[Dilip K. Arora] Fungal Biotechnology in Agricultu(BookFi)

FungaI BiotechnoIogy in Agricultural, Food, and Environmental Ap pIications edited by

Ddip K. Arora National Bureau of Agriculturally Important Microorganisms New Delhi, lndiu

Associate Editors Paul D. Bridge British Antarctic Survey Cambridge, United Kingdom

Deepak Bhatnagar U S . Department of Agriculture

New Orleans, Louisiana, U.S.A.

m MARCEL

DEKKER

MARCEL DEKKER, INC.

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MYCOLOGY SERIES

Editor

J. W. Bennett Professor Department of Cell and Molecular Biology Tulane University New Orleans, Louisiana Founding Editor

Paul A. Lemke

1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke 2. The Fungal Community: Its Organization and Role in the Ecosystem, edited by Donald T. Wicklow and George C. Carroll 3. Fungi Pathogenic for Humans and Animals (in three parts), edited by Dexter H. Howard 4. Fungal Differentiation:A Contemporary Synthesis, edited by John E. Smith 5. Secondary Metabolism and Differentiationin Fungi, edited by Joan W. Bennett and Alex Ciegler 6. Fungal Protoplasts, edited by John F. Peberdy and Lajos Ferenczy 7. Viruses of Fungi and Simple Eukaryotes, edited by Yigal Koltin and Michael J. Leibowitz 8. Molecular lndustrial Mycology: Systems and Applications for Filamentous Fungi, edited by Sally A. Leong and Randy M. Berka 9. The Fungal Community; Its Organization and Role in the Ecosystem, Second Edition, edited by George C. Carroll and Donald T. Wicklow 10. Stress Tolerance of Fungi, edited by D. H. Jennings 11. Metal lons in Fungi, edited by Gunther Winkelmann and Dennis R. Winge 12. Anaerobic Fungi: Biology, Ecology, and Function, edited by Douglas 0. Mountfort and Colin G. Orpin 13. Fungal Genetics:Principles and Practice, edited by Cees J. Bos 14. Fungal Pathogenesis: Principles and Clinical Applications, edited by Riclhard A. Calderone and Ronald L. Cihlar 15. Molecular Biology of Fungal Development,edited by Heinz D. Osiewacz 16. Pathogenic Fungi in Humans and Animals: Second Edition, edited by Dexter H. Howard 17. Fungi in Ecosystem Processes, John Dighton 18. Genomics ofPlants and Fungi, edited by Rolf A. Prade and Hans J. Bohriert I9. Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts, edited by James F. White Jr., Charles W. Bacon, Nigel L. Hywel-Jones, and Joseph W. Spatafora 20. Handbook of Fungal Biotechnology, Second Edition, edited by Dilip K. Arora 21. Fungal Biotechnology in Agricultural, Food, and EnvironmentalApplications, edited by Dilip K. Arora

Additional Volumes in Preparation Handbook of Industrial Mycology, edited by Zhiqiang An

Preface

The study of fungal biotechnology is proceeding at an unprecedented rate with an array of new tools to generate a wealth of disciplines and subdisciplines. By means of modern biotechnology, fungi have justified their practical application to varied domains of human enterprise, and thus promise considerable potential in the agricultural, food, and environmental spheres. The successful application of fungal biotechnological processes in these areas requires the integration of a number of scientific disciplines and technologies. These may include subjects as diverse as agronomy, chemistry, genetic manipulation, and process engineering. The practical use of newer techniques such as genetic recombination, bioinformatics, and robotics has revolutionized modern biotechnology-based agri-food industries, and created the enormous range of possible applications of fungi. Tremendous biodiversity of agriculturally important fungi exists—the benefit of which is not fully harnessed. The level of technology required to take full advantage may range from the simple introduction of a single fungus in biocontrol processes to the extensive manipulation of the organism that facilitates overproduction of a particular enzyme or metabolite. In modern agri-industry, fungi offer many established beneficial roles, particularly as biofertilizers, mycorrhizae, and biocontrol agents of pathogens, pests, and weeds. As pathogens, fungi represent a heavy negative impact on human health, agriculture, and environment. In agriculture, annual crop losses by phytopathogenic fungi in the field and also during postharvest exceed 200 billion Euros, and in the United States alone, over $600 million are spent annually on agricultural fungicides. The balance of beneficial and detrimental effects is reflected in many other areas of agriculture and horticulture. Fungi that inhabit tropical or temperate soils, as mycorrhiza, endophytes, phytopathogens, entomopathogens, or simple saprophytes, are significant resources in transformation of biological matter, and they offer many bioproducts including secondary metabolites, antibiotics, and catabolic enzymes of enormous potential. The world has won a crucial battle in the area of food security, but the war is still on. A total of 800 million people—that is, one of every six persons in the developing world—do not have access to food. One-third of all pre – school-age children in the developing countries face food insecurity. In the food and feed arena, fungi are historically important as mushrooms and fermented foods and in baking and brewing. Such roles are supplemented by the provision of fungi to offer food processing enzymes and additives, and more recently the development of protein-based foodstuffs from filamentous fungi. On the detrimental side, fungi cause extensive spoilage of stored and processed foodstuff. Through direct pathogenesis and biodeterioration of foods and other agricultural commodities, fungi cause considerable economic consequences as well. In these cases, techniques developed from biochemistry and molecular biology can be deployed to analyze the relevant processes, and to evolve tools for the detection, characterization, and tracking of the organisms involved. Although such endeavors may seem rather far removed from the traditional definition of fungal biotechnology, the information derived can be pivotal in understanding the underlying intricate processes, and arriving at suitable control measures. The utilization of fungi in the environment is a more recent development, and can have particular association with both food and agriculture, with fungal remediation of land having implications for biofertilizers, mycorrhizae, and food crop iii

Preface

development, among many other considerations. The degradative activities of fungi have also been harnessed in programs related to bioremediation of contaminated land, treatment of industrial wastes, and biotransformation of speciﬁc compounds. Many of the applications of fungal biotechnology in these areas rely not on identifying new activities but in harnessing and expanding roles that the fungi undertake normally in the environment. Several books on the role of fungi in agricultural, food, and environmental applications have appeared since the 1990s. However, subjects relating to these areas are so broad that no single book can provide all the available information. Consequently, this book complements the others by providing valuable information that is not available elsewhere. The book encompasses a broad range of information on biotechnological potential of entomopathogenic fungi, ergot alkaloids, fungi in disease control, the development of mycoherbicides, control of nematodes, control of plant disease, strategies for controlling vegetable and fruit crops, mycotoxigenic fungi, development of biofungicides, production of edible fungi, fermented foods, and high-value products such as mycoprotein, yeasts in the wine industry, the role of fungi in the dairy industry, molecular detection of fungi in food and feeds, antifungal food additives, the importance of fungi in forest and arid ecosystems, the role of fungi in the biomineralization of heavy metals, bioconversion of distillery waste, decoloration of industrial waste, and fungal degradation of cellulose, hydrocarbons, dye water, and explosives. Together with its companion publication, the Handbook of Fungal Biotechnology, Second Edition (Marcel Dekker, 2004), this incomparable book reigns as the top source on the role of fungi in agriculture, food technology, and environmental applications. The book will be useful for teachers and students, in both undergraduate and graduate studies, in departments of agricultural microbiology, food science, food technology, food engineering, microbiology, environmental sciences, botany, bioengineering, plant pathology, mycology, and, of course, biotechnology. In addition, the book will be useful for agri-food producers, research establishments, and government and academic units. No work of this magnitude can be accomplished without the support and contributions of many individuals. I am deeply indebted to my colleagues and associate editors who have assisted me throughout the production of this book. I appreciate the hard work of authors for their up-to-date discussions on various topics and immense persistent cooperation. My gratitude is expressed to my teacher J. L. Lockwood. I thank Ms. Sandra Beberman (Vice President) and Ms. Dana Bigelow (Production Editor) at Marcel Dekker, Inc., for their dedicated assistance and advice in editorial structuring at all stages of the production of this book. Dilip K. Arora

Contents

Preface Contributors I

iii ix

AGRICULTURAL BIOTECHNOLOGY 1

Biotechnological Approaches in Plant Protection: Achievements, New Initiatives, and Prospects Sally Ann Leong

Chemical Identiﬁcation of Fungi: Metabolite Proﬁling and Metabolomics Kristian Fog Nielsen, Jørn Smedsgaard, Thomas Ostenfeld Larsen, Flemming Lund, Ulf Thrane, and Jens Christian Frisvad

Isozyme Analysis in Fungal Taxonomy, Genetics, and Population Biology Stephen B. Goodwin

Molecular Methods for Identiﬁcation of Plant Pathogenic Fungi Maren A. Klich and Edward J. Mullaney

The Application of Molecular Markers in the Epidemiology of Plant Pathogenic Fungi Paul D. Bridge, Tanuja Singh, and Dilip K. Arora

Molecular Biology for Control of Mycotoxigenic Fungi Robert L. Brown, Deepak Bhatnagar, Thomas E. Cleveland, and Zhi-Yuan Chen

Biotechnological Potential of Entomopathogenic Fungi Travis R. Glare

Biotechnological Potential of Ergot Alkaloids M. Flieger, P. Mehta, and A. Mehta

Fungi as Plant Growth Promoter and Disease Suppressor M. Hyakumachi and M. Kubota

101

Challenges and Strategies for Development of Mycoherbicides Susan M. Boyetchko and Gary Peng

111

Contents

Biofungicides Beom Seok Kim and Byung Kook Hwang

123

Molecular Biology of Biocontrol Trichoderma Christian P. Kubicek

135

The Biological Control Agent Trichoderma from Fundamentals to Applications A. Herrera-Estrella and I. Chet

147

Biological Control of Fungal Diseases on Vegetable Crops with Fungi and Yeasts Zamir K. Punja and Raj S. Utkhede

157

Control of Postharvest Diseases of Fruits Using Microbes Wojciech J. Janisiewicz

173

Arbuscular Mycorrhizal Fungi in Plant Disease Control Lisette J. C. Xavier and Susan M. Boyetchko

183

Commercialization of Arbuscular Mycorrhizal Biofertilizer Pragati Tiwari, Anil Prakash, and Alok Adholeya

195

Control of Nematodes by Fungi Hans-B€orje Jansson and Luiz V. Lopez-Llorca

205

II FOOD AND FEEDS 19

Fungi in Food Technology: An Overview George G. Khachatourians

217

Fungi and Fermented Food T. B. Ng

223

Production of Edible Fungi R. D. Rai

233

Mycoprotein and Related Microbial Protein Products Juan Ignacio Castrillo and Unai Ugalde

247

Genetic Variability of Yeast in Wine Fermentation Amparo Querol, M. Teresa Fernandez-Espinar, and Eladio Barrio

257

Yeast in the Dairy Industry T. K. Hansen and M. Jakobsen

269

Flavors and Aromas Renu Agrawal

281

Antifungal Food Additives Purbita Ray and Michael B. Liewen

291

Molecular Detection of Fungi in Foods and Feeds Janos Varga

299

The Role of Spoilage Fungi in Seed Deterioration Naresh Magan, Vicente Sanchis, and David Aldred

311

Mycotoxins Fun S. Chu and Deepak Bhatnagar

325

Contents

vii

343

Genetics and Biochemistry of Mycotoxin Synthesis Jiujiang Yu

III

ENVIRONMENTAL BIOTECHNOLOGY

Cellulose Degradation by Fungi Justine M. Niamke and Nam Sun Wang

363

The Importance of Wood-Decay Fungi in Forest Ecosystems Nia A. White

375

The Biodegradation of Lignocellulose by White Rot Fungi Gary Ward, Yitzhak Hadar, and Carlos G. Dosoretz

393

Biomineralization of Heavy Metals T. C. Crusberg, S. S. Mark, and A. Dilorio

409

Decoloration of Industrial Wastes and Degradation of Dye Water Kirsten Schliephake, Warren L. Baker, and Greg T. Lonergan

419

Bioconversion of Distillery Waste Jozefa Friedrich

431

Degradation of Hydrocarbons by Yeasts and Filamentous Fungi John B. Sutherland

443

Biodegradation of Azo Dyes by Fungi John A. Bumpus

457

Fungal Degradation of Explosives J. L. Faull, S. C. Baker, S. Wilkinson, and S. Nicklin

471

Restoration of Mycorrhizae in Disturbed Arid Ecosystems Season R. Snyder and Michael F. Allen

481

Index

493

Contributors

Alok Adholeya

The Energy and Resources Institute, New Delhi, India

Renu Agrawal

Central Food Technological Research Institute, Mysore, India

David Aldred Cranﬁeld University, Bedford, United Kingdom Center for Conservation Biology, University of California, Riverside, California, USA

Michael F. Allen Dilip K. Arora S. C. Baker

Banaras Hindu University, Varanasi, India

School of Biological and Chemical Sciences, Birkbeck College, London, United Kingdom

Warren L. Baker Australia

Environment and Biotechnology Centre, Swinburne University of Technology, Melbourne,

Eladio Barrio University of Vale`ncia, Vale`ncia, Spain, Instituto de Agroquı´mica y Tecnologı´a de Alimentos, CSIC, Vale`ncia, Spain Deepak Bhatnagar U.S. Department of Agriculture– Agricultural Research Service, New Orleans, Louisiana, USA Susan M. Boyetchko

Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada

Susan M. Boyetchko Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada Paul D. Bridge* Birkbeck College, University of London, London, and Royal Botanic Gardens Kew, Surrey, United Kingdom Robert L. Brown U.S. Department of Agriculture–Agricultural Research Service, New Orleans, Lousiana, USA John A. Bumpus

University of Northern Iowa, Cedar Falls, Iowa, USA

Juan Ignacio Castrillo

University of Manchester, Manchester, United Kingdom

*Present afﬁliation: British Antarctic Survey, Cambridge, United Kingdom

Contributors

Louisiana State University, Baton Rouge, Lousiana, USA

Zhi-Yuan Chen

I. Chet The Weizmann Institute of Science, Rehovot, Israel Fun S. Chu University of Wisconsin, Madison, Wisconsin, USA Thomas E. Cleveland USA

U.S. Department of Agriculture– Agricultural Research Service, New Orleans, Lousiana,

Worcester Polytechnic Institute, Worcester, Massachusetts, USA

T. C. Crusberg A. Dilorio

Worcester Polytechnic Institute, Worcester, Massachusetts, USA

J.L. Faull

School of Biological and Chemical Sciences, Birkbeck College, London, United Kingdom

M. Teresa Ferna´ndez-Espinar University of Vale`ncia, Vale`ncia, Spain, Instituto de Agroquı´mica y Tecnologı´a de Alimentos, CSIC, Vale`ncia, Spain M. Flieger Dr. H.S. Gour University, Saugor, India; Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic Jozefa Friedrich

National Institute of Chemistry, Ljubljana, Slovenia

Jens Christian Frisvad BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark Travis R. Glare

AgResearch, Lincoln, New Zealand

Stephen B. Goodwin U.S. Department of Agriculture—Agricultural Research Service, and Purdue University, West Lafayette, Indiana, USA Yitzhak Hadar T. K. Hansen

The Hebrew University of Jerusalem, Rehovot, Israel The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

A. Herrera-Estrella

Centro de Investigacio´n y Estudios Avanzados, Unidad Irapuato, Irapuato, Me´xico

Byung Kook Hwang College of Life and Environmental Sciences, Korea University, Seoul, South Korea M. Hyakumachi Gifu University, Yanagido Gifu, Japan M. Jakobsen The Royal Veterinary and Agricultural University, Frederiksberg, Denmark Wojciech J. Janisiewicz Appalachian Fruit Research Station, U.S. Department of Agriculture –Agricultural Research Service, Kearneysville, West Virginia, USA Hans-Bo¨rje Jansson Universidad de Alicante, Alicante, Spain George G. Khachatourians

College of Agriculture, University of Saskatchewan, Saskatoon, Canada

Beom Seok Kim Institute for Structural Biology and Drug Discovery, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA Maren A. Klich

U.S. Department of Agriculture –Agricultural Research Service, New Orleans, Louisiana, USA

Christian P. Kubicek

Institute of Chemical Engineering, Vienna, Austria

M. Kubota Gifu University, Yanagido Gifu, Japan Thomas Ostenfeld Larsen

BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Contributors

Sally Ann Leong U.S. Department of Agriculture –Agricultural Research Service, and University of Wisconsin, Madison, Wisconsin, USA General Mills, Inc., Minneapolis, Minnesota, USA

Michael B. Liewen

Environment and Biotechnology Centre, Swinburne University of Technology, Melbourne,

Greg T. Lonergan Australia

Luis V. Lopez-Llorca

Universidad de Alicante, Alicante, Spain

Flemming Lund BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark Naresh Magan S. S. Mark*

Cranﬁeld University, Bedford, United Kingdom

Worcester Polytechnic Institute, Worcester, Massachusetts, USA

A. Mehta Dr. H.S. Gour University, Saugor, India; Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic P. Mehta Dr. H.S. Gour University, Saugor, India; Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic Edward J. Mullaney U.S. Department of Agriculture–Agricultural Research Service, New Orleans, Louisiana, USA T. B. Ng The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China University of Maryland, College Park, Maryland, USA

Justine N. Niamke

S. Nicklin DSTL, Sevenoaks, Kent, United Kingdom Kristian Fog Nielsen BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark Gary Peng

Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada

Anil Prakash Barkatullah University, Bhopal, Madhya Pradesh, India Zamir K. Punja Simon Fraser University, Burnaby, British Columbia, Canada Amparo Querol University of Vale`ncia, Vale`ncia, Spain, Instituto de Agroquı´mica y Tecnologı´a de Alimentos, CSIC, Vale`ncia, Spain R. D. Rai

National Research Centre for Mushroom, Solan, Himachal Pradesh, India

Purbita Ray General Mills, Inc., Minneapolis, Minnesota, USA Vicente Sanchis Universitat de Lleida, Lleida, Spain Kirsten Schliephake Melbourne, Australia Tanuja Singh

Environment and Biotechnology Centre, Swinburne University of Technology,

Banaras Hindu University, Varanasi, India

Jørn Smedsgaard BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark Season R. Snyder

Sapphos Environmental, Inc., Pasadena, California, USA

*Present afﬁliation: Cornell University, Ithaca, New York, USA

xii

Contributors

John B. Sutherland National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, USA Ulf Thrane BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark Pragati Tiwari Unai Ugalde

The Energy and Resources Institute, New Delhi, India

University of the Basque Country, San Sebastia´n, Spain

Raj S. Utkhede Paciﬁc Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada Ja´nos Varga

University of Szeged, Szeged, Hungary

Nam Sun Wang University of Maryland, College Park, Maryland, USA Gary Ward MIGAL-Galilee Technology Center, Kiryat Shmona, Israel Nia A. White University of Abertay, Dundee, Scotland, United Kingdom S. Wilkinson DSTL, Sevenoaks, Kent, United Kingdom Lisette J.C. Xavier Canada Jiujiang Yu

Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan,

U.S. Department of Agriculture –Agricultural Research Service, New Orleans, Louisiana, USA

Naresh Magan Cranﬁeld University, Bedford, United Kingdom Carlos G. Dosoretz Technion –Israel Institute of Technology, Haifa, Israel

1 Biotechnological Approaches in Plant Protection: Achievements, New Initiatives, and Prospects Sally Ann Leong U.S. Department of Agriculture – Agricultural Research Service, and University of Wisconsin, Madison, Wisconsin, USA

In the last century, the Green Revolution addressed the food needs of the human population through the development of high yielding and early maturing varieties that performed under favorable conditions of nutrition and moisture (Khush 2001). Prior to this time, increased production was dependent on expansion of land area for crop production. In recent years, yield gain through breeding has not kept pace with population growth (Serageldin 1999). Furthermore, genotype is not the only factor limiting productivity. Abiotic and biotic stress factors also contribute to losses in yield both pre and postharvest. For example, in Asia these technical constraints on rice production may reduce production by 23% (Evanson et al. 1996). Socioeconomic constraints also contribute to practices that affect yield. Ninety percent of the world’s rice is grown in Asia on small farms with limited resources. Thus, decisions are made based on economics rather than achieving technically optimum yields. Despite much research on the application of biotechnology to solve these technical production constraints, biotechnology has had limited impact to date on rice production in Asia (Houssain 1997). This has been due in part to the reluctance to adopt the use of genetically modiﬁed organisms (GMOs) in most countries of Asia. China is the only Asian country that has embraced biotechnology on any notable scale as a solution to technical production constraints in agriculture (Huang et al. 2002; Pray et al. 2002). For example, over four million smallholders have been able to increase yield and reduce pesticide costs and adverse health effects of applying pesticides by using transgenic Bt-cotton (Pray et al. 2002). Another constraint relates to the complexities of ownership rights that can delay and discourage the scientiﬁc application of biotechnology discoveries and their transfer to the market place (Kowalski et al. 2002). Finally, public concern about the safety of consuming GMOs has left tons of food aid containing transgenic corn untouched in

CHALLENGES FOR FOOD SECURITY IN THIS CENTURY AND BEYOND

Today we face many critical issues in agriculture: (a) an exponentially growing human population; (b) recurrent famine; (c) the destruction of natural landscapes such as tropical rain forests to extend agriculture to previously unused lands; (d) the exodus of human civilization from rural communities to cities; (e) the destruction of environmental quality resulting from exposure to agrochemicals, erosion of soils and salinization of soils as well as exhaustion and contamination of fresh water resources; (f) the loss of biodiversity through monocropping and the destruction of natural habitats; (g) the reliance of agricultural production, transport, and storage systems on fossil fuel; (h) the acquisition and concentration of agricultural wealth by multinational corporations; and (i) an issuant lack of knowledge by a growing proportion of human civilization on how to cultivate, prepare, and preserve food. The United Nations Food and Agriculture Organization predicts that agricultural productivity in the world will be able to sustain the growing human population by 2030 but hundreds of millions of people in developing countries will remain hungry and environmental problems caused by agriculture will remain serious (FAO 2002). By 2025,83% of the expected global population of 8.5.2 billion will be in the developing world (United Nations 2002). The social consequences are obvious. Food is a basic human need and right. How can we sustain the food needs of the earth’s biotic community in the 21st century and beyond while preserving environmental quality and the diversity and quality of life on earth (Time, August 26, 2002)? What solutions can biotechnology provide to address these problems (Khush and Bar 2001)? 1

Leong

Zimbabwe and Zambia despite the prediction that 13 million people are now at risk of famine due to severe drought in Southern Africa (Paarlberg 2002). Organic growers worldwide have rejected GMOs as an allied technology to disease and pest management. Late blight in potato is still very difﬁcult to control in these farming systems (Mader et al. 2002). Biotechnology as applied to plant protection against fungal pathogens has seen three phases of development over the last 20 years: (a) the application of molecular markers to markerassisted breeding and the map-based cloning of genes associated with disease resistance and the plant defense response as well as to study fungal pathogenesis and host recognition; (b) the development of routine methods for stable and transient transformation of plants and fungi with foreign genes; and (c) the application of New Biology approaches to study plant growth and development and mechanisms of plant response to abiotic and biotic stresses and fungal pathogenesis. The purpose of this short review is to provide a critical analysis of these recent biotechnological approaches to plant protection with emphasis on fungal pathogens.

TESTED STRATEGIES

2.1 2.1.1

Marker-Assisted Breeding and Map-Based Cloning of Genes Molecular Maps

With the advent of recombinant DNA technology came the application of cloned DNAs as probes to genomic DNA of the source organism and the revelation that different alleles could be detected between individuals based on restriction fragment length polymorphisms (Helentjaris et al. 1985) (Figure 1). Genetic maps based on these and other types of molecular markers (Table 1) were developed for many organisms including crop plants such as rice (McCouch et al. 1988; http://rgp.dna.affrc.go.jp/publicdata/geneticmap2000/index. html), lettuce (Kesseli et al. 1994), tomato (Tanksley et al. 1992), alfalfa (Brouwer and Osborn 1999), and Brassica spp. (Kole et al. 2002), among others. Likewise molecular

maps were developed for key fungal pathogens such as Magnaporthe grisea (Farman and Leong 1995; Nitta et al. 1997; Skinner et al. 1993; Sweigard et al. 1993), Phytophtora infestans (van der Lee 2001), and Leptosphaeria maculans (Pongam et al. 1988). These studies also began to reveal the complexities of these genomes in terms of repeated DNAs, their function as transposable elements (Goff et al. 2002; Hamer et al. 1989; Kachroo et al. 1997), their distribution within the genome (Goff et al. 2002; McCouch et al. 1988; Nitta et al. 1997; Yu et al. 2002), and their role in genome evolution and host recognition (Farman 2002; Farman et al. 2002; Kang et al. 2001; Song et al. 1997; 1998). Comparative maps were generated in plants by mapping markers across genera and showed considerable synteny within families of plants (Ahn and Tanksley 1993; Bennetzen and Freeling 1993; Chen et al. 1997; Dunford et al. 1995; Gale and Devos 1998; Hulbert et al. 1990; Saghai Maroof et al. 1996; Tanksley et al. 1992). The mapping of phenotypic markers, both native and induced by mutation, followed closely behind and yielded precise information on the chromosomal location of genes important to plant defense (Ronald et al. 1992; Wang et al. 1995) and fungal host speciﬁcity (Dioh et al. 2000; Smith and Leong 1994; Sweigard et al. 1993) and led to their cloning by chromosome walking (Cao et al. 1997; Farman and Leong 1998; Martin et al. 1993; Orbach et al. 2000; Song et al. 1995; Sweigard et al. 1995). The cloning of a plethora of disease resistance genes from many plant species has shown that they belong to a small number of structural classes (Brueggeman et al. 2002; Chauhan and Leong 2002; Dangl and Jones 2001; Meyers et al. 1999; Xiao et al. 2001) (Figure 2). By contrast, the predicted structures of fungal cultivar speciﬁcity genes are quite diverse (Bohnert et al. 2001; De Wit and Joosten 1999; Orbach et al. 2000; Sweigard et al. 1995). These studies have been complemented by the mapping of candidate genes such as the PR (pathogenesis-related) proteins in plants that were discovered from differential expression of RNA and protein during plant infection (Muthukrishnan et al. 2001) or resistance gene analogs based on the conserved structural features of disease resistance genes (Boyko et al. 2002; Chauhan et al. 2002; Faris et al.

Table 1 Molecular markers used in mapping of traits Marker

Figure 1 Cosegregation of a RFLP marker (R-23 16) with Pi-CO39(t) locus in homozygous F2 susceptible progenies. Genomic DNA of CO39 (R, resistant), 51583 (S, susceptible) and F2 progenies was digested with Dra1, blotted and probed with R-2316. Recombinant progenies show DNA fragments from both parents. Phosphoimage of Southern blot is shown.

RFLP RAPD APD CAP AFLP Microsatellite

CDNA-AFLP

Restriction fragment length polymorphism Random amplified polymorphic DNA Amplified polymorphic DNA Cleaved amplified polymorphism Amplified fragment length polymorphism Polymorphism based on different numbers on mono, di, tri, or tetranucleotide repeats cDNA amplified restriction fragment length polymorphism

Biotechnological Approaches in Plant Protection

Figure 2 Different classes (A – G) of plant disease resistance genes [reviewed in Chauhan and Leong (2002)): Genes in classes A and B are cytoplasmic proteins differing only in their N-terminal domains; Class C genes encode putative transmembrane molecules with an extracellular LRR domain; Xi21 is a transmembrane protein with an extracellular LRR domain; Pto is a cytoplasmic Ser/Thr kinase; RPW8 contains a putative N-terminal TM domain and a CC domain; Hm1 is a unique enzyme that detoxiﬁes a fungal toxin; Abbreviations for domains: TIR, Drosophila Toll/Human Interleukin-lreceptor; CC, Coiled-coil; NBS, Nucleotide binding site; LRR, Leucine-rich repeat; TM, Transmembrane; Ser/Thr, Serine/threonine kinase.

1999; Gebhardt and Valkonen 2001; Huang and Gill 2001; Li et al. 1999; Shen et al. 1998; Speulman et al. 1998). This analysis has been particularly well advanced in wheat and its relative Aegilops tauschii. Resistance and defense response genes in A. tauschii are localized in clusters primarily in distal/telomeric regions of the genome (Boyko et al. 2002) while in Chinese spring wheat defense response genes are localized in clusters and/or at distal regions of chromosomes (Li et al. 1999). In many cases, these genes or gene homologs have been correlated with loci that affect quantitative or single gene resistance in the respective plants. For example, QTLs with large effects in wheat were shown to contain RGAs or clusters of defense response genes such as catalase, chitinase, thaumatins, and an ion channel regulator (Faris et al. 1999). Similar results are emerging in the genomes of potato (Gebhardt and Valkonen 2001), Arabidopsis (Speulman et al. 1998) and pepper (Pﬂieger et al. 2001). Efforts are underway to functionally characterize 179 NBS-LRR-encoding genes that may encode disease resistance genes in the Arabidopsis genome (Figure 2). These have been organized into subclasses and their distribution mapped to the chromosomal sequence (Michelmore 2002; www.nibh-rs.ucdavis.edu). A publicly available, draft ordered sequence of the rice genome is anticipated by the end of 2002 (http://rgp.dna.affrc.go.jp/cgi-bin/statusdb/ seqcollab.pl) and will allow comparisons to be done across syntenic regions of grass genomes (http://www.gramene.org!). The unordered draft sequence of rice varieties Nipponbare

and 93-11 has revealed numerous NBS-LRR-containing sequences as well as sequences potentially encoding other minor classes of R genes and Arabidopsis genes known to control defense response signal transduction (Goff et al. 2002; Yu 2002). Preliminary studies based on conservation of RGAs in comparative maps of the grasses have shown evidence for some conservation but also redistribution of this class of genes among the grasses (Leister et al. 1998). Likewise a detailed comparison of a syntenic region between barley and rice did not reveal any candidate resistance genes in rice that could be the ortholog of Rpgl in barley (Han et al. 1999; Kilian et al. 1997).

2.1.2

Differential cDNA-AFLP Screens

Differential cDNA-APLP screening has been done to isolate hypersensitive response (HR)-speciﬁc genes to the Cladesporium fulvum elicitor Avr4 in tomato and has led to the isolation of a previously known and corresponding disease resistance gene cluster Cf-4 as well as numerous new candidate genes involved in the HR response (De Wit et al. 2002; Takken et al. 2001). This method is a robust and inexpensive way to identify differentially expressed genes involving the digestion of cDNAs with two different restriction enzymes and the ampliﬁcation of the resulting products after ligation to adapters for these enzymes. The sizes of the resulting amplicons are measured by gel

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electrophoresis and resulting fragments can be excised and sequenced. Comparison of this method with differential display has shown the cDNA-AFLP method to be superior (Jones and Harrower 1998). Using the cDNA-AFLP technique, Durrant et al. (2000) found a strong coincidence between the expression of genes involved in race-specific resistance and the wound response in tobacco cell cultures. Collectively, these candidate genes will provide additional markers for studies of disease resistance traits in the potato and tomato genomes. Infection-specific cDNA-AFLPs have also been identified in Arabidopsis thaliana inoculated with Peronospora parasitica (van der Biezen et al. 2000). Interestingly, most fragments were derived from the fungal pathogen showing the power of this method to study genes expressed in the pathogen, which in many cases may represent a minor component of the mass of the tissue studied. Previous genetic studies by Valent et al. (1991) have shown that infection of some grass hosts by M. grisea is a quantitatively inherited trait. The cDNA-AFLP approach would allow for the identification a unique set of cDNAAFLPs in each progeny showing varying degrees of pathogenicity and in some cases segregating with pathogenicity. This approach has been recently used to create genome-wide transcription maps of Arabidopsis and potato and study inheritance of the cDNA-AFLPs in segregating populations (Brugmans et al. 2002). Thus phenotypes can be directly associated with molecular genotypes and candidate gene fragments can be excised from gels for further analysis. We are using this approach in an attempt to identify major and minor genes controlling resistance to blast and drought tolerance in Eleusine coracana. Computational methods for relating the size of the AFLP restriction fragment products to the predicted restriction fragment products from sequenced cDNA libraries have been developed and used to identify putative, infection stagespecific, pathogenicity factors from the plant pathogenic nematode Globoderu rostochiensis without need for sequencing the gel fragments (Qin et al. 2001). For those organisms having fully sequenced, annotated full length cDNA libraries such as Arabidopsis (Seki et al. 2002), this approach provides for the rapid functional classification of the cDNA-AFLPs.

2.1.5

Identiﬁcation of QTL-Associated Genes

Very few QTL studies in plants have led to the cloning of a single gene within a QTL that is responsible for the variation seen [reviewed in Buckler and Thornsberry (2002)]. These few examples represent QTLs that had major effects on variation. Buckler and Thornsberry (2002) have proposed that association approaches should be also considered to provide improved resolution and to reduce the time of analysis as mapping populations are not needed since natural variation in a population is investigated instead. The resolution of association that can be obtained depends on the linkage disequilibrium (LD) structure of the population of organism being studied and some insight on candidate gene(s) to target. Studies on LD structure have shown that inbreeding plants

such as Arabidopsis have large LD structures on the order of 250 kb or 1 cM (Nordborg et al. 2002) while outbreeding plants like maize have very small LD structures in the order of kbs (Buckler and Thornsberry 2002). Using this approach, Thornsberry et al. (2001) were able to associate polymorphisms found in the Dwarf8 gene of maize with variation seen in flowering time. In Saccharomyces cereviseae whose entire genome sequence is known, a QTL for high temperature growth (Htg) commonly found in clinical isolates was rigorously analyzed to identify the responsible genes (Steinmetz et al. 2002). Using reciprocal hemizygosity tests, involving selective gene disruption of candidate genes in both parental genomes and then forming diploid hybrids among these strains, three genes were found to contribute to the phenotype in the QTL interval. However, the alleles of two genes came from one parent strain while that of the third came from the other parent. By contrast attempts to employ natural sequence variation or mRNA expression levels determined from several natural isolates of yeast did not provide a clue to which gene(s) in the interval contributed to the phenotype. Thus employing LD and association by decent to accelerate gene identification in an interval may not always succeed and genetic studies will be required to study inheritance and create reciprocal hemizygotes in targeted regions of the genome. The use of allele-specific gene silencing methods (see below) may allow this to be done in the F1 generation of plants while targeted gene disruption methods can be used in fungi such as Ustilago maydis that have a stable diploid phase and facile gene knockout system. Transformation of haploid fungi with wild type and disrupted, endogenous or alternative alleles of candidate genes might be a useful strategy for those fungi that cannot form stable diploids. In fact these strategies have been used to unravel the complex functions of the east and west alleles of b mating type locus of U. maydis (Gillissen et al. 1992; Kamper et al. 1995). Gene silencing has been used in fungi such as P. infestans in which silencing of the fungal elicitin INF1 increased virulence on Nicotiana benthamiana (Kamoun et al. 1998).

2.1.5

Candidate Gene Validation

It should be emphasized that candidate genes are simply “candidate” genes and that conﬁrmation of a gene’s function with a genetically and/or expression-deﬁned phenotype must be done by transformation and complementation tests (Farman and Leong 1998; Orbach et al. 2000; Song et al. 1995; Wang et al. 1999; Yoshimura et al. 1998). Recently gene silencing has also been successfully applied to study gene function in several plants (Azevedo et al. 2002; Baulcomb et al. 2002; Peart et al. 2002; Wesley et al. 2001). This method involves the cloning of a small fragment (, 200 nucleotides) of a gene into an expression vector and transforming the plant [reviewed in Baulcomb et al. (2002)]. The resulting small RNA is made double stranded and then is digested into small dsRNA fragments (siRNA, small interfering RNA), which are thought to guide RNAse to the

Biotechnological Approaches in Plant Protection

nascent wild-type gene transcript and causes it to be degraded thus leading to a net loss in the gene’s expression. Direct bombardment of plant cells with dsRNA is also possible (Schweizer et al. 2000). In the examples described earlier, the speciﬁc genes RAR1 in barley, and Rx, N , Pto, and EDS1 in N. benthamiana implicated in disease resistance signaling were silenced and found to be essential for the signaling process. This approach is being extended to the high throughput analysis of the HR candidate genes from tomato noted earlier (De Wit et al. 2002) as well as in a normalized cDNA library from N. benthamiana (Baulcomb et al. 2002). Interestingly, many genes in N. benthamiana were found in the preliminary round of analysis to affect the HR while not affecting pathogen growth while some genes were affected in both phenotypes when silenced. Moreover almost 1% of the genome appears to affect the HR response.

2.1.5

Integration of Molecular Biology with Classical Breeding

The application of molecular markers to traditional breeding has provided a powerful method to accelerate breeding as phenotypic tests are not essential and rapid DNA isolation methods using hole punch-sized pieces of leaf tissue are possible in young seedlings (Huang et al. 1997). Thus tightly cosegregating or gene(allele)-specific markers can be followed and confirmation of phenotype can be done on a selected set of plants within a population that are destined for further crossing. Phenotypic validation is essential as recombination, gene conversion or other confounding events can take place, even within the gene being studied, leading to an inaccurate scoring based on markers alone. As we learn more about the function of plant genes and specific alleles of these genes in disease resistance through mapping and functional tests, we can anticipate the increased application of molecular markers and gene chips (see later) to the breeding of disease resistance in plants. In particular, it will be interesting to know what contribution pathogenesis-related proteins, which are generically present in all plant genomes, make to quantitative resistance. Is expression more efficient in some genomes than others because of the gene’s placement in clusters and/or their specific transcription regulatory elements and/or their duplication or absence in some genomes and/or the efficacy of specific alleles? Likewise, what is the genetic and molecular basis of host specialization in the fungi? The location of many disease resistance and defense genes at the ends of chromosomes in wheat may affect their stability through recombination and chromosome breakage as well as their expression through unique chromatin organization (Faris et al. 2000). The telomeric location of AVR1-PITA in M. grisea has been shown to contribute to its instability leading to strains with increased virulence (Orbach et al. 2000). The BUF1 gene of M. grisea appears to be readily deleted in one parental chromosome by intrachromosomal recombination of repeated DNA flanking the locus as a result of mispairing of homologous chromosomes during meiosis (Farman 2002). In Alternaria alternata a conditionally

dispensable chromosome controls production of a hostspecific toxin (Hatta et al. 2002). Likewise Han et al. (2001) found that genes required for pathogenicity on pea are present on a dispensable chromosome of Nectria haematococca. Exploitation of the multitude of novel genotypes found in plant germplasm now available in gene banks will often require the methods of molecular mapping and candidate gene isolation described above to identify the genes that contribute to these unique phenotypes (Fulton et al. 1997; Tanksley and McCouch 1997; Xiao et al. 1998). This is true even for plant genomes for which the entire genome sequence is known, thus allowing candidate genes to be isolated in related genomes. For example, the short stature mutation sg1 found in green revolution rice variety IR8 encodes a mutant biosynthetic gene for gibberellin while the semidwarf phenotype in green revolution varieties of wheat is conferred by mutations in the gibberellin signaling pathway (Sasaki et al. 2002). In addition, many previously unidentified genes have been found in every genome that has been sequenced (Goff et al. 2002; Yu et al. 2002). In the case of disease resistance genes, the functions of only a few are known in each sequenced genome and not all LRR-containing coding sequences are likely to function in disease resistance. For example, a LRR receptorlike transmembrane protein kinase gene, that is gibberellin-induced and specifically expressed in growing tissues of deep-water rice, may function in hormone signaling (van der Knaap 1999). The genetic location of disease resistance genes must be determined in the genome that contains the functional gene if the reference genome sequence lacks a functional copy. These tenets also apply to the identification of fungal genes involved in plant recognition. Examples exist for the complete absence of a recessive gene in fungal strains that have lost cultivar specificity (van den Ackerveken et al. 1992; Farman et al. 2002). Furthermore, the recently released genomic DNA sequence of M. grisea having 7X coverage (http://www-genome.wi.mit.edu/annotation/fungi/ magnaporthe/) does not contain the AVRI-CO39 cultivar specificity gene (RS Chauhan, D Lazaro, and SA Leong, unpublished data). This genome sequence is thus useless without precise genetic mapping data for this AVR gene that can be used to identify in the reference, sequenced genome, a contiguous sequence spanning the genome between these markers. This sequence can then be used to develop new genetic markers and to probe libraries of a strain that does carry AVR1-CO39. In fact this AVR gene was originally cloned using a more laborious chromosome walking strategy in the genome of a strain carrying the functional gene (Farman and Leong 1998).

2.2

Transgenic Plants As a Tool for Plant Protection

Following clues from the molecular biology of plant-microbe interactions, many genes involved in disease resistance and

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the defense response have been cloned from a variety of plants (Dangl and Jones 2001). A number of these genes have been tested for their ability to control fungal pathogens in transgenic plants grown in the laboratory and to a more limited extent in the field. In addition, natural or synthetic antimicrobial peptides and genes for resistance to pathogenderived toxins have been introduced into plants. This work is thoroughly reviewed by Bent and Yu (1999); Rommens and Kishore (2000), and Melchers and Stuiver (2000). Only more recent studies providing significant new findings or extensions of this work will be considered here. Current efforts have continued to focus on introduction of disease resistance genes, natural and synthetic antimicrobial peptides as well as selected enzymes such as chitinase and b-glucanase into several crop plants and the laboratory and field evaluation of these plants for disease resistance. Little has been reported in the literature on the field performance of these transgenic plants, although about 5% of all permits for field testing of transgenic plants in the United States over the last decade have been for transgenic plants having fungal resistance (Information Systems for Biotechnology 2002). Moreover, most permits have been issued to companies and this proprietary work is not yet in the public domain. No transgenic plants having fungal resistance have been approved for use as food and/or feed (AgBios 2002). Despite this seemingly limited progress, some promising

achievements have been made in the last few years and many major resistance genes associated with fungal disease resistance have been cloned or at least tagged with molecular markers (Brueggeman et al. 2002; Chauhan and Leong 2002; Gebhardt and Valkonen 2001). Thus, we can expect many new developments with regard to this ﬁeld in the next decade. Furthermore, despite public concern about transgenic crops, the global adoption of transgenic crops continues to increase, particularly in the United States, where 74.8 million acres were planted in transgenic crops including corn, soybean, cotton, and canola in 2000 (Transgenic Crops 2002). This represented about 50% of the total soybean and cotton acreage planted in that year. The International Service for the Acquisition of Agri-Biotech Applications predicts that the world market for genetically engineered plants will be $8 billion in 2005 and $25 billion by 2010 (http://nature.biotech. com) (Figure 3). Plant pathogens cause $30 –50 billion dollars of loss annually in crop productivity (Baker et al. 1997) thus justifying this investment in biotechnological approaches to crop protection. The reduction in use of agrochemicals for disease control is another important incentive for this technology. Japanese growers spend more than $600 million a year to control diseases on rice (Bonman 1998). Already, the reduction in insecticide use in China through use of Bt transgenic crops has impacted farmer income and health (Huang et al. 2002; Pray et al. 2002).

Figure 3 Impact of genomics on the world economy.

Biotechnological Approaches in Plant Protection

2.2.1

Defense Pathways

Despite many projections made in the reviews listed earlier, a flurry of published reports has not followed. In many cases, this can be attributed to the observation being made first in Arabidopsis and not in a crop plant. However, this lag is being addressed with Arabidopsis genes to assess function in crop plants and through the identification of homologs of Arabidopsis genes. For example, the Arabidopsis NPR1 gene (Cao et al. 1998) when overexpressed in rice caused enhanced resistance to the rice pathogen Xanthomonas oryzae pv. oryzae (Chern et al. 2001) and the investigators were able to retrieve, in a two hybrid screen, a bZIP family of interactors showing that a similar pathway of signaling is likely present in rice as Arabidopsis (Zhang et al. 1999). In addition, Yoshioka et al. (2001) have shown that Arabidopsis will respond to the rice fungicide probenazole by induction of PR genes and show enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and P. parasitica Emco5. This response was dependent on a functional NPR1 gene and was compromised in NahG transgenic plants further supporting the connection of this pathway with generalized resistance to pathogens in both Arabidopsis and rice. Six NPR1 homologs are reported in the recently released Nipponbare genome sequence (Goff et al. 2002). It will be interesting to see how overexpression and silencing of these genes affects resistance of rice to key fungal pathogens of rice such as Rhizoctonia solani and M. grisea. Overexpression of the Arabidopsis ACD2 (accelerated cell death) gene leads to tolerance of susceptible Arabidopsis plants to P. syringae infection by reducing disease symptoms associated with cell death such as ion leakage, while allowing the bacteria to grow to similar levels as in susceptible plants (Mach et al. 2001). Fungal pathogens were not tested. Broader testing of other genes that have shown widespectrum disease resistance to bacterial, fungal, and viral pathogens when overexpressed such as Prf (Oldroyd and Staskawicz 1998) and Pto (Tang et al. 1999) in tomato has not been reported. Nor have further reports been made on constitutively active variants of Pto (Rathjen et al. 1999). Presumably, this approach can be used in other crop plants. Introduction of the bacterial blight resistance gene Xa21 into elite rice cultivars has lead to the expected resistance phenotype when inoculated with X. oryzae pv. oryzae; however, strains that are virulent on Xa21 were not tested nor were other pathogens (Tu et al. 1998). Performance of these lines was tested under natural field conditions without any apparent loss of yield performance (Tu et al. 2000). Disease resistance genes from one crop plant have now been successfully used in other crop species. For example, the Bs2 resistance gene from pepper confers resistance to X. campestris pv. vesicatoria in tomato in the laboratory as well as in preliminary field tests (Staskawicz et al. 2002; Tai et al. 1999). Work from my laboratory in conjunction with studies from the laboratories of Mark Farman at the University of Kentucky and Yukio Tosa at Kobe University has suggested that the Pi-CO39 (t) gene (Chauhan et al. 2002)

for resistance in rice to M. grisea carrying the AVR1-CO39 gene (Farman and Leong 1998) will be useful in other grass species such as perennial rye grass as functional copies of the AVRl-CO39 gene are found in the grey leaf spot pathogen (ML Farman personal communication). Coexpression of the C. fulvum Avr9 and tomato Cf-9 genes in Brassica napus was investigated as a method for inducing broad-spectrum resistance to fungal pathogens (Hennin et al. 2001). Induction of PR1, PR2, and Cxc750 was detected following injection of the Avr9 peptide obtained from intercellular fluids of B. napus transgenic plants expressing Avr9 into B. napus expressing the tomato Cf-9 gene. F1 plants and progeny from a cross of the Avr9 and Cf-9 plants were evaluated for resistance to fungi. Disease development was delayed at the site of infection of L. maculans and Erysiphe polygoni but enhanced at the site of infection of Sclerotinia sclerotiorum. Thus, heterologous expression of AVR-R gene pairs may be a useful strategy for control of fungal disease in a variety of plants. However, the finding that Arabidopsis resistance gene RPM1 requires another plant gene RIN4 in order to accumulate and interact with avrRpm1 or AvrB (Mackey et al. 2002) as well as the inability to show direct interaction of the products of Avr9 with Cf-9 (van der Hoorn et al. 2002; Luderer et al. 2001) suggests that this strategy must be used cautiously. This may explain the inability of van der Hoorn et al. (2002) to see a necrotic response in the nonsolanaceous plant lettuce with this gene combination. More recent reports on the use of the coexpression strategy for plant protection against fungi in solanaceous plants have not been made (Melchers and Stuiver 2000), however the coexpression of Avr9 and Cf-9 under control of nematode inducible promoters in tobacco has been studied (Bertioli et al. 2001). Surprisingly these plants underwent spontaneous necrosis in the absence of the nematode. Evidence for activity of the genes was found both in aerial and root tissue. The cloning of MLO locus of barley (Shirasu et al. 1999), which confers nonrace-specific resistance to Blumeria graminis f. sp. hordei, has been followed with investigations of its potential use for control of different fungal pathogens. Jarosch et al. (1999) found that in contrast to increased resistance conferred by recessive alleles of MLO to powdery mildew, these barley plants have increased susceptibility to penetration by M. grisea despite showing similar ability to wild type plants to respond to M. grisea elicitor. Likewise Kumar et al. (2001) showed that mlo plants were more susceptible to the necotrophic pathogen Bipolaris sorokiniana. These reports reveal the complexity of various fungal interactions with the host and the difficulty of using a single strategy to control multiple pathogens. Recent work on MLO has shown that it is a novel calmodulin-binding protein that is responsive to both abiotic and biotic stresses through down regulating the oxidative burst and cell death response (Kim et al. 2002a,b; Piffanelli et al. 2002). Binding to calmodulin is essential to full function of MLO (Kim et al. 2002a). A rice homolog of MLO that also interacts with calmodulin was isolated (Piffanelli et al. 2002). It will be interesting to see how silencing of MLO in rice

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plants affects interaction with biotrophic and necotrophic pathogens.

2.2.2

Antifungal Proteins and Peptides

Several new reports have appeared on the use of antifungal proteins such as Ag-AFP from Aspergillus giganteus, chitinase, b-glucanase and Ribozome-Inactivating Proteins (RIP) (Chareonpornwattana et al. 1999; Datta et al. 2002; Oldach et al. 2001), thaumatinlike protein (PR-5) (Datta et al. 1999), and human lysozyme (Takaichi and Oeda 2000) in plants for protection against fungal disease and show different levels of promise for these approaches. Chitinase and AFP appear to increase resistance in wheat, however, the results were not corroborated with levels of these proteins in transgenic plants (Oldach et al. 2001). Other efforts to introduce chitinases in wheat have led to gene silencing (Chareonpornwattana et al. 1999). The recent isolation of cDNA clones for novel acidic chitinases and b-1,3-glucanases from wheat spikes infected by Fusarium graminearum (Li et al. 2001) is exciting as these enzymes may be more effective in control of this pathogen in this tissue. Introduction of infection-related chitinase and rice thaumatinlike protein into rice has led to moderate control of sheath blight caused by R. solani (Datta et al. 2002). Field evaluation of these plants is underway. Finally, studies on carrot transformed with human lysozyme, which can cleave b-1,4 glycosidic bonds of peptidoglycan in bacterial cell walls and chitin in fungal cell walls, suggest that this approach may have promise for control of E. heraclei and A. dauci (Takaichi and Oeda 2000). Natural and synthetic peptides have been evaluated for control of pre and postharvest damage by fungi. Ali and Reddy (2000) studied four cationic peptides for antimicrobial activity in vitro and in plants. All were shown to have significant activity in the micromolar range against P. infestans and A. solani completely inhibiting growth of the fungi on potato tissues. Alfalfa antifungal peptide defensin from seeds of Medicago sativa was shown by Gao et al. (2000) to have significant activity against Verticillium dahliae in vitro, and transgenic potato plants expressing the peptide showed a reduced area under the disease progress curve compared to vector control plants. Moreover, resistance was correlated with the levels of peptide found in root samples. Similar results were obtained for transgenic potato plants expressing a N terminus-modified cecropin-melittin cationic peptide chimera (Osusky et al. 2000). The efficacy of the peptide against Phytophthora cactorum and F. solani infection was demonstrated in variety Desiree while not affecting plant growth or tuber morphology or size. Tubers remained resistant for more than one year and the peptide could be detected in this tissue. By contrast transgenic Russet Burbank plants showed significant morphological alterations and resembled lesion mimic plants, produced very small tubers, and showed less resistance to P. cactorum. Trangenic raw tubers were fed to mice without significant growth effects relative to untransformed tubers. Rajasekaran et al. (2001)

have shown that the synthetic antimicrobial peptide DE41 is active at the micromolar range against many important bacterial and fungal plant pathogens. Crude protein extracts from transgenic tobacco plants constitutively expressing D4E1 showed ability to reduce growth of A. flavus and V. dahliae while control plant extracts did not (Cary et al. 2000). Furthermore, the transgenic plants showed increased resistance to Colletotrichum destructivum. The D4El gene has been introduced into cotton and was shown to be present in cottonseed. Reduction of aflatoxin in cottonseed oil is a desired outcome. Dow AgroSciences LLC has licensed the technology and is collaborating with USDA-ARS scientists who developed the technology to further evaluate the efficacy of the transgenic plants (SeedQuest 2002). The effects of this peptide on plant growth or other nontarget organisms have not been reported. Finally, the antimicrobial peptide MS1-99, an analog of magainin 2, a defense peptide secreted from the skin of the African clawed frog, was expressed from the chloroplast genome of tobacco and showed significant ability to control many phytopathogenic bacteria and fungi (DeGray et al. 2001). Trangenic plant homogenates inhibited the fungi A. flavus, F. monilzjbrme and V. dahliae and anthracnose lesions were absent in transgenic plant infected with C. destructivum. Transformation of the chloroplast genome is an innovative approach to control the spread of the transgene as pollen will not carry the transgenic chloroplast. Evidence for pollen transfer of transgenes at the commercial field level is now available (Reiger et al. 2002). While the use of peptides has shown significant promise, thorough testing of the toxicity of plants producing these peptides will be important. Their potential ability to inhibit microflora that are essential to plant health as well as health of animals, humans, and birds needs careful evaluation. Feeding raw potatoes containing a cecropin-melittin cationic peptide chimera to mice was not a convincing test of the toxicity as the animals do not normally eat this food and lost weight on this diet until it was supplemented with normal feed (Osusky et al. 2000). More realistic tests are needed. The use of cooked potatoes should be tested. Furthermore, only short-term effects were studied. The survival and biological impact of large quantities of these peptides in the environment resulting from crop plant decay also needs to be evaluated. These issues are only beginning to be addressed in a multitrophic context for insect resistant transgenic plants (Groot and Dicke 2002). These issues need to be critically addressed at the time of risk assessment. Public concern has been spurred largely by a lack on confidence in transgene technology because of the lag time in responding to the large-scale effects that agrochemicals are having on human and environmental health despite early warnings by Carson (1962) many decades ago.

2.2.3

Phytotoxin Detoxiﬁcation

In planta studies of the hydroxylation and glycosylation of destructin B, a phytotoxin produced by A. brassicae, to a nontoxic product have shown a correlation between plant resistance with phytoalexin production and the efﬁciency of

Biotechnological Approaches in Plant Protection

these modiﬁcations of the toxin in Brassica spp. (Pedras et al. 2001). These data suggest that improved resistance can be engineered in or transferred within Brassica hosts of A. brassicae by enhancing hydroxylation of destruxin B. The analysis of the biosynthetic pathway of saponins, antimicrobial metabolites of plants, may allow the transfer of these genes to other plants. Mutants defective in the saponin avenacin in oat were studied and shown to deﬁne seven loci and to be compromised in their ability to resist fungal attack (Haralampidis et al. 2001). The sad1 gene was shown to encode b-amyrin synthase.

3 3.1

NEW INITIATIVES AND PROSPECTS: THE NEW BIOLOGY From Genome Sequence to Gene to Mutant to Function

Research in the last five years has been increasingly driven by the availability of whole genome sequences and cDNA libraries from many plants and microorganisms (National Center for Biotechnology Information, http://www.ncbi.nlm. nih.gov). The Arabidopsis genome sequence was released in 2001 (Arabidopsis Genome Initiative 2001) and the draft DNA sequence of two subspecies of rice was released this year (Goff et al. 2002; Yu et al. 2002). Likewise, several fungal genome sequences are now available for model organisms as well as plant pathogens (http://www-genome. wi.mit.edu/annotation/fungi/magnaporthe/; http://wwwgenome.wi.mit.edu/annotation/fungi/neurospora/; Friedrich et al. 2001; Turgeon and Yoder 2002). Consortia of scientists have been established to bring together fungal genomic resources for a research community. These include M. grisea (http://www.riceblast.org/) and Phytophthora (http://www. ncgr.org/pgc; Torto et al. 2002). Comparative analysis of several fungal genome sequences including that of C. heterostrophus, F. graminearum, and Botrytis cinerea, with those of Neurospora crassa and yeast has led to the identification of putative common essential genes, candidate fungal-specific genes, and candidate pathogenicity genes (Turgeon and Yoder 2002). Systematic analysis of these candidate pathogenicity genes through knock out studies is underway. In addition, genes for polyketide synthases and nonribosomal peptide synthetases are being identified and systematically disrupted since many secondary products from fungi are known to have functions as host-specific toxins.

