded when the role of fungal communities in driving nutrient cyclingin other, more transiently changing ecosystems is investigated. M.O. Gessner, V. Gulis,K.A. Kuehn, E. Chauvet and K. Suberkropp examine the role of fungi in plant litterdecomposition in two aquatic ecosystems in which fungal decomposers and plant litterdecomposition have been studied to the greatest extent: standing-dead shoots of emergentvascular plants in salt and freshwater marshes. Finally, in order to stimulate theapplication of fungal degradation of key plant cell wall macromolecules, which accumulatein enormous amounts annually, C. Gamauf, B. Metz and B. Seiboth illustrate thebiochemical and molecular genetic properties of different genera of fungi involved intheturnoverofthesecompounds.We hope that this volume will prove useful both for scientists who wish to updatethemselves in any of these research areas as well as to graduate students interested inobtaining a first, multifaceted overview. We are grateful to all individual contributorswho took the time and invested effort in collaborating with us on the updating of thisvolume, and especially that they all helped us to get this task finished within the expectedtime schedule.Wien, AustriaMarch 2007Christian P. KubicekIrina S. DruzhininaVolume Editors

Volume Preface to the First EditionIn their concept of The Mycota, Karl Esser and Paul Lemke (Series Editors) determinedthat a volume was needed to examine research on fungal populations and communities.WewereinvitedtoorganizeVolumeIV,Environmental and Microbial Relationships,and were instructed to concentrate on fungal responses to the physical environment,interactions with other fungi, microorganisms and invertebrates, and the role of fungiin ecosystem processes such as ecomposition and nutrient cycling. Individual chapterauthors were asked to concentrate on ecological themes that could be supported byselected studies in depth and to judge fungal systems for their promise as research tools.We were advised not to solicit exhaustive reviews to cover all ecological groups of fungi.Several authors were asked to emphasize the technology transfer of ecological information,showing how specific knowledge of the ecology and biology of fungi hasapplication in biological control, in enzymatic conversions of plant biomass, biodegradationof toxic organic pollutants, and the discovery of natural products. Here, we askedchapter authors to take into account the basic ecological underpinnings of such technologies.Initially, we intended to place chapters emphasizing biological control strategiesor industrial mycology in a section entitled “Technology transfer of ecological information.”However, it was found more desirable to integrate these specific chapters within theecological framework of the volume. We hope that this approach will better unite aspectsof ecological research classified as fundamental vs. applied mycology. The immediatechallenge for mycological ecology is to identify those examples where ecological studiesof fungi in nature provide basic information leading to the development of a particulartechnology. The book begins with a section entitled “Fungal life history and geneticstrategies.” Here, J.H. Andrews and R.F. Harris examine the interconversion of differentgrowth forms in a fungal life cycle, noting that the precise nature of the environmentalsignals which trigger these phases and how they are transduced by the organism areunknown. Fungal ecological genetics has become the most dynamic area of mycologicalresearch in the 1990s, largely due to the efforts of A.D.M. Rayner and colleagues at theUniversity of Bath. In the present volume, M. Ramsdale and A.D.M. Rayner outline someofthehighlightsofprogressmadeinrecentyearsandalsopresenttheirvisionforthefuture of fungal ecological genetics.The second section is concerned with the role of selected environmental factorsand ecosystem disturbance in determining the species structure of fungal communities.J.C. Zak and S.C. Rabatin begin by examining various experimental designs, approachesand methodologies for analyzing and describing fungal communities. They emphasizethe importance of scale in community ecology and argue that disturbance may bethe single most important process regulating the structure and functioning of fungalcommunities in nature. C.F. Friese, S.J. Morris and M.F. Alien evaluate disturbancedynamics over a wide array of scales and interacting factors, highlighted by a case linkingsite disturbance by harvester ants and the renewal of plant communities every 100 to1000 years. R.M. Miller and D.J. Lodge identify research needed to understand how fungirespond to disturbance created by management practices in agriculture and forestry.Disruption of mycorrhizal hyphal networks and the response of saprophytic hyphaehave impacts on soil structure and nutrient pools associated with the fungal hyphae.

XVIM. Wainwright and G.M. Gadd show how a more accurate assessment of the effects ofpollutants on the growth and activity of fungi in the environment has been made possibleby new methods for determining active fungal biomass and enlightened approaches to invitro experimentation. In reviewing examples of fungi living in extreme environments,N. Magan argues a critical need for experiments which better simulate fluctuating abioticparameters and their impact on the ecophysiology of these fungi. A knowledge of global,regional and endemic patterns of fungal distribution is important for the understandingof evolutionary processes and biodiversity patterns and E.J.M. Arnolds observes thatsuch information may be applied to the control and spread of crop pests as well as infungal conservation.The interactions of fungi with one another, with other microbes, nematodes andarthropods have produced a wealth of ecological information with the potential fordevelopment of biological control strategies. This is considered in the third section,“Fungal interactions and biological control strategies.” In reviewing fungal competition,P. Widden relates this knowledge to what is known about plant competition andthe predictive value of competition theory. Because many decomposer fungi have toreplace an existing microflora in order to colonize a substrate, interference competitioncan have an important impact on decomposition. P. Jeffries recognizes mycoparasitismas a widespread phenomenon and presents numerous examples of mycoparasitic interactions.Mycoparasitism is believed to have an important influence on competitiveinteractions involving fungi in nature, and in the biological control of phytoparasiticfungi. Trichoderma harzianum is presented as a model system of a typical mycoparasite.I. Chet, J. Inbar and Y. Hadar also examine research to improve plant resistanceto fungal pathogens by integrating cloned fungal chitinase with antifungal polypeptides.Again, Trichoderma is shown to be a useful model system. While recognizingthat mycoinsecticides have had little impact on insect pest control to date, A.K. Charnleysuggests a promising future for these entomopathogenic fungi. This optimism isbased on the current rate of progress in research on epizootology, mass production,formulation, application and mechanisms of pathogenesis. Fungal agents for controllingplant pathogenic nematodes often perform poorly or inconsistently because theyhave been released prematurely, without sufficient basic knowledge of their biology andecology. B. Kerry and B. Jaffee explain the importance relating basic information on themode of action and epidemiology of selected fungal biocontrol agents to methods ofmass production, formulation and application. In his examination of the potential ofmycoherbicides, D.A. Shisler emphasizes the importance of understanding phylloplanemicrobial dynamics in order to obtain consistent field efficacy of a mycoherbicide, includingstrategies for bolstering the pathogen at this same weak point. T. McGonigleshows how the selective grazing of soil and litter fungi by arthropods can have an impacton community structure. Fungi have responded in different ways to reduce the negativeeffects of predation. J.B. Gloer offers numerous examples showing how observations infungal ecology have generated hypotheses about fungal antagonism and defense which,in turn, have led to the discovery of novel bioactive fungal metabolites.The final section recognizes fungus-mediated decomposition and the nutrient mobilizingpotentials of fungi in both aquatic (marine and freshwater) and terrestrialecosystems. J. Dighton considers aspects of metal ion accumulation and enzymaticcompetence of saprotrophic fungi. Such basic information can be applied to the degradationof toxic organic compounds and the uptake and accumulation of metal ions incontaminated soils. J.R. Leake and D.J. Read review evidence that mycorrhizae in decomposinglitter have a direct role in recycling of organic nutrients. There is surprisinglylittle information on the extent to which processes of nutrient mobilization occur innature.M.O.Gessner,K.SuberkroppandE.Chauvetexaminetheroleoffungiinplantlitter decomposition in aquatic environments (e.g. salt marshes, mangrove swamps andstreams), and they observe that mechanisms controlling the allocation of resources

Volume Preface to the First EditionXVIIbetween mycelium and reproductive structures are not yet understood. In an effort toencourage lateral thinking, M.J.R. Nout and co-authors examine different approachesfor estimating fungal biomass in food fermentations and consider the appropriatenessof various approaches for fungal ecologists investigating litter decomposition. Likewise,the bioconversion of plant fibres to fuel, feed or precursors for chemical syntheses isexamined by R. Sinsabaugh and M.A. Liptak, scientists whose principal research interestis in the biochemistry of plant litter decomposition and the ecology of the decomposersin nature.We hope that not only professional biologists who wish to learn research directionsand opportunities in mycological ecology will find this volume interesting, but alsothat graduate students in mycology and microbial ecology will find it useful. Thanksare especially due to those individual contributors who produced outstanding originalfigures and to all for promptly responding to our various editorial requests.Peoria, Illinois, USALund, SwedenJanuary 1997Donald WicklowBengt SoderströmVolume Editors

ContentsLife History and Genetic Strategies1 EvolutionaryEcologyoftheFirstFungiJ.H. Andrews, R.F. Harris ........................................ 32 Molecular Approaches for Studying Fungi in the EnvironmentK. Brunner, S. Zeilinger, R.L. Mach ............................... 17Determinants of Fungal Communities3 Disturbance in Natural Ecosystems:Scaling from Fungal Diversity to Ecosystem FunctioningS.J.Morris,C.F.Friese,M.F.Allen ................................ 314 Fungal Responses to Disturbance: Agriculture and ForestryR.M. Miller, D.J. Lodge .......................................... 475 Fungi and Industrial PollutantsG.M. Gadd ...................................................... 696 Fungi in Extreme EnvironmentsN. Magan ....................................................... 857 BiogeographyandConservationE.J.M. Arnolds .................................................. 105Fungal Interactions and Biological Control Strategies8 Plant Disease Biocontrol and Induced Resistance via Fungal MycoparasitesA. Viterbo, J. Inbar, Y. Hadar, I. Chet ............................. 1279 Antagonism of Plant Parasitic Nematodes by FungiS. Casas-Flores, A. Herrera-Estrella ............................ 14710 Entomopathogenic Fungi and Their Role in Pest ControlA.K. Charnley, S.A.Collins ....................................... 15911 Bacterial Weapons of Fungal Destruction: Phyllosphere-TargetedBiological Control of Plant Diseases, with Emphasis on Sclerotinia Stem Rotand Blackleg Diseases in Canola (Brassica napus L.)W.G.D. Fernando, R. Ramarathnam, T. de Kievit ................... 189

XXContents12 Effects of Animals Grazing on FungiT.P. McGonigle .................................................. 20113 Fungal EndophytesP. Bayman ...................................................... 21314 Mycorrhizal Fungi: Their Habitats and Nutritional StrategiesM.Girlanda,S.Perotto,P.Bonfante ............................. 22915 Applications of Fungal Ecology in the Searchfor New Bioactive Natural ProductsJ.B. Gloer ...................................................... 257Decomposition, Biomass and Industrial Applications16 Nutrient Cycling by Saprotrophic Fungi in Terrestrial HabitatsJ. Dighton ...................................................... 28717 Fungal Decomposers of Plant Litter in Aquatic EcosystemsM.O. Gessner, V. Gulis, K.A. Kuehn, E. Chauvet, K. Suberkropp ....... 30118 Degradation of Plant Cell Wall Polymers by FungiC.Gamauf,B.Metz,B.Seiboth.................................... 325BiosystematicIndex ................................................. 341SubjectIndex ....................................................... 345

List of ContributorsM.F. Allen(e-mail: michael.allen@ucr.edu)Center for Conservation Biology, University of California,Riverside, CA 92521, USAJ.H. Andrews(e-mail: jha@plantpath.wisc.edu)Plant Pathology Department, University of Wisconsin,1630 Linden Drive, Madison, WI 53706, USAE.J.M. Arnolds(e-mail: arnolds21@hetnet.nl)Holthe 21, 9411 Beilen, The NetherlandsP. Bayman(e-mail: pbayman@uprrp.edu)Departamento de Biología, Universidad de Puerto Rico–Río Piedras,P.O. Box 23360, San Juan, PR 00931, USAP. Bonfante(e-mail: paola.bonfante@unito.it)Department of Plant Biology, University of Torino,Viale PA Mattioli 25, Torino 10125, ItalyExtra AddressIPP-CNR, Viale PA Mattioli 25, Torino 10125, ItalyK. Brunner(e-mail: brunner@mail.zserv.tuwien.ac.at)FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, AustriaS. Casas-Flores(e-mail: scasas@ipicyt.edu.mx)División de Biología Molecular,InstitutoPotosinodeInvestigaciónCientíficayTecnológica,Camino a la Presa de San José 2055, 78210 Tangamanga, San Luis Potosí SLP, MéxicoA.K. Charnley(e-mail: bssakc@bath.ac.uk)Department of Biology and Biochemistry, University of Bath,Claverton Down, Bath BA2 7AY, UK

XXIIList of ContributorsE. Chauvet(e-mail: echauvet@cict.fr)Laboratoire d’écologie fonctionnelle – EcoLab, UMR 5245 CNRS,University Toulouse 3, National Polytechnic Institute of Toulouse,29, rue Jeanne Marvig, 31055 Toulouse Cedex, FranceI. Chet(ilan.chet@weizmann.ac.il)Department of Plant Sciences, Weizmann Institute of Science,Rehovot 76100, IsraelS.A.CollinsDepartment of Biology and Biochemistry, University of Bath,Claverton Down, Bath BA2 7AY, UKJ. Dighton(e-mail: dighton@camden.rutgers.edu)Rutgers University Pinelands Field Station,P.O. Box 206, 501 Four Mile Road, New Lisbon, NJ 08064, USAW.G.D. Fernando(e-mail: D_Fernando@Umanitoba.ca)University of Manitoba, Department of Plant Science,66 Dafoe Road, Winnipeg, Manitoba R3T 2N2, CanadaC.F. Friese(e-mail: Carl.Friese@notes.udayton.edu)Department of Biology, University of Dayton,300 College Park, Dayton, OH 45469, USAG.M. Gadd(e-mail: g.m.gadd@dundee.ac.uk)Division of Environmental and Applied Biology,College of Life Sciences, University of Dundee,Dundee, DD1 4HN, Scotland, UKC. Gamauf(e-mail: ch_gamauf@gmx.at)FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, AustriaM.O. Gessner(e-mail: gessner@eawag.ch)Department of Aquatic Ecology,Eawag: Swiss Federal Institute of Aquatic Sciences and Technology,and Institute of Integrative Biology (IBZ), ETH Zurich,Überlandstrasse 133, 8600 Dübendorf, SwitzerlandM. Girlanda(e-mail: mariangela.girlanda@unito.it)Department of Plant Biology, University of Torino,Viale PA Mattioli 25, Torino 10125, ItalyJ.B. Gloer(e-mail: james-gloer@uiowa.edu)Department of Chemistry, University of Iowa,Iowa City, IA 52242, USA

List of ContributorsXXIIIV. Gulis(e-mail: v.gulis@ua.edu)Department of Biological Sciences, University of Alabama,Tuscaloosa, AL 35487-0206, USACurrent address:Department of Biology, Coastal Carolina University,Conway, SC 29528-6054, USAY. Hadar(e-mail: hadar@agri.huji.ac.il)Department of Plant Pathology and Microbiology,The Hebrew University of Jerusalem, Faculty of Agriculture,Rehovot 76100, IsraelR.F. Harris(e-mail: rfharris@wisc.edu)Soil Science Department, University of Wisconsin,1525 Observatory Drive, Madison, WI 53706, USAA. Herrera-Estrella(e-mail: aherrera@ira.cinvestav.mx)Laboratorio Nacional de Genómica para la Biodiversidad,Cinvestav Campus Guanajuato, Km 9.6 Libramiento Norte Carretera Irapuato-León,A.P. 629, Irapuato 36500, Guanajuato, MéxicoJ. Inbar(kobi.inbar@gmail.com)Department of Plant Sciences, Weizmann Institute of Science,Rehovot 76100, IsraelPresent address:P.O. Box 5592, Yavne 70600, IsraelT. de Kievit(e-mail: dekievit@ms.umanitoba.ca)University of Manitoba, Department of Microbiology,Winnipeg, Manitoba R3T 2N2, CanadaK.A. Kuehn(e-mail: kevin.kuehn@usm.edu)Department of Biological Sciences, The University of Southern Mississippi,Hattiesburg, MS 39406-0001, USAD.J. Lodge(e-mail: djlodge@caribe.net)International Institute of Tropical Forestry, USDA - Forest Service,P.O. Box 1377, Luquillo, PR 00773, USAR.L. Mach(e-mail: rmach@mail.zserv.tuwien.ac.at)FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, AustriaN. Magan(e-mail: n.magan@cranfield.ac.uk)Applied Mycology Group, Cranfield Health, Cranfield University,Barton Road, Silsoe, Bedford MK45 4DT, UK

XXIVList of ContributorsT.P. McGonigle(e-mail: mcgoniglet@brandonu.ca)Department of Botany, Brandon University,270-18th Street, Brandon, Manitoba R7A 6A9, CanadaB. Metz(e-mail: bmetz@mail.zserv.tuwien.ac.at)FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, AustriaR.M. Miller(e-mail: rmmiller@anl.gov)Biosciences Division, Argonne National Laboratory,9700 S. Cass Avenue, Argonne, IL 60439, USAS.J. Morris(e-mail: sjmorris@bradley.edu)Biology Department, Bradley University,1501 W. Bradley Avenue, Peoria, IL 61625, USAS. Perotto(e-mail: silvia.perotto@unito.it)Department of Plant Biology, University of Torino,Viale PA Mattioli 25, Torino 10125, ItalyR. Ramarathnam(umramara@cc.umanitoba.ca)University of Manitoba, Department of Plant Science,66 Dafoe Road, Winnipeg, Manitoba R3T 2N2, CanadaB. Seiboth(e-mail: bseiboth@mail.zserv.tuwien.ac.at)FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, AustriaK. Suberkropp(e-mail: ksuberkp@bama.ua.edu)Department of Biological Sciences, University of Alabama,Tuscaloosa, AL 35487-0206, USAA. Viterbo(e-mail: ada.viterbo@weizmann.ac.il)Department of Plant Sciences, Weizmann Institute of Science,Rehovot 76100, IsraelS. Zeilinger(e-mail: szeiling@mail.zserv.tuwien.ac.at)FB Molekulare Biochemie der Pilze, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, Austria

Life History and Genetic Strategies

1 Evolutionary Ecology of the First FungiJ.H. Andrews 1 ,R.F.Harris 2CONTENTSI. Introduction ........................ 3II. Biogeochemical Settingand Geological Timescale .............. 3III. Phylogenetic Evidenceand its Interpretation ................. 4IV. Eukaryote Evolutionand Fungal Phylogeny ................. 5V. Bioenergetic Analyses Appliedto Evolutionaryand Phylogenetic Relationships ......... 11VI. Conclusions ......................... 13References.......................... 14I. IntroductionOther than the origin of life, arguably the othermajor evolutionary benchmarks are the symbioticorigin and phylogenetic radiation of eukaryotes,and the inception of aerobiosis. Intertwinedwith these developments, and of particular interestto mycologists, is a third event, the origin of thefungi.Here, we review current thinking on the originof eukaryotes, and the position of the fungi withinthe so-called tree of life. To provide a contextfor this discussion, we begin with a consensusgeological timescale for dating the key geologicaland evolutionary events. This leads to an overviewofthetypesandstrengthofevidenceusedtoconstructphylogenies, followed by the phylogeniesthemselves summarized as a phylogenetic treestarting with the origin of life, and focusing oneukaryotes, fungi, and alphaproteobacteria (thepresumed ancient mitochondrial endosymbiont).The tree is expanded to include energy-generatingand other ecophysiological trophic traits of themajor taxa, which allows identification of the taxa1 Plant Pathology Department, 1630 Linden Drive, University ofWisconsin, Madison, WI 53706, USA2 Soil Science Department, 1525 Observatory Drive, University ofWisconsin, Madison, WI 53706, USAthat are closest trophically to the mitochondrialand eukaryotic root. We conclude by depicting theenergy-generating trophic relationships betweenfungi and alphaproteobacteria as a functionof a simple, quantitative, bioenergetic electronflow framework (linked electron donors andacceptors) based on integrated thermodynamicand biochemical principles. The framework allowsvisual comparison of dissimilatory reactions andpathways that can, and cannot, proceed underambient and standard environmental conditions.Further, it identifies energy connections betweenthe mitochondrial endosymbiont and aerobicand anaerobic fungi, and allows quantitativeassessment of hypotheses for the symbiotic originand evolutionary ecology of eukaryotes.II. Biogeochemical Settingand Geological TimescaleDiverse evidence suggests that Earth originatedabout 4.5 Gyr (Gyr = Giga or billion years ago) froma collision between planetismals, followed by coalescenceof the debris (Nisbet and Sleep 2001). Aftera period of cataclysmic chaos as Earth consolidatedand cooled, a lithospheric crust was formed, andtemperatures became conducive to the prolongedexistence of liquid water as part of a functional hydrosphere.This set the primordial stage for chemicalevolution toward systems supporting the organicprecursors of life, followed by evolution ofself-replicating, progenotic assemblages bridginginto prokaryotic and eukaryotic organisms. Thefour geological timescales (eons) are the:(a) Hadean (from origin of planet, ∼4.5 Gyr,totheorigin of life, ∼4.0 Gyr);(b) Archaean (4.0–2.5 Gyr);(c) Proterozoic (2.5–0.56 Gyr); and(d) Phanerozoic (0.56 Gyr–present).Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

4 J.H. Andrews and R.F. HarrisOur focus is on the late Archaean and Proterozoic(Nisbet and Sleep 2001).Life probably originated by the early Archaean(∼3.8 Gyr). Fossil evidence is strong (see sectionbelow on Phylogenetic Evidence) by the mid-Archaean (3.6–3.3 Gyr) forwidespreadbacterialbiofilms and stromatolites (“organosedimentarystructures produced by microbial trapping,binding, and precipitation, generally but notalways photosynthetic”; Nisbet and Sleep 2001).Geochemical markers, including those typical ofcyanobacteria, imply that oxygenic photosynthesiswas occurring by 3.5 Gyr, and by 2.8–2.2 Gyratmospheric oxygen levels were significant, asinterpretedfromoxidizedrock(NisbetandSleep2001; Bekker et al. 2004). This was sufficient tosupport aerobic respiration, a key event associatedwith the evolution of mitochondria, though highrates of oxygen utilization kept the levels at 1–2%of present-day concentrations until about 1.9 Gyr,when levels rose to at least 15% of present-dayvalues (Knoll 1992; Bekker et al. 2004). Of course,anaerobic environments persist to this day in habitatssuch as the gut tract and anoxic sediments.Unicellular eukaryotes (protists), arisingmore or less concurrently with mitochondria,were present by 1.5 Gyr, and possibly muchearlier (Knoll 1992; Nisbet and Sleep 2001; Dyallet al. 2004). Fossils interpreted as presumptiveeukaryotes are dated at 1.45 Gyr (Javaux et al.2001), and a well-established fossil benchmark fora multicellular red alga protist (Bangiophyceae),i.e., implying the presence of both mitochondriaand plastids, was recovered from 1.25–0.75 Gyrcarbonate (sedimentary) rocks in arctic Canada(Butterfield et al. 1990). A “probable fungus”(Tappania sp.) dated at 1.43 Gyr (Butterfield 2005)has been recovered from Australian rock.III. Phylogenetic Evidenceand its InterpretationPrimarilytwoformsofevidencehavebeenusedto reconstruct the history of life: paleontological(fossil or morphological), and molecular sequence(nucleotide or protein) information. The formeris dated by biophysical or geochemical methods(Schopf et al. 1983; Knoll 1992); the latter, by molecularclock assumptions (Wilson et al. 1987). (Thiscategorization excludes evidence of life inferredfrom geochemical profiles or signals; Knoll 1992;Nisbet and Sleep 2001.) While morphological andmolecular lines of data are complementary andhavegenerallybeeninbroadagreement(BakerandGatesy 2002; Smith and Peterson 2002; Donoghueand Smith 2004), there are notable exceptions thathave not been reconciled. Both approaches involvenumerous inferences and assumptions, and havesignificant constraints, alluded to briefly as follows.FossilsRobust fossil evidence depends on a readily interpretable,accurately dated specimen. The fossilprovides a minimum possible age for that taxon(and for sister taxa and preceding lineages; Berbeeand Taylor 1999, 2001; Smith and Peterson 2002).Departure from the ideal specimen occurs becauseof both geological and biological limitations. Evenamong paleontologists, there is considerable controversyand subjectivity in interpreting retrievedspecimens (Brasier et al. 2002). No rocks are yetknown from the estimated origin of the planet (theoldest rocks reported to date are about 4 Gyr;Sternet al. 1998). For body fossilization to occur, generallythe organism needs to be coated very quicklyby sediment. Sedimentation associated with theearly planet has been altered frequently, obscuringfossils, and under the best of conditions (shallowwater) sediments are only sporadically distributed(Schopf et al. 1983). Thus, the early fossil recordsare more or less obliterated, with the first evidencefor microbes appearing as stromatolites (3.5 Gyr,see above; Nisbet and Sleep 2001), and most ofthe macrofossils appearing over the past 0.6 Gyr(Schopf et al. 1983; Knoll 1992). Fossilization isfacilitated by mineralized, or otherwise resistantmaterials not possessed by many organisms; thosereadily fossilized may not occur in geographic regionsgeologically suitable for preservation (Schopfet al. 1983). Because of their size and composition,fungi are not as well suited as dinosaurs for fossilization!Furthermore, much of microbial evolutionis internal, reflecting changes in genetics, biochemistry,orultrastructure,ratherthaninexternalbody form (see Sect. IV., Eukaryote Evolution andFungal Phylogeny). This results in what has imaginativelybeen called “the Volkswagen syndrome”(Schopf et al. 1983), alluding to the many generationsof visually identical VW ‘beetles’. Finally,homoplasy (similarity in features among taxa forreasons such as convergence, i.e., other than commonancestry) may be a confounding factor, particularlyin interpreting morphological evidence

Evolutionary Ecology of the First Fungi 5(Givnish and Sytsma 1997; but see Baker and Gatesy2002).MoleculesWhile it is often asserted confidently that “sequencesdon’t lie”, the data and analyses leadingto inference of a particular sequence or phylogenyare not as objective as implied by this remark(Hillis et al. 1996; for fungi, see Berbee and Taylor1999). The molecular approach dates essentiallyfrom the work by Zuckerkandl and Pauling, amongothers (reviewed by Wilson et al. 1987; Donoghueand Smith 2004), showing that the rates of aminoacid replacement due to mutational change wereabout constant among lineages for the proteinscytochrome c and hemoglobin. This suggestedthat the times of divergence for pairs of speciescan be calculated. If fossil evidence is availablefor certain specimens to calibrate the clock, thenit can be used in principle to estimate divergencetimes for species for which no fossil evidence exists(Wilson et al. 1987), including fungi (Berbee andTaylor 2001). Despite much research showing thatthe clock ticks irregularly (Wilkins 2002; Thomaset al. 2006), it is still used widely as a phylogeneticinstrument. Among the early, retrospectivelyerroneousplacementsontheuniversal‘treeoflife’ were the amitochondriate eukaryotes, thehyperthermophilic bacteria, and probably, theroot of the tree (Gribaldo and Philippe 2004).Perhaps the largest assumption from molecularapproaches, which is only rarely even mentioned,is that the gene- or protein-based trees portrayedin countless diagrams actually equate with organismtrees, notwithstanding the fact that they arelabeled as such. Beyond this, there are two basicsorts of deficiencies (Gribaldo and Philippe 2004)arising from either reconstruction models or basicbiological processes. A single phylogenetic treeis usually specified by a model, yet trees are constructedby different rules (e.g., based on evolutionarydistance, or maximum parsimony, or maximumlikelihood) with different topologies and estimationsof ancestral relationships (Lutzoni et al.2001; Pace 2004). Early generation mathematicaldepictions made misleadingly simplistic assumptionsthat potentially caused the wrong tree to bespecified with strong support (Embley and Martin2006). Probably the most notorious artifact, termedlong-branch attraction, refers to clustering of thelongest branches due to variable evolution rates(i.e., the most rapidly evolving taxa, not necessarilythe most closely related, are grouped; Gribaldo andPhilippe 2004). Other errors arise from loss of mutationalsignal in excessive noise (mutational saturation);clustering of taxa due to DNA base composition(e.g., biased by similar G+C content); andvariable substitution rates within a protein acrosslineages (heterotachy; Gribaldo and Philippe 2004).When phylogenies from different proteins or genesconflict for the same group of species, this may bedue to artifacts, or to lateral gene transfer (Gribaldoand Philippe 2004). At least partial solutions tomostoftheseproblemsarenowavailable,includingmore sophisticated grouping functions in newermathematical models (Gribaldo and Philippe 2004;Embley and Martin 2006).Further discussion of this important issue isbeyond the scope of this chapter, but recognitionthat significant pitfalls exist for both morphologicaland molecular methods is important, so that theyare used and interpreted cautiously, preferably inunison (Givnish and Sytsma 1997; Baker and Gatesy2002).IV. Eukaryote Evolutionand Fungal PhylogenyEukaryotic EmergenceFigure 1.1 presents a stylized phylogenetic treestarting with the origin of life, and focusing oneukaryotes, fungi, and alphaproteobacteria (thehypothetical ancient mitochondrial endosymbiont).We have expanded the tree to includeenergy-generating and other ecophysiologicaltrophic properties of the major taxa relative tothose of alphaproteobacteria. The closest functionalmatches potentially identify the taxa closestto the mitochondrial eukaryotic root (either complementingor conflicting with the phylogeneticposition) and/or the taxa exposed to environmentalevolutionary pressures similar to those of theoriginal alphaproteobacterial endosymbiont.While it was once tacitly assumed that eukaryotessimply emerged from prokaryotes, the currentinterpretation is contentious and much morecomplex (Roger 1999; Martin and Russell 2003;Horner and Hirt 2004; Martin et al. 2003; Embleyand Martin 2006; de Duve 2007). (For convenienceand consistency with convention in virtuallyall the literature [e.g., Adl et al. 2005], werefer collectively to the bacteria and archaea asprokaryotes; we note that Pace [2006] has argued

6 J.H. Andrews and R.F. HarrisFig. 1.1. A view of eukaryote evolution from the originof life, recognizing trophic properties of the major lineagesand focusing on the fungi. This view integrates recent informationand concepts (Roger 1999; Richards and Cavalier-Smith 2005; Embley and Martin 2006; Pace 2006; Jameset al. 2006; Kurland et al. 2006; Steenkamp et al. 2006; deDuve 2007), with the trophic ancestral origin of life (Kurlandet al. 2006) amplified and expanded to include theancestral origin of eukaryotes and fungi, and the trophicproperties of the major lineages. Broken lines identify controversy(e.g., existence or not of amitochondriate Eukarya,and sisterhood or descendant relationship of Eukarya toArchaea). All trophic categories assigned to the differenttaxa are based on extant properties. Unless specified otherwise(footnotes 1–6 ), the unikont taxa are trophically simplemitochondrial organisms

Evolutionary Ecology of the First Fungi 7against continued use of this term.) In terms of energygeneration trophic properties, the alphaproteobacterialmitochondrial ancestor of eukaryotesis generally considered to have been chemotrophic(chemical energy), rather than phototrophic (primaryenergy light), organotrophic (organic electrondonor) or litho-organotrophic (organic andsupplementary inorganic sulfur e − donor), ratherthan lithotrophic (inorganic electron donor), andaerobic (O 2 electron acceptor for energy generation)or facultatively anaerobic (O 2 and alternativeelectron acceptor for energy generation) (Cavalier-Smith 2004; Richards and Cavalier-Smith 2005; Embleyand Martin 2006), although as recognized inFig. 1.1, extant alphaproteobacteria are more diversethan this. The fact that eukaryotes split intoeither chemotrophs or oxygenic phototrophs arguesagainst an anoxygenic phototrophic alphaproteobacteriumas the ancestral mitochondrial endosymbiont.The two most common scenarios for the emergenceofeukaryotesareeither(1)anucleus-bearing,amitochondriate, proto-eukaryote cell acquiringan alphaproteobacterial endosymbiont, andgiving rise to a mitochondrion-bearing eukaryote,or (2) a prokaryote (specifically archaeal) hostacquiring an alphaproteobacterial endosymbiontthat became a mitochondrion, with the recipientbecoming progressively more eukaryote-like(acquiring or evolving a nucleus, cytoskeleton,etc.; Dyall et al. 2004; Embley and Martin 2006;Margulis et al. 2006). Neither hypothesis is entirelycompelling, the former being deficient becauseno extant, primitively amitochondriate eukaryoticorganisms have yet been found (Embley andMartin 2006), the latter because extant prokaryotesare not phagocytic, and with one exception, are not1,3 Chytridomycota (para- or polyphyletic) include hydrogenosomalanaerobes such as Neocallimastix and Piromyces (Yarlett andHackstein 2005). The initial lineage is represented by Rozella.2 Microsporidia are composed of mitosomal obligate anaerobessuch as Tracipleistophora and Encephalitozoon (Van der Giezenet al. 2005).4 Glomeromycota (formerly Glomales of Zygomycota) are obligateendoparasites of photoautotrophs (Schüßler et al. 2001).5 Ascomycota include mitochondrial, facultatively anaerobic fermenterssuch as Saccharomyces (Tielens et al. 2002); denitrifiers andammonia fermenters such as Aspergillus and Fusarium (Takasakiet al. 2004); methylotrophic yeasts such as Pichia and Candida(Nakagawa et al. 2005) and mycelial fungi such as Trichoderma,Gliocladium,andPaecilomyces (Tye and Willetts 1977).6 Amoebozoa include mitochondrial aerobes such as Phalansterium,the potential ancestral heterotrophic aerobic uniciliateeukaryote (Cavalier-Smith 2004), and mitosomal anaerobic Archamoeba/Endamoebasuch as Entamoeba (Van der Giezen et al.2005).known to harbor symbionts (Cavalier-Smith 2002,2004; Embley and Martin 2006). While there is considerableevidence that eukaryotic informationalgenes have archaeal homologs, and that eukaryoticoperational genes have bacterial homologs(Embley and Martin 2006; Kurland et al. 2006),the Eukarya have unique “signature proteins”, asdo the other two domains (Hartman and Fedorov2002). Such commonalities may suggest early,failed experiments in symbiosis, or gene transferevents among the three major domains Bacteria,Archaea, and Eukarya (see below, and Pace 2004).Pace states that “molecular trees based on rRNAand other reliable genes show unequivocally thatthe eukaryotic nuclear line of descent is as old asthe archaeal line” (2005, p. 57). He further believesthat “the modern kind of eukaryotic cell [sic],with organelles, probably also arose early, morethan 3.5 Byr ago” ([sic; Byr = Gyr] Pace 2004, p.84). Consequently, we show in Fig. 1.1 the origin oflife and the universal ancestor as unspecified, withvarious evolutionary interconnections possible(dottedlines)amongthethreedomains.Fungal EmergenceProceeding up the tree, the most fundamentaldivision among the eukaryotes is the split betweenthe ancestrally uniciliate (unikont) and biciliate(bikont) lineages (Fig. 1.1; Cavalier-Smith 2002;Stechmann and Cavalier-Smith 2003; Richards andCavalier-Smith 2005; Embley and Martin 2006).Generally speaking, the unikonts include theanimals, fungi, and amoebozoa, while the bikontscomprise the plants, algae, and protozoans (Embleyand Martin 2006). Among organisms examinedto date, the genes for dihydrofolate reductase(DHFR) and thymidylate synthetase (TS) are fusedin bikonts, but separate in unikonts; additionally,protein trees and other gene fusion/duplicationdata support this major eukaryotic division (Stechmannand Cavalier-Smith 2003; Cavalier-Smith2004). From such evidence is drawn the presumptiveconclusion that the root of the eukaryote treeis between the bikonts and unikonts (Stechmannand Cavalier-Smith 2003; Cavalier-Smith 2004;Embley and Martin 2006).Fungi, animals, and various unicellular eukaryotes(the Choanozoa) together form the monophyleticOpisthokonta (Fig. 1.1), phenotypically diversebut linked mainly by a common molecularphylogeny (Steenkamp et al. 2006). This is an ancientlineage, perhaps at least 1 Gyr old (Knoll 1992;

8 J.H. Andrews and R.F. HarrisButterfield 2005). Depending on the classificationsystem, the microsporidia (obligate intracellularparasites mainly of animals) either are (Bullerwelland Lang 2005; Adl et al. 2005; James et al. 2006)or are not (Steenkamp and Baldauf 2004; Embleyand Martin 2006; Steenkamp et al. 2006) consideredwiththefungi,withwhichtheyareregardedtobeatleast very closely aligned (Hirt et al. 1999). A recentreconstruction of fungal evolution based on a sixgenephylogeny (James et al. 2006) shows the earliestdivergence within the kingdom Fungi containsthe microsporidia and the endoparasitic chytridRozella allomycis. The closest known relative to anancestral fungus is an amoeboid, phagotrophic Nuclearia(Fig. 1.1, and James et al. 2006).The ancestral fungus in Fig. 1.1 was likelya chemoheterotroph, and obtained its food asa parasite (or possibly as a commensal or mutualist),osmo/phagotrophically, with mitochondriaproducing energy by organotrophic or lithoorganotrophic,aerobic, and anaerobic metabolism(e.g., integration of Fig. 1.2a and b). Dependingon habitat selection pressures, the primordialosmo/phagotrophic fungus could early haveevolved into Rozella-type aerobic chytrids, andinto anaerobic microsporidia, by mitochondrialdegeneration into anaerobic mitosomes, and lossof phagotrophism. The chytrid line later evolvedinto Neocallimastix-type anaerobic chytrids, bydegeneration of mitochondria into hydrogenosomesand loss of phagotrophism, and then intoaerobic chytrids. Still later, the more advancedextant fungi evolved in part by physiologicallosses, and lateral gene transfer (LGT) gains withrespect to the ancestral fungus (Fig. 1.1).The general consensus from evidence basedon SSUrDNA sequences, protein trees, and limitedinformation from cellular structure and physiology,is that the animal and fungal kingdoms areeach monophyletic and the most closely related toone another (Steenkamp and Baldauf 2004; however,see comments below on geological age). TheChoanozoa, consisting of four classes, constitutea paraphyletic group, and the order of branchingof lineages within the opisthokonts is unclear(Steenkamp and Baldauf 2004). The choanoflagellates(aquaticuniflagellates)havebothanimal(sponges) and fungal similarities, suggesting thatthe Chytridiomycota, the oldest true fungal phylum,may have arisen from a chitinous thecatemember (Steenkamp and Baldauf 2004; Steenkampet al. 2006). Chytrids, which are aquatic (occasionallymarine) and to some extent soil-borne, haveFig. 1.2.a Bioenergetic electron flow diagram of mitochondrialandcytosolicdissimilatoryreactionsofextantaerobic and facultatively anaerobic fungi, and overlappingdissimilatory reactions of extant facultative alphaproteobacteria,under microaerophilic conditions. The e −transfer half-reaction couples are located in non-scalarline with decreasingly less negative e − transfer potential(Eh 7 ); standard activity neutral pH potentials (Eh 07 )areinparentheses. The potentials are expressed to 4 decimals toallow ΔG derivation identical to that using the ΔG 0 f of thecomponents in a completely mass-balanced equation. Thetotal available e − are in parentheses behind the compoundname. Dotted lines identify e − flow from e − donor to e −acceptor couples. In general, the number of e − transferredfrom/to a couple is standardized on a single or multiple 2e −basis. Appropriate prorating of the half-reaction couplesis needed to achieve transfer of the same number of e − , n,from the donor to the acceptor for a complete reaction. Theenergeticsofacompletereactionisafunctionofn,theFaradayconstant (F), and the difference in e − transfer potentialbetween the e − acceptor and e − donor couples, as shown inthe equation, with e − flowing downhill for an exergonic (energygenerating) reaction. The non-redox reactions showthe ambient (ΔG 7 ) and standard (ΔG 07 , in parentheses)energeticsof the reactions. Compounds enclosed by boxes areterminal metabolites. The enzymes or processes for a linkede − donor and e − acceptor, or non-redox reaction, arenumerically coded as follows. 1 NAD-linked glycolysis tophosphoenol pyruvate (PEP), 2 pyruvate kinase, 3F oxygenlinkedalcohol oxidase (fungi), 3a methanol dehydrogenase(α-proteobacteria), 4 NAD-linked formaldehyde dehydrogenaseand formate dehydrogenase, 5 NAD-linked acetatereduction to ethanol via acetyl CoA (reverse of reaction 6),6 NAD-linked alcohol dehydrogenase, CoA-acylating aldehydedehydrogenase, acetyl CoA synthetase, 7 NAD-linkednitrate reductase, 8 NAD-linked nitrite reductase, 9 NADlinkednitrate and nitrite reductase, 10 pyruvate transportinto mitochondria (fungi), 11 NAD-linked pyruvatedehydrogenase, 12 ATP production via acetate:succinateCoA-transferase, 13 NAD-linked acetate oxidation via TCAcycle conversion of oxaloacetate to succinate, 14 UQ-linkedsuccinate dehydrogenase, 15 fumarase, 16 NAD-linkedmalate dehydrogenase, 17 recycling of oxaloacetate backinto the TCA cycle, 18–24 respiratory e − carrier system,25 nitrate reductase, 26 nitrite/nitric oxide reductase,27 nitric oxide/nitrous oxide reductase, 28 denitrificationto nitrous oxide, 29 UQ-linked sulfide oxidasegenerally relatively sparse, coenocytic thalli, andare unique among the fungi in possessing uniflagellated(rarely polyflagellated) zoospores and gametes.Most extant species are saprobes, thoughseveral are parasites of plants, animals, or fungi.The unifying characteristics of the monophyleticgroup Fungi (Eumycota) include a nutritivemode based on absorptive heterotrophy(osmotrophy, rather than phagotrophy), andpossession of typically tube-like walls composedof β-glucan and chitin enclosing multi-nucleate

Evolutionary Ecology of the First Fungi 9protoplasm, with mitochondria or derivatives suchas hydrogenosomes or mitosomes present (Martinet al. 2003; Steenkamp and Baldauf 2004; Adl et al.2005). The so-called higher fungi, comprising theAscomycota and Basidiomycota, each of which ismonophyletic,formasistercladewiththemonophyleticGlomeromycota (as endomycorrhizae,obligate symbionts of photoautotrophs; Schüßler

10 J.H. Andrews and R.F. HarrisFig. 1.2.b Bioenergetic electron flow diagram of hydrogenosomaldissimilatory reactions of extant anaerobicfungi, and overlapping dissimilatory reactions of extantfacultatively anaerobic alphaproteobacteria (seeFig. 1.2a caption for explanation of figure structure).The codes for the enzymes or processes are as follows.1 NAD-linked glycolysis to phosphoenol pyruvate (PEP),2 pyruvate kinase, 3 phosphoenolpyruvate carboxykinase,4 pyruvate:formate-lyase, 5 NAD-linked alcohol dehydrogenase,6 NAD-linked lactate dehydrogenase, 7 NAD-linkedmalate dehydrogenase, 8 fumarase, 9 NAD-linked fumaratereductase, 10 and 11 pyruvate and malate import intohydrogenosome (fungi), respectively, 12 NAD-linked malicenzyme, 13 pyruvate:formate-lyase, 14 ATP production viaacetate:succinate CoA transferase, 15 NAD-linked hydrogenase,16a formic hydrogen lyase (alphaproteobacteria). Anasterisk means that the reaction is unique to fungi withinthe anaerobic eukaryoteset al. 2001; Steenkamp and Baldauf 2004; Jameset al. 2006; Steenkamp et al. 2006). The basalbranches are occupied by the Zygomycota andChytridiomycota, both of which are paraphyleticor polyphyletic, and for which the relationshipsand evolutionary pathways are unclear and controversial(Steenkamp and Baldauf 2004; Jameset al. 2006; Steenkamp et al. 2006). A general inferenceis that the primordial fungi were primarilyaquatic, and lacked aerial dispersal (James et al.2006). Numerous adaptations are apparent inthe life cycle as fungi colonized land (see below)along with animals and plants. Among these arean increase in filamentous habit optimized for

Evolutionary Ecology of the First Fungi 11absorptive nutrition based on saprotrophy orparasitism; loss of flagellated spores (on at leastfour separate occasions; James et al. 2006); andmultiple innovations for spore production, aerialdispersal, and survival.For reasons discussed above (see PhylogeneticEvidence and its Interpretation), estimation of thegeological history of the fungi based on fossil evidenceis often not in accord with inference frommolecular clock assumptions. Based on their owncalibrated molecular clock calculations and reviewof the literature, Berbee and Taylor (2001) estimatedthat the fungi diverged from the animallineage at about 1.0–0.9 Gyr, prior to terrestrialflora, though Butterfield’s (2005) claim of a “probablefungus” with characteristics similar to the zygomycetesdated at 1.43 Gyr from Australian rock,if correct, moves this back substantially. A majormulti-gene analysis of the plant–animal–fungi divergencetimesestimatedthesetobeapproximatelyconcurrent and relatively ancient at 1.58±0.09 Gyr(Wang et al. 1997). Subsequent radiation of the terrestrialfungi, roughly at 0.6 Gyr, likelyfollowedclosely the origin of land plants (Berbee and Taylor2001; see also Selosse and Le Tacon 1998), but alsoinvolved affiliations with algae before the evolutionof vascular plants (Heckman et al. 2001; Lutzoniet al. 2001; Yuan et al. 2005). The final major divergence,occurring perhaps at about 0.4–0.5 Gyr,was the splitting of the Ascomycota from the Basidiomycota(Berbee and Taylor 2001; Taylor et al.2004).Since much of microbial evolution is internal,rather than external, as alluded to at theoutset in this chapter, internal changes beyondultrastructure and physiology involve informational(replication, transcription, and translation)and operational (assimilatory biosynthesis, andenergy-generating dissimilatory metabolism)genes (Embley and Martin 2006). Energy generationis the province of mitochondria, and remnantmitochondria such as hydrogenosomes and mitosomes.LGT from the mitochondrion to the nucleushas occurred extensively over time, depending onecological role and environmental conditions (seebelow; Kurland and Andersson 2000; Bullerwelland Lang 2005). In addition, LGT from externalsourcestothenucleusalsohasoccurredfrequently(see below; Kurland and Andersson 2000; Gribaldoand Philippe 2004; Bullerwell and Lang 2005).Accordingly, any mitochondrial endosymbiontancestry of the dissimilatory processes carried outunder nuclear control in the cytosol is shroudedin history. However, LGT from the nucleus orexternal sources to the mitochondrion is much lesslikely (Kurland and Andersson 2000; Bullerwelland Lang 2005). Thus, genes retained in extantor remnant mitochondria may well be ancestralto the original mitochondrial endosymbiont.The general consensus is that extant eukaryoticaerobes and facultative anaerobes have evolutionarilyretained the inherited mitochondrialendosymbiont dissimilatory respiratory chainproperties within their mitochondria, but haveundergone extensive loss or intracellular transfersof other mitochondrial genes to the nucleus, andalso may have gained nuclear genes via externallateral gene transfer. Similarly, many extanteukaryotic anaerobes have apparently lost muchof their superfluous respiratory chain machinery,so that their mitochondria have evolved intoATP-generating, H 2 -producing hydrogenosomesor mitosomes (Embley and Martin 2006; Yarlettand Hackstein 2005). The only common denominatorof mitochondria, hydrogenosomes, andmitosomes is iron-sulfur cluster assembly (Vander Giezen et al. 2005; Embley and Martin 2006).From Fig. 1.1 it can be seen that, as a group, extantalphaproteobacteria have much more diversemetabolic properties than any of the eukaryotes.Standard logic in phylogenetics is that propertiesof ancestors can be inferred from commonalities ofthe descendants (Pace 1991). This leads to the furtherinference that more can be understood frommodern organisms that are more closely relatedto the earliest organisms than from those that areevolutionarily more distant (Pace 1991). Applyingthat rationale to physiology, the extant eukaryoticlineagewiththemostmetabolicoverlapwithextantalphaproteobacteria is evolutionarily closestto the endosymbiotic mitochondrial ancestor (Tielenset al. 2002) – fungi are that group (Fig. 1.1).V. Bioenergetic Analyses Appliedto Evolutionaryand Phylogenetic RelationshipsFigure 1.2a and b provide a mechanistic expansionof the energy-generating trophic relationshipsbetween fungi and alphaproteobacteria identifiedin Fig. 1.1, as a function of an operationally simple,quantitative, bioenergetic electron flow (linkedelectron donors and acceptors) framework basedon integrated thermodynamic and biochemical

12 J.H. Andrews and R.F. Harrisprinciples. This framework allows direct comparativevisualization of dissimilatory reactionsand pathways that can and cannot proceed underambient and standard environmental conditions,and identifies evolutionary energy connectionsbetween the mitochondrial endosymbiont andaerobic and anaerobic fungi. It also allows quantitativeassessment of hypotheses for the symbioticorigin of eukaryotes.Construction and interpretation of the electronflow diagrams are identified in the figure captionsand footnotes, and described in more detailelsewhere (Harris and Arnold 1995; Andrews andHarris 2000; Zwolinski et al. 2000). The focus ofFig. 1.2a and b is on comparison of the dissimilatoryreactions in fungal mitochondria and hydrogenosomes,respectively, as distinct from thecytosol, which overlap with the dissimilatory reactionsof extant alphaproteobacteria. In brief, theelectron transfer half-reactions shown in the figuresare dissimilatory electron donors and electronacceptors linked in diverse combinations; specificreactions and pathways of interest are codedfor the extant catalytic enzymes involved. The e −transfer half-reactions are ranked according to decreasinglyless negative e − transfer potential (decreasinge − donor/increasing e − acceptor potential)under ambient conditions. Qualitative energeticsare readily apparent by visual examination (downhillflow of e − identifying an exergonic reaction).If desired, quantitative energetics are readily derivedas ΔG=−nFΔE (Fig. 1.2a and b), where −ΔGrefers to the Gibbs free energy change of the reaction(with a negative sign denoting an exergonicreaction), n is the number of electrons transferred,F is the Faraday constant, and ΔE is the differencebetween the electron transfer potential of the electronacceptor and electron donor. This procedureisstraightforwardcomparedtothelabor-intensiveclassical construction of completely stoichiometricallybalanced equations, and calculation of ΔGasafunctionofenergyofformationandambientactivities of all reaction components (Amendand Shock 2001, cited by Martin and Russell 2003).The proton ionizable forms of the metabolites usedin the figures are those dominant at the ambientpH (7) as defined by the pKa; the Eh 07 data werederived from ΔG f 0 properties (Amend and Shock2001; Thauer et al. 1977), or for the respiratorychain e − carriers, obtained directly from the literature(Tielens et al. 2002; Voet and Voet 2005).Ambient activities used for the figures were generallyin the upper Km range, with the followingexceptions: (1) O 2 activity was set at an order ofmagnitude lower than the Km to provide highlymicroaerophilic conditions under which O 2 activitywould be marginal for enzyme accessibility; (2)oxaloacetate and H 2 activity, NAD(P)H/NAD(P) +ratios, were adjusted to levels needed for exergonicpathways. Operationally simple Eh adjustments fortenfold changes in ambient activities are given inthe figures.Overlapping with alphaproteobacteria (Gorrelland Uffen 1977; Garrity et al. 2005), the followingnoteworthy trophic properties of fungi can besummarized from Fig. 1.2a for aerobes and facultativeanaerobes (properties 1 and 2a–d), and fromFig. 1.2b for obligate anaerobes (property 3):(1) All aerobic fungi, together with other aerobiceukaryotes, have a classical mitochondrial respiratorychain (Fig. 1.2a, reactions 18–24) forenergy-conserving e − flow to O 2 (Tielens et al.2002).(2) Certain ascomycete fungi:(a) in common with amoeba and ciliated protozoans,can use hydrogen sulfide as a mitochondriale − donor (Fig. 1.2a, reaction 29)for detoxification, if not energy generation(Theissen et al. 2003);(b) in common with facultative protists, canuse intracellular organics as cytosolic e −acceptors for NAD + regeneration (e.g., acetatereduction to ethanol, Fig. 1.2a, reaction5) under O 2 -limiting conditions;(c) are the only eukaryotes capable of cytosolic,methylotrophicuseof1Ccompoundsas aerobic e − donors (Fig. 1.2a, reactions3F and 4; Tye and Willetts 1977; Nakagawaet al. 2005);(d) are the only eukaryotes capable of anaerobicmitochondrial respiration using nitratedenitrification to nitrous oxide as analternate e − acceptor (Fig. 1.2a, reactions25–28), and cytosolic nitrate reduction toammonium (Fig. 1.2a, reactions 6–9) asa mechanism for substrate-level ATP productionand NAD + regeneration (Tielenset al. 2002; Takasaki et al. 2004).(3) Anaerobic chytrids are unique within theeukaryotes in having hydrogenosomes thatgenerate energy by substrate-level ATP productionfrom malate oxidation to pyruvate,followed by pyruvate degradation to acetateand formate (pyruvate formate lyase pathway,PFL; Fig. 1.2b, reactions 12–14), accompanied

Evolutionary Ecology of the First Fungi 13by NAD(P)H-linked hydrogenase reductionof protons to H 2 (Fig. 1.2b, reaction 15;Yarlett and Hackstein 2005). The PFL pathwaycompares to the pyruvate-ferridoxin oxidase(PFO) pathway for pyruvate oxidation and H 2production by non-fungal hydrogenosomaleukaryotes (Yarlett and Hackstein 2005; Embleyand Martin 2006), a pathway that is minorand likely obtained by lateral gene transferover evolutionary time by alphaproteobacteriasuch as Rhodospirillum (Gorrell and Uffen1977).For anaerobic fungi, trophic categorization(property 3) of the Chytridiomycota as closest toalphaproteobacteria supports their phylogeneticplacement as the oldest fungal phylum (Fig. 1.1).In contrast, within aerobic and facultative eukaryotes,fungal ascomycetes are the closest toalphaproteobacteria based on energy-generationtrophic properties, and from this standpoint arethe closest to the mitochondrial root. This affinityis decreased if the cytosolic properties (2b, 2c andpart of 2d) are discounted, but not enough to affectthe outcome. However, this trophic identificationof ascomycetes as the oldest conflicts with thephylogenetic-based location of ascomycetes as theyoungest member of the fungal group (Fig. 1.1).A possible explanation is that the closest matchhypothesis is compromised by LGT and/or thepossibility that the ascomyetes involved wereexposed over time to environmental evolutionarypressures similar to those of the original alphaproteobacteriaendosymbiont, thereby retaining therelated, ecologically advantageous properties fromthe original alphaproteobacterium.Included in Fig. 1.2b (bottom right) is recognitionthat hydrogenosomal anaerobic eukaryotesoften form symbiotic relationships with chemoautolithotrophicpartners that make a living from theH 2 ,acetate,andbicarbonatewasteproductsoftheeukaryote. This maintains a low pool size of thesemetabolites, and results in beneficial dissimilatorye − transfer potentials favorable for exergonic reactionsby the eukaryotic partner. The quantitativelybased electron flow framework facilitates interpretationand prediction of whether such symbioticrelationships are thermodynamically feasible,or not, as a function of operational e − transferpotentials dictated by standard potentials andmetabolite pool sizes. Accordingly, in the exampleshown in Fig. 1.2b, the symbiotic relationshipwill fail for an H 2 pool size above about 10 5 mol/l(for this pool size, the Eh 7 for 2H + /H 2 would become∼ −0. 51 + 5 ∗ 0. 03 = − 0.36 V, whichistoonegative (uphill e − flow) for proton reduction byNADH/NAD + of Eh 7 = − 0.32 V).The symbiotic relationship in Fig. 1.2b is thebasis of the hydrogen hypothesis for the forcedriving the ancient endosymbiotic formationof protomitochondria from an H 2 -producingalphaproteobacterium endosymbiont and an H 2 -consuming archaeal methanogenic host (Embleyand Martin 2006). The viability of other currentand new hypotheses for primordial symbioticassociations may be supported, or discounted byuse of the bioenergetic electron flow framework.VI. ConclusionsEfforts to understand the two fundamental benchmarksin the history of life on earth – the originof living systems, and the origin of eukaryotes –have provoked arguably more thought and debatethan any other scientific question. With increasinglycomprehensive paleontological information,and substantial advances in molecular systematicsin the past two decades, more coherent scenariosare emerging and being subjected to critical test.However, like an exponential decline curve that infinitelyapproaches, but never reaches the axis, scientistscan approach, but will never achieve fullcomprehension of the primordial world, which isliterally lost in time.Therootoftheuniversaltreeandthenatureofthe universal ancestor are unclear, and likely to remainso, with relatively strong opinions supportedby relatively weak data (Cavalier-Smith 2002, 2004;Embley and Martin 2006; Kurland et al. 2006). Regardlessof their ultimate position in the tree oflife (perhaps now more shrub-like in form!), fungiand allies display incipient forms of key biologicaltrends, and hence are a particularly informativelineage. Fungi inhabit, as parasites, mutualists,commensals, or saprobes, most if not all terrestrialand aquatic habitats. They show, perhaps more sothan any other lineage, extreme phenotypic andgenotypic plasticity (Andrews 1991, 1992). This includesa system of balanced, coexisting genomeswithin one cell and one individual (the dikaryon),and life cycles that are variously either exclusivelyasexual, that bypass the sexual by unusual means(parasexuality), that alternate between sexual andasexual, or that can apparently run indefinitely as

14 J.H. Andrews and R.F. Harrisone or the other. They show interesting and occasionallycomplex life cycles, especially among theparasites, most notably the rust fungi. The phylogenetictransition from a relatively undifferentiated,motile, aquatic lifestyle (chytrids) to a differentiated,sessile,terrestriallifestylesetsinmotioncompetingforms of natural selection, one operating atthe cell lineage, the other at the level of the individual(Buss 1987). This in turn invokes isolatingmechanisms during ontogeny, and of self/non-selfrecognition systems characteristic of the eukaryotes(Buss 1987). These attributes are clearly quitedifferent from those of the bacteria or archaea.There is strong evidence that eukaryote cellsarose by symbiosis, and that the symbiont (protomitochondrion)was an alphaproteobacterium,though the nature of the recipient (archaebacteriumor proto-eukaryote) is unclear. Theconsensus now is that all eukaryotes contain mitochondriaof one sort or another (Martin et al. 2003;Embley and Martin 2006), and that certain earlyeukaryotes subsequently acquired other kinds ofsymbionts (cyanobacterial origin of plastids in thePlantae; Cavalier-Smith 2002; Dyall et al. 2004). Despitesome genetic homology and other commonattributes, the domains Archaea, Bacteria, andEukarya are quite different in ultrastructure, andin many cellular properties, including the cell wall(Cavalier-Smith 2002; de Duve 2007), not to mentionsizes and geometries. So, whatever the natureof the initial symbiosis, the eukaryotic descendantshave radiated into remarkably diverse extant formsunlike their ancestor. While numerical superiorityand genotypic diversity on Earth may be reflectedin the prokaryotes, the phenotypic diversity isrepresented overwhelmingly by the eukaryotes.This is testimony to the nature of the eukaryoticcell, environmental heterogeneity that set thestage for such radiation to occur, and energeticforces discussed further below. With the evolutionof progressively more sophisticated systems toharvest energy, especially light energy, togetherwith the use of oxygen as an electron acceptor,energy availability increased exponentially, andwith it, biodiversity.An important issue not addressed mechanisticallyby phylogenetics is what drove theevolutionary origins of the fungi and other earlyeukaryotes.Wehaveapproachedthisfunctionalquestion by adding the dimension of trophicproperty to the conventional phylogenetic tree(Fig. 1.1), and exploring the biochemical aspectsfurther by using bioenergetic electron flow diagrams(Fig. 1.2a, b). This shows that the Fungi areclearly closely related metabolically to the ancestralmitochondrial eukaryote and to the alphaproteobacterialendosymbiont. The Fungi are notablein being osmotrophs, chemotrophs, heterotrophs,organotrophs (some litho-organotrophs) withaerobic, facultatively anaerobic, or anaerobiccapabilities. Based on their osmotrophy andbiochemical versatility, in particular with respectto diverse energetic pathways, “a eukaryotic treewith fungi first would make sense”, according toMartin et al. (2003) who have updated a hypothesisof Cavalier-Smith from the early 1980s. Thoughthe trophic relationships are not always in closeaccord with phylogenetic placement, they addan important biological dimension in efforts tounderstand evolutionary patterns. 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2 Molecular Approaches for Studying Fungi in the EnvironmentK. Brunner 1 ,S.Zeilinger 2 ,R.L.Mach 1CONTENTSI. Introduction ........................ 17II. Immunological Detection of Fungi ....... 18III. PCR-Based Methods .................. 19A. QualitativeDiagnosticPCR.......... 20B. QuantitativePCR.................. 21C. Molecular Fingerprinting Technologies . 221. Denaturing Gradient GelElectrophoresis(DGGE) .......... 222. PCR-Restriction Fragment LengthPolymorphism (PCR-RFLP),Amplified Fragment Polymorphism(AFLP) and Random AmplifiedPolymorphicDNA(RAPD) ........ 23IV. Probe Hybridization Technologies ....... 24V. Conclusions ......................... 24References.......................... 25I. IntroductionThe accurate identification of fungi from differentecosystems is essential in the fields of medical science,plant pathology, environmental studies andbiological control. In phytopathology as well as animaland human pathology, the early identificationof the disease-causing agent is crucial for the timelyinitiation of countermeasures.Fungal community structure is rather difficultto be completely profiled in many environments,as individual ecosystems are generally complex,with fungi forming only one component of hugecommunity assemblages. Most fungal biology hasconcentrated on that part of the Fungal Kingdomwhich is culturable, visible to the naked eye, or discerniblemorphologically under the microscope.The very nature of physiological and biochemicalstudies requires organisms which can be cul-1 FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften,TU Wien, Getreidemarkt 9/166/5/2, 1060 Vienna, Austria2 FB Molekulare Biochemie der Pilze, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften, TU Wien,Getreidemarkt 9/166/5/2, 1060 Vienna, Austriatured, and species identification relies on isolation,cultivation, biochemical tests and morphology. Althoughculture-based approaches are still a cornerstonein fungal diagnostics, and have told us mostof what we presently know about fungal ecology,the methods used to isolate fungi tend to selectfor species able to grow only on particular media,and are therefore quite limited. Many fungalspecies are not culturable on given media, or fastgrowingspecies often obscure the slower growingones and, thus, the analysis might not reflectthetruefungalcommunityinasample(MacNeilet al. 1995). Furthermore, conventional identificationmethods are very time consuming, have to beperformed by skilled personnel, and can lead toproblems in identification – incorrect interpretation,diagnosis and disease treatment can be theconsequence. Furthermore, conventional methodsused for fungal detection are predominantly nonquantitative.To overcome the drawbacks of culture-basedidentification of organisms, throughout the lastdecade rapid screening technologies have beendeveloped and are nowadays established for almostall aspects of fungal identification. In contrastto conventional detection methods, samples canbe tested directly without any elaborate isolationand cultivation steps needed before proper classification.These novel detection methods includeimmunological tests, PCR-based technologies likerestriction length polymorphism (PCR-RFLP),random amplified polymorphic DNA (RAPD),amplified fragment length polymorphism (AFLP)and denaturing gel electrophoresis (DGGE), andan ever increasing number of diagnostic microarrays.These novel techniques are insensitive tomicrobial backgrounds and non-target organisms.The most recent technique enabling identificationof whole microbial communities, without theneed to cultivate any organisms, is metagenomics.Metagenomics is the whole-genome shotgunsequencing of pooled environmental DNA sam-Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

18 K. Brunner, S. Zeilinger, R.L. Machples. Although this method has been applied toenvironmental samples of diverse origin, and hassuccessfully revealed the composition of numerousprocaryontic communities, the application of thisnovel approach has not yet been established forfungal DNA.Immunological diagnostics rely on recognitionby antibodies, with specific antigens eitherpresented on the surface or secreted by the fungusas targets. As most of this methods are ELISA(enzyme-linked immunosorbent assays) based,they are simple to use and can be applied byunskilled personnel. Additionally, ELISA assaysallow quantification of the detected object andlaboratory-independent on-site testing. However,it can be difficult and expensive to raise antibodieswith the required specificity, and the applicationof this technique remained limited to a feworganisms. Although numerous detection assayshave been developed and commercially exploitedduring the 1990s, the increasing progress of a noveltechnique led to a replacement of ELISA tests.Polymerase chain reaction (PCR) became the mostreliable method for diagnostics and, since theend of the last century, almost no new immunologicaltests for fungal identification have beenpresented.The main advantage of PCR is that it is highlyspecific and sensitive enough to detect even minuteamounts of fungal DNA in environmental samples(Lee and Ward 1990). This method allows to distinguishbetween different species of a genus andeven within different populations (Lee and Ward1990; Ward 1995, 1998). Besides the high specificityand sensitivity, quantitative application (real-timePCR) has contributed to the breakthrough of PCRfor diagnostic applications. Related techniques likePCR-RFLP, RAPD, AFLP and DGGE, all based onPCR amplification of DNA, provide enough resolutionto separate and identify strains from wholefungal communities. To further enhance the detectionand discrimination capability of PCR, two-stepapproaches have been developed for the identificationof large numbers of fungal strains in a singletest cycle: for a start, DNA is amplified with universalprimers and, thereafter, hybridization withhighly specific probes distinguishes amplicons ofdifferent nucleic acid composition (Vanittanakomet al. 1998; Ingianni et al. 2001; Wu et al. 2002).Throughout the last decade, probe hybridizationtechnologies have developed into the modern microarrayapplications which have taken place alsoin fungal diagnostics. Generally, target DNA containingorganism-specific sequences is amplifiedby PCR with universal primers, labelled and hybridizedto the array. Microarrays allow the identificationof numerous organisms in a single run.Since the late 1990s, the use of fluorescent probeshas allowed the direct analysis of organisms in situ.This fluorescent in situ hybridization (FISH) hasbeen adapted for fungal identification, and investigationseven on unculturable organisms can beperformed.Even though the abovementioned up-to-datetechniques are commonly accepted as cultivationindependent, initial isolation and cultivation stepscannot be omitted completely. For ELISA tests, thetarget antigen has to be isolated in advance to allowthe production of suitable antibodies. To designspecific primer pairs for non-quantitative diagnosticPCR and real-time PCR or probes for varioushybridization techniques, diagnostic target DNAfragments from pure cultures must be sequenced.The essential knowledge about nucleic acid compositionresults in the inability to use these techniquesforanalysesofcomplexfungalcommunitiescomprising representatives of unknown genera orspecies. To overcome this bottleneck, methods likeRFLP, RAPD, AFLP, or the separation of mixtures ofPCR amplicons on a DGGE gel, have been adapted.These allow gaining an overview of the compositionof fungal populations in an ecosystem.II. Immunological Detection of FungiMost immunological tests are based on enzymelinkedimmunosorbent assay (ELISA), which wasdeveloped during the 1970s (Clark and Adams1977). This technique is simple to use, specific,allows high throughput testing, and can be usedquantitatively. One key factor is to develop antibodieswith the required degree of specificity.The development of antibodies against viruseshas generally been successful but this approachhasworkedlesswellforcomplexorganismssuchas fungi (McCartney et al. 2003). Another keyfactor for a successful detection is the abilityto recover sufficient amounts of antigens fromthe environmental sample, and the knowledgeabout degradation or retention time of the targetantigen in the sample. The dynamics of the antigenin the sample has to be well known to avoidmisinterpretation due to its rapid breakdown orlong retention time (Dewey et al. 1996).

Molecular Approaches for Studying Fungi in the Environment 19The use of monoclonal antibodies to detectfungi and to study fungal interactions with hostshas been applied mainly in two different fields,namely plant pathology and biological control. Iyerand Cousin (2003) developed an indirect ELISA todetect Fusarium contamination in foods. Antibodiesagainst F. graminearum and F. moniliforme proteinssucceeded in identifying 15 different Fusariumisolates – 13 species different from F. graminearumand F. moniliforme. The assay was tested forspecificity with 70 different moulds belonging to23 genera, and only two other strains gave a falsepositive signal.Otten et al. (1997) describe methods toimprove the use of immunoassays for the detectionand quantification of the fungal soil-borneplant pathogen, Rhizoctonia solani. Theauthorsused a monoclonal antibody which recognisesa catechol oxidase secreted by the hyphae ofRhizoctonia. The influence of different soil typeson the retention of the antigen was tested, anda thousand-fold reduction in sensitivity of theassay was determined for clay soil, comparedto sand and loam. This detection method hasthereafter been used to monitor the influenceof the soil-borne biocontrol agent Trichodermaon population dynamics of Rhizoctonia duringsaprophytic growth in compost-based soils(Thornton and Gilligan 1999). For the quantificationof Trichoderma during antagonisticinteraction, a monoclonal antibody-based ELISAhas been developed. The antibody MF2 detectsantigens of numerous species of the generaHypocrea/Trichoderma and Gliocladium but doesnot cross-react with common soil organisms(Thornton et al. 2002). This antibody binds to thehyphae and septa of its target organisms, and wasused to stain Trichoderma sp. while coiling aroundRhizoctonia during the mycoparasitic interaction.To specifically detect only actively growingmycelia, two different antibodies were producedwhich selectively recognise glycoproteins secretedfrom the growing tips of either Trichoderma orRhizoctonia (Thornton 2004). The authors demonstratedthat Trichoderma successfully competeswith the pathogen for nutrients and preventsthe saprophytic growth of Rhizoctonia, and nocross-reactivity was found with fungi naturallyoccurring in the soil. Other ELISA tests based onmonclonal antibodies are commercially availablefor the detection of Pythium, Phytophtora andSclerotinia (Ali-Shtayeh et al. 1991; Timmer et al.1993; Miller 1996; Miller et al. 1997).III. PCR-Based MethodsPCR is the exponential amplification of DNA withtwo short oligonucleotide primers which are complementaryto the 5 ′ and 3 ′ ends of the target sequencefragment. PCR is highly sensitive, and a fewtemplate strands can be amplified up to some microgramsof product DNA. This sensitivity, in combinationwith the specificity obtained by the twoprimers, makes PCR the most important techniquein molecular diagnostics nowadays. To further increasesensitivity and specificity, PCR-associatedtechniques like nested PCR have been developed.After performing the first conventional amplificationstep, a second PCR is added with two newprimers lying within the previously synthesizedDNA.NestedPCRallowsthedetectionofminuteamounts of DNA, several folds lower than normalPCR.The successful application of PCR-based diagnostictools generally requires three crucial steps:(1) the selection of a suitable DNA marker sequenceto accurately identify an organism, (2) extractionoftotalDNAfromthesampleand(3)amethodtoidentify the presence of the target sequence in theamplified DNA.Selection of a Specific Target DNA SequenceA proper selection of the target fragment to beamplified allows the detection of whole genera,inthecaseofprimersdesignedacrossconservedDNA regions, or the identification of a singlespecies even in a background of taxonomicallyrelated organisms. One of the main targets forthe development of diagnostic PCR assays are thegenes coding for the ribosomal RNA, which arepresentinallorganismsathighcopynumbers.Theabundance of this type of DNA facilitates detectionand, thereby, improves the sensitivity of the assay(White et al. 1990). The fungal nuclear ribosomalDNA consists of three genes encoding the 28S, 18Sand 5.8S subunits. These genes are separated byinternal transcribed spacer regions (ITS), and thisunit is repeated many times. The ITS region is ofparticular importance for fungal diagnostics asit consists of conserved areas and highly variablesequences as well. Fungal ITS regions can beisolated by universal primers (White et al. 1990)and, after sequencing, strain- or genus-specificprimers can be used for the identification offungi from various samples. Although Atkinset al. (2004) discriminated between two varietiesof the same fungus based on ITS sequences, in

20 K. Brunner, S. Zeilinger, R.L. Machgeneral, the variability of this area might not besufficient to distinguish between closely relatedstrains (Atkins and Clark 2004). Besides the rRNAand the ITS region, the β-tubulin-encoding geneis among the most prominent diagnostic genes(Fraaije et al. 1999, 2001; Hirsch et al. 2000; Atkinsand Clark 2004). The database of β-tubulin genesequencesisnotaslargeasthatforribosomalDNA but, for particular applications, diagnosticsbased on this gene can be useful. The detectionand real-time PCR quantification of Fusariumspeciesrelymainlyonamplificationofβ-tubulingene fragments (Ali-Shtayeh et al. 1991; Machet al. 2004; Reischer et al. 2004). If the sequence ofthe ITS regions or β-tubulin genes is not suitable,random parts of the genome can be searched forareas unique to taxa or species to be identified(Schesser et al. 1991; Mutasa et al. 1995). Primersdesigned within these arbitrary regions are oftenless discriminating because, for comparison, onlylittle information is available in databases. Thesetailor-made applications can often lead to falsepositive results due to recognition of non-targetorganisms.A very recent approach for species identificationis DNA barcoding. Barcoding rests on the ideaofa’universalproductcode’–afewnucleotidesonly which can unambiguously be attributedto a particular species. However, prior to usingthese species-barcodes, comprehensive publiclyavailable libraries of diagnostic sequences have tobe assembled. Although this system may facilitatespecies identifications in the near future, until nowonly a few specialised web databases can be consulted.A Fusarium barcode database is availableat http://fusarium.cbio.psu.edu, but the sequenceinformation provided is still limited. Most advancesin barcoding fungal genera have recentlybeen made for Hypocrea/Trichoderma. Druzhininaet al. (2005) provide the TrichOKey barcodeidentification system with a web interface locatedat www.isth.info. This system is based on a combinationof several diagnostic oligonucleotidesallocated within ITS1 and ITS2, and was developedon the basis of 979 sequences of 88 species.DNA Extraction Various protocols are availablefor extracting DNA from environmental samplessuch as soil or infected plant material. Variationsin yield and purity of DNA can have severe influenceon subsequent analytical techniques such asPCR. Usually, samples are suspended in buffer andmostprotocolsrelyoninsitulysisofthefungalcellwalls by mechanical forces like bead-beating orgrinding under liquid nitrogen, followed by purificationsteps like phenol/chloroform extractionor spin column centrifugation, and a final precipitationwith ethanol or isopropanol. However,these general protocols need often to be adaptedtotheparticularproblemsarisingfromthesamplematerial. DNA isolation from particular plantscanleadtotheco-extractionofinhibitorysubstances(Malvick and Grunden 2005), and commonsoil compounds including humic acids areknownasstronginhibitorsofPCR-polymerases(Watson and Blackwell 2000). To remove potentiallyunfavourable substances prior to subsequentanalyses, many commercial kits – tailored for particularapplications – are available nowadays fromQiagen, Mo-Bio Laboratories (both UK), Epicentre(WI) and Zymo Research (CA).A. Qualitative Diagnostic PCRConventional PCR is not quantitative but qualitative,withveryhighspecificity,andcanbeusedtodetect, identify and monitor fungi from a broadspectrum of environmental samples. Most fungaldiagnostic PCR applications have been developedto detect phytopathogens, mainly directly frominfected plant material, food and feed samples,or from infested soil. Numerous species-specificprimer pairs have been designed within recentyears, for almost all economically relevant plantpathogens (Fraaije et al. 1999, 2001; Wang andChang 2003; Weerasena et al. 2004; Jurado et al.2005; Rubio et al. 2005; Sanchez-Rangel et al. 2005;Vettraino et al. 2005; Chadha and Gopalakrishna2006). Moreover, PCR has successfully beenapplied to identify airborne fungi for various airsampling methods (Mukoda et al. 1994; Williamset al. 2001). To further improve PCR specificity andsensitivity, nested PCR approaches with two subsequentamplification steps have been developedfor fungal diagnostics from different samples, andDNA target amounts of a few attograms can bedetected reliably (Zhang et al. 2005; Klemsdal andElen 2006; Lockhart et al. 2006). As the samplethroughput of conventional PCR tests is notsufficient for particular applications, multiplexassays can be used as an alternative. Two or moreprimer pairs with the same optimal annealingtemperature are used, and several ampliconsare generated from a single run (Casimiro et al.2004; Zhang et al. 2005; Klemsdal and Elen 2006;

Molecular Approaches for Studying Fungi in the Environment 21Bezuidenhout et al. 2006; Lockhart et al. 2006).Although multiplex PCR allows the cost-efficientcreation of several amplicons in a single reaction,this technique is difficult to optimize for customapplications as the determination of the optimalconcentration for each primer is difficult and timeconsuming. The reaction conditions have to beoptimized accurately to allow efficient generationof all amplicons. Another multiplex approach wastested by Brandfass and Karlovsky (2006). A pair ofprimers was designed to amplify a fragment fromF. graminearum and F. culmorum simultaneously,and the mixture of amplicons was further resolvedby melting curve analysis.In addition to the simple species detection,PCR has been used to target biosynthetic pathwayspecificgenes. Sanchez-Rangel et al. (2005) demonstratedthat the presence of the F. verticillioidesfum1 gene correlates with fumonisin productionin most isolates. The presence of the tri5 geneindicates trichothecene production capability ofFusarium spp., and the tri7 gene can be used to discriminatedifferent chemotypes, namely nivalenoland deoxynivalenol producers. The moleculardetection of Fusarium mycotoxin biosynthesisrelatedgenes has only recently been reviewed(Mule et al. 2005). To target ochratoxin-producingPenicillium spp. in food- and feed-related sources,specific primers have been designed to detecta non-ribosomal peptide synthetase gene which isessential for the respective pathway (Geisen 2004).B. Quantitative PCRAs the lack of quantification of PCR productswas the main bottleneck of this new technology,competitive PCR (cPCR) was introduced to allowquantitative evaluation of the target signals. Thismethod involves the addition of another targetsequence to the assay. The sequence must berecognisedbythesameprimersbuttheproductsmust be of different length. Serial dilutions acrossa wide range are added to individual PCR reactions,and visually quantified on an agarose gel.If the bands derived from both target sequencesare of the same intensity, then the unknownamplified template must match the quantity ofthe added target sequence. Competitive PCR hasbeen applied successfully for the quantificationof the nematophagous fungus Verticillium clamydosporiumfrom soil samples. Increases in fungalgrowth were observed in the rhizosphere of potatocyst nematode-infested plants after 14 weeks usingcPCR (Mauchline et al. 2002). Providenti et al.(2004) tracked the environmental fate of T. reeseiover a 6-month period in soil in a growth chamber.Survival was tested in three different soils, and theeffect of plant rhizosphere was investigated usingthe cPCR technique (Nicholson et al. 1996, 1998).Although the accuracy of competitive PCR canbe very high (Chunming and Cantor 2004), nowadaysquantitative real-time PCR has been widelyaccepted as the gold standard for accurate DNAquantification. During a real-time PCR run, theaccumulation of the product is measured by an integratedfluorimeter. The threshold cycles (ct) determinedfor each sample correlate with the initialamount of DNA. Target DNA is quantified by theconstruction of a calibration curve which relatesthe amounts of calibration-DNA to a certain thresholdcycle. Using SYBR Green as intercalation dye isthe most economic way for real-time analysis. Althoughthis dye allows quantification with high accuracy,the degree of specificity can sometimes notbe sufficient to detect specific DNA in a backgroundof similar targets. To further increase specificityof assays, fluorescent probe-based techniques havebeen established. Molecular beacons (Tyagi andKramer 1996; Tyagi et al. 1998), scorpions (Thelwellet al. 2000) and TaqMan TM probes (Roche MolecularSystems, Pleasanton, CA) are nowadays stateof the art, and even sequences distinguished bysingle nucleotide polymorphisms can be discriminated(Tyagi and Kramer 1996). For more detailedreviews concerning DNA quantification with realtimePCR, the reader is referred to Bustin (2002),Ginzinger (2002), Klein (2002), Ong and Irvine(2002), Filion et al. (2003), Mackay (2004), Valasekand Repa (2005), Wong and Medrano (2005) andBustin et al. (2005). The main advantage of realtimePCR over competitive PCR is the detectionrangeofsixmagnitudes,comparedtoonlytwoforcPCR (McCartney et al. 2003). Most applicationsof quantitative real-time PCR have been implementedin the field of phytopathogen diagnostics,Fusarium being the most prominent representative.Most assays have focused on the detection ofmycotoxin-producing isolates from soil samples ordirectly from infected plant tissues (Schnerr et al.2001; Filion et al. 2003; Bluhm et al. 2004; Reischeret al. 2004; Dyer et al. 2006). Reischer et al. (2004)succeeded in the detection and quantification ofonly five copies of the tub1 gene of F. graminearumfrom wheat ears. To monitor the early infection processof Botrytis and Alternaria before any macro-

22 K. Brunner, S. Zeilinger, R.L. Machscopic symptoms become visible, a reliable assaywas established by Gachon and Saindrenan (2004).Biscogniauxia mediterranea is a fungal pathogenwhich causes severe damages to oaks after a long,symptomless endophytic period. A real-time assaybased on a TaqMan probe was used to identifythis pathogen before disease outbreak (Luchiet al. 2005). Boyle et al. (2005) investigated compatible,incompatible and non-host interactions ofdifferent species of the obligate biotroph Melamsporawith plants. Until recently, light microscopywas the method of choice to detect interaction betweenarbuscular mycorrhizal fungi, or betweenmycorrhiza and potential host plants. To describethe symbiotic contribution of different mycorrhizalstrains, a real-time PCR assay was developed. Thisdetection method allows discrimination and quantificationof different species of Glomus in variousrhizospheres (Alkan et al. 2006). Like conventionalPCR, real-time PCR can be carried out in a multiplexassay to allow quantification of more than onegene in a single run (Wittwer et al. 2001; Bluhmet al. 2004).C. Molecular Fingerprinting TechnologiesIn contrast to conventional PCR detection, whichis a highly adapted and sensitive identificationmethod for a single strain or for a few organisms,molecular fingerprinting techniques allow thefungal ecologist to rapidly profile whole populationsinanecosystem.Asmanyofthegenesused for diagnostic purposes have conserved andless-conserved regions, primers can be designedto amplify many or most species of the KingdomFungi. A further resolution of the amplifiedmixture of products can be performed in a subsequentstep based on nucleic acid composition orvariation of the amplicon length.1. Denaturing Gradient Gel Electrophoresis(DGGE)Denaturing gradient gel electrophoresis is a frequentlyused and established approach for the restrictionenzyme-independent detection of DNAsequence variations, such as single-base substitutions(Eng and Vijg 1997). DGGE exploits the principlethatsequencealterationscausechangesinthe melting temperature of double-stranded DNA,which can be analysed by a linearly increasing gradientof DNA denaturants established in polyacrylamidegels. Initially, the fragments move accordingto their molecular weight but, as they progressinto higher denaturing conditions, each (dependingon its sequence composition) reaches a pointwhere the DNA begins to melt. In practice, nearly allsingle-base substitutions in amplicons up to 500 bpjoined to a GC-clamp can be detected by PCR-DGGE-based analysis (Sheffield et al. 1989). DGGEprovides the means to investigate fungal communities,in particular shifts and changes in communitycomposition. This technique benefits from the abilitytoanalyseahighnumberofsamplesonasinglegel, and provides sufficient resolution to comparewhole fungal communities, rather than single isolates,without the need of precultivation.To develop tools for early and specific detectionof Fusarium langsethiae, and for distinguishing itfrom related species of section Sporotrichiella andDiscolor (F. poae, F. sporotrichioides, F. kyushuense,F. robustum, F. sambucinum and F. tumidum),sequence variations in their β-tubulin-encoding(tub1)genewereemployedtodesignaPCR-baseddenaturing gradient gel electrophoresis assay.DGGE reliably separated all these strains, evenfrom mixtures and in the presence of DNA fromtheir natural hosts Zea mais, Triticum aestivumand Avena sativa (Mach et al. 2004). Van Elsaset al. (2000) developed a DGGE application basedon nested PCR to assess the persistence of selectedfungi in soil and to analyse the response of thenatural fungal community to a spill of petrol.The primers for the first PCR were designed toamplify the rRNA genes of numerous membersof Ascomycetes, Basidiomycetes, Zygomycetesand Chytridiomycetes whereas the primers usedfor the second nested PCR produced ampliconsseparable on denaturing gradient gels. DGGEallowed the resolution of mixtures of PCR productsof different fungi into distinct band patterns.The fungi colonizing the rhizosphere of pineswere monitored during regeneration of woodsby a DGGE-based assay. Fungal diversity wasinvestigated by a denaturing gradient gel followingthe PCR amplification of ITS sequences. Thismethod successfully detected mycorrhizal andnon-mycorrhizal fungi (Anderson et al. 2003). Thefungal population dynamics in soil and in the rhizospheresof two maize cultivars grown in tropicalsoils were studied by a cultivation-independentanalysis of directly extracted DNA. A fragment ofthe 18S rRNA amplified from the total communityDNA was analysed by DGGE, and 39 differentisolates could be identified (Gomes et al. 2003).

Molecular Approaches for Studying Fungi in the Environment 232. PCR-Restriction Fragment Length Polymorphism(PCR-RFLP), Amplified FragmentPolymorphism (AFLP) and Random AmplifiedPolymorphic DNA (RAPD)PCR-RFLPTypically, PCR-RFLP markers are used to detectvariation of DNA-restriction fragment patterns inPCR products. Based on diversity in the restrictionpattern of a 1.3-kb tub1 fragment, a method to discriminatespecies belonging to the plant parasiteCeratocystis has been developed. In all, 200 isolatescomprising seven different species have beensuccessfully identified (Loppnau and Breuil 2003).AnotherapplicationforRFLPwasthemonitoringof populations of two Tricholoma populinum andT. scalpturatum, bothassociatedtoblackpoplar.Theanalyseswerebasedontherestrictionfragmentpattern of the ITS region (Gryta et al. 2006),and the authors revealed differences in the populationdynamics of the two species. On one hand,RFLP provides a rapid technique to screen microbialpopulations but, on the other hand, thismethod has to be optimized carefully to providereliable results. The greatest variation among profilesgenerated from the same DNA sample was reportedto be produced by using different Taq DNApolymerases (Osborn et al. 2000). Incomplete digestionby the restriction enzyme may, as a resultof the generation of partially digested fragments,lead to an overestimation of the overall diversitywithin a community.RAPDThe most common version of PCR-based fingerprintingtechniques is RAPD analysis, in which therandom amplification products are separated onagarose gels in the presence of ethidiumbromide,and visualized under ultraviolet light (Williamset al. 1990). The enormous attractions of thesearbitrary priming techniques are that there is norequirement for DNA probes or sequence informationfor the design of specific primers, since the procedureinvolves no blotting or hybridizing steps. Itis quick, simple, can be automated, and very smallamounts of initial DNA (10 ng per reaction) are sufficientfor amplification (Williams et al. 1990). Inmany cases, RAPD techniques are applied to fungalcommunities to identify an amplified fragmenthighly specific for a single representative memberorasubgroup.Basedonthesequenceofthesecharacteristicfragments, sequence-characterised amplifiedregion (SCAR) primers are designed forconventional PCR. These SCAR primers allow thedetection of underrepresented species in a backgroundof highly similar organisms. This techniquehas commonly been applied to monitor biocontrolfungi like Pochonia, Paecilomyces and Trichoderma(Hermosa et al. 2001; Rubio et al. 2005;Zhu et al. 2006), the plant pathogenic species ofFusarium (Moeller et al. 1999), and ochratoxinproducingspecies of Aspergillus (Taniwaki et al.2003; Pelegrinelli-Fungaro et al. 2004).AlthoughRAPDhasbeenusedtoidentifyanddistinguish species in several studies, it is now evermore widely recognised and critically discussedthat, to obtain reproducible band profiles on thegels, it is absolutely essential to maintain consistentreaction conditions. Numerous studies havereported the negative effects of altering differentparameters like the ratio of template DNA, primers,concentration of Taq polymerase and Mg concentration.AFLPMore recently, a new PCR-based technique hasbeen developed, amplified fragment lengthpolymorphism (AFLP; Vos et al. 1995), which is essentiallyan intermediate between RFLP and PCR.The initial step involves restriction digestion of thegenomic DNA, which is then followed by selectiverounds of PCR amplification of the restricted fragments.The amplified products are radioactively orfluorescently labelled, and separated on sequencinggels. Two studies used this novel technique toidentify ochratoxin-producing Aspergillus strainsfrom various environmental samples (Vos et al.1995; Castella et al. 2002) but their results did notalways show a correlation between AFLP genotypedetermination and ochratoxin production. However,based on the sequence of marker fragmentsidentified consistently in several studies, threehighly specific primer pairs have been developedfor A. ochraceus detection (Vos et al. 1995; Castellaet al. 2002; Schmidt et al. 2003, 2004). Gadkar andRillig (2005) developed an AFLP assay with precedentPhi29-polymerase chain reaction to monitorarbuscular mycorrhizal fungi, and succeededto resolve genomic DNA obtained from singlespores of several fungal species. Only recently,AFLP was used to identify characteristic amplifiedfragments of three industrially relevant fungi,A. niger, A. oryzae and Chaetomium globosum.Thesequence of the strain-specific marker fragments

24 K. Brunner, S. Zeilinger, R.L. Machwas used to design primers suitable to determinethefateandpersistenceofthesefungiinnaturalsoil microcosms (Gadkar and Rillig 2005).IV. Probe Hybridization TechnologiesThis technology uses radioactively or fluorescentlylabelled probes with sequence homology to thetarget DNA. Short oligonucleotides, or largefragments of several hundred base pairs, canbe used as DNA probes. Once bound to a DNAsample, which is immobilized an a membrane,the probe is detected by exposure to X-ray films.This technique, originally developed by Southern(1975), has been further improved and, today,oligonucleotide fingerprinting of rRNA genes(OFRG) is well established as a means of identifyingand discriminating genera or species obtainedfrom complex samples. Amplified rDNA fragmentsare arrayed on a membrane and are hybridizedwith numerous probes according to a particularalgorithm, which allows sorting of the spottedsamples into taxonomic clusters (Borneman et al.2001). This method was previously developed toexamine bacterial community composition buthas now been adapted for fungal samples. Valinskyet al. (2002) analysed 1,536 fungal rDNA clonesderived from soil samples. A large fraction oftheclonescouldbeidentifiedasmembersofthegenera Fusarium and Raciborskimycetes; the otherclones showed sequence similarity to probes characteristicfor Alternaria, Ascobolus, Chaetomium,Cryptococcus and Rhizoctonia. Similar assays weredeveloped to examine the occurrence of commonairborne fungi, based on the sequence variationof the 18S rRNA gene (Wu et al. 2003) or themitochondrial rDNA (Zeng et al. 2003).The next step in hybridization technology isto reverse the process: the use of immobilizedoligonucleotide probes allow testing a samplesimultaneously with a high number of probes.Generally, target DNA is PCR amplified, labelledand subsequently hybridized to the array. Thismethod can be used for the detection of very highamounts of organisms in a single assay, providedthat sufficient polymorphisms exist within theamplified fragment. For diagnostic DNA arrays,the rRNA genes and the ITS region are commonlyselected as targets. The discriminatory potential ofthe probe-oligonucleotides is crucial for successfulapplication, since many species may vary only inafewnucleotidesoreveninasinglenucleotidepolymorphism (SNP). As a consequence, theaccurate detection of SNPs is the aim of modernDNA-chip technology. Lievens et al. (2006) demonstratedthe utility of array technology to detectsingle mismatches at different positions of theoligonucleotide, focusing on the accurate discriminationof ITS fragments of various plant pathogenicfungi. The hybridization is carried out with a highexcess of amplified DNA, and the results can beimaged and analysed qualitatively and quantitatively.The first environmental application of thisnew method was the detection of bacteria but themethod was soon adapted to identify a range ofPythium and Phytophthora species (Levesque et al.1998). Within the scope of fungal detection, DNAarrays have mainly been developed to identify eitherplant pathogens from various environmentalsamples or mycotoxin-producing strains in foodsamples (Levesque 2001; Lievens et al. 2003, 2004,2005; Pelegrinelli-Fungaro et al. 2004; Nicolaisenet al. 2005; Kostrzynska and Bachand 2006; Zezzaet al. 2006). Nowadays, numerous arrays for thedetection of fungi have been developed by privateor public laboratories, and systems like OLISA TM(OLIgo Sorbent Assay, Apibio Biochips, Grenoble,France) are commercially available.A very recent technology, also based on probehybridization, is fluorescent probe hybridization.FISH is a cytogenetic technique which can be usedto detect and localize DNA sequences on chromosomes,as it hybridizes with the sample DNA atthe target site. The probe signal can then be seenthrough a fluorescent microscope, and the sampleDNA scored for the presence or absence of sequenceshomologous to the fluorescent probe. Althoughthis probing technique has often been usedfor detections of organisms in medical samples,environmental applications have been rare. Bakeret al. (2004) used a FISH-based test to identify andmonitor the fungal community of biofilms on acidicmine drainage water. Since the members of Ascomyceteswere morphologically indistinguishable,rRNA-specific fluorescent probes were designed totarget Dothideomycetes and Eurotiomycetes.V. ConclusionsWithin the last decade, many methods for themolecular detection of fungi in diverse ecosystemshave evolved. In the mid-1990s, identification

Molecular Approaches for Studying Fungi in the Environment 25based on immunological tests was state of the artbut this expensive technique was soon replacedby PCR-based detection systems. Nowadays, thereare many applications of PCR in diagnostics, mostof these focusing on rapid detection directly fromthe source material, without any time-consumingcultivation steps. This technique has changed theview of molecular analysis, as now even minuteamounts of organisms, some attograms of DNA,are detectable. PCR technology has opened up newways of investigations, in particular the analysis ofwhole fungal communities from complex sources.In contrast to conventional culture-based methods,even unculturable strains can be detected.The combination with other techniques, likeDGGE, RFLP, AFLP, or the use of random primers(RAPD) allow the identification of yet unknownisolates, although the information about theirDNA sequence is limited or inexistent.Recent reverse probing technologies (microarrays)emerged from the hybridization techniquedeveloped by Southern in 1975. These DNA chipscan be scaled up, so that hundreds of different organismsare detected simultaneously in the sample.Microarray technology has maybe advanced morethan other molecular analysis methods within thelast few years. In contrast to PCR-based technologies,arrays represent a closed system technology,and the oligonucleotide probes on the chip limit thedetection capability. 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Determinants of Fungal Communities

3 Disturbance in Natural Ecosystems:Scaling from Fungal Diversity to Ecosystem FunctioningS.J. Morris 1 ,C.F.Friese 2 ,M.F.Allen 3CONTENTSI. Introduction ........................ 31II. Ecosystem Disturbance:A Conceptual Framework .............. 32A. ConceptualModelOverview ......... 32B. Disturbance and Species Diversity . . . . . 32III. Fungal Community Dynamics:A Natural Disturbance Model ........... 36A. Disturbance Types and Characteristics . 36B. Biotic and Abiotic CharacteristicsoftheDisturbanceModel ........... 37IV. Scaling from Patchto Catastrophic Disturbance ............ 38A. Individuals....................... 38B. Community-LevelEffects ........... 39C. Ecosystem CharacteristicsandFeedbackLoops ............... 41V. Conclusions ......................... 42A. The Impact of DisturbanceontheFunctionalRoleofFungi....... 42B. FutureResearchDirections .......... 42References.......................... 43I. IntroductionTheuniquestructureandphysiologyoffungimakethem both sensitive and resilient in the face of naturaldisturbances. The type and scale of disturbancedeterminethedegreetowhichfungiwillsurvive a given disturbance. Disturbance, as definedby Pickett and White (1985), includes elementsof both scale and process. Specifically, “a disturbanceis any relatively discrete event in timethat disrupts ecosystem, community or populationstructure and changes resources, substrate availability,or the physical environment.” The structureand functioning of fungal communities are influencedby disturbances through changes in vegetation,substrate,environment,andanumberofother components characteristic of the ecosystem1 Biology Department, Bradley University, 1501 W. Bradley Avenue,Peoria, IL 61625, USA2 Department of Biology, University of Dayton, 300 College Park,Dayton, OH 45469, USA3 Center for Conservation Biology, University of California, Riverside,CA 92521, USAin which the fungal community is located. Manyof the descriptive studies of fungal communitiescome from the development of associations withparticular vegetation types, which is dependentupon the successional sere, or with specific decomposingsubstrates. These, in turn, comprise continuouslyandgraduallychangingsubstratesthathave demonstrable trajectories. This led to relativelydeterministic descriptions of fungal communitiesand their changing compositions (sensuClements 1916, 1936; Christensen 1981, 1989).Fungi play a number of important roles inecosystem dynamics. Fungi, as saprophytes, areamong the primary decomposers of substrates,and are essential for recycling nutrients to plants.Fungi are also essential as mutualists. Mycorrhizalfungi are involved in nutrient capture, and inexpanding the resources available to plants. Somefungi achieve this goal through increasing surfacearea explored in the soil, while others increaseactivities such as active decomposition (Cairneyand Burke 1998; Dighton et al. 2001) or parasitism(Johnson et al. 1997; Klironomos and Hart 2001).Pathogenic fungi are also essential components ofecosystems, and play key roles such as maintainingbiodiversity and mediating competition (Priceet al. 1986; Winder and Shamoun 2006). Asdisturbance at any scale will alter the biomass anddiversity of fungi in ecosystems, fungi involved inthese activities will be altered to differing degrees,basedonthephysicallocationoforganismsintheecosystem, and the type of disturbance (Snyderet al. 2002). With the differences in characteristicsof these groups in mind, we will attempt to discussfungi in general with examples from each of thesegroups.Unfortunately,thereisgreaterinformationavailableforsomeofthesegroupswithregardtodisturbances than for others. This has potentialto limit our discussion, and more importantly,our general understanding of the impacts ofdisturbance on fungi. It certainly brings to theforefront the necessity to understand the responseEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

32 S.J. Morris, C.F. Friese, M.F. Allenof these groups of organisms to disturbance, andto understand their impacts on ecosystems.Fungi have a unique physiology, morphology,and reproductive biology. This makes attempts todescribe their dynamics difficult in the rather conventionaltermsusedforhigherplantsandanimals.Fungi, because of the size of individual hyphae oryeast cells, and because they are predominantlystudied in the laboratory under a microscope,are viewed as microorganisms that primarilyrespond to minute quantities of substrates in theirenvironment. In mass and spatial extent, however,many fungi are macroorganisms, often extendingacross large patches. These patches may be ratherstatic in space, and exist over long time periods,such as the mat-forming ectomycorrhizal fungi, orthe Armillaria mycelial networks (E.B. Allen et al.1995). Nevertheless, because these large organismsare still made up of microscopic hyphae with thepotential to function independently, detailing theirlife-history characteristics means understandingboth the macroscopic and microscopic aspects oftheir existence. As a result of this dichotomy in thestructure of fungal “individuals”, any disturbance –from an individual gopher mound to a volcano –has important ramifications to the types of organismsand roles they play in ecosystem dynamics.For example, a large disturbance such as a volcaniceruption can destroy an entire fungal community,requiring subsequent reinvasion and establishmentsubject to classical models of succession (Allenet al. 1992, 2005b). In contrast, a gopher moundcan disrupt mycelial networks within multispeciespatches and within “individual” fungal types.This scenario allows for some recolonization fromexisting species, should the mycelial network rebuildfrom the remnant hyphal fragments. Each ofthese disturbance extremes directly and indirectlyregulates the composition of the plant communityand, in turn, the animal community. It is this rangeintheactivitythatwewilldescribe.Wewillthenattempt to develop a set of conceptual models thatwill allow us to begin to link our understanding offungal biodiversity and ecosystem functioning.II. Ecosystem Disturbance:AConceptualFrameworkA. Conceptual Model OverviewUnderstanding the factors influencing the diversity,distribution, and abundance of organisms ina community has long been a goal of ecologists andmycologists. Disturbance has been proposed to influencecommunity diversity and structure througha variety of mechanisms. A single disturbance type,e.g., gopher mounds, can enhance (Allen et al.1984, 1992), or retard (Koide and Mooney 1987)the rate of succession by altering soil resources,or species diversity. Disturbances may also affectcommunities by creating new sites for colonization(Platt 1975; Collins 1987), or creating newsubstrates (Cooke and Rayner 1984; Gams 1992).Ultimately, the ability of a disturbance to modifythesubstrateonwhichafungalcommunityexistswill determine the extent to which the communitywill change. This modification can be to the substratechemistry (e.g., differing litter types), or itcan be the result of disturbances that affect the environmentalfactors under which those substratescan exist, such as changes in soil moisture contentwith removal of the litter layer. If disturbances differentiallyaffect substrate and environment, thendisturbances caused by a variety of agents will alterfungal species diversity within a communityin different ways. We have developed a conceptualmodel of natural disturbance based on processesthat scale from patch to ecosystem levels (Fig. 3.1).Ouroverviewincludeselementsofthesedisturbancescales, and the factors that affect the variousstates of the disturbance cycle. The conceptualmodel integrates processes operating at the level ofpatchdisturbance,suchassoilenrichment,withthose at the ecosystem level, such as spatial variationsin natural disturbance density (e.g., fire, animaldigging), soils, microbial and plant communitystructure (Fig. 3.1). We believe that emphasis on thefeedbackbetweenpatchdynamicsandlocalecosystemprocessesisanimportantkeytoincreasingourunderstanding of the role of natural disturbance infungal community structure and ecosystem function.B. Disturbance and Species DiversityDisturbances influence the composition andspecies richness of communities through a varietyof mechanisms. Some disturbances result indecreases in fungal biodiversity, while others arehypothesized to release ecosystems from unproductivestates of “retrogression” (Wardle 2006).Within a short time scale, some disturbancesaffect the entire community simultaneously, suchas volcanic eruptions (Andersen and MacMahon

Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 331985; Allen et al. 1984, 2005b; Allen 1988) andcatastrophic winds (e.g., Dunn et al. 1983), whileother disturbances such as animal diggings (Koideand Mooney 1987; Allen et al. 1992; Friese andAllen 1993) or invasive species (Roberts andAnderson 2001; Eviner and Chapin 2003; Bohlen2006) influence only a relatively small proportionof the community at a time. Others initially startsmall, and expand over longer time scales, suchas for invasive species (van Mantgem et al. 2004),N deposition (Egerton-Warburton and Allen2000), and glacial expansions and retreats (Helmet al. 1999; Jumpponen et al. 2002). The scale andintensity of disturbances can, in turn, significantlyaffect the response of organisms and resultingsuccessional patterns (e.g., Bazzaz 1983; Sousa1984; Pickett and White 1985; Pickett et al. 1989;McClendon and Redente 1990; Egerton-Warburtonand Allen 2000).Large-scale disturbances affect fungal communitiesin different ways. Volcanoes, representingthe most extreme large-scale disturbance, cancompletely destroy fungal communities, leavingtopsoil buried under sterile tephra (Allen et al.1984; Hendrix and Smith 1986) or lava (Gemmaand Koske 1990), or even creating new lands(Henriksson and Henriksson 1974). Fungal communitiesin the most severely damaged areascan be destroyed completely. The recovery ofthese areas is driven by wind or small animalvectors capable of bringing new propagules fromsurviving areas, patches, or from source areasat differing distances. Materials deposited asa consequence of the eruption, such as tephraor lava, can have different characteristics, suchas altered bulk density, chemistry or pH, whichleads to the creation of new communities. On themost devastated area of Mt. St. Helens followingFig. 3.1. Conceptual framework illustratinghow ecological concepts ofhierarchy and scale can be used tointegrate and model the relationshipsof small-scale phenomena, such as theimpact of disturbance on microbialfunctional groups, to larger-scale spatialpatterns and processes (e.g., ecosystemdynamics) (based on Friese et al. 1997)

34 S.J. Morris, C.F. Friese, M.F. AllenFig. 3.2. Theoretical conceptual modeldepicting how patch-level disturbanceprocesses can affect microbialfunctional dynamics, which, in turn,can shape larger-scale patterns andprocesses in community- and ecosystem-leveldynamics (based on Frieseet al. 1997)the blast of 1980, Ascomycota were found tocolonize the tephra within the first year, followedby Basidiomycota (Carpenter et al. 1987).After 10 years, AM and ectomycorrhizal fungireestablished at the site, mediated by gophersand wind dispersal (Allen et al. 1992), althoughthe ectomycorrhizal fungi were initially poorlydeveloped.The rate at which reinvasion progresses at themost severely disturbed sites depends on the availabilityof sources of fungal inoculum. With the existenceof nearby or internal source patches of inoculum,fungal communities can begin reinvadingand establishing. The rates of reinvasion of mycorrhizalfungi onto the pumice plain of Mt. St. Helens,to Krakatau following the 1883 eruption (Allen1991), and from Hawaii (Gemma and Koske 1990)demonstrate that reinvasion of these fungi is dependenton the location and type of inocula, andupon the reinvasion of vegetation. Mt. St. Helens,by having mycorrhizae in the most damaged areasas soon as 1 year after the eruption, recovered morequickly than Krakatau, where facultatively mycorrhizalplant species were reported 3 years after theeruption. This is because the most severely damagedareas of Mt. St. Helens were surrounded byremnant vegetation patches, rather than water, ason the island of Krakatau. On the Hawaiian Islands,there was a rapid invasion of mycorrhizal speciesfrom adjacent kipukas, or isolated patches of vegetationthat remained untouched by disturbance(Gemma and Koske 1990). Volcanic eruptions suchas the ones above occur relatively frequently, andare considered predictable and therefore subject toselective evolutionary pressure. Presumably, dispersalstrategies and life histories of the plants onthese islands are adapted to these disturbances, anditisprobablethatthesameistrueforthefungi.Fire presents another example of large-scaledisturbance that has the ability to affect fungalcommunities in different ways. Following a severefire, there is an initial decrease in the number ofpropagules (Wright and Bollen 1961), and shift inthe diversity of fungi present in an area (Wicklow1973; E.B. Allen et al. 2003; Allen et al. 2005a;Bastias et al. 2006). Changes in soil pH and mineralizationrates caused by fire regulate the fungi thatcan initially establish the area (Gochenaur 1981).Following the initial decrease in fungal propagules,a rapid increase in fungal biomass occurs, often tomore than ten times the pre-fire value (Ahlgrenand Ahlgren 1965; Wicklow 1973). These species,

Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 35Fig. 3.3. Detailed model of the biotic and abiotic characteristicsof individual disturbance types. This model is a subsetof the complete conceptual model depicted in Fig. 3.2. Thedisturbance agent determines aspects such as the size, duration,and timing of an individual disturbance event. Allof these variables create a wide diversity of disturbancepatches, with unique biotic and abiotic characteristics. Ifeach disturbance type creates a unique set of biotic andabiotic characteristics, then it is hypothesized that the fungalcommunity will also be differentially affected withineach of these disturbance types (based on Friese et al.1997)commonly referred to as pyrophilous fungi, arecapable of taking advantage of the new resourcesmade available by the fire.Mycorrhizal species may decrease in number ordiversity following fire (Vilarino and Arines 1991;E.B. Allen et al. 2003; Tuininga and Dighton 2004;Allen et al. 2005a), or remain unaffected within theplant root (Molina et al. 1992). However, some ofthese effects are relatively shallow, and do not affectdiversity or density lower in the soil profile(Pattinson et al. 1999; Bastias et al. 2006). Fire canhaveanumberofeffectsonmycorrhizalspores,depending on the maximum ground temperaturereached while burning. Vilarino and Arines (1991)found that, following fire, the number and viabilityof AM spores decreased. They also determined thatfor at least one site, the dominant species of mycorrhizalfungus changed from Acaulospora laevisto Acaulospora scrobiculata. Percent root colonizationby arbuscular mycorrhizae increased over theyear following the burn, but did not reach the levelsfound before the fire. The depression in levels of soilcolonization following fire at this site was detectedfor longer than in other similar research, such asthat of Dhillion et al. (1988) on prairie soils. The authorssuggested that the temperatures reached onthese soils with a shrub and tree vegetation weregreater than those reached with a herbaceous vegetation,the former producing longer-lasting effects.Treseder et al. (2004) found that fire in Alaskanboreal forests had little impact on AM fungi, butrecolonization by ECM appeared to be delayed upto 15 years following the disturbance. Alternatively,in secondary forests in the Yucatan Peninsula witha very shallow, highly organic soil, a “hot” fire virtuallyeliminated all inoculum (E.B. Allen et al. 2003),whereas following a “cooler” fire, where some organicmatter persisted, the richness of fungi wasmuch higher. The two types of fires can cause significantlydifferent patterns of vegetation recovery(E.B. Allen et al. 2003; Allen et al. 2005a). Therefore,the frequency and intensity of a fire, which is determinedlargely by the structure of the plant community(e.g., forest vs. grassland), can determineboth the spatial and temporal patterns of fungalcommunity development.Small-scale disturbance can also affect fungalcommunities in a variety of ways. These disturbancesare often poorly characterized, becausethey create a mosaic of heterogeneous patcheswithin the landscape that are often functionallyand structurally different from those of the landscapethat surrounds them. Patches are definedecologically as discrete spatial patterns with easilyidentifiable boundaries (Pickett and White 1985).Disturbances such as mound building by animalscreate, or alter the patchiness of a landscape (Allen1988; Friese and Allen 1993; Snyder et al. 2002).These patch disturbances disrupt existing externalsoil mycelial networks, such as those described byFinlay and Read (1986) for ectomycorrhizal fungi,and by Friese and Allen (1991) for arbuscular my-

36 S.J. Morris, C.F. Friese, M.F. Allencorrhizal fungi (e.g., absorptive hyphal networks,and hyphal bridges). Disruptions in the soil hyphalnetwork will create openings for the colonizationandspreadofnewfungi,therebyincreasingfungalbiodiversity, just as occurs for colonial animals(Connell 1961) and higher plants (Allen andForman 1976). Gophers and ants are examples ofanimals that are capable of overturning soil, andmoving mycorrhizal propagules within that soilto new patches in the soil matrix. Additionally,gophers trap spores within their fur, and cantransport these fungi to new areas.Ingestion and excretion of viable propagulesat new locations by large mammals are also consideredsmall-scale disturbances that have the capacityto change the community composition ofa patch. The deposition of dung by elk, containingviable mycorrhizal spores from areas adjacentto the blast zone on the tephra at Mt. St. Helens,and this days following the blast, allowed the returnof fungal propagules to a biotically sterile area(Allen 1987). Chronic small-scale disturbances canalso be caused by large ungulates. Serengeti ecosystemsare heavily grazed, resulting in elevated nitrogenand dung applications to the soil (Seagleet al. 1992). This increases nitrogen levels, andmineralizable carbon sources for microorganisms.Studies of mycorrhizal distribution in this ecosystemdemonstrated an inverse relationship betweensoil fertility and the presence of mycorrhizal fungi(McNaughton and Oesterheld 1990). This relationshipis also associated with a smaller gradient of nutritionalstatus in the vegetation. The mycorrhizaeallow the plants to maintain a high nutritional statusacross a broad range of soil nutritional ranges,which ultimately results in better forage for the animals,and a return of nutrients to the soil in theform of urine and dung.III. Fungal Community Dynamics:A Natural Disturbance ModelA natural disturbance model that evaluates fungimust examine fungal individuals and communitieswithin the context of concomitant impacts ofthese organisms on ecosystem dynamics. Regardlessof disturbance size, fungal biomass and diversitywill be altered. The degree to which thisalters specific ecosystem characteristics is dependenton the disturbance event itself, the substratesthat exist following the disturbance, the subsequentmicroclimate and its effects on the substrates, andthe surrounding organisms. A disturbance eventdirectly alters the fungal community by destroyinghyphae and propagules of exposed species, althoughimpactsofanydisturbancewillbemodifiedby the intensity of the disturbance, and the seasonalityof the event (Gochenaur 1981). A “hot” fireduring a drought will devastate many litter fungi,whereas a “cool” one initiated during a wet seasonmayaffectonlysmoke-sensitivespecies,orthosethat exist exclusively on highly flammable materials.As the soil environment changes, e.g., decreasedsoil moisture in response to a lack of coverfrom direct radiation, the survival and growth ofmany hyphae are compromised (Boddy 1984). Lossof plant hosts, and changes in substrate qualityand quantity will further alter the fungal community.Changes in diversity do not have to be largeto impact a system. Change in the growth patternsofasingleimportantfungalornon-fungalspecies as a consequence of disturbance can ramifythrough an entire community. These changes canalter, among other things, the competitive balanceamong species within a community, or ecosystemdynamicsthroughchangestothetypesandactivitiesof enzymes available.An additional dilemma in determining the effectsof disturbance at the patch or ecosystem scaleis determining the extent of a fungal community(Cooke and Rayner 1984). These effects can bestudied if the interpretation that communities aremade of individuals with definable sets of speciesinteractions (MacMahon et al. 1978) is used to defineour concept of community. Large-scale disturbancescan alter an entire landscape (Turner andDale 1998), while small-scale disturbances may affectonly a patch within the community. For thisreason, the model developed here (Fig. 3.1) predictsthe effects of disturbance within the patch, yetin some circumstances the patch may encompassan entire community. Following disturbance, thechanges within the patch may affect the communityof which it was a part, or may become a communityof its own.A. Disturbance Types and CharacteristicsThere are many types of disturbance that will affecta fungal community. In the fungal literature, disturbancesare often subdivided into two main groups –enrichment, and destructive disturbances (Cookeand Rayner 1984). For understanding fungal com-

Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 37munity dynamics, these classifications are often toobroad, because disturbances at the scale that relatesto the fungal community can never be entirely enriching,or entirely destructive. For example, theeffect of a forest fire on a fungal community is dependenton the type and size of fungal communitybeing described, and the state that community is inwhen the fire occurs. The fire is destructive to thephylloplane fungal community, whereas soil fungi5to10cm deep may react to the fire as an enrichmentdisturbance. In areas prone to hurricanedamage, high winds increase the amount of leaf andbranch litter on the soil surface. However, the littercan decompose very rapidly, and overall, this decreaseslitter content, compared with the situationbefore a hurricane (Vargas and Allen, unpublisheddata). Further, the downed branch litter providesa large amount of fuel, often resulting in devastatingwildfires that reduce, or even totally consumeorganic matter (see Gomez-Pompa et al. 2003).Disturbances that alter plant and animal communitydynamics will resonate through the fungalcommunities. Wind storms strip leaves and blowdown canopy trees in a forest. These impacts areusually greater near the edge, which decreases habitatquality especially for small stands. These windevents provide substrate for saprophytic fungi, butthey decrease host density for mycorrhizal fungi. Incomplicated food webs, such as those that involvetruffle-eating flying squirrels, windthrow eventscan decrease food resources and canopy connectivity,which can diminish squirrel populations (Carey2000; Ransome et al. 2004), and ultimately impairsquirrel dispersal of mycorrhizal fungal sporescontained in the truffles. As fungi participate inmany different relationships in ecosystems, evena single disturbance, regardless of size or enrichment/destructivecapacity, has the potential to impactfungi directly or indirectly through the organismsupon which fungi depend. Ultimately, this willfeed back to alter fungal community dynamics.B. Biotic and Abiotic Characteristicsof the Disturbance ModelThe greatest effect of disturbance on the fungalcommunity will be through influences on key factorsthat impact the growth of fungal structures,and the germination of spores. These factors,denoted as biotic and abiotic characteristics inFigs. 3.2 and 3.3, include physical characteristics,resources, soil flora and fauna, and fungalpropagules or hyphal fragments (Boddy 1984;Gams 1992). The physical components of thefungal community affected by disturbance includelight, temperature, moisture, and pH (Gentry andStiritz 1972; Rogers and Lavigne 1974; Mandeland Sorensen 1982). The resources include water,oxygen, organic matter, and a variety of mineralnutrients for growth and sporulation, of whichsome may be required in higher quantities thanfor vegetative growth (Moore-Landecker 1990).Another key resource included here is the physicalenvironment within which organisms grow. Forfungi in most terrestrial ecosystems, the complexsystem of decomposing wood and litter on theforest floor, or simply litter in grassland systems,is an essential part of their ecosystem. The litterlayer can provide a fungal substrate, or it canmodify the soil quality below it. As such, litter layercomposition and characteristics, and the impactof disturbances on the litter layer can have severeconsequences for fungi in terrestrial systems.Plants and animals also regulate the compositionof fungi in soils, but often in a nonlinearmanner. Plants can provide both energy and carbonfor fungal growth, but they can also providemany inhibitory substances. Animals can remove(graze), disperse, provide housing, or provide substrate(defecate or die) for different members of thefungal community, depending on fungal requirements.Additionally, changes in more than one ofthese parameters can act synergistically, or antagonistically,to further change the emergent fungalcommunity (Snyder et al. 2002). In one case, the additionof CO 2 increased plant production, and C inputsto soil fungi. However, that input was matchedby increased grazing by soil animals (Allen et al.2005c). So, although the standing crop of fungalbiomass remained the same under elevated CO 2 ,the total throughput actually increased. Fire canalso alter fungal–animal relationships. For example,the loss of the litter layer, a physical factorand/or substrate, by environmental factors such asfire, results not only in substrate loss, but also in increasesin soil temperatures and decreases in waterholdingcapacity. As this happens, soil organismssuch as mites and collembolans will migrate moredeeplyintothesoiltoescapedrought,reducinggrazing on fungi, but also reducing fungal dispersalnear the surface (Klironomos and Kendrick 1995).Thus, changes in each of the above characteristicswill be dependent on the type and intensityof the disturbance, and the interaction among theresponse variables.

38 S.J. Morris, C.F. Friese, M.F. AllenThe spatial structure of systems, as a consequenceof environmental patterning or small-scaledisturbance, has the potential to impact our abilityto determine the effects of disturbance on organisms.For example, the existence of a pre-burnant mound has the potential to alter the impact ofa disturbance such as fire on an ecosystem. Alterationsmay be a consequence of different physicalstructures – for example, the movement of windacross the raised surface, or decreased burn heat asa consequence of increased mound moisture. Thedifficulty lies in separating the impacts of fire on thesystem from the impacts of pre-fire small-scale disturbance.Failure to detect changes in fire damagemay be the result of sampling a mound without incorporatingthe specific characteristics of a mound.Boerner et al. (2000) found that enzyme dynamicsrelated to bacterial and fungal activity in single-treeinfluence circles (see Zinke 1962) were altered byfire. Detecting changes in fungal dynamics followingdisturbance requires understanding of predisturbancespatial dynamics, and scale-appropriatepostdisturbance sampling efforts. Failure to understandthe impacts of patch-level disturbances, andof other agents capable of creating spatial structurein fungal communities will decrease the likelihoodof detecting postdisturbance impacts.IV. Scaling from Patchto Catastrophic DisturbanceDisturbances impact fungi at a number of levels.As discussed above, the smallest level reasonablyavailable for description of disturbance impacts isthepatch,andthelargestistheecosystem.Whileintheory large-scale disturbances can influence entirelandscapes, and the last ice age certainly disturbedentire biomes, our discussion focuses on individuals,communities, and impacts at the ecosystemlevel, as these are the most common levels currentlystudied. Landscapes can be discussed as large mosaicsof divergent ecosystems across a particular,extensive integrated distance, or they can be discussedas groupings of similar ecosystems at differentsuccessional stages recovering from similardisturbance regimes. This point is important, asadjacent ecosystems house communities that providepropagules for the reestablishment of similarcommunities. While not addressing this directlyhere, the availability of propagules similar tothose of predisturbance communities is an essentialcomponent of reestablishment that, over evolutionarytime,mayhaveproducedthemechanismsand community dynamics discussed below.A. IndividualsOur understanding of impacts of disturbance onfungal individuals or species has been limited bya lack of appropriate techniques for identifying either.While culture techniques allowed us to examinesome species, the availability of moleculartechniques has greatly increased our knowledgeand understanding of fungi as they exist in theirnatural environment. Studies prior to the commonuse of these techniques provide a great deal of theinformation on which our understanding of patchdynamics and the individual is based. So, whileit is anticipated that many of the questions generatedregarding individuals and the impacts ofdisturbance are only beginning to be addressed ingreaterdetail,ourdiscussionofindividualsofnecessitydepends on data derived from a number ofdifferent approaches.Patch-level disturbances often have greatestimpact at the individual level. They alter fungalmycelial networks, and disturb the hyphal networksof individual fungi as they grow in soil. Assuch, individuals will change a great deal as a consequenceof patch-level disturbances. Yet, this may,or may not impact fungal communities. The loss ofaspeciesfromtheecosystemwilloccuronlyiftheindividual impacted is rare, has poor sporulationcapacity or mechanisms, poor dispersal ability, isunable to germinate under new conditions, or is notrepresented in another, reasonably close resourcepatch. Differences in these characteristics amongfungi, and consequences for recolonization followingdisturbance have been explored in the past. Recently,Drew et al. (2006) detected differences in thedegree to which AM fungi can grow and colonizenewhosts.Intheirstudy,Glomus mosseae demonstratedgreater capacity for exploring a habitat tofind new hosts than did G. intraradices,suggestingsome fungi will be more successful in navigatingpatch disturbances than others. Each fungus existsin a system constrained by abiotic and bioticcharacteristics. Disturbances that alter these characteristicsmay result in the elimination of speciesfrom certain areas. This can occur across thedisturbance intensity continuum; however, at thepatch level, the fungus may be eliminated from theimmediate vicinity of the disturbance, but not from

Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 39thefungalcommunityatlarge.Inoneexample,alternate year cropping and fallowing of a wheatfieldin western Nebraska eliminated the dominantfungus of the native prairie, a small-spored Glomusfasciculatum. However, a larger-spored Glomusmosseae wasabletopersistthroughthefallowperiods, and increased relative to G. fasciculatum.Oneoutcomewasafungusthatwaslessvaluable,even detrimental, to wheat drought tolerance andproduction (Allen and Boosalis 1983).Further research on the impacts of disturbance,such as tillage, on agricultural soils suggests thatsoil disturbance decreases fungal biomass, and alterssoil structure (McGonigle and Miller 1996;Denef et al. 2001; Rillig and Mummey 2006). Whilethese impacts often occur at the patch level in naturalsystems, the degree to which they impact individualsand species has been little studied. It islikely that the level of patch disturbance by digginganimals and insects alters species composition atthe patch level, while maintaining fungal communitycomposition at the ecosystem level. Studiesthat have examined soil disturbances such as antmounds have detected some of these patterns inarid systems. Friese and Allen (1993) found bioticenrichment of microorganisms in ant nests at studysites in Colorado and Wyoming. In this case, disturbanceby harvester ant digging increased thetotal number of AM fungal propagules in the antnests, compared to adjacent soil from blue gramagrass (Bouteloua gracilis) atthesiteinColorado,and under shrubs (Artemisia tridentata) atthesite in Wyoming. The assemblages and dominantspecies of fungi also differed between mound andnon-mound sites, with more fungi (characteristicof mesic sites) occurring in ant nests (C.F. Frieseet al., unpublished data). Fungal species richnesswas higher in mound-associated material than insoil adjacent to mounds, or soil collected undershrubs at the Wyoming site, but not from soil adjacentto mounds at the Central Plains ExperimentalRange location(C.F. Friese et al., unpublished data).It appeared that for both locations, microenvironmentsselected a distinct assemblage of dominantfungi, with Fusarium spp. dominating the root material,and Aspergillus and Penicilllum species predominatingin seed cache soil (C.F. Friese et al.,unpublished data). However, Aspergillus fumigatushad high densities in offmound soil from theColorado site. Mucoraceous taxa, (i.e., Cunninghamella,Rhizopus,andSyncephalastrum)wereisolatedprimarily from mound material, suggestingthat the ant mounds may represent refugia for thesemore mesic-adapted fungi (C.F. Friese et al., unpublisheddata). These results suggest the greatestimpacts of these types of patch disturbances wereon individual species through changes in biomassand organic matter, whereas overall fungal communitydiversity across the site was impacted toa much lesser degree.B. Community-Level EffectsChange in species composition within the patchmay cause changes within the larger fungal community.The establishment of fungi within the patchcan affect the diversity and distribution of fungiwithin the community, or it can cause the establishmentof a new community that exists only withinthe patch. The exact outcome will be determined bythe characteristics of the disturbance, especially thesize of the disturbance, but also by the heterogeneityof the landscape prior to disturbance. A landscapematrix is established by a series of disturbancesof increasing scale. The patches are nestedwithin a habitat that can be relatively homogenousor heterogeneous, depending on the scale and intensityof other disturbances occurring across thelandscape. From an area of no disturbance, smallerscaledisturbances are overlaid by the increasingintensity of larger-scale disturbance. The impactof smaller-scale disturbances may alleviate the effectsof larger-scale disturbances, by acting as islandsof inoculum, or dispersal agents. This wasthecaseforthearbuscularmycorrhizal(AM)fungiat Mt. St. Helens. Disturbances by gopher diggingand elk droppings resulted in AM fungal inoculumreturning to a site completely decimated by the volcanoblast (Allen et al. 1984, 1992, 2005b). Initially,EM fungi were predominantly dispersed back ontothe site by wind (Allen 1987). Thus, across the pyroclasticflow zone, small AM fungal patches reformedalong animal pathways, whereas the EMplants initially established at random locations. Itwas the reestablishment of these individuals, or ofthe small eclectic group of fungi that existed in thedeposited inoculum, which led to recolonizationfollowing the most severe disturbance type. Similarly,Jumpponen (2003) found the fungal communityin the youngest soils adjacent to a recedingglacier to have dormant mycorrhizal fungi evenbefore the arrival of the plant community. As thesefungi are biotrophs dependent on plant hosts, airbornespore deposition preceded plant arrival providingsymbionts for arriving seeds.