3.2

source fungus to create a disruption of the sequence in the genome by gene replacement. This method has been adapted to a high throughput scheme and the resulting mutants are then tested for phenotypic alterations using a battery of tests. The success of this scheme is not dependent on access to the full genome sequence of a fungus. Agrobacterium tumefaciens T-DNA is also being used to directly mutagenize the genome of a number of fungi (De Groot et al. 1998; Mullins et al. 2001; Rho et al. 2001) as well as plants such as rice (An et al. 2001) and Arabidopsis (see the following: Krysan et al. 1999). M. grisea and F. oxysporum mutants altered in virulence have been identiﬁed (Mullins et al. 2001; Rho et al. 2001). The randomness of insertion in the target genome and the possibility that some mutants may be caused by secondary events needs to be thoroughly evaluated in fungi, nevertheless, this method provides a straightforward way of tagging genes in fungi and appears to be superior to restriction enzyme-mediated insertion (REMI) mutagenesis in which the transforming DNA containing a selectable marker is linearized and transformed with a restriction enzyme to promote insertion at restricted sites in the genome (Balhadere et al. 1999; Bolker et al. 1995; Lu et al. 1994; Sweigard et al. 1998). This method did not generate certain kinds of expected mutants presumably due to the lack of sites for this enzyme in the genes or the inaccessibility of the genes to enzyme because of their lack of expression under the conditions of growth and/or transformation. Mutants lacking a DNA insertion in the mutated gene have also been documented using this method. Thus reisolation of the mutant gene is not simple.

3.3

Transient Expression of Fungal Genes in Plants

Sequenced libraries of cDNAs from Phytophthora are being systematically tested for their ability to induce resistance or disease symptoms in tobacco and potato by transient expression using potato virus X (PVX) and A. tumefuciens T-DNA vectors as delivery systems (Takken et al. 2000; Torto et al. 2002). Agroinﬁltration has been used in several plant systems to study the interaction phenotype of products of avirulence genes and speciﬁc resistance genes (van der Hoorn et al. 2000; Tai et al. 1999). Often plants are scored simply for formation of a HR or necrosis. Whether this is truly representative of the natural response to infection is unclear. It will be interesting to see what kinds of the candidate genes are found and whether their disruption or silencing corroborates the heterologous expression tests described here.

Random Mutagenesis with Transposons 3.4

Another strategy for identiﬁcation of pathogenicity genes has been to introduce transposons in fungal genomic DNA cloned into cosmid vectors (Hamer et al. 2001). The insertion site of the transposon is sequenced to assess what function has been disrupted. Each tagged insert is then transformed into the

Microarrays

With the recent availability of massive amounts of DNA sequence data for many organisms, new technologies have emerged to study this information in a high through put and cost effective manner. Microarrays based on spotting of

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thousands of cDNAs on glass surfaces or synthesis of genespecific oligonucleotide microarray (OMA) on glass surfaces have allowed the parallel analysis of expression of large numbers of genes (Brown and Botstein 1999; Fodor 1997; Lashkari et al. 1997). For example, new understanding of: (a) stage-specific gene expression during sporulation in yeast (Chu et al. 1998); (b) the functional role of a set of yeast mutants each carrying a deletion in one of 5,916 open reading frames (96.5% of total) (Giaever et al. 2002); (c) the transcriptional hierarchy of metabolic genes in yeast (DeRisi et al. 1997); and (d) gene expression during meiosis (Primig et al. 2000) has resulted from the use of OMAs. Microarrays are currently being used to study global gene expression in Arabidopsis (Zhu et al. 2001a; see later) and rice (Zhu et al. 2001b). One caution in using these arrays is that they only provide information on gene expression (RNA abundance) while many cellular processes are regulated at the translational or posttranslational levels. Giaever et al. (2002) found that many genes required for fitness through gene knock out did not show increased expression using OMAs. Moreover, many genes that showed increased expression were not required for fitness. In addition, Primig et al. (2000) found very different expression profiles when they compared RNA from different yeast strains undergoing meiosis. The overlapping gene set could be correlated with genes previously known to have a role in meiosis. This result emphasizes the need to use different isolates to find the core set of genes that are needed for a biological process that shows differential gene expression.

3.5

Proteomics

Proteomics is the study of all proteins from a living organism. The most advanced approach is the use of mass spectrometry to study whole cell/tissue protein content and modifications (Haynes and Yates 2000). The method has also been used to analyze proteins in biological complexes (Link et al. 1999). Although this technology is very new and requires sophisticated equipment and technical expertise, it offers a more realistic view of the physiology of the cell at a given point in time. Many studies that have traced RNA expression as a means to identify important genes in a biological process have led to disappointing results when the gene(s) is disrupted (Basse et al. 2000; Giaever et al. 2002; Timberlake and Marshall 1988). Furthermore, redundancy in gene function can confound this type of study. Analysis of the proteome can distinguish which family member is made and modifications that it may have undergone. Initial studies on the identification of membrane proteins from arbuscular mycorrhizas formed between M. truncatula and Glomus versiforme led to the identification several protein from microsomes fractionated from mycorrhizal roots using 2D polyacrylamide gel electrophoresis and MALDI-TOF-MS (Mussa et al. 2002). Spectra of the four proteins identified were queried against the EST (expressed

sequence tag) database of M. truncatulu, and good matches were found with calreticulin, nonseed lectins and an ion channel. These early results are promising, as a homolog of the nonseed lectins is known to be part of the pea nodule where it is thought to function as a storage protein. A similar relationship at this location of the root might exist between the plant and fungus at the arbuscular membrane.

3.6

Plant Model Systems

Considerably more progress has been made toward our understanding of plant genes to date because of the higher investment that has been made in plant biology. From 1985 – 1995, the Rockefeller Foundation sponsored an international research program in rice biotechnology that led to the generation of rice molecular maps, transformation of rice, and the characterization of many basic biochemical pathways for abiotic stress and disease and insect resistance in rice. The United States National Science Foundation (NSF)-Arabidopsis 2010 and Plant Genome Initiatives have also had a major impact on plant science research in the last decade (Ausubel 2002). Many of the projects deal with how Arabidopsis responds to and resists pathogens such as: (a) the Arabidopsis RPM1 disease resistance signaling network; (b) expression proﬁling of plant disease resistance pathways; (c) functional and comparative genomics of NBS-LRR-encoding genes; (d) Functional genomics of quantitative traits. Expression level polymorphisms (ELPs) of QTLs affecting disease resistance pathways in Arabidopsis; and (e) the endgame for research genetics. Isolation and distribution of a knockout mutant for every gene in Arabidopsis. More information can be obtained at the web sites of these projects (Ausubel 2002). National Science Foundation Plant Genome has also funded allied work in other crops plants (httn://www.nsf.gov). This research represents the cutting edge of plant science using the latest tools: microarrays to study gene expression, T-DNA insertion mutants, large scale mutant hunts, molecular mapping, and structure-function analysis of genes at the whole genome level. High through put methods for creating mutants using gene silencing that allow gene libraries or cDNA collections to be cloned in a silencing vector using an in vitro recombinase should facilitate systematic functional analysis of plant genes in plant defense (Wesley et al. 2001). Conventional mutagenesis of plants carrying a construct such as a luciferase or GUS (b-glucuronidase) reporter gene under the control of a defense gene promoter are yielding new and interesting classes of mutants with constitutive broad-spectrum disease resistance (Maleck et al. 2002). These mutants showed strong resistance to P. parasitica isolates Noco2 and Emco5 and variable resistance to Elysiphe cichoracearum. In addition to T-DNA, endogenous and heterologous plant transposable elements are being used to systematically mutate the rice genome and isolate promoter elements via enhancer trapping elements (Greco et al. 2001; Hirochika et al. 2001). The transcriptome of Arabidopsis has been characterized during

Biotechnological Approaches in Plant Protection

systemic acquired resistance (Maleck et al. 2000). Analysis of 402 putative transcription factors from Arabidopsis using OMAs allowed the identiﬁcation of transcription factors that may be involved in regulation of various pathways responsive to environmental stress and bacterial infection (Chen et al. 2002). These systems level approaches to analysis of model plants are expected to yield an integrated view of how every gene contributes to the growth and development and defense of plants.

SUMMARY AND CONCLUSION

The tools of biotechnology have already had a significant impact on control of fungal plant pathogens. The use of molecular markers to create genetic maps, identify, transfer, and clone plant genes of importance to plant protection has had a spectacular beginning. With the availability of new types of markers such as cDNA-AFLP and OMAs as well as the genomic and cDNA sequences of many important fungal pathogens and model dicot and monocot plants, rapid progress in this area is anticipated in the near and distant future. These and other genome-wide approaches will lead to an integrated view of the defense response for different classes of fungal pathogens as well as the core of pathogenspecific genes required for successful plant infection of host plants. This information will lead to new technologies for plant protection based on rational design whether through molecular breeding or transgene or chemical approaches. At present, few promoters related to disease control from plants have been thoroughly characterized. Likewise, our understanding of the rapid turnover of some defense gene products, such as Rpm1, is very poor. What relationship gene transcription has to levels of protein expression and modification and degradation for components of the defense response is unknown. Moreover these relationships must be known at the cellular not tissue level. Knowledge of the complete cascade of players involved defense signaling for one specific pathogen elicitor/effecter is still unavailable. Clearly there is much to learn and much promise ahead based on the knowledge to be gained. Public confidence in these approaches will require improved communication of the scientific research community with the public sector and comprehensive risk assessment supported by high quality research. Biotechnology is not a solution but a tool to be used along with the many other tools that already exist for plant protection, such as crop rotation and other cultural practices. The appropriate and reasoned use of this technology is needed.

ACKNOWLEDGEMENT I thank Dr. Rajinder S. Chauhan for critically reading this manuscript and providing the images used for the ﬁgures. The work reported from my laboratory was supported by grants

from the Rockefeller Foundation, the Mcknight Foundation, USDA-ARS and the Graduate School of the University of Wisconsin to SAL and a fellowship from the Department of Biotechnology, Government of India to RSC.

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17 Yoshimura S, Yamanouchi U, Katayose Y, Toki S, Wang ZX, Kono I, Kurata NY, Yano M, Iwata N, and Sasaki T (1998). Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA 95(4):1663– 1668. Yoshioka K, Nakashita H, Klessig DF, and Yamaguchi I (2001). Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action. Plant J 25(2):149 –157. Yu J, Hu S, Wang J, Wong GK-S, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X, Cao M, Liu J, Sun J, Tang J, Chen Y, Huang X, Lin W, Ye C, Tong W, Cong L, Geng J, Han Y, Li L, Li W, Hu G, Huang X, Li W, Li J, Liu Z, Li L, Liu J, Qi Q, Liu J, Li L, Li T, Wang X, Lu H, Wu T, Zhu M, Ni P, Han H, Dong W, Ren X, Feng X, Cui P, Li X, Wang H, Xu X, Zhai W, Xu Z, Zhang J, He S, Zhang J, Xu J, Zhang K, Zheng X, Dong J, Zeng W, Tao L, Ye J, Tan J, Ren X, Chen X, He J, Liu D, Tian W, Tian C, Xia H, Bao O, Li G, Gao H, Cao T, Wang J, Zhao W, Li P, Chen W, Wang X, Zhang Y, Hu J, Wang J, Liu S, Yang J, Zhang G, Xiong Y, Li Z, Mao L, Zhou C, Zhu Z, Chen R, Hao B, Zheng W, Chen S, Guo W, Li G, Liu S, Tao M, Wang J, Zhu L, Yuan L, and Yang HYu (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79 – 92. Zhang Y, Fan W, Kinkema M, Li X, and Dong X (1999). Interaction of NPRl with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc Natl Acad Sci USA 96(11):6523 – 6528. Zhu T, Budworth P, Han B, Brown D, Chang H-S, Zou Z, and Wang X (2001a). Towards elucidating global gene expression in developing Arabidopsis: parallel analysis of 8300 genes. Plant Physiol Biochem 39:221– 242. Zhu T, Chang H-S, Schmeits J, Gil P, Shi L, Budworth P, Zou G, Chen X, and Wang X (2001b). Gene Expression Microarrays: Improvements and Applications Towards Agricultural Gene Discovery. JALA 6(6):95 –98.

2 Chemical Identiﬁcation of Fungi: Metabolite Proﬁling and Metabolomics Kristian Fog Nielsen / Jørn Smedsgaard / Thomas Ostenfeld Larsen / Flemming Lund / Ulf Thrane / Jens Christian Frisvad BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark

Commonly used macro- and micromorphological characters (rough conidia, fluffy mycelia, color, etc.) can be difficult to determine unequivocally and are difficult to link to gene sequences. The production of secondary metabolites can typically be linked to a particular gene sequence or gene cluster and is most likely to be regulated as a response to growth factors. However, there are far more metabolites than genes as demonstrated by modern metabolomics. Schwab (2002) estimates that there might be as many as 10– 100 metabolites for each gene in higher organisms. Therefore, a metabolite profile can provide an indirect method of detecting a large set of metabolite coding genes which are expressed at the same time. Metabolite profiling also allows detection of a specific gene cluster through the identification of different compounds from that pathway. Turner (1971) and Turner and Aldridge (1983) suggested subdividing secondary metabolites according to their biosynthetic origin. In this way a selected metabolite originating from a pathway, e.g., polyketide, terpene, diketopiperazine, and cyclopeptide is treated as representative of that particular pathway. This is important, as only a limited number of members of a biosynthetic pathway are expressed under a given set of conditions, e.g., external stimuli and growth conditions (Section 2.2). Mantle (1987) has reviewed secondary metabolite production by Penicillium species based on biosynthetic pathways. He suggested a renaming of original isolates according to new taxonomic systems and emphasized that frequent misidentifications have lead to errors, especially for isolates no longer available for the scientific community. Frisvad and Filtenborg (1983) first demonstrated the advantage of secondary metabolite profiling in fungal taxonomy with the genus Penicillium,

INTRODUCTION

The identification of filamentous fungi has always been considered difficult and many misunderstandings and misidentifications can bee found in the literature (Frisvad 1989; Mantle 1987). Phenotypic characters, e.g., morphology and growth on selected media have traditionally formed the basis for fungal taxonomy (Domsch et al. 1980; Mantle 1987; Pitt 1979; Raper and Fennell 1977; Raper and Thom 1949). Advancements in the developments of analytical methodology have allowed the use of “secondary” metabolite profiling for fungal identification and been used to revise the taxonomy within genera of Penicillium, Aspergillus, Fusarium, Alternaria, and their perfect states. The success of metabolite profiling in the classification of filamentous fungi relies on the fact that a major part of the fungal growth is expressed by the production of numerous diverse metabolites, most of which are excreted into the media. The extracellular metabolites have been termed the exome, a subgroup of the metabolome (all metabolites), and these are related to the genome as illustrated in Figure 1. The reasons why most filamentous fungi produce such a diverse profile of secondary metabolites are still unclear, but they are probably produced as a result of stimuli and are directed against, or support actions on, receptor systems (Christophersen 1996) or as outward directed (extrovert) differentiation products. Possible others functions, include chemical signaling between organisms (Christophersen 1996; Frisvad 1994a). Williams et al. (1989) described their functions as “. . . serve the producing organisms by improving their survival fitness . . . .” 19

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MYCOLOGY AND BIOSYSTEMATICS

Biosystematics can be divided into taxonomy (¼the theory of classification), nomenclature, identification and phylogeny. The species is the central concept in biosystematics. It is important to note that classification deals with the natural nonoverlapping hierarchical grouping of isolates into species, species into genera, etc, whereas identification deals with allocating new isolates to already existing classes in an effective, nonequivocal, and practical way. Figure 1 The profile of extra-cellular metabolites or the exome is linked to genomics through a mostly one-way connection from the genome. Far more metabolites than genes are found in most cells, and metabolite profiles are phenotypic characters as are most other characters used in classical mycology.

using a simple agar-plug-TLC technique. They later also included HPLC methods in their studies. Efficient identification based on metabolite profiling relies on combining this information with more classical tools and a´ priori knowledge. For general use it is important to know at least the genus of the fungus being studied and which growth media to use. The analytical methodology depends on the group/genus being studied, but can vary from the simple TLC approach to hyphenated LC-MS-MS. This combined approach is illustrated in Figure 2. It is important to note that efficient identification of fungi will, in most cases, require the use of profiles of metabolites from crude extracts, rather than single or selected metabolites. However, a limited number of species-specific metabolites can be used efficiently as markers for particular species in particular cases. This chapter focuses on the practical considerations in the use of metabolite profiles in identification. The text follows the general approach illustrated in Figure 2, and four different cases of various techniques are given.

2.1

The characters most often used in identification keys for filamentous fungi are based on arrangements, forms, sizes, and ornamentations of mature asexual and sexual structures. Another kind of latent characters used in identification keys are responses to abiotic factors such as macromorphology and colony growth rate or diameter at different temperatures, water activities, pH, redox potential, etc. (Pitt 1973). The problem with all these different tests is the potential large number needed (economy) and the need for standardization. Morphological features occasionally overlap or can be difficult to record precisely (Frisvad et al. 2000), as they may be dependent on the media used (for example different brands) and is particularly pronounced for colony diameters and colors. Standardization of incubation conditions (Pitt 1973) and the use of chemically defined media have been proposed to avoid these problems. Image analysis has also been explored in Penicillium (Do¨rge et al. 2000), here the information found in fungal colonies such a colony texture, diameter and color from very accurately recorded and calibrated images could identify terverticillate penicillia, and has recently been used for clones recognition (Hansen et al. 2003). In order to identify filamentous fungi it is necessary to identify the fungi to genus level before using traditional keys or chemotaxonomic methods. Filamentous fungi can be identified to genus level by the use of keys [e.g., Samson et al. (2000)]. Once the genus is known references to keys and taxonomic treatments can be found, but as new species are described each year, it may be difficult to obtain good and upto-date keys for large genera. Knowledge of the associated funga (mycobiota) of different habitats can be a major help in identifying the most common species (Filtenborg et al. 1996). For example the only Penicillia that can grow on citrus fruits are Penicillium italicum, P. ulaiense, and P. digitatum, so identification of greenish mould growth on citrus fruits will be relatively easy.

2.2 Figure 2 Efficient identification of filamentous fungi requires a synthesis of mycology, analytical chemistry and informatics although the metabolite profile can sometime give the full answer.

Classical Identiﬁcation

Cultivation and Media for Metabolite Proﬁling

Most fungi have evolved on solid matrices, and hence solid media are generally better than liquid media in terms of quantity and

Chemical Identiﬁcation of Fungi

number of metabolites produced. On agar media, any contamination is usually visible, and an agar plug technique can be used to sample different parts of the fungal colony and its surroundings. In general, agar media for optimal secondary metabolite and mycotoxin production have been based on media containing yeast extract. Yeast extract sucrose (YES) broth was introduced as a “semisynthetic” broth medium for aﬂatoxin production by Davis et al. (1966). It was later shown to be a very effective general secondary metabolite production medium when used with a crude yeast extract (DIFCO or SIGMA) and formulated as an agar medium (YES agar) by Frisvad (1981); Frisvad and Filtenborg (1983), and has been used for Penicillium, Aspergillus, Fusarium, Alternaria, and many other fungal genera (Andersen et al. 2002; Thrane 2001). Other media including Czapek yeast autolysate (CYA) agar, Potato dextrose (PD) agar can be used to supplement YES agar, depending on the genus being considered, as seen in Table 1. Some of these semisynthetic agar media can occasionally give problems as certain brands of yeast extract, malt extract, potato extract, agar, peptone, or tryptone, etc. may differ signiﬁcantly in composition, although this may be diminished by adding trace elements and magnesium sulphate (Filtenborg et al. 1990).

TECHNIQUES FOR METABOLITE PROFILING

Metabolite profiling for identification in its simplest form consists of three elements: getting hold of the metabolites (e.g., extraction), determining the compounds (the profile) (e.g., analysis), and data processing (e.g., chemometrics). This will include all relevant metabolites needed for reliable identification of a fungus, and as discussed in the previous

Table 1

section, profiles of metabolites rather than single metabolites should be used for identification. It is very important to note that metabolite profiles are strongly influenced by the analytical scheme used, thus full metabolite profiles can only be compared if they are produced by the same analytical protocol. Over the years, many specific techniques have been developed to determine a few selected metabolites, mostly known mycotoxins, from cultures and from complex samples such as food and feed. Some of these methods can be expanded to include a broad spectrum of metabolites, but dedicated profiling methods are required to efficiently obtain the best possible metabolite profiles and allow reliable identification. Developments in chromatography and mass spectrometry (MS) have greatly increased their resolution, sensitivity and productivity in analytical chemistry. There is, therefore, a set of tools available that allows fast metabolite screening from a very small amount of sample. As a result, a broad range of metabolites can now be determined in one analysis with high selectivity. Furthermore, several of the newer techniques have shown their potential as general rapid profiling methodology working directly on raw samples or extracts. These include MS, nuclear magnetic resonance, and FT-IR (including NIR), which eliminate a time consuming chromatographic step. However, currently it is not wise to fully eliminate chromatography and one should rather use systems based on complementary methods. In planning an identification from an analytical approach the following considerations must be made: (a) A´ priori knowledge about metabolites produced by the genus, (b) whether the general chemical classes are alkaloids, acids, neutrals, volatiles, as well as large or small molecules, (c) sample matrix or growth medium composition and interference, (d) number of samples to handle, (e) expected biological variation, and if large or small chemical diversity is expected, (f) sensitivity of analytical instrumentation, and (g) cost.

Recommended media for microscopy and chemotaxonomy of important fungal genera

Genus Penicillium subgenus: Furcatum, Penicillium, Aspergilloides Penicillium subgenus Biverticillium Aspergillus

Paecilomyces Stachybotrys Trichoderma Fusarium Alternaria

Teleomorphic state

Microscopy

Metabolite proﬁling

Eupenicillium

MEA

YES, CYA

Talaromyces

MEA

OAT, MEA, YES

Eurotium, Emericella, Neocarpenteles, Neosartorya, Petromyces, Neopetromyces, Chaetosartorya, Sclerocleista, Hemicarpenteles, Fennellia Byssochlamys Melanopsamma Hypocrea Gibberella, Nectria, Cosmospora, Haematonectria Lewia

MEA

YES, CYA

MEA CMA OAT, CMA SNA GAK

YES, PD PD, ALK YES, PD YES, PD DRYES

Media recipes, see text or our website http://www.biocentrum.dtu.dk/mycology/analysis/.

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Figure 3 The agar plug extraction method is a simple scheme by which extracts for metabolite proﬁling can be prepared rapidly using only a minute amount of solvent (Smedsgaard 1997a).

3.1

Extraction

The first step in metabolite profiling for fungal identification is to obtain the metabolites produced by the fungi growing on some sort of substrate, see Section 2.2 and Table 1. Depending on the group of metabolites of interest, two main schemes can be used to collect metabolites for a profile: (a) an extraction approach is used for the nonvolatile metabolites, as illustrated in Figure 3, in combination with HPLC and/or MS and (b) a headspace approach is used for the volatile metabolites as illustrated in Figure 4, in combination with GC or GC –MS.

Figure 4 Collection of volatile metabolites using adsorption techniques. A stainless steel petri dish lid with a standard 1/400 Swagelocke ﬁtting in the centre is used to hold the different adsorption tubes or with a septum to be used with a SPME syringe. Sampling is done by placing an adsorption tube, open only in the lid end, in the ﬁtting and leaving the lit on the cultures for a few hours. The tube or syringe is then analyzed.

Nearly all sample preparations start with either an extraction based on distribution of the analyte between two immiscible phases or by a gas phase sampling. One phase is the fungus (biomass) and/or growth medium, the other is the solvent or a purge gas. Some physical (thermal or mechanical) assistance may be needed to help the rapid distribution of metabolites between the two phases. There may be some overlap in the compounds determined by the two different approaches, e.g., some of the terpenes can be found in both extracts and by headspace analysis. The key point is to select extraction solvents with a high affinity for metabolites of interest. Adjusting pH can enhance the solubility of metabolites in the extraction solvent. Furthermore, choice of extraction solvent can also be used to favor extraction of a particular subgroup, and a selection of several solvents can be used to extend the range of metabolites. Many extraction protocols use grams of material and many milliliters of solvents, however these procedures can be miniaturized due to the very high sensitivity and selectivity of modern instrumentation. A simple method is the plug extraction method (Smedsgaard 1997a) illustrated in Figure 3, where a few 6 mm plugs (0.5– 1 cm2 area) are cut from plates of the fungus and extracted with about 500 ml of solvent. The extraction is performed ultrasonically to improve extraction efficiency and speed. If the raw extract is compatible with the subsequent analysis it can be injected directly, otherwise the solvent have to be evaporated and the sample redissolved in an appropriate solvent (Figure 3). Other analytical applications requires a more elaborate sample preparation particularly if GC –MS analysis is required. If fungi are to be identified in complex natural samples, e.g., foods or building materials a much more elaborate sample preparation protocol is needed. It will nearly always be necessary to remove interfering matrix compounds, by liquid–liquid extraction or by passing the crude extract through a disposable mini-column (solid phase extraction, SPE).

Chemical Identiﬁcation of Fungi

Sampling the volatile metabolites from fungi growing in culture can be done by two approaches: either by one of three different dynamic headspace (HS) methods using direct collection of volatiles present in the gas phase above the growing fungus or by extraction of volatiles present in the fungal biomass (or sample). Figure 4 illustrates the simple method to collect volatile metabolites by diffusive sampling described by Larsen and Frisvad (1994; 1995c). The volatile metabolites are collected by adsorption on an adsorbent, e.g., activated carbon black, a synthetic polymer like Tenax TA, or recently a coated fiber (Solid Phase Microscale Extraction, SPME). Carbon black tubes are normally desorbed using solvents, e.g., dichloromethane or diethylether. Tenax tubes and SPME fibers are analyzed by thermal desorption, the SPME fibers in a split/splitless injection port, the tubes using thermal desorption equipment.

3.2

HPLC

HPLC is by far the most common analytical technique used for metabolite profiling due to its versatility, relative ease of operation, and the broad spectrum of metabolites that can be determined directly. Basically, HPLC is an integration of a separation and detection system working with separation in a liquid phase. Metabolite profiling using HPLC is almost always done using gradient elution on reversed phase material (C8 –C18 phase or similar) with polar mobile phase. The most widely used mobile phases are water-acetonitrile or water-methanol containing some modifier, e.g., tri-fluoroacetic acid (TFA). The flow rates used for metabolite profiling depend on the columns, but in general analytical columns with diameters between 2 and 4 mm are used at flow rates from 0.2 to 1 ml/minute. Smaller diameter columns will give a better separation at the cost of lower absolute sensitivity, as a smaller amount of sample can be injected. As described in the introduction to this section identification of fungi from metabolite profiles can be done either by detection of specific metabolites or by using the full chromatogram as a profile. In both cases it is important to keep the analytical conditions as constant as possible. In the first case, the identification of metabolites can be determined from the retention times used together with standards. Possible spectral detection is described later. The effect of small, unavoidable shift, in retention time over time can be reduced by using a series of alkylphenones to calculate a retention index for each peak in the chromatograms (Frisvad and Thrane 1987). If the full profiles are to be used constant analytical conditions will reduce the alignment needed (Nielsen et al. 1998; 1999). In cases of metabolite profiling, detection of compounds eluting from the HPLC column is mostly done by UV detection, fluorescence detection (FLD), or MS. The UV detection accounts for the majority of the applications, however there is currently considerable growth in the use of MS (see section 3.4). In classical HPLC, compounds are detected by measuring the absorbance of the column eluent at a specific

wavelength. The chromatogram [e.g., plot of absorbance vs. time] shows peaks representing the compounds eluting from the column. This technique requires that the compounds have a chromophore that absorbs light at the selected wavelength and that the solvent is transparent (or at least does not have any significant absorption). Fluorescence can be measured with a selected pair of excitation/emission wavelengths in a similar fashion, if the compounds have a fluorophore. Several mycotoxins have distinct fluorophores, whereas others can be derivatized prior to detection. Identification of fungi can in some cases be achieved by identifying a set of specific metabolites produced, regardless of the analytical method. This requires that relevant metabolites to be identified with high certainty, and usually requires either use of an authentic standard and/or several spectral and chromatographic techniques. Spectral detection by a UV-spectrometer (DAD or PDA) is commonly used as these techniques greatly enhance the information available by providing structural information about each peak (compound). In HPLC – UV detection, a UV-spectrum is collected at regular intervals in such a way that between 10 –20 spectra are collected across a chromatographic peak. The resulting data file is illustrated in Figure 5 and it is important to understand the structure of these files to fully exploit the data of a full HPLC –UV chromatographic matrix. The chromatographic UV-traces at selected wavelengths are present at the time axis and a UV spectrum is present at a specific retention time. The full data matrix can be visualized as a chemical image of the sample and can also be used for fungal identification without identifying the metabolites (Nielsen et al. 1998; 1999; Thrane et al. 2001). Figure 5 shows that quite different looking chromatograms can be obtained from the same sample by selecting different wavelengths. This feature can be used to enhance the chromatographic resolution and to find specific metabolites, e.g., viomellein is at 18.69 minutes on the 400 nm trace in Figure 5 and overlaps with puberuline at 18.40 minutes on the 220 nm trace. These traces are all profiles of the sample and as such can be used alone or in combination, or to aid specific detection. As quantitative detection is based on Beers law it is necessary to use the same wavelength for quantifying standards and unknowns for each compound but it is not necessary to use the same wavelength for different compounds. Identification or characterization of compounds can be achieved from UV spectra if the compound has a characteristic chromophore structure (bond structure typical containing pelectrons systems), e.g., xanthomegnin, which is two anthraquinone systems, has a chromophore giving an absorption maximum around 410 nm. This is fortunately the case for many fungal metabolites but not for all (Cole and Cox 1981). The combination of retention time or preferably the retention index and a characteristic UV spectrum is quite reliable for identification of many fungal metabolites (Frisvad and Thrane 1987; 1993), however standards are needed for confirmation. The use of UV-spectra in combination with other detectors, e.g., FLD can greatly enhance the specificity.

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Figure 5 The HPLC-UV data matrix showing both information about composition in form of chromatographic traces and structural information in form of UV spectra. Analysis of a plug extract from Penicillium cyclopium, IBT 16932, grown on CYA collecting approx. one spectrum/sec, with a resolution of 4 nm.

For identifying fungi, it is not necessary to know the identity of all of the components in a chromatographic profile. If a peak has unique features in terms of retention time /index and chromophore it can be designated with a code and used along with the metabolites known from a particular species. The most efficient identification can be done if metabolites are grouped in chromophore families (known and unknown compounds), as compounds with similar UV spectra often belong to the same bio-synthetic pathway. Therefore, if just one member of a chromophore family is present it can be used as indication of that particular pathway is active in a fungus.

3.2.1

Case I. Ochratoxin A Determination in Aspergillus niger

Ochratoxins can easily be detected in the two penicillia, P. nordicum and P. verrucosum as well as in A. ochraceus and Petromyces alliaceus where they are good species markers. They can be detected using HPLC with UV detection with full scan UV spectral conﬁrmation. However, in the A. niger

complex, the two ochratoxin producing species A. niger (only 6% of the isolates produces ochratoxins) and A. carbonarius produce many interfering components that elute across the whole chromatogram (Figure 6) and obscures ochratoxin detection. The FLD gives a very high specificity and sensitivity (1 –20 pg on column), with the possibility of obtaining full scan fluorescence spectra. This is illustrated in Figure 6 where an A. niger isolate has been analyzed by HPLC with simultaneous UV and FLD, ochratoxins A and B are hidden in the UV chromatograms under peaks of tetracyclic components. The simultaneous detection of all ochratoxin analogues (a, b, A and B) serves as an extra confirmation.

3.3

Gas Chromatography and GC – MS

Gas chromatography (GC) can be used to analyze volatiles directly and numerous semivolatile components after derivatization, e.g., trichothecenes, amino acids, sugars,

Chemical Identiﬁcation of Fungi

Figure 6 Extract from an Aspergillus niger isolate analysed by HPLC-DAD-FLD. Ochratoxin A, B, a, and b, are easily seen in the lower ﬂuorescence trace (ex. 230 nm, em. 450 nm) with the ﬂuorescence spectra of ochratoxin A and a inserted. In the upper UV trace (210 nm), several tetracyclic compounds (UV spectra inserted) obscure the detection of Ochratoxin A, B, and a.

lipids, and sterols. The advantage of GC is its very high separation power in comparison to HPLC and its relative ease of operation. Furthermore, GC is easily interfaced to MS (GC– MS) or other spectral detectors, forming a powerful tool that can both deliver high separation power and give structural information in one run. The most important step in GC is the injection, which if performed poorly can have a severe effect on the separation power. The most commonly used techniques for liquids are split, splitless, on-column injection, thermal adsorption of trapped volatiles, and headspace (Grob 1993). Currently fused silica columns for gas –liquid chromatography are used due to the high resolution power which is needed to separate complex mixtures of volatiles such as mono- and sesquiterpenes. Different stationary phases as well as film thickness can be chosen depending on the polarity and volatility of the compounds to be separated. More volatile compounds require a thicker film column, whereas high separation power is best obtained by thin film columns (Grob 1993). The mass spectrometer can be used to: (a) provide structural information from fragmentation in electron impact ionisation (EI) easily searched in databases, (b) accurate mass using the modern time-of-flight (TOF) instrument, or (c) using ion-traps (or multistage MS –MS instruments) as very high selectivity detectors (or to get very detailed fragmentation

information). Both TOF and MS–MS instrumentation greatly increases the capability of the instruments, and the metabolite profiles that can be obtained from these instruments are generally not needed for fungal identification and they are therefore not discussed further. For metabolite profiling, the most important detection method is MS which in many cases will give a molecular ion (and thereby molecular mass) and a characteristic fragmentation pattern—the mass spectrum—from each compound eluting from the column. The limitations of MS in the identification of unknowns can be an insufficient information content of the mass spectrum to stereo and positional isomers in aromatic systems (Ramaswami et al. 1986). A great potential for MS is the ability to scan for a selected number of characteristic ions—selected ion recording (SIR or SIM) which can improve detection sensitivity from the ng level to the pg levels. Flame ionization detection (FID) is an important and robust GC detector, which basically detects carbon atoms in the sample with high sensitivity and over a large dynamic range, but with no structural information. If metabolite profiles are compared on two different columns of substantially different polarity, similarity in the retention times of metabolites on these two columns can be used for their indirect identification (Davies 1990).

Fourier transform infrared spectrometry (FTIR), can be used to detect compounds eluting from GC columns. Most functional groups, in particular the carbonyl group (CvO), have a unique absorption (ﬁngerprint) from vibration energy. The FTIR will therefore give an unambiguous identiﬁcation of functionality of the functional groups of compounds and it is possible to identify these by comparing spectra to those of known authentic compounds. The method is nondestructive and can therefore be used in combination with either FID or MS. GC–FTIR will often allow discrimination between structural- and stereo-isomers making the method a very powerful supplement to GC–MS. The major drawback of GC– FTIR is a lower sensitivity compared to GC-FID and GC-MS.

Nielsen et al.

3.3.1

Case II: Volatile Metabolites for Identiﬁcation Of Penicillia

Volatile metabolites are believed to play an important role in chemical interactions between fungi and other organisms. Recently the total volatile proﬁle of the endophytic fungus Muscodor albus was demonstrated to effectively inhibit or kill a number of other fungi and bacteria (Strobel et al. 2001). In general volatile production is correlated with spore formation ﬁtting well with a chemical ecological point of view. The insects, often adapted to the toxic metabolites produced by the fungi, can act as vectors for fungal spores for their further spread.

Figure 7 Chromatograms showing proﬁles of volatile sesquiterpenes produced by Penicillium roqueforti (IBT 16403) and P. carneum (IBT 6884). The two compounds produced in largest amounts by P. roqueforti are b-elemene and (þ)-aristolochene, whereas the three largest peaks in the P. carneum chromatogram represents geosmin, an unknown sesquiterpene and dodecanoic acid methyl ester.

Chemical Identiﬁcation of Fungi

Some important studies have shown that the production of some possibly species-specific sesquiterpenes could be related to the production of important mycotoxins such as aflatoxins (Zeringue et al. 1993) and trichothecenes (Jelen et al. 1995). Often fungal metabolites are only present in particular compartments of the organism such as in the conidia (or sclerotia), and these may have a role in protecting them against being eaten by other organisms. Microfungi such as the penicillia usually produce a species-specific set of volatile metabolites (Larsen 1998; Larsen and Frisvad 1995a). Often even very closely species such as P. roqueforti and P. carneum (Figures 7 and 8) can easily be differentiated by the volatiles they produce. Fungal volatile metabolites include small ketones, alcohols, esters, and hydrocarbons such as small alkenes, mono- and sesquiterpenes. The latter are by far the most relevant metabolites for identification purposes or for use as biomarkers (Larsen and Frisvad 1995b). The major sesquiterpenes produced by P. roqueforti are b-elemene, selenine, and patchuline (Larsen and Frisvad 1995b) together with aristolochene (Figure 8), recently reported by Demyttenaere et al. (2002). In general P. carneum produces relatively lower amounts of sesquiterpenes than P. roqueforti, however, the species has a much more pronounced moldy odor than P. roqueforti due to the production of the moldy smelling compound geosmin together with large amounts of isopentanol (not shown in Figure 8). It should be emphasized that the production of some volatile compounds is strongly related to medium composition, e.g., lipid degradation of fat rich media leads to the production of ketones and secondary alcohols, as seen in Camembert and especially Blue cheese production. The NIST and Wiley MS databases contains many spectra of mono- and sesquiterpenes (generated a 70 eV) for identification of single

Figure 8 Structures of the main fungal terpene metabolites obtained from the headspaces of Pencillium roqueforti and geosmin produced by P. carneum. 1) Limonene 2) b-elemene 3) g-patchulene 4) b-caryopyllene 5) b-myrcene 6) (þ )aristolochene 7) a-selinene 8) geosmin.

compounds, and a lot of spectral information can also be found in the atlas of Joulian and Ko¨nig (1998). A good review of methods for the identiﬁcation of sesquiterpenes can be found in Ko¨nig et al. (1999). As mentioned in Section 3, the sensitivity can be greatly enhanced by the use of SIR, and the method has been used together with SPME to investigate how early volatile metabolites can be detected (Larsen 1997). The SIR of four to seven of the most characteristic ions of mainly sesquiterpenes from cheese-associated fungi allowed the identiﬁcation to species level within two days, at which time they had not started to sporulate and were only white mycelia. Volatiles from a mixed culture of P. roqueforti and P. commune, inoculated in a ratio of 1000:1, could be used to detect both fungi within three days, showing the possibility of checking starter cultures for cross-contamination.

3.4

Atmospheric Pressure Ionization MS

The last decade has seen a tremendous development in biological MS, and it is currently one of the fastest growing analytical techniques in biotechnology. The MS is the determination of the mass to charge ratio of charged species of molecules or highly specific fragments of these (as in GC –MS). These can be either positively charged or negatively charged. In the atmospheric pressure ionization (API) LC–MS techniques, ions are formed at atmospheric pressure and transferred into the vacuum of the mass analyzer. There are two predominant ionization techniques: Electrospray ionization (ESI); and atmospheric pressure chemical ionization (APCI). In ESI the eluant from the column is sprayed though a narrow bore capillary to which a high voltage is applied (around 3 kV). This will produce a spray of highly charged droplets. The solvent is evaporated from the droplets by a heated gas, leading to shrinkage and disintegration to charged species through a complex process. The ions are formed either in the solvent before spraying or during the spray droplet shrinkage and the key parameters influencing the ion production are: solvent composition (surface tension, volatility, modifiers, pH, ion strength), source parameters (temperature, drying gas flow, potential), and interaction between analytes in the sample (Berkel 2000). The charged species are then sampled into the vacuum of the mass analyzer. In APCI the eluant from the HPLC column is sprayed through a co-axial capillary with a heated gas to evaporate the solvent. Evaporated solvent molecules are ionized by a corona discharge from a needle that is usually placed across the sampling orifice. Analyte molecules are ionized by chemical reaction in the gas phase at atmospheric pressure through a process much like the classical chemical ionization. The ions are sampled into the mass analyzer by a process similar to ESI (see Table 2). In general, ESI is the most versatile technique for a very broad range of bio-molecules and also the easiest to use, therefore ESI is also the most frequently used technique.

Nielsen et al. Table 2 Comparing some of the characteristics of the two major ionization techniques in LC-MS

Type of ions positive Negative ions Ionization in Solvents Fragments Multiple charged ions Clusters and complexes Mass range Typical ﬂow rate with HPLC

ESI

APCI

M þ Hþ, M þ Naþ, solvent adducts (M 2 H)2 Solvent (solvent gas interface) Some ions required None or few Yes Many High (multiple charged ions) 5 – 500 ml/min

Mþ, M þ Hþ M2, (M 2 H)2 Gas phase Apolar solvent possible Common No Few As the mass analyser 300 ml/min– 1.5 ml/min

Not all molecules are ionized (usually protonated) in the positive mode, and are detected much better as negative ions. Gas phase chemistry is however not like solvent chemistry, and many carboxylic acids are much more efficiently protonated in gas phase by positive ESI than determined as anions in negative ESI. Negative ESI will give many fewer adducts and clusters than positive ESI, see Table 3, and so it is therefore easier to interpret spectra. However, a higher sensitivity can be obtained for some classes of compounds in APCI than in ESI. The charge to mass ratio is determined using a mass analyzer, which is either: Quadrupol, time of flight (TOF), ion-trap, sector (electric and magnetic) or an ion-cyclotron (ICR). These analyzers can be grouped into scanning analyzers (mass filters) where ions of just one mass to charge ratio can pass at a time and nonscanning analyzers where all ions entering the analyzer are detected (Table 4). Mass resolution and accuracy are the two most important factors for the identification of compounds whereas sensitivity and scan speed are of most chromatographic importance. Accurate mass determination relies on both sufficient resolution to separate isotopes and a very stable mass to

Table 3 Quasi-molecular ions are often seen along with adducts in a low mass positive electrospray mass spectrum. This is highly solvent and parameter dependent. Very few adducts and clusters are seen in negative ESI Adducts Charging by Solvent adducts Dimers Multiple charged Fragments Others

H , Na , K H2O, CH3OH, CH3CN 2M þ Hþ, 2M þ H3Oþ, 2M þ Naþ Double charged is rarely seen below mass 800 – 1000 Da/e – H2O, Fe, Zn, and other metal complexes are sometime seen e.g. (M 2 Hþ) þ Fe3þ þ H3CCOO2 þ M ions can be seen from some types of compounds

charge determination. The performance of two common mass analyzers is shown in Figure 9. The TOF analyzer raw data (often called a continuum spectrum) show a resolution of approx 7500 (half height) and the quadrupole approx 900 (half height). Mass spectra are normally used as centroid (stick) spectra for mass determination where the stick is placed at the center of the continuum peak. This also reduces the disk space needed to store the spectra. Formula can be calculated from the mass, and with sufficient mass accuracy the number of possible structures is limited if sensible limits for composition are applied (Table 5). Combining a mass spectrometer with HPLC will allow the recording of mass spectra as peaks are eluted from the column. As was the case collecting UV-spectra, mass spectra are collected continuously with about 10 –20 spectra across a peak to produce a data matrix containing both chromatographic and mass spectral information see Figure 10. Ion traces are highly specific chromatographic profiles of the samples which depend on the mass accuracy. Figure 10 shows an example of high accuracy ion traces corresponding to the protonated mass of puberuline (444.2287 Da) and xanthomegnin (575.1187 Da) both using a window width of 15 ppm (6.7/8.7 mDa) showing only one peak in each; compare this to Figure 5. For each peak a mass spectrum can be retrieved, given structural information about the sample. In this case using an accurate TOF mass spectrometer also gives an estimate of peak formula. Comparing the HPLC-MS image in Figure 10 to the HPLC-UV image in Figure 5 there are significantly more details and higher specificity in the former of these two. The data shown on Figures 5 and 10 were acquired during the same run using a nondestructive UV detector in series with the MS to give the maximum information. There are some restrictions on the use of HPLC-MS which depend on the ionization techniques: the eluants must be volatile including modifiers (e.g., acids), the flow rate must be suitable for the interface and modifiers/ion strength must match the ionization technique (ESI þ /2 or APCI þ /2 ). In general, most reversed phase chromatographic solvent systems can be used, e.g., water, acetonitrile, and methanol with the modifiers acetic acid, formic acid, and ammonium

Chemical Identiﬁcation of Fungi

Table 4 Typical performance of the different mass analyzer although this may vary due to special designs. The sensitivity is relative to the other analyzers and dependent on the analytical approach

Quadrupol Iontrap TOF Sector FT-ICR-MS a

Resolution

Accuracy

Accuracy at 1000 Da

Sensitivity

Scan speed

Relative price

1000 – 2000 1000 . 10.000 . 50.000 . 100.000

100 – 300 ppm 100 – 300 ppm 2 – 5 ppma 1 – 5 ppma 0.05 – 0.1 ppma

0.1– 0.3 Da 0.1– 0.3 Da 2 – 5 mDaa 1 – 5 mDaa 0.05– 0.1 mDaa

medium medium high high high

high high Very high low Very high

low low Medium High Very high

Using internal mass correction.

Figure 9 Comparing mass resolution and accuracy of the puberuline M þ Hþ ion C27H30O3N3 mass 444.2287 Da/e) from analysis of a crude plug extract see the case below. A peak width at half height of about 0.06 Da and a precision of approx. 1 mDa (centroid data) is found by the TOF instrument (top) and a peak width of about 0.5 Da and a precision of approx. 30 mDa by the quadrupole instrument (bottom).

acetate. However, trifluoroacetic acid should particularly be avoided in APCI (both positive and negative), whereas it can be used in low concentration in positive ESI. It is very difficult to run ESI in pure organic solvent due to volatility, and the limit seems to be around 90–95% acetonitrile-water, however ACPI works well in a pure organic solvent. Metabolite profiling by HPLC –MS (ESI or APCI) are very efficient tools for identification and classification of fungi. As the specificity is very high, rapid chromatographic methods can be used. If a list of expected ions can be made, then the

Table 5 Number of structures possible for mass 444.230 Da with different mass accuracies Accuracy 1 ppm (0.44 mDa) 5 ppm (2.2 mDa) 50 ppm (22 mDa)

Number of structures 1 7 61

Composition limits C , 100 H , 500 O , 12 N , 10 DBE , 50

ion traces corresponding to these can easily be drawn. It is then a simple matter to interpret. In most cases it is not necessary to use high resolution/accurate mass spectrometers for fungal identification. In practical identification, a combination of several metabolites with different retention time is used as a mark for each species thereby limiting the number of misidentifications. However it may be necessary to use more than one ionization technique, as some components are difficult to ionize in positive ESI. If the goal is to get a full profile of all metabolites produced under specific conditions for metabolomics, then high resolution/accurate mass determination is a major advantage, as it also provides the molecular composition of the ions.

3.4.1

Case III: Direct Infusion MS

a. Metabolite Proﬁling in Taxonomy Of Penicillium Series Viridicata. An advantage of ESI mass spectrometry is that the analytical conditions can be optimized to limit fragmentation and cluster formation. In the ideal case, only protonated molecules are observed from each compound in a

Nielsen et al.

Figure 10 The HPLC-ESIMS data matrix showing both information about composition in form of chromatographic traces and mass information in form of mass spectra. Analysis of a plug extract from Penicillium cyclopium, IBT 16932, grown on CYA collecting approx. One spectrum/sec. With a mass resolution of 6000 and an accuracy , 5 ppm (Micromass LCT with lockspray). Table 6 Production of secondary metabolites by cereal associated Penicillia Speciesa Metabolite Xanthomegnin Viomellein Aurantiamine Viridamine 3-methoxyviridicatin Brevianamide A Ochratoxin A Citrinin Penitrem A Oxaline Terrestric acid Penicillic acid Verrucosidin a

III

0 0 þ 0 0 0 0 0 0 0 þ þ þ

þ þ þ 0 þ 0 0 0 0 0 0 þ 0

þ þ 0 0 0 0 0 0 0 0 þ 0 0

IV 0 0 0 0 þ 0 0 0 0 0 0 þ þ

VII

0 0 0 0 þ 0 0 0 0 0 0 þ 0

þ þ 0 þ 0 þ 0 0 0 0 0 þ 0

þ þ 0 0 þ 0 0 0 0 0 0 þ 0

VIII b

0 0b 0 0 0 0 0 0 þ þ 0 þ þ

IX 0 0 0 0 0 0 þ þ 0 0 0 0 0

I: Penicillium aurantiogriseum; II: P. freii; III: P. tricolor; IV: P. polonicum; V: P. aurantiocandidum; VI: P. viridicatum; VII: P. cyclopium; VIII: P. melanoconidium; IX: P. verrucosum and detected by TLC. b The metabolites can be detected by HPLC.

Chemical Identiﬁcation of Fungi

sample, thus injecting a mixture of compound will give a mass proﬁle of the sample. This approach was used by Smedsgaard in a study of the terverticilllate penicillia (Smedsgaard 1997b; Smedsgaard and Frisvad 1997). In that study, crude plug extracts from cultures were injected directly into the mass spectrometer in positive ESI mode with solvent and other parameters optimized to minimize fragmentation and cluster. Samples were used at low concentration to reduce matrix effects, thus avoiding “the winner takes it all effect.” About 10– 15 samples can be analyzed per hour by this approach, which have recently been used to study other organisms such as bacteria (Vaidyanathan et al. 2002) and actinomycetes (Higgs et al. 2001). Nine of the major species in the Penicillium series Viridicata (The Penicillium aurantiogriseum complex) were analyzed by direct injection on a quadrupole mass spectrometer (Smedsgaard and Frisvad 1996). Figure 11 shows crude extract spectra from three different species cultivated on CYA (Samson et al. 2000). The spectra show signiﬁcant difference between the species and ions corresponding to the protonated mass of major known metabolites. These spectra can be stored and searched in a spectral database using the software included with most instruments, although this software in general is designed for EI spectra (Smedsgaard 1997b). To prove the concept in more detail a cluster analysis of the full centroid mass spectra (normalized) from 45 isolates of the nine major Penicillium series Viridicata species (Figure 12) was performed. This cluster analysis is done directly on the centroid spectra with 1 Da

resolution using the correlation coefficient and UPGMA linkage. As it can be seen from Figure 12 most species cluster together, however P. aurantiocandium is found in the P. cyclopium and P. tricolor clusters. One P. viridicatum is grouped with P. aurantiogriseum and another is an outlier. The ions (metabolites) that are important to the segregation of the species can be found using Principle Component Analysis (PCA) as the PCA loadings (Figure 13). If we consider loadings above 0.04 or below 2 0.04, 47 ions are found in the plot from the first three principal components. Of these, 15 ions correspond to the protonated ions from 15 of the most important metabolites produced by these species (out of about 28 metabolites). Furthermore five ions correspond to the 13C isotope of major metabolites. Five to six characteristic metabolites are not found as they are not produced on these media, however they can be seen in the mass spectra from some of the known producers. Four significant ions of unknown structure are found from the loadings plot at mass 205, 235, 243, and 274 Da.

3.5

A Combined Approach: The Agar Plug-TLC Method

The agar plug method was ﬁrst introduced as a taxonomic tool for Penicillium in 1983 (Frisvad and Filtenborg 1983).

Figure 11 ESIþ mass spectra from injection of crude extracts of three Penicillium isolates.

Nielsen et al.

Figure 12 Cluster analysis of 45 mass spectra from direct infusion ESI þ MS of crude culture extracts of Penicillium species. Calculated using correlation coefﬁcient and UPGMA linkage.

The method uses a combined extraction—TLC analysis, where the extraction is done by adding a drop of solvent on top of a small plug cut from a colony. After a few seconds, the solvent remaining on top of the colony is applied to a TLC

Figure 13 Loadings from the ﬁrst three principal components from PCA analysis of direct infusion mass spectra collected from 45 isolates of Penicllium species from the Viridicata series.

plate by gently pressing the wetted side down on the plate. The advantage of the method is that it is simple, cheap equipment is required, sample throughput is high, and with many groups of fungi can be identiﬁed as illustrated on Penicillium series Viridicata (the P. aurantiogriseum complex) later. In the general Penicillium procedure plugs from CYA media are examined for intracellular metabolites, whereas the plugs from YES are examined for extra-cellular metabolites. A normal 20 by 20 cm plate accommodate 21 lanes from each side, and so 10 isolates can be analyzed (Figure 14). The TLC plates are eluted in saturated chambers, dried and examined in daylight and under UV light (366 nm and 254 nm) and spots appearing are noted (color, shape, etc.). The spots are marked gently with a pencil (or the plate is photographed). This procedure is repeated after: the whole plate is sprayed with AlCl3 and heated at 1308C for 8 minutes; the CAP side is sprayed with Ce(SO4)2; the TEF side is sprayed with ANIS and heated at 130 8C for 8 minutes. On our web site http://www.biocentrum.dtu.dk/mycology/ analysis/tlc/ houses a collection of pictures of TLC plates from 18 of the cost common Penicillium species.

Chemical Identiﬁcation of Fungi

Figure 14 The TLC-agar plug method. A 3 mm plug is cut from a colony and is placed directly on the TLC plate with the agar side down for the extra cellular metabolites or a drop of solvent is added to the mycelium side and the plug is placed with the wetted side down. The plate is eluted in two systems and the dried plate is examined under UV light before and after spraying.

3.5.1

Case IV: Identiﬁcation of Cereal Borne Penicillia by TLC

Cereals represent a habitat with a limited associated funga, and it is relatively easy to determine Penicillium species when kernels are placed on DG18 (Dichloran 18% Glycerol agar), DRYES (Dichloran Rose Bengal Yeast Extract agar) or DYSG (Dichloran Yeast Extract 18% Glycerol agar). When the Penicillium species are isolated and inoculated in 3-points on the identification media CYA, MEA (Malt extract agar), YES, and CREA (Creatine Sucrose agar) (Samson et al. 2000) it is relatively easy to observe that on MEA, most isolates have two stage branched rough stipes (terverticillate) and smooth conidia (excluding P. hordei), and that growth is inhibited on CREA. At this stage it is not possible to make a definite identification as these criteria fit with the following nine Penicillium species found on cereals: P. aurantiogriseum, P. freii, P. tricolor, P. polonicum, P. aurantiocandidum, P. viridicatum, P. cyclopium, P. melanoconidium (Penicillium series Viridicata), and P. verrucosum (Penicillium series Verrucosa). Agar plugs from twenty isolates on CYA and YES can be applied on a TLC plate (20 £ 20 cm) within 30 min, and the plate developed in TEF (Figure 14). The two secondary metabolites xanthomegnin and viomellein are always seen simultaneously as two brown spots under visible light in samples from CYA, and reduces the number of possible species to four, i.e. only four cereal-borne Penicillium species (Table 6). Aurantiamine, 3-methoxyviridicatin, bre-

vianamide A, viridamine, ochratoxin A, and citrinin are visible as colored spots under UV light (365 nm), usually in highest quantities on CYA. Aurantiamine and viridamine are both seen as blue spots, however viridamine is more light blue and has a lower Rf than aurantiamine. Viridamine will identify the fungus as P. viridicatum, and can be confirmed by the presence of brevianamide A as a yellow spot. Aurantiamine can be produced by two species, but simultaneously detection of 3-methoxyviridicatin as a blue spot under 255 nm light, and xanthomegnin and viomellein identifies P. freii. P. verrucosum is identified by a tailing yellow spot of citrinin and a blue green spot of ochratoxin A. The TLC plate should then be sprayed with AlCl3 and then heated at 1308C for 8 minutes. Penitrem A is the visible as a bluish black spot in daylight and oxaline as a yellow brown spot very close to the application point. If both metabolites are present on CYA the fungus is P. melanoconidium. The TLC plate should then be spayed with ANIS and heated to 1308C for 8 minutes. In cultures from YES yellow tailing spots in daylight identify terrestric acid that in combination with aurantiamine definitely identify the isolate as P. aurantiogriseum. A yellow brown spot under UV light on CYA detects verrucosidin, which in combination with 3-methoxyviridicatin identifies P. polonicum. P. tricolor is identified as producer of terrestric acid, xanthomegnin and viomellein. Except P. tricolor and P. verrucosum all these species produce penicillic acid seen as a bluish red spot under UV light after ANIS spray. In summary eight of nine species can be identified solely based on TLC results however the species, P. aurantiocandidum cannot be

Nielsen et al.

identiﬁed using TLC results alone, and must be combined with the poor sporulation on CYA.

3.6

Data Handling, Processing, and Chemometrics

Multivariate statistical methods (chemometrics, taxometrics) are ideal for evaluating chemotaxonomic data. Some of these methods are best used for unsupervised classification approaches such as cluster analysis, multidimensional scaling, correspondence analysis, and PCA, whereas other methods such as Partial Least Squares Discriminant (PLS-D) analysis and soft independent modeling of class analogy (SIMCA) are more suited for discriminant analysis and identification (Frisvad 1994b; So¨derstro¨m and Frisvad 1984). The immense quantities of data collected by modern analytic instruments dictates some form of automatic data handling and analysis (Nielsen et al. 1999) although problems such as handling of simultaneous UV, MS, and nuclear magnetic resonance data without component identification needs to be solved, as well as issues relating to the storage in searchable databases, and how data can be combined with other biodiversity information.

CONCLUSION AND SUGGESTIONS

Profiles of secondary metabolites provide powerful tools for fungal identification and can give insight in very large parts of the fungal genome, in addition of being functional and ecological characters. Metabolite profiling is currently being revolutionized by developments in MS as well as chemometrics and data handling which is necessary to cope with the immense quantities of data collected (@1 Gb/day). Chemical characters can be used directly in synoptic keys. They are also very suitable for databases as they can be accurately recorded using specific chemical methods. Natural classifications are often based on a polyphasic approach, where ideally many different ecologically relevant characters should be used.

ACKNOWLEDGEMENTS This project was supported by The Danish Technical Research Council under the project “Functional biodiversity in Penicillium and Aspergillus” (grant no. 9901295) and Center for Advanced Food Studies (LMC).

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Chemical Identification of Fungi eds. Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Amsterdam: Harwood Academic Publishers. pp 113 –125. Grob K (1993). Split and Splitless Injection in Capillary Gas Chromatography. Heidelberg: Hu¨thig. Hansen ME, Lund F, and Carstensen JM (2003). Visual clone identification of Penicillium commune isolates. J Microbiol Meth 52:221 – 229. Higgs RE, Zahn JA, Gygu JD, and Hilton MD (2001). Rapid method to estimate the presence of secondary metabolites in microbial extracts. Appl Environ Microbiol 67:371– 376. Jelen HH, Mirocha CJ, Wasowicz E, and Kaminski E (1995). Production of volatile sesqueterpenes by Fusarium sambucinum strains with different abiliteis to synthesize trichothecenes. Appl Environ Microbiol 61:3815 –3820. Joulian D and Ko¨nig WA (1998). The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. Hamburg: E.B.-Verlag. Ko¨nig A, Bu¨low N, and Saritas Y (1999). Identification of sesquiterpene hydrocarbons by gas phase analytical methods. Flavour Fragr J 14:367– 378. Larsen TO (1997). Identification of cheese-associated fungi using selected ion monotoring of volatile terpenes. Lett Appl Microbiol 24:463– 466. Larsen TO (1998). Volatiles in fungal taxonomy. In: Frisvad JC, Bridge PD, Arora DK eds. Chemical Fungal Taxonomy. New York: Marcel Dekker. pp 263 –287. Larsen TO and Frisvad JC (1994). A simple method of collection of volatile metabolites from fungi based on diffusive sampling from Petri dishes. J Microbiol Methods 19:297 –305. Larsen TO and Frisvad JC (1995a). Characterization of volatile metabolites from 47 Penicillum taxa. Mycol Res 99:1153 – 1166. Larsen TO and Frisvad JC (1995b). Chemosystematics of Penicillium based on profiles of volatile metabolites. Mycol Res 99:1167 –1174. Larsen TO and Frisvad JC (1995c). Comparison of different methods for collection of volatile chemical markers from fungi. J Microbiol Methods 24:135– 144. Mantle PG (1987). Secondary metabolites of Penicillium and Acremonium. In: Peberdy JF ed. Penicillium and Acremonium. New York: Plenum Press. Nielsen N-PV, Carstensen JM, and Smedsgaard J (1998). Aligning of single and multiple wavelength chromatographic profiles for chemometric data analysis using correlation optimised warping. J Chromatogr A 805:17– 35. Nielsen N-PV, Smedsgaard J, and Frisvad JC (1999). Full secondorder chromatographic/spectrometric data matrices for automated sample identification and component analysis by non-data-reducing image analysis. Anal Chem 71:727 – 735. Pitt JI (1973). An appraisal of identification methods for Penicillium species: novel taxonomic criteria based on temperature and water relations. Mycologia 65:1135 – 1157.

35 Pitt JI (1979). The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London: Academic Press. Ramaswami SK, Briscese P, Gargiullo J, and Geldern T (1986). Sesquiterpene hydrocarbons: From mass confusion to orderly line-up. In BM Lawrence, BD Mookherjee, BJ Willis, eds, Proceedings of the 10th International Congress of Essential Oils, Fragrances and Flavors, Washington-DC. pp 951 –980. Raper KB and Thom C (1949). A Manual of the Penicillia. Baltimore: Williams and Wilkins. Raper KB and Fennell DI (1977). The Genus Aspergillus. New York: Robert E. Kriger Publishing Company. Samson RA, Hoekstra ES, Frisvad JC, and Filtenborg O eds. (2000). Introduction to Food-and Air Borne Fungi, 6th ed. Utrecht: Centraalbureau voor Schimmelcultures. Schwab W (2002). Metabolome diversity: too little genes, too many metabolites. 1st International Congress on Plant Metabolomics 7– 11 April 2002, Wageningen. Smedsgaard J (1997a). Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures. J Chromatogr A 760:264 –270. Smedsgaard J (1997b). Terverticillate penicillia studied by direct electrospray mass spectrometric profiling of crude extracts. II. Database and identification. Biochem Syst Ecol 25:65 – 71. Smedsgaard J and Frisvad JC (1997). Terverticillate penicillia studied by direct electrospray mass spectrometric profiling of crude extracts. I. Chemosystematics. Biochem Syst Ecol 25:51– 64. So¨derstro¨m B and Frisvad JC (1984). Separation of closely related asymmentric penicillia by pyrolysis gas chromatography and mycotoxin production. Mycologia 76:408 –419. Strobel GA, Dirkse E, Sears J, and Markworth C (2001). Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiol UK 147:2943 – 2950. Thrane U (2001). Developments in the taxonomy of Fusarium species based on secondary metabolites. In: Summerell BA, Leslie JF, Backhouse D, Bryden WL, Burgess LW, eds. Fusarium. St. Paul: APS. pp 29– 49. Thrane U, Poulsen SB, Nirenberg HI, and Lieckfelt E (2001). Identfication of Trichoderma strains by image analysis of HPLC chromatograms. FEMS Microbiol Lett 203:249 – 255. Turner WB (1971). Fungal Metabolites. London: Academic Press. Turner WB and Aldridge DC (1983). Fungal Metabolites II. London: Academic Press. Vaidyanathan S, Kell DB, and Goodacre R (2002). Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification. J Am Soc Mass Spectrometr 13:118 –128. Williams DH, Stone MJ, Hauck PR, and Rahman SK (1989). Why are secondary metabolites (natural products) biosynthesised? J Nat Prod 52:1189 –1208. Zeringue HJ, Bhatnagar D, and Cleveland TE (1993). C15H24 Volatile compounds unique to aflatoxinogenic strains of Aspergillus flavus. Appl Environ Microbiol 59:2264 –2270.

3 Isozyme Analysis in Fungal Taxonomy, Genetics, and Population Biology Stephen B. Goodwin U.S. Department of Agriculture—Agricultural Research Service, and Purdue University, West Lafayette, Indiana, USA

examples of isozyme analysis for the major groups of fungi will be discussed to illustrate the many potential applications of this extremely useful and powerful technique.

INTRODUCTION

Isozyme analysis is a powerful technique for assaying genetic variation within and among populations. This versatile approach has been used on bacteria, fungi, plants, and most major groups of animals, from insects and other invertebrates to fish and mammals. The appeal of isozyme analysis stems from its ease of use, low cost, and speed. DNA-based technologies can provide a higher level of polymorphism with concomitantly greater resolution. However, when sufficient variation is present, isozymes provide the fastest, easiest answers to biological questions at a significantly lower cost compared to other available techniques. Isozyme analysis has a long history in the fungi. Differences among general protein patterns of species of Neurospora and Pythium were first noted during the early 1960s (Chang et al. 1962; Clare 1963). This approach developed into isozyme analysis with the application of methods for detection of specific enzyme activities. Since the mid 1960s, the technique has been applied to questions of fungal taxonomy, genetics, population biology, and species or strain identification (Micales et al. 1986; 1992) but it is only during the past 15 years that the true potential of the technique has been realized. The purpose of this chapter is to introduce the concept of isozyme analysis in fungi. Strategies for developing new isozyme systems and for addressing particular questions about fungal taxonomy, genetics, and population biology will be discussed. Particular emphasis will be placed on identifying common pitfalls and barriers to successful implementation of new isozyme systems, especially interpretation of banding patterns, preparation of tissue samples, and methods and approaches to data analysis. Specific

WHAT IS AN ISOZYME?

Isozymes are different forms of a single enzyme that perform the same or similar function. Each form has some small change that allows it to be distinguished from other forms (isozymes) of the same enzyme. The changes usually result from point mutations in the DNA that cause single amino-acid substitutions into the protein making up the enzyme. Changes that affect the charge or size of the ﬁnal enzyme may alter its mobility in an electric ﬁeld. Such changes often can be detected by electrophoresis, and this forms the basis for isozyme analysis. The terms “isozyme” and “allozyme” often are used interchangeably and can be a source of great confusion. However, the terms are not identical. Isozymes that have been analyzed genetically and are known to be alleles at a single genetic locus can be called allozymes. Therefore, all allozymes are also isozymes, but not all isozymes are allozymes. Isozyme is a more general term and should be used unless the genetic basis of the different enzyme forms is known for certain.

ISOZYME TECHNIQUES FOR FUNGI AND OOMYCETES

Many methods have been developed for separating enzyme variants and visualizing isozyme variation. The basic 37

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approach is to obtain crude extracts of total soluble proteins, separate the proteins by electrophoresis through some type of solid matrix (usually a gel), then visualize the isozymes by use of chemicals that produce a visual reaction in response to specific enzyme activity. General methods of isozyme analysis have been described elsewhere (Selander et al. 1971; Shaw and Prasad 1970; Soltis et al. 1983) and will not be repeated here. However, aspects of isozyme analysis that are specific to fungi will be discussed briefly. Additional information can be found in previous reviews of isozyme analysis and compilations of recipes specifically adapted for fungi (Micales et al. 1986).

3.1

Types of Tissue and Preparation of Samples for Isozyme Analysis of Fungi

Fortunately, virtually any type of fungal tissue can be used for isozyme analysis. This could include sections of sporocarps collected in the field or produced in culture, mycelia from plates or liquid cultures, or even conidia washed from lesions or cultures. The main requirement is that the tissue must be living so the enzymes are functional. Old cultures that are dead or dying will not give satisfactory results. This precludes dried herbarium material as the activity of most enzymes will be destroyed during the drying process. However, living material that has been lyophilized and stored at 2808C will maintain enzyme activity for many months and possibly for years. Lyophilization also may be a good option to save material until a sufficient quantity is available for analysis if only limited amounts of material can be collected at a time. Regardless of source or amount, the fungal material must be macerated to release the enzymes. Lyophilized material can be frozen quickly with a small quantity of liquid nitrogen and ground in a mortar after the nitrogen has evaporated. Small amounts of tissue can be ground in microcentrifuge tubes using a glass rod as a pestle. Disposable plastic pestles designed specifically for use in microcentrifuge tubes also can be used. Fresh tissue can be crushed with a disposable plastic pestle attached to a variable-speed electric drill. Adhering liquid and tissue should be wiped from the pestle between samples, but there is no need to clean the pestle completely as

a small amount of cross contamination will not affect the results due to the relatively low sensitivity of the technique. To maintain enzyme activity, all samples should be kept on ice whenever possible. Tissue extracts also can be frozen at 2808C for several weeks if necessary with little or no enzyme degradation. However, this should be tested for each system speciﬁcally before used on a large number of samples.

3.2

Gels of 12% hydrolyzed potato starch have been the mainstay of isozyme analysis for decades, Advantages are cost and throughput. A single starch gel can yield 3 –4 horizontal slices (possibly more), each of which can be stained for a different enzyme. Depending on the size of the filter-paper wicks used for sample loading and the width of the gel, 20– 50 or more samples can be loaded per gel. Thus, more than 200 data points (50 individuals times four enzymes) can be obtained per gel once the system is optimized, and an average of 80 or more is common. This high sample capacity is excellent for population genetics analyses that require large sample sizes for meaningful conclusions. Starch gels can be photographed easily for a permanent record. A newer approach is to use precast cellulose-acetate plates obtained from commercial suppliers. This technique has been used for many years in medical diagnostics but has only been applied to isozyme analysis in general since the mid 1980s (Hebert and Beaton 1993). Advantages of cellulose acetate are that the pre-cast gels can be purchased ahead of time and stored dry until needed, so there is no time, effort, or skill involved in pouring the gels; specially designed gel boxes and sample applicators can be purchased so a laboratory can be set up quickly; and the complete analysis can be run in an hour from start to finish, so many runs can be completed in the same day. Very small volumes are needed so extremely small amounts of tissue can be analyzed. The stained gels can be photographed or the gels themselves dried and saved for a permanent record. Isozymes also can be separated on many other types of solid matrix and approach, such as polyacrylamide gels or using isoelectric focusing. Polyacrylamide in particular is often used in an attempt to gain increased resolution. Although these systems often do reveal additional bands, the banding patterns can be difficult to interpret genetically. Furthermore, throughput and ease of use are lower, and costs often are higher compared to starch and cellulose acetate.

3.3

Figure 1 Isozymes of glucose-6-phosphate isomerase in 12 isolates of the barley scald pathogen, Rhynchosporium secalis. This haploid fungus yields a single band in each lane corresponding to one of the three putative alleles 87, 100, or 114.

Choice of Separation Matrix

Enzyme Activity Staining

Once the proteins have been separated on a gel, a set of staining protocols must be followed to reveal the isozymes. Speciﬁc enzyme activity staining is what makes isozyme analysis possible. Recipes and techniques for staining hundreds of enzyme activities are provided in numerous compilations (Micales et al. 1986; Selander et al. 1971; Shaw

Isozyme Analysis of Fungi

and Prasad 1970; Soltis et al. 1983) and are beyond the purview of this chapter. However, a brief description of the major types of staining systems is provided here. Three staining systems are used commonly for isozyme analysis: positive, negative, and fluorescent. The vast majority of isozyme visualization systems are based on positive staining. The general idea is that activity of a specific enzyme generates a colored precipitate at zones of enzyme activity. These isozymes are visualized as dark bands against a light background (Figure 1). Negatively stained isozymes are visible as light-colored bands against a dark gel. Common negative staining systems include those for the enzymes superoxide dismutase and catalase. A third class of enzyme staining system works by generating bands that fluoresce when exposed to ultraviolet light. Commonly used fluorescent staining systems include those for arylesterase and b-glucosidase.

INTERPRETATION OF ISOZYME DATA

The best guide for interpreting isozyme banding patterns is to perform a thorough genetic analysis. Unfortunately, for many fungi this may be difﬁcult or impossible. The purpose of this section is to provide enough information for accurate interpretation of isozyme banding patterns even without a genetic analysis. Accurate interpretation of isozyme banding patterns requires knowledge of the ploidy of the organism and the number of subunits required to form the active enzyme. Most enzymes should give a relatively simple banding pattern with only one to three bands per individual (Figure 1). Enzymes with relatively nonspeciﬁc stains (e.g., esterases)

often give complicated banding patterns that are difﬁcult or impossible to interpret without a genetic analysis. Unless crosses can be made easily those systems are best avoided.

4.1

For haploid fungi, each individual ideally will produce a single band on a gel. Such isozymes can be scored easily by assuming that each band corresponds to an allele at a single genetic locus. Each allele can be named by indicating its relative migration distance on the gel. The easiest approach is to designate the most common allele as number 100. Then all other alleles can be indicated by their migration distance relative to the 100 allele. For example, an allele migrating 14% faster than allele 100 would be allele 114, and one migrating 13% slower would be allele 87 (Figure 1). More than one band per haploid individual may indicate multiple loci. If alleles at each locus migrate within a limited, defined region of the gel then it may be possible to score each locus separately. Alleles at each locus can be named as indicated previously but with a locus designation, e.g., Gpi-1 100 and Gpi-2 100 to indicate the 100 alleles at two loci coding for the enzyme glucose-6-phosphate isomerase. Enzymes giving more complicated banding patterns in haploids should be avoided as accurate interpretation is almost impossible without thorough genetic analysis. “Null” alleles—those in which no enzyme activity is detected— should be regarded with suspicion as it is difficult to distinguish a null allele from a null caused by faulty technique. Furthermore, it is difficult to imagine a haploid being truly null for important enzyme activities. All potential null alleles should be verified thoroughly—most probably will turn out to be errors caused by poor enzyme extraction from particular individuals or other problems with technique.

4.2

Figure 2 Subunits with 2 2 and 2 3 charges combine at random to form an active dimeric enzyme. A, In this example, random combination of subunits yields three dimers with total charges of 24, 2 5, and 26 in a ratio of 1:2:1. B, The pattern that results on a gel after electrophoresis and staining. Direction of migration is indicated by the arrow.

Interpretation of Isozyme Banding Patterns in Haploids

Interpretation of Isozyme Banding Patterns in Diploids, Dikaryons, and Polyploids

Interpretation of isozyme banding patterns in diploids and dikaryons is much more complicated, and is aided by knowing whether a particular enzyme is mono- or multimeric. For monomeric enzymes, homozygous diploid individuals should produce a single band on a gel as described for haploids. Diploid individuals that are heterozygous at a locus for a monomeric enzyme will give two bands in a properly stained isozyme gel following electrophoresis. Each band can be interpreted easily as a different allele at a single genetic locus. Banding patterns are more complicated for enzymes composed of two or more subunits. In these cases the subunits must join together to form the active enzyme. Because dimeric enzymes (those composed of two subunits) are the most common, they will be the subject of this discussion. However, the concepts covered here are directly applicable to

enzymes composed of three or more subunits. To correctly interpret isozyme banding patterns produced by dimeric enzymes, it is necessary to understand how the subunits combine to form the active enzyme. In heterozygous diploid individuals, the subunits produced by the two alleles combine at random in the cytoplasm of the cell to form the active molecule. For the hypothetical case of a diploid with two alleles, one coding for a subunit with a 2 2 charge, the other with a 2 3 charge, there are four possible ways the subunits can be joined to form the active dimer (Figure 2A). Three types of dimer can result: two homodimers (when identical subunits combine) and a heterodimer (composed of two different subunits). In Figure 2A, each 2 2 subunit can join with another 2 2 subunit (to form a 2 2/2 2 homodimer) or with a 2 3 subunit (for a 2 2/23 heterodimer). Similarly, half of the 23 subunits will pair with a 2 2 subunit and half with a 23 subunit. Notice that for every 2 2/2 2 homodimer (total charge ¼ 2 4) there are two 2 2/2 3 heterodimers (total charge ¼ 2 5) and one 2 3/2 3 homodimer (total charge ¼ 2 6), for a ratio of 1:2:1. When these molecules are separated according to charge in an electric field, they will migrate to three regions on the gel, which after staining will be visualized as three distinct bands (Figure 2B). Notice that the middle (heterodimer) band is approximately twice as intense as either homodimer band, reflecting the 1:2:1 ratio in the numbers of each molecule. This pattern is unmistakable on isozyme gels (Figure 3). The pattern is slightly more complicated for trisomic or polyploid individuals possessing three alleles. In this case, the subunits pair at random as before, only now there are nine possibilities (Figure 4A). If the different alleles vary by single-step charge differences, for example with charges of 22, 2 3, and 24, then the 23/23 homodimer will have the same 26 charge as the 2 2/24 heterodimers and will migrate to the same place on the gel during electrophoresis. This will produce a five-banded phenotype in which the intensities of the bands should be in a ratio of approximately 1:2:3:2: 1 (Figure 4B). This pattern is seen in the US-8 genotype of the

Figure 3 Banding patterns of glucose-6-phosphate isomerase in a diploid. Heterodimers are seen as more intensely staining bands between two other bands. The gel shows interspeciﬁc hybrids between the oomycetes Phytophthora infestans and its close relative P. mirabilis. The P. infestans parent was heterozygous 86/122and the P. mirabilis parent was homozygous l08/108. Interspeciﬁc hybrids are 86/108 (lanes 1,2,4,7, and 8) or 108/122 (lanes 3,5,6, and 9). Lanes 10 (homozygous 108/108) and 11 (86/122) indicate self fertilization of the P. mirabilis and P. infestans parents, respectively.

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oomycete Phytophthora infestans at the Gpi locus, and was conﬁrmed by thorough genetic analyses (Goodwin et al. 1992). Other banding patterns can be produced by individuals containing three or more alleles separated by unequal charges. For example, an individual heterozygous for three alleles in uneven steps, e.g., 2 2, 23, and 25, would yield a sixbanded pattern in a 1:2:1:2:2:1 ratio. Band-intensity ratios in polyploids also can be affected by the number of copies of particular alleles at a locus. For example, an individual with three alleles, two of which are identical, e.g., 2 2, 24, and 24, should give rise to a three-banded pattern in a 1:4:4 ratio. This has been documented in the US-1 genotype of P. infestans which has two copies of the 100 and one copy of the 86 allele at the Gpi locus (Goodwin et al. 1992). Even more complicated banding patterns are produced by tetrameric enzymes and by multiple loci that share alleles. Interpretation of these patterns was covered in detail elsewhere (Micales et al. 1992). Fortunately, most enzymes are dimeric so the principles discussed previously will apply directly. An understanding of these basic principles combined with genetic analyses should allow unambiguous interpretation of virtually any isozyme pattern.

4.3

Choosing Among the Available Enzyme Systems

Every isozyme project begins with a choice of enzymes. Although it is not possible to predict which enzymes will

Figure 4 Formation of dimers in a triploid. A, The subunits can combine nine ways to form dimers with ﬁve possible charges. B, Separating these dimers on a gel and staining gives ﬁve bands in a ratio of intensities of 1:2:3:2: 1. The most intense band results from co-migration of 2 2/24 and 2 3/2 3 heterodimers. All six bands could be visible if the differences in charge among alleles were uneven. Direction of migration is indicated by the arrow.

Isozyme Analysis of Fungi

work with a particular fungal species, some guidance can be obtained from previous research. The best approach is to screen a few isolates on a large number of enzyme systems, then choose those enzymes which give well resolved, easily storable banding patterns. However, choosing which enzymes to try ﬁrst can be daunting. Enzyme testing results from 27 published surveys plus three unpublished surveys are summarized in Table 1. Among the 82 enzymes listed, 32 never worked and another 20 were successful in less than 50% of the surveys. The most promising enzymes to try in an initial survey would be the 20 enzymes that were tried four or more times and provided useful information in 50% or more of the studies surveyed (indicated in bold in Table 1).

APPLICATIONS OF ISOZYME ANALYSIS

Isozyme analysis can be applied to address a wide variety of biological questions in the fungi. These markers usually are considered to be selectively neutral so are ideal for looking at sources of inoculum for pathogenic fungi or gene flow among populations. The markers can indicate whether specific tissues are haploid or diploid, can aid strain or species identification, and can be used to identify hybridization either in the laboratory or in nature.

5.1

Taxonomy and Species Identiﬁcation

Isozyme analysis was first applied to fungal taxonomy during the 1960s (Clare 1963; Hall 1967; Meyer et al. 1964; Peberdy and Turner 1968). Since then it has been applied at many taxonomic levels from typing individual strains to delimitation and identification of species. The best enzymes for taxonomic purposes are those that are monomorphic within but different among taxa. A caveat is that isozymes cannot be used to infer phylogenetic relationships. This is because bands with the same migration rate on a gel in fact may not be identical. Furthermore, it is not possible to infer which allele is ancestral or to estimate the number of mutations that cause the isozymes to migrate differently; alleles of similar size may be more different evolutionarily than those with larger migration distances on a gel. Therefore, clustering algorithms such as neighbor joining (Saitou and Nei 1987) that allow for unequal rates of evolution on branches are not appropriate for isozyme data. Instead, isozyme data are analyzed usually by calculating a simple distance coefficient and drawing clusters with the Unweighted Pair Group Method with Arithmetic mean (UPGMA) (Michener and Sokal 1957). These analyses can be performed with several computer programs such as NTSYSpc (Rohlf 1998), POPGENE (http://www.ualberta.ca/ -fyeh/index.htm), PHYLIP (http://evolution.genetics. washington.edu/phylip.html), or PAUP* (http://paup.csit. fsu.edu/index.html). Cluster analyses always should be accompanied by bootstrap analysis or some alternative method of indicating the level of statistical support for particular groupings. Unfortunately, bootstrap analysis cannot

be performed with NTSYS, but it is available with some of the other computer programs as well as the program WinBoot (Yap and Nelson 1996). Specific applications of isozyme analysis to taxonomic questions include identifying strains of Trichoderma harzianum (Zamir and Chet 1985), varieties of Verticicladiella wageneri (Otrosina and Cobb 1987), or anastomosis groups within Rhizoctonia (Damaj et al. 1993). The technique also can be used to identify fungal cultures to species (Six and Paine 1997). Isozyme analyses have confirmed a high level of genetic differentiation among host-associated varieties of Leptographium wageneri (Zambino and Harrington 1989) and have revealed previously unknown genetic subdivision within various species of Phytophthora (Mchau and Coffey 1994; 1995). In the rust genus Puccinia, isozyme variation can distinguish among species and also among formae speciales on different hosts (Burdon and Marshall 1981). The most common use of isozyme analysis in fungal taxonomy is to divide isolates into species and to test how well biochemical species identification corresponds to classical taxonomy. Usually, species groups identified by isozyme analysis correspond quite closely with those identified morphologically (Hsiau and Harrington 1997; Oudemans and Coffey 1991a,b; St. Leger et al. 1992; SurveIyer et al. 1995). However, sometimes two or more taxa are found to be the same genetically (Oudemans and Coffey 1991b; Yoon et al. 1990) and are combined into a single species. The opposite also is a common result of isozyme analysis: single species frequently can be divided into two or more species based on previously hidden genetic differentiation uncovered by isozyme analyses (Altomare et al. 1997; St. Leger et al. 1992).

5.2

Genetics

In addition to simple Mendelian genetics (Bonde et al. 1988; Burdon et al. 1986; Hellman and Christ 1991; Shattock et al. 1986a; Spielman et al. 1990) and linkage analysis (May and Royse 1982b), isozymes can be used to distinguish hybrid from nonhybrid progeny in both intra- (May and Royse 1982a; Shattock et al. 1986b) and inter-specific crosses (Goodwin and Fry 1994), and to infer the ploidy level of vegetative hyphae (Goodwin et al. 1994; Shattock et al. 1986b). Dimeric enzymes are ideal for this kind of analysis (Figure 3). Isozymes also can be used to analyze parasexual genetics in fungi. In addition to laboratory genetics, isozyme analysis implicated somatic hybridization as the probable origin of a new forma specialis of cereal rust in Australia (Burdon et al. 1981) and identified naturally occurring hybrids among field isolates of Phytophthora species (Man in ‘t Veld et al. 1998). Estimates of relatedness based on isozyme analysis among strains of Agaricus brunnescens were used to aid the choice of parents in a mushroom breeding program (Royse and May 1982b). Most studies have shown normal Mendelian segregation of isozyme alleles (i.e., the isozymes can be considered allozymes), although instances of aberrant

3/4 5/9

3.1.3.2 4.2.1.3 3.5.4.2 2.7.4.3 3.4.11.12 1.4.1.1 1.1.1.1 1.2.3.1 3.1.3.1 3.2.1.1 3.1.1.2 1.10.3.3 2.6.1.1 1.4.3.16 3.1.1.1 1.11.1.6 1.10.3.1 2.7.3.2 3.4.11.1 3.4.12.18 1.8.1.4 3.4.15.1 4.2.1.1 1.2.1.2 3.1.3.11 4.1.2.13 4.2.1.2 1.1.1.48 3.2.1.23 2.7.1.2 1.1.1.47 1.1.1.49 5.3.1.9 3.2.1.21 2.4.1.11 3.2.1.31 1.4.1.2 1.4.1.3 2.6.1.2 1.6.4.2 1.2.1.12 1.2.1.13

Acid phosphatase Aconitate hydratase Adenosine deaminase Adenylate kinase Alanine aminopeptidase Alanine dehydrogenase Alcohol dehydrogenase Aldehyde oxidase Alkaline phosphatase a-Amylase Aryl (ﬂuorescent) esterase L -Ascorbate oxidase Aspartate aminotransferase (glutamate oxalate transaminase) Aspartate oxidase Carboxylesterase Catalase Catechol oxidase (tyrosinase) Creatine kinase Cytosol (leucine) aminopeptidase Cytosol nonspeciﬁc dipeptidase Dihydrolipoamide dehydrogenase (diaphorase) Dipeptidyl carboxypeptidase I Enolase Formate dehydrogenase Fructose-bisphosphatase Fructose-bisphosphate aldolase Fumarate hydratase Galactose dehydrogenase b-Galactosidase Glucokinase Glucose dehydrogenase Glucose-6-phosphate dehydrogenase Glucose-6-phosphate isomerase b-Glucosidase Glucosyl transferase b-Glucuronidase Glutamate dehydrogenase Glutamate dehydrogenase NADP Glutamate pyruvate transaminase Glutathione reductase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (NADPþ) 3/3 8/10 0/1 4/5 6/7 9/14 1/2 4/8 0/1 0/1 0/2 1/6 2/4 0/1 0/1 1/5 10/12 0/1 0/1 0/1 5/11 2/3 1/2 0/4 0/1

1/1 4/7 3/7 1/1 7/10 1/1 2/4

0/2 2/2 3/6 0/2 0/4 7/8 10/10 0/1

1/2 0/1 0/1

3/3

1/5

6/6 3/7 0/1 1/4 1/3 0/1 2/6

Basidd

1/1 0/4 0/3 2/6

0/3

Ascoc

E.C. no.b

Enzyme

1/2 0/1

0/1 1/3 1/1

0/1 5/6 5/5 2/4

1/3 0/3 2/5 0/1 0/1

1/4 2/2 0/1 0/1 5/7 3/5 1/3 0/1 1/1

0/1 0/4 0/2 0/3 0/1 1/2 0/1 3/5

0/2

1/3 3/5

Deute

0/1 0/1

1/1

0/1 0/1 2/2 4/4 0/1

1/1 1/2 1/3

1/2 4/4 1/2

0/1

1/1

0/1

2/2 1/1 1/1

Oomf

No. times useful/no. times testeda

77 56 50 20 33 33 13 0 20 0 86 0 65 0 50 90 0 0 67 75 47 0 50 0 25 31 44 0 0 0 0 71 94 29 0 0 56 100 60 25 13 0

Percent useful

1.8 1.0 2.0 2.0 2.0

1.5 2.3 3.5

2.0 2.5 1.7

3.0

2.3 1.9 2.1

2.6 1.7

2.3

1.8

2.0

1.6 1.9 2.0 1.0 3.0 1.0 5.0

Avg. no. of alleles

Table 1 Isozyme systems tested and success rates in various groups of fungi and oomycetes. The 20 enzymes that were tested four or more times and were useful in 50% or more of the surveys are indicated in bold

42 Goodwin

1.1.1.29 1.1.1.8 4.4.1.5 3.5.4.3 2.7.1.1 3.2.1.30 1.1.1.30 1.1.1.14 1.1.1.42 1.10.3.2 1.1.1.27 1.4.1.9 1.4.1.15 1.1.1.37 1.1.1.40 1.1.1.67 5.3.1.8 1.6.99.2 1.6.99.3 1.6.99.1 2.7.4.6 2.6.1.1 1.1.1.73 1.11.1.7 4.1.1.31 2.7.1.11 5.4.2.2 1.1.1.44 2.7.2.3 1.14.18.1 2.7.1.40 1.1.1.25 1.3.99.1 1.15.1.1 3.2.1.28 5.3.1.1 2.7.7.9 2.7.4.4 1.1.1.204 3.4.13.9 0/1 0/1 0/1 7/10 3/7 0/2 3/5 3/6 0/1 0/1

2/5

2/2 0/3

2/5

0/4 0/3 0/2

7/10 4/6 0/3 0/3

1/5 0/2 2/2

0/1 1/4

9/9 6/6

8/9 2/4 3/3 1/3 1/1

5/5

1/2 0/1 1/2 0/2

4/8 1/1 0/1 0/3 0/5

2/5 0/1

0/3 0/1

1/1 0/2

0/2 0/1 1/3 0/1 3/4 1/1 0/1 0/2 1/1

0/1 5/5 2/3 1/1

0/1

1/1

2/3 2/3

5/6 1/4

0/1 0/1 5/6 0/1 0/3 0/1

0/1 0/2 1/1 0/1 3/4

1/2

1/1

2/3

0/1

1/2 4/4

4/4 2/2 0/1 3/3 1/2

3/3

0/1 2/3

2/3

20 0 100 0 55 100 0 0 63 0 17 0 0 83 47 50 64 58 0 0 100 50 0 33 0 0 85 84 25 0 0 9 0 31 0 89 100 0 25 100 1.7 4.0

2.4 1.0

2.0

1.0

2.3 1.5 3.0

2.0

2.0 2.0

1.9 1.4 1.7 1.6 1.1

2.0

1.8

1.9 1.0

2.0

4.0

a Based on an analysis of 27 published enzyme surveys plus three surveys of septoria pathogens (G. Zhang and S. B. Goodwin, unpublished). The published surveys were those of: Altomare et al. (1997); Andrews et al. (1988); Burdon and Roelfs (1985a); Damaj et al. (1993); Gaur et al. (1991); Goodwin et al. (1993); Huss (1996); Leuchtmann and Clay (1989); (1990); Leung and Williams (1986); Linde et al. (1990); Nyasse´ et al. (1999); Old et al. (1984); Otrosina and Cobb (1987); Otrosina et al. (1992); Oudemans and Coffey (1991a); Riley et al. (1998); Royse and May (1982a); Six and Paine (1999); Surve-Iyer et al. (1995); Tooley and Fry (1985); Tuskan and Walla (1989); Vogler et al. (1991); Welz et al. (1994); Yoon et al. (1990); Zambino and Harrington (1989); Zhu et al. (1988). b Enzyme Commission number. International Union of Biochemistry and Molecular Biology (1992). Enzyme Nomenclature. Academic Press, Inc. c Ascomycates. d Basidiomycetes. e Deuteromycetes.

Glycerate dehydrogenase Glycerol-3-phosphate dehydrogenase (NADþ) Glyoxalase Guanine deaminase Hexokinase Hexosaminidase 3-Hydroxybutyrate dehydrogenase L -Iditol (sorbitol) dehydrogenase Isocitrate dehydrogenase (NADP1) Laccase L -Lactate dehydrogenase Leucine dehydrogenase Lysine dehydrogenase Malate dehydrogenase Malate dehydrogenase (NADPþ) (malic enzyme) Mannitol dehydrogenase Mannose-6-phosphate isomerase Menadione reductase NAD dehydrogenase NADPH dehydrogenase Nucleoside diphosphate kinase Nucleoside phosphorylase Octanol dehydrogenase Peroxidase Phosphoenolpyruvate carboxylase 6-Phosphofructokinase Phosphoglucomutase Phosphogluconate dehydrogenase Phosphoglycerate kinase Polyphenol oxidase Pyruvate kinase Shikimate dehydrogenase Succinate dehydrogenase Superoxide dismutase Trehalase Triose-phosphate isomerase Uridine diphosphoglucose pyrophosphorylase Uridine monophosphate kinase Xanthine dehydrogenase X-Pro dipeptidase

Isozyme Analysis of Fungi 43

Goodwin

segregation have been noted (Spielman et al. 1990). It is important to remember that no isozyme should be referred to as an allozyme until a proper genetic analysis has been completed.

5.3

Population Biology

Gene flow is the movement of individuals among populations and can be estimated indirectly through isozyme analysis as the number of migrants per generation (Slatkin and Barton 1989) or by analysis of “private” alleles—those limited to a single species or population. Different species usually have highly differentiated allele frequencies and a higher number of private alleles compared to individuals from a single panmictic population. The computer program POPGENE , among others, can perform this type of analysis. Applications of isozyme analysis to epidemiology have included monitoring the spread of specific genotypes of Phytophthora infestans (Goodwin et al. 1995; Legard and Fry 1996), determining the influence of various evolutionary forces on the composition of inoculated populations of the barley scald pathogen Rhynchosporium secalis (Goodwin et al. 1994), and estimating the size of and boundaries between individuals of the puffball Lycoperdon pyriforme on decaying logs (Huss 1993). The first real attempt to use isozymes to analyze the genetic structure of fungal populations was for Neurospora intermedia by Spieth (1975). That analysis revealed a high level of genetic variation within but low differentiation among populations. Spieth’s pioneering work was followed by additional studies within a decade and this has accelerated during the 1990s. Many of these studies have had similar results and demonstrated high gene diversity within but low differentiation among populations of a wide diversity of fungi (Andrews et al. 1988; Goodwin et al. 1993; Huss 1996; Tuskan et al. 1990). Other authors have found varying degrees of subdivision among populations, usually among host-associated forms (Harvey et al. 2001; Leuchtmann and Clay 1989; 1990) and, more rarely, among physiological races (Welz et al. 1994). Gene flow analysis of isozyme markers also can indicate when taxa have become sufficiently isolated reproductively to be considered separate species. Existence of separate species was shown conclusively for several closely related groups in the genus Phytophthora (Goodwin et al. 1999; Man in ‘t Veld et al. 2002; Nygaard et al. 1989) which could not have been discovered without the use of molecular markers. A similar approach identified distinct biological species within the mushroom Pleurotus eryngii (Urbanelli et al. 2002). Isozyme analyses of some populations of fungi and oomycetes have revealed extremely low levels of genetic variation reflective of a highly clonal population structure. This occurred for rice-infecting isolates of the rice blast fungus Magnaporthe grisea (Leung and Williams 1986), asexual populations of cereal rusts (Burdon and Roelfs 1985a,b), and worldwide populations of the oomycete

Phytophthora infestans (Spielman et al. 1991; Tooley et al. 1985). Populations of fungi infecting conifer hosts in the western United States also appeared to be highly clonal and specific clones are characterized by fixed heterozygosity (Otrosina et al. 1992; Vogler et al. 1991). Differences in levels of genetic variation among locations can help identify the center of origin of a species. The most diverse populations genetically should occur at or near the center of origin, as these populations will have had the longest time in which mutations can accumulate; derived populations usually will contain only a subset of the total species diversity. This idea has been combined with isozyme analysis to indicate that the edible mushroom Agaricus bisporus may be indigenous to North America (Kerrigan and Ross 1989) and that Phytophthora infestans, P. palmivora, and P. megakarya probably originated in central Mexico (Tooley et al. 1985), southeast Asia (Mchau and Coffey 1994), and central Africa (Nyasse´ et al. 1999), respectively. Very limited genetic diversity indicated that the plant pathogens Puccinia graminis f. sp. tritici, Phytophthora cinnamomea, and Dilophospora alopecuri had only very limited introductions into Australia (Burdon et al. 1982; Old et al. 1984; Riley et al. 1998), although the original source populations for the introductions could not be identified for certain.

CONCLUSIONS

Isozyme analysis is an extremely powerful and versatile technique that can answer many questions about fungal genetics, taxonomy, and population biology. Compared to alternative technologies, isozymes are cheap, fast, and easy to interpret. They only assay expressed genes and, by analysis of dimeric enzymes, can distinguish heterozygous individuals from physical mixtures of cultures—an advantage that is not shared by DNA-based markers. With newer types of electrophoresis systems, isozymes are suitable for analysis of small tissue samples and could be portable to remote locations. The main disadvantage of isozyme analysis is a lower level of polymorphism compared to DNA-based markers, but this usually can be overcome by increasing the number of enzyme systems assayed. The many advantages of isozyme analysis should ensure its place in the molecular toolbox for many years to come.

ACKNOWLEDGEMENTS Thanks to Kristi Brikmanis and Bryan Wallace for retrieving references and helping with much of the typing.

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4 Molecular Methods for Identiﬁcation of Plant Pathogenic Fungi Maren A. Klich / Edward J. Mullaney U.S. Department of Agriculture – Agricultural Research Service, New Orleans, Louisiana, USA

1 1.1

molecular sites concur with one another and with “trees” based on morphological and physiological data, one has fairly strong evidence that the phylogeny is accurate. Some fungi do not grow or do not sporulate in culture. Molecular methodologies allow identiﬁcation of these isolates by comparison of DNA sequence data from the unknown isolate with sequences from known species. Molecular methods have also been used to distinguish between closely related species with few morphological differences and to distinguish strains (or even speciﬁc isolates) within a species. In studies of fungal metabolites, especially mycotoxins, there has been no way of knowing whether a nonproducing strain is truly (genetically) incapable of producing the metabolite or if it could possibly produce it under different environmental conditions. Once the genes of a metabolic pathway have been cloned, they can be used to determine whether or not a strain possesses the genes for production of the metabolite, providing a better indication of the potential of a given strain to produce a metabolite.

INTRODUCTION Need for Molecular Methods in Fungal Identiﬁcation

For centuries, fungal identification has been based on morphological, physiological, and chemical characteristics of specimens. For the most part, these systems still work extremely well. They provide accurate species identification inexpensively, are not labor-intensive, and require little equipment beyond a microscope and chemical reagents. Since phenotype is the result of the expression of hundreds of genes, the higher level classifications based on morphology/ physiology are generally sound. A major drawback of the traditional identification methods is that they require some technical training in order to acquire the skills necessary to identify fungi or characterize strains. This training has become increasingly difficult to acquire, whereas molecular biology techniques are now taught widely in secondary schools and can be applied to a multitude of fields. Another drawback of the traditional methods is that they can take a week or more for fungal colonies to grow and develop the characters necessary for identification. In some cases, the necessary characters never develop. Several of the molecular methods provide identifications more quickly and do not rely on the presence of reproductive structures. Molecular biology has brought many powerful new tools to fungal taxonomists including the potential for rapid identification of isolates, methods for rapid determination of virulence or toxicity of strains, and the means to elucidate the relationships among fungal species. DNA sequence data provide large numbers of data points that can be compared among fungi and analyzed to determine sequence relatedness, which can be assumed to reflect phylogenetic relatedness among species. If “phylogenetic trees” from different

1.2

Assumptions and Limitations

The assumptions made in making fungal identifications using molecular methods are the same as those made using traditional methods. The concept of a species (or any other taxonomic level) is ultimately what those working in the field agree it to be. These decisions are based on available data, but there is always some degree of subjectivity in the process, especially when considering closely related species. None of the current molecular methods used in fungal identification can screen more than a minute portion of the genome. There is no single method that can be used to distinguish all species or all genera from one another. Generally, if the data provided by a molecular method support current taxonomic thought on a taxon, they are used. 49

Klich and Mullaney

A molecular method appropriate for identification of one species or genus (or part thereof) may not be useful for species in other genera or even for all portions of one genus. Molecular methods provide powerful new tools to aid in fungal identification, but they have not provided (and probably will never provide) a universal solution to problems associated with fungal identification. They are tools, not panaceas, for taxonomists. Currently molecular methods are more labor-intensive and more expensive than many of the traditional methods of fungal identification, but the field is evolving and more rapid and affordable methods will probably become available.

1.3

Scope

It would be beyond the scope of this paper to review all of the studies using molecular methods for identification of plant pathogenic fungi. Instead, we will provide an overview of the methods available for identification and then give one or two recent examples from the literature. These citations will give interested readers a starting point for finding related citations in the literature.

METHODOLOGIES

The majority of molecularly-based identification methods rely on the interaction of unique enzymes and nucleic acids. Two extensively utilized classes of enzymes are restriction endonucleases and DNA polymerases. Restriction endonucleases cleave DNA molecules at specific nucleotide sequences. Different endonucleases recognize and cut at different restriction recognition sequences. An example of two endonucleases and the specific sequence they recognize are Eco RI (GAATTC) and Bam HI (GGATCC). Eco RI cleaves between the guanine and the adenine, GÂATTC and Bam HI between the guanines G^GATCC. Restriction fragment length polymorphism (RFLP) is one molecular technique that requires restriction endonucleases to produce the unique DNA fragments which are the basis for this technique. Polymerases are enzymes that catalyze the formation of DNA or RNA from nucleotide precursors. All DNA polymerases also require a preexisting template. For example, various DNA polymerases are utilized for different applications [DNA polymerase I (Kornberg polymerase), is used to label DNA; Taq DNA polymerase is used to amplify DNA fragments in the Polymerase Chain Reaction (PCR); etc.]; and some have been engineered for enhanced features, e.g., Sequenasee which is used in DNA sequencing. In RFLP the assorted DNA fragments resulting from the restriction endonuclease digestion are resolved by gel electrophoresis. Ethidium bromide staining is then used to reveal the fragments under UV (260 nm) light. Southern blotting of the gel can transfer the DNA fragments to a support membrane. The DNA is then fixed to the membrane

and can be subjected to hybridization analysis. This enables identification of bands with sequence similarity to a labeled probe. Several printed reference laboratory manuals [e.g. Molecular cloning: A Laboratory Manual, 3rd Edition; Short Protocols in Molecular Biology, 4th Edition] and Internet web sites such as www.protocol-online.net and www.nwfsc.noaa.gov/protocols detail these techniques. In dot blots, the target nucleic acid sample is affixed directly to the membrane without electrophoresis followed by hybridization with the labeled probe. Detailed information on these protocols can also be obtained by utilizing a search engine such as Googlee to search the internet. DNA fingerprinting is an umbrella term that describes molecular identification techniques. As the term implies, the basic genetic material in the organism is utilized to determine its identity and relationship with other isolates. One of the earliest hybridization probes utilized was variable number tandem repeats (VNTRs) or minisatellite DNA (Jeffreys et al. 1985). The term “satellite” originated from buoyant density centrifugation studies in which the bulk of the sample DNA sediments as one main band, but smaller satellite bands were also observed. Satellites are composed of tandem repeats of DNA arranged in consecutive repeats. They sediment apart from the main band containing the bulk of the DNA due to their significantly different base composition. Minisatellites range in size from 1 kb to 20 kb. The VNTR are a type of minisatellite located in the noncoding regions of the genome. The size of the repeat units varies from 9 to 80 base pairs (bp). Smaller repeats consisting of only 1 –6 bp are designated microsatellites, or short tandem repeats (STR). The STR have a repetitive region of less than 150 bp. Sequences of both mini- and microsatellites have been utilized in DNA fingerprinting. In Meyer et al. (1991), the DNA of various filamentous fungi were digested separately with different restriction endonucleases and the resulting fragments separated by electrophoresis. Selected VNTR oligonucleotides were labeled with 32P and hybridized to the dried gel. Results confirmed that the method based on these minisatellite DNA probes could differentiate between species and strains of fungi. Microsatellites have been used to discriminate strains of the aflatoxigenic species Aspergillus flavus and A. parasiticus (Tran-Dinh and Carter 2000). The PCR technology provides researchers with a means to rapidly amplify small amounts of DNA, and frees them from the time-consuming task of having to isolate sufficient DNA for RFLP analysis. In PCR a thermostable polymerase is employed to enzymatically amplify a specific region of DNA sequence, defined by a set of two oligonucleotide primers. The target DNA is denatured and the primers are then annealed to the single-stranded DNA. The DNA polymerase then synthesizes the complementary DNA strand across the target region. This process is then repeated and the targeted DNA may be amplified a million-fold or more. Randomly Amplified Polymorphic DNA (RAPD) is a PCR technique that yields genetic markers without the need to obtain prior nucleotide sequence data (Williams et al. 1990). Random nucleotide sequences are annealed to the template

Molecular Methods for Identiﬁcation of Plant Pathogenic Fungi

DNA under low stringency. This is followed by PCR amplification and electrophoresis to produce a DNA fingerprint. The RAPD technique is relatively easy, fast, and requires only a minimum amount of starting material. However, the low stringency of the annealing process can produce PCR artifacts. Several methods have been developed to reduce the number of artifacts produced by PCR amplification. One method is by using two pairs of PCR primers, or nested primers (Plikaytis et al. 1990). In this technique, the first pair produces a PCR fragment. Then this fragment and the second pair of primers (nested primers) binding to DNA sequence a few bases internal to the first pair of primers, are used in a further amplification. If the first pair of primers amplified the correct locus, then the second pair of primers will produce a slightly smaller PCR fragment. This method requires a knowledge of the sequence immediately adjacent to the first pair of PCR primers in order to synthesize the other “nested” pair of primers. If the host DNA sequence is not known, Amplified Fragment Length Polymorphism (AFLP) will also reduce the number of PCR artifacts (Vos et al. 1995). In this technique, the target DNA is digested with restriction endonucleases to yield an assortment of different sized DNA fragments. Specific double-stranded adapter oligonucleotides are then ligated to these fragments. PCR primers specific to the adapter sequences with various selective 30 nucleotides are then utilized under high stringency PCR amplification and electrophoresis to produce a unique fragment profile. This technique is time-efficient and amplification is not completely random as in RAPD. When the sequence of the target DNA is known, several other PCR procedures for identification are available, such as PCR amplification of internal transcribed spacer (ITS) of ribosomal DNA (rDNA). An example of the application of this PCR method is Beck and Ligon (1995) who designed PCR primers to detect Stagonospora nodorum and Septoria triticic in wheat. These primers were derived from species specific DNA sequences of the ITS of the pathogen’s ribosomal DNA. The PCR amplification of ITS rDNA has also been employed to identify a wider variety of fungi that were potential pathogens and allergens (Makimura et al. 2001). Knowledge of the specific sequence of polymorphic loci permits high-stringency PCR and thus circumvents the problem of artifacts and low reproducibility associated with random-primer methods (Scott and Straus 2000). One method of site-specific polymorphisms they reviewed was based on the sequence variability found in the introns of single copy metabolic and structural genes. Glass and Donaldson (1995) tested several such oligonucleotide primers for their ability to amplify segments of DNA that span introns in a selection of these genes. They identified primer sets that provided a useful tool for phylogenic studies of filamentous ascomycetes and related fungi. These oligonucleotide primers were utilized to differentiate Fusarium species (Donaldson et al. 1995). The PCR fragments generated were digested with several 4 bp recognition restriction enzymes. The short recognition site,

four bases, of the restriction endonuclease increased the probability that a single base pair polymorphism could be detected. Two other methods of high-stringency PCR are microsatellite based PCR-amplification and the use of the small subunit ribosomal DNA (SSU rDNA) based primers for DNA fingerprinting (Scott and Straus 2000). For example, Gargas and DePriest (1996) describe a list of PCR primers used to amplify and sequence the small subunit of fungal nuclear rDNA. This information identifies primers for special applications (intron-spanning, intron specific, etc) and represents a valuable resource for further research. Groppe et al. (1995) synthesized oligonucleotides corresponding to regions of the sequence of a microsatellite of the endophytic ascomycete Epichloe¨ typhina. These were used for PCR amplification of DNA from different Epichloe¨ isolates. The DNA from most isolates produced a single PCR product. This study pointed to a potentially useful role for microsatellitecontaining loci as a molecular marker for population studies of Epichloe¨ and other unrelated fungi. DNA sequencing, while not yet extensively utilized because of the time and resources required, has been employed to a limited degree in species identification. Wang et al. (2001) recently reported on the use of mitochondrial cytochrome b gene to identify species of Aspergillus section Flavi. Mycelia of the Aspergillus isolates were harvested and their hyphae ruptured with glass beads and zymolyase. Their mitochondria were then collected by centrifugation and their mtDNA extracted. The sequence of the cytochrome b gene has also been used to distinguish species of Aspergillus section Fumigati (Wang et al. 2000) and for investigating the phylogentic relationships of other species of Aspergillus (Wang et al. 1998). Single-Strand Conformation Polymorphism (SSCP) is another PCR-based system that requires knowledge of the target DNA sequence to generate specific oligonucleotide primers. In this technique, the target DNA is concurrently labeled and amplified by PCR using a labeled substrate. The PCR product is then denatured and resolved by electrophoresis. Any changes (mutations, etc.) in the target DNA are detected as altered mobility of separated single strands in autoradiograms. Precise information about the exact change can then be obtained by eluting the targeted DNA from the gel and amplifying it again for sequence determination (Hayashi 1991). Heteroduplex analysis, like SSCP, is a recently developed technique that can detect a single base difference in target DNA (Keen et al. 1991). PCR amplification products from the isolates are combined after heat denaturation and then allowed to reanneal to form heteroduplexes. Any mismatched nucleotides, caused by substitutions, insertions, deletions, etc, will affect the DNA structure of the heteroduplex and lower its electrophoretic mobility. The heteroduplex is compared to duplexes with complete base complementarity by electrophoresis. Kumeda and Asao (2001) employed this technique for the detection of intraspecific variation in isolates of Aspergillus Section Flavi. In their heteroduplex panel analysis (HAP), fragments of the internal spacer (ITS)

Klich and Mullaney

regions of the rDNA gene of the different isolates were ﬁrst ampliﬁed. Heteroduplexes were then generated with the standard ITS reference fragment and then subjected to electrophoresis. The results of this HAP study corresponded well with the established taxonomy of the Section Flavi.