40 S.J. Morris, C.F. Friese, M.F. AllenThe composition of the postdisturbance communityis also dependent on the competitive interactionsamong residuals and immigrants. A gooddealofworkhasbeenundertakenoninteractionsof competitive (C), stress-tolerant (S), or ruderal(R) strategies in saprophytes following disturbance(Cooke and Rayner 1984). Immediately after a disturbance,the R-strategy is likely to predominatealthough, depending on the disturbance, manyS-strategists may remain as residuals. Presumably,the C-, and more S-strategies will predominatelater. Fungi such as Mucor and Rhizopus arepresumed to be ruderals because they exploitsimple carbohydrates rapidly (R-strategy). Alternatively,Phanerochaete grows slowly, but candegrade almost any type of substrate (S-strategy).Cephalosporium is an outstanding competitorbecause it expends a large amount of resources toproduce antibiotics that restrict access to its own resourcebase. However, these subdivisions are highlyartificial, and organisms exist rather along gradientsbetween these extremes. For example, a commonfungus of burned pine forests is Morchella.Is that because it tolerates the fires, and the harshconditions following fires (S), competes well withother residuals and immigrants by growing hyphaerapidly and utilizing resources in the early spring,before other saprophytes become active (C), producesa massive sporulating fungus that dispersesspores by wind and animals when released fromcompetition (R), or (most probably) has someeffective combination of all these strategies?Taylor and Bruns (1999) examined the ECMcommunity structure in a mature pine forest. Theydetected minimal overlap between the active mycorrhizalcommunity and the community presentas resistant propagules. This suggested that differencesin colonization strategies, such as the C, S,and R described above, and resource preferencescombined with resistant fungal structures allow diversityto be maintained in forest communities, sothat organisms can respond to environmental cuesand disturbances that have been historically part ofthe ecosystems. Other studies on mycorrhizal communitiesfurther suggest that community developmentis dependent on the type (enrichment vs. destructive),intensity, and frequency of disturbance.Lilleskov and Bruns (2003) found that differencesin the timing of root colonization by two ECM fungiwas altered by soil nutrient status. Rhizopogon occidentalis,an early successional species, colonizedrootsearly,andthenwasreplacedasadominantspecies by Tomentella sublilacina as the forest matured.Under high nutrient conditions, however,this replacement was delayed, suggesting interspecificinteractions between the two species were mediatedby soil nutrient content. As disturbancesalso act as a stress upon a system, the return timeof the stress can alter interspecific relationships.Puppi and Tartaglini (1991), evaluating the effectsof fire on Mediterranean communities, found thatalthough the communities were under similar environmentalconstraints, the vegetative and mycorrhizalcommunity structure differed. These differenceswere attributed to the recurrence time of fire.In the more disturbed community, AM fungi weremore common, whereas in the less disturbed community,EM were prevalent. EM were hypothesizedto be more stress-tolerant than AM. Alternatively,in many grasslands, the plants forming AM weretolerant of fire, whereas those forming EM tendednottobe.Thefungimaysimplybelockedtothehost strategy.Fungal pathogens have unique roles in disturbances.Pathogen density and diversity can beimpacted by disturbances. The role of pathogensin natural ecosystems, especially in relation tohistorical disturbance regimes, has not beenaddressed well in the literature. Increases in theloss of plants, especially forest trees, throughpathogen attack has stimulated focus on fungi aspathogens. This may be a consequence of recentattention in the face of economic loss, or maybe as a consequence of increased importance ofpathogensasregulatorsofcommunitydynamics.Allen et al. (2005a) suggested that altered weatherpatterns in the tropical dry forest they examinedmay have resulted in increased incidence ofan indigenous fungal pathogen. Hence, alteredhistorical disturbance regimes may change relativeabundance and impact of pathogens on hosts.Parker et al. (2006) found that fire suppressionresulted in increased levels of fungal pathogenactivity in North American forests. This activityis now being altered by large-scale wildfires, andthe reintroduction of more historically representativefire regimes using prescribed burns.However, these new disturbances are also causingincreases in pathogen activities. Reintroduction ofnatural disturbance regimes are needed to reducepathogen activities, yet the way to achieve thesereductions may require greater understandingof the impacts of fire on the physiology andstructure of these pathogens in their naturalhabitat. Changes in historical disturbance patternsand physical characteristics of the environment,

Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 41such as altered temperature and moisture, may beresulting in increases in pathogen activity.Fungal pathogens also make interesting contributionsas disturbances to plant communities.Fungi such as root rot fungi increase the likelihoodof canopy gaps in forests (Bendel et al. 2006)directly through impacts on trees. These fungiincrease substrate for saprophytes, and decreasehabitat for mycorrhizal species. Other fungalpathogens have been found to aggravate the severityof natural disturbances. Papaik et al. (2005)found that beech bark disease does more damageto trees by decreasing the resistance of beech todisturbance events such as windthrow than it doesby directly impacting the plant it infects.C. Ecosystem Characteristicsand Feedback LoopsChanges in fungal community structure will havethe greatest impact on the ecosystem if functioningchanges with composition, or if the fungi affectplant diversity (e.g., Renker et al. 2004). Changes inprimary production, quality of material produced,decomposition rates, or nutrient pool conversioncan affect the stability, productivity, and ultimatelythe functioning of ecosystems (Chapin et al. 2002).Consequently, disturbances that modify soil processesor the vegetation will alter the correspondingecosystem dynamics. Ecosystem-level feedbackloops (Figs. 3.1 and 3.2) can influence patch structurethrough effects on disturbance types, characteristics,and the biotic and abiotic characteristicsof the patch. Ecosystem dynamics will affectsmall-scale disturbance by influencing such thingsas animal types and densities, and large-scale disturbancesby fuel loads and litter layer thickness.All scales of ecosystem disturbance, ranging fromlandslides to animal burrowing and to hyphal grazingby microarthropods, can disrupt critical pointsin the hyphal network that exist in the soil. Alteringthese hyphal network points changes nutrientavailability and transfer to plant hosts, which, inturn, alters ecosystem productivity.The most significant impacts of disturbanceto fungal communities will be through changesin structure. It is predicted that a disturbed sitetends to return to a community structure thatdoes not entirely resemble the predisturbancestate (Gochenaur 1981). The existence of a newassemblage of species, even of the same speciesof different age or density, may restrict the abilityof the community to return to a predisturbancestate. This was observed in the simple experimenton cultured Penicillium and Aspergillus by Armstrong(1976), which demonstrated that althoughAspergillus would exclude Penicillium in platedcultures of the same age, if Aspergillus spores wereplated with Penicillium spores of a younger culture,then both would be maintained. While terrestrialecosystems have a great deal more complexity thanthis two-species model, it is likely that changesinduced by disturbance have differential effects onthe species present, which will ultimately affect thecomposition of the postdisturbance community.Mycorrhizal fungi may provide the linksnecessary to evaluate the impact of communitystructure, and of changes in community structureon ecosystem function (Read and Perez-Moreno2003). The effects of individual fungal specieson plant communities are expressed throughtheir impacts on aboveground productivity anddiversity. Fungal species can increase productivitythrough increased resource acquisition. Theycan alter plant diversity directly by presence orabsence, through mechanisms of specificity and byaltering the outcome of aboveground competition.They can cause changes to litter and tissue qualitythrough differences in nutrient acquisition. Theassociations that link belowground communitystructure to aboveground dynamics are established,maintained, and disrupted by disturbancesthat alter fungal community dynamics.Understanding the impacts of changes in communitiesof saprophytic fungi on ecosystem functionis also essential for understanding the overallimpacts of a given disturbance type. Modelinghas been used recently to examine linkagesbetween communities and ecosystem dynamics.While these models have been important for understandingglobal change scenarios, they have alsoallowed independent evaluation of key componentsof belowground communities in ecosystem dynamics.Hunt and Wall (2002) modeled effects of speciesloss on biodiversity. They found that deletion ofsaprophytic fungi and bacteria caused changes tonet primary productivity. As they were modelinga large number of belowground groups, and detectedlittle change with deletions of other groups,these results emphasize the importance of saprophytesas determinants of ecosystem characteristics.It also emphasizes that changes in this groupas a result of disturbance has the potential to alterlarger-scale functioning. There is currently a greatdeal of interest in tying community structure to

42 S.J. Morris, C.F. Friese, M.F. Allenfunction at the ecosystem level. The response offungal communities to disturbance events mightprovide good systems to evaluate these linkages.V. ConclusionsA. The Impact of Disturbanceon the Functional Role of FungiThis chapter was designed to demonstrate that disturbancemay be the single, most important processregulating the structure and functioning of fungalcommunities. This is due to the unique physiology,morphology, and reproductive biology offungi. While microbial ecologists are beginning todescribe the importance of individual disturbanceevents, we know far less about the interactionsof small- and large-scale perturbations set withina larger landscape of single or multiple plant communities.This distinction becomes of even greaterimportance in the light of the global dimensions ofanthropogenic influences on ecosystems, such asN fertilization, exotic species migrations, habitatfragmentation, and global climate change. We suggestthat understanding the roles of disturbancein fungal communities, and the feedbacks fromthe fungal communities to ecosystem functioning,are crucial to understanding the results from theselarger global concerns. Developing models for linkingthe range of scales that comprise disturbancedynamics depends on linking two distinct types ofstudies. First, we must begin to build an array ofcase studies from particular ecosystems in whichwe know the natural history of the fungi, and howthese natural histories contribute to existing communitycomposition and functioning. Second, wemust develop a conceptual framework that integratesall of these various case studies into a comprehensiveview of how communities work, andwhat factors regulate them. Finally, we must continuouslyreevaluate those conceptual models todevelop a quantitative model of the complex rolesof fungi within landscapes that are undergoing anthropogenicand natural change.Whileitwouldbeeasiesttoevaluatedisturbanceas a static entity, it is unfortunately a movingtarget. As anthropogenic impacts alter naturalecosystems, we are also altering disturbanceregimes, and impacts of disturbance events.Ecosystems are comprised of organisms thathave interacted with their biotic and abioticenvironments over exceedingly long time scales.The systems examined today are a consequence oforganisms responding to the stresses experiencedon predictable time scales. As anthropogenicdisturbances have escalated only over the last200 years, we cannot adequately predict the consequencesof a given disturbance based on historicaldata, because we do not know if the system we areexamining is similar to that which existed in thepast. Ecologists use the terms “resistance” and “resilience”toevaluatethedegreetowhichasystemresists change in the face of disturbance, or the degreeto which it returns to the predisturbance state.In theory, a system facing a predictable disturbancewould respond in a resistant, or resilient fashion.However, with added unpredictable stresses such aschronic N deposition, herbicide use, global warming,and atmospheric pollution, systems may beless resistant or resilient to disturbances that theymay have easily recovered from in the past. Loss ofevolutionary history within ecosystems not onlydecreases our ability to understand the complexaffects of disturbance on fungal communities, butit also has great potential to damage the ecosystemsthat currently exist within our biosphere.B. Future Research DirectionsAs a result of the widespread interest in anthropogenicchanges to the earth’s atmosphere, and theeffects that these changes may have on the biosphere,it is important to study and “tease out”a better understanding of how small-scale microbialprocesses (such as nutrient mobilization andimmobilization) fit into the larger, global picture.The diversity and biomass of microbial communitiesis a direct indicator of the extent of the functionalrole that these organisms play in the dynamicsof different ecosystems. If anthropogenic changealters the structure and biodiversity of microbialcommunities, then it is also likely that their criticalfunctional roles in ecosystem and global-level nutrientcycling are also impacted. As is the case withothergroupsoforganisms,itisasimportanttounderstandhow the functional role of fungi is affectedby various forms of human impact on the environment(e.g., Meyer 1993; Read 1993; M.F. Allen et al.1995). One approach to evaluate this impact wouldbe to examine the fungal communities present atecological restoration sites that are at least 10–20 years old, and compare the microbial characteristicsof these “established” restoration sites tothose of their references sites. Differences at these

Disturbance in Natural Ecosystems: Scaling from Fungal Diversity to Ecosystem Functioning 43sites would allow one to evaluate the impacts thathuman alteration of system characteristics has onfungal community dynamics. Future research onfungal community dynamics should also focus onlinking the issues of fungal biodiversity and functionalitywith both natural disturbance and anthropogenicchange. This research direction is criticalfor us to completely understand and explain the importanceof fungi in ecosystem dynamics. Attemptsto explore and integrate all of the above factors arecrucial if we are ever to gain a comprehensive understandingof the functional role of fungi in diverseecosystems, and the biosphere as a whole.Acknowledgements. The authors wish to thank ShivcharnDhillion and Tom Crist for technical assistance in the areasof field/laboratory work, and input on the development ofconceptual models. 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4 Fungal Responses to Disturbance: Agriculture and Forestry 1R.M. Miller 2 ,D.J.Lodge 3CONTENTSI. Introduction ........................ 47II. Disturbance as a General Phenomenon ... 48III. Fungi as Control Pointsin Management Practices .............. 49A. TheHabitatsoftheFungus .......... 49B. EffectsofDisturbanceonFungi....... 49C. Contributions of FungitoNutrientCycling ................ 50D. Contributions of Fungito the Hierarchical ViewofSoilAggregation ................ 51IV. Fungi and Agriculture ................ 53A. Tillage and Crop Rotation EffectsonFungi......................... 53B. Role of Fungi in Soil Restorations . . . . . 55V. Fungi and Forestry ................... 56A. Nutrient Additions in Forest Systems . . 56B. Effects of Air PollutionandFertilizationonFungi........... 58C. Effects of Invasive Exotic Earthwormson Northern Forests and Forest Fungi . . 59D. Forestry Practices for PulpwoodandLumberProduction............. 59E. EffectofSitePreparationonFungi .... 59F. Effects of Woody DebrisonEcosystemProcesses............. 61G. Effect of Opening the Canopyand Moisture Fluctuations on Fungi . . . 61VI. Conclusions: Future Role of Fungiin Sustainable Practices ............... 61References.......................... 62I. IntroductionSoils are the most diverse and complex habitat onthis planet, which have been formed by biological,chemical and physical processes, all persistingin parallel (Young and Crawford 2004). It has only1 This paper was written and prepared in part by a U.S. Governmentemployee on official time, and is therefore in the public domain andnot subject to copyright.2 Biosciences Division, Argonne National Laboratory, 9700 S. CassAvenue, Argonne, IL 60439, USA3 International Institute of Tropical Forestry, USDA - Forest Service,P.O. Box 1377, Luquillo, PR 00773, USAbeeninthelastdecadethatanappreciationofthecomplexityofsoilsisbeingrealizedinthestudyof soil fungal communities and the processes theyinfluence. Much of the past research on fungal responsesto land management practices has beendescriptive, being concerned with the compositionand richness of fungal species. Such an approachmay inform us on how a particular managementpractice or disturbance may affect fungal communitystructure; they contribute little to our understandingof the role of fungi in nutrient cycling andaccumulation of organic matter. With the dawn ofmetagenomics, the opportunities for truly integratingfungal diversity with function should be soonrealized.Studies using trophic structure and food-webapproaches to understanding the effects of disturbancesof tillage, crop rotation, and silviculturalpractices on the soil biota are identifying preciselyhow important fungi are for maintenance of a sustainablesoil system (e.g., Wardle 1995; Wall andMoore 1999; Hedlund et al. 2004; Wardle et al.2004; Moore et al. 2005); e.g., by integrating fungalresponses with tillage practices, particularly informativelinkages of fungi to processes associatedwith the dynamics of soil organic matter (SOM)have been identified (e.g., Hendrix et al. 1986; Hedlundet al. 2004; Moore et al. 2005). Also, investigationshave corroborated the importance of fungiin the hierarchical model of soil structure (Tisdalland Oades 1982; Oades 1984) by demonstrating thestructural role of hyphae, and the annealing propertiesof the polysaccharides and glycoproteins thatthey exude to form and maintain a stable aggregatestructure (e.g., Miller and Jastrow 1990; Tisdall1991; Degens 1997; Rillig and Mummey 2006).Although there are many studies concernedwith management practices in agriculture andforestry, a neglected area of research is theintegration of fungal responses with these practices,especially as the responses relate to soilstructure, nutrient cycling and organic matterEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

48 R.M. Miller and D.J. Lodgeaccumulation in soils (Miller and Jastrow 2000;Ritz and Young 2004; Six et al. 2004, 2006). Onereason for this neglect is that plant and fungalresponses and disturbance responses associatedwith land management practices are often studiedat different spatial and temporal scales. Even so,from a conceptual viewpoint this research hasdemonstrated that fungi are a major contributorto system processes and functions that occur atvarious hierarchical organizational levels, indicatinglinkages and feedbacks between fungi andsystem responses (O’Neill et al. 1991; Miller andJastrow 1994; Langley and Hungate 2003; Johnsonet al. 2006; Six et al. 2006). The difficulty lies inour ability to focus questions and to measureresponses or processes that function as controlpoints.Fungi are increasingly recognized as playinga critical role in mediating ecosystem responsesto anthropogenic disturbances (Gadd 1993; Cairneyand Meharg 1999; Staddon et al. 2002; Rillig2004; Six et al. 2006). Anthropogenic disturbancesinclude unintentional stresses, as well asstresses resulting from management practices associatedwith agriculture and silviculture. In addition,the responses of fungal-dominated ecosystemsto chronic inputs of atmospheric nitrogen,and shifts in fungal-to-bacterial ratios and concomitantshifts in rates of nutrient cycling in responseto introduced invasive earthworm speciesare major areas of concern (Lawrence et al. 2002;Lilleskov et al. 2002; Frey et al. 2004). Responsesof soil microbial communities to alien earthwormsin North American forests resemble responses totillage in agriculture where invasive earthwormsappear to be responsible for decreases in fungalto-bacterialbiomass ratios. While these are onlya few examples from among many disturbances ofanthropogenic origin, they help illustrate the hypothesizedmechanisms of response and point toareas where more research is needed.In this chapter, we will elaborate on the impactsof the various management practices associatedwith agriculture and forestry as they influencefungal structure and function. In addition, we willaddress examples of the impact of chronic stressin forest ecosystems from increasing nitrogen inputsand introduced soil fauna. Contributions offungi to nutrient cycling, organic matter accumulation,and the formation of soil structure will bediscussed. Furthermore, we will discuss the hierarchicalnature of soils, and how this nature influencesa systems response to disturbance. We willuse examples from agroecosystems, soil restorations,and forest systems.II. Disturbanceas a General PhenomenonDisturbance is a common feature of most systems,occurring at all levels of ecological organizationand at numerous temporal and spatial scales(DeAngelis et al. 1985; Zak 1992). For our discussion,we define “disturbance” as the physical orchemical phenomena that disrupt communitiesand ecosystems. Disturbances may be eitheranthropogenic or natural, but it is the biota andthe variation in terms of disturbance severity,frequency, and scale that result in different pathwaysof ecosystem response (rather than being thesource of the disturbance; Waide and Lugo 1992).Some disturbances disrupt the physical structureof communities, such as soil tillage, bioturbationfrom introduced earthworms, clearcut harvestingof trees, and storm damage; other disturbancesinvolve chemical additions, such as acid rain, fertilization,salinization, heavy metals, or biocides.Fungal communities may be directly affected bythese physical or chemical disturbances. Fungi alsorespond to the indirect effects of disturbance, suchas the mortality of litter decomposers that resultsfrom the increased drying frequency of the forestfloor following disturbance to forest canopies, andrepression of certain fungal enzyme systems byexcess nitrogen (e.g., Sinsabaugh et al. 2002, 2005).Substrata for decomposition can be viewed asdiscrete or continuous. Fungi of continuous substratainhabit a niche that continually receives newresources, whereas fungi of discrete substrata inhabitagivensubstrate.Inhisclassicalstudyonecological groupings of soil fungi, Garrett (1951)states that the succession of fungi on a substratecauses a progressive deterioration in the capacityof the substrate to support further growth. He furtherstates that the substrate comes directly to thesoil microorganism, i.e., roots grow through soiland die in it; dead leaves fall upon the soil. The formerexample views a substrate as discrete, whereasthe latter views a substrate as being continuous.Accordingly, disturbance can be a perturbation toboth substrate and fungus.For a substrate, disturbance disrupts its deliveryrate, its quality and its accessibility, whereas forthe fungus disturbance represents the physical or

Fungal Responses to Disturbance: Agriculture and Forestry 49chemical disruption of the mycelial network. Forexample, tilling of soil breaks and damages the filamentousnetwork of fungal hyphae inhabiting thesoil. In turn, the loss of the hyphal network removesor damages the physical mechanism responsible forstabilizing soil macroaggregate structures. Thesemacroaggregate structures physically protect particulateorganic matter within them. The hyphalnetwork disruption increases the potential for disruptionof the macroaggregate structure, exposingthe once-entrapped particulate organic matterto rendering by the microbial community (Angerset al. 1992; Jastrow and Miller 1998; Six et al. 2004).III. Fungi as Control Pointsin Management PracticesAlong with Archaea and bacteria, fungi are themost numerically abundant organisms in the terrestrialecosystem, and are the primary decomposersof organic residues in soil. Although fungimay be numerically less abundant than bacteria,fungi can account for as much as 78–90% by weightof the soil microbial biomass (Kjoller and Struwe1982; Lynch 1983). These fungi may be free-livingsaprotrophs, pathogens, or in mutualistic associationswith plant roots. Studies of grassland andagricultural soils indicate that fungal biomass typicallyoutweighs bacterial biomass in undisturbedgrassland communities and reduced tillage agroecosystems(Beare et al. 1992; Bardgett et al. 1996;Frey et al. 1999; Bailey et al. 2002), especially whenlow-quality residues are a primary input source(e.g., Bossuyt et al. 2001). It has been suggestedthat the size and composition of microbial communitiesin soils are primarily controlled by thequality, quantity, and distribution of substrata, allof which are influenced by land management practices(Anderson and Domsch 1980; Schnürer et al.1985; Giller et al. 1997; Six et al. 2006). Differencesin fungal-to-bacterial ratios likely reflect changesin microbial composition in response to managementeffects on the retention of litter and its quality,e.g., fungi typically dominate under reduced orno-till conditions (e.g., Hendrix et al. 1986; Beareet al. 1992; Six et al. 2006). Furthermore, many ofthe discrepancies reported in the literature may beexplained by where bacteria and fungi tend to livein soil; e.g., a primary reason for fungal biomassbeing larger than bacterial biomass in surface litterisrelatedtotheabilityoffungalhyphaetotraversethe gap between surface litter and soil more readilythan do bacteria (Holland and Coleman 1987;Beare et al. 1992; Frey et al. 2000).A. The Habitats of the FungusIn an attempt to better conceptualize soil systemsinto biologically relevant regions on the basis oftheir spatial and temporal heterogeneity, the conceptof “sphere of influence” has been proposed(Coleman et al. 1994; Beare et al. 1995). The fiveareas of concentrated activity in soils include:(1) The detritusphere, composed of the litter, fermentation,and humification layers above thesoilsurfacethathaveconsiderableroot,mycorrhizaland saprotrophic fungal biomass, andgrazing of that biomass by the soil fauna;(2) The drilosphere, which is that portion of thesoil that is influenced by the activities of earthwormsand their casts;(3) The porosphere, which is a region of waterfilms occupied by bacteria, protozoa, and nematodes,and of channels between aggregatesoccupied by microarthropods and the aerialhyphae of fungi;(4) The aggregatusphere, the region where the activityof microbes and fauna is concentratedin the voids between microaggregate and evenmacroaggregates; and(5) The rhizosphere, or the zone of soil influencedby roots, associated mycorrhizal hyphae, andtheir products.The spheres are formed and maintained by biologicalinfluences that operate at different spatial andtemporal scales. Moreover, each sphere has distinctproperties that regulate interactions among organismsand the biogeochemical properties that theymediate (Coleman et al. 1994; Beare et al. 1995).B. Effects of Disturbance on FungiSoils are spatially heterogeneous because of theirbiologically mediated properties (Beare et al. 1995;Young and Crawford 2004). A driving force for creatinga spatially heterogeneous environment is theprocess of bioturbation (Hole 1982). These biologicallymediated disturbances result in creation ofa spatially heterogeneous environment, a very differentoutcome to system processes than is the casefor disturbances associated with land managementpractices, such as tillage or clearcut harvesting thatusually result in a loss of spatial heterogeneity.

50 R.M. Miller and D.J. LodgeFor example, in the drilosphere, i.e., the regionof soil influenced by the activities of earthwormsand their casts, the grazing activities of microarthropodsand millipedes change the size anddistribution of litter on the soil surface, therebyincreasing the surface area for fungal colonizationand mixing the fragments with other debris (Beareet al. 1995). Macrofauna redistribute the litter inand upon the soil, creating a patchwork of bothsubstrata and refugia for soil fungi, bacteria, andfauna (Lee and Pankhurst 1992). In the porosphere,the physical rearrangement of soil particles bygrowing roots and earthworm burrowing createsmacropores that influence the preferential flowof water and nutrients. In the aggregatusphereand detritusphere, the relationship between soilorganisms and bioturbation is considerable. Largeamount of faunal feeding occurs on surface litterand associated fungal hyphae, resulting in theaccumulation of particulate and fecal aggregatesin surface soils. Earthworm casts also accumulateinthesurfacesoils;however,theprimaryagentofaggregate stabilization is through the depositionof bacterial and fungal residues, and by hyphalentanglement of particles (Chenu 1989; Tisdall1991; Degens 1997; Guggenberger et al. 1999). Thecontinued inputs of microbial gums and glues, aswell as the production of filamentous hyphae, arenecessary for the long-term maintenance of thesoil macroaggregate structure.Conventional land management practices usuallyresult in a loss of the spatial heterogeneity ofsoil. With more sustainable practices, the goal ofmanagement is to create a more spatially heterogeneoushabitat; e.g., tillage results in the physicaldisruption of the more transient fungal hyphal network.Furthermore, tillage mixes surface residuesvertically within the soil profile while usually doinglittle direct damage to the aggregate structure(Angers et al. 1992); however, without the physicalcontinuity of the hyphal network, the rewetting ofdried aggregates will cause their disruption or slaking.Another phenomenon associated with plowingis a temporary flush in C and N mineralization,which appears to be directly related to the exposureof organic residues to soil biota as a resultof slaking (e.g., Elliott and Coleman 1988; Guptaand Germida 1988; Van Veen and Kuikman 1990;Kristensen et al. 2000). Some of the nutrient flushassociated with tillage is attributed to disruption ofthe hyphal network. The amount of nutrient flushdepends on (1) the overall amount of organic matterin the soil, (2) the quality of organic residuessequestered within the aggregated portion of thesoil, and (3) the amount of microbial biomass andits activity (Six et al. 2004).C. Contributions of Fungi to Nutrient CyclingIn agricultural and forest ecosystems, the primaryrole of saprotrophic fungi is as decomposerswhere their activities contribute to C and nutrientcycling (Kjoller and Struwe 1982). SOMdynamics is influenced by saprotrophic fungithrough their regulation of the decompositionof plant and microbial residue, the productionof polysaccharides, and the stabilization of soilaggregates (Six et al. 2004, 2006). Fungi areespecially good decomposers of nutrient-poorplant polymers. Furthermore, saprotrophic fungipossess several growth habits that enable them togrow in nitrogen-deficient environments (Paustianand Schnürer 1987). These habits include:(1) Lysis and reassimilation of nitrogen from degeneratedhyphae (Levi et al. 1968),(2)Directedgrowthtolocallyenrichednutrientsites (Levi and Cowling 1969; St. John et al.1983; Boddy 1993), and(3) The translocation of cytoplasm to hyphalapices from mycelium in nitrogen-depletedregions (Cooke and Rayner 1984).Fungal hyphae may also translocate mineral nitrogento nitrogen-poor substrata where the absoluteamount of nitrogen in a decomposing substrateincreases during the early stages of decomposition(Aber and Melillo 1982; Holland and Coleman1987; Frey et al. 2000). Also, the lateral andupward movement of 15 N-label inorganic nitrogenfrom mineral soil to decomposing litter has beendemonstrated (Frey et al. 2000). Furthermore, thenet immobilization of nitrogen in surface litter canbe relieved by the application of fungicide (e.g.,Beare et al. 1992; Frey et al. 2000). In addition tomineral N transfer to litter by saprotrophic fungi,a fungal-mediated litter-to-soil C transfer has beendemonstrated where litter C enters the soil viafungal mycelia and becomes stabilized within themacroaggregate soil structure (Frey et al. 2003).Similar bidirectional translocation of nutrients byrhizomorphs of wood-decay fungi have also beenobserved (Lindahl et al. 2001).Fungal biomass represents a significant poolof available nutrients in soils, and its turnoverhas important consequences for carbon andnutrient cycling (Langley and Hungate 2003; Zhu

Fungal Responses to Disturbance: Agriculture and Forestry 51and Miller 2003; Six et al. 2006). Soil microbialbiomass, e.g., of a temperate grassland, contains1–2 and 2–5 tha −1 for bacteria and fungi, respectively(Killham 1994). The availability of hyphalcomponents and cell products in the form of cellwalls, cytoplasm, and extracellular polysaccharidesrepresents a relatively labile organic poolin soils. It has been estimated from a survey ofarable soils that the biomass of the microflora inagricultural soils has about 108 and 83 kg ha −1of N and P, respectively (Anderson and Domsch1980). In a forest stand composed of spruce andpine, the amount of ectomycorrhizal biomassfoundassociatedwithmantlesandmyceliumisestimated to be 700–900 kg ha −1 (Wallander et al.2001). The amount of N in ectomycorrhizal fungalbiomass is 187 kg N ha −1 (Wallander et al. 2004).Hence, fungal mycelia represent a major soil sinkfor nutrients.As reported above, the mycorrhizal funguscan represent a considerable portion of the fungalbiomass, with reports of 20–30% of the microbialbiomass carbon being composed of mycorrhizalfungal biomass in grassland and agroecosystems(Miller et al. 1995; Olsson et al. 1999; Leake et al.2004). The importance of mycorrhizal fungi innutrient cycling is that, as symbionts, they providea direct physical link between primary producersand decomposers. Plant processes influenced bymycorrhizal fungi include host functions such asphotosynthesis, nutrient uptake, and water usage.Mycorrhizalfungalinfluencesonhostfunctionscan affect nutrient accumulation and alter nutrientratios in plant tissues. In addition, mycorrhizaecan affect plant nutrient uptake by affectinga host’s growth rate and by influencing mineralion uptake (Smith and Read 1997).The contributions of externally produced arbuscularmycorrhizal (AM) fungal hyphae to nutrientcycling are considerable (Zhu and Miller2003; Rillig 2004; Leake et al. 2004; Staddon 2005).Although studies of carbon allocation to belowgroundstructures are few, they indicate that externalAM hyphae represent approximately 20% of thelabeled extraradical organic carbon pool (Jakobsenand Rosendahl 1990). Annual production ofexternal hyphae in prairie soils is estimated to be28 mcm −3 of soil, with a calculated annual hyphalturnover of 26% (Miller et al. 1995). Recently, acceleratormass spectrometry microanalysis of 14 Cwasused to quantify the turnover rate of extraradicalhyphae in plants grown in a controlled environment(Staddon et al. 2003). A surprising outcomeof this study is that the turnover rate of hyphae attachedto plant roots averaged only 5–6 days, suggestingthat carbon flow from host plants to hyphaein soil might quickly be respired back to theatmosphere. More importantly, the findings suggesta rapid pathway for atmospheric carbon toenter the soil carbon cycle (however, see Olssonand Johnson 2005). Since AM hyphal cell walls arecomposed primarily of chitin, a carbohydrate thatis rather recalcitrant to decomposition, the rapidturnover of live hyphae would still allow for theaccumulation of hyphal residues that could remainwithin the soil matrix for a considerable period oftime (Zhu and Miller 2003).Turnover rates appear to be faster for extraradicalhyphae of mycorrhizal fungi than for intraradicalhyphae of roots (Staddon et al. 2003). Thismay be true for the relatively thin-walled, smalldiameterhyphae, but a substantial portion of theAM hyphal network is composed of thick-walledrunner or arterial hyphae (Friese and Allen 1991;Read 1992). These runner hyphae are likely to belonger lived and more recalcitrant than the thinnerwalledhyphae that are probably directly involvedin nutrient acquisition. Moreover, a considerableproportion of the hyphae extracted from soil is eithernonviable or highly vacuolated (Schubert et al.1987; Sylvia 1988; Hamel et al. 1990), suggestingsubstantial persistence of these hyphae. Becausefungal cell walls also contain chitin, they may bea relatively passive source of nitrogen, too (Langleyand Hungate 2003).D. Contributions of Fungito the Hierarchical Viewof Soil AggregationAn important component of a successful soil managementstrategy is the creation and maintenanceof the soil aggregate structure (Jastrow and Miller1998; Miller and Jastrow 2000; Six et al. 2004). Thedegree to which a soil has been degraded will determinethe extent of formation versus maintenanceof soil aggregates. The importance of aggregatedsoils in soil management comes not only from therole of aggregates in controlling soil erosion, butalso because aggregates facilitate the maintenanceof nutrient cycles. In arable systems, the nutrientreserve of a soil is typically maintained by inputsfrom crop residues and from fertilizers. In grasslandand forest systems, the nutrient reserve is typicallymaintained by inputs from litter. In temperate

52 R.M. Miller and D.J. Lodgeforest systems, litter accumulates mainly from leaffall, while root inputs are more important in grasslandsand the few tropical forests that have beenstudied. Unless organic inputs are protected withinthe soil macroaggregates, the accumulation of organicmatter and the concomitant buildup of soilnutrients is usually minimal (Elliott and Coleman1988; Six et al. 2004). Without the physical protectionafforded within macroaggregates, organicmatterandassociatednutrientsmayberapidlylostvia both mineralization and erosion (Elliott 1986;Jastrow and Miller 1998; Six et al. 2004).The hierarchical view of soil aggregation isbased on the spatial and temporal actions of variousorganic and mineral binding agents (e.g. Tisdalland Oades 1982; Oades 1984; Jastrow and Miller1998; Six et al. 2004). These binding agents can begrouped into three classes based on age and the degreeof rendering of organic inputs. The first class iscomposed of microbial and plant-derived polysaccharidesthat can decompose quite rapidly and arereferred to as transient binding agents. The secondclass, referred to as temporary agents, is composedmainly of living or dead fibrous roots and hyphae,and can normally persist through a growing seasonor even longer in perennial systems. The thirdclass, referred to as persistent binding agents, iscomposed of decayed or more rendered materialshaving humic acid moieties in association withclays and/or amorphous mineral complexes. In thehierarchical model of soil aggregation, fungal hyphaeplay an essential role as temporary stabilizingagents of soil macroaggregates.The characteristics that allow fungal hyphaeto contribute to the formation and stabilization ofsoil macroaggregates are related to a wide rangeof factors including soil characteristics, vegetationtype, management practices, and characteristics ofthe fungus itself (Degens 1997; Miller and Jastrow2000; Six et al. 2004). For example, because of theirphysical dimensions, fungal hyphae can grow andramify through and within pores the size of thosefound between soil macroaggregates. In addition,hyphae are believed to persist for longer periodsof time in soil because of their filamentous nature,coarse branching habit, and rather large diameters.Tisdall and Oades (1979) reported that AM hyphaecould stabilize aggregates up to 22 weeks after theirhost plants had died. By comparison, saprotrophichyphae, with their more regular branching habitand typically thin walls, appear to have a more transienteffect on aggregation, often lasting no morethan a few weeks. Another factor enabling AM fun-galhyphaetocontributetosoilaggregatestabiliza-tion is their obligate association with plant roots.BecauseAMhyphaehavedirectaccesstophotosyntheticcarbon, they represent a conduit for hostcarbon into the soil, bypassing the decompositionprocess. Additionally, AM hyphae have been foundto produce a very stable glycoprotein, called glomalin,which may act as a longer-term binding agent(Wright and Upadhyaya 1996; Rillig and Mummy2006).Many of the early investigations of AM hyphaeand aggregation were conducted in sandy soils orsand dune systems (Nicolson 1959; Miller and Jastrow2000). These studies indicate hyphae are theprimary mechanism for binding sand particles intoaggregated units (Degens et al. 1996). However, forsoils where organomineral binding agents are importantto stabilizing soil aggregates, the contributionsof AM hyphae go beyond the entanglementmechanism described for sandy soils. Rather, inloamy textured soils of alfisols, mollisols, and vertisols,AM hyphae have been found to contributeto the maintenance of a hierarchically arranged aggregatedsoil structure composed of both macroaggregateand microaggregate structures (Miller andJastrow 2000). In oxisols, where oxides are the dominantbinding agent of soil particles, aggregatesdo not appear to be hierarchically organized. Thisattributedoesnotprecludetheentanglementorenmeshment of primary and secondary particlesby saprotrophic and AM hyphae, but the role ofthe fungus may be secondary to the oxide-bindingmechanism. For soils with relatively high clay content,the shrink–swell capacity of the soil may overrideor minimize the contributions of hyphae tosoil aggregation. Hence, when viewed over a widerange of soil types, the contributions of fungi tosoil stabilization depend largely on broad texturalcharacteristics, and whether the structure of a soilis hierarchical in construction, among other factors(Table 4.1).To date, little information exists for ectomycorrhizalfungi contributing to soil aggregate stabilization,although saprotrophic basidiomyceteshave been demonstrated to contribute to aggregatestabilization (Caesar-TonThat and Cochran 2000).It has been suggested that ectomycorrhizal hyphaemay create a very stable soil structure by producingstronger bonds with clays than do other hyphaltypes (Emerson et al. 1986). What is knownis that ectomycorrhizal hyphae have the ability toextend considerable distances into the soil; exudepolysaccharides and organic acids; and enmesh soil

Fungal Responses to Disturbance: Agriculture and Forestry 53Table. 4.1. Fungal hyphal and abiotic influences on soil structure for contrasting soil textures (modified from Miller andJastrow 2000)Abiotic and biotic propertiesInfluence in soil typeSand Loam ClayShrink–swell capacity Minimal Important MaximumAbiotic aggregation Minimal Important MaximumFungal hyphal effects Important Important Minimalparticles between them (Skinner and Bowen 1974;Foster 1981). Furthermore, the release of organicacids by ectomycorrhizal hyphae may be a factor inthe dissolution of clays (Leyval and Berthelin 1991).IV. Fungi and AgricultureBecause of the various management practices usedin agroecosystems, fungi experience vastly differentdisturbance regimes. The impacts of these disturbancesare expressed directly, and by their effectson food-web structure and function (Hendrixet al. 1986; Paustian et al. 1990; Hedlund et al. 2004;Moore et al. 2005). Under conventional managementpractices, the activities of the soil biota, includingsaprotrophic and mycorrhizal fungi, havebeen largely marginalized by the use of agrochemicalssuch as fungicides, herbicides, pesticides, andfertilizers, which suppress pests or bypass nutrientcycles. With stronger societal pressures to reducethe use of agrochemicals and fertilizers, however,an ever increasing reliance on processes influencedby soil biota, and especially fungi, will emerge.Several areas of research with fungi offerpromise for reducing the use of agrochemicalsand intensive tillage regimes. These areas includethe contributions of mycorrhizal fungi in plantproduction and soil aggregation, the use of fungias biological agents for control of plant pathogensas well as insect and nematode pests (not discussedin this chapter), and the management of nutrientcycling through better use of saprotrophic andsymbiotic processes (Elliott and Coleman 1988;Bethlenfalvay and Linderman 1992; de Leij et al.1995; Cavagnaro et al. 2006; Six et al. 2006).A. Tillage and Crop Rotation Effects on FungiAgricultural practices such as tillage, crop rotation,crop residue retention, and fertilizer use all affectthe ecological niches available for occupancy bythe soil biota. Simply put, an agricultural field isbasically an experiment in natural selection wherethose organisms best adapted to those habitatsand niches gradually replace those individuals notso well adapted (Rovira 1994). Hence, soils managedby conventional, reduced or no-till practiceshave distinctly different soil biotic communities. Ofthese practices, tillage most disrupts the soil fungalcommunity (Table 4.2), resulting in a reductionof the soil’s macroaggregate structure (e.g., Guptaand Germida 1988; Beare et al. 1997; Kabir et al.1997; Wright et al. 1999) and a reduction in fungalbiomass (Gupta and Germida 1988; Frey et al.1999). The loss or reduction of this basic structuralcomponentofsoilresultsinthedestructionofmany of the ecological niches suitable for soil fungiand bacteria (Young and Ritz 2000). The practiceof tillage serves many purposes, including preparationof seed bed, mechanical weed control, acceleratedmineralization of nutrients from organicmatter, and improved water capture and storage inthe soil profile (Cook 1992). Unfortunately, tillagealso sets the stage for soil erosion and loss of organicmatter.A major control point in conservation tillage isthe management of crop residues. In conventionaltillage practices, plowing results in the mixingof soil profiles and the burial of crop residues;whereas in no-tillage systems the soil is not plowedand residues are placed on the soil surface asmulch. These differences in soil disturbance andresidueplacementcaninfluencethecompositionand activity of the fungal community (Hendrixet al. 1986; Beare et al. 1993; Six et al. 2006).Using a detritus food-web approach, it has beendemonstrated that no-tillage systems favored thefungal component of the soil microflora, resultingin the buildup of fungivorous microarthropods,nematodes, and earthworms (e.g., Hendrix et al.1986; Beare et al. 1992; Wardle et al. 2004). Alternatively,conventional tillage practices favoredthe bacterial component, resulting in the buildupof a completely different group of organisms by

54 R.M. Miller and D.J. LodgeTable. 4.2. Fungal and microbial properties of soil aggregatesize classes from a native grassland soil and adjacent soilsubjected to cultivation for 69 years (data from Gupta andGermida 1988)Aggregate size class Fungal biomass Microbial biomassLength Biovolume C N(mm) (mg −1 ) (mm 3 g −1 ) (μgg −1 ) (μgg −1 )Native soil>1.00 874 3.74 1538 1390.50–1.00 1276 12.89 1862 1550.25–0.50 2163 10.85 1463 1330.10–0.25 508 2.06 1161 1241.00 180 0.98 886 860.50–1.00 166 1.12 946 920.25–0.50 543 3.63 859 900.10–0.25 144 1.70 655 82

Fungal Responses to Disturbance: Agriculture and Forestry 55the hyphal network in the soil, and either kills portionsof the hyphae or breaks the hyphae into lessinfectiveunits (Neate 1994). More recent studiesindicate a soil’s structure mediates the growth ofRhizoctonia, where hyphal growth is preferentialin gaps found between soil aggregates (Otten et al.2004). It appears that invasion of the fungus intohost populations operates at two scales – at the microscopicscale, the fungus preferentially explorescertain pathways, i.e., gaps in the soil that influencethe mode of growth, and at a larger scale a criticaldensity of host is necessary, allowing the fungus toswitch from noninvasive to invasive growth (Ottenand Gilligan 2006).Tillage and crop rotation practices can also influencethe AM fungus (Abbott and Robson 1994;Kabir 2005). Many studies have demonstrated thatcropping and tillage practices influence the mycorrhizalfungi (e.g., Dodd et al. 1990; Johnsonet al. 1991; Kabir et al. 1998; Jansa et al. 2003).Tillage results in maximum disturbance to the fungalpropagula and the extraradical hyphal network,reducing crop nutrient uptake (O’Halloran et al.1986; Kabir et al. 1998; Jansa et al. 2003). The use offallow rotations can also result in the expression ofphosphorus deficiency, even though there may beno decrease in available phosphorus (Thompson1987). The mechanism for the deficiency responseappears to be a reduction in AM fungal propagulaassociated with the fallow practice.Although most crops are dependent upon mycorrhizalfungi, roots of crops belonging to thechenopod and crucifer families usually do not possessmycorrhizal fungi. These two families includecrops such as spinach, sugar beet, canola, rapeseed,and mustards. When such crops are used in rotations,they tend to lead to a reduction in mycorrhizalpropagula (Gavito and Miller 1998; Thompson1991). Hence, the yield of mycorrhiza-dependentcrops after a nondependent crop may decline, if theneeds of that crop for symbiont-supplied nutrientscannot be fulfilled.Cropping and tillage practices have beendemonstrated to result in a loss of specific groupsof AM fungal species (Sieverding 1991; Doudset al. 1995; Boddington and Dodd 2000; Jansaet al. 2003). Cropping and tillage can also selectfor less-effective mycorrhizal fungi. Rotationswith sod crops are necessary to eliminate declinesymptoms associated with the proliferation ofa certain mycorrhizal fungi when grown withtobacco as the host crop (Hendrix et al. 1995).Furthermore, those AM fungi that proliferateunder tillage conditions may be less beneficial,or even detrimental to those crops in which theyproliferate (Johnson et al. 1992).B. Role of Fungi in Soil RestorationsDepending on the degree of degradation of a soil, animportant component of a successful restoration isthe reestablishment of a nutrient reserve (Bradshawet al. 1982). This reserve is initiated by the combinationof atmospheric deposition, weathering, anddetrital inputs from vegetation, or by additions oforganic amendments and fertilizers. Unless theseorganic inputs are stabilized, accrual of organicmatter and microbial biomass, and the concomitantbuildup of nutrient reserves in soils are usuallyminimal. As described above, organic residuesare generally protected or stabilized within soilsthrough the formation of soil aggregates. Hence,a major goal of any soil restoration should be toestablish conditions that favor formation of stablesoil macroaggregates, thereby facilitating an importantstep in the creation of a nutrient reserve(Miller and Jastrow 1992a, b; Six et al. 2004).Mycorrhizal fungal dynamics appears to bea good indicator for determining the consequencesof different crop rotations on soil stability. Studiesin Australia indicated that 50 years of crop rotationdecreased the amount of stable macroaggregates,and simultaneously decreased the lengths of rootsand AM fungal hyphae in the soil, comparedwith soils from long-term pasture and naturalsites (Tisdall and Oades 1980). The study foundthat the positive association between the amountof macroaggregates and the length of externalmycorrhizal hyphae was related to the type ofcrop rotation and the frequency of fallow rotations(Fig. 4.2). A similar relationship was also foundbetween macroaggregates and root length. Thisstudy indicated that frequent use of fallow in croprotations can significantly decrease the amountof soil held as macroaggregates. Conversely, thelonger a soil has a cover crop, the greater theamount of soil held as macroaggregates. Thestudy also suggested that the loss of stable soilmacroaggregates associated with fallow rotationsmay be caused, at least in part, by a reduction inthe mycorrhizal fungus population that is broughton by both tillage and fallow disturbances.Using a series of prairie reconstructions in thecentralUnitedStatesasameansofinvestigatingtheaggradative phase of a soil indicates that a stable

56 R.M. Miller and D.J. LodgeFig. 4.2. The relationship between extraradical hyphaeand percentage of water-stable aggregates fora soil under different crop rotations (PP=old pasture,PPW=pasture-pasture-wheat, WW=wheat every year,WPF=wheat-pasture-fallow, PFW=pasture-fallow-wheat,WF=wheat-fallow, FW=fallow-wheat, PPPWWP andWPPPPW=2 years wheat and 4 years pasture, respectively;redrawn from Tisdall and Oades 1980)soil aggregate structure can develop rapidly underprairie and pasture vegetation (Jastrow 1987;Miller and Jastrow 1990; Jastrow et al. 1998). Thesoils of the study area had been under cultivationfor over 150 years; however, within 8 years afterbeingplantedtoprairiespecies,theproportionsof stable macroaggregates had approached that ofa nearby prairie remnant (Jastrow 1987). The rapidrecovery of soil aggregates was most likely due toa well-developed microaggregate structure that remainedrelatively intact during cropping, in combinationwith a rapid reestablishment of a relativelydense root and hyphal network after the cessationof tillage (Miller and Jastrow 1992b; Jastrow andMiller 1998). Both total root length and the lengthof roots colonized by AM fungi increased with timefrom disturbance (Cook et al. 1988). Furthermore,root and soil hyphal lengths were associated withincreases in the proportion of soil held as waterstablemacroaggregates (Miller and Jastrow 1990;Jastrow et al. 1998).The conversion of these soils from tillage-basedagriculture to prairie also enhanced microbialbiomass, with fungal biomass increasing ata proportionally great rate than bacterial biomass(Allison et al. 2005). Moreover, extramatrical AMfungal biomass responded more strongly to theprairie conversion than did saprotrophic fungalbiomass. Finally, in addition to modifications inhyphal length and biomass, the composition of theAM fungal community changed with the cessationof tillage and reconstruction of the prairie (Millerand Jastrow 1992b). Using spore biovolume asa measure of AM species contributions revealedthat soils under conventional tillage practices weredominated by Glomus constrictum and G. etunicatum.However, with the cessation of cultivation,Gigaspora gigantea replaced Glomus as the dominantfungal group by the fifth growing season. Thespore biovolume of Glomus species was negativelyassociated with recovery time since disturbance,external hyphal length, and percent of soil heldas macroaggregates; in contrast, Gigaspora waspositively associated with extraradical hyphallength and macroaggregation. Other investigatorshave also found Gigaspora to be more effectivethan Glomus in producing aggregates (Schreinerand Bethlenfalvay 1995). These trends suggestthat AM fungi may differ in their sensitivitiesto disturbance and in their abilities to produceextraradical hyphae, both of which can influencethe development of soil aggregates.V. Fungi and ForestryForests that are managed for recreation, timber,pulpwood, or other forest products differ widely intree diversity, their dominant mycorrhizal fungalassociates, the seral stage that is being managed,climate, seasonality, and decomposability of litterfall. For example, tree plantations and many nativetemperate and boreal forests are dominated by oneor a few tree species, whereas many tropical forestsare speciose; a single hectare of lowland Amazonianforest in Ecuador was reported to have over470 tree species greater than 10 cm in diameter atbreastheight(Valenciaetal.1994).Such underlyingdifferences among forest ecosystems will greatly influencehowfungirespondtodisturbancesrelatedto forestry practices.A. Nutrient Additions in Forest SystemsFertilization and atmospheric inputs of nitrogencaninfluencetherateatwhichfungaldecomposersrecycle nutrients from organic matter, but sucheffects may differ among forest types. Rates ofleaf decomposition differ among tree species andforest ecosystems, primarily because of limitations

Fungal Responses to Disturbance: Agriculture and Forestry 57on the growth and activity of fungal decomposersimposed by differences in concentrations ofmineral nutrients in the fallen leaves, climate, andabundance of lignin and other recalcitrant or toxicsecondary plant compounds (e.g., Meentemeyer1978; Swift et al. 1979; Aber and Melillo 1982; Vogtet al. 1986). Low nitrogen concentrations in fallenlitter are often more limiting to nutrient cycling intemperate forests, whereas the availability of phosphorusis often more important in lowland tropicalforests (Vitousek and Sanford 1986), althoughphosphorus concentrations can also be importantin temperate coniferous systems (Dyer et al. 1990).Many successional forest trees produce highlydecomposable leaf litterfall containing higherconcentrations of nutrients and labile carbon andlower concentrations of secondary chemicals thando species from later successional stages (Marks1974; Bazzaz 1979; Grime 1979). Therefore, theeffects of episodic fertilization or chronic additionsof nitrogen via air pollutants on litter decomposerfungi and the rate of decomposition may dependon the forest species composition, and may becorrelated with climate.In a late successional subtropical wet forest inPuerto Rico, complete fertilization accelerated decompositionof fine roots that had very low nutrientconcentrations, thereby lowering the deadroot standing stocks after 9 months (Parrotta andLodge 1991). Fertilization apparently also accelerateddecomposition of leaf litter in the same experiment,as indicated by a higher turnover rate.Production of leaf litter was significantly increasedby fertilization, while standing stocks of litter didnot differ among treatments at low elevation; litterfallwas greater, and litter standing stocks lowerin fertilized than in control plots at high elevation(Zimmerman et al. 1995).Despite higher rates of leaf decomposition,certain decomposer fungi were apparently negativelyaffected by fertilization in the above study.Superficial and interstitial mycelia of decomposerbasidiomycetes completely disappeared from thelitter layer in fertilized plots, and the diversityand abundance of their fruitifications were alsoreduced, compared with control plots (D.J. Lodge,S. Cantrell, and O. Oscar, unpublished data),suggesting that the fungal decomposer communitymay have changed in response to repeated fertilization.Similarly, Heinrich and Wojewoda (1976)found that fertilization of forest plots in Poland significantlydecreased the number of fruiting bodiesof basidiomycetes that decompose SOM and wood.Shifts in the composition of the fungal decomposercommunity in response to fertilization may reflectdifferential sensitivity among species to saltstress (Castillo Cabello et al. 1994), or reducedcompetitive advantage of fungi that use hyphalcords and rhizomorphs to translocate nutrientsinto nutrient-depauperate food bases (Boddy 1993;Lodge 1993). The disappearance of cord-formingbasidiomycetes may have a negative impact on theecosystem in terms of loss of SOM and nutrients.Absence of cords formed by basidiomycetes in thelitter layer had previously been found to significantlyincrease the rate of litter export from steepslopes during storms, and subsequent soil erosionfrom the exposed surfaces (Lodge and Asbury1988). Thus, there may be contrasting responses tofertilization in different ecosystem-level processesthat are mediated or influenced by fungi. Instandard forestry practices, fertilization generallyoccurs only at planting or post-thinning at 10 to15 years (Barrett 1962; Oliver 1986), so negativelong-term impacts on litter fungi are probably lessthan in the experiments cited above.Addition of mineral nutrients that are limitingtothegrowthoffungaldecomposersmightreasonablybe expected to consistently increase the rate ofdecomposition, but divergent responses occur indifferent forest ecosystems (Sinsabaugh et al. 2005).Although higher nitrogen and phosphorus concentrationswere correlated with higher initial ratesof decomposition of coniferous litter in Sweden,later decomposition and N-mineralization were inhibited(Berg et al. 1982, 1987). As noted in a reviewby Berg (1986), later decomposition may beslowed by ammonium and amino acid repressionof fungal ligninolytic enzymes, and by the complexingof nitrogen with phenolic compounds toform particularly recalcitrant products (Stevenson1982; Nommik and Vahtras 1982). In nitrogen additionexperiments used to simulate atmosphericinputs of N from air pollutants in northern USAhardwood forests, activity of enzymes that are importantin delignification (phenol oxidase and peroxidase)decreased along with decomposition ratesin low-quality (high lignin) litter, while oxidativeenzyme activity (e.g., cellobiohydrolase) and ratesof decomposition increased in high-quality (lowlignin) litter (Carriero et al. 2000; DeForest et al.2004; Waldrop et al. 2004; Sinsabaugh et al. 2005).Suppression of white-rot basidiomycete fungi ortheir enzyme activity has been implicated in alteringforest floor biogeochemical processes thatinfluence ecosystem level responses to increases in

58 R.M. Miller and D.J. Lodgenitrogen (Fog 1988; Berg and Matzer 1997; Carrieroet al. 2000).Sinsabaugh et al. (2005) have suggested that notall ecosystem-specific responses in forests to nitrogenadditions can be attributed to suppression ofwhite-rot basidiomycete fungi and their enzymes.Although white-rot basidiomycetes may be the organismsprimarily responsible for delignificationof leaf litter in some ecosystems, some soft-rotfungi, such as Aspergillus wentii, degrade ligninfaster when nitrogen is more abundant (Fog 1988).Furthermore, Sinsabaugh et al. (2005) noted thatnot all white-rot fungi exhibit N-dependent expression,and that changes in specific enzyme activitiesoccur in systems that vary widely in microbialcommunity composition and fungal abundance(DeForest et al. 2004; Gallo et al. 2004; Waldrop et al.2004). Nitrogen effects on litter decomposition mayresult from an uncoupling of polysaccharide andpolyphenol degradation (Sinsabaugh et al. 2002),and hydrolytic enzymes such as cellulases andphosphatases may also play a role (Sinsabaugh et al.2005). Berg and Ekbohm (1991) hypothesized thatnitrogen and phosphorus initially stimulate microbialdecomposition and nitrogen mineralization,thereby increasing the rate of release of nitrogencompounds that react with aromatic compoundsto form resistant residues. Söderström et al. (1983)found that annual fertilization of Scots pine plantationscaused slowing of the long-term decompositionrate, and a 50% reduction in microbial respirationand abundance in humus. The addition ofnitrogen through fertilization or air pollutants maytherefore be expected to increase the rate of totaldecomposition in forests where litter contains lowconcentrations of both nitrogen and polyphenoliccompounds, but may slow complete decompositionin forests where litter contains high concentrationsof polyphenolics and low concentrations of nitrogen(Berg and Ekbohm 1991). Palm and Sanchez(1990) found that leguminous leaves that naturallycontained high concentrations of both nitrogenand polyphenolic compounds decayed more slowlyin tropical agroecosystems than predicted fromtheir nitrogen concentrations alone, indicatingthat the negative interaction between nitrogen andpolyphenolics is a widespread phenomenon.Some studies using experimentally elevatedlevels of nitrogen input have found decreasedfungal-to-bacterial ratios (Waldrop et al. 2004),while others have not (DeForest et al. 2004). Thekey processes that initiate the chain of events thatdetermine carbon flow and fate in forest ecosystemsoccur in the litter layer (Zak et al. 2006).MethodssuchasPLFAthathavebeenusedtodateto determine changes in overall microbial communitystructure in forest litter (e.g., ratios of bacteria,fungi and protozoans; DeForest et al. 2004) are notable to detect changes in basidiomycete versus softrotfungi, or changes in fungal species dominancethat may be more relevant to ecosystem-levelresponses to elevated nitrogen inputs. Othermethods, such as real-time PCR, will be neededto determine how fungal community compositionshifts contribute to the radical changes in carboncycling observed in some forest ecosystems.B. Effects of Air Pollutionand Fertilization on FungiAtmosphericnitratedeposition,primarilyfromautomobileexhaust and power plants, has increased5- to 20-fold in northeastern USA and parts of Europe(Galloway 1995). In addition to the ecosystemprocess-level effects on fungal activity of increasednitrogen inputs noted above in Section V.A, therehave been significant changes in ectomycorrhizalfungal communities in European forests. The declinein fruiting bodies of ectomycorrhizal fungi inEurope (Derbsch and Schmitt 1987; Termorshuizenand Schaffers 1987; Jakucs 1988; Arnolds 1989; Fellner1989, 1993; Nauta and Vellinga 1993) has beenattributed to direct and indirect effects of air pollution(Arnolds 1991). While ectomycorrhizal fungihave decreased, fruiting by wood-decay fungi hasincreased in central Europe (Fellner 1993). Applicationof nitrogen fertilizer was found to causesimilar declines in the diversity or abundance (orboth) of ectomycorrhizal fungal species (Fiedlerand Hunger 1963; Heinrich and Wojewoda 1976;Schlechte 1991). Ectomycorrhizal and ericoid mycorrhizalfungi are thought to confer special advantagesto plants in the uptake of nitrogen from organicresidues in environments in which the availabilityor uptake of nitrogen is limited (Read 1991),which may help explain why chronic additions ofnitrogen from air pollutants are associated withdeclines in ectomycorrhizal fungi and their associatedhost trees.Some fungal responses to air pollution may berelatedtosoilpH.Bååth et al. (1979) found thatactive hyphae of soil fungi were decreased by applicationof artificial acidified rain. Not all ectomycorrhizalfungi have been negatively affected by airpollution. For instance, Fellner (1993) found that

Fungal Responses to Disturbance: Agriculture and Forestry 59certain Cortinarius species in the subgenus Dermocybethat are favored by acidic conditions were increasingin highly acidified forests. Schlechte (1991)reported that moderate amelioration of soil pHthrough application of dolomitic limestone hada stabilizing effect on ectomycorrhizal fungal communitiesin chronically acidified acid forests. However,addition of some forms of lime has been foundto adversely affect the abundance or diversity of ectomycorrhizalfungal fruiting bodies (Hora 1959;Fiedler and Hunger 1963; Heinrich and Wojewoda1976).C. Effects of Invasive Exotic Earthwormson Northern Forests and Forest FungiThe effects of invasions of exotic earthworms intopreviously undisturbed North American forests isof considerable concern for the maintenance offorest fungi and the nutrient cycling process theymediate (Bohlen et al. 2004a). Invasions by nonnativeearthworms, such as Lumbricus terrestrisand Dendrobaena octaedra, haveresultedinradicallosses of forest floor (Hazelhoff et al. 1981;Alban and Berry 1994; Bohlen et al. 2004b), resultingin altered nutrient availability to trees (Paréand Bernier 1989; Lawrence et al. 2002) throughchanges in nutrient cycling (Walbridge et al. 1991;Alban and Berry 1994; Scheu and Parkinson 1994a,b; Bohlen et al. 2004a, b). In addition, earthworminvasions in North America have increased susceptibilityof forests to erosion through the loss of soilcover (Hazelhoff et al. 1981; Alban and Berry 1994;Bohlen et al. 2004a). Forest floor is a key componentthat is critical for maintaining the stabilityof many forest ecosystems, for example, throughprotection against soil erosion, and for facilitatingforest regeneration after disturbance (Bormannand Likens 1979). The presence and extent of negativeeffects of exotic earthworm invasions varywith the characteristics of forest stands, includingforest type, soil characteristics, and history ofprevious disturbance (Bohlen et al. 2004a). Suarezet al. (2004) found that broadleaf forest plots invadedby L. terrestris had more total P at 0–12 cmdepth than did reference plots, but more of the Pwas fixed by Al and Fe hydroxides, and was thusunavailable. Walbridge et al. (1991) also noted thepotential for negative effects of mineral soil fromworm casts on phosphorus availability in the organichorizon. In forests with typically higher developmenton strongly weathered soils that haveelevated phosphorus-fixing capacity, most of theavailable phosphorus, and thus most of the P uptakeby trees via mycorrhizal fungi, occurs in theorganic forest floor, rather than the mineral soil(Wood et al. 1984; Yanai 1992). Lawrence et al.(2002) found that mycorrhizal fungal colonizationrates and percentage of colonized root length declinedsignificantly in organic forest soil horizonsinvaded by earthworms, indicating they can affectthe primary means of nutrient uptake by trees.Paré and Bernier (1989) found that stands of sugarmaple in southern Canada had impaired phosphorusnutrition where earthworms were abundant.The activities of exotic earthworms that removeforest floor litter and process it below ground willundoubtedly be detrimental to litter decomposerfungi, especially basidiomycete fungi that form littermats and are thus critical for preventing erosionlosses on steep forest slopes, but this aspect has notbeen studied.D. Forestry Practices for Pulpwoodand Lumber ProductionIn sustainable pulpwood forestry, monospecificstands are often planted and harvested on short rotations,using tree species from an early seral stagesuch as pines or other conifers, certain eucalyptus,white birch, aspen, and poplars. Such even-agedmanagement practices have been predominantin the United States for pulpwood and lumbersince 1950 (Oliver 1986). In such cases, clearcutharvesting sometimes followed by burning of slashis often used to regenerate the species. Thoughsuch forestry practices are often viewed as severeby environmentalists, these management practicesoften mimic natural disturbances to which thoseearly seral stage tree species and their associatedfungi are adapted (Oliver 1986). For example,some forest types, especially certain pines, aredependent on fire for their establishment andmaintenance of their integrity (Oliver 1986).E. Effect of Site Preparation on FungiThe effects of different intensities of site preparationand silvicultural practices on microbialbiomass and microbial nutrient stores beforereplanting of pine were studied in the southeasternUnited States by Vitousek and Matson (1984).They compared (1) whole-tree versus stem-onlyharvest; (2) chopping the harvest debris versus

60 R.M. Miller and D.J. Lodgeshearing and piling of all debris and forest floorinto wind rows, followed by disking of the mineralsoil; and (3) application of herbicide. Vitousek andMatson (1984, 1985) found that microbial nutrientimmobilization was effective in preventing leachinglosses of nitrogen when the forest floor wasleft intact (chopping) but not when the forest floorwas removed (shearing/piling/disking). Lossesof nitrate to stream water and groundwater vialeaching (Borman and Likens 1979; Vitousek andMelillo 1979; Robertson and Tiedje 1984), andatmospheric losses through denitrification occurin forests that have been cleared, but microorganismsin the forest floor and soil immobilize andconserve most of the nitrogen that is mineralizedin response to the disturbance (Marks and Borman1972; Vitousek and Matson 1984, 1985). Largewoody debris (slash) and whole-tree harvestinghad little effect on microbial immobilizationof nitrogen, at least during short-term study(2 years), but herbicides may have been toxicto microorganisms, thereby increasing leachinglosses of nitrogen (Vitousek and Matson 1985).Although burning of coarse woody debris(slash) is considered an appropriate managementpractice for regeneration of Douglas fir in thePacific Northwest (Oliver 1986), broadcast burningfollowing clearcuts (Harvey et al. 1980) and partialcuts (Harvey et al. 1997) has been found to greatlydecrease the abundance of active ectomycorrhizaein the forest floor, and levels of ectomycorrhizalfungal inoculum became insufficient for regenerationif replanting was delayed until the next dryseason. Broadcast burning has been misapplied toold-growth true fir forests, resulting in mortalityofyoungtrees,predispositionofthefirstofungithat decay stems (Oliver 1986), and destructionof the litter fungal community. Highly decayedcoarse woody debris is important for maintenanceof ectomycorrhizal fungi in the northern RockyMountains during the hot dry summers (Harveyet al. 1980, 1997). In addition to affording a moistrefugium to mycorrhizae during drought, ectomycorrhizalfungi apparently obtain part of theirnitrogen from organic sources including rottingwood (Read et al. 1989). Thus, removal of coarsewoody debris from forests by whole-tree harvestingor burning of woody debris following harvestmay have negative effects on ectomycorrhizalfungi and tree nutrition, especially in areas withseasonal drought (Harvey et al. 1980, 1997).Others have also reported that the severityof a particular management activity can affectthe persistence of ectomycorrhizal fungi in thenorthwestern United States (e.g., Schoenbergerand Perry 1982; Perry et al. 1982; Amaranthusand Perry 1987). Moreover, studies of youngerclearcut stands indicate that prompt regenerationmaybeimportanttosecureadequateformationofindigenous fungi, suggesting mycorrhizal nurserystock may be more useful on older and moreseverely burned sites (Pilz and Perry 1984).Studies of site preparation effects on mycorrhizalfungi from tropical forest systems arepractically nonexistent. The few studies that havebeen conducted demonstrate disturbances due tosite preparation reduce AM fungal propagule levelsand species composition, compared to undisturbedforest levels (Alexander et al. 1992; Mason et al.1992; Wilson et al. 1992). In a Cameroon study,mycorrhizal spore loss appeared to be related tothe severity of disturbance, with decreases beinggreatest in stands with vegetation completelyremoved. Reduced spore numbers in manuallycleared stands were not as severe as in thosecleared by mechanical methods, and plantingof the stands with Terminalia seedlings allowedfor their recovery, although mycorrhizal speciescomposition remained altered (Mason et al. 1992).Similar findings were found in a Terminaliaplantation in the Côte d’Ivoire, where even thoughsite preparation effects on spore numbers were lesseasily differentiated, manual clearing of vegetationresulted in weaker spore reduction than didmechanical means (Wilson et al. 1992). Also, sporenumbers recovered more rapidly in the manuallycleared stand; AM fungal richness increasedgreatly under both methods of clearcutting, althoughspecies balance under manual clearcuttingwas closer to that of the undisturbed forest. Thesestudies suggest selective harvest practices dominimal damage to the mycorrhizal community,in that they support extensive mycelial networks,which for natural systems are the primary sourceof infection for tree seedlings (Alexander et al.1992). The reduced levels for mycorrhizal roots,hyphae, and spores in heavily logged systems aredue to the mortality of root fragments containingmycorrhizal hyphae. Hence, clearing practices thatallowforthecontinuedexistenceofamycelialnetwork may be preferable ecologically andsilviculturally (Table 4.3).Stumps of felled trees, roots in the soil, andother woody residues left from forestry operationsoften provide essential resources for pathogenicfungi and those that cause root or stem decay

Fungal Responses to Disturbance: Agriculture and Forestry 61Table. 4.3. The most probable number (MPN propagula per100 g fresh soil±SE) of AM fungi in the top 15 cm of soilfor Malaysian forest sites under different logging intensities(n = 5; data from Alexander et al. 1992)Logging intensityUndisturbedSelectively loggedHeavily loggedHeavily logged (eroded)MPN a307.5±13.8a291.2±7.8a75.3±2.6b6.0±1.5ca Values followed by the same letter are not significantlydifferent (p

62 R.M. Miller and D.J. Lodgeter represents the gleaning from a wide variety ofsources ranging from decomposition studies, to detritalfood-web investigations, and to research onsoil aggregation, all of which suggest an importantrole for fungi in managed systems. Moreover, obstaclesto identifying the contribution of fungi inmanaged systems have been just as much methodologicalas conceptual.Although our ability to quantify the variousfungal components (especially their activities) isstill limited, major conceptual breakthroughs haveoccurredonhowweviewsoils.Twoimportantconceptual advances are the hierarchical theory ofsoil structure (Tisdall and Oades 1982), and the“spheres of influence” view of soil systems (Colemanet al. 1994; Beare et al. 1995). In both of theseconceptual views of soils, the contributions of fungiare considerable. Furthermore, the perturbationsimposed by management practices have played animportant role in the development of these views.Fungal responses to disturbance offer an opportunityto test these two compelling theories of soilstructure and function.A necessary step in developing sustainablemanagement practices in agriculture and forestrywill require identifying practices that allow forcontrolled manipulations of the fungal community.Although our ability to manipulate fungi is ratherlimited, such manipulations are not impossible.Research is needed to better understand theresponse of both the saprotrophic hyphae andthe mycorrhizal hyphal network to disturbancesassociated with different management practices.Of crucial importance is the development of managementpractices that maximize nutrient uptakebut are not detrimental to the litter layer and thesoil aggregation process. Specifically, researchis needed on how disruption of the mycorrhizalhyphal network via tillage and rotation (e.g.,fallow or non-mycorrhizal host crops) affects inagriculture, and harvesting and site preparation(e.g., clearcut or slash removal) affect in forestry interms of the soil aggregate structure. Research alsoneeds to be directed at developing managementpractices that take advantage of the nutrient poolsassociated with the fungal hyphae. These kindsof studies would represent important steps incontrolling the soil’s labile nutrient pools.Acknowledgements. The support of RMM for the preparationof this chapter was by the U.S. Department of Energy,Office of Science, Office of Biological and EnvironmentalResearch, Climate Change Research Division, under contractno. W-31-109-ENG-38.ReferencesAbbott LK, Robson AD (1994) The impact of agriculturalpractices on mycorrhizal fungi. In: Pankhurst CE,DoubeBM,GuptaVVSR,GracePR(eds)Soilbiotamanagement in sustainable farming systems. 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5 Fungi and Industrial PollutantsG.M. Gadd 1CONTENTSI. Introduction ........................ 69II. Predicted Effects of Pollutantson Fungal Populations ................ 70III. Fungi and Xenobiotics ................ 70IV. Effects of Acid Rainand Airborne Pollutantson Fungal Populations ................ 72V. Effects of Toxic Metals on Fungi ......... 73A. Effects of Metals on Fungal Populations 74B. Mycorrhizal ResponsesTowardsToxicMetals............... 75C. Metal and Metalloid TransformationsbyFungi......................... 751.MetalMobilization............... 762.MetalImmobilization ............ 763.Organometal(loid)s.............. 77D. Accumulation of Metalsand Radionuclides by Macrofungi . . . . . 77E. Accumulation of RadiocaesiumbyMacrofungi.................... 77F. Fungi as Bioindicators of Metaland Radionuclide Contamination . . . . . 78VI. Conclusions ......................... 79References.......................... 79I. IntroductionFungi may be exposed to a wide variety of organicand inorganic pollutants in the environment. Sincefungi play a major role in carbon, nitrogen, phosphorusand other biogeochemical cycles (Wainwright1988a,b;Gadd2006),theimpairmentoffungalactivity could have important consequences forecosystem function. It is obviously desirable thatmore is known about the impact of pollutants onthese organisms. Unfortunately, while it is easy tospeculate on the likely effects of pollutants on fungi,it is often far more difficult to demonstrate such effects.Studies on pollutant effects on fungal populationsare difficult, largely because of the inadequacy1 Division of Molecular and Environmental Microbiology, Collegeof Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland,UKof many of the techniques which are available tostudy fungi, and the complexity of microbial communities.However, an appreciation of the effectswhich pollutants can have on fungi can be obtainedby a combination of the following measurements:(1) pollutant concentration, composition and distribution(2) pollutant bioavailability (3) pollutantconcentrations which cause a toxic or physiologicalresponse in vitro, (4) effects of the pollutant onfungal population/community size and compositionand (5) secondary changes resulting from pollutioneffects on fungal populations, e.g. impact onleaf litter decomposition. While pollutant concentrationand composition may be determined usingstandard analytical techniques, with varying degreesof difficulty depending on the pollutant andthe environmental matrix, analyses of pollutantbioavailability and speciation remain challengingproblems.The effect of pollutants on fungal population/communitysize and composition isparticularly difficult to assess. Many earlier studiesused the dilution plate count method to assesschanges in fungal community composition. Theshortcomings of this technique have been criticisedat length and are now well known. To overcomeproblems relating to the use of plate counts,biomarkers such as phospholipid fatty acid (PLFA)composition, and extraction and analysis of DNAare now routinely used, though no method(s) areexempt from problems. Another problem is that itis unlikely that a meaningful picture of how fungirespondtopollutantsintheenvironmentcanbegained from determining responses to pollutantsadded to solid or liquid growth media in laboratoryexperiments. The effects of toxic metals on soilfungi growing in vitro, for example, is markedlyinfluenced by the composition of the medium used:metalsarelikelytobemoretoxictofungiinlowcarbonmedia than in carbon-rich media wherethe production of large amounts of extracellularpolysaccharides and chemical interactions withEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

70 G.M. Gaddthe medium will tend to reduce metal availability.Medium components may also complex metalsout of solution, making them unavailable (Gaddand Griffiths 1978). Finally, interactions betweendifferent pollutants and their breakdown productsmay have a major influence on the toxicity ofa pollutant in the natural environment.This chapter will outline some of the main effectsof organic and inorganic pollutants on fungi,and will include discussion of effects at the cellularand community levels as well as their applied andenvironmental significance.II. Predicted Effects of Pollutantson Fungal PopulationsEnvironmental pollution might be expected to leadto both toxic (destructive) and enrichment disturbanceon fungal populations (Wainwright 1988b).Although toxic disturbance is likely to predominate,instances will occur where both types of disturbanceare found together. Toxic disturbance offungal populations is likely to be particularly damagingto ecosystem function, while the rarer enrichmentdisturbance may occasionally produce beneficialeffects on soil processes.Toxic disturbance is likely to lead to a reductionin fungal numbers and species diversity, as well asbiomass and activity changes which may detrimentallyinfluence fundamentally important processessuch as litter decomposition. The resultant degreeof toxic disturbance will depend upon both toxicantconcentration and its availability to the fungalpopulation, as well as to the susceptibility of the individualsinvolved. Toxicants may be selective andaffect only a few species, or they may have a moregeneralised effect. Selective inhibition may haveless of an impact on overall soil fungal activity thanmight be imagined, since susceptible species canbe replaced by more resistant fungi, some of whichmaybemoreactiveinagivenphysiologicalprocessthan the original population. While concentrationeffects are generally emphasised, it is surprisinghow often the question of toxicant bioavailabilityis avoided in studies on the effects of pollutantson microorganisms. In soils, for example, bioavailabilityof a pollutant will generally depend uponfactorssuchas(1)adsorptiontoorganicandinorganicmatter, (2) chemical speciation, (3) microbialtransformation and/or degradation and (4) leaching.Anotherfactorofimportanceinrelationtotheeffects of toxicants on soil fungi concerns nutrientavailability. Fungi are generally thought to bealreadystressedbythelowlevelsofavailablecarbonpresent in most soils and other environments(Wainwright 1992). They will grow slowly, if at all,under these conditions, and may be more susceptibleto pollutants than when growing under highnutrient conditions.Fungal populations are unlikely to remainstatic when confronted with a toxic agent, andresistant populations are likely to develop whichwill be a major factor in determining populationresponses to the pollutant. On the other hand,a number of studies have shown that fungi isolatedfrom metal-contaminated soils show less adaptationto toxic metals, such as copper, than mightbe expected (Yamamoto et al. 1985; Arnebrantet al. 1987). Mowll and Gadd (1985) also foundno differences in the sensitivity of Aureobasidiumpullulans to lead when isolates from contaminatedand uncontaminated phylloplanes were compared.Enrichment disturbances may also be eitherselective or non-selective. Non-selective enrichmentdisturbance might theoretically result fromthe input into the ecosystem of a pollutant whichis widely used as a nutrient source. Since suchenrichmentisrare,mostexamplesofthisformof disturbance will be selective. Reduced formsof sulphur are, for example, likely to enrich thesoil for S-oxidizing fungi, while phenolics andhydrocarbons may favour species capable ofutilizing these compounds.III. Fungi and XenobioticsSome fungi have remarkable degradative properties,and lignin-degrading white-rot fungi, such asPhanerochaete chrysosporium, can degrade severalxenobiotics including aromatic hydrocarbons,chlorinated organics, polychlorinated biphenyls,nitrogen-containing aromatics and many otherpesticides, dyes and xenobiotics. Such activities areof bioremedial potential where ligninolytic fungihave been used to treat soil contaminated withpentachlorophenol (PCP) and polycyclic aromatichydrocarbons (PAHs) (Singleton 2001). In general,treatment involves inoculation of the contaminatedsoil followed by nutrient addition, irrigation, andaeration and maintenance by general land farmingprocedures. Correct preparation of the fungalinoculum can be crucial: fungi may be grown on

Fungi and Industrial Pollutants 71lignocelluosic substrates prior to introductioninto the soil (Singleton 2001). Treatment cantake weeks to months or longer, depending onthe level of contamination and environmentalfactors. In many cases, xenobiotic-transformingfungi need additional utilizable carbon sourcesbecause, although capable of degradation, theycannot utilize these substrates as an energy sourcefor growth. Therefore, inexpensive utilizablelignicellulosic wastes such as corn cobs, straw andsawdust can be used as nutrients for enhancedpollutant degradation (Reddy and Mathew 2001).Wood-rotting and other fungi are also receivingattention for the decolourization of dyes andindustrial effluents, various agricultural wastessuch as forestry, pulp and paper by-products,sugar cane bagasse, coffee pulp, sugar beet pulp,apple and tomato pulp, and cyanide (Barclay andKnowles 2001; Cohen and Hadar 2001; Knapp et al.2001).Polycyclic aromatic hydrocarbons (PAHs)enter the environment via many routes, includingfossil-fuel combustion, vehicle exhaust emissions,gas and coal tar manufacture, wood-preservationprocesses, and waste incineration (Harvey 1997;Pozzoli et al. 2004). Many PAHs are toxic towardsmicroorganisms, plants and animals; PAHs of lowmolecular weight and high water solubility are themost toxic (Cerniglia and Sutherland 2006). PAHsdisappear relatively slowly in the environmentthrough physical, chemical and biological processes,some of which are mediated by bacteria andfungi. PAH recalcitrance of soils and sedimentsincreases with molecular weight, but severalother physico-chemical and biological factors cancontribute to this, e.g. lack of PAH-degradingmicroorganisms, nutrient deficiency, low bioavailability,preferential utilization of more easilydegradable substrates, and the presence of othertoxic pollutants or breakdown products (Cernigliaand Sutherland 2006). Other related factors whichaffect PAH biodegradation in soil include soiltype, pH, temperature, oxygen concentration,irradiation, as well as the solubility, volatility andsorption properties of the PAHs (Huesemann et al.2003; Lehto et al. 2003; Rasmussen and Olsen2004). Bioremediation by mixed communities maybe enhanced by bacteria which produce degradativeenzymesaswellasbiosurfactants(Straubeet al. 1999; Cameotra and Bollag 2003). Aerobicbiodegradation of PAHs by soil microorganismsuses monooxygenase, peroxidase and dioxygenasepathways; the first and third of these pathways areutilized by bacteria while the first and second arefound in fungi.Many fungi can metabolize PAHs (Cernigliaand Sutherland 2001, 2006; Sutherland 2004; Verdinet al. 2004). Since fungi cannot generally use PAHsas the sole carbon and energy source (Cernigliaand Sutherland 2001), they must be suppliedwith nutrients to allow co-metabolism. A smallnumber of yeasts and filamentous fungi have beenreported to use some PAHs, including anthracene,phenanthrene, pyrene and benzo[a]pyrene, ascarbon and energy sources (Romero et al. 2002;Lahav et al. 2002; Saraswathy and Hallberg 2002;Veignie et al. 2004). Some fungi co-metabolizePAHs to trans-dihydrodiols, phenols, quinones,dihydrodiol epoxides, and tetraols but seldomdegrade them completely to CO 2 (Casillas et al.1996; Cajthaml et al. 2002; da Silva et al. 2003).The transformation of PAHs by ligninolytic,wood-decaying fungi involves several differentenzymes. The enzymes produced by white-rotfungi which are involved in PAH degradationinclude lignin peroxidase, manganese peroxidase,laccase, cytochrome P450, and epoxide hydrolase(Haemmerli et al. 1986; Bezalel et al. 1996;Cerniglia and Sutherland 2006). Ligninolyticfungi metabolize PAHs via reactions involvingreactive oxygen species to phenols and quinones(Pickard et al. 1999; Steffen et al. 2003), and thesemay be further degraded by ring-fission enzymes(Cerniglia and Sutherland 2006).Severalwood-decayingfungi,e.g.Bjerkandera,Coriolopsis, Irpex, Phanerochaete, Pleurotus andTrametes spp., have been investigated for bioremediationof PAH-contaminated soils (Baldrian et al.2000; Novotný et al. 2000; Cerniglia and Sutherland2006). Laboratory trials have demonstratedtheir ability to degrade complex mixtures of PAHs,such as those in creosote and coal tar, but actualbioremediation of contaminated soils usingthese fungi has met with varying success (Pointing2001; Cerniglia and Sutherland 2001; Canet et al.2001; Hestbjerg et al. 2003). Non-ligninolytic fungi,including Cunninghamella, Mucor, Fusarium andPenicillium spp.,havealsobeenconsideredforPAHbioremediation (Colombo et al. 1996; Pinto andMoore 2000; Ravelet et al. 2001; Saraswathy andHallberg 2002).Biodegradation may require the presenceof mixed bacterial and fungal communities,although less is known about the pathways ofPAH degradation by co-cultures (Juhasz andNaidu 2000). The evolution of 14 CO 2 from

72 G.M. Gadd14 C-phenanthrene in soil was enhanced almosttwo-fold (from 19.5 to 37.7%) when P. chrysosporiumwas added to the indigenous soil microflora(Brodkorb and Legge 1992). Boonchan et al. (2000)combined Penicillium janthinellum with eitherStenotrophomonas maltophilia or an unidentifiedbacterial consortium. The fungus couldpartially degrade pyrene and benzo[a]pyrenebut could not use either as a carbon source;S. maltophilia could use pyrene as a carbonsource and co-metabolize benzo[a]pyrene. Thefungal–bacterial combinations grew on pyrene,chrysene, benz[a]anthracene, benzo[a]pyreneand dibenz[ah]anthracene, converting 25% ofthe benzo[a]pyrene to CO 2 in 49 days. Thewhite-rot fungus P. ostreatus and the brown-rotfungus Antrodia vaillantii enhanced the degradationof fluorene, phenanthrene, pyrene andbenz[a]anthracene in artificially contaminatedsoils (Andersson et al. 2003). Unlike P. ostreatus,which inhibited the growth of indigenous soilmicroorganisms, A. vaillantii stimulated soilmicrobial activity.Ligninolytic fungi partially oxidize PAHsby reactions involving extracellular free radicals(Majcherczyk and Johannes 2000), making thePAHs more water-soluble so that they are able toserve as substrates for bacterial degradation (Meulenberget al. 1997). Partial oxidation increasesPAH bioavailability at most contaminated sites(Mueller et al. 1996; Meulenberg et al. 1997), andPAH-contaminated soils may contain large populationsof PAH-transforming bacteria (Johnsenet al. 2002) and fungi (April et al. 2000; Saraswathyand Hallberg 2002). Combinations of severalmicroorganisms are usually better able to degradebenzo[a]pyrene and other high-molecular-weightPAHs than pure cultures (Kanaly et al. 2000).IV. Effects of Acid Rain and AirbornePollutants on Fungal PopulationsAlthough acid rain is generally regarded as a longrangepollution phenomenon, high concentrationsof mineral acids will pollute ecosystems close topoint source emissions (Tabatabai 1985; Francis1986; Helander et al. 1993). Acid rain effects willalso impinge on the availability and effects of otherpollutants such as toxic metals, which may accompanyatmospheric dispersal and/or be releasedfrom soil components as a result of increased acidity(Wainwright et al. 1982). Baath et al. (1984)showed that soil biological activity, as determinedby respiration rate, was significantly reduced followingtreatment with simulated acid rain. Myceliallengths (FDA active) were also reduced by the treatment,while plate counts showed no response. Fritze(1987), on the other hand, showed that urban airpollution had no effect on the total length of fungalhyphae in the surface horizons of soils supportingNorway spruce (Piceaabies). Bewley and Parkinson(1985) showed that the contribution which fungimake to the total respiration of a soil was reducedby acid rain. In contrast, Roberts et al. (1980) concludedthat the addition of acid rain to forest soilsdid not affect the normal 9:1 balance of fungal tobacterial respirations. These studies clearly illustratehow difficult it is to generalise about the effectsof atmospheric pollutants on soil microorganisms.Among higher fungi, simulated acid rain has beenshown to increase the dominance of some ectomycorrhizalfungi, while decreasing species diversityamong saprophytic species (Sastad and Jenssen1993). Shaw et al. (1992) also showed that fumigationwith sulphur dioxide or ozone had no effecton mycorrhizal populations. Acid treatments havebeen shown to impair the decomposition of bothdeciduous leaves and conifer needles (Baath et al.1984; Prescott and Parkinson 1985). Small-scale inhibitoryeffects were common, although stimulatoryeffects were also observed. Pollution in theform of alkaline dust from iron and steel works wasshowntoleadtoadoublingofthetotallengthoffungal hyphae (Fritze 1991; Fritz and Baath 1993).The addition of lime has been shown to variouslydecrease soil fungal populations (Nodar et al. 1992)or to have no measurable effect (Persson et al. 1989).The measurement of leaf litter and cellulosedecomposition provides a means of assessing theimpact of atmospheric pollutants on soils. However,in the absence of a means of partitioning therelative impact of the toxicants on fungi, bacteriaand soil animals, such methods provide a measureof the effects of the pollutants only on the totalsoil community. Atmospheric pollutants from cokingworkscan,forexample,reducepopulationsofsoil microarthropods, a response which retards therateoflitterdecompositionindeciduouswoodlandsoils (Killham and Wainwright 1981).Few examples of the effects of enrichment disturbanceby air pollutants on fungal populationscan be found in the literature. However, some fungihave been reported to utilize atmospheric pollutiondeposits from coking works as a nutrient source,

Fungi and Industrial Pollutants 73as well as being able to oxidize the reduced sulphurwhich these particles contain (Killham andWainwright 1982, 1984).V. Effects of Toxic Metals on FungiTheabilityoffungitosurviveinthepresenceofpotentially toxic metals depends on a number ofbiochemical and structural properties, includingphysiological and/or genetical adaptation, morphologicalchanges and, finally, environmentalmodification of the metal in relation to speciation,availability and toxicity (Fig. 5.1; Gadd andGriffiths 1978; Gadd et al. 1984; Gadd 1992, 2007).Terms such as “resistance” and “tolerance” areoften used interchangeably in the literature, andmay be arbitrarily based on the ability to grow ona certain metal concentration in laboratory me-dia (Baath 1991; Gadd 1992). “Resistance” is probablymore appropriately defined as the ability ofan organism to survive metal toxicity by means ofa mechanism produced in direct response to themetal species concerned; the synthesis of metallothioneinand γ-glutamyl peptides in response toCu and Cd respectively providing perhaps the bestexamples (Mehra and Winge 1991). Metal tolerancemay be defined as the ability of an organism to survivemetal toxicity by means of intrinsic propertiesand/or environmental modification of toxicity(Gadd 1992). Intrinsic properties which can determinesurvival include possession of impermeablepigmented cell walls and extracellular polysaccharide,and metabolite excretion, especially wherethis leads to detoxification of the metal species bybinding or precipitation (Gadd 1993a). However,such distinctions are often difficult to recognisebecause of the involvement, in fungal survival inresponse to metal toxicity, of several direct and in-Fig. 5.1. Diagrammatic representation of the interactionsof toxic metals and radionuclides with fungi in the terrestrialenvironment. The dotted line shows direct effects ofmetal species on fungi; this may sometimes occur and ismore likely for metal species, such as Cs + ,whicharehighlymobile. Release of metal/radionuclide species from deadand decomposing animal, plant and microbial biomass isnotshownbutwillbeanimportantpartofmetalcycling.Fungal roles in metal solubilization from naturally occurringsubstrates and/or industrial materials are indicated(see Burgstaller and Schinner 1993). For more detailedinformation regarding physiological and cellular interactions,see Mehra and Winge (1991) and Gadd (1993a); fororganometal(loid) transformations, see Gadd (1993b)

74 G.M. Gadddirect physicochemical and biological mechanisms(Gadd and White 1985).Biological mechanisms implicated in fungalsurvival (as distinct from environmental modificationof toxicity) include extracellular precipitation;complexation and crystallization; the transformationof metal species by, e.g. oxidation, reduction,methylation and dealkylation; biosorption to cellwalls, pigments and extracellular polysaccharide;decreased transport or impermeability, efflux; intracellularcompartmentation; and finally, precipitationand/or sequestration (Gadd and Griffiths1978; Gadd et al. 1984; Gadd 1990, 1992; Mehraand Winge 1991).A. EffectsofMetalsonFungalPopulationsA range of fungi from all the major groups maybe found in metal-polluted habitats (Gadd 1993a).In general terms, toxic metals may affect fungalpopulations by reducing abundance and speciesdiversity, and selecting for a resistant/tolerant population(Jordan and Lechevalier 1975; Babich andStotzky 1985; Arnebrant et al. 1987). However, theeffect of toxic metals on microbial abundance innatural habitats varies with the metal species andthe organism present, and also depends on a varietyof environmental factors, making generalisationsdifficult (Gadd and Griffiths 1978).General reductions in fungal “numbers” (as assessedby the dilution plate count in many earlierstudies) have often been noted in soils pollutedwith Cu, Cd, Pb, As and Zn (Bewley and Stotzky1983; Babich and Stotzky 1985). However, numericalestimates alone may provide little meaningfulinformation unless possible changes in fungalgroups and species are considered, and the problemsassociated with plate counting are in any casewell known. Frostegard et al. (1993) analysed thephospholipid fatty acid (PLFA) composition of soilin order to detect changes in the overall compositionof the microbial community and provide morereliable information on fungal populations thancan be produced using plate counts. Two soils wereamended with Cd, Cu, Ni, Pb and Zn and analysedafter 6 months. PLFA 18:2ω6 isregardedasan indicator of fungal biomass, and this increasedwith increasing metal contamination for all metalsexcept Cu, possibly reflecting the well-known mycotoxicityof Cu. However, in forest soils, such anincrease in PLFA 18:2ω6wasnotobservedbecauseof masking by identical PLFAs derived from plantmaterial (Frostegard et al. 1993).Several studies have shown that microbial populationresponses to toxic metals are characterisedby a shift from bacteria, including streptomycetes,to fungi (Mineev et al. 1999; Kostov and Van Cleemput2001; Olayinka and Babalola 2001; Chanderet al. 2001a, b; Khan and Scullion 2002). However,other studies have shown a higher metal sensitivityof the fungal component of the microbial biomass(Pennanen et al. 1996). What seems clear is that allnutritional groups of fungi (saprotrophs, biotrophsand necrotrophs) can be affected by toxic metals.Ruhling et al. (1984) found that the soil respirationrate, fluorescein diacetate active mycelium (FDA)and mycelial standing crop were all reduced withincreasing copper concentration in soils proximalto a brass mill. Nordgren et al. (1983, 1985) alsoshowed that fungal biomass and soil respirationdecreased by ∼75% along an increasing concentrationgradient of metal pollution. A relativedecrease in an indicator fatty acid for arbuscularmycorrhizal fungi and an increase for other fungihave been reported for zinc-polluted soil (Kellyet al. 1999). Toxic metals (Cd, Cr, Cu, Ni, Pb andZn) led to a decrease in the number of arbuscularmycorrhizal fungi and low colonization of plantroots and, as a result, changes in mycorrhizalspecies diversity (Del Val et al. 1999; Moynahanet al. 2002; Mozafar et al. 2002). Toxic metals alsoreduce plant root colonization by ectomycorrhizalfungi and ectomycorrhizal species composition(Hartley et al. 1999; Markkola et al. 2002). The mostfrequent soil saprotrophic microfungi isolatedfrom heavily metal-polluted habitats in Argentina,Czech Republic and Ukraine were reported to bespecies of Penicillium, Aspergillus, Trichoderma,Fusarium, Rhizopus, Mucor as well as Paecilomyceslilacinus, Nectria invertum, Cladosporium cladosporioides,Alternaria alternata and Phomafimeti (Kubatova et al. 2002; Massaccesi et al. 2002;Fomina, Manichev, Kadoshnikov and Nakonechnaya,unpublished data). Melanized fungi, such asCladosporium sp., Alternaria alternata and Aureobasidiumpullulans, were often isolated from soilcontaining high concentrations of copper and mercury(Zhdanova et al. 1986), and can be dominantmembers of the mycoflora of metal-contaminatedphylloplanes (Mowll and Gadd 1985). Dark septateendophytes were found to be dominant fungiamong isolates from roots of Erica herbacea L. inPb-, Cd- and Zn-polluted soil (Cevnik et al. 2000).Metal pollution of plant surfaces is widespreadbut many filamentous and polymorphic fungi appearto be little affected (Bewley 1979, 1980; Be-

Fungi and Industrial Pollutants 75wley and Campbell 1980; Mowll and Gadd 1985).On polluted oak leaves, Aureobasidium pullulansand Cladosporium species were the most numerousorganisms (Bewley 1980). In fact, numbers ofA. pullulans showed a good positive correlationwith lead, whether derived from industrial or vehicularsources, and this fungus was frequentlythe dominant microorganism present (Bewley andCampbell 1980; Mowll and Gadd 1985).In conclusion, elevated concentrations oftoxic metals can affect both the qualitative andquantitative composition of fungal populations,although it is often extremely difficult to separatetheir effects from those of other environmentalpollutants. It is apparent that certain fungi canexhibit considerable tolerance towards toxicmetals and can become dominant microorganismsin some polluted habitats. However, whilespecies diversity may be reduced in certain cases,resistance/tolerance can be exhibited by fungifrom both polluted and non-polluted habitats.Physico-chemical properties of the environment,including changes associated with the metalpollution, may also influence metal toxicity andthereby affect species composition (Gadd 1984,1992, 1993a; Baath 1989).B. Mycorrhizal Responses Towards Toxic MetalsPlant symbiotic mycorrhizal fungi can accumulatemetals from soil components, and this may haveconsequencesformetalnutritionofthesymbiosisas well as increased or decreased toxicity. Sinceplants growing on metalliferous soils are generallymycorrhizal, an important ecological role for thefungus has frequently been postulated, althoughsuch a role, e.g. phytoprotection, is often difficultto confirm (Meharg and Cairney 2000).Ericaceous plants appear to be entirely dependentonthepresenceofericoidmycorrhizasforprotection against copper, the fungus preventingmetal translocation to plant shoots (Bradley et al.1981, 1982). Arbuscular mycorrhizas (AM) frommetal-contaminated sites are often more metal tolerantto, e.g. Cd and Zn, than other isolates, suggestinga benefit to the plant via increased metal resistance,nutrient uptake, etc.; in some cases, however,AM plants do not necessarily require fungalcolonization for survival (Griffioen 1994). It is oftenpostulated that mycorrhizas provide a barrierto the uptake of potentially toxic metals (Wilkins1991; Hetrick et al. 1994; Wilkinson and Dickinson1995; Leyval et al. 1997; Meharg and Cairney2000), though this has not been confirmed in everycase.Further,insomeinstances,AMmaymediateenhanced accumulation of essential metals which,unless regulated, may lead to phytotoxicity (Killhamand Firestone 1983). It is generally concludedthat local conditions at metal-contaminated sitesmay determine the nature of the relationship betweenthe plant and the AM fungus, since detrimental,neutral or beneficial interactions have allbeen documented (Meharg and Cairney 2000).For ericaceous mycorrhizas, clear host protectionis observed for host plants, e.g. Calluna sp.,Erica sp. and Vaccinium sp. growing on pollutedand/or naturally metalliferous soils (Bradley et al.1981, 1982). Further, ericaceous plants are generallyfound on nutrient-deficient soils, and it is likelythe mycorrhiza additionally benefit the plants byenhanced nutrient uptake (Smith and Read 1997).A protective metal-binding effect of ectomycorrhizalfungi (EcM) has been postulated frequently(e.g. Leyval et al. 1997), though other workers pointout the lack of clear evidence (Denny and Wilkins1987; Colpaert and Van Assche 1987, 1993; Dixonand Buschena 1988).C. Metal and Metalloid Transformationsby FungiFungi can transform metals, metalloids (elementswith properties intermediate between those of metalsand non-metals: the group includes arsenic, seleniumand tellurium) and organometallic compoundsby reduction, methylation and dealkylation.These are all processes of environmental importance,since transformation of a metal or metalloidmay modify its mobility and toxicity. For example,methylated selenium derivatives are volatileand less toxic than inorganic forms while reductionofmetalloidoxyanions,suchasseleniteortelluriteto amorphous elemental selenium or tellurium respectively,results in immobilization and detoxification(Thompson-Eagle and Frankenberger 1992;Morley et al. 1996). The mechanisms by whichfungi (and other microorganisms) effect changesin metal speciation and mobility are important survivaldeterminantsaswellascomponentsofbiogeochemicalcycles for metals and many other elements,including carbon, nitrogen, sulphur andphosphorus (Fig. 5.1; Gadd 1999, 2007).Metals and their compounds interact withfungi in various ways depending on the metal

76 G.M. Gaddspecies, organism and environment, while fungalmetabolism also influences metal speciation andmobility. Many metals are essential, e.g. Na, K,Cu,Zn,Co,Ca,Mg,MnandFe,butallcanexerttoxicity when present above certain thresholdconcentrations (Gadd 1993b). Other metals, e.g.Cs,Al,Cd,HgandPb,havenoknownbiologicalfunction but all can be accumulated by fungi(Gadd 1993b, 2001a, b). Metal toxicity is greatlyaffected by environmental conditions and thechemical behaviour of the particular metal speciesin question. Despite apparent toxicity, manyfungi survive, grow and flourish in apparentlymetal-polluted locations and a variety of mechanisms,both active and incidental, contributeto tolerance. Fungi have many properties whichinfluence metal toxicity, including the productionof metal-binding peptides, organic and inorganicprecipitation, active transport and intracellularcompartmentalization, while fungal cell wallshave significant metal-binding abilities (Gadd andGriffiths 1978; Gadd 1993b; Fomina and Gadd2002).1. Metal MobilizationMetal mobilization from solids, e.g. rocks, minerals,soil, ash, mine spoil and other substrates,can be achieved by chelation by excreted metabolitesand siderophores, and methylation which canresult in volatilization. Fungi can solubilize mineralsby means of proton efflux, the production ofFe(III)-binding siderophores, and as a result of respiratorycarbon dioxide accumulation. In addition,other excreted metabolites with metal-complexingproperties, e.g. amino acids, phenolic compoundsand organic acids, may also be involved (Fig. 5.1).Fungal-derived carboxylic acids provide a sourceof protons for solubilization and metal-complexinganions (Gadd 1999, 2001a; Burgstaller and Schinner1993; Gadd and Sayer 2000). Many metal citrates arehighly mobile and not readily degraded. Oxalic acidcan act as a leaching agent for those metals whichform soluble oxalate complexes, including Al andFe (Strasser et al. 1994). Solubilization phenomenacan have consequences for mobilization of metalsfrom toxic metal-containing minerals, e.g. pyromorphite(Pb 5 (PO 4 ) 3 Cl), contaminated soil andother solid wastes (Sayer et al. 1999). Fungi canalso mobilize metals and attack mineral surfacesby redox processes. Fe(III) and Mn(IV) solubilityis increased by reduction to Fe(II) and Mn(II) respectively.Reduction of Hg(II) to volatile elementalHg(0) can also be mediated by fungi (Gadd 1993a,b).The removal of metals from industrial wastesand by-products, contaminated soil, low-gradeores and metal-bearing minerals by fungal “heterotrophicleaching” is relevant to metal recoveryand recycling and/or bioremediation of contaminatedsolid wastes, and perhaps the removal ofunwanted phosphates. The ability of fungi, alongwith bacteria, to transform metalloids has alsobeen utilized successfully in the bioremediationof contaminated land and water. Selenium methylationresults in volatilization, a process whichhas been used to remove selenium from the SanJoaquin Valley and Kesterson Reservoir, California,involving evaporation pond management andprimary pond operation (Thompson-Eagle et al.1989; Thompson-Eagle and Frankenberger 1992).2. Metal ImmobilizationFungal biomass provides a metal sink, either bysorption to biomass (cell walls, pigments and extracellularpolysaccharides), intracellular accumulationand sequestration, or precipitation of metalcompounds onto and/or around hyphae (Fig. 5.1).Fungi are effective biosorbents for a variety of metalsincluding Ni, Zn, Ag, Cu, Cd and Pb (Gadd1990, 1993b), and this can be an important passiveprocess in both living and dead biomass (Gadd1990, 1993b; White et al. 1995; Sterflinger 2000). Thepresence of chitin, and pigments such as melanin,strongly influences the ability of fungi to act asbiosorbents (Mowll and Gadd 1985; Manoli et al.1997; Fomina and Gadd 2002). In a biotechnologicalcontext, fungi and their by-products have receivedconsiderable attention as biosorbents formetals and radionuclides (Gadd and White 1992;Gadd 2002). However, attempts to commercializebiosorptionhavebeenlimited,primarilyduetocompetition with commercially produced ion exchangemedia of high specificity.Fungi can precipitate a number of inorganicand organic compounds, e.g. oxalates, oxidesand carbonates (Grote and Krumbein 1992;Arnott 1995; Gadd 1999; Gharieb and Gadd 1999;Verrecchia 2000), and this can lead to formationof biogenic minerals (mycogenic precipitates).Precipitation, including crystallization, will immobilizemetals but also leads to release of nutrientslike sulphate and phosphate (Gadd 1999). Fungican produce a variety of metal oxalates witha variety of different metals and metal-bearing

Fungi and Industrial Pollutants 77minerals, e.g. Cd, Co, Cu, Mn, Sr, Zn and Ni(Gadd 1999), which may provide a mechanismwhereby fungi can tolerate toxic metal-containingenvironments. A similar mechanism occurs inlichens growing on copper sulphide-bearing rocks,where precipitation of copper oxalate occurswithin the thallus (Purvis 1996).Many fungi precipitate reduced forms of metalsand metalloids in and around fungal hyphae,e.g. Ag(I) can be reduced to elemental silver Ag(0);selenate [Se(VI)] and selenite [Se(IV)] to elementalselenium; tellurite [Te(IV)] to elemental tellurium[Te(0)] (Gharieb et al. 1995, 1999).3. Organometal(loid)sOrganometals (compounds with at least onemetal–carbon bond) can be attacked by fungi, withthe organic moieties being degraded and the metalcompound undergoing changes in speciation.Degradation of organometallic compounds can becarried out by fungi either by direct biotic action(enzymes) or by facilitating abiotic degradation –for instance, by alteration of pH and excretion ofmetabolites. Organotins, such as tributyltin oxideand tributyltin naphthenate, may be degradedto mono- and dibutyltins by fungal action, inorganicSn(II) being the final degradation product.Organomercury compounds may be detoxified byconversion to Hg(II) by fungal organomercurylyase, the Hg(II) being subsequently reduced toHg(0) by mercuric reductase, a system analogousto that found in mercury-resistant bacteria (Gadd1993a).D. Accumulation of Metals and Radionuclidesby MacrofungiElevated concentrations of toxic metals andradionuclides can occur in the fruiting bodies ofhigher fungi sampled from polluted environments(cf. Fig. 5.1). This phenomenon is of significance inrelation to the use of macrofungi as bioindicatorsof metal pollution, and because of human toxicityresulting from the consumption of wild fungi.In general, levels of Pb, Cd, Zn and Hg foundin macrofungi from urban or industrial areasare higher than from corresponding rural areas,although there are wide differences in uptakeabilities between different species and differentmetals (Tyler 1980; Bressa et al. 1988; Lepsovaand Mejstrik 1989). Cadmium is accumulatedto quite high levels in macrofungi, averagingaround 5 mg kg dry wt −1 , although levels of up to40 mg kg dry wt −1 have also been recorded (Byrneet al. 1976). Laccaria amethystina caps exhibitedtotal As concentrations of 100–200 mg kg dry wt −1(Stijve and Porette 1990; Byrne et al. 1991).Accumulation of 110 Ag and 203 Hg was studied inAgaricus bisporus and concentration factors (metalconcentration in mushroom:metal concentrationinsubstrate)werefoundtobeupto40and3.7respectively, with the highest Ag and Hg contentsrecorded being 167 and 75 mg kg dry wt −1 respectively(Byrne and Tusek-Znidaric 1990). As well asfruiting bodies, rhizomorphs (e.g. of Armillariaspecies) can concentrate metals up to 100 times thelevel found in soil. Concentrations of Al, Zn, Cuand Pb in rhizomorphs were 3,440, 1,930, 15 and680 mg kg dry wt −1 respectively, with the metalsprimarily located in extracellular portions (Rizzoet al. 1992).E. Accumulation of Radiocaesiumby MacrofungiFollowing the Chernobyl accident in 1986, therewere several studies on radiocaesium (mainly137 Cs) accumulation by fungi. Free-living andmycorrhizal basidiomycetes can accumulateradiocaesium (Haselwandter 1978; Elstner et al.1987; Byrne 1988; Dighton and Horrill 1988;Haselwandter et al. 1988; Clint et al. 1991; Dightonet al. 1991; Heinrich 1992); these organisms appearto have a slow turnover rate for Cs, and comprisea major pool of radiocaesium in soil (Clintet al. 1991). Mean activities of 25 Ukrainian, sixSwedish and ten North American collections were4660, 9750 and 205 Bq kg dry wt −1 respectively(Smith et al. 1993). Deviations in the 137 Cs: 134 Csratios attributable to Chernobyl have revealedconsiderable accumulation of pre-Chernobyl Csin macrofungi, probably as the result of weaponstesting (Byrne 1988; Dighton and Horrill 1988). Itappeared that about 20% of the 137 Cs in EasternEurope (Moscow area, Belarus, Ukraine) wasof non-Chernobyl origin (Smith et al. 1993).Radiocaesium accumulation in basidiomycetesappears to be species-dependent, with influencesexerted by soil properties. Significantly higheractivities may be found in mycorrhizal speciescompared to saprotrophic and parasitic fungi(Smith et al. 1993). Smith et al. (1993) foundthat many prized edible mycorrhizal fungi may

78 G.M. Gaddcontain unacceptably high levels of 137 Cs,i.e.levelsexceeding 1000 Bq kg dry wt −1 . It has also beendemonstrated that the fungal component of soilcan immobilize the total Chernobyl radiocaesiumfallout received in upland grasslands (Dightonet al. 1991), although grazing of fruiting bodies byanimals may lead to radiocaesium transfer alongthe food chain (Bakken and Olsen 1990).F. Fungi as Bioindicators of Metaland Radionuclide ContaminationAs mentioned above, higher fungi growing at contaminatedsites can show significantly elevated concentrationsof metals in their fruiting bodies, andsome experiments have demonstrated a correlationbetween the quantities of metals in a growth substrateand the amounts subsequently found in thefruiting bodies (Wondratschek and Roder 1993).The concept of bioindicators has been usually discussedin terms of reaction indicators and accumulationindicators. Reaction indicators may compriseindividual organisms and/or communitieswhich may decline or disappear (sensitive species)or show increases (tolerant species). For accumulationindicators, the indicator organism is analysedfor the pollutant. Some organisms, in theory, cantherefore serve as both reaction and accumulationindicators.As described above, alteration of macrofungalcommunitiesbymetalpollutionhasfrequentlybeen recorded. Ruhling et al. (1984) noted a declinefrom about 40 species per 100 m 2 to about15 species near the source of metal contamination(smelter emissions), with only Laccaria laccataincreasing in frequency at more polluted locations.Other higher fungi which are apparentlytolerant of high metal pollution include Amanitamuscaria and several species of Boletus;someRussulaspecies, on the other hand, appear metal sensitive(Wondratschek and Roder 1993).Fungi possess several advantages over plants asmetal accumulation indicators. The fruiting bodiesmay accumulate greater amounts of metals than isthe case for plants, while the large area of myceliumensures contact with and translocation from a largearea of soil. Furthermore, fruiting bodies mayproject above the ground for only a short period,thereby minimising contamination from aerial orwet deposition of metal pollutants. Sporophoresare also easily harvested, and amenable to rapidchemical analysis (Mejstrik and Lepsova 1993).However, it is debatable whether a sufficientlyclear relationship exists between indicator speciesand the metal pollution under consideration.For mercury, wide variations in metal content offruiting bodies occur in different species sampledatthesamesite,rangingoverasmuchasthreeorders of magnitude, with some species showingextremely high Hg accumulation values. Mercuryconcentrations in fungi generally occur in therange 0.03–21.6 mg kg dry wt −1 ,althoughconcentrationsgreater than 100 mg kg dry wt −1 have beenrecorded from polluted sites. Despite this, severalmacrofungi have been suggested as being suitablebioindicators of mercury pollution (see Mejstrikand Lepsova 1993; Wondratschek and Roder 1993;Table 5.1).A wide variation in Cd content has alsobeen recorded in macrofungi, with ranges ofreported values from

Fungi and Industrial Pollutants 79(Tyler 1980). However, with both Cu and Zn, thereis a tendency for metal concentrations in fruitingbodies to be independent of soil concentrations,which reduces their value as bioindicators (Gastet al. 1988).It is clear that many factors contribute tothe wide variations in recorded metal contentsof macrofungal fruiting bodies, even in thesame species sampled at the same site. Despitenumerous studies, most investigations tend to becontradictory and provide little useful information(Wondratschek and Roder 1993). Apart fromorganism-related factors, environmental factorsare of paramount importance in relation tometal accumulation by higher fungi, and includephysico-chemical soil properties such as moistureand temperature, all of which influence metalavailability as well as the physiological activityof the fungus. It can be concluded, therefore,that a perfect fungal bioindicator does not exist,although macrofungi may be useful in determiningthe extent of a polluted or unpolluted area.VI. ConclusionsIt is clear from the above that fungi are of importancein the transformation of both organic andinorganic pollutants in the natural environment.While pollutants may exhibit toxicity, and causechanges in fungal community composition, fungipossess a range of mechanisms which confer resistanceor tolerance, many of these resulting inpollutant transformation to less-toxic forms. Suchactivities are part of natural biogeochemical cyclesfor major elements such as C, N, O, P andS but also metals, metalloids and radionuclides,as well as having applications in the bioremediationand natural attenuation of polluted habitats.However, pollutant interactions are complex andgreatly influenced by environmental factors. Whilethe theoretical response of fungi to pollutants canreadily be speculated upon, some effects are difficultto demonstrate and quantify because of theinadequacy of several common techniques usedto study fungal populations and their activities.Despite this, newly developed approaches usingmolecular biology and biomarkers are allowinga better understanding of community structureand responses to environmental factors, includingpollutants. Growth media containing low and,therefore, more realistic concentrations of availablecarbon should also be used if in vitro techniquesare employed to help determine the effectsof pollutants on fungal growth. However, it is clearthat because of the complexity of the fungal growthform, their multiplicity of biological responses andinteractions with pollutants, coupled with the complexityof the terrestrial (and other) environments,a wealth of knowledge still awaits discovery.Acknowledgements. The author gratefully acknowledgesresearch support from the Biotechnology and BiologicalSciences Research Council, the Natural Environment ResearchCouncil, and British Nuclear Fuels plc.ReferencesAndersson BE, Lundstedt S, Tornberg K, Schnürer Y,Öberg LG, Mattiasson B (2003) Incomplete degradationof polycyclic aromatic hydrocarbons in soilinoculated with wood-rotting fungi and their effecton the indigenous soil bacteria. Environ Toxicol Chem22:1238–1243April TM, Foght JM, Currah RS (2000) Hydrocarbondegradingfilamentous fungi isolated from flare pitsoils in northern and western Canada. Can J Microbiol46:38–49Arnebrant K, Baath E, Nordgren A (1987) Copper toleranceof microfungi isolated from polluted and unpollutedforest soil. Mycologia 79:890–895Arnott HJ (1995) Calcium oxalate in fungi. In: Khan SR (ed)Calcium oxalate in biological systems. CRC Press, BocaRaton, FL, pp 73–111Baath E (1989) Effects of heavy metals in soil on microbialprocesses and populations (a review). Water Air SoilPollut 47:335–379Baath E (1991) Tolerance of copper by entomogenous fungiand the use of copper-amended media for isolationof entomogenous fungi from soil. Mycol Res 95:1140–1152Baath E, Lundgren B, Soderstrom B (1984) Fungal populationsin podzolic soil experimentally acidified tosimulate acid rain. Microbial Ecol 10:197–203Babich H, Stotzky G (1985) Heavy metal toxicity to microbemediatedecological processes: a review and potentialapplication to regulatory policies. Environ Res 36:11–137Bakken LR, Olson RA (1990) Accumulation of radiocaesiumin fungi. Can J Microbiol 36:704–710Baldrian P, in der Wiesche C, Gabriel J, Nerud F, Zadraûil F(2000) Influence of cadmium and mercury on activitiesof ligninolytic enzymes and degradation of polycyclicaromatic hydrocarbons by Pleurotus ostreatus in soil.Appl Environ Microbiol 66:2471–2478Barclay M, Knowles CJ (2001) Cyanide biodegradation byfungi. In: Gadd GM (ed) Fungi in bioremediation.Cambridge University Press, Cambridge, pp 335–358Bewley RJF (1979) The effects of zinc, lead and cadmiumpollution on the leaf surface microflora of Loliumperenne L. J Gen Microbiol 110:247–254

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6 Fungi in Extreme EnvironmentsN. Magan 1CONTENTSI. General Introduction ................. 85II. Thermotolerance .................... 85A. Mechanisms of Thermotolerance . . . . . 87III. Psychrophiles ....................... 88A. SurvivalatLowTemperatures ........ 89IV. Water Relations of Fungi ............... 91A. ConceptofWaterAvailability ........ 91B. Fungal Growth and Water Potential . . . . 93C. AdaptationtoWaterPotential ........ 94D. Yeast Physiology and Osmotic Stress . . . 94E. XerophilicFilamentousFungi ........ 95V. Anaerobic Fungi ..................... 95VI. Acidophiles and Alkalophiles ........... 97A. Acidophiles ...................... 97B. Alkalophiles...................... 98C. Mechanisms of SurvivalinExtremepHEnvironments ........ 98VII. Irradiation and Fungi ................. 98VIII. Conclusions ......................... 100References.......................... 100I. General IntroductionIn nature, fungi are ubiquitous, having evolved overtime to occupy a wide range of ecological niches.To occupy these niches, they must be involved inprimary resource capture which is determined bythe ability to germinate and become establishedrapidly and to produce the necessary extracellularenzymes in the immediate environment. Their activitywill be further impacted upon by prevailingabiotic factors such as temperature, water availability,gas balance and pH. It has been suggested thatboth primary and secondary resource capture arein a state of flux and determined predominantly byprevailing biotic and abiotic interactions (Maganand Aldred 2006). Thus, niche occupation is determinedby a complex of interacting factors. Certainspecies may occupy similar general niches becauseof their ecologically similar behaviour within1 Applied Mycology Group, Cranfield Health, Cranfield University,Barton Road, Silsoe, Bedford MK45 4DT, UKa community. The abiotic stress factors may bea long-lived or a permanent feature of a habitatwhereas other disturbance factors may be transient,having a short-lived impact.Basedonworkinplantcommunities,ithasbeen suggested that fungi employ different primarystrategies to survive and prosper in differentenvironments. These are so-called combative(C-selected) strategies, which maximise occupationand exploitation of resources in relativelynon-stressed and undisturbed conditions; stress(S-selected) strategies, which have involved thedevelopment of adaptations allowing survival andendurance under continuous environmental stress;and ruderal (R-selected) strategies, characterisedby a short life span and high reproductive potentialwhich often enable success under severely disturbedbut nutrient-rich conditions. These threestrategies can merge to give secondary strategies(C-R, S-R, C-S, C-S-R) which form part ofa continuum with transition zones between them(Cooke and Whipps 1993). The main attributesof these three primary groups are summarized inFig. 6.1.In this chapter, we are specifically concernedwith fungi which may use S-selected strategies forgrowth and survival in a range of so-called extremeenvironments. It should, however, be rememberedthat under natural conditions fungal activity in terrestrialecosystems will be influenced by interactionsbetween abiotic and biotic factors both spatiallyand temporally. Community structure anddominance can thus vary during succession in specifichabitats.II. ThermotoleranceFungi have the ability to grow over a widerange of temperature conditions. Fungi, andmicroorganisms generally, have been classifiedEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

86 N. MaganFig. 6.1. Summary of attributes of fungiin relation to the three major ecologicalstrategies (from Cooke and Rayner1984)Fig. 6.2. Optimum, minimum and maximumtemperatures and range of conditionsfor growth of some thermotolerantand thermophilic fungias psychrophiles, mesophiles, thermotolerantand true thermophiles. A thermophilic fungusis defined as one which has minimum growth at20 ◦ C or above and a maximum growth at 50 ◦ C orabove. Optima for thermophilic fungi thus occur inthe range 40–50 ◦ C.Figure6.2showstheoptimal,maximum and minimum temperature range forsome thermotolerant and thermophilic fungi.Whiletherearewell-knownexamplesofbacteriawhich are able to grow in a variety of naturalenvironments including hot springs and geyserswhere temperatures can reach 100 ◦ C,eukaryotesare much more sensitive because, at temperaturesabove 65 ◦ C, their membranes becomeirreparably damaged. However, many mesophilicthermotolerant fungi do exist; for example, somedeuteromycetes isolated from thermal springshave maximum growth temperature of 61.5 ◦ C(Tansey and Brock 1973). One must, however,distinguish between the ability to actively growas a thermophile at such high temperatures andsurvival. Often, thermotolerant species are found

Fungi in Extreme Environments 87Fig. 6.3. Heat resistance of ascosporesand conidia of different filamentousfungi and yeast cells (from Baggermanand Samson 1988). The D- and z-valueswere determined in phosphate buffer(0.2 ml/l, pH 5.5) containing sucrose(400 g/l)as components of communities of fungi colonizinga range of damp organic substrates, particularlyhay, straw-based composts and moist-temperatureand tropical cereals, bird nests and tropical soils.They thus form important components of thesuccession of fungi colonizing a wide variety ofsubstrata.Most vegetative yeast cells, fungal myceliumand asexual conidia are killed by exposure to 80 ◦ Cfor only 1 min. By contrast, sexually produced ascosporesof some food-spoilage fungi are able tosurvive readily even for 1 h exposure at this temperature.Such fungi include Talaromyces flavusand Neosartorya fischeri var. glaber (Beuchat 1988;Baggerman and Samson 1988). Heat resistance hasusually been described by the determination of twotypes of values, D and z. The D-value is defined asthe decimal reduction time, and indicates the periodof time required to reduce a certain number ofliving organisms by a factor of 10 under standardtemperature and other environmental conditions.If the D-values at different temperature are plottedon a logarithmic scale, then a straight-line graphshould be obtained, the slope of which is knownas the z-value. This defines the increase in temperature( ◦ C) necessary to decrease or increase theD-value by a factor of 10. Figure 6.3 shows the heatresistance of ascopsores, conidia and some yeastcells at different temperatures. These data clearlydemonstrate the gradation of sensitivity of yeastsand spores of filamentous fungi to increasing temperature.It should, however, be borne in mind thatthe D- and z-values will also be influenced by pH,water availability and the actual nature of the substratum.A. Mechanisms of ThermotoleranceThere have been a number of hypothesis proposedfor explaining the basis of thermophily. Crisan(1973) suggested four main possibilities: (1) lipidsolubilization; (2) rapid resynthesis of essentialmetabolites; (3) molecular thermostability and(4) ultrastructural thermostability. The latter maybe of particular importance because there is thepossibility that solubilization of cellular lipids canoccur at high temperature to the extent that cellslosetheirintegrity.Anincreaseintemperaturemayresult in cellular lipids containing more saturatedfatty acids which have a higher melting point thanthose present in mesophiles. They would thusbe able to maintain cellular integrity at highertemperatures than is the case for mesophiles,which contain markedly less saturated lipids. It hasalso been suggested that the increased fluidity ofsaturated lipids at high temperatures may enablemetabolic activity and cell functioning to enableactive growth at >40 ◦ C.Recently, attention has been focused on theability of organisms generally to produce a specificrange of proteins, so-called heat shock proteins,when exposed to extremes of environmentalfactors, particularly temperature. However, verylittle work has been carried out on heat shockproteins in fungi. These have to a large extentbeen carried out on Saccharomyces cerevisiae, Neurosporacrassa, Aspergillus nidulas, Achlya ambisexualisand Schizophillum commune.Inalmostallcases, studies have involved exposure of strains ofthese fungi to elevated temperatures of, for example,45–55 ◦ C for a period of 1–3 h. Suchcondi-

88 N. Magantions have been found to reduce growth but notaerial hyphal development, and often resulted inthe appearance of a number of proteins resolvableby SDS-PAGE which were newly synthesised or ofwhich the synthesis increased. In the basidiomyceteS. commune, it was also found that proteolytic processeswere significantly affected by such exposure(Higgins and Lilly 1993).In the last 5 years, intensive investigations intothe activities of heat shock proteins have producedsignificant advances in understanding their cellularrole. It has been discovered that many of these (heatshock) proteins are in fact essential proteins whichare synthesised normally by cells at temperaturesoptimal for growth. Sometimes, this has been accompaniedby concomitant production of the lowmolecularsugar alcohol, glycerol, which will beconsidered in more detail in Section IV. Detailed informationof the type of heat shock proteins and thecellular effects of heat shock have been extensivelyreviewed by Plesofsky-Vig and Brambl (1993). Thephysiology and secretion of thermophilic enzymesby different thermophilic fungi have been reviewedmore recently (Maheshwari et al. 2000). The adventof whole-genome sequences for some fungi, e.g. Aspergillusfumigatus, means that by using microarraysit is possible to examine the physiological pathwaysinvolved and also the number of genes whichare up- and down-regulated during heat shock proteinproduction. This will provide a more thoroughunderstanding of the gene clusters involved in thisprocess.III. PsychrophilesThereisawiderangeofnaturalhabitatswherelow temperatures occur continuously or intermittentlydue to seasonal effects. These regions includeoceans, the tundra and sub-Arctic regions. For example,although the oceans have a stable temperatureof

Fungi in Extreme Environments 89Table. 6.1. Examples of growth, enzyme activity and viability of fungi at low temperatures (modified from Robinson 2001)Taxon/species Test temperatures/type of experiment Site Reference54 mitosporic fungi Growth at 0–25 ◦ C, mainly psychrophiles/in vitro, artificial mediaPhoma herbarum Faster growth at 2.5 than 20 ◦ C/sand+glucose-mineral agar31 mitosporic fungi All grew at 4 ◦ C, most optimaat 15–20 ◦ C/synthetic defined mediaHumicola marvinii Growth at –2.5 ◦ C, optimum at 15;no growth at 20–22 ◦ CEnzyme productionPhialophora hoffmanni Occurs at 1 ◦ C, optima between 18–20 ◦ C/in vitro pectinase activityGeomyces pannorum Occurs at −4 ◦ C,optimumat5 ◦ C/in vitro cellulase activityVerticillium lecanii Active at 5, 15 and 25 ◦ C/in vitro chitinase activityArcticBergeroet al. (1999)Devon Island, Canada Widden andParkinson(1978)Antarctic, Macquarie Island Kerry (1990)Signey Island, AntarcticArctic sitesArctic sitesAntarctic moss sampleWeinsteinet al. (1997)Flanagan andScarborough(1974)Feniceet al. (1998)in such a very specialized ecological environment.Table 6.1 summarizes some of the fungi which havebeen isolated from such environments and the typeof experiments carried out with these.As mentioned above, there are many climaticregions where very cold winter temperaturesare followed by quite warm temperatures in thesummer months, e.g. sub-Arctic zones. Here, overwinteringand survival are of critical importancefor effective competition in the following season.A large number of phyllosphere, endophytic andsoil fungi are able to survive very severe wintertemperatures of

90 N. Maganin electrolyte concentration, and effects on thelipid-protein membrane, thereby affecting osmoticresponsiveness; leakage of intracellular solutesthrough the plasma membrane; and intracellularice formation during fast cooling (>−3 ◦ C/min).Thus, cryo-injury is dependent on the rate ofcooling, cell type, and the internal and externalcomposition of the cells and their substrate.Compounds are, however, often producedwithin the cell to enable survival and growth atlow temperature and, indeed, other stresses. Ofparticular importance for the survival of fungi isthe production of osmoregulators or compatiblesolutes which are often synthesized when thefungus is placed under general environmentalstress, but particularly involving water availabilityorlow-temperaturestress.Thesewillbereferredto in more detail in a section below and includearabitol, erythritol, glycerol, proline and trehalose.Trehalose has, for example, been demonstratedto effectively improve cryo-tolerance of Saccharomycescerevisiae, baker’s yeast, to withstandtemperatures of −20 to −70 ◦ C. They are able toreduce the amount of ice formation at low temperature,thereby reducing water loss from cells.However, during freezing significant shrinkage ofthe cell membrane can occur, which can influencesubsequent survival. For example, it has beenshown that the diameter of hyphae of Phytophthoranicotianae was reduced by up to 60%, compared tountreated controls. This can cause direct physicalinjury by dissolution of the cell membrane as iceformation results in an increase in volume of about10%. Cryo-protectants have the effect of preventingsuch significant shrinkages, and reduce ice crystalformation. This can be achieved by exogenousaddition of cryo-protectants to fungi prior tofreezing for culture collection purposes (Smith1993). The effects of cooling rates and storagetemperatures on the recovery of strains have beenstudied in detail to determine the best methods ofpreservation (see Smith 1993). Studies of possibleantifreeze proteins (AFPs) are scarce, except foragroupofsnowmoulds(Typhula species; Snideret al. 2000). Isolates of T. incarnata, T. ishikariensisand T. phacorrhiza showed antifreeze activity inall fractions at 4 and 10 ◦ C. However, no antifreezeactivity was found in isolates with an optimumgrowth at 14 ◦ C (e.g. Microdochium nivale). Theantifreeze found in T. phacorrhiza was shown tooriginate from protein molecules. Interestingly,the ice crystal structures associated with snowmould species showed growth patterns differentfrom those previously observed, which suggestedthat these AFPs may bind to different planes ofthe ice crystal lattice than is the case for the AFPsisolated from fish, insects and plants.In the 1980s, the use of cryogenic light microscopyto examine the effect of freezing and thawingon the fungal mycelium and propagules provedparticularly useful. Studies of Penicillium expansumshowed that shrinkage of hyphae occurred atslow cooling rates, and intracellular ice formationat faster rates of cooling. Interestingly, hyphal septawerefoundtoformnobarrierstosuchicenucleation.Comparison of P. expansum with P. nicotianaeshowed that the former was extremely resistantto such freezing and thawing whereas thelatter was sensitive and failed to recover from suchtreatment (Smith et al. 1986). Extensive and rapidshrinkage of mycelium occurred at all cooling ratesup to −120 ◦ C/min. Culture age, growth phase, andnutrientstatusofthemediumhaveallbeenfoundto influence these effects on fungi. There do appearto be two distinct groups of fungi: those whichshow shrinkage at slow rates of cooling but less athigh rates, accompanied by intracellular ice formation;and those which shrink at all cooling rates,with no intracellular ice formation. These two subdivisionscut across taxonomic groupings. For example,P. nicotianae (oomycete), Aschersomia alleyrodis(hyphomycete) and Lentinus edodes andVolvariella volvacea (basidiomycetes) all respondwithout any ice nucleation occurring.Some contrasting effects have been observedwith these groups of fungi. For example, fungi suchas P. nictianae and P. expansum react by shrinkageof the mycelium and loss of plasma and nuclearmembranes and cytoplasm between the cell walland membrane. On subsequent thawing, the hyphaeoften re-expand to their original shape andsize and are able to grow again. For other fungi, e.g.L. edodes, the hyphae do not return to their originalsize or shape. However, such species still retainviability. Smith (1993) has suggested that the membraneis a critical structure in tolerance to freezingand in thawing cycles. Because it is not very elasticand therefore does not fold easily, material mustbe lost from the structure during shrinkage due tofreezing. Thus, rapid shrinkage can cause damageto the hyphal cells, although quite a number of fungican survive during rapid cooling, even if intracellularice formation occurs (Morris et al. 1988).Generally, information is to a large extent availableon the impact of freezing and thawing in relationto cryo-preservation. However, more data are

Fungi in Extreme Environments 91required on the ecology of psychrophilic fungi innatural habitats, what type of niche overlap mayexist between different species, and their competencetosurvivesuchconditionsandtoactivelygrowwherethecyclemayinvolvebothslowandfast cooling or thawing.Comparisons of Hebeloma spp. from Arcticand temperate regions have indicated that substantialaccumulation of trehalose occurred in theArctic species when grown at low temperature(Tibbet et al. 1998a). Similarly, Humicola marviniand Mortierella elongate, psychrophilesisolatedfrom Signy Island, Antarctica and grown at 5 and15 ◦ C, accumulated trehalose intracellularly toa significantly greater extent (75% more for thelatter species) at 5 than 15 ◦ C (Weinstein et al.2000).Studies by Weinstein et al. (1997) also suggestedthat sugar alcohols such as mannitol as wellas trehalose were increased in isolates of H. marvini,compared with the non-psychrophilic speciesH. fuscoatra. They quantified glycerol, erythritol,mannitol and arabitol as well as trehalose, glucoseand fructose. Table 6.2 shows a comparison betweenthe results from this study. The significantsynthesis of mannitol in the psychrophilic speciesand the relative increase in trehalose clearly reflectits ecological niche. In H. fuscoatra, bycontrast,sugars are largely accumulated which have no rolein cryo-protection.Recent studies have also focused on the capacityof these fungi to produce various enzymesinvolved in decomposition and mycorrhizal associationswith plants. Work by Weinstein et al. (1997)showed that H. marvinii was capable of solubilizinginorganic phosphate and produce proteases. Studiesby Tibbett et al. (1998a, b, 1999) have carriedout a detailed examination of 12 ectomycorrhizalstrains of Hebeloma species from the Arctic tundraTable. 6.2. Comparison of polyol and sugar accumulation(mg/100 mg dry wt) after 8 weeks at 15 ◦ C in Humicola marviniifrom Antarctic soils and H. fuscoatra from a temperateclimatic region (Weinstein et al. 1997)Polyol/sugar H. marvinii H. fuscoatraGlycerol 0.35 0.80Erythritol 0.27 0.11Arabitol 0.21 Not detectedMannitol 41.01 0.51Trehalose 7.76 4.51Glucose 0.18 8.07Fructose Not detected 5.03(Svalbard) and from Alaska, and compared thesewith some from France and Scotland. At

92 N. MaganTable. 6.3. Water activity, equilibrium relative humidity(ERH) and water potentials at 25 ◦ CWater activity ERH (%) Water potential(−MPa)1 100 00.99 99 1.380.98 98 2.780.97 97 4.190.96 96 5.620.95 95 7.060.9 90 14.50.85 85 22.40.8 80 30.70.75 75 39.60.7 70 40.10.65 65 59.30.6 60 70.3olism than is bound water, but the ease with whichit can be removed depends on the water contentof the substrate. The degree of binding also varieswith the type of substrate, and thus total water contentis not a good indicator of water availability formicrobial growth.Scott (1957) suggested that water activity (a w )would best describe the water availability for microbialactivity, a w being the ratio between thevapour pressure of water in a substrate (P) andthat of pure water (Po) at the same temperatureand pressure; thus, a w =P/Po.Thea w of pure wateris 1.00. A substrate containing no free water hasa smaller vapour pressure than does pure water andits a w is consequently less. An alternative measureof a w is that of water potential (Ψ), which is oftenused in soil microbiology and is measured in pascals(Pa). This is the sum of the osmotic, matrixandturgorpotentialsandisrelateddirectlytoa wby the following formula:Water potential(Ψ) = RT|Vlog n a w (+P)whereRistheidealgasconstant,Ttheabsolutetemperature, P the atmospheric pressure and V thevolume of 1 mole of water. The advantage of Ψ isthat it is possible to partition osmotic and matriccomponents and their influence on growth andphysiological functioning of microbes. The relationshipbetween a w and Ψ is shown in Table 6.3.In cells, the Ψ of the environment normally almostalways equals that of the cell: Ψenv. = Ψcell. In mostcases, the Ψ is a function of the osmotic component.In addition, because fungi have a very rigidcell wall, this prevents swelling of the cytoplasm,and the total Ψ is the combination of the osmoticand turgor pressure of the cell wall.Microorganisms which are able to tolerate andactively grow under conditions of water stresshave been described by various terms. The mostcommon have included halophilic, osmophilic,osmotolerant, xerotolerant or xerophilic. The twomost appropriate terms for fungi are probably“osmophilic”, which describes specialized groupsof yeasts which are able to grow in high-saltenvironments, and “xerophilic” (from the Greek,dry-loving). Pitt (1975) defined a xerophile asafunguswhichisabletogrowinsomephaseofitslife cycle at − 22.4 MPa (0.85 a w ), and this has nowbecome generally accepted.Fig. 6.4. Idealised relationship between growth and waterpotential: curve a solute potential, curve b matric potential,and showing Ψopt., Ψmax., Ψmin. (from Griffin 1981)Fig. 6.5. Diagrammatic representation of the effect of wateractivity(Yaxis) and temperature (Xaxis)ongrowthofPenicilliumversucosum. The values on the isopleths representgrowth rates in mm/day. Dotted line is absolute minimumfor germination

Fungi in Extreme Environments 93B. Fungal Growth and Water PotentialThe effect of Ψ experimentally has been usuallydetermined by measurement of growth responsesto different steady-state osmotic and matric potentialsin vitro by modifications of media usingionic or non-ionic solutes (e.g. NaCl, KCl, glycerol,and polyethylene glycol, 200–6000 mol wt). Thishas enabled data to be obtained on optimum, maximumand minimum conditions of Ψ for growthand survival. Figure 6.4 shows a general exampleof the pattern of relative growth rate in relation toΨ of a growth medium. Osmotolerant yeasts suchas Zygosaccharomyces rugosus, Z. rouxii, Torulopsishalonitatophila and Saccharomyces mellis or S. cerevisiaeall grew in this manner with media modifiedwith PEG 200 (Anand and Brown 1968). For theseyeasts, the Ψopt. is quite close to the Ψmax. StudiesofarangeofsoilChytridiomycotinaspecies hasrecently shown that species and strains of thesefungi can tolerate about 5% salt ( − 4.2 MPa) incertain complex media (Gleason et al. 2006). Generally,growth decreased and eventually ceased asΨ was increased (drier conditions). Although thesefungi are quite sensitive, compared to the xerotolerant/xerophilicspecies described below, this doessuggest that these lower fungi will survive wettingand drying fluxes in soil.For some xerophilic fungi, the Ψopt. may bevery different from the Ψmax. For example, Eurotiumspp., Xeromyces bisporous and Chrysosporiumfastidium have Ψopt. values at 25 ◦ C of −7. 0,−5. 6 and − 2.8 MPa respectively (Pitt and Hocking1977; Magan and Lacey 1984a). However, it shouldbe noted that optimum Ψ for growth will also beinfluenced by interaction with other environmentalfactors, particularly temperature, pH and gascomposition (Magan and Lacey 1984a, b; Maganet al. 2004). Interactions between temperature andΨ have been studied in detail, and Figure 6.5 showsexamples of the effect of Ψ×temperature interactionson the growth rates of a xerotolerant species.The isopleths represent points of similar rates ofminimum, maximum and optimum growth temperatureand Ψ for growth (mm/day). This showsa two-dimensional relationship between two veryimportant environmental factors which can influencethe ecological competence and competitiveability of different fungi (Magan and Lacey 1984c).It has been found that usually germination occursat a slightly lower Ψmin. than that for growth (Maganand Lacey 1984a). Indeed, Ayerst (1969) andSmith and Hill (1982) found the reciprocal of germinationtime to be significantly correlated withlinear growth rates. However, others have surprisinglyfound that for some Fusarium spp. growthoccurred over a wider Ψ range than for germination.It is particularly important to consider not onlysingle- but also two- and three-way interactionsbetween environmental factors which might affectcommunity structure within an ecosystem. Thishas seldom been examined in detail. However, usingthe stored grain ecosystem as a model, attemptshave been made to predict the dominance of individualfungi, niche overlap, and temporal changesin community structure (Magan and Lacey 1984c,1985; Magan et al. 2004; Marin et al. 2004). Thesestudies showed that a w ,temperature,andsubstratenutrient status all have a profound influence on antagonism,competitiveness and dominance of individualfungi. The production of secondary metabolites,mycotoxins, is also similarly affected and cancontribute to the success of individual fungi bypre-emptive exclusion from a common resource.However, for many mycotoxigenic species, the twodimensionalprofiles for germination and growthhave been found to be wider than for mycotoxinproduction (Sanchis and Magan 2004).Scott (1957) suggested that the optimum Ψ forgrowth of osmotolerant fungi was dependent onthe predominant solute used in the medium. However,a range of filamentous spoilage fungi havebeen found by Pitt and Hocking (1977) and Andrewsand Pitt (1987) to grow similarly with differentsolutes. For some soil fungi, distinct differencesin germination and growth optima havebeen found for germination and growth in relationto ionic/non-ionic osmotic solutes and matricpotential (PEG 6000) alterations (Magan 1988).Nevertheless, Scott’s (1957) suggestion that absolutegrowth rate was related to solute type has beenborne out in a number of studies. The radial growthrates of fungi over a range of Ψ have been found tovary considerably for species of decomposer fungi,with lowerΨmin.forgrowthwithglycerolassolutethan with KCl to modify the medium, perhaps becauseof the role of glycerol as a compatible soluteat lowered Ψ (Luard 1983; Magan and Lynch 1986;Marin et al. 1999). Recent studies by Hallsworthet al. (2003) have suggested that chaotropic solutes(e.g. phenyls, urea, ethanol and benzyl alcohols)can impose water stress, and have shown that thismay be another form of water stress, in additionto osmotropic responses of microorganisms. However,very few studies have compared these types of

94 N. Maganstress, and assessed whether physiological mechanismsovercoming these are different.C. Adaptation to Water PotentialGrowth under water stress, due to osmotic ormatrix-potential effects, requires the maintenanceof cell turgor for cell functioning, and growth andreproduction to occur. A shift to a high osmotic/water potential affects nutrient uptake, proteinbiosynthesis and a number of enzyme activities.To enable internal cell functioning, particularlyof essential enzymes, fungi produce so-calledcompatible solutes, often polyhydric alcohols or organicacids. The polyols include glycerol, arabitol,erythritol and mannitol. The low-molecular weightpolyol, glycerol, is particularly important as it isable to protect hydrated biopolymers and allowsstructural integrity under low-water potential conditions.Glycerol is more important than the otherpolyols because it produces a lower a w at a givenmolar concentration, followed by arabitol, erythritoland then the higher-molecular weight polyol,mannitol. The physiology of osmophilic yeasts andof filamentous xerophilic fungi will be consideredbelow in more detail, as the mechanisms foradaptation to water stress can be different.D. Yeast Physiology and Osmotic StressIn yeasts, polyols are the main compatible solutes,glycerol being the predominant polyol and arabitolthe minor one (Edgeley and Brown 1978). However,there are differences in the way yeast speciesuse glycerol as a compatible solute. Sensitive isolatesof S. cerevisiae have been found to both synthesizeand secrete glycerol, thereby maintainingapprox. the same ratio between intra- and extracellularglycerol concentrations (Edgeley and Brown1978). Experiments in media containing 10% salt(approx. − 7.0 MPa water potential) showed almosta 40-fold increase in the enzyme glycerol-3-phosphate dehydrogenase. By contrast, the osmotolerantyeast Zygosaccharomyces rouxii retainsa higher proportion of the same synthesized glycerolwithin the cell, indicative of a lower permeabilityof the plasma membrane. Van Zyl and Prior(1990) demonstrated that, in Z. rouxii, glycerolwas actively transported into the cell via a carriermediatedsystem with a high specificity for glycerol.An active glycerol transport system has alsobeen demonstrated for another osmotolerant yeast,Debaryomyces hansenii, whichshowsanincreasein the production and accumulation of glycerol incells at lowered water potentials (Andre et al. 1988;Larsen et al. 1990), although the importance of thecarrier system has not yet been determined. Recentstudies on yeasts which are being used as biocontrolagents suggest that different polyols are accumulatedby taxonomically different species. Forexample, Candida sake was shown to accumulatesignificant amounts of erythritol under water stressconditions (Teixido et al. 1998). By contrast, studieswith a xerophilic yeast Pichia anomala showedthat arabitol and the cryo-protectant trehalose weresynthesized to much higher levels than under nonwaterstressconditionswhenarangeofsoluteswereused (e.g. glucose, glycerol, NaCl, proline; Mokiouand Magan 2002).Studies with S. cerevisae and Z. rouxii haveshown that the trigger for glycerol synthesis maybe K + depletion, because of the transient rapid decreasewhen these yeasts are transferred from hightolow-osmotic potential media. By contrast, in theosmotolerant yeast D. hansenii, Na + is excludedand K + accumulated, so that the internal K + :Na +ratio is much higher than that of the medium.However, glycerol accumulation is still probablymore important in overcoming such stress. Workhas been concentrated on the way in which yeastcells may sense and respond to osmotic stress.Mager and Varela (1993) have proposed the hypothesisthat change in external osmolarity is probablysensed at the plasma membrane as a result of disturbanceof ion gradients (e.g. Na + ,K + ,H + ). ThisFig. 6.6. Impact of increased water stress imposed by NaClon the relative accumulation of sugars and sugar alcohols byaxerophilicAspergillus flavus strain (adapted from Neschiet al. 2004)

Fungi in Extreme Environments 95results in a loss in turgor pressure and a complexseries of molecular events including the protein kinasecascade, leading to modification of enzymeactivities and changes in gene expression. Part ofthis process may include synthesis of trehalose andcertain heat shock proteins to help in recovery processesof the cell. At the same time, polyols such asglycerolareproducedtorestoretheturgorpressurein the yeast cell.E. Xerophilic Filamentous FungiIt is interesting to note that, compared with thewealth of information on physiological adaptationof yeasts to water stress, much less work has beencarried out on filamentous fungi. Lower filamentousfungi have been found to lack polyols underwater stress but rather to accumulate organiccompounds such as proline in response to loweredwater potentials. Luard (1982b) demonstrated thatMucor hiemalis, Phytophthora cinnamomi andPythium debaryanum all synthesized proline whengrown on media containing either ionic or nonionicsolutes to modify water potential. Althoughthese fungi are not xerophilic and are quite sensitiveto water stress, proline may act in a similar wayto glycerol in yeasts, enabling enzymes to functionefficiently. However, while osmotic tolerance ofchytrids has been demonstrated, no informationis available on the mechanisms of tolerance andwhether high- or low-molecular weight polyolsare synthesized by these species. For other filamentousfungi, work by Luard (1982a, b) has beenparticularly useful in understanding the relativeimportance of different polyols in enabling growthat low water potentials. This showed that the type ofsolute present in a medium may influence the majorpolyol accumulated in the mycelium of fungi.For example, the marine fungus Dendryphiellasalina accumulates glycerol, mannitol and inositolwhen grown on media containing NaCl, MgCl andinositol to modify water potential. Luard showedthat, for xerophilic fungi such as Chrysosporiumfastidium and xerotolerant Penicillium chrysogenum,glycerol was the major polyol accumulatedin the mycelium, with lower concentrations ofarabitol and erythritol. As water potential wasreduced, mannitol levels decreased, suggestingthat it may function as a carbohydrate source orenergy reserve for glycerol production. Studies byHocking (1986) have also shown that xerophilicfungi accumulate glycerol during active growthphases but that, when sporulation occurs, glyceroldepletion can occur rapidly, suggesting that it couldbe acting as an energy reserve for the productionof conidia. More recent work on non-xerophilicentomopathogenic fungi has demonstrated that,depending on the C:N ratio and solute used formodifying water potential, significant accumulationsof glycerol, mannitol and erythritol canoccur in conidia of B. bassiana, M. anisopliae andP. farinosus (Hallsworth and Magan 1994a, b), andcan provide an ecological advantage in colonizinginsects in the environment, particularly at lowereda w (Hallsworth and Magan 1995, 1996).Recent studies have examined whether matricstress actually does represent a bigger hurdle toovercome than does solute stress, by quantificationof polyol accumulation in xerophilic mycotoxigenicfungi such as the Aspergillus ochraceus andA. flavi groupaswellaspathogenssuchasFusariumgraminearum (Ramos et al. 1999; Neschi et al.2004; Ramirez et al. 2004). Whereas growth wasmore sensitive to matric water stress, with slowergrowth rates and narrower ranges of matric potentialfor growth, the synthesis of polyols was notsignificantly different. These studies suggest thatglycerol and sometimes erythritol are accumulatedin significant amounts in the mycelial biomass andalso in the spores under both solute and matric waterstress.Figure6.6showstheeffectofincreasingwater stress on relative accumulation of sugars andsugar alcohols in biomass of an Aspergillus flavusisolate. These results suggest that Griffin’s (1981)original view that these types of stress have verydifferent impacts on the physiology of such speciesmay not hold for some xerophilic species. With theavailability of genomic arrays, it may now be possibleto confirm the similarity or differences betweenthe impacts of solute and matric stress on the physiologyof these fungi, by comparing up- and downregulatedgenes under both conditions. This wouldalso provide a better understanding of the functionof these biosynthetic pathways in relation to activegrowth and survival in different ecosystems and inrelation to secondary metabolite production. Thesemay further enable species to maintain a competitiveedge in naturally fluctuating environments.V. Anaerobic FungiIn natural substrata, fungi are often exposed toatmospheric conditions which are aerobically not

96 N. Maganbacteria and 10 6 anaerobic fungi per gram. Thus,ideal. Thus, many fungi are microaerophilic andcontents commonly contain 10 6 protozoa, 10 10 Recently, a beta-glucosidase gene was cloned fromable to actively colonize substrates under lowoxygenconditions. Such environments includeaquatic habitats, wood, and man-made grainecosystems where elevated CO 2 is sometimes usedas part of controlled-atmosphere storage systems.Dematiaceus hyphomycetes, and some Aspergillusand Penicillium spp., are good examples of suchfungi.Manyotherfungiareconsideredtobefacultativeanaerobes. Such fungi include Saccharomycescerevisiae, and members of the Chytridiomycetesand Oomycetes, e.g. Blastocladiella ramosa andAqualinderella fermentans (Held 1970; Gleasonand Gordon 1989). Gleason and Gordon (1988)showedthat,offourzygomycetesstudied(Mucorgenevensis, Benjaminiella poitrasii, Mycotyphamicrospora and Dispira cornuta), the former twowere strict anaerobes growing under nitrogen.The others grew under microaerobic conditionsunder nitrogen. For such fungi to actively grow,anaerobic-specific exogenous supplies of nutrientssuch as fatty acids, sterols and vitamins arenecessary (Bull and Bushell 1976; Gibb and Walsh1980; Gleason and Gordon 1988).In 1975, true anaerobic fungi, morphologicallysimilar to Chytridiomycete fungi, were found in therumen of sheep (Orpin 1975). Since then, severalgenera of anaerobic fungi have been isolated andidentified from the rumen of herbivores. Thesefungi appear to generally have a vegetative stageand sporulate by the production of zoosporeswhich can be mono- or polyflagellate. Because oftheir unusual morphological characters, includingthe presence of hydrogenosomes and fine structure,they have been placed in a separate familyaptly called the Anaeromycetales. They are notparasites but symbiotic, as they utilize nutrientsandalsoprovidenutrientstotheherbivorebytheproduction of volatile fatty acids in the animal(Trinci et al. 1994).in this ecosystem, active competition occurs forthe readily utilizable sugars which may be presentin the structural components of plant material(Orpin and Ho 1992; Trinci et al. 1994). Effectivecompetition by anaerobic fungi is also partiallyinfluenced by the pH of the rumen and its contents.The pH is usually in the range 5.7–7.0 and optimumgrowth of anaerobic fungi occurs at pH 6.0–7.0(Orpin 1975) whereas the temperature range isrelatively narrow and fluctuates only between37–41 ◦ C, depending on the herbivore species.One of the factors which has made these fungisuch effective inhabitants of this special habitatis their ability to rapidly catabolize plant material,predominantly cellulose and hemicellulose,to actively grow and multiply in the rumen. Ithas been suggested that approx. 8% of the rumenand hindgut microbial biomass is of fungal originand contributes about 30% to cellulose degradation.The life cycle thus consists of two mainstages: a vegetative stage when attached to fragmentsof digested plant material, and a sporulationstage when motile zoospores are released. Thesezoospores are very important in enabling anaerobicfungi to have a competitive advantage overother organisms. The zoospores, via a chemotacticresponse, are able to rapidly utilize the nutrients,particularly glucose, sucrose and fructose, infreshly ingested plant material. They are able to attachthemselves to the material, enabling them toencyst rapidly. The hemicellulose and cellulose areutilized efficiently whereas the more recalcitrantlignified tissue is often less effectively metabolized.Metabolism in anaerobic fungi has been studiedpredominantly in Neocallimastix patriciarum,and operates predominantly via a mixed-acid fermentationresulting in the production of volatilefatty acids and lactate. Other compounds producedinclude ethanol and succinate and, of course, CO 2Fungi such as Neocallimastix frontallis, and hydrogen. There appears to be a close interrelationshipPiromonas communis and Spaeromonas communisare confined to the herbivore rumen and thusneed to compete effectively with a range of othermicroflora and fauna, including anaerobic andbetween some bacteria, particularlymethanogens, which can result in cross-feeding offermentation products and more effective breakdownof the plant material.facultative anaerobic bacteria, anaerobic protozoa, Biotechnological exploitation of anaerobicand each other. The rumen is a very complexecosystem and it is worthwhile comparing thepopulations of different organisms in the rumento obtain a perspective on the possible interactionsand competitive nature of the different componentsin this type of environment. The rumenfungi has now become important because oftheir ability to perform mixed-acid fermentation,which is normally carried out by bacteria. Thus,genes from anaerobic species such as Piromycessp. E2 have been inserted into yeasts such asSaccharomyces cerevisiae for ethanol production.

Fungi in Extreme Environments 97the anaerobic species Orpinomyces PC-2; it hasasignificantlyimprovedconversionofcelluloseto fermentable sugars (Li et al. 2004). Becauseanaerobic fungi are capable of producing a rangeof enzymes including cellulases, cellodextrinase,xylanases, glycosidases and aryl esterases, whichall enable efficient utilization of plant materialintheherbivorerumeninacompetitivehabitat,this fungal ecological niche is being exploited forbiotechnological applications. Orpin (1993) andTrinci et al. (1994) have detailed the importanceof the production of these enzymes by anaerobicfungi, and which places them at a competitiveadvantage in this very specialized ecological niche.VI. Acidophiles and AlkalophilesThere are distinct ranges of hydrogen ion concentrationover which biochemical and chemicalprocesses in cells will effectively and efficiently becompleted. Because the hydrogen ion concentrationaffects the ionic state and therefore the possibleavailability of inorganic ions and metabolites to theorganism, it is critical in determining the metabolicactivity of cells. Very high concentrations (acidic)or very low concentrations (alkaline) of hydrogenionswillhaveaprofoundeffectontheactivityandthus ability of organisms to effectively live in an environment.They function best at pH values closeto neutrality. For example, extreme pH can resultin the primary and secondary structure of proteinsbeing irreversibly damaged. Thus, even if the externalpHweretobeextreme,theinternalcellularpHmust be maintained at close to neutrality for efficientcellular functioning. However, the possessionof an osmotic barrier to the external environmentcan maintain the cytoplasmic components at a pHdifferent to that of the surrounding substrate. It isthis ability which has enabled fungi to become establishedin both extremely low- (close to 1) andhigh-pH (11) environments (Longworthy 1978).A. AcidophilesAcidophiles have been defined as organisms whichareabletogrowdowntopH1.0andareableto actively grow at pH < 4.0. Although acidophilicmicroorganisms are predominantly bacteria, particularlythiobacilli (Ingledew 1990), a range ofyeasts and filamentous fungi have been found togrow in very low-pH substrates. Good examples oflow-pH environments include geothermal regionswith high hydrogen sulphide emissions, coal refusetips, and acidic copper mine wastes. Most yeastsgrow optimally at pH 5.5–6; some, including Candidakrusei, Rhodotorula mucilaginosa and Saccharomycesexigua, can grow at pH 1.5–2.0 (Reccaand Mrak 1952; Battley and Bartlett 1966). Speciesof Saccharomyces, S. ellipsoideus, S. guttulata andS. cerevisiae were demonstrated to actively grow atpH 2.5, 2.0 and 1.9 respectively (Pfaff et al. 1978).The most acidophilic filamentous fungireported to date are Acontium velutium anda Cephalosporium sp. which were isolated fromlaboratory media containing 2.5 N H 2 SO 4 (Starkeyand Waksman 1943). Many other filamentous fungihave been shown to be able to grow at very low pHvalues. Many Aspergillus, Eurotium, Fusarium andPenicillium spp. can grow down to pH values of2.0 but also have pH optima of up to 10. Acontiumpullulans was isolated at pH 2.5 from acidiccoal waste and acid streams by Belly and Brock(1974). Recent studies have attempted to examinemetabolically active eukaryotic communities inacidic mine drainage systems by using molecularprobes such as fluorescent in situ hybridization(FISH), and 18S rRNA and beta-tubulin genephylogenies and probes for different genera (Bakeret al. 2004; Lopez-Archilla et al. 2004). Thesestudies have demonstrated that species such asDothideomycetes and Eurotiomycetes andanumberof other ascomycete fungi are present in acid minedrainage. Such culture-independent techniquesare essential to obtain a better perspective onthe fungal community structure in such extremeenvironments.Detailed studies on intracellular pH homeostasisin xerophilic fungi (e.g. Aspergillus niger) areproviding a better understanding of the capacityfor such fungi to tolerate and remain active underextreme acidic conditions. By using real-time NMRand 31 P, it was possible to examine immobilizedbiomass in alginate pellets when exposed to differentacidic conditions (Hesse et al. 2002). VacuolarH + influx was observed in response to extremecytoplasmic acidification, suggesting that homeostasiswas operating in this organelle in A. niger.Interestingly, NMR spectra of citric acid-producingbiomass showed that, even in the presence of a verylow pH of 1.8 and a high acid-secreting capacity, thepH levels in the cytosol and vacuoles were maintainedat pH 7.5 and 6.4 respectively. This providesevidence for the adaptation capacity of such fungiunder extreme acidic stress.

98 N. MaganB. AlkalophilesAkaline environments include soda lakes,desert soils and alkaline springs where the pHcan often be consistently at about pH 10. In manycases, the presence of ammonium carbonate,potassium carbonate, sodium borate or sodiumorthophosphate is responsible for the alkalinenature of these environments. Alkalotolerantorganisms have been defined as those which growoptimally at approx. pH 7 but are able to activelygrow at pH values of up to 9–9.5. Alkalophilicorganisms are defined as those which do not growat pH < 8.5 or have optimum growth at two pHunits above neutrality (Kroll 1990).Many fungi are able to grow over a very widerange of pH values, and often between pH 2 and11. However, many of these fungi, from 15 differentgenera including species of Botrytis, Colletotrichum,Cladosporium, Fusarium, Penicilliumand Paecillomyces, are most probably alkalotolerant.For example, Paecillomyces lilacimus wasdescribed as being alkaliphilic and able to growvery well between pH 7.5–9.0. True alkaliphilicChrysosporium spp. were isolated and describedfrom bird nests, having a pH maximum for growthof pH 11. These fungi are specialized keratinolyticorganisms living in a very specialized environment.Among the yeasts, Exophila alcaliphila, Candidapseudotropicalis and Saccharomyces fragilis havebeen described as being alkalotolerant. However,few examples of truly alkaliphilic yeasts exist.C. Mechanisms of Survival in Extreme pH EnvironmentsThe hydrogen ion (H + ) is a very special cationbecause it is a proton with no electrons. In solution,itbecomeshydratedtoformthehydroniumion(H 3 0 + ). At acid pH, this predominates whereas, atalkaline pH, the hydroxyl ion (OH − )isdominant.The protons and the membrane transport andbioenergetic processes are critical to the abilityof acidophiles and alkalophiles to colonize suchspecialized environments. The ability to occupythesenichesisdeterminedlargelybytheabilityof microorganisms, including fungi, to havepH controlling systems. This involves efficienttrans-membrane transport systems, so that solutesneeded to achieve intracellular modifications canbe effectively utilized to maintain the membranepotential with respect to the outside environment.This entails the efficient control of proton movementinto and out of the cells, and the meeting ofnecessary energy requirements. It is also linkedwith the control of osmotic pressure, because ofthe involvement of cations and anions. However,practically no work has been carried out on themechanisms involved in yeasts or filamentousfungi, having been carried out predominantly withacidophilic and alkalophilic bacteria. This aspecthas been extensively reviewed by Longworthy(1978), Ingledew (1990) and Kroll (1990).Mechanisms of internal pH control have beenreported for bacteria such as Streptococcus faecaliswhere the control has been found to be completelydue to the action of ATPase in increasing protonpump efficiency. For example, in extreme acidicconditions, the internal pH falls quickly, and ATPis used to rapidly pump protons out of the bacterialcells via ATPase to increase the internal pHof the cell. In alkalophilic bacteria such as Bacillusalcalophilus,sodium(Na + ) is utilized to reversethe pH gradient under extreme alkaline conditions.Adaptations of bacteria to enable growth in theseenvironments include the possession of flagella,modifications of cell walls and membranes, and inbiochemical activity including respiration and oxidativephosphorylation. The extensive studies onbacteria need to be extended to yeasts and filamentousfungi to enable a more clear understanding oftheir occupation of such ecological niches. However,the possible impact of pH must not be seen inisolation – organism activity will also be influencedby interactions between pH, water availability andosmotic potential, and temperature.VII. Irradiation and FungiThe two types of radiation which fungi are exposedto are firstly, non-ionizing radiation due tosolar radiation and ultraviolet (UV) light and secondly,ionizing radiation from natural and manmadesources. The most important component ofnon-ionizing radiation in the environment is UV-Blight. Because of depletion of the ozone layer, exposureto UV-B light (290–320 nm)hasincreasedandits impacts on plants and microorganisms is receivingmore attention. Man-made irradiation sourcesinclude gamma-irradiation, which has been usedfor a long time as a method of preserving food,particularly that intended for human consumption(IAEA-FAO 1978). Microwave radiation has also

Fungi in Extreme Environments 99been used increasingly to enable more rapid dryingof substrates and the destruction of pests and microorganisms,including fungi. However, specificgroups of fungi which, for example, occupy environmentswhere they are exposed particularly tonatural radiation from UV light, such as plant surfaces(phyllosphere), often have dark pigmentationof both the mycelium and spores to increase survivalpotential.Work on the effects of UV-B radiation onsaprophytesandpathogensofplantshasmorerecently received particular attention. For example,Ayres et al. (1996) showed that yeasts suchas Sporobolomyces roseus and Crytococcus spp.had different sensitivities to UV-B light. S. roseuswas less sensitive than Cryptococcus sp. Sporegermination of cereal plant pathogens such asSeptoria nodorum, butnotS. tritici, wasinhibitedby low doses of UV-B light. Interestingly, isolatesfrom warmer climatic regions in North Africawere found to be less sensitive to UV-B fluxes thanwere UK isolates (Ayres et al. 1996). Thus, differentstrains of the same fungus may have evolved invery different ways in temperate and subtropicalenvironments, where exposure to UV-B occurs forlonger periods of time. While radiation damagesDNA of microorganisms to some extent, some areable to repair radiation-induced damage relativelyquickly to maintain essential metabolic functions.Thus, some fungi may protect themselves fromnaturalradiationby,e.g.pigmentation;othersmayhave developed rapid mechanisms for repair ofdamaged DNA.Man-made radiation from sources such asgamma rays or microwaves splits water into freeradicals which damage DNA and are highly toxicto a wide range of fungi. Therefore, the higher thewater content, the more free radicals producedand the higher the level of DNA damage (Kiss andFarkas 1977). As mentioned above, a relativelylarge amount of information exists on the effecton fungi alone or those present on agriculturalsubstrates exposed to various irradiation doses.Saleh et al. (1988) demonstrated that ten species offungi from the genera Alternaria, Aspergillus, Cladosporium,Curvularia, Fusarium and Penicilliumwere inactivated by doses of gamma-irradiationbetween 0.6 and 1.7 kGy. However, dematiaceousfungi with a high level of pigmentation were moreresistant than moniliaceous species when tested inaqueous suspensions or on enriched agar media(Saleh et al. 1988). Other workers have found thatthe concentration required for the destruction offungi present on substrates, e.g. temperate andtropical grains, varied with both grain type andwater content (Cuero et al. 1986; Ramakrishna1991). On maize, barley and wheat, Penicilliumand Aspergillus spp. were killed by doses of 0.3and 1.2 kGy respectively, whereas 12 kGy wasnecessary to eliminate all filamentous fungi andyeasts (Cuero et al. 1986; Ramakrishna 1990;Hamer 1993). However, there are concerns aboutthe efficacy of irradiation on mycotoxins. Thereareconflictingdatawithsomestudiesreportingthat an increase in mycotoxin formation occursafter irradiation. It has been suggested that fungalspecies in food matrices surviving irradiation maygrow more rapidly in the absence of competitors.Other views are that irradiation can make morenutrients available and this could have an influenceon the ability of surviving species to colonize suchmatrices rapidly (Farkas 1989; Monk et al. 1995;Shea 2000).Microwave energy has also been used particularlyin the agricultural and food industries fordisinfection processes. Exposure of fungi directlyor of fungi contaminating different agriculturalsubstrata has been carried out (More et al. 1992;Cavalcante and Muchovej 1993). The microwaveenergy used is dependent on the frequency (often1250 MHz)andthetimeperiodofexposure.Forinstance,a 30-s exposure at different power levels canrepresent energy inputs of between 2.25 and 18 kJ.As for gamma irradiation, moisture content of themedium can significantly influence resistance orsensitivity to the chosen energy level. In naturallycontaminated sorghum grain, between 9–18 kJof energy completely destroyed Eurotium spp.,Aspergillus candidus, A. niger, Penicillium spp.and species of Cladosporium and Alternaria (Moreet al. 1992). Interestingly, germination of sporesof Aspergillus flavus, A. niger, Colletotrichum spp.,Fusarium oxysporum and Bipolaris sorokinianaon slides directly at 6, 9 and 18 kJ for periods of0–7 min significantly reduced viability. Singlecelledspores were more sensitive to microwaveexposure than multi-celled spores, and darkpigmentedspores (A. niger, B. sorkiniana) wereless affected by exposure to such concentrations ofmicrowaves than were hyaline spores (Cavalcanteand Muchovej 1993).It may be that dark pigmentation formssites which absorbed ionising or non-ionisingradiation, thereby preventing more direct damageto DNA. However, bacterial mutants devoid ofpigments were not found to be more sensitive

100 N. Maganthan wild types protected by pigments (Nasimand James 1978). It is now believed that DNArepair mechanisms are predominantly responsiblefor radiation resistance. The mechanisms ofDNA repair involve both photo and dark repair,depending on whether visible light is involved.The reader is referred to the review by Strike andOsman (1993) for more detailed information onDNA repair mechanisms.VIII. ConclusionsThis review has covered a wide range of highly complexstressed ecosystems which contain specializedcommunities of fungi. For some extreme environments,e.g. water, temperature stress, a wealth of informationis available. The rapid development andavailability of molecular-based techniques have allowedsignificant advance to be made in more detailedanalyses of fungal communities based ondirect measurements, as opposed to culture techniquesonly. The unravelling and public availabilityof whole genomes of yeasts and filamentous fungi,such as Aspergillus fumigatus, A. flavus, A. nidulansand now some Fusarium species, will enable muchmore detailed knowledge to be obtained on thefunction of gene clusters under different ecophysiologicalconditions which simulate or mimic naturalfluxes in extreme environments, and will enablethe switches and triggers involved in germination,growth, survival, secondary metabolite productionand competitiveness in such highly complexecosystems to be understood. An understandingof the functioning of fungi in these extreme environmentscould also provide a wealth of new enzymes,pigments and pharmaceutical leads whichcould be exploited biotechnologically.ReferencesAnand JC, Brown AD (1968) Growth patterns of so-calledosmophilic and non-osmophilic yeasts in solutions ofpolyethylene glycol. J Gen Microbiol 52:205–212Andre L, Nilsson A, Adler L (1988) The role of glycerolin osmotolerance of the yeast Debaryomyces hansenii.Arch Microbiol 139:110–116Andrews S, Pitt JI (1987) Further studies on the water relationsof xerophilicfungi, including the characterisationof some halophiles. J Gen Microbiol 133:233–238Ayerst G (1969) The effects of moisture and temperature ongrowth and spore germination in some fungi. J StoredProd Res 5:127–141Ayres PG, Rasanayagam MS, Paul ND, Gunesekera T(1996) UV-B effects on leaf surface saprophytes andpathogens. In Frankland JC, Magan N, Gadd GM(eds) Fungi and environmental change. CambridgeUniversity Press, Cambridge, pp 32–50Baggerman WI, Samson RA (1988) Heat resistance of fungalspores. In: Samson RA, van Reenen-Hoekstra ES(eds) Introduction to food-borne fungi. Centraalbureauvoor Schimmelcultures, Baarn, pp 262–267Baker BJ, Lutz MA, Dawson SC, Banfield PLB (2004)Metabolically active eukaryotic communities inextreme drainage. Appl Environ Microbiol 70:6264–6271Battley EM, Bartlett EJ (1966) A convenient pH-gradientmethod for the determination of the maximum andminimum pH for microbial growth. Antonie Leeuwenhook32:245–255Belly RT, Brock TD (1974) Widespread occurrence of acidophilicstrains of Bacillus coagulans in hot springs.J Appl Bacteriol 37:175–177Bergero R, Girlanda M, Varese GC, Intili D, Luppi AM (1999)Psychrooligotrophic fungi from arctic soils of FransJoseph Land. Polar Biol 21:361–368Beuchat LR (1988) Thermal tolerance of Talaromyces flavusascospores as affected by growth medium and temperatureand age and sugar content in the activationmedium. Trans Br Mycol Soc 90:359–364Bull AT, Bushell ME (1976) Environmental control of fungalgrowth. In: Smith JE Berry DB (eds) The FilamentousFungi vol II. Biosynthesis and metabolism. Arnold,London, pp 1–31Cavalcante MJB, Muchovej JJ (1993) Microwave irradiationof seeds and selected fungal spores. Seed Sci Technol21:247–253Cochrane VW (1958) Physiology of fungi. Wiley, New YorkCooke RC, Rayner ADM (1984) Ecology of saprotrophicfungi. Longman, LondonCooke RC, Whipps JM (1993) Ecophysiology of fungi. Blackwell,LondonCrisan EV (1973) Current concepts of thermophilism andthe thermophilic fungi. Mycologia 65:1171–1198Cuero R, Smith JE, Lacey J (1986) The influence of gammairradiation and sodium hypochlorite sterilisation onmaize microflora and germination. Food Microbiol3:107–113Edgley M, Brown AD (1978) Response of xerotolerant andnon-tolerant yeasts to water stress. J Gen Microbiol104:343–345Farkas J (1989) Microbiological safety of irradiated foods.Int J Food Microbiol 9:1–15Fenice M, Selbmann L, Di Giambattista R, Federici F (1998)Chitinolytic activity at low temperature of and Antarcticstrain (A3) of Verticillium lecanii.ResMicrobiol149:289–300Flanagan PW, Scarborough AM (1974) Physiologicalgroups of decomposer fungi on tundra plant remains.In: Holding AJ, Heal OW, MacLean Jr SF, Flanagan PW(eds) Soil organisms and decomposition in tundra.Tundra Biome Steering Committee, Edmonton, pp159–181Furhman JA, Anzam F (1980) Bacterioplankton secondaryproduction estimates in coastal waters of BritishColumbia, Antarctica, and California. Appl EnvironMicrobiol 39:1085–1095

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7 Biogeography and ConservationE.J.M. Arnolds 1CONTENTSI. Introduction ........................ 105II. Mapping of Fungi .................... 105A. Methods......................... 105B. MapsandScales................... 106III. Distribution Patterns ................. 107A. GlobalDistribution ................ 107B. LocalEndemics ................... 111C. ContinentalPatterns ............... 112D. NationalandRegionalPatterns....... 113E. LocalPatterns .................... 114F. RecentChangesinDistributionPatterns 115IV. Expansion of Distribution Areas ........ 115A. IntroductionsofPlants ............. 115B. IntroductionsofFungi.............. 116C. Decline and Extinction of Fungi . . . . . . 116V. Conservation of Fungi ................ 117A. RedListing....................... 118B. Threatened Fungi and Their Habitats . . 119C. Mycological ReservesandNatureManagement............ 120D. Harvests of Wild Edible MushroomsandLegalProtection ............... 120VI. Conclusions ......................... 121References.......................... 122I. IntroductionBiogeography is the study of distribution patternsof organisms in space and time, including the studyof factors determining these patterns. These factorscomprise actual conditions such as climate,soil, the availability of hosts and substrates, anddispersal capacity of species as well as historicalconditions, including geological and evolutionaryprocesses. Biogeographical studies on fungi are relativelyscarce, due mainly to methodological problems.A bibliography on distribution maps of fungihas been published by Kreisel in Feddes Repertoriumfrom 1970 onwards. Information on distributionpatterns is important for our understandingof evolutionary processes and patterns of biodiversitybut also in such practical disciplines as control1 Holthe 21, 9411 Beilen, The Netherlandsof crop pests, plantation forestry and nature conservation.In view of the increasing disturbance ofecosystems by human activities and the growingpublic focus on applied research, conservation offungi has become a topic of rising interest.II. Mapping of FungiA. MethodsMapping of fungi is basically a simple procedure:the collecting of records of a certain taxon in a certainarea from different sources and plotting theseon a topographical map. However, in practice, thiswork is hampered by a number of complicationsproper to fungi (Kreisel 1985; Redhead 1989;Pringle and Vellinga 2006):1. Problems in detection of mycelia Ideally, mappingof fungi should be based on observationsof mycelia, since mycelia are functionally themost important part of a fungus. However,mycelia are usually living within the substrateand can only rarely be identified in the fieldusing morphological characters, e.g. somegenera forming mycelial strands, such as thehoney fungus. In addition, ectomycorrhizalfungi may be identified by characteristic structuresonroottipsofhostplants(HortonandBruns 2001). Recently, molecular techniqueshave been developed to identify mycelia insoil samples (Landeweert et al. 2003). Mostsaprotrophic microfungi can be isolated andidentifiedonlybyspecialtechniques(Gams1992).The above methods of detection depend on theanalysis of small soil samples in the order of100 cm 3 . Therefore, these methods are effectiveonly in small-scale plots, e.g. a forest stand. Becauseof the extremely small size of the samplesrelativetothevolumeofthesubstrate,itisveryEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

106 E.J.M. Arnoldsunlikely that less-common species be detectedby these techniques.2. Problems in detection of sporocarps Sporocarpscan be observed and identified much moreeasily than mycelia, in particular of macrofungi.Therefore, most mapping programmesare based on inventories of these propagationstructures. Complications are the ephemeralcharacter of most sporocarps and the strongannual fluctuations in sporocarp production.In addition, some groups of ascomycetesand basidiomycetes produce hypogeoussporocarps which are difficult to detect forthat reason (Lawrynowicz 1990). A morefundamental objection against this approachis that the distribution of sporocarps may notnecessarily reflect the distribution of mycelia.Some species may be common in a vegetativestate but rarely produce sporocarps, and viceversa. For instance, at the community levelit has been demonstrated that 70% of theectomycorrhizal root tips in a Sitka spruceplantation were occupied by the corticioid fungusTylospora fibrillosa (Burt) Donk whereasthe bulk of ectomycorrhizal sporocarps wereproduced by agarics (Taylor and Alexander1991).3. Defective taxonomic knowledge Many groupsof fungi are in need of critical revision, and numeroustaxa still have to be described. For instance,in well-investigated Europe the numberof known species of the agaric genus Entolomaincreased between 1992 and 2004 by 39%, from246 to 342 (Noordeloos 2004). In addition, taxonomicconcepts are still changing, also becauseof new molecular techniques. Amongthe 50 species mapped in Europe by Lange(1974), selected because they were “well definedand easy to identify”, at least four speciesare now regarded as species complexes, e.g.Armillaria mellea (Vahl.: Fr.) Kumm. This fungusproved to comprise five biological specieswith different distribution patterns in Europe(Kile et al. 1991). Problems on conspecificitybecome more complex when distant areas arecompared, such as North America, Europe andEast Asia (Redhead 1989).4. Availability of data Distributional data comefrom herbarium collections, records in the literature,databases and unpublished observationsinnotebooks,etc.Reliabledataarescantyin most parts of the world. Even in relativelywell-investigated Europe, the data are ofteninappropriate to provide realistic distributionpatterns (Lange 1974).5. Accessibility of data Even if data are available,it may be problematic to trace all relevant information.This problem can be solved by establishmentof central databases for records offungi, at present realized mainly at a nationallevel, e.g. in Germany, Great Britain, Sweden,The Netherlands, Belgium and Australia.Indeed, the gaps in our knowledge are still enormousbut the situation at present is not so hopelessthat “writing an essay on the geographical distributionof fungi is to attempt to accomplish an impossibletask” (Pirozynski 1968).In practice, two different, although not clearlyseparated, approaches exist to mapping of fungi.In the monographic approach, specialized taxonomistscollect records on a particular group offungi, usually only herbarium collections and incombination with a taxonomic revision (Kreisel1967; Demoulin 1971; Lawrynowicz 1990). Thedistribution patterns are therefore reliable in a taxonomiccontext but, at the same time, relativelyinaccurate because of the limited data. The secondapproach concerns mapping programmes, whichare usually carried out on a national or regionalscale. Such programmes attempt to collect as manyrecords as possible on the occurrence of selected(but not necessarily taxonomically related) species(Lange 1974) or all species in a certain area(Krieglsteiner 1991; Nauta and Vellinga 1993).The data come from different sources, includingliterature records and unpublished observations,and are collected by many mycologists, bothprofessional and amateur. As a result, the mapsare less reliable from a taxonomic point of viewbut more accurate because of the larger number ofobservations.B. Maps and ScalesDistributional studies are carried out and presentedat different geographical scales: (1) globalpatterns may reveal relations to macroclimate,host distribution and historical factors (Fig. 7.1);(2) continental patterns may, in addition, berelated to mesoclimate (e.g. mountain ranges)and large-scale edaphic patterns, e.g. zonal soils(Figs. 7.2, 7.3 and 7.4); (3) regional patterns,often studied within the political borders ofacountry,mayexpressmoresubtledifferencesofthe landscape – for instance, patterns of alluvial

Biogeography and Conservation 107Fig. 7.1. World distribution of Phaeosphaeria nodorum (E. Müller) Hedjar (CAB International Mycological Institute 1992)deposits in river valleys or human influence byagriculture or forestry (Figs. 7.5, 7.6); (4) localpatterns are studies within a single landscape orstand, and may be related to microclimate, anddistribution patterns of plant communities andindividual plants (Fig. 7.7).Results of biogeographical studies can be presentedon different kinds of maps (Kreisel 1985):1. Outline maps, where borders of the knowndistribution areas are indicated with a line(Fig. 7.1). One disadvantage here is that theoriginal records are not indicated. Consequently,it is impossible to detect possibledifferences in density inside a given area andto provide an alternative interpretation of thedistribution.2. Dot maps, where each record or locality is indicatedby a separate sign (Fig. 7.2). This methodis more accurate and objective than an outlinemap, the accuracy being slightly influenced bythe size of the dot (Lange 1974). However, whenresearch efforts are not evenly spread over themapped area – which is often the case – concentrationsofdotsmaybeartefacts,markingrather the activities of mycologists than theabundance of the fungus (Kreisel 1985). Dotmapscanbecombinedwithoutlinemaps.3. Grid maps, where the records are plotted ina topographical grid with units of a given constantsize (Figs. 7.3, 7.5 and 7.6). The accuracyof grid maps is intermediate between that ofdot maps and outline maps, and depends onthe mesh size of the grid. It varies with the sizeof the investigated area: usual grid units are,for instance, 50×50 km for Europe (Fig. 7.4),12×13 km for Germany (Fig. 7.5) and 4×4 kmfor Belgium (Fig. 7.6). The applied grid maybe a national grid indicated on topographicalmaps of a country (Fig. 7.5) or an internationalgrid – for instance, in Europe the UTM grid(Figs. 7.4, 7.6), also used for the mapping ofvascular plants and invertebrates. The lines ofthe grids can be deleted thereafter (Fig. 7.4).The different grids used in different Europeancountries hamper an easy integration of dataon a continental scale.III. Distribution PatternsA. Global DistributionMaps on the world distribution of plant pathogenicfungi are published by CMI and revised at irregularintervals – for example, the map of Phaeosphaerianodorum (E. Müller) Hedjar, an ascomyceteoccurring on various grasses, including cereals(Fig. 7.1). Nowadays, it has a subcosmopolitandistribution, following cultivated host plantsalmost everywhere. The original range is difficultto trace, however, as in many other pathogens on

108 E.J.M. ArnoldsFig. 7.2. Examples of distribution patterns of agaricsin North America. a Marasmius epiphyllus (Pers.:Fr.) Fr. with boreotemperate distribution. b Marasmielluscandidus (Bolt.: Fr.) Sing. with bicoastal distribution.c Marasmius plicatulus Pech with westerntemperate distribution. d Marasmius pyrrhocephalusBerk. with eastern temperate distribution (Redhead1989)

Biogeography and Conservation 109Fig. 7.3. World distribution of Lycoperdon echinatum Pers.:Pers. (in Europe) and its vicariant L. americanum Demoulin(in America). Each rectangle represents one degree latitude× one degree longitude (Demoulin 1987)cultivated crops. Data on the global distributionof non-pathogenic fungi depend largely on theavailability of world monographs. General ideason distribution patterns of fungi have evolvedin the course of time (Demoulin 1971). In the19th century, it was thought that most fungalspecies inhabit small areas, comparable to thoseof phanerogams. In contrast, in the first decadesof this century, it was generally assumed that mostfungi are (sub)cosmopolitan. Since 1930, these oppositeviewpoints have merged into the notion thatfew species are truly cosmopolitan but that, nevertheless,distribution areas of fungi are larger thanthe ranges of most vascular plants (Diehl 1937).In addition, distribution areas of soil-inhabitingand marine microfungi seem to be generallylarger than those of larger fungi (Pirozynski 1968;Volkmann-Kohlmeyer and Kohlmeyer 1993).Data on global patterns in some monographedgroups of macrofungi are summarized in Table7.1. The subdivision into distribution types isnecessarily strongly simplified. Ryvarden (1991)stressed the large distribution areas of genera ofpolypores, being mostly either (sub)cosmopolitan(23%), circumpolar (26%) or pantropical (17%).Relatively few genera are endemic to one continentwhere they are usually widely distributed, incontrast to many genera of phanerogams withvery restricted distribution areas. Redhead (1989)stated that transatlantic disjunction patterns are,in general, exhibited by vascular plants at thegeneric or family level but by agarics at the specieslevel, just as they are for lichenized ascomycetesand bryophytes. Hallenberg (1991) drew similarconclusions on the basis of distribution patterns ofCorticiaceae. In Lentinus Fr., most species (78%)are tropical but only very few species (3%) arepantropical or (sub)cosmopolitan (5%; Pegler1983a). Pantropical species seem to be lacking inThelephora Ehrh.: Fr. whereas 39% of the speciesare endemic to Southeast Asia (Corner 1968).The proportions of supracontinental distributionareas are also low for Bovista Pers.: Pers. andScleroderma Pers.: Pers., although these puffballsproduce enormous amounts of airborne spores(Kreisel 1967; Guzman 1970). The number of(sub)cosmopolitan species among Ascobolus Pers.and Saccobolus Boud. is very high (Van Brummelen1967). This may be due to their specialized ecology:most species grow on dung, and ascospores mustpass through the intestines of grazing animals.On the other hand, dispersal of the heavy sporesover large distances seems unlikely. Many speciesmay, in fact, have spread by introduction of cattle

110 E.J.M. ArnoldsFig. 7.4. Distribution of Lycoperdon echinatum Pers.: Pers.in Europe accordimg to UTM grid. Each dot representsasquareof50× 50 km. The dashed line indicates the distributionarea of Quercus robur L. (Demoulin 1987)to other continents. In contrast, the proportion ofEuropean species is also very high; this may be dueto undercollecting in other parts of the world.Distribution patterns of fungi are, in general,so similar to the ranges of phanerogams, albeit atdifferent taxonomic levels, that they are likely to bedetermined by the same environmental and historicalfactors. Some data supporting the importanceof geological phenomena are:1. Some species and genera are common tosouthern South America and Australia orNew Zealand, e.g. mycorrhizal symbionts ofNothofagus. They may be considered as remnantsof populations fragmented by the breakup of Gondwanaland approximately 100 millionyears ago (Horak 1983; Ryvarden 1991).2. A number of species show a disjunct distributionin Europe and (eastern) North America,or two closely related vicariant species exist inthese areas. These patterns may be explainedby the opening of the Atlantic in the Eocene(Demoulin 1973).The species diversity of most taxonomic groups isconsiderably larger in North America than in Europe,which may be caused by different possibilitiesfor reaching refugia during Pleistocene glacial periods(Redhead 1989).The ideas on historical events are not in conflictwith palaeontological evidence. Fossil basidiomycetesare known from the Middle Pennsylvanian,approximately 300 million years b.p. (Dennis1970).An alternative hypothesis for the explanationof supracontinental ranges of fungi is the existenceof efficient long-distance spore dispersal (Redhead1989; Hallenberg 1991; Ryvarden 1991). Argumentsagainst this hypothesis are (1) the large majorityof spores are deposited in close proximity to the

Biogeography and Conservation 111Fig. 7.5. Distribution of Lycoperdon echinatumPers.: Pers. in Germany. Each square represents12 × 13 km (Krieglsteiner 1991)source; (2) most spores are not viable after a stay inhigher atmospheric strata; (3) the chance of reachingan appropriate substrate and microhabitat issmall in many cases; (4) many basidiomycetes mustestablish a dikaryon; the chance that two compatiblecolonies are formed close to each other is extremelysmall; (5) wind dispersal by spores doesnot necessarily imply genetic exchange between allopatricpopulations.Consequently, also for most fungi, dispersalis likely to be restricted by geographic barriers,such as oceans and mountain ranges. Circumstantialevidence is given by some pathogenic fungiwhich were introduced to other continents andcaused catastrophic epidemics in newly availablehost plants. Apparently, they were unable to spreadso far without human assistance. In the cosmopolitan,wood-inhabiting fungus Schizophyllum commune,very limited intercontinental gene flow wasfound, although populations from various continentsare still able to interbreed (James et al. 1999).B. Local EndemicsMany species and even genera of vascular plantsare restricted to small areas in the order of 1 to1000 km 2 . Concentrations of such local endemicsare found in isolated areas surrounded by effectivebarriers against dispersal – for instance, remoteislands and isolated mountains. The percentage oflocal endemic plant species may exceed 50%. Thereis no evidence that this is also true for fungi.The mycoflora of some islands has beenadequately described, e.g. of the Lesser Antillesby Pegler (1983b), but comparable information onsurrounding areas is not available. It is generallythought that local endemics are non-existent orscarce among fungi, except for species restrictedto endemic host plants. However, the situation israther paradoxical, since many species of fungiare known only from their type locality and,consequently, are potential local endemics. In themonographs listed in Table 7.1, the percentage

112 E.J.M. ArnoldsFig. 7.6. Distribution of Lycoperdonechinatum Pers.: Pers. in Belgium accordimgto UTM grid. Each squarerepresents 5×5 km (except in the correctionarea). The dotted line representsthe altitude of 400 m (Demoulin1987)of species known from one locality ranges onlyfrom 10% in Scleroderma to 28% in Ascobolus andBovista. Even the monograph of the striking genusHygrophorus sensu lato (Agaricales) in relativelywell-investigatedNorthAmericaincludes96taxaout of 244 (39%) which are known only fromthetypelocality.Amongthese,21specieswerecollected exclusively before 1920 (Hesler and Smith1963).The following questions arise: are these speciestruly endemic, do they fruit sporadically, and havesome simply escaped the attention of mycologists?Only more intensive field research can provide theanswer. It is striking that some fungi are subsequentlyrecordedquitefarawayfromtheoriginallocation. For instance, Bovista verrucosa (G.H.Cunn.) G.H. Cunn. was described by Kreisel (1967)on the basis of two collections, one from Piedmont,Italy (1857), and one from South Australia (1922).Squamanita odorata (Cool) Bas is a very remarkableagaric, discovered in The Netherlands in 1915and observed there since then at approximately 30localities. It was long thought to be endemic toThe Netherlands but it seems to be spreading nowin West Europe. It was reported from Denmarkin 1948, Germany (1963), Norway (1968), Switzerland(1989) but, remarkably, also from the northwesternUnited States in 1951. Such patterns maybe caused by introductions but evidence is lacking.Other examples of odd disjunct distributionpatterns were given by Pirozynsky (1968).C. Continental PatternsContinental patterns of fungi have been studiedmainly in Europe, North America and, more recently,in Australia (Grey and Grey 2005). The secondCongress of European Mycologists initiateda mapping programme for 100 selected species ofmacrofungi. Maps of 50 species were published byLange (1974). Lange distinguished seven climatologicallydetermined distribution patterns. In addition,the occurrence of some species appearedto be determined by edaphic factors. Only onespecies showed a more or less disjunct distribution,viz. Phallus hadriani Vent.: Pers., with centresin the coastal dunes of West Europe and continentalsteppe areas in Central Europe. Some species wererestricted to sub-Arctic and alpine regions in NorthEurope, others to the Central European mountains,although there are many similarities in climate andsoil. These differences may be caused by isolationof these areas since the Pleistocene glaciationsbut also actual environmental factors (e.g. differencesin soil temperatures in summer) may playarole.Redhead (1989), in his overview of the distributionof Canadian fungi, provided dot mapsof 78 species in North America. He distinguished13 main types of distribution patterns, largelycoinciding with those of phanerogams. Four typesare shown in Fig. 7.2. Many fungi appear to berestricted to parts of the continent east and west

Biogeography and Conservation 113InAustralia,dotmaps of100 targetspecies wererecently published by Fungimap, a national organizationcoordinating the activities of volunteers(Grey and Grey 2005). The maps show distinctivepatterns of species restricted to native eucalyptforests (e.g. Boletellus obscurecoccineus (Höhn.)Sing.), coastal rainforests (e.g. Cymatodermaelegans Jungh.), southern temperate Nothofagusforests (e.g. Cortinarius metallicus (Bougher et al.)Peintner et al.), and arid areas in the centre of thecontinent (e.g. Podaxis pistillaris (L.: Pers.) Fr.).Patterns of some introduced species (e.g. Amanitamuscaria (L.: Fr.) Lam.) are presented, too.D. National and Regional PatternsFig. 7.7. Spatial distribution of five Russula species in 32subquadrats (5 × 5 m) in a forest of Castanopsis cuspidataand Pasania edulis near Fukuoka, Japan. The numbers of theleft indicate the abundance of basidiocarps, over a periodof 2 years (Murakami 1987)of the Rocky Mountains. This mountain rangeand the prairie area of the Midwest may be aneffective barrier for numerous species. Somespecies occupying the entire boreal zone showsubtle differences in morphology and/or ecology.Redhead (1989) suggested that these differencesmay be the results of survival of eastern andwestern populations in different refugia duringPleistocene glaciations. In Europe, Murat et al.(2004) reconstructed the postglacial colonizationroutes of Tuber melanospermum in France, usingpolymorphism in ribosomal DNA.In principle, studies on regional distribution patternsare less complicated than those on large-scaledistribution: data are easier to collect and subjectto less taxonomic and nomenclatural problems. Regionalstudies have been carried out mainly in Europeand published in a wide variety of journalsand books. In some countries, systematic mappingprogrammes have been initiated, e.g. in Germany,Great Britain, The Netherlands and also in Australia.The firstdistributionatlas onmacrofungi hasbeen published in Germany (Krieglsteiner 1991,1993; Fig. 7.5). It contains grid maps (without accompanyingtext) of 5,500 taxa, based on about3,000,000 records. An annotated atlas of 370 selectedmacrofungi was published in The Netherlands(Nauta and Vellinga 1995).As an example, distribution maps of differentscales are presented of the saprotrophic, wellcharacterizedpuffball Lycoperdon echinatum sensulato (Figs. 7.3, 7.4, 7.5 and 7.6). They may illustratesomeproblemsencounteredwithmappingand the interpretation of the results. The worlddistribution of L. echinatum sensu lato covers temperateareas in Europe and eastern North America(Fig. 7.3). However, Demoulin (1971) discoveredthat the North American populations are morphologicallydifferent from the European ones, and describedthe new species, Lycoperdon americanum.Consequently, what seemed to be a species withadisjunctareaprovedtobetwovicariantspecies.The distribution of L. echinatum in Europe isalmost restricted to the nemoral zone, dominatedby deciduous, broad-leaved trees, such as Quercusrobur L., of which the area is indicated on the map(Fig. 7.4). It is tempting to interpret this patternin terms of ecological dependence on deciduous

114 E.J.M. ArnoldsTable. 7.1. Percentages of species or genera of some groups of macrofungi in different parts of the worldReference Ryvarden Pegler Corner Kreisel Guzman Van Brummelen(1991) (1983a) (1968) (1967) (1970) (1967)Taxonomic Polypores Lentinus Thelephora Bovista Scleroderma AscobolusgroupBasidiom. Basidiom. Basidiom. Basidiom. Basidiom. Ascom.Principal Wood Wood Soil Soil Soil DungsubstratePrincipal Saprotroph Saprotroph Mycorrhizal Saprotroph Mycorrhizal SaprotrophwayoflifeNecrotrophSaprotrophTaxonomic Genera Species Species Species Species SpeciesunitsNumber 132 63 49 46 20 61of taxaKnown from 9 12 13 13 10 17one locality1.(Sub)cosmopolitan 23 5 4 2 5 282. Temperate/mediterraneana.Bipolar – – – 4 15 –b.Boreocircumpolar 26 5 6 2 5 –c.Southern 1 2 – 11 – –temperated. Eurasia 1 – 2 11 – –e. Europe 2 3 10 2 – 23f.Asia 2 3 6 13 – 1g. Europe – – 4 4 20 20and N. Americah.E.Asia 3 – 4 – – –and N. Americai.N.America 3 4 10 22 25 103. Tropicala.Pantropical 17 3 – 4 – 5b.Paleotropical 4 11 – – – –c.Africa 5 14 2 7 15 –d. S.E. Asia 4 13 39 2 – 1e. Tropical America 7 24 8 9 15 7f. Australia/ 1 13 2 7 – 5Pacificforests but, within the nemoral zone, the speciescan be found also in conifer plantations. Consequently,it is more likely to be limited by climatologicalfactors, in particular temperature. The speciesseems to be much more common in north-westernEurope than in the southwest, southeast and eastbut this might very well be an artefact due to undercollectingin these regions (Demoulin 1987).ThedistributioninBelgium,onthebasisofa4×4 km grid (Fig. 7.6), provides again more detailedinformation. The species is lacking in thenorth and west of the country as well as in the Ardennesmountains above 400 m. Demoulin (1987)suggested that climatological factors caused its absencein the Ardennes. However, L. echinatum isfound in adjacent western Germany in many areasabove 500 m (Fig. 7.5). It seems that its regionaldistribution is primarily determined by soil conditions,since L. echinatum is absent or scarce in allareas with acid, sandy or peaty soils with humusof the mor type: western Belgium, north-westernGermany and also The Netherlands. Rather, it iswidespread in areas with forests on calcareous,loamy soils producing mull humus. On the otherhand, the occurrence of L. echinatum in mountainsabove 1000 m on the Mediterranean island of Corsica,also noted by Demoulin (1987), seems to be determinedby the climate and not by soil conditions.E. Local PatternsDistribution patterns of fungal species in a standare usually not regarded as the subject of biogeo-

Biogeography and Conservation 115graphic research but, rather, as part of mycocoenologicalstudies. For instance, Jansen (1984) studiedthe distribution of macrofungi in oak forestsin The Netherlands using a grid of 40 subplotsof 5×5 m within 1000 m 2 plots. She was able todemonstrate correlations between the occurrenceof certain plants and fungi. Murakami (1987) didthesameforRussula speciesin32subplotsof25m 2in a Japanese Castanopsis-Pasania forest (Fig. 7.7).He demonstrated that the three dominant species(R. densifolia Gill., R. laurocerasi Melz., R. lepidaFr.) showed little spatial overlap whereas twoother species (R. castanopsidis Hongo and R. lilaceaQuel.) were found fruiting in much lower numbersscattered over the plot. The analysis of the distributionof individual mycelia or genets in stands isconsidered to belong to the domain of populationbiology.F. Recent Changes in Distribution PatternsStudies on changes in distribution patternsdepend on the availability of accurate data ongeographical ranges from different periods. Suchdata are known on a global scale only for somepathogenicfungionplants.Changesinregionaldistribution patterns or frequencies of macrofungihave been investigated by (1) repeated mapping,(2) comparison of representative samples of thelocal mycoflora over the years, (3) comparison ofsporocarp counts in selected stands or plots indifferent periods and (4) in some cases, data on thesupply of edible fungi to local markets (Arnolds1988a, b; Arnolds and Jansen 1992). Direct comparisonbetween numbers of records or occupiedgrid units of a certain species during differentperiods (methods 1 and 2) is usually inadequateto draw conclusions on increase or decrease. Theuse of correction factors was described by Arnoldsand Jansen (1992) and Nauta and Vellinga (1993).IV. Expansion of Distribution AreasExpansionoftherangeoffungimaybecausedbynatural colonization of new areas by propagules,introduction of host plants outside their naturalrange, and introduction of fungi outside their naturalrange. Introductions may be intentional or accidental.In practice, it is often difficult to trace theorigin of species because of lack of reliable historicaldata (Pringle and Vellinga 2006).Few well-documented examples exist ofspontaneously expanding fungi. A strikingphenomenon is the recent increase of somewood-inhabiting Aphyllophorales in the NorthwestEuropean lowland, for instance, Fomesfomentarius (L.: Fr.) Fr., Pycnoporus cinnabarinus(Jacq.: Fr.) Donk (Kreisel 1985; Thoen et al. 1998),Plicaturopsis crispa (Pers.: Fr.) D. Reid (Arnoldsand Van den Berg 2001) and Schizopora flavipora(Cooke) Ryvarden (Keizer 1990). The causes ofthese extensions of range are unknown. Therehave been suggestions attributing them to climatologicalchange, changes in forestry practices,and decline of tree vitality.Another group of increasing macrofungi inEurope comprises agarics such as Strophariarugosoannulata Farlow, S. aurantiaca (Cooke) P.D.Orton and Psilocybe cyanescens Wakef (Kreisel1985; Arnolds and Van den Berg 2005). Thesespecies are characteristic of mixtures of fertile soiland wood chips. Such habitats were rare in the pastbut are increasing, in particular in urban areas. Themost spectacular example of this group is Agrocyberivulosa Nauta, a species first found in The Netherlandsin 1999 and formally described only in 2004.Nowadays, it is one of the most common speciesin this habitat, rapidly spreading in West Europe.The invasive character of this species suggests thatit may be originally introduced, which may be trueforotherspeciesofthisecologicalgroupaswell.In Europe, also some ectomycorrhizal fungiare recently spreading northwards, e.g. the sub-Mediterranean Amanita caesarea (Scop.: Fr.) Pers.in Belgium, probably as result of global warming(Fraiture and Walleyn 2005).A. Introductions of PlantsThe artificial extension of ranges of plants by introductioncreates a new potential environment forassociated parasites, symbionts and saprotrophs.Fungi may colonize the new area spontaneouslyby propagules or may be introduced together withplant material. On the other hand, the lack of compatiblemycorrhizal symbionts canlimitthe spreadingof alien plants (Richardson et al. 2000).All over the world, many trees are plantedon a large scale outside their original range. Innorthwest Europe, for instance, Picea abies (L.)Karsten and Larix decidua Miller are widely usedin forestry. These trees are native to North Europeand the Central European mountains. Only partof the host-specific ectomycorrhizal symbionts

116 E.J.M. Arnoldsfollow their tree outside its natural range, thenumber of species decreasing with increasingdistance from the original habitat. For instance, inPicea plantations in The Netherlands, only 12 hostspecificsymbionts are found, most of them beingrare, whereas over 50 species are native to CentralEurope. Only six Larix symbionts occur in easternGermany and The Netherlands (Kreisel 1985). Inthese cases, dispersal is probably not inhibited bygeographical barriers but rather by environmentalconditions. Local strains of fungi with a broadhost range are apparently better adapted to theseconditions, and occupy their niches.Also North American trees were introducedinto Europe on a large scale, e.g. Pseudotsuga menziesii(Mirbel) Franco, Picea sitchensis (Bong.) Carriereand Quercus rubra L. Only very few specificsymbionts of these trees have found their way intoEurope, which may at first sight indicate the effectivenessof the Atlantic as a barrier for spore dispersal.However, all introduced Pseudotsuga symbiontsremain local and rare. Also in this case, localstrains take the niches of the original symbionts.Some ectomycorrhizal trees have been introducedin areas where suited native symbioticfungi are lacking. Successful forestry depends inthese cases either on accidental import of ectomycorrhizalfungi with nursery trees or on artificialinoculation of nursery plants with selected strains(Grove and Le Tacon 1993; Richardson et al. 2000).Diez (2005) demonstrated that successful forestationwith Eucalyptus in Spain depends on theaccidental introduction of some ectomycorrhizalsymbionts from Australia. Native fungi were neverfound in association with Eucalyptus. IntroducedPinus spp. in New Zealand, South Africa andmany tropical countries are associated withectomycorrhizal species from the original areas inEurope and North America (Dunstan et al. 1998).The spreading of some ectomycorrhizal fungioutside their original area is well documented –for instance, of Amanita muscaria in Australiaand New Zealand (Sawyer et al. 2001; Bagley andOrlovich 2004) and of Amanita phalloides outsideEurope (Pringle and Vellinga 2006).B. Introductions of FungiMany pathogenic fungi have been introduced accidentallyinto new areas, sometimes with detrimentaleffects on valuable crops. A well-knownexample is the introduction of Phytophthora infestans(Mont.) de Bary, the cause of late blightin potato, in Europe around 1845, resulting in thenotorious famine in Ireland where 1,000,000 peopledied. The coffee rust, Hemileia vastatrix Berk.&Br.,arrived in Ceylon in 1869 and destroyed 200,000 haof coffee plantation in 20 years; it converted theBritish from coffee drinkers into tea drinkers. It issometimes suggested that such epidemics are typicalof monocultures. However, the introductionof Cryptonectria parasitica (Murr.) Barr from EastAsia into North America around 1900 almost exterminatedthe American chestnut, Castanea dentataBorkh., an important component of native forests.Almost equally detrimental was the introductionfrom China into The Netherlands around 1915 ofCeratocystis ulmi (Buisman) C. Moreau, causingwilting of elm trees (Dutch elm disease; Campbelland Madden 1990).These catastrophic introductions have led onthe one hand to quarantine procedures to preventunwanted introductions, on the other to the useof some fungi for biological control of introducedweeds. Successful examples are the suppression ofthe weed Chondrilla juncea L. by the rust fungusPuccinia chondrillina Bubak & Syd. in Australia andthe USA, and of Ageratina riparia on Hawaii by anunidentified Cercosporella-likefungus(EvansandEllison 1990).Less is known about accidental introductionsof saprotrophic and mycorrhizal fungi (see alsoSect. IV.A.). Most known cases of successfulintroductions concern Phallales, whichcombinea striking appearance with limited spore-dispersalcapacity (spread by insects) and a preferencefor disturbed habitats – for instance, in EuropeMutinus ravenelii (Berk. & Curt.) E. Fischer andM. elegans (Mot.) E. Fischer from North Americaand Anthurus archeri (Berk.) E. Fischer from thesouthern hemisphere (Kreisel 1985). The extensionof the latter species was described in detail byParent et al. (2000). Some edible mushrooms wererecently purposefully introduced into plantationsoutside their original range, in particular truffles(Yun and Hall 2004).C. Decline and Extinction of FungiThe dramatic loss of natural and semi-naturalhabitats on earth, caused by human activities,is reflected in the rarification or extinction ofmany vascular plants and animals. It is inevitablethat fungi are subject to the same environmental

Biogeography and Conservation 117Fig. 7.8. Distribution of Sarcodon imbricatus in theNetherlands in the periods 1890–1949 (left), 1950–1972(centre) and 1973–1985 (right). Dotted Acid, Pleistocenesands; hatched calcareous, Holocene sands (Arnolds 1989)changes. Nevertheless, to my knowledge, nota single species has been reported to be definitelyextinct on a global scale. This is certainly in partan artefact caused by limited research efforts inthis field, and the methodological complicationsoutlined above. How do we know that a rarespecies has really disappeared when its ephemeralsporocarps have been collected at intervals of manyyears at scattered localities (see also Sect. III.B.)?Nevertheless, it is probably true that fungi areless vulnerable to extinction than vascular plantsbecause (1) there are few species endemic to smallareas; (2) their dispersal capacity is, in general,larger, at least on a continental scale; (3) it is hardlypossible to purposefully exterminate fungi, e.g. byharvesting of sporocarps, as has been the case forsome plants and larger animals.However, decline and extinction of regionalpopulations of macrofungi were observed in severalparts of Europe. Evidence of a considerable declineinsomeecologicalandtaxonomicgroupsoffungi has stimulated efforts for fungal conservation(see section below). Here, only one example of a regionallynearly extinct fungus is described in somedetail in order to illustrate some of the methodologicalproblems encountered. It concerns the declinein The Netherlands of Sarcodon imbricatus (L.: Fr.)P. Karst., an obligate ectomycorrhizal species ofconiferous trees, producing large epigeous sporocarps.Thespecieswasreportedascommonintheeastern Netherlands up to the 1950s, became rarein the next decade and was recorded from only fourlocalities in the period 1973–1985 (Arnolds 1989;Fig. 7.8). Nowadays (2006), it is found at only onelocality. The maps are only a weak reflection of itstrue decline, because of the enormous increase inmycofloristic research in The Netherlands: from thedecade 1980–1989, over 200,000 records on macrofungiare available and, from all the years before1950, only 11,000. The decrease of this species isbetter expressed as a proportion of the total numberoffungalrecords,decreasingfrom0.21%before1949 to less than 0.005% since 1980.V. Conservation of FungiConservation of fungi has only recently becomean issue of major concern (Winterhoff andKrieglsteiner 1984; Arnolds 1991a). Research inthis field is restricted mainly to macrofungi, andgeographically to Europe and North America.Motives for the increasing attention to fungalconservation on both continents are the rapid declineof some habitats rich in rare and specializedspecies, and concern on the sustainability of masscollecting of edible wild mushrooms. In Europe,extensive evidence exists for severe decrease ofsome species in certain areas (Arnolds 1988a,

118 E.J.M. Arnoldsb, 1991b). In addition, international concern ondecrease of biodiversity in general, culminatingin 1992 in the United Nations Conference on theEnvironment and Development in Rio de Janeiroand the Convention on Biological Diversity (’Rioconvention’), has promoted interest in the fate offungi and other micro-organisms.It is generally accepted that the most importantstrategy for conservation of fungi is in situ conservationof their natural environment (Staley 1997;Moore et al. 2001). For species threatened with extinction,exsitu cultivationmay offera(temporary)conservationstrategy,e.g.forPleurotus nebrodensis(Inzenga) Quël., an endemic species in Sicily(Venturella and La Rocca 2001).The most important nongovernmental organizationfor global nature conservation is the InternationalUnion for Conservation of Nature (IUCN).Itis,forinstance,responsiblefortheeditionofglobal Red Data Lists of endangered organismsand the formulation of criteria for such lists (IUCN2005). The IUCN founded a specialist group forfungi in 1990 with representatives of most continents.Unfortunately, this group has not been veryactive since. In Europe, conservation issues areinitiated mainly by professional and amateur mycologists,often united into mycological societies(Moore et al. 2001). Intensive cooperation takesplace in a permanent European Council for Conservationof Fungi (ECCF), erected during the ninthCongress of European Mycologists in 1985. Thecouncil organizes specialist meetings devoted toconservation, at regular intervals (Senn-Irlet 2005).A. Red ListingOne of the main tools for the conservation of fungiis the publication of Red Data Lists, originally anacronym for ‘Rarity, Endangerment and DistributionData lists’. They consist of enumerations ofspecies which are considered to be threatened intheir long-term survival in a given area. The aimsof Red Lists are (1) to inform mycologists in orderto stimulate research on threatened species and inareas where many such species are present; (2) tofacilitate the use of mycological data by natureconservationists and environmental planners forprotection and management of nature areas; (3) toinform decision-makers and politicians, in orderto develop measures and laws in favour of fungalconservation; (4) to provide data for the selectionof species for monitoring programmes; (5) toprovide data for the selection of species to beprotected by law.Several categories of threatened species qualifyfor a Red List: (1) Extinct species which have notbeenobserved during a considerable period inspiteof extensive field surveys; (2) Critically Endangeredspecies, facing an extremely high risk of extinction;(3) Endangered species, facing a very high risk ofextinction; (4) Vulnerable species, facing a highrisk of extinction. Besides these, some othercategories are distinguished: Near-Threatened,when the species is likely to meet one of the threatcategories in the near future; Least Concern, whena species does not qualify for one of the above categories;Data Deficient, when there is inadequateinformation to make a direct or indirect assessmentof its risk of extinction. Three main parametersare used to determine the threat status of a species:(1) decline of the population; (2) geographicalrangesizeand(3)populationsize.Thesehaverecentlybeen elaborated for each Red List category inoften complicated, quantitative parameters (IUCN2005). There are special directives for applicationof these criteria at regional levels (IUCN 2005).TheIUCNapproachtotheRedListingprocedureis designed mainly for animals with discreteindividuals and well-defined populations. Applicationto fungi is possible only after adaptation ofthe criteria. A global list of threatened fungi doesnot exist and is unlikely to appear in near future.Virtually all species would qualify as Data Deficienton a worldwide scale. National Red Lists of fungihave been published to date in 25 European countries,in some countries already in several editions.A recent survey was published by Arnolds (2001).Most lists are restricted to macrofungi, since microfungiqualify almost automatically as data deficient.However, some microfungi are considered in somecountries, mostly species associated with threatenedhost plants (Denchev 2005). In such cases, thequalificationofthefungusasthreatenedisusuallybased on indirect evidence.Most of the Red Lists are still based on expertjudgement, rather than on numerical data andquantitative criteria, but some more recent lists useemended IUCN criteria. The number of species incorporatedranges between 17 in the former USSRto 1,655 in The Netherlands. It is evident that thesevaluesarebynomeansrepresentativeoftherealthreatstotheMycotainvariouscountries.Indicativeis the difference in the number of listed speciesin comparable countries such as Latvia (38 species)and Lithuania (740 species). It is clear that the crite-

Biogeography and Conservation 119ria and aims of the various lists are widely different.A total number of 4,400 species of macrofungi isincluded in one or more of the national lists, about30% of the estimated total number of Europeanspecies. These species belong to saprotrophic fungion soils (35%), ectomycorrhizal species (31%),saprotrophs on wood (28%), parasitic fungi ontrees (2%) and even some fungi on dung (1%). It isevident that the number of threatened species ona continental scale is much lower. A first attempt toa Red List for Europe was made by Ing (1993), enumerating278 species. His selection was based onthe number of national lists in which a species is included.However, this is not a satisfactory criterionbecause the threat to species with a restricted distributionarea is thereby underestimated. A revisedEuropean Red List, using quantitative IUCN criteria,is in preparation by ECCF (Senn-Irlet 2005).Outside Europe, Red Lists of fungi have beenpublished in the USA for the states Oregon andIdaho (Molina et al. 2001).B. Threatened Fungi and Their HabitatsFrom a comparison of Red Lists, it appearsthat some ecological and taxonomic groups offungi deserve special attention regarding theirconservation.1. Species largely restricted to pristine andold-growth forests, mainly lignicolous fungion large logs and other woody debris, includingmanypolypores.Thisiscertainlythemost widespread and important threatenedecological group throughout the world, as theresult of increasing logging. Also sustainabletimber harvests with selective cutting of largetrees only may do much harm to the vastgroup of fungal species dependent on largelogs and other coarse woody debris. In Europe,most of these species are at present restrictedto forest reserves in remote parts of Scandinaviaand the Central European mountains(Kotiranta and Niemela 1993; Parmasto 2001).In the north-western USA, conservation ofold-growth forests and their characteristicorganisms is a major political issue whichhas lead to extensive mycological researchefforts (Molina et al. 2001). A monitoringprogramme has been initiated to survey221 indicator species, subdivided into fourcategories in terms of frequency (uncommonor rare) and the possibilities of predisturbancesurveys (Castellano et al. 2003). The flagship ofthreatened fungi is here Oxyporus nobilissimusW.B. Cooke, a very rare polypore with giganticbasidiomes on huge stumps of Abies andTsuga, and endemic to this region. Federallaws stipulate that 240 ha of habitat will bepreserved at each known fruiting location ofthis species (Pilz and Molina 1996). In thetropics, almost no information is available onthreatened fungi but the rapid loss of tropicalforests must necessarily go hand in hand withunnoticed loss of mycological biodiversity.Numerous species may even become extinctbefore they are ever discovered and described.2. Fungi characteristic of peat bogs, fens andother wetlands, often associated with Sphagnumspp. or other bryophytes, e.g. Armillariaectypa (Fr.) Herink, listed on Red Lists in10 European countries. These habitats arewidespread in the northern hemispherebut, in most areas, they are subject to peatexcavation, drainage activities, reclamationand/or afforestation. In Europe, extensiveundamaged bogs are at present found mainlyin Scandinavia, the Baltic states and Russia.3. Saprotrophic fungi characteristic of coastaland inland sand dunes, sometimes also steppe,including many gasteromycetes, for instance,of the genera Geastrum Pers.: Pers., TulostomaPers.: Pers. and Disciseda Czern. These arethreatened throughout Europe by sand excavation,recreational activities, afforestation,and natural succession on remaining relics(Winterhoff and Krieglsteiner 1984).4. Saprotrophic fungi of old, nutrient-poor,unfertilized meadows and hayfields, includingmany species of Hygrocybe (Fr.) Kumm,Entoloma (Fr.) Kumm., Dermoloma (J. Lange)Herink, Clavariaceae and Geoglossaceae, arestrongly threatened in most parts of Europe.The distribution of grassland relics rich inHygrocybe species is well documented inThe Netherlands (Arnolds 1988a, b), GreatBritain (Newton et al. 2003) and Denmark(Raid 1985). The decline of grassland fungiin Sweden was described by Nitare (1988),who used Geoglossaceae as indicator species.Grassland fungi are threatened on the onehand by intensification of grassland use (e.g.use of dung and fertilizers), on the otherby abandonment and afforestation, bothprocesses being promoted by EEC agriculturalpolicy.

120 E.J.M. Arnolds5. Many ectomycorrhizal fungi, associated withboth frondose and coniferous trees, havestrongly declined since 1960 in some denselypopulated and industrialized parts of Europe.Reports on decline come mainly from theCzech Republic (Fellner 1993), parts of Germany(Derbsch and Schmitt 1987) and TheNetherlands (Arnolds 1988a, 1991b; Nauta andVellinga 1993). The decrease in ectomycorrhizalsporocarps is most prominent in forestson acidic soils poor in nutrients and humus,including well-known edible species suchas Cantharellus cibarius Fr.: Fr. and Boletusedulis Bull.: Fr. (Derbsch and Schmitt 1987;Jansen and Van Dobben 1987). The decrease insporocarps may or may not coincide with a reductionof mycorrhizal rootlets (Dighton andJansen 1991). The decline of ectomycorrhizalfungi is generally attributed to the effects ofair pollutants on trees (reduced photosynthesis)and soil (acidification and nitrogenaccumulation; Jansen and Van Dobben 1987;Arnolds 1991b; Dighton and Jansen 1991).Recently, a strong reduction in the emission ofacidifying agents in some regions has lead topartial recovery of the ectomycorrhizal flora(Arnolds and Van den Berg 2001; Fellner andLanda 2003).C. Mycological Reservesand Nature ManagementTheerectionofnaturereservesbyconservationauthorities is very rarely primarily based on thesignificance of its mycoflora. Because it is generallyaccepted that fungi are optimally conserved whentheir habitats are preserved (Staley 1997), this situationis not necessarily dramatic. However, somehabitats appear to be mycologically very importantwhereas their flora and fauna are not of particularinterest. It is an important task for mycologists toidentify these cases and to draw special attention totheir fate. In Great Britain, The Netherlands and theUSA, various methods have been developed to defineand recognize important fungal areas (Jalinkand Nauta 2001; Molina et al. 2001; Newton et al.2003). In addition, the European organization forconservation of wild plants, Planta Europa, currentlyincludes data on fungi in its programme forthe selection of important plant areas.An important and often neglected aspect offungal conservation is appropriate management ofhabitats in and outside nature reserves. In completelynatural habitats, such as primeval forestsand bogs, every form of human interference isusually disadvantageous for maintenance of biodiversity,including fungi. However, in exploitedforests, semi-natural habitats and cultural landscapes,some kind of management may be usefulor even necessary. In production forests, a certainproportion of living trees and coarse dead woodmight be left behind after cutting in order to favourthe survival of mycorrhizal and wood-inhabitingfungi. Management of poor grasslands by cuttingor grazing is essential to prevent natural successionto forests (Nitare 1988; Keizer 2003). In polluted areas,removal of contaminated litter may help to restorethe ectomycorrhizal flora in forests. Adequatemanagement of roadsides may create refugia forendangered grassland fungi and ectomycorrhizalspecies (when planted with trees) in cultural landscapes.Surveys of the relations between managementand fungi are given by Keizer (1993, 2003).D. Harvests of Wild Edible Mushroomsand Legal ProtectionPicking of mushrooms for private consumption isatraditionsincecenturiesinmanypartsoftheworld. Only in the last decade of the 20th centuryhave commercial harvesting and trade of wildmushrooms become a booming business, and a majorsource of income for some rural communities ineastern and southern Europe, western North America,Africa and China (Arora 2001; Chiu and Moore2001; Boa 2004). At the same time, it has becomea major source of concern for mycologists and natureconservationists who query whether this harvestis sustainable in the long run. This question isnot yet definitely answered.Some authors stress that normal harvestshave been intensive in many regions but withoutdemonstrable decrease of mushroom production(Arora 2001). They believe that the generallylong-living mycelia, enormous production ofspores, and great dispersal capacity of fungi guaranteethe long-term survival of these organisms,provided that appropriate habitats are maintainedand harvesting methods are not destructive.This opinion is supported by research in twoforest plots in Switzerland, where all epigeoussporocarps were removed with weekly intervalsduring 29 years. No significant effects were foundon species diversity and sporocarp production of

Biogeography and Conservation 121any species (Egli et al. 2006). A similar experimenton picking of Cantharellus formosus (Corner)during13yearsinOregon(USA)alsoshowedan absence of significant effects (Norvell 1995).However,tramplingofthesoil,anormalside-effectduring collecting, reduced sporocarp productiontemporarily by about 30%, apparently by damageto primordia (Egli et al. 2006). Productivityrecovered after termination of trampling of thesoil. Destructive harvesting occurs only whenthe top soil is thoroughly disturbed by raking ordigging in search of, e.g. truffles and very young(and very precious!) sporocarps of Tricholomamatsutake (S. Ito & Imai) Sing. and allies, withdetrimental damage to mycelia.Other authors use additional argumentsagainst unlimited gathering of (edible) fungi.Some stress the possible importance of sufficientspore production for the colonization of new areas.Sporocarps are food not only for humans but alsofor some wild animals, and they may occupy animportant link in food webs in the forest. An interestingexample is the importance of hypogeoussporocarps for the survival of squirrels in thenorth-westernUSA,intheirturnthestaplefoodfor the endemic and endangered Great spottedowl (Strix occidentalis; Molina et al. 2001). Manyother possible interactions, e.g. with numerousspecialized insects feeding on sporocarps, are stillto be discovered. In addition, not only scientific butalso ethical and esthetical arguments play a role inthe debate, such as the possibility for perceptionof undisturbed nature by naturalists and walkers.In many European countries, harvesting ofmushrooms is regulated in some way or another.However, the restrictions differ from one countryto another. In general, no extraction of any naturalmaterial is allowed in national parks and otherstrict nature reserves. This is also true for the USA.In some countries, e.g. the Czech Republic andHungary, some species, including edible ones, areprotected by law, so that collecting is prohibitedeverywhere and at any time. In The Netherlands,no national regulations exist; in some municipalities,however, any collecting of any mushroomis prohibited whereas, in others, no legislationexists. In Switzerland, regional regulations existthat restrict collecting to certain quantities andcertain periods. Data on the effectiveness of theseand other regulations are not available.In Europe, international legislation is becomingincreasingly important for nature conservation,also on a national scale, e.g. by the Bern convention,including lists of protected species and protectionof their habitats, the Habitat directive andthe Natura 2000 programme, offering protection tomany valuable sites. Most groups of plants and animals,including some bryophytes and lichens, arecoveredbythislegislationbutfunginot,andthisdespite attempts by ECCF to add some macrofungito the species lists protected under the Bern convention.There is still a long way to go before conservationoffungiisaswellregulatedasthatofmorepopular and well-known groups of organisms.VI. ConclusionsBiogeographical information on fungi is becomingincreasingly important for the understanding ofevolutionary processes and biodiversity patternsas well as for control of spread of crop pests and fornature conservation. However, this kind of informationis still scanty, scattered over the literatureand, therefore, often difficult to obtain. Gatheringknowledge on distribution patterns of fungi isoften hampered by the biological properties ofthese organisms and by methodological problems.Relatively little is known on global distributionpatterns as well as the frequency and significanceof local endemism. Programmes for mappingof (macro)fungi exist in only some Europeancountries.Expansion of ranges of fungi has been documentedmainly for plant pathogens, usually dueto accidental introductions and sometimes withprofound effects on ecosystem functioning or cropproduction. Relatively few examples exist of saprotrophicor mycorrhizal fungi invading other continents.On the other hand, in some areas, in particulardensely populated parts of Europe, a dramaticdecline of many species has been recorded.These changes in the mycoflora are caused mainlyby the destruction of (semi)natural habitats andenvironmental pollution. In particular, ectomycorrhizalspecies appear to be sensitive to acidificationand eutrophication of forest ecosystems. Itis to be expected that similar losses of biodiversityare currently taking place in other parts of theworld, in particular the tropics, but escaping ourattention. The exploration and documentation ofbiodiversity of fungi in these areas are thereforeurgently needed. Effective conservation of fungican be achieved only within the context of integralecosystem protection and management.

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Fungal Interactions and Biological Control Strategies

8 Plant Disease Biocontrol and Induced Resistancevia Fungal MycoparasitesA. Viterbo 1 ,J.Inbar 1 ,Y.Hadar 2 ,I.Chet 1CONTENTSI. Introduction ........................ 127A. Antibiosis........................ 128B. Competition...................... 128C. Mycoparasitism................... 128II. Mycoparasites as Biocontrol Agents ...... 129A. BiotrophicMycoparasites ........... 1291. Sporidesmium sclerotivorum ....... 1292. Ampelomyces quisqualis .......... 130B. NecrotrophicMycoparasites ......... 1301. Pythium nunn .................. 1302. Talaromyces flavus ............... 1313. Corniothyrium minitans .......... 1314. Gliocladium and Trichoderma spp. . . 132C. Induced Systemic Resistanceby Trichoderma spp. ............... 134D. Mycoparasitismin Suppressive Environments(SoilsandComposts)............... 135III. Hyphal Interactions in Mycoparasitism . . . 136A. Biotrophs........................ 136B. Necrotrophs...................... 137IV. Molecular Aspects and Genetic Engineeringin Mycoparasitism ................... 140V. Conclusions ......................... 141References.......................... 142I. IntroductionDue to the adverse environmental effects ofpesticides that create health hazards for humanand other nontarget organisms, including thepests’ natural enemies, these chemicals havebeen the object of substantial criticism in recentyears. The development of safer, environmentallyfeasible control alternatives has therefore becomea top priority. In this context, biological controlis becoming an urgently needed component ofagriculture.1 Department of Plant Sciences, Weizmann Institute of Science,Rehovot 76100, Israel2 Department of Plant Pathology and Microbiology, The HebrewUniversity of Jerusalem, Faculty of Agriculture, Rehovot 76100,IsraelBiological control of plant pathogens is definedas the use of biological processes to lower inoculumdensity of the pathogen, with the aim of reducingits disease-producing activities (Baker and Cook1974).Biological control may be achieved by both directand indirect strategies. Indirect strategies includethe use of organic soil amendments that enhancethe activity of indigenous microbial antagonistsagainst a specific pathogen. Another indirectapproach, cross-protection, involves the stimulationof plant self-defense mechanisms againsta particular pathogen by prior inoculation of theplant rhizosphere with a nonvirulent strain or othernonpathogenic rhyzo-competent bacteria or fungi.Successful protection resulting in induced resistancehas been documented for viruses, bacterialpathogens, and fungi (van Loon et al. 1998; Harmanet al. 2004; Haas and Defago 2005).The direct approach involves the introductionof specific microbial antagonists into soil or plantmaterial (Cook and Baker 1983). These antagonistshave to proliferate and establish themselves inthe appropriate ecological niche in order to beactive against the pathogen. Antagonists are microorganismswith the potential to interfere withthegrowthand/orsurvivalofplantpathogens,and thereby contribute to biological control. Antagonisticinteractions among microorganisms innature include parasitism or lysis, antibiosis, andcompetition. These microbial interactions serveas the basic mechanisms via which biocontrolagents operate. An elucidation of the mechanismsinvolved in biocontrol activity is considered tobe one of the key factors in developing usefulbiocontrol agents. Of the numerous biocontrolagents examined, only a few have been subjectedto a thorough analysis of the mechanisms involvedin the suppression of the pathogen. In this chapter,we shall use examples to briefly demonstrate thedifferent mechanisms, and will concentrate onmycoparasitism.Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

128 A. Viterbo et al.A. AntibiosisHandelsman and Parke (1989) restricted the definitionof antibiosis to those interactions that involvea low-molecular-weight diffusible compound, or anantibiotic produced by a microorganism that inhibitsthe growth of another microorganism. Thisdefinition excluded proteins or enzymes that killthe target organism. Baker and Griffin (1995) extendedthe scope of the definition to “inhibition ordestruction of an organism by the metabolic productionof another.” This definition includes smalltoxic molecules, volatiles, and lytic enzymes.The production of inhibitory metabolites byfungal biocontrol agents has been reported in theliterature over the last five decades (Bryan andMcGowan 1945; Dennis and Webster 1971a, b;Ghisalberti and Sivasithamparam 1991). Gliocladiumvirens is a common example of the role ofantibiotics in biological control by fungal antagonists.Gliovirin is a diketopiperazine antibioticthat appears to kill the fungus Pythium ultimumby causing coagulation of its protoplasm.Howell and Stipanovic (1983) obtainedgliovirin-deficient mutants of G. virens via ultravioletmutagenesis. These mutants failed to protectcotton seedlings from P. ultimum damping-offwhen applied to the seeds, whereas the normalparent strain protected the seedlings. Moreover,a gliovirin-overproducing mutant provided controlsimilar to that of the wild type, althoughit exhibited a much lower growth rate. A combinationof G. virens treatment of cotton seedwith reduced levels of the fungicide metalaxylprovided diseased suppression equal to that ofa full fungicide treatment (Howell 1991). Anextensive review on antibiosis and production ofTrichoderma secondary metabolites is provided inHowell (1998). Baker and Griffin (1995) concludedthat the impact of antibiosis in biological control isuncertain. Even in cases where antifungal metaboliteproduction by an agent reduces disease, othermechanisms may also be operating. Synergismamong various lytic enzymes, and between enzymesand antibiotics has been shown to be verycritical for the activity of many biocontrol agents(reviewed by Woo et al. 2002).B. CompetitionMany plant pathogens require exogenous nutrientsto successfully germinate, penetrate, and infecthost tissue (Baker and Griffin 1995). Garrett (1965)concluded that the most common cause of death ina microorganism is starvation. Therefore, competitionfor limiting nutritional factors, mainly carbon,nitrogen, and iron, may result in biological controlof plant pathogens.Research over the years has concentrated oncompetition by bacterial biocontrol agents, mainlyfor iron (Fe). However, fungal antagonists have receivedvery little attention. Sivan and Chet (1989)found that a strain of T. harzianum (T-35) that controlsFusarium spp. on various crops may operatevia competition for nutrients and rhizosphere colonization.The potential of microorganisms, which areapplied as a seed treatment, to proliferate and establishalong the developing root system has beennamed rhizosphere competence (Ahmad and Baker1987). When T-35 conidia were applied to soil enrichedwith chlamydospores of F. oxysporum f. sp.melonis and f. sp. vasinfectum, andamendedwithlow levels of glucose and asparagine, the abilityof the chlamydospores to germinate was reduced.This inhibitory effect could be reversed by addingan excess of glucose and asparagine or of seedlingexudates to the soil. After its application as a seedtreatment, this strain effectively colonized the rhizosphereof melon and cotton, and prevented colonizationof these roots by F. oxysporum. Thus,competition for carbon and nitrogen in the rhizosphere,as well as rhizosphere competence itselfmay be involved in the biocontrol of F. oxysporumby T. harzianum strain T-35 (Sivan and Chet 1989).C. MycoparasitismMycoparasitism is defined as a direct attack ona fungal thallus, followed by utilization of its nutrientsby the parasite. The term hyperparasitismis sometimes used to describe a fungus that isparasitic on another parasitic pathogenic fungus.Barnett and Binder (1973) divided mycoparasitisminto: (1) necrotrophic (destructive) parasitism, inwhich the relationships result in death and destructionof one or more components of the host thallus,and (2) biotrophic (balanced) parasitism, in whichthe development of the parasite is favored by a living,rather than a dead host structure.Necrotrophic mycoparasites tend to be moreaggressive, have a broad host range extending towide taxonomic groups, and are relatively unspecializedin their mode of parasitism. The antag-

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 129onistic activity of necrotrophic mycoparasites isattributed to the production of antibiotics, toxins,or hydrolytic enzymes in proportions that causethe death and destruction of their host. Biotrophicmycoparasites, on the other hand, tend to havea more restricted host range and produce specializedstructures to adsorb nutrients from their host(Manocha 1990).The parasitic relationships between fungi andtheir significance in biological control are the subjectsof this chapter. Both types of mycoparasitismare described and discussed in the scope of theircontribution to biological control. We will concentrateon the morphological, biochemical andmolecular aspects of mycoparasitism in relation tobiological control. The ecological aspects of thisphenomenon are discussed in Jeffries (1997).II. Mycoparasites as Biocontrol AgentsMany comprehensive reviews on mycoparasitismin biological control in general have been publishedover recent years (Chet 1990; Deacon 1991;Elad and Chet 1995; Benitez et al. 2004). Due totheir nature, only a few examples of biotrophic mycoparasitesas biocontrol agents exist (Ayers andAdams 1981; Sztejnberg et al. 1989; Adams 1990).Necrotrophic mycoparasites, being more common,saprophytic in nature, and less specialized in theirmode of action, are easier to study. As a result, themajority of the mycoparasites used as biocontrolagents in greenhouse or field trials to date havebeen necrotrophs. In this chapter, we will emphasizethose examples in which the research that hasbeencarriedoutisbothappliedandbasicinnature.A. Biotrophic Mycoparasites1. Sporidesmium sclerotivorumSporidesmium sclerotivorum is a dermatiacesushyphomycete that was isolated from field soil byUecker et al. (1980). In nature, the fungus has beenfoundtobeanobligateparasiteonsclerotiaofSclerotinia sclerotiorum, S. minor, S. trifoliorum,Sclerotium cepivorum, andBotrytis cinerea (Ayersand Adams 1981). In response to chemicalsreleased by the host’s sclerotia, macroconidia ofS. sclerotivorum in the soil germinate and thegerm tubes infect the sclerotia. When volatilecompounds secreted by sclerotia of Sclerotiniaminor, Sclerotinia sclerotiorum, and Sclerotiumrolfsii were tested to determine if they could stimulategermination of conidia of S. sclerotivorum,none of the chemicals alone or a combination ofall chemicals induced germination (Fravel et al.2002). The hyphae penetrate the intercellularmatrix of the conidia, which is composed mainly ofβ-glucans (Ayers et al. 1981). The production andactivity of haustoria by the mycoparasite stimulatethe host sclerotia to increase their glucanase, andprobably other enzyme activities, resulting in thedegradationofglucanintoavailableglucose(Bullocket al. 1986). The mycoparasite establishes itselfin the sclerotia, where its mycelium grows out intothe surrounding soil to infect additional sclerotiaand to produce new macroconidia (Adams et al.1984). The interaction between S. sclerotivorumand Sclerotinia minor depends on both the hostand parasite density (Adams 1986). The infectionprocessisfavoredbysoilpH,waterpotentialandtemperature (20–22 ◦ C) (Adams and Ayers 1980).Under field conditions, a single application ofan S. sclerotivorum preparation at a concentrationof 10 2 or 10 3 macroconidia g– 1 soil causeda 75–95% reduction in the number of sclerotia ofS. minor perplot.Controloflettucedropcausedby S. minor in these plots varied from 40–83% infour consecutive lettuce crops (Adams and Ayers1982). These results were significant but not economicallyimportant (Adams 1990). Adams (1990)concluded that “one of the biggest obstacles to practicalbiological control is the large quantity of theagent necessary to achieve biological control whenapplied directly to soil in the field.” He thereforesuggested two alternatives: (1) to add sclerotia ofS. minor or a nonpathogenic Sclerotinia that is alsoahostofS. sclerotivorum that are already infectedby the mycoparasite; (2) to apply a low dosage of themycoparasite preparation to a diseased crop, andthen immediately incorporate the treated crop intothe soil. This latter procedure ensures that a highpercentage of the mycoparasites will be present inthe soil in close contact with the sclerotia of thepathogen. Although the author assumes that thesealternatives are easier and more practical, neitherhas been explored to any significant extent (Adams1990). Field studies were later conducted (Del Rioet al. 2002) to evaluate the effectiveness of S. sclerotivorumto control Sclerotinia stem rot of soybean.Experimental plots were infested with S. sclerotivorummacroconidia at a rate of 0, 2, or 20 spores percm 2 . Two years later, the disease was completelysuppressed in all plots. S. sclerotivorum was retrievedfrom all infested plots at all locations 2 years

130 A. Viterbo et al.after infestation with sclerotia of S. sclerotiorum asbait. This paper constitutes the first report describingthe biocontrol of a disease on field crops thatmay be employed economically.2. Ampelomyces quisqualisAmpelomyces quisqualis, a hyperparasite onErysiphales, has been reported as a biocontrolagent of powdery mildews (Sztejnberg et al. 1989).An isolate of A. quisqualis obtained from an Oidiumsp. infecting Catha edulis in Israel proved to beinfective to several powdery mildew fungi belongingto the genera Oidium, Erysiphe, Sphaerotheca,Podosphaera, Uncinula, and Leveillula. In fieldtrials, A. quisqualis parasitized the powderymildews of cucumber, carrot, and mango, andreduced the disease. A. quisqualis was tolerant tomany fungicides used to control powdery mildewsand/or other plant diseases. Treating powderymildew of cucumber (cv. Hazera 205) with spores ofA. quisqualis alone significantly decreased diseaseseverity and increased cucumber yield by approximately50%. Combining the fungicide pyrazophoswith the mycoparasite resulted in a larger increasein cucumber yield (Sztejnberg et al. 1989).Treating powdery mildew-infected zucchinileaves with A. quisqualis increased the rates ofphotosynthesis from 3.8 μmol CO 2 m −2 s −1 inuntreated plants to 10.2, compared to 12.8 in uninfectedhealthy plants (Sztejnberg and Abo-Foul1990). Electron micrographs of leaf sections of diseasedcucumber plants revealed marked deteriorationon the morphological organization of chloroplastmembranes. Chloroplasts of A. quisqualistreatedplants seemed undamaged, like those of untreatedplants (Abo-Foul et al. 1996). Fluorescencemeasurements (e.g., low-temperature fluorescenceemission spectra, and room-temperature fluorescencetransients) indicated a disease-correlatedincrease in levels of uncoupled chlorophyll (Abo-Foul et al. 1996). A simple, inexpensive mediumbased on potato dextrose broth (PDB) was developedfor mass production of infective spores ofA. quisqualis in fermentation for biological control(Sztejnberg et al. 1990), which was later developedin the commercial biofungicide AQ10.The interaction between the hyperparasiteA. quisqualis and its host fungi was studied byHashioka and Nakai (1980) and Sundheim andKrekling (1982). The infection process of thecucumber powdery mildew Sphaerotheca fuligineaby A. quisqualis was studied by scanning electronmicroscopy. Within 24 h after inoculation, thehyperparasite had germinated, and the germ tubeshad developed appressorium-like structures atthe point of contact with the powdery mildewhost. Both conidia and hyphae were parasitizedby penetration. Within 5 days of inoculation,the hyperparasite had developed pycnidia withconidia on the powdery mildew hyphae andconidiophores (Sunhdeim and Krekling 1982).Hashioka and Nakai (1980) used both transmissionand scanning electron microscopy to study thehyphal extension and pycnidial development of themycoparasite A. quisqualis Ces. inside the hyphae,and conidiophores of several species of powderymildew fungi belonging to Microsphaera, Erysiphe,and Sphaerotheca. The mycoparasite cells grewnormally inside the host cells, despite gradualdegeneration of these latter cells. The invadinghyphal cells of the mycoparasite migrated into theneighboring host cells by constricting themselvesthrough the host cell’s septal pore. The mycoparasiteextended hyphae inside the conidiophoresof the hosts, and formed pycnidia consisting ofa unicellular outer layer and interior cells thatlater differentiated into conidiogenous structures(Hashioka and Nakai 1980). Recent studies basedboth on morphological and life cycle parametersand ribosomal DNA internal transcribed spacerregion 1 sequence analysis have shown that isolatespreviously attributed to the genus Ampelomyceswere actually isolates of Phoma spp. Phomaglomerata can colonize and suppress developmentof powdery mildew on oak, and may have utility asa mycoparasitic agent (Sullivan and White 2000).B. Necrotrophic Mycoparasites1. Pythium nunnPythium nunn is a mycoparasite isolated from soilsuppressivetoaplantparasiticPythium sp. Whenthis mycoparasite was introduced into soil conduciveto Pythium sp., the competitive saprophyticability of this isolate was suppressed. An inverse relationshipwas found between propagule densitiesof the plant pathogen and of the antagonist P. nunn(Lifshitz et al. 1984b).The modes of hyphal interaction between themycoparasite P. nunn and several soil fungi werestudied by both phase-contrast and scanning electronmicroscopy (Lifshitz et al. 1984a). In the zoneof interaction, P. nunn massively coiled around

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 131and subsequently lysed hyphae of P. ultimumand P. vexans without penetration. In contrast,P. nunn penetrated and eventually parasitizedhyphae of R. solani, P. aphanidermatum, Phytophthoraparasitica, andP. cinnamomi,formingappressorium-like structures. However, P. nunnwas not mycoparasitic against F. oxysporum f.sp. cucumerinum or Trichoderma koningnii,andwas destroyed by T. harzianum and T. viride. Theauthors concluded that P. nunn is a necrotrophicmycoparasite with a limited host range anddifferential modes of action among susceptibleorganisms (Lifshitz et al. 1984a).Lysis and penetration of the host cell wall atthe site of interaction with the mycoparasite weredemonstrated by Elad et al. (1985). CalcofluorWhite M2R binds to the edges of polysaccharideoligomers (Kritzman et al. 1978). Using thisreagent, the appearance of fluorescence indicatedlocalized lysis of the host cell wall by P. nunn.The cell walls of Oomycota are composed ofβ-glucan, cellulose, and less than 1.5% chitin.Basidiomycota and Ascomycota contain mainlyβ-glucan and chitin but no cellulose. P. nunnproduced large amounts of β-1-3-glucanase andchitinase in liquid cultures containing cell walls ofpathogenic fungi belonging to the class Basidiomycota.This mycoparasite produced cellulase but nochitinase when grown on culture containing cellwalls of two pathogens belonging to the Oomycota(Elad et al. 1985). These extracellular hydrolyticenzymes were detected in P. nunn when grownindualculturewithsixhostfungibutnotwithten nonhost fungi, indicating specificity in theantagonistic activity of P. nunn (Baker 1987).2. Talaromyces flavusTalaromyces flavus (the perfect stage of Penicilliumdangeardii; synonym:P. vermiculatum)isamycoparasiteof several soil-borne plant pathogenicfungi including R. solani (Boosalis 1956), S. sclerotiorum(McLaren et al. 1986) and Verticilliumspp. (Fahima and Henis 1990). Laboratory investigationsusing light and electron microscopy indicatethat T. flavus is a destructive hyperparasite ofS. sclerotiorum. In dual culture, hyphae of T. flavusgrew toward, and coiled around the host hyphalcells. The coiling effect intensified as the hyphaeof T. flavus branched repeatedly on the host surface.Tips of the hyphal branches often invaded thehost by direct penetration of the cell wall withoutformation of appressoria. Infection of host cells byT. flavus resulted in granulation of the cytoplasmand collapse of the cell walls (McLaren et al. 1986).Direct invasion of R. solani hyphae via the productionof penetration pegs by T. f lavus was observedby Boosalis (1956). These pegs developedfrom either a mycelium coiling around the host hyphaeor from a hypha in direct contact with thehost. Fahima and Henis (1990) applied T. flavusas an ascospore suspension to soil naturally infestedwith Verticillium dahliae, the causal agentof Verticillium wilt in eggplant. Twelve weeks aftertransplanting, 77% disease reduction was achieved,compared with the untreated control.Scanning electron micrographs showed heavyfungal colonization and typical T. flavus conidiaonthesurfaceofthemicrosclerotiaburiedinthetreated soil, but not in control soils. Transmissionelectron micrographs of microsclerotia incubatedwith T. flavus on agar revealed parasitism involvinginvasion of some host cells by means of small penetrationpegs; the host cell walls were lysed mainlyat their site of contact with the parasite hyphal tips.Further colonization of the microsclerotial cells occurredsimultaneously with the degradation of theinvaded host cell contents, rather than the cell walls(Fahima et al. 1992). It was suggested that mycoparasitismof V. dahliae microsclerotia by T. flavushyphae may be involved in the biological control ofVerticillium wilt disease. Fravel and Keinath (1991),however, claimed that T. flavus is known to producecompounds that mediate antibiosis, which is thereforesuspected of being involved in the control ofVerticillium wilt of eggplant and potato. Similarly,McLaren et al. (1986) observed that hyphal cells ofS. sclerotiorum eventually collapse as a result of infectionby T. flavus, but host cell walls remain intact.They suggested that cell wall-degrading enzymesmay not play a major role in the control of S. sclerotiorumby T. flavus, and that antibiotics producedby the parasite may be involved in the deteriorationof the host’s hyphae (McLaren et al. 1986). Ina recent work (Duo-Chuan et al. 2005), two chitinases(CHIT41 and CHIT32) were isolated fromT. flavus and were shown to be able to decomposechitin in the cell walls of V. dahliae, S. sclerotiorumand R. solani, thus indicating that these enzymesmayplayanimportantroleinthemycoparasiticbehavior of T. flavus.3. Corniothyrium minitansCorniothyrium minitans has been found to beanaturalmycoparasiteofsclerotiaoftheplant

132 A. Viterbo et al.pathogenic fungus S. sclerotiorum. In Canada,Huang (1977) found that sclerotia of S. sclerotiorumin roots and stems of sunflower, at the endof the season, became infected with the parasiteC. minitans. This infection actually provided naturalbiological control of this pathogen in the field.Applying C. minitans to the seed furrow in fieldtrials, in soil naturally or artificially infested withS. sclerotiorum, produced 42–78% disease controlof sunflower wilts over 2 successive years (Huang1980). C. minitans is a destructive parasite that killsboth hyphae and sclerotia of S. sclerotiorum. Byusing scanning electron microscopy, it was shownthat hyphae of C. minitans grow intracellularly inthe infected sclerotia (Phillips and Price 1983; Tu1984). Phillips and Price (1983), based on transmissionelectron microscopic studies, concludedthat penetration of the rind cells of S. sclerotiorumsclerotia by C. minitans is due to physical pressure,rather than enzymatic lysis of the cell wall. Ina later study, Huang and Kokko (1987) found, bytransmission electron microscopy, that there wasdestruction and disintegration of the sclerotialtissues, caused by penetration of the parasitichyphae. Evidence from cell-wall etching at thepenetration site suggests that chemical activityis indeed required for hyphae of C. minitans topenetrate the thick, melanized rind walls. Themedullary tissue infected by C. minitans showedsigns of plasmolysis, aggregation and vacuolizationof the cytoplasm, and dissolution of the cellwalls. The authors concluded that cell wall-lysingenzymes, responsible for the degradation ofS. sclerotiorum hyphae, may also play a significantrole in the dissolution and degradation of thesclerotial rind wall at the penetration site andother affected areas (Huang and Kokko 1987). Glucanase,chitinase, cellulase, and xylanase enzymeactivities were recently reported in sclerotiacontainingcultures of C. minitans (Kaur et al.2005).Infection of S. sclerotiorum hyphae by the hyperparasiteC. minitans has been reported by severalworkers (Huang and Hoes 1976; Tu 1984). However,researchers are not in complete agreementon the mode of hyperparasitism. Using light microscopy,Huang and Hoes (1976) observed thathyphal tips of C. minitans invade hyphae of S. sclerotiorumby direct penetration, without formingany special structure. Host cytoplasm disintegratesand cell walls collapse as a result of infection. Microconidiaand intrahyphal hyphae were producedby S. sclerotiorum in infected colonies.Production of appressoria by C. minitans whenit comes into contact with the undamaged hyphaeof S. sclerotiorum in dual culture on potato dextroseagar (PDA) was observed by Tu (1984). He statedthat hyphal penetration by the hyperparasite sometimesoccurswithouttheformationofappressoria,but only on damaged host cells.Huang and Kokko (1988), using scanningelectron microscopy, confirmed previous reportsfrom light microscopic studies that hyphal tipsof C. minitans invadethehosthyphaebydirectpenetration, without developing appressoria, andthat indentation of the host cell wall at the pointof penetration is often evident. No functionaldistinction between main branch and side branchhyphae of the hyperparasite was found, and tips ofeither type of hypha are capable of invading hosthyphae by direct penetration.4. Gliocladium and Trichoderma spp.The morphological borders between Trichodermaand Gliocladium are blurred. Therefore, in recentyears molecular methods have been applied as anaidtoresolvingthetaxonomyandsystematicofTrichoderma and Gliocladium. Gliocladium virensis now generally recognized as belonging to thegenus Trichoderma (Gams and Bisset 1998).Particular attention has been paid to speciesidentification of the genus Hypocrea/Trichodermathat has proved problematic when traditionalmethodsareused.Anupdateonthetaxonomyand phylogeny of the 88 taxa (which occur as 14holomorphs, 49 teleomorphs and 25 anamorphsin nature) of Hypocrea/Trichoderma,confirmedbya combination of morphological, physiological andgenetic approaches, is presented in Druzhininaand Kubicek (2005).Several species of Gliocladium have beenreported to be hyperparasites of many fungi. Thebiology, ecology, and potential of this genus forbiological control of plant pathogens have beenextensively reviewed in a comprehensive treatiseby Papavizas (1985). Huang (1978) reported thatG. catenulatum parasitizes S. sclerotiorum andFusarium spp. It kills the host by direct hyphalcontact, causing the affected cells to collapse ordisintegrate. Pseudoappressoria are formed by thehyperparasite, but hyphae derived from these donot penetrate the host cell walls. Vegetative hyphaeof all species tested, and macroconidia of Fusariumspp. are susceptible to this hyperparasite, butchlamydospores of Fusarium equiseti are resistant.