RECENT APPLICATIONS IN PLANT PATHOLOGY

Most recent studies in molecular identification have used PCR in some form. This is not surprising given the power of this tool for analyzing DNA. As the use of PCR methodologies in plant disease diagnosis was reviewed by Henson and French (1993), Mills (1996), our emphasis will be on studies conducted since these reviews. The number of studies on various plant pathogenic genera generally reflects the relative importance of these genera in plant pathology. One of the genera receiving the most attention from molecular biologists, for both understanding phylogeny and pathogenicity, has been Fusarium. Recent work on this genus has been reviewed elsewhere (Nicholson 2001). Applications of molecular techniques in plant pathology have provided methods for identification of isolates of plant pathogens, and identification of pathogens directly from plant materials such as leaves, seeds, or roots. These procedures may be applied at almost any taxonomic level, but usually address taxa at species level and below [e.g. races of a given pathogen]. Molecular methods have also proved useful for distinguishing nontaxonomic categories such as virulence or toxicity.

3.1

Identiﬁcation of Pathogens In Vitro

Information from some recently published studies using molecular methods to identify plant pathogenic fungi from

culture is summarized in Table 1. Many of these studies were designed to develop methods to distinguish between closely related taxa within a single genus or species for which morphological or physiological characters overlap or take too long to develop. There are several advantages in isolating the fungus of interest in culture before conducting molecular identiﬁcations. First, one knows immediately whether or not the pathogen is viable in the plant. Second, working with pure cultures lessens the possibility of errors such as accidentally creating a PCR product from the wrong fungus. There are also disadvantages to this approach. First, it takes longer time because the pathogen must be isolated before it is analyzed. Second, some true (obligate) pathogens cannot be cultured in the laboratory. Finally, metabolites such as mycotoxins may remain in the crop even after the fungus dies and these may be missed if no viable fungus is present.

3.2

Identiﬁcation of Pathogens Directly from Plant Parts

Examples of studies using molecular methods to identify pathogens directly from plant parts are given in Table 2. Most of these involve identification of fungal species using RAPDs or PCR of the ITS-rDNA. Developing methods for direct isolation of specific fungal DNA from plant tissues is more difficult than isolating DNA from a pure fungal strain, but the potential impact of the former methods is tremendous. These assays have demonstrated the presence of the pathogens in asymptomatic plants (Doohan et al. 1998). Some of these procedures take only seven to 24 h to perform (Lee et al. 2001; Lovic et al. 1995), compared with several days to a week for traditional methods or methods requiring that the fungus be isolated prior to DNA

Table 1 Examples of studies on molecular identiﬁcation of fungal plant pathogens in vitro Fungal genus Alternaria Botrytis Claviceps Colletotrichum Elsinoe Fusarium Fusarium Gaeumannomyces Gibberella (Fusarium) Gibberella (Fusarium) Macrophomina Rhizoctonia Rhynchosporium Tilletia Venturia

Host

Level of discrimination

Method

Citation

Umbellifers Onion Sorghum Alfalfa Citrus Tomato Cucumber Turf-grass Banana/corn Banana/corn Bean, cornþ Various Barley Wheat Pear

Species Species subgroup Species/populations Species Species Virulence within race Forma specialis Variety Species/toxicity/host Species Population Anastomosis grp subsets Species Species Species

RAPD PCR-ITS/rDNA AFLP and RAM AFLP RAPD RAPD RAPD PCR-ITS/rDNA RAPD PCR-ITS/rDNA AFLP PCR-ITS/rDNA PCR-ITS/rDNA TaqManPCR-MtDNA PCR-ITS/rDNA

Pryor and Gilbertson 2002 Nielsen et al. 2001 Tooley et al. 2000 O’Neill et al. 1997 Hyun et al. 2001 Mes et al. 1999 Vakalounakis and Fragkiadakis 1999 Goodwin et al. 1995 Jimenez et al. 2000 Jimenez et al. 2000 Mayek-Perez et al. 2001 Carling et al. 2002 Lee et al. 2001 Frederick et al. 2000 Le Cam et al. 2002

Molecular Methods for Identiﬁcation of Plant Pathogenic Fungi

Table 2 Examples of studies on molecular identiﬁcation of fungal plant pathogens in vivo Fungal genus Alternaria Fusarium Fusarium Leptosphaeria Melampsora Monosporascus Monilinia Mycosphaerella Peronosclerospora Phakopsora Pythium Rhynchosporium a b

Host

Method

Level of discrimination

Carrot Wheat Wheat Crucifers Willow Cucurbits Stone fruits Banana/plantain Sorghum Soybean 3 speciesb Barley

PCR-ITS/rDNA RAPD RAPD PCR/GenBank M77515a RAPD PCR-ITS/rDNA dot blots and PCR PCR-ITS/rDNA dot blots-genomic DNA TaqMan-PCR-ITS/rDNA PCR-ITS/rDNA PCR-ITS/rDNA

Species Species Species/variety Virulence Stem/leaf variants Species Species Species Species Species Species Species

Citation Konstantinova et al. 2002 Parry and Nicholson 1996 Doohan et al. 1998 Taylor 1993 Pei et al. 1997 Lovic et al. 1995 Boehm et al. 2001 Johanson and Jeger (1993) Yao et al. 1990 Frederick et al. 2002 Kageyama et al. 1997 Lee et al. 2001

Seed cultured in liquid medium. Cucumber, sugar beet, and Chinese cabbage.

extraction. Moreover, fungi that do not grow in pure culture may be studied with these techniques. On the other hand, amplifying DNA of nonviable fungi would lead to falsepositives for disease potential, but would nevertheless provide useful information for researchers interested in mycotoxins.

CONCLUSIONS

Molecular methodologies have been used successfully to address a number of problems in identification of plant pathogenic fungi. Not all of the molecular identification methods have been fully utilized, but this will probably change in the future. Practical application of molecular methodologies is increasing as the instruments become less expensive and the protocols less complex. The demand for rapid, accurate identification of plant pathogens is growing. In addition to the need for such methods in field diagnosis and import/export issues, the threat of plant pathogens as potential bioterrorist weapons has added impetus to the need for rapid identification. The lack of a universally applicable technique that would differentiate all fungi makes rapid identification difficult unless preliminary morphological or physiological information is available. New methods are being developed (Schaad et al. 2002) which hold great promise for rapid identification of many plant pathogens.

ACKNOWLEDGEMENTS We would like to thank Richard Baird (Mississippi State University) for providing reprints on the molecular biology of plant pathogens, and Deepak Bhatnagar (USDA ARS, New Orleans, LA) for inviting us to write this review. We thank Joan Bennett (Tulane University, New Orleans, LA), Hurley Shepherd, and Robert Brown (both of USDA ARS, New Orleans, LA) for useful comments on the draft of the manuscript.

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54 Groppe K, Sanders I, Wiemken A, and Boller T (1995). A microsatellite marker for studying the ecology and diversity of fungal endophytes (Epichloe¨ spp.) in grasses. Appl Environ Microbiol 61:3943 –3949. Hayashi K (1991). PCR SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl 1:34 – 38. Henson JM and French R (1993). The polymerase chain reaction and plant disease diagnosis. Annu Rev Phytopathol 31:81 –109. Hyun J-W, Timmer LW, Lee S-C, Yun S-H, Ko S-W, and Kim K-S (2001). Pathological characterization and molecular analysis of Elsinoe isolates causing scab diseases in citrus in Jeju Island in Korea. Plant Dis 85:1013 – 1017. Jeffreys AJ, Wilson V, and Thein SL (1985). Hypervariable minisatellite regions in human DNA. Nature 314:67 –73. Jimenez M, Rodriguez S, Mateo JJ, Gil JV, and Mateo R (2000). Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA. Syst Appl Microbiol 23:546 – 555. Johanson A and Jeger MJ (1993). Use of PCR for detection of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka leaf spots in banana and plantain. Mycol Res 97:670 – 674. Kageyama K, Ohyama A, and Hyakumachi M (1997). Detection of Pythium ultimum using polymerase chain reaction with species-specific primers. Plant Dis 81:1155 –1160. Keen J, Lester D, Inglehearn C, Curtis A, and Bhattacharya S (1991). Rapid detection of single base mistmatches as heteroduplexes on Hydrolink gels. Trends Genet 7:5. Konstantinova P, Bonants PJM, van Gent-Pelzer MPE, van der Zouwen P, and van den Bulk R (2002). Development of specific primers for detection and identification of Alternaria spp. in carrot material by PCR and comparison with blotter and plating assays. Mycol Res 106:23 –33. Kumeda Y and Asao T (2001). Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus section Flavi strains. Appl Environ Microbiol 67:4084 – 4090. Le Cam B, Devaus M, and Parisi L (2002). Specific polymerase chain reaction identification of Venturia nashicola using internally transcribed spacer region in the ribosomal DNA. Phytopathology 91:900– 904. Lee HK, Tewari JP, and Turkington TK (2001). A PCR based assay to detect Rhynchosporium secalis in barley seed. Plant Dis 85:220 – 225. Lovic BR, Vladez VA, Martyn RD, and Miller ME (1995). Detection and identification of Monosproascus spp. with genus-specific primers and nonrandioactive hybridization probes. Plant Dis 79:1169 –1175. Makimura K, Hanazawa R, Takatori K, Tamura Y, Fujisaki R, Nishiyama Y, Abe S, Uchida K, Kawamura Y, Ezaki T, and Yamaguchi H (2001). Fungal flora on board the Mir-Space Station, identification by morphological features and ribosomal DNA sequences. Microbiol Immunol 45:357 – 363. Mayek-Perez N, Lopez-Castaneda C, Gonzalez-Chavira M, GarciaEspinosa R, Acosta-Gallegos J, Martinez de la Vega, and Simpson J (2001). Variability of Mexican isolates of Macrophomina phaseolina based on pahtogenesis and AFLP genotype. Physiol Mol Plant Pathol 59:257 –264. Mes JJ, Weststeijn EA, Herlaar F, Lambalk JJM, Wijbrandi J, Haring MA, and Cornelissen BJC (1999). Biological and molecular characterization of Fusarium oxysporum f.sp. lycopersici

Klich and Mullaney divides race 1 into separate virulence groups. Phytopathology 81:156 –160. Meyer W, Koch A, Niemann C, Beyermann B, Epplen JT, and Borner T (1991). Differentiation of species and strains among filamentous fungi by DNA fingerprinting. Curr Genet 19:239 –242. Mills PR (1996). Use of molecular techniques for the detection and diagnosis of plant pathogens. BCPC Symp Proc 65:23 – 32. Nicholson P (2001). Molecular assays as aids in the detection, diagnosis and quantification of Fusarium species in plants. In: Summerell BA, Leslie JF, Backhouse D, Bryden WL, Burges LW eds. Fusarium Paul E. Nelson Memorial Symposium. St Paul, MN: APS Press. pp 176 – 192. Nielsen K, Justesen AF, Jensen DF, and Yohalem DS (2001). Universally primed polymerase chain reaction alleles and internal transcribed spacer restriction fragment length polymorphisms distinguish two supbroups in Botrytis aclada distinct from B. byssoidea. Phytopathology 91:527 –533. O’Neill NR, van Burkum P, Lin JJ, Kuo J, Udi GN, Kenworthy W, and Saunders JA (1997). Application of amplified restriction fragment length polymorphism for genetic characterization of Colletotrichum pathogens of alfalfa. Phytopathology 87:745 –750. Parry DW and Nicholson P (1996). Development of a PCR assay to detect Fusarium poae in wheat. Plant Pathol 45:383 –391. Pei MH, Whelan MJ, Halford NG, and Royle DJ (1997). Distinction between stem- and leaf-infecting forms of Melampsora rust on Salix viminalis using RAPD markers. Mycol Res 101:7 – 10. Plikaytis BB, Gelber RH, and Shinnick TM (1990). Rapid and sensitive detection of Mycobacterium leprae using a nestedprimer gene amplification assay. J Clin Microbiol 28:1913 – 1917. Pryor BM and Gilbertson RL (2002). Relationships and taxonomic status of Alternaria radicina, A. carotiincultae, and A. petroselini based upon morphological, biochemical and molecular characteristics. Mycologia 94:49– 61. Schaad NW, Opgenorth D, and Gaush P (2002). Use of a protable real-time fluorescent PCR system for one-hour on site diagnosis of Peirces disease of grape in early season asympotmatic vines. Phytopathology 92:721– 728. Scott J and Straus N (2000). A review of current methods in DNA fingerprinting. In: Samson RA, Pitt JI eds. Integration of Modern Taxonomic Methods for Penicillium and Aspergillus classification. Reading, U.K. Harwood Academic Publishers. pp 209 –224. Taylor JL (1993). A simple, sensitive, and rapid method for detecting seed contaminated with highly virulent Leptosphaeria maculans. Appl Environ Microbiol 59:3681 – 3685. Tooley PW, O’Neill NR, Goley ED, and Carras MM (2000). Assessment of diversity in Claviceps africana and other Claviceps species by RAM and AFLP analyses. Phytopathology 90:1126 – 1130. Tran-Dinh N and Carter D (2000). Characterization of microsatellite loci in the aflatoxigenic fungi Aspergillus flavus and Aspergillus parasiticus. Mol Ecol 9:2170– 2172. Vakalounakis DJ and Fragkiadakis GA (1999). Genetic diversity of Fusarium oxysporum isolates from cucumber: differentiation by pathogenicity, vegetative compatability and RAPD fingerprinting. Phytopathology 89:161 –168.

Molecular Methods for Identification of Plant Pathogenic Fungi Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, and Zabeau M (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acid Res 23:4407 –4414. Wang L, Yokoyama K, Miyaji M, and Nishimura K (1998). The identification and phylogentic relationship of pathogenic species of Aspergillus based on the mitochondrial cytochrome b gene. Med Mycol 36:153– 164. Wang L, Yokoyama K, Miyaji M, and Nishimura K (2000). Mitochondrial cytochrome b gene analysis of Aspergillus fumigatus and related species. J Clin Microbiol 38:1352 – 1358.

55 Wang L, Yokoyama K, Takahasi H, Kase N, Hanya Y, Yashiro K, Miyaji M, and Nishimura K (2001). Identiﬁcation of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene. Int J Food Microbiol 71:75– 86. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, and Tingey SV (1990). DNA polymorphisms ampliﬁed by arbitrary primers are useful as genetic markers. Nucleic Acid Res 25:6531. Yao CL, Fredericksen A, and Magill CW (1990). Seed transmission of sorghum downy mildew: detection by DNA hybridization. Seed Sci Technol 18:201 –207.

5 The Application of Molecular Markers in the Epidemiology of Plant Pathogenic Fungi Paul D. Bridge* Birkbeck College, University of London, London, and Royal Botanic Gardens Kew, Surrey, United Kingdom

Tanuja Singh / Dilip K. Arora† Banaras Hindu University, Varanasi, India

plant involves relatively long periods of intimate interaction without apparent damage to the host, and where the pathogen can persist in asymptomatic hosts for many years (Stanosz et al. 1997). The effects of fungi on living plants vary considerably. At one extreme, damage may be limited to small lesions on leaves or stems [e.g., caused by some Alternaria species, see Ellis (1968)], while at the other extreme the plant may be rapidly killed [e.g., by some Verticillium species, see Pegg (1984)]. Much work has been done to control fungal disease through selection and breeding programs, the genetic modification of both host and pathogen, and the introduction of resistant varieties [see Stukely and Crane (1994)]. The success of these efforts depends largely on the understanding of genetic variability in the fungal population and monitoring this variation in nature. Many aspects of the biology of the fungi have important consequences at the population level. This particularly applies to the mode of reproduction (i.e., the relative contributions of sexual and asexual, outcrossing and selfing mechanisms), and to hyphal anastomosis between genetically different individuals (Brasier 1991; Glass and Kuldau 1992; Milgroom et al. 1996). A further consideration is that some fungi are predominantly haploid in their vegetative phase, some are diploid, and some are dikaryotic. In the case of pathogenic fungi, genetic variability can be introduced through a variety of mechanisms, either during sexual reproduction or independently of it (Kistler and Miao 1992). Such variability is significant as it can influence

INTRODUCTION

Fungi are present in a variety of forms, in almost every habitat, where they are often specific in their occurrence on particular types of host (or substrate) and ecological niche. Fungi may also become partners with higher plants and enter complex biological relationships with the host (Clay and Kover 1996; Thrall and Burdon 1997). The term pathogen is defined as “a parasite able to cause disease in a host or range of hosts” (Kirk et al. 2001), and pathogenic fungi can occur on all plants. In this chapter this definition of plant pathogenic fungi will be limited to those that cause diseases of living plants, and therefore does not include the fungi involved in the spoilage of stored plant materials that are often referred to as causing post harvest “diseases.” The plant pathogenic fungi consist of a large group of genera and species from diverse areas of the fungal kingdom. Recent developments in the understanding of the evolution of eukaryotic organisms have meant that a number of important plant pathogenic organisms have been reclassified, and are no longer considered as fungi sensu stricto. These include the economically important genera of Phytophthora, Pythium, and other Oomycetes, that are now placed in the Straminipila (Dick 2001). Plant pathogenic fungi have a significant influence on crop productivity. Devastating fungal diseases such as corn smut, potato blight, and black stem rust of wheat can destroy many economically important crops. This situation becomes more critical when the interaction between pathogen and the host

Present afﬁliations: *British Antarctic Survey, Cambridge, United Kingdom. † National Bureau of Agriculturally Important Microorganisms (NBAIM), New Delhi, India.

Bridge et al.

the host-pathogen interaction as genetic ﬂexibility allows the fungi to readily adapt to changing environmental conditions, including the introduction of new host genotypes. Understanding the epidemiology of plant pathogenic fungi depends upon on the ability to unambiguously identify sexually produced individuals and asexually produced clones. Classical identiﬁcation traits, such as morphological, physiological, and disease characters, lack the required sensitivity and accuracy needed for identifying individuals within a population, and this has prevented detailed population studies for many years. Recent developments in molecular techniques now allow population studies on plant pathogenic fungi, and these can be performed with great sensitivity and accuracy. An almost unlimited number of polymorphic loci can be used to detect individual genotypes for the direct assessment of genetic variation in a given fungal population. Application of molecular markers has also allowed the investigation of evolutionary processes in a large number of agriculturally important fungi (Mitchell and Brasier 1994; Milgroom et al. 1996; Valent and Chumley 1991; Vilgalys and Cubeta 1994), and the number and scope of these studies is rapidly expanding.

REASONS FOR DETERMINING EPIDEMIOLOGY

As is the case for human and animal diseases, a knowledge of the epidemiology of plant disease can provide important information for its treatment and control, and can lead to the development of forecasting systems [see Shaw 2001; Zadoks and Schein 1979]. In the case of fungal plant diseases, particularly those affecting agricultural crops, the two main areas that need to be determined are the mechanism of spread of the disease, and the speciﬁcity and host range of the infecting organism. Fungi are transmitted to, and between, plants by a number of different mechanisms. Many fungi are spread through the soil, some growing from previously infected debris in or above the soil. Others can be transmitted as spores and other propagules through water droplets, or directly as airborne particles. Some plant pathogens may exist on secondary hosts, such as Fusarium oxysporum (Armstrong and Armstrong 1958) or on weeds [see Terry and Parker (2001)]. If these hosts are present in or near a crop, they may then act as a reservoir that allows a crop disease to be carried over successive plantings. A few plant pathogenic fungi such as Ophiostoma species [see Brasier (1991)] can be spread by insects and other vectors, and some such as Sclerospora graminicola (Shetty et al. 1980) remain in the seeds of infected plants, and cause disease in subsequent generations. If the mechanism of transmission is known, this knowledge may be important in the development of control or treatment strategies. Such knowledge is particularly important in the selection of planting material, the preparation and maintenance of planting areas, and the establishment of crop successions and rotation [see Maude (1996)].

In addition to knowledge of the mode of transmission of a plant pathogen, it is also important to be able to determine exactly what is being transmitted in terms of fungal populations. In some cases a single fungal species may consist of a number of different host specific populations. An example of this is the vascular wilt pathogen F. oxysporum, where around 170 different forms (referred to as special forms) have been identified. Each of these special forms shows preferential or specific pathogenicity to different hosts, and so F. oxysporum special form cubense will cause vascular wilt of banana, but would not be expected to cause significant disease on oil palm. Therefore, in order to monitor what fungi are present, and may pose a risk to a crop, it is necessary to know specific details of their pathogenicity. This situation becomes further complicated if there are subpopulations within the pathogen that show differential pathogenicity, either in terms of the degree of damage or the particular cultivars attacked. Such populations are generally described as races, and the ability to differentiate these can make a significant difference to their control. A further factor in considering populations in fungal plant pathogens is whether the population is comprised of meiotic or mitotic forms. Some pathogenic fungi, such as Fusarium species, occur almost exclusively in a mitotic (or imperfect) form. In this state, the variability within the population can be assumed to be relatively low, particularly if the disease is the result of a single introduction. Some fungal pathogens, such as Phytophthora and the rusts and smuts, are however present on the plant in a meiotic (or perfect) form, and this allows for variability to be introduced into the population at each generation [for example see Duncan et al. (1998)].

TYPES OF MOLECULAR MARKERS

Detection of fungi on the basis of visual examination of morphology is highly selective and species-specific identification of fungi and spores is therefore difficult. Molecular techniques present several advantages over the traditional ones and, most importantly, nucleic acid sequences unique to particular organisms can generally be found. As these techniques do not rely on phenotypic examination, gene expression is not required and identification times can be reduced significantly. In the fungi, molecular markers can be derived from both variable and conserved regions of the nuclear and mitochondrial genome, and different markers have been used to define populations at all levels from an individual isolate upwards. Some methods have the potential for the detection of specific genomic DNA sequences directly from initial plant samples, thereby eliminating the requirement to isolate and culture the fungus. Specific molecular markers, probes, and primers have commonly been developed from a variety of DNA sequences including randomly cloned genomic DNA fragments and specific regions such as genes coding for ribosomal RNA (rRNA), virulence factors, and insertion sequences.

Epidemiology of Plant Pathogens

3.1

Ribosomal DNA

The nuclear genomes of fungi have a number of particular features. They are relatively small (approximately 13 –93 million nucleotide base pairs), and in comparison to higher plants and animals they have a much lower percentage of redundant DNA (about 10 –20%) (Lu 1996). Around 30% of the entire fungal genome consists of duplicated regions and genes (Mewes 1997). These repetitive sequences provide potential targets for molecular markers due to their high copy number. Ribosomal DNA (rDNA), speciﬁcally the regions coding for the rRNA subunits and their associated spacers, is one of the most commonly used DNA regions for fungal molecular markers [see Bridge and Arora 1998; Bruns et al. 1991; www.mendel.berkeley.edu/boletus/boletus.html; www. biology.duke.edu/fungi/]. The nuclear-encoded rRNA genes (rDNA) and spacers occur as a gene cluster (typically of 8–12 kb) that is multiply repeated (see Figure 1). The basic unit consists of the genes for the small ribosomal subunit, the 5.8S subunit, and the large subunit. The three genes are separated by two internally transcribed spacers (ITS), and the repeated gene clusters are separated by an intergenic spacer (IGS) that in many, but not all fungi, also contains the gene for the 5S subunit. [see Hillis and Dixon (1991)]. Several restrictions sites are conserved in fungal rDNA, and this makes them convenient sequences for cloning [see other reviews by Gargas and DePriest (1996); Hibbett (1992)]. The rRNA cluster has proved to be a good region for deriving molecular markers for many fungi [see Bruns et al. (1991); Hibbett (1992)]. The subunit genes have both conserved and variable domains, and can be used for comparisons of genera, the spacer regions are considerably more variable and can be used for comparisons of species or in some cases speciﬁc pathogenic forms (see Table 1). As the cluster is universal and multiply repeatedly, it is a good target for molecular studies. Originally these studies involved obtaining RFLPs with probes hybridized to total genomic DNA digests, and many

studies of populations and species of plant pathogens were carried out in this way [e.g., Jabaji-Hare et al. (1990), Manicom et al. (1990)]. More recently these studies have largely been replaced by polymerase chain reaction (PCR) based studies, particularly as the rRNA cluster can often be detected in old or contaminated environmental samples [see Bruns et al. (1990)]. The varying levels of specificity of the different DNA regions in the gene cluster also mean that it is possible to amplify fungal DNA directly from samples of infected plant material (Bridge et al. 2000; Gardes and Bruns 1993). Both the ITS and the IGS regions have been used to develop species-specific primers for plant pathogen detection in plant material [e.g., Brown et al. (1993); Moukhamedov et al. (1993)]. It is becoming increasingly common in rRNA cluster studies to obtain sequences of all the regions of interest. Although knowledge of the complete sequences provides a large amount of information, useful information may be obtained from simple restriction digestions of rRNA amplification products. This approach generally produces relatively simple patterns containing 1 –4 bands, and in certain cases these patterns, or individual bands, may be specific for particular pathogens [e.g., Chen (1992)]. It is not possible to list all the work done using ITS and IGS regions to develop molecular markers. One example of this was the study of Mazzola et al. (1996) who developed an oligonucleotide primer set that consistently and selectively amplified a 511 bp fragment in the ITS region that could be used to differentiate between Rhizoctonia solani and R. oryzae. It should, however, be remembered that RFLP analysis is essentially a one-tailed analysis of variation; and although different patterns indicate that two organisms are different, a common pattern is based on only the position of a few restriction sites. Therefore, identical RFLP band patterns do not imply that the rest of the sequence is identical.

3.2

Protein Coding Genes

There are numerous gene sequences that have been examined in the systematics and phylogeny of plant pathogenic fungi. These include genes for the production of actin, tubulin, elongation factors, cytochromes, proteases, and many others [e.g., Glass and Donaldson (1995); Mehmann et al. (1994); Schoch et al. (2001)]. These genes are generally highly conserved between distant organisms; but can contain short introns that can be very variable in insertion position and number [see Edelmann and Staben (1994)]. This variation in introns can be useful as a molecular marker among closely related organisms and this has been investigated for a number of plant associated fungi including Fusarium, Ascochyta, and Phoma (see later).

3.3

Figure 1 Ribosomal rRNA gene cluster arrangement.

Fingerprinting Methods

For the purposes of this chapter DNA ﬁngerprinting methods will be limited to those that have been used with plant

Bridge et al.

Table 1 Features of commonly used molecular markers Marker RAPD Simple repetitive PCR sequences; micro- and mini-satellite probes and primers AFLPs mtDNA RFLPs ITS/IGS region RFLPs ITS region sequencing rRNA gene sequences Major structural/functional protein genes

Taxonomic level resolved

Affected by meiosisa

Individuals, subspeciﬁc groups Individuals, subspeciﬁc groups

Yes Yes

Individuals, subspecific groups, some closely related species Subspecific groups, closely related species Closely related species, some subspecific groups Some subspecific groups, closely related species Species, genera, families, phyla Species, genera, families, phyla

Yes No Not generallyb Not generally No No

For many fungi the effects of meiosis on markers have not been speciﬁcally considered. Yes and no entries refer to general assumptions. One reason for the selection of rRNA gene cluster was that it was resistant to crossover. However, there is at least one report of presumed recombination in the rDNA of fungi (see text).

pathogenic fungi to generate largely random PCR fragments from the total genome. These techniques have also been referred to as total genome profiling. One of the oldest and most widely used of such PCR methods is random amplified polymorphic DNA (RAPD) analysis (Welsh and McClelland 1990; Williams et al. 1990). Essentially RAPD analysis relies on the reduction in specificity of the PCR process at reduced temperatures. Total genome DNA extractions are used, and these are amplified with single, short (usually decamer) primers at a reduced annealing temperature. These conditions result in less stringent binding of the primer to the target DNA, and allow the amplification of a number of generally small regions of DNA. These are separated by electrophoresis to give a profile of bands. The RAPD analysis has been criticized as it is not always entirely reproducible, but it has been used extensively for profiling populations of plant pathogens [e.g., Bentley et al. (1995); Cooke et al. (1996)]. In many cases these studies have shown correlations between band patterns and host, disease type or geographical origin, and band patterns have also been used to differentiate between forms of the same fungus causing different disease symptoms [e.g., Pei et al. (1997)]. Another common PCR fingerprinting method that has been used for plant pathogenic fungi is amplification of sequences based on simple repetitive primers. In this method, single short repetitive primers are used at moderate annealing temperatures in order to amplify largely repetitive fragments of the genome. One target site for this method is the flanking region of genes that can contain variable numbers of such repeats. Repeat motifs that have been used for primers have varied from simple 2 –3 base repeats such as (CA)8 and (CAG)5 (Freeman and Rodriguez 1995; Latge et al. 1998) to more complex sequences including the M13 bacteriophage universal sequencing primer [see Bridge et al. (1997)]. Another method of fingerprinting that has been used with some fungi is based on a group of short repetitive DNA sequences that have been found dispersed throughout the genome of diverse bacterial species [see van Belkum et al. (1998)]. Primers specific to these repetitive sequences produce multiple products in PCR with fungal

genomic DNA, and these can then be separated to provide simple fingerprints. Three particular unrelated families of such repetitive DNA sequences, BOX (54 bp), ERIC (124 bp), and REP, (35–40 bp) have also been used to characterize subspecific populations of different plant pathogenic fungi [see Arora et al. (1996); Toda et al. (1999)]. A relatively recent development in fingerprinting fungi has been the introduction of amplified fragment length polymorphism (AFLP) analysis [see Vos and Kuipper (1997); Vos et al. (1995)]. In this technique, total DNA is digested with restriction enzymes, and then short artificial oligonucleotides (linkers) are ligated to the restriction enzyme sites. Specific primers are then designed that show a particular degree of specificity to the linker sequences, and large fractions of the total DNA can then be amplified as fragments. The AFLP analysis generates many bands, and electrophoresis is usually undertaken in large polyacrylamide gels, it is however, possible to undertake more restricted studies that generate fewer bands and that can be analyzed in smaller electrophoresis systems [e.g., Mueller et al. (1996)]. At the conclusion of RAPD and AFLP analyses PCR bands of interest can be extracted, purified, and sequenced to produce sequence characterized amplified regions (SCARs). The sequence information obtained from SCARs can then be used to develop more specific PCR primers for detection methods [e.g., Dobrowolski and O’Brien (1993); Leclerc-Potvin et al. (1999)].

3.4

Other Total Genome Approaches

Two other methods that have been used to characterize populations of plant pathogenic fungi are the analysis of overall repetitive DNA, and chromosome size and number. When total fungal DNA is digested with frequent cutting restriction enzymes, a large number of fragments of many different sizes are generated, and these appear in a gel as a “smear.” Brighter staining bands can be seen in these smears where there are multiple copies of fragments of the same size.

Epidemiology of Plant Pathogens

These bands are, in part, the result of multiple copy DNA such as the rRNA genes and mitochondrial DNA (mtDNA). Differences in the patterns of these bands can be a quick, simple way for differentiating some closely related fungi, and this approach has been used for race designation in F. oxysporum sp. f. pisi [see Coddington et al. (1987)]. Unlike plants and animals, fungal chromosomes can be very variable, and in many fungal species different isolates may show differences in the number and size of their chromosomes. This has been investigated in some isolates of Colletotrichum and special forms of F. oxysporum. In Colletotrichum variation was found in the number and size of the smaller (type B) elements, and in F. oxysporum sp. f. cubense variation in chromosome sizes was found between different races and vegetative compatibility groups [see Masel et al. (1993); Ploetz (1990)].

3.5

Mitochondrial Sequences

The mitochondrial (mt) genome has been used extensively in the investigation of population structures in the plant pathogenic fungi. In fungi the mitochondrial genome is a circular structure of between 17 and 121 Kb [see Zimmer et al. (1984); Curole and Kocher (1999); Grossman and Hudspeth (1985); Lu (1996)]. The fungal mitochondrial genome makes up between 1–20% of the DNA occurring in fungal cells, and generally contains a high proportion of sequences that lack a coding function. In addition it may contain many repeat sequences and introns, and these features can allow for considerable variation in mitochondrial sequences between closely related organisms [see Clark-Walker (1992); Clark-Walker et al. (1987); see chapter numbers 11 and 12 in this book]. As it is present in multiple copies, mtDNA can be a good target for molecular methods. In most cases mitochondrial DNA is inherited unilinearly during meiosis but recombination may occur in some fungi (Wolf 1996). In addition, mtDNA can be transferred independently of the nuclear genome during unstable vegetative fusion (Collins and Saville 1990). The mtDNA can contain GC rich palindromic repeats, but overall, simple GC sequences are relatively rare in fungal mitochondria, and this has been used in differential DNA restriction protocols to generate presumptive mitochondrial RFLPs [e.g., Kouvelis and Typas (1997); Lacourt et al. (1994)]. The mitochondrial genome contains both variable and conserved regions and so sequence information may be used at a variety of taxonomic levels [see Zhou and Stanosz (2001)]. In some cases closely related species may have very different mitochondrial genomes, and one example of this is in the yeast Saccharomyces, where mtDNA varies from 24 to 78 Kb between different species [see Grossman and Hudspeth (1985)]. Analysis of mtDNA variation has been used at a variety of different population and systematic levels, and these have correlated with subspecies, vegetative incompatibility groups, different populations and individuals (Gordon and Okamoto 1992; Jacobson and Gordon 1990; Miller et al. 1999).

3.6

Inserted Elements

Both nuclear and mitochondrial genomes in fungi may contain a wide range of inserted elements [e.g., see Edelmann and Staben (1994); chapters 11 and 12]. At the very simplest these may be very short sequences left after transposon insertion and removal, and at the more complex they include a wide range of different transposons [see Daboussi (1997); Daboussi and Langin (1994)]. Inserted elements, particularly introns, have been investigated as potential molecular markers at both the population level and the higher phylogenetic level (DePriest 1993; Neuve´glise et al. 1997).

3.7

Application of Individual Markers

The suitability and resolution of individual markers will depend very much on the fungus being considered, and a marker that is useful for differentiating species in one group of fungi, may be considerably more conserved or variable in other fungal groups. One example of this is the use of presumptive mtDNA RFLPs (AT rich DNA). The mtDNA RFLPs have been found useful for determining species and subspecific populations in some species of Aspergillus, whereas in the species Metarhizium anisopliae and Verticillium lecanii the same approach identifies numerous subspecific groupings (Kouvelis and Typas 1997; Typas et al. 1998; Varga et al. 1994). In the plant pathogen F. oxysporum sp. f. cubense mtDNA RFLPs have been used to distinguish between different races and supported the theory that the recently determined race 4 was not derived from the existing race 1 or 2 (Thomas et al. 1994). In the basidiomycete Ganoderma boninense mtDNA RFLPs have been found useful for defining individuals (Miller et al. 1999), and in some Phytophthora species they have been used for determining parental lines (Whittaker et al. 1994). Differences in the level of variability seen with the same marker from different taxa is not restricted to mtDNA, and appears to be a feature of most markers investigated for population and species level investigations. One example of this is the degree of ITS sequence difference seen between isolates of a single species, or between closely related species. As an example there is generally up to around 5% ITS sequence variation within individual species of Colletotrichum, and a maximum of about 23% variation between species (Sreenivasaprasad et al. 1996). In Rhizoctonia solani, up to 30% variation in the ITS sequences has been reported between isolates of the same anastomosis group (Kuninaga et al. 1997).

3.8

Combining Markers

In general terms the use of different markers can give rise to a hierarchic system, with particular techniques giving more, or less, resolution than others [see Bruns et al. (1991)], and so it

Bridge et al.

may be possible to select an appropriate marker for the situation under study. An example of the way in which a broadly hierarchic arrangement of markers can be used for the study of fungal plant pathogens is detailed later with G. boninense. This approach will however not always generate consistent results, and one example of this is the group of fungal plant pathogens known as the “Ascochytacomplex” that occurs on beans, peas, and other legumes. In this case there are a number of distinct species currently assigned to either the genus Ascochyta or Phoma (see Table 2). Most of these species can be deﬁned individually from their ITS sequences, and some can in turn be subdivided on the basis of their mtDNA RFLPs. When a single part of the mitochondrial genome is considered there is less variability, and the species can be arranged in three groups. The groupings obtained from RFLPs derived from the b-tubulin gene are less consistent and group some species together, while also showing subspeciﬁc groups in others (Fatehi 2000).

3.9

Selection of Molecular Markers

Two of the most important factors that need to be considered in the selection of molecular markers in any study are the taxonomic rank under consideration, and the life cycle of the fungus. As detailed above, a marker that is particularly useful at a certain taxonomic rank for one species may not be useful at the same rank for another species. One explanation for this is that different systematists or plant pathologists have had different species concepts, and so the terms species and subspecies may not be directly comparable between different fungal genera. In some genera, such as Fusarium, there has been a tradition of placing different pathogenic forms in the special form category, whereas in others, such as Colletotrichum or Phoma, there has been a general history of describing new species. Such differences in species concepts may reﬂect evolutionary ages, or may reﬂect levels of

variation in other characters. Whatever the reason, there can be significant differences in the degree of variation seen in the molecular markers chosen. Fungi occur in asexually (imperfect, anamorphic) and sexually (perfect, teleomorphic) reproducing forms, and in some cases both forms are present at the same time (holomorphic). In the imperfect state cell division is solely by mitosis, whereas in the perfect state recombination and meiosis will occur. Recombination and meiosis can have a significant effect on results obtained from some molecular markers (see Table 1). Isoenzyme markers could be expected to be subject to allelic variation under such circumstances, as would many DNA fingerprinting markers. The degree to which a marker will be affected will vary considerably, and one example is the comparison of sibling haploid lines derived from a single dikaryotic fruit body. In these circumstances, the haploid progeny have arisen by meiosis and may show different isoenzyme or DNA fingerprints from the parental material. This has been investigated in the oil palm pathogen G. boninense where both RAPD (Pilotti et al. 2000) and simplified AFLP (Figure 2) fingerprints differed both between siblings and between siblings and parent. This variation can then be further compounded through subsequent mating and recombination. Some molecular markers can be expected to be consistent despite meiosis and recombination. DNA sequences of major structural and functional proteins will be resistant to recombination events, and the rRNA gene cluster is one region generally considered to be maintained under such conditions (Hillis and Dixon 1991). There are however some indications that this is not always the case, and there is at least one report that in some fungi, not only can the rRNA region be affected by crossover, but also that this may occur at a higher frequency than predicted (Selosse et al. 1996). Nonnuclear markers may be recombination insensitive, and mtDNA has been used to demonstrate a single hereditary line, where the mtDNA was inherited unilinearly (Whittaker et al. 1994). It should be remembered however that this will

Table 2 Features of Ascochyta complex species on legumes Species P. exigua A. rabiei A. fabae A. fabae f. sp. lentis A. pisi P. medicaginis var. pinodella A. pinodes P. subboltshauseri

a b

mt SSU rRNA size

b-tubulin gene RFLPa

ITS sequenceb

mtDNA RFLPs

749 bp 749 bp 660 bp 660 bp 660 bp 660 bp 645 bp 645 bp 645 bp 645 bp 645 bp

D A A C C C B B E F G

1 1 2 3 4 5 6 6 7 7 7

Multiple, distinct

Letters A –G designate 7 different RFLP patterns obtained by digestion of a PCR ampliﬁed fragment of the b-tubulin gene. Numbers 1–7 designate 7 different RFLP patterns obtained by digestion of the complete ITS1/5.8 s/ITS2 region.

Multiple, distinct Multiple, distinct Single, distinct Single, distinct Multiple, distinct Multiple, distinct Single, distinct

Epidemiology of Plant Pathogens

Figure 2 Simple sequence repetitively primed molecular ﬁngerprints for 4 lines of Ganoderma derived from a single bracket. Lane 1, molcular size markers; lanes 2 and 3 monokaryotic culture derived from basidiospore a; lanes 4 and 5, monokaryotic culture derived from basidiospore b; lanes 6 and 7, monokaryotic culture derived from basidiospore c; lanes 8 and 9, dikaryotic culture obtained from b and c.

not always be the case, as not all fungi have unilinear mitochondrial inheritance, and in some cases mitochondrial recombination will occur during biparental inheritance (Borst and Grivell 1978). The range and type of variation associated with molecular markers can provide many different tools that can be used for determining the epidemiology of plant pathogenic fungi. At one level, recombination insensitive markers may be available for the detection of a particular taxon in the environment, such as species and pathogen speciﬁc probes and primers. At a lower level, recombination sensitive markers may be used to follow individuals or lines, or to determine if a disease is spread by spores or through vegetative growth.

ANALYSING DATA

Different molecular markers will generate results in different forms. Simple RFLPs and some fingerprinting methods will produce generally simple band patterns, usually of the order of between one and 20 bands. These patterns can be translated into a simple binary form where each band obtained in the analysis is considered as an independent character, and is scored as present or absent. In most cases, these binary records have then been compared by the use of one or more distance or association calculation, and represented as some form of dendrogram. There is a wide range of coefficients available for such comparisons. These include coefficients that do not consider matching negative characters, and others that provide for a double weighting of common bands to reflect the presence of common restriction sites or primer sequences at each end of the bands [e.g., Nei and Li (1979)].

It should be remembered that some of these coefficients have been described independently on more than one occasion, and others can be related to each other by simple transformations. For example Nei and Li’s genetic distance is equivalent to 1-Sorensen’s coefficient, and Sorensen’s coefficient is mathematically identical to Dice’s coefficient [see Bridge and Saddler (1998); Sneath and Sokal (1973)]. Similarly, association coefficients can be related to distance measures, and taxonomic distance can be defined as the square root of 1 minus the simple matching coefficient. It is therefore important if more than one measure is used to ensure that those selected are independent. Although cluster analysis methods are a common way of showing relationships within and between fungal populations, this methodology does however have some limitations. One obvious limitation with any tree diagram is that all the isolates must be linked, as there is no provision for an isolate that is not related. A second limitation is that cluster analysis is a good technique for showing the membership of a group, it is less precise in showing relationships between groups. This is a particular failing of average linkage based systems, but is also true of most other clustering approaches [see Abbott et al. (1985)]. A further limitation to cluster analysis is the tendency of isolates that are unrelated to the main population to form a separate cluster together, even though they may be only distantly related to each other. Such clusters are sometimes described as sharing only the single property that they are not related to the main population. One way in which some of these limitations can be overcome is by using an ordination-based method such as principle component analysis (PCA). In these methods correlated variance between characters is combined to produce a further set of axes that are essentially made up of additive components of correlated individual characters. Each axis represents a proportion of the total variance in the data, and the placement of isolates is by plotting their positions in relation to the first 2 or 3 axes [see Alderson 1985; Dudzinski 1975). A refinement of PCA is principle coordinate analysis (PCO). PCO has been shown to be appropriate for binary data, such as obtained from band patterns, and unlike PCA, does not require the use of strictly metric coefficients (Gower 1966; Sneath and Sokal 1973). Unlike a cluster analysis, this does not produce a series of groups, but a scatter-plot where similar isolates may be placed near each other. These ordination methods can behave differently from cluster analysis, and typically are better at representing between group relationships than close within group relationships. One further aspect of PCA is that under some circumstances it may filter random variation from a complex data set, as any correlated variation will tend to be included in the first few axes [see Bridge (1998)]. Band analysis methods are essentially the same for simple and complex patterns. It however becomes necessary to consider band reading software for the very complex patterns that may be produced by techniques such as AFLP, as the large number of bands produced cannot be easily scored by

Bridge et al.

eye. There are a range of band reading and matching software packages available, and most of these convert band patterns in a gel into densitometric traces where peak presence, height, and shape correspond to band presence, intensity, and thickness, respectively. These packages commonly have manual and automated routines for correcting gels for shift and stretch events, and routines for including standard size markers and other reference bands. Trace data can be readily converted to quantitative values as “x,y co-ordinates” and this is suitable for largely distance based analyses. Quantitative data can also be compared for overall pattern similarity through methods such as correlation coefficients, and concentration independent calculations such as cosine theta [see Feltham and Sneath (1979)]. Analysis of sequence data is more complicated as the likelihood of certain events may also be included in the analysis. The first stage in comparing DNA sequences is to align them to each other. Alignment routines will always seek the best alignment of the sequences being studied, and so the addition or deletion of sequences to a data set will require a new alignment to be made. In determining an alignment, and calculating a measure of difference between the sequences involved it is also necessary to consider the effects of transversions, transitions, and gaps. The bases in DNA strands pair through purine/pyrimidine bonds, and so when aligning sequences, a change from purine to purine or pyrimidine to pyrimidine (transition) may be considered of less importance than a change from purine to pyrimidine (transversion). The importance given to transitions and transversions can therefore be varied to reflect their relative importance, and this may also depend on the particular sequences being considered. When aligning sequences it may be necessary to insert a gap in some sequences to align where insertion/ deletion events have occurred. Again, the relative importance of inserting a gap, and also of extending that gap can be adjusted according to the perceived significance of the event in the sequences under consideration [see Thompson et al. (1994)]. It is common with sequence analysis to use phylogenetic techniques to produce trees [see Swofford and Olsen (1990)]. While these approaches are suitable when considering different species and genera, they are less appropriate for comparisons of closely related populations. Analysis of DNA sequence data is an area that is currently receiving further attention, and some of these developments are described in more detail in chapter 33.

FOLLOWING PLANT DISEASE: A CASE STUDY

Molecular markers have been used in a wide range of studies with plant pathogenic fungi (see earlier). Although these can be reviewed, the volume of the literature available is considerable, and so a single case study is presented here that illustrates how the molecular epidemiology of a plant pathogen can be related directly to agricultural practice.

One series of studies that has shown the range and limitations of molecular markers in following plant disease epidemiology is the investigation of basal stem rot (BSR) of oil palm by G. boninense. BSR was first recognized in West Africa in 1915, and as oil palm was distributed through out the world, it was recorded in many other countries. The first report in SE Asia was in 1931, and since that time BSR incidence has increased to the point where the lack of techniques for management of the disease is considered a major constraint to oil palm production in SE Asia [see Ariffin et al. (2000)]. Ganoderma species attack a variety of tropical perennial crops including rubber, tea, and pineapple. In these instances the Ganoderma appears to be largely transmitted through the soil, possibly in plant debris, and spreading infection patches may be evident in fields. The species G. boninense occurs as a saprophyte on dead palms, particularly coconuts, but appears to be pathogenic only to oil palm. For some years transmission of G. boninense in oil palm was believed to be through the soil, as for other species, and disease control was attempted through practices that included digging large pits around infected palms (Turner 1981). The first attempt to use molecular methods to investigate the epidemiology of BSR in oil palm was made in the 1990s, when initial studies were made with iso-enzyme profiles (Miller et al. 1995). Although some enzyme systems initially appeared useful for differentiating species, in G. boninense it was found that in general iso-enzyme profiles were either consistent, or showed considerable variation. Ganoderma is a basidiomycete that forms polyporpoid brackets on the outer surface of infected palms. The brackets are dikaryotic, and basidiospores are produced by meiosis. The mycelial form found in infected tissue is also generally dikaryotic, and some of the isoenzyme variability may therefore be due to recombination events from the original fusion of monokaryotic basidiospores. However, pectinase isoenzyme analysis identified a characteristic enzyme profile that was consistent for nearly all Ganoderma isolates obtained from palm hosts (Miller et al. 1995; 2000). Given the known involvement of pectin and pectin degradation in plant pathology, this finding may indicate a common mode of action for all of the palm associated Ganoderma species. The first DNA based method to be investigated in these studies was analysis of RFLPs derived from presumptive mtDNA (AT rich DNA). This relatively simple technique gave rather unusual results, in that different RFLPs were obtained from different cultures, suggesting considerable heterogeneity in the mitochondrial genome (Miller et al. 1999). The RFLP profiles proved to be consistent among single spore isolates from a single basidiome, and so were considered to provide “parental line” fingerprints, characteristic of the dikaryon. These RFLPs could therefore be used to define sibling families. This assumption was supported by monokaryon and dikaryon intercompatibility studies (Pilotti et al. 2000). Subsequent investigation of molecular fingerprinting methods including RAPDs and AFLP supported the bulk of the iso-enzyme studies and gave different profiles for isolates

Epidemiology of Plant Pathogens

derived from single spores from the same basidiome (Bridge et al. 2000; Pilotti et al. 2000). When these molecular methods were applied to isolates obtained from single plantations and planting blocks it was found that nearly all of the isolates differed from each other, including isolates obtained from adjacent palms. This finding was again supported by intercompatibility studies (Miller et al. 1999; 2000; Pilotti et al. 2000). These results could not have come about as a result of simple mycelial spread in the soil, as vegetative spread could be expected to result in at least some palms being infected by the same isolate. It was therefore concluded that infection could be due to one of two mechanisms, either singly or in combination. The first was that there might have been mycelial spread from multiple inoculum sources, with virtually no cross infection. This would account for the molecular variability recorded, but would also require each infection to be the result of different infected debris. The second possibility was that infections were due to new dikaryons formed from fusion of monokaryons from individual spores [see Sanderson and Pilotti (1997); Sanderson et al. (2000)]. Although the molecular markers studied showed sufficient variability to identify individual isolates for local epidemiology, they did not show sufficient conservation to allow the wider detection of the pathogen in the environment. BSR was considered to be due to the single species G. boninense, and although there is considerable uncertainty regarding species concepts in Ganoderma, some information is available on sequences within the rRNA gene cluster. Initial studies have shown that the ITS regions are relatively similar across the genus, with most variation being found in the 30 terminal region of the ITS2 sequence (Moncalvo et al. 1995a,b). The ITS sequences obtained from multiple isolates of G. boninense showed very little sequence variation, and a short sequence of 16 bases in the ITS2 region was found to be unique to the species. A PCR primer has been derived from this region, and the combination of this and the universal rRNA primer ITS3 allowed the specific amplification of G. boninense sequences from cultures, specimens, and infected palm material (Bridge et al. 2001). Current surveys being undertaken by the Oil Palm Research Association in Papua New Guinea with the PCR based diagnostic are detecting G. boninense in the internal tissues of recently cut frond bases of young oil palms (Bridge et al. 2000; 2001). This finding suggests that such cut surfaces may provide an entry route for spores. This mode of infection has been reported many times before for fungal pathogens of woody trees, and the initial results from the oil palm research suggest that the developmental state of the oil palm and other factors may also be important in the establishment of infection.

CONCLUSIONS

The tools of molecular evolutionary biology and genomics are making it possible to use genetic variation in pathogens and hosts to prevent and treat plant pathogenic fungi. There is a

wide range of molecular features that can be used as markers at the population level for studying plant pathogenic fungi. The different markers will often reflect different levels of variation within and between plant pathogenic taxa, and may also reflect changes resulting from meiosis and recombination. It is therefore possible to study the spread and dynamics of fungal populations on crop plants, and to determine the role of populations of the same fungus occurring on secondary hosts, in the soil or on debris. Molecular markers can also be used to determine the genetic integrity of host or variety specific groups, and can provide information on differences between pathogenic races. This information is fundamental to understanding the spread of fungal plant diseases, and is important in developing disease control strategies. The choice of marker will depend on the particular fungus under study, and the correct choice of markers may also provide information as to the role of spores or particular mating types in epidemiology. Variation in fungal pathogen genotype is the basis for developing methods to identify these pathogens using PCR. Recently, strains/species specific molecular markers/primers have been developed for several plant pathogenic fungi. There is unfortunately no single marker system that can be guaranteed to provide the desired level of discrimination for all fungi, and some initial screening of different methodologies may be required before a full study can be initiated.

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6 Molecular Biology for Control of Mycotoxigenic Fungi Robert L. Brown / Deepak Bhatnagar / Thomas E. Cleveland U.S. Department of Agriculture – Agricultural Research Service, New Orleans, Lousiana, USA

Zhi-Yuan Chen Louisiana State University, Baton Rouge, Lousiana, USA

for Industry: Fumonisin Levels in Human Foods and Animal Feeds” in the November 9, 2001, Federal Registry. More than 50 countries have established or proposed regulations for controlling aflatoxins in foods and feeds, and at least 15 countries have regulations for levels of other mycotoxins (Haumann 1995). The FDA has set limits of 20 ppb, total aflatoxins, for interstate commerce of food and feed and 0.05 ppb of aflatoxin M1 for sale of milk. Because of both food and feed safety concerns and the establishment of regulatory limits on DON and aflatoxins, it is estimated that over $1.5 billion in crop losses occur annually due to contamination of corn, cottonseed, peanut, and treenuts with aflatoxins and of wheat and barley with DON (Robens 2001). An association between mycotoxin contamination and inadequate post harvest storage conditions has long been recognized. However, studies have revealed that seeds are contaminated with mycotoxins primarily at the preharvest stage [reviewed in Lisker and Lillehoj (1991)]. Therefore, many current research strategies focus on preharvest control of mycotoxins [reviewed in Brown et al. (1998)]. Maintaining good cultural and management practices that promote the general health of crops can reduce but not eliminate preharvest mycotoxin contamination. For example, insect resistant germplasm, such as corn transformed with the gene encoding Bacillus thuringiensis crystal protein (Bt maize), reduced levels of fumonisins (Dowd 2000). Irrigation of peanut essentially prevents aflatoxin contamination of this crop, probably by preventing drought stress, known to induce contamination in peanut (Cole et al. 1985). However, optimization of management practices to control mycotoxins is not always possible due to production costs, geographic location, or the nature of the production system for the

INTRODUCTION

Mycotoxins are fungal metabolites that can contaminate foods and feeds, and exhibit toxic effects in higher organisms (Sharma and Salunkhe 1991) that consume the contaminated commodities. The regulatory guidelines and advisory limits issued by the United States Food and Drug Adminstration (FDA) on some contaminated commodities can facilitate severe economic losses to the growers. Therefore, mycotoxin contamination of foods and feeds is a serious food safety problem affecting the competitiveness of U.S. agriculture, both domestically and worldwide. Mycotoxins that significantly impact agriculture include aflatoxins produced by Aspergillus flavus and A. parasiticus, trichothecenes (in particular deoxynivalenol or DON) produced by Fusarium spp., ochratoxins produced by A. ochraceus and Penicillium viridicatum, and fumonisins produced by F. verticillioides (synonym, moniliforme, as used in some literature cited in the present article) (Brown et al. 1998). Cyclopiazonic acid produced by A. flavus, can also be included on this list of significant mycotoxins. Aflatoxins, potent liver toxins, and carcinogens comprise the most widely studied mycotoxins (CAST 1979; Diener et al. 1987; Payne 1998), because of established results in their ability to induce animal diseases, particularly liver cancer in humans [reviewed in Eaton and Groopman (1994)]. However, other mycotoxins such as DON, are of particular concern for the brewing industry which has cutoff levels as low as 0.5 ppm for DON in barley used in malting (Robens 2001). In addition, recognizing the potential for fumonisins to cause animal or human health problems (Marasas 1996), the FDA has now announced the availability of a final guidance document entitled “Guidance 69

Brown et al.

particular crop vulnerable to mycotoxins. In addition, even the best management practices are sometimes negated by biotic and abiotic factors that are hard to control and by extremes in environmental conditions. The complex epidemiology of A. ﬂavus on corn (Wicklow 1991) can drastically affect the outcome of measures to control aﬂatoxin contamination on this crop. Therefore, there is an urgent need for development and utilization of strategies involving state-of-the-art technologies to control preharvest mycotoxin contamination. The current article highlights recently published and high-impact research involving molecularbased technologies that has been accomplished and that enhances a host plant resistance strategy for controlling mycotoxin contamination.