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 133Phillips (1986) studied aspects of the biologyof G. virens and its parasitism of sclerotia ofS. sclerotiorum in soil. G. virens parasitizedand decayed sclerotia of S. sclerotiorum, S. minor,Botrytis cinerea, Sclerotium rolfsii, andMacrophomina phaseolina on laboratory media,and caused a reduction in the survival of sclerotiaof S. sclerotiorum in soil. However, parasitism ofthe mycelium was not detected.AstrainofG. virens isolated from the parasitizedhyphae of R. solani by Howell (1982) significantlysuppressed damping-off in cotton seedlingsby this pathogen and by Pythium ultimum. Treatmentwith G. virens more than doubled the numberof surviving cotton seedlings grown in soil infestedwith either pathogen. G. virens parasitizedR. solani by coiling around, and penetrating the hyphae.P. ultimum was not parasitized by G. virens,but was strongly inhibited by antibiosis. Treatmentof soil infested with propagules of R. solani or P. ultimumwith G. virens resulted in a 63% reductioninthenumberofviableR. solani sclerotia after3 weeks of incubation, whereas oospores of P. ultimumwere unaffected. Strains of G. virens wereseparatedintotwodistinctgroups,PandQ,onthebasis of secondary metabolite production in vitro(Howell et al. 1993).Gliovirin was very inhibitory to P. ultimum,but exhibited no activity against R. solani, andstrains that produced it (P group) were more effectiveseed-treatment biocontrol agents of diseaseincited by P. ultimum.Conversely,gliotoxinwasmore active against R. solani than against P. ultimum,andstrainsthatproducedit(Qgroup)weremore effective seed treatments for controlling diseaseincited by R. solani. Based on these results,the authors suggested that it may be necessary totreat seeds with a combination of strains in orderto broaden the disease control spectrum.Howell (1987) isolated mutants of G. virens,obtained by irradiation with ultraviolet light, thatshowed no mycoparasitic activity. The selected mutantsretained the same antibiotic complement asthe parent strains. Peat moss-Czapek’s broth culturesof parent and mutant strains were similarlyeffective as biocontrol agents of cotton seedling diseaseinduced by R. solani, and as antagonists ofR.solanisclerotia in soil. In the light of these results,Howell (1987) concluded that mycoparasitism isnot a major mechanism in the biological control ofR. solani-incited seedling disease by G. virens.In addition, Pachenari and Dix (1980) concludedthat G. virens need not make intimatecontact with Botrytis allili to cause severe internaldisorganization of host cells, coagulation ofcytoplasm, vacuolation, and loss of contents fromorganelles. Cultures of B. allili parasitized by G. roseumcontained considerable β-(1-3)-glucanaseand chitinase, and the cytoplasm coagulatedwithout physical contact. G. virens isolate G1-21was grown on various solid and liquid media:wheat bran and peanut hull meal (PHM), as wellas spent glucose tartrate broth (GTB), Czapek-Doxbroth (CDB), and potato dextrose broth (PDB)(Lewis et al. 1991). Aqueous extracts of these mediacaused leakage of carbohydrates and electrolytesfrom hyphae of the soil-borne plant pathogenR. solani, and its mycelial weight was reduced. Sizefractionation experiments indicated that it wasa combination of factors associated with G. virens,rather than a single one, which induced thisphenomenon. Gliotoxin was detected in culturefiltrates from G. virens grownonbranandPHMmedia. Gliotoxin preparations induced leakageof carbohydrates and electrolytes from R. solani,and caused a concomitant reduction in mycelialweight, which suggests the action of a leakagefactor (Lewis et al. 1991). The authors speculatedthat hydrolytic enzymes such as β-1-3-glucanase,β-1-4-glucanase, chitinase, and protease, shownto be produced by isolates of G. virens (Robertsand Lumsden 1990), have the potential to act onR. solani cell walls and membranes. The role ofextracellular chitinase in the biocontrol activityof Trichoderma virens was later examined usinggenetically manipulated strains of this fungus. TheT. virens strains in which the chitinase gene (cht42)was disrupted (KO) or constitutively overexpressed(COE)wereconstructedthroughgenetictransformation.Biocontrol activity of the KO and COEstrains were significantly decreased and enhanced,respectively against cotton seedling disease incitedby Rhizoctonia solani when compared with thewild-type strain (Baek et al. 1999).More than 60 years ago, Weindling (1932) wasthe first to demonstrate the mycoparasitic natureof fungi from the genus Trichoderma. He suggestedtheir potential use as biocontrol agents of plantpathogenic fungi. However, the first report on a biologicalcontrol experiment using Trichoderma spp.under natural field conditions came 40 years later,by Wells et al. (1972) who used T. harzianum grownon an autoclaved mixture of ryegrass seeds and soilto control Sclerotium rolfsii Sace. Since then, moreTrichoderma isolates have been obtained from naturalhabitats, and used in biocontrol trials against

134 A. Viterbo et al.several soil-borne plant pathogenic fungi underboth greenhouse and field conditions (Chet 1990;Harman and Lumsden 1990; Harman 2006).A seed treatment was developed by Harmanet al. (1980) to reduce the amount of Trichodermaadded to the soil to control soil-borne plantpathogenic fungi. T. hamatum conidia appliedin the laboratory, to seeds of pea and radish asa Methocel slurry, provided protection to seedsand seedlings from Pythium spp. and R. solani,respectively, almost as effectively as fungicideseed treatment. Establishment of the mycoparasiteand long-term action were demonstrated, as thepropagules of T. hamatum increased approximately100-fold in soils planted with treated seeds.Population densities of R. solani and Pythium spp.were lower in soils containing T. hamatum than insoils lacking this antagonist. Replanting these soilsonce, or even twice with untreated seeds yieldedlower disease incidence than in soils originallyplanted with untreated seeds. Addition of chitinor R. solani cell walls to the coating of seedspreviously treated with a conidial suspensionincreased both the ability of T. hamatum to protectthe seeds against Pythium spp. or R. solani, andthe population density of Trichoderma in the soil.T. hamatum with chitin, but without R. solanicell walls, effectively reduced damping-off causedby Pythium spp., compared to seed treatmentcontaining only T. hamatum (Harman et al.1980). Sivan et al. (1984) applied a peat-branmixture (1:1 v/v) preparation of T. harzianum(isolate 315) to either soil or rooting mixture,and efficiently controlled damping-off inducedby Pythium aphanidermatum in pea, cucumber,tomato, pepper, and gypsophila. Several isolatesof T. harzianum and T. hamatum were found toantagonize and control Macrophomina phaseolinain beans and melon (Elad et al. 1986). Isolates ofT. harzianum and T. hamatum antagonized andcontrolled Rosellinia necatrix in almond seedlings(Freeman et al. 1986). Sztejnberg et al. (1987) combinedsublethal soil heating with an application ofT. harzianum to yield better control of R. necatrixthan that achieved by either treatment alone.Sivan and Chet (1986) isolated a new Trichodermaharzianum isolate (T-35) from the rhizosphereof cotton plants grown in fields infestedwith Fusarium. In a further study, the isolate wastested in biological control trials over two successivegrowing seasons against Fusarium crown rotof tomato in fields naturally infested with F. oxysporumf. sp. radicilycopersici(Sivan et al. 1987).T. harzianum was applied as a seed coating oras a wheat branpeat (1:1, v/v) preparation introducedintothetomatorootingmixture.Trichoderma-treatedtransplants were better protectedagainst Fusarium crown rot than untreated controlswhen planted in MB-fumigated or nonfumigatedinfested fields. The total yield of tomatoes inthe T. harzianum-treated plots increased as muchas 26.2% over the controls. Integrated control ofVerticillium dahliae in potato by T. harzianum andthe fungicide Captan was reported by Ordentlichet al. (1990).C. Induced Systemic Resistanceby Trichoderma spp.Some Trichoderma rhizosphere-competent strainscolonize entire root surfaces with morphologicalfeatures reminiscent of those seen during mycoparasitism(Yedidia et al. 1999). Penetration of theroot tissue is usually limited to the first or secondlayers of cells, and occurs only in the intercellularspaces. Trichoderma strains capable of establishingsuch interaction induce metabolic changesin plants that increase resistance to a wide rangeof plant-pathogenic microorganisms and viruses(Harman et al. 2004; Fig. 8.1). This response seemsto be broadly effective for many plants, which indicatesthat there is little or no plant specificity.At least three classes of substances that elicitplant defense responses have been identified.These elicitors include proteins, peptides, andlow-molecular-weight compounds (Harmanet al. 2004; Viterbo et al. 2004). The systemicresponse in plants occurs through the jasmonicFig. 8.1. Induced resistance toward the leaf pathogenCochliobolus heterostrophus in maize. Seedling roots wereinfected with germinated Trichoderma spores (10 5 ml −1 )48 h prior to pathogen leaf infection (800 spores). Thesymptoms were recorded 72 h after infection

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 135acid/ethylene signaling pathway in a way similarto the rhizobacteria-induced systemic resistance(van Loon et al. 1998; Shoresh et al. 2005). Severalstudies have shown that root colonization byTrichoderma strains results in massive changes inplant gene expression patterns and metabolome.Changes in plant metabolism lead to the accumulationof antimicrobial compounds. In cucumber,root colonization by T. asperellum strain T-203causes an increase in phenolic glucoside levels inleaves, which are strongly inhibitory to a rangeof bacteria and fungi (Yedidia et al. 2003). Theprotection afforded by the biocontrol agent isassociated with the accumulation of mRNA oftwo defense genes: the phenylpropanoid pathwaygene phenylalanine ammonia lyase (PAL) andthe lipoxygenase pathway gene hydroxyperoxidelyase (HPL) (Yedidia et al. 2003). Increased levelsof other defense-related plant enzymes, such asperoxidases, chitinases, and β-1,3-glucanases, havebeen recorded in Trichoderma-treated cucumberseedlings upon pathogen challenge (Shoresh et al.2005). This potentiation in the gene expressionenables Trichoderma-treated plants to be moreresistant to subsequent pathogen infection. TheMAPK signal transduction pathways, both of theplant and of Trichoderma,areimportantfortheinduction of systemic resistance (Viterbo et al.2005; Shoresh et al. 2006).D. Mycoparasitismin Suppressive Environments(Soils and Composts)Suppression of soil-borne plant pathogens occursin environments such as field soils, and soilsamended with organic matter or compost as theorganic component in media for container-grownplants. A review on this topic for field soils wasrecently published by Stone et al. (2004).Pathogen suppressiveness has been definedby Cook and Baker (1983) as “soils in which thepathogen does not establish or persist, establishesbut causes little or no damage, or establishes andcauses disease for a while but thereafter the diseaseis less important, although the pathogen maypersist in the soil.”Baker and Cook (1974) divided suppressionmechanisms into two broad categories defined asgeneral and specific. General suppression is a resultof total microbial activity. In contrast, specificsuppression applies when bacteria or fungi, individuallyor as a group, are responsible for the suppressioneffect. Mycoparasitism, the focus of thischapter, is a major mechanism of specific suppression.For example, Chet and Baker (1981) reportedon a soil which was suppressive to R. solani of carnationnear Bogota, Colombia. This soil containedhigh levels of organic matter (35%), was highlyacidic (pH 5.1), and its main microbiological componentwas the antagonistic fungus T. hamatum ata population density of 8 ×10 5 propagules g −1 .Thelevel of this mycoparasite in mineral-conducive soilwasfourordersofmagnitudelower.In another, related study, Henis et al. (1978)showed the effect of successive plantings onthe development of suppression. A soil sownwith radish every week became suppressive toR. solani by the fourth sowing, and was even moresuppressive by the fifth and subsequent sowings.The population of T. harzianum,antagonistictoR. solani, increased with successive sowings ofradish (Liu and Baker 1980), possibly in responseto increases in the amount of R. solani in thesoil resulting from its parasitism of the radishseedlings. The addition of T. harzianum spores toa conducive soil at the same density as that foundin the suppressive soil caused the conducive soil tobecome suppressive. Trichoderma spp. were alsoreported to be responsible for the suppression ofthe take-all disease caused by Gaeumannomycesgraminis. Low pH conditions were found tobe favorable to Trichoderma, and to enhancesuppression (Simon and Sivasithamparam 1990).A practical approach to utilizing suppressionin agriculture is the use of suppressive composts,mainly in container media. Composting is thebreakdown of organic waste material by a successionof mixed populations of microorganismsin a thermophilic aerobic environment. The finalproduct is compost or humus, which is the stabilizedorganic matter populated by microorganismscapable of suppressing soil-borne plant pathogens.Disease-suppressive effects of composts havebeen investigated intensively over the past twodecades, and were recently reviewed by Nobleand Coventry (2005) and Zinati (2005). Compostof a wide variety of waste materials (hardwoodorpinebark,municipalsludge,grapemarc,orcattle manure) is an economically and ecologicallysound alternative to pesticides.The mechanisms of suppression in compostsdo not differ substantially from those describedfor soils, and can be either general or specific.Physiological profiling, and the use of DNA-based

136 A. Viterbo et al.techniques such as denaturing gradient gel electrophoresis(DGGE) may lead to an improvedunderstanding of the changes in microbial communitiesassociated with disease control resultingfrom compost amendment of soil, sand, or peat.Nelson et al. (1983) identified specific strains offour Trichoderma spp. and isolates of Gliocladiumvirens as the most effective fungal hyperparasitesof R. solani present in bark compost. A few of the230 other fungal species also showed activity, butmost were ineffective. Kwok et al. (1987) describedsynergistic interactions between T. hamatum andFlavobacterium balustinum. Several other bacterialstrains, including Enterobacter, Pseudomonas,and Xanthomonas spp.,alsointeractedwiththeTrichoderma isolate in suppression of Rhizoctoniadamping-off (Kwok et al. 1987). Composted grapemarc was effective in suppressing disease causedby S. rolfsii in beans and chickpeas (Gorodecki andHadar 1990). Hadar and Gorodecki (1991) placedsclerotia of S. rolfsii on composted grape mare toisolate hyperparasites of this pathogen. Viabilityof sclerotia decreased from 100% to less than 10%within 40 h. It remained close to 100% for sclerotiaplaced on a conducive peat mix. Penicillium spp.and Fusarium spp. were observed by scanningelectron microscopy to colonize the sclerotia. Trichodermapopulations in the grape mare compostwere at very low levels (10 2 cfu g– 1 dry weight).The hyperparasites present in this compost aretherefore quite different from those isolatedfrom tree bark compost, where Trichoderma andGliocladium isolates predominate.In conclusion, suppression of soil-borne plantpathogens in field soil or container media isbrought about by antagonistic microorganisms.Such systems could be a source for mycoparasitiesto be used in biocontrol, or to be incorporatedinto integrated disease control programs. Theinoculation of composts with biological controlagents may improve the efficacy and reliability ofdisease control obtained.III. Hyphal Interactionsin MycoparasitismA. BiotrophsPiptocephalis virginiana is a haustorial biotrophicmycoparasite that parasitizes fungi belongingto the order Mucorales exclusively (Manocha1981). Attachment of a biotrophic mycoparasiteto its host surface is considered to be an essentialprerequisite step for further penetration of thehost by the parasite (Manocha and Chen 1990).P. virginiana attaches to the surface of boththe compatible Choanephora cucurbitarum andMortierella pusilla, andtheincompatiblePhascolomycesarticulosus hosts, but not to the surfaceof the nonhost Mortierella candelabrum (Manocha1985; Manocha et al. 1986). Comparative researchwas performed by Manocha and his coworkers inan attempt to unravel the molecular basis for specificityand recognition in this system. Cytologicaland biochemical investigations were carried outto study the structure and chemical compositionof cell walls of host and nonhost species (Manocha1981, 1987). The germ tubes of the biotrophicmycoparasite P. virginiana were found to attachto the cell-wall surface of the host, but not to thatof the nonhost (Manocha 1985; Manocha et al.1986). This attachment could be specifically inhibitedby chitobiose and chitotriose. The authorstherefore suggested a possible involvement ofcarbohydrate-binding proteins in the specificityof this interaction. A comparison of protein andglycoprotein profiles of cell-wall extracts revealedmarked differences between host and nonhostspecies. Two high-molecular-weight glycoproteinswere observed only in the extract of host cell walls,being absent in that of the nonhost (Manocha 1985;Manocha et al. 1986). Further isolation and characterizationof the host cell surface proteins revealedthat attachment and appressorium formation bythe parasite germ tubes could be inhibited by treatinghost cell-wall fragments with 0.1 M NaOH orpronaseE.Furthermore,thetwopurifiedglycoproteinswere able to agglutinate both nongerminatedand germinated spores of the mycoparasite.Arabinose, glucose, and N-acetylglucosaminecould totally inhibit this agglutination. Theseglycoproteins were suggested to be two subunitsof a carbohydrate-binding agglutinin presenton the host cell surface, and to be involved inagglutination and attachment of the mycoparasitegerm tubes (Manocha and Chen 1991).Using fluorescein isothiocyanate-labeledlectin-binding techniques, Manocha et al. (1990)were able to show differences in the distributionpattern of glycosyl residues at the level of the cellwall between fungi that are hosts and those thatare nonhosts of the mycoparasite P. virginiana,and at the protoplast level between compatible andincompatible hosts.

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 137Thecellwallsofthecompatiblehosts(C. cucurbitarumand M. pusilla)andtheincompatiblehost(P. articulosus), as well as that of the mycoparasiteitself, contain glucose and N-acetylglucosamine.In the nonhost (M. candelabrum), however, othersugars such as fucose, N-acetylgalactosamine,and galactose could also be detected. These lattersugars could be detected on both the host andthe parasite surface after mild treatment withproteinase or when grown in liquid medium.The researchers speculated that the failure ofthe mycoparasite to attach to the host cellsafter proteinase treatment or in liquid culturemay be due to the appearance of galactose andgalactosamine at the host cell surface. The ideathat N-acetylglucosamine and glucose may beinvolved in the attachment of P. virginiana to itshost cell surface was supported by the observationthat pretreatment of the mycoparasite germ tubeswith N-acetylguclosamine or glucose inhibitedtheir attachment to the host cells. In addition, thegerm tubes attached to agarose beads coated withglucose or with N-acetylglucosamine, but not withN-acetylgalactosamine (Manocha et al. 1990).The protoplast surfaces of compatible hostscontained all of the above-listed sugars, andthese protoplasts could attach to the germ tubeof the mycoparasite. Only lectins specific forN-acetylglucosamine and glucose were boundat the protoplast surface of the incompatiblehost; these protoplasts did not attach to themycoparasite germ tubes. Indications were foundfor different factors being responsible for attachmentand for appressorium formation, aspretreatment of the mycoparasite with glucoseand N-acetylglucosamine inhibited its attachmentto the host cell surface, but had no obvious effecton appressorium formation. On the other hand,appressorium formation was inhibited by heattreatment of host cell-wall fragments that stillpermitted attachment (Manocha et al. 1990).The authors therefore suggested a model forthe recognition between P. virginiana and itshost fungi that operates at two levels at least:the cell wall, and the protoplast surface. At thecell-wall level, the attachment probably involvescarbohydrate-binding agglutinins that recognizespecific sugar residues on the host but not on thenonhost cell wall. After the initial recognition andattachment, at the protoplast level, the parasitedistinguishes compatible from incompatible hosts.The mechanism of this distinction is not clear.Yet, it seems that protoplast membrane sugarsare not a major factor in recognition at thislevel (Manocha et al. 1990). Immunofluorescencemicroscopy was used to detect, in the mycoparasiteP. virginiana, the presence of a complementaryglycoprotein that binds specifically to the hostcell surface glycoproteins. This technique revealedsurfacelocalizationoftheproteinonthegermtubes of P. virginiana. Fluorescence was alsoobserved at the surface of the germinated sporesand hyphae of the host M. pusilla, after treatmentwith complementary protein from P. virginiana,and with primary antibody prepared against thecomplementary protein (Manocha et al. 1997).B. NecrotrophsAs early as 1932, Weindling reported the coiling ofTrichoderma spp. hyphae around hyphae of otherfungi. These strains were later shown to actuallybe a species of Gliocladium (Webster and Lomas1964). Dennis and Webster (1971a, b, c) publishedan extensive report on the antagonistic propertiesofspeciesgroupsofTrichoderma.Thehyphalinteractionbetween Trichoderma and plant pathogenicfungi was first comprehensively studied in theirwork. Since then, numerous studies on the hyphalinteraction and coiling phenomenon of Trichodermaaround its host hyphae have been carriedout with the use of light and electron microscopy(Chet et al. 1981; Elad et al. 1983a; Baker 1987; Inbarand Chet 1992; Omero et al. 1999; Rocha-Ramirezet al. 2002; Fig. 8.2).The destructive mode of parasitism in Trichodermaappears to be a process consisting of severalconsecutive events initiated by attraction anddirected growth of Trichoderma toward its host,probably by chemotropism. Positive chemotropismwas found in Trichoderma (Chet et al. 1981), asit could detect its host from a distance and begintobranchinanatypicalway.Thesebranchesgrew toward the pathogenic host fungi. Similar behaviorwas also found in Pythium nunn (Lifshitzet al. 1984a), P. oligandrum (Lewis et al. 1989),and in Gliocladium spp. (Huang 1978). This eventis presumably a response of the antagonist to thechemical gradient of an attractant coming from thehost. However, no specific stimuli other than aminoacids and simple sugars have thus far been detected(R. Barak and I. Chet, unpublished data). Hence,the specificity of the phenomenon is not clear. Apparently,it is not an essential step for mycoparasitism,although it may hold some advantage for

138 A. Viterbo et al.Fig. 8.2. A mycoparasitic relationship. Scanning electronmicrograph of T. harzianum hyphae coiling around thoseof the plant pathogenic fungus S. sclerotiorum. Bar 10 μmthe antagonist. Subsequently, contact is made, andin some cases, Trichoderma coils around or growsalong the host hyphae and forms hook-like structures,presumably appressoria, which probably aidin penetrating the host hyphal cell wall (Chet et al.1981; Elad et al. 1983b). The coiling phenomenonand appressoria formation have been reported forother mycoparasites as well (Tu 1984; Lifshitz et al.1984a). However, Deacon (1976) concluded that inthe case of P. oligandrum,coilingoftheantagonistaround its host hyphae indicates temporary hostresistance, rather than susceptibility. Nevertheless,in Trichoderma, this reaction was found to be ratherspecific, and Trichoderma attacks only a few fungi.Moreover, Dennis and Webster (1971c), using plasticthreads of a diameter similar to that of P. ultimumhyphae, concluded that the coiling of the Trichodermais not merely a thigmotropic response.The Trichoderma hyphaenevercoiledaroundthethreads, but rather grew over or followed them ina straight course. This led to the idea that there isa molecular basis for the specificity. However, despitethe fact that first observations and reports ofthis phenomenon were published decades ago, weareonlynowonthevergeofbeingabletounderstandit. Reviews dealing with cellular interactionsin fungi (Tunlid et al. 1992; Manocha and Sahai1993), and the specificity of attachment of fungalparasites to their hosts (Manocha and Chen 1990)have been published. The physiology and biochemistryof biotrophic mycoparasitism in particularhave been extensively reviewed by Manocha (1990).Attachment and “recognition” between the mycoparasiteand its host appears to be essential, anda crucial stage for successful continuation of theprocess. Lectins are sugar-binding proteins or glycoproteinsof nonimmune origin that agglutinatecells and/or precipitate glycoconjugates (Goldsteinet al. 1980). First discovered in plants and later inother organisms, they are involved in interactionsbetween the cell surface and its extracellular environment(Barondes 1981). Indeed, lectins werefoundtobeproducedbysomesoil-borneplantpathogenic fungi such as R. solani and S. rolfsii(Barak et al. 1985, 1986), and by different membersof the Sclerotiniaceae (Kellens et al. 1992).Therefore, a role for lectins in the recognitionand specificity of attachment between Trichodermaand its host fungi was suggested. However, no conclusiveevidence to support this hypothesis wasavailable at the time. In an attempt to test this hypothesis,Inbar and Chet (1992) used a novel approachbased on the binding of lectins to a surfaceof nylon fibers. This biomimetic system imitatesthe host hyphae, and enables an examination oftheroleoflectinsinmycoparasitism.Inertnylonfibers were chemically activated to enable the covalentbinding of the lectins (Inbar and Chet 1992).Concanavalin A, a plant lectin that is similarto the lectin of S. rolfsii (LSR) in its carbohydratespecificity (cf. they are both specific to D-glucoseandD-mannose),wasusedfirsttoestablishthesystem.The Trichoderma recognized the LSR-treatedfibers as a host, and attached and coiled aroundtheminapatternsimilartothatseenwithrealhosthyphae (Inbar and Chet 1992; Fig. 8.3). In contrast,in the untreated control, no interaction could beobserved – the Trichoderma grew uninterruptedlyover and along the fibers, exactly as outlined byDennis and Webster (1971c). These findings providedthe first direct evidence for the role of lectinsin mycoparasitism. The researchers were able toshow that inert nylon fibers coated with fungallectins mimic the real host hyphae, and can stimulatethe parasite to coil around them.A novel lectin was isolated and purifiedfrom the culture filtrate of the soil-borne plantpathogenic fungus S. rolfsii (Inbar and Chet 1994).Agglutination of E. coli cells by the purified lectin

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 139Fig. 8.3. Biomimetic systems for simulating the interactionbetween Trichoderma and plant pathogenic fungi. a Scanningelectron micrograph of T. harzianum hyphae coilingaround inert nylon fibers coated with a surface lectin fromthe plant pathogenic fungus S. rolfsii.Bar10μm. b Appressoriumformation by T. harzianum grownonnylonfiberscoated with the S. rolfsii lectin. Bar 10 μmcouldbeinhibitedbytheglycoproteinsmucinandasialomucin. Proteases, as well as β-1,3-glucanase,werefoundtobetotallydestructivetotheagglutinationactivity, indicating that both protein andβ-1,3-glucan are necessary for agglutination. Usingthe biomimetic system, it was apparent that thepresence of the purified agglutinin of the surfaceof the fibers significantly induces mycoparasiticbehavior in T. harzianum, compared with theuntreated ones or with those treated with nonagglutinatingextracellular proteins from S. rolfsii(Inbar and Chet 1994).It was later demonstrated that induction ofchitinolytic enzymes in Trichoderma is elicited bythe recognition signal (i.e., lectin–carbohydrate interactions).It was postulated that recognition is thefirst step in a cascade of antagonistic events triggeringthe parasitic response in Trichoderma (Inbarand Chet 1995).The same biomimetic system was used to testthe involvement of signal transduction pathwaysin the induction of coils in T. harzianum (Omeroet al. 1999). Two activators of G protein-mediatedsignal transduction induced coiling of hyphaearound nylon fibers. The peptide toxin mastoparanincreased coiling more than twofold in comparisonwithcontrols.Theactivatorfluoroaluminate(A1F4) had a similar effect, whereas aluminumions alone were ineffective; cAMP increased coilingabout threefold. Although the two G-proteinactivators, mastoparan and fluoroaluminate, havevery different modes of action, they share theGa subunit as a target. Based on these results,it was proposed that a signal for mycoparasiticbehavior from the host cell surface is transducedby heterotrimeric G protein(s) and mediated bycAMP.Rocha-Ramirez et al. (2002) isolated a T. atrovirideG-protein alpha-subunit (Ga) gene (tga1).Transgenic lines overexpressing tga1 showedadelayedsporulation and coiled at a higher frequency,compared to the wild type. Likewise, transgeniclines that expressed an activated mutant proteinwith no GTPase activity did not sporulate andcoiled at a higher frequency. Lines that expressedan antisense version of the gene were hypersporulatingand coiled at a much lower frequency in thebiomimetic assay. The loss of tga1 in these mutantscorrelated with the loss of GTPase activitystimulated by the peptide toxin Mas-7. The applicationof Mas-7 to growing mycelia raises intracellularcAMP levels, suggesting that tga1 canactivate adenylyl cyclase. In contrast, cAMP levelsand cAMP-dependent protein kinase activity dropwhen diffusible host signals are encountered andthe mycoparasitism-related genes ech42 and prb1are highly expressed. These results demonstratedthat the product of the tga1 gene is involved in bothcoiling and conidiation.Penetration and degradation of the host cellwall under the coiling and interaction sites are evidentby visual observation, fluorescent indicators,and enzymatic studies. Using scanning electronmicroscopy, lysed sites and penetration holeswere found in hyphae of R. solani and S. rolfsiifollowing removal of Trichoderma spp. hyphae(Elad et al. 1983b). The cell walls of Basidiomycotaand Ascomycota contain chitin and laminarin(P glucan) but no cellulose. Oomycota containβ-glucans and cellulose and relatively small

140 A. Viterbo et al.amounts of chitin (

Plant Disease Biocontrol and Induced Resistance via Fungal Mycoparasites 141the control of a constitutive viral promotor, andp35SR2,amarkerforselectionaftertransformation,encoding for acetamidase. Two transformantsshowed increased constitutive chitinase activity(specific activity 11 and 5 times higher than therecipient; Fig. 8.4), and excreted a protein ofca. 58 kDa, the expected size of S. marcescenschitinase, when grown on synthetic medium.Antagonistic activity of the transformants wassignificantly higher than that of the wild-typeT. harzianum, as evaluated by testing their abilityto overgrow the plant pathogen S. rolfsii in dualculture (Haran et al. 1993). The major advantage ofsuch genetic manipulations is the ability to isolategenes from one strain and introduce them intoother varieties of fungi, bacteria, or plants. Thisenhances the potency of biocontrol agents andmakes a single strain consistently effective againstmore than one plant pathogenic fungus, withoutthe hazardous effects of chemical pesticides.This approach was taken by Broglie et al. (1991)who, in a pioneering work, produced seedlings constitutivelyexpressing a bean chitinase gene underthe control of the cauliflower mosaic virus 35S promoter.The timing of the natural host defense mechanismwas modified to produce fungus-resistantplants with increased ability to survive in soil infestedwith the fungal pathogen R. solani,delayingthe development of disease symptoms.Since then, genetic manipulations of valuablecropplantswithoneormorecellwall-degradingenzymes from mycoparasitic fungi have been considereda potent tool for improving plant resistanceFig. 8.4. Chitinase-specific activity of crude enzyme (unitsper mg protein) excreted by the wild-type T. harzianum (wt)and transformants (401 and 402), after 5 days on syntheticmedium. Columns headed by different letters are significantlydifferent (p = 0. 05) according to Duncan’s multiplerange test (Haran et al. 1993)to fungal pathogens. Transgenic apple plants expressingT. atroviride endochitinase and exochitinase,singly or in combination, were produced andscreened for resistance to Venturia inaequalis,thecausal agent of apple scab (Bolar et al. 2001). Plantsexpressing both enzymes at the same time weremore resistant, demonstrating for the first time inplanta synergism between the two enzymes. Constitutiveexpression of Trichoderma endochinasecan be exploited to enhance resistance to fungalpathogens in important forest tree species. Theech42 from T. harzianum was introduced into foresttrees, black spruce (Picea mariana) andhybridpoplar (Populus nigra x Populus maximowiczii),by Agrobacterium-mediated transformation.In vitro assays demonstrated that the transgenicpoplarshadincreasedresistancetotheleafrustpathogen Melampsora medusae. Seedlings of transgenicspruce lines showed an increased resistanceto the spruce root pathogen Cylindrocladium floridanum(Noël et al. 2006).V. ConclusionsMycoparasitism is a quite common, and yet excitingphenomenon. It appears to play an importantrole in biological control, even though it should bepointed out that mycoparasitism is only one specificaspect in the whole complex system of biologicalcontrol of plant diseases. For example, thedirect effects of root-colonizing Trichoderma spp.on plants are at least as important as the directeffects on pathogens – or perhaps more so. Thesefungi have profound impacts on plant growth anddevelopment, and they also induce resistance toa variety of classes of plant pathogens.Mycoparasitism is a complex process thatincludes the following steps: (1) chemotrophicgrowth of the antagonist toward the host;(2)recognitionofthehostbythemycoparasite;(3) attachment; (4) excretion of extracellularenzymes; and (5) lysis and exploitation of the host.Mycoparasitism occurs under appropriate ecologicalconditions. The population and activity ofthe mycoparasite can be increased by relatively specificsubstances,suchaschitin.Thegenecodingforchitinase is only one example of genes with mycoparasiticactivity. Other potential genes are thosecoding for β-1,3-glucanase, protease, and lipase.Engineering various chitinases together withother genes that may act as antifungal agents

142 A. Viterbo et al.mayleadtobetterprotectionofplantsagainstpathogenic fungi. It may therefore be possibleto improve mycoparasitism, and to enhance theplants resistance response by integrating clonedchitinase with different lytic enzymes and otheravailable antifungal polypeptides. By understandingthe mode of action of biocontrol mycoparasiticfungi, we should be able to manipulate the fungalagent, the plant, and their interactions to achievemore effective and safer plant resistance to variousbiotic and abiotic stresses.Acknowledgements. 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9 AntagonismofPlantParasiticNematodesbyFungiS. Casas-Flores 1 ,A.Herrera-Estrella 2CONTENTSI. Introduction ........................ 147II. Biological Control of Nematodes ........ 148III. Virulence Factors .................... 149A. SerineProteases................... 150B. Chitinases ....................... 153C. OtherEnzymes ................... 154D. Toxins or Inhibitory Metabolites . . . . . . 154IV. Improvement of Nematode-Trapping Fungiby Genetic Engineering ................ 155V. Concluding Remarks .................. 155References.......................... 155I. IntroductionThecontinuinggrowthofthehumanpopulationand the consequent foodstuffs demand require theimplementation of novel alternatives to increasefood production. A reduction in crop damagecaused by pests and diseases will be the bestchoice to solve food problems worldwide. Viruses,bacteria, fungi and nematodes are responsible formajor losses in economically important crops.Plant parasitic nematodes represent a seriousproblem in agriculture around the world. Theglobal loss per year in agricultural productiondue to damage generated by nematodes has beenestimated as 100 billion US dollars worldwide(Nordmeyer 1992).Nematodes have successfully establishedin nearly all-ecological niches. Plant parasiticnematodes obtain their food from the plant foliageand roots. The nematodes that obtain their foodfrom plant roots are migratory ectoparasites,migratory endoparasites, semi-endoparasites andsedentary endoparasites. Many nematodes thatdirectly or indirectly affect plant growth have1 División de Biología Molecular, Instituto Potosino de InvestigaciónCientífica y Tecnológica, Camino a la Presa de San José2055, Lomas 4a sección, CP 78216, San Luis Potosí SLP, México2 Laboratorio Nacional de Genómica para la Biodiversidad, CinvestavCampus Guanajuato, Km 9.6 Libramiento Norte CarreteraIrapuato-León, A.P. 629, Irapuato 36500, Guanajuato, Méxicodeveloped parasitic strategies that make themmore efficient in exploiting their source of food(Sijmons et al. 1994). The damage provoked bynematodes cannot be recognized at first sight,because their ecological niche is mostly undersoil. Plants infected by nematodes resemble thosesuffering from water or nutrients stress, loosingvigor and showing chlorosis.Plant parasitic nematodes affect the plant directlyby altering the morphology of the root systemas a result of their feeding activities or by invasionof the plant tissue. This damage can be generatedby the migratory stages of endo- and ectoparasiticnematodes. The most specialized level has beenreached by the sedentary endoparasitic nematodesthat invade the root, and partially reorganize theroot function to satisfy their own demand of nutrients(Jung and Wyss 1999). In order to carry outthese modifications, nematodes transform some oftheir cells into highly specialized feeding structuressuch as the stylet and feeding tubes, which providea permanent source of nutrients enabling them tosettle essentially indefinitely in the place of infection(Jung and Wyss 1999). The nematode penetratesthe root tissues only by means of its stylet andinjects secretory fluids, produced in esophagealglands; these fluids modify the plant cytoplasmprior to food removal. Some nematode species feedon the root tips of their host plants, which becometransformed into terminal galls. Galls containnecroticcellsandenlargedmultinucleatedcellsthatare metabolically highly active andessential fornematode development, growth and reproduction.The most dramatic alterations in root architectureare generated by cyst and root-knot nematodes.The most important nematodes in agriculture arethe root-knot nematode (Meloidogyne), and thecyst nematodes (Heterodera and Globodera), dueto the extensive damage they cause to crops. Rootknotnematodes are generally polyphagous andeach species can infect a large variety of plantsspecies, from grasses to trees, by generating gallsEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

148 S. Casas-Flores, A. Herrera-Estrellaintheroot.Duetotheirbroadhostrange,thesenematodes cannot be controlled by crop rotation.In contrast, cyst nematodes are highly host-specificparasites, and can be effectively controlled by croprotation using non-host plants.Since the origins of agriculture, man has useddiversestrategiestoeliminatepeststhatattackcropplants. Centuries ago, the Chinese used natural pestenemies to eliminate these, introducing ants in cultivatedlands in order to eradicate hornets, wormsand insects (Doutt 1964). Similarly, in the 19th century,AmericanandEuropeanscientistsusednaturalpredatorsandpathogenstoprotectcropsandforests, obtaining successful results (Doutt 1964).The work of those researchers lost impact at thebeginning of the past century, with the discoveryof chemical pesticides that resulted more efficient,cheaper, and with wider action spectra (Spiegeland Chet 1998). Since the second half of the lastcentury, the use of pesticides has increased alarminglywith the aim of increasing agricultural production.Today the most common plant parasiticnematodes are controlled with chemical nematicides,cultural practices and by the use of resistantcultivars. Chemical nematicides can directly or indirectlyreduce the density of nematode populationsin soil (Johnson and Feldmesser 1987). Thesechemicals are applied as fumigants; root dips, foliarlyor as seed treatments, and can be formulatedas gases, volatile liquids, emulsifiable concentrates,etc. (Akhtar 1997). Most nematicide fumigants alsoshow phytotoxic activity (Akhtar 1997). The use ofchemical pesticides has provided good solutions,butonlyintheshortterm.In spite of the “successful” use of chemical pesticidesto efficiently control plant pests, it has beendetermined that these compounds are highly hazardousto human health and the environment. Anotherdisadvantage of chemical pesticides is theirpersistence in the environment, which favors theselection of resistant pests, leading to the use ofmore toxic pesticides. These actions have generatedconcern around the world. Consequently, there isaheightenedscientificinterestintheestablishmentof integrated pest management strategies in orderto reduce the application of chemical pesticides;these should be more effective, and less pollutant,such as traps, and other means of biological control.The term biological control, in the classicalsense, was defined by De Bach (1964) as the actionof parasites, predators or pathogens in maintainingthe population density of another organism ata lower average than would occur in their absence.There is a plethora of natural enemies of plantpests, including bacteria, protozoa, predatory nematodes,andfungi,whichcouldbeusedtoreducepopulations of phytophagous nematodes. Duringthe second half of the 20th century, few researchareas in plant pathology attracted more interestthan the use of microorganisms to control plantpathogens. The great interest generated by the useof biological control against plant pathogens is a responseto the growing concern of society regardedtheunconstraineduseofchemicalpesticides.II. Biological Control of NematodesBiological control agents frequently use severalmodes of action such as antibiosis, parasitism,and competition for nutrients and space. It isimportant to highlight that, in addition to theirantagonistic activity, a good control agent shouldhave the ability to survive in the habitat whereit is going to be applied. Nematodes have a largevariety of natural enemies, including bacteria,protozoa, other nematodes, insects, mites, andfungi (Stirling 1991; Stirling and Smith 1998).Some antagonists of plant parasitic nematodeshavebeenshowntobeusefulasbiologicalcontrolagents (Stirling 1991; Stirling and Smith 1998).Natural enemies of nematodes are often foundin nematode-suppressive soils. Additionally, theintroduction of natural enemies in soil has resultedin an efficient method for the biological controlof nematodes. Most research on the interactionof nematodes with their natural enemies hasbeen focused mainly on plant parasitic nematodespecies, due to their economic importance as croppests (Akthar 1997).The fungal antagonists of nematodes includenematode-trappingxs fungi, predacious fungi, endoparasiticfungi, egg parasitic fungi, cyst parasiticfungi and fungi that produce nematotoxic metabolites(Mankau 1980). Furthermore, these types offungi play a key role in recycling elements suchas carbon, nitrogen, and other important elementsfrom the biomass found in soil, these activities positioningthem as important microbial decimatorsin trophic chains.General parasites such as the nematodetrappingfungi can attack plant parasitic nematodesand free-living nematodes. Since thedescription of the nematode-trapping fungusArthrobotrys oligospora in 1988 (Zopf 1888),

Antagonism of Plant Parasitic Nematodes by Fungi 149Fig. 9.1. Nematode captured by theconstricting rings of the predatoryfungus Arthrobotrys anchonia. Notethat the ring cells “cushion” around thebody of the victim, but have not yetconstrictedthebody.Thisisaveryearlystage after capture (scanning electronmicrograph reprinted with permissionfrom Barron)nematode-trapping fungi have become the moststudied group with a high potential for use inbiological control (Kerry 2000). Members ofthis group produce diverse structures to capturetheir preys (Barron 1977, 1979), such as hyphaltraps, adhesive trapping nets or constricting rings(Fig. 9.1). Traps can be spontaneously produced, ortheir formation induced by nematodes or peptides(Nordbring-Hertz 1973; Barron 1977). Similarly,adhesive conidia of endoparasitic nematophagousfungi strongly attract nematodes, whereas nonadhesiveconidia do not (Jansson 1982). Adhesiveconidia of M. coniospora adhere specifically to theFig. 9.2. Scanning electron micrograph of the head region ofa nematode, fixed in glutaraldehyde, heavily infected withconidia. Arrow Adhesive bud of conidia, bar 2 μm (takenfrom Jansson et al. 1984)head of female P. redivivus, and the head and tailof the male (Fig. 9.2; Jansson et al. 1984).III. Virulence FactorsNematophagous fungi are classified, according totheir role as biological control agents, into freelivingnematode parasites, and egg parasitic fungi(Kerry and Jaffe 1997). These can also be classifiedas facultative and obligate parasites. It is consideredthat facultative parasitic fungi use the nematodesas a supplementary source of nutrients, rather thana primary source. However, the construction ofnematode-trapping nets by nematophagous fungishows that nematophagy is an important trophicstate (Jaffe 1992; Jaffe et al. 1992). The egg parasiticfungus P. chamydosporia grows much betteron nematode-infested roots than on healthy rootsor in the soil (Kerry 2000), and shows a genetic variabilityclosely related to the host from which theywere isolated (Morton et al. 2003). These observationssuggestthatnematodesmaybemoreimportantto these fungi than being simply an eventualsourceofnutrients.Inthecaseofobligateparasites,nematode infection initiates through the ingestionof spores or their attachment to the cuticle (Mortonet al. 2003). Some endoparasites produce zoosporesthat are attracted to the nematodes before adhesionand encystment on the cuticle surface.The parasitic fungi of free-living nematodesare similar to the egg parasitic fungi in some aspects.Nematode-trapping fungi grow in the soiland in the rhizosphere where they form nets ofhyphae with trapping structures to capture nema-

150 S. Casas-Flores, A. Herrera-Estrellatodes. Some species produce consistent constrictingrings, whereas others have adhesive hyphae.The outer-most layer of a nematode is constitutedby carbohydrates and proteins, and is consideredto be of great importance in recognition eventswith the host plants and their antagonists (Birdand Bird 1991). The cuticle surface influences thespecific interaction between the nematode and thefungus, including nematode-trapping fungi (Mendozade Gives et al. 1999). Nematophagous fungipenetrate the nematode cuticle via trapping organsin predacious fungi, or conidia in endoparasiticfungi. The nematodes not only serve as prey,but also induce the formation of trapping structuresof the fungus (Barron 1977; Nordbring-Hertz1977). Cuticle penetration takes place only in livingnematodes, with dead individuals being invadedthrough the mouth, anus, or other naturalopenings (Nordbring-Hertz and Stalhammar-Carlemalm 1978).The initial phase of penetration is believed to beassociated with recognition mediated by a lectincarbohydrateinteraction (Nordbring-Hertz 1983).Lectins located on fungal traps or adhesive conidiabind specifically to carbohydrates on the nematodecuticle. Further studies on the role of lectins in thisinteraction led to the proposal that the recognitionevent allows the release of enzymes, to proceed withthe attachment of the fungus onto the nematode(Tunlid et al. 1992). However, disruption of the genethat encodes the lectin in A. oligospora did notaffect the virulence of the fungus on the nematode(Balogh et al. 2003). Nevertheless, the possibilitythatanotherlectincouldcompensateforthelossof the product of the disrupted gene can not bediscarded.It has been suggested that after the recognitionevent, the fungus immobilizes the nematodeandsecretesextra-cellularenzymesatthepointof contact that allow posterior parasitism (Tunlidet al. 1994). The endoparasitic fungi D. coniosporaand Hirsutella rhossiliensis, through their adhesivespores, attack the anteroposterior part of thebody of juvenile nematodes. Similarly, when the hyphaefromnematophagous fungi reach the eggshell,they form appresoria from which extra-cellular enzymesare secreted (Lopez-Llorca and Robertson1992). The formation of appresoria depends on therecognition of the host surface. Surface hydrophobicityis considered an important recognition factor(Lopez-Llorca et al. 2002).Several studies have revealed that extra-cellularenzymes play an important role as virulence factorsin entomopathogenic fungi and nematophagousfungi (St. Leger 1995; Clarkson and Charnley 1996).In the case of nematophagous fungi, these enzymesareinducedbythepresenceofnematodeeggs.Theset of enzymes of which the production is stimulatedbyeggshasbeenfoundtobedirectlyrelatedto the structure of the eggshell, which is formed byseveral layers (Wharton 1980), including a chitinouslayer composed of a protein matrix (50–60%)embedding chitin microfibrils. The main enzymesinduced are chitinases and proteases, and are consideredvirulence factors; some appear to be importantdeterminants of host specificity (Åhmanet al. 1996).A. Serine ProteasesAlkaline serine proteases are produced by a widevariety of fungi that digest proteins under diversenutritionalconditions.Theroleofserineproteasesin invertebrate pathogenesis was initially characterizedin the entomopathogenic fungi Metarhiziumanisopliae (St. Leger et al. 1987), and Bauveriabassiana (Bidochka and Khachatourians 1987).A 30-kDa serine protease (Pr1) was found to playan important role in the infection process (Mortonet al. 2004). Proteases from entomopathogenicfungi sharing characteristics with Pr1, includingsize, reaction to inhibitors and substrate utilization,called Pr1-like, were purified and characterizedfrom the nematophagous fungi Paecilomyceslilacinus (Bonants et al. 1995), P. rubescens (Lopez-Llorca 1990), and P. chlamydospora (Segers et al.1994). Consequently, research on proteases fromentomopathogenic fungi has been closely followedby scientists studying the role of proteases in theinfection process in nematodes.Thefirstreportonproteaseproductioninnematophagousfungi came from nematode-trappingspecies (Schenck et al. 1980). The extra-cellularprotease P32 from the egg parasite Verticilliumsuchlasporium was the first protease purifiedand characterized from a nematophagous fungus(Lopez-Llorca 1990). A similar protease, alsonamed P32, was immunolocalized in appresoriaof the fungus P. rubescens, which infects eggsof the beet cyst nematode Hetrodera schachtii.These results pointed to the involvement of thisprotease in the infection process (Lopez-Llorcaand Robertson 1992). In further research, the useoftheproteaseinhibitorsPMSFandDFPreducedegg penetration by the fungi Lecanicillium lecanni

Antagonism of Plant Parasitic Nematodes by Fungi 151and P. chlamydosporia, showing the relevanceof proteases at the early stages of the infectionprocess (Lopez-Llorca et al. 2002).Those fungi that infect nematode eggs formspecialized structures called appresoria; thesestructures adhere to eggshells through mucigens,where the infection develops to penetrate theeggshell (Morton et al. 2004). The fungus Pochoniachlamydosporia produces an alkaline serine protease,VCP1, during the infection of nematode eggs.The incubation of nematodes eggs with purifiedVCP1 resulted in the removal of the outer vitellinmembrane from eggs of Meloidogyne incognita(Segers et al. 1994). Subsequent infections of theseeggs by P. chlamydosporia extensively degradedthe eggshell, to the degree of generating large holesin the structure, with no evident appresoriumformation (Morton et al. 2004). Contrasting resultswere obtained when eggs of G. pallida were treatedwith VCP1, which might be due to the differentcomposition of nematode eggshells (Morton et al.2004). Based on these observations, it may beconcluded that the outer vitellin membrane isa barrier that helps protect against infections,and which is overcome by the action of secretedproteases; furthermore, the vitellin membranemaybeinvolvedinhostrecognition(Mortonetal.2004).Further research on the role of proteases inthe process of infection by nematode egg parasiticfungi showed that incubation of M. incognitaeggs with P. lilacinus culture filtrates disrupts eggdevelopment. This effect was not observed witheggs containing mature juveniles, but hatchingappeared to be stimulated (Bonants et al. 1995). Inyet another example, a serine protease belongingto the subtilisin family, designated PII, whichimmobilizes the active stages of Panagrellus redivivusand hydrolyzes its cuticle, was described forthe fungus A. oligospora (Tunlid et al. 1994). Theenzyme is expressed under starvation conditionsandisrepressedbyprimaryglucoseandnitrogensources, which are more easily assimilated than thenematode cuticle (Åhman et al. 1996). Similarly,infection of nematode eggs by D. coniosporawas blocked by the addition of the proteaseinhibitor chymostatin, indicating the possible roleof chymotrypsin-like proteases in the infectionprocess (Jansson and Frimen 1999).Data obtained on the role of a neutral serineprotease designated Aoz1 from the nematophagousfungus A. oligospora point in the same direction.The purified protein showed a molecular mass ofapproximately 38 kDa. The expression of this proteinis enhanced by addition of gelatine to the culturemedium. Treatment of nematodes with purifiedenzyme showed dramatic structural changesin the nematode cuticle (Minglian et al. 2004). Sequenceanalysis from the cDNA and genomic clonesrevealed 97% similarity with PII from A. oligospora(Minglian et al. 2004). These data suggest that Aoz1is likely a PII ortholog (Minglian et al. 2004).Recently, the neutral serine protease Mlxfrom the nematophagous fungus Monacrosporiummicroscaphoides waspurifiedandcloned(Wanget al. 2006). The protease could immobilize thenematode Penegrellus redivivus in vitro anddegrade its purified cuticle, suggesting that Mlxcould serve as a virulence factor during infection(Wang et al. 2006).Almost all identified proteases from entomopathogenicand nematophagous fungi belong tothe K subtilisin family, a large family of endopeptidasesfound only in fungi and bacteria. Proteinsbelonging to this family share a high degree ofhomology at the sequence level, showing onlyminor deletions or insertions in the sequence ofits members (Siezen and Leunissen 1997; Fig. 9.3).The protease-encoding genes that have beencloned and sequenced are: the serine protease PIPfrom P. lilacinus (Bonants et al. 1995), the PII andAozl genes from A. oligospora (Åhman et al. 1996;Minglian et al. 2004), the Vcp1 gene from P. chlamydosporia(Segers et al. 1994), the Ver112 gene fromLecanicillium psalliotae (Yang et al. 2005), and theneutral serine protease-encoding gene Mlx fromM. microscaphoides (Wang et al. 2006).Sequence analysis of the promoters of theseprotease-encoding genes has allowed the identificationof regulatory elements described in otherfungal systems that participate in responses tospecific nutritional conditions. The promotersequence from these genes include TATA boxes,GATA boxes, and CREA boxes that are involved innitrogen and carbon catabolic repression (Screenet al. 1997).Sequence analysis of these proteases shows thetypical characteristics of subtilisins, such as thethree catalytic amino acids Asp-His-Ser (Siezenand Leunissen 1997; Fig. 9.3). In addition, few variationswere detected in the sequences, and thesedifferences might be related to observed differencesin substrate specificity (St. Leger et al. 1991). Thisgroup of proteases has a wide spectrum of peptidicsubstrates, but with preferences for specificsubstrates. Furthermore, Morton et al. (2003) ob-

152 S. Casas-Flores, A. Herrera-EstrellaFig. 9.3. Multiple sequence alignment of proteases fromdifferent nematophagous fungi containing the conservedcatalytic domains, performed by means of the ClustalWprogram. A thin line (top) indicates the region spanning thesignal peptide, followed by the propeptide indicated witha thicker line. A high level of similarity among the differentsources of proteases can be observed. Residues in darkindicate amino acids identical among all sequences. Theputative catalytic triad (D-H-S) of the subtilase active siteis marked by asterisks. TheGenBankaccessionnumbersAAX54903, ABF72192, CAD20584, AAA91584, AAM81583,BAD44716, CAA32820, Q68GV19, CAA63841, AF516146,and AAW21809 correspond to the following organismsand proteases, respectively: Arthrobotrys conoides (cuticledegradingprotease), Dactylella avrietas (cuticle-degradingprotease), Cordyceps chlamydosporia (VCP1), Paecilomyceslilacinus (pSP-3), Dactylaria parvispora (cuticledegradingprotease), Monacrosporium megalosporium(cuticle-degrading protease), Tritirachium album (cuticledegradingprotease), Lecanicillium psaliotae (Ver12),Arthrobotrys oligospora (PII), Arthrobotrys oligospora(Aoz1), and Monacrosporium microscaphoides (Mlx)

Antagonism of Plant Parasitic Nematodes by Fungi 153served strong variations at the sequence level andfor substrate utilization in isolates from P. chlamydosporiain different nematode hosts.B. ChitinasesChitinases have been detected in a great variety oforganisms that do not contain chitin, such as bacteria,higher plants and vertebrates, and also in thosethat contain chitin, such as insects, crustaceans andfungi (Herrera-Estrella and Chet 1999). Chitin isa structural polymer found in the cell wall of fungi,and constitutes the exoskeletons of invertebrates; itis an important component of the middle layer ofnematode eggshells. Chitin is degraded by the combinedaction of endo- and exo-chitinases. All fungianalyzed to date produce intracellular chitinasesnecessary for the apical growth of hyphae (Takayaet al. 1998), while extra-cellular chitinases play animportant role in parasitism (Chet et al. 1997). Thechitinases and proteases of the biological controlagents Trichoderma spp. are very similar to thoseof nematophagous fungi, and have been shown tohave a great potential for their use in the biologicalcontrol of nematodes (Sharon et al. 2001).Nematophagous fungi that parasitize eggsmust penetrate the eggshell during the infection(Lysek and Krajci 1987; Lopez-Llorca and Duncan1988). As mentioned above, the structure of theeggshell is formed by several layers, includinga chitinous one (Wharton 1980). This is thethickest layer, and is likely to be the major barrierfor infection (Bird and Bird 1991).The first report for chitinase activity innematophagous fungi was in Verticillium spp.,isolated from infected nematode eggs, both inscreening on solid media with colloidal chitinand in liquid media (Dackman et al. 1989).More recently, several chitinases produced bythe egg parasitic fungi, P. chamydospora andP. rubescens, have been purified. Culture filtratesof P. rubescens show higher activity of N-acetylb-D-glucosamidasethan culture filtrates ofP. chlamydospora (Bird and Bird 1991). This mightbe due to the fact that eggshells from their preferredhosts are different. Globodera eggshells arethicker than those of Meloidogyne,andP. rubescensFig. 9.4. Scanning electron microscope(SEM) images of eggs of the nematodeGlobodera, treated with variousenzymes and enzyme combinations. aUntreatedeggs(control).b Treated withpurified chitinase from V. suchlasporium(CHI43). c Close-up of b to show scarson eggshell. d Treatment with V. suchlasporiumprotease (P32). e Treatmentwith both P32 and CHI43. f Close-up ofe to show extensive peeling of eggshell(modified from Tikhonov et al. 2002)

154 S. Casas-Flores, A. Herrera-Estrellaappears to prefer Globodera eggs. G. pallida eggswere treated with the purified chitinase CHI43,provoking scars on the egg surface; the addition ofthe protease P32 to the mix generated peeling onthe eggshell membrane (Fig. 9.4; Tikhonov et al.2002). These results show the importance of bothenzymes in the infection, and contrast with thefailure of purified VCP1 to produce similar effectson Globodera (Morton et al. 2004).Unfortunately, current knowledge on the roleof chitinases in the infection process is still verylimited, especially at the level of gene expression.Fortunately, the situation is likely to change in thenearfuture,withthestudyofmorefungalgenomes.In this sense, recently, the cDNAs from an acidicchitinase gene, chi1, and a basic chitinase gene,chi2, fromVerticillium lecanii were isolated andthe deduced protein sequence analyzed. The chi1cDNA encodes a predicted protein of 370 aa,whilethe cDNA of chi2 encodes one of 423 aa. Thebasicchitinase gene (chi2) was successfully expressedin Pichia pastoris, where CHI2 was shown to bea functional enzyme that can hydrolyze chitinoussubstrates (Lu et al. 2005).C. Other EnzymesMost studies of the chemical composition of thenematode cuticle have been performed with themammal parasitic nematode, ascarids (Schencket al. 1980). The ascarid cuticle is a three-layered,fibrous structure that contains collagen and keratinof types unique to the Nematoda (Bird 1971).Collagens are among the most complex of proteins,and are slowly degraded in natural soils and waters(Weiss 1976). Collagenolityc enzymes have beenisolated from vertebrate (Gross and Lapiere 1962;Gross and Nagai 1965) and invertebrate (Phillipsand Dresden 1973) animal tissues and from bacteria,but reports of production of collagenase byfungi are relatively rare (Hurion et al. 1979). Collagenasewas defined as an enzyme that catalyzesthe hydrolysis of collagen and gelatin, rather thanother protein substrates (Mandl et al. 1953).During the infection of nematodes, nematophagousfungi must penetrate the nematodecuticle; it is believed that collagenase is an importantenzyme involved in the pathogenicityof nematophagous fungi (Dackman et al. 1992;Tunlid et al. 1994). One of the first attempts todetermine whether nematode-trapping fungiproduced extra-cellular collagenase and keratinasewas made by Schenck et al. (1980), who observedthat a group of eight nematode-trapping fungiproduced collagenase in the growth media ofall tested species. All the tested species couldsecrete extra-cellular collagenase with acceptablecollagen-hydrolyzing activities. In contrast,keratinase activity was not found in the culturemedia.More recently, Tosi et al. (2002) reported resultsof a screening process comparing the Antarcticnematophagous fungus Arthrobotrys tortor withother Arthrobotrys species in the production ofextra-cellular collagenases. To carry out this research,they used the nematode Caernorhabditiselegans. Collagenase activity was determined usinginsoluble collagen from bovine Achilles tendon,and measuring the amount of solubilized hydroxyprolineproduced. The results showed thatthe total amount of collagenase produced by theAntarctic strain of A. tortor was about three-foldhigher than that observed for the other species.In the Antarctic strain, collagenase was shown tobe a constitutive enzyme. The level of collagenaseproduction in nematode-trapping fungi could berelated to their virulence. Certain organisms producingcollagenase are highly invasive; presumably,thesecollagenasescontributetotheirvirulence.D. Toxins or Inhibitory MetabolitesIn order to eliminate their competitors, many microorganismsproduce toxic metabolites such asantibiotics. Toxins are also important for parasiticmicroorganisms, because they facilitate infectionby debilitating the host (Morton et al. 2004). Organismsthat can produce metabolites similar tothose of nematicides have been investigated andconsidered as possible biocontrol agents.Nematophagous fungi are not an exception inthe production of toxins. The fungus P. lilacinusproduces acetic acid that paralyzes juvenile nematodes(Djian et al. 1991). Fusarium equiseti producescompounds that reduce hatching of rootknotnematode eggs and immobilizes infective juveniles(Nitao et al. 1999). Fungal filtrates from severalfungi grown in malt extract broth were toxicto infective juveniles and eggs (Chen et al. 2000).A metabolite with nematicidal activity against infectivejuveniles, phomalactone, was isolated fromP. chlamydosporia (Khambay et al. 2000). Researchinto finding new metabolites with nematicidal activityby nematophagous fungi is practically a new

Antagonism of Plant Parasitic Nematodes by Fungi 155field to be exploited. Genetic and molecular analysison the metabolic pathways to synthesize thesemetabolites could help to manipulate the productionof these compounds in order to use them infields infested by nematodes.IV. Improvement of Nematode-TrappingFungi by Genetic EngineeringAlthough many microbial antagonists of nematodeshave been found and tested for their activityagainst nematodes, they have not led to the developmentof commercial products as cost-effective aschemical nematicides (Oka et al. 2000).The most common strategy to control plantparasitic nematodes by the use of nematodetrappingfungi has been to mass-produce thefungus that infects the nematodes on solid substrates,followed by application to the soil. Thereare several reports of successful biological controlof plant parasitic nematodes by means of nematophagousfungi; however, these are perceivedas inefficient, compared to chemical nematicides.Based on this has emerged the demand for newstrategies in order to combat phytophagousnematodes, one of these being the generation ofimproved fungal strains.Improvement of biological control agents hasinvolved the overexpression of lytic enzymes. In anattempt to obtain improved strains from the entomopathogenicfungus A. oligospora, Åhman et al.(2002) investigated the potential roles of protease IIin host infection by generating several PII mutants,including mutants in which the corresponding genehad been disrupted, and transfromants that overexpressedit. Deletion of the PII gene had a limitedeffect on pathogenicity, including decreased percentagesof adhesion and immobilization of nematodes,while overexpression of the gene resulted ina higher capacity to kill nematodes. Other interestingobservations were that the deletion mutantproduced less traps, while the multicopy transformantsproduced more (Åhman et al. 2002).V. Concluding RemarksDespite the great amount of knowledge accumulatedon the structural characteristics of specializedstructures produced by nematode-trappingfungi during interaction with their hosts, and evenon the life cycle and ecology of this organisms,our understanding of the host-fungus interactionis poor at the molecular level, and it is necessaryto gain further knowledge in this domain. All studieson lytic enzymes reviewed in this chapter undoubtedlyindicate that they are important virulencefactorsintheinfectionprocess.However,asmentioned above, present understanding of theirmode of action and their regulation at the molecularlevel is still limited. More research on virulencefactorsandtheirregulationisnecessaryinorderto better understand the mechanisms underlyingthenematodeinfectionprocessbyfungi,whichinturncouldbeusedtogenerateimprovedbiologicalcontrol fungi against nematodes by geneticengineering. It must be noted that lytic enzymesare unlikely to be the only factors involved in theinfection process. In addition, the analysis of differentisolated structures such as knobs, trappingnets, appresoria, and other structures will help tounderstand their role in the parasitic process asinfection structures. Identification of new potentialvirulence factors is important, and new technologiessuch as functional genomics, proteomics,andmetabolomicsshouldenableustoidentifytheplayers involved in the infection process and to elucidatethe signals that switch on the process in thefungus.ReferencesÅhman JB, Ek B, Rask L, Tunlid A (1996) Sequence analysisand regulation of a gene encoding a cuticle-degradingserine protease from the nematophagous fungusArthrobotrys oligospora. Microbiology 142:1605–1616Åhman JB, Johansson T, Olsson M, Punt PJ, Van Den HondelC, Tunlid A (2002) Improving the pathogenicity ofa nematode-trapping fungus by genetic engineeringof a subtilisin with nematotoxic activity. Appl EnvironMicrobiol 68:3408–3415Akhtar M (1997) Current options in integrated managementof plant-parasitic nematodes. Chapman and Hall, LondonBalogh J, Tunlid A, Rosen S (2003) Deletion of a lectin genedoes not affect the phenotype of the nematodetrappingfungus Arthrobotrys oligospora. Fungal Genet Biol39:128–135Barron GL (1977) The nematode-destroying fungi. Topicsin Mycobiology 1, Canadian Biological Publications;GuelphBarron GL (1979) Observations on predatory fungi. CanJ Bot 57:187–193Bidochka MJ, Khachatourians GG (1987) Purification andproperties of an extracellular protease produced by theentomopathogenic fungus Beauveria bassiana. ApplEnviron Microbiol 53:1679–1684

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10 Entomopathogenic Fungi and Their Role in Pest ControlA.K. Charnley 1 , S.A.Collins 1CONTENTSI. Introduction ........................ 159II. Taxonomy .......................... 159III. Infection Process .................... 160A. FungalInvasionoftheHost.......... 160B. Host Response to Fungal Invasion . . . . . 162IV. Epizootology of Fungal Diseases in Insects 162V. Pest Control ......................... 163A. Approaches to the Use of InsectPathogenicFungiforPestControl..... 163B. CurrentUse ...................... 1651.StatusofMycoinsecticides......... 1652.ConstraintsonEfficiency.......... 1703. Integration in Pest ManagementSchemes....................... 171C. Development of a Mycoinsecticide . . . . 1711.IsolateSelection................. 1712.ProductionandFormulation....... 1723.Application .................... 1754.Safety......................... 1765.Registration.................... 177VI. Future Developments ................. 178A. PotentialTargets .................. 178B. Constraints on the Commercial UseofEntomopathogenicFungi ......... 178C. StrainImprovement................ 179VII. Conclusions ......................... 180References.......................... 181I. IntroductionSynthetic chemical pesticides have been themainstay of insect pest control for over 50 years.However, insecticide resistance, pest resurgenceand concern over the environmental impact ofagricultural inputs give urgency to the search foralternative, biologically based forms of pest control.The impact on insect populations of naturalepizootics caused in particular by fungal and viralpathogens demonstrates the potential of microbialpest control. Seminal attempts towards the end ofthe 19th Century to use the Ascomycota pathogenMetarhizium anisopliae for pest control (describedin Gillespie 1988) inspired more recent extensive1 Department of Biology and Biochemistry, University of Bath,Claverton Down, Bath BA2 7AY, UKefforts to harness entomopathogenic fungi forbiocontrol. This review outlines the current stateof knowledge of insect fungal pathogens as itrelates to their present use and future potential asmycoinsecticides.II. TaxonomyRelationships between fungi and insects may bemutualistic, through commensal to obligatelypathogenic. The term entomogenous is often usedto encompass all types of association betweeninsects and fungi, with disease-causing fungibeingreferredtoasentomopathogenic.Afurtherdistinction can be made between fungi which areaggressively pathogenic, like Metarhizium anisopliae,andopportunists,likethewoundpathogenMucor haemalis (McCoy et al. 1988; Samson et al.1988; Tanada and Kaya 1993). The system ofclassification adopted here accords with that in theIndex Fungorum (www.speciesfungorum.org).Entomopathogens are to be found in mosttaxonomic groupings in the fungal kingdom,apart from the higher Basidiomycota. The phylumOomycota, in the kingdom Chromista (Leipe et al.1994), originally classified as fungi, includes themosquito pathogen Lagenidium giganteum. Ofthe fungi, the most common insect pathogens inphylum Chytridiomycota are found in the genusCoelomomyces, e.g. C. psorophorae, a mosquitopathogen with an obligate copepod secondaryhost. Entomophthorales, e.g. genera Conidiobolus,Entomophaga, Erynia and Pandora, are widespreadmembers of the phylum Zygomycota. Mummifiedaphids stricken by fungi of this group are familiarfeatures of cereal crops in temperate regions(Fig. 10.1f). Among the members of the phylum Ascomycota,Cordyceps spp. have fruiting structuresor perithecia which can dwarf the cadavers of theirinsect victims (Fig. 10.1b). The most widespreadinsect pathogenic fungal genera are found inEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

160 A.K. Charnley, S.A.Collinsthe order Hypocreales of the phylum Ascomycota,viz. Beauveria and Metarhizium. Beauveriabassiana (Fig. 10.1g, h) and Metarhizium anisopliae(Fig. 10.1g) have broad host ranges, thoughconsiderable specificity occurs among individualisolates. Subspecies M. aniopliae var. majus andM. anisopliae var. acridum are specific for Scarabidbeetles and grasshoppers/locusts respectively(Samson et al. 1988; Driver et al. 2000). Other importantentomopathogens in the phylum Ascomycotainclude Lecanicillium (Fig. 10.1d) (formerlyVerticillium), Nomuraea (both Incertae sedis: Ascomycota)(Fig. 10.1c), Paecilomyces (Eurotiales:Ascomycota) and Aschersonia (Hypocreales: Ascomycota)(Fig. 10.1a). The fungi described aboveare all destructively pathogenic. Laboulbeniales(Ascomycota), on the other hand, are biotrophic.They remain largely external to their hosts, gainingnutrition via a penetrant haustoria while apparentlycausing little harm. Most Trichomycetes(Zygomycota) have a commensal existence in theguts of their Dipteran hosts (Misra 1998).III. Infection ProcessUnique among entomopathogens, fungi do nothave to be ingested and can invade their hostsdirectly through the exoskeleton or cuticle. Therefore,they can infect non-feeding stages such as eggsand pupae. The site of invasion is often betweenthe mouthparts, at intersegmental folds or throughspiracles, where locally high humidity promotesgermination and the cuticle is non-sclerotised andmore easily penetrated (Hajek and St Leger 1994;Clarkson and Charnley 1996). Members of theHypocreales such as Metarhizium spp. and Beauveriaspp. are opportunistic hemibiotrophs witha parasitic phase in the live host and saprotrophicphase during post-mortem growth on the cadaver.These fungi may use toxins to help overcomehost defences (Gillespie et al. 2000; Freimoseret al. 2003a). By contrast, Entomophthorales arebiotrophs with little or no saprotrophism; they killby tissue colonisation without the help of toxins(Charnley 2003; Freimoser et al. 2003b).A. Fungal Invasion of the HostM. anisopliae and B. bassiana have hydrophobicspores which appear to bind to insect cuticle bynon-specific interactions, though failure to adhereto particular insect species may help to defineisolate host range (Holder and Keyhani 2005).Zoospores of Lagenidium giganteum are hostselective. Cuticle-degrading enzymes are presenton the surface of conidia of M. anisopliae and,therefore, there is the potential for the fungus tomodify the cuticle surface to aid attachment. Hostand fungal lectins have been implicated also inthe process of attachment. Germination in vitroof nutrient-dependent spores of M. anisopliae andB. bassiana is consequent upon a non-specific,accessible source of carbon and nitrogen, thoughin vivo isolate specificity may depend on responseto qualitative and quantitative differences in availablenutrients on the host cuticle. More selectivepathogens appear to have more specific requirements,e.g. strains of M. anisopliae which arespecialist for Scarabid beetles. Ability to withstandantifungal compounds in the cuticle, such as shortchain fatty acids, is a prerequisite for successfulinvasion (see Boucias and Pendland 1991). Theimportance of signal exchange between host andpathogen is seen in particular in the cues whichcause the fungus to stop horizontal growth on theFig. 10.1. a Aschersonia aleyrodis (Ascomycota: Hypocreales),sporulating on cadavers of the glasshouse whiteflyTrialeurodes vaporariorum (Homoptera: Aleyrodidae)(with permission of HRI at the University of Warwick,UK). b Stroma of Cordyceps sp. (Ascomycota: Hypocreales:Clavicipitaceae) emerging from a locust (Orthoptera:Acrididae). They each contain many ascospores (withpermission of H. Evans, CABI Bioscience, Egham, UK).c Dead caterpillar (Lepidoptera) covered in the greenconidia of Nomuraea rileyii (Ascomycota: Hypocreales)(with permission of HRI at the University of Warwick,UK). d Scanning electron micrograph of conidia ofLecanicillium longisporum (Ascomycota: Incertae sedis)(with permission of HRI at the University of Warwick, UK).e A germinating conidium of Metarhizium anisopliae var.acridum (Ascomycota: Hypocreales), on the surface of thewing of a desert locust, Schistocerca gregaria (Orthoptera:Acrididae). The germ tube terminates in an appressorium.f Pandora neoaphidis (Zygomycota: Entomophthorales)on the aphid, Macrosiphum euphorbiae (Homoptera:Aphididae). Note white halo of spores on the leaf aroundthe dead aphids (brown, centre-right). g Dead larvaeof the vine weevil, Otiorhynchus sulcatus (Coleoptera:Curlionidae); the one on the left is covered in conida ofBeauveria bassiana (Ascomycota: Hypocreales), the one onthe right is covered in conidia of Manisopliae var. anisopliae(Ascomycota: Hypocreales) (with permission of HRI atthe University of Warwick, UK). h Beauveria bassiana(Ascomycota: Hypocreales) sporulating on cadavers ofblowflies, Phormia regina (Diptera: Calliphoridae) (withpermission of D. Steinkraus, University of Arkansas, USA)

Entomopathogenic Fungi and Their Role in Pest Control 161

162 A.K. Charnley, S.A.Collinssurface of the cuticle, produce an appressoriumand initiate penetration. Differentiation of thegerm tube to produce the holdfast structure, orappressorium, is most completely understood forM. anisopliae. Isolate ARSEF2575 requires lowconcentrationsofacomplexcarbonandnitrogensource,andahardsurface.Metarhizium isolatesfrom Homoptera form appressoria in media (highconcentrations of simple sugars) which are repressivefor isolates from Coleoptera. This is probablyan adaptation to parasitism, as the cuticle ofplant-sucking bugs (Homoptera) is contaminatedwith sugars from their copious excreta (St Legeret al. 1992a). The specificity for locusts of at leastone isolate of M. anisopliae var. acridum appears inpart to be due to cues for appressorium formation(Wang and Leger 2005; Fig. 10.1e).Numerous light and electron microscopestudies on the invasion of host cuticle by entomopathogenicfungi are consistent with theinvolvement of both enzymes and mechanicalpressure. Insect cuticle comprises between 60–70%protein; therefore, it is perhaps not surprisingthat recent work has implicated proteases inparticular in the penetration process (for review,see Charnley 2003).Oncethefungusbreaksthroughthecuticleandunderlying epidermis, it may grow profusely in theblood, in which case insect death is probably the resultof starvation or physiological/biochemical disruptionbrought about by the fungus. Alternatively,insecticidal secondary metabolites may contributetothedemiseoftheinsectand,inthiscase,extensivegrowth of the fungus may occur only on the cadaverof the host (Charnley 2003). For many fungi,the reality is probably somewhere between thesetwo extremes. The absence of fungal proteases, soprominent during cuticle penetration, is a markedfeature of this stage of mycosis (Wang et al. 2005).Few studies have examined the effect of fungal infectiononhostphysiology/behaviour.Thisisunfortunatebecause, from the point of view of cropprotection, sub-lethal or prelethal effects of mycosismay be just as useful as the death of the host.Detrimental effects of mycosis on food consumption,egg laying and flight behaviour have beenrecorded (Nnakumusana 1985; Seyoum et al. 1995).The life cycle is completed when the fungussporulates on the cadaver of the host. Under appropriateconditions, particularly high relative humidity(RH),the fungus will breakoutthrough the bodywall of the insect, producing aerial spores. This mayallowhorizontalorverticaltransmissionofthediseasewithin the insect population. Resting spores(chlamydospores) produced within the dead insectmay enable the fungus to survive for long periodsunder adverse conditions (Samson et al. 1988). Additionally,in the locust pathogen M. anisopliae var.acridum, conidia may be produced in internal airspaces as the cadaver dries out under low humidity(Prior, personal communication).B. Host Response to Fungal InvasionHost response has been extensively reviewed(Vilcinskas and Gotz 1999; Gillespie et al. 2000).The cuticle is not only the first but also themajor barrier to host invasion. Structural featuressuch as sclerotisation impede penetration, whileenzyme inhibitors and tyrosinases, which generateantimicrobial melanins, are frontline defencesagainst weak pathogens. Blood-borne defencesseem to have little impact on virulent fungalpathogens. Phagocytosis by individual blood cellsand cooperative behaviour between haemocytesubpopulations viz. encapsulation and granulomaformation are often not recorded after the initialincursion. This has been attributed to a failure ofthe insect’s non-self recognition system, in somecases brought about by toxic fungal metabolites,in others due to the removal of immunogeniccomponents from fungal cell walls (or even thewalls themselves) in the blood of infected insects(see Charnley 2003). The important contributionof antifungal peptides in defence against nonadaptedpathogens is also recognised (Kim andKim 2005).IV. Epizootology of Fungal Diseasesin InsectsAlthough epizootics of insect fungal diseasesare comparatively common, our understandingof population-level interactions between entomopathogenicfungi and their hosts is based onlyon a restricted number of studies (Carruthersand Soper 1987; Goettel et al. 2005). Naturaldisease development and spread are affectedby the characteristics of the host and pathogenpopulations, the environment, and the impact ofhuman activities (particularly in agroecosystems).Properties of the pathogen population which areimportant include virulence, dispersal, and survivalin the host’s environment, inoculum density

Entomopathogenic Fungi and Their Role in Pest Control 163and spatial distribution. Host population factorswhich need to be considered are susceptibility,density, movement and spatial distribution. Abioticenvironmental factors such as temperature,moisture and sunlight may determine whetherinfection can occur. Germination and sporulationare particularly dependent on moisture.Temperature may also be limiting for disease,particularly when short generation time for thehost is favoured by a temperature which is aboveor below the optimum for the pathogen. Possiblecomplex interactions between virulent and avirulentisolates were suggested by the outcome ofan experimental dual infection in the leaf-cuttingant Acromymex echintior (Hughes and Boomsma2004). The normally avirulent Aspergillus flavusdominated when inoculated with M. anisopliae– probably the outcome of immunosuppressionachieved by Metarhizium toxins.Some of the more detailed epizootology studiesof insect pathogenic fungi come from agroecosystems.Examples include Nomuraea rileyi infectionof Anticarsia gematalis on soybean (Ignoffo1981) and Entomophthora muscae on the onionfly Delia antiqua (Carruthers and Haynes 1986).Forests are more diverse stable habitats than agroecosystems;consequently, the insect–pathogen interactionsmay be more complex. The spruce budwormChoristoneura fumiferana is a major defoliatorin balsam and spruce trees in eastern NorthAmerica. Among a range of pathogens which attackthis insect, the fungi Erynia radicans and Entomophagaaulicae produce the highest mortality(Perry and Whitfield 1984). Disease incidence dependsoninsectageandpositioninthetreecanopy;the role of abiotic factors is not completely understood.Humid, tropical forests have a rich, variedentomopathogenic mycoflora (Evans 1982), includingin particular Cordyceps spp., and it has beensuggested that these fungi have a significant role inthe regulation of insect populations because of thestable microclimates in such habitats.Rangelands are more stable than agriculturalsystems but more uniform in structure thanforest ecosystems. Grasshoppers are often thedominant phytophagous insects in such habitats.Entomophaga grylli mycoses cause high mortalityamong populations of Camnula pellucida andMelanoplus bivittatus in western North America(Pickford and Riegert 1963).Soil is a complex habitat which harboursalargefaunaandflora.Metarhizium anisopliae isone of the most frequent mycopathogens of soilinsects in temperate regions, particularly of beetles(Keller and Zimmermann 1989). Epizootics havebeen found on wireworms (Agriotes spp.) andlarvae of Amphimallon solstitialis. M. anisopliae,B. bassiana and Paecilomyces sp. are commonlyisolated from temperate soils. The ubiquity inparticular of the former two species must reflecttheir broad host ranges. Beauveria brongniartii isprimarily a pathogen of cockchafers, Melolonthaspp., and other Scarabidae. Investigations of thepopulation dynamics of cockchafers in easternSwitzerland showed that B. brongniartii is themain regulating factor (Zimmermann 1992). Soilcan also function as a reservoir for fungi whichgenerally infect insects on aerial parts of plants.It has been shown experimentally that sporesof the lepidopteran pathogen N. rileyi adhere toleaves of plant seedlings as they emerge throughthe soil (Ignoffo et al. 1977). Fungi may persistin soil as mycelium within mummified cadavers,conidia, resting spores (e.g. Entomophthorales)or pseudosclerotia. Temperature, pH, water andorganic content can affect fungal survival (Kellerand Zimmermann 1989).Aquatic ecosystems present different problemsto entomopathogenic fungi. In comparison withterrestrialhabitats,fluctuationsintemperatureandsunlightmaybelessimportantwhereasfluctuationsin pH, salinity, currents and dissolved solutesmay affect pathogen persistence. Althoughsome pathogens may infect aquatic insects usingspecialist-shaped spores or motile zoospores to aidhost location, e.g. Lagenidium giganteum, othersconfine themselves to the aerial adults, therebyavoiding the problems presented by the aquatic environment(Lacey and Undeen 1986).V. Pest ControlA. Approaches to the UseofInsectPathogenicFungiforPestControlSeveral strategies have been adopted for the useof insect pathogenic fungi in pest control. Themost cost effective is permanent introduction(also known as ‘classical control’). This involvesthe establishment of a disease in a populationwhere it is does not occur normally, in order toobtain long-term or permanent suppression of thepest. Although there is a history of this approach,there are comparatively few examples wheredetailed population studies have determined the

164 A.K. Charnley, S.A.Collinseffectiveness of introductions. Milner et al. (1982)introduced an Israeli isolate of Zoophthora radicansinto alfalfa fields in Australia in an attemptto control an accidentally introduced pest, thespotted alfalfa aphid Therioaphis trifolii maculata.The fungus became established and spread fromthe release site in subsequent seasons, causing highpercentage mortality among aphid populations.Entomophaga maimaiga was introduced fromJapan into eastern North America in 1910–1911to control the gypsy moth Lymantria dispar. Thefungus did not appear to have become established.However, in 1989 and 1990, it caused extensivemortality among gypsy moth in 10 states in theUSA (Hajek et al. 1995). It is possible that itwas present but unrecognised for many years,as symptoms of mycosed gypsy moths resemblethose of moths infected with a baculovirus. Restingspores of the fungus have been used successfullyto introduce the fungus into new areas.Promoting natural fungal epizootics byadopting appropriate cultural and crop protectionpractices is an alternative way of harnessingentomopathogenic fungi for pest control. A classicexample is the development of early harvestingstrategies for alfalfa to maximise developmentand spread of natural infections of Zoophthorasp. among alfalfa weevils (Hypera postica)(Brownand Nordin 1986). Steinkraus et al. (1995) showedthat natural infections of Neozygites fresneii (Entomophthorales)could reduce or even eliminatethe need for chemical control of aphids on cotton.They were able to predict epizootics a weekin advance and advise farmers to refrain fromchemical sprays at crucial times.Epizootics of fungal pathogens on crop pestsoften occur too late to be of economic value. Applicationof an additional inoculum can acceleratethe process. When this results in secondaryspread of disease, the process is termed ‘inoculativeaugmentation’, otherwise the strategy is called‘inundative augmentation’; in reality, these are thetwo extremes of a continuum. Augmentation nowalso encompasses the application of a fungus ina situation where mycosis may not naturally occur.Mass production for inundative control is knownas the ‘microbial pesticide’ or ‘mycoinsecticide’ approach(Tanada and Kaya 1993). The developmentof mycoinsecticides has received the most attentionin recent times and is focussed on here.Mycoinsecticides may perform inconsistentlyfor a variety of reasons but particularly dueto unfavourable environmental conditions (seebelow). Therefore, strategies have been developedto increase efficiency and accelerate kill, e.g. bycombining fungi with sub/low lethal doses ofchemical pesticides. This approach is based on theassumption that, weakened by another stressor,the insect will succumb more readily to mycosis. InChina, combinations of B. bassiana and certain insecticideshave been recommended for applicationagainst crop and forest pests (Feng et al. 1994). Mostexperimental studies with mixtures suggest additiveeffects of the ingredients. However, synergyoccurred between imidacloprid and B. bassianaagainst the Colorado beetle (Furlong and Groden2001) and the caterpillar Spilarctia obliqua(Purwar and Sachan 2006). The same insecticidewas antagonistic with B. bassiana against Bemisiaargentifolii (James and Elzen 2001). B. bassianaand Bacillus thuringiensis toxins were synergisticagainst the Colorado beetle (Wraight and Ramos2005). An alternative is to use the two treatmentssequentially, rather than in combination. Earlyseason use of Bbassianafollowed by insecticidesgavegoodcontrolofbeetleandcaterpillarpestson crucifer crops (Vandenberg et al. 1998).The alternative to the use of a chemical stressoris to combine entomopathogenic fungi with othermicrobial pathogens (see Zimmermann 1994).Though there are few examples where this strategyhas been tried, it is given credence by observationsof mixed infection in field-collected insects.Laboratory and glasshouse trials established theefficacy of a combination of M. anisopliae andentomopathogenic nematodes against the beetleHoplia philanthus (Ansari et al. 2004). In Columbia,there is a product called Micobiol Completo, whichis a mixture of B. bassiana, M. anisopliae, N. rileyi,P. fumosoroseus and the bacterium B. thuringiensisat 1 ×10 9 spores of each pathogen per g of product,for control of larvae and adult Lepidoptera,Coleoptera, Hemiptera and Diptera, and mites(Alves et al. 2003). However, it seems unlikely thatcombinations will be used widely, as this strategycomplicates application procedures as well asincreasing the costs of pest control and initialdevelopment (particularly registration).Limitation of fungal isolates to particular climaticconditions could theoretically be remediedby co-formulation of isolates, from the same or differentspecies, with different ecological tolerances.This receives some support from the literature, e.g.Inglis et al. (1997). Interestingly, rarely is synergyobserved and often one isolate dominates over theother (Wang et al. 2002; Rao et al. 2006).

Entomopathogenic Fungi and Their Role in Pest Control 165B. Current Use1. Status of MycoinsecticidesCrop protection is still dominated by chemical pesticides.Sales of biopesticides, at ca. US$600 million,accountforonly2%oftheglobalcropprotectionmarket (Anon 2005). Mycoinsecticides accountedfor a small fraction of the biopesticide market, themajor share being taken by products based on thetoxicogenic bacterium Bacillus thuringiensis (Bt).The controversial but successful introduction ofcrops,e.g.cotton,maize,potatoandrice,geneticallyengineeredtoproduceBttoxinheraldsanewera for insect control (James 2005).Other than for transgenic crops, biopesticideproduction is dominated by small to medium-sizedcompanies (SMEs). Large multinational agrochemicalcompanies who, encouragingly, invested in biologicalforms of pest control in the 1980s havesincepulledout;anexceptionisthecurrentinvolvementof some Japanese companies, e.g. Certisis the biopesticide arm of Mitsui & Co Ltd. However,it is clear that concerns over insecticide resistance,the impact of synthetic chemicals in environmentallysensitive areas, cost of registering chemicalsfor high-value but specialist crops, and the growinginterest in ‘organic’ food ensure a market forbiological forms of pest control including productsbased on entomopathogenic fungi.Anyattempttoproduceadefinitivelistofrecommendablecommercial producers is hinderedby the difficulty in obtaining realistic information,rather than company hype. A particular problemis gauging the size of an operation. The companiesand products detailed in Table 10.1. appear tobe the most important in the countries in whichthey originate. Taken as a whole, the list indicatesa considerable level of interest and involvement inmycoinsecticide use.Fig. 10.2. a Vertalec, a productbased on Lecanicillium longisporum,targeted against glasshouse aphids(see Table 10.1.). b Initial brochure ofthe multi-donor funded LUBILOSAproject on developing Metarhiziumanisopliae for locusts and grasshoppercontrol. c Logo of the product,based on Metarhizium anisopliae var.acridum, developed for locust controlby LUBILOSA (see Table 10.1.). d Mycotrol,a product based on Beauveriabassiana, forcontrolofanumberofpests. Wettable powder formulationand a plate culture of the fungusare also shown (see Table 10.1.).E Botanigard, a product based onBeauveria bassiana, for control ofa number of pests (see Table 10.1.)

166 A.K. Charnley, S.A.CollinsTable. 10.1. Commercial production of mycoinsecticides aCountryof the companyAustria Melocont®PilzgersteProduct Company/*registrationother than home countryAustralia BioCane TM Becker Underwood Pty Ltd.(www.beckerunderwood.com)Target Active ingredient/productionKwizda Agro GesmbH Cockchafers BeauveriabrongniartiiGreybackcanegrubBioGreen TM ditto Red-headedcockchaferGreen Guard TM ditto Australian plagueBelgium PreFeRal WG Biobest N.V. (www.biobest.be),*Finland, France, Luxemburg,The Netherlands, Norway,Poland, SwedenBolivia Probiomet Probioma(www.probioma.org.bo)locust, winglessgrasshoppersGreenhousewhitefly(all stages)MetarhiziumanisopliaeMetarhiziumanisopliaeM. anisopliaevar. acridum,aerial conidia,solid statePaecilomycesfumosoroseusProbiovert ditto Whitefly, scales VerticilliumlecaniiProbiobass ditto Variety ofbeetle pestsBrazil Metarhizium PM Biocerto (www.biocerto.com.br) Range oflarval pestsBoveril wp Itaforte(www.itafortebioprodutos.com.br)Formulation and shelf life Label cropsBarley kernels,7.5 ×10 9 spores g −1 ,12 months at 2 ◦ CGranules,ongrain,2×10 9 g −1 ,6monthsat5–10 ◦ CGrassland, forest,horticulture cropsSugarcaneGranules Pasture and turfUltra-low volume, 12 monthsunder recommended storageWater-dispersible granule,2 ×10 9 g −1 ,6 monthsat2–6 ◦ CAgricultural areas,pastures, crops,forage cropsnon-crop areasOrnamentals,cucumberand tomatoesCaterpillars M. anisopliae Range includingornamentals,rice, fruit treesBeauveriabassianaRange includingvegetables, fruittrees, coffee plantsRange includingvegetables, fruittrees, coffeeM. anisopliae Rice granules Sugarcane, pastureWhitefly B. bassiana Sugarcane, coffeeMetarril wp ditto Sucking insects M. anisopliae Sugarcane, pastureVertirril WP ditto Whitefly, aphids V. lecanii Coffee, citrusChina Kiloca TM Chonqing Chongda Bio-TechDevelopment Co. Ltd.(www.cccbt.com)Tianli Nature and Man Group(www.jxtianren.com)Columbia Bassianil Edafon(www.controlbiologico.com)Locusts M. anisopliae var.acridumDendrolimuspunctatusVarious beetlepestsMetabiol ditto Various beetlepests5 ×10 10 g −1 , oil formulation Pasture andfield cropsB. bassiana Pine forestsB. bassiana 3 months, at 4–10 ◦ C Banana, sweetpotato, citrusM. anisopliae 3months,at20 ◦ C RiceVertilec ditto Whitefly, scales V. lecanii 3months,at20 ◦ C Tomatoes

Entomopathogenic Fungi and Their Role in Pest Control 167Table. 10.1. (continued)Countryof the companyProduct Company/*registrationother than home countryBassiana Semicol Ltd.(www.semicol.com.co)Target Active ingredient/productionVarious,including whiteflyCebiopest ditto Various,including whiteflyB. bassianaB. brongniartiiAnisafer ditto M. anisopliaeV. lecaniiVercani ditto Various,including whiteflyCuba Bibisav-2 Inisav (www.inisav.cu) B. bassiana 3 months, at 10-20 ◦ C,France Betel Betel Reunion SA(Head Company Arysta Lifescience),*ReunionGuatemala Met-92 Agricola el Sol(www.agricolaelsol.com)Formulation and shelf life Label crops1 ×10 9 conidia g −1 ,bait compositionSugarcane beetle B. brongniartii Clay microgranules SugarcaneVarious insects M. anisopliae WP, 3 months at 20–25 ◦ C Grass, sugarcane,banana, coffeeTeraboveria ditto Various insects B. bassiana WP, 3 months at 20–25 ◦ C Grass, sugarcane,banana, coffee,fruitZeroQK ditto Cockroaches M. anisopliae Contains mineral talc BuildingsSucking insects V. lecanii WP, 1 ×10 8 g −1 ,India Bio-Catch T. Stanes & Co(www.tstanes.com/products.html)8 months at 20–25 ◦ COrnamentals, fieldvegetables, turfBio-magic ditto Beetles M. anisopliae ditto Variety includingPriority ditto Mites P. fumosoroseus ditto, 8 months at 20–25 ◦ C dittoMultiplex Baba Multiplex(www.multiplexgroup.com)Sucking insectson vegetablesand ornamentalscereals, vegetables,fruit crops,greenhousesB. bassiana Liquid and powder Variety includingvegetables,ornamentals,indoor andoutdoor nurseriesMultiplex Mycomite ditto Red spider mite P. fumosoroseus WP Coffee and teaplantationsM. anisopliae Liquid and powderMultiplexMetarhiziumditto Beetle pestsand termitesMealikil Agri-Life (www.somphyto.com) Mealibugsand scale insectsV. lecanii WP, 1 ×10 8 g −1 ,6 monthsunder recommended storagePacer ditto termites M. anispoliae WP, 8 ×10 8 g −1 ,12 months Buildings,interiors,agricultural fields,orchards

168 A.K. Charnley, S.A.CollinsTable. 10.1. (continued)Countryof the companyProduct Company/*registrationother than home countryRacer ditto Caterpillars onavarietyofcropsBiovert Rich Plantrich (www.plantrich.com) Sucking insectsincluding aphids,and whiteflyTarget Active ingredient/productionFormulation and shelf life Label cropsB. bassiana 1 ×10 8 g −1 ,6 monthsunder recommended storageRice, cotton,vegetables, chillies,oil seeds, pulses,tea, cardamomVerticillium sp. 2 ×10 9 g −1 Variety includingspices, citrus andhorticulture cropsPacihit Rich ditto Thrips, whitefly Paecilomyces sp. 2 ×10 9 ml −1 Variety includingBioguard Rich ditto Variety ofcaterpillarsB. bassiana 2 ×10 9 ml −1Biomet Rich ditto Variety of insects M. anisopliae 2 ×10 9 ml −1 ,liquidformulationIndonesia Bevaria Bio Brahma Nusantara,Jakarta, BaratB. bassianaBiometeor ditto M. anisopliaeNirAma ditto P. fumosoroseusItaly Beavaria brong Agrifutur s.r.l Cockchafers B. brongniartii Barley kernels, 7.5 ×10 9conidia g −1 ,12 monthsat2 ◦ CJapan Biolisa Kamikiri Nitto Denko Cerambycidbeetles in citrusMexico Bea-Sin Plant Health Care(www.phcmexico.com.mx)orchardsVarious insects B. bassianaMeta-Sin ditto Various insects M. anisopliaeThe Netherlands Mycotal Koppert B.V. (www.koppert.nl),*Finland,Italy,Norway,Switzerland,Turkey, UK (Denmark Pending)Vertalec (Fig. 10.2a) ditto, *Finland, Japan, Norway,Switzerland, UKNew Zealand Vertikil Crop Solutions Ltd.(parent company MilleniumMicrobes)(www.milleniummicrobes.com)Whitefly, thrip LecanicilliummuscariumB. brongniartii Non-woven fibre bands,impregnated conidiaWettable powder, 1 ×10 10 g −1 ,6 months underrecommended storageAphids L. longisporum Blastospores, WP, 1 ×10 9 g −1 ,6monthsat2–6 ◦ CThrips, whitefly Two strains WP, 1 ×10 10 g −1 ,appliedinof L. muscarium waterwithsomevegetableoilspices, banana,coconut, arecanutVariety includingsugarcane, tea,coffee, spicesVariety includinggrassland,horticulture, fruitVarious, includingglasshousevegetables andornamentalsGlasshouse cropsVegetables, flowersunder glass.Outdoor cropsinclude kiwifruitand cucurbits

Entomopathogenic Fungi and Their Role in Pest Control 169Table. 10.1. (continued)Countryof the companyProduct Company/*registrationother than home countryVertiblast ditto Aphids, potatoPsyllidBeaugenic ditto Sucking insects,particularly thripsSouth Africa Bb Plus Biological Control Products SA(Pty) Limited (BCP)(www.biocontrol.co.za)Target Active ingredient/productionRed spider mitesand aphidsFormulation and shelf life Label cropsL. longisporum, Wettable powder, 1 ×10 9 g −1 ,blastospores liquid dittoB. bassiana WP, 1 ×10 10 g −1 , ditto OutdoorsB. bassiana WP, 2 ×10 10 g −1 ,9monthsat4 ◦ CBb Weevil ditto Banana weevil B. bassiana Dusting powder, 2 ×10 10 g −1 ,9monthsat4 ◦ CGreen Muscle TM ditto, *throughout Africa Desert locust M. anisopliaevar. acridumSwitzerland Engerlingspilz Andermatt BioControl AG,Grossdietwil(www.biocontrol.ch)USA BotaniGard® ES(Fig. 10.2e),BotaniGard® 22WPMycotrol O(Fig. 10.2d)Laverlam Int’l Corporation(www.bioworksbiocontrol.com),*EuropeOil formulation, 5 ×10 10 g −1 ,1yearat4 ◦ C or 6 monthsin oil formulationCockchafer B. brongniartii Conidia on barley kernels,A wide varietyof soft-bodiedinsectsditto, *Denmark, Italy, Sweden Sucking insects,including whitefly,Naturalis TM L Troy Biosciences Inc. TM(www.troybiosciences.com),*OECD Greece, Italy, Mexico,Spain, SwitzerlandaphidsVarious, includingwhitefly, weevils,caterpillarsNaturalis TM HG ditto Various, includingleaf-feedingcaterpillars,Colorado potatobeetlesVariouscaterpillarsand bugsBassi ditto Sucking inectsand weevilsVenezuela Meta-Ven San Pedro Agrobiológicos(www.agrobiologicos.com.ve/)>1 year at 2 ◦ CB. bassiana 2 ×10 11 g −1 ,18 months at 27 ◦ CB. bassiana ES, 2 ×10 10 g −1 ,18 months at 27 ◦ CB. bassiana Flowable formulation,Protected cropsBananasUnspecifiedMeadow turfA wide varietyof greenhouseand field cropsOrganicagricultureVarious:2.3 ×10 7 ml −1 glasshouseand outdoorsB. bassiana Liquid formulation,M. anisopliae+ B. bassianaB. bassiana+ P. fumosoroseusVarious:2.3 ×10 7 ml −1 vegetables,ornamentals, lawnsWPWPa WP=wettable powder, ES=emulsifiable concentrate, Verticillium=has not been renamed Lecanicillium unless the identityof the species is certain. This table was compiled from a search of the worldwide web, information direct from companiesand additional information supplied by CPL Business Consultants

170 A.K. Charnley, S.A.CollinsProducts are based on a restricted numberof species, primarily M. anisopliae, B. bassiana,B. brongniartii, P. fumosoroseus, Lecanicilliumlongisporum, L. muscarium. Some companies haveretained the name V. lecanii.Thefungiinquestionwould probably now be classified as L. longisporumor L. muscarium. However, unless the identity iscertain, the original names have been retained inTable 10.1. Most production is based on solid or diphasicsystems where conidia are concerned. Blastosporesare produced by liquid fermentation, e.g.Vertalec (Fig. 10.2a) and Vertiblast. There are someproducts which have more than one active ingredient,either two isolates of the same fungus (Vertikil)or several different species (Meta-Ven and Bassi).Thereislittlereliableinformationonthesituationin Russia. This is unfortunate because Boverin,aproductbasedonB. bassiana and used againstthe Colorado beetle Leptinotarsa decemlineataand the codling moth Cydia pomonella, usedtobe an oft-quoted success story. There may be over100 companies/organisations producing microbialpesticides in India. The ones cited in Table 10.1.appear to be the most significant. Elsewhere, inAsia AppliedChem in Thailand make Metazan(M. anisopliae)andBuverin(B. bassiana).Throughout South America, particularlyin Brazil, Peru, Columbia, Cuba, Mexico andArgentina and, to a much lesser extent, in Ecuador,Uruguay, Panama, Paraguay, Dominican Republic,Guatemala, Honduras, Nicaragua, Costa Rica andTrinidad and Tobago, small to medium commercialproducers, research stations and universities areinvolved with producing mycoinsecticides (Alveset al. 2003; Ray Quinlan, personal communication).These are largely based on M. anisopliaeand B. bassiana but there is some use of P. fumosoroseus,V. lecanii, Nomuraea rileyi, Sporothrixinsectorum, E. virulenta. Another oft-quotedproduct, ‘Metaquino’, used in Brazil for the controlof spittle bugs, Mahanarva posticata,onsugarcaneis produced by a local authority/NGO project.There are more than 60 workshops/cottagestyleproducers of unregistered mycoinsecticidesin China. New regulations require that producersof biopesticides must not only register their productsbutalsohaveaproductlicenseandacompanylicense to produce pesticides (Yuxian Xia, personalcommunication). Registration requirementsformycoinsecticidesarethesameasforchemicalpesticides. At the moment, only the two companiesdetailed in Table 10.1. have satisfied the newregulatory requirements. Entomopathogenic fungihave been used more widely on forest pests thanon crop pests (Feng 2003). The registered productsare aimed at pests in these two ecosystems.The most influential mycoinsecticide developmentproject was the multidonor LUBILOSAprogramme (Fig. 10.2b). Initiated in 1989, itinvolved research institutes from the UK, TheNetherlands and the republics of Benin and Niger.Theaimwastodevelopfungiasbiocontrolagentsfor grasshoppers and locusts. Twelve years laterthis programme, which involved 40 scientists ata cost of US$17 million, resulted in a commercialproduct, Green Muscle TM (Fig. 10.2c). It gives70–90% control of grasshoppers and locusts assoon as 5–15 days after application but, underless favourable conditions, at 14–20 days. It isrecommended for control of these pests by FAO(Bateman 1997; Jenkins et al. 1998). Lessons informulation and application, in particular, learnedfrom LUBILOSA have influenced much subsequentresearch and development, e.g. Green Guard TM forcontrol of locusts in Australia (see Table 10.1.).2. Constraints on EfficiencyA need for high humidity for disease initiationandspreadhasoftenbeenconsideredthemajorconstraint on the use of fungi for insect control,though this may not be as important with somepreparations as previously thought. Furthermore,RH in microenvironments, e.g. on a leaf surface dueto transpiration, may be higher than that measuredby equipment on a larger scale (Vidal et al. 2003).The inclusion of moisture-retaining substancesin aqueous formulations and the use of oil-basedformulations (see below) may help to overcome therequirement for a high environmental RH. Abilityof the disease to spread within a population isnot necessarily an issue with an efficiently appliedvirulent mycoinsecticide; however, one key advantageover chemical pesticides is lost if an epizooticor, at least, vertical inter-generational spread doesnotoccur.Developmentofmycosisisaffectedbyextremes of temperature (32 ◦ C)butoften the fungus is only delayed. In tropical regions,more equitable night temperatures may provide anopportunity for a fungus to initiate disease. However,insects such as locusts, which have the abilityto regulate their body temperature behaviourally,exploit this fungal weakness; maintenance of a highset point (behavioural fever) prevents mycosisand limits mycoinsecticide efficiency in the field(Blanford et al. 1998). Fungi are sensitive to UV.

Entomopathogenic Fungi and Their Role in Pest Control 171Experimentally, it has been possibly to alleviatethis problem by the addition of protectants tothe formulation (Hunt et al. 1995). In practice,theingredientsmaybetoocostly,thoughneweroil formulations themselves provide some UVprotection (Alves et al. 1998) and field persistenceis often longer than anticipated from UV studies.Mycoinsecticides should be compatible withother crop protection measures. Several studieshave shown that fungicides, herbicides and insecticidescan prevent germination and/or mycelialgrowth of entomopathogenic fungi in vitro (e.g.Moorhouse et al. 1992). However, pest control byfungi is often not affected by chemical pesticides,as long as there is a ca. 7-day gap between the twoapplications (Anderson and Roberts 1983; Moorhouseet al. 1992).Fungal pathogens act generally in a densitydependentfashion against their hosts and have relativelyslow kill; thus, they are not good candidatesfor pest control in crops with low damage thresholds.Specificity is often perceived to be an advantageof microbial pesticides generally. However,specificity can be a problem when there is a pestcomplex and no single pathogen can give control(Powell and Jutsum 1993; Ravensberg 1994).Plants have evolved chemical and physicaldefences against pathogens and herbivorous insects.However, these attributes can also influencesusceptibility of insects to their own pathogens.Whilst some of the defensive phytochemicalsmay be sufficiently stressful to cause an increasein the susceptibility of generalist feeders topathogens, some adapted insects can sequesterthese chemicals and may thereby acquire someprotection from their own pathogens (Gallardoet al. 1990). Interestingly, the entomopathogenNeozygites tanajoae produced more conidia whenexposed to volatiles from leaves damaged by thecassava mite, Monoychellus tanajoe, thantocleanair (Hountondji et al. 2005). Pea aphids were moresusceptible to the Entomophthoralean fungus Pandoraneoaphidis on pea cultivars with reduced waxblooms, because spores adhered more effectivelyto leaf surfaces with less wax (Duetting et al. 2003).3. Integration in Pest Management SchemesAlthough mycoinsecticides can provide standalonepest control, they may be regarded as oneweapon in an armoury of techniques employedin integrated pest management schemes (Dent2000). A virulent pathogen could have indirectdetrimental effects on existing natural control, e.g.by reducing the availability of hosts for parasiticinsects. However, use of fungi can help maintainbiocontrol on protected crops in the Northern Europemid-season when arthropod natural enemiesfalter due to adverse environmental conditions,and also help with pest clearance at the end of theseason (Jacobson et al. 2001).C. Development of a Mycoinsecticide1. Isolate SelectionThere are some 700 species of entomopathogenicfungi known from 85 genera. However, comparativelyfew have been investigated as potential mycoinsecticides.Natural epizootics caused in particularby fungi in the order Entomophthorales occurfrequently in natural and agricultural terrestrialecosystems (Samson et al. 1988). This has led toanumberofattemptstouseEntomophthoraleanfungi, e.g. for pest control of aphids, with varyingdegrees of success. Problems have included the inabilitytoculturecertainspeciesinvitroandthefactthat the most stable spore form is the resting spore,which is not infective and not produced by the mostpathogenic isolates (Latgé 1986). The genera Cordycepsand Torubiella also contain some virulent butobligate insect pathogens. As a consequence, mostdevelopment work has focused on certain Ascomycota,particularly M. anisopliae, B. bassiana, N. rileyi,Aschersonia aleyrodis and L. longisporum,andOomycota, e.g. Lagenidium giganteum, whicharemore readily cultured in vitro.Isolates are often selected on the basis of laboratorybioassay using cultured insects under optimalconditions. However, lead isolates need to bechecked in a commercial setting. Immersion of testinsects in a conidial suspension as part of a bioassayrepresents a temporary exposure to high inoculum.Prolongedexposureofinsects,e.g.insoil,mayprovide a lethal dose from a sub-lethal bioassayconcentration through the accumulation of sporeswith time (Ferron 1985). Laboratory assays do notallow for avoidance of the pathogen biologically,ecologically or behaviourally.There is no consensus about whether isolatesoriginating from the target host (homologous isolates)or isolates from other hosts (heterologousisolates) of an established pathogen, or an exoticfungus (a species which is not present in the geographicalarea of application), are likely to providethe most suitable candidate for a mycoin-

172 A.K. Charnley, S.A.Collinssecticide. One aspect of the debate is whether anadapted pathogen (homologous isolate) evolves towardsa balanced relationship with its host whichprecludes high virulence. There is evidence fromwork on human viral and bacterial pathogens thatthis may not always be the case (Ewald 1993); similarly,in a screen for fungal pathogens of the desertlocust, Schistocerca gregaria, themajorityofisolatesof Metarhizium spp. with high virulence camefrom this or related Acridids (Prior 1992). However,in other studies heterologous isolates have provedthe most virulent (e.g. see Vestergaard et al. 1995).Sincethereareconcernsoverthepossiblenontargeteffects of exotic fungi (e.g. see Lockwood1993), it may be wise to search and screen isolatesfrom the country where the mycoinsecticide is tobe deployed, in order to facilitate commercial registration(Prior 1992). Passage of the key isolatethrough the target host may select for increasedvirulence (Quesada-Moraga and Vey 2003).Studies have shown correlations between virulenceand attributes such as spore size, speedof germination and attachment to cuticle (P. fumosoroseusagainst the diamond-back moth, Altreet al. 1999), and toxin production (B. bassianaagainst locusts, Quesada-Moraga et al. 2006). Thus,a rational approach to strain selection has beenadvocated. Barranco-Florido et al. (2002) selectedV. lecanii isolates based on better growth associatedwith low water activity, and high chitinase and proteaseactivitiesaswellasCO2 production (measureof growth). However, the reality seems to be thatsuch studies may only go some way to explainingwhy a particular isolate is virulent, rather than providinga basis for a priori selection.The relevance of isolate virulence to pest controlis clear. However, high sporulation, stabilityduring bulk storage, and epizootic potential maybe equally important. Selection for ability to operateover a range of abiotic conditions, includingsoil chemistry, light, pH, likely to be encounteredin practice while having little impact on naturalenemies and other non-targets is a ‘biorationalapproach’ which has clear advantages (Yeo et al.2003). Since natural habitats have highly variabletemperatures, incorporation of cycling temperatureregimesintoselectionprogrammesisaninsightfuldevelopment (Lecuona et al. 2005).2. Production and FormulationLarge-scale in vivo cultivation of a fungus inlaboratory-reared or field-derived insects is usuallyemployed only for obligate pathogens whichdo not grow readily outside their hosts. This maybe a viable method of inoculum production fora programme of ‘introduction’ (Soper and Ward1981). However, it is unlikely to be economicfor large-scale mycoinsecticide use. The methodadopted for in vitro cultivation must take intoaccount that:1. Theinoculumproducedmusthaveoptimumvirulence and retain viability over an extendedperiod in storage and after application in thefield.2. Serial in vitro transfer can lead to loss ofvirulence (Brownbridge et al. 2001). Use ofsingle spore isolates can help to alleviate thisproblem and ensure uniformity of the product.However, genetic change through chromosometransformation, transposable elements,cytoplasmically transmitted genetic elementsand the parasexual cycle may promote drift(Couteaudier et al. 1994) and loss or reductionof pathogenicity factors (Shah and Butt 2005).Storage of the original culture under liquidnitrogen and periodic passage of the fungusthrough the host may be required to maintainvirulence of a product.3. The culture medium should balance the needsof cost effectiveness in terms of yield per unitoutlay with the production of a highly virulent,stable inoculum.4. The production system may affect the propaguletype and virulence, which may impact onkillingpower,shelflife,environmental stability,and the formulation and application strategies.5. The production system may have to be scaleduptoproducecost-effectivetreatmentonthousandsorevenmillionsofhectares(Lisanskyand Hall 1983; Bradley et al. 1992; Jenkins andGoettel 1995).6. Conditions for maximum productivity may notbe the best for producing good-quality sporesfor field application (Tarocco et al. 2005).Three types of production system have been employed:submerged (liquid) fermentation, surfacecultivation, and diphasic fermentation (see, e.g. reviewby Deshpande (1999).Submerged liquid fermentation would be thepreferred option because existing large-scaledeep-tank fermentation equipment could be used,and the process is most easily controlled and canbe much faster than other methods. The majordrawback is that dimorphic fungi like M. anisopliae,L. longisporum, Paecilomyces farinosus and

Entomopathogenic Fungi and Their Role in Pest Control 173B. brongniartii typically produce blastospores,rather than true conidia in liquid culture. In vitroproducedblastospores are similar to the in vivoyeast phase which enables many fungi to developand spread quickly in the haemolymph of the host.However, the wall structure of blastospores is oftensimilar to that of the mycelium and, being unpigmented,in vitro-produced blastospores are oftenunstable with limited shelf life and field stability.Yields of spores from submerged cultures are notas good as from aerial production systems (see below),e.g.5×109 ml −1 of V. lecanii(Latgé et al. 1986)and 1.1 ×10 9 ml −1 Paecilomyces fumosoroseus(Jackson et al. 2003). In fact, 5 ×10 12 ml −1 producedin a short time frame (45 h)mayberequiredfor commercial viability (Bradley et al. 1992).Despite constraints on their use, blastospores formthe basis of some commercial formulations, e.g.Vertalec (Fig. 10.2a) produced by Koppert, andVertiblast produced by Crop Solutions Ltd (seeTable 10.1.). Furthermore, blastospores are morevirulent than conidia of P. fumosoroseus againstthe silverleaf whitefly (Jackson et al. 2003). Certainstrains of B. bassiana (Feng et al. 1994), M. anisopliaevar. acridum (formerly M. flavoviride)(Jenkins and Prior 1993; Leland et al. 2005) andHirsutella thompsonii (Van Winkelhoff and McCoy1984) will conidiate in liquid culture in appropriatemedia, and yields of 1.5 ×10 9 ml −1 have beenachieved with M. anisopliae var. acridum –beinghydrophilic, however, these spores will requiredifferent formulation to the hydrophobic aerialconidia. Resting spores but not infective spores ofEntomophthora spp. are formed in liquid culture(Latgé 1986), as are the sexually derived oosporesof Lagenidium giganteum (Kerwin et al. 1986).Several methods have been developed for producingmycelia in submerged cultures. Myceliumis applied in the field where it will sporulate, producinginfective conidia. In the ‘Marcescent process’,first developed for Entomophthorales (Mc-Cabe and Soper 1985) and then adapted for Ascomycota(Rombach et al. 1988), mycelium is driedin a sugar desiccation process, milled and thenstored at low temperature prior to use. Fluid beddriedmycelial granules of M. anisopliae with a shelflife of at least a year at 4 ◦ C can provide good longtermcontrol of the vine weevil O. sulcatus whenincorporated into compost (Stenzyl et al. 1992).Dried mycelia fragments have been successfullyfield tested (Rombach et al. 1987) and a product,BIO1020, based on dried mycelial granules, hasbeen developed by Bayer AG in Germany (Stenzelet al. 1992). However, none of these mycelialpreparations have been developed into commercialproducts.In surface cultivation,theuseofsolidsubstrateis the most common method of production (seeKrishna 2005), though pH, temperature, nutrientstatus,andaerationmaybemoredifficulttocontrolthan in submerged liquid culture (Feng et al.2000). Large-scale production has been carriedout on agricultural, brewing or other wastes, e.g.Dalla Santa et al. (2005), though such media canbe too variable and of low immediate metabolicavailability. The high surface-area-to-volumeratio of small cereal grains such as sorghumand rice leads to better nutrient absorption,gas exchange and heat transfer. Aeration canbe improved by the use of a rotating drum;however, premature germination and reducedyield can occur when conidia are dislodged fromconidiophores (Lisansky and Hall 1983). The sizeof the initial inoculum may influence spore yield,and procedures need to be optimised to minimisehyphal growth and maximise sporulation. Kanget al. (2005) used a packed-bed bioreactor withrice and straw to produce B. bassiana for control ofthe diamond-back moth. The yield was 4.9 ×10 8conidia per g without support and 1.1–1.2 ×10 10conidia per g on polypropylene foam. New designsfor solid-state fermentation bioreactors have beendeveloped experimentally, including that of Ye et al.(2006). The latter comprised upright multitrayedconidiation chambers which produce 2.4 ×10 12Beauveria conidia per kg rice. Mycotech Corporation(now owned by Laverlam International)used a computer-controlled, forced-aerationsolid-state fermenter to produce B. bassiana atarateof3×10 13 per kg of starting material in 1 lof fermenter space (Wraight et al. 2001). Giventhe capital outlay with sealed, automated bioreactors,such systems are only cost effective withhigh output; otherwise, simpler labour-intensivesystems are employed (Jenkins et al. 1998), n.b. thecottage-style industries which have proliferated incertain parts of the world.In diphasic fermentation, in most cases solidstatefermentation is preceded by liquid fermentationin order to provide a mycelial inoculum. Fungusis grown in fermentation tanks to the end of logphase. The resulting mycelium is then applied to eithera nutrient or non-nutrient surface. The latterallows fine tuning of the nutrients provided to promoteoptimum spore production. A large surfacearea to volume is important (Jenkins et al. 1998).

174 A.K. Charnley, S.A.CollinsSolid, absorbent non-nutrient surfaces such as claygranules, vermiculite, sponge or clothe are used.Grain and particularly white rice are commonlyused as nutrient support surfaces. The secondstagefermentation of B. bassiana as Boverin hasbeen based on the use of shallow layers of sterilisedliquid media in polypropylene plastic bagsinoculated with mycelium (Bradley et al. 1992).Such an approach does not appear to be in currentuse. Mycotech have developed a two-step solidstatefermentation method for producing conidiaof B. bassiana. In this system, inoculum is producedin liquid fermenters and then placed in bioreactors(seelastsection)whereitisabsorbedontoastarchbasedsolid substrate.Harvesting the fungus from liquid culture isusually a matter of centrifugation, then rapid controlleddrying to prevent bacterial growth, thoughexcessive temperatures can reduce viability. Followingsolid-substrate cultivation, spores can bewashedoffthesubstrateordriedinsitutoasuitablemoisture content, then milled. Water contenthas an important bearing on conidial storage characteristicsand temperature tolerance; increasingthe level of desiccation can increase temperaturetolerance of M. anisopliae and M. anisopliaevar. acridum conidia (Moore et al. 1995). Dryharvesting may be achieved using apparatus likethe cyclone MycoHarvester (DropData at InternationalApplication Research Centre, ImperialCollege http://www.dropdata.net/mycoharvester/,and ACIS http://www.acis.co.uk/index.htm).Thefungusneedstobeformulatedtohelpstabilisethe product during storage and to facilitatedelivery to the insect target in the field (Burges1998; Wraight et al. 2001). An 18-month storageperiod is ideal for economic use. Mechanicalharvesting and dry storage of unformulated conidiacan prolong viability (71%, 12 months, 4 ◦ C,B. bassiana; Chen et al. 1990, quoted in Feng et al.1994). Blastospores are more difficult to formulatebecause of their instability. Freeze-drying has beenused (3 months, B. bassiana, Belova 1978). Lactoseandsucrose-enhanced desiccation tolerance ofP. fumosoroseus blastospores during freeze-dryingwhile whole milk in a starch–oil formulationallowed storage at −20 ◦ C for a year without lossof viability (Jackson et al. 2006). Spray-driedblastospores of M. anisopliae var. acridum were68% viable after 1 year at 20 ◦ C (Stephan andZimmermann 1998). M.anisopliaevar. acridum(= flavoviride) conidia harvested in the lightpetroleum fraction oil Edelex or groundnut vegetableoil, then diluted with Shellsol K, deodorisedkerosene (Edelex) or an antioxidant (groundnutoil), retained 60% viability after storage at 17 ◦ Cfor 30 months, as long as they were dried bythe addition of non-indicating silica gel to theformulation (Moore et al. 1995). In dry or wettablepowder formulations, the main ingredient maydilute the ‘active ingredient’ to a concentrationwhich can be handled more easily. Phyllosilicates(clays) are the most commonly used, as they arerelatively inert and cheap. They can promoteconidial viability over extended periods (Ward1984). Coating of ingredients may include stickers,humectants, UV protectants, an emulsifier forwater-based spraying of hydrophobic spores andnutrients, though inclusion of nutrients may bea natural consequence of the production process.Either way, nutrients in the formulation may allowsaprophytic growth and sporulation on foliage,increasing inoculum potential, and enhancingthe chances of secondary pickup and verticalinfection, e.g. aphid formulation Vertalec® ofL. longisporum (Fig. 10.2a).Many of the currently available mycoinsecticidescomprise conidia in wettable powders whichcan be delivered in water using simple hydraulicsprayers. The development of oil-based formulationsin the 1990s was a significant development.Vegetable (e.g. sunflower, soybean), mineral andparaffinic oils (e.g. Shellsol K, Ondina) are compatiblewith hydropobic conidia of Beauveria andMetarhizium, can be applied from ULV sprayersand do not evaporate (Bateman 1997; Burges 1998).Emulsifiable adjuvant oils (EAO) (paraffin- or vegetableoil-based) allow conidia and oil to be mixedin water, thereby reducing the amount of oil whichhas to be used (Polar et al. 2005). An adjuvant ofemulsified vegetable oil (‘Addit’) increases the efficiencyof the whitefly formulation Mycotal® ofL. muscarium at low RH. Plastic-lined foil (trilaminate)bags, with small packets of silica gel to ensuredry conditions, allowed storage of M. anisopliaevar. acridum foroverayearat30 ◦ C,thoughrapidrehydration of these dry spores is lethal (Jenkinset al. 1998). Dry powder formulations retained 70%viabilityafter7yearsat5 ◦ C (Dave Moore, personalcommunication).Use of mycelial preparations presents differentproblems for formulation. Pure dry myceliumtreated with maltose or sucrose produced moreconidia after storage (Pereira and Roberts 1990).Bayer’s BIO1020, consisting of mycelial granules,was stored vacuum packed after fluid-bed drying

Entomopathogenic Fungi and Their Role in Pest Control 175andretainedtheabilitytoformconidiaafterrehydrationfollowing 12-month storage at 4 ◦ C.Incorporationof mycelium into alginate pellets, with orwithout additional nutrient sources, has been triedsuccessfully with B. bassiana (Pereira and Roberts1991), providing enhanced shelf life and environmentalstability particularly against solar radiationafter application. Cornstarch or cornstarch oilformulation also enhanced conidial production bymycelium after several months storage (Pereira andRoberts 1991).3. ApplicationThe timing of application may be important. Suitableweather conditions (high humidity, equitabletemperature) may occur during late evening/earlymorning.Thepestshouldbeatthemostsusceptiblestage (the most juvenile stages are often the mostsusceptible and easiest to control). The method ofapplication depends on the nature of the inoculumand the niche of the pest insect.Conidia or blastospores of Ascomycota such asB. bassiana, M. anisopliae and L. longisporum canbe suspended in a liquid or mixed with a powdercarrier and sprayed with conventional machineryused for the application of synthetic chemicalinsecticides. High-volume hydraulic sprayersareusedtoapplyL. longisporum spores in wateragainst aphids on chrysanthemums in glasshouses.Secondary spore pickup, rather than just directhit, is critical to success, which makes sedentaryinsects like Aphis gossypii more difficult to control.Repeat low-dose treatments may be helpful (Helyerand Wardlow 1987), though regular use of largevolumes of water on a crop may promote plantfungal disease. Koppert recommend 2–3 applicationsof Mycotal 7 days apart to control whiteflieson cucumber. Improved targeting against aphids,which prefer abaxial leaf surfaces, can be providedby electrostatic sprayers. The equipment impartsachargetodroplets,therebyincreasingabaxialdeposition and aphid control (Sopp et al. 1989).A more recent study was less encouraging of thisapproach (Saito 2005).Mist blowers and helicopters have been usedsuccessfully to apply B. brongniartii to swarmingadults of the cockchafer Melolontha melolonthain Switzerland (2 ×10 14 spores ha −1 ). Ultra-lowvolume(ULV) application of water- or mineraloil-based formulations on the ground or by aircrafthas been used on >0.8 million hectares in Chinaforthecontrolofvariousforestandcropinsects(Ying 1992). Bateman et al. (1993) have shown inlaboratory experiments that mineral or vegetableoil formulations of M. anisopliae and M. anisopliaevar. acridum were more virulent than water-basedformulations against the desert locust, Schistocercagregaria, and decreased reliance on high environmentalRH. These formulations in ULV sprays havebeen used successfully in field trials against thebrown locust Locustana pardalina in South Africa(Bateman et al. 1994), the variegated grasshopperZonocerus variegatus in West Africa (Lomer et al.1993) and Australian plague locust (Hunter et al.2001). ULV oil locust formulations of M. anisopliaeneed to be sprayed as droplets of 40–120 μm,with500–10,000 spores per drop at a concentrationof 5 ×10 12 spores per litre and rate of 1 l per ha(Bateman and Chapple 2001). Only a few dropletsare needed to produce a dose which kills in2–3 weeks. Diseased insects are usually soslow that they are taken by predators beforedeath from mycosis (Dave Moore, personalcommunication).Soil-borne pests can be treated either prophylacticallyor curatively with fungal spores. Priorincorporation of conidia of M. anisopliae intocompost can give yearlong protection of Impatienswallerana against the vine weevil (Moorhouseet al. 1993). The success of drenches of aqueousspore suspensions is influenced by the depth towhich spores percolate, the volume of the drench,adsorption to soil particles, and movement ofthe insect which will facilitate uptake of a lethaldose. Direct drilling of M. anisopliae conidia usingexisting (crop sowing) machinery to a depth of20–25 mm in pasture gave long-term control of theredheaded cockchafer Adorphorus couloni in trialsin Tasmania (Rath 1992).A novel method of pest control was suggestedby the observation that B. bassiana exists as anendophyte in certain genotypes of maize (Vakili1990). Bing and Lewis (1991) prepared a granularformulation of B. bassiana by spraying a suspensionof B. bassiana (1.1 ×10 8 conidia g −1 )ontocorn grits in a rotating drum of a Gustafon minimixer.Handheld inoculators were used to apply0.4 g of the granules to whorls of plants (10 7 ×4.55conidia plant −1 2.5 ×10 12 conidia ha −1 ). Conidiaof B. bassiana applied in this way moved withinthe plants and provided season-long control of theEuropean cornborer, Ostrinia nubilalis.Nofurtherprogress has been made with this system, thoughestablishing Beauveria as an endophyte in cottonexpressing a Bt toxin gene implies the possibility of

176 A.K. Charnley, S.A.Collinssynergy between these forms of pest control (Lewiset al. 2001).Baits and traps provide ways of bringing theinsects to a source of inoculum, rather than thereverse. Application of conidia of B. bassiana tobranhasgivensomesuccessintrialsagainstrangelandgrasshoppers in Canada (Johnson and Goettel1993). Sex pheromone has been used to attractmale diamond-back moths Plutella xylostella totraps where they are infected with Zoophthora radicans.Fungus is carried by contaminated mothsto susceptible larvae, thereby initiating or enhancingan epizootic (Furlong et al. 1995). Traps lacedwith conidia of M. anisopliae were commerciallyavailable in the USA for cockroach control. Suchautodissemination methods are target specific, reducethe amount of fungus required, and protect itwhile it is in the trap (Shah and Pell 2003).Experimental infection of aphids with L. longisporumis enhanced in the presence of aphid alarmpheromone or a sub-lethal dose of the insecticideimidacloprid, which increase movement and thelikelihood of acquiring a lethal dose of spores froma leaf (Roditakis et al. 2000).Preparations of dried mycelial pellets whichneed to be rehydrated prior to the production ofinfective conidia are particularly suitable for applicationto soil (Stenzyl et al. 1992). Direct drilling ofcereal grains with sporulating fungus is also used(see Table 10.1.).4. SafetyIt is sometimes assumed a priori that microbialpesticides must be considerably safer to humansand their environment than synthetic chemicalinsecticides. However, quite correctly, registrationprocedures in most countries require that mycoinsecticides,like chemical insecticides, are safetytested. Risk assessment frameworks are still evolving.Current registration protocols are often basedon those devised for synthetic chemical insecticidesand are not appropriate for insect pathogens(Hokkanen et al. 2003a). Risk assessments need toaddress the following: define the ecological contextin which the mycoinsecticide will be operating andchoose the most appropriate non-target species totest; determine host specificity; assess the abilityof the fungus to disperse from the area of treatment;assess the ability of the fungus to becomeestablished; determine direct effects of the funguson non-target species; determine the indirecteffects on non-targets (including the possibilityof competitive displacement); check specificallyfor mammalian allergenicity; finally, collate theinformation into a ‘risk assessment’ (Goetteland Jaronski 1997). The possible side effects ofentomopathogenic fungi may be summarised asinfections, toxicosis and allergies in non-targetanimals or humans. However, more subtle effectsmay occur, such as competitive displacement of nativeentomopathogens if non-indigenous isolatesare used, and depletion of other natural enemiesbecause of the decline in host population. Amongnon-target arthropods, mycoses of honeybees,bumblebees, parasitoids and predatory beetleshave been shown in laboratory tests and fieldexperiments (Hokkanen et al. 2003b; Vestergaardet al. 2003). The absence of natural epizooticsof candidate fungi among pollinators suggeststhat the risks are low, while several studies haveshown the compatibility of fungi with parasitoidsand predators in integrated control programmes.For example, Jaronski et al. (1998) found a lowimpact on natural enemies of ‘above label’ dosesof B. bassiana applied against whitefly on cotton.Interestingly, a number of experimental studieshave shown avoidance behaviour by predators(Meyling and Pell 2006) and parasites (Lord 2001)when faced with conidia from insect pathogens.Some studies have shown mortality of non-targetinvertebrates in the field. However, they are lowerthan those found in laboratory tests and can beminimised with appropriate application methods,doses and timing (Vestergaard et al. 2003). Thekey point appears to be that laboratory assays withfungi like B. bassiana, B. brongniartii, M. anisopliaeand P. fumosoroseus canresultinmortalityamong beneficials which is largely absent in fieldapplications for ecological or behavioural reasonsor absence of stress. Judicious choice of isolates willalso help; Butt et al. (1998) showed that honeybeescouldbeusedtovectorM. anisopliae to causeinfection of pollen beetles without adverse effect.A further aspect of risk evaluation is the needto monitor the fate and impact of an introducedfungus (Bidochka 2001). This requires techniquesto be able to identify a genotype in an environmentcontaining related organisms (Goettel and Jaronski1997). A variety of approaches have been takento produce suitable molecular markers (Bidochka2001). RFLP analysis, allozymes and cloned DNAprobes enabled differentiation between two closelyrelated Entomophthoralean pathogens, E. aulicaeand E. maimaiga, in populations of the gypsy mothin eastern USA. The dominance of the introduced

Entomopathogenic Fungi and Their Role in Pest Control 177E. maimaiga during epizootics was elegantly shown(see Bidochka 2001). A single, molecular signaturewould be much more efficient (see, e.g. Castrilloet al. 2003); the approach taken by Hu and St Leger(2002) involved tracking an isolate of Metarhiziumexpressing a green fluorescent protein gene (seenext section).A particular safety problem occurs in areasof China and India where the silkworm, Bombyxmori, isrearedinlargenumbersinthesilkindustry.Beauveriosis caused by native isolates ofB. bassiana is potentially a natural, severe problemand thus mycoinsecticides are kept away from areaswhere the industry is prevalent.Very occasionally, vertebrate infections havebeen reported with entomopathogenic fungi,though none have been associated with their use aspest control agents. None of the entomopathogenicfungi currently in use or under considerationare invasively pathogenic to humans. However,immunocompromised individuals are open toopportunistic infections which very occasionallyhave included entomopathogens (see, e.g. Revankaret al. 1999), though none of the fungi underdevelopment can grow efficiently at 37 ◦ C.Many Hypocreales produce toxic secondarymetabolites in vitro which have detrimental effectson whole animals and cultured cells. The extentto which these chemicals contribute to the diseaseprocess is not know, though several studies haveimplicated cyclic peptides in the pathogenesisof B. bassiana and M. anisopliae (see Charnley2003). The amount of the cyclic peptide, destruxin,produced by M. anisopliae in the inoculum orduringmycosisinaninsectisverysmalland,thus,environmentally damaging levels are unlikely tooccur (Strasser et al. 2000). Furthermore, studiessuggest that known toxins such as destruxins donot enter the food chain or contaminate the watersupply following field application (Vey et al. 2001).Several groups have been investigating the bestway to determine the toxicity of mycotoxins duringsafety assessments (Skrobek et al. 2006), thoughrisk of exposure appears to be more importantto those charged with regulating products (DaveChandler, personal communication). Choosingan isolate which produces no or little toxinwould minimise this risk. Quality control duringproduction is also essential to ensure there is nocontamination from toxicogenic spoilage fungisuch as Aspergillus flavus.Hypersensitive reactions to fungal antigens derivedfrom hyphae, spores or from metabolites areperhaps the most likely health hazards to humans.Experiments with mice, rats and guinea pigs suggestthat the main route of sensitisation is the respiratorysystem, and people involved with largescaleproduction are most at risk (e.g. Ward et al.2000).5. RegistrationThe cost, complexity and between-country inconsistencyof registration are major constraints onthe development and use of biopesticides. However,pragmatism is becoming the order of theday. Most registration schemes for biopesticideshave followed the chemical pesticide model in requiringstudies on efficacy, toxicity, and impactagainst non-target and beneficial species (Thomaset al. 2000). However, recognition of the problemsof small companies, the additional environmentalbenefit of the use of biopesticides, and the intrinsicallybenign nature of biocontrol agents haveresulted in the introduction of reduced registrationrequirements. Within the European Union,a recent development of Objective 3 of the Directive91/414/EEC ‘Plant Protection Products Regulations’advocates ‘reducing the levels of harmful activesubstances by substituting the most dangerouswith safer (including non-chemical) alternatives’(NAT/156 2003). In addition, the thematic strategyon the sustainable use of pesticides by the EC Commissiongives clear encouragement for biopesticidedevelopment.An interesting development in the EU is theintroduction of tailored requirements for biopesticideregistration, rather than the use of a simplifiedversionofthoserequiredforsyntheticchemicals.Thus, 2001/36/EC, for example, asks for thelife cycle of the microorganism and its infectiveness,relationships to known human and animalpathogens, stability and ability to produce toxins(Hamer 2003). On the back of the EU initiative,UK PSD launched a new system in 2006 which hashalved the registration charge for a biopesticide(PSD 2006). The US EPA has a tiered approach tothe registration process. If the results of the first tiertests indicate no adverse effects, then data from theother tiers are not required. Furthermore, there isno need for proof of efficacy (Cole 2004). The reductionin data requirements has led to significantcost savings, and biopesticides are now often registeredinlessthan1year,comparedtoanaverageofover 3 years for conventional insecticides (Redbond2004).

178 A.K. Charnley, S.A.CollinsVI. Future DevelopmentsA. Potential TargetsEntomopathogenic fungi have potential for controlof some but not all insect pests, and it is importantto identify appropriate targets for mycoinsecticidedevelopment. Fungi, in contrast to bacteriaand viruses, invade their hosts by actively penetratingthe exoskeleton (cuticle). Therefore, fungiare particularly important natural pathogens ofsucking insects such as aphids, whitefly, thrips andleafhoppers, since the feeding strategy of these insectstends to preclude acquisition of pathogenswhich are infectious per os. Larval and adult beetlesare frequently hosts to fungal infections but appearto have comparatively few bacterial and viralpathogens. Thus, fungi are often the pathogens ofchoice for bug and beetle pests (Samson et al. 1988).Certain ecological niches lend themselves particularlywell to the deployment of mycoinsecticides.The habitats in question have in commonthat chemical control is difficult or inappropriate(concerns over human or environmental health)and the environments are conducive to fungal infection(e.g. high RH). The overwhelming interesthas been in exploring the potential for M. anisopliaeand B. bassiana.Some examples of recent investigations arework on thrips (Ekesi and Maniania 2003), theSunn Pest (Eurygaster integriceps, apentatomidbug),amajorinsectpestofwheatandbarleyinWest and Central Asia (Parker et al. 2003), andthericewaterweevil,apestofriceinNorthandSouth America (Chen et al. 2005). However, failureof research in the 1980s, on fungal control of thebrown plant hopper Nilaparvata lugens,themajorrice pest in SE Asia (see, e.g. Aguda et al. 1987), toresult in a commercial product is a reminder ofthe difficulty of treating a large-acreage, low-valuecrop economically with a biopesticide. Of insectswhich target animals and humans, rather thancrops, work on tsetse flies (Maniania et al. 2003)and mosquitoes (Blanford et al. 2005) can behighlighted. Interestingly, the B. bassiana isolateused against mosquitoes was not the most virulent.Instead, one which formed the basis of an existingagricultural mycoinsecticide was employed, as itwas thought that this would make registrationeasier. Parasitic mites (Smith et al. 2000), ticks(Samish et al. 2004), blowflies (Wright et al. 2004),reduviid bugs such as Triatoma (Lazzarini et al.2006) and the bee parasite Varroa destructor (Shawet al. 2002) have all been investigated as potentialtargets for mycoinsecticides.Of beetle pests, the Asian longhorned beetle,Anoplophora glabripennis, whichisalreadyusedto control B. brongniartii in its native Japan (seeTable 10.1.), is being considered for treating introducedpopulations in the USA and Canada. Studieson stored product pests (Throne and Lord 2004),fire ants (Brinkman and Gardner 2004), moundbuildingtermites (Milner 2003) and Brassica rootflies (Delia radicum and Delia floralis) (Eilenbergand Meadow 2003) show the variety of potentialtargets for mycoinsecticides. A Metarhizium-basedproduct aimed at cockroaches and developed in theUSA was never effective enough to become a commercialreality.B. Constraints on the Commercial Useof Entomopathogenic FungiDevelopment of mycoinsecticides would have occurredmore quickly with greater investment frommultinational agrochemical companies. The commercialview of insect pathogens has been that theyare too specific, too expensive, difficult to formulate,too erratic, have a short shelf life and are difficultto patent (Lisansky 1999; Butt et al. 2001).This has led on the whole to the development ofmicrobial insecticides by small to medium-sizedcompanies for niche markets, viz. where chemicalsdo not work well (through resistance or thewithdrawal of registration, for environmental reasons,ofeffectiveproducts,e.g.protectedcrops),orare environmentally unacceptable and have beenbanned (e.g. forests in North America). In addition,these niches have environments which promoteactivity of mycoinsecticides, viz. protectionfrom temperature extremes, UV and desiccation,and crops which can sustain some damage withouteconomic loss. The successful development of fungifor use against locusts and grasshoppers (the productGreen Muscle; Fig. 10.2c), which came fromthe LUBILOSA project (Fig. 10.2b; Bateman 1997),needed significant public investment. Green Musclegave long-term control through low-level recyclingwith low impact on natural enemies in inhospitableterrain over large areas; by comparison,resurgence occurred in plots where organophosphateinsecticides were used.Implementation of re-registration schemesfor existing chemical pesticides and governmentbackeduse reduction schemes in a number of

Entomopathogenic Fungi and Their Role in Pest Control 179countries, e.g. Denmark, has reduced the numberof chemical products available and may openup more niche markets for microbials, includingfungi. Growing public demand for food with lowor no chemical residues increases the pressure forbiological alternatives to synthetic pesticides. Thecost of registration in the past has been a majorconstraint on the development of mycoinsecticides.However, as indicated in Sections V.C.5 and VI,there are grounds for optimism with new initiativesboth in Europe and the USA in this domain.The rate of application of a mycoinsectide requiredto give adequate control is critical to commercialsuccess.Therateisinfluencedbythesigmoidaldose-response of insects to doses of sporesfrom entomopathogenic fungi. This results in the‘Allee effect’, i.e. there is a threshold number ofspores before an infection can be initiated (Deviand Rao 2006). Whatever the reason for this, itincreases the rate required for effective pest control;10 13 conidia ha −1 is the current bench mark(Bradley et al. 1992). Good control of the vine weevilon glasshouse ornamentals was achieved experimentallyusing an equivalent dose of 1.6 ×10 14conidia ha −1 (Moorhouse et al. 1993). The recommendedrate for the use of L. muscarium as Mycotalagainst glasshouse whitefly is 3 ×10 13 conidia ha −1on cucumber. B. bassiana as Boverin was usedat 6 ×10 12 –2.2 ×10 13 spores ha −1 against the Coloradobeetle. Since Mycotal contains 10 10 conidiag −1 and Boverin contains 6 ×10 9 –1.2 ×10 10 conidiag −1 , these rates represent as much as 10 kg ormore of product per hectare (Bradley et al. 1992).Improvements in application technology, e.g. ULVspraying of oil formulation, have resulted in muchlower rates being used. Only 100 gha −1 is recommendedfor the locust mycoinsecticide Green Muscle(Fig. 10.2c), and as little as 25 gha −1 can beeffective (Dave Moore, personal communication).C. Strain ImprovementAs our understanding of the epizootology ofdisease and the biochemical basis of pathogenicity/virulenceof entomopathogenic fungi improves,and techniques are developed for their geneticmanipulation, it will be possible to devise strategiesfor strain improvement. Characteristics whichcould be addressed include: enhanced shelf life andenvironmental stability (e.g. UV resistance, temperaturetolerance), improved sporulation duringmass production, ability to initiate infection at lowhumidity, expansion of the host range, acceleratedkill (reduced LT 50 ), and increased killing power(reduced LD 50 ).Metarhizium spp. and Beauveria spp. arefacultatively saprophytic, with soil-dwelling (rhizosphere)and pathogenic stages. Thus, epizooticpotential may be improved by engineering traitswhich increase saprophytic potential. To thisend, Wang et al. (2005) used ESTs and cDNAmicroarrays to explore gene expression duringgrowth on a plant root exudate. Intriguingly, thetranscriptome in this medium (nutrient poor) wassimilartothatonSDAbroth(nutrientrich)butvery different to that on either host (Manducasexta)cuticleorhaemolymph.Culture conditions can influence the characteristicsof fungal spores and can be manipulatedto increase mycoinsecticide efficiency. Blastosporesof B. bassiana from nitrogen-limited cultures hadhigher concentrations of carbohydrate and lipidand were significantly more virulent (lower LT 50 )towards the rice green leafhopper than were blastosporesfrom carbon-limited cultures (Lane et al.1991). Growth of B. bassiana, M. anisopliae andP. farinosus on agar-based media with low wateractivity or with a high concentration of glycerol encouragedaccumulation of polyols in conidia whichwere more pathogenic at lower RH than those producedon control media (Hallsworth and Magan1994, 1995).Genetic modification of entomopathogenicfungi to improve efficiency of pest control iscomplicated by the fact that the leading candidatesare largely Ascomycota with no easily manipulatedsexual stage. The parasexual cycle and protoplastfusion have been used to cross isolates of M. anisopliaeand L. longisporum (see review by Healeet al. 1989). However, rarely have the progeny hadimproved characteristics – indeed, the reverse canbe the case. It has been suggested that disruptionof clusters of pathogenicity genes is the cause.However, Riba et al. (1994) successfully crossedthe non-entomopathogenic B. sulfurescens, whichproduces an entomotoxic glycoprotein, with anatoxigenic pathogenic isolate of B. bassiana.Stable, partial diploid, hypervirulent, toxigenicrecombinants ensued.Strain selection and parasexual crossing maybe the most effective method of obtaining environmentaltolerance, as these traits are probablycontrolled polygenically. The alternative, direct geneticmanipulation would provide enhanced targetingfor single genes or gene clusters. Genetic en-

180 A.K. Charnley, S.A.Collinsgineering needs the establishment of transformationand cloning systems, which have been achievedfor M. anisopliae, B. bassiana and P. fumosoroseus(Bernier et al. 1989; St Leger et al. 1992b; Smithsonet al. 1995; dos Reis et al. 2004; Lima et al. 2006).The growing literature on mechanisms ofpathogenesis and the development of techniquesto genetically modify insect pathogenic fungisuggest strategies for rational strain improvement.It will be possible to increase isolate virulenceand/or extend the host range by altering thetiming and release of virulence factors, increasecopy number of virulence genes, and introducespecificity genes from other isolates or toxin genesfrom other organisms (St. Leger and Screen 2001).Indeed, St Leger et al. (1996) have inserted extracopies of the pr1 gene (encoding the proteasePR1a) from M. anisopliae into the genome ofM. anisopliae such that the gene was constitutivelyoverexpressed in the tobacco hornworm, Manducasexta. This resulted in the activation of the host’sprophenoloxidase system. In comparison with insectsinfectedwithwildtype,thecombinedeffectsof PR1 and the reaction products of phenoloxidasecaused a 25% reduction in time to death and a 40%reduction in food consumption by insects infectedwith engineered fungus. Transforming M. anisopliaeto express an insect-specific scorpion toxinhas also enhanced virulence against mosquitoesand caterpillars (M. sexta) (St. Leger, personalcommunication). An additional strategy for thecontrol of vector-borne diseases is to use funginot only to control the insect but as a vehicle forintroducing anti-parasite genes, e.g. M. anisopliaeversus mosquitoes which transmit Plasmodium,the causative agent of malaria (Blanford et al. 2005).Molecular biological studies on other entomopathogenssuch as baculoviruses, though muchmore advanced than those on fungi, have stillnot resulted in the commercial production anduseofGMforms.Thisisdespitethefactthatbaculoviruses expressing insect-specific toxinsfrom mites and spiders have been shown incarefully regulated, small-scale field trials to bemore effective than wild type and environmentallysafe (Kamita et al. 2005). Any attempt to assessthe likely environmental impact of a geneticallyengineered fungus is hampered by a basic lackof understanding of the population structure ofnaturally occurring fungal genotypes (Hajek et al.2000). Hu and St Leger (2002) used a M. anisopliaeisolate expressing green fluorescent protein (GFP)gene alone or with extra copies of a homologousprotease gene pr1 in an EPA-approved field releaseinto a plot of cabbage plants. This study showed:theusefulnessofGFPformonitoringfungi;noevidencefor transmission to non-target organisms;genetically modified organisms were stable for>1 year under field conditions; no displacement ordepression of native culturable, fungal microfloraand maintenance of the Metarhizium in therhizospherewithdeclineinthenon-rhizospheresoil. Before further use could be contemplated,however, detailed assessment of potential environmentalrisk of a candidate, genetically modifiedmycoinsecticide would be necessary along the linesof that advocated by Snow et al. (2005) to minimisethe likelihood of negative ecological effects.VII. ConclusionsMycoinsecticides have had comparatively littleglobal impact on insect pest control to date. However,the pressing need for alternatives to chemicalpesticides, and progress in research on epizootology,mass production, formulation, applicationand mechanisms of pathogenesis and host defencesuggest optimism for the future of mycoinsecticides.Furthermore, the drive to pesticide-freeproduction in some sectors is a helpful development,e.g. the British Tomato Growers Associationhas a commitment to pesticide-free production inthe next 10 years, which is re-enforced by retailerdrive for zero-pesticide residues. The US EPA’sintroduction of comparative risk assessment forpesticide registration, as part of a policy to promotereplacement of existing products with saferalternatives, has reduced conventional pesticideregistrations and increased that of biologicals and‘low-risk chemicals’ in the last 6 years. Despitethese and other initiatives to encourage the developmentof biological alternatives to pesticidesmarket (see Sect. V.C.5.), entry costs for SMEs formicrobials are still onerous. In The Netherlands,thereisaprogrammewhichsubsidisesuptohalfof the registration costs of biopesticides. Likewise,in the USA, the IR4 programme subsidises costsfor agents designed for small-market use. There isa strong argument for more widespread use of publicmoney to subsidise biopesticide development,as this is in both national and international interest.It is to be hoped that more governments will recogniseand act in this way and thus promote activelythe development and use of mycoinsecticides.

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11 Bacterial Weapons of Fungal Destruction:Phyllosphere-Targeted Biological Control of Plant Diseases,with Emphasis on Sclerotinia Stem Rotand Blackleg Diseases in Canola (Brassica napus L.)W.G.D. Fernando 1 , R. Ramarathnam 1 ,T.deKievit 2CONTENTSI. Introduction ........................ 189II. Biological Control .................... 190III. Phyllosphere Biocontrol ............... 191A. Chemical and Physical EnvironmentoftheLeafSurfaces ................ 191B. Bacterial AdaptationsinthePhyllosphere ................ 191IV. Bacterial Phyllosphere Biocontrolof Plant Diseases, with Special Emphasison Sclerotinia Stem Rot and Blackleg ..... 191A. TimingofApplication .............. 192B. MechanismsofBiocontrol........... 1921.Antibiosis...................... 1922. Pre-Emptive ColonizationofTargetSite ................... 1943.AntifungalVolatiles.............. 1944.InducedSystemicResistance....... 195V. Conclusions ......................... 196References.......................... 196I. IntroductionCanola, also known as oilseed rape, has worldwideproduction, with China, Canada, Australiaand the European Union leading the production.In Canada, the 10-year average is 11.3 million acresharvested, making it the largest single producerof canola (Canola Council of Canada 2006). Developedin 1974, a rapeseed variety to qualify ascanola has to fit the following definition: “an oilmust contain less than 2% C22:1, and the solid componentof the seed must contain less than 30 micromolesofanyoneormixtureof3-butenylglucosinolate,4-pentenyl glucosinolate, 2-hydroxy-3-butenylglucosinolate, and 2-hydroxy-4-pentenyl glucosinolatepergramofairdry,oilfreesolid”(Adolpheet al. 2002). Canola is currently more valuable thanpeanut, cottonseed and sunflower as a source of1 University of Manitoba, Department of Plant Science, 66 DafoeRoad, Winnipeg, Manitoba R3T 2N2, Canada2 University of Manitoba, Department of Microbiology, Winnipeg,Manitoba R3T 2N2, Canadavegetable oil (Sovero 1993). It is the third most importantsource of vegetable oil.Sclerotinia stem rot, caused by Sclerotiniasclerotiorum (Lib.) de Bary, and blackleg, causedby Leptosphaeria maculans (Desm.) Ces & De Not(anamorph, Phoma lingam (Tode: Fr./Desm.)),are the two most economically important diseasesof canola. Both diseases lead to significantlyhigh yield loss in western Canada, with up to5–100% (Manitoba Agriculture 2002) reportedfor stem rot and 50% for blackleg (Petrie et al.1985). The primary inoculum of both diseasesoriginates from overwintering structures. Forstem rot, the overwintering sclerotia germinatecarpogenically to produce apothecia (Willets andWong 1980) which, in turn, produce ascospores,the primary inoculum which germinates andinfects senescing petals. The infected petals,which act as a nutrient source for the germinatingascospore, fall onto the leaves where thefungus infects through mechanical pressure. Theinfection then moves from the leaves to the stem(Martens et al. 1994). In severe cases of infection,the sclerotia develop in the stem and are cycledback into the soil during harvest. For blackleg,ascospores are the primary and main source ofinfection. They are produced in pseudothecia inthe infected stubble. Seedling infection is achievedby penetration of the cotyledons or young leavesthrough stomata or natural openings. The initialinfection of the tissue is biotrophic, but mostof the hyphal front becomes necrotrophic. Thenecrotrophic infection leads to the productionof the asexual structures (pycnidia) in the deadtissue (Hammond et al. 1985; Hammond andLewis 1987). During a rain event, the pycnidiosporesare spread by splashing and, thus, arethoughttoinfectotherleavesandneighbouringplants. The colonization of the intercellular spacesfollows the initial infection, which leads to thecolonization of the xylem or the spaces betweenthe xylem parenchyma and the cortex in theEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

190 W.G.D. Fernando, R. Ramarathnam, T. de Kievitpetiole. This intercellular growth is systemic,biotrophic and visually symptomless (Hammondet al. 1985). The fungus eventually invades andkills the cells of the stem cortex, resulting ina blackened canker which completely girdles thebase of the stem. Thus, the disease is named“blackleg”.Control of sclerotinia stem rot by traditionalmethodshasnotbeenveryeffective.Itisdifficulttobreed for resistance against S. sclerotiorum, sinceresistance is governed by multiple genes (Fulleret al. 1984). Control based on crop rotations is unrealisticdue to the persistence of survival structures(sclerotia) in the soil for long periods andbecause Sclerotinia has such a wide host range(Nelson 1998). These factors necessitate the use offungicides, which have been known to have adverseeffects on non-target organisms (Rose 1995;Gilmour 2001; McGrath 2001). Several strategiesbased on, for example, crop rotation, stubble management,chemical control, sanitation and resistantcultivars have been advised for blackleg control(Guo et al. 2005). With the introduction of cultivarswith major gene resistance to L. maculans,a high potential exists for increased prevalence ofthe aggressive isolates of L. maculans, orevolutionof new virulent pathotypes of the pathogen(Mayerhofer et al. 1997). The report on the appearanceofthemoreaggressivepathogenicitygroup3(PG3) and 4 (PG4) isolates in western Canada andthe North Central USA, where PG2 is the predominantgroup, is a good example of this phenomenon(Fernando and Chen 2003; Chen and Fernando2005; Bradley et al. 2005). With respect to chemicalcontrolwithfungicides,theperceivedhealthand environmental risks of using these chemicalshas led to increased interest in alternative diseasemanagement strategies (Jacobsen and Backman1993).A clear understanding of the life cycle andmode of infection of both pathogens gives us anopportunity to design alternative disease controlstrategies, such as biological control, for the managementof these pathogens and their diseases.For sclerotinia stem rot, as most inoculums areascosporic, a few germinating sclerotia can leadto significant infection levels in the field (Davies1986). Also, ascospores can travel long distancesfrom neighbouring fields to infect petals (Venette1998). Therefore, there is a need for research intothe biocontrol of S. sclerotiorum on canola, specificallyon limiting petal infection by ascospores.Foliar applications of biocontrol agents (BCA) areimportant in the Sclerotinia/canola system, as theascospores generally infect senescing petals at theflowering stage (Turkington and Morrall 1993). Asthis is a narrow window to protect the plant frominfection, biological control may work well in controllingthe germination of ascospores on petal surfaces.Similarly, the narrow susceptibility period ofcanola to blackleg favours the use of biocontrolas a viable disease control strategy. The disease isdestructive to the canola crop only when the infectionoccurs early in plant development, from thecotyledon to the six-leaf growth stage (West et al.2001). If the plant can be protected during this mosthighly susceptible period, then the disease couldbe managed to minimise yield loss. Therefore, biologicalcontrol represents a promising, alternativecontrol strategy which can be implemented in theintegrated management of sclerotinia stem rot andblackleg diseases of canola.II. Biological ControlBaker and Cook, in 1974, defined biological controlas “the reduction of inoculum density or diseaseproducing activities of a pathogen or parasite in itsactive or dormant state, by one or more organisms,accomplished naturally or through manipulationof the environment, host, or antagonist, or by massintroduction of one or more antagonist”. Bacteriahave proved to be excellent sources of antagonists,owing to their multiple mechanisms of disease control.Earlier work on the biocontrol of plant diseasesfocused more on the root-colonizing rhizobacteria,with special emphasis on their ability to producesecondary metabolites, such as siderophores.Siderophores efficiently sequester iron and deprivethe pathogen of this vital element, essential formetabolic functioning and the process of pathogenesis(Kloepper et al. 1980). Over the past twodecades, numerous studies have established therole of other mechanisms such as antifungal antibiotics,enzymes and volatiles produced by bacteriain plant disease control (Weller et al. 1988; Whipps1997). Other than their direct role in pathogen control,bacteriaalsoproducemetaboliteswhichenhanceplant growth (e.g. root growth), or trigger theinduction of systemic resistance which acts in theform of immunization, thereby preventing plantdisease (Van Loon et al. 1998). With the abovementionedmultiple mechanisms of disease control,bacteria serve as excellent antagonists.

Bacterial Weapons of Fungal Destruction 191III. Phyllosphere BiocontrolA. Chemical and Physical Environmentof the Leaf SurfacesThere is limited knowledge on the chemical characteristicsof the leaf surface which make it suitablefor microbial growth. The unevenness of the thickwaxy cuticle, along with the presence of veinsand trichomes, affect the microclimate of the leafsurface. Factors such as rain, dew and leaf exudatesresult in the transport of nutrients from plantcells, and other microbes or pollutants which helpbacteria to maintain their metabolic activity andpopulation size (Suslow 2002). On the leaf surface,bacteria acquire their carbon and nitrogen sourcesmainly from the exudates, which are comprised ofsugars such as sucrose, glucose and fructose. Inaddition, other organic acids, alcohols and aminoacids are present on the leaves. The concentrationof nutrients on the leaves is very low, ranging from1–20 μg/leaf (Tukey 1971). Wilson and Lindow(1994a, b) have clearly shown that the growthof bacteria on the leaf surface is limited by theavailability of carbon-containing nutrients underphysical environmental conditions conducive forbacterial multiplication.The microclimate of the leaf, under highly variableenvironmental conditions such as for humidity,temperature, leaf wetness and ultraviolet radiation,poses a major challenge for colonization bybacteria. Leaf surfaces are subjected to lack of freemoisture and rapid fluctuations in relative humidityor temperature (Beattie and Lindow 1995, 1999).The ultraviolet radiation present in sunlight causessevere damage to bacterial cells (Sundin and Jacobs1999). An additional challenge faced by these bacteriais the diurnal change from cool nights of amplemoisture in the form of dew to higher temperaturesand drier conditions which cause osmotic shockduring the daytime.B. Bacterial Adaptations in the PhyllosphereBacteria have evolved mechanisms to overcomethese constraints and establish themselves underthe harsh conditions present in the phyllosphere.Bacteria arrive through airborne, waterborne andvector-borne deposition from sources of plant originsuch as tree buds, seeds and residues of previouscrops (Manceau and Kasempour 2002). Thebacteria multiply on the leaf surface, when free wateris available, and form aggregates which enhancetheir ability to survive when ecological conditionsbecome less favourable on the surface. Aggregationoccurs along the anticlinal walls of epidermalcells and around the trichomes, where leachingof nutrients is higher (Backman et al. 1997).Larger bacterial aggregates are present at the baseof trichomes, probably due to their ability to retainwater, and secrete mucilage and essential nutrients,such as sugars and amino acids (Simon1997; Ascensao and Pais 1998). Bacteria increasetheir fitness in the phyllosphere by the expressionof phenotypic features such as flagellar motility,extracellular polysaccharide production, productionof biosurfactants, phytohormone production,and phytotoxins and siderophore production (Lindow1991; Beattie and Lindow 1995, 1999). Bacteriaalso adopt an endophytic mode of life style whenthey colonize the inside of leaf tissues to escapethe harsh conditions occurring on the leaf surfacewhen the environment becomes dry (Whitesidesand Spots 1991). Epiphytic bacteria adapt themselvesto an endophytic mode of survival by multiplyingin the apoplast and escaping plant defencesystems (Kazempour 1998). Other than epiphyticbacteria, saprophytic bacteria develop mechanismsof tolerance to stress, such as pigmentation whichis often correlated with UV tolerance, to maintainpopulation levels high enough to survive in thephyllosphere (Wilson et al. 1999).IV. Bacterial Phyllosphere Biocontrolof Plant Diseases,with Special Emphasison Sclerotinia Stem Rotand BlacklegPseudomonas spp. and Bacillus spp. mediate cropprotection by exerting multiple mechanisms of inhibitoryactivity such as the production of extracellularenzymes (Dunlap et al. 1996; Pleban et al.1997), competition (Lugtenberg et al. 1999), inducedsystemic resistance, ISR (Yan et al. 2002; Ryuet al. 2003), and antibiosis (Silo-Suh et al. 1994;Raaijmakers et al. 2002).One of the most successful examples ofphyllosphere biocontrol is the suppression ofErwinia amylovora by epiphytic bacteria, and thereduction of fire blight disease in apple and pear.Pseudomonas fluorescens A506 occupies the samesitesonstigmaasthosecolonizedbyE. amylovora,

192 W.G.D. Fernando, R. Ramarathnam, T. de Kievitand utilizes nutrients important for the growthof the pathogen (Wilson and Lindow 1993). Onthe other hand, Pantoea agglomerans producesantibiotics (Ishimaru et al. 1988; Vanneste et al.1992; Kearns and Hale 1996; Wright and Beer1996), in addition to other growth-limiting nutritionalsubstrates and thereby competes for sites,suppressing E. amylovora on stigmas (Hattinghet al. 1986; Wilson et al. 1992). Bacillus has beenone of the key genera used for the phyllospherebiocontrol of foliar diseases. Bacillus spp. formendospores, which are structures capable ofsurviving desiccation, heat, oxidizing agents andUV radiation (Setlow 1995). These characteristicsoffer ecological fitness and also enable long-termstorage and relatively easy commercialization ofBacillus-based products. Control of bean rust byBacillus subtilis (Baker et al. 1985), and controlof Alternaria on tomato (Stevens et al. 1996)and Cercospora on peanut by Bacillus cereus(Kokalis-Burelle et al. 1992) are good examples.Biological control of foliar pathogens at thephyllospherehasbeenlessexploited,asisevidentinthehugeliteratureavailableonthebiocontrolof root pathogens. Harsh conditions in the phyllosphere,which make for difficult establishmentand survival of the biocontrol agent, is one of themain reasons for the lack of attempts on phyllospherebiocontrol. Yet, there are some host-diseasesystems in which phyllosphere biocontrol has beensuccessfully used in disease control. The followingsection deals with the timing of application of thebacterial biocontrol agent (BCA), and the variousmechanisms exerted by the bacterial BCA for thephyllosphere control of plant disease, such as antifungalantibiosis, efficient colonization, volatileproduction and induced systemic resistance. Thesectionalsolaysemphasisonthebiocontrolofsclerotiniastem rot and blackleg of canola by bacterialBCAs.A. Timing of ApplicationA clear understanding of the pathogen life cycle,the growth stage at which the host is susceptible toinfection by the pathogen, and the mechanism ofhost infection would help in the application of theBCAattheappropriateplaceandtime.Also,ifthereis a narrow window of infection by a pathogen,this favours the use of BCAs as a control strategy,as the antagonist need only survive, establish itspopulation and prevent pathogen infection duringthat short critical period. The successful useof biocontrol relies on an effective delivery systemand subsequent survival of bacteria in the infectioncourt. Since the primary infection of S. sclerotiorumon canola occurs through ascosporic infectionof senescing petals (Adams and Ayers 1979),biocontrol studies performed in our laboratory employdirect delivery of bacteria onto the petals, inthe form of one or two spray applications duringthe blooming stage (Fernando et al. 2007). Resultsfrom two field trials conducted during 2003 and2004 indicate that either one or two applicationsof bacterial strains suppressed stem rot under fieldconditions. Applications of Pseudomonas chlororaphisstrain PA23 and Bacillus amyloliquefaciensstrain BS6 twice at 30% and 50% bloom significantlyreduced the percent canola stem rot incidenceinbothtrials.Theresultsarecomparableto those obtained for the application of the fungicideRovral-Flo, and are significantly different fromthose for the pathogen-inoculated control.Similarly, bacterial application at the cotyledonstage, i.e. the stage most susceptible to pathogeninfection, plays an important role in the preventionof blackleg infection (Ramarathnam 2006). Thisphenomenon was clearly established in our fieldstudy where B. amyloliquefaciens strain DFE16 appliedat the cotyledon stage suppressed the diseaseas efficiently as the fungicide which was tested.Bacteria seem to prevent early infection of thecotyledon leaves, reducing the chances for systemicinfection, and girdling and cankering of the stem.The need for BCA application at the cotyledonstage is further strengthened by our finding thatthe same bacteria applied only at the 3–4 leaf stagefailed to suppress the disease, suggesting infectionof the plant could have occurred prior to theapplication of the bacteria. The cotyledon stage ofcanola is a narrow window of infection potentialand, therefore, it represents an opportunity to beexploited for biocontrol of blackleg.B. Mechanisms of Biocontrol1. AntibiosisDirect antagonism of the pathogen throughantibiosis is one of the mechanisms by whichdisease-suppressive bacteria achieve disease control.Antibiosis is mediated through the productionof a chemically heterogeneous group of organic,low-molecular weight compounds (Raaijmakerset al. 2002) which, at low concentrations, are

Bacterial Weapons of Fungal Destruction 193deleterious to the growth or metabolic activities ofother microorganisms (Fravel 1988; Thomashow etal 1997). Restriction of growth, due either to limitationin nutrients or to high cell density, leads to theinitiation of secondary metabolism and antibioticproduction, which helps the organisms remaincompetitive in their environment (Vining 1990).Stigma colonization by E. amylovora is the crucialinitial step in the development of most fireblight infections in apple and pear trees. Suppressionat this point of the disease process by antagonistsof E. amylovora, suchasPantoea agglomerans(Erwinia herbicola) strain Eh1087, has resultedin significant success. The ability to producea phenazine antibiotic, D-alanylgriseoluteic acid(AGA), helps the antagonistic strain to outcompeteE. amylovora in the colonization of the stigma (Giddenset al. 2003). In competition experiments on thestigmas of apple flowers, E. amylovora was significantlyless successful against the P. agglomeransAGA-producing wild type than against the AGAnon-producingmutant. Further, it was found thatAGA production significantly enhanced the competitivenessof Eh1087 when it was applied at thesame time or 24 h before the pathogen. This studyclearly established the role of this antibiotic in theecological fitness of the producing strain, and itsrole in disease control.Pseudomonas chlororaphis strains PA23 (PA23)and DF190 (DF190) are strong antagonists of S. sclerotiorumand L. maculans respectively. The bacteriawere identified as producers of the antifungalantibiotics phenazine and pyrrolnitrin (Ramarathnamand Fernando 2006; Zhang et al. 2006).Control of Gaeumannomyces graminis var. triticiin wheat by phenazine produced by P. chlororaphisPCL1391 (Chin-A-Woeng et al. 1998), and controlof Rhizoctonia solani in cotton by pyrrolnitrin producedby P. fluorescens BL915 (Ligon et al. 2000)are well-known examples of an established role forthese antibiotics in biocontrol. The production ofphenazines and pyrrolnitrin corresponds to the antifungalactivity of PA23 culture extracts in the inhibitionof sclerotial and/or spore germination ofseveral plant pathogens, including Fusarium oxysporum,R. solani, Sclerotium rolfsii, Macrophominaphaseolina, Alternaria solani and Botryodiplodiatheobromae (Kavitha et al. 2005). Production ofmultiple antibiotics, with overlapping or differentdegreesofactivity,mayaccountforthesuppressionof specific or multiple plant pathogens (Raaijmakerset al. 2002). Characterization of a PA23Tn5mutant, called PA23-63, revealed a Tn insertion inphzE, which forms part of the phenazine biosyntheticcluster. Despite producing no phenazines,this strain exhibited wild-type levels of antifungaland biocontrol activity against S. sclerotiorumand L. maculans (Poritsanos 2005; Ramarathnam2006). Our findings indicate that phenazine productionis not essential for PA23 biocontrol of thesetwo pathogens. Mutant PA23-63 produces pyrrolnitrinat levels equal to that of the wild type (Paulitz,personal communication). Therefore, we believethat pyrrolnitrin is mainly, but not exclusively, responsiblefor the antibiosis-mediated biocontrol ofS. sclerotiorum, the stem rot pathogen (Poritsanos2005), and L. maculans, the blackleg pathogen ofcanola (Ramarathnam 2006). The primary antifungalmechanism of pyrrolnitrin is the interferenceof the osmotic signal transduction pathway, andthe secondary mechanism is thought to be inhibitionof respiration, evident at a high dosage ofthe antibiotic in N. crassa and other fungi (Okadaet al. 2005). This explains the antifungal activityof pyrrolnitrin over a wide range of basidiomycetes,deuteromycetes and ascomycetes (Ligonet al. 2000).Another Pseudomonas BCA isolated by ourlaboratory, Pseudomonas sp. strain DF41 (DF41),demonstrated strong antagonistic activity againstS. sclerotiorum. By means of Tn5 mutagenesisstudies, a DF41 antifungal-deficient mutant wasisolated harbouring a Tn insertion in a gene with91% identity to the syrB gene of the P. syringae syringomycinbiosynthetic cluster (C. Berry, W.G.D.Fernando, T.R. de Kievit, unpublished data). A Tn5insertion in this gene completely abolishes antifungalactivity, suggesting a molecule similar tosyringomycin, probably a cyclic lipopeptide, mayberesponsiblefortheantibiosisinstrainDF41.Bacillus spp., especially B. subtilis, B. cereus andB. amyloliquiefaciens, are reported to be effectivefor the control of plant diseases caused by soilborne,foliar and post-harvest fungal pathogens(Silo-Suh et al. 1994; Raupach and Kloepper 1998;Shoda 2000; Janisiewicz and Korsten 2002; Chiouand Wu 2003; Tjamos et al. 2004). Bacillus cereusstrain DFE4 (DFE4), and B. amyloliquefaciensstrains BS6 (BS6) and DFE16 (DFE16) exhibitedagar-diffusible antifungal activity, and greenhouseand field suppression of sclerotina stem rot andblackleg of canola (Fernando et al. 2005; Ramarathnam2006). All three Bacillus strains harbourbiosynthetic genes for the lipopeptide antibioticsiturin A, bacillomycin D and surfactin. Moreover,cell extract analysis confirmed the production

194 W.G.D. Fernando, R. Ramarathnam, T. de Kievitof iturin A, bacillomycin D and surfactin bythese BCAs (Ramarathnam 2006). BacillomycinD has been reported to exhibit strong antifungalactivity towards aflatoxin-producing fungi such asAspergillus flavus (Moyne et al. 2001). Iturin A isknown to exhibit strong antifungal activity andpotential for biocontrol (Yoshida et al. 2002; Choet al. 2003). The multiple antibiotics producedby these Bacillus spp. could possibly explain thebroad spectrum of antifungal activity exhibited bythesebacteriaoverarangeofpathogens.2. Pre-Emptive Colonization of Target SiteCompetence of the BCA and synchronization of itsactivity, in both time and space, with the pathogenare key factors determining the efficiency of a BCA(Folman et al. 2003). Earlier studies by Bull et al.(1991) on the colonization of roots by P. fluorescensin the suppression of G. graminis var. tritici,andbyParke (1990) on the successful colonization of thepea spermosphere in the prevention of Pythiuminfection are good examples which stress the importanceof colonization of BCAs at the target site asa prerequisite for suppression of plant pathogens.Pre-emptive stigma colonization by the antagonisticP. agglomerans strain Eh107 resulted in a populationwhich was resilient to subsequent invasionbythepathogen.Similarly,thecolonizationof the stigma by the pathogen, prior to the antagonist,prevented the establishment of the antagonist.This clearly established the importance ofpre-emptive colonization and utilization of the nutrientsrequired by the pathogen (Giddens et al.2003). This experiment suggested that niche exclusionhas a dominant influence on the dynamics ofbacterial populations on stigmas, and also in thesuppression of the fire blight disease.The time of inoculation of the BCA playsa very important role in the suppression of bothsclerotinia stem rot and blackleg disease of canola.The application of the Pseudomonas sp. strainDF41 (Savchuk and Fernando 2004), strains PA23or BS6 (N. Poritsanos, C. Selin, W.G.D. Fernando,S. Nakkeeran and T.R. de Kievit, unpublished data)24 h prior to the pathogen, or co-inoculation withthepathogenwerecrucialinthesuppressionofstem rot disease under greenhouse conditions.Antagonistic bacteria, when applied 24 and 48 hafter ascospore application, did not control thedisease. The same phenomenon was also applicablein the blackleg suppression assays done oncotyledon plants of canola in the greenhouse.The inoculation of the bacteria PA23, DF190 andDFE4, 24 and 48 h prior to the application ofpycnidiospores of L. maculans on wounded canolacotyledons, led to significantly lower levels ofdisease, which was manifested in a low, resistantinteraction phenotype (IP) ratings (

Bacterial Weapons of Fungal Destruction 195the management of overwintering structures insoil. Pathogen-infested stubble and sclerotia aresubstrates for the production of sexual and asexualspores which cause primary infection of the crop.Destruction of the overwintering structures wouldgreatly limit primary inoculum production andtheestablishmentofthediseaseinthecrop.4. Induced Systemic ResistanceThe efficiency of BCAs can be improved throughelucidation of the mechanism(s) of their action.Plants may be protected against pathogens byway of endogenous defence mechanisms triggeredin response to the attack of either an insector pathogen (Heil 2001). Induced resistance bybacterial antagonists in several crops is associatedwith the enhancement of lignification,and stimulation of host-defence enzymes andsynthesis of pathogenesis-related (PR) proteins(Hammerschmidt and Kuc 1995). Our resultssuggest that two applications of strain PA23 inducedresistance against S. sclerotiorum infection(Fernando et al. 2007). An application of PA23 atboth 30 and 50% bloom, followed by challengeinoculation with Sclerotinia ascospores, inducedsignificantly higher chitinase and β-1,3-glucanseactivity. In contrast, the activity was less in thehealthy control, ascospore-inoculated control, andPA23 treatments. Accumulation of these hydrolyticenzymes reached the highest level 4 days after inoculation(DAI) for chitinase (2.5-fold increase overinoculated control), and 6 DAI for β-1,3-glucanase(threefold increase over inoculated control),followed by a slow decline thereafter. One chitinaseisoform (34 kDa)wasdetectedbywesternblottingusing tobacco chitinase antiserum in PA23-treatedplants with or without challenge inoculation byS. sclerotiorum. Expressionwasverylowintheinoculated control. The PR proteins chitinase andβ-1,3-glucanase both inhibit fungal pathogens(Mauch et al. 1988). The enhanced accumulationof PR proteins (chitinase and β-1,3-glucanase) andoxidative enzymes (peroxidases) in PA23-treatedcanola leaf tissues may contribute to the reductionof Sclerotinia infection in pathogen-inoculatedplants. Since fungi have chitin and glucan as cellwall components (Sing et al. 1999), increasedactivity of chitinase and β-1,3-glucanase in canolaplants exposed to biocontrol bacteria may preventthe establishment of pathogens. Similarly, phyllosphereinoculation studies have been carriedout earlier for induced systemic resistance (ISR)with B. mycoides strain Bac J for the control ofCercospora leaf spot in sugar beet (Bargabus et al.2002), and P. putida WCS358r and P. fluorescensWCS374r for the control of bacterial wilt causedby R. solanacearum in Eucalyptus urophylla (Ranet al. 2005). Inoculation of B. mycoides strain Bac Jinduced the activity of chitinase, β-1,3-glucanaseand peroxidase, and significantly reduced Cercosporaleaf spot in sugar beet (Bargabus et al.2002). Also, the bacteria were not isolated from thepathogen-inoculated leaves, which demonstratedthe role of ISR in disease suppression. B. subtilisstrain BacB formed cell aggregates which inducedsystemic resistance against Cercospora leaf spot,even though the cell number on the leaf reducedsignificantly (Collins et al. 2003). Disease controlcould not be attributed to either antibiosis orparasitism in this study.Plant growth promoting rhizobacteria (PGPR)strains with ISR activity can be active againsta wide range of pathogens (Raupach and Kloepper1998). Application of fluorescent pseudomonadsstrengthens the host cell wall structures andresults in the restriction of pathogen invasion ofhost-plant tissue (Chen et al. 2000). RhizobacteriamediatedISR leads to enhanced sensitivity of theinduced tissue to jasmonic acid (JA) and ethylene(ET), rather than increasing their production(Pozo et al. 2005). Conrath et al. (2002) term thisphenomenon “priming”, which is the enhancedcapacity of induced tissues for rapid and effectiveactivation of cellular defence response uponchallenge by pathogen infection. This has beenproved with analysis of local and systemic levels ofJAandETinplantsexpressingISR,whereinducedresistance was not associated with detectablechanges in their production (Pieterse et al. 2000).Priming has been well established towards diseasereduction in tobacco, where cells showed fasterand stronger lipid peroxidation and proteinphosphorylation in response to fungal elicitorsafter preconditioning by MeJA treatment (Duberyet al. 2000).In contrast to the ISR in mature canola elicitedby strains PA23 and BS6, no such induction wasobserved in the suppression of blackleg in canolacotyledons. Lipopolysaccharides (LPS) of thebacterial cell wall and iron-chelating siderophoreshave clearly been shown to elicit systemic resistancein plants (Whipps 2001). In addition,antifungal antibiotics have also been reported toinduce systemic resistance (SR). Audenaert et al.(2002) demonstrated that phenazine-1-carboxylic

196 W.G.D. Fernando, R. Ramarathnam, T. de Kievitacid and a siderophore, pyochelin, producedby Pseudomonas aeruginosa strain 7NSK2 bothcontributed to the ability of this isolate to induceSR in tomato against Botrytis cinerea. Amongthe lipopeptide antibiotics produced by Bacillusspp., treatment of potato tuber cells with purifiedfengycins resulted in the accumulation of someplant phenolics involved in, or derived fromphenylpropanoid metabolism (Ongena et al. 2005).Production of siderophores and antifungal antibioticsby strains PA23 (Poritsanos 2005; Zhang et al.2006), DF190, DFE4 and DFE16 (Ramarathnam2006) have been established. These bacteria weretested for their ability to induce SR in canolacotyledons. Living bacterial cells and their culturebroth extract were tested through split and localinoculation assays. Split inoculation determinedthe ability of the bacteria to induce SR, andthe local inoculation tested for direct antifungalactivity on the pycnidiospores of L. maculans.Boththe bacteria and their culture broth extract failedto suppress the pycnidiospores of L. maculans,and the blackleg lesion on cotyledons in thesplit inoculations. The bacteria and the brothextracts suppressed the blackleg disease lesionon cotyledons when inoculated locally with thepathogen spores. This clearly indicated a lackof systemic induction by the bacteria and brothextracts, but localized inhibition of the pathogen.To further ascertain the mechanism involvedin the localized suppression of the disease, theactivity of PR enzymes was studied. None of thebacterial treatments (except for DF190-inducingβ-1,3-glucanase) had significant induction ofchitinase, β-1,3-glucanase or peroxidase (Ramarathnam2006). The lack of localized PR-enzymeactivity, but suppression of the pycnidiospores ofL. maculans, suggest the potential role of directantifungal activity of the bacterial cells.V. ConclusionsIn the quest for safer, more environmentallyfriendly alternatives to pesticides, BCAs emergeas attractive candidates for the management ofplant diseases. Thus far, natural bacterial isolateshave shown inconsistent performance in the field.Poor field performance can be attributed in partto varying environmental conditions which, inturn, influences expression of essential biocontrolfactors. Thus, if BCAs are ever to become realisticalternatives to chemical pesticides, it is essentialthat we understand, at a molecular level, bothbiotic and abiotic factors regulating the expressionof antifungal metabolites. By employing transcriptomicsand proteomics, we can advance ourunderstanding of gene/protein expression notonly in the BCAs and fungal pathogens but alsoin the plant host during complex interactions inthe phytosphere. This will provide us with a fullerpicture of beneficial plant–microbe interactions.Defining factors which influence antibiosis, ISR,as well as predation and niche exclusion willultimately lead to better-performing biocontrolproducts. Notably, this knowledge can be used tocreate genetically engineered strains exhibitingimproved biocontrol capacity. It is expected thatgenetically modified strains will be the biocontrolproducts of the future, able to rival chemicalfungicides in managing plant diseases.ReferencesAbawi GS, Grogan RG (1979) Epidemiology of plant diseasescaused by Sclerotinia species. Phytopathology 69:899–904Adams PB, Ayers WA (1979) Ecology of Sclerotinia species.Phytopathology 69:896–899Adolphe D, Daun J, Fitzpatrick K, Gruener L, MacykD, Mayko J, Peters L, Wilkins D (2002) Canola(http://www.canola-council.org, accessed 12 December2002)Ascensao L, Pais MS (1998) The leaf capitate trichomes ofLeonotis leonurus: histochemistry, ultrastructure andsecretion. Ann Bot 81:263–71Audenaert VK, Pattery T, Cornelis P, Hofte M (2002) Inductionof systemic resistance to Botrytis cinerea in tomatoby Pseudomonas aeruginosa 7NSK2: role of salicylicacid, pyochelin, and pyocyanin. Mol Plant Microbe Interact15:1147–1156Backman PA, Brannen PM, Mahaffee WF (1997) Bacteriafor biological control of plant diseases. In: Rechcigl NA,Rechcigl JE (eds) Environmentally safe approaches tocrop disease control. Lewis, New York, pp 95–109Baker KF, Cook RJ (1974) (eds) Biological control of plantpathogens. W.H. Freeman, San Francisco, CABaker CJ, Stavely JR, Mock N (1985) Biocontrol of beanrust by Bacillus subtilis under field conditions. PlantDisease 69:770Bargabus RL, Zidack NK, Sherwood JW, Jacobsen BJ (2002)Characterization of systemic resistance in sugar beetelicited by a non-pathogenic, phyllosphere-colonizingBacillus mycoides, biological control agent. Physiol MolPlant Pathol 61:289–298Beattie GA, Lindow SE (1995) The secret life of bacterialcolonist on leaf surfaces. Annu Rev Phytopathol33:145–172

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12 Effects of Animals Grazing on FungiT.P. McGonigle 1CONTENTSI. Introduction ........................ 201II. The Fungivores in Soil and Litter Systems . 202A. GeneralConsiderations............. 202B. ASurveyofRelevantGroups......... 2021.Acari ......................... 2022.Collembola .................... 2033.Annelida ...................... 2034.Nematoda ..................... 2035.Others ........................ 203III. Some Limitations to the Studyof Fungivore Grazing ................. 204A. GutContents ..................... 204B. FoodPreferenceTests .............. 204C. EnumerationofHyphae............. 204IV. Grazing and Community Structure ...... 204A. Changes in Species RichnessandCommunityDiversity........... 2041. Grazing on a Dominant Fungus . . . . . 2042. Grazing on a FungusWhichisnotDominant........... 205B. Replacement of some Species by Others 206C. ModifyingFactors................. 2061.SelectivityofGrazing............. 2062.IntensityofGrazing.............. 2073. Responses of the Grazed Fungi . . . . . 207V. Conclusions from Laboratory Studiesand Extrapolation to the Field .......... 209References.......................... 209I. IntroductionThis review considers the impact which fungivoregrazing can have on communities of fungi in soiland litter. The fungi are represented by large numbersof species (Christensen 1989) and considerablebiomass (Kjøller and Struwe 1982) in soil and littersystems. Up to 75% of the soil fauna biomass residesin the fungivore trophic category (McGonigle1995), and so grazing by soil fauna on fungal tissuesis expected to play a significant role in shaping thefungal communities of field systems.1 Department of Botany, Brandon University, 270-18th Street, Brandon,Manitoba R7A 6A9, CanadaIn order to assess the impact of grazing of fungion community structure, it is useful initially tobriefly consider fungi in relation to general principlesof community ecology. The individuals withinaspeciesandatagivenlocationcompriseasinglepopulation. Discussion of the concept of the individualwith respect to fungi has been made elsewhere(Rayner and Todd 1982). The indeterminategrowth of many plants has led to the quantificationof modules of plant growth, rather than ofindividuals (Harper 1977). Fungi can be treatedsimilarly where recognizable structures such assporophores are visible (Shaw 1985). However, fruitbodies may represent only less than 1% of the unseenbut living vegetative fungal biomass (Frankland1982). Communities are collections of populationswhich are present inside defined limitsin space, and within specified taxonomic or functionalgroups (Begon et al. 2006). Communities canbe described most simply in terms of species richness,which is the number of species present. Diversityreferstotherelativeabundanceofeachofthespecies being considered (Begon et al. 2006). Fora given species richness, the diversity of the communitywill be determined by the relative abundancesof the species present; high abundance ofa small number of species equates to low diversitywhereas greater evenness, or equitability ofthe species abundances, reflects higher diversity.Fungivore grazing can be expected to affect speciesrichness by promoting the elimination or the introductionof fungal species. In addition, the grazingof fungi can be expected to affect relative abundanceand community diversity.Characterization of fungal communities has insome cases evaluated species richness, althoughthis is in itself a mammoth undertaking (Swift 1976;Christensen 1981). Even prolonged isolation studiesappear to be unable to complete the species listfor a given sampling location; Christensen (1989)found that after more than one thousand isolateshad already been taken from one location, eachEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

202 T.P. McGonigleincrement of 100 additional isolates consistentlyyielded approximately 10 species new to the site.When isolation frequencies are used as a measureof relative fungal abundance, fungal communitiesfollow a lognormal distribution (Lussenhop 1981);this is consistent with communities for other typesof organisms in other kingdoms (Begon et al. 2006).In theory, the lognormal distribution reflects theway in which species abundances are the result ofthe interplay of a variety of independent factors(May 1975).Swift (1976, 1982) considered the questionof niche diversification among sympatric fungalspecies. Although many fungi show a surprisinglybroad range of occurrence, there is some specializationfor different resource types; these includevariousplantpartslikeleavesandtwigs,andthedifferent plant species they originate from. Thereis also the opportunity for specialization amongthe different microenvironments within resourcetypes, such as between the vein and mesophyll ofaleaf,andamongthevariouscarbonsubstratestherein.Reviews of fungivore grazing have consideredthe processes of comminution of substrate, dispersalof fungi, and the direct impact of feeding itselfon fungal community structure (Visser 1985) andnutrient cycling (Ingham 1992; McGonigle 1995).Comminution of substrate stimulates microbial activityand accelerates decomposition (Swift et al.1979). The dispersal of fungi through the movementsof grazing animals was clearly establishedin earlier studies (Brasier 1978; Wiggins and Curl1979). Enhanced dispersal will encourage a communityto fulfil the potential it intrinsically hasfor a poor or rich species compliment, playing animportant role in the establishment of microbialcommunities. However, in this review it is the biologicalinteractions which occur after dispersal,and which are involved in the determination ofthe fungal community structure, which are considered.The effect of the disruptive physical action ofgrazing animals on fungi, such as by the processof trampling, is not easy to distinguish from theeffects resulting from ingestion of the fungal material.The breaking-up of fungal material by animalbody movements has been suggested (Wicklow andYocum 1982) to be one of the reasons why fungalcommunities change in response to grazing.This review will initially outline the relevantfeatures of the various types of invertebrate fungivoresin soil and litter, and proceed to considersome limitations encountered in the study of grazingonfungi.Exampleswillthenbegiventoshowthe ways in which grazing can affect fungal communitiesthrough effects on species richness, communitydiversity, and replacement of some species byothers. In the final section before the conclusions,various features of the fungivores and fungi whichcan modify the outcome of grazing will be discussed.Specifically, these modifying features arethe selectivity and intensity of grazing, and theability of the fungus being grazed to respond tothat grazing.II. The Fungivores in Soiland Litter SystemsA. General ConsiderationsThe soil fauna is composed of a great diversityof species distributed among many phyla. Of particularimportancewithregardtofungivoryarethree phyla: the Annelida, the Arthropoda and theNematoda. The Enchytraeidae within the Annelidadisplay extensive fungivory, and gut passagein Lumbricidae also affects fungi. The arthropodclasses Arachnida and Insecta contain the groupsAcari and Collembola respectively, each of whichhas many fungivorous members. Among the nematodes,the Dorylaimida and Tylenchida containan abundance of fungivores.Using the terminology of Moore et al. (1988),animals feeding on hyphae can be divided into twocategories: engulfing fungivores and fluid-feedingfungivores. These feeding modes are mostly consistentfor fungivores within defined taxonomicboundaries.B. A Survey of Relevant Groups1. AcariMites are very abundant in soil and litter, and manyof them consume fungi. Chitin-rich cell walls of engulfedhyphaepassthroughmitegutwithoutbeingdigested whereas the fungal disaccharide trehaloseis utilized (Hubert et al. 2001). Soil mitescomprise four major groups. Many members of theCryptostigmata, or oribatid mites, are generalistfeeders (Swift et al. 1979) and will ingest decayingplant material, fungal hyphae, and algae. However,there is some specialization within the group:members of the family Phthiracaridae feed onlyon plant residues and are able to digest cellulose

Effects of Animals Grazing on Fungi 203but not the fungal storage carbohydrate trehalose;by contrast, the Oppiidae and Eremaeidae specializeon fungi and cannot digest cellulose (Luxton1972). Some Cryptostigmata readily feed on nematodes(Rockett 1980). The well-known accumulationof 15 Ninconsumersrelativetothematerialsthey ingest has been applied to the questionof niche diversification within the Cryptostigmata.Schneider et al (2004) determined that 15 N/ 14 Nratiosamong species of oribatids below beech andoak indicated trophic-level separation within theoribatid community. Members of the Gamasinain the Mesostigmata are predatory, with differentgenera showing varying degrees of specializationin their choice of prey (Moore et al. 1988). TheAstigmata show a diverse range of feeding activity:members of the family Acaridae feed by engulfinghyphae,aswellastakinglivenematodesand protozoa, whereas some members of the familyHistiostomatidae specialize in the ingestion ofa slurry of decaying residues mixed with microbes(Walter and Kaplan 1990). The Prostigmata havefluid-feeding fungivores in several families, e.g. Tydaeidaeand Tarsonemidae; however, Prostigmataalso contain families of predatory mites, such as theBdellidae and Stigmaeidae (Moore et al. 1988). Inforest systems, the Cryptostigmata are most abundant(Hogervost et al. 1993) whereas, in desert systems,the fungivorous Prostigmata are the moreimportant group (Santos and Whitford 1981).2. CollembolaThe majority of Collembola are primarily fungivorous,and can be divided (Wallwork 1976) intotwo distinctive life forms as follows: those at thesoil surface or in litter layers are usually large, pigmentedindividuals with well-developed eyes. TheCollembola of deeper layers are more often small,weakly pigmented and with reduced eyes. Based onanalysis of gut contents, the two life form groupsshow little difference in the extent to which they engagein fungal feeding (Takeda and Ichimura 1983).Collembola collected from the field often have nogut contents, which may be due to intermittent cessationof feeding caused by the moulting cycle ofthe animals (Christensen 1990).3. AnnelidaGut contents of enchytraeids contain a mixture offungal hyphae, plant residues and soil materials,with fungal material typically representing onethird of those gut contents (Dash et al. 1980). Whenoffered fungal baits, the gut content of enchytraeidswith fungal material can increase to between50–70% of the total (Dash and Cragg 1972). The gutcontents of some enchytraeid species can containtwiceasmuchfungalmaterialaswouldbeexpectedon the basis of random ingestion of substrate(O’Connor 1967). Estimates of fungivory in enchytraeidsrange from 25% (Persson et al. 1980) to asmuch as 80% (Whitfield 1977). Feeding biologyof the various enchytraeids needs further workbeforetheroleofthisgroupinthefunctioningof soil systems is adequately appraised (Didden1993). The burrowing earthworm Lumbricusterrestris L. feeds on surface litter, although fungion the litter are a major component of their diet(Tiunov and Scheu 2000).4. NematodaBased on their anterior morphology, free-living soilnematodes can be identified as fungivores, bacterivores,predators or omnivores (Twinn 1974). Thediet of omnivorous nematodes can consist of algae,fungal spores, protozoa and other nematodes(Swift et al. 1979). In addition, there are also somefree-living soil nematodes which feed on root epidermalcells and root hairs (Yeates et al. 1993).Fungivorous nematodes pierce hyphae with theirstylets, and feed on the fluid protoplasm of the fungususing a pumping action (Freckman and Baldwin1990). Empty hyphal walls remain behind. Underculture conditions, the impact of grazing byfungivorous nematodes can be severe, killing allaerial hyphae and reducing growth on agar relativeto that seen in the absence of the nematodes (Shaferet al. 1981).5. OthersVarious other groups in the soil fauna also eat fungi.Among the larvae of dipterous flies, those in thefamily Sciaridae graze fungi in dung deposited onthe soil surface (Wicklow and Yocum 1982), membersof the Phoridae are mycelial feeders (Tibbleset al. 2005), and larvae of the families Chironomidaeand Mycetophilidae are mainly fungivorous(Swift et al. 1979). Interestingly, mycetophilids areattractedtothezonesofantagonismatthecontactbetween fungal individuals (Boddy 1983). Theisopod Oniscus asellus L. was able to reduce fungalstanding crop to one third of that in controlleaf–fungusmicrocosms(HanlonandAnderson

204 T.P. McGonigle1980). A selection of genera of soil amoebae areable to feed on hyphae (Bamforth 1988), but theimpact of this on populations of soil fungi is unclear(Chakraborty et al. 1983).III. Some Limitations to the Studyof Fungivore GrazingSeveral approaches are used to investigate grazing,including direct observation, examination of gutcontents, and monitoring through time the hyphaepresent. Food preference tests can be used to ascertainthe palatability of different fungi to the faunaltaxa of interest.A. Gut ContentsExamination of gut contents can provide valuableinformation on dietary choices of fauna among theavailable foods. However, some food types moreeasily retain their structural integrity and are recognizablein the gut for a longer time. Walter (1987)found that nematodes observed to be consumedby a selection of mites, which are normally consideredmycophagous, were not detectable in gutboluses because of the lack of sclerotization of thenematode body.B. Food Preference TestsCollembola have been seen during feeding evaluationtests to switch from feeding predominatelyon one palatable fungus to feeding days lateralmost exclusively on a simultaneously offered butdifferent palatable fungus (Visser and Whittaker1977). Mixed diet seems to be important forCollembola (Scheu and Simmerling 2004) andfungivorous nematodes (Ruess et al. 2000) to avoidover-consumption of toxic metabolites.C. Enumeration of HyphaeMeasurement of fungal biomass through timepresents difficulties, because estimates of theproportion of hyphal length which is active varyaccording to the staining method used (Schubertand Mazzitelli 1989; Hamel et al. 1990).IV. Grazing and Community StructureIn various sections below, studies on grazingof plants are referred to in order to providea theoretical framework in which we can considergrazing on fungi. Comparisons have been drawnpreviously between grazing in fungivore–fungusand herbivore–plant systems (Wicklow 1981;Visser 1985; Shaw 1992).A. Changes in Species Richnessand Community Diversity1. Grazing on a Dominant FungusDarwin (1859) reported that when a previouslymown 90-cm by 120-cm turf plot was left to grow,thenumberofplantspecieswithinitwasreducedfrom 20 to 11 because of the proliferation of somespecies. Thus, mowing can maintain increasedspecies richness. Crawley (1983) argues thatboth mowing and grazing of herbaceous systemsfunction in an essentially similar way, by theselective removal of more of some species than ofothers. Mammal grazing exclosures on grasslanddid not change species richness but increasedproliferation of a few herb species, showing thatgreater diversity and more equitability amongspecies had previously been maintained by grazing(Tansley and Adamson 1925). However, undervery intense grazing which fell just short ofuncovering bare soil, plant species richness itselfwas reduced (Tansley and Adamson 1925). Thesestudies (Darwin 1859; Tansley and Adamson1925) established that grazing can increase speciesrichness or increase community diversity by thesuppression of species which would otherwise bemore dominating. Alternatively, when the intensityof grazing is sufficiently high, then species richnesscan be reduced.Whenonefungusismoreproductivethananother,selective grazing on the more abundant funguscan suppress what would otherwise have beenthedominantmemberofthecommunity.Theoutcomeof grazing in this situation will be a communityof greater diversity. An example from Newell(1984a, b) is the suppression of the otherwise dominantMarasmius androsaceus (L. ex Fr.) Fr. undergrazing pressure from the collembolan Onychiuruslatus Gisin, leading to more extensive developmentof the sympatric species Mycena galopus(Pers ex Fr.) Kummer (Fig. 12.1). In the study ofNewell (1984a, b), two species of fungi were con-

Effects of Animals Grazing on Fungi 205Fig. 12.1. a Growth of Mycena galopus and Marasmius androsaceusin culture as a function of temperature. M. galopusis the slower-growing species (data of Newell 1984a).b Effect of collembolan grazing on the percentage of pineneedles colonized by M. galopus and M. androsaceus in microcosms.Grazing is seen to suppress M. androsaceus andallow greater proliferation of M. galopus (data of Newell1984b)sidered. However, the principles involved can beapplied in theory to larger communities. Whereincreased representation of several otherwise suppressedspecies occurs at the expense of one dominant,the result will be greater equitability amongspecies. An analogous effect may occur with theredistribution of nutrient resources among membersof herbaceous communities through arbuscularmycorrhizal plant-to-plant connections (Grimeet al. 1987).on seemingly co-dominant fungi can be seen inParkinson et al. (1979), where the collembolanOnychiurus subtenuis Folsom was introduced intoaspen leaf microcosms which had been inoculatedwith two fungi isolated from an aspen littersystem. When the two fungal isolates were kept inseparate microcosms, grazing by Collembola hadlittle impact (Fig. 12.2). However, when the fungiwere grown together, grazing by the Collembolaacted to polarize the system by the suppressionof isolate sterile dark 298 and promotion ofisolate basidiomycete 290 (Fig. 12.2). It was noted(Parkinson et al. 1979) that basidiomycete 290 wascompletely unpalatable to O. subtenuis in feedingchoice tests. Ek et al. (1994) demonstrated thatgrazing by O. armatus Tullb. was able to suppressthe development of saprotrophic fungi in the genusPaecilomyces much more strongly when Pinus contortaDougl. ex Loud. seedlings in the microcosmswere mycorrhizal with Paxillus involutus (Batsch)Fr., compared to when they were non-mycorrhizal(Fig. 12.3). This example underlines the importanceof the interplay between any direct impact ofgrazing, and the interactions between the variousfungi present. Deletion competition for resourcesin the grazed system shifted in favour of Paxillusand away from Paecilomyces, leading to almostcomplete suppression of the latter. However, in thenon-mycorrhizal situation, grazing was only ableto cause a delay in the development of Paecilomyces(Fig. 12.3).2. Grazing on a Fungus Which is not DominantSelective grazing on a fungus which is of equalaggressiveness compared to a neighbour can actto polarize the community, making the ungrazedfungus more abundant relative to the grazedneighbour. Fungivore selection of a fungus alreadyshowing relatively low production within the communitywill act to reinforce or further exaggeratethe dominance of the ungrazed fungus. In bothcases, the trend will be towards a community ofreduced diversity. One example of selective grazingFig. 12.2. Effect of collembolan grazing on the numbers ofleaf cores colonized by the isolates sterile dark 298 and basidiomycete290. In one series, the fungi were inoculated ontoleaf cores so as to keep the fungi in separate microcosms. Inthe other series, the leaf cores were inoculated with the fungimixed in the same microcosms. Collembola were added at0, 20 and 50 per microcosm. Grazing after 10 days is seen topromote the competitive success of basidiomycete 290 oversterile dark 298 (data of Parkinson et al. 1979)

206 T.P. McGonigleFig. 12.3. The number of sporulating colonies of saprotrophicfungi seen per microcosm after 67 and 88 days ofdevelopment of Pinus contorta. Seedlings were either nonmycorrhizal(NM) ormycorrhizal(M) withPaxillus involutus,and they were with or without 50 Collembola addedper microcosm. The majority of the saprotrophic fungi belongedto the genus Paecilomyces. Bars with different letters(a–c) are significantly (P

Effects of Animals Grazing on Fungi 207are available (Giannakis and Sanders 1989; Ruesset al. 2000). Selectivity in feeding has also beenshown for oribatid mites, several species of whichprefer ectomycorrhizal and ericoid mycorrhizalhyphae to those of saprotrophs (Schneider et al.2005). Earthworm impact on fungi seems also tobe selective in terms both of the fungal speciestaken (Moody et al. 1995) and the survival of fungiduring gut passage (Moody et al. 1996; Tiunov andScheu 2000).Microarthropods show a strong preferenceamong available saprotrophic fungi, grazing particularlythe dark pigmented forms (Kaneko et al.1995; Varga et al. 2002). This preference for darkpigmented forms is pronounced for Collembola(Maraun et al. 2003), Oribatida (Schneider andMaraun 2005) and Astigmata (Hubert et al. 2004).Further, some preference is shown among speciesof dark pigmented fungi (Schneider and Maraun2005). Selection of dark pigmented forms makessense, given that these fungi form 30–60% ofisolates from soil and are a high-quality food(Maraun et al. 2003).Crawley (1983) explains that removal of speciesin proportion to their abundance will not be ableto affect the structure of a community; for speciesrichness or community diversity to be influencedrequires selective removal of greater quantities ofsome species. It seems that grazing of fungi is almostalwaysfoundtoinvolveselectivefeeding,andit can therefore be expected to affect fungal communitystructure.2. Intensity of GrazingGenerally, intense levels of grazing lead to eliminationof some species. Wicklow and Yocum (1982)imposed a range of grazing intensities by varyingthe number of Lycoriella mali Fitch. dipteran flylarvae added to 2-g dry-mass-equivalent samplesof rabbit dung. In the absence of grazing, 14 fungalspecies were recovered from each sample. However,the number fell to between six and nine species persample when the grazing intensity was above 10 larvaeg −1 dung (Fig. 12.5). This reduction in speciesrichness was due to the loss of what appeared tobe grazing-sensitive species. Over and above theimpact of grazing seen in Fig. 12.5, some fungalspecies had a reduced frequency of occurrencein the more highly grazed microcosms, althoughother species were unaffected (Wicklow and Yocum1982). Reduction of species richness in response toFig. 12.5. Number of fungal species recovered from rabbitdungmicrocosms in response to the number of fungivorousdipteran larvae added. Increasing grazing intensity reducesspecies richness, although no further consistent reductionis seen above 12.5 larvae g −1 dung (data of Wicklow andYocum 1982)high intensities of grazing have been seen in thefungi of aquatic systems (Bärlocher 1980).3. Responses of the Grazed FungiAny negative impact of grazing on the vigour offungi, through loss of biomass or damage to tissues,mightbeoffsetbycompensatorygrowthproducedin response to that grazing. Indeed, where senescentparts of the mycelium are removed and this, inturn, is able to permit re-growth from an otherwisefunctionally dormant hyphal system, grazing canhave a positive, rather than a negative impact on thegrazed fungus. Grazing of Botrytis cinerea Pers exFr. by Folsomia candida doubled the respiration ofthe fungus (Hanlon 1981), which was attributed toremoval of senescent parts of the colony. Removalofoldhyphaeallowednewfungalgrowthwherenutrientavailability permitted (Hanlon 1981). A similareffect on respiration was shown for grazingof Onychiurus quadriocellatus Gisinonthefungigrowing on Diplopod faecal pellets (van der Driftand Jansen 1977). One collembolan per pellet decreasedfungal standing crop, yet increased fungalrespiration by 10% over controls (van der Drift andJansen 1977).The impact of grazing on fungal biomass isnonlinear. Low grazing intensities can stimulatefungal production whereas higher levels reducethat production. Such responses for enchytraeidfungivores have been likened (Hedlund andAugustsson 1995) to the process of grazing optimizationas described for mammalian herbivores(McNaughton 1979). A density of 0.1–0.4 Collem-

208 T.P. McGoniglebola g −1 soil appears to be a threshold, above whichthe quantity (Bakonyi et al. 2002) and function(McGonigle 1995) of arbuscular mycorrhizal fungiis restricted. The detrimental impact of higherCollembola densities on arbuscular mycorrhizaeis severance of the hyphae (Klironomos andUrsic 1998). Folsomia candida, at a density of0.25 animals g −1 dry soil, increased hyphae ofFusarium oxysporum Schlect. to 4.8 mg −1 dry soil(Moore 1988), from 3.2 mg −1 dry soil in ungrazedcontrols. However, at a density of 1.0 g −1 dry soil,these Collembola reduced the hyphae to 1.2 mg −1dry soil (Moore 1988).Based on a modelling approach, Bengtssonet al. (1993) concluded that fragmentation offungal thalli into patches, such as might becaused by grazing, would be expected to promotecompensatory growth by the fungi. The effect ofgrazing on fungal respiration appears to consist oftwo temporal phases of fungal response. Grazingof Onychiurus armatus reduced respiration ofMortierella isabellina Oudem during the 2-dayperiod in which the Collembola were in contactwith the mycelium, but fungal respiration wasstimulated relative to ungrazed controls in a subsequent5-day grazing-free interval (Bengtssonand Rundgren 1983).Mortierella isabellina adopts an alternate andfaster-growing mode of more aerial hyphae inresponse to grazing, possibly in an attempt bythe fungus to escape the grazer (Hedlund et al.1991). Morphological responses to grazing are alsopronounced in wood-decomposing basidiomycetefungi (Fig. 12.6). Utilization of wood is limitedmostly to white-rot basidiomycetes and xylariaceousascomyctes, both able to degrade lignin, aswell as brown-rot basidiomycetes able to utilizecell-wall polysaccharides in the presence of lignin;these constitute a relatively limited number offungi providing a grazing resource of immensepotential, given global wood production (Swiftand Boddy 1984). Wood-decay fungi formingoutgrowths from one wood-resource block to thenext are particularly susceptible to grazing whenthe strength of the resource base is reduced interms of time spent on the source block or in termsof a small size of the source block (Harold et al.2005). Variability in grazing impact also arisesbecause of different levels of grazing intensity andfor different species of Collembola (Kampichleret al. 2004). However, a general pattern emerges ofaresponsetograzingintheformoffastergrowthin some sections of the outgrowth mycelium, comparedtoarrestedgrowthinothersections.Thisresponse is interpreted as a strategy adopted by thefungus to escape local grazing (Kampichler et al.2004) and forage for additional wood resources tocolonize (Tordoff et al. 2006).Fig. 12.6. Outgrowth from wood blocks of the wood-decayfungus Hypholoma fasciculare a without and b with grazingby Collembola. Grazing leads to marginal fans connected tothe wood resource by cords (data of Tordoff et al. 2006)

Effects of Animals Grazing on Fungi 209Table. 12.1. A summary of responses noted by Riffle (1971)for fungi cultured individually and grazed by the nematodeAphelenchoides. The 43 fungi studied were subdivided byRiffle (1971) into five groups (I to V), according to theresponse of fungus and nematode to grazingGroup Fungal linear Sub-culture Nematode Number of Examplegrowth rate viability density fungal speciesdevelopedI Unchanged Good High 11 Flammulina velutipes (Curt. ex Fr.) KarstII Unchanged Good Low 12 Suillus variegatus (Fr.) O. KuntzeIII Reduced Reduced High 5 Amanita rubescens ([Pers] Fr.) S. F. GreyIV Reduced Lost Low 7 Boletus edulis Bull. ex Fr.V Reduced Lost High 8 Armillaria mellea (Vahl ex Fr.) KummerFollowing introduction of low densities of thefungivorous nematode Aphelenchoides into agarcultures of various fungi, Riffle (1971) noted a varietyof responses by both the fungus and grazer(Table 12.1). Fungal characteristics are clearly importantin determining the outcome: Riffle (1971)noted that members of group I (Table 12.1) werefast growers of which the rate of mycelium productionexceeded the rate of mycelium consumptionby nematodes, while those in group IV were slowgrowingfungi; group II were suspected of producinganti-feeding metabolites (Riffle 1971). The diversityof responses seen in Table 12.1 for fungalproductivity and viability after imposing a standardizedgrazing regime emphasizes the complexityof ways in which grazing can modify fungalcommunity structure. Depending on the type offungus, grazing can be seen to have no effect or,alternatively, a strong negative effect on the grazedfungi. Simultaneously, for both the unaffected andgrazing-damaged fungi, examples are to be seenwhere the nematode population is stimulated toreach high densities, and in other cases not. Thesedifferent types of responses of the grazer populationcan, in turn, be expected to feed back in differentways to modify fungal community structurethrough different levels of subsequent grazing.V. Conclusions from Laboratory Studiesand Extrapolation to the FieldGrazing of fungi has been shown under controlledconditions to have the capacity to change fungalcommunity structure. Feeding on fungi whichwould be dominant in the absence of grazingwill enhance diversity. Alternatively, grazing onco-dominant or subordinate fungi will act topolarize the fungal community, and diversity willbe reduced. Grazing can facilitate the transitionfrom one successional stage of fungi to the next.The impact of grazing is modified by three factors:(i) grazing must be selective to impact communitystructure, (ii) high intensities of grazing lead tothe loss of fungal species from the communityand (iii) responses of the fungi to grazing can insome cases negate or exceed any negative impactof grazing.There is an abundance of animals in soil andlitter which eat fungi. As might be expected, fungivoresare found to be particularly associated withmycelia in the field. For example, Cromack et al.(1988) reported the biomass per unit soil mass ofCollembola, oribatid mites and nematodes to be2.0, 1.5 and 1.2 times greater in ectomycorrhizalfungal mats than in adjacent non-mat soil respectively.Microcosms have shown grazing can modifysaprotrophic fungal communities when isolatedfrom active mycelium after removal of spores bywashing (Tiunov and Scheu 2005). The potentialclearly exists for grazing by fungivores to modifyfungal communities extensively in the field.ReferencesBakonyiG,PostaK,KissI,FabianM,NagyP,NosekJN(2002) Density-dependent regulation of arbuscularmycorrhiza by collembola. Soil Biol Biochem34:661–664Bamforth SS (1988) Interactions between Protozoa andother organisms. Agric Ecosystems Environ 24:229–234Bärlocher F (1980) Leaf-eating invertebrates as competitorsof aquatic hyphomycetes. Oecologia 47:303–306Begon M, Townsend CR, Harper JL (2006) Ecology: fromindividuals to ecosystems, 4th edn. Blackwell, MaldenBengtsson G, Rundgren S (1983) Respiration and growth ofa fungus, Mortierella isabellina, inresponsetograzingby Onychiurus armatus (Collembola). Soil BiolBiochem 15:469–473

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13 Fungal EndophytesP. Bayman 1CONTENTSI. Introduction ........................ 213II. Definitions and Interactions ............ 214III. How Endophytes are Studied ........... 214A. MicroscopyinPlanta............... 214B. IsolationinPureCulture ............ 215C. PCR-BasedMethods ............... 215D. BiochemicalMethods .............. 215IV. Clavicipitaceous Endophytes of Grasses . . . 215A. Systematics, Evolution and Life Cycles . . 2161. Clavicipitaceae and Key Genera . . . . . 2162.LifeCyclesandEvolution.......... 2163. Coevolution and InterspecificHybridization .................. 217B. Secondary Metabolites and Their Effects 217C. Effects of Mutualistic Grass Endophyteson Plant Hosts and Other Organisms . . 2181.EffectsonHosts................. 2182.EffectsonPlantCommunities...... 2183. Effects of EndophytesonInsectHerbivores ............. 2184. Effects of EndophytesonMammalianHerbivores ........ 219V. Endophytes of Maize .................. 219VI. Endophytes of Trees .................. 219A. Transmission and Sources of Inoculum . 220B. Specificity ....................... 2201.SpeciesSpecificity ............... 2202.OrganandTissueSpecificity ....... 2203. Site Specificity and Spatial Variation . 221C. EffectsofTreeEndophytes........... 2211. Endophytes as Latent PathogensorMisplacedPathogens........... 2212.EffectsonHostPlants ............ 2223.EffectsonInsectHerbivores ....... 2224.EffectsonPlantPathogens......... 222VII. Endophytes of Tropical Plants .......... 222VIII. Endophytes of Nonvascular Plantsand Lichens ......................... 223IX. Uses in Agriculture and Biotechnology . . . 223A. Clavicipitaceous Fungi of Grasses . . . . . 223B. OtherEndophytes ................. 223C. Bioprospecting.................... 224X. Conclusions ......................... 224References.......................... 2241 Departamento de Biología, Universidad de Puerto Rico–RíoPiedras, P.O. Box 23360, San Juan, PR 00931, USAI. IntroductionFungi are important in mysterious ways and mysteriousin important ways. Part of their charm isthat much of their lives is hidden from our view –hidden by the microscopic size of their hyphae andby their substrata – yet they play key roles in nature,in agriculture, in biotechnology, and in the lives ofmany other organisms.Much of the mystery and importance of thefungi can be found in the endophytes. They are (bydefinition) hidden in plant tissues, but they haveimportant implications for communities, agricultureand biodiversity. In addition, certain endophytesare an excellent model system for studyinginteractions between organisms.No plant is an island. Each plant is a community,including diverse types of microorganisms.One guild of plant-associated microorganisms, theendophytic fungi, is the topic of this chapter. Allplants and plant-like protists have endophytes, includinglichens and nonvascular plants (see Stoneet al. 2000) and algae (Stanley 1992).Some endophytes are hitchhikers with no noticeableeffect on their plant host, but others haveintimate relations with their hosts and profoundeffects on their function and survival. The importanceof endophytes remained largely hidden until1975, when Charles Bacon and colleagues (Baconet al. 1975) demonstrated that endophytes of pasturegrasses caused toxic syndromes in cattle. Thisstimulated research in many aspects of the biologyof endophytes, both basic and applied.This chapter reviews the principal guildsof endophytes, their relationships with theirhosts, and their importance for other organismsand human affairs. It focuses on the guilds ofendophytes that have been most extensivelystudied: clavicipitaceous endophytes of grassesand endophytes of trees. The chapter is intendedto be illustrative, rather than comprehensive. ItsEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

214 P. Baymanpurpose is to stimulate young investigators toconsider endophytes in their research, becausemany interesting endophytes and aspects ofendophytes are probably still hidden.II. Definitions and InteractionsThe term ‘endophyte’ was coined by de Bary in1866 to describe fungi that colonize internal tissuesof stems and leaves (cited from Wilson 1995).DeBary’sdefinitionhassincebeenmodifiedmanytimes. Two widely accepted definitions follow:“Endophytes colonize symptomlessly the living,internal tissues of their host, even though theendophyte may, after an incubation or latency period,cause disease” (Petrini 1991).“Endophytes are fungi or bacteria which, forall or part of their life cycle . . . cause unapparentand asymptomatic infections entirely within planttissues . . . ” (Wilson 1995).Both of these definitions (but not de Bary’s)encompassendophytesofroots,whichareubiquitous(Schulz et al. 2006). Latent stages of infectionsby pathogens are included in these definitions,but mycorrhizal fungi are considered to beexcluded because they are partly external and oftensymptomatic (Saikkonen et al. 1998). Unlikethe definitions of mycorrhizae or pathogens, thesedefinitions of endophytes are based primarily onlocation, rather than type of symbiotic interaction.In contrast, symbioses (e.g. parasitism,competition, mutualism, etc.) are defined bywhich partners benefit from the interaction. Thebest-known endophytic fungi are mutualists:Neotyphodium species that infect cool-seasongrasses (see Sect. IV. below). They are of greateconomic importance, and a superb model systemto study the biology of interactions. BecauseNeotyphodium is an example of mutualism, thereis a widespread misconception (in the case ofpeople who do not work with endophytes) orfantasy (in the case of those of us who do) thatmost other endophytes are mutualists as well.However, in most cases a mutualistic relationshiphas not been demonstrated.Endophytic interactions form a continuum,and the interaction between a given microorganismand host may change over time (Saikkonenet al. 1998; Schulz and Boyle 2003). For example,a mutualistic endophyte may become pathogenicand vice versa, depending on the environment andthe condition of the host plant. For this reason, thedifferent types of symbiosis best describe an interactionat a particular point in time, rather thana species of microorganism (for most endophytes).Even a mutualistic interaction may be viewed asa balanced antagonism, where each partner triesto maximize the benefit obtained from the otherwithout loss of its own resources (Saikkonen et al.1998; Schulz et al. 1999). To further complicatematters, whether an endophytic interaction appearsmutualistic, parasitic or commensal maydepend on the scale at which it is viewed (Carroll1995; Wilson 2000). Of course, these problems donot mean that endophytes are less interesting orless worthy of study; rather, they mean that oneshould be cautious about categorizing them.Like mycorrhizae, endophytism is an ancientform of symbiosis, perhaps dating to the first landplants or even before. However, endophytism hasarisen so many times independently, involving differentfungi and plants, that the idea of a singleevolutionary history does not make sense; rather,there are many different histories. An exceptionis the evolution of endophytism in the Clavicipitaceae,discussed below.III. How Endophytes are StudiedSince endophytes are (by definition) internal andasymptomatic, their detection and identificationcan be difficult. Four sets of techniques are used:microscopy in planta, isolation in pure culture,DNA- (and RNA-) based methods, and biochemicalmethods. Results may depend on choice of technique;each has advantages and limitations, andeach may bias results in favour of certain taxa.A. Microscopy in PlantaThe definitive way to detect endophytes and determineinfection frequency is by direct microscopicobservation. Microscopy is also useful for locatingendophytes in specific tissues. For example, Rhabdoclineparkeri and Phyllosticta abietis are bothendophytes of Douglas fir needles, but the formerinfects epidermal cells and the latter spongy mesophyllcells (Stone 1987, 1988). This difference in locationmay be important for their interaction withthe plant or with other organisms. However, thestandard culturing and molecular techniques cannotdetect such differences.

Fungal Endophytes 215Nevertheless, microscopy in planta has a disadvantage:identification of the fungi is very difficultbecause hyphae of many different taxa look alike.Detection with taxon-specific probes or antibodiesallows identification of certain target organisms butnot others.B. Isolation in Pure CultureThe great majority of studies of fungal endophytesaredonewithsurface-sterilizedplantpiecesincubatedon culture media. Plant pieces are usuallysurface-sterilized in ethanol and/or sodiumhypochlorite, and incubated on a general purposemedium (e.g. potato dextrose agar or malt extractagar) amended with antibiotics. Methods of isolationhave been reviewed extensively (Schulz et al.1993; Bills 1996). These methods are simple, inexpensive,and allow detection of a wide range offungi. However, the choice of surface-sterilizationmethod and culture medium will bias the results infavour of certain organisms. Fungi that are obligatebiotrophs and do not grow in culture media will beoverlooked, and weedy fungi that grow quickly onagar will be overrepresented. Distinctions betweenepiphytes and endophytes depend on the assumptionthat surface sterilization kills the former andnot the latter. These limitations have stimulated theuse of molecular techniques to identify endophytes.Sampling strategy has a great impact on whichendophyteswillgrowoutofleafpiecesonagar.In general, using small leaf pieces reveals greaterendophyte richness than when using large leafpieces. Because leaf-to-leaf variation is considerable,several leaves should be sampled. Manystudies on endophyte diversity are not rigorous insampling strategy, which often makes it difficult tocompare results between studies. These samplingissues have been discussed at length (Carroll 1995;Lodge et al. 1996; Wilson 2000; Gamboa et al. 2002;Bayman 2006).C. PCR-Based MethodsPCR-based methods are increasingly being used todetect and identify endophytic fungi. A variety oftechniques are available, including DNA sequencing,RFLPs and variations, DGGE and RAPDS. Inmany such studies, fungi are first isolated in pureculture and DNA is then extracted. This approachisexcellentforidentification,butitcannotdetectunculturable fungi or escape the biases inherentin culturing.To date, relatively few studies on endophyteshave used direct amplification (also called environmentalPCR). This approach can serve to detectfungi that do not grow in culture. But howmany endophytic fungi are unculturable? A studyin loblolly pine compared culturing vs. direct amplification,and identified fungi by DNA sequencing(Arnold et al. 2007). Direct amplification detectedmore species than culturing, but culturing detectedmore orders of fungi. Also, more species of Sordariomyceteswere detected by culturing than by directamplification. These results are surprising: inepiphytic bacteria (and many other guilds of bacteria),direct amplification reveals more diversityat all levels (Yang et al. 2001).Direct amplification of endophytes from planttissueswillprobablyleadtothediscoveryofnew,high-level clades of fungi. However, PCR-basedmethods and culturing are complimentary, as theloblolly pine study suggests. To maximize diversityof fungi identified and economy of materials,endophyte diversity studies should include bothdirect amplification and culturing (Bayman 2006).Even as large-scale sequencing and metagenomicsinvestigations become more common and morerevealing, much of the currently hidden diversityin fungi may be revealed by culturing.D. Biochemical MethodsErgosterol content can be used to detect and quantifyfungal infection of plants, as can fatty acidsand other biochemical markers. To date, these havebeen used mostly for clavicipitaceous endophytesof grasses. Immunoblot assays and other assaysbased on monoclonal antibodies provide convenientand specific tools for detection of Neotyphodiumin grass stems (Richardson and Logendra1997; Clay et al. 2005; Koh et al. 2006). However,such tools are expensive to develop, and are applicableonly to fungi for which monoclonal antibodieshave been developed.IV. Clavicipitaceous Endophytesof GrassesClavicipitaceous fungi (that is, in the FamilyClavicipitaceae) in grasses were first describedover 100 years ago (see Wilson 1996). However, the

216 P. Baymanimportance of these endophytes was not realizeduntil 1975, when Charles Bacon et al. demonstratedtheir association with toxicity syndromes in cattle.In the 30+ years since Bacon’s discovery, thestory of grass–endophyte relationships has beenpursued from many different perspectives, severalof which are discussed below.For graduate students and new investigators,there are several advantages of working in a fieldof study this young: the literature is recent, and injournals that are still available; the major playersare still active as of this writing, and one can talkwith them at meetings; and many interesting symbioses,problems and potential applications haveyet to be studied. For example, Neotyphodium endophyteswere recently found in wild wheat speciesin Turkey (Marshall et al. 1999).A. Systematics, Evolution and Life Cycles1. Clavicipitaceae and Key GeneraThe Clavicipitaceae (Ascomycota) are characterizedby perithecia produced in stromata, asci withapical thickenings, and filamentous, multicelluarascospores. They are biotrophic parasites ofinsects, fungi, and monocotyledonous plants.The most conspicuous genera are Claviceps andCordyceps, pathogens of cereals and insects respectively.The stromata of both genera are used inmedicine. Claviceps has influenced human historymany times (Matossian 1989; Pendell 2005). TheClavicipitaceae of interest as endophytes are about30 species of Balansia, Myriogenospora, Atkinsoniella,Balansiopis, Epichloë and Neotyphodium(Clay 1988). Neotyphodium species (formerlyplaced in Acremonium) are anamorphs of Epichloë.Balansia includes epiphytes and endophytes ofgrasses and sedges in the Americas and Asia. Theendophytic species of Balansia are found on warmseasongrasses with C 4 photosynthesis in the Americas,and appear to have evolved from epiphytes(White et al. 2000; see below). Hyphae are epicuticularin epiphytes and intercellular in endophytes.They are easy to culture on a variety of standard media,though many require vitamins (Bacon 1985).Theswitchtoendophytismmayhaveconferredseveral advantages, including easier absorption ofnutrients from the host, protection from the environment,and protection from surface-feeding insectsand parasitic fungi (White et al. 2000). Studieson the evolution of endophytism in this groupare very interesting, partly because they integratemolecular phylogeny, morphology, life history, hostrange and secondary metabolism into a single story(White 1994; Clay and Schardl 2002).Epichloë and Neotyphodium infect cool-seasongrasses native to Europe and North America withC 3 photosynthesis. Stromata of Epichloë alwaysform around a grass inflorescence and adjoiningleaf sheath; the blade of the leaf emerges from thestroma (White et al. 2000). Hyphae are mostlyintercellular. The stromata produce conidia (whichfunction as spermatia) and receptive hyphae; theconidia are transferred between stromata by flies.The mating system is heterothallic, at least inE. typhina (White and Bultman 1987). However,some of these fungi have lost the ability to makethe perfect (Epichloë)stage,asdescribedbelow.2. Life Cycles and EvolutionThe relationship between Clavicipitaceae andgrasses is believed to be 40 million years old (Clayet al. 2005). The presumed ancestral conditionin Clavicipitaceae is seen in Claviceps, whichinfects and replaces grass ovaries and has bothintercellular and intracellular hyphae (Bacon andWhite 2000). It does not infect vegetative organsand produces disease symptoms rapidly, and istherefore not considered endophytic.A more derived relationship with the hostis seen in Myriogenospora, Echinodothis, Atkinsonellaand some Balansia species. These fungiare mostly epiphytic and epicuticular, producingstromata on stems, leaves or inflorescences. Ifthese fungi infect meristems, they may perenniate.Some of these fungi inhibit sexual reproductionin the host plant, by physically preventing thedevelopment of the inflorescence and perhaps byother means as well. Preventing seed set in thehost may leave more resources available for theirown sexual reproduction (Bacon and White 2000).The more derived species are endophytic andintercellular, showing epiphytic growth only whenthey emerge to produce stromata.Themostderivedgroupofspeciesisentirelyendophytic and intercellular. They cause systemicinfections and are transmitted vegetatively andvertically through the seeds, and usually produceno spores. They produce the Neotyphodium(= Acremonium) stage in culture but do notproduce stromata or perithecia. Compared tosome of the pathogenic species, the situation isreversed here: the grass can reproduce sexually butthefunguscannot.Thesespeciesaremutualists.

Fungal Endophytes 217Finally, some species are inconsistent, sometimesallowing the host to set seed and sometimesreplacing inflorescences with their own stromata.These species are of great evolutionary interest, becausethey may represent transitional forms betweenpathogenic species that reproduce sexuallyand are horizontally transmitted, and the mutualisticspecies that reproduce asexually and are verticallytransmitted (Schardl et al. 1994, 1997; Schardland Clay 1997).Most of the following discussion focuses on thesystemic, endophytic and asexual species of Neotyphodium.They are the best studied, because theyinfect economically important grasses and becausethey are an excellent model of mutualistic interactionsand coevolution.The mutualistic Neotyphodium species are sointimately associated with their hosts that it is conceivablethey may eventually lose their independentidentity and become organelles: they are verticallytransmitted through the hosts’ seeds. They arenever found in nature as free-living organisms(although they grow well in culture). They have lostthe capacity for sexual reproduction, which limitstheir ability to adapt to new environments. And,they are widespread (although endophyte-freeplants are found in populations of infected grasses).3. Coevolution and Interspecific HybridizationGene genealogies of Epichloë and several tribes inthe Pooideae are concordant, suggesting the fungiand their hosts may have coevolved (Schardl et al.1997). Further evidence of coevolution comes fromexperiments of cross-inoculation of endophytes.Most Epichloë and Neotyphodium isolates produceno noticeable defence response when inoculatedonto their natural hosts. Inoculation onto non-hostgrasses, however, often produces a response, includingdeath of endophyte cells or host tissues(Lane et al. 2000). The absence of such responsesonthenaturalhostsmaybetheresultofcoevolution.Epichloë and Neotyphodium comprise a numberof biological species, each isolated from the othersby intersterility and often by differences in hostrange and geographical distribution (Schardl et al.1997; Schardl and Wilkinson 2000). However, a hybridorigin has been postulated for several species.This is interesting and surprising because some ofthese species lack a sexual stage, and because interspecifichybridization is relatively rare in fungi(Burnett 2003).The initial evidence of hybridization camefrom isozyme studies, in which most Neotyphodiumisolates had single bands but othershad double bands (Leuchtmann and Clay 1990).Further evidence came from phylogenetic treesbased on β-tubulin sequences, in which someisolates contained two copies that belonged todifferent branches of the tree, suggesting a hybridorigin (Schardl et al. 1994; Tsai et al. 1994).The most interesting case is N. coenophialum,a ubiquitous and important endophyte of tall fescue(Festuca arundinaceae) (SchardlandWilkinson2000). Tall fescue itself is a (polyploid) hybridof F. pratensis (which has its own endophyte, N. uncinatum)andF. glaucescens. When the hybrid tallfescue first arose, it kept the N. uncinatum endophyteof F. pratensis. N. uncinatum later hybridizedin separate events with two Epichloë species toproduce N. coenophialum. Thisstoryiscomplex,but it is the simplest explanation for the multipleβ-tubulin sequences in N. coenophialum and iscompatible with host phylogeny.These fungal hybridization events presumablyoccurred in host plants infected with multiplespecies of endophytes, at least some of which wereasexual species. This suggests that the hybridsmay have come from parasexual (or somatic)recombination events. In turn, hybridization mayhave eliminated the possibility of future sexualreproduction, due to meiotic irregularities.The previous (1997) edition of The Mycota volumeIVincludesaninterestingchapterontheevolutionof mutualism in clavicipitaceous endophytes(Shardl and Clay 1997).B. Secondary Metabolites and Their EffectsThe Clavicipitaceae are famous for alkaloid production;the most flamboyant are the ergot alkaloidsof Claviceps purpurea used in medicine andthe production of LSD. The alkaloids produced byendophytic species are less scandalous but of enormousecological and agricultural interest.Intermsofchemicalecology,grassesandendophytesare a perfect match: grasses make relativelyfew secondary metabolites. Secondary metabolitesare important in plant defences against herbivoresandpathogens,sothegrassesarerelativelyundefendedcompared to other plant families. Theirclavicipitaceous endophytes provide chemical defences(Clay and Schardl 2002).Four types of alkaloids are produced. Two appearto be most effective against mammalian herbi-

218 P. Baymanvores and two against insects (see Bush et al. 1997;Lane et al. 2000):1. Ergot alkaloids, especially the lysergic acidamide derivative ergovaline: they are commonin tall fescue, perennial ryegrass and othergrasses. Concentrations are usually 50% of grass specieswith mutualist endophytes.C. EffectsofMutualisticGrassEndophyteson Plant Hosts and Other OrganismsThe mutualistic endophytes lend themselves to experimentsthat are simple and elegant in design.Endophyte-free plants are easy to produce, so experimentscan compare plants of the same cultivarwith and without infection.1. Effects on HostsThe frequency of endophyte infection often increasesin grass populations over time. For instance,old populations tend to have higher infectionrates than recently established populations(see Clay 1988). This suggests that the endophytesconfer an adaptive advantage on their hosts, eventhough they grow at the expense of host metabolism.Endophyte infection increases growth rate ofperennial ryegrass and tall fescue (see Clay 1988).The increase is significant for many growth parameters:shoot production, seed production, numberof tillers, etc. Neotyphodium endophytes also increasedrought tolerance in grasses, by means of osmoregulationand stomatal regulation (see Baconand Hill 1996). These mechanisms have allowedperennial ryegrass to colonize large areas of thesouth-eastern United States that would otherwisebe too hot and dry. Production of plant hormonesmay be part of the mechanism of action.The species that abort inflorescences in theirhostsobviouslylimithostsexualreproduction,but they also stimulate vegetative growth (see Clay1988). Similarly, infection by Balansia cyperi increaseddryweightandnumberoftubersinpurplenutsedge (Cyperus rotundus) (but decreased thesize of tubers; Stovall and Clay 1988). Since purplenutsedge is one of the world’s worst weeds and usestubers for vegetative reproduction, the presence ofthe endophyte may have serious consequences foragriculture.2. Effects on Plant CommunitiesBoth experimental studies and field surveys showthat the mutualistic Neotyphodium endophytesconfer competitive advantages on their hosts. Inplots seeded with infected and endophyte-free tallfescue, plant species richness was significantlylower for infected plots (Clay and Holah 1999). Themain mechanism for these competitive advantagesis herbivore deterrence (see below), but they mayalso reflect allelopathic effects of alkaloids (Clayet al. 1993; Bush et al. 1997).Endophytes also affect decomposition ratesof grasses: endophyte-infected tall fescue decomposedmore slowly than endophyte-free plants(Lemons et al. 2005). The presence of endophytesalso altered the composition of detritivorecommunities, particularly Collembola.3. Effects of Endophytes on Insect HerbivoresEffects of clavicipitaceous fungi on various herbivoreshavebeendemonstrated.Themostnotoriouscase is Claviceps purpurea on rye, where the herbivoresare unsuspecting humans.