MYCOTOXIN PREVENTION THROUGH ENHANCEMENT OF HOST RESISTANCE IN CROPS

Preharvest host resistance is a widely explored strategy for combatting fungal attack. By far, most studies aimed at the incorporation of antifungal resistance against mycotoxigenic fungi have been applied toward improvement of resistance against preharvest aflatoxin contamination in corn [reviewed in Brown et al. (1998)]. With corn, the strategy of enhancing host resistance to aflatoxin contamination through breeding has gained prominence because of: (a) the successful identification of germplasm resistant to aflatoxin contamination [reviewed in Brown et al. (1999)] and (b) the significant advances in the identification of natural resistance mechanisms and traits (Brown et al. 1998; 1999; Chen et al. 2001). However, these investigations indicated that resistance to aflatoxin contamination involves multiple chromosome regions and several genes (Davis and Williams 1999). Therefore, attempts to select for resistance traits in the development of commercial corn varieties, while maintaining desirable agronomic characteristics, have been slowed due to a failure to identify expressed genes and proteins involved in resistance. This is especially needed since resistance, thus far identified is in poor genetic backgrounds. Therefore, research is needed to elucidate the biochemical mechanisms that confer resistance in corn kernels and other crops that are vulnerable to aflatoxin contamination. These resistance mechanisms could then be used to enhance germplasm through marker-assisted breeding and/or genetic engineering, two methods for employing the identified traits towards the development of resistant commercially-acceptable crops (Brown et al. 1999). Gaining an understanding of the natural resistance mechanisms in corn could serve as “nature’s lesson” about the specific requirements for seed-based resistance against fungal attack. This information will help efforts to incorporate and enhance resistance in other crops vulnerable to aflatoxin contamination such as cottonseed, peanut, and tree nuts, and will perhaps even help efforts to

enhance resistance against other groups of mycotoxigenic fungi.

2.1

Development of Aﬂatoxin-Resistance Screening Tools

Several screening tools have been developed and used to facilitate corn breeding for developing germplasm resistant to fungal growth and/or aflatoxin contamination (King and Scott 1982). Inoculation methods employed with corn include the pinbar inoculation technique (for inoculating kernels through husks with A. flavus conidia), the silk inoculation technique, and infesting corn ears with insect larvae infected with A. flavus conidia. (King and Scott 1982; Tucker et al. 1986). Two resistant inbreds (Mp420 and Mp313E; Scott and Zummo 1988; Windham and Williams 1998) were discovered and tested in field trials at different locations, using the pinbar technique, and released as sources of resistant germplasm. A rapid laboratory kernel screening assay (KSA) was developed and used to study resistance to aflatoxin production in mature kernels (Brown et al. 1993; 1995). The results of this study indicated the presence of two levels of resistance: at the pericarp and at the subpericarp level. The subpericarp level of resistance was shown to require a viable embryo (Brown et al. 1993). KSA studies further demonstrated a role for pericarp waxes in kernel resistance (Guo et al. 1995; 1996) and highlighted quantitative and qualitative differences in pericarp wax between resistant and susceptible genotypes (Gembeh et al. 2001; Russin et al. 1997). This research was all based on the prior identification, during field studies, of a resistant corn breeding population, GT-MAS:gk (Widstrom et al. 1987). The KSA also confirmed sources of resistance among 31 inbreds tested in Illinois field trials (Brown et al. 1995; Campbell and White 1995), thus demonstrating that the KSA can be used, at least initially, to rank corn for its field resistance to aflatoxin contamination. Subsequently, the KSA was used as a preliminary screen for resistance to aflatoxin contamination in kernels of maize inbreds selected for ear rot resistance in West and Central Africa (Brown et al. 2001a). The KSA has advantages over traditional field screening techniques (Brown et al. 1995), mainly because of the rapidity of the assay. However, field trials are irreplaceable for confirmation of resistance. Recently, the KSA was improved by including a method to quantify fungal biomass using the b-glucuronidase (GUS) or green fluorescent protein (GFP) (Du et al. 1999; Windham and Williams 1998; Windham et al. 1999) reporter genecontaining A. flavus tester strains. A. flavus tester strains were genetically engineered with a gene construct consisting of the GUS reporter gene linked to an A. flavus b-tubulin gene promoter for monitoring fungal growth (Brown et al. 1995; 1997) or with the reporter gene linked to an aflatoxin biosynthetic pathway gene which could also provide a quick and economical way to indirectly measure aflatoxin levels

Molecular Biology for Control of Mycotoxigenic Fungi

(Brown-Jenco et al. 1998; Payne 1997). Thus, it is now possible to accurately assess fungal infection levels and to predict the corresponding aﬂatoxin levels in the same kernels, as a result of fungal infection. This technology might be applied to screening for resistance to mycotoxin contamination by other mycotoxigenic fungi. Recently, an F. verticillioides strain, containing a GUS reporter gene was used in the KSA to demonstrate that this fungus is inhibited in aﬂatoxin-resistant genotypes (Brown et al. 2001b). This indicates that some resistance mechanisms may be generic for ear rotting/mycotoxigenic fungi.

2.2

Identification of Resistance-Associated Proteins (RAPs) and Natural Compounds in Corn That Inhibit Aspergillus flavus Growth and Aflatoxin Contamination

Developing resistance to fungal infection in wounded as well as intact kernels would go a long way toward solving the aflatoxin problem (Payne 1992). Studies demonstrating subpericarp (wounded-kernel) resistance in corn kernels have led to research for identification of subpericarp resistance mechanisms. Examinations of kernel proteins of several genotypes revealed differences between genotypes resistant and susceptible to aflatoxin contamination (Guo et al. 1998). Imbibed susceptible kernels, for example, showed decreased aflatoxin levels and contained germination-induced ribosome inactivating protein (RIP) and zeamatin (Guo et al. 1997). Both zeamatin and RIP have been shown to inhibit A. flavus growth in vitro (Guo et al. 1997). In another study, two kernel proteins were identified from a resistant corn inbred (Tex6) which may contribute to resistance to aflatoxin contamination (Huang et al. 1997). One protein, 28 kDa in size, inhibited A. flavus growth, while a second, over 100 kDa in size, primarily inhibited toxin formation. When a commercial corn hybrid was inoculated with aflatoxin and nonaflatoxin-producing strains of A. flavus at milk stage, one induced chitinase and one b-1,3-glucanase isoform was detected in maturing infected kernels, while another isoform was detected in maturing uninfected kernels (Ji et al. 2000). In another investigation, an examination of kernel protein profiles of 13 corn genotypes revealed that a 14 kDa trypsin inhibitor protein (TI) is present at relatively high concentrations in seven resistant corn lines, but at low concentrations or is absent in six susceptible lines (Chen et al. 1998). The mode of action of TI against fungal growth may be partially due to its inhibition of fungal-amylase, limiting A. flavus access to simple sugars (Chen et al. 1999b) required not only for fungal growth, but also for toxin production (Woloshuk et al. 1997). The TI also demonstrated antifungal activity against other mycotoxigenic species (Chen et al. 1999a). The identification of these proteins may provide markers for plant breeders, and may facilitate the cloning and introduction of

Figure 1 Strategy for enhancing host plant resistance to aflatoxin contamination. The research approach being used to identify and employ resistance factors, such as resistanceassociated proteins (RAPs) that are identified in aflatoxinresistant corn lines. After resistant germplasm is identified, various tools are used to characterize the expression of resistance, such as KSA-based studies, GUS/GFP reporter constructs, and seed physiology studies. These can lead to RAP identification protocols such as proteome analysis. Genes corresponding to RAPs can be cloned, and clones then used for QTL studies, plant transformation, or marker-assisted breeding.

antifungal genes through genetic engineering into other aflatoxin-susceptible crops (Figure 1). A recent investigation into corn kernel resistance (Chen et al. 2001) determined that both constitutive and induced proteins are required for resistance to aflatoxin production. It also showed that one major difference between resistant and susceptible genotypes is that resistant lines constitutively express higher levels of antifungal proteins compared to susceptible lines. The real function of these high levels of constitutive antifungal proteins may be to delay fungal invasion, and consequent aflatoxin formation, until other antifungal proteins can be synthesized to form an active defense system.

2.2.1

Brown et al.

Identiﬁcation of RAPs Through Proteome Analysis

To increase protein resolution and detection sensitivity by 10–20 fold and, thus, enhance ability to identify more RAPs, a proteomics approach was recently employed. The increased reproducibility, reliability, and accuracy of 2-D gel electrophoresis is due to advances in technology, such as immobilized pH gradient (IPG) gel strips and sophisticated computerized 2-D gel analysis software (Appel et al. 1997; Go¨rg et al. 1998). Endosperm and embryo proteins from several resistant and susceptible genotypes have been compared using large format 2-D gel electrophoresis, and over a dozen such protein spots, either unique or 5-fold upregulated in resistant lines, have been identified, isolated from preparative 2-D gels and analyzed using ESI-MS/MS after in-gel digestion with trypsin (Chen et al. 1999a; 2000; 2002). These proteins can be grouped into three categories based on their peptide sequence homology: (a) storage proteins, such as GLB1, GLB2, and late embryogenesis abundant proteins (LEA3, LEA14); (b) stress-responsive proteins, such as aldose reductase (ALD), a glyoxalase I protein (GLX1), and a 16.9 kDa heat shock protein, and (c) antifungal proteins, which include TI. Thus far, no investigation has been conducted to determine the possible direct involvement of stress-related proteins in host fungal resistance. Heretofore, most RAPs identified have had antifungal activities. However, increased temperatures and drought, which often occur together, are major factors associated with aflatoxin contamination of maize kernels (Payne 1998). Other studies have found that drought stress imposed during grain filling reduces dry matter accumulation in kernels. This often leads to cracks in the seed and provides an easy entry site to fungi and insects. Possession of unique or of higher levels of hydrophilic storage or stress-related proteins, such as the aforementioned, may put resistant lines in an advantageous position over susceptible genotypes in the ability to synthesize proteins and defend against pathogens under stress conditions. Therefore, the necessary requirements for developing commercially-useful, aflatoxin-resistant maize lines may include, aside from antifungal proteins, a high level of expression of stress-related proteins. Further studies including physiological and biochemical characterization, genetic mapping, plant transformation using RAP genes, and marker-assisted breeding should clarify the roles of stress-related RAPs in kernel resistance.

2.2.2

Natural Compounds That Affect Mycotoxin Biosynthesis

Several compounds have been identified in corn which may have regulatory effects on the aflatoxin and trichothecene biosynthetic process. The compound 4-acetyl-benzoxazolin2-one (ABOA), which was isolated from maize lines tolerant to F. graminearum, strongly inhibited acetyl-deoxynivalinol production at 5 mM, aflatoxin production at 2 mg/ml, and feeding by maize weevils at 1000 ppm (Miller et al. 1996).

Steryl esters from maize significantly increased aflatoxin production by some A. flavus strains at 0.3 and 1.0 mg/ml (Norton and Dowd 1996). Anthocyanins and related flavonoids, some of which occur naturally in maize kernels, inhibited aflatoxin production by more than 50% at 0.76 mM (Norton 1999). More highly glycosylated forms of the anthocyanins tended to be less effective in inhibiting aflatoxin production (Norton 1999). Carotenoids containing an alphaionone type ring tended to be more effective inhibitors of aflatoxin production by A. flavus, with some having an I50 of about 6 mM (Norton 1997). Although most strains of A. flavus exposed to beta-carotene at 50 mg/ml had aflatoxin production inhibited by 90% or more, some peanut derived strains were less sensitive (Wicklow et al. 1998). In vitro studies indicated plant peroxidase could greatly enhance the ability of plant chemicals to inhibit spore germination and hyphal growth of F. graminearum (Dowd et al. 1997). A. flavus was considerably more resistant to quinone products potentially produced by plant peroxidases compared to F. graminearum and F. verticillioides (moniliforme) (Dowd et al. 1997). In addition, volatile compounds from corn and cotton, which are products of the lipoxygenase pathway, were shown to have effects upon aflatoxin biosynthesis and fungal development in vitro [reviewed in Bhatnagar et al. (2001)].

2.3

Plant Breeding Strategies for Enhancing Host Resistance to Mycotoxigenic Fungi

Several resistant inbreds among the 31 tested in Illinois field trials (Campbell and White 1995) and highlighted through the KSA (Brown et al. 1995), have been incorporated into an aflatoxin-resistance breeding program whose major objective is to improve elite Midwestern corn lines such as B73 and Mo17. In this program, the inheritance of resistance of inbreds in crosses with B73 and/or Mo17 was determined (Hamblin and White 2000; Walker and White 2001; White et al. 1995b; 1998), and in the case of several highly resistant inbreds, genetic dominance was indicated. Overall, results indicated that selection for resistance to Aspergillus ear rot and aflatoxin production should be effective, and that development of resistant inbreds for use in breeding commercial hybrids should be successful (White et al. 1995a). Chromosome regions associated with resistance to A. flavus and inhibition of aflatoxin production in corn have been identified through Restriction Fragment Length Polymorphism (RFLP) analysis in three “resistant” lines (R001, LB31, and Tex6) in the Illinois breeding program, after mapping populations were developed using B73 and/or Mo17 elite inbreds as the “susceptible” parents (White et al. 1995b; 1998). In some cases, chromosomal regions were associated with resistance to Aspergillus ear rot and not aflatoxin inhibition, and vice versa, whereas other chromosomal regions were found to be associated with both traits. This suggests that these two traits may be at least partially under separate genetic control. Also, it was observed that

Molecular Biology for Control of Mycotoxigenic Fungi

variation can exist in the chromosomal regions associated with Aspergillus ear rot and aflatoxin inhibition in different mapping populations, suggesting the presence of different genes for resistance in the different identified resistance germplasm. The RFLP technology may provide the basis for employing the strategy of pyramiding different types of resistances into commercially viable germplasm, while avoiding the introduction of undesirable traits. Another Quantitative Trait Loci (QTL) mapping program was undertaken using a mapping population created from a resistant inbred Mp313E and a susceptible one, Va35 (Davis and Williams 1999), and regions on chromosomes, associated with resistance to aflatoxin contamination, were revealed. Other work using this technology is attempting to pyramid insect and fungal resistance genes into commercial germplasm (Guo et al. 2000; Widstrom et al. 2003). Breeding strategies to enhance resistance to A. flavus infection are being carried out in other crops vulnerable to aflatoxin contamination such as peanut and tree nuts. Promising sources of resistant peanut germplasm have been identified from a core collection representing the entire peanut germplasm collection (Holbrook et al. 1995), although resistance screening has proven to be a difficult task with this crop (Holbrook et al. 1997). Promising peanut germplasm has less than acceptable agronomic characteristics, and is thus being hybridized with lines with commercially acceptable features. Resistant lines also are being crossed to pool resistances to aflatoxin production. Thus, some success has been achieved in identifying resistant peanut germplasm, and field studies are being conducted by various researchers to verify this trait. Among tree nuts, strategies for controlling preharvest aflatoxin formation by breeding for host resistance have been studied mainly in almonds (Gradziel et al. 1995). The approach has been to integrate multiple genetic mechanisms for control of not only Aspergillus spp. but also insects. Resistance to fungal colonization has been shown to be present in the undamaged seed coat of several advanced breeding selections and is further being pursued through breeding/genetic engineering of resistance to A. flavus growth in kernel tissues. Genotypes are also under development that produce low amounts of aflatoxin following fungal infections (Gradziel and Dandekar 1999). Naphthoquinones in walnut hulls delayed germination of A. flavus conidia and were capable of inhibiting growth of the fungus at higher concentrations (Mahoney et al. 2000; Molyneux et al. 2000). These compounds also appeared to have a regulatory effect on aflatoxin biosynthesis and may be involved in resistance to aflatoxin contamination of walnut. Results seen here could lead to breeding applications to enhance resistance in walnut to aflatoxin contamination using naphthoquinone derivatives as selectable markers. Investigations have also been conducted with figs and pistachios to identify the mode of infection of the crops by A. flavus and develop strategies to identify germplasm with agronomically desirable characteristics and resistance to fungal infection (Doster et al. 1995). However, until more is

known about the nature of selectable resistance markers associated with reduced aflatoxin contamination in crops other than corn, breeding for insect resistance, or better management of insects which vector aflatoxigenic fungi may be a more viable immediate approach to manage aflatoxin contamination. Recent studies indicate that naturally occurring resistance may reduce invasion of crops by other economically important mycotoxigenic fungi. For example, resistance to head blight in wheat varieties was correlated with a reduction in contamination with DON (Bai et al. 2001). Further investigations utilizing differentially resistant wheat germplasm may lead to the identification of selectable resistance markers useful in breeding for reduced DON contamination in wheat.

2.4

Genetic Engineering Strategies to Enhance Host Resistance to Mycotoxin Contamination

Plant breeding for resistance is practical when a large germplasm pool exists with differential resistance in the crop, such as exists in corn. However, genetic engineering for resistance may be essential for crops such as cotton which seems to have little resistance to aflatoxin contamination of its seed (Cotty 1989). Extensive research has focused upon identifying genes encoding antifungal proteins effective against mycotoxigenic fungi. Bacterial chloroperoxidase (CPO) (Wolffram et al. 1988) and its gene have been evaluated both in vitro in laboratory assays (Jacks et al. 1999) and in vivo in enhancing fungal disease resistance in transgenic tobacco plants (Rajasekaran et al. 2000b). In in vitro bioassays using A. flavus as the test organism, CPO greatly reduced the viability of A. flavus conidia (Jacks et al. 1999) and transgenic tobacco expressing the CPO gene demonstrated significant resistance to attack by Colletotrichum destructivum (Rajasekaran et al. 2000b). In another study, a small lytic peptide, D4E1, demonstrated broad spectrum antimicrobial activity and convincing inhibitory activity against A. flavus in vitro (Rajasekaran et al. 2001), thus indicating the possibility of transforming plants with the gene encoding D4E1 to reduce infection of seed with toxigenic fungi. In further substantiation of this strategy, the D4E1 gene when transformed into tobacco was shown to greatly enhance resistance to C. destructivum (Cary et al. 2000). Cotton is being transformed with CPO and D4E genes with the hope that aflatoxin contamination of cottonseed can be reduced (Chlan et al. 1999; Rajasekaran et al. 1999; 2000a). Mechanisms of mycotoxin biosynthesis and regulation have been investigated extensively (Bhatnagar et al. 2002; Cleveland and Bhatnagar 1992; Desjardins and Proctor 2001). The goal is to identify weak links that can be exploited to control mycotoxin contamination through genetic engineering of plants. The finding that trichothecenes contribute to the virulence of F. graminearum on wheat and maize has

identified such a weak link. If production of trichothecenes increases pathogen virulence, then increased plant resistance to the toxin should increase plant resistance to the pathogen. Three genes that increase plant resistance to trichothecenes have recently been identified, and whether such genes also can increase plant resistance to F. graminearum is under investigation. Two trichothecene resistance genes are fungal genes that encode proteins that reduce the toxicity of trichothecenes. TRI101 from F. sporotrichioides encodes trichothecene 3-O-acetyltransferase which converts trichothecenes to less toxic derivatives (Kimura et al. 1998; McCormick et al. 1999). PDR5 from yeast encodes a multidrug resistance transporter protein that transports trichothecenes extracellularly and is similar to TRI12, a trichothecene biosynthetic gene (Alexander et al. 1999; Balzi et al. 1994). Transgenic expression of either TRI101 or PDR5 increased resistance of tobacco to trichothecenes (Muhitch et al. 2000). Wheat and barley lines expressing TRI101 and PDR5 are being tested for resistance to F. graminearum (Okubara et al. 2000). Trichothecenes are potent inhibitors of protein synthesis and are believed to bind to the 60S ribosomal protein L3 (RPL3). A rice gene encoding RPL3 was modified to change amino acid 258 from tryptophan to cysteine, a change that confers trichothecene resistance to yeast. Transgenic expression of the modified Rpl3, increased resistance of tobacco to trichothecenes (Harris and Gleddie 2001). Maize, wheat, and barley lines expressing the modified Rpl3 gene are being tested for resistance to F. graminearum (Harris and Gleddie 2001). No analogous weak link in the aflatoxin biosynthetic pathway has been discovered that can be exploited in a similar host resistance strategy, nor has a clear role for aflatoxin in fungal virulence been demonstrated. However, the aflatoxin biosynthetic pathway and the gene cluster comprising genes that govern this pathway, including a key regulatory gene (aflR), have been characterized (Bhatnagar et al. 2002). Also, the regulation of these genes during invasion of the host plant is being investigated using a genomics approach. This approach is based upon the fact that certain plant-derived natural products apparently have regulatory effects on aflatoxin biosynthesis [as recently reviewed in Bhatnagar et al. (2001) and reported in recent publications cited in this article: Miller et al. 1996; Norton 1999; Norton and Dowd 1996; Wicklow et al. 1998]. Genetic engineering may provide innovative solutions to prevent the accumulation of fumonisins in Fusarium-infected maize. One approach currently under development is detoxification of fumonisins by enzymes introduced into maize via genetic engineering. Enzymes that detoxify and degrade fumonisins have been identified from Exophiala spinifera, a black yeast found on moldy maize kernels. The initial steps in fumonisin detoxification are ester hydrolysis followed by oxidative deamination to produce derivatives that lack the free amino function that is believed to be important for toxicity (Blackwell et al. 1999). Genes encoding the deesterification and deamination enzymes have been cloned

Brown et al.

and are being expressed in transgenic maize to evaluate their effect on fumonisin accumulation and ear rot symptoms (Duvick 2001). The gene encoding the antifungal protein, TI, previously shown to be correlated with corn kernel resistance, was transformed into and expressed in both tobacco and cotton. Fungal growth inhibition assays of transgenic tobacco expressing TI protein showed efficacy against A. flavus, but not at the levels observed with extracts from tobacco transformed with genes encoding CPO or D4E1 (Cary et al. 2000; Rajasekaran et al. 2000b). The gene encoding TI also has been transformed into cotton, but no inhibitory activity has yet been noted in extracts from transgenic plants. It is well documented that insect injury can provide a port of entry by mycotoxigenic fungi and that crops containing the B. thuringiensis (Bt) gene encoding an insecticidal protein, have shown reduced levels of mycotoxin contamination (Dowd 2000). Currently, a binary vector is being used in this laboratory to express both the antifungal D4E1 gene and a synthetic anti-insecticidal gene, cryIA(c), of B. thuringiensis in tobacco and cotton. Successful expression of these genes under independent promoters should provide both fungal and insect resistance in cotton, thus potentially reducing the amount of fungal entry through insect injury sites as well as retarding the growth of the aflatoxin producing fungus in cotton bolls and seed.

CONCLUSIONS

Since it is unlikely that preharvest mycotoxin contamination of crops will be reduced significantly through careful cultural practices, control of these problems will likely be dependent upon the development and introduction into the commercial market, of germplasm, resistant to the growth of mycotoxigenic species, and/or biosynthesis of toxins by these species. The identification of resistance traits in corn and other crops can, through marker-assisted breeding, facilitate a more rapid development of resistant, commercially-acceptable germplasm. Genetic engineering provides a tool especially useful for introducing resistance genes into crops with little natural genetic diversity (e.g., cotton), and for testing the efficacy of putative resistance genes. Studies identifying compounds that affect mycotoxin biosynthesis offer hope to researchers. Limiting fungal growth in crops is an important aspect of host resistance, however, obtaining zero growth of fungi capable of exploiting a variety of different substrates, such as the facultative pathogen A. flavus, may be difficult to achieve. Therefore, the identification of a natural compound that blocks mycotoxin biosynthesis might be the closest we come to discovering a “magic bullet.” Nevertheless, the investigations discussed in this chapter, using molecular-based technologies to identify and characterize various resistance mechanisms in crops susceptible to mycotoxin contamination, and against different mycotoxigenic fungi, are building a foundation which can lead to the implementation of a successful gene pyramiding

Molecular Biology for Control of Mycotoxigenic Fungi

strategy to produce mycotoxin-resistant, commerciallyattractive crops.

ACKNOWLEDGEMENT We sincerely thank the editors for inviting us to contribute to this edition of Handbook of Fungal Biotechnology.

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75 Brown RL, Chen Z-Y, Menkir A, Cleveland TE, Cardwell K, Kling J, and White DG (2001a). Resistance to aflatoxin accumulation in kernels of maize inbreds selected for ear rot resistance in West and Central Africa. J Food Prot 64:396– 400. Brown RL, Cleveland TE, Woloshuk CP, Payne GA, and Bhatnagar D (2001b). Growth inhibition of a Fusarium verticillioides GUS strain in corn kernels of aflatoxin-resistant genotypes. Appl Microbiol Biotechnol 57:708– 711. Brown-Jenco CS, Obrian GR, Sloan S, and Payne GA (1998). Identification of the DNA binding site for the Aspergillus flavus AFLR in the NOR-1 promoter. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, St. Louis, MO, p.102. Campbell KW and White DG (1995). Evaluation of corn genotypes for resistance to Aspergillus ear rot, kernel infection, and aflatoxin production. Plant Dis 79:1039 –1045. Cary JW, Rajasekaran K, Jaynes JM, and Cleveland TE (2000). Transgenic expression of a gene encoding a synthetic antimicrobial peptide results in inhibition of fungal growth in vitro and in planta. Plant Sci 154:171181. CAST (1979). Aflatoxins and other mycotoxins: an agricultural perspective. Counc. Agric. Sci. Technol. Rep., Ames, IA. Chen Z-Y, Brown RL, Lax AR, Guo BZ, Cleveland TE, and Russin JS (1998). Resistance to A. flavus in corn kernels is associated with a 14 kDa protein. Phytopathology 88:276– 281. Chen Z-Y, Brown RL, Lax AR, Cleveland TE, and Russin JS (1999a). Inhibition of plant pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli. Appl Environ Microbiol 65:1320 –1324. Chen Z-Y, Brown RL, Russin JS, Lax AR, and Cleveland TE (1999b). A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus aamylase. Phytopathology 89:902– 907. Chen Z-Y, Brown RL, Damann KE, and Cleveland TE (2000). Proteomics analysis of kernel embryo and endosperm proteins of corn genotypes resistant or susceptible to Aspergillus flavus infection. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Yosemite, CA, p. 88. Chen Z-Y, Brown RL, Cleveland TE, Damann KE, and Russin JS (2001). Comparison of constitutive and inducible maize kernel proteins of genotypes resistant or susceptible to aflatoxin production. J Food Prot 64:1785 – 1792. Chen Z-Y, Brown RL, Damann KE, and Cleveland TE (2002). Identification of unique or elevated levels of kernel proteins in aflatoxin-resistant maize genotypes through proteome analysis. Phytopathology 92:1084 – 1094. Chlan C, Rajasekaran K, and Cleveland TE (1999). Transgenic cotton. In: Bajaj YPS ed. Biotechnology in Agriculture and Forestry Series. Vol. 46. Heidelberg: Springer Verlag. p 283. Cleveland TE and Bhatnagar D (1992). Molecular strategies for reducing aflatoxin levels in crops before harvest. In: Bhatnagar D, Cleveland TE eds. Molecular Approaches to Improving Food Quality and Safety. New York, NY: Van Nostrand Reinhold. pp 205 –228. Cole RJ, Sanders TH, Hill RA, and Blankenship PD (1985). Mean geocarposphere temperatures that induce preharvest aflatoxin contamination of peanuts under drought stress. Mycopathologia 91:41– 46. Cotty PJ (1989). Effects of cultivar and boll age on aflatoxin in cottonseed after inoculation with Aspergillus flavus at simulated exit holes of the pink bollworm. Plant Dis 73:489 –492. Davis GL and Williams WP (1999). QTL for aflatoxin reduction in maize. Maize Genet Conf 41:22.

76 Desjardins AE and Proctor RH (2001). Biochemistry and genetics of Fusarium toxins. In: Summerell BA, Leslie JF, Backhouse D, Bryden WL, Burgess LW eds. Fusarium. St. Paul, MA: The American Phytopathological Society. Diener UL, Cole RJ, Sanders TH, Payne GA, Lee LS, and Klich MA (1987). Epidemiology of aflatoxin formation by Aspergillus flavus. Annu Rev Phytopathol 25:249– 270. Doster MA, Michailides TJ, and Morgan DP (1995). Aflatoxin control in pistachio, walnut, and figs: identification and separation of contamination nuts and figs, ecological relationships, and agronomic practices. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Atlanta, GA, p. 63– 64. Dowd PF (2000). Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensis corn under controlled and open field conditions: utility and limitations. J Econ Entomol 93:1669 –1679. Dowd PF, Duvick JP, and Rood T (1997). Comparative toxicity of allelochemicals and their enzymatic oxidation products to maize fungal pathogens, emphasizing Fusarium graminearum. Nat Toxins 5:180 –185. Du W, Huang Z, Flaherty JE, Wells K, and Payne GA (1999). Green fluorescent protein as a reporter to monitor gene expression and food colonization by Aspergillus flavus. Appl Environ Microbiol 65:834 – 836. Duvick J (2001). Prospects for reducing fumonisin contamination of maize through genetic modification. Environ Health Perspect 109(supp 2):337– 342. Eaton DL and Groopman JD (1994). The Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance. San Diego, CA: Academic Press, Inc. p 544. Gembeh SV, Brown RL, Grimm C, and Cleveland TE (2001). Identification of chemical components of corn kernel pericarp wax associated with resistance to Aspergillus flavus infection and aflatoxin production. J Agric Food Chem 49:4635 –4641. Go¨rg A, Boguth G, Obermaier C, and Weiss W (1998). Twodimensional electrophoresis of proteins in a immobilized pH 41-2 gradient. Electrophoresis 19:1516 – 1519. Gradziel TM and Dandekar A (1999). Endocarp ventral vascular tissue development appears to be the Achilles heel for almond susceptibility to insect damage and aflatoxin contamination. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Atlanta, GA, p. 5. Gradziel T, Dandekar A, Alhumada M, Hirsh N, Driverm J, and Tang A (1995). Integrating fungal pathogen and insect vector resistance for comprehensive preharvest aflatoxin control in almond. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Atlanta, GA, p. 5. Guo BZ, Russin JS, Cleveland TE, Brown RL, and Widstrom NW (1995). Wax and cutin layers in maize kernels associated with resistance to aflatoxin production by Aspergillus flavus. J Food Prot 58:296 –300. Guo BZ, Russin JS, Cleveland TE, Brown RL, and Damann KE (1996). Evidence for cutinase production by Aspergillus flavus and its possible role in infection of corn kernels. Phytopathology 86:824– 829. Guo BZ, Chen Z-Y, Brown RL, Lax AR, Cleveland TE, Russin JS, Mehta AD, Selitrennikoff CP, and Widstrom NW (1997). Germination induces accumulation of specific proteins and antifungal activities in corn kernels. Phytopathology 87:1174 –1178.

Brown et al. Guo BZ, Brown RL, Lax AR, Cleveland TE, Russin JS, and Widstrom NW (1998). Protein profiles and antifungal activities of kernel extracts from corn genotypes resistant and susceptible to Aspergillus flavus. J Food Prot 61:98– 102. Guo BZ, Widstrom NW, Cleveland TE, and Lynch RE (2000). Control of preharvest aflatoxin contamination in corn: fungus – plant –insect interactions and control strategies. Recent Res Dev Agric Food Chem 4:165 –176. Hamblin AM and White DG (2000). Inheritance of resistance to Aspergillus ear rot and aflatoxin production of corn from Tex6. Phytopathology 90:292– 296. Harris LJ and Gleddie SC (2001). A modified Rpl3 gene from rice confers tolerance of the Fusarium graminearum mycotoxin deoxynivalenol to transgenic tobacco. Physiol Mol Plant Pathol 58:1 – 9. Haumann F (1995). Eradicating mycotoxins in food and feeds. Inform 6:248– 256. Holbrook CC, Wilson DM, and Matheron ME (1995). An update on breeding peanut for resistance to preharvest aflatoxin contamination. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Atlanta, GA, p. 3. Holbrook CC, Wilson DM, Matheron ME, and Anderson WF (1997). Aspergillus colonization and aflatoxin contamination in peanut genotypes with resistance to other fungal pathogens. Plant Dis 81:1429 – 1431. Huang Z, White DG, and Payne GA (1997). Corn seed proteins inhibitory to Aspergillus flavus and aflatoxin biosynthesis. Phytopathology 87:622– 627. Jacks TJ, Delucca AJ, and Morris NM (1999). Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores. Mol Cell Biochem 195:169 – 172. Ji C, Norton RA, Wicklow DT, and Dowd PF (2000). Isoform patterns of chitinase and b-1,3-glucanase in maturing corn kernels (Zea mays L.) associated with Aspergillus flavus milk stage infection. J Agric Food Chem 48:507 –511. Kimura M, Kaneko I, Komiyama M, Takatsuki A, Koshino H, Yoneyama K, and Yamagushi I (1998). Trichothecene 3-O-acetyltransferase protects both the producing organism and transformed yeast from related mycotoxins. J Biol Chem 273:1654 – 1661. King SB and Scott GE (1982). Field inoculation techniques to evaluate maize for reaction to kernel infection by Aspergillus flavus. Phytopathology 72:782– 785. Lisker N and Lillehoj EB (1991). Prevention of mycotoxin contamination (principally aflatoxin and Fusarium toxins) at the preharvest stage. In: Smith JE, Henderson RS eds. Mycotoxins and Animal Foods. Boca Raton, FL: CRC Press. p 689 – 719. Mahoney N, Molyneux RJ, and Campbell BC (2000). Regulation of aflatoxin production by naphthoquinones of walnut (Juglans regia). J Agric Food Chem 9:4418 –4421. Marasas WFO (1996). Fumonisins: history, world-wide occurrence, and impact. In: Jackson LS, DeVries JW, Bullerman LB eds. Fumonisins in Food: Advances in Experimental Medicine and Biology. Vol. 392. New York: Plenum Publishing Corporation. pp 1– 17. McCormick SP, Alexander NJ, Trapp SC, and Hohn TM (1999). Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferaase, from Fusarium sporotrichioides. Appl Environ Microbiol 65:5252 –5256.

Molecular Biology for Control of Mycotoxigenic Fungi Miller JD, Fielder DA, Dowd PF, Norton RA, and Collins FW (1996). Isolation of 4-acetyl-benzoxazolin-2-one (4-ABOA) and diferuloylputricine from an extract of gibberella ear rotresistant corn that blocks mycotoxin biosynthesis and the insect toxicity of 4ABOA and related compounds. Biochem Syst Ecol 24:647– 658. Molyneux RJ, Mahoney N, Campbell BC, McGranahan G, and McKenna J (2000). Anti-aflatoxigenic activity of walnut constituents. Proceedings of the USDA-ARS Aflatoxin/ Fumonisin Elimination Workshop, Yosemite, CA, p. 64. Muhitch MJ, McCormick SP, Alexander NJ, and Hohn TM (2000). Transgenic expression of the TRI101 or PDR5 gene increases resistance of tobacco to the phytotoxic effects of the trichothecene 4,15-diacetoxyscirpenol. Plant Sci 157:201 – 207. Norton RA (1997). Effect of carotenoids on aflatoxin B1 synthesis by Aspergillus flavus. Phytopathology 87:814 – 821. Norton RA (1999). Inhibition of aflatoxin B1 biosynthesis in Aspergillus flavus by anthocyanidins and related flavonoids. J Agric Food Chem 47:12301235. Norton RA and Dowd PF (1996). Effect of steryl cinnamic acid derivatives from corn bran on Aspergillus flavus, corn earworm larvae, and driedfruit beetle larvae and adults. J Agric Food Chem 44:2412 –2416. Okubara PA, Hohn TM, Berka RM, Alexander NA, Wang Z, Hart LP, and Blechl AE (2000). Optimizing the expression of candidate anti-Fusarium protein genes in hexaploid wheat. Proceedings 2000 National Fusarium Head Blight Forum, Michigan State University, p 39 –43. Payne GA (1992). Aflatoxin in maize. Crit Rev Plant Sci 10:423– 440. Payne GA (1997). Characterization of inhibitors from corn seeds and the use of a new reporter construct to select corn genotypes resistant to aflatoxin accumulation. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Memphis, TN, pp 66– 67. Payne GA (1998). Process of contamination by aflatoxinproducing fungi and their impact on crops. In: Sinha KK, Bhatnagar D eds. Mycotoxins in Agriculture and Food Safety. New York, NY: Marcel Dekker. pp 279 – 306. Rajasekaran K, Cary JW, Jacks TJ, and Cleveland TE (1999). Inhibition of fungal growth by putative transgenic cotton plants. USDA-ARS Aflatoxin Elimination Workshop, Atlanta, GA, p. 64. Rajasekaran K, Cary JW, Jacks TJ, Stromberg KD, and Cleveland TE (2000a). Inhibition of fungal growth in planta and in vitro by transgenic tobacco expressing a bacterial nonheme chloroperoxidase gene. Plant Cell Rep 19:333 –338. Rajasekaran K, Hudspeth RL, Cary JW, Anderson DM, and Cleveland TE (2000b). Highfrequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures. Plant Cell Rep 19:539545. Rajasekaran K, Cary JW, Jacks TJ, and Cleveland TE (2001). Antimicrobial assays with transgenic cottons. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Yosemite, CA, p. 144. Robens, JF (2001). The Costs of Mycotoxin Management to the USA: Management of Aflatoxins in the United States http://www.apsnet.org/online/feature/mycotoxin/table1.html. Russin JS, Guo BZ, Tubajika KM, Brown RL, Cleveland TE, and Widstrom NW (1997). Comparison of kernel wax from corn

77 genotypes resistant or susceptible to Aspergillus flavus. Phytopathology 87:529 –533. Scott GE and Zummo N (1988). Sources of resistance in maize to kernel infection by Aspergillus flavus in the field. Crop Sci 28:505– 507. Sharma RP and Salunkhe DK (1991). Introduction to mycotoxins. In: Sharma RP, Salunkhe DK eds. Mycotoxins and Phytoalexins. Boca Raton, FL: CRC Press. pp 3 –11. Tucker DH, Jr, Trevathan LE, King SB, and Scott GE (1986). Effect of four inoculation techniques on infection and aflatoxin concentration of resistant and susceptible corn hybrids inoculated with Aspergillus flavus. Phytopathology 76:290– 293. Walker RD and White DG (2001). Inheritance of resistance to Aspergillus ear rot and aflatoxin production of corn from CI2. Plant Dis 85(3):322 – 327. White DG, Rocheford TR, Kaufman B, and Hamblin AM (1995a). Further genetic studies and progress on resistance to aflatoxin production in corn. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, Atlanta, GA, p. 7. White DG, Rocheford TR, Kaufman B, and Hamblin AM (1995b). Chromosome regions associated with resistance to Aspergillus flavus and inhibition of aflatoxin production in maize. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop Atlanta, GA, p. 8. White DG, Rocheford TR, Naidoo G, Paul C, Hamblin AM, and Forbes AM (1998). Inheritance of molecular markers associated with, and breeding for resistance to Aspergillus Ear Rot and aflatoxin production in corn using Tex6. Proceedings of the USDA-ARS Aflatoxin Elimination Workshop, St. Louis, MO, pp. 4 –6. Wicklow DT (1991). Epidemiology of Aspergillus flavus in corn. In: Aflatoxin in corn: New perspectives. North Central Regional Research Publication 329, Research Bulletin 599, Iowa State University, Ames, Iowa: Iowa Agriculture and Home Economics Experiment Station. p 315. Wicklow DT, Norton RA, and McAlpin CE (1998). B Carotene inhibition of aflatoxin biosynthesis among Aspergillus flavus genotypes from Illinois corn. Mycoscience 39:167– 172. Widstrom NW, McMillan WW, and Wilson D (1987). Segregation for resistance to aflatoxin contamination among seeds on an ear of hybrid maize. Crop Sci 27:961– 963. Widstrom NW, Butroń A, Guo BZ, Wilson DM, Snook ME, Cleveland TE, and Lynch RE (2003). Control of preharvest aflatoxin contamination in maize through pyramiding resistance genes to ear-feeding insects and invasion by Aspergillus spp. Eur J Agron, in press. Windham GL and Williams WP (1998). Aspergillus flavus infection and aflatoxin accumulation in resistant and susceptible maize hybrids. Plant Dis 82:281– 284. Windham GL, Williams WP, and Davis FM (1999). Effects of the southwestern corn borer on Aspergillus flavus kernel infection and aflatoxin accumulation in maize hybrids. Plant Dis 83:535– 540. Wolffram C, van Pee K-H, and Lingens F (1988). Cloning and highlevel expression of a chloroperoxidase gene from Pseudomonas pyrrocinia. FEBS Lett 238:325 – 328. Woloshuk CP, Cavaletto JR, and Cleveland TE (1997). Inducers of aflatoxin biosynthesis from colonized maize kernels are generated by an amylase activity from Aspergillus flavus. Phytopathology 87:164 –169.

7 Biotechnological Potential of Entomopathogenic Fungi Travis R. Glare AgResearch, Lincoln, New Zealand

fungus and applied it in the ﬁeld for sugar-beet weevil control. Despite the early discovery of their potential, it was only recently that entomopathogenic fungi have been utilized successfully in biocontrol programs. The fungi are often effective as natural control agents, but their activity is very dependent upon environmental conditions. Many have restrictive temperature ranges for germination, infection, and sporulation, or high humidity requirements for sporulation and spore germination. In some cases, the infective stage is not robust and, as many of the most promising candidate fungi for pest control have lost the ability to form persistent stages such as resting spores, storage and application can be problematic. Variation within species or clusters of species has not been well understood, thus strain selection has not often been attempted or not been possible. The application of biotechnology to the study and development of entomopathogenic fungi has the potential to overcome some of these limitations. Biotechnology has contributed to all areas in the development of entomopathogenic fungi as biocontrol agents, from identiﬁcation to formulation. This chapter reviews the contribution of biotechnology to the development of entomopathogenic fungi.

INTRODUCTION

Fungi have been known to attack insects and mites for thousands of years. Although the causative agent of fungal disease of insects was not always understood, insects infected with fungi were recorded by the Chinese in the seventh century (Tanada and Kaya 1993) and drawings of Cordyceps infections abound in early 18th and 19th century literature. The first experimental demonstration of a microbe as a disease-causing organism was by Agostino Bassi, published in 1835 –1836, in Italy, with the silkworm pathogenic fungus, Beauveria bassiana. He demonstrated that the fungus causes insect death and could be transmitted to other silkworms. It was not long after the first demonstration of the devastating impact of an entomopathogenic fungus on a beneficial insect that it occurred to researchers that disease may be a useful method for control of insect pests. Pasteur is credited with the early proposition that fungi could be used to control a pest insect. He proposed that a fungus could be used against Phylloxera in grapevines, a pest eventually controlled using copper solutions. However, it was the Nobel Prize winning researcher, Elie Metchnikoff, who first developed a fungus as a practical control agent for application to a pest. Working in Russia from 1878, Metchnikoff developed the fungus Metarhizium anisopliae for control of the cereal cockchafer, Anisoplia austriaca, then a devastating pest. Metchnikoff carried out the first successful infection experiments with larvae of A. austriaca and the sugar beet weevil, Cleonus punctiventris, and initiated mass production of the fungus for field experiments (Zimmermann et al. 1995). With the mass production of M. anisopliae, Metchnikoff applied a biotechnological approach to entomopathogenic fungi for the first time, a precursor to the development of biopesticides. The first actual field application of M. anisopliae in Russia was left to Krassilstchik (1888) who mass produced the

BIOPESTICIDE POTENTIAL OF ENTOMOPATHOGENIC FUNGI

There are a number of methods for using entomopathogenic fungi against insect pests. Eilenberg et al. (2001) recognized four main strategies: (a) classical biological control, the intentional introduction of an exotic strain for long term, unmanaged control, (b) inoculative biocontrol, the intentional release of endemic strains for long-term unmanaged biocontrol of endemic pests, (c) inundative biological control, 79

Vertalec

L. giganteum Laginex Nomuraea rileyi AGO Biocontrol Nomuraea 50 P. fumosoroseus AGO biocontrol Paecilomyces PFR-97 biological insecticide V. lecanii Ago Biocontrol Verticillium 50 Mycotal

Green muscle Metarhizium Schweizer Taenure

Dispel Ostrinil B. brongniartii AGO Biocontrol Beauveria 50 Engerlingspilz M. anisopliae Ago Biocontrol Metarhizium 50 BIO 1020 BioGreen BioCane Green guard

B. bassiana AGO Biocontrol Bassiana Biorin BotaniGard and Mycotrol Beauveria Schweizer

Species and product name

Natural plant protection Ago Biocontrol Andermatt Biocontrol, AG Ago Biocontrol Bayer AG/ BioCare Technology Pty. Ltd. BioCare Pty. Ltd. Seed Grain and Biotechnology Australia Pty. Ltd. Biological Control Products SA PTY Eric Schweizer Seeds Ltd.

Coleoptera, Homoptera, Lepidoptera, Diptera Melolontha melolontha Lepidoptera, Coleoptera, Homoptera, Orthoptera Vine weevil A. couloni (red-headed cockchafer) Greyback canegrubs Locust and grasshopper

Ago Biocontrol Thermo Trilogy Corporation

Ago Biocontrol Koppert Biological Systems B.V.

Coleoptera, nematodes Whiteﬂies, aphids, thrips, spidermites

Homoptera, Diptera Whiteﬂies, some activity against thrips

Koppert Biological Systems B.V.

Ago Biocontrol

Lepidoptera

Aphids

AgraQuest, Inc.

All mosquito larvae

Earth BioSciences Inc.

Biotech International Emerald BioAgriculture (ex. Mycotech Corporation) Eric Schweizer Seeds Ltd.

Lepidopteran caterpillars Homoptera/Heteroptera, thrips, Coleoptera, Lepidoptera and Orthoptera Turf/grassland, fruit growing, viticulture and horticulture Podborers Corn earworm O. nubalis

Locusts and grasshoppers Turf/grassland, fruit growing, viticulture and horticulture Root weevils, grubs, ticks, immature thrips, white ﬂies

Ago Biocontrol

Company

Coleoptera, Homoptera, Lepidoptera, Diptera

Targets

Table 1 Biopesticides based on entomopathogenic fungi (Milner 2000; Reddy et al. 2001; Shah and Goettel 1999)

Netherlands, UK, Switzerland, Finland, Norway, Denmark Netherlands, UK, Switzerland, Finland, Norway, Denmark

Colombia, USA

European Union, USA

Colombia

Colombia, USA

http://www.taensa.com/products-taenure. html

South Africa Switzerland

Germany, USA Australia Australia Australia

Colombia

Switzerland

Columbia

India France

Switzerland

India www.biotech-int.com USA

Columbia

Main countries and reference

80 Glare

Biotechnological Potential of Entomopathogenic Fungi

the use of fungi to limit pests when control is achieved exclusively by the mass release of the organism, and (d) conservation biological control, modiﬁcation of the environment to enhance fungal infection. Inundative biological control usually relies on the development of biopesticides based on pathogenic microbes, which is the most obvious application of biotechnology to entomopathogenic fungi.

2.1

Biopesticides Based on Fungi

Application of fungi in mass inoculations against insect pests began with Krassilstchik in 1888. Indeed, such was the optimism at that time Krassilstchik confidently predicted “the idea of controlling insects by means of artificially induced epidemics, an idea expressed some 20 years ago by scholars, has become a practically feasible one, which in the future will be perfected and broadly utilized” (Krassilstchik 1888). Unfortunately, progress has been much slower than what was predicted. While progress in the early 1900s was promising, the discovery and application of effective chemical pesticides in the 1930–1940s reduced interest in the use of insect pathogens. Insect pathogenic fungi were more difficult to use, and it was not until environmental and health problems associated with the use of chemical insecticides became apparent in the 1960s that interest in fungal biopesticides again increased. Currently, most biopesticides based on entomopathogenic fungi in the market include either B. bassiana or M. anisopliae (Table 1). These two species, the so-called muscardine fungi, have broad host ranges, although individual strains may be restricted in the number of insect species that they can attack. These species are relatively easy to produce, as they produce vast amounts of asexual conidia in culture as well as on insects. They are generally considered to have low mammalian toxicity and few nontarget impacts have been reported (see Section 6). A number of biopesticides have been based on the white muscardine fungus, B. bassiana (Table 1). The better known products are those of Emerald BioAgriculture (a merger between Mycotech Corporation and Auxein Corporation) such as BotaniGardw and Mycotrolw. BotaniGard is a liquid emulsion formulation of B. bassiana conidia while Mycotrol is based on powdered conidia. There are a number of other products based on Beauveria spp. registered around the world. In France, Ostrinile, based on B. bassiana has been produced for many years for corn earworm (Ostinia nubalis) control, while in India the biopesticide Dispel is sold for control of podborers (Reddy et al. 2001). Similarly, a number of biopesticides are based on the green muscardine fungus, Metarhizium spp. Biopesticides based on Metarhizium spp. have had a long (if not always successful) history. In the 1980s, Bayer Corporation produced a biopesticide, Bio1020, which was a formulation of M. anisopliae with excellent shelf life and application potential. It was primarily developed for control of black vine weevil (Reinecke et al. 1990), but was tested against a number of other pests [e.g., Tabata (1992)]. However, the product was not commercially successful for a

number of reasons (Reinecke et al. 1991) and was unavailable for many years. Recently, Bio1020 has reappeared in the market as Taenuree, sold by Earth BioSciences (http://www.taensa.com/products-taenure.html). A recent success story for biopesticides has been the development of novel strains of M. anisopliae var. acridium for locust control in several countries. Initially, a strain of this fungus was developed in Africa under a program called LUBILOSA, which led to the biopesticide “Green Muscle” (Lomer et al. 2001). This program has inspired development of indigenous strains of M. anisopliae var. acridium in other countries. For example, in Australia the success of the LUBILOSA program has been duplicated with the development of Green Guarde based on an Australian isolate of M. anisopliae var. acridium (Milner 2000). Other biopesticides based on Metarhizium are sold around the world. In Australia, an isolate of M. anisopliae has also been developed as a commercially available biopesticide for the control of sugarcane scarabs, particularly the grayback canegrub, Dermolepida albohirtum. BioCanee is effective when applied at 33 kg/ha (1 £ 1010 conidia/m) before fillingin of the planting furrow (Samson et al. 1999), giving 50–60% control of grayback larvae (Logan et al. 2000). In Columbia, a product based on several entomopathogenic fungi (Micobiol) has been tested against Prodiplosis longifila (Diptera: Cecidomyiidae) infesting tomatoes, but was not as effective as conventional control products (Delgado et al. 1999). Several other entomopathogenic fungi have been developed as commercially available biopesticides (Table 1). Fungi such as Paecilomyces and Verticillium are similar in action to Metarhizium and Beauveria. However, a more unusual fungus for development as a biopesticide is the aquatic active, Lagenidium giganteum. This Oomycete fungus is active against mosquito larvae and has been developed into the biopesticide Laginexe in California and is now sold by AgraQuest, Inc. In field trials against the mosquito Culex quinquefasciatus, Laginexe compared favorably with Vectobace (based on Bacillus thuringiensis israelensis), in terms of persistence of control (Hallmon et al. 2000). No biopesticides are currently produced using any species of the Entomophthorales, which is a large order containing mainly entomopathogenic fungi. These fungi, which typically forcibly discharge their primary conidia, often cause large-scale epizootics among insects. This suggests huge potential for development of this group of fungi as mass applied biopesticides. However, problems in production and stabilization of the fragile conidia or the more durable resting spores have not been overcome, and economic products are not feasible at this time. Biopesticide production has increased in many Central and South American nations and some are not strictly commercial. For example, in Cuba where, as a result of the trade embargo, it has been difficult to obtain cheap chemical pesticides, a biopesticide production industry has grown to fill the gap. Under the Cuban Ministry of Agriculture, decentralized laboratories provide insects, nematodes, and

Glare

entomopathogens (bacteria, fungi, and viruses) throughout Cuba’s 15 provinces (Rosset and Moore 1997). These “Centres for the Production of Entomophages and Entomopathogens” (CREEs) have facilitated the rapid adoption of IPM systems in crops previously managed under pesticidebased systems. Several fungal entomopathogens are produced for a number of pests, including B. bassiana for control of coleopteran pests and Verticillium lecanii for whiteﬂy, Bemisia tabaci. In 1994, 781 metric tonnes of B. bassiana, 196 of V. lecanii and 142 of M. anisopliae were produced by the production centers. Similarly, biopesticides based M. anisopliae are produced by some Central American sugar plantations for the control of pests. The sugar companies have their own production facilities for Metarhizium and Beauveria [e.g., Badilla (2000) and Grimm (2001)]. These localized production facilities produce sufﬁcient quantities of fungal inoculum for control of pests such as the coffee berry borer, the diamondback moth, and spittlebugs.