Fungal Endophytes 219Several studies have found that endophyteinfectedgrasses significantly reduce survivorshipand growth in insects, e.g. fall armyworm (Clayet al. 1993). However, endophytes and alkaloidscan have different effects on closely related insectspecies. A single insect species may have very differentresponses to different species of endophyteinfectedgrasses. Many insects are relatively unaffectedby ergot alkaloids at concentrations foundin plants (Clay et al. 1993; see Richardson 2000). Incontrast,peramineismuchmoretoxicthanergotalkaloids to the Argentine stem weevil, a majorpest of perennial ryegrass (see Bush et al. 1997).4. Effects of Endophyteson Mammalian HerbivoresIn the 20th century, millions of hectares in thesouth-eastern United Sates were converted fromnative vegetation to tall fescue, a European grassthat is productive and nutritious for livestock (seeBush et al. 1997). However, livestock grazing on tallfescue occasionally develop a variety of toxic syndromes,which are correlated with the presence ofendophytes (Bacon et al. 1975, 1977). A similar relationshipwas discovered in New Zealand in sheepgrazing on endophyte-infected perennial ryegrass(Latch et al. 1984). The ergot alkaloids are implicatedin many such syndromes, and have diverseeffects: they can cause weight loss, elevated bodytemperature, vasoconstriction, and reduced milkproduction in livestock (see Bush et al. 1997). Ergotalkaloids also reduce prolactin and melationinlevels in blood, have an affinity for dopamine receptors,and stimulate cAMP production.Reproductive effects are also well known. Consumingendophyte-infected forage led to reducedconception, increased spontaneous abortions, andaltered gestation times in cattle, sheep and horses(see Richardson 2000). Reproductive effects onrodents have also been found. Effects on spermproduction and testosterone levels have also beenshown.Endophyte alkaloids are feeding deterrents forgrazers. Since alkaloids tend to have a bitter taste,it is possible that herbivores can detect the presenceof alkaloids directly. Cattle reduce forage consumptionwhen presented with endophyte-infectedgrasses.Lolitrems are implicated in neuroreceptorbinding and inhibition of membrane channels.Although lolines are thought to be more toxic toinsects than to mammals, they are immunosuppressivein mice and may cause vasoconstriction.The list of toxicity syndromes in grazing animalscaused by clavicipitaceous fungi is expectedto keep growing.V. Endophytes of MaizeMaize has an intimate and interesting relationshipwith endophytes. Fusarium moniliforme(= Gibberella fujikuroi or G. moniliformis) causesserious diseases of maize including seed rot, stalkrot, root rot, and kernel or ear rot (see Bacon andHinton 1996). F. moniliforme produces five groupsof mycotoxins, including fumonisins. Some ofthese mycotoxins are cytotoxic, mutagenic, immunosupressiveand estrogenic in animal studies,and have been implicated in human oesophagealcancer (see Bacon and Hinton 1996).However,thesameisolatesofF. moniliformethat cause disease in some maize lines are endophytesin others. While the pathogenic F. moniliformeis horizontally transmitted, the endophyticF. moniliforme is vertically transmitted through theseed. This is potentially dangerous because infectionis very common: Fusarium can almost alwaysbe isolated from maize seeds, infections are usuallyasymptomatic, and fumonisins can often bedetected in seeds (John Puhalla, personal communication;see Kuldau and Yates 2000).Fusarium moniliforme and maize are sometimesmutualists. Endophytic infection byF. moniliforme can protect against infection byF. graminearum (= G. zeae), often a more aggressivepathogen (van Wyck et al. 1988), and againstinfection by other pathogens as well (see Kuldauand Yates 2000).All these aspects of the F. moniliforme–maizeinteraction are reminiscent of the clavicipitaceousendophyte–grass interaction. Fusarium mycotoxinsare a more direct threat to human healththan the clavicipitaceous alkaloids because of theimportance of maize in the human diet. However,the ecology of the interaction has not been studiedas extensively. Fusarium endophytes are found ina wide range of other plants as well (Kuldau andYates 2000).VI. Endophytes of TreesMost studies on endophytes of trees have focusedon fungal ecology and systematics (Carroll 1995).Questions, techniques, levels of taxonomic resolu-

220 P. Baymantion and sample size vary greatly among these studies,so it can be difficult to compare results amongstudies. However, some patterns have emerged.A. Transmission and Sources of InoculumTransmission of endophytes in trees is mostly horizontal.In some plants, endophytes can be isolatedfrom seeds but the infection is not passed on to theseedling (Wilson and Carroll 1994; Bayman et al.1998). Most endophytes are probably transmittedas wind-, water- or animal-borne spores. In mostcases, it is not clear where and when they sporulate,how they overwinter, or how infection occurs.In this sense, the endophytic habit is a paradox(Bayman 2006). To propagate and disperse to newhosts horizontally, most endophytes must (strictlyspeaking) become something other than an endophyte:either sporulate on the surface, sporulate ondead tissue, or be exposed by damage to the hostorgan (mechanically or by an animal vector).However, a few cases of dispersal are well documented.For example, Rhabdocline parkeri,anendophyteof Douglas fir needles, produces conidia onmidge galls on needles, and produces conidia andascospores on fallen needles (Stone 1987; Carroll1995). New needles are infected by rainwater containingspores. Similarly, Discula quercina,acommonendophyte of Oregon white oak, sporulates onfallen leaves and probably on the bark of its host(Wilson and Carroll 1994). Rainwater collected belowtrees contained ascospores, and endophytefreetrees inoculated with the rainwater developedD. quercina infections. Filtered rainwater withoutspores (as a control) did not cause infection.Newly emerged leaves are usually endophytefree,though there are exceptions (Rodrigues 1994).Infection levels often increase with leaf age, whichis consistent with horizontal transmission. Seasonalchanges in infection frequency have beenfound in some endophytes but not in others (seeWilson 2000). Infection frequency is often correlatedwith rainfall. Insect wounds can be infectionsites for some endophytes but not for all (Faeth andHammon 1997).B. Specificity1. Species SpecificityHow specific are fungal endophytes for host plantspeciesandviceversa?Thereisawiderangeofinteractions, from highly specific to generalist.Most plants have diverse endophyte floras,including a few dominant taxa and many raretaxa – a typical log-normal distribution (Lodgeet al. 1996). Some of the dominant taxa are oftenfound only in one or a few host species, and canbe considered host-specific. Examples includeDiscula umbrinella and Hypoxylon fragiforme inEuropean beech, Lophiodermium piceae in spruce,and Phyllosticta multicorniculata in balsam fir(see Petrini 1996); others are mentioned elsewherein this chapter. Many other taxa can be isolatedfrom a wide range of hosts, and some are usuallyweedy fungi that can be isolated from many othertypes of substrata as well. These weedy fungi (e.g.Aspergillus, Penicillium and Cladosporium) areoften assumed to be accidental endophytes, forwhom most endophytic infections are dead ends.When endophyte communities of different treespecies at the same site are compared directly, theyusually differ – for example, of pine and beech, oakand willow (Petrini and Fisher 1988, 1990). Amongspecies of pine, endophyte communities were moresimilarinrelatedspeciesthaninmoredistantlyrelated species (Hata and Futai 1996). However, intropical trees the picture is not as clear (see below).When a single fungal species infects varioushost species, it may comprise host-specific races(or cryptic species). For example, although D. umbrinellawas isolated from chestnut, oak and beech,conidia of isolates from beech were capable of infectingonly beech leaves (Toti et al. 1992).On the plant side, intraspecific variation maybe reflected in differences in endophyte floras. Inhybrid cottonwood trees (Populus fremontii x P. angustifolia),frequency of twig endophytes was correlatedwith contribution of P. fremontii to thegenome (Bailey et al. 2005). Endophyte frequencywas negatively correlated with tannin concentrationin twigs, which is lower in P. fremontii than inP. angustifolia. Endophyte genotype and host genotypewere correlated in Venturia ditricha infectionsof birch (Ahlholm et al. 2002). These examples suggestthat plant evolution can affect relationshipswith endophytes.2. Organ and Tissue SpecificityIn Gynoxis oleifolia, a tropical tree, several endophyteswere specific to roots, bark or leaves (Fisheret al. 1995). In some conifers and rainforest trees,fungalcommunitiesinleafbladesweredifferentfrom those in petioles (Carroll et al. 1977; Petrini

Fungal Endophytes 2211996; Lodge et al. 1996). Differences were also seenin different parts of palm leaves (Rodrigues 1994).Different endophytes may lead separate liveswithin a single leaf blade. Rhabdocline parkeri andPhyllosticta sp. both infect Douglas fir needles, butthe former grows in epidermal and hypodermalcells and the latter grows between mesophyll cells(Stone 1987, 1988). This shows that a leaf is a heterogeneousand complex environment for organismsthesizeoffungi,andmayhaveimplicationsforhowendophytes interact. Unfortunately, few other studieshave mapped endophytes at the level of specifictissues.Endophyte and epiphyte communities of singleleaves may differ. Of the five most frequentgenera in coffee leaves, Guignardia, Xylaria andColletotrichum were significantly more frequent asendophytes, while Botryosphaeria and Pestalotiawere significantly more frequent as epiphytes (Santamaríaand Bayman 2005). These differences areremarkable, considering that the two communitieslive less than a millimetre apart. Since most endophytespresumably arrive at the surface of the leaf,the epiphyte community probably plays a role indetermining which new arrivals are successful atcolonizing.3. Site Specificity and Spatial VariationEndophytes of a single host species may vary considerablyfrom site to site. Environmental conditions,especially rainfall, influence infection frequencyand community composition (Rodrigues1994; see Carroll 1995, Wilson 2000 for reviews).On a local scale, differences among endophyte communitiesof a single host species may increase withdistance, as was shown for cacao trees in Panama:similarity of endophyte communities among leavesdecreased significantly as distance between treesincreased (Arnold et al. 2001; Arnold and Herre2003).Individual leaves on a single tree may differ inendophyte community composition (Lodge et al.1996; Gamboa and Bayman 2001). If the endophytesaffect tree performance, then this variabilitymay complicate other studies that assume uniformitywithin a single tree. This variability can beexplained by the island theory of biogeography anda recent offshoot, the neutral theory of biodiversity(Hubbell 2001). If each leaf is viewed as an island, itsendophyte community size and composition willbe determined by its size, age and distance fromsource communities (Wilson 2000; Bayman 2006).Assumingthesourcecommunityismuchlargerand richer than the community of a single leaf, it isnotsurprisingthatindividualleaveswillvary.Island biogeography has been used to explaindistribution of epiphytic microorganisms onleaves, but not endophytes (Kinkel et al. 1989;Andrews and Harris 2000). Both epiphytes andendophytes would be good model systems forthese questions because sample sizes can be verylarge. Spatial variation within and between treepopulations, trees and leaves has been reviewed byCarroll (1995) and Wilson (2000).C. Effects of Tree Endophytes1. Endophytes as Latent Pathogensor Misplaced PathogensThe distinction between endophytes andpathogens is often blurred. Endophytes maybe latent or opportunistic pathogens. Once establishedin plant tissues, they can remain quiescentuntil a change in the environment or a decline inhost defences allows them to become pathogenic.Others may be pathogens that have colonized thewrong host and are thus unable to cause disease.Endophytes and pathogens of western whitepine have been compared using a phylogenetic approach(Ganley et al. 2004). Two thousand endophyteswere identified by sequencing the nuclearribosomal ITS and BLAST searches. It was foundthat 90% of endophytes belonged to a single family,Rhytismataceae, whichalsoincludesthreemajorpathogens of western white pine. However, noneof the endophytes were clearly conspecific withthe pathogens. Rather, most were closely related topathogens of other species of pines. This suggeststhat either they are specialized as endophytes (e.g.pathogens that have lost the ability to cause disease)or they are pathogens that have infected a non-hostspecies and are therefore unable to cause disease.The authors found the first explanation more likely,based on host and endophyte phylogeography.There is experimental evidence that an endophytemay be derived from a pathogen by loss ofpathogenicity. A nonpathogenic Colletotrichummutant capable of infecting plants was derivedfrom a pathogen in the laboratory (Redman et al.1999). However, many endophytes are still capableof producing phytotoxic secondary metabolites. Infact, endophytes produced more such compoundsin vitro than pathogens of the same hosts (barleyand larch) (Schulz et al. 1999). The authors

222 P. Baymansuggested that these endophytes were still capableofcausingdisease,butthattheplanthostswereable to limit their growth.2. Effects on Host PlantsEffectsofendophytesarehardertodemonstrateintreesthaninherbaceousplantsthatcanbemoreeasily manipulated. For example, it is harder to generateendophyte-free leaves of trees to use as controls.However,effectsofendophytesonleaveshavebeen demonstrated. For example, endophytes ofgorse and oak leaves promoted senescence (Fisheret al. 1986; Wilson 1993). Also, abscised oak leaveshad lower levels of the endophyte Ophiognomoniacryptica than leaves still attached to the tree (Faethand Hammon 1997). Endophytes probably influencecoloration of senescing leaves as well.Effects of endophytes on photosynthesis havebeen demonstrated, but are not always significant.For example, Colletotrichum musae in bananadecreased photochemical capacity compared toendophyte-free plants (Rodrigues Costa Pintoet al. 2000).Endophytes may help host plants survive heatand drought. In the herb Dichanthelium lanuginosum,plantswithaCurvularia endophyte survivedhigh soil temperature and water stress betterthan endophyte-free plants (Redman et al. 2002).This plant lives in areas where soil temperaturescan reach 57 ◦ C,sothepresenceoftheendophytemay increase plant fitness, as with Neotyphodiumin grasses (see Sect. IV.). Such symbioses are of increasingimportance, since they might help plantsadapt to global climate change (Rodriguez et al.2004).3. Effects on Insect HerbivoresMany studies have searched for effects of endophyteson insect herbivores of tree leaves. (In contrast,very few have looked at other types of herbivores.)Results have been interesting but inconsistent;few studies have conclusively demonstrateda negative effect on insects (see Carroll 1995). A surveyof the literature on effects of tree leaf endophyteson insect herbivores found no consensus: ofcorrelative studies, about equal numbers of studiesfound a negative correlation between endophytesand insects, a positive correlation, or no correlation(Wilson 2000). Of experimental studies, somefound endophytes had negative effects on insects,inhibiting growth, survivorship or oviposition, butothers found no effect. Given the diverse nature ofendophytes, the heterogeneity of endophyte–plantinteractions, and the technical difficulties of thesestudies, such mixed results are not surprising.4. Effects on Plant PathogensA protective effect of endophytes against plantpathogens has been demonstrated in cacao(Arnold et al. 2003). Endophyte-free seedlingswere inoculated with a mixture of seven commonendophytes of cacao. These seedlings and controlseedlings were then inoculated with Phytophthorainfestans, a serious pathogen of cacao. Endophytecontainingleaves had significantly smaller lesionsand lower mortality than did control leaves. Thedifference was more dramatic in old leaves thanyoung leaves, presumably because young leavestend to contain more chemical defences againstpathogens.VII. Endophytes of Tropical PlantsIt is thought that endophytes of tropical plants arean important component of global fungal biodiversity(Hawksworth and Rossman 1997; Frolichand Hyde 1999). Why? First, the tropics are richin undescribed plant species. Second, the ratio ofplant-associated fungal species to plant species hasbeen estimated at 6:1 in Britain. (Britain is used asa point of reference because its floras are well sampled.)If this ratio holds for the tropics, it wouldimply a vast number of undescribed endophytespecies.However, it is not clear what the fungus:plantratio is. The plant species richness in the tropicsmay select against highly specific plant-associatedmicroorganisms (May 1988), in which case the ratiowould be lower than 6:1. This controversy has stimulatedresearch on biodiversity and host specificityof tropical endophytes. Results are more mixedthan for temperate trees: while some studies havefound evidence of host specificity (Arnold et al.2001), others studies have not (e.g. Bayman et al.1997; Cannon and Simmons 2002).Taxonomic studies support the view thatmany of the ‘missing fungi’ are endophytes,especially in tropical plants. Many new speciesof endophytes and saprophytes of palms havebeen described by Kevin Hyde and coworkers inHong Kong (Hyde 2001). Based on the number offungi they could identify from a single palm tree,

Fungal Endophytes 223they argue that the 6:1 ratio of fungal species toplant species is too low. They propose a 33:1 ratio,which would greatly increase the extrapolatedtotal number of fungal species (Frölich andHyde 1999).Comparisons of endophyte communities inneighbouring plant species usually show quantitative,rather than qualitative differences. Forthis reason, it may be more accurate to speakof endophyte host preferences, rather than hostspecificity (Lodge 1997), at least for tropical plants.Specificity aside, the most frequent endophytesin the tropics often differ from those of temperateplants. Xylaria and its anamorphs have beenisolated as endophytes in a wide range of tropicalplants, including important crops, but in temperateareas they are saprotrophs and wood-rotters ratherthan endophytes (Rodrigues and Petrini 1997;Rogers 2000; Bayman 2006). Xylaria spp. are ofspecial interest because they produce several typesof bioactive secondary metabolites. Like manytropical endophytes, Xylaria is difficult to fruitin culture, making identification difficult. Studieson tropical endophytes often group sterile fungitogether in morphospecies, on the basis of morphologyin culture (Arnold et al. 2000; Gamboaand Bayman 2001). However, a comparison ofmorphospecies vs. DNA sequencing showed thatmorphospecies data are a fairly accurate way toestimate the number of species in a sample (Arnoldet al. 2000). Other genera such as Guignardia fitthe general pattern described here for Xylaria.VIII. Endophytes of Nonvascular Plantsand LichensEndophyte floras of lichens are as rich as thoseof leaves of vascular plants, if not richer (Miadlikowskaet al. 2005). Over 500 morphospecies wereisolated from 17 lichen thalli (Petrini et al. 1990).Mosses and liverworts also harbour endophytes,including Xylaria (Davis et al. 2003). Endophytesof marine algae are common but largely unexplored(Stanley 1992). Some endophytes of marine algaeare conspecific with terrestrial fungi, and it is notclear if they are accidental endophytes or adapted tothe marine, endophytic habit. In the case of Acremoniumisolated from the brown alga Fucus serratus,endophytic isolates formed a clade distinctfrom terrestrial isolates, suggesting that they arenot accidental (Zuccaro et al. 2004).IX. Uses in Agricultureand BiotechnologyEndophytes have many potential uses in agricultureand biotechnology. This section focuses onthree uses: manipulation of clavicipitaceous fungiof grasses for purposes of biocontrol; manipulationof other endophytes for biocontrol; and naturalproducts discovery or bioprospecting.A. Clavicipitaceous Fungi of GrassesEndophyte-infected turfgrasses are commerciallyavailable; their increased resistance to insectsdecreases the need for insecticides. For pasturegrasses, the ideal biocontrol agent would producealkaloids toxic to insects (lolines and peramines)but not those toxic to mammals (ergot alkaloidsand lolitrems; see Sect. IV.B. above). A Neotyphodiummutant unable to make ergovaline(a major ergot alkaloid) has been produced byknockout of a peptide synthetase gene (Panaccioneet al. 2001). Such designer endophytes wouldhave many advantages as biocontrol agents: theyare stable, are not transmitted horizontally tonon-target hosts, and are transmitted vertically toprogeny (Clay 1989). Artificial inoculation of fungiinto non-host plants is another way of producingendophyte–host combinations with desirableproperties, though it tends to be hit-or-miss (Clay1988). Clavicipitaceous endophytes have also beensuggested as surrogates for genetic transformationof plants, since it is sometimes easier to transformthe endophyte than the host (Clay 1988).B. Other EndophytesBacterial endophytes have been exploited more extensivelythan fungal endophytes for control ofplant diseases (Azevedo et al. 2000). For example,Bacillus subtilis is effective for controlling mycotoxinproductionbyFusariumin corn, and its usehas been patented (Bacon et al. 2001). Curtobacteriumflaccumfaciens shows promise for control ofcitrus variegated chlorosis (Lacava et al. 2004), andgenetically modified bacteria can be introducedinto plants, allowing for inoculation with improvedstrains.Nonetheless, inoculation with endophyticfungi can reduce the frequency and severity ofdisease, compared to endophyte-free plants. Thistechnique has demonstrated a protective function

224 P. Baymanforendophytesagainstbrownrustofwheat(Pucciniatriticina, Dingle and McGee 2003), tan spotof wheat (Pyrenophora tritici-repentis, Istifadahand McGee 2006), and Phytophthora infestans oncacao (Arnold et al. 2003; see Sect. VI.C.4. above).The mechanisms of protection are not clear butcould include antagonism between the endophyteand pathogen, parasitism of the pathogen by theendophyte, competition for nutrients, or inductionof nonspecific host defences. Manipulation ofendophytes and endophyte populations has greatpromise for control of plant diseases.C. BioprospectingEndophytes are a rich source of new natural products(Tan and Zou 2001). The best-known exampleis the production of taxol by a previously unknownendophyte of the Pacific yew, Taxomyces andreanae(Stierle et al. 1993). Other fungal endophytes werelater shown to produce taxol as well (Strobel andDaisy 2003). Until this discovery, the yew was theonly organism known to produce taxol. These discoverieswere exciting because at that time yewbark was the only known source of taxol, an importantanti-cancer drug. Taxol is present in barkat such low concentrations that many trees hadto be felled to meet demand. An organic semisynthesishas replaced both bark and endophytesas the commercial source of taxol. However, thereare still many undescribed endophytes capable ofproducing many new natural compounds. Recentlyisolated secondary metabolites from fungal endophytesare being explored as insecticides, antitumordrugs, antioxidants and immunosuppressants(Strobel and Daisy 2003).X. ConclusionsMany researchers who work with plants are completelyunaware of endophytes, but endophytesmay affect their results. Plant molecular systematistsoccasionally amplify and sequence endophytegenes, rather than plant genes – for example, inpines (Liston and Alvarez-Buylla 1995), bamboos(Zhang et al. 1997) and spruces (Camacho et al.1997). In pines, phylogenetic trees based on thecontaminant sequences reflected the predictedphylogeny of the pines (possibly as a result ofcoevolution), so contamination went undetectedfor some time. In other cases, sequences depositedas plant sequences in databases have turned out tobe fungal. Furthermore, variation in endophytesand their metabolites could be confounding factorsin studies of plant breeding, plant physiology,plant ecology, and animal nutrition.Endophytes embody the mystery and importanceof the fungi. They are a taxonomically diversegroup that have little in common, except a hiddenway of life. They pose interesting questions onmany levels, from natural products chemistry tocommunity ecology, and lead us to address manyfundamental questions about relationships.Acknowledgements. I thank Miguel Angel Gamboa for contributionsto this chapter.ReferencesAhlholm JU, Helander M, Henriksson J, Metzler M, SaikkonenK (2002) Environmental conditions and host genotypedirect genetic diversity of Venturia ditricha, a fungalendophyte of birch trees. Evolution 56:1566–1573Andrews JH, Harris RF (2000) The ecology and biogeographyof microorganisms on plant surfaces. Annu RevPhytopathol 38:145–180Arnold AE, Herre EA (2003) Canopy cover and leaf age affectcolonization by tropical fungal endophytes: ecologicalpattern and process in Theobroma cacao (Malvaceae).Mycologia 95:388–398ArnoldAE,MaynardZ,GilbertG,ColeyPD,KursarTA(2000) Are tropical fungal endophytes hyperdiverse?Ecol Lett 3:267–274Arnold AE, Maynard Z, Gilbert G (2001) Fungal endophytesin dicotyledonous neotropical trees: patterns of abundanceand diversity. Mycol Res 105:1502–1507Arnold AE, Mejía L, Kyllo D, Rojas E, Maynard Z, HerreEA (2003) Fungal endophytes limit pathogen damagein a tropical tree. Proc Natl Acad Sci USA 100:15649–15654Arnold AE, Henk DA, Eells RL, Lutzoni F, Vilgalys R (2007)Diversity and phylogenetic affinities of foliar fungalendophytes in loblolly pine inferred by culturing andenvironmental PCR. Mycologia (in press)Azevedo JL, Maccheroni W Jr, Pereira JO, Araújo WL (2000)Endophytic microorganisms: a review on insect controland recent advances on tropical plants. ElectronJ Biotechnol http://www.ejbiotechnology.info/content/vol3/issue1/full/4/ index.htmlBacon CW (1985) A chemically defined medium for thegrowth and synthesis of ergot alkaloids by species ofBalansia. Mycologia 77:418–423Bacon CW, Hill NS (1996) Symptomless grass endophytes:products of coevolutionary symbioses and their rolein the ecological adaptations of infected grasses. In:Redlin SC, Carris LM (eds) Endophytic fungi in grassesand woody plants. APS Press, St Paul, MN, pp 155–178Bacon CW, Hinton DM (1996) Symptomless endophytic colonizationof maize by Fusarium moniliforme.CanJBot74:1195–1202

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14 Mycorrhizal Fungi: Their Habitats and Nutritional StrategiesM. Girlanda 1 ,S.Perotto 1 ,P.Bonfante 1CONTENTSI. Introduction ........................ 229II. Mycorrhizal Fungi Interactwith both Soil and Plants .............. 230A. Fungal DiversityinPlant–FungalInteractions......... 230B. Fungal DiversityinSoil–FungalInteractions .......... 233III. Nutritional Strategies of Mycorrhizal Fungiat the Soil–Root Interface .............. 235A. MineralNutrients ................. 2351.UtilisationofPhosphorus ......... 235a) AMFungi .................... 235b) ECMFungi ................... 2362.NAcquisition................... 237a) ECMFungi ................... 237b) AMFungi .................... 239B. Carbon.......................... 2391. Uptake of Plant-Derived Cat the Symbiotic InterfacebyAMandECMFungi ........... 2402. Fate of Plant-Derived CinAMandECMMycelia .......... 241IV. At the Interface Between Several Host Plants:Common Mycelial Networks (CMNs),a Unifying Phenomenonin ECM and AM Fungi ................. 242A. Evidence for the OccurrenceandFunctionofCMNs.............. 242B. What may be the Ecological SignificancefortheHostPlants? ................ 2431. InteractionsBetweenAutotrophicPlants........ 2432. Interactions Between AutotrophicandHeterotrophicPlants.......... 244C. . . . and what Ecological SignificancefortheMycorrhizalFungi?........... 246V. Conclusions ......................... 246References.......................... 246I. IntroductionMycorrhizal fungi are specialised root symbionts,engaging in intimate association with a great diversityof plants (Smith and Read 1997). The best1 Department of Plant Biology, University of Torino, Viale PA Mattioli25, Torino 10125, Italyunderstood function of such fungi in the symbiosisis improvement of plant mineral nutrientacquisition, in exchange for some photosynthate,resulting in positive host growth responses. However,this symbiosis has a multifunctional character(Newsham et al. 1995) because mycorrhizal fungimay perform many other significant roles, includingprotection of the plant from biotic and abioticstress, for instance, by altering host environmentaltolerances to water deficit or pollutants, or reducingsusceptibility to soil-borne pathogens.Historically, the variety of mycorrhizal associationsestablished between plants and fungihas been placed into seven categories (ectomycorrhiza,arbuscular endomycorrhiza, ericoidendomycorrhiza, orchid endomycorrhiza, ectoendomycorrhiza,arbutoid mycorrhiza, monotropoidmycorrhiza) based primarily on structural characteristicsof the symbiotic interfaces and thetaxonomic identity of the symbionts (Smith andRead 1997). Classification based on functionalcriteria has also been proposed, distinguishing‘balanced’ and ‘exploitative’ mycorrhizal associations(Brundrett 2002, 2004). Although theassociation is generally assumed to be mutualistic,with bilateral nutrient exchange between the plantand fungal partners, host responses ranging frompositive to negative may in fact be observed,with mycorrhizal fungi sometimes functioning ascommensals, necrotrophs or antagonists of hostor non-host plants, their roles varying during thelifespan of the association. Conversely, exploitationofthefungalpartnerbythehostplantwithoutanyapparent benefit in return also occurs in peculiarmycorrhizal associations. Mycorrhizal outcomesare indeed conditional, depending upon complexinteractions between environmental, developmentaland genotypic factors, and the association istherefore best outlined as occupying a wider rangeon the symbiotic continuum, including commensalismand antagonism (Johnson et al. 1997;Brundrett 2002, 2004; Egger and Hibbett 2004).Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

230 M. Girlanda, S. Perotto, P. BonfanteThe first periods of research on mycorrhizahave been dominated by a cataloguing, reductionistapproach (Read 2002) that has allowed a basicframework to be established, the functioning ofsingle pairs of symbionts in terms of nutrient exchange.Traditionally perceived as being restrictedto the root niche, mycorrhizal fungi thrive in fact atthe interface between two distinct habitats, a biotic(host roots) and abiotic (soil) habitat. Although alreadyin 1973 the ‘mycorrhizosphere’ concept (thesoil volume under the influence of mycorrhizalroots, extending beyond the area directly accessibleto roots; Rambelli 1973) had recognized the keyrole of mycorrhizal fungi in linking plants to soil,only later has consideration of these fungi shiftedfrom an almost exclusive focus on their relationshipwith the plant host to the realization of their environmentalroles. Thus, greater attention has beenpaid to the functioning and importance of the extraradicalmycelium of mycorrhizal fungi and itsinteractions with the biotic and abiotic environmentand, due to innovative combinations of methods(for instance, root-free compartments coupledwithisotopeprobing),thelastdecadehasyieldedvaluable insights on the activities of this dynamicand functionally diverse component of the symbiosis(Leake et al. 2004a). At the same time, progressin molecular identification and characterization offungal symbionts has revealed a much more diverseand complex picture of the multifunctional natureof mycorrhizal fungi. Martin et al. (2007) have reviewedthe perspectives opened by environmentalgenomics, which combine community/populationstructure and function studies applying genomics.The fungal and plant genes, regulation of theirexpression, and biochemical pathways for nutrientexchange between symbiotic partners are nowcoming under intense study (see, e.g. Graham andMiller 2005), and will eventually be used to definethe ecological nutritional role of the fungi.Another promising tool is stable isotope measurements,which rely on calculating ratios of heavyto light isotopes of key biological elements (e.g.13 C: 12 C, 15 N: 14 N). This method has been increasinglyapplied to explore fungal functioning (Hobbie2005). The study of natural abundance of stableisotopes in fungal structures, fungal-derivedcompounds and the environment, as well as theuseofstableisotopetracers(thefateofwhichisfollowed into different ecosystem components, includingfungi;cf.stableisotopeprobing;see,e.g.Dumont and Murrell 2005) will help us to link fungalidentitytofunction,andtoimproveourun-derstanding of fundamental ecosystem processesdriven by fungi (Hobbie 2005).Since these topics have been the objects of manyrecent reviews, the aim of this chapter is to shortlysummarize the most relevant aspects of mycorrhizalfungi as a “tie that binds” (Read 1997) the hostplant to the biotic and abiotic environment, witha focus on the two main types of mycorrhizal symbionts,i.e. arbuscular endomycorrhizal (AM) andectomycorrhizal (ECM) fungi.II. Mycorrhizal Fungi Interactwith both Soil and PlantsA. Fungal Diversityin Plant–Fungal InteractionsThe mycorrhizal habit has arisen multiple times infungal evolution. Based on evidence from paleobotanicalstudies and the analysis of rDNA-basedphylogenies, the most ancient mycorrhizal fungiare those forming arbuscular mycorrhizas. Theirearliest fossil record dates back to the Ordovician,460 million years ago (Redecker et al. 2000a),but molecular clock-based inferences estimatetheir origin at much earlier (Tehler et al. 2000;Berbee and Taylor 2001; Schüßler et al. 2001a, b),possibly as far back as between 1200 and 1400 Maago (Heckman et al. 2001). Such an ancient originanticipates emergence of vascular terrestrialplants, and the thallus of bryophyte-like precursorshad associations resembling modern arbuscularmycorrhizas even before roots evolved (reviewedin Brundrett 2002). Thus, the hypothesis has beenput forward that symbiosis was a key factor inthe colonization of land by plants (Taylor et al.1995; Phipps and Taylor 1996). Modern arbuscularmycorrhizal (AM) fungi comprise a number ofancient lineages (Redecker et al. 2000b; Schüßleret al. 2001a; Schüßler 2002), which based on morphological,biochemical and ecological traits, canunequivocally be separated from all other majorfungal groups. Based on 18S rDNA sequences, AMfungi appear to form a monophyletic (althoughweakly supported) group, which was given phylumstatus (Glomeromycota; Schüßler et al. 2001b). TheGlomeromycota possibly diverged from the samecommon ancestor as the dikaryomycetes (AscoandBasidiomycota; Gehrig et al. 1996; Tehler et al.2000; Schüßler et al. 2001b). Information fromdifferent genomic loci, which is being increasingly

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 231collected (see, e.g. Helgason et al. 2003; Corradiet al. 2004; Da Silva et al. 2006), is needed toprovide further support to the phylum, and toclarify evolutionary patterns within the group. Informationderiving from multigene trees will alsoimprove our understanding of species boundariesin AM fungi. Based on a traditional, morphologicalspecies concept, the Glomeromycota areviewed as a species-poor group, with about 150species recognized by conventional spore-basedmorphology (Morton and Benny 1990). Since thesefew morphospecies colonize about two-thirds ofmodern plants (Smith and Read 1997), arbuscularmycorrhizas appear to be not only the mostubiquitous and abundant terrestrial symbiosisbut also nonspecific, with most AM fungi readilycolonizing almost any susceptible plant species(Molina et al. 1992). The few AM fungi usedin laboratory experiments indeed exhibit broadhost spectra. However, several recent reports onthe occurrence of natural root-colonising AMfungi, identified by their rDNA sequences infield-collected roots (Helgason et al. 1998, 2002;Husband et al. 2002; Vandenkoornhuyse et al. 2002,2003; Öpik et al. 2003; Rosendahl and Stukenbrock2004; Johnson et al. 2004; Scheublin et al. 2004;Öpik et al. 2006), have revealed challenging AMfungal richness and specificity (Sanders 2002,2003, 2004). Whereas some plant species enterinto specialised relationships with only a few AMfungi, others appear to associate with as many as20 different AM fungi, thus hosting very diverseAM fungal communities that include currentlyunknown taxa. Possible relationships betweenplant responsiveness to mycorrhiza and theirselectivity towards specific AM fungi remain to beelucidated (van der Heijden 2002), but it has beensuggested that the AM fungal genetic diversityobserved may be, at least partially, accounted for bydiversification in functional traits, e.g. efficiency infunctions such as mineral nutrient acquisition, soilbinding, protection from pathogens, or sensitivityto abiotic and biotic environmental factors (Fitter2005). AM fungal morphospecies or sequencegroups have indeed been shown to be functionallydiverse (Hart and Reader 2002a, b; Munkvold et al.2004; van der Heijden et al. 2004; Avio et al. 2006;Koch et al. 2006).On the other hand, the possibility that someof the sequence diversity found in these studiesmay be within – rather than among – individualscannot be ruled out (Pawlowska and Taylor 2004;Rosendahl and Stukenbrock 2004). Uncommonlypolymorphic sequences of rDNA genes have beenfound within single AM fungal spores, whichharbour hundreds of nuclei (Lloyd-MacGilpet al. 1996; Pawlowska 2005), in contrast withthe situation found in other organisms, wherehomogenization of rDNA repeats is carried outthrough concerted evolution, a recombinationdrivenprocess (Dover 1982). No evidence forsexual reproduction is available either in ancestralor in modern AM fungi, leading to the suggestionthat they may represent one of the oldest groupsof clonally reproducing eukaryotic organismson Earth (Judson and Normark 1996). The highintrasporal variability found in these fungi fornormally conserved genes has provoked intensecontroversy over their genetic structure, and theprocesses governing it (reviewed in Pawlowska2005). While one possible explanation involvesdistribution of the intrasporal rDNA variationamong different, therefore genetically distinctnuclei (heterokaryosis; Kuhn et al. 2001; Bever andWang 2005; Hijri and Sanders 2005), an alternativehypothesis envisages such variation as being containedin each individual nucleus (homokaryosis),with polyploid genome organization to accommodateintranuclear rDNA polymorphism and tobuffer these apparently asexual organisms againstthe effects of accumulating mutations (Pawlowskaand Taylor 2004, 2005). The reproductive mode(clonal vs. recombining) of AM fungi in natureremains elusive (Pawlowska 2005), and we stillknow too little about the basic genetics andgenome organization of AM fungi to resolve thesedifferent scenarios. Approaches such as multiplexand global PCR amplification for individualspores (Gadkar and Rillig 2005; Stukenbrock andRosendahl 2005), and genome sequencing of representativeAM fungi such as Glomus intraradices(Martin et al. 2004) will hopefully provide newclues to widen our understanding of reproductionand genetics in these fungi.In contrast with the situation of arbuscular mycorrhiza,established exclusively by a specific fungalgroup, the ectomycorrhizal (ECM), symbiosisinvolves a diverse range of fungi (a large numberof homobasidiomycetes, some ascomycetes, anda handful of zigomycetes belonging in the genusEndogone; Smith and Read 1997). Unlike AM fungi,ECM fungi show conventional sexual mechanisms,with possibly a few exceptions such as the apparentlysterile, widespread ascomycete Cenococcumgeophilum (LoBuglio et al. 1996; Shinohara et al.1999).

232 M. Girlanda, S. Perotto, P. BonfanteMost likely, the ECM symbiosis emergedlong after the arbuscular association. Whereasdivergence between Endogonales and Glomerales(the AM fungi) would have occurred at leastabout 600 Ma ago, well before any evidence ofvascular plants, Asco- and Basidiomycota wouldhave diverged from one another in the Palaeozoic,at least 500 Ma ago (Berbee and Taylor 2001).Although the only true ECM fossils are from recentMiddle Eocene materials (LePage et al. 1997), it islikely that most ECM fungi (basidiomycetes) wentthrough rapid diversification during the period ofangiosperm radiation in the Cretaceous and in theEocene-Oligocene transition (Bruns et al. 1998;Bruns and Shefferson 2004), perhaps assistingplant migration from the Tropics to the poorertemperate regions (Smith and Read 1997). Theectomycorrhizal assemblage is therefore clearlypolyphyletic, since a common ECM precursor forall of these fungal groups should have evolvedearlier than the Glomeromycota, and prior toland plant evolution (Bruns and Shefferson 2004).ECM ascomycetes belong in at least four distinctlineages (LoBuglio et al. 1996; Percudani et al.1999), and several studies have provided evidencethat the ECM habit has developed convergentlyin multiple lineages of homobasidiomycetes,evolving repeatedly from saprotrophic ancestors(Bruns et al. 1998; Hibbett et al. 2000; Moncalvoet al. 2000). Multiple reversion to a free-living,saprotrophic lifestyle has also been postulated,consistently with theoretical predictions that mutualismis inherently unstable (Hibbett et al. 2000).It is, however, unclear what ecological advantages,in terms of acquisition of open niches, reversionto saprotrophism would confer, since saprotrophicbasidiomycetes had been well established priorto the evolution of the ECM habit (Bruns andShefferson 2004). Whatever the directions of theevolutionary patterns, the polyphyletic originsof ECM fungi suggest considerable functionaldiversity in these fungi (Brundrett 2002), whichis indeed indicated by physiological studies(e.g. Abuzinadah and Read 1986; Coleman et al.1989; Arnebrant 1994; Keller 1996; Dickie et al.1998; Read and Perez-Moreno 2003). Convergentevolution is likely to have occurred also on theplant side, since the ECM trait is present inunrelated groups such as the Pinaceae and at least12 independent groups of angiosperms (Brunsand Shefferson 2004). Although specialists occurin both fungi and plants, the majority of ECMfungi and plants are capable of association withmultiple partners (e.g. Trappe 1962; Molina andTrappe 1982; Molina et al. 1992; Horton and Bruns1998; Horton et al. 1999; Cullings et al. 2000). Suchcapability sets the stage for the establishment ofimpressively diverse ECM fungal communities,which are being described belowground by meansof molecular identification (e.g. Dahlberg 2001;Horton and Bruns 2001; Taylor 2002; Chen andCairney 2002; Dickie et al. 2002; Landeweert et al.2003; Tedersoo et al. 2003; Anderson and Cairney2004; Izzo et al. 2005; Koide et al. 2005; Saari et al.2005; Genney et al. 2006; Toljander et al. 2006).Not only is sequence-based identification helpingto dissect ECM community structure but it is alsoincreasingly broadening the set of known ECMtaxa even in well-investigated environments –such as temperate forests – where it has revealedunsuspected ECM capability in ascomycetous (see,e.g. Vrålstad et al. 2000; Tedersoo et al. 2006) and(hetero)basidiomycetous (Selosse et al. 2002a;Urban et al. 2003; Bidartondo et al. 2003) fungi.An unexpected finding from these studies(sometimes coupled to resynthesis experiments)has been the recognition of both ecto- andendomycorrhizal competence in the same fungus.Simultaneous formation of ectomycorrhiza intree hosts and ectoendomycorrhiza in plants ofthe subfamilies Arbutoideae and Monotropoideae(family Ericaceae) has long been known (Kamienski1881; Björkman 1960; Smith and Read 1997),and often arbutoid and monotropoid ectoendomycorrhizaeare, in fact, not considered as a distinctmycorrhizal category (Egger and Fortin 1988; Yuet al. 2001; Brundrett 2004). By contrast, fungiable to establish ericoid (i.e. Cadophora (syn.Phialophora) finlandia, amemberoftheso-calledHymenoscyphus ericae aggregate; Vrålstad et al.2002) or orchid (i.e. Sebacina and Tulasnellaspecies) endomycorrhiza have only recently beenproven to be also able to develop typical ectomycorrhizain trees (Selosse et al. 2002a, b; Taylor et al.2002; Bidartondo et al. 2003, 2004; Urban et al.2003; Villarreal-Ruiz et al. 2004; Weiss et al. 2004).This triangular relationship suggests that thesefungi are part of a common guild (Vrålstad 2004),setting the intriguing scenario of mycobiontmediatedinterplant interactions (see below). Italso reveals fungal phenotypic plasticity in theproduction of distinct interfaces within differenthosts, and suggests a decisive role of the hostplant in determining the ecto- or endomycorrhizalcharacter of the association. Symbiotic interfacesare complex compartments involved in nutrient

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 233exchange between partners, and likely result fromspecific morphogenetic processes involving temporallyand spatially controlled activity of genesand proteins. A fungus capable of developing suchdifferent interfaces as those characterizing ectovs.endomycorrhizae (cf. fungal hyphae remainingexternal to plant cell walls or breaching cell wallsrespectively, but remaining separated from thecell cytoplasm by a plant-derived membrane andan interfacial matrix; Peterson and Massicotte2004) represents an interesting model to study thedevelopment of the structurally different ecto- andendomycorrhizal organs.Recent molecular ecology investigations havealso revealed a continuum between mycorrhizalfungi and other nonpathogenic root associates,the wide and diverse group of non-mycorrhizalroot endophytes mostly referred to as DSE (darkseptate endophytes) or DSM (dark sterile mycelia;Schulz and Boyle 2006). These conidial or sterileseptate fungal endophytes, with known or likelyaffinities with distinct ascomycetous lineages,grow asymptomatically in the roots either in thepresence or absence of ecto- or endomycorrhizalmycobionts (Jumpponen and Trappe 1998; Sieberand Grünig 2006; Girlanda et al. 2006a). They maybe as abundant and consistent as mycorrhizal fungi(Jumpponen and Trappe 1998; Girlanda et al. 2002;Mandyam and Jumpponen 2005) and, by virtue ofsuch a constant association with roots, they canbe qualified as true root symbionts (Schulz andBoyle 2006). Their peculiar structural interactionwith roots, achieved by hyphae growing interandintracellularly in the root cortex and microsclerotia(Schulz 2006; Schulz and Boyle 2006),differs from the specialised interfaces that arediagnostic of mycorrhizae (Brundrett 2006). Thefungal partner likely profits from a stable nutrientsource and some protection from abiotic stressesand, although DSE exhibit variable life historystrategies, several examples have been reportedof nutritional and non-nutritional benefits to thehost plant, thus indicating a possible mutualisticcharacter of the association (Mandyam andJumpponen 2005; Schulz 2006; Schulz and Boyle2006). Fungi with endophytic behaviour in someplants are capable of forming typical mycorrhizalstructures in other hosts (Bergero et al. 2000, 2003;Cairney 2006; Schulz 2006; Bayman and Otero2006; Rice and Currah 2006; Girlanda et al. 2006a);on the other hand, classical mycorrhizal fungimay have endophytic phases in non-host plants,non-mycorrhizal plants, or plants with dual – AMand ECM – mycorrhizal associations (Brundrett2006), thus prompting contrasting interpretationsof root endophytism. One view (Brundrett 2002,2004, 2006) contrasts mycorrhizal and endophyticassociations, and proposes the latter as possibleevolutionary precursors of the former, hence postulatinga continuum of association types, startingwith endophytic occupation of roots by fungi andconcluding with mycorrhizal associations withsynchronised nutrient transfer. An alternative viewincludes root endophytes in a broader conceptof symbiotic mycorrhizal associations, spanningfrom mutualism to parasitism (Johnson et al.1997), and considers them as nonconventional mycorrhizalsymbionts (Jumpponen 2001; Mandyamand Jumpponen 2005). Data available on DSEfunctions thus far are admittedly scant, and westill know little to correctly appreciate the degreeof ecological and functional overlap between rootendophytes and mycorrhizal symbionts. Whateversuch a relationship, the possibility of multiple(endophytic and mycorrhizal) interaction for thesame root coloniser reinforces the idea of plantcontrol over the character of root associations(Girlanda et al. 2006a).B. Fungal Diversity in Soil–Fungal InteractionsThe broad diversity of mycorrhizal fungi ismirrored by their interactions with the soil environment,engaged by the extraradical mycorrhizalmycelium (ERMM). Progress in our understandingof crucial processes such as transport ofplant-derived carbon, production of extracellularenzymes, mineralization of nitrogen andphosphorus, uptake and transport of nutrients,weathering of minerals, soil aggregation, andinteractions with other soil organisms has longbeen hindered by difficulties in observing andstudying the dynamic and functionally diversemycelial systems, extending beyond the colonisedroot into the opaque soil matrix without disturbingand destroying them. Only in the past decadehave studies started to focus on the extent andfunctioning of ERMM in the field, and previouslyintractable aspects of biomass, structure andfunction of ERMM have been addressed withestimation of biochemical markers (e.g. ergosteroland specific phospholipid fatty acids as a signaturefor fungal membranes), and observations carriedout in either mycorrhizal root-organ cultures(Fortin et al. 2002) or thin-layer soil microcosms

234 M. Girlanda, S. Perotto, P. Bonfantecoupled with radioactive and stable isotope tracers(these methods, and the insights gained from theirapplication, have been critically covered in severalrecent reviews, e.g. Olsson et al. 2002; Treseder2004; Leake et al. 2004a; Cairney 2005; Rillig andMummey 2006). Of particular importance hasbeen the use of mesh barriers to provide root-freecompartments into which mycorrhizal myceliumcan grow, which have allowed us to distinguishthe biomass and effects of ERMM from those ofroots both in laboratory and, increasingly, in fieldexperiments. Mesh bags buried in soil have, forinstance, been used to estimate ERMM biomassin field settings and, although the difficulty indistinguishing ECM from saprotrophic myceliaremains a major problem with such systems,discrimination between the contributions of thetwo fungal components has been based on eitheranalysis of the carbon isotopic composition ofmycelia extracted from the mesh bags, comparisonwith ‘closed’ cores (i.e. PVC pipes that preventedthe in-growth of mycorrhizal fungi), or theuse of acid-washed sand to allow preferentialdevelopment of mycorrhizal mycelia (see, e.g.Wallander et al. 2001; Högberg and Högberg 2002;Nilsson and Wallander 2003; Querejeta et al. 2003;Hendricks et al. 2006). Mesh-walled soil-filledcores, either left undisturbed or rotated to severthe hyphal connections with roots, have also beenused to quantify in situ nutrient uptake by theERMM (Schweiger et al. 1999; Schweiger andJakobsen 2000; Johnson et al. 2001, 2002a, b), andtransfer of C from the host plant.Emerging from such experimental studies isthe dynamic and functionally diverse nature ofERMM, its great contribution to soil biomass,carbon and nutrient fluxes, and global C, P andN cycles. ERMM, which may comprise up to 85%of the total mycorrhizal fungal biomass (Colpaertet al. 1992; Wallander et al. 2001), often constitutes20–30% of total soil microbial biomass (e.g. Millerand Kling 2000; Olsson and Wilhemsson 2000;Högberg and Högberg 2002). By exceeding tens ofmetres per gram of soil (e.g. Miller et al. 1995; Ek1997), it also provides extensive conduits for C andnutrient fluxes through the soil.ERMM morphology and growth patterns aswell as nutrient capturing mechanisms (see alsoSect. III.A.) indicate different foraging strategiesfor AM and ECM fungi (Olsson et al. 2002). AMfungi form a uniformly distributed mycelium insoil, growing as aseptate thick-walled runner hyphaeassociated with lateral absorbing thin-walledhyphae. In the patchy soil environment, AM hyphalproliferation has been observed near potentialhost roots as well as in response to severaltypes of organic materials, although organic substratesare not generally exploited as a C sourceby the AM external mycelium (Green et al. 1999;Ravnskov et al. 1999). By contrast, the ECM usuallyform denser hyphal fronts than AM fungi, and theyrespond to both organic material and inorganicnutrients by increased growth. They exhibit thewidest and most active set of enzymes for foragingon complex organic material, and also seem to bemore active in the exudation of organic acids. Significantmorphological differentiation is often observedin ECM species that produce the most extensiveERMM, with the formation of hydrophobic hyphalaggregates (hyphal cords or rhizomorphs) involvedin long-distance transport, and hydrophilicdistal mycelium involved in the uptake of solublenutrients. Olsson et al. (2002) hypothesized thatsuch differences between AM and ECM ERMM mayreflect distinct evolutionary strategies, aimed at optimalsearch for potential new host roots in AMfungi, and at optimised nutrient capture in competitionwith other mycelia and translocation intothe host roots in ECM fungi, possibly as a result ofdifferences in size and persistence of energy supplyby the host species.Consideration of the functional importance ofERMMhasalsostimulatedanewapproachtothecharacterization of the diversity of natural mycorrhizalfungal communities. Some ECM producelittle mycelium, apart from the mycorrhizal mantle,whereas others produce extensive external mycelialfans and strands. These differences most likelyreflect distinct functional patterns of explorationand nutrient exploitation (Agerer 2001). Becausethey are relatively easy to culture, and amenableto laboratory manipulation, ECM fungi producingfairly extensive ERMM (such as Suillus, Paxillus,Pisolithus, Rhizopogon species) have been mostpopular for use in the microcosm studies on whichso many of our notions of ECM functioning arebased (Read and Perez-Moreno 2003; Leake et al.2004a). Such species do not appear to dominate inthe field, as judged by DNA-based characterizationof ECM root tips (see, e.g. Horton and Bruns 2001),but they may be nevertheless functionally veryimportant in carbon and nutrient cycles. Onlyrecently have DNA-based identification methodsbeen applied to ECM mycorrhizal mycelium insoil, and ERMM-based diversity data compared tothe picture obtained from molecular identification

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 235of mycorrhizal roots (e.g. Chen and Cairney 2002;Dickie et al. 2002; Landeweert et al. 2003; Wallanderet al. 2003; Hunt et al. 2004; Koide et al. 2005;Kjöller 2006; Genney et al. 2006). These studies areopening a new window on the diversity and spatialdistribution of ECM fungi in soil, having revealed,for instance, foraging at different spatial scales bydifferent species, segregation of some mycorrhizasand corresponding ERMM in different soil layers,and unexpected high frequency of ERMM ofsmooth-type mycorrhizae (Genney et al. 2006).Just as the molecular identification of ECM rootshas allowed us to shift from an ‘aboveground’ viewbased on surveys of sporocarps, which mostlyreflects fruiting patterns, to a ‘belowground’ viewproviding information on the outcome of competitionamong the fungi for host roots (Horton andBruns 2001), the ‘mycelial’ perspective on the ECMfungal community will be invaluable to establishfunctional differences between mycorrhizal fungiin the field (Leake et al. 2004a). This perspectiveappears to be more appropriate if we are primarilyconcerned with functions of mycelia such as wateror nutrient uptake, or competition for physicalspace (Koide et al. 2005). The need to relate fungaldiversity in the bulk soil to that in the plantroots clearly exists also in the case of AM fungi,and mycelium-based studies are eagerly awaitedfor these mycorrhizal symbionts (Johnson et al.2005a).III. Nutritional Strategiesof Mycorrhizal Fungiat the Soil–Root InterfaceThe extraradical mycorrhizal mycelium (ERMM)performs water and nutrient capture from the soiland is an important sink for host carbon. Withinthe root, carbohydrates and mineral nutrients arethen transferred across interfaces that are borderedby plant and fungal plasma membranes (Smithand Smith 1990). Although the molecular mechanismsare not clearly understood, such bidirectionaltransfer is generally recognized as passiveefflux into the symbiotic interfaces and then activeuptake by the receiver organism (Smith and Read1997).Whereas details of molecular mechanisms forcarbon and P exchange between AM symbiontshave emerged rapidly from genetic studies on nutrienttransport and metabolic pathways (Harrison1999), dissection of host–fungal interactionsfor ECM fungi has been more problematic (Martinet al. 2001).A. Mineral NutrientsThe success of mycorrhizal fungi in time and spacemostly relies on the nutritional benefits they conferto their plant hosts: they take up phosphate (Pi)and other macronutrients as well as microelementsand water from the soil, and transfer them to theplant. Despite the fact that mycorrhizal fungi playa crucial role in N, P and C cycling in ecosystems,their detailed function in nutrient dynamics is stillunknown. Consistent with their wide genetic variability,mycorrhizal fungi differ in their functionalabilities, thus offering distinct advantages to thehost plant. Some fungi are particularly effective inscavenging organic N, and associate with plantsfor which acquisition of N is crucial (Peter et al.2001); others are more effective at P uptake andtransport. An important goal is therefore to developapproaches (i.e. the identification of 13 Cand15 N isotopic signatures) by which the functionalabilities of the symbiotic fungal communities areassessed directly in the field.1. Utilisation of PhosphorusPhosphate is an essential nutrient for all livingcells, and plants have developed different strategiesto ensure and enhance Pi acquisition (i.e. bymodifying root architecture and extension to explorelarger portions of soil, and by secreting organicacids or phosphatases that allow the release ofbound Pi; Marshner 1995). The alternative adoptedby most land plants is to use the Pi “catering service”,as Requena (2006) has defined Pi uptakeguaranteed by the symbiotic association with mycorrhizalfungi.a) AM FungiOrthophosphate uptake is greatly enhanced duringAM association (Smith and Gianinazzi-Pearson1988). Current explanations for this increased efficiencyare that (i) AM fungal mycelium exploresthe soil more efficiently than the root itself, andspreads beyond the phosphate depletion zone, (ii)it takes up phosphate from the soil and transfersit along the hyphae, and (iii) it delivers the nutrientto the plant root cells. Mycorrhizal plantscan therefore acquire Pi either directly from the

236 M. Girlanda, S. Perotto, P. Bonfantesoil through plant-specific phosphate transporters(PT) or through uptake and transport systems ofthe fungal symbiont. Both systems can work simultaneouslybut – at least for some plants – thereis a preferential uptake via fungal hyphae. Recentdata demonstrate how this fungus-mediated uptakerequires the coordination of gene expressionin both symbionts, such as up-/down-regulation ofthe high-affinity PT genes in both partners, andup-regulation of secreted acid phosphatase genesby the host (Ezawa et al. 2005).In the extraradical mycelium, Pi is absorbedby active PTs operating at the fungus–soil interface(Harrison and Van Buuren 1995; Maldonado-Mendoza et al. 2001; Benedetto et al. 2005), andaccumulates in the vacuoles of extraradical hyphaeas inorganic polyphosphate (polyP), a linear polymerof Pi linked by high-energy bonds (Ezawa et al.2004). Its synthesis and accumulation in AM hyphaeoccur rapidly but the molecular and biochemicalbases of such processes are not yet known. SincepolyP chains are accumulated in acidic compartmentssuch as the vacuoles, they are supposedlytransferred by means of a motile tubular vacuolarnetwork (Uetake et al. 2002) in the intraradicalcompartment; Pi ions resulting from polyPhydrolysis are assumed to be released by membranepassive carriers into the periarbuscular space(Ezawa et al. 2002). Mycorrhiza-specific PTs possiblyresponsible for plant Pi uptake in arbusculecontainingcells have recently been characterizedin many plants: potato, tomato, rice, maize, barleyand Medicago truncatula (Rausch et al. 2001; Harrisonet al. 2002; Paszkowski et al. 2002; Glassopet al. 2005; Nagy et al. 2005). The importance ofsuch mycorrhizal-inducible PTs is indicated by thepresence of multiple PTs in Solanaceae, suggestinga gene duplication event at least in these plants(Nagy et al. 2005). A combination of genetic, physiologicaland molecular approaches demonstrateshow the mycorrhiza-inducible LePT3 and LePT4are expressed also in mutant plants (rmc) wherethe block to fungal colonisation is not complete(Poulsen et al. 2005), suggesting that these PTs canbe considered reliable markers for a functional Piuptake. Fungal PT genes have been so far isolatedexclusively from Glomus species (G. versiforme,G. intraradices and G. mosseae), and encode forhigh-affinity proton-coupled transporters (Harrisonand Van Buuren 1995; Maldonado-Mendozaet al. 2001; Benedetto et al. 2005). As expected,they are predominantly detected in the extraradicalmycelium, even though G. mosseae PT, differentlyfrom G. intraradices PT, shows a significantexpression also in the mycorrhizal roots.Taken as a whole, these data provide a rathercomplete picture of the molecular mechanisms thatoperate in order to guarantee the fungus-to-plantphosphate transfer. However, many questions,mostly concerning the way in which the fungusaccumulates polyP and then releases it, as well asthe cost paid by the plant for such symbiotic route,remain unanswered (Requena 2006).b) ECM FungiIn the case of ectomycorrhiza, the host plants relyheavily on the fungus for Pi and N uptake becauseECM fungi may be able to take up complexor immobilised forms of these nutrients that theroot cannot absorb. Due to the production of extracellularacid and alkaline phosphomono- andphosphodi-esterases, phosphatases and phytases,ECM fungi can solubilize insoluble forms of Pi, suchas Al and Ca phosphates and inositol hexaphosphates(Cumming and Weinstein 1990; Lapeyrieet al. 1991; Leake and Read 1997; Nilsson and Wallander2003). The ability of some ECM fungi tomobilise Pi directly from minerals, through excretionof organic chelators such as organic acids(mainly citric and oxalic acids), has also becomeincreasingly apparent (e.g. Wallander 2000a, b; vanBreemen et al. 2000; Landeweert et al. 2001; Wallanderet al. 2002). Weathering of rock substratesthrough physical and chemical mechanisms enablesectomycorrhizal plants to utilize essential nutrientsfrom insoluble mineral sources, and playsa fundamental role in mineralogical processes, includingpedogenesis and biogeochemical cyclingof nutrients in forest systems (Burford et al. 2003).Once dissolved, Pi is taken up by ECM hyphae andthen translocated to the host roots, its absorptionand efflux being likely regulated by intracellularPi and inorganic polyphosphates (polyP) pools.Although several low- and high-affinity Pi transportershave been identified in the genome sequenceof Laccaria bicolor (F. Martin, unpublisheddata) and other symbiotic fungi, the molecular processescontrolling Pi uptake in ECM fungi remainunknown.Many ECM basidiomycetes also appear tohave retained some of the saprophytic abilitiesof their decomposer relatives and, thereby, canaccess a range of organic sources of N and P fromthe soil, including partially decayed tree litter,pollen and nematodes (Read and Perez-Moreno

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 2372003). Based on the efficiency of nutrient recoveryfrom such detrital materials by ECM tree seedlingsin soil microcosms, and estimates of the annualproduction of these nutrient sources in borealforests, it has been suggested that as much as 15%of P and 12% of N supplied to trees in these forestecosystemsmaycomeonlyfromnutrientuptakefrom these sources by the ECM ERRM (Read andPerez-Moreno 2003).2. N AcquisitionInadditiontotheirfunctioninPnutrition,AMandespeciallyECMfungiplayapivotalroleintheuptake of N by plants.Whilemycorrhizaresearchhasemphasizedtherole of symbiosis in facilitating capture of mineralnutrients in ionic form, attention has shifted sincethe mid-1980s to analysing the mycorrhizal fungalabilities to release N and P from detrital materialsof microbial and plant origins, which arethe primary sources of these elements in terrestrialecosystems (Read and Perez-Moreno 2003). The resultsobtained over the last decade have providedmuch support to the hypothesis (Read 1991) that,on a global scale, mycorrhizal fungi may be makingsignificant contributions to ecosystem nutrientcycling (Read and Perez-Moreno 2003). In theforest ecosystems of Eurasia and North America,soilacidity,ahighC:Nratioinlittermaterial,andseasons marked by low temperatures and surfacedrought restrict nitrification and ammonificationprocesses, and hence N availability to plants (Attiwilland Adams 1993; Francis and Read 1994; Smithand Read 1997; Perez-Moreno and Read 2001a,b). The amount of free N in soil water is alsolimited by the tight association of ammonium tohumic substances (Yu et al. 2002). In such environments,where accumulation of nutrient-poorrecalcitrant litter and paucity of mobile N maythreaten the fitness of autotrophs, selection hasfavoured associations with ECM fungal symbiontsthat are physiologically equipped – due to theirwell-developed saprotrophic capabilities – to facilitatecapture of these elements from their locallypredominant organic sources (Read 1991; Read andPerez-Moreno 2003; Read et al. 2004). The significanceof AM colonisation in plant N acquisition, anaspect formerly overlooked (Smith and Read 1997),is now becoming increasingly clear in both agriculturaland natural ecosystems (Johansen et al.1996; Hodge et al. 2001). However, nitrogen availableto both AM and ECM plants should not beregarded as a single pool open to free competition,co-occurrence of ECM and AM fungi beingallowed by exploitation of different niches in thesame ecosystem.a) ECM FungiExploitation of organic N ECM fungi have biochemicaland physiological attributes that makethem highly efficient in scavenging organic sourcesof N and P in surface soil horizons (Read et al. 2004).Being localised mostly in the upper, organically enrichedsoil horizon, the ECM ERMM is indeed ideallyplaced for nutrient acquisition from organicpools (Smith and Read 1997). ECM fungi may usesoluble amino acids, and some of these fungi havehighly developed proteolytic capabilities enablingthem to directly access macromolecular N (Abuzinadahand Read 1989). Absorption of soluble orprotein-derived amino acids by ECM fungi is mediatedby high-affinity amino-acid uptake systems.Functional complementation of a yeast strain deficientin amino-acid transporters has allowed identificationof amino-acid transporters from A. muscaria(Nehls et al. 1999) and H. cylindrosporum(Chalot et al. 2002; Wipf et al. 2002), which appearto be involved in both uptake of amino acids fromthe soil solution and retention of amino acids underN-deprivation conditions. While amino acidsare readily assimilated by most ECM fungi andused as a N source, the fate of the carbon containedin these materials remains uncertain. Negligibletransferofglycine-derived13 Cfromrootstoshoots in either mycorrhizal or non-mycorrhizalplants was found when seedlings of Pinus sylvestriswere fed with double-labelled ( 15 Nand 13 C) glycineas their sole N source, in contrast with the considerablequantities of 15 Nobservedinthemycorrhizaltips, roots and shoots (Taylor et al. 2004).Recently, di- and tripeptide transporters were alsoisolated by yeast functional complementation usinga Hebeloma cylindrosporum cDNA library, andwere shown to mediate dipeptide uptake (Benjdiaet al. 2006).Whereas model proteins have been used todetermine the potential of ECM fungi and theirplant associates to mobilise N from polymericmacromolecules, investigations have subsequentlyfocused on the use of detrital material of plant andanimal origin (e.g. tree litter and nematode necromass),which presumably represent the majorpotential sources of N and P in the field (Read andPerez-Moreno 2003; Read et al. 2004). The ECM

238 M. Girlanda, S. Perotto, P. BonfanteERMM efficiently colonise nutrient patches in thesoil of thin-layer microcosms, and convert suchmaterial into more readily usable forms (reviewedin Leake et al. 2004a; Read et al. 2004). Many ECMfungi have been shown to deploy a broad spectrumof ‘saprotrophic’ enzyme activities, and to bedirectly involved both in the mobilisation of N andP from the organic polymers in which they aresequestered and in the degradation of the polymersthemselves, thus challenging the conventional viewthat mycorrhizal fungi are fundamentally differentfrom saprotrophs and lack the ability to directlyparticipate in decomposition processes (Leakeet al. 2004a). Such a situation sets the stage forintense competition, especially for the more labileN and P sources, between mycorrhizal and saprotrophicfungi in forest soils, in particular otherlarge fungi such as the wood decomposers. Soilmicrocosm studies (reviewed in Leake et al. 2002,2004a; Read et al. 2004; Cairney 2005) have revealedthat mycelial systems of ECM and saprotrophicwood-decomposer fungi can be antagonistic toeach other, both in growth and functioning. Thisantagonism can lead to significant transfer ofnutrients between the two trophic groups upon interaction(Lindahl et al. 1999). Although symbioticfungi may be favoured over saprotrophs by thehost’s continuous provision of carbon compounds,competition for lignocellulose seems unlikely,since actual ligninolytic capabilities are mostlymodest in ECM fungi (Chen et al. 2001). Interactionsbetween mycorrhizal fungi and saprotrophsmay also be beneficial for nutrient acquisition.For instance, nutrient recovery by the ECM fungusPisolithus tinctorius, from highly recalcitrantprotein–tannin complexes that most ECM fungitested to date have little or no enzymatic capacityto hydrolyse, is facilitated by pre-treatment of thecomplex by saprotrophs (Wu et al. 2003).Through their participation in the removal of Nand P from organic polymers, ECM fungi inevitablyincrease the C:N and C:P ratios of the residual materialsand thus will contribute to C retention in soil,potentiallystronglyaffectingthecarbonsource–sink relationships upon which global climate systemsultimately depend (Read and Perez-Moreno2003).Scavenging of inorganic nitrogen (NO − 3 and NH+ 4 )AMandECMmyceliaarealsoeffectivescavengersof inorganic forms of N, either NH + 4 or NO− 3 .ECM mycelia are particularly effective in theuptake of ammonium. Phyllosilicate weatheringhas been demonstrated for Pisolithus,whichgainsaccess to NH + 4 and Ca+ 2 ions trapped in betweenthe vermiculite layer in pots probably by meansof soluble exudates (Paris et al. 1995). Rapid andeffective uptake of inorganic N by ECM has alsobeen demonstrated in forest soils, as indicated bythe lower NH + 4 and NO− 3 levels in a conifer forestwith active mycorrhizal networks, compared toplotswithplastictubesinsertedtoexcludeECMhyphae (Nilsson and Wallander 2003).The molecular bases of NO − 3 and NH+ 4 uptakehave been investigated, and transporters andassimilating enzymes have been characterized inECM fungi such as Pisolithus, Laccaria and Tuber(Jargeat et al. 2003; Javelle et al. 2003). NH + 4 absorbedby ERMM, or derived from NO − 3 reduction,may be rapidly assimilatedintoaminoacids,mostlyglutamine, and is subsequently incorporated intomycelial proteins or translocated to the host, glutaminebeing regarded also as the main translocationform in ECM mycelia (Martin and Botton1993). In Tuber, the glutamine synthase gene ishighly expressed both during fruitbody ripening(Lacourt et al. 2002) and N starvation (Montaniniet al. 2003). Nitrate and high-affinity ammoniumtransporters are differentially expressed at least1.5-fold in response to N deprivation (Montaniniet al. 2006).Incorporation of mineral N into amino acidsentails a significant C cost for the fungus, as demonstratedby respiration increases observed followingthis nutrient uptake (e.g. Ek 1997). Recent studiesin ECM and AM mycorrhiza have highlighted thepotential for direct transfer of ammonia from fungalto plant cells at the symbiotic interface (Chalotet al. 2006). Together with the expression of putativeammonium exporter genes in the ectomycorrhizalfungus Amanita muscaria, expressionanalysisof a high-affinity ammonium importer fromPopulus tremula×tremuloides (PttAMT1.2)hasrevealedthat PttAMT1.2 expression is root-specific,is affected by N nutrition, and strongly increasesin a N-independent manner upon ectomycorrhizaformation, thus suggesting that ammonium couldact as a direct N source delivered by the fungus inECM symbiosis (Selle et al. 2005). A close relationshipcould exist between C availability and the formof N transferred from an ectomycorrhizal fungusto the host plant: under C availability, the largeflux of C compounds towards the fungal compartmentwould ensure the assimilation of inorganicN and the further release of organic nitrogen bythe fungus whereas, under C depletion, the synthe-

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 239sis of organic N might be strongly down-regulated,and ammonium would be transferred to the plant(Chalot et al. 2006).b) AM FungiAlthough Pi acquisition has received most attention,nitrogen is also an important elementthe uptake of which is improved during rootcolonisation by AM fungi. AM mycelia take up andassimilate NH + 4 ,NO− 3 and amino acids but the impactof such metabolisms on plant nutrition is stillunknown (Smith and Read 1997). NH + 4 directlyabsorbed by ERMM mycelia, or derived fromNO − 3 reduction, is rapidly assimilated throughthe glutamine synthetase/glutamate synthase(GS/GOGAT) cycle found in ectomycorrhizal fungi(Martin et al. 2007). By measuring mRNA levels forkey enzymes, such as the putative nicotinamideadenine dinucleotide (NAD)-dependent glutamatedehydrogenase (GDH) gene, Govindarajulu et al.(2005) found support that inorganic N assimilationalso operates in the extraradical myceliumof AM fungi via the GS/GOGAT pathway. Themechanisms involved in N transport along thefungal hyphae, and then the delivery to the plantare still unclear. Very recently, Jin et al. (2005)supplied 15 N- and/or 13 C-labelled substrates toGlomus intraradices on Ri T-DNA-transformedcarrot roots growing in two-compartment Petridishes. They investigated the levels and labellingof free amino acids in the extra- and intraradicalmycelium in mycorrhizal roots by gas chromatography/massspectrometry and high-performanceliquid chromatography, and demonstrated thatarginine was the predominant free amino acid intheAMERMM.Theaminoacidrepresentsthemajor form of stored N, which is then transportedto the intraradical mycelium. However, NH + 4 isthe most likely form of N transferred to host cellsfollowing its generation from arginine breakdown(Jin et al. 2005).Hodge et al. (2001) demonstrated that AMs alsoacquire nitrogen directly from organic material,a feature that – for a long time – has been consideredcharacteristic of ECM fungi. There is clearlya need to understand the mechanisms involved insuch organic N mobilisation by AM fungi, and todetect the molecular basis of such events. A putativeamino-acid permease has been recently detectedin Glomus mosseae (Cappellazzo et al., personalcommunication), its expression being exclusivelylocated in the extraradical hyphae and N dependent.As a further step, suppressive subtractive hybridization(SSH) and reverse Northern dot blotwere performed on extraradical structures of Glomusintraradices grownoncarrothairyrootsinordertoisolategenesresponsivetolowversushighorganic N concentrations. Genes involved in defenceand signalling transduction pathways havebeen identified to be activated following a 48-htreatment with a 2 μM amino-acid pool. This novelset of data indicates that G. intraradices extraradicalstructures perceive organic N limitation in thesurrounding environment, leading to a specific responseat transcriptional level, and supports therole of N as a signalling molecule in AM fungi (Cappellazzoet al. 2007).B. CarbonIn order to compete with other organisms in thepatchy and changing soil environment, mycorrhizalfungi rely on a continuous supply of organic Cfrom the plant. Whereas saprotrophic soil microorganismsare typically C limited – their C sourcesare spatially and temporally heterogeneous – mycorrhizalfungi gain direct access to a plant carbohydratesupply that is unparalleled amongst soilmicrobial populations in terms of both quality andquantity.AM fungi receive all of their carbohydratesfrom the host plant. By contrast, many ECM fungilikely acquire C both via host photosynthesis andassimilation following the degradation of soil carbonpolymers. As mentioned above, ECM fungishow saprotrophic capabilities that allow them toattack structural polymers in the organic detritus(reviewed in Leake et al. 2002; Read and Perez-Moreno 2003; Read et al. 2004; Lindahl et al. 2005),thus possibly reducing the amount of C neededfrom the host plant.Quantitative estimates of the amount of C allocatedto AM and ECM fungi by plants (reviewedin Leake et al. 2004a) range between about 2–20%of net fixation for AM and 7–30% for ECM fungi,although large variations may occur dependingon individual plant–fungus combinations, amountof fungal biomass, and environmental conditions(Jakobsen 1999; Lerat et al. 2003a, b). Fungal C drainrepresents a cost of the symbiosis, but experimentswith AM fungi indicate that the actual charge of mycorrhizalfungi to their host plants may be negligiblebecause mycorrhizal colonisation can increasethe rate of photosynthesis, alleviate shoot N and P

240 M. Girlanda, S. Perotto, P. Bonfantelimitation, and cause a substantial increase in leafarea, a response that maximizes the area availablefor CO 2 assimilation (Wright et al. 1998a; Graham2000; Jakobsen et al. 2002; Simard et al. 2002; Readand Perez-Moreno 2003). The fungus-mediated increasein photosynthesis does not appear to be anindirect result of enhanced mineral nutrition inmycorrhizal plants, and does not result in increasedhost biomass production (Wright et al. 1998a; Staddonet al. 1999; Jifon et al. 2002; Miller et al. 2002).Thus, it has been hypothesized that up-regulationof photosynthesis may be due to the carbohydratedemand by the mycorrhizal fungus (Wright et al.1998b; Miller et al. 2002): mycorrhizal colonisationof the root, by increasing its sink strength, wouldstimulate the process of C assimilation.In vivo NMR, coupled with 13 C labelling,radiorespirometry and transcriptome profilingstudies, are beginning to unravel some details ofC transport processes at the molecular level inmycorrhizal symbioses (Pfeffer et al. 1999, 2001;Bago et al. 2000, 2002; Lammers et al. 2001; Nehlset al. 2001; Ferrol et al. 2002a; Jakobsen et al.2002; Simard et al. 2002). In both AM and ECMmycorrhiza, the primary transport carbohydratein plants, sucrose, can be used as a C sourceby fungi only provided it is hydrolyzed by cellwall-bound invertases, with fungal absorption ofglucose preceding that of fructose (Nehls et al.2001; Simard et al. 2002).1. Uptake of Plant-Derived C at the SymbioticInterface by AM and ECM FungiTransfer of C from plant to fungus in an establishedmycorrhiza may be regulated by factors associatedwith either symbiont (Jakobsen et al. 2002).In AM fungi, in vitro 13 C-NMR studies haveshown that intraradical hyphae, but not externalhyphae, import exogenously supplied hexoses,mainly in the form of glucose (Soliaman andSaito 1995; Shachar-Hill et al. 1995). While thereis good evidence that arbuscules are the siteof fungus-to-plant P transfer, the location of Ctransfer from plant to fungus remains unclear.The arbuscule could be the site of both hexoseand phosphate transfer, and up-regulation ofgenes involved in sucrose hydrolysis (e.g. sucrosesynthases and soluble acid invertase) has beentaken as evidence that photoassimilate is directedtowards arbuscule-containing cells (Blee and Anderson1998; Ravnskov et al. 2003). However, thereis as yet no evidence for a fungal hexose transporterexpressed on the arbuscular membrane(Smith et al. 2001). The plasma membranes of thearbuscular branches indeed lack ATPase staining,which might exclude these as a site for active sugaruptakebythefungus(whichrequiresaprotongradient generated by an H + -ATPase). By contrast,the intercellular interfaces show high levels ofATPase activity, thus qualifying as the principalsite of fungal hexose uptake (Gianinazzi-Pearsonet al. 1991, 2000). Five ATPases have been identifiedfrom G. mosseae and two from G. intraradices(Ferrol et al. 2000, 2002b). Expression of oneof these genes occurs in the intraradical phaseof G. intraradices, indicating active transportprocesses in these hyphae, but the specific locationof these transcripts remains to be determined.An alternative possibility involves passive uptakemediated by specific sugar carrier(s) in the fungalmembrane, with the sugars concentration gradientbetween the fungus and the cortical apoplast beingmaintained by rapid conversion, in the fungus, totranslocation and storage compounds (Bago et al.2000; Jakobsen et al. 2002). The same mechanismscould operate not only in arbuscules but also inintercellular hyphae or coils.In ECM, fungal uptake of hexoses has beensuggested to take place at the plant–fungusinterface (Hartig net) because the ECM representsamajorcarbohydratesink.Themechanismsrelyon the fungal conversion of glucose and fructoseto fungal metabolites, thereby maintaining a concentrationgradient from the plant to the ECMfungus, and allowing for continued fungal uptakeof plant-derived hexoses (Simard et al. 2002).The poplar monosaccharide transporter genePttMST3.1 has been shown to be up-regulated bymycorrhiza formation, suggesting that root cellsareabletocompetewithfungalhyphaeforhexosesfrom the common apoplast during symbiosis(Grunze et al. 2004). Degenerate PCR primersfrom ESTs have been used to isolate a numberof candidate genes for ECM fungal transportprocesses. In Hebeloma cylindrosporum, screeningof genomic and cDNA libraries has revealed 38tags corresponding to genes encoding carriers orchannels that may play a role in nutrient uptakefrom the soil solution, from the host plant apoplast,or in transport towards the host plant (Chalotet al. 2002). So far, only one hexose transportersystem (AmMst1) has been identified from anECM fungus, A. muscaria (Nehls et al. 2000, 2001;Wiese et al. 2000). Increased monosaccharideconcentrations at the fungus/plant interface have

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 241beenfoundtoup-regulateexpressionofmonosaccharidetransporters in the fungus (Rieger et al.1992; Nehls et al. 1998, 2001), resulting in enhancedphotoassimilate sequestration by the fungus thatwill trigger additional photoassimilate supply bythe host to the fungus, ultimately increasing ratesof photosynthesis (reviewed in Simard et al. 2002).The results suggest active transport across fungalmembranes, but the form of hexose transferred aswell as the function and localization of transportproteins in mycorrhizal tissues are poorly known.Although it is assumed that most movement of Cfrom root to fungus is by way of sugar transport(Smith and Read 1997), there is evidence ofbidirectional movement of amino acids betweensymbionts (Lewis 1976), with carbon in aminoacids usually moving from fungus to root by way ofthe glutamine/glutamate shuttle (see Sect. III.A.2.),but sometimes also moving as glutamine fromroot to fungus (Lewis 1976).The identification of novel fungal transportgenes in combination with the developmentof transformation techniques for mycorrhizalfungi (see, e.g. Harrier et al. 2002; Pardo et al.2002; Marmeisse et al. 2004; Grimaldi et al. 2005;Kemppainen et al. 2005; Rodriguez-Tovar et al.2005; Muller et al. 2006) will greatly advance ourunderstanding of how carbon and nutrient releaseand transport events at the apoplastic interfaceare coordinated and controlled by the fungus andhost.2. Fate of Plant-Derived C in AM and ECM MyceliaOnce within the fungal mycelium, plant-derived Cmeets several fates. In both AM and ECM symbioses,sugars are used for translocation and storagesynthesis, for the production of the large extraradicalmycelium, and for support of the respiratorydemands.Knowledge of the nature of the substancestransported from the intraradical to the extraradicalmycelium has advanced considerably. Whatdifferentiates AM fungal C metabolism from ECMmetabolism is the accumulation of lipids as theprimary storage reserve. Lipids constitute by farthe largest C pool in AM fungi, and are synthesisedin the intraradical structures before beingtranslocated to the external mycelium (Bago et al.2000, 2002). A recent report confirms that fattyacid synthase activity of AM fungi is expressedexclusively in the intraradical mycelium (Trépanieret al. 2005). In AM intraradical mycelium, hexoseacquired from the root is converted into trehaloseand then glycogen (Bago et al. 2000). Glycolysis,the TCA cycle, and the pentose phosphate pathwaysare functioning in the intraradical hyphae(Pfeffer et al. 1999), which synthesize and storelarge amounts of triacylglycerides (TAGS; Pfefferet al. 1999; Bago et al. 2002). Since the extraradicalmycelium is unable to take up exogenous hexose(Pfeffer et al. 1999), and no storage lipid productionoccurs in it, the carbohydrate requirementis likely met via translocation of TAGS fromintraradical hyphae, and their conversion via theglyoxylate cycle (Bago et al. 2002). Glycogen isthe other main C compound exported into theextraradical mycelium (Bago et al. 2003). Translocationof carbon compounds from intraradical toextraradical hyphae appears to occur in tubularvacuoles along the entire length of the hyphae(Ashford and Allaway 2002). The incorporationof mineral N into amino acids by the fungus, andtheir transfer to the plant would return some of theC skeletons originally supplied by the plant. Thesepostulated metabolic and transfer pathways matchthose that are known to occur in ECM mycelium(Martin et al. 1998).TheamountsofCallocatedtotheextraradicalAM and ECM mycelia by plants have been determinedin some studies (reviewed in Leake et al.2004a). Microcosm and field experiments with eitherAM (Johnson et al. 2002a, b) or ECM (Ek 1997;Bidartondo et al. 2001; Leake et al. 2001; Wu et al.2002) plants have indicated that C allocation to theexternal fungal network may reach approx. 30%of net C fixation. Such experiments are corroboratedby field studies showing that daily variationsin the δ 13 C isotopic signature of recently fixed C intreesiscloselyfollowed,withinafewdays,bytheisotope signature of root and rhizosphere respiration,which must include the external mycorrhizalmycelium (Ekblad and Högberg 2001).Pulse labelling with 13 CO 2 combined with theuse of root-excluding hyphal compartments (reviewedin Leake et al. 2004a) has indicated thatthere are two distinct fates for C within the extraradicalmycorrhizal mycelium. A major part hasshort residence time in the hyphae, being rapidlytranslocated throughout the network and quicklyrespired. The fast turnover portion will also includesome C that is transferred back to host plants (e.g.as amino acids generated from the hyphal uptakeof inorganic N). Some of the C enters more stable,longer-term pools, for example, as structuralcomponents of the mycelium and hyphal walls. In

242 M. Girlanda, S. Perotto, P. Bonfanteaddition to their contribution to biomass, AM andECM hyphae may produce metabolites, either secretedinto the environment or contained in thehyphal walls (and then secondarily arriving in thesoil via hyphal turnover and decomposition), suchas the AM-secreted protein glomalin; accumulatingin the soil, these have been implicated as animportant mechanism in soil aggregation, and contributea substantial amount of the more stable soilorganic C (Rillig and Mummey 2006).Given the considerable portion of soil microbialbiomass accounted for by the mycorrhizal extraradicalmycelium (see Sect. II.B.), this fungalnetwork provides a major pathway for C movementfrom plants to soil, with an outstanding impacton C cycling at the ecosystem scale (Rillig2004). Such a contribution is beginning to be recognizedin quantitative terms (Leake et al. 2004a).In addition, through the extraradical mycorrhizalmycelium, carbon can also access other plants inthe environment, thus setting the stage for the possibilityforareversalofCfluxbetweenplantsandfungi.IV. At the Interface Between SeveralHost Plants: Common MycelialNetworks (CMNs),a Unifying Phenomenonin ECM and AM FungiA. Evidence for the OccurrenceandFunctionofCMNsA breakthrough in mycorrhizal ecology has beenthe discovery that individual mycelia of either ECMor AM fungi can interlink different host plants, thusestablishing common mycelial networks (CMNs,often referred to as the ‘wood-wide web’ in the caseof ECM mycelia) that connect plants, belonging tothe same or different species, and providing potentialpathways for interplant transport of mineralnutrients and C (Simard et al. 2002; Simard andDurall 2004; Taylor 2006; Selosse et al. 2006).Although occurrence of such networks hadbeen postulated as early as 1881 (Kamienski1881, 1882), only careful laboratory observationsstudies since 1969 (reviewed by Simard and Durall2004) provided physical and functional evidenceof hyphal interplant linkages. Transparent rhizoboxes(thin-layer soil microcosms) have beeninstrumental to visualize plant-to-plant mycelialconnections and to apply isotope probing andimaging (cf. the same techniques used to assess thetransport of C and nutrients, growth, and spatialand temporal foraging activities of extraradicalmycorrhizal mycelia; see Sect. II.B.). Thesetechniques have demonstrated the concurrentexistence of interplant mycorrhizal networks andtransfer of elements from one root to another (e.g.McKendrick et al. 2000a; Wu et al. 2001).In the field, direct visualization of CMNs ishamperedbytheopaquenatureandstructuralcomplexity of soil as well as by intermingling ofmycorrhizal and non-mycorrhizal mycelia. Sharingof mycorrhizal fungal species by different hostplants growing together in the field, a feature ofboth ECM and AM symbioses (see Sect. II.A.),provides potential for the formation of commonmycelial networks in plant communities (Kennedyet al. 2003), although only sharing of the samefungal individual (genotype, or genet) may allowestablishment of CMNs. Many ECM fungi spreadvegetatively below ground from root to root, anda single genet can colonise large areas. Genotypingusing either basidiome and rhizomorphic tissue,or DNA extracted directly from soil (reviewed byLeake et al. 2004a; Cairney 2005), has indeed revealedthat although genet size varies considerablybetween species and, for a given species, is stronglyaffected by factors such as disturbance and forestage, individual genotypes of some ECM taxa mayspan up to tens of metres, presumably by progressivelyinfecting root tips, thus providing high potentialfor the formation on CMNs. Only recently,however, was the capability of a single fungal ECMgenotype to colonise multiple host trees conclusivelydemonstrated by using microsatellite markerson ECM root tips to identify the genotypes ofboth the fungal symbiont and host tree (Lian et al.2006). Similarly, data referred to Mediterraneandark septate endophytes (see Sect. II.A.) have indicatedthat the same DSE genotype may be sharedbetweenanericoidandanectomycorrhizalhost(Bergero et al. 2000), setting the stage for a role ofDSE in interactions between ecto- and endomycorrhizalplants (Girlanda et al. 2006a). Since it islikely that, in diverse plant communities, virtuallyall mycorrhiza-compatible plants will join commonmycelial network but that not all plant species willshare the same fungal partners (species or individualsof the same genet), complex overlapping host–fungus species interactions probably occur (Leakeet al. 2004a). Formation of larger mycelia from thefusion of individual ones offers another mecha-

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 243nism potentially enabling the formation of hyphalinterplant linkages. The capability of anastomosisbetween self or genetically closely related hyphae(as determined by vegetative incompatibility systems;see, e.g. Esser 2006) has long been known forseptate mycelia, and it has recently been shown toallow networks originating from plants of differentspecies, genera and families to become interconnected(Giovannetti et al. 2004). Although burial ofmembranes on which the hyphae grow may revealmuch about mycelial branching and hyphal anastomosispatterns (Balaz and Vosatka 2001), the actualextent of anastomosis formation in the fieldremains undetermined, as does the spread of individualAM mycelia in natural settings. Genet sizestudies also cannot ascertain whether these myceliaare actually continuous or have fragmented as ramets,i.e. spatially discontinuous mycelial units of thesame genotype, a functionally important aspect becauseit will define, at least in part, the spatial limitswithin which the mycelia can effect movements ofC and minerals through soil (Cairney 2005).Evidence of a functional role of CMNs in nutrienttransport and exchange derives from isotopelabelling studies. These indicate that mineral resourcessuch as N and, to a lesser extent, P move betweenplants via both AM and ECM CMNs (Simardet al. 2002; Tuffen et al. 2002; He et al. 2003, 2005,2006). Carbon also moves within both ECM and AMCMNs, but actual net transport of C between interconnectedplants remains controversial (Robinsonand Fitter 1999; Simard et al. 2002; Simard and Durall2004; Taylor 2006; Selosse et al. 2006). For AM,many laboratory studies have shown C movementfrom one mycorrhizal plant to another but nonehave demonstrated net transfer (Perry 1999), the labelledC remaining associated with the fungus. Forinstance, by using pulse labelling in a monoxenicculture system with Ri T-DNA-transformed carrotroots colonised by Glomus intraradices growingin subdivided Petri plates (to avoid CO 2 recyclingandtoeliminatelabelmovementfromonerootsystemto another by diffusion), Pfeffer and colleagues(2004) found that carbon provided to a donor rootas labelled glucose moved to recipient roots viaa common AM fungal network, but remained infungal compounds. Such data indicate that carbontaken up by the mycorrhizal fungus in associationwith one mycorrhizal root does not become nutritionallyavailable to other roots. These findings onAM have raised serious doubts as to whether thedetection of a carbon label in mycorrhizal rootsrepresents a nutritionally meaningful transfer forthe plant or, indeed, any transfer at all (Fitter et al.1998; Robinson and Fitter 1999; Wu et al. 2001;Pfeffer et al. 2004).In contrast to the laboratory experiments, carbonexchange between plant species pairs sharingeither ECM (Simard et al. 1997) and AM fungi(Lerat et al. 2002) has been demonstrated in fieldexperiments, where labelled C pulsed to a donorplant was found in both the roots and shoots of thereceiver plants. In these experiments, comparisonsbetween mycorrhizal and non-mycorrhizal controlplants (plants that did not tap into the CMN underexamination) suggest that transfer occurred predominantlyvia the CMNs, as indicated by smallor negligible transport to the incompatible controlplants (Simard et al. 1997; Lerat et al. 2002; Simardand Durall 2004).B. What may be the Ecological Significancefor the Host Plants?1. Interactions Between Autotrophic PlantsThe possibility of substantial interplant C transferhas led to the hypothesis that such transfers mayinfluence interactions in plant communities, thussuggesting the need for a radical reappraisal of conventionalconcepts of competition in plant ecology(Leake et al. 2004a).Field labelling experiments (Simard et al. 1997;Lerat et al. 2002) have shown that although bidirectionalcarbon transfer occurs between interconnectedplants, a net C gain (up to 10%) can be foundin one plant over that of its connected partner, thusconfirming previous laboratory reports (Newman1988; Miller and Allen 1992). Such a net flux hasbeen suggested to be governed by a source–sinkrelationship, established, for example, by shadingof one of the paired plants (Francis and Read 1984;Finlay and Read 1986; Simard et al. 1997). Similarly,one-way P transport has been shown to beregulated by fertilization of donor plants with phosphorus(e.g. Ritz and Newman 1986), and N hasbeenobservedtobetransferredfromN 2 -fixing mycorrhizalto non-N 2 -fixing mycorrhizal plants (Heet al. 2003). Such unidirectional net transfers arelikely to affect a range of interplant interactions,such as plant competition, plant diversity, plantcommunity dynamics, and patterns of seedling establishment(Simard et al. 2002; Simard and Durall2004; Taylor 2006; Selosse et al. 2006). A more evendistribution of carbon among plants, as a resultof belowground transfer, is likely to reduce dom-

244 M. Girlanda, S. Perotto, P. Bonfanteinance and to allow co-occurrence of less aggressivespecies, thus contributing to maintain biodiversity,and therefore ecosystem productivity, stabilityand sustainability. Involvement in ecologicalsuccession has also been hypothesized (e.g. Hortonet al. 1999), also in invasion by exotics outcompetingnative species (e.g. Carey et al. 2004).Resource sharing between adult high-canopytrees and understorey seedlings, which wouldcounterbalance low light influx and inefficientphotosynthesis by juveniles, has been proposed asa mechanism to explain the improved establishmentfrequently observed for ECM seedlings inclose proximity to existing plants of the same anddifferent species (Taylor 2006). Despite numerousstudies on seedling establishment, no transfer ofnutrients or C from nurse plants to seedlings hasbeen demonstrated so far, and juvenile plants mayactually benefit only indirectly from CMNs. Forexample, the costs to the seedling of supportingmycorrhizalfungiwithcarbonmaybeoffsetbythelarger nurse plant. Even without carbon transfer,this situation would represent a clear benefit toseedlings, which would simply tap into a mycelialnetwork already developed and supported by thesurrounding vegetation (van der Heijden 2004;Simard and Durall 2004; Taylor 2006). This hypothesiscanbecorroboratedbytheobservationthat,based on natural variations in stable carbon isotoperatios ( 13 C), overstorey trees appear to supply,partly or wholly, the nutrient-absorbing myceliaof their alleged competitors, the understoreytrees (Högberg et al. 1999). Furthermore, CMNscouldsimplyallowmorerapidplantmycorrhizalcolonisation, with benefits arising merely fromincreased mycorrhization, which may or may notrequire network connection (Simard and Durall2004; Bever and Schultz 2005; Taylor 2006).The extent to which interplant element transfersoccur routinely between interlinked greenplants in the field and over what scale, as well astheir significance in plant ecology under field situationstherefore remain contentious. Although thereseems little doubt that there is considerable potentialfor their existence, the general significance ofthese networks in plant ecology remains unknown.Only fragmentary evidence is thus far available,experimental limitations (including the use ofC isotope pulses, since the short application-toharvestperiod of labels would not provide enoughtime for large amounts of carbon to move throughfungi or for carbon to move from fungal tissuesinto plants; Carey et al. 2004) constrain the scopeof the conclusions reached and, in most cases, theimportance of the observed CMN-mediated carbontransport for the total plant C budget has yet tobe quantified. However, the outcome of any plant–fungus combination most often appears to be verycontext-dependent, and differences in CMN feedbacksamongspeciesorexperimentalconditionscould be influenced by factors such as soil fertility,plant–fungus interactions (providing variouscarbon sink strengths), and the age of adult plants(Selosse et al. 2006). Other soil organisms, such asmycophagous collembolans and earthworms, mayalso significantly impact CMN-mediated transfers(Tuffen et al. 2002; Johnson et al. 2005b), thusproviding another possible explanation for someof the contradictions reported between differentnutrient transfer studies (Selosse et al. 2006). Inautotrophic plant nutrition, therefore, the ecologicalrelevance of CMN-derived C, at least undersome circumstances (e.g. specific growth stages orenvironmental conditions), remains a fascinatingpossibility that cannot be entirely ruled out and,when coupled with differential effects of hostplant–mycorrhizal fungus combinations, couldcontribute to our understanding of the influenceof mycorrhizal fungi over plant competitiveperformances in mixed natural communities.2. Interactions Between Autotrophicand Heterotrophic PlantsWhereas the evidence for nutritionally significantcarbon movement from the fungus to the plant remainsdubious in autotrophic plants communities,an undisputed example of interplant CMNmediatednet C transfer involves achlorophyllousplants. By lacking photosynthetic pigments,achlorophyllous plants behave as heterotrophsand deploy alternative strategies to acquireorganic carbon for growth (Leake 1994). Someachlorophyllous species are direct epiparasiteson photosynthetic plants whereas others acquireorganic carbon through mycorrhizal association.Leake (1994) introduced the term “mycoheterotrophy”to describe this peculiar strategy, whichrelies on the ability of fungi to fetch organiccompounds from the environment. This strategyarose repeatedly in angiosperm evolution, leadingto over 400 mycoheterotrophic (MH) species in 87genera derived from multiple independent lineagesof green plants (Leake 1994). Recent studies onthe identity and diversity of mycorrhizal fungiassociated with MH plants belonging to distant

Mycorrhizal Fungi: Their Habitats and Nutritional Strategies 245taxa have outlined common features and providedkey information on their nutritional strategies(Taylor et al. 2002; Leake 2004; Bidartondo 2005).Most fungal partnerships of mycoheterotrophs arecharacterized by extreme specificity, contrastingwith the generally broad specificity pattern of mostmycorrhizal associations (see, e.g. Bidartondoand Bruns 2002; Taylor et al. 2002; Leake 2004).They involve ECM (Selosse et al. 2002a, b; Tayloret al. 2002) or AM fungi (Bidartondo et al. 2002)capable of forming simultaneous mycorrhizaon adjacent green plants that are the ultimatesource of C for the mycoheterotroph. By takingcarbon through their mycorrhizal partner, MHplants behave both as epiparasites on the greenmycoheterotrophic (MH) plants that support theshared fungal partners with sugars (Leake 2004),and as “cheaters” towards the mycorrhizal fungus(Bidartondo 2005). The potential of mycorrhizalfungi for the formation of multiple interfaces withdifferent neighbouring plants (see Sect. II.A.) ismost likely the premise for the invasion of bothECM and AM by MH epiparasites (Leake 2004); inaddition, the photosynthetic plant interacts onlyindirectly with the epiparasitic plant, and cannotselect against the epiparasitic plant without selectingagainst a fungal mutualist (Bidartondo 2005).Thesignificanceoftheunusuallyhighspecificityof MH plants, featuring exclusive associations withasingle(oranarrowrangeof)fungalspeciesevenwhen surrounded by numerous potentially alternativefungal symbionts, is far from clear (Gardes2002; Taylor et al. 2002; Leake 2004; Taylor 2004)but has been related to their strategy as cheatingparasites, since parasitism tends to favour specificitybyselectionforresistanceandevolutionaryarms-races (Taylor and Bruns 1997; Taylor 2004).Isotope-labelling experiments, coupled withmorphological observations and biomass measurementsin experimental microcosms withand without mycelial interconnections betweenautotrophs and mycoheterotrophs, have actuallydemonstrated C flow via a shared ECM myceliumfrom photosynthetic plants to either a Monotropa,MH orchid or liverwort (Björkman 1960; Mc-Kendrick et al. 2000a; Bidartondo et al. 2003).However, the physiological mechanism for thefungus-to-plant transfer remains unknown. Twodistinct pathways for C transport from fungi toplants have been proposed: (i) the turnover of fungalbiomass, particularly intracellular hyphae, anappealing option in cases such as the Orchidaceaewherethereismassiveintracellularcolonisationfollowed by hyphal collapse; and (ii) the transfer offungal compounds through ephemerally disruptedmembranes at the fungal–plant interface, morelikely in the Monotropoideae where there isminimal intracellular penetration (Bidartondo2005). Whether digestion of intracellular hyphaeby the plant may account for the entire amount of Ctransferred from the photosynthetic to MH plant,as well as for the rate of the process, remains uncertain.Carbon flux from AM fungi to MH plantsalso awaits demonstration (Bidartondo 2005).Whatever the mechanism involved, MH plantsexemplify both the ability of mycorrhizal networksto transport substantial quantities of C andnutrients and the potential importance of thesenetworksforthecontrolofspeciescompositionofnatural communities (Leake et al. 2004a). Evidencehas indeed been provided that in the absence oftheir critical fungal partners, MH plants fail togerminate and to establish (see, e.g. McKendricket al. 2000b), and the distribution of a singlemycorrhizal fungus can forcefully constrain theestablishment and resulting distribution of a MHmonotrope (Leake et al. 2004b).Alongside plants that are obligately nonphotosyntheticthroughout their lifetime, others exhibitan MH strategy during specific life stages.This is, for instance, the case of the early developmentalstages of germination of most orchids,and the gametophyte stages of many ferns and lycophytes,which can therefore be defined as ‘initiallyMH’ plants (Leake et al. 2004a). Some greenorchids also exhibit prolonged stages of adult dormancy,consisting of periods of one or more yearswhere no sprouts are produced and no photosynthesisoccurs. A recent study on the genus Cypripedium,which exhibits this phenomenon, indicatesthat mycorrhizal specificity towards Tulasnellaceaeis fairly high (Shefferson et al. 2005). Studiesof natural isotope abundance have indicatedthat fully MH plants have distinctive stable isotopesignatures (enrichment in both 13 Cand 15 N) relativeto co-occurring autotrophic plants (Gebauerand Meyer 2003; Trudell et al. 2003), most similarto the signatures of ECM fungi. Such a distinctiveheavy-isotope enrichment has also beenfound in some forest green-leaved orchids previouslyassumed to be autotrophs, thus indicatingthat these plants, although photosynthetic, dependat least in part upon fungal C (Gebauer and Meyer2003). The term ‘mixotrophy’ has been proposed(Selosse et al. 2004) to indicate this dual (photosyntheticand mycoheterotrophic) strategy in the or-

246 M. Girlanda, S. Perotto, P. Bonfantechid genera Cephalanthera, Epipactis and Limodorum,which accompanies ectomycorrhizal associationand, hence, connection to a CMN linking tosurrounding trees (Bidartondo et al. 2004; Selosseet al. 2004; Julou et al. 2005; Girlanda et al. 2006b).Green forest orchids that grow in deeply shadedforest habitats likely experience reduced photosyntheticefficiency in a shaded environment. ForC. damasonium, the amount of carbon obtainedfrom fungi was estimated (by variation in stableisotope abundance) to span a quite wide range,from ca. 85% (Gebauer and Meyer 2003) to 30–50%(Bidartondo et al. 2004; Julou et al. 2005), suggestinga strong influence of the environment on therelative contribution of autotrophic photosynthesisand heterotrophism to the carbon metabolism ofmixotrophic orchids. These findings parallel a variableextent of mycobiont specificity in green forestorchids (Bidartondo et al. 2004; McCormick et al.2004; Julou et al. 2005), implying that the degree ofspecificity to a given fungal symbiont may mirrorthe degree of heterotrophy and nutritional dependencyfor carbon, possibly as a function of environmentalconditions. By supplying mycobiont extraradicalmycelium with 13 C-labelled glycine andshoots with 14 CO 2 , bidirectional transfer of C wasrecently demonstrated between the green forest orchidGoodyera repens and its fungal symbiont Ceratobasidiumcornigerum (Cameron et al. 2006).Taken together, these findings suggest thatwhilst achlorophyllous plants are necessarilybound to heterotrophy, and mycoheterotrophylikely represents an extreme ‘cheating’ type ofmycorrhiza (Bidartondo et al. 2002), a continuumofCtransferfromfungitoplantspossiblyexists,with some chlorophyllous plants obtaining part oftheir carbon heterotrophically. Although evidencefor interplant C transfer between fully autotrophicspeciesisnotthatcompellingasyet,themultipleevolutionary origins ofmycoheterotrophy as well asthe occurrence of mixotrophic plants occupying anintermediate position in a likely gradient betweenfull autotrophy and full mycoheterotrophy suggestthat plant-to-plant carbon transfer via mycorrhizalfungimaybeamuchmoreuniversalphenomenonthan usually recognized (Leake et al. 2004a).C. . . . and what Ecological Significancefor the Mycorrhizal Fungi?Within this framework, hardly anything is knownabout the significance of CMNs in fungal ecology,a situation that reflects our limited knowledgeof the biology of these microorganisms. Even inwell-characterized situations such as those involvingfully MH plants, the characteristics of fungitargeted by successful mycoheterotrophs remaina matter of speculation (Bidartondo 2005), as dopossible benefits derived by the fungus from theassociation (e.g. see Bidartondo et al. 2000). Thegreat majority of the work on CMNs has focused onthe benefits and consequences for the abovegroundplant community (Fitter et al. 1999), while the ‘mycocentric’perspective on CMNs (i.e. an invertedmycorrhizosphere, or networks of fungi linked byshared trees) has been neglected in terrestrial ecology,largely biased towards a ‘phytocentric’ view(Selosse et al. 2006).V. ConclusionsIn the last decade, application of modern techniquesand innovative combinations of methodologicalapproaches has yielded valuable new insightson the roles played by mycorrhizal fungi innatural communities, and the mechanisms underlyingsuch fungal processes. A picture has emergedof greater than expected plasticity of these symbioticorganisms, which have therefore multiple potentialof impacting their hosts from the molecularto the ecosystem level. By facing the challengeposed by natural complexity, mycorrhizal researchhasthusfeaturedsubstantialadvancestowardsecologicalrelevance (Read 2002). Due to their key positionat the plant–soil interface, mycorrhizal fungimust be taken into account in the study of ecosystemimpacts of multifactorial global changes includingelevated atmospheric gas concentrations,increasedatmosphericdepositionofnutrientssuchas nitrogen, climatic variations, and invasions byexotic species (see, e.g. Rillig et al. 2002). Furtherefforts to place the mycorrhizal function within thisbroader context will be invaluable in predicting thefuture impacts of global environmental change onterrestrial ecosystems.ReferencesAbuzinadah RA, Read DJ (1986) The role of proteins in thenitrogen nutrition of ectomycorrhizal plants. I. Utilizationof peptides and proteins by ectomycorrhizalfungi. New Phytol 103:481–493Abuzinadah RA, Read DJ (1989) The roles of proteins in thenitrogen nutrition of ectomycorrhizal plants. 4. Theutilization of peptides by birch (Betula pendula L.)infected with different mycorrhizal fungi. New Phytol112:55

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15 Applications of Fungal Ecologyin the Search for New Bioactive Natural ProductsJ.B. Gloer 1CONTENTSI. Introduction ........................ 257A. Fungal Natural Products:From Mycotoxins to Antibiotics . . . . . . 257B. Chemical Ecology: A General GuidetoNaturalProductDiscovery ........ 259II. Observations in Fungal Ecology Associatedwith the Production of Bioactive Metabolites 261A. Competitive or Antagonistic Interactions 261B. Resistance of Key Fungal StructurestoFungivoryorMicrobialAttack...... 2651. Claviceps Ergot ................. 2652.SclerotiaofOtherFungi........... 2663.Ascostromata................... 2674.OtherFungalStructures .......... 267C. Fungi that May Confer Host ResistancetoHerbivoryorDisease............. 268D. Fungi that Cause Diseases or DamagetoHostSpecies.................... 2691.PlantPathogens................. 2692. Entomopathogenic andNematophagousFungi............ 2703. Mycoparasitic and Fungicolous Fungi 270III. Perspectives and Future Directions ...... 274References.......................... 276I. IntroductionA. Fungal Natural Products:From Mycotoxins to AntibioticsAmong the most fascinating and important propertiesoffungiistheirabilitytoproduceatremendousvariety of so-called secondary metabolites that displaya broad range of biological activities (Demainet al. 2005). Fungi are widely known for theproduction of compounds that have a deservedlynegative reputation due to their activities as carcinogensor mammalian toxins (mycotoxins). Suchcompounds include aflatoxins, ochratoxins, citreoviridin,trichothecenes, fumonisins, and variousindole-derived tremorgenics (Miller and Trenholm1 Department of Chemistry, University of Iowa, Iowa City, IA 52242,USA1994; Cole and Schweikert 2003). In general, mycotoxinsare not inherently more toxic than naturalproducts from bacteria, plants, or other sources,buttheyaremuchmoreproblematicbecauseoftheir widespread occurrence as contaminants offood for humans and livestock, as well as indoorenvironments (Jarvis 2003; Jarvis and Miller 2005).Knowledge of mycotoxin chemistry is essential toefforts to monitor and reduce the levels of exposureto such compounds.On the other hand, numerous important pharmaceuticalshave also been discovered throughstudies of fungal chemistry (Masurekar 2005;Demain et al. 2005). This dichotomy is indicativeof the diversity of bioactive compounds that fungican produce. Despite a decrease in resourcesinvested in industrial natural product discoveryresearch in the USA over the last 25 years, naturalproductsingeneralcontinuetobeamongthemostimportant therapeutic agents and lead compoundsin medicine (Cragg and Newman 2000; Newmanet al. 2000, 2003). While the extent of their rolesvaries from one therapeutic area to another, naturalproducts have been particularly important inthe development of effective therapies for cancer,malaria, bacterial and fungal infections, and CNSand cardiovascular diseases (Newman et al. 2000).For example, over 60% of all drugs in clinicaltrials against cancer as of 2003 are either naturalproducts, or are derived directly or indirectly fromnatural product leads (Cragg and Newman 2005).This is particularly impressive when one considersthe many other intensively investigated approachesto the discovery of anticancer therapeutics. Naturalproducts from various sources show both provenutility and promise in agriculture as well (Dukeet al. 2003; Rimando and Duke 2006).Fungi are particularly prolific sources ofbiologically active natural products (Peláez 2005).Many billions of dollars in annual sales of pharmaceuticalsof various types can be traced tofungal natural product chemistry (Demain et al.Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

258 J.B. Gloer2005). Antibacterial agents such as penicillinsand cephalosporins are perhaps the best knownexamples, but a variety of other compounds withdistinctive pharmacological activities have alsobeen discovered as fungal metabolites.A textbook example is provided by mevinolin(= mevacor = lovastatin), the lead compound inthedevelopmentoftheso-calledstatinclassofcholesterol-lowering drugs. Lovastatin was at onepoint a billion-dollar drug, and is still a usefulproduct, though it is now off-patent. A slightstructural modification of lovastatin affordedZocor® (= simvastatin), an even more lucrative“blockbuster drug” product with over US$5 billion/yearin sales (until it also went off-patentin 2006). Other medically important compoundsinclude cyclosporin, mycophenolic acid, and theergot alkaloids. Many other fungal metaboliteshave been discovered as potential pharmaceuticalsor leads thereto, with a wide range of pharmacologicallyrelevant activities in mechanism-basedand whole-organism assays (Caporale 1995; Masurekar2005). A recent commercialized exampleis provided by Cancidas® (= caspofungin; Keatingand Figgitt 2003), a member of the pneumocandin/echinocandinclass of cyclic peptide fungalmetabolites. Cancidas® was introduced by Merckin 2001 upon approval as a treatment for refractoryinvasive aspergillosis, and represented the firstnew class of systemic antifungal agents to enter themarket in 25 years. This example also illustratesthe intriguing fact that fungi can serve as sourcesof useful antifungal agents. In fact, a survey ofthe literature on bioactive fungal metabolitesduring the period from 1993–2001 (718 references)revealed that antifungal activity was the mostcommon biological effect reported (Peláez 2005),although, as noted by the author, this is likely dueat least in part to the modest resources neededfor such assays. Fungi are effective in producingcompounds with activity in assays relevant toother therapeutic areas as well, such as HIV(Singh et al. 2005). Fungal products also showconsiderable potential as natural agrochemicals(Gardner and McCoy 1992; Anke and Sterner 2002;Peláez 2005; Liu and Li 2005), with importantexamples including nodulisporic acids (insecticides/antiparasitics),strobilurins (fungicides),and various phytotoxins (herbicides).The practical advantages of fungi as sourcesof useful natural products are well documented.Fungal metabolites are renewable resources,and methods for large-scale production of importantfungal metabolites can be developedusing established techniques. Modification ofmetabolite structure and dramatic improvementsin metabolite production efficiency can be accomplishedthrough strain mutation, mediumvariation, and optimization of culture conditions(Masurekar 2005). For example, manipulation ofmetabolite-producing cultures eventually resultedin a 6,000-fold improvement in penicillin production(Demain 1992), and a 900-fold improvementin compactin production (Chakravarti and Sahai2004). Thus, although these steps are not trivial,promising metabolites isolated from fungi canbe made available on a practical scale throughapplication of existing technology. Of course, suchcompounds can also be used as models in thedevelopment of synthetic or semisynthetic derivativesor analogs that may have improved activityor other more desirable properties, as has been thecase for penicillins, cephalosporins, and lovastatin(Demain et al. 2005). In addition, considerableadvances are being made in development oftechniques for exploiting the genetics of microbialbiosynthesis (Khosla and Keasling 2003; Du et al.2003; Keller et al. 2005; Wang et al. 2005). Thediscovery of fungi that express new biosyntheticpathways of interest can provide valuable new “rawmaterial” for research in this area.Of course, the odds against finding a trulyuseful agent are daunting. On average, it requiresapproximately 12 years and is estimated to costover US$800 million to develop and bring tomarket a new drug, and thousands of candidatecompounds are dropped for every one that ultimatelyreaches the market (Anonymous 1995;DiMasi et al. 2003; Barton and Emanuel 2005).Even so, the potential payoff, and the trackrecord of fungi as sources of useful compoundshave fostered continued industrial interest infungal natural products chemistry within manyscreening programs. In recent years, it has becomeincreasingly difficult to find new bioactive naturalproducts from microbial sources because of theextensive screening efforts that have already takenplace. In fact, the need to dereplicate cultures,i.e., to weed out well-known metabolites thatare responsible for positive results in a bioassay(Harris 2005; Dinan 2005; Hansen et al. 2005), isa source of tremendous expense and frustration(Corley and Durley 1994), and is viewed by manyas a significant negative feature of continuedscreening efforts. Part of this problem stems fromlong-term reliance on screening of sheer numbers

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 259of actinomycetes and common fungi isolatedmainly from soil samples as sources of bioactivemetabolites, while other, less widely studied nichegroups have been largely neglected. Inattentionto the specific types of organisms chosen forscreening and the habitats from which they areisolated, together with disinterest in fungi thatare slow-growing, more difficult to isolate, and/ordifficult to adapt to standard liquid fermentationprotocols, combine to exacerbate the problem.Thus, many taxonomic and ecological groups offungi have not been systematically explored foruseful secondary metabolites, despite literatureevidence that directly or indirectly indicates theirpotential in this area.The fungi provide almost limitless potential formetabolic variation. Fungi rank second only to theinsects in estimated species biodiversity. Conservativeestimates suggest that there are likely to beover 1.5 million fungal species, of which only ca.5% have been described (Hawksworth 1991, 2001).This is over five times the number of predictedplant species, and 50 times the estimated numberof bacterial species (Hawksworth 1991). In recentyears, an element of urgency has been conferredupon studies of the chemistry of certain fungi forthe same reasons often cited to rationalize appealsfor accelerated studies of plant chemistry, i.e., concernsaboutthelossofbiodiversity(Balandrinetal.1993). Many endangered plant and insect speciesareassociatedwithspecificfungalflora,andlossofthosespecieswouldalsoresultinaconcomitantloss of fungal species.The importance of seeking isolates for industrialscreening programs from relativelyunexplored niche groups or substrates has beenrecognized (Monaghan and Tkacz 1990; Miller1991; Dreyfuss and Chapela 1994; Bills 1995; Caporale1995), and a number of programs have madesignificanteffortstoexpandtheirscopeinordertoinclude such isolates. However, the primary objectiveof this chapter is to provide specific examplesof how observations in fungal ecology can belinkedtothesearchforbioactivenaturalproductsthat may be useful in medicine or agriculture.Thischapterisanupdatedversionofonethatappeared in the previous edition in this seriesa decade ago (Gloer 1997). Examples from theliterature will again be cited, with most taken frommore recent literature. As before, emphasis will beplaced on results from our own research programthat demonstrate the potential value of this kindof approach as a complement to those typicallytaken in random screening programs. Detailedchemistry results from our program that weredescribed in the 1997 volume will not be repeatedhere, although overall summaries will be provided.Instead, descriptions of results from our laboratorywillfocusonhighlightsofexplorationsundertakensince the appearance of the earlier edition.B. Chemical Ecology: A General Guideto Natural Product DiscoveryIt is widely accepted that natural products play importantroles in the ecology of many different typesof organisms. It is also well known that studies ofthese types of ecological phenomena can lead tothe discovery of novel natural products that havebioactivities of potential practical importance.Examples include compounds produced by plantsthat serve as chemical defenses against herbivores(Harborne 1987), and metabolites producedby marine invertebrates that deter attacks bypredators (Harper et al. 2001). Many instances ofthe involvement of chemical agents in attack anddefense are known among other groups as well,such as insects and amphibians (Daly 1998). Bycontrast, relatively little is known of the chemicalecology of microbial ecosystems. Principles ofchemical ecology would suggest that slow-growingfungi inhabiting competitive niches, or those thatproduce long-lived physiological structures, wouldexperience considerable evolutionary pressureto produce antagonistic secondary metabolitesthat could play roles (offensive or defensive) intheir ecology. Even so, as noted above, manycompetitive niches have not been widely sampled,and slower-growing fungi are often overlooked ordiscarded by screening programs.As suggested by a number of researchers, thereis ample evidence to suspect that the search forbioactiveagentsfromfungimaybeaidedbyapplicationof ecological rationale (Wicklow 1981; Monaghanand Tkacz 1990; Dreyfus and Chapela 1994;Caporale 1995; Gloer 1995a, b; Anke et al. 1995; Eckermanand Graham 2000). Fungi commonly thrivein competitive environments, and it is often hypothesizedthat some of their secondary metaboliccapabilities may be influenced by selection pressuresexerted by other organisms. However, the majorityof known bioactive fungal natural productshave been discovered through industrial programsthat involve mass random screening of uncharacterizedisolates using laboratory fermentation

260 J.B. Gloerconditions. Choices of isolates for chemical investigationare based strictly on bioassay results, asare metabolite isolation procedures. Most modernindustrial programs utilize an extensive array ofspecialized, often proprietary biological assays foractivity against specific molecular, cellular and/orwhole-organism targets. Advances in assay miniaturizationand robotics permit extremely highthroughput in many of these screens. Because ofthe sheer numbers of samples needed to satisfy thecapacityoftheseassays,suchprogramscanrarelyprovide support for ecological or taxonomic investigationsof the organisms being screened, let alonestudies of the possible roles of their metabolites.The most important fungal secondary metabolitediscoveries in recent years have been due tothe creative development and implementation ofbioassays, rather than to chemical studies of rare orspecially selected types of fungi. For example, oncean assay for inhibitors of HMG-CoA reductase wasselected and developed, many fungi were foundthat produced lovastatin and/or close analogsthereof (Chakravarti and Sahai 2004). Similarly,assays for 1,3-β-D-glucan synthase inhibitors haveproven to be effective in leading to discovery ofvarious members of the powerful echinocandinclass of antifungal agents (Barrett 2002; Wiederholdand Lewis 2003). Many other architecturallycomplex fungal metabolite-types with potentbiological effects (e.g., trichothecenes, destruxins,cytochalasins, paspalinines, cyclosporin A,mycophenolic acid) have been found to occuras metabolites of disparate fungal taxa. On theother hand, a considerable number of bioactivemetabolites have been reported only from a singlespecies or a single isolate. The latter cases providesupporting arguments for studies targetedtoward rare fungal taxa or relatively unexploredecosystems. Secondary metabolite profiling ofisolates being surveyed can be valuable in quicklyrecognizing known compounds and providinguseful taxonomic information, and can also affordimportant clues about which species might beproducing unknown metabolites (Frisvad 1989;Larsen et al. 2005). However, bioassay-guidedfractionation remains the paradigm of naturalproduct discovery in most laboratories.Examples of fungal chemicals likely to beassociated with ecological phenomena includetoxic mushroom metabolites that are presumedto play roles in defending fruiting bodies fromfungivores, phytotoxins from plant pathogenicfungi that play roles in disease processes, insecticidalmetabolites responsible for the toxiceffects of entomopathogenic fungi, and the ergotalkaloids, chemical defenses found in the ergot ofClaviceps spp.andincertaingrassesasmetabolitesof fungal endophytes. Chemical investigations ofsuch sources could be viewed as “ecology-based”approaches leading to the discovery of fungalmetabolites with somewhat predictable typesof bioactivities. Studies of such phenomena arecomplementary to random screening because theyincorporate rationale into the process of selectingfungi for chemical study. This kind of approach isparticularly well suited to academic laboratoriesbecause resources for efficient mass screeningon-site in an appropriately wide variety of assaysare not typically available in academia. Realistically,throughput limitations in most academiclaboratories, coupled with longer turnaround timefor results and a somewhat more diverse (e.g.,educational) mission, preclude true direct competitivenesswith industrial in-house microbialchemistry programs. Ultimately, collaborationswith industry are essential if compounds are to befully evaluated. However, application of rationaleat the beginning of the screening process limitsthenumberoforganismsthatneedtobeinvestigated,reduces costs, facilitates dereplication, andpermits evaluation (and subsequent result-basedmodification) of a hypothesis. Our own work hasillustratedthatsuchrationalecanbeveryeffectivein leading to the isolation of new (and patentable)fungal metabolites with antifungal or antiinsectanactivity, as well as other effects. Although we havenot yet demonstrated through our own work thatsuch an approach can lead to marketable products,application of ecological rationale in the search fornovel bioactive fungal products may well result infindings with practical utility.There are many elements of natural productscience and efforts to apply ecological principlesto the search for bioactive fungal metabolitesthat seem somewhat incongruous with typicalapproaches to commercial product development.Even if one assumes that some metabolites haveevolved because they play a role in the life cycle ofthe producing species, the natural targets of thesemolecules are likely to be different from those ofgreatest interest in medicine. Such molecules arealso unlikely to have built-in properties suitable fordesirable pharmacokinetics. Furthermore, whilefocus in the search for new pharmaceutical oragrochemical agents is typically placed on a desirefor discovery of extremely potent “magic-bullet”

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 261compounds, natural functions that secondarymetabolites might serve do not necessarily requirehigh levels of potency or specificity, nor do theyeven require single entities. Synergistic effectsoccur commonly in nature, but can be difficult tosort out, and are much more complicated to takeadvantage of in pharmaceutical development. Itcould be argued that defensive or antagonisticeffectsexertedbycocktailsofmoderatelyactivecompounds with differing modes of action wouldbe more effective against predator or competitororganisms because evolution of resistance to suchmixtures would be more difficult. It is interestingto note that some recently marketed pharmaceuticalproducts (including Vytorin®, a successor toZocor® as a cholesterol-lowering drug) are actuallymixtures of compounds that have different modesof action. Despite these incongruities, applicationof ecological considerations to the search for newbioactive natural products provides rationale fortargeting certain organisms or types of organismsthat reduces the level of randomness in the process,while leading to new information about potentialnatural roles of secondary metabolites, as well asnew tools for ecological studies.II. Observations in Fungal EcologyAssociated with the Productionof Bioactive MetabolitesA. Competitive or Antagonistic InteractionsCompetitive and/or antagonistic interactionsamong fungal species constitute a general categoryof effects that are widely recognized to occur innature (Shearer 1995), and also are among themost straightforward to attribute to secondarymetabolites. Antagonism between species ofnaturally competing fungi has been reported invirtually every fungal ecosystem. Examples includecoprophilous (Wicklow 1992a, b), carbonicolous(Wicklow and Hirschfield 1979a), lignicolous(Strunz et al. 1972; Boddy 2000), phylloplane(Fokkema 1976), rhizosphere (Carroll 1992),marine (Strongman et al. 1987), and freshwateraquatic fungi (Shearer and Zare-Maivan 1988). Ithas long been proposed that such interactions areimportant factors in determining the organization,composition, and pattern of succession withinthese ecosystems (Webster 1970; Wicklow 1981).The mechanism of antagonism often appears toinvolve the production of a chemical agent (oragents)byonespeciesthatinhibitsthegrowthof another, but many such reports have notbeen followed up by studies of the chemistryassociated with these phenomena. Chemicalstudies of this nature have significant appeal, sincethe metabolites responsible for these effects areessentially natural antifungal agents. The needfor new antifungal agents in both medicine andagriculture continues to grow. Although effectivetopical antifungal agents are relatively abundant,many types of topical fungal infections recur aftercessation of treatment, and others (e.g., nail infections)are particularly difficult to treat effectively(Baker et al. 2005). More importantly, very fewdrugs are available that are therapeutically usefulin the treatment of systemic fungal infections(Richardson and Marriott 1987; Balkovec 1998;Wong-Beringer and Kriengkauykiat 2003). Fungaldiseases have become increasingly common, andthere are several risk groups of growing population(e.g., AIDS and chemotherapy patients)that are particularly susceptible to opportunisticfungal infections (Koltin 1990; Wong-Beringerand Kriengkauykiat 2003). Moreover, resistanceto existing antifungals has become problematic(Anderson et al. 2003). New agriculturally usefulfungicides are also being continually sought. It canbe argued that fungi are logical sources to explorein search of agents that regulate fungal growth,interact with important fungal receptors, or modulatethe activities of key fungal enzymes. Indeed,several particularly promising antifungal leadshave been isolated from fungal sources, includingechinocandins, pneumocandins, sordarins, andpapulacandins, and, as noted above, there areantifungal agents in use in both clinical andagricultural settings that are based on compoundsoriginally obtained from fungal sources.Our initial chemical studies of antagonistic interactionsinvolved investigations of coprophilous(dung-colonizing) fungi (Malloch 1981). Thesefungi were targeted in part because of theirwell-documented successional patterns on naturalsubstrates (i.e., replacement of early-occurring,fast-growing colonists by later, slower-growingcompetitors; Harper and Webster 1964; Webster1970; Angel and Wicklow 1983), along with sporadic,but tantalizing reports of antagonism amongsuch species that were presumed to be due to theproduction of antagonistic agents (Harper andWebster 1964; Ikediugwu and Webster 1970a, b;Singh and Webster 1973;Wicklow and Hirschfield

262 J.B. Gloer1979b). From an evolutionary perspective, it seemslogical that slower-growing colonists might evolvemechanisms that help to eliminate fast-growing,nutrient-consuming competitors from the localsubstrate. Additional support for the concept oftargeting coprophilous fungi was based on theirtaxonomic distinctiveness that is, in turn, associatedin a significant way with their adaptationsto this niche. Few coprophilous species had beenpreviously studied from a chemical standpointprior to our work, and it is, of course, appealingforanaturalproductchemisttoexploreasourcethat has not been previously investigated, since theprobability of finding new agents would likely behigher under such circumstances.Based on these and other considerationsoutlined in greater detail in the earlier volume(Gloer 1997), we targeted certain types of mid- tolate-successional coprophilous fungi as potentialsources of antifungal agents. In petri plate competitionassays carried out in our laboratories, over60% of the coprophilous isolates surveyed displayedinhibitory effects toward competitor fungiat a distance. The vast majority of these culturesrepresented genera and/or species for which nochemistry had been previously reported. Chemicalstudies focusing on the antagonistic isolates wereundertaken, and this work afforded a variety of newbiologically active natural products, many of whichdisplayed antifungal effects. Our early chemistryresults in this area have been reviewed previously(Gloer 1997), but studies of coprophilous fungiin our laboratory have continued to afford newbioactive metabolites, and selected representativesencountered in more recent years are included inFig. 15.1. Studies of Sporormiella vexans affordeda set of new aromatic metabolites called sporovexins(e.g., 1), along with a previously unreportedpreussomerin analog (2; Soman et al. 1999a).Preussomerins are intriguing and unusual structuresthat display a variety of biological effects, includingantifungal activity. The first representativesof this class were originally reported by our groupfrom a different coprophilous fungus (Preussia isomera;Weber and Gloer 1991). Podospora communisproduced a series of at least eight novel polyketidederivedmetabolites with furanone, cyclopentanoid,furanofuran, and furanocyclopentanoidstructures (e.g., 3–5; Che et al. 2004a, 2005). Thevariety of structural types and different ring systemsarising from an apparent common biogeneticprecursor was particularly impressive in this in-Fig. 15.1. Some representative new metabolites from coprophilous fungi (1–9)

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 263stance. Bombardioidea anartia affordedaseriesofnew anti-Candida furanones called bombardolides(e.g., 6), as mixtures of geometric isomers thatcould not be separated (Hein et al. 2001). Furtherexamples include cyclic peptides (e.g., 7), additionalpolyketides (e.g., 8), and terpenoid-derivedmetabolites (e.g., 9; Che et al. 2001, 2002b).Some of the new compounds encounteredin this project showed potent activity againstcompetitor fungi (MIC values < 5 μg/ml), and/oractivity against more medically relevant organismssuch as Candida albicans or Aspergillus flavus.Naturally, known compounds were also encountered,and some of the new metabolites are closelyrelatedtocompoundsthatwerepreviouslyknown,but others contain some quite novel structuralfeatures.Inmanyinstances,thesefindingsarosefrom studies of fungal species for which noprior chemistry had been described. Given thehigh incidence of antifungal activity and newchemistry among these isolates, the probability ofencountering more promising activity or usefullead structures through further studies of thechemistry of coprophilous fungi seems high.Indeed, other research teams have also had successin targeting coprophilous fungi as sources ofbioactive metabolites (Ondeyka et al. 1998; Maciaset al. 2001; Segeth et al. 2003; Singh et al. 2003;Ridderbusch et al. 2004; Weber et al. 2005; Lehret al. 2006).Although the results above demonstrate thatantifungal activity is a logical and effective primarytarget assay, agents with antifungal activity oftenexhibit other potentially useful biological effects.For example, cyclosporin was originally isolatedfrom a Tolypocladium sp. as an antifungal agent(Masurekar 2005). Cyclosporin is now a vitally importantimmunosuppressive drug, and a valuablepharmacological tool.The studies summarized above involved onlyasmallsubsetoforganismsisolatedfromonlyone of many niches in which interference competitionamong fungal competitors has been reported.Naturally, the success of this project led usto consider exploration of antagonistic fungi fromother ecosystems. Another underexplored ecologicalgroup that parallels the coprophilous niche asa reasonable choice in many respects consists ofthe freshwater aquatic fungi. Freshwater aquaticfungi are similarly underexplored from a chemicalstandpoint, have been reported to show antagonisticactivity toward competitors in some instances(Shearer and Zare-Maivan 1988), and are taxonomicallyand morphologically distinctive (Goh andHyde 1996). Initial investigations reviewed previouslyled to the discovery of several new bioactivenatural products (Gloer 1997). Further studiesof antagonistic freshwater aquatic fungal isolatesin our laboratory have continued to affordnovel metabolites with unusual chemical structuresand/or significant biological activities.While a small number of other research groupshave also published in this area (Schlingmann andRoll 2005; Dong et al. 2005), some recent examplesfrom our own ongoing work are shown in Fig. 15.2.An isolate of Massarina tunicata was particularlyprolific, producing multiple new metabolites representingdifferent polyketide-derived structuralclasses (Oh et al. 1999a, 2001, 2003) exemplifiedby structures 10–12. Dendrospora tenella produceda series of new antibacterial diphenyl ether derivatives(e.g., 13; Oh et al. 1999b). Ophioceras venezuelensewas found to produce a series of monocyclictetrahydropyran-type compounds (ophiocerins A–D; e.g., 14, 15; Reátegui et al. 2005). Surprisingly,despite their simplicity and abundance in culture,these compounds had not been previously encounteredas natural products. This species also affordeda new africanene sesquiterpenoid (16) thatwasofinterest not only because it has a rare skeleton,but also because it is the first member of this terpenoidgroup to be encountered from a microbialsource.ThenewspeciesAnnulatascus triseptatusproduces a series of simple, yet previously unreported,polyketide-derived pyrones and furanones(annularins; 17–19), some of which show antibacterialactivity (Li et al. 2003a). The fused pyrone–furanone system in annularin F (18)hadnotbeenpreviously reported. An unidentified member ofthe family Tubeufiaceae afforded several new altenueneanalogs (e.g., 20; Jiao et al. 2006a), while investigationsof an isolate of Decaisnella thyridioidesyielded a series of five new palmarumycin-typecompounds called decaspirones (e.g., decaspironeA, 21) withsignificantactivityagainstA. flavus(Jiao et al. 2006b). Studies of the aero-aquatic fungusHelicodendron giganteum ledtothediscoveryof three more new antifungal metabolites (heliconols;e.g., 22) possessing another unusual ringsystem (Mudur et al. 2006a).Many of the fungal isolates explored during thecourse of this project represent rare and/or previouslyundescribed genera and species. Our resultsto date, only a portion of which have been published,include the first secondary metabolite structureseverdescribedfrommembersofthegenera

264 J.B. GloerFig. 15.2. Some representative new metabolites from freshwater aquatic fungi (10–22)Anguillospora, Annulatascus, Decaisnella, Helicodendron,Kirschteiniothelia, Pseudoproboscispora,Dendrospora, Ophioceras, Massarina, Aniptodera,and Pyramidospora.Analogous studies of other niche groups thatcommonly display interspecies antagonism can besimilarly fruitful. Studies of wood-decay fungi thatshow antagonistic effects have afforded a numberof agents that could be useful in controlling economicallyimportant wood-rotting fungi (Strunzet al. 1972; Ayer and Miao 1993; Ayer and Kawahara1995). In other cases, individual niche groups aretargeted for chemical studies mainly because of thedistinctiveness of the niche, rather than because ofspecific indications of antagonistic effects. This isexemplified by the rapidly increasing number of reportsof chemistry from marine fungal isolates (Linet al. 2001; Proksch et al. 2003; Rowley et al. 2003;Bugni and Ireland 2004; Oh et al. 2005; Bhaduryet al. 2006; Boot et al. 2006; Cueto et al. 2006; Koeniget al. 2006), and from other, narrower niche groups,such as mangrove (Lin et al. 2001, 2002; Chen et al.2003) and lichenicolous (Hawksworth et al. 1993;Bills et al. 2000; Seephonkai et al. 2002; He et al.2005; Lin et al. 2005) fungi.It is interesting to consider whether antifungalmetabolites produced by antagonistic fungalspecies occur under natural conditions, since thiswould be a requirement in order for them to playsome role in the ecology of the producing species.Studiesofthiskindofissueareunfortunatelyrare,but there are exceptions. For example, it has beendemonstrated that the antifungal agent sordarin(Odds 2001), produced by a coprophilous fungus,is indeed produced in the natural substrate (Weberet al. 2005). It has also been shown that somespecies produce active metabolites under a certainset of laboratory culture conditions only when chal-

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 265lenged with microbial competitors (Oh et al. 2005).However, it is difficult to demonstrate true rolesfor such compounds in nature with confidence.Detailed individual case studies would be particularlyvaluable in shedding light on the ecologyof interspecies interactions, and relating ecologicalobservations directly to chemistry produced by individualorganisms under specific circumstances.Nevertheless, efforts in this area must be balancedwith the broader interest in discovery of potentiallyuseful new chemistry. Because our primary focus isonthesearchfornewchemistryofpotentialvaluein medicine or agriculture, it is unfortunately impracticalfor us to investigate in depth the roles ofthe metabolites we encounter in most instances.However, the following section describes a projectwherein the potential roles of metabolites were investigatedin somewhat greater detail.B. Resistance of Key Fungal Structuresto Fungivory or Microbial AttackThe vast majority of known fungal metabolites areproduced in liquid fermentation cultures underconditions very different from those encounteredby the fungi in nature. Although little is knownabout possible functions these compounds mayhave within the producing organisms, there isfrequently a close correlation between secondarymetabolite production and morphological differentiationin liquid culture (Bennett 1983).Interestingly, many fungi produce morphologicalstructures under natural conditions (or on solidsubstrates in the laboratory) that are not generallyformed in liquid cultures. Such structures includevarious fruiting bodies, sclerotia, and stromata.If there is secondary chemistry associated withsuch structures, it is reasonable to expect that suchchemistry may not manifest itself under conditionsunder which these bodies are not formed.Our own studies in this area focused onimportant fungal survival structures (sclerotiaand ascostromata) that are exposed to potentialpredators (fungivorous insects) under naturalconditions. Our results indicated that fungalsclerotia and ascostromata often contain uniqueantiinsectan metabolites that may help to protectthem from predation (see below). This work wascharacterized by a particularly high incidence ofpreviously undescribed natural products, even incases where the producing fungi had significanthistories of prior chemical investigation.Aconsiderablebodyofdirectandcircumstantialevidence for the production by fungi of chemicaldefenses against predation had been previouslycompiled and reviewed (Wicklow 1988a). Fungaltoxins are often implicated as possible defenses ina general sense, but relatively few detailed studies ofthe chemistry involved have been reported. Thereare many reports of grazing preferences amongfungivores. For example, mycelia or fruiting structuresof certain fungi are known to be avoidedby fungivorous insects or arthropods (Curl et al.1985; Shaw 1992; Wicklow 1992a). The presence ofbioactive secondary metabolites is often invokedor implicated in these phenomena, and agents responsiblefortheobservedeffectshaveoccasionallybeen identified (Wicklow 1988a; Bernillon et al.1989; Koshino et al. 1989). Some fungivores appearto have evolved detoxification mechanisms allowingthem to consume, and sometimes specializein consuming, toxin-producing fungi (Wicklow1988a; Dowd and VanMiddlesworth 1989; Shaw1992). There is also evidence that some advantagesmay be conferred on certain fungi upon moderategrazing by fungivores (e.g., inoculum dispersal;Stevenson and Dindal 1987; Shaw 1992). Manyother instances of selective grazing or refusal offungi by individual mycophagists could be cited.1. Claviceps ErgotConsidering the observation that the concentrationof plant defensive metabolites is often highestin reproductively important plant parts (Rhodes1985), it seems logical to initiate discussion of fungal“chemical defenses” by considering physiologicalstructures that are particularly important tofungal survival. One precedent provides a particularlyuseful introduction. The ergot alkaloids comprisea class of medicinally useful compounds thatwere originally isolated from the ergot (sclerotia) ofClaviceps purpurea (Mantle 1978; Masurekar 1992),a parasitic fungus found on many species of cerealplants.Thisclassofcompoundsexhibitsawidearrayof physiological activities, and many ergot alkaloidshave found medicinal uses (Floss 1976; Stadlerand Giger 1984). Chemical studies of Claviceps werenot based on a general interest in sclerotial metabolites,but rather were stimulated by the long-termimplication of ergots in poisonings of humans andlivestock. It is especially significant that the ergotalkaloids were originally found only in the sclerotiaof the fungus (Mantle 1978). Claviceps sclerotiaare not formed in liquid fermentation cultures, and

266 J.B. Gloermany of the fungal ergot alkaloids would not havebeen discovered through screening of liquid culturesalone. The medicinal importance of the ergotalkaloids has led to the gradual development by thepharmaceutical industry of Claviceps strains thatproduce some of the compounds in liquid fermentations,but even in these cultures, alkaloid productionis associated with “sclerotial-like” cells (Mantle1978). It has been proposed that the evolutionarydevelopment of the ergot alkaloids may have beenguided at least partly by selection pressures exertedby herbivores (Kendrick 1986; Wicklow 1988a).2. Sclerotia of Other FungiThere is, of course, no reason to believe thatproduction of bioactive sclerotial metabolites islimited to Claviceps. Several literature reportsafford circumstantial evidence that sclerotiaproduced by other fungi contain biologically activemetabolites. These fungi include species of Sclerotinia,Sclerotium, Verticillium, Macrophomina,and Aspergillus (Tanda et al. 1968; Morrall et al.1978; Wicklow and Cole 1982; Wicklow et al. 1988).Sclerotia, in general, are specially adapted,multicellular structures produced by certain fungias a survival mechanism (Willets 1971, 1978;Coley-Smith and Cooke 1971). These durableresting bodies can survive periods of dry, nutrientpoorconditions that other fungal parts cannotwithstand. They then serve as vital sources ofprimary inoculum for the fungi when conditionsagain become favorable for growth. Generally,sclerotia survive under more severe conditionsand for longer periods than any other kinds offungal bodies, sometimes remaining viable insoil for periods of several years. Sclerotia are byfar the largest fungal propagules, ranging in sizefrom 30 μm to several cm, depending on species,and their germination frequently results in thegeneration of very large quantities of inoculum.Thus, sclerotia represent a substantial metabolicinvestment for the producing fungi.It is interesting to consider how the presenceof bioactive metabolites could impact on sclerotialsurvival (Wicklow 1988a). Sclerotia typicallyform in fungus-infected plant tissues, and are separatelydispersed onto the soil surface or remainattached to decaying plant parts. Soil is heavilypopulatedwithinsectsandotherinvertebrates(Kevan1965), many of which are known to consumefungi (Wicklow et al. 1988). A dormant (or germinating)sclerotium would represent a substantialnutrient reward for an insect predator, especiallysince sclerotia possess a much higher nutrient contentthan the unorganized mycelium (Willets 1971).Sclerotia damaged by insect larvae are much moresusceptible to microbial decay than are undamagedsclerotia (Baker and Cook 1974). Thus, if sclerotiacommonly contain metabolites that somehow limitfeeding by insects, this property could clearly influencethe longevity of these important fungal bodies.Based on parallel observations in plant chemicalecology (e.g., allocation of defensive metabolitesto seeds; Rhodes 1985), there is reason to suspectthat selection pressures exerted by insect predationmay have led to the evolution of sclerotial chemicaldefenses (Wicklow and Cole 1982; Wicklow et al.1988).Our efforts in this area were initially stimulatedby the fact that the sclerotia of Aspergillusflavus are avoided by the common detritivorousbeetle Carpophilus hemipterus, an insect that feedson the conidia and mycelia of the same fungus(Wicklow et al. 1988). In light of this observation,we investigated the secondary metabolites ofA. flavus sclerotia, and encountered a number ofantiinsectan compounds not found in the conidiaor mycelia of the fungus (Gloer et al. 1988; Wicklowet al. 1988). These compounds were not producedin simple liquid shake cultures of A. flavus, andmost of them had not been previously reported(Gloer et al. 1988). Furthermore, the most potentantiinsectan metabolite was nontoxic to vertebratesat 300 mg/kg (Cole et al. 1981), but was found tobe present in sclerotia at levels that would effectivelydeter feeding in C. hemipterus.Thesefindingsled us to initiate general studies of the chemistryof Aspergillus sclerotia as sources of new antiinsectannatural products (Gloer 1995a, b). Assaysfor activity against C. hemipterus and the importantcrop pest Helicoverpa zea (corn earworm) wereemployed to guide isolation procedures. H. zea isunlikely to be ecologically relevant to the sclerotiaof Aspergillus spp., but the discovery of agents withpotent activity against H. zea could be importantfrom a more practical perspective.Most of the results from this project weresummarized in detail in the earlier volume in thisseries (Gloer 1997). Briefly, our bioassay-guidedchemical studies of sclerotia afforded over 70new metabolites and approximately 30 knowncompounds, the majority of which display somedegree of activity against insects (Gloer 1995b).Indole diterpenoids, a unique class of structurallycomplex fungal metabolites that display a broad

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 267array of biological activities (Parker and Scott2005), were particularly common among thesesamples, although other metabolite-types werealso encountered. In most cases where detailedanalyses were performed, the compounds ofinterest were found to be concentrated at least tosome degree in the sclerotia. The success rate infinding new compounds was exceptionally high,despite extensive prior studies of the chemistry ofAspergillus spp. A few additional new Aspergillussclerotial metabolites have been published sincethe previous review (Oh et al. 1998; Whyte et al.2000). In addition, these studies were extendedtoalimitednumberofPenicillium spp. that arealso known to produce sclerotia, and some newbioactive compounds were obtained in theseinvestigations as well (Belofsky et al. 1998a; Joshiet al. 1999a).The existence of antiinsectan sclerotialmetabolites does not conclusively prove thatthey have a role in chemical defense – thesestudies provide only circumstantial evidence.However, the success of these investigations arguesfor chemical studies of other fungal structuresthat serve similar functions. These results alsodemonstrate that assumptions about the identityof compounds causing ecological effects shouldnot be based on prior knowledge of the chemistryof a fungal species or genus. Given the vast priorknowledge about Aspergillus metabolites, suchassumptions might have seemed warranted in thecase of Aspergillus sclerotia,butwouldnothavebeen valid based on the results described above.3. AscostromataOther fungi produce morphological structuresthat are analogous to sclerotia in function. Certainascomycetes, for example, produce ascostromata,which are proposed to play ecological rolessimilar to those of sclerotia (Wicklow and Cole1984; Horn and Wicklow 1986). Studies of suchstructures have shown that they also can containbioactive metabolites. Examples include ascomataof Epichloe, Petromyces, Eurotium, Eupenicillium,Daldinia, andTalaromyces spp. (Wicklow 1988a;Koshino et al. 1989; Nozawa et al. 1994; Belofskyet al. 1995; Wang et al. 1995; Ooike et al. 1997a, b;Suzuki et al. 1999; Stadler et al. 2001).We therefore undertook what we viewed asa logical extension of the project described aboveby expanding our studies to include the sclerotioidascostromata produced by species of Eupenicillium.Initial bioassays showed an incidence ofantiinsectan activity comparable to that observedamong Aspergillus sclerotial extracts, although thepotencyofactivitywassomewhatloweronaverage.Studies of several Eupenicillium spp. affordedinteresting results, including several more newcompounds with antiinsectan activity. Interestingly,several of the compounds encountered wereeither identical to those obtained from sclerotiaof Aspergillus spp. (e.g., aflavinine analogs andother indole alkaloids; Belofsky et al. 1995; Wanget al. 1995), or structurally similar, and in caseswhere analysis of ascostromata vs. other fungalmaterial was carried out, the compounds wereagain heavily concentrated in the ascostromata.Based on these findings, it appears that at leastsome of the ascostroma-producing Eupenicilliumspp. have evolved (or retained) chemical defensesystems similar to those of Aspergillus. Studiesofother Eupenicillium spp.ledtoisolationofotheractive metabolite types, some of which displayedantiinsectan effects (Belofsky et al. 1998b; Wanget al. 1998).4. Other Fungal StructuresOther fungal structures such as spores, cleistothecia,or fruiting bodies may also be chemicallydefended. Selective chemical studies of very smallstructures are complicated by the difficulty inseparating them in quantity from other fungalmaterial, while studies of fruiting bodies are oftenhindered by an inability to form them in laboratorycultures, or to reliably predict when and wherethey will occur in nature. Some researchers haveproposed defensive roles for metabolites found infruiting bodies of basidiomycetes (Sterner et al.1985; Shaw 1992). The amanitins and phalloidins,well-known toxic peptides found in fruiting bodiesof the genus Amanita, are often cited as examplesof possible chemical defenses. These compoundsare toxic to a variety of insects, although someinsect species, either coincidentally or throughselection processes, have evolved the capacity todetoxify or tolerate these metabolites (Jaenikeet al. 1983; Shaw 1992). Metabolites of ediblemushrooms may also display potentially usefulbiological activities. The strobilurins are producedby a common edible mushroom, and have proventobeimportantasfungicidesandleadsthereto(Anke 1995; Peláez 2005). These compounds havealso been proposed to play an ecological role,and are reportedly produced on their natural

268 J.B. Gloersubstrate (Anke 1995), although most, if not all,of the strobilurins have been isolated from laboratorycultures. Other researchers have discussedthe potential value of mycophagy to the fungibecause of its importance as a mechanism ofspore dispersal (Shaw 1992), citing examples ofmetabolites that attract fungivores. These reportsare illustrative of the complexity of interpretingaspects of fungus–fungivore interactions as theymight relate to secondary metabolite production.Sporadic reports suggest that smaller structuresmay also contain unique bioactive metabolites.Brevianamide A was found to be localized in thepenicillus of Penicillium brevicompactum andproposed to deter feeding in fungivorous arthropods(Wicklow 1986). Ascomata of Chaetomiumbostrychodes were reportedly avoided by certainfly larvae, and this behavior was presumed tobe due to the presence of secondary metabolites(Wicklow 1988a).C. Fungi that May Confer Host Resistanceto Herbivory or DiseaseIt has been proposed that the capability of fungito produce mycotoxins evolved and has beenretained partly because they render fungal substrates(e.g., fruits, seeds, etc.) unpalatable toherbivores (Janzen 1977; Kendrick 1986; Wicklow1988a). While this could be viewed as a defense ofsubstrate resources from competing consumers, itis too general a concept to be particularly helpfulin targeting fungi for chemical investigation, anddoesnotlenditselftofieldinvestigations.Ontheother hand, there are documented instances wheresecondary metabolites play roles in mutualismsbetween certain fungi and their hosts. One ofthe best known examples of such a system is theproductionbygrassendophytesoffungalergotalkaloids that influence feeding by herbivores (Clay1988; Carroll 1992; Scott 2001, 2004). Evidence forparallel effects in other systems is also available.For example, fungal endophytes found in coniferneedles produce metabolites that confer somelevel of resistance to herbivory by insects (Miller1991; Findlay et al. 1995). Numerous industrialand academic groups have targeted endophyticfungi in general for inclusion in their screeningprograms (Caporale 1995; Lee et al. 1995), andseveral reviews of endophyte secondary metaboliteshave appeared recently (Tan and Zou 2001;Strobel 2002; Schulz et al. 2002; Gunatilaka 2006).Although some researchers have argued thatother endophytic fungi are likely to play roles inthe ecology of the producing species, at least insome instances, the in-depth studies required todemonstrate this are seldom carried out, as thetargetingofendophytesisinmostcasesviewedprimarily as a means of enhancing the diversityof species being screened for activity, rather thananyacknowledgmentthatsuchfungimightplaydefensiverolesthroughmetaboliteproduction.Arelated,intriguingareaofresearchinvolveseffortsbysomegroupstoexplorethechemistryof endophytes from specific plant hosts that aretargeted because the plants produce compoundsof industrial importance. The main objective ofsuch work is to find fungal strains that producecompounds isolated from the plant, thereby enablingproduction of the compounds of interestby fermentation processes, and also perhaps enablingmore ready access to the corresponding secondarymetabolic genes. Reports of the importantplant metabolites taxol, podophyllotoxin, andcamptothecin as trace constituents of endophytesfrom the corresponding plant hosts have appeared(Li et al. 1998; Puri et al. 2005; Eyberger et al. 2006).Recently,wehaveinitiatedpreliminarystudiesof a subgroup of endophytes that do not appear tohave been chemically explored by others to a significantextent. This work stems from an interestin the antagonistic properties of Acremonium zeae;an endophyte of corn that is widespread in agroecosystemsin the USA and elsewhere. A. zeae is oneof the two most common seed-borne endophytesof pre-harvest maize, typically producing symptomlesskernel infections (Wicklow 1988b; Arinoand Bullerman 1994), but the significance of its occurrenceis not understood (Munkvold et al. 1997).Patterns of fungal occurrence within plant seedscan sometimes be indicative of interactions amongthe corresponding fungal species (Wicklow 1988a).Based on several experimental observations,including exhibition of antagonistic activitytoward other fungi in our laboratories (includingAspergillus flavus), our colleague D.T. Wicklowhypothesized that A. zeae produces antifungalmetabolites that could interfere with mycotoxigenic,destructive fungal pathogens of maize.Chemical investigations led to the discovery thatisolates of A. zeae from across the USA producea series of unusual antifungal and antibacterialantibiotics (e.g., pyrrocidine A, 23; Fig. 15.3;Wicklow et al. 2005). Although the lead compoundsin the series were published first by an

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 269Fig. 15.3. Structure of pyrrocidine A (23), a bioactivemetabolite from the corn endophyte Acremonium zeaeindustrial group from another fungal source (Heet al. 2002), we continued our studies of the naturaloccurrence and potential significance of theseA. zeae metabolites, and have found that theyoccur commonly as major metabolites of isolatesof A. zeae from various geographical locations,and that the most active component shows potentactivity against mycotoxigenic fungi, as well asfungal and bacterial pathogens of corn.There are many other endophytes that areknown to occur and grow asymptomaticallyin healthy crop tissues, including latent plantpathogens (Sinclair and Cerkauskas 1996), andmostofthesedonotappeartohavebeenstudiedchemically. There have been several reportsof specific grass/cereal endophytes that showantagonism against plant pathogenic fungi, butfor which no fungitoxic compounds have beendescribed (Siegel and Latch 1991; Stovall and Clay1991; Christensen 1996; Danielsen and Jensen1999). On basis of our results with A. zeae,weviewthis group of endophytes as another potentiallyimportant set of targets for chemical investigation,not only because of their apparent potential toproduce antagonistic (antifungal) metabolites,or because of interest in roles they might play inagroecosystems, but also because knowledge ofthe chemistry of fungi that occur widely in cropplants could be of importance to human health,even if these fungi do not produce obvious plantdisease symptoms.D. Fungi that Cause Diseases or Damageto Host SpeciesThe concept of using biological control strategiesin crop protection has been studied intensivelyfor many years (Gardner and McCoy 1992; Powell1993). These strategies involve the use of certainfungi as mycoherbicides (Boyette 2000; Ghorbaniet al. 2005), mycoinsecticides, or mycoparasites(Harman 2006) to control weeds, insects, or fungalpathogens, respectively. Such approaches involvethe deployment of a microbial disease agent effectiveagainst the pest, rather than (or as an adjunctto) the application of a chemical pesticide of somesort. Ultimately, damage to the pest is often causedby toxin(s) produced by the pathogen, and the useof measured and properly formulated quantitiesof the natural products themselves could providean alternative control strategy. Use of the microorganismitself is appealing because it serves to selectivelydirect the toxin(s) to the target. Some toxinproducingmicroorganisms, most notably the bacteriumBacillus thuringiensis, haveproventobeparticularly effective in such applications. However,there are many specialized hurdles that mustbe overcome to implement an effective biocontrolstrategy (Powell 1993), and the use of biocontrolagents will not necessarily eliminate theproblem of resistance (Gardner and McCoy 1992).From a chemistry standpoint, knowledge of anymetabolites involved in biocontrol effects is importantas a means of avoiding unwanted side-effects,and precedents indicate that studies of fungi withmycopesticidal properties are likely to lead to discoveryof pesticidal compounds. Some examples ofsuch results are provided below.1. Plant PathogensPlant pathogenic fungi are well known as producersof diverse compounds with phytotoxic effectson host plants (Turner and Aldridge 1983; Harborne1993; D’Mello and MacDonald 1998). Theeffects of the toxins are often principal causes ofsymptoms associated with the corresponding plantdisease. In many cases, fungal phytotoxins producedamage that fosters fungal invasion and colonizationoftheplant.Typically,thecompoundsaregeneralphytotoxins (Ballio 1991), but in some cases,a coevolutionary process has led to at least somedegree of host selectivity (Walton and Panaccione1993; Wolpert et al. 2002). The host-selective toxinsare particularly interesting from a chemical standpointbecause they tend to have structural featuresthatdistinguishthemfromcommonlyencounteredfungal metabolites that might show more generaltoxicity. Notable examples include victorin, HCtoxin,and certain Alternaria toxins (Walton andPanaccione 1993; Wolpert et al. 2002; Masunaka

270 J.B. Gloeret al. 2005). Plant pathogenic fungi as a group couldbe viewed as logical sources to explore in search ofbiologically active metabolites in a general sense,since they have already demonstrated the capacityto produce bioactive compounds with distinctivechemical structures. Some fungal metabolites withphytotoxic effects are known to exhibit medicallyrelevant activities as well, including antitumor andantibiotic effects. It is interesting to note that mevinolinis a rather potent herbicide (Hoagland 1990).From a more specific viewpoint, fungipathogenic to weeds have been proposed asrational sources of herbicides. Indeed, phytotoxinswith novel and unusual structures have beenisolated from weed pathogens, and subsequentlyshown to display herbicidal activity toward weeds(Hoagland 1990, 2001; Amalfitano et al. 2002;Evidente et al. 2004). This would seem to be a particularlyworthy avenue of investigation in view ofthe fact that other microbial natural products, suchas bialaphos (produced by a Streptomyces sp.),have already been used successfully as commercialherbicides (Hoagland 2001).2. Entomopathogenic and Nematophagous FungiA number of entomopathogenic and nematophagous(nematode-trapping) fungi have beenconsidered as biocontrol agents (Sun and Liu2006), and the physiological effects associatedwith some fungi from these categories have beenlinked to fungal toxins (Wicklow 1988a). Suchtoxins could serve as leads to the development ofnew insecticides or nematocides. In view of thispossibility, together with the association of a significantcomponent of predicted fungal diversitywith estimates of insect diversity, it is surprisingthat relatively few studies of entomopathogenicfungi as sources of bioactive metabolites havebeen reported. Several distinctive classes ofinsecticidal metabolites have been encounteredthrough early studies of such species, includingthe beauverolides, destruxins, and viridoxins(Turner and Aldridge 1983; Gupta et al. 1993).Further studies have appeared more recently, andare thoroughly summarized in a recent review(Isaka et al. 2005). Some of these compoundsdisplay both dietary and topical activity againstinsects. Metabolites from nematopathogenic ornematophagous fungi are even more uncommon;however, a related approach involves investigationof nematophagous fungi as sources of nematocidaltoxins that cause paralysis or mortality of thenematode prey (Kwok et al. 1992; Stadler et al.1994; Anderson et al. 1995; Anke et al. 1995).3. Mycoparasitic and Fungicolous FungiMycoparasitic fungi act as parasites of others, andthe invaded organism often suffers negative effectsfrom this interaction that are likely to be caused inat least some cases by fungal toxins. Fungi encounteredas colonists of others (often termed fungicolousor mycophilic isolates) may also produceantifungal metabolites, even though a true parasiticrelationship may not have been demonstrated.Some mycoparasitic fungi that have been used orproposed as biocontrol agents because of their antifungaleffects (e.g., Trichoderma, Verticillium spp.)have been shown to produce agents that inhibit thegrowth of other fungi (Cardoza et al. 2005). Examplesinclude peptide antibiotics, phenolics, and terpenoids(Huang et al. 1995a, b; Morris et al. 1995).Antifungal metabolites and compounds with otherbiological activities have also been reported fromother fungicolous and mycoparasitic species (Ayeret al. 1980; Tezuka et al. 1994, 1997; Wagner et al.1995, 1998; Breinholt et al. 1997, 1998; Schneideret al. 1997; Fabian et al. 2001; Feng et al. 2003; Wilhelmet al. 2004).Our own interest in this area was in part anoutgrowtharisingfromtheprojectsummarizedabove that involved studies of sclerotial metabolites.Our initial investigations were stimulated bythe recognition that Aspergillus sclerotia collectedfrom soil were often colonized by other fungi. Inmany cases, when these sclerotia were subjectedto standard germination conditions, many of thosecolonized by others were nonviable. This raised thequestion of whether such “attacking” species mightbe producing compounds that were inhibitory orperhaps fungicidal to Aspergillus. Given the medicalimportance of Aspergillus spp. as opportunistichuman pathogens, it seemed logical and particularlyappealing to investigate the concept ofwhether species that attack Aspergillus sclerotiamight serve as sources of new antifungal naturalproducts specifically having anti-Aspergillus effects.On the basis of this rationale, sclerotia ofA. flavus were buried in soil for extended periods,recollected, and examined for the presence offungal colonists. Many isolates were obtainedand screened, and a sizable number of theseproduced fermentation extracts that showedactivity against A. flavus. Chemical studies of the

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 271active extracts afforded a variety of antifungalmetabolites, including a number of compoundswith interesting new structures (Fig. 15.4). Oneof the first sclerotium-colonizing species investigatedin detail was an isolate of Humicolafuscoatra.Initialstudiesofthisorganismaffordedfive known compounds – three representativesof the monocillin/monorden class, as well astwo cerebroside analogs (Wicklow et al. 1998).Interestingly, monocillins had been previouslyisolated from Monocillium nordinii, aparasiteofa tree rust, and are thus among the few previousexamples of antifungal metabolites reported frommycoparasitic fungi (Ayer et al. 1980). This resultprovided an immediate, intriguing parallel toour findings from the sclerotial project, in thatwe again observed metabolic similarities amongdifferent fungi with similar adaptive requirements.The occurrence of cerebrosides in this organismwas also interesting because they are known topotentiate the activity of other antifungal agentsin assays against Candida albicans (Sitrin et al.1988). Continued studies of this H. fuscoatraisolate led to the identification of several other,unrelated compounds (Joshi et al. 2002), includingthe new triterpenoid glycoside fuscoatroside(24), which displayed potent activity in assaysagainst A. flavus, and a new, relatively simple, butpreviously unreported amide (fuscoatramide; 25).Studies of a sclerotium-colonizing isolate ofMortierella vinacea afforded three new benzenoidscalled mortivinacins A–C (e.g., 26, 27; Soman et al.1999b). Mortivinacin A (26) contains a thiol esterfunctionality that is rarely encountered in fungalsecondary metabolites. Another rich source of activemetabolites obtained as a colonist of A. flavussclerotia was an isolate of Gliocladium catenulatum.This species is known to be mycoparasitic, asare certain other Gliocladium spp. Ten compoundswere isolated, including new verticillin analogs(28–30) andanewglisopreninanalog(31; Joshiet al. 1999b). The anti-Aspergillus activity was ultimatelyascribed to a series of unusual polyketidediglycosides (e.g., 32; Joshi 1999), although thesame structures were published at approximatelythesametimebyanothergroupfromasoilisolateof G. catenulatum as cytotoxic agents and referredto as TMC-151-A, E, F, and G (Kohno et al. 1999).Each of these metabolites contains a large, highlymethylated polyketide linked to two different,unusual monosaccharide units. These compoundswere quite potent in assays against A. flavus.Threemore intriguing new antifungal compounds wereobtained from a sclerotium-colonizing isolate ofPodospora curvicolla, amemberofwhatisconsidereda typically coprophilous genus (Che et al.2004b). Curvicollides A–C (e.g., 33) accountedfor most of the antifungal activity of the extract,showing activity in our assays against A. flavus andFusarium verticillioides at one-tenth the potency ofnystatin. These compounds have no close knownrelatives, and are unusual in that they appear toconsist of two different polyketide units linked viathe central γ-lactone ring.These efforts were successful and reasonablyproductive in leading to new antifungal metabolites,and other researchers have demonstrated thatmycoparasites that colonize the sclerotia of otherfungi can also produce metabolites active againstthe host (McQuilken et al. 2003). However, we feltthat the strategy of burying sclerotia in soil for longperiods as “bait” for fungal colonists to survey forantifungal metabolites was not particularly appealingduetothetimerequired,thelabor-intensivenature of the corresponding field work, and limitationsthat seemed likely to arise from the use ofa single host-type. Consideration of other optionsled us to widen the scope of this work to includefungi that colonize other long-lived physiologicalstructures, such as basidiomycete fruiting bodies.Of course, some such structures are much longerlivedthan others, but many wood-decay fungi, forexample, form stromata that last for considerableperiods. Such bodies are widespread and macroscopic,making them relatively easy to find and collect,and would comprise a diverse array of differenthost-types that seem likely to lead to a diversecollection of colonizing fungal taxa. Initial collectionsof fungal sporocarps were made in the upperMidwest USA. Isolation of fungal colonists fromtheir surfaces, followed by fermentation, bioassay,andchemicalanalysisagainledtothediscoveryofa variety of new compounds, many with antifungaleffects, thereby validating the concept and eventuallyleading to further collections in the southeasternUSA and in Hawaii that have afforded manyadditional new bioactive natural products. Thesestudies remain a major focus of our current researchprogram.Aside from interesting new chemistry, aswill be highlighted below, this effort has also ledto some intriguing contributions to mycology.As one might expect, these include discovery ofa number of new taxa representing a variety ofgenera. However, some unanticipated findingsalso arose. One observation made early in this

272 J.B. GloerFig. 15.4. Some representative new metabolites from fungi isolated as colonists of Aspergillus flavus sclerotia (24–33)Fig. 15.5. Some representative new metabolites from fungicolous Penicillium isolates (34–40)

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 273project was that Penicillium spp. were encounteredwith considerable frequency. Given the ubiquitousnature of Penicillium spp. in the environment, itwas not particularly surprising that such speciesmight be encountered among those occurring ascolonists of the stromata of other fungi. However,it became clear upon continued investigation thatsome of the Penicillia being encountered appearedto be different from known species. Upon carefulinvestigation of these isolates, accompanied byanalysis of ITS and 26S rDNA sequences (ca. 1,200bases; Peterson et al. 2004), it was discoveredthat many of them represented new species.This was an unexpected finding, and leads oneto speculate about whether there is a subset ofPenicillium species that have some predilectionfor colonization of other fungi. Encouragingly,chemical studies of some of these fungicolousPenicillium isolates have afforded interestingresults (Fig. 15.5). While some of the new speciesproduce previously known Penicillium metabolites,Penicillium thiersii, anewspeciesisolatedfrom a Hypoxylon stroma collected in Wisconsin,produces a range of new compounds, includingthiersinines (e.g., 34; Li et al. 2002) thiersindoles(e.g., 35; Li et al. 2003b), thiersilones (e.g., 36; Li2003), and decaturins and new oxalicine analogs(e.g., 37, 38) that were similar to further newmetabolites encountered during this project fromanother new species, P. decaturense (Zhang et al.2003; Li et al. 2005). These metabolites includestructures that incorporate several new or rarelyencountered ring systems. A number of thesemetabolites show potent activity against the croppest Spodoptera frugiperda, while compound36 displays antifungal activity against A. flavus.Although not a new species, a Hawaiian isolate ofPenicillium griseofulvum obtained as a colonist ofawood-decayfungusaffordedasetoffivenewcompounds called penifulvins (e.g., penifulvin A;39) that incorporate a novel dioxafenestrane-typering system (Shim et al. 2006a, b). Compound 39showed significant antiinsectan activity againstthe fall armyworm Spodoptera frugiperda,causinga 74% reduction in growth rate relative to controlswhen tested at a 160 ppm dietary level. Theseunusual compounds appear to be derived fromsilphinene sesquiterpenoid precursors with anoxidative cleavage step occurring at some pointin the biosynthesis, and this hypothesis wassupported by the isolation of a new silphineneanalog (40) from the same extract (Shim et al.2006b).Other fungicolous isolates obtained fromvarious sporocarps collected through these studieshave also been quite prolific as sources of newbioactive natural products. Only some recent examplesthat have already been published are citedhere (Fig. 15.6). Polyketide-derived compoundspossessing three additional new ring systems(cladoacetals, e.g., 41 and malettinins, e.g., 42, 43)were obtained from two unidentified fungicolousisolates (Höller et al. 2002; Angawi et al. 2003a,2005). One of these colonists is a novel, but as yetunnamed hyphomycete resembling Cladosporium,and another is a Mycelia sterilia.MalettininA(42)contains a relatively rare, naturally occurringtropolone unit, and shows potent activity againstA. flavus (MIC 6 μg/ml). Lowdenic acid (44) isa similarly potent antifungal obtained from a newVerticillium species (Angawi et al. 2003b). Studiesof an isolate of Verticillium lecanii encounteredas a colonist of a basidiocarp of the mushroomAmanita bisporigera led to the isolation of severalnew pyridine derivatives (vertilecanins; e.g., 45;Soman et al. 2001). V. lecanii is known as aninsect pathogen, and the extract showed activityagainst Helicoverpa zea, but the antiinsectan constituentswere not isolated. Several antibacterialsesquiterpenoid metabolites (e.g., 46) andacyclopentenonederivative (47) wereobtainedfroma Phoma sp. isolated from stromata of Hypoxylonsp. (Che et al. 2002a), while further antibacterialsesquiterpenoids (e.g., 48)werealsoobtainedfroma culture of Pestalotiopsis disseminata isolatedfrom stromata of an unidentified pyrenomycete(Deyrup et al. 2006). A Phaeoacremonium sp.isolated from stromata of Hypoxylon truncatumafforded two new isobenzofuranoids (49, 50), with49 being somewhat distinctive among membersof this class in that it retains the aromaticityof the furan moiety, rather than the benzenering. However, the antifungal activity of thefermentation extract was ascribed to the presenceof more common sorbicillin-type compounds(Reátegui et al. 2006). An isolate of Sporormiellaminimoides was found to produce a pair ofantifungal agents (IC 50 vs. A. flavus ca. 6 μg/ml;MIC ca. 10 μg/ml) with intriguing structures (51,52) ofpresumedpolyketideorigin(Muduretal.2006b).Many additional new compounds have alreadybeen isolated and identified from further fungicolousisolates in our laboratory, but have not yetbeen reported. In summary, this niche is proving tobe a particularly prolific one from the standpoint of

274 J.B. GloerFig. 15.6. Some representative new metabolites from other fungicolous fungal isolates (41–52)newchemistry,andmanyoftheagentsencountered(both new and previously known) display antifungalactivity, as was proposed at the outset of theproject.III. Perspectives and Future DirectionsThe results reviewed here do not prove that fungihave evolved chemical defenses, or that the antagonisticagents produced by certain fungi rendera competitive advantage. Carefully controlled studiesare needed to determine whether these compoundsare truly significant in the life historiesof the producing species. Even so, it is clear thatobservations in fungal ecology can be employedto generate strategies that have proven effective inthe discovery of novel bioactive fungal metabolites.From a mycological perspective, these findings helpto validate the application of fundamental principlesof chemical ecology to studies of fungi, provideadditional tools with which to study the ecology ofthe fungi involved, foster the discovery of new taxa,and supply information that could be useful in exploringgenetic relationships.Investigations based on observations in fungalecology are appealing from a chemistry standpointbecause it is more attractive to seek bioactivemetabolites with a hypothesis in mind than toscreen organisms at random. As demonstratedhere, such studies can lead to the discovery offascinating new chemistry, and often provideunexpected analytical or structural challenges.Although unambiguous identification of naturalproducts, even previously reported metabolites,can be a lengthy process, continuing technicalimprovements in methodology for isolation andstructure determination (Harris 2005) now allowidentification of milligram- or sub-milligramquantities of complex, unknown natural productsin many cases.

Applications of Fungal Ecology in the Search for New Bioactive Natural Products 275The frequency of occurrence of novel compoundsin our studies to date continues to be relativelyhigh. This is due in part to the assay systemsemployed. The use of “ecologically relevant”,whole-organism assays, in addition to more sophisticatedmedically and agriculturally relevanttest systems, would understandably lead to somediscoveries not likely to be made by those employingonly the latter assays. However, the less randomselectionoforganismsforstudy,andtherelativelyunexplored nature of many of these fungi and theirinteractions are also contributing factors. Strategiesof this type cannot replace random screeningprograms in the search for new bioactive naturalproducts, but can offer insights that could increasethe diversity of compounds encountered in suchprograms.Forexample,these results argue stronglyfor employment of solid-substrate fermentation instudies of fungal metabolites, for consideration ofhabitat and ecological characteristics when selectingfungiforstudy,andforallowinglongerfermentationtimes to access metabolites produced byslower-growing species.Thereisnoshortageofcurrentandfuturechallengesthat fungal natural products chemistry canhelp to confront. Adequate treatments are still conspicuouslylacking for many viral diseases, fungalinfections, and human cancers. The long-termfailure to find effective therapeutic agents in theseareas does not mean that further, more effectiveagents cannot be found among natural sources.Discovery of suitable drugs may simply await thedevelopment of appropriate assays or the discoveryof appropriate molecular targets. A newer, developingchallenge is posed by the increasing occurrenceof antibiotic resistance among bacterial infections,as well as the emergence of drug-resistant tuberculosisand malaria. The development of new agentseffective against such diseases, particularly thosehaving novel modes of action, is becoming an urgentpriority. On the agricultural front, the demandfor new pesticides is compounded by the fact thatproblematic insect pests, e.g., are developing resistanceto many commonly used commercial pesticides.In addition, the use of some effective pesticideshas been curtailed due to concerns about undesirableenvironmental impact. Similar concernsabout other agrochemicals imply that new alternativesare needed, and that products of naturalorigin would be particularly appealing (Rimandoand Duke 2006).Another promising avenue of investigation canmake use of advances in molecular biology and biochemistry.Knowledge of metabolite biosynthesishas been extended to an awareness of genes linkedto regulation and production of the enzymes associatedwith the biosynthesis of some importantfungal metabolites (Calvo et al. 2004; Demain et al.2005; Keller et al. 2005; Wang et al. 2005). Somefungal genes involved in antibiotic production areknown to be clustered, as is the case with manyactinomycetes (Demain 1992; Cary 2004). Potentialapplications of this technology include the recognitionand cloning of genes involved in metabolitebiosynthesis, improvement in metabolite productiontechniques, production of novel hybridmetabolites, and the use of fungal DNA librariesto aid in the discovery process.The rapidly increasing capacity of biologicalscreening systems has outpaced the capability ofeven the most prolific microbiology programs toprovide adequate numbers of samples for assay.Thepressureforshort-termresults,togetherwithcompetition from other approaches to discoveryof bioactive lead compounds (e.g., combinatorialchemistry, molecular modeling-based design)played a significant role in the termination and/orcontraction of several industrial natural productsprograms in the USA over the past 25 years.Considering the diversity of fungi (and othersources) that remain unexplored, it is unfortunatethat some institutions have had to abandon a lineof research that has been instrumental to theirsuccess. Obviously, such occurrences increase thepressure on remaining natural products researchprograms to come up with promising leads, whiledecreasing the total effort underway in the field.At this stage, there is still no substitute for naturalproducts chemistry as a source of truly novellead structures, and it appears that recognition ofthis fact is beginning to stimulate a resurgence ofinterest in this field (Rouhi 2003). The unsurpassedchemical diversity of natural products is widelyrecognized, and there is considerable interestin construction of natural product libraries forgeneral screening purposes (Bindseil et al. 2001).Interestingly, a current mainstream objective incombinatorial chemistry involves the developmentof so-called natural-product-like syntheticchemical libraries (Shang and Tan 2005; Messeret al. 2005). In any event, practical successes aremore important than ever to continued supportfor fundamental studies of fungal taxonomy,ecology, and natural products chemistry. In thisclimate, the value and importance of cooperationbetween mycologists and chemists cannot be

276 J.B. Gloeroveremphasized. The results described and citedin this chapter are illustrative of the potentialrewards of such collaborative efforts.Acknowledgements. Generous support for our research inthis area over the past 10 years from the National Institutesof Health (AI 27436 and GM 60600) and the National ScienceFoundation (CHE-0315591, CHE-0079141, and CHE-9708316) is gratefully acknowledged. The author also wishesto express his appreciation to Drs. D.T. Wicklow, P.F. Dowd,D. Malloch, and C.A. 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Applications of Fungal Ecology in the Search for New Bioactive Natural Products 283Wicklow DT (1992a) The coprophilous fungal community:an experimental system. In: Carroll GC, Wicklow DT(eds) The fungal community: its organization and rolein the ecosystem, 2nd edn. Marcel Dekker, New York,pp 715–728Wicklow DT (1992b) Interference competition. In: CarrollGD, Wicklow DT (eds) The fungal community:its organization and role in the ecosystem, 2nd edn.Marcel Dekker, New York, pp 265–274Wicklow DT, Cole RJ (1982) Tremorgenic indole metabolitesand aflatoxins in sclerotia of Aspergillus flavus Link: anevolutionary perspective. Can J Bot 60:525–528Wicklow DT, Cole RJ (1984) Citreoviridin in standing corninfested by Eupenicillium ochrosalmoneum.Mycologia76:959–961Wicklow DT, Hirschfield BJ (1979a) Competitive hierarchyin post-fire ascomycetes. Mycologia 71:47–54Wicklow DT, Hirschfield BJ (1979b) Evidence of a competitivehierarchy among coprophilous fungal populations.Can J Microbiol 25:855–858Wicklow DT, Dowd PF, TePaske MR, Gloer JB (1988) Sclerotialmetabolites of Aspergillus flavus toxic to a detritivorousmaize insect (Carpophilus hemipterus, Nitidulidae).Trans Br Mycol Soc 91:433–438Wicklow DT, Joshi BK, Gamble WR, Gloer JB, Dowd PF(1998) Antifungal metabolites (monorden,monocillin IV, and cerebrosides) from Humicolafuscoatra Traaen NRRL 22980, a mycoparasite ofAspergillus flavus sclerotia. Appl Environ Microbiol64:4482–4484Wicklow DT, Roth S, Deyrup ST, Gloer JB (2005) A protectiveendophyte of maize: Acremonium zeae antibioticsinhibitory to Aspergillus flavus and Fusarium verticillioides.Mycol Res 109:610–618Wiederhold NP, Lewis RE (2003) The echinocandin antifungals:anoverviewofthepharmacology,spectrumandclinical efficacy. Expert Opin Inv Drugs 12(8):1313–1333Wilhelm C, Anke H, Flores Y, Sterner O (2004) New peptaibolsfrom Mycogone cervina. J Nat Prod 67:466–468Willets HJ (1971) The survival of fungal sclerotia underadverse environmental conditions. Biol Rev 46:387–407Willets HJ (1978) Sclerotium formation. In: Smith JE, BerryDR (eds) The Filamentous Fungi vol III. Developmentalmycology. Wiley, New York, pp 197–213Wolpert TJ, Dunkle LD, Ciuffetti LM (2002) Host-selectivetoxins and avirulence determinants: what’s in a name?Annu Rev Phytopathol 40:251–285Wong-Beringer A, Kriengkauykiat J (2003) Systemic antifungaltherapy: new options, new challenges. Pharmacotherapy23:1441–1462ZhangY,LiC,SwensonDC,GloerJB,WicklowDT,DowdPF(2003) Novel antiinsectan oxalicine alkaloids fromtwo undescribed fungicolous Penicillium spp. Org Lett5:773–776

Decomposition, Biomass and Industrial Applications

16 Nutrient Cycling by Saprotrophic Fungi in Terrestrial HabitatsJ. Dighton 1CONTENTSI. Introduction ........................ 287II. Derivation of Nutrients from Rock:Soil Formation ...................... 287III. Leaf Litter Decomposition ............. 289A. PhylloplaneFungalAction........... 290B. Litter DecompositionandNutrientMineralization ......... 291C. NutrientImmobilization............ 293D. Interactions Between FungiandOtherMicrobes................ 295IV. ImpactofPollutantsonNutrientCycling. . 296V. Conclusions ......................... 297References.......................... 297I. IntroductionIn his introduction to Carroll and Wicklow(1992), Rayner (1992) proposes that the potentialrole of fungi in regulating energy and nutrientfluxes through natural ecosystems is likely to beimmense, via their involvement in soil development,decomposition and uptake of nutrientsby plants. This is, in part, due to the structuraladaptations of fungal mycelia that have indeterminategrowth, presenting a large surface area totheir environment for enzyme production andrecovery of end products of enzymatic activity.They have the ability to translocate nutrients andenergy from one location to another through theirhyphal structures (Cairney 2005) and, due theirpersistence, can endure stressful changes in theenvironment, which are less well tolerated by otherorganisms (Zak 1993; Zak et al. 1995). However,fungi do not work alone in the environment andmore research is needed to understand the role ofconsortia of organisms in mediating processes inecosystems. A wider view of the ecosystem servicesthat fungi provide is given by Dighton (2003).1 Rutgers University Pinelands Field Station, P.O. Box 206, 501 FourMile Road, New Lisbon, NJ 08064, USATerrestrial ecosystems present a wide diversityof habitats ranging from polar arctic conditions,through temperate coniferous and deciduousforests and grasslands, heathlands and tropicalforests to deserts. Within one single chapter, it isnotpossibletodescribetheroleoffungiinnutrientcycling in all of these systems; rather, I will providesome general principles that are common to all terrestrialsystems, and that will be modified by thespecific constraints of the system under consideration.A scheme for nutrient cycling in a forestecosystem is given in Fig. 16.1. The saprotrophiccondition of fungi is, possibly, the fundamental lifestrategy where dependence on excreted enzymesis enhanced by the presentation of a large surfacearea of hyphae to the environment, from which endproducts of decomposition are absorbed. However,other fungal groups, particularly necrotrophs andmycorrhizae, although specialized in their function,retain this general modus operandi and canbe considered facultative saprotrophs when conditionsdictate. As such, functional groups otherthan saprotrophs will be included in the followingdiscussion where necessary.II. Derivation of Nutrients from Rock:Soil FormationIt is more common to think of the abiotic forces offreeze-thaw,waterdissolutionandwinderosionasthe primary factors for producing the fine mineralparticles from parent rock to create soil (Brady andWeil 1999). However, the action of plant roots isalso important in both the production of organicacids to dissolve rock and hydrostatic pressure tofracture rock. The same can be said for fungalhyphae, which act either alone or in consortiumwith other organisms to effect similar processes,but at a smaller spatial scale. In particular, fungiin the symbiotic association with bacteria and al-Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

288 J. DightonFig. 16.1. Diagrammatic representation of nutrient cycling in woodland ecosystems and the role of fungi in that process(after Dighton and Boddy 1989)gae, namely lichens, are an important contributorto early soil development. Indeed, Rayner (1992)says that lichens “clothe what might otherwise bebare parts of the planet”.Approximately 8% of terrestrial ecosystems arelichen dominated and, in many of these systems,the ground cover by lichens is often very high, upto 100% (Honegger 1991). Many of these systemsare climatically extreme or oligotrophic (nutrientimpoverished) environments, and it is here thatlichens become important actors in the formationof soils. These fungal tissues, in association withsymbiotic algae and non-photosynthetic bacteria(Banfield et al. 1999), play a major role in soil biogenesis.Hydration of oxides of iron and aluminum is animportant process in rock degradation; for example,hematite (Fe 2 O 3 ) is converted into ferrihydrate(Fe 10 O 15 9H 2 O) (Brady and Weil 1999). Hydrolysisis important in the release of essential nutrients forplant growth. For example, potassium is releasedfrom microcline, a feldspar, where dissolution allowsthe dissociation of anions and cations fromcomplex materials, and gypsum dissolves to releasecalcium and sulfate ions. Crustose lichens are oftenthefirstorganismstocolonizeoutcropsofbarerock.Theyareabletoscavengewaterandnutrientsfrom the atmosphere (Lange et al. 1994) thatsupport their slow rate of growth, and by whichthey are able to tolerate complete desiccation. Duringtheir growth on the surface of rocks and in rockcrevices, the organic acids they produce (oxalic, citric,lichenic and tartaric acids) solubilize the rockand assist in its physical breakdown. This actionof lichens has been reported to cause significantdamage to both buildings and sculptures made ofrock (Chen et al. 2000), by hyphal penetration intothe parent rock beneath the aerial structure of thelichen (Ascaso and Wierzchos 1995).Fungi alone produce organic acids that are capableof breaking down rock. Ascaso and Wierzchos(1995) cite studies by Eckhardt (1985) thatshow that yeasts and filamentous fungi, such asAspergillus niger,aloneareinvolvedinrocksolubilization,releasing cations from amphibolite, biotiteand orthoclase. Penicillium and yeasts were alsofoundtobeabletodissolvecalcium-richrocks,such as limestone, marble and calcium phosphate

Nutrient Cycling by Saprotrophic Fungi in Terrestrial Habitats 289(Chang and Li 1998). Solubilization of fluorapatitehas also been shown in pure cultures of Aspergillusniger (Nahas et al. 1990). Small holes (3–10 μm diameter)in feldspars and hornblende (Jongmanset al. 1997) were attributed to the production ofmicromolar concentrations of organic acids (succinic,citric, oxalic, formate and malate) secretedby saprotrophic and ectomycorrhizal fungi associatedwith the overlying pine forest ecosystem. Thinsections of feldspars observed under the microscopehave revealed fungal hyphae bearing crosswalls in hyphal-generated tunnels in the rock (Hofflandet al. 2002, 2004). In addition, Connolly et al.(1998) showed that the white rot wood decay fungus,Resinicium bicolor, could solubilize strontianitesand to release the strontium contained within.Even in the absence of nutrient-rich substrates,fungi seem capable of obtaining energy and nutrientsfrom minimalist media and scavenging fromthe atmosphere. Such fungi are known as oligotrophs,and are reviewed by Wainwright (2005).In a similar manner to lichens, Hirsch et al.(1995) showed a loose relationship between fungi,bacteria and coccal cells (thought to be algae)that together form an endolithic communityin sandstone and granite that produce organicacids for rock dissolution. Typical saprotrophicfungal species present included Alternaria,Aspergillus, Aureobasidium, Candida, Cladosporium,Paecilomyces, Phoma, Penicillium andSporobolomyces.The saprotrophic capabilities of mycorrhizalfungi have also been shown to play a role inthe dissolution of parent rock material in moreestablished soils. Azcon et al. (1976) showed thatinteractions between bacteria and arbuscularmycorrhizae of lavender facilitated acquisition ofphosphorus by the host plant from rock phosphate,due to a synergism between the bacteriaand mycorrhizal fungi and the differences inbehavior between the two mycorrhizal fungalspecies. Berthelin and Leyval (1982) showed thatin measures of maize growth (biomass), uptake ofpotassium, calcium and magnesium (derived fromthe breakdown of biotite) was similar betweenmycorrhizal fungi and rhizospheric bacterialcommunities,andtherewasnosynergisticeffectof the combination of mycorrhizae and bacteria.This suggests that arbuscular mycorrhizalfungi alone were capable of rock breakdown.Mojallala and Weed (1978) showed that mycorrhizalsoybeans used weathered potassiumfrom the biotites, phlogopite and muscovite, butpotassium release was insufficient to sustain theenhanced growth of the mycorrhizal plants. Plantacquisition of nutrients from insoluble or poorlysoluble sources is also enhanced by consortia ofmycorrhizae, saprotrophic fungi and bacteria.Singh and Kapoor (1998) showed that mungbean plants in association with a consortiumof the arbuscular mycorrhizal fungus Glomusfasciculatum, fungal saprotroph Cladosporiumherbarum and the bacterium Bacillus circulansgrown under natural soil conditions could betterobtain phosphorus from rock phosphate than eachorganism alone. Similar interactions were foundby Vanlauwe et al. (2000) in the savanna zone ofNigeria.April and Keller (1990) and Gobran et al. (1998)demonstrated the importance of rhizospheric microbial(fungal and bacterial) communities in therhizosphere of forest trees for forest biogeochemistry.The abundance of weatherable minerals nearthe root surface was consistently less than in thebulksoil,duetoincreasedhydrogenionandcarbondioxide content and the presence of complexingorganic acids produced by these rhizosphericcommunities. By observing the solubilization oflimestone, marble and calcium phosphate by sevenectomycorrhizal, two saprotrophic fungal generaand Pseudomonas fluorescens, Chang and Li (1998)suggestedthatsynergisticactivitieswereimportantin mediating calcium availability in soils. Other interactionsbetween ectomycorrhizae and nutrientelement solubilization include potassium from biotiteand microcline in Scots pine (Wallander andWickman 1999), rock dissolution and K, Mg and Ca(Landeweert et al. 2001) and NH 4 -N from Mioceneshales and, possibly, other rocks (Thompson et al.2001).To these embryonic soils, organic matter addedby the death of fungi, lichens, bacteria, algae, etc.makes a more ‘complete’ soil in which other soilorganisms can live. Here, the release of nutrientsbydecompositionaswellasenhancedsoilstructuresupport the growth of cryptogamic and vascularplants, which further add their own floristicand chemical diversity to the decomposition cycle,leading to a more complex soil structure.III. Leaf Litter DecompositionThe nutrient component of soil comes partly fromthe dissolution of parent rock, and secondarily

290 J. Dightonfrom the decomposition of dead plant and animalremains. Dead plant parts (above- and belowground)are returned to the soil where the activitiesof bacteria, saprotrophic fungi and soil faunadegrade the complex organic components. Extracellularenzyme activity of heterotrophic microorganisms,and the recovery of resulting solubilizednutrients is rarely 100% efficient, resulting in mineralizedlabile nutrient elements becoming availablefor plant uptake in soil solution (fertility).The activities of fungal extracellular enzymes hasbeen reviewed by Sinsabaugh and Liptak (1997)and Sinsabaugh (2005). The activity of these saprotrophicfungi is neatly summarized by Forsytheand Miyata (1984) . . . “under the silent, relentlesschemical jaws of the fungi, the debris of the forestfloor quickly disappears”.Part of the structure of different soils is relatedto the balance between rates of decomposition andmineralization and the rate of input of dead plantparts to soil. Where decomposition is very slow, organicmatter accumulates and peaty soils develop.In contrast, where plant litter turnover rates arehigh, resulting soils have a more mineral profilewith low organic matter content. Much of theregulation of plant litter decomposition is relatedto the relative availabilities of carbon and nutrientelements within the plant resource. Melillo et al.(1982) showed that the carbon to nitrogen ratioin plant resources and, particularly, the lignin:Nratio could predict the rate of decomposition oflitter, which, in turn, influences the saprotrophicfungal community capable of decomposingthem.The process of decomposition starts in thephylloplane when leaves senesce, and progressivelycontinues in the soil in the litter and organichorizons, where fungi interact with bacteria andsoil fauna (Ponge 1991, 2005).A. Phylloplane Fungal ActionIn wet tropical forests, some 7% of the total expectedleaf litter fall never reaches the forest floor,but is trapped in the canopy of the trees or inthe canopy of the understory shrub community(Hedger et al. 1993). Plant litter trapped in thecanopies is held there by fungal hyphae and, particularly,rhizomorphs formed by species of the generaMarasmius and Marasmiellus, which effect thedecomposition of the plant litter. The balance betweenthe amount of leaves trapped is a function ofleaf weight, tensile strength of the retaining fungalstructures, and weight loss due to decomposition.As the leaves decompose, mineralization will releasenutrients that will wash to the forest floor inthroughfall rain. Thus, the formation of ‘soil’ in thetree canopy is a reality and, probably, has a significantimpact on the fertility of the tropical forestecosystem. Under crop canopies, Beare et al. (1989)showed enhanced microbial activity and litter decompositionof low-resource quality materials inthe presence of throughfall rainfall.The magnitude of effects of rainfall volume andleaching rates from canopy plant parts in wet tropicalforests is much greater than that of other ecosystems.However, even in temperate forest ecosystems,the changes occurring in the stemflow waterchemistryisenoughtoprovideasuitablehabitatfor epiphytic lichen communities. The extensivebiomass of the epiphytic lichen, Ramalina menziesii,(590 kg ha −1 )onblueoak(standingcropleafbiomass of 958 kg ha −1 )hadahigherdepositionoftotal N, organic N, Ca, Mg, Na and Cl in throughfallrain than was the case for trees without lichens(Knops et al. 1996). Trees with lichens had a lowerthroughfall of SO 4 , and the concentrations of NO 3 ,NH 4 , K and total P were not different because lichenlitter reduced the decomposition of oak leaf litter,Table. 16.1. Resource quality and rates of decompositionof contrasting plant litters in relation to ecosystem succession(from left to right, these components become moreimportantcontributorstothedecompositionpool),andthe increased dependency of consortia of soil organisms toeffect their decomposition (after Heal and Dighton 1986)Lower Herbaceous Angiosperm Coniferous Woodplants plants leaves leavesCellulose (%) 16–35 20–37 6–22 20–31 36–63Lignin (%) 7–36 3–30 9–42 20–58 17–35C:N ratio 13–50 29–160 21–71 63–327 294–327Decay (% year −1 ) 20 30–70 40–60 3–50 1–90Faunal Enchytraidae Enchytraidae, Oligochaeta, Acari, Insecta,importance Enchytraidae Oligochaeta, Collembola, Collembola, otherDiptera Acari Oligochaeta Arthropoda

Nutrient Cycling by Saprotrophic Fungi in Terrestrial Habitats 291Table. 16.2. Regression analysis (regression coefficients) ofdecomposition (mass loss) and nitrogen release rate in relationto determinants of leaf litter resource quality (afterVanlauwe et al. 1997). Asterisk indicates a significant regression(a = 0. 05)N mineral-izationLittermass lossC:N ratio 0.74* 0.61Lignin:N ratio 0.68 0.42Polyphenol:N ratio 0.54 0.76*(Lignin+polyphenol):N ratio 0.77* 0.68*such that the release of N and P were reduced by 76and 2%, respectively.B. Litter Decompositionand Nutrient MineralizationTherateatwhicharesourceisdecomposedisdependenton its chemical composition (Heal andDighton 1985; Heal et al. 1997), edaphic factors(available moisture and temperature), and the colonizationof the resource by appropriate saprotrophicorganisms (Table 16.1). Many of these factorsare discussed by Cooke and Rayner (1984). Theinput of different types (chemical composition and,hence, resource quality) of plant litter varies withecosystem type (Dickinson and Pugh 1974; Cadishand Giller 1997). The general consensus is that thecarbon:nitrogen and lignin:nitrogen ratios can beused as determinants of the resistance of resourcesto decomposition, and ultimate mineralization ofnutrients (Melillo et al. 1982). Where the C:N orlignin:N ratios are high, there are reduced ratesofdecompositioncomparedtoresourcescontaininglower ratios. However, other secondary chemicalsproduced by plants, particularly polyphenolsand tannins, also inhibit rates of decomposition ofplant material by soil microorganisms (Harborne1997). Vanlauwe et al. (1997) showed that bothrates of decomposition (mass loss) and nitrogenmineralization rates are strongly correlated to the(lignin+polyphenol):N ratio (Table 16.2).Due to the variability in chemical compositionof plant and animal remains, not all materials canbeutilizedbyallfungalspecies.Fungalspeciesdifferin their access of simple or complex forms ofcarbohydrate and mineral nutrients. Decompositionis a product of enzyme activity, where thetypes of enzymes required are dependent on thesubstrates (chemical constituents) of the resource.Sinsabaugh and Liptak (1997) give a description ofthe various ectoenzymes produced by fungi, andtheir biochemical effects on organic resources inplant litters. The ability of different species of fungito produce specific enzymes dictates, in part, thesuccession of fungi colonizing resources. In additionto enzymatic competency, there are other factors,suchasrelativegrowthrates,theproductionof antibiotic secondary metabolites, and environmentalconstraints, which influence the ability ofspecific fungi to colonize resources in the face ofcompetition against other fungi (Cook and Rayner1984; Frankland 1992, 1998; Lockwood 1992; Wicklow1992). Linkins et al. (1984) discussed some ofthe factors affecting the activity of extracellular cellulase,particularly the positive influence of temperature,and the cellulose to lignin ratio. Celluloseappears to become unavailable for microbial usewhen the cellulose:lignin ratio declines below 0.5.Theroleofplantlitterqualityonthepatternoffungal colonization of resources has been discussedin Dickinson and Pugh (1974). Here, many examplesof the change in species composition of fungalcommunities as different plant substrates undergothe cascade of decay are described. In general, thereappears to be a succession of fungi utilizing differentresources within the litter. The classic assumptionis that the initial colonizers use soluble carbohydratesources (sugars), and are later replacedby fungal species having greater enzymatic competencethat are able to break down organic sources ofcarbon such as cellulose, and lignin. However, thereare few clear distinctions in the succession and, infact, many of the species overlap in time and space.The successional trends of fungi colonizing decomposingplant material have been described in moredetail for the litter of the fern Pteridium aquilinumby Frankland (1992) in her discussion of fungal successions(Frankland 1998). She describes changesfrom lesion-forming Rhizographus and Aureobasidiumon standing dead litter, through the colonizationby basidiomycetes in relation to the rateof loss of cellulose and lignin, and the consequentialdecrease in C:N ratio from some 200:1 to 30:1.By microscopic observation of small samples offorest floor leaf litter, Ponge (1990, 1991) characterizedthe colonization of Pinus sylvestris needlesinto four stages (Table 16.3). Early stages of decompositionare characterized by minor changesto the leaf structure by fungi common in the phylloplane.Subsequent fungal colonization is associatedwith browning of the leaf, and decompositionof relatively available resources. This is fol-

292 J. DightonTable. 16.3. Fungal succession in relation to changes in resources during decomposition of Scots pine needles (after Ponge1991)Stage of Fungal species Litter characteristicsdecompositionsPhase ILophodermium Browning of cellulose walls, cytoplasm missing and replaced by hyphaeand CeuthesopraPhase II Verticillum, Marasmiusand CenococcumMelanized hyphae through stoma. Hyaline hyphae invade xylem tracheidsand resin ducts. Lignocellulose walls disruptedPhase III Cenococcum Invades fecal pellets of mites and enchytraeids as soil fauna, bacteriaand algae invadePhase IV Cenococcumand HyphodontiaPenetration by mycorrhizal fungi. Needles become hollowlowedbygreaterinvasionoftheleaftissuebydecomposingmicro-fungi and basidiomycete fungi,culminating in the entry of soil arthropods, invasionby mycorrhizal fungi, and the establishmentof a greater diversity of decomposer organisms.Within any group of fungi, however, there are considerabledifferences in efficiency of decompositionabilities. In a comparison of larch litter decompositionbetween 15 basidiomycete and 16 ascomycetefungi, Osono et al. (2003) showed litter weight lossof –2.0 to 7.8% by ascomycetes, and −0. 8 to 14.2%by basidiomycetes. Thus, the generalization thatbasidiomycetes have greater enzyme diversity isnot always correct, although the results suggesteda stronger relationship to N mineralization due tolignin decomposition by basidiomycetes, in contrastwith ascomycetes. A review of fungal successionduring decomposition can be found in Ponge(2005).These observations help to validate the modelproposed by Swift et al. (1979) where changesfrom ‘sugar’ fungi to basidiomycetes in relation tochangesinavailableresourcesandtheinfluenceof climatic stresses are presented. The modelsuggests that during initial decomposition, thecarbohydrate component is used as an energysource until such a time that the C:nutrient ratioapproaches that of the decomposer organism(around 15:1 for P, and 6:1 for N in fungi). Onlythen is there net conversion of organic nutrient toinorganic nutrient – net nutrient mineralization.In general, initial resource structure is chemicallyheterogeneous, thus supporting a variety of fungalspecies. As decomposition proceeds, only recalcitrantchemicals are left, requiring a specializedsubset of fungal species that are capable of producingthe enzymes necessary to degrade the complexresources. Hence, diversity is reduced. However,the colonization of decomposing plant materialin relation to resource quality has been presentedonly in reference to the chemical composition ofthe whole leaf. As fungal hyphae are of a small diameter(∼5 μm), their patterns of growth, enzymeexpression and the subsequent changes in leaflitter chemistry occur at a scale of resolution muchsmaller than that of a whole leaf. In recent studies(Mascarenhas et al. 2000; Dighton et al. 2001) usingmicroscopic Fourier transform infrared (FT-IR)spectroscopy, real-time micro-scale (100×100 μm)changes in leaf surface carbohydrate chemistryresulting from fungal colonization are beginningto be revealed at the level of individual hyphae.At the ecosystem level, plant tissue chemistryentering the decomposer pool changes during seralsuccession of vegetation from herbaceous to forestecosystems (Heal and Dighton 1985). The initialseral stages are marked by an addition of highqualityresources to the decomposer community,consisting mainly of cellulose and a high C:N ratioand low lignin content. Following forest canopyclosure, woody resources and more recalcitrant leaflitters dominate (Attiwill and Adams 1993). Theselitters have high lignin content and low C:N ratiosand, therefore, decompose at a slower rate. Inaddition to changes in the dominance of fungalspecies or group with ecosystem succession, thedegree of interaction between fungi and animalsincreases. There are increasingly intimate associationsbetween fungi and fauna in the exploitationof more recalcitrant plant residues (Table 16.1).In forested systems, much dead wood remainsin the canopy prior to recruitment to the forestfloor. This standing dead material may have a differentfungal community than wood on the forestfloor. The work of Boddy and Rayner (1983) on oakwood in canopies showed that 12 basidiomycetefungal species dominated in the community.Of these, Phellinus ferreus, Sterium gausapatum

Nutrient Cycling by Saprotrophic Fungi in Terrestrial Habitats 293and Vuilleminia comendens were pioneer speciesof partially living branches, Phlebia adiata andCoriolus versicolor were secondary colonizers,and Hyphoderma setigerum and Sterium hirsutumrelatedtoinsectactivity.Inwood,theinteractionsbetween fungi can be most clearly observed. Thezones of interaction between adjacent, competingfungal colonies have been mapped in three dimensionsusing wood as a resource (Rayner 1978;Rayner and Boddy 1988). Clear demarcation zonesare set up when genetically incompatible strains orspecies meet in a relatively homogenous resource.In an environment where resources are patchilydistributed, such as mixed litter on the forest floor,the colonization of individual resource units ismore difficult to map. The colonization patternof individual straw resource units by a range offungal species was correlated to relative growthrates of the fungi on agar (Robinson et al. 1993a).These rates of growth allowed four species to beranked in combative order. Mixtures of fungalsignificantly reduced growth rate of less combativefungal species, where enhanced respiration ofmixed species communities (Robinson et al. 1993b)resulted from competition, rather than substrateutilization. Thus, the cascade of decompositionisrelatedtocolonizationofasubstratebyfungibased on their enzymatic competence in relationto the chemical resources available, and also onthe outcome of interaction with other potentialcolonizers of that resource.Sinsabaugh et al. (1993) demonstrated thatmost extracellular enzymes involved in wooddecomposition are derived from fungal activity.Using standardized wood as a resource, theyshowed that the production of lignocellulaseenzyme did not differ between different locationsin a temperate forest ecosystem. However, therate of immobilization (mainly fungal) of totalnitrogen and total phosphorus into decomposingwood ranged from 2.2–4.4 μgg −1 wood for P,and 43–139 μgg −1 for N at the time when 80%mass loss was achieved. The spatial variabilityof this parameter was much greater than thatfor lignocellulase, but much less than for acidphosphatase and N-acetylglucosaminase activity.The process of decomposition is governed by theproduction of enzymes, which are, in turn, regulatedby the availability of nitrogen or phosphorus.Thus, where nutrient elements are less available,the fungi expend greater amounts of energy toproduce enzymes to sequester the nutrients fromorganic sources. These results suggest a largedegree of edaphic (soil condition) control overenzyme expression, which is closely related tothe availability of inorganic N and P suppliesin soil water. Thus, Sinsabaugh et al. (1993) andSinsabaugh (2005) developed a model that containsboth fungal (microbial) and soil nutrient controlsover the expression of enzymes. Particularly,Sinsabaugh (2005) describes a successional loopthrough which the community composition ofsaprotrophic fungi and their enzyme expressionsare tied to resource quality in organic matterdecomposition. Use of models like this can helpus to better understand the complexities ofdecomposition and nutrient cycling processesby allowing hypothesis development, leading tothe design of experiments that can logically altersingle or multiple parameters to investigate thekey processes and organisms that are responsiblefor driving ecosystem processes.Lodge and Asbury (1988) demonstrated thatthe ability of fungal hyphae and cords to bind leaflitter together on the forest floor is important inpreventing down-slope loss of leaf litter in tropicalforest ecosystems. The potential loss of organicmatter, containing nutrients for plant growth, isprevented by the action of a number of, mainly, basidiomycetefungi that bind the leaf litter together.Species of Collybia, Marasmiellus, Marasmius andMycena are the main fungi involved in forest floorlitter trapping. The effect of litter binding by fungiincreases with increasing ground slope. Lodge andAsbury (1988) concluded, from field manipulationexperiments, that loss of litter was reduced by 35%from shallow slopes (

294 J. Dightoning simple carbohydrates, have lower investment inbiomass than longer-lived basidiomycetes, growingon woody resources; by implication, the potentialaccumulation in basidiomycetes is greater (Franklandet al. 1982).Where the C:nutrient ratio of a resource is veryhigh, as in wood, the Swift et al. (1979) model proposesinitial fungal immobilization and import offree nutrient during initial stages of decompositionuntil the fungal resource C:nutrient contentis equivalent to that of the fungus. Fungal immobilizationof nutrients can be considerable and isof economic concern in ‘nitrogen lock-up’ in postharvestforest residues. To alleviate this competition,there has been the establishment of burningprotocols to rid the site of both woody debris and,incidentally, leaf litter and the nutrients they contain(Dighton 1995). A greater understanding of theinteractions between nutrient availability, temporarynutrient immobilization, and alternative applicationsfor post-harvest residues could lead toa more rational use of residues to provide sustainableforestry without the loss of nutrients from theecosystem from burning, and without the need forexogenous nutrients in the form of fertilizers (Joneset al. 1999). For example, Stark (1972) showed thathyphae had 193 to 272% greater N content, and 104to 223% greater P content than the pine needle litteron which they were found, suggesting immobilizationof these elements into fungal biomass. Datafrom Lodge (1993) show similar, but lower rates offungal accumulation of nutrients in tropical forestecosystems (N 1.6%, P 22%, K 3.7% and Ca 2%)from leaf litter. Fogel and Hunt (1983) estimatedthat saprotrophic fungal biomass accounted for 2%of the standing stock of a temperate Douglas-firforest ecosystem, and Clinton et al. (1999) showedsignificant nutrient accumulation in fungal fruitbodiesof a Nothofagus forest from both forest floorand dead woody material (Table 16.4).Table. 16.4. Accumulation of nutrients into fungal fruitbodiesin a Nothofagus forest from either forest floor ordead woody material, expressed as a percentage of resourcenutrient calculated as mg kg −1 (after Clinton et al. 1999)Nutrient % from forest floor % from dead woodN 402 2066P 400 2500K 628 19,000Mg 800 400Ca 7 89Marumoto et al. (1982) suggested a longerturnover time for fungi than bacteria in their experimentaldecomposition study of killed bacteria,fungi, and combined bacteria and fungal cells.Using 14 Cand 15 N labeling techniques for each ofthe cells, they showed that the rate of carbon loss,as CO 2 , was similar between microbe sources, butthat the rate of mineralization of nitrogen as bothNH 4 and NO 3 -N was slower in the decompositionof fungal cells. In tropical ecosystems, fungalbiomass can attain values of 5–15 mg g −1 litter(Lodge 1993), from which nutrients can be slowlyreleased upon death and subsequent decomposition.Here, fungi may be important retainers ofnutrients that would otherwise readily leach fromthe soil due to high precipitation volumes. Due tothehighnutrientcontent,decompositionoffungalfruitbodies may be faster than that of surroundingforest floor material, providing localized spots ofhigh rates of mineral nutrient release. However,the duration of immobilization of those nutrientsinto fungal hyphae before translocation to thefruitbody could be an important aspect of the controlfungi have on the rates and timing of release ofpulses of nutrients within the forest ecosystem. Intemperate zones, the spring and fall abundance offruitbody production may coincide with the highroot growth and high nutrient demand by treesduring these periods, a correlation that has yet tomade by observation and measurement.Fungi as non-discrete organisms are able totranslocate elements within the fungal thallus(Cairney 1992), allowing for the spatial redistributionof elements. For example, if an elementwere always translocated away from dying regions,translocation would increase the length of time ofimmobilization into fungal components. Olssonand Jennings (1991) demonstrated that translocationof 14 Cand3 2 PthroughhyphalsystemsofRhizopus, Trichoderma and Stemphylium occurredby diffusion. Real-time rates of translocationof carbon within the fungal thallus have beenshown to react to provide directional flow to thebuilding phases of the hyphae (Olsson 1995). Thisacropetal translocation is through cytoplasmicflow and diffusion in the cytoplasm and apoplasm.In contrast to this diffusion model of C and Ptranslocation, Gray et al. (1995) demonstratedthat translocation of 137 Cs through hyphae ofSchizophyllum commune was slower than diffusion,suggesting incorporation of the elementinto structural components of the cytoplasm orhyphal wall. This presents a plausible mechanism

Nutrient Cycling by Saprotrophic Fungi in Terrestrial Habitats 295Table. 16.5. Velocities of translocation of elements within saprotrophic basidiomycete mycelia (after Cairney 2005)Fungus Element/material Velocity (cm h −1 )Armillaria mellea Glucose ( 14 C) 3.2A. mellea Glucose ( 3 H) 1.2A. mellea P 2.5Armillaria spp. Cs 14.2Armillaria spp. Cs 17.9A. gallica Cs 0.6A. ostoyae Cs 0.8Serpula lacrimans Glucose ( 14 C) 24.8Phanerochaete velutina Glucose ( 14 C) 336P. velutina Aminoisobutyric acid ( 14 C) 0.05Schizophyssum commune Cs 0.0002S. commune Aminoisobutyric acid ( 14 C/ 32 P) 0.0018Pleurotus ostreatus Aminoisobutyric acid ( 14 C/ 32 P) 0.0018for long-term accumulation of radiocesium inbasidiomycete fungi (Dighton and Horrill 1988;Yoshida and Muramatsu 1994).Solute translocation through wood-colonizingbasidiomycetes is important in facilitating colonizationof low-resource quality substrates. Wellsand Boddy (1990) showed that 75% (Phanerochaetevelutina) and 13% (Phallus impudicus)of the phosphorus added to a decomposed woodresource is translocated to newly colonized woodresources through mycelial cord systems frompreviously colonized and partially decomposedwood. The maximum rate of P translocation is7225 nmol P cm −2 day −1 through cords. Thesecords are formed only in unsterile soil, suggestingthe trigger for cord formation is derived fromother organisms. In field experimental manipulations,Wells and Boddy (1995a) showed thattranslocation could be conducted over distancesof up to 75 cm between decomposing resourceson the forest floor. Translocation of phosphorusin mycelial cords is temperature dependent, withgreater rates of movement at higher temperatures(Wells and Boddy 1995b). A change from wetto dry soil conditions induces a thickening ofthe cord system of Phanerochaete velutina anda reduction in the translocation of phosphorusto a new wood resource; wetting appears to haveno effect on cord structure or P movement (Wellset al. 2001).Velocity of translocation of materialwithinbasidiomycetemyceliacanbehigh(Cairney2005), although few studies have looked at nutrientelements other than phosphorus or cesium(Table 16.5). Changes in the source–sink relationshipswithin the mycelium, and the degree ofimportance of translocation in non-basidiomycetefungiinrelationtonutrientcyclingareasyetrelatively unexplored.D. Interactions Between Fungiand Other MicrobesIt is important to remember at the outset that saprotrophicfungi involved with decomposition and nutrientcycling in soil do not perform that function inisolation. Plant and animal remains may be comminutedby soil fauna and subjected to enzymeattack by bacteria and actinomycetes. Interactionsbetween these organisms are important in determiningthe rate of decomposition and diversity ofsoil biota. The decomposition process is also dynamic– for example, the same suite of organismsis not present on the plant or animal remains (resource)forthedurationoftheprocessofdecomposition.Itwillbeseenthatdifferentfungihavedifferentenzymatic capabilities, so their appearanceon a resource will be dictated by (i) their ability toutilize the resource, (ii) their rate of arrival at theresource either by growth or by transport as spores,etc., and (iii) their ability to compete against otherfungal species with similar physiological competence.The interactions between fungi and bacteriain the decomposition of leaf litter may also notalways be synergistic. In an incubation study ofbeech leaves, Møller et al. (1999) showed that thecellulolytic fungus Humicola sp. caused double thecarbonutilizationfromleavesthanincombinationwith a mixed inoculum of soil bacteria. However,mixed species fungal inoculum significantly increasedβ-N-acetylglucosaminidase and endo-exo-

296 J. Dightoncellulaseactivitycomparedtothepresenceofbacteriaalone, but the presence of bacteria decreasedtheseenzymeactivitiesinthepresenceofHumicolarelative to Humicola alone. The quality of resultantdissolved organic carbon from decomposition alsodiffered between fungal and fungal/bacterial decompositionof the leaf material.As complexity in the system increases, the influenceof trophic interactions alters the effectivenessof fungi to effect decomposition, and the subsequentmobility of nutrients. Nieminen and Setälä(2001) constructed microcosms of increasing complexityby sequentially adding fungi, bacteria andbacteria-feeding nematodes. Fungi alone had lowerdecomposition efficiency (higher respiration perunit biomass) when alone in the system, and effectedlower C loss due to a smaller total biomassin the system, compared to more complex associations.Thus, generalizations of the role of fungi innutrient cycling need to be modified when placedin a ‘real world’ context of fungal interactions withother soil biota. Further additive trophic interactionstudies of this kind need to be done in orderto better understand individual functional groupsand their interactions.IV. Impact of Pollutantson Nutrient CyclingChanges in the environment caused by greenhousegases and pollutants have been shown to directlyand indirectly influence fungal communities andtheir activity in the decomposition process. Newshamet al. (1992a, b) and Boddy et al. (1996)showed that, although there was change in the fungalcommunity on tree leaf litter exposed to sulfurdioxide, the leaf occupancy by fungi was notaltered. These changes in fungal species had littleeffectonrateofdecompositionoftheleaflitter,probablyduetotherapidleachingofsulfurfrom the system. In contrast, Dursun et al. (1996a,b) showed reduced hyphal extension and activity(measured as respiration) of fungi in leaf litter exposedto sulfur dioxide.Enhanced carbon dioxide in the atmospherealters plant chemistry. For example, Norby et al.(2001) show that lignin concentrations of leaf litterincrease, whilst N concentrations decline. Thesechangesinleaflitterchemistryinfluencetherateof litter decomposition, nutrient mineralizationand affect the fungal communities that colonizethem (Treseder 2005). The effect of UV-B light(as enhanced by ozone depletion in the atmosphere)significantly reduced spore germinationandhyphalgrowthofsomephylloplane-andleaflitter-inhabiting fungi (Aspergillus fumigatus, Penicilliumhordei, P. janczewskii, P. purpurogenum)but not others (Verticillium sp., Mucor heimalis,Cladosporium cladosporioides, Leptosphaeriaconiothyrum, Trichoderma viride, Ulocladiumconsortiale and Marasmius androsaceus) (Moodyet al. 1999). Changes in the species compositioncould affect the decomposition of leaf litter andsubsequent nutrient mineralization.Information on the role of fungi in nutrientcycling may also be gained by the interactionsof fungi and heavy metals and radionuclides.Although these chemicals do not form the majornutrient elements that are essential for growth,their behavior may indicate the pathway of otherelements within fungal-driven processes. Much ofthe interaction between fungi and heavy metalshas recently been summarized by Fomina et al.(2005). The presence of heavy metals has beenshown to alter growth patterns of foraging hyphae,which may influence their effectiveness indecomposition because hyphal aggregation shiftsbetween assimilative and non-assimilative statesas hyphal growth crosses ‘hostile’ chemical regions(Ritz 1995; Rayner et al. 1995). By the excretion offungal acids and enzymes, fungi can change boththe valency states and mobility of heavy metals,altering their toxicity to both fungi and otherorganisms in the decomposer food web. Ionizingradiation from radionuclides has been shown toaffect the community composition of soil microfungi(Zhdanova et al. 1995, 2005) where tolerantspecies are capable of decomposing ‘hot particles’containing radionuclides (Zhdanova et al. 1991),possibly by sensing the ionizing radiation anddirecting their growth toward it (Zhdanova et al.2004). The fate of the radionuclides released bysuch action are unknown, but it is possible, basedon information from radiocesium accumulationin saprotrophic fungi (Dighton et al. 1991) andmycorrhizal fungi (Oolbekkink and Kuyper 1989;Haselwandter and Berreck 1994; Guillette et al.1994), that long-term accumulation in fungaltissues may result (Dighton and Horrill 1988).It is interesting to note that the role of melaninhas been implicated in both the accumulationandsensingofthepresenceofheavymetalsandradionuclides (Zhadnova et al. 2004; Fomina et al.2005).

Nutrient Cycling by Saprotrophic Fungi in Terrestrial Habitats 297V. ConclusionsAmong the ecosystem services that fungi provide(Dighton 2003), plant litter decomposition andmineral nutrient cycling are among the mostprominent processes regulated by saprotrophicfungi. Despite many years of research into this roleof fungi, many of the subtle interactions with otherorganisms in the ecosystem have yet to be elucidated.Much of the role of these fungi is modified byenvironmental pollution and other environmentalchanges (global warming, etc.). Traditional techniquesfor measuring nutrient cycling as mediatedbyfungiwillonlytellussomuch.Emergingtechnologiesare poised to allow us to investigate newavenues of nutrient cycling. Among these is the useof natural abundance and labeling with radionuclidesto follow the fate of nutrients and carbon inecosystems, providing a new way of investigatingfluxes in situ. Significant strides forward have beenmade in fungal ecology (see review by Hobbie2005), in which isotopic discrimination betweensaprotrophic and mycorrhizal fungi can showwhere these two functional groups obtain bothcarbon and mineral nutrients. For example, saprotrophicfungi are several parts per mille more enrichedin 14 C compared to ectomycorrhizal fungi,and wood-decomposing fungi have a higher δ 15 Nsignature than leaf-litter decomposers (Hobbie2005). Together with greater understanding of thecommunities of fungi involved in nutrient cyclingand the physiology of the individual members ofthe community, these techniques are likely to leadto significant improvements in our understandingof the role of fungi in providing ecosystem services.Traditional concepts of nutrient cycling rely onanunderstandingofleaflitterandwooddecomposition.If we consider the greater dynamics ofecosystems, we can see that other, more transientcomponents of the ecosystem may play an equallyimportant role in driving nutrient cycling and impactingfungal communities. Writing the final partof this chapter during a gypsy moth outbreak in ourlocal forest leads me to contemplate the followingas potentially fruitful areas of future study:1. What are the consequences of episodic litter inputsto the decomposer community (insectfrass, plant parts other than leaves that provideshort-term pulses of input, e.g., bracts,flowers)?2. How do short-term pulses of interruption toecosystem stability (fire, rainfall, drought) influencethe rates of decomposition, fungal communities,and nutrient cycling?3. How do food web dynamics change during suchtimesasmentionedaboveinrelationtofungalmediation of nutrient cycling?4. How important are fungal mycelia and fruitbodiesin the temporary and long-term storageand release patterns of nutrients within diversenatural and agro-ecosystems?These are but a few of the questions that still needto be addressed to complete our understanding ofhow we may best manage both natural and managedecosystems to maintain diversity, effect conservation,and optimize our use of ecosystems.ReferencesApril R, Keller D (1990) Mineralogy of the rhizosphere inforest soils of the eastern United States. Biogeochemistry9:1–18Ascaso C, Wierzchos J (1995) Study of the biodeteriorationzone between the lichen thallus and the substrate.Cryptogamic Bot 5:270–281Attiwill PM, Adams MA (1993) Nutrient cycling in forests.New Phytol 124:561–582Azcon R, Barea JM, Hayman DS (1976) Utilization of rockphosphate in alkaline soils by plants inoculated withmycorrhizal fungi and phosphate solubilizing bacteria.Soil Biol Biochem 8:135–138Banfield JF, Barker WW, Welch SA, Taunton A (1999) Biologicalimpact of mineral dissolution: application of thelichen model to understanding mineral weathering inthe rhizosphere. Proc Natl Acad Sci USA 96:3404–3411Beare MH, Blair JM, Parmelee RW (1989) Resource qualityand trophic responses to simulated throughfall: effectson decomposition and nutrient flux in a no-tillageagroecosystem. Soil Biol Biochem 21:1027–1036Berthelin J, Leyval C (1982) Ability of symbiotic and nonsymbioticrhizospheric microflora of maize (Zea mays)to weather micas and to promote plant growth andplant nutrition. Plant Soil 68:369–377Boddy L, Rayner ADM (1983) Ecological roles of basidiomycetesforming decay communities in attachedoak branches. New Phytol 93:177–188BoddyL,FranklandJC,DursunS,NewshamK,InesonP(1996) Effects of dry-deposited SO 2 and sulphiteon saprotrophic fungi and decomposition of treeleaf litter. In: Frankland JC, Magan N, Gadd GM(eds) Fungi and environmental change. CambridgeUniversity Press, Cambridge, pp 70–89Brady NC, Weil RR (1999) The nature and properties ofsoils. Prentice Hall, Upper Saddle River, NJCadish G, Giller KE (1997) Driven by nature: plant litterquality and decomposition. CABI, Wallingford

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17 Fungal Decomposers of Plant Litter in Aquatic EcosystemsM.O. Gessner 1 ,V.Gulis 2 ,K.A.Kuehn 3 ,E.Chauvet 4 ,K.Suberkropp 2CONTENTSI. Introduction ........................ 301II. Fungal Decomposers in Saltand Freshwater Marshes ............... 303A. MarshesasFungalHabitat........... 303B. Fungal DiversityonEmergentWetlandPlants ......... 303C. Fungal Biomass and Production . . . . . . 304D. EnzymaticCapabilities ............. 307E. Respiratory Activitieson Standing-Dead Wetland Plants . . . . . 308III. Fungal Decomposers in Streams ......... 309A. StreamsasFungalHabitat........... 309B. Fungal Diversity on Decomposing Litter 309C. Fungal Biomass and Production . . . . . . 311D. Responses of Fungal DecomposerstoDissolvedNutrients.............. 313E. EnzymaticCapabilities ............. 314F. Significance of Fungal DiversityforLeafDecomposition............. 315IV. Importance of Fungal Decomposersin Aquatic Ecosystems ................ 315A. Fungal Biomass and ProductionattheEcosystemScale.............. 315B. Fungal vs. Bacterial BiomassandProduction ................... 316C. DecompositionBudgets............. 317V. Conclusions ......................... 318References.......................... 319I. IntroductionFungi are ubiquitous in the biosphere – from glacialforefields in high mountain to the deep sea (Jumpponen2003; Gessner and Robinson 2003; Damare1 Department of Aquatic Ecology, Eawag: Swiss Federal Instituteof Aquatic Science and Technology, and Institute of IntegrativeBiology (IBZ), ETH Zurich, Überlandstrasse 133, 8600 Dübendorf,Switzerland2 Department of Biological Sciences, University of Alabama,Tuscaloosa, AL 35487-0206, USA3 Department of Biological Sciences, The University of SouthernMississippi, Hattiesburg, MS 39406-0001, USA4 Laboratoire d’écologie fonctionnelle - EcoLab, UMR 5245 CNRS,University of Toulouse 3, National Polytechnic Institute of Toulouse,29 rue Jeanne Marvig, 31055 Toulouse Cedex, Franceet al. 2006) and from hot springs to the polar icecaps (Jones et al. 2000; Gunde-Cimerman et al.2003; Magan, this volume). One of their basic functionsin natural ecosystems is the decompositionof plant matter, such as leaves, wood and fruits(Harley 1971). The view that fungi are instrumentalin plant litter decomposition has been a longstandingparadigm in terrestrial ecology (Dighton,this volume) but, for aquatic ecosystems, this notionhas not gained wide acceptance. Nevertheless,there is now strong evidence that fungi are criticallyimportant, if not the predominant, decomposers ofplant litter in marine and freshwater ecosystems(Newell 1993; Newell and Porter 2000; Gessner andVan Ryckegem 2003; Bärlocher 2005; Gulis et al.2006b).Fungal decomposers in aquatic ecosystems areparticularly prominent at the interfaces betweenlandandwater,wheredensegrowthofhigherplants typically results in abundant litter supplywhen plants or plant parts senesce and die.These environments comprise coastal marineareas, inland wetlands such as mires (peatlands),freshwater swamps and marshes, including littoralzones of lakes and rivers, and forest ponds andstreams, which receive plant litter from adjacentriparian vegetation. Plant matter decomposingin these environments may be constantly submerged,periodically or occasionally flooded,or permanently exposed to air as in the case ofstanding-dead shoots of emergent plants in saltand freshwater marshes. Since different typesof plant matter may vary greatly in chemicalcomposition, physical structure, particle size, andtiming when they become available to microbialcolonization, a range of opportunities exist forfungal development within these habitats. Incontrast to terrestrial environments, the continualavailability of water and, in many cases, theabundant supply of dissolved nutrients oftencreate conditions that are particularly favourablefor fungal growth and activity.Environmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

302 M.O. Gessner et al.Todemonstratethatfungiactasimportantdecomposersin a given environment, several conditionsshould be met:1. Fungi must be present in the natural system,where they may be detected by directobservation or indirect methods involvingchemical indicator molecules (Newell 1992;Gessner and Newell 2002; Tsui and Hyde 2003;Foster et al. 2004; Graça et al. 2005). Culturingtechniques also may be useful, althoughthey carry a greater danger of introducingbias.2. Identified species must be able to grow andreproduce under conditions prevailing in thenatural habitat, i.e. the available substrate andunder the environmental conditions defined byabiotic factors such as humidity, salinity, oxygenconcentration, and temperature.3. They should elaborate the enzymes necessaryto degrade plant tissues and to produce themin amounts sufficient to cause significant litterdegradation.4. These activities should result in mass loss oforganic matter or, when species are growingonly in mixed assemblages, in an accelerationof mass loss.5. Finally, the fungi should be successful in competingwith other organisms present in the systemand thus either rapidly colonize a resourceandgrowatacompetitiverateorbeabletooustestablished species.Ultimate proof of fungal participation in decompositionconsists in demonstrating fungus-specificdegradative activity. This may be indicated whenactivities of a fungus grown on litter in microcosms(Hicks and Newell 1984; Suberkropp 1991; Tretonet al. 2004) are similar to the activities observed insitu. Careful application of antibiotics and fungalinhibitors (Padgett 1993), coupled with the kindof measurements given above, may also be useful,although this approach is loaded with potential pitfalls(Oremland and Capone 1988). More powerfuland currently promising methods appear to be thequantification of mRNA and/or enzymes, both ofwhich are likely to provide important insights inthe future as transcriptomics and proteomics areeventually applied to microbial assemblages associatedwith decomposing plant litter.Decomposition of plant remains involvesa range of biotic and abiotic transformationsthat result in the formation of carbon dioxideand other mineral substances, dissolved organicmatter (DOM), and fine particulate organic matter(FPOM), but also in the biomass production ofmicrobial decomposers, such as fungi (Gessneret al. 1999). The rates of all these processes aregoverned by the response of decomposers toenvironmental conditions (external factors) andthe intrinsic quality of litter (internal factors), andthey are modulated by biotic interactions withinand between different groups of decomposersand with other components of aquatic food webs.Outcomes can be divided into those affecting thedecomposition process (Fig. 17.1, bottom left)and those affecting fungal performance (Fig. 17.1,bottom right). Outcomes pertaining to fungalperformance relate to life-history patterns, oftenapparent as shifts in fungal community structure,mycelial growth, and allocation of resources toreproduction. Outcomes relevant at the ecosystemlevel include litter mass loss, nutrient dynamicsincluding immobilization and mineralization, andgeneration of litter transformation products suchas FPOM and DOM. Thus, plant litter decompositionmay be viewed from an ecosystem processperspective or from a decomposer point of view(Fig. 17.1).Fig. 17.1. Schematic representation of a fungus-dominatedlitterdecompositionsystemasviewedfromaprocess(lowerleft cases) and a fungal (lower right cases) perspective

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 303This chapter will address both perspectives.Emphasis will be placed on two aquatic ecosystemsin which fungal decomposers and plant litterdecomposition have been studied to the greatestextent: standing-dead shoots of emergent vascularplants in salt and freshwater marshes (Sect. II.), andleaf litter that falls into forest streams from adjacentriparian vegetation (Sect. III). In addition, theimportance of fungi as producers of biomass willbe addressed at the ecosystem level; a comparisonwill be made with the biomass and production ofbacteria, the other potentially important microorganismsassociated with decomposing plant material;moreover, the role of fungi in decompositionbudgets will be evaluated (Sect. IV.).II. Fungal Decomposers in Saltand Freshwater MarshesA. MarshesasFungalHabitatSalt and freshwater marshes are among the mostproductive ecosystems on the planet (Mitsch andGosselink 2000). Emergent vascular plants, such asthesmoothcord-grassSpartina alterniflora Loisel,the reed Phragmites australis (Cav.) Trin. ex Steud.and cattails (Typha spp.), are conspicuous featuresin these wetlands and often account for the greatestfraction of the total annual plant biomass produced(Dai and Wiegert 1996; Wetzel and Howe 1999).Since herbivore consumption is typically low (

304 M.O. Gessner et al.A particularly marked shift in sporulating fungaltaxa appears to coincide with the transition ofdecay of standing-dead shoots to decompositionof fallen or collapsed plant parts on the sediment(Van Ryckegem et al. 2007).In addition to temporal shifts in communitystructure, fungi may exhibit distinct spatial distributionpatterns on standing-dead shoots (Pughand Mulder 1971; Apinis and Taligoola 1974; Poonand Hyde 1998; Van Ryckegem and Verbeken 2005).Different taxa may occupy specific plant parts, suchas leaf blades, leaf sheaths, or the nodes and internodesof culms. In addition, fungal taxa associatedwith P. australis in tidal marshes showeddistinct vertical distribution patterns in the plantcanopy, which appeared to be a primary factordetermining fungal community composition (VanRyckegem and Verbeken 2005; Van Ryckegem et al.2007). These small-scale distribution patterns onshoots may be a result of small-scale spatial variationin environmental conditions and/or differencesin the resource quality of plant litter, such asvarying amounts of recalcitrant compounds withindifferent plant tissues.In salt marshes, observations of fungal reproductivestructuresassociatedwithstanding-deadS. alterniflora revealed that ascomycete species ofPhaeosphaeria, Mycosphaerella and Buergenerulaare typically the most frequently encountered(Gessner 1977; Newell 1993, 2001a; Newell et al.2000). Studies using molecular methods todescribe fungal communities on decomposingwetland plants, such as ITS rDNA sequencing andterminal restriction fragment length polymorphism(T-RFLP), largely concur with observationsbased on traditional microscopic techniques(Buchan et al. 2002, 2003; Lyons et al. 2005;Torzilli et al. 2006). In particular, on leaf blades ofstanding-dead Spartina shoots in a South-EasternU.S. salt marsh, analysis of ascospore expulsionrates, ITS clone libraries and T-RFLPs provideda similar picture of fungal community composition,with P. spartinicola, Mycosphaerella sp. andP. halima being the dominant taxa encountered(Buchan et al. 2002). Overall, these data suggestthat fungal communities associated with Spartinaare not particularly complex, with a single speciesaccounting for most of the fungal biomass (Newellet al. 1989) and reproductive output (Newelland Wasowski 1995; Newell 2001a) in somesalt marshes. This degree of species dominanceappears to be in contrast with the often muchmore diverse fungal communities associated withstanding-dead plants in freshwater marshes (seeabove), although strong dominance has also beenfound in freshwater marshes (Neubert et al. 2006).Depending on characteristics of the habitat (i.e.degree and regularity of inundation by tides), fungion standing-dead shoots in salt marshes may showvertical distribution patterns. Typical terrestrialfungi have often been observed on upper portionsof standing-dead S. alterniflora shoots not exposedto tidal inundation (Gessner 1977), whereas marinetaxa were most commonly observed on lowerportions of plant shoots that were regularly submergedby tides (Gessner 1977). Similar observationshave been reported for fungal communitieson both standing-dead and collapsed plant partsof P. australis in a brackish tidal marsh (Poon andHyde 1998; Van Ryckegem and Verbeken 2005; VanRyckegem et al. 2007). Distinct fungal communitiessporulated in different microhabitats (e.g. middleor basal canopy of standing-dead shoots), withgreater numbers of terrestrial species associatedwith upper shoot portions. Flooding height and frequencyinfluenced the vertical species distribution,presumably in response to not only water availabilitybut also salinity.C. Fungal Biomass and ProductionIn addition to the wealth of qualitative evidenceshowing pervasive fungal colonization of emergentwetland plants, the productivity and functional roleof fungi has been assessed in several systems (e.g.Newell and Porter 2000; Gulis et al. 2006b). Historically,the lack of suitable methods to quantifyfungal biomass and rates of biomass productionhas been a major impediment to obtaining suchdata. A particular problem has been the intimateassociation of fungi with decomposing plant tissue(Newell 1992) – hyphae penetrate the planttissue, rather than adhere to its surface (Fig. 17.2;Newell et al. 1996b). Consequently, earlier estimatesof fungal biomass based on measurements of hyphallength (Table 17.1), in particular after clearingof leaves, are likely to be severe underestimates(Newell 1992; Gessner and Newell 2002). However,these methodological problems have been largelyovercome by the use of indicator molecules suchas ATP, chitin, certain fatty acids in phospholipidsand, particularly, the membrane lipid, ergosterol,which is likely to provide the most accurate estimates(Newell 1992; Gessner and Newell 2002).ATP is not specific for fungi but can be used as a re-

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 305Table. 17.1. Some estimates of fungal biomass associated with decomposing plant litter in streams aFungal No. of streams Litter type b Method Referencebiomass(mg g −1detrital mass)0.12 1 LB(1) Biovolume c Iversen (1973)8–49 1 LB(3) Biovolume d Findlay and Arsuffi (1989)23 1 LB(1) ATP Findlay and Arsuffi (1989)15–111 8 d LB(1) ATP Suberkropp and Chauvet (1995)47–83 2 LB(2) ATP Suberkropp et al. (1993)127–158 2 LB(2) Ergosterol Suberkropp et al. (1993)61–155 1 e LB(7) Ergosterol Suberkropp et al. (1993),Gessner and Chauvet (1994)78–226 4 LB(1) Ergosterol Methvin and Suberkropp (2003),Carter and Suberkropp (2004)54–73 4 RCL Ergosterol Methvin and Suberkropp (2003),Carter and Suberkropp (2004)1–175 10 WV Ergosterol Simon and Benfield (2001),Stelzer et al. (2003), Gulis et al. (2004)2–25 4 WS Ergosterol Díez et al. (2002),Spänhoff and Gessner (2004)24–86 2 RCWS Ergosterol Gulis et al. (unpublished data)a Hyphal lengths were converted mycelial biomass by assuming an average hyphal diameter of 3 μm and a density of500 fg μm −3 (cf. Findlay and Arsuffi 1989; Newell 1992). ATP was converted fungal biomass assuming that fungal ATPaccounted for 90% of the total ATP (cf. Findlay and Arsuffi 1989), at an average ATP concentration of 1.75 mg g −1 mycelialdry mass (Suberkropp 1991; Suberkropp et al. 1993). Ergosterol was converted to fungal biomass assuming an averageconcentration of 5.5 mg g −1 dry mass (Gessner and Chauvet 1993), unless more specific data were availableb LB, leaves in litter bags, with the number of leaf types in parentheses (maximum fungal biomass from decompositionexperiments is given); RCL, randomly collected naturally occurring leaf litter (mean annual fungal biomass); WV, woodveneers and WS, wood sticks (range of fungal biomass from decomposition experiments); RCWS, randomly collectedwood sticks (mean fungal biomass)c Hyphal length determined after clearing of whole leaf materiald Hyphal length determined after grinding and collecting leaf fragments on membrane filterse Different sites or years or both in the same streamliable index of fungal biomass because, in terms ofbiomass, fungi commonly outweigh bacteria associatedwith plant litter (see Sect. IV.B.), and majorsources of ATP other than bacteria are usually absent(Golladay and Sinsabaugh 1991; Suberkroppet al. 1993). Phospholipid fatty acid (PLFA) profilinghas been used to quantify fungi in terrestrialhabitats (e.g. Klamer and Bååth 2004) but hasnot yet been applied to fungi in aquatic habitats.Beyond the use of these biomass indicators, measurementof [ 14 C]acetate incorporation into ergosterolfacilitates the determination of in situ fungalgrowth rates and production to assess the dynamicsof fungal biomass accrual and loss (Newell andFallon 1991; Gessner and Newell 2002).Application of these and other quantitativemethods has provided compelling evidence thatfungi are a key component of microbial assemblageswithin standing-dead shoots of emergentwetland plants, suggesting an overall importantcontribution to carbon and nutrient cycling inmarsh ecosystems (Gulis et al. 2006b). In pioneeringstudies involving a range of methods,fungal biomass in leaves of S. alterniflora rangedfrom 1.8% (microscopic determination of mycelialbiovolume) to as much as 20% (immunosorbentassay, ELISA) of the total organic matter (Newellet al. 1989). Estimates based on the determinationof ergosterol concentrations were intermediate(5%) but would have been about twice as highif a more realistic conversion factor (see Newell1994) had been used. A large proportion of thisbiomass can be in the form of ascomata, whichsometimes account for as much as 31% of the totalfungal biomass in decomposing Spartina leaves(Newell and Wasowski 1995).Assessment of multiyear patterns of fungalbiomass associated with salt-marsh plants corroboratesthese earlier observations. Fungi associatedwith both standing-dead S. alterniflora (Newell2001b) and Juncus roemerianus (Newell 2001c)accumulated substantial levels of biomass, with

306 M.O. Gessner et al.Fig. 17.2. Transmission electron micrograph of a crosssectionof a yellow poplar leaf (Liriodendron tulipifera L.)that had been decomposing in a hardwater stream for3weeks.Theleafwasfixedin2%glutaraldehydein10mMNa cacodylate, pH 7, immediately after it was removedfrom the stream. It was then post-fixed in 2% osmiumtetroxide, dehydrated, embedded and sectioned. Note themasses of fungal hyphae within leaf cells. Four hyphae areindicated by arrows.Scalebarindicates2μmestimated peak values exceeding 5% of the totalorganic mass of dead plant matter. On both plantspecies, fungal biomass was generally higherduring the winter and spring and low during thesummer, and S. alterniflora supported ∼1.5-foldhigher fungal biomass than J. roemerianus. InS. alterniflora leaves, ergosterol concentrationswere negatively correlated with rainfall and tidalheight (Newell 2001b), which supports findingsfrom manipulative field experiments suggestingthat prolonged water saturation has a negativeeffect on fungi associated with standing-deadwetland plants (Newell et al. 1996a).Similar to the consistent temporal pattern overseveral years, a large-scale comparative study alongthe eastern coast of North America revealed no significantlatitudinal differences in fungal biomass inleaves of standing-dead Spartina or Juncus in saltmarshes between 29 and 43 ◦ Nlatitude(Newelletal.2000). This suggests that substantial fungal productionderived from the aboveground primaryproductionofthesesalt-marshplantsistherule.However, Samiaji and Bärlocher(1996)foundmuchlower fungal biomass associated with S. alternifloraleaves in the Bay of Fundy (Canada) at 45 ◦ N, indicatingthat harsher environmental conditions atmore northern latitudes may begin to limit fungalbiomass accrual in standing-dead shoots.Substantial fungal biomass accumulationshave also been reported from standing-deadshoots decaying in freshwater marshes (Newellet al. 1995; Bärlocher and Biddiscombe 1996; Kuehnand Suberkropp 1998a; Gessner 2001; Findlay et al.2002a; Newell 2003; Welsch and Yavitt 2003). Forexample, in Erianthus giganteus, a reed-like grass,fungal biomass increased gradually during shootsenescence and early decomposition to reach 7and 4% of total detrital mass in leaves and culmsrespectively (Kuehn et al. 1999). However, fungalbiomass varied spatially along the culm axis of thisplant, resulting in a maximum concentration inthe lower portion of only about 1.5%.The observed accrual of substantial fungalbiomass in shoots of wetland plants is consistentwith high rates of biomass production that fungican exhibit in this type of habitat. Newell (2001b)reported production rates of 70–329 μgg −1 organicmass h −1 on decomposing leaf blades ofS. alterniflora over a 3-year period, correspondingto growth rates of ∼0.1–0.3% h −1 .Higherrateswere generally observed in winter and springand lower rates during the summer, consistentwith patterns of fungal biomass accrual. Similardynamics were observed for fungi associated withJ. roemerianus (Newell 2001c), where rates rangedfrom 66–366 μgg −1 h −1 and were lower in summerthan in spring and autumn. High rates of fungalproduction are not restricted to salt marshes insubtropical climate and, as with fungal biomassaccrual, no significant latitudinal difference inproduction rates was observed in either Spartinaor Juncus (Newell et al. 2000), suggesting thatappreciable fungal production is a common featureof standing-dead plant shoots in salt marshes.On S. alterniflora, fungal production rates werenegatively correlated with the C:N but not theC:P ratio of leaves (Newell 2001b), indicating thatnitrogen may be the critical nutrient controllingfungal growth in this system (e.g. Newell et al.1996a). In J. roemerianus, however, no significantrelationship was observed between rates of fungalproduction and C:N or C:P ratios of the plantmaterial.Fungal production rates in freshwater wetlandscan also be comparable to those measured in saltmarshes (Newell et al. 1995; Kuehn et al. 2000;Findlay et al. 2002a; Su et al. 2007). For example,fungal production rates on leaves of standing-deadT. angustifolia ranged from 12 to 359 μgg −1 h −1(Fig. 17.3), corresponding to growth rates of∼0.02–0.2% h −1 (K.A. Kuehn et al., unpublished

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 307data), very similar to the ranges reported byNewell (2001b, c). Fungi associated with Typhastems showed similar seasonal patters in biomassproduction but rates were only 11–108 μgg −1 h −1(corresponding growth rates ∼0.05–0.4% h −1 ),consistent with a much lower fungal biomass instems (Fig. 17.3). Similar fungal production rateswere observed for Typha and Phragmites stems(between 2 and 70 μgg −1 h −1 ) in a tidal freshwatermarsh (Findlay et al. 2002a). These data indicatethat fungal production rates on standing-deadshoots of emergent wetland plants may varysignificantly, depending on both environmentalconditions and the intrinsic quality of plant litter.Detachment of leaf blades from their parentplant, or collapse of standing-dead shoots to thesediments or overlying surface waters often lead todistinct changes in the biomass and productivityof the fungi associated with this plant material. Ina study with J. effusus,fungalbiomassandproductionrates declined rapidly following submergenceof plant material colonized during the standingdeadphase (Kuehn et al. 2000). Similar declines offungal biomass have been observed for fungi on thesalt-marsh grass S. alterniflora (Newell et al. 1989),and the emergent freshwater plant P. australis inboth lakes (Tanaka 1991; Komínková et al. 2000)and a tidal freshwater marsh (Van Ryckegem et al.2007). These concordant patterns suggest that fungiFig. 17.3. Fungal biomass (a) and temperature trend andfungal production (b) associated with standing-dead shootsof T. angustifolia in a temperate littoral marsh (K.A. Kuehnet al., unpublished data). Symbols indicate means±1 SE(n = 6)colonizing standing-dead shoots of wetland plantsare poorly adapted to the abrupt changes in environmentalconditions associated with the transitionof plant material from an aerial, standing-deadto an aquatic or semi-aquatic decay phase. However,despite the strong decline in fungal biomassfollowing collapse or detachment of the plant material,fungi continued to account for a major portionof total microbial biomass (Newell et al. 1989; Sinsabaughand Findlay 1995; Komínková et al. 2000;Kuehn et al. 2000; Su et al. 2007).D. Enzymatic CapabilitiesFungi associated with emergent wetland plants canproduce a variety of extracellular enzymes thatare involved in the degradation of plant cell walls(Gessner 1980; Torzilli 1982; Pointing and Hyde2000). Much of the current knowledge comes fromlaboratory-based studies of fungi isolated from decomposingshoots of the salt-marsh grass S. alterniflora.These isolates have typically been foundto produce enzymes that degrade cellulose andhemicelluloses, including those containing xyloseand arabinose (Gessner 1980; Torzilli 1982). Mixedresults have been obtained concerning the abilityof isolates to degrade pectin. Gessner (1980)found that only five of 20 isolates tested producedpolygalacturonases. Torzilli (1982) detected pectinolyticactivity when assays were carried out atpH 8 (pectin lyase) for all three species tested,but only one species produced pectinolytic activitywhen assayed at pH 5 (polygalacturonase). Whenfourfungalspecieswereprovidedwithisolatedcellwalls from S. alterniflora,allofthemgrew,andfiltratesfrom these cultures caused release of reducingsugars, indicating that these fungi were able todegrade polysaccharides embedded in native cellwalls (Torzilli 1982). Three species grown in culturealso caused losses of both the cellulose andhemicellulose fractions but not the lignin fractionof Spartina tissue (Torzilli and Andrykovitch 1986).Although less efficient than typical terrestrialwhite-rot fungi (Basidiomycota), there is evidencethat fungal decomposers associated with emergentwetland plants are often capable of degradinglignin. When provided with Spartina lignocellulosein which the lignin had been specificallyradiolabelled, the ascomycete Phaeosphaeriaspartinicola caused a loss of 6% (3.3% mineralizedand 2.7% solubilized) in the lignin fraction within45 days (Bergbauer and Newell 1992). In the same

308 M.O. Gessner et al.time interval, P. spartinicola caused 26% loss intotal lignocellulose (22% mineralized and 4%solubilized). Further, transmission electron micrographsof decaying Spartina leaves collected in thefield revealed symptoms of soft rot from each offour ascomycete species examined, demonstratingthe fungal ability to degrade cell wall materialin its native state (Newell et al. 1996b). In linewith this enzymatic and microscopic evidence,a high diversity of DNA sequences encodinglaccase, a key enzyme in lignin degradation, hasbeen demonstrated within the natural fungalcommunity on S. alterniflora (Lyons et al. 2003).Laccase sequences amplified directly from decayingleaf blades were dominated by sequencescharacteristic of P. spartinicola, Mycosphaerella sp.and P. halima, which were previously identifiedas the principal fungal colonizers of standingS. alterniflora leaves (Newell 1993; Buchan et al.2002, 2003). Thus, dominant fungi colonizingstanding-dead S. alterniflora have the enzymesneeded to degrade lignocellulosic tissues and canelaborate these enzymes when growing in theirnatural habitat. Fungi on other emergent wetlandplants, both in salt and freshwater marshes, mayhave similar enzymatic capabilities, given thefrequent occurrence of Ascomycota, includingtheir anamorphs, on plant matter in these habitats.E. Respiratory Activities on Standing-DeadWetland PlantsIf fungi are the dominant component of microbialassemblages associated with standing-dead wetlandplants, as comparative estimates of fungal andbacterial biomass and productivity suggest (seebelow), then general microbial activities, such asCO 2 release resulting from microbial respiration,should largely be attributable to fungi (e.g. Kuehnet al. 2004). The rationale behind the argumentsand conclusions in this section are based on thispremise.As in most terrestrial ecosystems, moistureavailability is the single most important factorlimiting microbial activity on and, thus, mineralization(CO 2 evolution) of dead emergent wetlandplants (Gallagher et al. 1984; Newell et al. 1985;Kuehn and Suberkropp 1998b; Kuehn et al. 2004).Fungiarewelladaptedtothissituationduetotheirability to respond quickly (within 5 min or less)to wetting, permitting them to take advantage ofeven short favourable periods in an environmentcharacterized by strongly fluctuating environmentalconditions (Newell et al. 1985; Kuehnand Suberkropp 1998b; Kuehn et al. 1998, 2004).During desiccation stress, these fungi surviveby accumulating intracellular compatible solutes(polyols and trehalose; Kuehn et al. 1998). As a resultof these adaptations, microbial assemblagesassociated with standing-dead wetland plants,particularly fungi, are capable of mineralizinga significant portion of the plant carbon beforethe collapse of leaves or shoots to the sediment(Kuehn et al. 2004).Respiratory activities of microbial assemblagesassociated with standing-dead S. alterniflora andJ. roemerianus shoots fluctuate rapidly after exposureto wetting or drying conditions (Newell et al.1985). During periods of desiccation (i.e. water content100 μg CO 2 -C g −1 h 1 )andaremaintained at high rates until exposure to dryingconditions (Newell et al. 1985). Frequent wettingof standing S. alterniflora shootshadanegativeeffecton fungal growth and ascomata production ofthe dominant fungal species (P. spartinicola)colonizingleaves. This counter-intuitive result suggeststhat fungi on S. alterniflora shoots are specificallyadapted to fluctuating water availability, and aredependent upon the cyclic episodes of desiccationand wetting for optimal growth and reproduction(Newell et al. 1996a).Similar respiratory patterns have been reportedfor microbial assemblages associated withstanding-dead plant shoots in freshwater marshes(Kuehn and Suberkropp 1998b; Kuehn et al. 1998,1999, 2004; Welsch and Yavitt 2003). For example,rates of CO 2 evolution from standing-dead P. australisexhibited a pronounced diel periodicity,with the highest rates occurring at night whencooling increased relative humidity (to the pointof dew formation) and thus water potentials of theplant material (Fig. 17.4). By contrast, respiratoryactivities virtually ceased during the day as a resultof desiccation. This indicates that diel fluctuationsin water availability play a key role in controllingmicrobial metabolic activities during the standingdecayphase of emergent vascular plants (Kuehnet al. 2004). Results of this study in a temperatewetland were remarkably similar to earlier observationsfrom J. effusus in a subtropical freshwatermarsh (Kuehn and Suberkropp 1998b), suggesting

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 309that pronounced diel shifts in microbial carbonmineralization of standing-dead wetland plantsmay be a geographically widespread phenomenon.As with fungal biomass and production(Fig. 17.3), large differences in microbial respirationpatterns have been observed amongplant litter types (species and organ) in terms ofmicrobial colonization and metabolic response towater availability. Respiration rates associated withdifferent P. australis shoot fractions varied considerably(Kuehn et al. 2004). Maximum respirationrates from standing-dead leaf blades were 24–42%higher than those from leaf sheaths under the sameenvironmental conditions, and maximum respirationrates from standing-dead culms were alwaysanorderofmagnitudelower.Thesedifferencesinrespiration rates were consistent with differencesin water absorption patterns, known structuralcharacteristics (e.g. lignocellulose concentration),and degree of fungal colonization among shootfractions (Kuehn et al. 2004). Maximum rates ofmicrobial respiration were positively correlated(r = 0. 72, p

310 M.O. Gessner et al.Zygomycota, Ascomycota and Basidiomycota), inaddition to the fungus-like Oomycota (KingdomStramenopila), can be detected on submergedleaves or wood by means of both traditionaland molecular techniques (e.g. Tsui and Hyde2003; Nikolcheva and Bärlocher 2004; Sheareret al. 2007). Casual observations suggest thatChytridiomycota, Zygomycota and Oomycotaprobably play a minor role as decomposers of plantlitter, while Ascomycota and their anamorphs,particularly hyphomycetes, assume the greatestimportance (Bärlocher 1992). Using phyla-specificprimers for the ITS region of rDNA, Nikolcheva andBärlocher (2004) found that fungal communitiesof submerged leaves and wood were consistentlydominated by Ascomycota in terms of speciesnumbers and abundance, followed by Basidiomycota(on wood) and Chytridiomycota (in winter).With such a molecular approach, the Ascomycotaalso included most of the mitosporic fungi oranamorphs commonly detected by conventionalmethods, i.e. aquatic and terrestrial hyphomycetes,the majority of which appear to have ascomycetousaffinities (Webster 1992). Although a diverseassemblage of Ascomycota (over 500 species) isassociated with wood in freshwaters, sexual (teleomorphic)stages of Ascomycota are uncommonon decomposing leaves in streams (Shearer 1993;Cai et al. 2003). Terrestrial hyphomycetes are partof the phylloplane microbiota in plant canopiesand thus colonize leaves before they enter streams(Bärlocher 1992). Their role in decompositiononce leaves have fallen into streams is not certain,but their activity appears to be limited at the lowwinter temperatures that prevail in temperate regionsafter leaf fall (Bärlocher 1992; Maltby 1992).Dematiaceous and other hyphomycetes that do notproduce intricately shaped conidia are less commononleavesinstreamsbutarefrequentlyisolatedfrom submerged wood (Goh and Tsui 2003).The most active fungal decomposers of leaflitter in streams are the aquatic hyphomycetes(Webster and Descals 1981; Bärlocher 1992;Suberkropp 1998b). The group includes at least320 species (Descals 2005). Aquatic hyphomycetesare well adapted to the stream environment(Bärlocher 1992; Suberkropp 1992b; Gessner andVan Ryckegem 2003), and produce tetraradiate, sigmoidor variously branched conidia that have beeninterpreted as traits that facilitate attachment to thesubstrate in flowing water (Webster and Descals1981; Webster 1987; Descals 2005; Dang et al. 2007).Thesefungiareabletoquicklycolonizeephemeralresources, such as leaf litter in streams, to growand rapidly produce spores, and thus to completetheir life cycle within a few weeks. At the low watertemperatures prevailing after leaf fall in temperateclimates, they are also able to outcompete otherfungi, mainly of terrestrial origin (Bärlocher 1992).Colonization of leaf litter by aquatic hyphomycetesis initiated by the impacting andtrapping of conidia on leaf surfaces after the leavesenter a stream. Subsequent germination is rapid,within 2–6 h in most species (Read et al. 1992).Once established, the fungal hyphae extend insidethe leaf matrix (Fig. 17.2), so that significantquantities of mycelial mass are built up withina few weeks after leaf colonization (Fig. 17.5a, seebelow). A striking feature of aquatic hyphomycetelife cycle is that mycelial growth is closely followedby the production of conidiophores, which maystart to release conidia in as little as 6–10 days afterleaves are submerged. This has been demonstratedboth in microcosm experiments (Suberkropp 1991;Gulis and Suberkropp 2003b; Treton et al. 2004)and under field conditions where sporulation ratesof natural communities often peaked earlier thanfungal biomass (Fig. 17.5b; Suberkropp et al. 1993;Baldy et al. 1995; Gulis and Suberkropp 2003c;Ferreira et al. 2006a). Sporulation rates rapidlyFig. 17.5. Fungal biomass (a) and sporulation rates ofaquatic hyphomycetes (b) associated with alder and oakleaves decomposing in a Portuguese woodland stream (datafrom Ferreira et al. 2006a). Symbols indicate means±1 SE(n = 4to6)

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 311increase to maxima and then decline (Fig. 17.5b).Maximum rates can reach seven conidia μg −1litter dry mass day −1 , although rates of at least anorder of magnitude lower are frequently observed(Bärlocher 1982; Suberkropp et al. 1993; Gessnerand Chauvet 1994; Suberkropp and Chauvet 1995;Bärlocher et al. 1995). The maximum rates ofsporulation are controlled by both internal factors,such as litter quality (Gessner and Chauvet 1994;Ferreira et al. 2006b), and external factors such astemperature, alkalinity, pH and nutrient availabilityin stream water (e.g. Jenkins and Suberkropp1995; Suberkropp and Chauvet 1995; Sridhar andBärlocher 1997; Chauvet and Suberkropp 1998;Gulis and Suberkropp 2003c; see Sect. III.D. fornutrient effects).Identification of many aquatic hyphomycetesis facilitated by characteristic conidial shapes. Diversityand community structure of these fungiis therefore often inferred from the relative abundancesof released conidia that are captured eitherafterincubationofleafmaterialinthelaboratory,by filtering stream water or by examining foamcollected from streams, in which conidia tend tobe trapped (Suberkropp 1992b; Gessner et al. 2003;Bärlocher 2005). This approach is based on the assumptionthat sporulation rates correlate with thebiomass of fungal species inside a leaf. Althoughthis is not necessarily the case, much of the currentknowledge on aquatic hyphomycete communitiesin streams has been derived from these types ofstudies. Individual leaves are colonized by typically4–10 and up to 23 species (Shearer and Webster1985; Suberkropp 1992b). On a stream scale,richness varies dramatically from just a few to >70species, depending on collection effort and streamcharacteristics (Bärlocher 2005). A range of factorsaffect the composition of aquatic hyphomycetecommunities. These include latitude and altitude,season, water chemistry (pH, alkalinity, concentrationsof inorganic nutrients, degree of pollution),composition of riparian vegetation, possibly interspecificcompetition, competition with and predationby invertebrates, and type of substrate. Thesefactors are discussed in greater detail elsewhere(Bärlocher 1992, 2005; Suberkropp 1992b; Gessnerand Van Ryckegem 2003).Alternative approaches to studying aquatichyphomycete communities make use of immunologicalor molecular techniques. Monoclonalantibodies raised against individual species anddetected by enzyme-linked immunosorbent assay(ELISA) or immunofluorescence enable in situidentification and, to some extent, quantificationof mycelial biomass of individual species(Bermingham et al. 1996, 1997). Even though thetechnique is highly specific and gives different insightinto species abundances than the traditionalapproach based on counting conidia, it has notbeen developed to a point where it is practicalfor ecological investigations. Antibodies need tobe available for each species in a community butto date they have been developed for only fourspecies. DNA-based approaches to analyse aquatichyphomycete communities include developmentof fluorescently labelled oligonucleotide probesfor in situ detection of fungal mycelia (FISH)(Baschien et al. 2001; McArthur et al. 2001) andPCR-based techniques. Denaturing gradient gelelectrophoresis (DGGE) and T-RFLP analysesof amplified fungal DNA from submerged leaflitter indicate that dominant phylotypes belong toaquatic hyphomycetes. These analyses also suggesthigher species richness during the initial stages ofdecomposition than have been detected with thetraditional microscopic identification of conidia,andsomedeclineinthenumberofphylotypesas decomposition progresses (Nikolcheva et al.2003, 2005). These results corroborate previousconclusions that aquatic hyphomycetes replacephylloplane/terrestrial fungi during early stagesof decomposition (Bärlocher and Kendrick 1974;Suberkropp and Klug 1976), and they also suggestthat some germinated aquatic conidia areunable to establish long-lasting viable colonies(Nikolcheva et al. 2005), pointing to a possible roleof interspecific competition in structuring fungalcommunities on decomposing leaves in streams.C. Fungal Biomass and ProductionFollowing submergence of leaves in streams, fungalbiomass usually increases gradually during a fewweeks to months and then levels off or decreasesslightly (Fig. 17.5a). The rate of fungal biomass accrualand maximum values attained largely dependon plant litter quality and stream water chemistry,and can vary dramatically among systems(Table 17.1). From a stoichiometric perspective, thelower C:N or C:P ratio of fungal biomass in comparisonto leaf litter, and especially wood, shouldresult in better fungal growth on substrates highin N and P (Stelzer et al. 2003; Gulis et al. 2006b).Indeed, slower fungal biomass accrual on low-Noak than high-N alder leaves (Fig. 17.5a; Gessner

312 M.O. Gessner et al.and Chauvet 1994; Nikolcheva et al. 2003; Guliset al. 2006a), on wood than on leaves (Nikolchevaet al. 2003; Stelzer et al. 2003), and generally lowerlevels attained on wood (Table 17.1) support thisidea. However, both oak leaves and wood have highlignin concentration, which could be more importantin determining fungal activity and decompositionrates than C:N or C:P ratios. In line with thisargument, initial lignin rather than N or P concentrationofleaflitterwasstronglycorrelatedwithlitter decomposition rate in a comparative studyacross seven leaf species, as were maximum fungalbiomass and sporulation rate of aquatic hyphomycetes(Fig. 17.6), suggesting that leaf litterdecomposition was controlled through a kineticcarbon limitation of fungal growth (Gessner andChauvet 1994).Estimates of fungal growth rate and production(Suberkropp and Weyers 1996; Gessner andChauvet 1997) give a better understanding of carbonflow from plant litter through fungal compartmentthan estimates of fungal biomass alone, sincelosses of fungal biomass as conidia or mycelialfragments, through respiration or as a result ofdetritivore feeding and hyphal death can be extensive.Growth rate (or ratio of daily productionto biomass, P:B) and production of fungi colonizingleaves during decomposition in litter bags peakvery early following leaf submergence in streamswhen fungal biomass is still relatively low, andgradually decrease as decomposition progresses(Suberkropp 1995; Weyers and Suberkropp 1996;Baldy and Gessner 1997; Suberkropp 2001; Baldyet al. 2002). Growth rates attain maxima of 0.01to 0.42 day −1 in decomposition experiments withlitter placed in mesh bags, and vary from 0.01to 0.17 day −1 from randomly collected leaf litterwhere the stages of decomposition are unknown(Suberkropp 1997; Methvin and Suberkropp 2003;Carter and Suberkropp 2004). In decompositionexperiments with litter bags, fungal production hasbeenfoundtopeakat0.6–16mg g −1 organic littermass day −1 (Suberkropp 1995, 2001; Weyers andSuberkropp 1996; Baldy and Gessner 1997; Pascoaland Cássio 2004; Pascoal et al. 2005), very similarto the range (0.8–10 mg g −1 day −1 )observedonrandomly collected leaves in streams (Suberkropp1997; Methvin and Suberkropp 2003; Carter andSuberkropp 2004). While average fungal biomassin randomly collected leaves is relatively constantthroughout the year, fungal growth rates and productionon a leaf mass basis (mg g −1 organic littermass day −1 ) are seasonal and peak in summer,probably in response to elevated temperature. Fungalbiomass and daily production calculated on anareal basis (gm −2 of stream bed) peak in autumnand winter following leaf fall in temperate regionsand sharp increases in the amounts of litter instream channels (Suberkropp 1997; Methvin andFig. 17.6. Relationships between decay coefficients of litterfrom seven deciduous leaf species and concentrations of refractoryleaf constituents (a), maximum sporulation ratesof aquatic hyphomycetes on leaves (b), and maximum fungalbiomass in leaves expressed as ergosterol concentration(c) (data from Gessner and Chauvet 1994). Symbols indicatemeans±1SE(n = 3)

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 313Suberkropp 2003; Carter and Suberkropp 2004).Similar data on fungi associated with submergedwood are scarce, but a recent estimate from randomlycollected wood sticks suggests that bothfungal growth rate and production (mg g −1 detritalmass d −1 ) are about an order of magnitude lowerthan those on leaves in the same streams, whilebiomass is only about twofold lower (Gulis et al.,unpublished data).Increases in fungal biomass associated withleaf litter often correlate with increases in nitrogenconcentration, suggesting that fungi immobilizeN from the stream water (Gulis et al. 2006b).Phosphorusconcentrationofleaflitteralsooftenincreases during decomposition, concomitant withfungal biomass accrual (e.g. Robinson and Gessner2000; Gulis et al. 2006a; Stallcup et al. 2006). Theseincreases in fungal biomass and elemental concentrationsenhance the palatability and nutritionalquality of litter as a food source for stream invertebrates(Bärlocher 1985; Suberkropp 1992a).D. Responses of Fungal Decomposersto Dissolved NutrientsA variety of factors influence fungal activityand decomposition of plant litter in aquaticecosystems. The most important ones are plantlitter quality (e.g. concentrations of nutrients andrefractory and inhibitory plant constituents), bioticparameters (e.g. fungal community structure,presence of detritivores), and environmental variables(e.g. temperature, pH, oxygen availability,and dissolved nutrient concentrations) (Fig. 17.1).The critical importance of dissolved nutrients inregulating fungal activity and fungal-mediateddecomposition in streams was recognized abouta decade ago (Suberkropp and Chauvet 1995),following earlier discovery that elevated nutrientconcentrations can stimulate litter decomposition(e.g. Elwood et al. 1981), and has received much attentionin recent years (e.g. Robinson and Gessner2000; Rosemond et al. 2002; Gulis and Suberkropp2003c; Ferreira et al. 2006b). Stream fungi canobtain inorganic nutrients (e.g. N and P) fromboth the plant litter they grow in and stream water(Suberkropp and Jones 1991; Suberkropp 1995).Since plant litter is typically low in N and P (i.e. C:Nand C:P are much higher than those of mycelium),fungi are often nutrient-limited in oligotrophicstreams, and their activity is significantly higherin streams with high dissolved nutrient concentrations(Suberkropp and Chauvet 1995) or followingexperimental nutrient addition (Grattan andSuberkropp 2001; Gulis and Suberkropp 2003c;Ferreira et al. 2006b). Strong positive correlationsbetween dissolved nitrogen and/or phosphorusconcentrations and fungal biomass, sporulation ofaquatic hyphomycetes, respiration and/or exponentialdecayratesofleaveshavebeenobservedin various streams (e.g. Suberkropp and Chauvet1995; Niyogi et al. 2003; Gulis et al. 2006a). Inaddition to this correlational evidence, microcosmstudies have clearly shown stimulation of fungalactivity and litter decomposition by dissolved Nand/or P (Sridhar and Bärlocher 1997; Suberkropp1998a; Gulis and Suberkropp 2003a, b). The mostconvincing results on the importance of dissolvednutrients, however, came from whole-stream nutrientenrichment experiments that demonstratedstimulation of microbial activity, and accelerationof leaf and wood decomposition in a variety ofstreams in different geographic settings (Gulis andSuberkropp 2003c; Stelzer et al. 2003; Gulis et al.2004; Benstead et al. 2005; Ferreira et al. 2006b;Stallcup et al. 2006).A few studies have not found evident effects ofnutrient addition on either fungal activity or litterdecomposition. This could happen when backgroundlevels of dissolved nutrients in streams arehigh and therefore not limiting to fungi (Royerand Minshall 2001; Simon and Benfield 2001), orwhen a non-limiting nutrient such as N is experimentallyadded to streams (Newbold et al. 1983)when another nutrient such as P is limiting (Elwoodet al. 1981). Furthermore, fungal activity innutrient-poor streams may be co-limited by N andP and, thus, the addition of either nutrient alonehas no effect (Tank and Webster 1998; Grattan andSuberkropp 2001).The shape of the dose-response curve betweendissolved nutrient concentrations in water andfungal activity or litter decomposition rate dependson the range of concentrations examined. Studiesin streams with low to moderate N and P concentrationsstrongly support a linear relationship(Suberkropp and Chauvet 1995; Niyogi et al. 2003).However, as the range of dissolved nutrient concentrationsis increased to include high-nutrientstreams, the relationship with fungal biomass,sporulation rate of aquatic hyphomycetes, microbialrespiration, and leaf litter decompositionrather follows a Michaelis-Menten-type saturationmodel (Fig. 17.7; Rosemond et al. 2002; Ferreiraet al. 2006b; Gulis et al. 2006a, b; Baldy et al. 2007).