2.2

Production, Formulation, and Application

Production of entomopathogenic fungi has not advanced greatly beyond the use of simple grains as substrates for the Deuteromycete fungi, such as Metarhizium and Beauveria. For many other entomopathogenic fungi, especially among the Entomophthorales, growth in culture is difﬁcult or has yet to be achieved. Both liquid and solid substrates have been substantially investigated (Burgess 1998). Two-stage systems, where both liquid and solid substrates are used, have occasionally proved successful. For example, fermentation to produce hyphae to use as starter cultures is now a widespread practice. There are number of advantages to using liquid cultures as starter cultures: (a) the competitive ability of the fungus is enhanced, reducing the risk of contamination from other microbes, (b) growth is more rapid in the early stages, (c) the liquid culture can be screened for contamination prior to use, and (d) the liquid ensures even coverage of the solid substrate (Jenkins et al. 1998). Liquid starter cultures are commonly used to begin solid substrate production. However, experience with M. anisopliae in our laboratory is probably typical of many other laboratories, where inoculation of rice grains with fermenter broth of M. anisopliae hyphal bodies gave no improvement in production over the use of conidia from plate cultures (Glare et al. unpublished data). Production on grains is generally in the range of 108 –1010 conidia/g of dry substrate [e.g., Feng et al. (1994)], taking between 2 and 3 weeks to reach maturity at optimal temperatures. Interestingly, Metarhizium and Beauveria sporulate better when the substrate is relatively poor in nutrient content. When grains were supplemented with sugars and yeast additives, less conidia per gram of substrate was obtained than with grains alone (Nelson et al. 1996). Similarly, in Brazil, M. anisopliae has been found to produce conidial yields of 5 –15 times higher using rice bran/rice husk substrate mixtures than yields

usually obtained for rice grains, with viabilities of higher than 85% (Dorta et al. 1990). The production of Green Musclee M. anisopliae for locusts in Africa used a two-stage production system with fermenter production of inoculum used to inoculate rice (Cherry et al. 1999). The process requires relatively low capital investment, but has high labor costs. As with production of most fungi, high variability in yield was reported between batches, and this variability was only partly accounted for by temperature and duration of incubation (Cherry et al. 1999). A method that showed some promise in the 1980s was the preparation of dried mycelium. Hyphal bodies were harvested by filtration, washed with water to remove culture medium residue, and then coated with a sugar solution before drying. This method was used with M. anisopliae and B. bassiana (Pereira and Roberts 1990). They found that conidial production was similar to other methods after storage for up to 4.5 months at 48C and could be superior to other methods with respect to storage at room temperature, however no products at present use this technology. The Emerald Bio production plant (previously Mycotech) in Butte, Montana, represents the technological end of the production of entomopathogenic fungi. Largely utilized for the production of B. bassiana, it is a “state of the art” dedicated facility, with in-line sterilization and large temperature controlled growth facilities. The actual production method is a trade secret, but is based on fermented starter cultures and solid substrate growth and sporulation. This highly technical facility contrasts with the numerous low technology “factories” producing fungi for insect control in China and much of Latin America. Compatibility between production, formulation, and application techniques is vital for the successful use of microbial biopesticides. The LUBILOSA program for locust control used Metarhizium in oil formulations and ULV spraying, which required lipophilic conidia for easy suspension in oils (Jenkins et al. 1998). While production of submerged conidia was seen as having many advantages, the resulting conidia were hydrophilic and lost viability quickly. Therefore, production on grains remains the standard with the locust products. For many years, approaches to the use of entomopathogenic fungi involved point release (“classical biological control”) or simple application of conidia, formulated in water with wetting agents. However, appropriate formulation can advance entomopathogenic fungi from curiosity to effective biocontrol agents. It has been an area that has benefited from the application of biotechnology. Formulation has been important in terms of improved survival during storage, persistence in the field (such as UV and desiccation tolerance), and ease of application. The LUBILOSA program, where M. anisopliae var. acridium was developed into a biopesticide for locust control in Africa, is an excellent example of formulation overcoming environmental constraints. As locusts live in hot, dry climates and M. anisopliae conidia require high humidity to germinate, it seems impossible that an entomopathogenic fungus could

Biotechnological Potential of Entomopathogenic Fungi

successfully control the pest. However, formulating Metarhizium conidia in nonevaporative diluents such as oils allowed the conidia to attach and germinate on susceptible locusts. M. anisopliae oil formulations are especially useful at low relative humidities (Bateman 1997). There have been several interesting studies on formulating hyphal material from members of the Entomophthorales. These fungi, because of the fragile nature of the mycelium and conidia, pose a much greater formulation problem than most of the Deuteromycetes, which has contributed to their lack of commercial success. McCabe and Soper (1985) patented a process of drying the mycelium of Zoophthora radicans and coating it with sugar, as a method for long-term storage. More recently, Shah et al. (1998) demonstrated algination as a method for formulating Erynia neoaphidis mycelium. An important area of formulation and production is the drying of conidia of entomopathogenic fungi. Moore et al. (1996) have shown that survival of conidia of M. anisopliae was highest at low (, 5%) relative humidity, therefore, this is an important aspect of producing a stable product. Use of appropriate application techniques that are suited for the application of biopesticide to the target pest is an obvious, but often neglected aspect of biopesticide use. Advances in chemical pesticide applications have slowly ﬁltered through to use with biopesticides, such as ultra-low volume (ULV) application of M. anisopliae for locust control (Lomer et al. 2001). Nonevaporative diluents such as oil are required to take advantage of ULV spraying. Rotary atomizers have been used for low volume oil formulations and ULVs for less than 5 l/ha. Electrostatically-charged ULV sprayers have been investigated for better coverage on leaf undersides (Sopp et al. 1989). Generally, application of fungal-based biopesticides has been with conventional equipment and research has focused on spray coverage, droplet size, and placement (i.e., penetration to the underside of leaves). Hydraulic spray systems have been used to apply water-based formulations on crops, air-blast and air-assist technologies are primarily used for low volume applications in ﬁelds and orchards. The best success has been with large numbers of droplets with high spore content per droplet (Goettel et al. 2000). Introducing large amounts of fungal inoculum into the soil and securing an even spread remains a problem. Many methods have been tested for application of fungal containing granules or conidia on grains to soil, including using seed drills for subsurface application, and hand application. The problems of spread of conidia after application to soil has lead to the Melolontha and researchers are developing an area wide approach based on augmentative applications of Beauveria brongniartii for long term suppression of pest populations (Hajek et al. 2001).

2.3

Novel Strategies for Biopesticide Use

In some cases, preexisting application technology may not be well suited to the requirements of a biological agent. One

approach that takes advantage of the biological nature of entomopathogenic fungi is the “lure and infect” approach, best demonstrated by research on Z. radicans for control of diamondback moth. Furlong et al. (1995) have shown that using pheromone lures to attract moths to traps containing sporulating Z. radicans can result in contamination and spread of the fungus through the target population. Such an approach has been investigated for use with scarab beetles in the Azores (Klein and Lacey 1999). Autodissemination of entomopathogenic fungi for control of Popillia japonica in the Azores used a trapping system of commercially available attractants with M. anisopliae. The viability of conidia in traps after 6 days was found to be about 35%, but the basic process was successful for introducing fungi into pest populations. Another approach has been bait stations, such as those used with termites (Rath 2000). The entomopathogenic fungus is placed in a trap together with a food-based bait, and the insect becomes contaminated when it enters the trap. The general approach is similar for lure-and-infect and bait stations: attract the insect to an inoculum source, rather than broadcast application to secure contact between pest and disease. Use of attractants is not restricted to luring to a single trap. Smith et al. (1999) investigated the use of vegetable fat pellets formulated with pheromone and B. bassiana to control the larger grain borer, Prostephanus truncates. Significantly higher numbers of beetles were attracted to pellets containing pheromone than those without pheromone incorporated. The pellets containing pheromone and fungus could be stored for several weeks, indicating this may be a useful strategy to increase the utility of entomopathogenic fungi. Development of biopesticides for social insects has been problematic because the method by which social insects defend against disease is mainly behaviorally-based rather than biologically-based. For example, hymenopteran wasps such as Vespula spp. have well-developed hygienic behaviour which includes removing all suspected material from a nest before contamination of nestmates occurs. Vespula do not reuse nests and, therefore, disease in one season does not result in disease in another season. Behavioral defense against disease requires novel application and formulation methods for any chance of success for entomopathogenic fungi. Similarly, termites are highly susceptible to entomopathogenic fungi, including M. anisopliae and B. bassiana but many factors such as avoidance of conidia, the removal and burial of fungus-killed termites, together with defensive secretions and inhibitory components in termite frass (Rath 2000), and grooming to remove spores (Milner and Glare, unpublished observations) reduce field efficacy. Boucias et al. (1996) used a low sublethal dose of a neurotoxin, imidacloropid to disrupt the grooming behaviour of termites, which then became highly susceptible to the fungus B. bassiana. One proposal is to use more than one pathogen to increase the utility of entomopathogenic fungi. It is often common in the field to find more than one pathogen exerting influence on a pest, such as both a nucleopolyhedrovirus and the fungus Entomophaga maimaiga infecting gypsy moth (Malakar et al.

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1999). The possibility of combining multiple species or strains in a single biopesticide to overcome limitations inherent in the single strain approach is intriguing. For example, Inglis et al. (1997) have investigated the use of both M. anisopliae and B. bassiana for control of locusts and grasshoppers, to overcome the temperature limitations of both species. There are potentially many methods whereby the efﬁcacy of biopesticides could be enhanced by combinations, such as those described earlier, but the economics of producing multiple pathogens for a single product are usually too limiting.

BIOACTIVES FROM ENTOMOPATHOGENIC FUNGI

While the focus on the practical use of entomopathogenic fungi has been on biocontrol using whole organisms, either as inoculative or inundative agents (Eilenberg et al. 2001), these fungi are known to produce a number of toxins and enzymes. Some of these extracellular metabolites have been studied with the aim of using them as bioactives against insect pests. This biotechnological approach to utilizing entomopathogenic fungi can be demonstrated by the discovery and formulation of spinosyns, insecticidal toxins produced by an actinomycete. From the discovery of a strain of Saccaropolyspora spinosa in the Carribbean, Dow Agrow Sciences have successfully developed a number of “green chemistry” insecticide products, such as Successe and Naturalytee. It may be possible to utilize active components from entomopathogenic fungi in a similar or novel fashion. It is not surprising that entomopathogenic fungi produce extracellular enzymes and toxins. These compounds are required to both assist penetration of the host cuticle and overcome other host defenses, while excluding competing microbes. Proteases produced by entomopathogenic fungi to degrade cuticle and assist entry into the host are similar to proteases used by insects to degrade their own cuticle during molting (Samuels and Paterson 1995). A number of enzymes are known from entomopathogenic fungi, such as the proteases, lipases, and chitinases that assist in cuticular breakdown. These enzymes can be thought of as bioactives and there has been increasing interest in use of these enzymes in pest control. Screen and St Leger (2000) have reported on the occurrence of typsins and chymotrypsins in M. anisopliae. The novel chymotrypsin (CHY1) is similar to bacterial chymotrypsins. Because paralogous genes for the chymotrypsins are not found in genome sequences for yeast, gram eubacteria, archaebacteria, and mitochondria they hypothesis that chy1 arose from horizontal gene transfer. Entomopathogenic fungi also produce insecticidal toxins. The early literature on toxins from entomopathogenic fungi was reviewed by Roberts (1981) and more recently by Strasser et al. (2000). Several metabolites from entomopathogenic Deuteromycetes are well known and described. For example, Beauveria spp. are known to produce beauvericin, a

depsipeptide metabolite which has shown toxicity to a number of invertebrates (Roberts 1981). Not all Beauveria can produce beauvericin, but it has been isolated from Paecilomyces fumosoroseus mycelium. B. bassiana is also reported to produce beauverolides, isarolides, and bassianolides, all cyclotetradepsipetides. Metarhizium strains are also well known for producing toxic metabolites, the best described of these are the destruxins. These cyclodepsipeptides are toxic to a number of insects, but susceptibility varies considerably, ranging up to 30 times between silkworm larvae and Galleria (Roberts 1981). Hirsutellin A is produced by Hirsutella thompsonii and is not proteolytic, but was toxic to a range of insects (Mazet and Vey 1995). Aspergillus species are occasionally insect pathogens and are known to produce many insecticidal metabolites. However, the occurrence of aflatoxin production in many Aspergillus that infect insects has restricted interest in this group, although it is by no means necessary that insecticidal strains produce aflatoxins in any appreciable amount (Roberts 1981). Not all entomopathogenic fungi produce toxins in the disease process. In some cases, toxins are suspected, but not conclusively demonstrated. Injection of culture filtrates of some entomopathogenic Entomophthorales into Galleria sp. resulted in blackening similar to that found in fully infected larvae [e.g., Roberts (1981)]. Some of the lower fungi, such as Coelomomyces and the Entomophthorales, may possess only weak toxins, if any at all. It is more likely they overcome hosts by utilizing the nutrients and invading vital tissue (Roberts 1981). Some entomogenous fungi produce antibiotics. As entomopathogenic fungi must compete for utilization of cadavers with numerous resident and environmental bacteria, it is not surprising that a number of antibiotics are produced by the various strains and species. Hirsutella and the allied genus Cordyceps also produce a number of metabolites that may be weak toxins or antibiotics. Krasnoff and Gupta (1994) described an antibiotic, phomalactone, from the H. thompsonii var. synnematosa that was also toxic to apple maggots, Rhagoletis pomonella (Dipt., Tephritidae). Phomalactone was inhibitory to other entomopathogenic fungi (Beauveria, Tolypocladium, and Metarhizium). Cordyceps-infected caterpillars are a traditional medicine in parts of Asia. This may be partly based on the production by Cordyceps of a weak antibiotic, cordycepin. Zabra et al. (1996) reported that metabolites from Z. neoaphidis had antibacterial activity. In the future, bioactives from entomopathogenic fungi may have a role in pest insect control, either formulated as pesticides, or through transgenic expression. Direct toxicity may not be the only aim, as some toxins or metabolites have antifeedant type activities (e.g., http://www.item.ba.cnr.it/biopesti.htm).

MOLECULAR GENETICS OF ENTOMOPATHOGENIC FUNGI

The use of molecular techniques to manipulate entomopathogenic fungi to overcome some of the limitations discussed

Biotechnological Potential of Entomopathogenic Fungi

earlier has been proposed for many years. In comparison with advances made in manipulation of viruses and bacteria, progress with the fungi has been slow, which is not surprising given the multigene nature of fungal insect diseases. Most progress has been made with the Deuteromycete muscardine fungi, B. bassiana and M. anisopliae.

4.1

Transformation Systems

Modification of entomopathogenic fungi has long been contemplated, but rarely reported. A limited number of studies have reported successful insertion of foreign genes into entomopathogenic fungi. A precursor to manipulation of entomopathogenic fungi using molecular techniques has been the development of transformation systems. There are several aims of transforming entomopathogenic fungi. These techniques enable gene disruption methods to be applied, which can lead to greater understanding of the genetics of disease processes, or the ability to introduce DNA into fungi may allow the modification of cell processes, potentially allowing improvements in the use of entomopathogenic fungi for insect control. The first transformation of an entomopathogenic fungus was reported by Goettel et al. (1990), where M. anisopliae was transformed to be benomyl tolerant using pBENA3, a plasmid containing the benA3 allele from Aspergillus nidulans. Since then, there have been other reports on transformation of the Dueteromycete entomopathogens using a variety of methods. St Leger et al. (1995) used eletroporation and biolistic delivery to transform M. anisopliae with the plasmids (pNOM102 and pBENA3) containing the b-glucuronidase and benomyl resistance genes. The cotransformants showed normal growth rates and retained their pathogenicity to insects (Bombyx mori). Polyethylene glycol (PEG)-mediated transformation of protoplasts is another method for transformation of entomopathogenic fungi, as used with the P. fumosoroseus and P. lilacinus (Inglis et al. 1992) using benomyl as the selective agent. More recently, a heterologous transformation system for B. bassiana and M. anisopliae was developed based on the use of the A. nidulans nitrate reductase gene (niaD) (Sandhu et al. 2001). The niaD stable mutants of B. bassiana and M. anisopliae were selected by treatment of protoplasts with ethane methane sulfonate (EMS) and regenerated on chlorate medium.

4.2

Strain Improvement Through Biotechnology

Improvements in strains of entomopathogenic fungi have been attempted through selection as well as molecular methods. Selection of fungal strains with altered acyclic sugar alcohol (polyol) and trehalose content of the conidia may improve the endogenous reserves to enhance viability and desiccation tolerance. Cultures of B. bassiana, M. anisopliae, and P. farinosus grown under different conditions to obtain

conidia with a modified polyol and trehalose content resulted in conidia with increased intracellular levels of glycerol and erythritol that germinated more quickly than unselected conidia and at lower water activity (Hallsworth and Magan 1995). Conidia with increased trehalose germinated more slowly but stored for longer than unselected conidia. Another approach is to use genetic modification to “improve” strains and overcome limitations. This type of approach is in its infancy for entomopathogenic fungi, but there have been some interesting studies indicating the utility of the process. Two of the more promising studies on the potential of biotechnology to improve entomopathogenic fungi were published by Couteaudier et al. (1996) and Vaiud et al. (1998). They demonstrated that protoplast fusion between a strain of B. bassiana from Leptinotarsa decemlineata with an insecticidal toxin-producing strain of B. sulfurescens resulted in recovery of some di-auxotrophic mutants with enhanced activity (faster kill) against L. decemlineata and the caterpillar Ostrinia nubilalis. The stability of the virulence following passage through the insect –host and stability of molecular structure for two of the fusion products suggested that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi (Vaiud et al. 1998). The most studied genes in the entomopathogenic fungi are the protease genes of M. anisopliae, particularly the Pr1 gene. This was the first protease gene from an entomopathogenic fungi implicated in disease and was isolated by St Leger et al. (1992). Pr1 has sequence similarity to proteinase K, but was more effective than that enzyme at degrading cuticle. It is similar to the subtilisin subclass of serine endopeptidases. Modification of pr1 gene expression in M. anisopliae resulted in melanisation and cessation of feeding 25 –30 h earlier than wild-type disease in caterpillars (St Leger et al. 1996). V. lecanii, B. bassiana, Tolypocladium niveum, and P. farinosus also produced Pr1-type enzymes during nutrient deprivation (St Leger et al. 1991). Southern analysis demonstrated that genes with significant homologies to Metarhizium pr1 were present in the entomopathogens A. flavus and V. lecanii but not Z. radicans (St Leger et al. 1992). More recently, 11 subtilisin proteases (Pr1s) were identified from one strain of M. anisopliae (St Leger et al. 2001). Recently, intended field release of a modified M. anisopliae strain was reported (St Leger 2001). The strain has the pr1 cuticle degrading protease gene under control of a constitutive promoter. The gene overproduction did not alter the host range, but resulted in a strain with a reduced median lethal time to kill. It also reduced the ability of transformants to sporulation. The pr1 gene expression was under dual control of a general carbon catabolite repression/depression mechanism and a carbon source induction mechanism to control expression. Overexpression of extracellular chitinase, an enzyme important in the cuticular penetration of insects by entomopathogenic fungi, has also been demonstrated for M. anisopliae var. anisopliae (Screen et al. 2001). They expressed a chitinase gene from M. anisopliae var. acridium under control of an Aspergillus regulatory element to express

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in noninducible conditions. While successful expression was achieved, there was no altered virulence to the caterpillar, Manduca sexta, compared to the wildtype fungus. Genetic manipulation of entomopathogenic fungi has a long way to go before transgenic pest control strains become available, if such technology is ever acceptable to regulators and the community. However, strain modiﬁcation continues to provide a wealth of data on disease processes.

MOLECULAR IDENTIFICATION AND TRACKING

In addition to improving our understanding of the genetics of disease caused by entomopathogenic fungi, molecular techniques have also been used to aid in identification, classification, and environmental monitoring of fungi. It is now almost a standard practice to perform some form of genetic characterization of fungi to specifically identify strains used for biocontrol purposes. The utility of molecular techniques, however, has been demonstrated beyond simple strain identification. There are a number of reviews on the use of molecular characterization for entomogenous fungi in the literature [e.g., Driver et al. (1998) and Glare (2002)]. An example of the importance of molecular characterization was the clarification of the taxonomic position of M. anisopliae strain IMI 330189 used in the LUBILOSA program for locust control. Often described as a M. flavoviride strain because of the morphology of the conidia and some other features, the correct classification was debated. Recently, Driver et al. (2000) published a revision of the subspecific relationships between M. anisopliae and M. flavoviride strains, based largely on the sequence of the ITS-5.8s regions of rRNA. They demonstrated that IMI 330189 and related strains formed a discrete clade on the M. anisopliae branch of the Metarhizium trees, a finding that appears to have been well received by those working on locust control. Driver et al. (2000) named the subspecies M. anisopliae var. acridium, as well as describing several other subspecies, some of which can only presently be distinguished by ITS sequencing. This demonstrates a problem with molecular characterization, as fungal species cannot be solely erected on sequence data and requires supporting morphological or biological descriptions. Molecular markers have been used to characterize the genotypes of individual fungal strains by examining gene products, but new techniques allow direct examination of variability at the DNA level. Pulsed field gel electrophoresis has been used to study karyotype variation in other fungi, and could be used with entomopathogens. Several studies have examined the number of chromosomes and mapped genes on those chromosomes. Viaud et al. (1996) studied the level of chromosome length polymorphism among nine isolates of B. bassiana to obtain a more extensive knowledge of the genomic organization. While extensive use of molecular characterization has proved useful, there are currently no standard techniques or agreement on even how many regions of the genome should be sampled to provide taxonomic data.

While this is not a problem for strain identification or comparison, it reduces the ability to compare between studies. Many of the molecular studies on entomogenous fungi have used the nuclear ribosomal DNA, but there are a number of other DNA regions used, such as mitochondrial DNA (mtDNA) restriction fragment length polymorphisms. The MtDNA has been used to estimate intraspecies variation in V. lecanii and M. anisopliae isolates. The contribution of molecular techniques to the development of entomopathogenic fungi has been enormous. The techniques have been used to clarify evolutionary relationships [e.g., Driver et al. (2000) and Jensen et al. (1998)]. Molecular techniques have also allowed development of theories of evolution around these often obligate pathogens. Generally, studies on the entomogenous fungi using conserved mitochondrial or nuclear regions have failed to find a link between fungal species and host species. For example, Bidochka et al. (2001) found that habitat rather than host selection drives population structure of M. anisopliae. There have been exceptions, such as B. bassiana strains from Sitona weevils (Maurer et al. 1997) and some Entomophthorales [e.g., Jensen and Eilenberg (2001)]. In the order Entomophthorales, sequencing of the small subunit rDNA has been used to examine phylogenetic relationships (Jensen et al. 1998). The molecular studies supported the use of spore discharge characteristics as an identifying characteristic for Entomophthorales. The role of horizontal gene transfer in microbial evolution has been the topic and studies by St Leger et al. (2001) have found some evidence for the involvement of horizontal gene transfer in evolution of fungal parasitism, finding similarity between genes in M. anisopliae and Streptomyces bacteria. Monitoring of specific strains of entomopathogenic fungi in the field after release is crucial for advancing and understanding of biopesticide ecology. It has often been difficult to conduct ecological studies on fungal persistence and spread after application, because there has been a lack of simple methods for isolation and specific strain characterization of these fungi. The molecular characterization of strains of entomogenous fungi has improved the ability to track specific fungi in the field. Specific identification of the B. brongniartii strain used for control of the scarab pest, Hoplochelus marginalis, in the ReUnion Islands was based on introns (insertions) in the 28s gene of the rDNA (Neuve´glise et al. 1997). Genetic modification is also a method to allow tracking following release of a strain into the environment or in a host. For example, a b-glucuronidase gene has been inserted in M. anisopliae to allow detection of hyphae in infected hosts (St Leger et al. 1995) and the expression of a green fluorescent protein-encoding gene for tracking purposes (St Leger 2001).

SAFETY OF ENTOMOPATHOGENIC FUNGI

Biopesticides based on entomopathogenic fungi are now available, with a range of different species and strains used.

Biotechnological Potential of Entomopathogenic Fungi

The growing use of these insect pathogens has raised interest in the safety of microbial pesticides, above the level previously required for registration purposes. This increased scrutiny of environmental and mammalian safety of entomopathogenic fungi is part of a worldwide move to more awareness of potential negative impacts of biotechnology. Few of the entomopathogenic fungi are thought to pose a direct threat to human health. There are exceptions, such as the entomophthoralean fungus, Conidiobolus coronatus, but it is unlikely any development of the potentially hazardous strains would be contemplated. Many products have required mammalian toxicology packages to be submitted during the registration process to demonstrate safety. Generally, the entomopathogenic Deuteromycetes are considered to have low risk of mammalian toxicity [e.g., Donovan-Peluso et al. (1980) and Shadduck et al. (1982)]. Recent papers on Metarhizium and Beauveria have raised some issues regarding mammalian safety of immunocomprised individuals (Burgner et al. 1998; Henke et al. 2002). In addition to viewing bioactives from entomopathogenic fungi as potentially useful, there has been consideration of their effect as potential hazards in registering entomopathogenic fungi. There are some results showing activity against human cell lines, such as tumor cell lines for P. tenuipes cytotoxic components (Nam et al. 2001). Production of toxic secondary metabolites has caused problems in the registration of some fungi in Europe. Strasser et al. (2000) summarizes data on specific secondary metabolites (destruxins, efrapeptins, oosporein, beauvericin, and beauveriolides) produced by the genera Beauveria, Metarhizium, and Tolypocladium. They found that fungal bioactives posed no obvious risk to humans, although the number of detailed studies is limited. Some studies have indicated low-level activity against animals of selected bioactives such as destruxins of Metarhizium have an intraperitoneal injection LD50 of 1 –16 mg/kg in mice. However, the levels of metabolites produced during insect infection were much lower than in culture. There is a growing body of research on nontarget impacts of fungal-based insecticides [e.g., Goettel et al. (2001) and Hokkanen and Hajek (2002)], which have not found increased environmental risk from their use. The present evidence is that mycoinsecticides are very safe in production and use from both an environmental and mammalian toxicity viewpoint (Goettel et al. 2001). However, the formulations being developed require stringent testing to ensure their superior safety compared with comparable chemical pesticides (Moore and Prior 1993). While the fungi themselves have generally not been found to be a risk through testing and natural exposure, the development of novel formulations and strain combinations will require careful evaluation to ensure no unexpected effects occur. This could be especially true of nontarget impacts. Similarly, any development of genetically modified strains will have to be carefully studied for environmental and mammalian safety. Regulations in all countries are becoming more stringent on these issues, especially for genetically modified organisms.

CONCLUSIONS

Biotechnological approaches to the study and development of entomopathogenic fungi have advanced the field in recent years. Improvements in formulations allowing new biopesticides to succeed in unexpected conditions (such as locust control in Africa), strain selection, and identification have advanced not only biopesticide formulation, but understanding of disease processes and ecology. More specific identification systems have allowed better monitoring of biopesticide applications as well as development of phylogenetic classification. Despite some success, commercial use of entomopathogenic fungi is restricted by high cost, inadequate or inconsistent efficacy, limited mass production capability, and poor shelf life. However, entomopathogenic fungi have several advantages over other microbes for formulation in biopesticides as many species have a robust spore stage, capable of survival in products for many months or years. Some are easy to grow on simple media and can be formulated using a number of simple procedures. They can often kill more than one target pest, although limited in host range enough for registration purposes. With continuing improvements in formulation and application technology, it is likely that many more niche biopesticides will come to market, especially with the increased markets due to a rise in organic production and the reduction in the number of chemical pesticides available. There are a number of new techniques and applications that will aid in the further development of entomopathogenic fungi. Application of molecular biological techniques to entomopathogenic fungi also holds the promise of strain improvement through genetic manipulation, or assist in strain improvement without genetic modification. For example, through techniques such as protoplast fusion and chromosome exchange, using knowledge of desired chromosomal gene location, may enable superior strain qualities to be combined in single isolates. Determining the underlying genetics of host specificity, the toxins and enzymes involved in the disease process, and genetics of fungal processes such as sporulation and germination are all under study around the world. Advances in these areas may allow greater use to be made of entomopathogenic fungi. The potential of entomopathogenic fungi lies not just in their application as biopesticides based on the live fungus, but also in the isolation and development of bioactives from these fungi. Toxins, enzymes, and antibiotics are all produced by entomopathogenic fungi and, as techniques for their isolation and expression increase, the potential for exploiting bioactives is enhanced. In some cases these bioactives are not toxins, but may exert other useful effects, such as antifeeding activity. There is also potential in novel strategies for biopesticide use such as mixtures of behaviour-modifying chemicals for enhancing control of social insects with pathogens. While the history of biopesticide development from entomopathogenic fungi is littered with more failures than

success, the future seems brighter. As the knowledge from several commercially successful products and new technologies are applied to biopesticide development, we can expect to see more novel biocontrol methods applied to insect pests in the future using fungal species.

ACKNOWLEDGEMENTS I thank Drs Maureen O’Callaghan Mary Christey and Trevor Jackson for comments on the manuscript, and Lois McKay for assistance with compiling the references.

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89 Rath AC (2000). The use of entomopathogenic fungi for control of termites. Biocontrol Sci Technol 10:563 – 581. Reddy CN, Singh VS, Singh Y, and Dureja P (2001). Bioefficacy of insecticides, biopesticides and their combinations against podborers in pigeonpea. Indian J Entomol 63:137 – 143. Reinecke P, Andersch W, Stenzel K, and Hartwig J (1990). BIO 1020, a new microbial insecticide for use in horticultural crops. Brighton Crop Prot Conf Pests Dis 1:49 –84. Reinecke P, Andersch W, Stenzel K, and Hartwig J (1991). Probleme bei der Entwicklung mickrobieller Pflanzenschutzmittel am Beispiel von BIO 1020. Nachrbl Dtsch Pflanzenschutzd 43:98– 100. Roberts DW (1981). Toxins of entomopathogenic fungi. In: Burges HD ed. Microbial Control of Pest and Plant Diseases 1970– 1980. New York: Academic Press. pp 441 –460. Rosset P and Moore M (1997). Food security and local production of biopesticides in Cuba. ILEIA Newslett 13:18. Samson PR and Milner RJ (1999). Metarhizium-based pesticides for Queensland canegrubs. Proc 7th Aust Conf Grassland Inv Ecol: 92– 98. Samuels RI and Paterson IC (1995). Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi. Comp Biochem Physiol B Biochem Mol Biol 110:661 –669. Sandhu SS, Kinghorn JR, Rajak RC, and Unkles SE (2001). Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans. Indian J Exp Biol 39:650– 653. Screen SE and St Leger RJ (2000). Cloning, expression, substrate specificity of a fungal chymotrypsin: evidence for lateral gene transfer from an actinomycete bacterium. J Biol Chem 275:6689– 6694. Screen SE, Hu G, and St Leger RJ (2001). Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta. J Invertebr Pathol 78:260– 266. Shadduck JA, Roberts DW, and Lause S (1982). Mammalian safety tests of Metarhizium anisopliae: preliminary results. Environ Entomol 11:189– 192. Shah PA and Goettell MS eds. (1999). Directory of microbial control products and services. Microbial control division. Soc Invertebr Pathol: 31. Shah PA, Aebi M, and Tuor U (1998). Method to immobilize the aphid-pathogenic fungus Erynia neoaphidis in an alginate matrix for biocontrol. Appl Environ Microbiol 64:4260 – 4263. Smith SM, Moore D, Karanja LW, and Chandi EA (1999). Formulation of vegetable fat pellets with pheromone and Beauveria bassiana to control the larger grain borer, Prostephanus truncatus (Horn). Pestic Sci 55:711 –718. Sopp PI, Gillespie AT, and Palmer A (1989). Application of Verticillium lecanii for the control of Aphis gossypii by a lowvolume electrostatic rotary atomiser and a high-volume hydraulic sprayer. Entomophaga 34:417– 428. St Leger RJ (2001). Notification of intent to release a transgenic strain of Metarhizium anisopliae (document submitted to FIFRA). www.epa.gov/pesticides/biopesticides/otherdocs/ release_notification.htm (16/08/01). St Leger RJ, Staples RC, and Roberts DW (1991). Changes in translatable mRNA species associated with nutrient deprivation and protease synthesis in Metarhizium anisopliae. J Gen Microbiol 137:807 –815.

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8 Biotechnological Potential of Ergot Alkaloids M. Flieger / P. Mehta / A. Mehta Dr. H.S. Gour University, Saugor, India, and Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic

recorded from outside this genus (Flieger et al. 1997; Kozlovsky 1999).

INTRODUCTION

Ergot alkaloids belong to the group of compounds produced by fungi, which are referred to as secondary metabolites. They are produced by a number of fungi mainly of Claviceps spp. but they have been also found in other fungi and higher plants. Ergot (sclerotium of the pyrenomycete Claviceps purpurea) develops in florets of grasses and sedges. In early days, the medieval midwives used to collect the fungus from naturally infected plants and used it in the induction of childbirth and in the control of postpartum bleeding. The role of ergot has undergone important changes from a dreaded toxic parasite to an important source of biologically effective substances. C. purpurea is, apart from yeast, the first fungus, which was biotechnologically exploited without its existence known. The beginning of modern ergot research dates back to the extraction of the first alkaloid mixture from sclerotia in 1875, isolation of ergotoxine (mixture of ergocornine, ergocristine, and ergokryptine) in 1907, and the discovery of the first clinically used compound, ergotamine in 1918. At the beginning of fifties the chemistry, biosynthesis, physiology, biochemistry, genetics, biotechnology, and therapeutical applications of ergot alkaloids have been extensively studied (Berde and Sturmer 1978; Mukherjee and Menge 2000; Rehacek and Mehta 1993; Tudzynski et al. 2001). The present review gives an overview of biotechnological potential of ergot alkaloids with a perspective for the future.

Ascomycetes Eurotiales: Aspergillus fumigatus, A. ﬂavus, A. japonicus, A. tamarii, A. versicolor, A. nidulans, A. oryzae; Penicillium aurantiovirens, P. camembertii, P. chermesinum, P. fumigatus, P. clavigerum, P. concavorugulosum, P. crustosum, P. griseofulvum, P. kapuscinskii, P. palitans, P. patulum, P. roqueforti, P. rubrum, P. rugulosum, P. sizovae, P. viridicatum Hypocreales: Balansia claviceps, B. epichloe, B. obtecta, B. strangulans, Epichloe typhina, Neotyphodium coenophialum, N. lolii, Hypomyces aurantius, Sepedonium sp. Basidiomycetes Corticium caeruleum, Lenzites trabea, Pellicularia ﬁlamentosa Zygomycetes Cunninghamella blakesleana, Mucor hiemalis, Rhizopus arrhizus, R. nigricans Higher plants Convolvulaceae Argyreia nervosa, Ipomoea argyrophylla, I. rubro-coerulea, I. piurensis, Rivea corymbosa, Stictocardia tiliifolia

STRUCTURE

The ergot alkaloids constitute the largest known group of nitrogenous fungal metabolites and over 80 alkaloids have been isolated from diverse natural material. The common part of chemical structure of the most ergot alkaloids is a tetracyclic ergoline ring system (Figure 1), which is biosynthesized from tryptophan (Taber and Vining 1959), and mevalonic acid (Groger et al. 1961). The ergot alkaloids

SOURCES

In nature, the ergot alkaloids are formed primarily by various species of Claviceps. However, ergot alkaloids have also been 91

Mehta et al.

natural substances having the unique structure called cyclol (Figure 4) that consists of three aminoacids. The ergopeptines are further divided to (a) ergotamine, (b) ergoxine, (c) ergotoxine, and (d) b-ergoannine group (Flieger et al. 1997).

Figure 1 Structure of ergoline.

can be separated into three main structural groups: (a) clavine alkaloids, (b) simple lysergic and paspalic acid derivatives, and (c) peptide alkaloids (ergopeptines). The clavine alkaloids are tricyclic (secoergolines) or tetracyclic (ergolines) compounds usually substituted with methyl, hydroxyl or hydroxymethyl group in position C-8 and in many cases have a double bond in positions 8,9 (D8,9-ergolenes) or 9,10 (D9,10-ergolenes) of the ergoline skeleton (Figure 2). The important feature of all D9,10-ergolenes (including simple derivatives of lysergic acid and ergopeptines) is an easy isomerization on C(8) resulting in formation of two isomers. Clavine alkaloids represent the largest group of ergot alkaloids due to the action of various enzymes, which direct agroclavine and/or elymoclavine from the main biosynthetic route agroclavine – elymoclavine – paspalic acid – lysergic acid. Such type of shunt activity is particularly evident in microorganisms that lack the complete pathway and are unable to synthesize substituted lysergic acid (Vining 1980). The simple derivatives of paspalic and lysergic acid (Figure 3) are mostly amides, in which the amide part is either small peptide or a simple alkylamide. Amides of lysergic and paspalic acids found in ergot are ergometrine, lysergic acid 2-hydroxyethylamide, lysergic acid amide (ergine), paspalic acid, and 10-hydroxy-paspalamide (Flieger et al. 1993). Ergopeptines are derivatives of lysergic acid and are the only

BIOGENESIS

Ergot alkaloids are derived from tryptophan, mevalonic acid, and methionine (Birch et al. 1960; Groger et al. 1960; Taylor and Ramsted 1960). The origin of the ergoline part from tryptophan and dimethylallylpyrophosphate was established at an enzymic level. The cell free biosynthesis of chanoclavines-I and II, agroclavine, and elymoclavine from precursors has been reported by Sajdl and Rehacek (1975) and Cavender and Anderson (1970). Tryptophan has also been found to be a factor in the induction and depression of enzymes catalyzing alkaloid formation (Krupinski et al. 1976). The first enzyme of alkaloid biosynthesis is dimethylallyltryptophan synthase (DMATS) (Heinstein et al. 1971) and the encoding gene was cloned from C. fusiformis and C. purpurea (Tsai et al. 1995; Tudzynski et al. 1999). Recent data of Tudzynski et al. (2001) show that in C. purpurea, all genes involved in alkaloid biosynthesis are organized in a cluster and are regulated by phosphate and pH level. This finding verified the previously published data on the negative role of phosphate in the induction of enzymes catalyzing the alkaloid synthesis (Krupinski et al. 1976) and on inhibition of alkaloid synthesis by high concentration of phosphate in the culture medium (Mehta 1984; Pazoutova et al. 1983). Gene for peptide synthetase homologous to that of C. purpurea was also detected in Epichloe and Neotyphodium species (Annis and Panaccione 1998; Panaccione et al. 2001). Ergot alkaloid synthesis requires changes in differentiation. In C. purpurea the formation of conidia is inversely dependent on the synthesis of alkaloids for those saprophytic strains, which partially retain parasitic development, i.e., differentiation of the sphacelial phase to the conidial or the

Figure 2 General structure of secoergolenes (1), D8,9-ergolenes (2), and D9,10-ergolenes (3).

Biotechnological Potential of Ergot Alkaloids

Figure 4 General structure of ergopeptines. Figure 3 General structure of lysergic (1) and paspalic (2) acid derivatives.

sclerotial phase or both (Mehta 1984). In strains with not clearly separated conidial phase, vegetative growth, and the alkaloid phase, the conidia formation occurs simultaneously with alkaloid synthesis. The morphological development of the mycelium in submerged Claviceps cultures shows significant differences between high-yielding and degenerated cultures (Flieger et al. 1982). The character of fat hyphae and sclerotia-like cells of submerged mycelium is reminiscent of the plectenchymatic structure of parasitic cultures and have been found to influence the alkaloid production in C. fusiformis (Dickerson et al. 1970). Cultivation conditions convenient for primary metabolism are not suitable for high alkaloid production (Taber and Vining 1963). To obtain overproduction of alkaloids, high citrate or Krebs cycle intermediate level in the medium is required (Pazoutova et al. 1981). The inability of submerged Claviceps cultures to grow on hexoses in absence of Krebs cycle intermediates comes from the parasitic way of life where the level of citrate and malate in the host plant phloem sap is high. The rate of oxidative metabolism of saccharides and the activity of alkaloid synthesis were proved to be proportional (Pazoutova et al. 1981). Another important fact is that clavine alkaloids are extracellular products with high solubility in the culture medium and feedbacks regulate their own biosynthesis (Flieger et al. 1988). Ergopeptines are mostly intracellular and are accumulated in lipid droplets (oleosomes) with no influence on the metabolism (Neumann et al. 1979).

5 5.1

INDUSTRIAL PRODUCTION Industrial Production of Ergot Alkaloids

The overall word annual production of ergot alkaloids was estimated at about 20,000 kg (Cvak 1999) in which the production of ergopeptines and their dihydroderivatives forms less then one third. The rest of the production is

concentrated on production of lysergic acid and other precursors of semisynthetic ergot preparations (lysergol, elymoclavine, ergine, and other simple derivatives of lysergic acid). In the last decade the production of ergopeptines (Table 1) remained confined due to their limited therapeutic use while the production of semisynthetic ergot preparations is gaining importance due to the development of new drugs with new and more specific therapeutical applications (Berde and Sturmer 1978; Eich and Pertz 1999; Pertz and Eich 1999). It is evident from the data (Table 1) that lysergic acid is the main precursor of semisynthetic ergot preparations, which can be obtained by chemical decomposition of ergopeptines or simple derivatives of lysergic acid. So far, very limited amount of ergot preparations have been synthesized from clavine alkaloids. Field production of ergot alkaloids is still an important source of ergopeptines. In the last decades, the major effort was devoted to selection of strains producing defined spectrum of alkaloids. Recently, the average yield of ergot reached the level of 1000 kg/ha with content of alkaloids above 1% (Cvak 1999).

5.2 5.2.1

Saprophytic Cultivation of Claviceps History

The first saprophytic cultivation of Claviceps on artificial nutritional media dates back to 1922 (Bonns 1922). The first attempt for the industrial production of ergot alkaloids was isolation of clavine alkaloids from submerged cultures of different Claviceps spp. (Abe and Yamatodani 1954; 1955; Abe et al. 1952; 1956). Later, conditions for saprophytic production of simple derivatives of lysergic acid were developed using different strains of C. paspali (Arcamone et al. 1960; 1961). It took only 5 years more when new isolate of C. purpurea was found to produce ergotamine under submerged conditions (Amici et al. 1966). Since that time all types of ergot alkaloids for direct use as therapeutic agents or precursors for the preparation of semisynthetic drugs can be obtained by fermentation.

Uterotonic, antimigraine, vasoconstrictor, hemostatic Uterotonic, oxytoxic Antimigraine, symphatolitic, vasoconstrict Cerebral and peripheral vasodilator Sympatholitic, peripheric vasodilator Antiparkinsonian, prolactine inhibitor, cerebral vasodilator Dopamine agonist, antiparkinsonian, prolactine inhibitor Cerebral vasodilator Serotonine antagonist, antimigraine, prolactine inhibitor Uterotonic, oxytocic Serotonine antagonist, antimigraine Serotonine antagonist, prolactin inhibitor, antiparkinsonian Dopamine agonist, prolactine inhibitor, antiparkinsonian Dopamine agonist, prolactine inhibitor, antiparkinsonian Dopamine agonist, prolactine inhibitor, antiparkinsonian 1000 – 1500 100 – 200 1500 – 2000 1000 – 1500 1000 – 1500 500 1000 10000 50 150 50 30 10 50 30 Ergotamine Ergometrine Dihydroergotamine Dihydrotoxine Dihydroergocristine Dihydro-a-ergokryptine Bromokryptine Nicergoline Metergoline Methylergometrine Methysergide Lisuride Terguride Pergolide Cabergoline

Therapeutic use Annual production (kg) Substance

Table 1 Annual production, therapeutic use, and source of recently used ergot preparations

Field production, submerged production, synthetic Field production, submerged production, synthetic Hydrogenation of field/fermented ergotamine Hydrogenation of field/fermented ergot mixtures Hydrogenation of field/fermented ergocristine Hydrogenation of field/fermented a-ergokryptine Bromination of a-ergokryptine Synthesized from lysergic acid/lysergol Synthesized from lysergic acid amide/dihydrolysergol Synthesized from lysergic acid Synthesized from lysergic acid Synthesized from lysergic acid/ erginine Synthesized from lisuride Synthesized from dihydrolysergol Synthesized from dihydrolysergic acid

Mehta et al.

Source

5.2.2

Strain

As with other fermentation processes the key to successful production of ergot alkaloids is in obtaining the proper strain of the fungus. There are three main processes used for the preparation of Claviceps strains for saprophytic culture. (a) Plating of plectenchymatic tissue from the surface sterilized sclerotia on an agar growth medium (Mantle 1969), (b) Plating of honeydew drops containing conidia formed at early stage of Claviceps infection (Janardhanan and Husain 1984; Pazoutova et al. 2002), and (c) Trapping of sexual ascospores ejected from fruiting bodies on germinated sclerotia. By this method monosporic culture can be obtained (Vasarhelyi et al. 1980).

5.2.3

Strain Improvement

The classical methods (selection pressure, mutagenesis, and recombination) used for the strain improvement are, to some extent, more complicated with Claviceps due to incomplete information on cell nucleus. Strains used for saprophytic cultivation might be heterokaryotic and homokaryotic (Didek-Brumec et al. 1991; Mantle and Nisbet 1976). Recently it was found that the number of chromosomes in C. purpurea is variable so that haploid as well as aneuploid nuclei may be encountered (Hu¨sgen et al. 1999). Mutagenesis followed by subsequent selection of strain is an important technique in increasing the yield of alkaloids (Didek-Brumec et al. 1987). An ergocristine producing C. purpurea strain showed 180-fold increase in alkaloid production after eightstep mutation-selection with different mutagens (Kobel and Sanglier 1978). Mutagenesis of sporulating strain results in monosporic isolates. To increase the mutation frequency the protoplasts prepared from spores of selected strains were used (Olasz et al. 1982; Zalai et al. 1990). More complicated situation is with mutagenesis of asporogenic strains. Hyphal fragments are rather unsuitable for mutagenesis due to the higher number of nuclei. Even protoplast formation from young mycelium and subsequent regeneration without any mutagenic treatment yielded strains with different properties (Schumann et al. 1987). Protoplast fusion technique is beginning to find useful applications either in producing improved mutant strains by intraspecific crosses or in formation of novel spectrum of products by interspecific hybrids (Socic and Gaberc-Porekar 1992). Relevant structural and regulatory genes of the alkaloid biosynthesis in C. purpurea form a cluster of about 50 kbp in length (Tudzynski et al. 2001), therefore, isolation and cloning of the entire pathway to more rapidly growing fungus would be difficult.

5.2.4

Maintenance Improvement and Long-Term Preservation

Degeneration, loss of production capabilities, is a general problem of high-yielding strains of Claviceps (Kobel 1969). Conservation and systematically performed selection of the

Biotechnological Potential of Ergot Alkaloids

isolates is the only way to eliminate the biological effects given by the transfer of cultures, ageing, and other inﬂuences. Methods applied for long-term preservation were recently reviewed (Hunter-Cervera and Belt 1996). For preservation of sporulating cultures two main methods are applied: deepfreezing and maintaining of cultures on rye grains or agar slants placed in refrigerator. Nonsporulating strains due to higher sensitivity to conservation procedure are frequently preserved as cultures on the agar plates. The universal technique applied for preservation is keeping of lyophilized cultures and cultures frozen, in liquid nitrogen (Baumert et al. 1979).

5.3

Fermentation

All technologies developed for industrial scale have the same aims: maximal production of desired ergot alkaloids, minimum amount of accompanying contaminants (other alkaloids, toxins, etc.), the shortest possible time of fermentation, minimized cost of medium, energy, equipments, and labor. For the production of ergot alkaloids, different fermentation technologies have been employed (a) stationary cultivation on liquid or solid medium (Kybal and Vlcek 1976; Trejo-Hernandez and Lonsane 1993), (b) submerged cultivations (Kobel and Sanglier 1986) also adapted to semicontinuous or continuous processes (Kopp and Rehm 1984). In some cases the immobilized microorganisms were used for production of ergot alkaloids under condition of submerged fermentation (Komel et al. 1985; Kopp and Rehm 1983; Kren 1991).

5.3.1

Stationary Surface Cultivation

In the beginning of sixties, the development of processes for production of ergot alkaloids under conditions of stationary cultivation on liquid media started (Adams 1962; Kybal et al. 1960; Molnar et al. 1964; Rochelmeyer 1965). The stationary surface cultivation on agar slants was commonly used for preparation of starting cultures. When transformed to industrial scale this technology showed some limitations mostly due to difficulties with control of aseptic conditions of large surfaces. Vlcek and Kybal (1974) developed technology for stationary cultivation of C. purpurea in plastic bags partially filled with inoculated liquid medium. This procedure was used for production of ergotoxins and later adapted to production of asexual spores of C. purpurea for field production.

5.3.2

Submerged Cultivation

Submerged fermentation in laboratory scale, i.e., shaker cultivation, is a primary step in getting knowledge of production microorganism physiology, biosynthesis, sporulation, stability, and inﬂuence of medium composition on production of alkaloids. In industrial scale, submerged fermentation in shaker culture is mostly used for preparation

of sufficient amount of inoculum for further cultivation step. During the inoculum preparation, an optimal state of the culture for biosynthesis of ergot alkaloids in the production step can be established (Socic et al. 1985; 1986). Usually, the whole industrial process consist of four steps i.e., (a) shaker culture, (b) preinoculating fermentation, (c) seed fermentation, and (d) production fermentation followed by downstream processing (Malinka 1999). Industrial production of clavine alkaloids, agroclavine, and elymoclavine by submerged fermentation of different strains of Claviceps sp. was reported in a number of patents (Kren et al. 1988; Rehacek et al. 1986; Trinn et al. 1990), reaching the maximum production of alkaloidal mixture about 6 g/l (Pazoutova and Tudzynski 1999; Pazoutova et al. 1987). In contrast to high production and well elaborated procedure of submerged cultivation the industrial production of clavine alkaloids does not receive much attention due to limited amount of procedures leading to preparation of desirable final products (cf. Table 1). Production of lysergic acid and its simple derivatives is of high technological and industrial interest due to relatively simple procedure of chemical modifications to semisynthetic ergot preparations. The main producers of these alkaloids are strains of C. paspali. The basic studies on biosynthesis, physiology, and production of simple derivatives of lysergic acid were done on relatively limited number of strains including C. paspali 31 (Rosazza et al. 1967), C. paspali ATTC 13892 (Socic et al. 1986), and C. paspali MG-6 (Bumbova-Linhartova et al. 1991). The concentration of produced alkaloids reached nearly 3 g/l of fermentation broth (Pertot et al. 1990) with the strain C. paspali L-52. One of the very interesting features of C. paspali strains is their ability to convert clavine alkaloids added to the cultivation medium to the simple derivatives of lysergic acid (Mothes et al. 1962). Flieger et al. (1989a, b) and Harazim and Malinka (1989) used this capability of C. paspali CCM 8061 and developed technology of aggressive bioconversion of clavine alkaloids to simple derivatives of lysergic acid with total production of nearly 6 g/l in batch cultivation and about 3 g/l in industrial fermentor. Different strains of C. purpurea, as the only producers of ergopeptines, were described for their submerged production. Ergotamine producing strains and their cultivation are the best-studied processes due to direct therapeutical use of ergotamine. Industrial technologies were developed for the following strains: (a) F.I. 32/17 producing 2 g/l of ergotamine and a-ergokryptine mixture (Amici et al. 1966), (b) IBP 47, IMET PA135 producing 1.5 g/l [mixture of alkaloids containing 75% of ergotamine (Baumert et al. 1979)], (c) L-4 (ATTC 20103) producing 1.5 g/l of ergotamine (Komel et al. 1985). Another type of ergopeptines produced by submerged cultivation belongs to group of ergotoxines, i.e., ergocristine, ergocornine, and a-ergokryptine. Between many published procedures and industrial technologies (Malinka 1999) the process developed for strain C. purpurea L-17 resulted in relatively high production of ergotoxines (2.4 g/l). This strain was further studied and intermediary metabolism

Mehta et al.

and production of secondary metabolites were correlated (Gaberc-Porekar et al. 1992).

5.3.3

Solid Substrate Fermentation

Robinson et al. (2001) has recently proposed solid substrate fermentation (SSF) for the production of enzymes and secondary metabolites. The production of ergot alkaloids by C. fusiformis using SSF procedure was found to be 3.9 times higher than that obtained by submerged liquid fermentations (SLF) (Hernandez et al. 1993). One of the reasons could be the necessity of use of antifoam chemicals and the shear stresses caused by stirring in SLF. Also, better air circulation can be achieved in SSF thus further increasing the ergot alkaloid yields (Balakrishnan and Pandey 1996). The SSF has been shown to produce a more stable, requiring less energy in smaller fermentors with easier downstream processing measures. Also, by removal, the cost and trouble associated with antifoaming chemicals and by maximizing yield production, SSF may be seen as a viable option for industrial scale production of ergot alkaloids.

modiﬁed ergopeptines was described by Bianchi et al. (1982) and Crespi-Perellino et al. (1992). The growth of the fungus and alkaloid formation in submerged batch fermentation was described by mathematical model, which can be further used in automatic process control and optimization. Grm et al. (1980) proposed for C. purpurea growth model based on morphological features of a cell population during the fermentation process. Votruba and Pazoutova (1981) proposed another model accentuating the antagonistic effects of phosphate on growth and alkaloid production. A mathematical simulation of different technological alternatives of clavine alkaloid production was done on this basis (Pazoutova et al. 1981). The activation– inhibition kinetics of clavine alkaloids production was evaluated for two C. fusiformis strains (Flieger et al. 1988) and it was found that feed-back inhibition can be eliminated by combination of fermentation and separation units in a closed loop. Increased efﬁciency (more than 100%) of the fermentation process was also found when inducers of cytochrome P-450 were used (Rylko et al. 1988).

6 5.4

Optimization, Control, and Modeling of Ergot Alkaloids Fermentation

The following facts should be taken into account for optimization and scale-up of fermentation processes for the production of ergot alkaloids (Kobel and Sanglier 1978), (a) the production strains should be continually tested to maintain the optimal quality of selected production strain and thus maximally eliminate the biological effects given by transfer of cultures, ageing, and other external influences, (b) long cultivation period in absence of antibiotics require very high standard of sterility in operation and equipment, (c) balanced aeration and stirring are required due to the sensitivity of the Claviceps cultures to the stress and high oxygen tension, and (d) the use of antifoam agents could cause considerable loss in alkaloid yield. Recently the application of oxygen vectors to C. purpurea cultivation was published (Menge et al. 2001). The classical problem of the large-scale fermentations is an optimal supply of oxygen to growing Claviceps sp. High oxygen demand in the exponential growth phase can be met by addition of different hydrocarbons (Gilmanov et al. 1996) or perfluorocarbons (Menge et al. 2001) to shake flask and/or stirred reactors. Perfluorocarbons were successfully applied also in cultivations of other microorganisms (Lowe et al. 1998). Besides these technological aspects, other techniques to optimize the fermentation process were described. The influence of nutrients, addition of ergot alkaloid precursors, mainly tryptophan and its derivatives, were described in many studies (Erge et al. 1984; Floss 1976). As a result of feeding, prolonged idiophase of the fermentation process was found (Milicic et al. 1987) and this process seems to be one of the perspectives in modern alkaloid production (Socic and Gaberc-Porekar 1992). Precursor controlled production of

CONCLUSIONS

The biotechnological relevance of ergot alkaloids is due to their therapeutical use, unquestionable. On the other hand, they play very important role as toxins in agricultural industry as products of endophytic fungi of the genus Neotyphodium and their production is coupled with serious problems of livestock grazing infected grasses. These two examples show the importance of molecular genetics of alkaloid biosynthesis. It could help, on one side, to develop new strategies for rational designing of ergot alkaloid based drugs, and, on the other one, to understand and control the production of ergot alkaloids by endophytic fungi. The following points need more attention for modern production of ergot alkaloids and its semisynthetic derivatives: (a) shortening of the initial nonproductive phase of alkaloid fermentation, (b) immobilization of cells, (c) construction of plasmids which can carry selectable markers, (d) the development of transformation systems with drug resistance markers, (e) study of membrane processes and vacuoles in the productive organism, (f) use of mathematical modeling for description of phenomenon observed during culture growth and alkaloid production, and (g) further development of solid state fermentation and application of oxygen vectors.

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Mehta et al. Menge M, Mukherjee J, and Scheper T (2001). Application of oxygen vectors to Claviceps purpurea cultivation. Appl Microbiol Biotechnol 55:411 –416. Milicic S, Kremser M, Gaberc-Porekar V, Didek-Brumec M, and Socic H (1987). Correlation between growth velocity and biosynthesis of ergot alkaloids in Claviceps purpurea batch fermentation. Appl Microbiol Biotechnol 27:117 –120. Molnar G, Tetenyi P, Udvardy, Nagy E, Wack G, and Wolf L (1964). Verfahren zur biosynthetischen Herstellung eines Mutterkornalkaloidgemisches. CH pat 455820. Mothes K, Winkler K, Gro¨ger D, Floss HG, Mothes U, and Weygand ¨ ber die Umwandlung von Elymoclavin in F (1962). U Lysergsaürederivate durch Mutterkornpilze (Claviceps). Tetrahedron Lett 21:933– 937. Mukherjee J and Menge M (2000). Progress and prospects of ergot alkaloid research. Adv Biochem Eng Biotech 68:1 –20. Neumann D, Losecke W, Maier W, and Groger D (1979). Localization of alkaloids in sclerotia and suspension cultures of Claviceps purpurea (Fr.) Tul. Biochem Physiol Pflzen 174:504 – 508. Olasz K, Gaal T, and Zalai K (1982). Improvement of Claviceps purpurea by mutagenic treatment of protoplast. Acta Biochim Biophys Acad Sci Hungar 17:126. Panaccione DG, Johnson RD, Wang J, Young CA, Damrongkool P, Scott B, and Schardl CL (2001). Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte. Proc Natl Acad Sci 98:12820 – 12825. Pazoutova S and Tudzynski P (1999). Claviceps sp. PRL 1980 (ATCC 26245), 59 and Pepty 695/ch-I: their true story. Mycol Res 103(8):1044 – 1048. Pazoutova S, Votruba J, and Rehacek Z (1981). A mathematical model of growth and alkaloid production in submerged culture of Claviceps purpurea. Biotech Bioeng 23:2837 – 2849. Pazoutova S, Slokoska LS, Nikolova N, and Angelov TI (1983). Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains. Eur J Appl Microbiol Biotechnol 16:208– 211. Pazoutova S, Flieger M, Rylko V, Kren V, and Sajdl P (1987). Effect of cultivation temperature, clomiphene and nystatin on the oxidation and cyclization of chanoclavine in submerged cultures of the mutant strain Claviceps purpurea 59. Curr Microbiol 15:97 – 101. Pazoutova S, Johnson N, and Rajasab AH (2002). Heteropogon triticeus, a new host of Claviceps sorghi in India. J Phytopathol 150:1 – 4. Pertot E, Gaberc-Porekar V, and Socic H (1990). Isolation and characterization of an alkaloid-blocked mutant of Claviceps paspali. J Basic Microbiol 30:51 –56. Pertz H and Eich E (1999). Ergot alkaloids and their derivatives as ligands for serotoninergic, dopaminergic, and adrenergic receptors. In: Kren V, Cvak L eds. Ergot the genus Claviceps. The Netherlands: Harwood academic publishers. pp 411 – 440. Rehacek Z and Mehta P (1993). Biological effects of ergot alkaloids. In: Rai B, Arora DK, Dubey NK, Sharma PD eds. Fungal Ecology and Biotechnology. Meerut, India: Rastogi Publications. pp 275 –287. Rehacek Z, Pazoutova S, Kren V, Rylko V, Kozova J, and Sajdl P (1986). Producing strain of Claviceps purpurea (Fr.) Tul 59 CC 5/86. CS pat 252603 (In Czech).