314 M.O. Gessner et al.Therefore, the linear responses observed in earlierexperiments are likely to represent only the risinglimb of the saturation model.Nutrient stoichiometry of plant litter may modifythe response of fungi to dissolved nutrients.A stimulating effect of exogenous nitrogen, forexample, would be less pronounced when fungaldemands can be met by nitrogen sources withindecomposing plant material. Consistent with thisidea, the effect of stream water nutrients appearsto be greater on wood, which has very high C:Nand C:P ratios, than on leaves (Stelzer et al. 2003;Gulis et al. 2004; Ferreira et al. 2006b). However,nutrient availability, whether external or internal,wouldbelesscriticalwhenlabilecarbonisinlimitedsupply, as may often be the case in leaf specieswith high concentrations of refractory carbon compoundssuch as lignin (Gessner and Chauvet 1994).Thus, the regulation of fungal activity and plant litterdecomposition by dissolved nutrients may varyaccording to the relative impact and interactions ofFig. 17.7. Relationship between nitrate concentrations instream water and percentage of initial litter mass convertedinto aquatic hyphomycete conidia (a) orlitterdecompositionrate (b). Data are fitted to a Michaelis-Mentensaturation-type model: V=V max [S]/(K m +[S]), where V maxis the maximum parameter value, K m is the nutrient concentrationat which half of the maximum parameter value isachieved, and [S] is the nutrient concentration. Open symbolsdenote high-nutrient leaf litter (alder) and closed symbolsindicate low-nutrient balsa wood veneers (data fromFerreira et al. 2006b)a range of controlling factors, both external onesrelated to the environment and factors intrinsic tothe decomposing plant material.E. Enzymatic CapabilitiesAquatic hyphomycetes produce a variety ofextracellular enzymes that degrade the structuralpolysaccharides of leaves (Chamier 1985;Suberkropp 1992b). Enzymes that hydrolyzecellulose (endoglucanases, exoglucanases andexoglucosidase) and hemicelluloses (xylanases,xylosidase and arabinosidase) are produced bya number of species in culture growing on puresubstrates or leaf material. Aquatic hyphomycetesalso typically produce several enzymes that degradepectin (Suberkropp and Klug 1980; Chamierand Dixon 1982). Pectin degradation leads to thesoftening and maceration of plant tissue, resultingin the release of mesophyll cells (Chamier 1985;Suberkropp 1992b). Both polygalacturonase andpectin lyase depolymerize pectin, and both areproduced by aquatic hyphomycetes, but the latterappears to play a greater role in leaf maceration(Suberkropp and Klug 1980; Jenkins andSuberkropp 1995). Since aquatic hyphomycetesalso produce enzymes to degrade proteins andlipids (Zemek et al. 1985; Zare-Maivan and Shearer1988; Abdullah and Taj-Aldeen 1989), it appearsthat most plant polymers can be metabolized bythe majority of these fungi.Lignin may be an important exception.Evidence from laboratory studies indicates thataquatic hyphomycetes can degrade lignin-likesubstrates (Fisher et al. 1983; Zemek et al. 1985;Zare-Maivan and Shearer 1988; Abdullah andTaj-Aldeen 1989), and some freshwater fungi havebeen reported to solubilize lignin in wood (Bucheret al. 2004). However, ligninolytic capabilities ofaquatic hyphomycetes appear to be limited, andthe general difficulty to assess lignin degradationstill hinders our understanding of this processunder natural circumstances (Chamier 1985).The general picture that emerges is, aside fromquantitative difference in activity (Suberkroppet al. 1983), an apparent lack of specializationamong species in terms of enzymatic capabilities.This indicates that aquatic hyphomycetes area rather homogenous and generalist group withrespect to nutritional niche breadth (Suberkropp1992b), even though some substrate preferencesand quite distinct communities associated with

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 315leaves vs. wood have been reported (Gulis 2001;Bärlocher 2005; Ferreira et al. 2006b).Little information is available on fungalenzyme activities associated with plant litter instreams, because fungi are not the only microbescolonizing this material. Nevertheless, sincefungi typically dominate microbial biomass andproduction on leaf litter (see Sect. V.B.), theyare likely to make a substantial contribution tothe enzymatic activities associated with decomposingleaves in streams. For example, Golladayand Sinsabaugh (1991) found that exocellulaseactivity on maple leaves was closely correlatedwith fungal biomass, suggesting this hydrolyticactivity was due to fungi. Similarly, the activityof four lignocellulose-degrading enzymes onwood showed generally positive relationships withfungal biomass (Tank et al. 1998). However, inanother study, leaf-associated activities of threehydrolytic enzymes (xylanase, endocellulase andgalacturonase) were lower in a hardwater thana softwater stream, whereas leaf softening anddecay were faster, and fungal biomass accrualand sporulation of aquatic hyphomycetes werehigher in the hardwater stream (Jenkins andSuberkropp 1995). It was therefore concludedthat the hydrolytic enzymes examined were poorindicators of decomposition. Pectin lyase activity,by contrast, was higher in the hardwater stream,concomitant with faster leaf breakdown andgreater fungal activity (Jenkins and Suberkropp1995). These and similar results by Griffith et al.(1995) suggest that pectin degradation mediatedby fungi is a key mechanism promoting leafdecomposition in streams (Suberkropp and Klug1980; Chamier and Dixon 1982).2005; Duarte et al. 2006). This points to a highdegree of functional redundancy among aquatichyphomycetes.However, mixed cultures of two early colonizersenhanced decomposition by 73% comparedto values expected from decomposition rates ofsingle-species cultures (Treton et al. 2004). Thisoutcome, in contrast to results from multispeciesexperiments (Dang et al. 2005; Duarte et al. 2006),is strong evidence of niche complementarity resultinginfasterlitterdecomposition.Inasimilarvein,Bärlocher and Corkum (2003) reported a tendencytowards faster decomposition with increasing fungalrichness (1–5 species), although mixed communitiesnever caused greater mass loss than themost effective species alone. Raviraja et al. (2006)also found that both species richness and identitiesaffected leaf mass loss in microcosms, althoughagain the most effective fungal species degradedleaves faster than did species mixtures.There is also evidence that richness of aquatichyphomycete communities can indirectly enhancedecomposition through a positive effect on resourcequality for invertebrate detritivores (Lecerfet al. 2005). Further, ecosystem processes otherthan litter decomposition (e.g. fungal biomass production)maybeenhancedbydiversecommunities(Duarte et al. 2006). Lastly, even when averagerates of decomposition are independent of speciesrichness, variability of rates has been found tostrongly decline with increasing fungal richness,as predicted from theoretical models (Dang et al.2005). All else being equal, this should lead tohigher predictability of litter decompositionrates when aquatic hyphomycete communities instreams are diverse.F. Significance of Fungal Diversityfor Leaf DecompositionIn view of possible consequences of species extinctionto ecosystem processes, the effects of fungaldiversity on litter decomposition in streams havebeen examined in several studies. Results from fieldsurveys suggest that species-poor fungal communitiesin streams affected by forestry practices orwater pollution do not result in altered leaf decompositionrates (Raviraja et al. 1998; Bärlocher andGraça 2002). Similarly, varying species richness ofaquatic hyphomycetes in microcosms had no effecton average leaf decomposition rates in mixedcommunities with up to eight species (Dang et al.IV. Importance of Fungal Decomposersin Aquatic EcosystemsA. Fungal Biomass and Productionat the Ecosystem ScaleWhen periodic estimates of fungal biomass orproductionpergramoflitterareaccompaniedby data on the amount of plant litter present perm 2 of habitat, then fungal importance can beestimated at the ecosystem scale. Such estimatesof fungal production in streams range from 16 to193 gm −2 year −1 , and are generally comparablewith estimates of bacterial and macro invertebrate

316 M.O. Gessner et al.production (Suberkropp 1997; Methvin andSuberkropp 2003; Carter and Suberkropp 2004;Gulis et al. 2006b). Fungal production on an arealbasis correlates well with the mean annual amountof leaf litter in streams. Amounts of benthic litter,in turn, are a function of litter input, downstreamtransport, and decomposition by microbes andinvertebrates. Small woodland streams receivehigh litter input per m 2 of stream bed because theyare intimately linked to their riparian zones, andalso often retain litter effectively during high flows,becausetheyareshallowandtendtohaveroughstream bottoms and other retention structures.Accordingly, annual fungal production per m 2in these streams is particularly high (Gulis et al.2006b).Consistent with generally lower fungal activityon submerged wood than on leaves, fungal productionon wood (randomly collected naturally occurringsticks, 5–40 mm in diameter) in two headwaterstreams was estimated at 9–11 gm −2 year −1(Gulis et al., unpublished data). Depending on sticksize and stream water nutrient concentration, thistranslates into 2–13% of wood carbon assimilatedby fungi per year, which is considerably lower thantheestimatedamountsofleafcarbonassimilatedbyfungi. However, taking into account the longer residencetimes of wood compared to leaves, the importanceof wood-colonizing fungi in many streams islikely to be significant as well.Fungal production associated with standingdeadplants in marshes is also sizeable andfurther points to the quantitative significanceof fungi at the ecosystem scale. For example, ina subtropical coastal salt marsh, fungal biomasson standing-dead shoots of S. alterniflora rangedfrom 9 (summer–autumn) to 37 gCm −2 of marsharea (winter–spring) (assuming 43% C in fungaldry mass). Estimated annual fungal productiontotalled 230 gCm −2 year −1 ,equivalenttoroughly40% of the annual plant production (Newell2001b). This estimate is based on the assumptionthat fungal communities of standing-deadS. alterniflora shoots are metabolically active (i.e.released from water stress) for 12 h per day (seeSect. II.E. above). Even if this were an overestimate,it indicates that conversion of plant biomass tofungal biomass can be substantial.Substantial fungal production has also beenobserved in freshwater wetlands. Annual fungalbiomass and production associated with leaf bladesand stems of standing-dead T. angustifolia shootsin a north-temperate lake littoral marsh was 70 and45 gCm −2 year −1 respectively (K.A. Kuehn et al.,unpublished data). This production estimate takesinto account the diel periodicity in water availability(i.e. dew formation) that regulates microbialactivities (see Sect. II.E. above). Substantial additionalfungal production can occur on submergedlitter in freshwater marshes. An annual productionof nearly 100 gCm −2 has been estimated in the submergedlitter layer of another littoral marsh dominatedby P. australis in a temperate lake (Buesingand Gessner 2006). Thus, all systems studied sofar (i.e. submerged leaf litter in streams, and bothsubmerged litter and standing-dead shoots in saltand freshwater marshes) have revealed very highpotential for fungal production, suggesting a greatimportance of fungi in food webs and organic matterturnoverattheecosystemscale.B. Fungal vs. Bacterial Biomass and ProductionStudies in diverse streams (Sanzone et al. 2001;Findlay et al. 2002b) and salt and freshwatermarshes (Sinsabaugh and Findlay 1995; Newelland Porter 2000) suggest that fungal biomassexceeds bacterial biomass on coarse submergedorganic particles such as leaves, wood and otherplant litter, whereas bacteria assume greaterimportance on finer organic particles and possiblyon decaying floating-leaved macrophytes (Mille-Lindblom et al. 2006). In streams, fungi typicallyaccount for 88–99.9% of the microbial biomass(i.e. the combined fungal and bacterial biomass)developing on decomposing leaves (e.g. Findlayand Arsuffi 1989; Baldy et al. 1995; Weyers andSuberkropp 1996; Baldy and Gessner 1997; Hieberand Gessner 2002; Gulis and Suberkropp 2003a).Given these ratios of fungal and bacterial biomass,and the experimentally demonstrated preferenceof stream detritivores for fungal-colonized leafpatches (Arsuffi and Suberkropp 1985; Suberkropp1992a), fungi appear to play a much greater rolethan bacteria in altering the palatability and foodquality of decaying leaf litter in streams, andprovideamuchlargerfractiontothenutritionof invertebrate detritivores (Suberkropp 1992a).Fungi appear to dominate microbial communitiesalso on submerged wood in streams (67–97% interms of biomass; Findlay et al. 2002b; Stelzeret al. 2003) but information is still very limited atpresent.Fungal dominance of microbial biomass (typically>90%) associated with standing-dead plant

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 317shoots and submerged litter in freshwater marshes(Sinsabaugh and Findlay 1995; Newell et al. 1995;Komínková et al. 2000; Kuehn et al. 2000; Findlayet al. 2002a; Su et al. 2007) and salt marshes (Newell1992, 1993; Newell and Porter 2000) is well established.For example, microbial biomass associatedwith naturally standing-dead shoots of the freshwatersedge, Carex walteriana, was dominated byfungi, with bacterial biomass often less than 0.5%that of fungi (Newell et al. 1995). Bacterial biomassincreased significantly once standing-dead plantmaterial fragmented and fell to the sediment surface.However, despite the change in decay conditions,fungal biomass still accounted for 97% of thetotal microbial biomass (Newell et al. 1995).Bacteriamayhavehighergrowthratesandshorter turnover times than fungi, suggesting thatcomparisons between both groups are more meaningfulon the basis of production than biomass.However, outcomes of both types of comparisonshave generally been similar. In particular, fungalproduction greatly exceeded bacterial production(1–627×) associated with leaves in streams in allstudies when both microbial groups were followedsimultaneously (Suberkropp and Weyers 1996;Weyers and Suberkropp 1996; Baldy et al. 2002;Pascoal and Cássio 2004; Pascoal et al. 2005). Thisconsistent finding further emphasizes the keyimportance of fungi colonizing leaf litter in streamecosystems. One exception from the generalpattern is an experiment with fresh green leavescollected in summer where fungal and bacterialproduction estimates were comparable (Baldy andGessner 1997).Similar findings have been reported for fungicolonizing standing-dead shoots and submergedlitter in salt and freshwater marshes, where fungalproduction accounted for >93% of the totalmicrobial production (Newell et al. 1995; Newelland Porter 2000; Kuehn et al. 2000; Findlay et al.2002a; Su et al. 2007). By contrast, bacterial productionoutweighed fungal production (>8:1) onsubmerged P. australis litter in a littoral marsh ofa lake (Buesing and Gessner 2006). The inverse relationshipbetween fungi and bacteria in this marshwas due to a particularly high bacterial production(average of 660 gCm −2 year −1 ), rather thanalowfungalproduction(93gCm −2 year −1 ), and itis possible that this very high bacterial productionwas an overestimate caused by the high concentrationof leucine used to determine protein synthesisrates as a measure of bacterial production (Gillieset al. 2006).C. Decomposition BudgetsEstimates of the different fates of decomposingplant material in addition to conversion into fungaland bacterial biomass have been made in severalaquatic ecosystems. However, most budgets consideringthese fates are partial and have been calculatedfor a particular period, usually advanceddecomposition stages. Consequently, they do notreflect the dynamic changes that characterize theentire decomposition sequence. Since much of thefungal biomass produced during litter decompositionis transient and eventually lost as CO 2 or inother forms (Gessner et al. 1999; see below), fungioften appear more important when budgets are calculatedat the time of maximum fungal biomass,rather than at final decomposition stages when theremaining mycelial biomass is relatively low (e.g.in streams, 0.5–3.9% of the initial organic littermass; Gessner et al. 1997). Aquatic hyphomyceteson leaves in streams channel a substantial proportionof their production (1.0–7.3% of initial organiclitter mass) into the formation of conidia(Findlay and Arsuffi 1989; Suberkropp 1991; Baldyet al. 1995; Hieber and Gessner 2002; Ferreira et al.2006b), and two species grown on leaves in microcosmseven allocated 46 and 81% of their productionto conidia, equivalent to 7 and 12% of leaf massloss respectively (Suberkropp 1991).Estimates of fungal reproductive output arealso available for fungi growing on standing-deadSpartina shoots in a salt marsh. Like aquatic hyphomycetesin streams, these salt-marsh fungi allocatesubstantial amounts of fungal biomass to reproductivestructures (ascomata of Phaeosphaeriaspartinicola and Mycosphaerella sp.). During periodsofleafwetness,anaverageof59ascosporesperhourwerefoundtobereleasedpercm 2 of theupper two thirds of decaying leaf blades attachedto standing-dead shoots. This value was conservativelyestimated to represent 7.5 g fungal biomassper m 2 of salt marsh per year, and nearly 5% of thetotal mycelial production in these leaves (Newell2001a). Since fungal spores typically contain highconcentrations of nutrients (Dowding 1976), sporereleaseislikelytobeamoresignificantpathwayof N and P loss from decomposing leaves than ofcarbon loss.CO 2 fluxes from standing-dead plant shootsas a result of microbial (presumably, mostlyfungal) respiration can represent an importantpathway of carbon flow in wetlands (Kuehn andSuberkropp 1998b; Kuehn et al. 2004). Taking

318 M.O. Gessner et al.into account diel fluctuations in respiration rates(see Sect. II.E.) and estimates of litter standingcrops, daily fluxes from standing-dead J. effususwere estimated at 1.4–3.6 gCm −2 (Kuehn andSuberkropp 1998a), which generally exceededCO 2 fluxes from sediments in the same wetland(0.12–2.4 gCm −2 day −1 ; see Roden and Wetzel1996). CO 2 fluxes from standing-dead P. australisshoots were lower (0.05–0.57 gCm −2 day −1 )butstill within the range of those from wetlandsediments in north-temperate climates (Kuehnet al. 2004). As a result, fungi and, to a smallerextent, other microorganisms could mineralizeasignificantportionofJ. effusus leaf (∼28%), andPhragmites leaf (∼8%) and sheath (∼29%) annualproduction under standing-dead conditions (Guliset al. 2006b). CO 2 fluxes from decomposingleaf litter in streams are also sizeable, and wereestimated in decomposition experiments to rangefrom 17 to 56% of total leaf carbon losses (Elwoodat al. 1981; Findlay and Arsuffi 1989; Baldy andGessner 1997; Gulis and Suberkropp 2003c).Estimates of the fraction of litter assimilated byfungi can be calculated as the sum of fungal productionand respiration, or from fungal productionwhenfungal growth efficiency is known.Forstreamfungi, growth efficiency ranges from 24 to 60%(Suberkropp 1991; Gulis and Suberkropp 2003b).This translates into a fungal assimilation of 5 to97% of the annual leaf input (Gulis et al. 2006b). Estimatesfor freshwater marshes suggest that at least∼10% of the annual aboveground Typha production(K.A. Kuehn et al., unpublished data) and 15%of aboveground Phragmites production (Buesingand Gessner 2006) go into the production of fungalbiomass. Both estimates consider only part ofthe fungal production per m 2 of marsh, becauseeither the standing-dead or submerged decompositionphase was ignored. Total fungal productiontherefore is likely to be much higher in both cases.One of the fates of leaves degraded by fungiin streams is the conversion to dissolved andfine particulate organic matter (DOM and FPOM;Suberkropp and Klug 1980; Gessner et al. 1999;Baldy et al. 2007). The ratio of released DOM toFPOM is variable but typically greater than one(Gessner et al. 1997), and the amounts of the tworeleased fractions combined (FPOM+DOM; 36%of leaf mass loss in Findlay and Arsuffi 1989, and8% in Baldy and Gessner 1997) may be on theorder of the fraction released as CO 2 (40 and 17%respectively). Greater release of DOM compared toFPOM (barely detectable) has been reported fromPhragmites leaves, with DOM representing some39% of the initial leaf mass (Komínková et al. 2000).FPOM and DOM can also be generated by feeding,and defecation or excretion by leaf-shreddingmacroinvertebrates (Wallace and Webster 1996).However, even where detritivore-mediated leafconversion to other forms of organic matter andCO 2 is high (e.g. >50% of total leaf mass loss vs.14–18% for fungi; Hieber and Gessner 2002), fungimaysignificantlycontributetolitterconversioninan indirect way by stimulating litter consumptionby detritivores (Suberkropp 1992b).V. ConclusionsA diversity of aquatic habitats occurs at land–waterinterfaces where the productivity of plants is oftenhigh and large amounts of plant matter enterthe detrital pool. Environmental conditions (e.g.temperature, salinity, nutrient availability) varywidely within and across these systems where differenttypes of plant matter from both aquatic andterrestrial sources are decomposing. The diversityof fungi present and potentially active in thesesystems is high. However, given the paucity of datafor many systems, the overall importance of fungias decomposers across aquatic ecosystems remainsdifficult to assess. Identification of the fungipresent, by either traditional or molecular methods,is a prerequisite but not sufficient to ascertainan important functional role of these organismsin ecosystems. However, quantitative data arebecoming increasingly available to evaluate the significanceof fungi as agents of decomposition andnutrient cycling, producers of biomass, and mediatorsof organic matter transfer in aquatic food webs.In a few types of aquatic ecosystems, particularlythe marshes and streams discussed in thischapter, the role of fungi as decomposers of organicmatter and producers of biomass has beendemonstrated to be substantial. Fungi are clearlythe key decomposers of standing-dead emergentplants in freshwater wetlands and salt marshes, andof terrestrial leaf litter in streams. The dominantspecies in these ecosystems possess the enzymaticpotential necessary to degrade the structural compoundsof litter, although fungal lignin degradationin streams is not well documented. Fungal biomassassociated with decomposing plant material caneasily exceed 10% of total litter mass in these systems,and typically outweighs bacterial biomass.

Fungal Decomposers of Plant Litter in Aquatic Ecosystems 319Comparisons of fungi and bacteria on a productionbasis generally yield similar results. Fungalbiomass production at an ecosystem scale variesamong systems and sites but can approach and evensurpass 100 gCm −2 year −1 .Evidencefromvarioussites suggests, furthermore, that fungal activity canbe responsible for a large proportion of leaf massloss during decomposition, leading to the mineralizationof plant organic matter to CO 2 as well asconversion into DOM and FPOM.Fungal activity and, consequently, leaf decompositionrates are regulated both by internal (e.g.litter nutrient concentration and carbon quality)and external (e.g. temperature, dissolved nutrientconcentrations) factors. As fungi grow in leaflitter, their production is partitioned between themycelium and reproductive structures. A significantfraction of biomass is ultimately channelledinto spores. 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18 Degradation of Plant Cell Wall Polymers by FungiC. Gamauf 1 ,B.Metz 1 ,B.Seiboth 1CONTENTSI. Introduction ........................ 325II. Structure and Compositionof Plant Cell Walls .................... 326III. The Degradation of the Plant Cell Wall .... 327A. BiodegradationofCellulose.......... 327B. Biodegradation of Hemicelluloses . . . . . 3291.Xylans ........................ 3302.Xyloglucan..................... 3303.(Galacto-)glucomannan........... 331C. BiodegradationofPectins ........... 331D. AccessoryEnzymes ................ 334IV. Biodegradation of Lignin .............. 335A. Brown-RotFungi.................. 335B. White-RotFungi .................. 336V. Conclusions ......................... 338References.......................... 338I. IntroductionThe main producers of primary biomass on earthare plants, through the photosynthetic fixationof carbon dioxide into organic matter. Fungi areheterotrophic organisms which depend on preformedorganic compounds as energy and nutrientsource for the biosynthesis of their own cellularcomponents. They are today the most importantand widespread group of organisms responsiblefor the recycling of plant material back into theecosystem and are therefore essential componentsof the global carbon cycle. A large group of fungiis in particular specialized to degrade the complexcell wall produced by plants. One of the reasonsfor this seems to be the “side by side” evolutionof plants and fungi. Symbiotic associations offungi and photosynthetic plant hosts have mostprobably facilitated the colonization of land. Thefirst fossil land plants and fungi appeared 480 to460 million years ago. These fossil fungi are foundclosely associated with rhizomes, the primitive1 FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik,Umwelttechnik und Technische Biowissenschaften,TU Wien, Getreidemarkt 9/166/5/1, 1060 Vienna, Austriaroot systems of the first vascular land plants. However,the time when colonization occurred remainsspeculative; molecular clock estimates suggestcolonization of land about 600 million years agowhereas protein sequence analyses indicate thatland plants appeared as early as 700 million yearsago (Simon et al. 1993; Remy et al. 1994; Heckmanet al. 2001). Today, symbiotic associations are stillfound in the arbuscular mycorrhizal fungi, whichare partners of 70% or more of today’s higherplants (Smith and Read 1997). During their furtherco-evolution, the fungal partner took advantageof the increasing availability of plant materialsand adapted to the decomposition of these plantsby either a parasitic or saprotrophic way oflife.Saprotrophic or saprobic fungi grow on deadmaterial and are particularly important for thebreakdown of various polymers in the plant cellwall.However,fungiarealsoadaptedtogrowasparasites or pathogens on living plants, and canbe subdivided into biotrophic and necrotrophicfungi. Biotrophic fungi feed from living cells withoutkillingthehostplantwhereasnecrotrophicparasiteskill the host organism as part of their attack.A number of economically important plants are attackedby fungi, leading to crop losses either byplant decomposition or by the production of toxicsubstances.Theimportanceoffungiintheglobalcyclingof carbon, the significance of enzymes producedby fungi to degrade the plant cell wall, andthe biotechnological applications of fungi andtheir enzymes have promoted research interesttowards understanding their role in the degradationof plant material. Today, the use of fungallignocellulose-degrading enzymes for total hydrolysisof plant biomass to sugars is under intensivestudy. The sugar monomers formed thereby couldserve as raw material in the bioproduction ofchemicals and fuels by microbes. Productionof bio-ethanol derived from agricultural wasteEnvironmental and Microbial Relationships, 2nd EditionThe Mycota IVC. P. Kubicek and I. S. Druzhinina (Eds.)© Springer-Verlag Berlin Heidelberg 2007

326 C. Gamauf, B. Metz, B. Seibothmaterials could partly replace fossil fuels. Furtherapplications of the enzymes and organisms canbe found in various industries including the food,feed,textile,andpulpandpaperindustries.Thegeneral use of sustainable and environmentallyfriendly technologies which have introducedlignocellulolytic enzymes at different stages ofpulp and paper manufacturing as pretreatment forpulping (biopulping), bleaching (biobleaching) orwastewater treatment has led to considerable energysavings and an overall reduction of pollutantsin wastewater from these industries.The fungi best studied with respect tobiopolymer-degrading enzyme formation arespecies of Aspergillus, Trichoderma and Penicilliumor – as an example of lignin degradation – thewhite-rot fungus Phanerochaete chrysogenum. Innature, most of these fungi produce high amountsof extracellular enzymes due to an efficient secretorymachinery. Some are easy and inexpensive togrow in large bioreactors, and suitable for geneticrecombination technologies. Industrial strainsof Aspergillus and Trichoderma can yield morethan one hundred grams of these extracellularenzymes per litre (Durand et al. 1988; Berka et al.1991; Cherry and Fidantsef 2003) and are thereforealso used in the production of recombinantproteins and enzymes. Indeed, it is not surprisingthat these fungi are among the first for whichagenomesequencebecameavailable.Therecentadvances in the sequencing and annotation ofdifferent fungal genomes of Aspergillus nidulans,Aspergillus niger, Hypocrea jecorina or Phanerochaetechrysogenum (http://genome.jgi-psf.org;http://www.broad.mit.edu/annotation/fgi/) givea first impression of the hidden enzymaticpotential of these organisms.This review summarizes our current knowledgeon the diversity of the plant cell walldegradingenzymes of these saprobic fungi.As an example for the complex degradation ofwood, lignocellulolytic degradation in brownandwhite-rot decay is highlighted in more detail.A complete listing of all enzymes involved in thebreakdown of plant cell wall materials and whichhave been characterized in the last few years isbeyond the scope of this review but web-baseddatabases are available. One example is the CAZydatabase, which describes the families of structurallyrelated catalytic and carbohydrate-bindingmodules of enzymes which degrade, modify orcreate glycosidic bonds (www.cazy.org/CAZY/;also see Coutinho and Henrissat 1999).II. Structure and Compositionof Plant Cell WallsPlant cells are usually enclosed by a cell wall whichprovides rigidity to the cell for structural andmechanical support, maintains and determines cellshape, counterbalances osmotic pressure, determinesthe direction of growth and, ultimately, thearchitecture and form of the plant. In addition, theplant cell wall protects against pathogens and otherenvironmental factors. The predominant polymersin plant cell walls are polysaccharides such ascellulose, hemicelluloses and pectins. Togetherwith the aromatic polymer lignin and proteins,these polysaccharides can form a complex andrigid structure termed lignocellulose.The middle lamella, the primary wall, and thesecondary wall are the three major regions to bedistinguished. The middle lamella is the outermostlayer and is composed primarily of pectin. The primarywall is expanded inside this middle lamellaand consists of several interconnected matricescomposed of polysaccharides and (glyco)proteins.In these matrices, cellulose microfibrils are alignedat all angles and cross-linked via hemicellulosictethers to form the cellulose–hemicellulose network(e.g. xyloglucan and galactoglucomannan).This network is embedded in the gelatinouspectin matrix composed of homogalacturonan,rhamnogalacturonan I and rhamnogalacturonanII (O’Neill and York 2003). Two different typesof primary cell walls are distinguished (Carpitaand Gibeaut 1993): type I is found in all dicotyledons,non-graminaceous monocotyledons andgymnosperms, and typically contains xyloglucanand/or glucomannan and 20–35% pectin. Type IIis found in the monocotyledons of the Poaceaefamily and is rich in arabinoxylan but contains lessthan 10% pectin.Some plants synthesize a secondary wall whichis constructed between the plant cell and the primarywall. The secondary wall mainly providessupport and is comprised primarily of celluloseand lignin. The cellulose microfibrils are generallyaligned in the same direction but, with each additionallayer, the orientation of the microfibrilschanges slightly. The secondary wall is altered duringdevelopmentbysuccessiveencrustationanddepositionof cellulose fibrils and other components.Non-structural components of the secondary wallrepresent generally less than 5% of the dry weightof wood, and include compounds extractable withorganic solvents which can be either polar (e.g. phe-

Degradation of Plant Cell Wall Polymers by Fungi 327nols and tannins) or apolar (e.g. fats and sterols),water-soluble compounds (e.g. sugars and starch)as well as proteins and ashes. Whereas the primarycell wall structure is of similar type for all speciesstudied to date, species and cell-type-specific differencesare typical for the secondary cell wall.The polysaccharides found in the cell wall areeither linear polymers composed of a single type ofglycosyl residue (e.g. cellulose is composed of 1,4-linked β-glucosyl residues), polymers with a regularbranching pattern (e.g. xyloglucan and rhamnogalacturonanII) or, as in the case of rhamnogalacturonanI, substituted with a diverse range of arabinosyland galactosyl-containing oligosaccharideside chains. Understanding the structures of thesepolymers and determining their primary structuresremains a major challenge, especially becausetheir biosynthesis is not template driven (O’Neilland York 2003).III. The Degradationof the Plant Cell WallDue to the overall structural and chemical complexityof many plant cell walls, a complete breakdownof the different components is, in nature,brought about only by a wide range of organismsacting in a consortium, following a characteristicdecomposition sequence which starts with organismscolonizing living plants and ends with theproduction of highly persistent soil humus. Thefungi found in these generalized decomposition sequenceslive in complex and diverse communitiesand are often specialized to degrade only certaintypes of polymers, reflecting their genetic and enzymaticcapabilities. The efficient breakdown ofthe plant cell wall by fungi is linked to their hyphalgrowth, which provides penetrating power,and to highly specialized extracellular plant cellwall-degrading enzyme systems. The enzymaticdecomposition of plant cell walls is normally synergistic:individual, highly specialized enzymes operateas components of multi-enzyme systems toefficiently degrade specific polymers. For the synthesisand export of these enzymes, a sophisticatedgene regulation system and highly productive secretorymachinery has developed. All these characteristicsenable fungi to successfully compete withother microorganisms in their environment, andthey are today the main agents of decomposition interrestrial and aquatic ecosystems.The diversity of substrates has contributed tothe difficulties encountered in enzymatic studies.The degradation has to occur extracellularly, sincethe substrates are usually large polymers whichare also often insoluble or even crystalline. Twoprincipal types of extracellular enzymatic systemsfor the degradation of the polymeric fraction havedeveloped: the hydrolytic system, which degradesthe polysaccharides mainly by hydrolases, anda unique oxidative ligninolytic system whichdepolymerizes lignin. Even for the degradationof the chemically simple polysaccharide cellulose,however, several enzymes are necessary. In general,three classes of enzymes can be distinguished: (i)exo-acting enzymes, which release mainly monoanddimers of the ends of the polymeric chain,(ii) endo-acting enzymes, which cleave in themiddle of the sugar chain and (iii) enzymes (oftenexo-acting) which are specialized in cleaving theresulting oligosaccharides into their monomers.Substituted polysaccharides require additionalsets of enzymes for a complete hydrolysis. Theresulting di- and monosaccharides are then readilytaken up into the cell by different permeases andfurther degraded by a wide range of specializedcatabolic pathways. In contrast to polysaccharides,lignin is a complex heteropolymer with nostereochemical regularity and must, therefore,involve a non-specific and non-stereoselectivemechanism.A. Biodegradation of CelluloseCellulose constitutes the most abundant biopolymeron earth, and accounts for about 50% of theprimary biomass production by plants due to photosyntheticfixation of carbon dioxide. It is estimatedthat approximately 4 ×10 9 tons of celluloseare formed annually (Eriksson et al. 1990).Cellulose is a linear and highly ordered polymerconsisting of about 8,000–12,000 β-1,4-linkedd-glucopyranose units. The polymeric chains arepacked together by hydrogen bonds to form highlyinsoluble microfibrils. Although cellulose hasa tendency to crystallize shortly after biosynthesis,less-ordered amorphous regions can also occur inplant tissues. The complete hydrolysis of celluloseto d-glucose requires a minimal set of threesynergistically acting enzymes: cellobiohydrolases(1,4-β-d-glucan cellobiohydrolases; EC 3.2.1.91),which act processively from the ends of thecellulose chain to generate mainly the glucose

328 C. Gamauf, B. Metz, B. SeibothFig. 18.1. Enzymatic degradation ofcellulose. Different cellobiohydrolases(CBH) act on either the reducingor non-reducing end of the chain.Endoglucanases (EG)hydrolyzeinternalglycosidic bonds, thereby providingadditional sites for the CBHs. Finally,smaller oligomers and the dimercellobiose are cleaved by β-glucosidases(BGL) into d-glucose. Swollenin supportsthe enzymatic degradation ofcellulose by disrupting the microfibrilstructuredisaccharide cellobiose. Based on their primarysequence, the cellobiohydrolases are classifiedin different glycosyl hydrolase (GH) families.Cellobiohydrolases of GH family 6 are characterizedby an inverting mechanism and cleavefrom the non-reducing cellulose ends. By contrast,GH 7 family members cleave cellobiose from thereducing end by a retaining mechanism. Endoglucanases(1,4-β-d-glucan 4-glucanohydrolase; EC3.2.1.4) attack the cellulose chains internally in theamorphous regions, thereby generating additionalsites for the attack of the cellobiohydrolases.Finally, β-glucosidases (EC 3.2.1.21) degradeoligosaccharides and cellobiose to d-glucose(Fig. 18.1; Beguin 1990; Teeri 1997).Alargenumberoffungiareabletogrowonamorphous cellulose or water-soluble derivatesbut relatively few are able to produce a completeenzyme system necessary to hydrolyze crystallinecellulose (Mandels and Weber 1969). Numerousgenes encoding cellulases have been isolated andthe respective enzymes studied in detail. One ofthe best studied cellulolytic fungi is Hypocrea jecorina(Trichoderma reesei), which was discoveredduring World War II on the Solomon Islands asa severe degrader of cellulosic material of the USArmy (Kubicek and Harman 1998). Numerouscellulase-encoding genes have been isolated froma wide variety of fungi. Detailed analyses of theenzymes and gene regulation are available for,e.g. Aspergillus niger, Hypocrea jecorina andPhanerochaete chrysogenum (de Vries 2003; Aroet al. 2005).A structural comparison of the different cellulasesshows that these proteins comprise – besidesthe domain responsible for the actual enzymaticreaction – often several other conserved functionaldomains. One of these domains is involved insubstrate binding and is usually about 40 aa in size.This domain can be found N- or C-terminal andwas originally described as the cellulose-bindingdomain (CBD). Removal of this domain leads toenzymes which are still able to cleave glycosyllinkages from smaller oligosaccharides but thebinding to cellulose and, therefore, the action oncrystalline cellulose is impaired. Later, these CBDswere also found in other carbohydrate-degradingenzymes, e.g. in the H. jecorina mannase, acetylxylan esterase, and in the Humicola xylanase;to date, more than 300 putative sequences havebeen identified. Therefore, such domains withcarbohydrate-binding activity were renamed ascarbohydrate binding modules (CBMs). CBMsare structurally similar, and their carbohydratebindingcapacity can be attributed to severalamino acids constituting the hydrophobic surface.Extensive data, classification and applications ofthese CBMS can be found in the Carbohydrate-Binding Module Family Server (http://afmb.cnrsmrs.fr/CAZY/fam/acc_CBM.html)and in a recentreview by Shoseyov et al. (2006). The CBMdomain is often spatially separated from the

Degradation of Plant Cell Wall Polymers by Fungi 329catalytic core domain by a linker region. Thislinker region is rich in prolines, serins and threonines,and the latter two amino acids are highlyO-glycosylated.The catalytic domain structure of the cellobiohydrolaseII of Hypocrea jecorina was the firstcellulase crystal structure resolved at the atomiclevel (Rouvinen et al. 1990) and has explained whythe enzyme is able to attack the cellulose chainsonly from the end and not from the middle. Thecrystal structure shows a tunnel-shaped active sitewhich is so tight that it can incorporate only onecellulose chain. Despite a similar overall structure,endoglucanases have in general a more open activesite and allow, therefore, an attack of the cellulosechainsfromthemiddle.Theactivesitetopologyof such polymer-degrading enzymes shows thattheseenzymeshaveextendedactivesiteswhichprovide binding sites for a number of sugar units.Thesesubsitespositionthesubstratetightlyandcorrectly with respect to the catalytic aminoacids.In addition to these classical cellulase enzymes,novel types of proteins have recently beendescribed which are involved in plant cell walldegradation: Swollenin (Swo1) from H. jecorinashows amino acid similarity to plant expansins.Expansins induce the extension of isolated cellwalls (McQueen-Mason et al. 1992) and inducenon-hydrolytic activity on cell wall polymers, e.g.pectins and xyloglucans, which are tightly boundto the cellulose microfibrills (McQueen-Masonand Cosgrove 1995). Swollenins are able to disruptthe cellulose microfibrils without any hydrolyticactivity and would, in this way, make the cellulosefibres more accessible for the cellulases to actupon (Saloheimo et al. 2002). Comparisons of theannotated genomes from cellulolytic and hemicellulolyticorganisms with EST data generatedunder appropriate conditions have led to theidentification of new proteins involved in cellulosedegradation (Foreman et al. 2003; Schmoll et al.2003; Schmoll and Kubicek 2005). One exampleis the isolation of a novel type of endoglucanasewhich can be attached to the membrane viaa glycosylphosphatidylinositol anchor. Regulationof cellulases and hemicellulases has been reviewedby de Vries et al. (2002) and Aro et al. (2005).B. Biodegradation of HemicellulosesHemicelluloses are heterogeneous polymers andthe second most abundant natural polysaccharide,accounting for around 20–30% of plant cell wallbiomass (Eriksson et al. 1990). These plant cellwall polysaccharides have a backbone of 1,4-linkedβ-d-pyranosyl residues, with the exception ofarabinogalactan, and are solubilized by aqueousalkali (O’Neill and Selvendran 1985; O’Neill andYork 2003). The backbone can consist of xylosyl-,glucosyl-, galactosyl-, arabinosyl- or mannosylresidues and, depending on the dominant sugar,these are trivially named xylans or arabinogalactans,for example, if both sugars occur innear-equal amounts. The chemical diversity of thehemicelluloses produced by the plants requiresa larger set of enzymes which either act onFig. 18.2. Enzymatic degradation ofhemicelluloses. The main chain of xylanis degraded by endo-1,4-β-xylanases(EXY). Accessory enzymes necessaryfor side group removal are β-xylosidase(XYL), α-glucuronidase (xylan-α-1,2-glucuronosidase, GLU), feruloyl esterase(FES), acetyl xylan esterase (AXE) andα-l-arabinofuranosidase (arabinoxylanarabinofuranohydrolase, ABF)

330 C. Gamauf, B. Metz, B. Seiboththe main chain or attack the side chains. Themain chain is internally cleaved by endo-actingenzymes whereas exo-acting enzymes liberate therespective monomers. In a synergistic degradationpattern, additional enzymes attack the sidechains, leading to the release of various monoanddisaccharides; in this way, the main chainbecomes more accessible for the endo-acting enzymes.Most extensively studied is the enzymaticdegradation of xylan, which involves endoxylanases,β-xylosidases and accessory enzymes(Fig. 18.2).1. XylansXylans are a highly heterogeneous group characterizedby a β-1,4-linked β-d-xylosepyranose backboneand including arabino-, glucurono- and glucuronoarabinoxylans.Xylans are abundant in thewalls of cereals (Poaceae) and in hardwood, e.g. thesecondary walls of woody plants, and are minorcomponents of the walls of dicotyledons and nongraminaceousmonocotyledons (Darvill et al. 1980;Ebringerova and Hienze 2000). Xylans found in cerealsare highly substituted with single residuesor short side chains of α-1,2- or α-1,3-linked l-arabinofuranose residues and are therefore commonlyreferred to as arabinoxylans (Izydorczykand Biliaderis 1995). Glucuronoxylans are typicalhardwood xylans and contain large amounts of α-1,2- and α-1,3-linked 4-O-methyl-α-d-glucuronicacid and acetyl groups at O-2 or O-3. In softwood,glucuronoarabinoxylans are found which are substitutedwith a higher content of α-1,2-linked 4-O-methyl-α-d-glucuronic acid than in hardwoodand, in addition, contain α-l-arabinosefuranosebut no acetyl groups. The l-arabinose residues maybe esterified at O-5 with feruloyl or p-coumaroylresidues, and a number of other minor residueshave been detected, too.The hydrolysis of the xylan backbone involvesendo-1,4-β-xylanases (endo-1,4-β-d-xylanxylanohydrolases; EC 3.2.1.8) and β-xylosidases(1,4-β-d-xylan xylohydrolase; EC 3.2.1.37). Endoxylanasescleave the main sugar chain dependingonthetypeofxylan,thedegreeofbranching,and the presence of different substituents (Polizeliet al. 2005). The main hydrolysis products aresubstituted or non-substituted oligomers whichare further converted by β-xylosidases into tri-, diandmonomers. Endoxylanases can be classifiedaccording to their end product into debranchingand non-debranching enzymes, based on theirability to release l-arabinose from arabinoxylan(Wong et al. 1988). Some enzymes cut randomlybetween unsubstituted d-xylose residues whereasthe cleavage site of some endoxylanases is dependenton the neighbouring substituents of the sidechains. β-Xylosidases can be classified accordingtotheirrelativeaffinitiesforxylobioseorlargerxylooligosaccharides,and release β-d-xylopyranoseby a retaining mechanism from the non-reducingend. β-Xylosidases are in general highly specificfor small unsubstituted d-xylose oligosaccharides,and the activity decreases with increasingpolymerization of the substrates. Accumulationof the short oligosaccharides would inhibit theaction of the endoxylanases but the hydrolysisof these products by β-xylosidases removes thispossible cause of inhibition, thereby increasingtheefficiencyofxylanhydrolysis(Andradeetal.2004). Similar to cellulases, most of the genesencoding endoxylanases and β-xylosidases havebeen characterized in different Aspergillus, Trichodermaand Penicillium spp. as well as in Agaricusbisporus and Magnaporthe grisea.2. XyloglucanXyloglucan is quantitatively the predominanthemicellulosic polysaccharide of dicotyledons andnon-graminaceous monocotyledons, constitutingup to 20% of the plant cell wall. Xyloglucans arestrongly associated with cellulose microfibrillsand support the structural integrity of the cellwall. The backbone is composed of 1,4-linkedβ-d-glucopyranose residues which are substitutedby d-xylopyranose via an α-1,6-linkage.Xyloglucans are classified as XXXG or XXGGtype, depending on the number of backboneresidues with branches; e.g. XXXG have three consecutivebackbone residues which are substitutedwith d-xylopyranose, and a fourth unbranchedbackbone residue. XXXG-type glucans are presentin numerous plant species whereas the XXGG typeoccurs in solanaceous plants. Sugars bound tothe d-xylopyranose include α-1,2-l-fucopyranose,β-1,2-d-galactopyranose, α-1,2-l-galactopyranoseor α-1,2-l-arabinose residues. Some of theseresidues can also contain O-linked acetyl groups(O’Neill and York 2003). Some of the endoglucanasesactive against cellulose are also active onxyloglucans; in addition, xyloglucan hydrolases(EC 3.2.1.151) which are specific for xyloglucanhave been reported (Pauly et al. 1999; Hasper et al.2002; Grishutin et al. 2004).

Degradation of Plant Cell Wall Polymers by Fungi 3313. (Galacto-)glucomannanMannan consists of a β-1,4-linked β-dmannopyranosebackbone whereas, in glucomannans,the backbone comprises both β-1,4-linkedβ-d-glucopyranose and β-d-mannopyranoseresidues which are randomly distributed. It can besubstituted by α-1,6-linked α-d-galactopyranoseresidues which can be substituted further byα-1,2-linked α-d-galactopyranose. These polysaccharidesare usually referred to as galactomannansand galactoglucomannans (Brett and Waldren1996). Galactomannans and galactoglucomannansare the major hemicellulose structuresof softwoods, and glucomannan of hardwood(Aspinall 1980; Stephen 1982). The d-glucoseor d-mannose residues are partially substitutedwith acetyl residues linked to O-2 orO-3. Thebackbone is degraded by endo-1,4-β-mannanases(Mannan endo-1,4-β-mannosidase, EC 3.2.1.78)and β-mannosidases (EC 3.2.1.25). The abilityof the endo-1,4-β-mannanases to degrade thesepolymers depends on the number and positionof the side chain substituents. The enzymesreleasing the glucose and galactose residues actin synergism with endo-1,4-β-mannanases andβ-mannosidases. β-Mannosidases split off the β-d-mannose residue from the non-reducing end ofthe manno-oligosaccharides and are characterizedby a retaining mechanism.C. Biodegradation of PectinsPectins are a complex and heterogeneous groupof polysaccharides characterized by a significantcontent of α-1,4-linked d-galacturonic acids. Theyare found mainly in the middle lamella and inthe primary cell wall, their proportion rangingfrom 5–10% in grasses to 30% in dicotyledons.The carbohydrate composition and, hence, thestructure vary depending on the species and celltype. Pectin is made up of several distinct domainswhich, depending on the side chains, are calledeither “smooth” or “hairy” regions (Pérez et al.2000; Ridley et al. 2001).The “smooth” region, or homogalacturonan(HG), consists of linear chains of α-1,4-linkedd-galacturonic acid residues which can carrymethyl esters at the terminal carboxyl groupand acetyl esters at the O-2 or O-3 position.Homogalacturonan with a high degree of methylesterification is referred to as pectin whereaspectic acid (pectate) has a low degree of esterification.The esterification of the uronic acidgroupresultsintheeliminationofthenegativecharge, which is of great significance for the gellingprocessofpectin,sincethecomplexesbetweenthe carboxyl groups and Ca 2+ ions in additionto borate and uronyl esters are involved in this(Vincken et al. 2003). Additionally, the number ofmethyl- and acetyl esters has a strong influenceon the susceptibility to cleavage by the differentpectinolytic enzymes. Rhamnogalacturonan I+IIand xylogalacturonan (XGA) are known as “hairy”regions due to the abundant and often branchedside chains. Rhamnogalacturonan I distinguishesitself from the other domains in that its backboneconsists of d-galacturonic acid and l-rhamnosein a [1->2)-α-l-Rha-(1->4)-α-d-GalA-(1->] nlinkage. Whereas d-galacturonic acid can besubstituted with either methyl- or acetyl esterssimilar to those in homogalacturonan, 20–80% ofthe l-rhamnose residues are substituted at the O-4position. The substituents contain l-arabinoseand d-galactose, and vary in size from monomersup to branched, heterogeneous oligomers. Theycan be terminated with α-l-fucose and (4-Omethyl)-β-d-glucuronicacid. The arabinan chainhas an α-1,5-linked l-arabinose backbone whichcan be substituted with α-1,3-linked l-arabinoseresidues. Two types of arabinogalactan sidechains have been identified. Type I consists ofachainofβ-1,4-linked d-galactopyranose whereastype II contains a backbone of β-1,3-linked d-galactopyranose residues which can be substitutedwith β-1,6-linked d-galactopyranose residues.Both are occasionally substituted with l-arabinoseat O-3. In addition, ferulic acid and p-coumaricacids have been identified in the pectic hairyregions attached to O-2 of l-arabinose and O-6 ofd-galactose.Rhamnogalacturonan II consists of a shortbackbone of α-1,4-linked d-galacturonic acidwhich is substituted either at the O-2 or O-3position (Vidal et al. 2000). The side chains havebeen found to be either dimers or branchedoligomers, and to contain rare sugars such asd-apiose and l-fucose in addition to l-arabinose,d-galactose and l-rhamnose.Thebackboneofanothersubstructurefoundinhairy regions, the xylogalacturonans, is similar tothat of the homogalacturonans but a major part ofthe d-galacturonic residues carry β-d-xylose substituentsat the O-3 position. It has so far beenfound only in reproductive tissues, including soybeanseed, apple fruit and pine pollen (Schols et al.

332 C. Gamauf, B. Metz, B. Seiboth1995). Whereas the composition and structure ofthe individual subunits are mostly established, themanner in which they make up the pectin polymeris still under investigation. For a long time,pectin was thought to consist of linear chains of homogalacturonaninterspersed with hairy regions.Recently, however, Vincken et al. (2003) proposeda new model in which rhamnogalacturonan I aloneforms the backbone, substituted with homogalacturonanand the abovementioned arabinan andgalactan side chains.To efficiently degrade pectin, fungi have developeda broad spectrum of pectinolytic enzymes.The term “pectinases” usually refers to enzymeswhichactonthepectinbackbone.Thedegradationof pectin has, besides in saprobic fungi, been mostextensively studied in plant pathogenic fungi(Botrytis cinerea, Fusarium oxysporum, Sclerotiumsclerotiorum). Enzymatic depolymerization ofpectin weakens the cell wall and exposes the othercell wall polymers to degradation by other plant cellwall-degrading enzymes. As a protection againstthis enzymatic attack, numerous plants have developedspecific plant defence proteins – the so-calledpolygalacturonase-inhibiting proteins (PGIPs),whichreducespecificallythehydrolyticactivityoffungal polygalacturonases (Cervone et al. 1990).PGIPs are leucine-rich repeat glycoproteins associatedwith the cell wall of both monocoyledonsand dicotyledons (D’Ovidio et al. 2004).Pectinases can be subdivided, in terms of theirreaction mechanism, into hydrolases and lyasesand further according to their substrate specificityinto, e.g. polygalacturonases and rhamnogalacturonases.Pectin lyases (EC 4.2.2.10), pectatelyases (EC 4.2.2.2) and rhamnogalacturonanlyases (EC 4.2.2.-) cleave polysaccharide chainsvia a β-elimination mechanism resulting in theformation of a Δ-4,5-unsaturated bond at thenewly formed non-reducing end.The three-dimensional structure of severalpectinases has been determined. The resultsshow that – in contrast to the cellulases andhemicellulases, which are characterized by a highdiversity of protein structures – pectinases sharea common conserved structure. Although theiroverall sequence similarity is low, pectinases possessa central core consisting of parallel β-strandsforming a large, right-handed helix defined asparallel β-helix (Jenkins and Pickersgill 2001).Whereas the catalytic mechanism differs betweenthe hydrolases and lyases, the substrate-bindingsites are all found in a similar location within a cleftformed on the exterior of the parallel β-helix. Thisstructure facilitates the binding and cleaving ofthe buried pectin polymers in the undamagedcell wall. The parallel β-helix fold confers thestability needed by these enzymes for efficientaggressive action in a variety of hostile extracellularenvironments. An exception to this rule isthe rhamnogalacturonan lyase from A. aculeatus,which displays a unique arrangement of thesethree distinct modular domains (McDonoughet al. 2004).In saprobes, the pectinolytic system has beenstudied in great detail in Aspergillus spp. includingA. niger (de Vries and Visser 2001). Polygalacturonases(PGAs) are the most extensively studiedclassofpectinasesandhavebeenisolatedfromvarious saprotrophic and plant pathogenic fungi.Endopolygalacturonases catalyze the hydrolyticcleavage of α-1,4 d-galacturonic bonds within thechain (EC 3.2.1.15), and exopolygalacturonases(Galacturan 1,4-α-galacturonidase. EC 3.2.1.67)cleave from the non-reducing end. Both endoandexopolygalacturonases belong to glycosidehydrolase family 28, and have similar reactionmechanisms and substrate specificities, but theirlevel of sequence identity is surprisingly low (Henrissatand Bairoch 1993; Biely et al. 1996; Markovicand Janecek 2001). Among the endopolygalacturonases,some enzymes cleave only once perchain (single attack or non-processive) whereasothers attack the pectin polymer multiple timesper strand (processive behaviour). Single-attackpolygalacturonases generally produce longerfragments, which are only gradually degradedinto dimers, trimers or short oligomers, providingpossible sites for exopolygalacturonases. Digestionwith processive enzymes, on the other hand, resultsin an accumulation of these short oligomers fromthe very beginning of the reaction. The circumstancesunder which the one or the other pathwayof degradation is preferred are not completelycleartodate,andmayberelatedtothebiologicalsignificance of the resulting fragments. A factorwhich significantly influences the activity of polygalacturonasesis the number (and distribution) ofmethyl- and acetyl ester groups. In general, mostendo- and exoPGAs prefer substrates with a lowdegree of esterification, although some exceptionsexist (Parenicova et al. 2000). In most cases, theactivity of a methyl/acetyl esterase is required toprepare the pectin molecule for PGA digestion.Genes encoding PGAs are organized into familiesthemembersofwhichexhibitahighdegreeofpoly-

Degradation of Plant Cell Wall Polymers by Fungi 333morphism (Annis and Goodwin 1997; Markovicand Janecek 2001). Most fungi produce multipleisozymes with a wide range of enzymatic properties,substrate specificities and pH optima, whichmay reflect the complexity of the pectin moleculein plant cell walls and the need for enzymes capableof cleaving the homogalacturonan backbone ina variety of structural contexts. Another structuralfeature which may determine the functionaldiversificationoftheseenzymesisthepresenceorabsence and type of N-terminal extension, whichhas been suggested to influence their substratespecificity and to play a role in their interactionwith particular regions of the pectin polymer(Parenicova et al. 2000; Gotesson et al. 2002).The degree of esterification is also importantfor the functional classification of lyases. Pectatelyases prefer substrates with a low degree of methylesterification, which therefore have a more acidiccharacter, and are strictly dependent on Ca 2+ forcatalysis. Pectin lyases, on the other hand, favourhighlymethyl-esterifiedsubstratesanddonotrequireCa 2+ ions (Jurnak et al. 1996).The pectinolytic enzyme system of A. nigerserves as an example of how a microorganism candegrade a pectin molecule. A. niger produces sevenpolygalacturonases, two of them (PgaA and PgaB)constitutively. These two are most active on pectinscontaining 22% methyl esters (Parenicova et al.2000), thus making them suitable for an initial attackonthenativesubstrate.Enzymessubsequentlyinduced at this early stage during growth on pectinare pectin methyl esterase PmeA, an exopolygalacturonasePgxA, and pectin lyases (PelA and PelD;de Vries et al. 2002). The action of the methyl esteraserendersthesubstrateaccessibletopolygalacturonases,which are expressed at a later stage afterremoval of the methyl esters, while the pectinlyases contribute to the breakdown of the still esterifiedpolymer. The exopolygalacturonase cleaves d-galacturonic acid monomers from the homogalacturonanpoly- and oligomers, which may serve asinducers for the other pectinases (Fig. 18.3).Similar to homogalacturonans, the degradationof the rhamnogalacturonan I backbone iscatalyzed by hydrolases and lyases (Fig. 18.3).Endorhamnogalacturonases have been isolatedfrom A. acculeatus and A. niger (de Vries andVisser 2001), and were shown to hydrolyze theα-1,4 glycosidic bonds in saponified hairy regions.Theresultingfragmentsweretetra-andhexamers of the backbone, which partly werestill substituted with d-galactose. This suggeststhat – similar to some exoPGAs ability to cleaveXGA (van den Broek et al. 1996) – endorhamnogalacturonasesare tolerant towards monomericFig. 18.3. Enzymatic degradationof rhamnogalacturonan I and homogalacturonan.The main chain ofrhamonogalacturonan I (shown above)is degraded by rhamnogalacturonanhydrolase (RGA) and rhamnogalacturonanlyase (RGL).The side chainsare degraded by rhamnogalacturonanacetyl esterase (RGAE), endoarabinase(ABN), endo-β-1,6-galactanases (GAL)and exogalactanases (GAX). Terminalmonosaccharides are removed by α-l-arabinofuranosidases (arabinoxylanarabinofuranohydrolases, ABF) andβ-galactosidases (BGA). The mainchain of homogalacturonan (below) isdegraded by endopolygalacturonases(PGA), exopolygalacturonases (PGX),pectin lyases (PLY), pectate lyases (PEL).Pectin methyl esterase (PME)andpectinacetyl esterase (PAE) act on the sidegroups

334 C. Gamauf, B. Metz, B. Seibothsubstituents. Depending on the monosaccharidecleaved from the non-reducing end of therhamnogalacturonan, two kinds of exorhamnogalacturonaseshave been described: rhamnogalacturonanα-d-galactosyluron-hydrolases andrhamnogalacturonan α-l-rhamnohydrolases. Allrhamnogalacturonan hydrolases were classified asmembers of GH family 28. Rhamnogalacturonanlyases cleave the rhamnogalacturonan backbonevia β-elimination. Unlike the hydrolases, they acton the Rha-(1->4)-α-d-GalA bond, resulting inthe formation of Δ-4,5-unsaturated d-galacturonicacid residues at the non-reducing end.D. Accessory EnzymesMost of the plant cell wall-degrading enzymesdescribed above act on the backbone of the respectivepolysaccharide, but usually their activity isimpaired by monomeric substituents or larger sidechains which are present in hemicelluloses andpectins. To ensure an efficient and complete breakdownof such polysaccharides, these substituentshave to be removed and degraded by differentfamilies of accessory enzymes. These accessoryenzymes work in synergism with the enzymesattacking the main chain and often depend on eachother for an efficient breakdown of the substrate.The substrate specificity of these enzymes varies;some of the enzymes can hydrolyze the intact polymerwhereas others show maximum activity onlyin the presence of shorter breakdown products (cf.Puls and Schuseil 1993; Tenkanen and Siika-aho2000). A detailed list of the enzymes involved inthe degradation of hemicelluloses and pectinasesand their mode of action has been reviewed forAspergillus spp. by de Vries and Visser (2001), andonly some of the major enzymes are listed here(Figs. 18.2 and 18.3).Side groups in xylans are generally small(mono-, di- and trimers) but can consist of severaldifferent sugars and acids (e.g. acetic acid, l-arabinose, ferulic acid, d-galactose, d-glucuronicacid) and, consequently, multiple enzymes arerequiredtomakethebackbonefullyaccessiblefor the xylanases. α-l-Arabinofuranosidases (EC3.2.1.55) remove terminal l-arabinose residues butdifferences in the specificity towards α-1,2-, α-1,3-or α-1,5-arabinosidic bounds and towards thesubstrata themselves have been observed. Whereasseveral representatives are also able to releasel-arabinose from pectins and xylans, others – alsocalled arabinoxylan arabinofuranohydrolases –are strictly specific for l-arabinose bound toxylan. In addition, some arabinofuranosidases areinhibited by the presence of d-glucuronic acidresidues adjacent to the targeted l-arabinose. Thisd-glucuronic acid and its 4-O-methyl ethers areremoved by α-glucuronidases (EC 3.2.1.139) andby xylan-α-1,2-glucuronosidases (EC 3.2.1.131).In addition to carbohydrate substituents, twotypesofestersarefoundinxylans–thosewithferulicandthosewithaceticacid–whichareboth hydrolyzed by specific esterases. Severaltypes of feruloyl esterases (EC 3.1.1.73) have beendescribed, their activity varying with the presenceof additional methoxy or hydroxyl substituents onthe ferulic acid’s aromatic ring, and with the typeof linkage (O-2, O-5 or O-6) to the carbohydratechain. Acetyl xylan esterases (EC 3.1.1.72) participatein the breakdown of the xylan backboneby removing acetyl ester groups from O-2 andO-3 of the d-xylose chain, thereby facilitating theaction of the endoxylanases. As is the case witharabinofuranosidases, synergistic effects withmain chain cleaving enzymes have been reported(Kormelink et al. 1993; Tenkanen 1998; de Vrieset al. 2000). So far, only two types of substituentshave been described in galacto(gluco)mannans:d-galactose mono- and dimers, and acetyl esters.The former are removed from the backbone byα-galactosidases (EC 3.2.1.22) whereas the latterare hydrolyzed by acetylglucomannan esterases(EC 3.1.1.-). Both reactions result in an increasedactivity of the endomannases and β-mannosidases(Tenkanen 1998; de Vries et al. 2000). For theremoval of α-1-6-linked d-xylose side groupsof the xyloglucan, specific α-d-xylosidases arerequired.Pectin contains two different types of substituents.On the one hand, acetyl esters at the O-2or O-3 and methyl esters at the carboxy group arebound to the d-galacturonic acid residues in thesmooth regions. On the other hand, polymericside chains consisting mainly of l-arabinoseand d-galactose are found in the hairy regions.Pectin methyl esterases (EC 3.1.1.11) and pectinacetyl esterases (EC 3.1.1.-) have been isolatedfrom different fungal species and they showa prominent synergism with polygalacturonases(de Vries et al. 2000). Similar results were achievedwith rhamnogalacturonan acetyl esterases (EC3.1.1.-) and rhamnogalacturonase or rhamnogalacturonanlyase (de Vries et al. 2000). Thed-galactose or l-arabinose side chains are often

Degradation of Plant Cell Wall Polymers by Fungi 335substituted with several other carbohydrates oracids. These chains are cleaved by endoarabinases(EC 3.2.1.99) and exoarabinases (EC 3.2.1.-), andby endo-β-1,4-galactanases (EC 3.2.1.89), endo-β-1,6-galactanases (EC 3.2.1.-) and exogalactanases(EC 3.2.1.23). Terminal monosaccharides are removedby α-l-arabinofuranosidases (EC 3.2.1.55)and β-galactosidases (EC 3.2.1.23) but additionalenzymes (e.g. α-l-fucosidases, α-glucuronidases)are required to completely degrade the side chains.IV. Biodegradation of LigninNext to cellulose, lignin is the most abundantpolymer in nature and accounts for 15 to 36% ofthe lignocellulosic material. It forms an extensivelycross-linked network within the cell wall, andconfers structural support and decreases waterpermeability. It protects the other, more easilydegradable cell wall components and is thereforethemainobstacleforanefficientsaccharificationofcellulose and hemicellulose. The aromatic polymeris synthesized from the three substituted phenylpropanoidalcohols coniferyl (guaiacyl propanol),synapyl (syringyl propanol) and p-coumaryl(p-hydroxyphenylpropanol). The softwood ofgymnosperms contains mainly coniferyl alcohols,some p-coumaryl but no sinapyl alcohol whereas,in the hardwood of the angiosperms, coniferyland sinapyl alcohols are found in equal amounts(46%), with a minor proportion of p-coumaryl(8%). The lignin polymer is synthesized by thegeneration of free phenoxy radicals, which isinitiated by plant peroxidases-mediated dehydrogenationof the three precursor alcohols. Theresult of this polymerization is a highly insoluble,complex-branched and amorphous heteropolymerjoined together by different types of linkages suchas carbon–carbon and ether bonds. The chemicalcomplexity and structural variability of the ligninpolymer makes it resistant to breakdown byconventional enzymatic hydrolysis and thereforethe initial attack is oxidative, non-specific, nonhydrolyticand extracellular (Kirk and Farrell 1987;Higuchi 1990; Hatakka 1994).Fungi are the most efficient group of organismsable to decompose or, at least, alter the woodstructure. Based on macroscopic characteristics,different types of decay have been distinguished,including white, brown and soft rot. White-rot basidiomycetesare the major group of wood rots todayand the only group which can completely degradelignin into CO 2 and H 2 O. They can overcomedifficulties in wood decay, including the low nitrogencontent of wood (a C:N ratio of about 500:1) andthepresenceoftoxicandantibioticcompounds.Besidesis group, brown-rot fungi are also able to degradewood extensively. Some ascomycetes also colonizewood in contact with soil but alter the lignincomponent only slightly. Their action leads to a decreasein the mechanical properties of wood, givingrisetoso-calledsoftrot,aprocesswhichofteninvolves bacteria. Soft-rot fungi can degrade woodunder extreme environmental conditions (extremewetnessorfrequentdryness)whichprohibittheactivityof other wood-degrading fungi. Soft-rot fungiare relatively unspecialized (hemi-)cellulolytic ascomycetesin the genera Chaetomium, Ceratocystisand Philaophora,andsomebasidiomycetescanalso cause a soft rot-type of decay pattern. In softrot,decaybyfungiiscloselyassociatedwithpenetrationby the fungal hyphae, because the enzymescannot diffuse through the plant cell wall. Two distincttypesofsoftrotarecurrentlyrecognized.Type1 is characterized by longitudinal cavities formedwithin the secondary wall of wood cells, and type 2by anerosionofthe entire secondary wall (Martinezet al. 2005). Although many white rots and brownrots secrete oxidative and hydrolytic enzymes, it isgenerally recognized that their enzymes are unableto diffuse through healthy wood and that smaller,non-proteinaceous molecules are involved in theinitiation of decay.A. Brown-Rot FungiBrown-rot fungi degrade mainly cellulose andhemicellulose of coniferous softwoods and partiallymodify the lignin mainly by demethylation(Eriksson et al. 1990). They attack cellulose inwood, which promotes rapid loss of mechanicalstrength – ultimately, extensively brown-rottenwood consists almost entirely of modified lignin.The term “brown rot” refers to the characteristicsof this decayed wood: a reddish-brown materialconsisting of oxidized lignin, which cracks intocharacteristic brick-like pieces. Representatives ofbrown-rot basidiomycetes comprise Schizophyllumcommune, Fomes fomentarius, Serpula lacrimansand Gloeophyllum trabeum. They are also themajor cause of decay of woods in commercialuse, and have an important role in coniferousecosystems through their contribution to humus

336 C. Gamauf, B. Metz, B. Seibothformation. These fungi grow mainly in the celllumen of the woody cells, and the degradationisnotlocalizedtothefungalhyphaebutfoundat greater distances from these. The extracellularenzymes formed are too large to penetrate healthycell walls and therefore – as noted above –degradation of cellulose by brown-rot fungimust involve diffusible low-molecular agents.Thedegradationprocessisstillnotcompletelyunderstood but it has been suggested that thebrown-rot fungi use both an oxidative and hydrolyticattack. Although some brown rots possesscellobiohydrolases, they generally lack the abilityto hydrolyze crystalline cellulose enzymatically.However, crystalline cellulose can be disruptedwhen classical endoglucanases act together with anoxidative degradation system such as extracellularreactive oxygen species. Reactive oxygen species(ROS), such as hydroxyl radicals (•OH) and theless reactive peroxyl (ROO•) and hydroperoxyl(•OOH) radicals, have been discussed as theagents which initiate degradation (Hammel et al.2002). There is a well-established pathway for thegeneration of these radicals via the Fenton reaction(H 2 O 2 +Fe 2+ +H + →H 2 O+Fe 3+ +•OH). In ordernottodestroythefungalhyphaeandtoactinthelignified parts of the secondary cell wall, the •OHproduction has to occur at a distance from thehyphae, and the fungal reductants should be stableenough to diffuse before they react to reduce Fe 3+and O 2 to Fe +2 and H 2 O 2 .Theproductionof•OHradicals can take place in several ways and differentsystems are being discussed, including secretedhydroquinones, cellobiose dehydrogenases, lowmolecular-weight glycopetides and phenolatechelators.The principle of the quinone redox cyclingfor •OH production is that the fungus reducesthe quinone extracellularly to its hydroquinonewhich then reacts with Fe 3+ to give Fe 2+ anda semiquinone radical. The semiquinone reducesO 2 to •OOH, which is a source for H 2 O 2 ,andis in this way recycled to quinone. Gloeophyllumtrabeum produces extracellular quinonesincluding 2,5-dimethoxy-1,4-benzoquinone and4,5-dimethoxy-1,2-benzoquinone which canreduce Fe 3+ and O 2 rapidly under physiologicalconditions, thereby generating both Fe 2+and H 2 O 2 .Moreover,thefunguswasshowntoreduce the resulting dimethoxyquinones backto hydroquinones, possibly by the action of anintracellular quinone reductase. Another nonenzymaticsystem includes phenolate or catecholechelators (Goodell 2003). These were isolatedfrom culture filtrates of the brown-rot fungusGloeophyllum trabeum and termed Gt chelator.They have a high affinity for the binding of ironandhavetheabilitytoreduceFe 3+ to Fe 2+ .Thetwocompounds 4,5-dimethoxy-1,2-benzenediol and2,5-dimethoxy-1,4-benzenediol were identified inthe Gt chelator fraction, and also their oxidizedbenzoquinone forms (see above). •OH radicalscan also be produced by the extracellular flavohaemoproteincellobiose dehydrogenase (CDH).CDH production has been reported for all typesof wood-rotting fungi (Zamocky et al. 2006), andCDH can act as cellobiose oxidase by reducingO 2 to H 2 O 2 . However, Fe 3+ is a better electronacceptor than O 2 and, thus, CDHs are actually Fe 3+reductases. Glycopeptides, implicated in wooddegradation, have been isolated from G. trabeumand Tyromyces palustris. They reduce Fe 3+ to Fe 2+and bind Fe 2+ (Goodell 2003; Enoki et al. 2003). Inthe presence of H 2 O 2 ,theglycopeptidegeneratesone-electron oxidation and possesses the abilityto oxidize NADH in the presence of oxygen, andthereby produces H 2 O 2 .Most brown rots secrete oxalic acid, which isa strong chelator of Fe 3+ and Fe 2+ but also reducesthepH.ThepHofwooditselfisgenerallyintherange 3–6, and is lowered to pH values between2.5 and 1.7. The reduction of pH is important forthe function of the extracellular enzymes and hasbeen identified as a key factor in several hypothesesrelated to molecular weight degradation systems, asdiscussed in the reviews listed above.B. White-Rot FungiWhiterotsarethemostfrequentlyfoundwoodrottingorganisms and are mainly basidiomycetesbut also some ascomycetes are able to cause whiterot. They are characterized by their ability tocompletely degrade lignin, hemicelluloses andcellulose, thereby often giving rise to a celluloseenrichedwhite wood material. Two differentwhite-rot patterns have been described:(i) Simultaneous (non-selective) delignificationattacks mainly hardwood and degrades cellulose,lignin and hemicellulose simultaneously.The cell wall is attacked progressively fromthe cell lumen towards the middle lamella.Degradation is associated with the fungalhyphae and substantial amounts of undecayedwood remains. Basidiomycetes (e.g. Tram-

Degradation of Plant Cell Wall Polymers by Fungi 337etes versicolor, Irpex lacteus, Phanerochaetechrysosporium, Heterobasidion annosum andPhlebia radiata) and some ascomycetes (e.g.Xylaria hypoxylon) perform this type ofdegradation.(ii) Selective delignification, or sequential decay,is found in hardwood and softwood. The initialattack is selective for lignin and hemicellulose,and the cellulose is attacked later. Ligninis degraded in the middle lamella, which is dissolvedby a diffusion mechanism, and in thesecondary wall. This type of degradation is performedexclusively by various basidiomycetes(e.g. Ganoderma australe, Phlebia tremellosa,C. subvermispora, Pleurotus spp. and Phellinuspini).Many white-rot fungi cause both types of rot, andthe amount of simultaneous or selective decayedwood varies even among different strains of thesame species and depends also on the substrate(Eriksson et al. 1990; Martinez et al. 2005). To date,P. chrysosporium is the most intensively studiedwhite-rot fungus (Cullen and Kersten 2004; Martinezet al. 2004).White-rot fungi degrade lignin via an oxidativeprocess involving peroxidases and laccases(phenol oxidases) which act non-specifically bygenerating lignin free radicals, which then undergospontaneous cleavage reactions. Peroxidases, suchas the lignin peroxidases (LiPs; EC 1.11.1.14),manganese peroxidases (MnPs; EC 1.11.1.13)and the versatile peroxidase (VP), have beendescribed as true ligninases due to their highredox potential which enables them to oxidizenon-phenolic aromatic substrates constitutingup to 90% of the lignin structure. Peroxidasesrequire the presence of H 2 O 2 as acceptor. LiPsare often produced as isoenzymes with a hemegroup in their active centre, and oxidize phenolicand non-phenolic compounds (Hammel et al.1986; Kersten 1990; Hatakka 1994; Eggert et al.1997). The catalytic, oxidative cycle of LiP issimilar to those of other peroxidases. LiP becomeshighly oxidized when H 2 O 2 isreducedtoH 2 O,and a two-electron reaction allows the activatedenzyme to oxidize two substrate units before it isreduced to the peroxidase resting state once again.Veratryl alcohol is oxidized to the short-livedVA cation radical which may oxidize the lignindirectly or pass on the charge to other, more stablecarriers which can act as diffusible mediators.Since enzymes such as LiPs are too large to enterthe plant cell, direct degradation is carried out onlyin exposed regions of the cell lumen (simultaneousdelignification). Microscopic studies of selectivelignin biodegradation revealed that white-rotfungi remove the polymer from inside the cell wall,which can be performed only by indirect oxidationmediated by LiP low molecular-weight diffusiblecompounds capable of penetrating the cell wall.MnPs are closely related to LiPs. They have thesame catalytic cycle involving a two-electronoxidation of the heme by H 2 O 2 , followed by twosubsequent one-electron reductions. MnPs oxidizeMn +2 to Mn +3 , which is stabilized by organicacids such as oxalate, fumarate and malate.Mn +3 acts as diffusible oxidizer on phenolicsubstrates and oxidizes non-phenolic substratavia lipid peroxidation reactions (Martinezet al. 2005).The more recently discovered versatile peroxidase(VP) combines the enzymatic properties ofLiP and MnP and oxidizes both Mn +2 and veratrylalcohol. It oxidizes hydroquinone in the absenceof exogenous H 2 O 2 when Mn +2 is presentin the reaction as well as dimethoxybenzenes. Thecrystal structures have been resolved for both LiPand MnP (Piontek et al. 1993; Poulos et al. 1993;Sundaramoorthy et al. 1994). The prosthetic group(iron protoporphyrin IX) of LiPs is accessible onlythrough a narrow pore (Piontek et al. 2001). Thecatalytic cycle is common to other peroxidases.However, the position of the iron-binding histidineresidue in ligninolytic peroxidases is displaced fartheraway from the heme iron, and, which leadsto an increased redox potential, and the existenceof specific binding sites for substrate oxidation areunique (Martinez 2002).The substrate-binding sites have been identifiedfor LiP, MnP and VP, and explain the dual catalyticproperties of VP. Mn 2+ oxidation occurs ata binding site near the cofactor which enables directelectron transfer. By contrast, veratryl alcoholis oxidized at the surface of the protein by a longrangeelectron transfer mechanism. The rationaleof the existence of this electron transfer mechanismis related to the fact that many of the aromaticsubstrates cannot penetrate inside the LiP/VP and,therefore, these substrates are oxidized at the enzymesurface, and electrons are transferred to theheme by a protein pathway (Sundaramoorthy et al.1997; Doyle et al. 1998; Gold et al. 2000).Laccases are multicopper phenoloxidases andgenerally larger than peroxidases. They performfour one-electron oxidations by reducing O 2 to

338 C. Gamauf, B. Metz, B. SeibothH 2 O, and are only able to directly oxidize phenolsand aromatic amines. The phenolic nucleusis oxidized by removal of one electron, generatingphenoxy-free-radical products, which can leadto polymer cleavage. Due to their low redox potential,non-phenolic substrates have to be oxidizedby other mediators. Metabolites such as 3-hydroxyanthranilate can mediate oxidation in Pycnoporuscinnabarinus (Eggert et al. 1997), and alsolignin degradation products can act as such redoxcharge transfer molecules (ten Have and Teunissen2001).Other extracellular enzymes involved in woodlignin degradation are H 2 O 2 -generating enzymeswhich are essential for the peroxidases. Fungaloxidases which can reduce O 2 to H 2 O 2 include glyoxaloxidase, glucose 1-oxidase, methanol-oxidaseand aryl-alcohol oxidase (Zhao and Janse 1996).Flavin is often used as cofactor, with the exceptionof, e.g. copper-containing glyoxal oxidasefrom P. chrysosporium.Cellobiosedehydrogenaseoxidizes soluble cello- and mannodextrine anduses a wide spectrum of electroacceptors (Fe 3+ ,Cu 2+ , quinone and phenoxy radicals). Proposedroles of CDH in the ligninolytic system whichhave been discussed comprise (i) the reductionof aromatic radicals formed by ligninolyticenzymes, thereby preventing repolymerizationand supporting lignin degradation, (ii) theproduction of •OH radicals via a Fenton-typereaction to modify cellulose hemicellulose andlignin and (iii) a cooperation with the manganeseperoxidases to make the abundant non-phenoliccomponents of lignin accessible for MnP andlaccases. 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Biosystematic IndexAcari, 202Acremonium, 216Adorphorus couloni, 175Agaricus,78arvensis,78bisporus, 77, 330campestris,78edulis,78haemorrhoidarius,78xanthodermus,78Agrocybe rivulosa, 115Alternaria, 289alternata,74solani, 193Amanita, 209caesarea, 115muscaria, 78, 113, 116, 237, 238, 240phalloides, 116rubescens,78strobiliformis,78Ampelomyces quisqualis, 130Angiosperms, 232, 244Annelida, 202, 203Anoplophora glabripennis, 178Anthurus archeri, 116Antrodia vaillantii,72Aphelenchoides, 209Aphis gossypii, 175Archaea, 7Armillaria, 61, 77, 209ectypa, 119mellea, 106Arthrobotrys, 303Arthropoda, 202Aschersonia aleyrodis, 160, 171Ascobolus, 109, 112Ascomycota, 9, 11, 139, 230–233, 303, 310Aspergillus, 7, 74, 220, 289, 326, 332, 334aculeatus, 332flavus, 163, 177fumigatus, 296niger, 288, 289, 328, 332, 333wentii,58Astigmata, 203Aureobasidium, 289, 291pullulans, 70, 75, 303Bacillus, 191, 193amyloliquiefaciens, 192-193cereus, 193circulans, 289subtilis, 192-193, 223thuringiensis, 164, 165Balansia, 216cyperi, 218Basidiomycota, 9, 11, 230, 232, 236heterobasidiomycetes, 232homobasidiomycetes, 231, 232Bdellidae, 203Beauveria, 174, 175bassiana, 160, 174, 175, 177–180brongniartii, 163, 70, 175-176, 178sulfurescens, 179bassiana, 170, 171, 176Bjerkandera,71Boletus, 78, 209obscurecoccineus, 113edulis, 120Bombyx mori, 177Botryodiplodia theobromae, 193Botryosphaeria, 221Botrytis, 207cinerea, 196, 332Bovista, 109, 112verrucosa, 112Brassica napus, 189Buergenerula, 304Cadophora (syn. Phialophora) finlandia, 232Calluna,75Candida, 7, 289Cantharellus formosus, 121Carex walteriana, 317Castanea dentata, 116Cenococcum geophilum, 231Cephalanthera, 246damasonium, 246Ceratobasidium cornigerum, 246Ceratocystis, 335ulmi, 116Cercosporella, 116Ceriporiopsis subvermispora, 337Chaetomium, 335Chironomidae, 203Chytridiomycota, 8, 10, 13Cladosporium, 75, 206, 220, 289cladosporioides, 74, 296herbarum, 289, 303Claviceps, 216purpurea, 218Clavicipitaceae, 216Coelomomyces, 159

342 Biosystematic IndexCollembola, 202, 203, 206Colletotrichum, 221musae, 222Collybia, 293johnstonii,61Conidiobolus, 159Coprinus comatus,78Cordyceps, 159, 160, 171, 216Coriolopsis,71Coriolus versicolor, 293Corniothyrium minitans, 131Cortinarius,59metallicus, 113Cryptonectria parasitica, 116Cunninghamella,71Curtobacterium flaccumfaciens, 223Curvularia, 222Cydia pomonella, 170Cymatoderma elegans, 113Cyperus rotundus, 218Cypripedium, 245Dactylaria, 303Delia floralis, 178radicum, 178Dendrobaena octaedra,59Dermocybe,59Dermoloma, 119Dichanthelium lanuginosum, 222Disciseda, 119Discula quercina, 220umbrinella, 220Dorylaimida, 202Encephalitozoon, 7Enchytraeidae, 202Endogone, 231Entomophthorales, 171Entamoeba,7Entoloma, 106, 119Entomophaga, 159aulicae, 163, 176grylli, 163maimaiga, 164, 200, 201Entomophthora muscae, 163Epichloë, 216typhina, 216Epicoccum, 206nigrum, 303Epipactis, 246Erianthus giganteus, 306Erica,75EricalesArbutoideae, 232Ericaceae, 232Monotropoideae, 232, 245Eremaeidae, 203Erwinia amylovora, 191Erynia, 159radicans, 163Eucalyptus urophylla, 195Eurygaster integriceps, 178Flammulina, 209Folsomia, 206–208Fomes fomentarius, 115, 335Fusarium, 7, 61, 71, 74, 208graminearum, 219moniliforme, 219oxysporum, 193, 332Gamasina, 203Gaeumannomyces graminis,54Ganoderma australe, 337Geastrum, 119Gibberella fujikuroi, 219Gigaspora spp., 56Gliocladium, 7, 132Globodera, 147, 153Gloeophyllum trabeum, 335, 336Glomerales, 232Glomeromycota, 9, 230–232Glomus, 56, 236constrictum,56etunicatum,56fasciculatum, 289intraradices, 231, 236, 239, 240, 243mosseae, 236, 239, 240versiforme, 236Goodyera repens, 246Guignardia, 221GymnospermsPinaceae, 232Gynoxis oleifolia, 220Hebeloma cylindrosporum, 237, 240Hemileia vastatrix, 116Heterobasidion annosum, 337Heterodera, 147Histiostomatidae, 203Humicola, 295, 296, 328Hygrocybe, 119Hymenoscyphus ericae, 232Hyphoderma setigerum, 293Hypholoma, 208Hypocrea, 132jecorina, 328, 329Hypoxylon fragiforme, 220Irpex, 71lacteus, 337Juncus, 306effusus, 303, 307, 308, 318roemerianus, 305, 306Laccaria, 238amethystina,77bicolor, 236accata,78Lagenidium giganteum, 159, 163, 171Larix decidua, 115Lecanicillium, 160longisporum, 160, 170, 171, 174–176, 179muscarium, 170, 174, 179Lentinus, 109Leptinotarsa decemlineata, 170Leptosphaeria, 189, 303

Biosystematic Index 343coniothyrum, 296Limodorum, 246Littorina, 206Locustana pardalina, 175Lolium, 218Lophiodermium piceae, 220Lumbricus, 203terrestris,59Lumbricidae, 202Lycoperdon,78americanum, 113echinatum, 113perlatum,78Lycoriella, 207Lymantria dispar, 164Macrophomina phaseolina, 193Manduca sexta, 180Marasmiellus, 290, 293, 303Marasmius, 204, 290, 293androsaceus, 296oreadus,78Medicago truncatula, 236Meloidogyne, 147Melolontha melolontha, 175Metarhizium, 172, 174, 177, 178anisopliae, 159, 160, 170, 171, 175, 176, 178–180var. acridum, 175var. majus, 160flavoviride, 173Mesostigmata, 203Monotropa, 245Mortierella, 208Mucor, 71, 74haemalis, 159heimalis, 296Mutinus ravenelii, 116elegans, 116Mycena, 204, 293pura,78Mycetophilidae, 203Mycosphaerella, 304, 308, 317Nectria invertum, 74Nematoda, 202, 203Neocallimastix,7,8Neotyphodium, 216lolii, 218Nilaparvata lugens, 178Nuclearia,8Nomuraea rileyi, 160, 163, 170, 171Oniscus, 203Onychiurus, 205, 207, 208Oomycota, 139Oppiidae, 203Orchidaceae, 245Ostrinia nubilalis, 175Otiorhynchus sulcatus, 160Oxyporus nobilissimus, 119Paecilomyces, 7, 205, 289arinosus, 173fumosoroseus, 170, 172, 173, 176, 180lilacinus,74Pandora, 159neoaphidis, 160, 171Panellus copelandi, 303Pantoea agglomerans, 192, 193Paxillus, 205, 206, 234Penicillium, 71, 74, 206, 220, 288, 289, 326hordei, 296janczewskii, 296janthinellum,72purpurogenum, 296Pestalotia, 221Phaeosphaeria, 304halima, 304, 308nodorum, 107spartinicola, 304, 307, 308, 317Phalansterium,7Phallus hadriani, 112Phanerochaete,71chrysogenum, 326, 328chrysosporium, 70, 72, 337, 338Phellinus pini, 337weirii,61Philaophora, 335Phlebia adiata, 293radiata, 337remellosa, 337Phoma, 130, 206, 289fimeti,74typharum, 303Phoridae, 203Phthiracaridae, 202Phragmites, 307, 318australis, 303, 304, 307–309, 316–318Phyllosticta, 221multicorniculata, 220Phytophthora,61Phytophthora infestans, 116, 222, 224Picea, 206abies, 115Pichia,7Pinus, 206sylvestris, 237Piromyces,7Pisolithus, 234, 238tinctorius, 238Pleurotus, 71, 337nebrodensis, 118ostreatus,72Plicaturopsis crispa, 115Plutella xylostella, 176Podaxis pistillaris, 113Populus fremontii, 220tremula x tremuloides, 238Prostigmata, 203Pseudomonas, 191aeruginosa, 196chlororaphis, 193fluorescens, 191, 195Psilocybe cyanescens, 115Puccinia chondrillina, 116triticina, 224Pycnoporus cinnabarinus, 115, 338Pyrenophora tritici-repentis, 224

344 Biosystematic IndexPythium nunn, 130Ralstonia solanacearum, 195Ramalina menziesii, 290Resinicium bicolor, 289Rhabdocline parkeri, 220Rhizoctonia solani, 54, 55, 193Rhizographus, 291Rhizopogon, 234Rhizopus, 74, 294Rhodospirillum,13Rhytismataceae, 221Rozella,8allomycis,8Russula,78castanopsidis, 115densifolia, 115laurocerasi, 115lepida, 115lilacea, 115Saccharomyces, 7Saccobolus, 109Sarcodon imbricatus, 117Schistocerca gregaria, 160, 172, 175Schizophyllum commune, 111, 335Schizopora flavipora, 115Sciaridae, 203Scleroderma, 109, 112Sclerotinia sclerotiorum, 189Sclerotium rolfsii, 193sclerotiorum, 332Sebacina, 232Serpula lacrimans, 335Solanaceae, 236Spartina, 304–307, 317alterniflora, 303, 304–308, 316Sporidesmium sclerotivorum, 129Sporobolomyces, 289Sporothrix insectorum, 170Squamanita odorata, 112Stemphylium, 294Stenotrophomonas maltophilia,72Sterium hirsutum, 293Stigmaeidae, 203Stropharia aurantiaca, 115rugosoannulata, 115Suillus, 234variegatus, 209,Talaromyces flavus, 131Tappania,4Tarsonemidae, 203Taxomyces andreanae, 224Terminalia,60Thelephora, 109Torubiella, 171Tracipleistophora,7Trametes,71versicolor, 337Trichoderma, 7, 74, 128, 153, 206, 294, 326, 330reesei, 328viride, 296Tricholoma matsutake, 121Tsuga mertensiana,61Tuber, 238melanospermum, 113Tulasnella, 232Tulasnellaceae, 245Tulostoma, 119Tydaeidae, 203Tylenchida, 202Tylospora fibrillosa, 106Typha, 303, 307, 318angustifolia, 306, 307latifolia, 303Tyromyces, 336Ulocladium consortiale, 296Vaccinium, 75Varroa destructor, 178Venturia ditricha, 220Verticillium, 153, 296lecanii, 170, 172Vuilleminia comendens, 293Xylaria, 221hypoxylon, 337Zonocerus variegatus, 175Zoophthora radicans, 164, 176Zygomycota, 10, 231Endogonales, 232

Subject Indexacid rain, 58, 72actinomycetes, 259, 275, 295aggregatusphere, 49, 50agriculture, 223, 265agrochemicals, 258, 275air pollution, 58airborne pollutants, 72algae, 288, 289Allee effect, 179alphaproteobacteria, 3, 5, 7, 11–14amino acid, 237–239, 241glutamine, 238, 241glycine, 237, 246reduction, 239amitochondriate, 7ammonium, 237–239antibiosis, 128, 192direct antagonism, 192limitationinnutrients,193low-molecular weight, 192antifungal agents, 258, 260, 261, 264antifungal volatiles, 194benzothiazole, 194decimate ascospore production, 194good soil amendments, 194nonanal, 194reduce apothecial formation, 194sclerotial control, 194aquatic ecosystems, 301aquatic hyphomycetes, 309–311, 313–315, 317arbuscula mycorrhiza, 74, 75, 229, 230, 231, 233–236, 237–243, 245arbuscule, 236, 240arbutoid mycorrhiza, 229, 232Arthropoda, 202ascostromata, 265, 267bacteria, 7, 49, 56, 71, 74, 287, 289, 295, 296, 316bacterial, 289aggregates, 191endophytic mode, 191extracellular polysaccharide production, 191flagellar motility, 191pigmentation, 191production of biosurfactants, 191baculoviruses, 180balsam fir, 220barcode DNA, 20barley, 236barrier, 113BCAapplication, 192application at the cotyledon stage, 192appropriate place and time, 192bacteria onto the petals, 192host is susceptible, 192pathogen life cycle, 192bikont, 7bioassay, 171, 260bioavailability, 70biodegradation, 71biodiversity, 31, 32, 221, 259neutral theory, 221bioenergetic electron flow, 11, 13diagram, 8, 14biogenic minerals, 76biogeochemical cycles, 75biogeography, 105, 221bioindicator, 78, 79biological control, 223, 127, 190antagonist, 190antifungal antibiotics, 190bacteria, 190enzymes, 190mechanism, 190siderophores, 190systemic resistance, 190volatiles, 190biomarkers, 69biomass, 49, 304, 311, 315, 316bacterial, 56, 71, 316fungal, 49–51, 56, 61, 304, 311, 315, 316microbial, 49–51, 56, 59, 61biomimetic system, 139biopesticide, 165, 177, 178bioprospecting, 223bioremediation, 71biosorption, 74, 76biosynthesis, 258, 275biotechnological application, 325enzyme, 325fungi, 325biotechnology, 223biotrophic, 128birch, 220BLAST, 221blastospores, 173, 174brown-rot fungi, 72, 335C:N ratio, 290, 291C:nutrient ratio, 294canola, 189

346 Subject Indexblackleg, 189stem rot, 189vegetable oil, 189carbohydrate binding module, 328carbohydrates, 235, 239–241fructose, 240glucose, 240, 243hexose, 240, 241monosaccharide, 240, 241sucrose, 240carbon 234, 235, 238, 239, 241–245CAZy database, 326cellulose, 203, 291, 326, 327-binding domain, 328to lignin ratio, 291chelators, 236defense, 260, 265, 266ecology, 259, 266, 274chitin, 202chitinases, 89, 131-135, 140-142, 147, 150, 153, 154, 172,195-197Choanozoa, 7, 8coevolution, 217commercial producers, 165, 170common mycelial networks, 242, 243, 246community, 32, 218, 304structure, 204, 311competition, 31, 128compost, 19, 87, 135-136, 173, 175conservation, 105of fungi, 117convergence, 4copper, 70coprophilous, 271fungi, 261–264cottonwood, 220Cryptostigmata, 202cultivation, 172diphasic fermentation, 173submerged liquid fermentation, 172surface cultivation, 173dark septate endophytes, 233, 242decline and extinction of fungi, 116decomposers, 31, 301, 303, 309decomposition, 218, 301, 317wood, 313delignification, 336, 337selective, 337simultaneous, 336dereplication, 260destruxin, 177detritusphere, 49, 50DGGE, 17, 18, 21, 25, 136, 215, 311diel periodicity, 308, 316disease, 54, 61laminated root rot, 61take-all, 54disjunct distribution, 110distributionatlas, 113maps, 105, 113patterns, 105, 107, 109, 110, 304changes in pattern, 115continental patterns, 106, 112expansion areas, 115global patterns, 106, 109local patterns, 107, 114national patterns, 113regional patterns, 106, 113distributional data, 106DOM, 318dot maps, 107Douglas fir, 60, 214, 220, 221, 294drench, 175drilosphere, 49, 50earthworm, 48, 50, 59invasive, 48, 59ectoendomycorrhiza, 229ectomycorrhiza, 72, 229, 231–246ectomycorrhizal fungi, 74, 75electrostatic sprayers, 175elicitors, 134endemics, 111endophyte, 175, 213, 222, 260, 268, 269bacteria, 223evolution, 216infection, 220inoculum, 220life cycles, 216transmission, 220endospores of Bacillus, 192endosymbiont, 3, 7, 13environmental pollutionacidification, 120forests on acidic soils, 120grassland, 119nitrogen accumulation, 120old-growth forests, 119peat bogs, 119pristine, 119sand dunes, 119enzyme, 150, 154, 287, 291, 293, 296, 307, 314, 327α-L-arabinofuranosidase, 334, 335α-galactosidase, 334α-glucuronidase, 334β-N-acetylglucosaminidase, 295β-galactosidase, 335β-glucosidases, 328β-mannosidases, 331β-xylosidases, 330acetyl xylan esterase, 334acetylglucomannan esterase, 334acid phosphatase, 293arabinoxylan arabinofuranohydrolase, 334aryl-alcohol oxidase, 338cellobiohydrolases, 327cellobiose dehydrogenase, 336, 338collagenase, 154endo-β-1,4-galactanase, 331, 335endo-β-1,6-galactanase, 335endo-acting, 327endo-cellulase, 295endoarabinase, 335endoglucanases, 328, 330endopolygalacturonase, 332endorhamnogalacturonase, 333

Subject Index 347endoxylanases, 330exo-acting, 327exo-cellulase, 295exoarabinase, 335exogalactanase, 335exopolygalacturonase, 332, 333exorhamnogalacturonase, 334feruloyl esterase, 334glucose 1-oxidase, 338glyoxal oxidase, 338invertases, 240keratinase, 154laccase, 337lignin peroxidase, 337lignocellulase, 293manganese peroxidase, 337methanol-oxidase, 338N-acetylglucosaminase, 293pectate lyase, 332, 333pectin acetyl esterase, 334pectin lyase, 332, 333pectin methyl esterase, 333, 334pectinase, 332peroxidase, 337phenol oxidase, 337phosphatase, 235, 236phosphoesterase, 236phytase, 236protease, 151rhamnogalacturonan acetyl esterase, 334rhamnogalacturonan lyase, 332, 334rhamnogalacturonase, 332serine protease, 150, 151subtilisin, 151xylan-α-1,2-glucuronosidase, 334xyloglucan hydrolases, 330epiparasites, 245epiphyte, 216, 221ergosterol, 233, 304, 305ergot, 265, 266ericoid mycorrhiza, 229, 242eukaryotes, 3-5, 7, 11-14, 86amitochondriate eukaryotes, 5european beech, 220European Council for Conservation of Fungi, 118European Union, 177extinction, 116extraradical mycorrhizal mycelium, 230, 233–235, 237–239,241Fenton reaction, 336fermentation, 265, 275ferns, 245fingerprint methods, 22–24AFLP, 17, 18, 23, 25DGGE, 17, 18, 21, 25, 136, 215, 311RAPD, 17, 18, 23, 25, 215RFLP, 17, 18, 23, 25, 176, 215, 304, 311foliar application, 190biocontrol agents, 190cotyledon, 190petal, 190six-leaf growth, 190food preference tests, 204food-web, 53formulation, 174fossil, 4, 5, 11, 71, 110, 230, 232, 325, 326FPOM, 302,318, 319freshwateraquatic fungi, 263marsh, 303fungalpopulations, 70, 72, 74-to-bacterial ratio, 48, 49, 58fungi asbiotrophs, 325endoparasites, 149entomopathogens, 150necrotrophs, 325nematophags, 149, 150saprotrophs, 325fungicolous, 270, 273genet, 115, 242, 243geneticmanipulation, 141modification, 179geographic barriers, 111geological timescale, 3Archaean, 3, 4Hadean, 3Phanerozoic, 3Proterozoic, 3, 4gliovirin, 128, 133glomalin, 52glycogen, 241grass, 216maize, 219ryegrass, 219tall fescue, 219grazing intensity, 207green fluorescent protein, 177, 180Green Muscle, 169, 170, 178grid maps, 107gut contents, 204Hartig net, 240heavy metals, 48, 296hemicellulose, 96, 307, 314, 326, 329, 331, 334-338arabinogalactan, 329galactoglucomannans, 331galactomannans, 331glucomannans, 331mannan, 331xylans, 330gypsy moth, 297herbivores, 218, 222heterokaryosis, 231heterologous isolate, 171homokaryosis, 231homologous isolate, 171, 172homoplasy, 4hot particles, 296hybridization, 18, 24, 25, 217AFLP, 17, 18, 23, 25DGGE, 17, 18, 21, 25, 136, 215, 311FISH, 18, 24, 97, 311RAPD, 17, 18, 23, 25, 215

348 Subject IndexRFLP, 17, 18, 23, 25, 176, 215, 304, 311hydrogen hypothesis, 13hydrogenosomes, 11, 12hyperthermophilic bacteria, 5immobilization, 293, 294, 313nitrogen lock-up, 294immunological methods, 17, 18, 25ELISA, 18, 19, 305, 311indole diterpenoids, 266induced systemic resistance, 134, 195enhancement of lignification, 195stimulation of host-defence enzymes, 195synthesis of pathogenesis-related (PR) proteins, 195infectioncotyledons, 189petals, 189stem, 189interaction, 214antagonism, 224International Union for Conservation of Nature, 118introductions of fungi, 116invasion, 160appressoria, 162cuticle, 160enzymes, 160germination, 160haemocyte, 162proteases, 162ITS, 19, 20, 22-24, 221, 273, 304, 310lateral gene transfer, 8. 11, 13lead, 70leaf surface, 191lectin, 138legal protection, 120lichens, 77, 223, 288–290lignin, 57, 58, 291, 307, 312, 314, 335lignin:N ratio, 290, 291lignocellulose, 238lipids, 241fatty acids, 241phospholipid fatty acids, 233triacylglycerides, 241litter, 49, 301quality, 49, 50litter/substrate quality, 57liverwort, 245LUBILOSA, 178lycophytes, 245lytic enzyme, 140maceration, 314macrofungi, 77, 78maize, 236management of habitats, 120mapping, 106of fungi, 105programmes, 106, 113melanin, 76, 162, 296metalimmobilization, 76mobilization, 76pollution, 74metalloidtransformations, 75metalloids, 75, 77, 79arsenic, 75, 77selenium, 75, 76, 77tellurium, 75, 77metals, 69, 70, 72–79silver, 76, 77aluminium 76, 77calcium, 76cadmium, 73-78cobalt, 76, 77copper, 73, 74, 75, 77, 78chromium, 74cesium, 73, 76, 77iron 76mercury 74-78potassium, 76magnesium 76manganese 76, 77sodium, 76nickel, 74, 76, 77lead, 74, 76–78strontium, 77zinc, 74–78microbial insecticides, 178microclimate, 191humidity, 191leaf wetness, 191temperature, 191ultraviolet radiation, 191microscopy, 214microsporidia, 8mineralization, 290, 294, 296, 309mitochondria, 3,-5, 8, 11-14,mitosomes, 11molecular clock, 4, 11molecular markers, 176monotropoid mycorrhiza, 229, 232morphospecies, 223mosquitoes, 180mutualists, 31, 214mycelial preparations, 174mycoheterotrophic, 244–246cheaters, 245, 246mixotrophy, 245, 246mycoheterotrophy, 244, 245mycoinsecticides, 165, 170 179Green MuscleTM, 169, 170, 178LUBILOSA, 170mycoparasites, 269, 270, 271mycoparasitism, 128mycophilic, 270mycorrhiza(e), 51, 59, 62, 214, 287, 289arbuscular, 51, 52, 55, 56, 60, 289ectomycorrhiza, 51, 52, 53, 58–60, 289, 297extraradical hyphae, 51, 55, 56extramatrical hyphae, 56intraradical hyphae, 51mycorrhizal fungi, 78, 289, 297mycorrhizosphere, 230mycotoxins, 177, 257, 268natural product, 257, 262, 263, 265, 266, 271, 275

Subject Index 349antiinsectan, 266, 267, 273nature reserves, 120necrotrophic, 128necrotrophs, 287nematodes, 49, 53, 147-155, 164, 202-209, 236, 296nitrate, 238, 239nitrogen, 48, 50, 56–58, 60, 61, 234, 235, 237–239, 241, 243non-target invertebrates, 176nutrient, 47, 48, 50, 51, 53, 55–57, 59–62, 313dissolved, 313enrichment, 313limiting, 313Opisthokonta, 7orchid, 245, 246mycorrhiza, , 232, 229Oregon white oak, 220organic acids, 288, 289citric, 288, 289formate, 289malate, 289oxalic, 288, 289succinic, 289tartaric, 288organometals, 77organomercury compounds, 77organotins, 77oribatid mites, 202outline maps, 107PA23 induced resistance, 195palm, 222parasexual, 217pathogen suppressiveness, 135pathogenesis, 180pathogens, 222pectin, 307, 314, 326, 331homogalacturonan, 331rhamnogalacturonan, 331xylogalacturonan, 331peptides, 237perithecia, 216pest control, 163classical, 163combinations, 164imidacloprid, 164inoculative augmentation, 164inundative augmentation, 164mycoinsecticide, 164pesticide, 275pharmaceuticals, 257phosphate, 235, 236, 239, 240phosphate transporters, 236polyphosphate, 236phosphorus, 57–59, 61, 234, 235, 237, 243photosynthesis, 222, 239–241, 244, 246achlorophyllous plants, 244, 246phylloplane, 290, 291phyllosphere biocontrol, 191antibiotics, 192phylogenetic tree, 3, 5eukaryote tree, 7long-branch attraction, 5phylogenies, 3phylogeography, 221phytochemicals, 171phytotoxins, 269, 270plant cell wall, 326composition of, 326degradation of, 327plant diseasebrown rust of wheat, 224citrus variegated chlorosis, 223tan spot of wheat, 224plant litter, 301plant pathogenic fungi, 269, 270plant pathogens, 127, 148pollutants, 69, 70, 296enhanced carbon dioxide, 296ozone, 296sulfur dioxide, 296UV-B light, 296polycyclic aromatic hydrocarbons, 70, 71polygalacturonase-inhibiting protein, 332poplar, 240porosphere, 49, 50potato, 236prairie, 56pre-emptive colonization, 194competence, 194niche exclusion, 194successful colonization, 194synchronization, 194productsBotaniGard® ES, 169Green Muscle, 169, 170, 178Vertalec, 168productionbacterial, 316fungal, 304, 311, 315, 316plant, 316prokaryotes, 7proteins, 237, 238protists, 4, 12PSD, 177pyromorphite, 76radiocaesium, 77, 78, 294-296radionuclides, 73, 76, 77, 79, 296, 297ratiosC:N, 290, 291, 306, 313, 314C:nutrient, 294C:P, 306, 313, 314cellulose:lignin, 291lignin:N, 290, 291rDNA, 230, 231reactive oxygen species, 336recombination, 217Red Data List, 118reproduction, 304, 317respiration, 308, 309, 317, 318rhizomorphs, 234, 242, 290cords, 293, 295rhizosphere, 49, 289rice, 236risk assessment, 176rock, 287–289amphibolite, 288

350 Subject Indexbiotite, 288, 289biotites, 289feldspar, 288feldspars, 289fluorapatite, 289granite, 289hornblende, 289limestone, 289marble, 289microcline, 288, 289muscovite, 289orthoclase, 288phlogopite, 289sandstone, 289shales, 289strontianite, 289salt marsh, 303sclerotia, 265–267, 270secondary chemicals, 291polyphenols, 291tannins, 291secondary metabolites, 217, 257, 259–261, 268, 271alkaloid, 217ergot, 217ergovaline, 218loline, 218lolitrems, 218lysergic, 218peramine, 218taxol, 224toxic syndromes, 219toxicity, 219seed treatment, 134seedlings, 244selective grazing, 206senescence, 222soft-rot, 58soil, 47, 50, 56aggregate, 47, 49–56, 62erosion, 51, 53, 57, 59organic matter, 47–50, 52–57, 61pH, 59structure, 47, 51–54, 56, 62spruce, 220stable isotope analyses, 230, 234, 237, 239, 241, 244–246isotopic signatures, 235sterile dark fungi, 207stoichiometry, 306, 313, 314stromata, 216, 273stromatolites, 4succession, 32, 291, 303suppressive environment, 135swollenin, 329symbiotic (mycorrhizal) interface, 232, 233, 235, 238, 240synergism, 128, 164imidacloprid, 164synergistic effects, 261T-RFLP, 304, 311tannins, 238trampling of the soil, 121trehalose, 203, 241trophic properties, 7trophic traits, 3chemotrophs, 14heterotrophs, 8, 14litho-organotrophs, 14organotrophs, 8, 14osmo/phagotrophically, 8osmotrophs, 8, 14phagotrophy, 8unikont, 7water availability, 308WCS374r, 195weathering, 236, 238western white pine, 221wetland, 301, 303white-rot, 58fungi, 71, 336wood, 305, 309, 311–314, 316-decay fungi, 271, 273wood-wide web, see common mycelial networksxenobiotics, 70yew, 224

Environmental and Microbial Relationships

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