Biotechnological Potential of Ergot Alkaloids Robinson T, Singh D, and Nigam P (2001). Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl Microbiol Biotech 55:284 –289. Rochelmeyer H (1965). Verfahren zur Gewinnung von Mutterkornalkaloiden in saprophytischer Kultur. DE pat. 1492109. Rosazza JP, Kelleher WJ, and Schwarting AE (1967). Production of lysergic acid derivatives in submerged culture. IV. Inorganic nutrition studies with Claviceps paspali. Appl Microbiol 15:1270 – 1283. Rylko V, Flieger M, Sajdl P, Rehacek Z, Malinka Z, Harazim P, and Stuchlik J (1988). Process for production of ergot alkaloids by Claviceps with induced increase of productive ability. CS pat appl. 4725 – 4788. Sajdl P and Rehacek Z (1975). Cyclization of chanoclavine-I by cell free preparations from saprophytic Claviceps strains. Folia Microbiol 20:365– 367. Schumann B, Maier W, and Groger D (1987). Characterization of some Claviceps strains derived from regenerated protoplasts. Z Naturforsch 42c:381 – 386. Socic and Gaberc-Porekar (1992). Biosynthesis and physiology of ergot alkaloids. In: Arora DK, Elander RP, Mukerje KG eds. Handbook of Applied Mycology, Fungal Biotechnology. New York: Marcel Dekker. pp 475 –515. Socic H, Gaberc-Porekar V, and Didek-Brumec M (1985). Biochemical characterization of the inoculum of Claviceps purpurea for submerged production of ergot alkaloids. Appl Microbiol Biotechnol 21:91 –95. Socic H, Gaberc-Porekar V, Pertot E, Puc A, and Milicic S (1986). Developmental studies of Claviceps paspali seed cultures for the submerged production of lysergic acid derivatives. J Basic Microbiol 26:533– 539. Taber WA and Vining LC (1959). Tryptophan as a precursor of ergot alkaloids. Chem Ind London: 1218 – 1219. Taber WA and Vining LC (1963). Physiology of alkaloid production by Claviceps purpurea (Fr.) Tul. Correlation with changes in mycelial polyol, carbohydrate, lipid and phosphorous containing compounds. Can J Microbiol 9:1 –14. Taylor EM and Ramsted E (1960). Biosynthesis of lysergic acid in ergot. Nature 188:494 –495.

99 Trejo-Hernandez MR and Lonsane BK (1993). Spectra of ergot alkaloids produced by Claviceps purpurea 1029c in solid state fermentation system. Inﬂuence of the composition of liquid medium used for impregnating sugar-cane pith bagasse. Process Biochem 28:23 –27. Trinn M, Manczinger L, Polestyukne NA, Kordik G, Pecsne RA, Zalai K, Beszedics G, Ferenczy L, Nagy L, Robicsek K, and Szegedi M (1990). Process for the elymoclavine production and preparation of new strain. HU pat 209325 (In Hungarian). Tsai H-F, Wang H, Gebler JC, Poulter CD, and Schardl CL (1995). The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis. Biochem Biophys Res Commun 216:119 – 125. Tudzynski P, Holter K, Correia T, Arntz C, Grammel N, and Keller U (1999). Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol Genet 261:133 – 141. Tudzynski P, Correia T, and Keller U (2001). Biotechnology and genetics of ergot alkaloids. Appl Microbiol Biotech 57:593– 605. Vasarhelyi G, Tetenyi P, Zambo I, Kinicky M, Balassa-Barkanyi I, Kiss J, Pecs-Razso A, and Zsoka-Somkuti E (1980). Strain of Claviceps purpurea, virulent on rye, for ergot alkaloids, especially ergocristine production and process for preparation of it. DE pat 3006989 (In German). Vining (1980). Conversion of alkaloid and nitrogenous xenobiotics. In: Economic Microbiology. New York: Academic Press. pp 523 –573. Vlcek V and Kybal J (1974). Equipment for cultivation of microorganisms. CS pat 172552 (In Czech). Votruba J and Pazoutova S (1981). Modelling of the optimal conditions for the maximum alkaloid synthesis. In: Abstract book, FEMS Symposium on Overproduction of Microbial Products, Hradec Kralove, Czechoslovakia, August 9– 14, 1981, p 104. Zalai K, Kordik G., Manczinger I, Pecsne RA, Beszedics G, Polestyukne NA, Ferenczy I, Olasz K, Szegedi M, and Trinn M (1990). Process for production of ergolen compounds, mainly ergometrine, and process of colour selection. HU pat 209324 (In Hungarian).

9 Fungi as Plant Growth Promoter and Disease Suppressor M. Hyakumachi / M. Kubota Gifu University, Yanagido Gifu, Japan

plant-pathogenic fungi, however, some fungi that are not normally considered as pathogens can also inhibit plant growth. These fungi have been termed indefinite pathogenic fungi, and in one study, isolates of Eupenicillium javanicum, Penicillium janthinellim, P. citreonigrum, and P. citrinum obtained from roots of zinnia plant caused a 23 –57% inhibition of the growth of the same plant (Yuen and Schroth 1986). Gamliel and Katan (1991) reported that almost all the fungi isolated from the rhizosphere and roots of tomato inhibited the growth of the plant. In contrast other soil-borne fungi, such as Trichoderma sp., Rhizoctonia solani, and others, can promote significant plant growth. Most of these PGPF have a high rhizosphere competence as a character. Because the genera found to be PGPF are common soil-borne fungi, there is a possibility that fungi having a similar role of PGPF exist widely in natural ecosystems. Some examples of plant growth promotion by PGPF are shown in Table 1. Most of these studies were quantified from the relative dry weights of root or above-ground part of treated plant seedlings with PGPF compared to nontreated ones over periods as short as 4 weeks. In some cases, significant growth promoting effects of PGPF were observed as increased yield of plants grown in fields over longer periods of 14 weeks or more (Shivanna et al. 1994).

INTRODUCTION

Reduction of the use of fertilizers and fungicides in agricultural production is necessary to help maintain ecosystems and to develop sustainable agriculture. The use of both bio-fertilizers and biocontrol systems can have minimal affect on the environment and such strategies have been widely researched. In soils, numerous microorganisms co-exist in association with plant roots. Some microorganisms live speciﬁcally in rhizosphere or on plant root surfaces, and these can have many effects on performance of the plant and may also affect the structure of the plant community. A unique microﬂora is particularly present around the plant root surface, where various substances are secreted. Most of the microorganisms distributed around plant root surface have a role in the decomposition of organic matter and some may suppress deleterious microorganisms, which could inhibit plant growth. Some of the root-associated microorganisms can promote plant growth, and they have been called “plant growth-promoting rhizobacteria” (PGPR; Kloepper et al. 1980) or “plant growth-promoting fungi” (PGPF; Hyakumachi 1994). The PGPR and PGPF are known to suppress some plant diseases. Similar effects are also observed in plants treated with mycorrhizal fungi, which have a symbiotic relationship with most plant species. Endophytes can also promote plant growth and these have recently been considered as potential biological control agents. In this chapter, fungi as known as PGPF, mycorrhizal fungi, and endophytic fungi, which act as plant growth promoters and disease suppressors are considered.

2 2.1

2.1.1

PGPF in Trichoderma

Isolates of Trichoderma harzianum and T. koningii have been shown to enhance seedling emergence in tomato with increased shoot and root dry weights when compared to nontreated control plants (Table 1) (Windham et al. 1986). These species also gave rise to increased shoot and root dry weights in tobacco (Table 1) (Windham et al. 1986). Isolates of T. viride have been reported to increase tomato plant height (Windham et al. 1986). Chang et al. (1986) have shown that isolates of T. harziamum enhanced seedling emergence in chilli pepper and promoted growth of tomato, chilli pepper,

FUNGI AS PLANT GROWTH PROMOTER PGPF

Many fungi isolated from soil can inhibit plant growth. Generally, the inhibition of plant growth is mostly caused by 101

R. nigricans F. roseum Phoma sp.

R. solani AG4

Sterile dark fungus Sterile red fungus

T. ciride Sterile black fungus

T. koningii

T. harzianum

Fungus

Cucumber

Lettuce Cotton Tomato Tomato Wheat Bean

Carrot

Tomato Tobacco Red pepper Periwinkle Bentgrass Tomato Tomato Tobacco Ryegrass Tomato Wheat Rye Wheat Wheat Rye Ryegrass Radish

Crop

40% 40% 30% 10 – 60% 10 – 60% 10 – 60% 13.4 – 19.8% 28.4 – 36.0% 80.0 – 97.7% 55.0 – 150.5% 58.4% 28.7% 42% 54% 46 – 77% 23 – 25% 11 – 52% 2.1 times 1.8 times

9.1 times 2.6 – 3.2 times 5.1 times 2.7 times 4.4 times

2.1 – 2.8 times 7.9 times

Growth promoting effect Increased dry weight, enhanced germination Increased dry weight, enhanced germination Enhanced germination Enhanced germination Increased dry weight and plant height Increased dry weight, enhanced germination Increased dry weight Increased dry weight, enhanced germination Increased dry weight and plant height Increased plant height Increased shoot dry weight Increased shoot dry weight Increased shoot dry weight Increased shoot fresh weight Increased shoot fresh weight Increased shoot fresh weight Increased shoot fresh weight Increased shoot dry weight Increased shoot fresh weight Increased shoot dry weight Increased shoot fresh weight Increased yield Increased shoot dry weight Increased shoot dry weight Number of grain Increased yield (Green house) Increased yield (Field) Increased fresh weight (6 weeks) Increased fresh weight (10 weeks)

Table 1 Growth promotion on plants treated with plant growth-promoting fungi (PGPF)

Windham et al. (1986) Windham et al. (1986) Chang et al. (1986) Chang et al. (1986) Hyakumachi (1994) Windham et al. (1986) Hyakumachi (1994) Windham et al. (1986) Hyakumachi (1994) Windham et al. (1986) Speakman and Kruger (1984) Speakman and Kruger (1984) Narita and Suzui (1991) Dewan and Sivasithamparam (1989) Dewan and Sivasithamparam (1989) Dewan and Sivasithamparam (1989) Sneh et al. (1986) Sneh et al. (1986) Sneh et al. (1986) Sneh et al. (1986) Sneh et al. (1986) Sneh et al. (1986) Lindsey and Baker (1967) Lindsey and Baker (1967) Shivanna et al. (1994) Shivanna et al. (1994) Shivanna et al. (1994) Hyakumachi (unpublished) Hyakumachi (unpublished)

References

102 Hyakumachi and Kubota

Plant Growth Promoter

and cucumber. Isolates of T. harziamum have also been used to enhance ﬂowering of periwinkle and to increase the number of ﬂowers per plant in chrysanthemum.

2.1.2

PGPF in Mycelial Fungi

Plant growth promotion has been obtained from isolates of mycelial fungi that do not produce any spores. These unidentified fungi have been termed sterile black fungus (SBF), sterile dark fungus (SDF), and sterile red fungus (SRF), and have been isolated from corn roots, wheat roots, and wheat and rye grass roots, respectively. Unidentified isolates considered to be SBF (Speakman and Kruger 1984) and SDF (Narita and Suzuki 1991) were shown to increase shoot dry weight in wheat and similarly isolates termed to SRF increased shoot wet weight in wheat (Dewan and Sivasithamparam 1989). Growth promotion by these unidentified fungi has been reported in other plants and isolates of SBF have been reported to increase shoot dry weight in barley (Speakman and Kruger 1984). Fungi considered as SRF have been reported to promote plant growth of rye, brome grass, chick pea, lupine, medic, pea, ryegrass, and clover, all of which are used as typical rotation crops with wheat, and resulted in an increased shoot fresh weight (Dewan and Sivasithamparam 1989). The mycelial isolates used in these studies have not been identified, although isolates of SRF are thought to be Basidiomycetes because of the presence of clamp connection. Strains of SBF and SDF are easily isolated from herbal plants as well as woody plants and their relationships to endophytic fungi are being currently considered.

2.1.3

PGPF in Rhizoctonia

A particular nonpathogenic strain of R. solani has shown growth promotion and significantly increased yield for various crops in field experiments (Sneh et al. 1986). These included increased wet and dry weights of radish roots and carrot roots, and increased weights of cotton fiber and wheat grains. In similar experiments with potato, although increases were observed in shoot and tuber weight until 63 –70 days after transplanting, there was no increase in yield at the time of harvest. Some isolates of binucleate Rhizoctonia have been found to be PGPF (Harris et al. 1993; Villajuan-Abgona et al. 1996).

103

nutrient-deficient one. The effect of PGPF was also observed in soil that had been converted to nutrient-rich by amendment with NPK fertilizer. The duration of the plant-growth promoting effect of PGPF in the treated plants is an important factor for the earlier application. Increased growth responses were observed in wheat treated with PGPF isolates during the seedling stage (2 weeks after sowing), vegetative stage (4 weeks), preflowering stage (10 weeks), and seed maturation stage (14 weeks) (Hyakumachi 1994; Shivanna et al. 1994). All of the isolates used increased plant height, and also significantly increased the ear-head length, weight, seed number, and biomass (Table 1) (Shivanna et al. 1994). In order to develop applications, it is important to isolate PGPF strains that (a) have high affinity for plants and can colonize their rhizosphere, (b) show high levels of plant growth promotion, and (c) offer consistent performance in field trials. Although an isolate with a wide host range is an ideal candidate, there is a requirement for isolates that show high specific effectiveness with an individual host plant.

2.2

Several hypotheses have been put forward for the mechanisms of plant growth promotion by PGPF, including (a) hormone production, (b) substrate degradation (mineralization), and (c) suppression of deleterious microorganisms.

2.2.1

Other PGPF

Isolates of Rhizopus nigricans and Fusarium roseum have been reported to increase shoot dry weight in tomato (Lindsey and Baker 1967). Soil conditions such as pH, water, nutrient and organic content, together with the presence of other micro-organisms are important considerations for the introduction of beneﬁcial micro-organisms into soil. Hyakumachi (1994) reported the plant growth promotion effect of PGPF occurred in sterilized or nonsterilized nutrient-deﬁcient and rich soils, potting soil, and most conspicuously in the

Hormone Production

Culture ﬁltrates of certain fungal species promote plant growth, due to the production of plant growth hormones by these fungi (Ram 1959). Growth promotion has been seen in some plants after treatment with mycelial exudates from PGPF strains of Trichoderma and SRF, and a gibberellin-like substance was reported to be involved (Gillespie-Sasse et al. 1991; Windham et al. 1986). Some strains of Phoma species have been found to produce abscisic acid, and this compound is also reported to promote plant growth. However, in general terms, these appear to have little relationship between the production of plant growth hormones and the ability of PGPF to promote plant growth.

2.2.2 2.1.4

Mechanisms of Plant Growth Promotion by PGPF

Mineralization

Close relationships have been shown between the reduction of barley grain weight due to PGPF, the subsequent growth promotion effect of PGPF, and their cellulase and starch degration activity (Hyakumachi 2000). Production of NH4-N and NO3-N in soil can also be increased by amendment with PGPF-infested barley grains. The total amount of nitrogen in PGPF infected-barley grains remains the same despite which PGPF isolate is used, however, the amount of NH4-N varies depending on the isolate, with the highest level being seen in grains infected

104

Hyakumachi and Kubota

with Phoma. The NH4-N levels later decreased in the order Phoma . Fusarium . Penicillium . Trichoderma . control: The amount of NH4-N was about 7.8 times higher in Phoma infested-barley grains than that of control (Hyakumachi 2000). Hyakumachi (2000) also demonstrated correlations between reduction of barley grain weight and cellulase activity, starch degradation activity of starch, and the dry weight of bentgrass. These results suggest that the mineralization of organic substrates by PGPF relates to the plant-growth promoting effect of those PGPF. The PGPF may therefore provide the plant with necessary mineral nutrients in an easily assimilating form.

2.2.3

Suppression of Deleterious Microorganisms

A remarkable plant growth promotion effect has been reported for ﬁeld-grown cucumbers, and this was attributed to the suppression of indigenous pathogenic Pythium spp. in the soil by PGPF (Hyakumachi 1994). The suppression of deleterious microorganisms by PGPF may therefore be one of the mechanisms of plant-growth promotion.

2.3

Mycorrhiza

As a deﬁnition of mycorrhizae, Smith and Read (1997) proposed “a symbiosis in which an external mycelium of a fungus supplies soil derived nutrients to a plant root.” Mycorrhizae are further divided into six types based on anatomical characteristics, which are: (a) arbuscular mycorrhizae (AM), (b) ectomycorrhizae, (c) orchid mycorrhizae, (d) ericoid mycorrhizae, (e) monotropoid mycorrhizae, and (f) arbutoid mycorrhizae. Some plants have requirement for mycorrhizae in order to complete their life cycle. Mycorrhizae may inﬂuence host plant survival in regeneration niches and mycorrhizae can also increase seed production, seed quality, and host and offspring vigor. Some types of mycorrhizae enhance host plant resistance against severe environmental conditions. The most widely studied and most-commonly encountered mycorrhizal systems are the ectomycorrhizae and arbuscular mycorrhizae.

2.3.1

Ectomycorrhizae

About 5000 fungi, Asco- and Basidiomycetes, are known to form ectomycorrhizal association with about 2000 species of woody plants (Kendrick and Berch 1985). Roughly 5% of the vascular plants are known to develop ectomycorrhizae and these associations are typically seen by the intercellular development of Hartig nets. Ectomycorrhizal fungi are known to enhance the uptake of water and nutrients by the host plant, and to promote plant growth. Growth effects have been observed in a broad range of forest trees, such as Douglas ﬁr, pine, and eucalyptus, with ectomycorrhiza associations forming in nurseries and in the ﬁeld. Laccaria laccota, Pisolithus tinctorius, Suillus plorans, Hebeloma cylindrosporum, and H. crustuliniforme have been used as soil

inoculations and their growth promoting performances were dependent on the host plant. Large increases in the growth of pines have been recorded in field experiments. For instance, 25–100% increases in growth have been reported for three pine species inoculated with Pisolithus tinctorius on five reforestration sites in the southern United States (Marx et al. 1977). Inoculation with Paxillus involutus has been associated with a marked increase in stem diameter and volume, especially with sessile oak at Bouxie`res where the volume almost doubled over 7 years (Garbaye and Churin 1997). The increase in growth resulting from inoculation with mycorrhizal fungi has been attributed to improved nutrition of the host plant in most cases. Ectomycorrhizal fungi are able to absorb and accumulate phosphorus, nitrogen, potassium, and calcium in the fungal mantles more rapidly and for longer periods of time than nonmycorrhizal feeder roots. Ectomycorrhizal fungi improve the efficiency of phosphorus uptake principally through the development of extramatrical hyphae, which increase the absorptive surface and effective rooting density of the plant. Ectomycorrhiza are likely to enhance N uptake where the fungus and host plant differ in their capacity to absorb and assimilate NO3-N. Mycorrhizal fungi generally have a preference for NH4-N, although a number of species can also utilize NO3-N (Plassard et al. 1991).

2.3.2

Arbuscular Mycorrhiza

Arbuscular mycorrhizal (AM) associations are due to Glomales, an order of Zygomycetes (Morton and Benny 1990). The order consists of 7 genera, Glomus, Entrophospora, Acaulospora, Archaeospora, Paraglomus, Gigaspora, and Scutellospora. Arbuscular mycorrhizal fungi develop arbuscules or hyphal coils within host plant cortical cells, and have a wide host range including many agricultural and horticultural crops worldwide. Growth promotion has been seen in many AM-associated plants including maize (Baltruchat 1987), tomato (Mohandas 1987), asparagus (Pedersen et al. 1991), Boston fern (Ponton et al. 1990), and gerbera (Wang et al. 1993). Despite the morphological differences between ecto- and arbuscular mycorrhizae, there appear to be many common features in their growth-promoting effects. Arbuscular mycorrhizal fungi develop extraradical hyphae that grow into the surrounding soil, increasing the potential of the root system for nutrient and water absorption, and improving the soil structure for better aeration and water penetration. One of the mechanisms of growth promotion by AM fungi involves the transport of phosphorus by AM fungi from the soil to the plant. Direct measurements of phosphorus transfer by AM fungal hyphae have been made by Jakobsen (1994) and Schweiger et al. (1999). Colonization of roots by AM fungi modiﬁes the growth response of the plant and increases supplies of phosphorus (Abbott et al. 1995), however, some studies have shown that effectiveness, in terms of plant growth promotion, is not related to the extent of host root colonization (Jensen

Plant Growth Promoter

1982; Sanders and Fitter 1992). Efﬁcient phosphorus uptake has been found to be more closely related to the quantity of mycelium partitioned into the extraradical phase of the fungi (Abbott and Robson 1985; Morin et al. 1994). Jakobsen et al. (2001) reported that the phosphorus transport capacity of AM fungi is related not only to colonization rate, but also to the transport character of AM fungi themselves. The AM fungi cause few changes to root morphology, but the physiology of the host plant may change signiﬁcantly. Tissue concentrations of growth-regulating compounds and other chemical constituents change, photosynthetis rates increase, and the partitioning of photosynthate to shoots and roots changes (Bethlenfalvay 1992). Allen et al. (1980) demonstrated differences in cytokinin content between Bouteloua gracilis plants with and without associated Glomus fasciculatus. They also reported quantitative and qualitative changes in GA-like substances in the leaves and roots of AM-associated plants (Allen et al. 1982). Incleases in auxin, cytokinin, GA and B-vitamin production have also been reported in plants associated with ectomycorrhizal fungi (Crafts and Miller 1974; Slankis 1973; Strzelczyk et al. 1977).

2.4

Endophyte

A widely accepted definition of an endophyte is; “endophytes symptomlessly colonize the living, internal tissues of their host, even though the endophyte may, after an incubation or latency period, cause disease” (Petrini 1991). This definition includes virtually any microbe that colonizes the internal tissues of plants. For example, some plant-pathogenic fungi, such as the smut fungi, can be defined as endophytes unless the plant shows symptoms after the infection (Stone et al. 2000). Endophytes are generally known to enhance plant tolerance to environmental stresses, damage from harmful insects, and diseases caused by pathogens and nematodes. There are a few studies on the plant-growth promoting effect of endophytic fungi. Yetes et al. (1997) observed a slight but significant increase in plant weight, shoot height, and shoot diameter in Fusarium moniliforme-infected plants, 28 days after planting compared to uninoculated control plants. Pinus contorta inoculated with Phialocephala fortinii increased uptake of phosphorus and nitrogen, that resulted in enhanced growth of inoculated plants compared with noninoculated plants (Jumpponen and Trappe 1998).

3 3.1

FUNGI AS DISEASE SUPPRESSOR PGPF

Almost all the PGPF reported so far have shown a pronounced suppressive effect against soil-borne diseases. One example of this is the suppression by Trichoderma harzianum of damping-off disease on barley, cucumber, radish, and tomato caused by Pythium ultimum (Ahmad and Baker 1988). Nonpathogenic R. solani AG4 has been reported to suppress

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damping-off disease caused by virulent R. solani and R. zeae by 76 –94% on cotton, radish, and wheat. The sterile fungi, SBF, SDF, and SRF, have been shown to decrease the occurrence of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici (Dewan and Sivasithamparam 1989; Narita and Suzuki 1991; Speakman and Kruger 1984). The PGPF isolated from zoysiagrass rhizosphere have been shown high suppressive ability against soil-borne diseases caused by Pythium aphanidermatum, P. irregulare, R. solani, Sclerotium rolfsii, Fusarium oxysporum f. sp. melonis, F. o. f. sp. cucumerinum, G. graminis var. tritici and Cochliobolus sativus (Hyakumachi 1994). When cucumber plants inoculated with PGPF isolates from zoysiagrass rhizozphere, disease suppression was observed against the air-borne pathogen, Colletotrichum orbiculare (Meera et al. 1994). In this work the PGPF were applied to plant roots and leaves were used for pathogen inoculation thereby the PGPF and pathogen were physically separated. The result therefore suggests that induced systemic resistance is involved as one of the mechanisms for disease suppression by PGPF. The PGPF isolates from zoysiagrass rhizosphere, Trichoderma, Fusarium, Penicillium, Phoma, and sterile fungi, all provided signiﬁcantly protection to air-borne anthracnose caused by C. orbiculare, bacterial angular leaf spot caused by Pseudomonas syringae pv. lacrymaus, and soil-borne Fusarium wilt by F. oxysporum f. sp. cucumeris (Koike et al. 2001). In the case of Fusarium wilt, a split-root system was used to ensure physical separation of PGPF and pathogen, and to assess induced resistance.

3.2

Mycorrhizae

Initial evidence for the role of ectomycorrhizal fungi in disease suppression was provided by a number of ﬁeld observations that showed mycorrhizal-associated seedlings or trees of both angiosperms and gymnosperms were more resistant to root pathogens than their nonmycorrhizal counterparts (Marx 1973). Ectomycorrhizal roots of various Pinus spp. and Sitka spruce (Picea sitchensis) seedlings were resistant to infection by a Rhizoctonia sp. that could readily infected nonmycorrhizal feeder roots (Levisohn 1954). Richard et al. (1971) suggested that the presence of ectomycorrhizal fungus, Suillus granulatus in the substratum completely prevented any negative effect of endophytic Mycelium radicis-atrovirens on Picea mariana seedlings. Hashimoto and Hyakumachi (2001) also suggested that the ectomycorrhizal fungi suppressed the deleterious effect of endophytic M. radicis-atrovirens on Betula platyphylla var. japonica seedlings. Arbuscular mycorrhizae associations have been shown to reduce damage caused by soil-borne plant pathogens. Although few AM isolates have been fully studied, some appear to be more effective than others. Furthermore, the degree of protection varies with the pathogen involved, and can be modiﬁed by soil types and other environmental

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conditions. Trotta et al. (1996) reported that the AM fungus, Glomus mosseae, reduced adventitious root necrosis and necrotic root apices caused by Phytophthora nicotianae var. parasitica by 63 –89%. The AM associations are also known to limit the damage by bacterial pathogens and pathogenic root nematodes (Garcia-Garrido and Ocampo 1989; Hussey and Roncadori 1982), however, the results are not consistent.

3.3

Endophyte

The enhanced resistance for disease shown by some endophyte-infected plants is generally considered to result from the production of defense compounds by the endophyteplant infection. An example of this is the inoculation of maize kernels by endophytic Fusarium monilimorme, which is reported to protect against infection by pathogenic F. graminearum (Van Wyck and Scholts 1988). Nonpathogenic, endophytic strains of F. oxysporum isolated from suppressive soils have been used as biological control agents for manage diseases caused by pathogenic Fusarium species on watermelon, cucumber, celery, and other crops (Larkin et al. 1996; Schneider 1984). In each case, these fungi were endophytes of the hosts they protected. Endophytic Heteroconium chaetosprira is reported to almost completely suppress clubroot formation in Chinese cabbage caused by Plasmodiophora brassicae (Narisawa et al. 1998).

4 4.1

MECHANISMS OF DISEASE SUPPRESSION BY FUNGI Antagonism

Many reports have shown that the growth-promoting effect of PGPF is due to their ability to suppress harmful microorganisms in the soil. It is generally accepted that hyperparasitism, antibiosis, and competition are all involved in the antagonistic activities of PGPF. The mechanisms of disease suppression by PGPF isolated from zoysiagrass are shown in Table 2. The isolates of PGPF did not show hyperparasitism to other fungi. In some cases involving Trichoderma there was a relation between antibiotic activity and disease suppression, however in most cases, disease suppression was closely related with the ability to compete for infection courts or nutrient on the surface of the plant root. The production of antagonistic substances is thought to be one of the mechanisms of protection provided by ectomycorrhizal fungi. As an example of this, antibacterial activities have been demonstrated for Paxillus involutus and Hebeloma crustuliniforme in pure culture (Marx 1973) and for Cenococcum graniforme in mycorrhizal symbiosis (Krywolap et al. 1964). The antibiotic effect of mycorrhizal fungi was attributed to the production of organic acids as demonstrated for P. involutus (Duchesne et al. 1989). Olsson et al. (1996) demonstrated that presence of the ectomycorrhizal mycelium decreased bacterial activities as

Table 2 Mechanisms of disease suppression against pathogenic fungi and bacteria by plant growth-promoting fungi (PGPF) isolated from zoysiagrass Pathogenic fungi R. solani

P. irregulare

S. rolfsii

F. oxysporum f. sp. cucumerinum

C. orbiculare

P. syringae pv. lachrimans

PGPF

Hyperparasitism

Antibiosis

Competition

Induced resistance

T. harzianum Phoma sp. F. equiseti T. harzianum Phoma sp. F. equiseti T. harzianum Phoma sp. F. equiseti T. harzianum Phoma sp. F. equiseti T. harzianum Phoma sp. F. equiseti P. simplicissimum T. harzianum Phoma sp. F. equiseti P. simplicissimum

2* 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

^ 2 2 ^ 2 2 ^ 2 2 ^ 2 2 ^ 2 2 2 ^ 2 2 2

þ þ þ þ þ þ þ þ þ 2 þ 2 2 2 2 2 2 2 2 2

NT** NT NT NT NT NT NT NT NT þ þ þ þ þ þ þ þ þ þ þ

* þ /2: Effective/not effective; ** NT: Not tested.

Plant Growth Promoter

measured by using the thymidine incorporation technique. Zak (1964) suggested that ectomycorrhizal fungi may: (a) utilize surplus carbohydrates in the root thereby reducing the amount of nutrients stimulatory to pathogens, (b) provide a physical barrier, i.e., the fungal mantle, to penetration by pathogen, (c) secrete antibiotics inhibitory to pathogens, and (d) support along with the root, a protective microbial rhizosphere population. Marx (1969) suggested that inhibitors produced by symbiotically infected host cortical cells may also have a function as inhibitors of the infection and spread of pathogen in ectomycorrhizal roots. Competition for colonization sites, direct antibiosis, nutritional aspects, and plant defense reaction have all been considered as possible mechanisms in disease suppression by AM fungi (Azcon-Aguilar and Barea 1996). However, these mechanisms are still poorly understood. Antifungal activities are thought to be involved in mechanisms of disease suppression by endophytic fungi. The endophytic fungi Neotyphodium coenophialum and N. lolii have been shown to form an inhibition zone under dual culture with the pathogenic fungi, Colletotrichum graminicola, Rhizoctonia cerealis, R. zeae, etc. (Siegel and Latch 1991). These results suggest that these endophytic fungi produce antifungal substances. Volatile compounds were collected from both endophyte-infected and endophyte-free tall fescue, and the sheath of endophyte-infected plants was found to produce high levels of 1-octen-3-ol, a characteristic fungal toxic volatile compound derived from lipid peroxidation in fungi, which was absent in endophyte-free plants (Yue et al. 2001). Hydroxamate siderophore synthesis by P. fortinii, a typical dark septate fungal endophyte, was reported by Bartholdy et al. (2001). Iron is an essential micronutrient for almost every organism and siderophore synthesis by P. fortinii may be expected to play a key role in iron nutrition for the plant, resulting in a lack of available iron for pathogens.

4.2

Induced Resistance

Induced-systemic resistance has been observed on cucumber plants treated with PGPF from zoysiagrass (Hyakumachi 1997; Koike et al. 2001; Meera et al. 1994). Almost all of these PGPF could induce resistance against anthracnose in cucumber. In contrast, Ishiba et al. (1981) reported that only 1.9 – 2.4% of the soil fungi isolated from cucumber rhizosphere, were able to induce systemic resistance against anthracnose in cucumber plants. Different types of PGPF have been isolated from all over the world and it would be interesting to know if any of these have as high performance of induced systemic resistance as PGPF isolated from zoysiagrass. Recently, induced systemic resistance caused by binucleate Rhizoctonia and Trichoderma has been reported in plants. Some isolates within these fungi have growthpromoting ability and others have been used as biological control agents. The induced resistance on cucumber caused by PGPF isolated from zoysiagrass can last as long as 9 weeks

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under glass house conditions and for up to 6 weeks under field conditions (Meera et al. 1995). Lignin deposition is known as one of the mechanisms of induced systemic resistance (Hammaerschimidt and Kuc´ 1982). Koike et al. (2001) reported that lignification of cucumber seedling hypocotyls was induced by culture filtrates of PGPF, following challenge inoculation with C. orbiculare. The result showed enhanced lignin deposition in cucumber after infection by C. orbiculare as compared to the control. The elicitor activity of culture filtrates of PGPF has been evaluated by chemiluminescence to determine the emission of active oxygen species from tobacco callus and cucumber fruit disks (Koike et al. 2001). The oxidative burst is characterized by a rapid and transient generation of active oxygen species immediately following fungal elicitor treatment. From these results, the . 12,000 MW fraction and both . 12,000 MW fraction and lipid fraction from the culture filtrate elicited the highest superoxide generation, respectively. A high correlation between superoxide generation ability and lignification ability was reported. Localized and induced-systemic resistance against Phytophthora parasitica caused by the AM fungi, Glomus mosseae, has been observed in tomato roots (Cordier et al. 1998). The phenomena were demonstrated by use of a split-root experimental system. Decreased pathogen development in mycorrhizal and nonmycorrhizal parts of the root system was associated with an accumulation of phenolics and plant cell defense responses. G. mosseae-containing cortical cells in the mycorrhizal tissues were immune to the pathogen infection and exhibited a localized resistance response with the formation of cell wall appositions reinforced by callose adjacent to intercellular hyphae. The systemically induced resistance in nonmycorrhizal root parts was characterized by elicitation of host wall thickenings containing nonesterified pectins and PR-1a protein in reaction to the intercellular hyphae of the pathogen. Systemic resistance was also characterized by the formation of callose-rich encasement material around P. parasitica hyphae that were penetrating root cells and PR-1a protein was detected in the pathogen wall only in these tissues. None of these cell reactions were observed in nonmycorrhizal pathogen-infected root systems, where disease development resulted in host cell death. Increased chitinase activities have also been reported in AM symbiosis as part of the induced defense reaction by these mycorrhizal fungi. Pozo et al. (1999) studied b-1,3-glucanase in tomato roots which were either colonized by AM fungi and/or infected by the pathogen Phytophthora parasitica. b-1,3-glucanase activity was higher in mycorrhizal roots compared to the nonmycorrhizal roots. Nonmycorrhizal roots infected by P. parasitica showed high levels of activity but the pathogen did not induce b-1,3-glucanases in AM colonized roots. There was strong evidence to suggest that these hydrolases are antifungal proteins. Increased chitinase activities have also been reported in ectomycorrhizal symbiosis (Albrecht et al. 1994; Sauter and Hager 1989).

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CONCLUSIONS

The original purpose of isolating beneﬁcial microorganisms from soil, especially from the rhizosphere of plants, was to obtain microorganisms, which showed a growth promotion effect in plants. In addition to this effect, subsequent studies have investigated direct suppressive effects on pathogens. Due to the large amount of research undertaken over the years, we now know that this view is limited. This chapter has focused on PGPF, mycorrhizal fungi, and endophytic fungi, all of which show efforts as plant growth promoters and disease suppressors, and the mechanisms of mineralization, hormone production, antagonism, and induced resistance have been considered. These mechanisms are commonly involved in plant-growth promotion or disease suppression by PGPF, mycorrhizal fungi, and endophytic fungi to some degree depending on the fungi studied. These fungi are symbiotic to plant roots, and so they offer advantages for keeping the plant healthy for long durations. In addition, mineralization and hormone production by these fungi have the potential to substantially improve agricultural productivity and to reduce environmental costs. The reduction in nitrogen fertilizer usage due to the use of these fungi can be expected to substantially reduce nitrate pollution of ground and surface water. In addition to these antagonistic activities, induced resistance in plants treated with these fungi broadens the potential range of pathogens that may be controlled. This resistance coupled with long-term colonization should provide important new tools for highly economical pest control with minimal environmental pollution.

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10 Challenges and Strategies for Development of Mycoherbicides Susan M. Boyetchko / Gary Peng Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada

detail some of the limitations associated with bioherbicides (Auld and Morin 1995; Makowski 1997; Mortensen 1998), including biological, environmental, and technological constraints. Critics have attributed these constraints to the lack of further development of many biocontrol agents. This review will provide an update on the status of mycoherbicide research, summarize some of the challenges encountered, and provide some thoughts on potential new approaches that may be used to address these challenges in order to advance the development of promising mycoherbicide candidates.

INTRODUCTION

The application of biological control for the management of weed populations has generally been viewed as an environmentally sound additional approach to chemical herbicides (Boyetchko et al. 2002; Mortensen 1998; Rosskopf et al. 1999). Bioherbicides are often described as the intentional use of plant pathogens that are mass-produced, formulated, and applied at high inoculum rates in a similar fashion as chemicals. Although a variety of microbial agents may be used, host-specific fungal pathogens often referred to as mycoherbicides, have been studied more extensively for biocontrol of weeds. In comparison, classical biological control involves the importation of natural enemies and relies on the natural survival, dissemination, and self-perpetuation of the living agent for control of weeds below ecological thresholds. The classical approach is often considered more appropriate for low management systems such as pasture and rangeland where site disturbance is minimal, while bioherbicides are ideal for single-season management of agricultural and forest weeds where site disturbance is the norm. Despite many economic, social, and environmental benefits ascribed to biological control, it is reasonable to ask why more bioherbicide or mycoherbicide products are yet to become widely available in the marketplace. Many researchers would argue that there has been a great deal of progress, with several additional microbial agents identified as potential bioherbicides and innovative improvements in mass-production, formulation, and application of living organisms. Despite the various accomplishments by researchers worldwide (Boyetchko et al. 2002; Charudattan 2001), the question remains whether we have made significant advancements in bioherbicide research that would facilitate increased adoption of this technology. Several reviews have discussed in great

STATUS OF BIOHERBICIDES

Several recent reviews have provided an overview on various bioherbicide projects being conducted around the globe (Boyetchko 1999; Boyetchko et al. 2002; Charudattan 2001; Rosskopf et al. 1999). Eight bioherbicides have been registered in various countries over the last two decades with several other microbial candidates in various stages of evaluation and development (Table 1). Devinew and Collegow, the ﬁrst mycoherbicides registered in the United States are currently marketed by Encore Technologies (Minnetonka, MN), while Stumpoutw, a wood-decaying fungus used to control resprouting of Acacia spp., is commercially available in South Africa. Chondrostereum purpureum is a wound pathogen that reduces regrowth of competing hardwood tree species and is marketed as BioChone by Koppert Biological Systems in the Netherlands. A Canadian strain of the pathogen is also currently undergoing registration approval through the Canadian Pest Management Regulatory Agency and U.S. EPA and will be sold as Chontrolw by MycoLogic (W.E. Hintz, MycoLogic, Inc., personal communication). Another mycoherbicide 111

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Table 1 Examples of mycoherbicide agents at various stages of development and commercialization Pathogen (Trade Name w or e)

Target weed

Country

C. gloeosporioides f. sp. aeschynomene (Collegow) P. palmivora (Devinew) C. laeve (Stumpoutw) C. purpureum (BioChone) C. gloeosporioides f. sp. malvae (Mallet WP)a P. canaliculata (Dr. Biosedgew) C. purpureum (Chontrolw) A. destruens (Smolderw)

Northern Jointvetch Stranglervine Black & golden wattle Hardwood tree species Round-leaved mallow Nutsedges Hardwood tree species Dodder

USA USA South Africa Netherlands Canada, USA USA Canada, USA USA

Status Commercially available

Registered, not commercially available Precommercial development

a Originally registered in Canada as BioMalw by Philom Bios; licensed to Encore Technology for registration as Mallet WP in Canada and the United States. Mycoherbicide not being further developed due to technical difﬁculties in mass production.

currently undergoing review is Alternaria destruens, under the name Smolderw, for control of dodder. Other examples of mycoherbicides have been discussed in greater detail by Boyetchko (1999), Boyetchko et al. (2002), and Rosskopf et al. (1999). Charudattan (2001) has also compiled a comprehensive list of bioherbicide projects worldwide. While the number of commercial bioherbicides appears to be limited, Charudattan (1991) calculated a success ratio of 20:1 for bioherbicide development compared to the success rate of less than 1% for chemical herbicide compounds evaluated and developed by chemical companies. This success ratio begins to look even more encouraging for bioherbicides when developmental costs are taken into consideration. Charudattan (2001) estimated that the resources and capital required for a chemical company to conduct research and development and register chemical herbicides is approximately US$ 50 million in comparison to US$2 million for bioherbicides. Nevertheless, a very small portion (i.e., less than 1%) of the commercially available weed control products are represented by bioherbicides and investment in these microbial-based products has largely been by small-tomedium sized enterprises. Government and university institutions have invested infrastructure and expertise in this area and it has been through such efforts that this technology has been transferred to industry.

CHALLENGES IN BIOLOGICAL CONTROL

Several reviews have provided a suggested list of desirable characteristics for a bioherbicide candidate in order for it to be successful (Charudattan 1991; Makowski 1997; Mortensen 1998). Generally, these traits include: (a) a narrow host range, (b) ease of use, (c) genetic stability, (d) ability to mass produce inoculum cost-effectively with long shelf life, and (e) ability to be fast-acting with predictable ﬁeld performance and provide sufﬁcient weed control comparable to chemical herbicides. Many of these traits, along with the term bioherbicide (mycoherbicide), may create unrealistic expectations that all bioherbicides should eradicate weed

populations, similar to chemical herbicides (Auld and Morin 1995). The challenges that have limited the advancement of bioherbicides have been categorized into four constraints: (a) biological, (b) environmental, (c) technological, and (d) commercial. While the commercial consideration is important, this review will focus on addressing the other three constraints. Researchers can make pragmatic decisions on the selection of an appropriate target weed that may have impact on the market decisions by industry to invest in the development of bioherbicide agents, but the regulatory environment for registration of such products is often affected by political will and/or policy of individual governments.

3.1

Biological Factors

Weeds are inherently variable by nature, with many weed species possessing several biotypes. For this reason, the genetic diversity of weed populations can present several challenges when researchers evaluate specific isolates or strains of fungal pathogens (Auld and Morin 1995; Boyetchko et al. 2002). Such is the case with yellow nutsedge that was found to have extensive genetic variability within and between populations and, therefore, greater variability in susceptibility to the bioherbicide agent Puccinia canaliculata, compared to purple nutsedge populations (Horak et al. 1987; Okoli et al. 1997). The success of Colletotrichum gloeosporioides f. sp. aeschynomene as the mycoherbicide Collegow was attributed to the uniform susceptibility of the weed northern jointvetch (Templeton et al. 1984). A better understanding of the target weed population structure will contribute to the effective search for bioherbicide agents with consistent performance on genetically diverse weed species (Boyetchko et al. 2002). Another consideration is the view that all bioherbicide candidates must be specific to one particular weed species. The advantage of a highly hostspecific bioherbicide candidate is the assurance that nontarget, beneficial plant species, or crops closely related to the weed will not be affected (Makowski 1997; Mortensen 1998). However, a strict host-range requirement may not be

Mycoherbicides

economically feasible because the majority of agroecosystems are comprised of multispecies weed communities (Frantzen et al. 2001). Plurivorous pathogens may be used safely under certain circumstances when they can be separated sufficiently from nontarget hosts in space and time (De Jong et al. 1999). Plant architecture and morphology have played a role in the success or failure of bioherbicide agents. The majority of weed species selected as targets for biological control have been broad-leaved weeds using foliar fungal pathogens (Charudattan 1991; 2001). Grasses are considered more difficult to control because the meristem is covered by a leaf sheath thereby prohibiting direct attack by the pathogen (Greaves and MacQueen 1992). Grass weeds are also closely related to many crops (e.g., cereals) in which they occur, making selectivity of mycoherbicide agents more challenging (Wapshere 1990). A particularly important factor with perennial weeds is their regeneration via rhizomes and stolons, which makes long-term weed control difficult (Greaves and MacQueen 1992). This often results in the weed out-growing the disease caused by foliar-applied bioherbicide candidates. However, there are many prospective soilborne fungal and bacterial agents that may be used as pre-emergent bioherbicides to control Poaceae and perennial weed species (Boyetchko et al. 2002). Other physical barriers such as leaf hairs and waxy cuticle layers may act as impediments to infection and establishment of fungal pathogens on the phyllosphere (Auld and Morin 1995). Although high inoculum applications have been used to overcome biological constraints resulting from low infection efficiency or virulence of the pathogen, these rates may not be technologically feasible due to plugging of spray equipment or economically viable from a production standpoint.

3.2

Environmental Factors

Two major limiting factors that have an impact on mycoherbicides are temperature and moisture requirements (Auld and Morin 1995; Makowski 1997; Mortensen 1998). TeBeest et al. (1992) considered temperature to be less important than moisture in most cases because many fungal pathogens will infect plants over a broad range of temperatures. However, there is often an interaction between temperature and moisture that has a greater effect than temperature alone. Free moisture and leaf wetness duration can signiﬁcantly affect the ability of the fungal pathogen to germinate, produce penetration structures, and ultimately cause plant infection. This requirement of leaf wetness duration often increases when temperatures are in suboptimal ranges. Early evaluations of potential mycoherbicide candidates have often been conducted where dew periods in excess of 12 h are provided to ensure a high rate of infection on the weed (Lawrie et al. 1999; McRae and Auld 1988; Morin et al. 1990a). Green and Bailey (2000a,b) reported that A. cirsinoxia for control of Canada thistle required at least 8 h

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of continuous leaf wetness. The pathogen infected the weed under a broad range of temperatures (10 –308C) when free water or high relative humidity was present, but long durations of intermittent leaf wetness was detrimental to survival of germlings and, therefore, not conducive to high bioherbicidal activity. Some pathogens such as rust fungi are wind-dispersed and generally require less moisture than water-disseminated pathogens such as Colletotrichum spp. where spores are contained in a mucilaginous matrix (Hasan and Wapshere 1973; TeBeest 1991). Makowski (1997) further reiterated that evaluating the impact of environmental parameters under controlled conditions may provide clues about potential performance of bioherbicide candidates, but further investigations under variable ﬁeld conditions where these factors are difﬁcult to control are more complicated.

3.3

Technological Factors

The feasibility of commercializing bioherbicide agents has often been dependent on the ease and economics of massproducing and formulating large amounts of viable, stable, and highly efficacious microbial propagules (Auld and Morin 1995; Mortensen 1998; Slininger et al. 1998). It is generally believed that submerged liquid fermentation is the most efficient commercial mass-production method for most biocontrol agents (Jackson et al. 1996). Although general guidelines or common fermentation ingredients for medium composition are available in public domains (Stanbury et al. 1995), most commercial fermentation protocols are custom designed for a specific organism and details are generally treated as trade secrets. However, when a company licenses a bioherbicide technology, the ability to mass-produce the agent economically and market it at a cost that is affordable to farmers, represents a strong determining factor in its development and commercialization potential. Lack of reliable field performance due to inadequate formulation and application technology has often been cited as a major reason for the lack of progress in bioherbicides (Greaves et al. 2000; Peng et al. 2001). It has been claimed that suitable formulation technology may help address some of the environmental constraints, particularly moisture requirements that often hinder the advancement of bioherbicide candidates beyond the discovery and evaluation phase. Propagules of foliar-applied fungal agents generally require free water to germinate and penetrate weeds. This leaf wetness requirement and its interaction with the ambient temperature often determine the outcome of a mycoherbicide application (Zhang and Watson 1997). Often, researchers evaluate mycoherbicide candidates by spraying till runoff, thereby overestimating the potential of many bioherbicide candidates at the early stage of evaluation (Lawrie et al. 1999). Less stringent dew requirement may be an advantage for foliar agents, especially in semiarid climates such as the Canadian prairies, where rainfall is infrequent at critical periods (e.g., in the spring). Intermittent dew occurs more often than

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continuous dew, but germinated fungal spores under short dew periods are more sensitive to desiccation and UV irradiation (Green and Bailey 2000b), therefore, survival of the germlings is going to be the key to successful infection under intermittent dew.

these processes may impact the others. At any stage of evaluation, refinements or modifications to these processes, even minor, may result in significant improvements in mycoherbicide performance.

4.1

APPROACHES FOR OVERCOMING CONSTRAINTS

Most mycoherbicide programs are initiated through surveys to discover fungal pathogens exhibiting bioherbicidal potential, followed by a series of biological and ecological assessments to determine the feasibility of mycoherbicide candidates. However, a pragmatic approach of selecting appropriate mycoherbicide candidates is required and should be considered as a continuum amongst several factors that will ultimately influence the field performance of the fungal pathogen (Figure 1). While nutritional and physical factors are vital during fermentation, down-stream processing is equally important for an efficient massproduction system. Selection of appropriate formulation technology is influenced by fermentation processes and should be based on critical limitations such as shelf life and environmental constraints encountered with mycoherbicide development. Formulation ingredients can affect delivery and application of the mycoherbicide agent. If these ingredients result in the inability to deliver the fungal pathogen to the target weed (e.g., high viscosity and ultimate plugging of equipment), effective weed control will not be achieved. Often, mass-production, formulation, and application can be interrelated, therefore, changes in one of

Selection and Improvement of Bioherbicide Agents

On average, chemical companies screen more than 60,000 compounds before a new active ingredient of pesticide can be determined. The number required for screening biocontrol agents should be less due to a relatively smaller range of variations amongst naturally occurring fungal populations. However, if we are to identify “nature’s best,” a systematic approach is essential during the exploration and discovery phase to thoroughly evaluate the biodiversity. This diversity provides excellent opportunities for ﬁnding fungal strains with potential suitable traits for biocontrol (Avis et al. 2001; Weidemann and TeBeest 1990). Substantial variations may exist amongst different strains of a fungal species in terms of its virulence and responses to environmental variables (Sands et al. 1997; Tessmann et al. 2001). To be effective, critical traits for selection should be clearly identiﬁed and sensitive bioassays developed. Pathogen strains with high levels of virulence may exist in nature at low frequencies due to higher extinction rates (Yang and TeBeest 1992). Results by Yang and TeBeest (1992; 1993) indicated that pathogens showing high virulence along with important epidemiological traits such as rapid infection rates and dispersal are more likely to be candidates of a successful mycoherbicide agent. The success

Figure 1 Strategic framework for evaluation and development of mycoherbicides.

Mycoherbicides

of C. gloeosporioides f. sp. aeschynomene for control of northern jointvetch was partially attributed to its ability to easily spread as an endemic pathogen. Yang and TeBeest (1995) further demonstrated a rapid rate of mortality of the weed as the number of pathogen lesions per plant increased from a single lesion. Therefore, more aggressive and virulent isolates of a pathogen with high infection efficiency, shorter latent periods, and better sporulation from diseased tissues should be selected from amongst the pathogen population. Chemical and physical methods have been used to create fungal mutants with acquired new traits such as elevated antibiotic production (Graeme-Cook and Faull 1991) or increased biocontrol efficacy (Palani and Lalithakumari 1999). Stability or low reversion frequency was observed with some mutants but, in general, stability can be a concern with chemical and physical mutagenesis (Wibowo et al. 1999). Ziogas et al. (1995) reported UV-induced mutants of Nectria haematococca with variable tolerance to fungicides that showed the same level of fitness as wild types as expressed by the rate of growth and virulence on squash seedlings. Mutagenesis is apparently a quick way of creating new fungal strains with variable traits. However, efficient bioassay systems based on the understanding of critical constraints are needed for an effective selection strategy. It is not uncommon for induced mutants to have lower competitiveness than the wild type due to reduced infectivity or reproductivity (Yang and TeBeest 1995), but judicious use of this technique may help develop new mycoherbicide strains that overcome critical hurdles such as those demonstrated with the plurivorous pathogen Sclerotinia sclerotiorum (Miller et al. 1989).

4.2

Fermentation/Mass Production

There are potentially three fermentation systems that may be used for mass production of mycoherbicide agents: submerged liquid culture, solid substrate fermentation, and two-phase system (Auld 1993a). At the industrial level, liquid fermentation is the most common method for economical production of microbial inoculum. Two commercial mycoherbicides, Collegow and Devinew, are manufactured this way in the United States (Churchill 1982; Stowell 1991). By understanding the importance of nutritional and environmental factors on induction of fungal sporulation, spore yield, and bioherbicidal efficacy, rational approaches can be taken to develop the most efficient liquid production procedures for mycoherbicide agents. Jackson and Bothast (1990) reported that carbon concentrations and C/N ratios are the key to sporulation of the mycoherbicide agent C. truncatum. Carbon concentrations ranging from 0.4 to 1.5% gave highest spore yields while higher concentrations (2 –4%) inhibited sporulation. Similarly, a C/N ratio at 15:1 was more favorable for sporulation than 40:1 or 5:1. In a further study, it was revealed that nutritional aspects impacted not only spore yield, but also spore efficacy of C. truncatum in controlling hemp sesbania (Jackson et al. 1996). Morin et al. (1990b) also observed that

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sporulation of Phomopsis convolvulus was completely inhibited when the C/N ratio was reduced from 1:1 to 1:5 using modified Richards medium, but the effect on spore efficacy was not reported. By understanding these impacts, fermentation procedures can be fine-tuned to maximize the production and potency of mycoherbicide agents. One of the drawbacks with liquid fermentation is that down-stream processing can be more complicated and costly. Large centrifuges are normally required to spin off spores and often a large number of spores can be imbedded in the mycelial biomass (Auld 1993a). Following recovery from the fermentor, it is usually necessary to dry the spores for longterm storage but retaining spore viability during the drying process may not be easy. Generally, spores should be dried rapidly and gently, but the drying conditions will vary with each organism. Drying methods frequently used include freeze, air, spray, or fluid-bed drying, or a combination of these methods (Churchill 1982). Solid substrate fermentation is used less commonly in commercial production of microbes except for the mushroom spawn industry. Often defined nutrients are added with liquids or solid materials such as vermiculite or paper pellets (Auld 1993a). Various cereal grains have been used to produce fungal inoculum and it is relatively easy to quantify and disperse the inoculum on these solid substrates (Boyette et al. 1991). In some places nutritive solid substances such as nutshells or straw may be available locally at low cost. Higher labor costs, difficulties in maintaining sterility, lack of control of cultural conditions, and recovery of spores from the substrate are inherent problems with solid substrate fermentation (Churchill 1982). Pfirter et al. (1999) evaluated a variety of solid substrates and found that Stagonospora convolvuli, for control of field bindweed, sporulated the best on cous-cous (cracked hard wheat) followed by maize semolina, yielding 5 £ 108 spores/g substrate and 3 £ 108 spores/g, respectively. Morin et al. (1990b) also reported the production of 7 £ 108 conidia/g with P. convolvulus using pot barley grain as a solid substrate. They also compared liquid and solid fermentation methods and found that conidia produced using both systems were morphologically similar and there were no differences in pathogenicity. Particle size, moisture content, and temperature appear critical for successful solid substrate production. A mycoharvester developed at CABI Bioscience (www.dropdata.net/mycoharvester) appears to be a simple device for collecting spores of the mycoinsecticide fungus, Metarhizium anisopliae, produced on rice grains. This device has also been attempted to reduce inoculum impurity of the mycoherbicide Pyricularia setariae (Gary Peng, unpublished data). By reducing the proportion of large particles in the inoculum, the mycoherbicide can be applied at high spore concentrations and low carrier volumes using common spray equipment (Peng et al. 2001). A two-phase system produces mycelium in deep tank fermentation followed by sporulation in shallow open trays. This method may be particularly useful for fungal agents that cannot be manipulated to sporulate in submerged culture, but this system is labor-intensive and expensive, and additional handling of the material may lead to

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contamination of the final product (Rosskopf et al. 1999). Liquid fermentation produces a large amount of biomass efficiently and sporulation in a “dry phase” may circumnavigate costly down-stream processing issues. Walker (1980) used A. macrospora as a model system to first produce fungal mycelium in a liquid, then homogenize and mix it with vermiculite, followed by thinly spreading the homogenate on a solid surface. Using a similar system, Walker and Riley (1982) successfully produced spores of A. cassiae, a mycoherbicide agent for control of sicklepod. One area of fermentation that can often be overlooked is the feasibility of scale-up from shake flask laboratory volumes to productionplant level. Optimization of fermentation conditions and media components can be readily achieved in the laboratory while pilot-plant scale fermentation can help to effectively verify these selected parameters (Kwanmin 1989). Some sophisticated pilot-scale devices with consistent designs to large-scale facilities are now available. These units are particularly useful for scale-up studies and provide a wide range of pH, agitation speeds, impeller designs, aeration rates, choices of regulated incoming gases, variations in baffling and background pressures, and temperatures (Churchill 1982). However, conditions required to reach optimal yield, costs, and efficiencies in production scale fermentation can be more difficult to achieve. Some biological factors that need to be considered are culture stability, number of generations, and mutation rate while chemical factors include pH, water quality, and fermentation medium quality. Physical factors that should be evaluated include aeration, agitation, pressure, temperature, and medium sterilization. Kwanmin (1989) indicated that factors that may not have been important at a smaller scale could have significant impact on the operation and design of fermentation processes at the production plant level.

4.3

Formulation

It is believed that many of the environmental challenges, particularly long dew period or leaf wetness requirements, can be tackled to a large extent with formulation technologies (Boyetchko et al. 1999; Greaves et al. 2000; Green et al. 1998). Formulation is essentially the blending of microbial propagules with a range of carriers or adjuvants to produce a form that can be effectively delivered to target weeds. For microbial agents, formulation may enhance pathogen survival and infection as well as extend propagule stability and product shelf life. Depending on the type of organism, mode of action, and available spray equipment, formulation ingredients vary substantially. For instance, foliar-applied agents may be exposed to rain-wash, UV irradiation, and desiccation prior to germination and penetration (Rhodes 1993). Therefore, various adjuvants with adhesive, sunblocking, or humectant properties have been suggested to alleviate the negative impact by these factors (Schisler et al. 1995; Womack and Burge 1993). Formulations that increase moisture-retaining properties, reduce the rate of evaporation

and/or enhance the rate of infection of the mycoherbicide agent should be explored to address dew limitations. In the literature, emulsions and hydrophilic polymers are reported most frequently to improve the performance of foliar-applied mycoherbicide agents. Formulation research has focused particularly on desiccation and dew requirements of fungal agents during the infection process and incremental to drastic improvements have been seen in different studies (Auld 1993b; Connick et al. 1990; Lawrie et al. 2000; Shabana 1997). There is a growing belief that innovations in formulation will be a vital component to the success of the next generation of bioherbicides, especially for foliar-applied products (Greaves et al. 1998). For best results, formulations should predispose weeds to infection by pathogens and buffer pathogen propagules against environmental extremes while promoting disease development. Nutrient supplements, including simple sugars, amino acids, pectins, salts, and plant extracts have been added to formulations to stimulate the infection process and protect germinating propagules, but these nutritional effects are often agent specific (Bothast et al. 1993; Schisler et al. 1995; Womack and Burge 1993). Exogenous nutrients may stimulate germination and growth of many fungi, but frequently appressorial initiation is even more important to plant penetration and infection. Oversupply of nutrients can lead to excessive growth of germlings, delaying, or even reducing appressorial formation and penetration (Takano et al. 1997). Tremendous efforts have been made on developing various emulsions to alleviate moisture constraints, thereby enhancing field performance. Invert emulsions showed the most impressive results by reducing or even eliminating the need for dew with several fungal agents (Connick et al. 1991b; Yang et al. 1993). Bioherbicidal control of hemp sesbania using C. truncatum in an invert emulsion was significantly enhanced under field conditions (Boyette et al. 1993). Invert emulsions consist of water droplets suspended in oil and evaporation of the trapped water is dramatically reduced and microbial propagules held in the water are, therefore, protected (Daigle et al. 1990; Womack and Burge 1993). Despite the apparent improvement, invert emulsions can be very complex, difficult to apply using existing spray equipment due to extreme viscosity and may exhibit phytotoxicity in many plant species (Boyette 1994; Womack et al. 1996). Although these invert emulsions have been used to expand the host range of mycoherbicides (Yang and Jong 1995), this change may also affect nontarget crops. High oil content also makes invert emulsions more costly, especially when high spray volumes are required. Being nonevaporative, oils were considered a compatible carrier with ultra-low volume application techniques for the mycoinsecticide M. anisopliae under extremely dry conditions (Bateman and Alves 2000). In contrast to invert emulsions, oil suspension emulsions are considered to be more practical because they have significantly lower oil content and can be applied with most existing spray equipment (Green et al. 1998). Fungal propagules can be first suspended in oils, then mixed with a much larger volume of water containing an emulsifier to make stable emulsions

Mycoherbicides

(Auld 1993b). Klein et al. (1995) used suspension emulsions of C. orbiculare made from two vegetable and mineral oils that were mixed and applied with water ranging in concentrations from 0.5 to 10% for control of Xanthium spinosum under field conditions. These formulations enhanced weed control in several field trials compared to water as a carrier sprayed at similar application volumes. Oils may have variable effects on propagule germination and performance of mycoherbicide agents. Paraffin oils were toxic to spores of Ascochyta pteridis, a mycoherbicide candidate for bracken, Pteridium aquilinum (Womack et al. 1996), while unrefined corn oil enhanced spore germination of C. truncatum in emulsions containing 10 –50% of oil (Boyette 1994; Egley and Boyette 1995). The mechanisms by which the oil suspension emulsions enhance mycoherbicidal activity are not well understood. Emulsions may help maintain the stability and infectivity of fungal propagules prior to onset of dew (Green et al. 1998). Spray retention is likely enhanced, reducing the spore dose required for effective weed control. Moisture retention has also been demonstrated with hydrophylic polymers such as Kelginw HV, MV, LV, Kelzanw xanthan gum, Gellan gum, N-Gele, Metamucilw, and Evergreenw500 (Shabana et al. 1997). These polymers enhanced viability, germination, and efficacy of the mycoherbicide agents A. cassiae and A. eichhorniae. Humectants such as psyllium (e.g., Metamucilw) are known to have high moisture retention properties and reduce the rate of moisture loss (Greaves et al. 2000). Coformulation of the mycoherbicide agent, A. caulina, with the skinning agent polyvinyl alcohol and Metamucilw enhanced control of Chenopodium album under reduced dew conditions. For mycoherbicides applied to the soil, encapsulation of the fungi in solid matrices is more suitable than liquid formulations. Calcium alginate has been used to mix fungal spores with a variety of carriers such as kaolin clay, ground oatmeal, soy flour, and cornmeal (Boyette and Walker 1986; Walker and Connick 1983; Weidemann and Templeton 1988). Conidial production and field efficacy can be enhanced by amending the mixture with various nutrients (Daigle and Cotty 1992; Weidemann 1988). “Pesta” has also been used as a type of granular formulation where fungal propagules are entrapped in a wheat-gluten matrix consisting of semolina flour, kaolin, and fungal biomass (Connick et al. 1991a). Further development of this process has resulted in the formation of uniform granules using a twin-screw extruder and by controlling the moisture content through fluid-bed drying (Connick et al. 1998).

4.4

Application Technology

Delivery and retention of sufﬁcient number of fungal propagules on weeds can be very challenging. Most initial studies on mycoherbicide efﬁcacy spray the inoculum to the weed foliage till runoff using aerosol sprayers. This spray method generally applies excessive volumes (up to 3000 l/ha) that can maximize the retention and exaggerate the potential

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of biocontrol agents (Greaves et al. 2000). Disregard for appropriate methods of application can contribute to poor or inconsistent field performance (Smith and Bouse 1981). In most field crops, application volumes over 600 l/ha are considered high (Matthews 1992), and the trend is generally toward lower volumes. Use of high spore concentrations can potentially reduce application volumes without compromising the efficacy of weed control (Peng et al. 2001). Increased propagule number may help improve the efficiency of foliar coverage by reducing the proportion of “empty” droplets (Jones 1998), but also pose high requirements on quantity and quality of the inoculum necessary to achieve the desired level of weed control. Mycelium clumps or other impurities in extremely concentrated formulations can also easily plug up the spray system. Inoculum concentration, carrier volume, and other spray parameters need to be studied jointly to optimize spray results (Jones and Burges 1998). Nordbo et al. (1993) suggested use of fast travel speed to improve spray retention on vertical leaf surfaces. In a recent study, Peng et al. (2001) observed that a finer droplet spectrum combined with more horizontal trajectories enhanced retention efficiency on green foxtail. Richardson (1987) and Spillman (1984) made similar suggestions on application of herbicides. These retention characteristics have also been discussed by Jones (1998) and Reichard (1988), and have been used to explain improved spray results in a number of herbicide studies (Knoche 1994). It needs to be recognized that there are limitations with manipulation of certain spray parameters. For instance, too fine a spray may not be practical in every case depending on the size of mycoherbicide propagules and due to potential spray drift concerns (Jones 1998). Interpretation of droplet size spectra for optimal dose transfer of biopesticides is required, and this will vary with the mycoherbicide agent, particularly in relation to the spore size and morphology (Bateman 1999). Although fine tuning of application parameters can improve spray characteristics, it is more important to determine if these improvements can be translated into meaningful enhancements of weed control efficacy.

4.5

Herbicide Synergy

As stand-alone products, mycoherbicides have achieved limited success in the marketplace. Technologies such as formulation and application methods can improve the performance of biocontrol agents, but more noticeable enhancements in weed control have been observed with the combined application of mycoherbicide agents and synergistic herbicides (Peng et al. 2000; Sharon et al. 1992; Wymore et al. 1987). Limited information seems to suggest that synergistic interactions are herbicide and pathogen speciﬁc. Use of glyphosate on Canada thistle assisted weed control by A. cirsinoxia only marginally, especially under ﬁeld conditions (Bailey et al. 2000). Peng et al. (2000) compared two groups of herbicides, bentazon and metribuzin, for

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interaction with a fungal pathogen on scentless chamomile, a noxious weed in the Canadian prairies. Preliminary results revealed significant fresh weight reduction by up to 150% with applications of herbicides plus the pathogen compared to herbicides alone. Tank-mixing of C. coccodes with thidazuron (N-phenyl-N0 -1,2,3-thiadiazol-5-yl-urea) also increased the mortality of velvetleaf when compared to the application of the mycoherbicide alone in field trials (Wymore et al. 1987). The application of a sublethal dose of glyphosate with A. cassiae resulted in an increase in susceptibility of the weed sicklepod (Sharon et al. 1992). As a result, equivalent control was achieved with five times less inoculum of the mycoherbicide agent. The herbicide was believed to interfere with the shikimate acid pathway that is involved in the elicitation of phytoalexins, low molecular weight antimicrobial compounds involved in a plant’s defense response. Subsequent interference with the plant’s defense mechanism resulted in greater susceptibility of the target weed to the mycoherbicide (Hoagland 1996). This synergistic interaction appears to be an attractive mechanism to enhance the effectivity and feasibility of mycoherbicide agents. According to Hoagland (1996), several benefits may be captured with the application of microbe/herbicide synergy: (a) when defense capabilities of weeds are lowered using herbicides, weeds become more susceptible to pathogen attack, (b) the quantity of mycoherbicide agent or the application rate of herbicides may be reduced, and (c) host range of a given mycoherbicide agent may be expanded with the use of selected chemical synergists.

CONCLUSION

The identification of efficacious biological control agents is only the beginning in the development of mycoherbicide products. Continuing strain selection is essential to ensure that “nature’s best” is employed. Likely there will always be limitations associated with naturally occurring organisms, therefore, enabling technologies such as fermentation, formulation, and application technology will be instrumental in determining whether a highly efficacious agent can be developed into an economically feasible mycoherbicide product. The ultimate goal is to incorporate mycoherbicides into agricultural production systems. More efforts should be directed into combining biocontrol agents with other weed control options including chemical herbicides, cultural practices, and use of the multiple pathogens to enhance the effectiveness and flexibility of integrated weed management systems and to reduce the chemical load on crops and in the environment (Boyetchko et al. 2002; Rosskopf et al. 1999). The challenge is to critically evaluate the merits of individual mycoherbicide candidates and to make realistic decisions whether they have all the essential characteristics required for successful application in agroecosystems. Based on discussions presented in this review, the following research priorities are suggested for development of mycoherbicide candidates:

(a) Assessment of natural strain variation and biodiversity within the pathogen population based on critical epidemiological characteristics, including environmental adaptation, virulence, dispersal, and infection efficiency. (b) Evaluation of key areas for efficient scale-up mass-production based on fundamental elements relating to nutritional and physical requirements of specific fungal agents that facilitate selection of economical fermentation ingredient substitutes, along with down-stream processing procedures that are compatible with production methods. (c) Selection of appropriate formulation technologies (i.e., liquid or solid-matrices) based on the mode of attack and critical efficiencies of candidate agents. Over-simplification of formulation by using single ingredients will not likely address the complex challenges that fungal organisms will encounter in the environment, including moisture constraints, temperature extremes, and UV irradiation. (d) Critical factors in application technology related to placement and penetration of the crop canopy to the target weed in order to maximize application efficiency. These factors include leaf-wetting properties and ability to penetrate physical barriers (e.g., waxy cuticles and leaf hairs), retention and dispersal on the leaf surface, optimum dose transfer in various liquid droplets or solid-based granules, and selection of application equipment such as nozzle types and angle position or soil-application placement (e.g., within furrow application, sidebanding, etc.). Application parameters should also be evaluated jointly with formulation ingredients. (e) Integration of mycoherbicide agents into crop production systems using several weed management tools (e.g., synergy with chemical herbicides, combinations with other biological control agents, or weed control options) to optimize weed control effectiveness. Judicious use of mycoherbicides as one of the components in an integrated weed management system will enhance their value and practicality for control of multispecies weed communities in agroecosystems.

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11 Biofungicides Beom Seok Kim Institute for Structural Biology and Drug Discovery, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA Byung Kook Hwang College of Life and Environmental Sciences, Korea University, Seoul, South Korea

exposed to agricultural environment, thus leading to the low residual level less harmful to the natural ecosystem (Tanaka and Omura 1993). Microbial metabolites can be exploited in a number of different ways for the development of new fungicides. They can be directly used as fungicide products or as leads for the design of novel synthetic products. Alternatively, they can be used to highlight novel mode of action available as a new screening target. The recent successes in fungicide development came mainly from the discovery of potent lead compounds followed by chemical modifications that gave additional useful features fungicides. Potent antifungal activity is not the only factor to decide whether the microbial metabolite can be used as a commercial fungicide. Along with the chemical stability in the field, it should also have residual activity enough to reduce the application time to the economical level and low volatibility sufficient to stay on the surface of host plants. Overall, it is very unlikely that a newly discovered microbial metabolite might possess all of the desired properties. Therefore, moving away from the viewpoint of antifungal metabolites as final products, microbial metabolites are currently reexploited as a source of enormously diverse chemical library that can supply lead compounds for development of fungicides. As seen in the example of the successful development of methoxyacrylates that are expected to be a major fungicide class in the future, it is not surprising that natural products are facing a revival as lead compounds for fungicide development. The advances in molecular, biological, and chemical techniques made it possible to reinvestigate microbial metabolites from a totally different point of view. The increasing knowledge about the complex multidisciplinary

INTRODUCTION

At present, approximately 200 different fungicides have been introduced into agriculture and horticulture worldwide. Despite the enormous advances in chemical management of fungal diseases, some of the important plant pathogens such as vascular wilt, anthracnoses, take-all of wheat, and other root infections remain uncontrolled by current fungicidal chemicals (Knight et al. 1997). The build-up of resistant strains of target pathogens and the increasing public concern about synthetic fungicides have intensiﬁed the need for better and safer compounds in terms of novel modes of action, low rates of use, and low toxicological and environmental ¯ mura 1993). As the risk (Godfrey 1994; Tanaka and O environmental and commercial requirements for new fungicides become more demanding, it is increasingly difﬁcult to discover new class of compounds to justify the effort and the costs of development. In order to get a chance to discover new fungicides that meet the mentioned characteristics, the exploitation of biologically active natural products is becoming mainstream in antifungal agent research. Microbial metabolites represented by antibiotics have a number of chemical and biological merits as fungicides. Microorganisms are capable of synthesizing versatile chemical structures with diverse biological activities beyond the scope of synthetic organic chemistry (Porter 1985). An unexpected and newly found chemical structure is more likely to have new fungicidal activity and mode of action, especially showing no cross-resistance to the commercial fungicides (Fru¨h et al. 1996). Biodegradability is the next property of microbial metabolites that cannot be overlooked. They degrade usually within a month or even a few days, when 123

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mystery of antifungal activity enables us to design a rationalized screening system based on the mode of action. Along with the innovative screening systems, the powerful instruments available for puriﬁcation and structural elucidation of natural products have made it possible to adopt a high throughput approach to natural product screening (Bindseil et al. 2001). In this chapter, we will review (a) microbial metabolites currently used as fungicides, (b) on-going efforts to discover lead compounds from diverse microbial sources, and (c) fungal speciﬁc targets to be used for screening of potential antifungal leads. In the later part of this review, we will discuss (c) trends in biofungicide research and interdisciplinary approaches to diversify their chemical library, which may yield novel antifungal compounds in the future.

MICROBIAL METABOLITES IN USE AS BIOFUNGICIDES

The most important antifungal metabolites in commercial use are listed below, which are applied to control fungal diseases on rice, vegetable, and fruits. The relative importance of the microbial compounds, when compared to synthetic fungicides, might have been underevaluated because of several reasons such as the limitation in their spectrum of activity and in certain instances, the development of resistance. Nevertheless, the excellent activity of these biofungicides inspired to launch the screening programs for antifungal microbial metabolites, which resulted in profound chemical libraries of natural products (Godfrey 1994; Knight et al. 1997). Blasticidin S, the first microbial fungicide available for plant protection, has been used practically for the control of rice blast disease caused by Magnaporthe grisea. Blasticidin S is a nucleoside antibiotic discovered from metabolites of Streptomyces griseochromogenes (Takeuchi et al. 1957). It potently inhibits the mycelial growth and conidial germination of M. grisea. The successful use of the compound encouraged further screening of microbial fungicides that eventually brought out kasugamycin, polyoxin, validamycin, and mildiomycin. Kasugamycin is an amino-sugar compound discovered in the metabolites of Streptomyces kasugaensis and Streptomyces kasugaspinus (Umezawa et al. 1965). It has in vitro antimicrobial activity against yeast and some plant pathogenic fungi including M. grisea. In vivo data showed that kasugamycin efficiently suppressed the development of M. grisea mycelia on rice plants by both preventive and curative treatments. However, it did not appear to inhibit the spore germination. Polyoxins were isolated from the culture broth of Streptomyces cacaoi var. asoensis (Suzuki et al. 1965). The excellent in vitro activity and in vivo efficacy led to its commercial use for the control of fungal diseases of fruit trees and vegetables such as black spot of Japanese pear caused by

Alternaria kikuchiana and gray mold diseases caused by Botrytis cinerea (Isono et al. 1965). Validamycin A produced by Streptomyces hygroscopicus var. limoneus was effective in controlling rice sheath blight caused by Rhizoctonia solani (Iwasa et al. 1970). Validamycin A was found to be a pro-drug, which is converted within the fungal cell to validoxylamine A, an extremely strong inhibitor of trehalase. This mode of action gives validamycin A a favorable biological selectivity, because the hydrolysis of the disaccharide trehalose does not occur in the vertebrates. The structural elucidation and total synthesis of validamycin A were achieved by Ogawa and coworkers (Suami et al. 1980). Mildiomycin is an aminoacylated nucleoside produced by Streptomyces rimofaciens (Harada and Kishi 1978). It was discovered by the method established to assay the control efﬁcacy of antifungal agents against powdery mildew. Mildiomycin has been known to act as an inhibitor of the fungal protein biosynthesis. Its low toxicity on vertebrates allows it to be an environmentally favorable crop protection agent.

MICROBIAL METABOLITES AS ANTIFUNGAL LEADS

The merits of natural products as fungicides can be a disadvantage in some respects. Their specific activity often resulted in a narrow antifungal spectrum with a limited application and the development of resistance strains under high selection pressure. Their biodegradability can also make them fragile, which results in short residual activity under harsh field conditions. These might be the reasons why the microbial metabolites used as commercial fungicide per se still commands less than 1% of total fungicide market ¯ mura 1993). Recently, a breakthrough in (Tanaka and O biofungicide research came from the semisynthetic approach using microbial metabolites as lead compounds. In particular, a far more promising and effective strategy for the development of new biofungicides is to use knowledge of the structure of antifungal compounds as the starting point for the synthesis of the compounds with optimized physical, biological, and environmental properties. The activity of natural products can in principle be improved by chemical modification. However, this approach relies heavily on the ready availability of sufficient quantities of the natural starting materials and the development of appropriate synthetic methodology. The biofungicides that were developed in this way are fenpiclonil and fludioxonil (Nyfeler and Ackermann 1992) and synthetic derivatives of antibiotic strobilurins such as b-methoxyacrylate azoxystrobin and kresoxim-methyl (Anke et al. 1977; Godfrey 1994). Such a derivative synthesized from microbial metabolites not only enhanced control efficacy but also improved properties such as photochemical stability, low cytotoxicity, and phytotoxicity. These successes encouraged the fungicide

Biofungicides

researchers to search versatile lead compounds from diverse microbial sources with novel mode of action.

3.1

Recent Success in Fungicide Development from Antifungal Leads

Since strobilurin A and oudemansin A were found to be fungicidal metabolites in Basidiomycete fungi Strobilurus tenacellus (Anke et al. 1977) and Oudemansiella mucida (Musilek et al. 1969), respectively, a number of structurally related compounds were reported to have fungicidal activity. Each member of this family incorporates a methyl b-methoxyacrylate group linked at its a-position to a phenylpentadienyl unit, and all the compounds except strobilurin A carry either one or two additional substituents on the benzene ring that render structural complexity (Figure 1). Their mode of action on mitochondrial respiration, binding at a speciﬁc site on cytochrome b, is not shared by any other known class of fungicides (Sauter et al. 1995). The unique mode of action may not provide a chance of crossresistance between b-methoxylacrylates and other fungicides. Although strobilurin A has excellent in vitro activity against a range of fungi, it did not show any useful in vivo activity in the greenhouse. This was due to its photochemical instability and relatively high vapor pressure, which cause it to disappear rapidly from a leaf surface. Through a series of synthetic program to solve these problems, azoxystrobin (Godwin et al. 1992) and kresoxim-methyl (Ammermann et al. 1992) were

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developed as commercial fungicides that overcame the problems of the lead compounds (Clough et al. 1995). Azoxystrobin has a methyl b-methoxyacrylate toxophore, like strobilurin A, whereas kresoxim-methyl has a methyl methoxyiminoacetamide structure (Figure 1). Axoxystrobin has a wide antifungal spectrum against all four taxonomic groups of fungi and strong preventative activity, including inhibition of fungal germination (Heaney and Knight 1994). Kresoxim-methyl is also a broad-spectrum fungicide with strong antifungal activity against powdery mildew and apple scab (Brunelli et al. 1996). Considering its novel mode of action and amenability for synthetic approach, strobilurins are expected to be a major fungicide in near future. Pyrrolnitrin is another example of a microbial metabolite used as a lead compound. Pyrrolnitrin, a secondary metabolite of Pseudomonas pyrrocinia, which has a very simple structure, is thought to play a significant role in biocontrol activity of the bacterium (Arima et al. 1964). Although it showed excellent in vitro and in vivo activity in the greenhouse against B. cinerea and M. grisea, the diseasecontrol efficacy in the fields was poor, because it rapidly decomposed when exposed to sunlight. In the extensive synthetic programs using pyrrolnitrin as a template, feniclonil (Nevill et al. 1988) and fludioxonil (Gehmann et al. 1990) were developed as seed-dressing agents against numerous fungal pathogens. The replacement of the chloro substituent in the 3-position of the pyrrole by a cyano group led to a remarkable enhancement in stability (Figure 1). Its biological activity also was optimized by appropriate substitution on the phenyl ring. Their improved photostability over pyrrolnitrin conferred the possibility as a foliar fungicide active against B. cinerea, Monilinia spp. and Sclerotinia spp. (Nyfeler and Ackermann 1992).

3.2 3.2.1

Figure 1 Chemical structures of biofungicides in practical use and their lead compounds.

Screening of Potential Leads from Diverse Microbial Sources Streptomyces, the Largest Reservoir of Diverse Chemical Structures

Actinomycetes have been a major supplier of natural products (Huck et al. 1991; Lee and Hwang 2003). In particular, Streptomyces is a prolific producer of versatile structures of antibiotics. Most of antibiotics developed for agricultural uses including pesticides were isolated from Streptomyces strains ¯ mura 1993). Among antifungal antibiotics (Tanaka and O recently discovered from Streptomyces spp., polyketidespiroketal spirofungins, macrolide cineromycins, and oligomycin A, antimycin type kitamycins, aflatoxin inhibitor aflastatins, aminoacetophenone family heptaene antibiotics, and novel nikkomycin analogs were found to have potent antifungal activity (Bormann et al. 1999; Hayashi and Nozaki 1999; Holtzel et al. 1998; Kim et al. 1999b; Ono et al. 1998; Schiewe and Zeeck 1999; Vertesy et al. 1998). Streptomyces have the ability to synthesize diverse compounds covering the chemical structures generated by

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the eukaryotic organisms such as fungi, algae, and plants. Streptomyces kurssanovii was found to have the ability to synthesize fumaramidmycin, which is structurally very similar to fumarimid and coniothriomycin produced by the fungi Sordaria sp. and Conithyrium sp. (Maruyama et al. 1975). Although the frequency of rediscovery of known compounds is relatively high, it should also be noted that Streptomyces strains continue to provide a larger number and wider variety of new antibiotics than any other microbial sources (Okami and Hotta 1988). Many macrolide antibiotics, for example, have already been introduced from a variety of Streptomyces spp., however, new macrolide compounds are still being discovered to be potent antifungal agents. Faeriefungin, a polyene type macrolide, isolated from S. griseus showed strong in vivo activity against asparagus (Asparagus officinalis L.) pathogens Fusarium oxysporum and Fusarium moniliforme (Smith et al. 1990). More recently, the antifungal substances, phenylacetic acid and sodium phenylacetate, active against Phytophthora capsici and M. grisea were isolated from the culture filtrates of S. humidus (Hwang et al. 2001). Streptomyces is a sole microbial source for a certain type of antibiotics such as members of manumycin type that contain a multifunctional mC7N unit as a central structural element. A manumycin type antibiotic SW-B has recently been purified from the culture of Streptomyces flaveus strain A11 (Hwang et al. 1996). The strain was isolated from cave soil in Korea by an extensive screening program for the Streptomyces strain antagonistic to P. capsici. The structure of manumycin SW-B was determined to be 2,4,6-trimethyl deca-(2E,4E)-dienamide (molecular formular C13H23NO) with the molecular weight of 209.178 (Figure 2). SW-B showed a high level of inhibitory activity and broad antifungal spectrum against several plant pathogenic oomycete and fungi such as P. capsici, M. grisea, Colletotrichum cucumerinum, and Alternaria mali. Hyphal growth of P. capsici and M. grisea was inhibited by more than 50% at 10 mg ml21 and by 90% at 50 mg ml21. The simplicity of the chemical structure and its broad antifungal spectrum provide the possibility as a lead compound for fungicide development.

3.2.2

Rare Actinomycetes, New Resource of Microbial Metabolites

Since rare actinomycetes have the properties such as slow growth, poor sporulation, and instability in preservation, it seems difﬁcult to isolate them without applying the selective isolation methods. Most of their metabolites, therefore, were not subjected to the antifungal screening. However, although the antifungal agents from these non-Streptomyces groups of actinomycetes have not yet been developed into commercial fungicides, they are expected to be useful microbial sources for diversifying chemical library of metabolites. The genus Micromonospora, only a minor component in the actinomycete population in soil, has been recognized as one of the important sources for antimicrobial metabolites.

Figure 2 Potential antifungal leads from microbial sources.

Micromonospora spp. was known to be distributed widely in soils of various geographical regions (Vobis 1991). Since gentamicin, an aminoglycoside antibacterial antibiotic, was isolated from M. purpurea and M. echinospora (Weinstein et al. 1964), Micromonospora spp. has been shown to produce diverse antibiotic substances such as aminoglycosides and macrolides (Betina 1994). In a screening program for antifungal antibiotics useful for plant disease control, Micromonospora coerulea strain Ao58 was isolated from sea-mud soils, which showed strong antifungal activity against P. capsici, M. grisea, C. gloeosporioides, and R. solani (Kim et al. 1998; Kim et al. 1999a). From the culture extracts, the antibiotic streptimidone (Figure 2) was purified using various chromatographic procedures. Streptimidone was known as an inhibitor of the protein synthesis on yeast, but little has been known about its efficacy as an antifungal agent against filamentous fungi. In the tests for antifungal spectrum, remarkable antifungal activities were observed against some plant pathogenic fungi P. capsici, M. grisea, Didymella bryoniae, and B. cinerea. In vivo tests showed its potent control efficacy against phytophthora blight on pepper plants, gray mold on cucumber leaves, and leaf blast on rice leaves. The compound effectively inhibited the development of these plant diseases on their host plants at the concentration of

Biofungicides

100 mg ml21, at which the commercial fungicides showed similar control efficacy against the diseases. No phytotoxicity was observed on any of the host plants at the concentrations of 500 mg ml21. Recently, two structurally related compounds isolated from rare actinomycetes were found to have potent antifungal activity against plant pathogenic fungi. Daunomycin and spartamycins were isolated from Actinomadura roseola and Micromonospora spartanea, respectively (Kim et al. 2000b; Nair et al. 1992). Both compounds have similar anthracycline aglycone moiety attached to one or three glycosides (Figure 2). Daunomycin noted for anticancer activity showed substantial in vitro antimicrobial activity against P. capsici, R. solani, B. cinerea, Cladosporium cucumerinum, Cylindrocarpon destructans, D. bryoniae, S. cerevisiae, and Gram positive bacteria. In particular, daunomycin showed strong inhibitory effect on the mycelial growth of P. capsici and Phytophthora development on pepper plants. In vivo efficiency against Phytophthora infection in pepper plants was somewhat less effective than that of the commercial fungicide metalaxyl. Spartamycins produced by M. spartanea were isolated from a potted soil with A. officinalis L. plants (Nair et al. 1992). Between the two spartamycin analogs A and B, the latter showed better antimicrobial activity against several microorganisms. The minimum inhibitory concentration (MIC) of spartamycin B on Aspergillus, Cladosporium, Cryptococcus, Rhodotorula, and Candida albicans ranged from 0.2 to 1 mg ml21. However, spartamycin B was not effective against the Staphylococcus aureus, Escherichia coli and Citrobacter spp. In view of antifungal activity and structural similarity of the anthracycline antibiotics, their analogs having different glycoside moieties may be worthwhile to examine their antifungal activity against various plant pathogenic fungi.

3.2.3

Other Microorganisms

Pseudomonas aeruginosa strain B5 was isolated from peppergrowing soils in Korea, which showed substantial inhibitory activity against P. capsici and other plant pathogenic fungi. From the culture broth of the antagonistic bacterial strain B5, one of the antibiotic substances active against P. capsici was purified and identified as a glycolipid antibiotic rhamnolipid B (Kim and Hwang 1993). Rhamnolipids containing rhamnose and b-hydroxy-decanoic acid were first found in Pseudomonas pyocyanea (the old name of P. aeruginosa) (Bergstro¨m et al. 1946). Recently, complete nuclear magnetic resonance signal assignments of rhamnolipid B based on intensive spectral analysis provided the evidence of 1,2-linkage of 3-[3-[-L -rhamnopyranosyl-(1 ! 2)-a-L rhamnopyranosyloxy] -decanoyloxy]-decanoic acid (Moon et al. 1996). The glycolipid antibiotic rhamnolipid B has the characteristic structure of biosurfactants, which is comprised of a hydrophilic portion (rhamnose moiety) and a hydrophobic portion (b-hydroxydecanoate moiety). The biosurfactant property was supposed to render the

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rhamnolipid the ability to intercalate into and to disrupt the zoospore plasma membrane, because zoospores are surrounded only by plasma membrane without typical cell wall (Stanghellini and Miller 1997). This hypothesis is well supported by the further finding that rhamnolipid B had no lytic activity on zoospore cysts surrounded with the cell wall (Kim et al. 2000a). In vitro growth inhibition assay performed in the microtiter dishes showed potent antifungal activities against Cercospora kikuchi, C. destructans, C. cucumerinum, Colletotrichum orbiculare, M. grisea, and P. capsici. In particular, rhamnolipid B had a high level of antifungal activity (10 mg ml21 of MIC) against P. capsici. In the microscopic study, most of the zoospores became non-motile in the presence of 25 mg ml21 of rhamnolipid B, subsequently lyzing within 1 min after treatment. Rhamnolipid B also was effective in inhibiting the germination of zoospore and the hyphal growth of P. capsici. The average hyphal length of germlings at the 50 mg ml21 was reduced by 55% of that in the untreated control. These results suggest that rhamnolipid B has not only the lytic effect on zoospores of P. capsici but also inhibitory effect on the growth of the oomycete. Zoospores have been implicated in the spread of the oomycete pathogen through irrigation water and rainwater (Hwang and Kim 1995b; Ristaino et al. 1993). The lytic effect of rhamnolipid B on zoospores may provide a merit as a preventive control agent against phytophthora blight in pepper-growing fields, which eliminate and/or reduce zoospore density and long-distance dispersal of the pathogen. In the recirculating hydroponic cultural system of crops, rhamnolipid B has been demonstrated to be very effective in controlling the dispersal of plant diseases caused by zoosporic oomycete pathogens (Stanghellini et al. 1996). Bacillus subtilis is known to produce diverse antifungal peptides represented by inturins. A series of fungicidal metabolites, named rhizocticines, were identified from B. subtilis ATCC6633 (Figure 2) (Fredenhagen et al. 1995). These peptides showed control efficacy against B. cinerea on apples and vines in the greenhouse. The proteolytic digestion test of the compound revealed that L-2-amino-5-phosphono3-(Z)-pentenoic acid was the actual structure active against B. cinerea. The antifungal activity was proven to be stereo specific, since the corresponding 3-(E) compound did not show any antifungal activity. The mixture of rhizocticines A, B, and D also showed control efficacy against gray molds on grapes in the field.

POTENTIAL TARGETS FOR DISCOVERY OF ANTIFUNGAL LEADS

Unlike the arena of development of antibacterial agents, in relative terms, where bacterial specific targets are abundant, it seems difficult to develop antifungal agents with a specific mode of action. Since, fungi as eukaryotic organism, have metabolism similar to those of mammal and plant hosts, most

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of antifungal agents discovered to be potentially active against plant pathogenic fungi have failed to survive during the testing process for practical usage. The following discussions will be of potential antifungal leads directed to fungal speciﬁc targets, with the examples of antifungal agents recently developed for clinical and agricultural uses.

4.1

Cell-Wall Biosynthesis

Fungal cell wall is a crucial target for antifungal agents. Enzymes responsible for the biosynthesis of fungal cell wall include chitin and glucan synthases (Douglas et al. 1997; Georgopapadakou 1997). Antifungal agent echinocandins have been discovered as inhibitors of fungal cell wall biosynthesis (Denning 1997). They are noncompetitive inhibitors of b-1,3-glucan synthase, an enzyme complex in the cell wall of many pathogenic fungi. b-1,3-Glucan synthase is a fungal specific enzyme that polymerizes UDP-glucose into b-1,3-glucan polymers that comprise the major scaffolding of the fungal cell wall (Kang and Cabib1986). Echinocandins and its synthetic analog containing the fatty acid side chain, cilofungin, exhibited comparable fungicidal activity with a narrow antifungal spectrum (Fromtling 1994). Recently, marked improvements in antifungal activity against clinical pathogens have been achieved by synthetic variations made in the lipid side chains of echinocandins. LY30336 and caspofungin are the examples of recently developed echinocandin analogs (Espinel-Ingroff 1998), which are generally more active in vitro against a variety of yeast and filamentous fungi. They are licensed by Lilly and Merck, respectively, for clinical usage. Such a novel mechanism of action, antifungal potency and relatively broad-spectrum activity of echinocandins provide the possibility that the inhibitors of b-1,3-glucan synthase may be available for the development of biofungicides effective against fungal diseases (Pfaller et al. 1998). Chitin, b-1,4-N-acetylglucosamine polymer, plays a major structural and strengthening role in fungal cell walls. Chitin is microfibrills consisting of hydrogen-bonded polysaccharide chains that may be covalently cross-linked to other polysaccharide, mainly glucan. It has been demonstrated that chitin synthase inhibitors and chitinase showed antifungal activity when applied to growing cells (Lorito et al. 1993). Nikkomycins are analogs of UDP-N-acetylglucosamine produced by Streptomyces spp. They have potent activity against chitin synthase by acting as specific competitive inhibitors (Hunter 1995). The potency of an inhibitor of chitinase synthase may depend on not only the isoform’s relative effectiveness in building a cell wall, but also its affinity to a given enzyme. Recent research on chitin synthase revealed that the multiple chitin synthase genes of fungi have different sensitivities to the inhibitors (Munro and Gow 1995). Therefore, new antifungal compounds with higher activity and specificity to chitin synthase may be generated from diverse chemical pool of microbial metabolites.

4.2

Sterol Biosynthesis

The biosynthesis of sterols is an essential metabolism that produce essential constituents of cellular membranes. Most of fungi contain ergosterol as a predominant sterol (Mercer 1991). Recent advances in our understanding of mode of action of sterol biosynthesis inhibitors (SBI) launched a novel approach to finding inhibitors of sterol biosynthesis, which could lead to new agricultural fungicides (Barrett-Bee and Ryder 1992). The antifungal effects of SBI have brought out a great commercial success in the synthetic fungicide market. The SBI fungicides covering about the half of the market is now practically applied to protect fruits, vegetables, and vines from plant diseases. The major SBI are the inhibitors of 14-demethylation which correspond to many antifungal compounds, referred to as azole compounds, with a wide spectrum of intrinsic activity against ascomycete, basidiomycete and deuteromycete pathogens (Aoki et al. 1993). The discovery of restricticins and lanomycin led to the introduction of a new target for screening of the antifungal natural products. The two structurally related compounds were first isolated from the cultures of Penicillium restrictum (Schwartz et al. 1991) and Pycnidiophora dispersa (O’Sullivan et al. 1992), respectively (Figure 2). Both restricticin and lanomycin showed potent antifungal activity through inhibition of lanosterol C14-demethylase, one of the main steps in ergosterol biosynthesis. It is interesting to note that the structure of restricticin does not have a (phenylethyl)triazole moiety found in all azole antifungal agents in the market, possibly causing adverse impacts in efficacy and resistance (Tuite 1996). However, restricticin needed to be improved in its chemical stability, because the compound was found to be unstable due to the lability of the glycin ester side chain toward base-mediated hydrolysis and the tendency of the triene functionality to undergo decomposition (Barrett-Bee and Ryder 1992). Along with the advances in the screening for inhibitors of other steps in sterol biosynthesis, more SBI sufficient for practical uses may be discovered from microbial metabolites.

4.3

Acetyl-CoA Carboxylase (ACC)

Discovery of soraphen A from myxobacteria was an important event in antifungal metabolite development, because of not only enlarging microbial diversity as a source of antifungal compounds but also introducing fungal ACC as a novel target for antifungal agent screening (Gerth et al. 1994). Acetyl-CoA carboxylase catalyzes carboxylation of acetyl-CoA to malonyl-CoA at the expense of ATP. While the functional units of ACC are usually separate proteins in prokaryotes, they form a multifunctional enzyme complex in eukaryotes. This may be the reason why soraphen A is inactive to bacteria. Soraphen A which is mainly responsible for antifungal activity of Sorangium cellulosum strain Soce26 effectively controlled powdery mildew (Erysiphe graminnis

Biofungicides

f. sp. hordei) in barley, snow mold (Gerlachia nivalis) in rye, apple scab (Venturia inaequalis) on apple and gray mold (B. cinerea) on grape (Reichenbach and Ho¨fle 1995). Soraphen A has no effect on ACC of plants, thus inducing no phytotoxicity in the field (Vahlensieck et al. 1994). In contrast, ACC from rat liver was strongly inhibited by the soraphen (Pridzun et al. 1995). Due to the risky side effects on experimental animals, soraphen A has not been practically used for control of plant diseases. However, the results of soraphen research strongly suggest that fungal ACC could be a target site for antifungal agent screening. Considering the numerous diversity of natural products related to the specificity of ACC, novel biofungicides from microbial metabolites that block specifically the activity of fungal ACC may be developed in the future.

4.4

Nucleic Acid Metabolism

One of the areas that can be exploited as antifungal targets is nucleic acid metabolism. The synthesis of nucleic acids involves numerous biochemical reactions ranging from the initial synthesis of purine and pyrimidine precursors to the ﬁnal polymerization of ribonucleoside and deoxyribonucleoside 50 -triphosphates into RNA and DNA. A large number of compounds have been known to be inhibitors of nucleic acid metabolism in fungi. However, few of these compounds have been used as agricultural and clinical antifungal agents. Recently, antibiotic tubercidin produced by Streptomyces violaceoniger was discovered to have antifungal activity against plant pathogenic fungi (Hwang and Kim 1995a; Hwang et al. 1994). It was highly active against P. capsici, Botryosphaeria dothidea, and R. solani. Tubercidin is an adenosine analog that interferes nucleic acid synthesis including de novo purine synthesis, rRNA processing, and tRNA methylation (Suhadolnik 1979). The potent in vivo activity of tubercidin against P. capsici was compared with that of systemic fungicide, metalaxyl, which is one of the best-studied acylalanine targeting on the synthesis of ribosomal RNA. Treatment with tubercidin on day 1 before inoculation of zoospores prevented phytophthora blight at 500 mg ml21. Tubercidin was effective as much as metalaxyl, irrespective of application time and concentrations, although its antifungal activity did not persist as long as metalaxyl in pepper plants. The potent antifungal activity of tubercidin against P. capsici suggests that possible targets for the antifungal agent screening may be present in nucleic acid metabolic pathway.

4.5

Protein Biosynthesis

Protein biosynthesis is available as a set of molecular targets for antibacterial agent development. The antibiotics such as chloramphenicol and streptomycin have been demonstrated to inactivate or alter the accuracy of the bacterial ribosome (Cundliffe 1990). However, the use of fungal protein

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biosynthesis as an antifungal target has been more challenging, because of the high degree of structural and functional identity of the components of the protein biosynthetic machinery between fungi and higher eukaryotes. As in the cases of cycloheximide, trichodermin, and hygromycin B, their activities on the fungal ribosome appear to be identical to those on the mammalian ribosome (Tuite et al. 1995). It was, therefore, of considerable interest to find out a specific antifungal agent targeting on fungal protein biosynthesis. Sordarins were found to have highly specific inhibitory activity against the elongation factor 2 involved in the translation of several fungal species (Justice et al. 1998). Sordarins were originally isolated from the terrestrial ascomycete Sordaria araneosa (Hauser and Sigg 1971). The fungal specific activity of sordarins is quite interesting, because the elongation factor 2 is a highly conserved protein. Recently, a mutant strain analysis revealed that sordarins had additional interactions with the ribosome itself (Justice et al. 1999), indicating that the selectivity of these compounds was governed by multiple points of interaction between the compound and the ribosome. Using a high throughput screening (HTS) targeting on protein synthesis in Candida spp., an analog of sordarin has recently been demonstrated to be an effective in vitro inhibitor with apparent selectivity for fungal protein synthesis (Kinsman et al. 1998).

FUTURE TRENDS IN BIOFUNGICIDE RESEARCH

During the last two decades, there were numerous efforts focusing on the isolation and identification of a wide range of biologically active natural products. As a result, hundreds of thousands to millions of compounds became available for the evaluation of their value as potential lead compounds. The concept of a HTS was developed to screen a large number of chemical libraries, which overcome the limitation of conventional in vitro and in vivo assay. The HTS is made possible by the advance in assay system, which was designed to target a specific biochemical event in fungal metabolism. A direct measure of the activity of the compound at the target of interest can be done without complications arising from other metabolic events. These approaches can enhance the possibility to discover new and useful biofungicides by supplying unique bioassay system. This innovative procedure was already applied in developing new fungicides such as sordarins mentioned earlier. The target-directed screening will be fortified by DNA sequence information that is exponentially increased in recent years by a number of fungal genome projects. The genomic information can provide a wealth of new targets to be validated and screened for new antifungal leads (DiDomenico 1999). Along with the innovations in screening systems, the efforts to diversify the chemical library of microbial metabolites has been continued through combinatorial

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approaches. Recently, the ability to synthesize a large number of chemical libraries from core structure of antibiotics was greatly enhanced by the advance of rapid combinatorial/ parallel synthesis method (Caporale 1995). The diversity and numbers of distinct compounds in combinatorial library enhance the possibility of finding a chemical structure with the desired properties. Combinatorial libraries can be synthesized in many different ways as reviewed by Dolle (1999). However, most of the successes in combinatorial chemistry have been accomplished by using small libraries to improve the properties of a specific toxophore. The successful optimization of azole and oxazolidinone lead compounds suggested a promising future of combinatorial chemistry in biofungicide research (Trias 2001). As another approach to diversify the chemical library of microbial metabolites, combinatorial biosynthesis was proposed to generate “unnatural” natural products, which use genetic information and DNA recombination techniques to alter the biosynthetic pathway of the microorganism to produce the designed chemical structure. This can also be done by introducing hybrid enzyme or/and swapping with heterologous biosynthetic machinery involved in the synthesis of other antibiotics (Cropp et al. 2002; Reynolds 1998). More recently, previously unknown chemical structures were generated by interchanging enzyme subunits or making hybrid enzymes of type I polyketide synthases (PKSs) (Kim et al. 2002; McDaniel et al. 1999; Yoon et al. 2002). Most of the combinatorial biosynthesis researches have been done on PKSs, especially in Streptomyces. Recently, the biosynthetic gene clusters of antifungal antibiotic pyoluteorin and 2,4-diacetylphloroglucinol also were identified from plant-associated pseudomonads, the well-known biological control agents (Bender et al. 1999). These biosynthetic gene clusters are expected to be used for the template of combinatorial biosynthesis for biofungicide development, although a number of questions about their enzymological functions still remains to be elucidated.

CONCLUSIONS

As the environmental and commercial requirements for new fungicides become more demanding, the merits of biofungicides over synthetic fungicides become more important than ever. Recently, a breakthrough in biofungicide research was made by semisynthetic approaches using antifungal microbial metabolite as the starting point. As seen in the examples of fenpiclonil, ﬂudioxonil and synthetic derivatives of antibiotic strobilurins such as b-methoxyacrylate azoxystrobin and kresoxim-methyl, this approach is a promising and effective strategy for the development of new biofungicides with desired chemical and biological characteristics. These successes encourage fungicide researchers to construct versatile chemical library of microbial metabolites that can be used for development of new fungicides. Recently, a number of antifungal compounds have been discovered from diverse microbial sources including Streptomyces, rare

actinomycetes, other eubacteria and fungi, which may be available for antifungal leads. The advances in the screening system directed to fungal speciﬁc targets have rendered more chances to get success in biofungicide development. A number of useful targets have been discovered from the fungal metabolism related to nucleic acid, protein, sterol, and cell-wall biosynthesis. The recent successful example of sordarin analogs show that better understanding of biochemical events in fungal cells would uncover more useful targets for the screening of antifungal leads. Combinatorial approaches in chemical and biochemical synthesis were suggested to diversify the chemical library of microbial metabolites, which can make it easier to discover the optimized antifungal compound with desired physical and biological properties. These new trends in developing novel biofungicides will be more facilitated and strengthened by innovative multidisciplinary approaches in the future.

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12 Molecular Biology of Biocontrol Trichoderma Christian P. Kubicek Institute of Chemical Engineering, Vienna, Austria

INTRODUCTION

Plant diseases, caused primarily by fungal and bacterial pathogens, produce severe losses to agricultural and horticultural crops every year. These losses can result in reduced food supplies, poorer quality agricultural products, economic hardship for growers and processors, and, ultimately, higher prices. For many diseases, traditional chemical control methods are not always economical nor are they effective, and fumigation as well as other chemical control methods may have unwanted health, safety, and environmental risks. Biological control involves the use of beneﬁcial microorganisms to attack and control plant pathogens and the diseases they cause. It offers an environmentally friendly approach to the management of plant disease and can be integrated into an effective integrated disease management system. Thus, biological control can be an important component in the development of a more sustainable agriculture. Trichoderma species have been investigated as biological control agents for over 70 years, but it is only relatively recently that strains have become commercially available. The previous considerations have stimulated researchers to gain a better knowledge of biocontrol by this fungus, and to understand their mechanisms of control. In view of the actuality of this research ﬁeld, there are numerous recent articles available which review the current state of knowledge of Trichoderma biocontrol (Chet et al. 1998; Harman and Bjo¨rkman 1998; Hjeljord and Tronsmo 1998; Monte 2001 see also the atricle by A. HerreraEstrella and I. Chet, this volume). In this article, the current state of biological knowledge on Trichoderma strains capable of biocontrol on a molecular level will be summarized.

TRICHODERMA BIOCONTROL TAXA AND STRAINS

The genus Trichoderma currently consists of more than 40 known taxa, which are usually cosmopolitan, (although some species display a geographic bias: Kubicek et al. 2002; Kullnig et al. 2000), and typically soilborne or wood decaying Teleomorphs of Trichoderma occur in the genera Hypocrea, Podostroma, and Sarawakus of the Hypocreaceae (Gams and Bissett 1998; Rossman et al. 1999). The latter two genera thereby most likely being synonyms of Hypocrea (GJ Samuels, personal communication). Rossman (1999) proposed that necrotrophy (on basidiomycetes) is the original habitat of these Hypocrea spp. and their lignicolous properties have developed later when the species were following their hosts into their habitat (wood and decaying wood in soil). Rossman et al. (1999) claim that the Hypocrea spp., which are found on decaying wood, actually are necrotrophic on the fungi in the wood. Several of the individual teleomorphic and anamorphic partners have been detected recently, and examples relevant to biocontrol are given in Table 1. The more than 100 species of Hypocrea with Trichoderma anamorphs which Doi and Doi (1986) described constitute unexplored source of potential biocontrol agents. Most of the isolates of the genus Trichoderma, which have been found to act as biocontrol agents, have been classiﬁed as T. harzianum Rifai, leading to the fact that T. harzianum is generally synonymized as a “biocontrol agent.” However, most of the Trichoderma strains used for biocontrol were identiﬁed at the species level exclusively on the basis of morphological and phenotypical characters, showing high convergence in many cases (Kullnig-Gradinger et al. 2003). Therefore, reports of a pronounced genetic variability of T. harzianum isolates by analyzing carbon 135

136

Kubicek

Table 1 Teleomorphs known for Trichoderma taxa used in biocontrol Anamorph T. harzianum T. atroviride T. virens T. asperellum T. parceramosum T. longibrachiatum

Teleomorph H. lixii (former H. nigricans) H. atroviride H. virens Not known Not known Not known

source utilization patterns (Manczinger and Polner 1985), secondary metabolite production (Okuda et al. 1982), isoenzyme polymorphism (Grondona et al. 1997; Stasz et al. 1989), RAPD profiles (Fujimori and Okuda 1994; Gomez et al. 1997; Muthumeenakshi et al. (1994); Turoczi et al. 1996; Zimand et al. 1994), RFLP patterns (Bowen et al. 1996; Muthumeenakshi et al. 1994), rDNA sequence (Grondona et al. 1997; Muthumeenakshi et al. 1994) and karyotype (Gomez et al. 1997) must be treated with caution. On the basis of a rigorous comparison of a pool of seventeen bonafide “T. harzianum” biocontrol strains with the neo-ex type strain of T. harzianum, Hermosa et al. (2000) showed that they actually comprised of four different species i.e., T. harzianum, T. atroviride, T. longibrachiatum and T. asperellum. Consistent results were also reported by Kullnig (2001), who by sequence analysis of the internally transcribed spacer regions of the rDNA (ITS1 and ITS2), the small subunit of the mitochondrial DNA (mtSSUrDNA), and part of the coding region of the 42-kDa endochitinase encoding gene ech42- reassessed the species identity of eight T. harzianum isolates, which are being used by several laboratories for key investigations on the genetics, biochemistry, and physiology of biocontrol. Thereby the strains T. harzianum CECT 2413, T-95, T-22, and T-11 were confirmed as T. harzianum, “T. harzianum” ATCC 74058, IMI 206040, ATCC 36042 identified as T. atroviride, and “T. harzianum” T-203 assessed as T. asperellum. As outlined above, there may be other species capable of biocontrol as well, T. virens being the most prominent example. In addition, molecular proof for identity of other species as biocontrol agents has been presented for T. ghanense (previously T. parceramosum; Arisan-Atac et al. 2002) and T. stromaticum (Samuels et al. 2000).

IN SITU MOLECULAR TOOLS FOR BIOCONTROL STRAINS

Even if the species identity is not a concern, the ability to recognize the strain which was introduced into the ﬁeld is of interest. Appropriate molecular tools have thus recently been introduced for identifying Trichoderma strains in the environment, and to follow their fate after introduction into

the soil in situ. To monitor the behavior of a given strain in the soil, Bae and Knudsen (2001) cotransformed T. harzianum with genes encoding green fluorescent protein (GFP), beta-glucuronidase (GUS), and hygromycin B resistance (hygB). One of the resulting strains was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. In addition, no significant differences were detected in colonization levels between wild-type and cotransformant strains; and the authors concluded that GFP proved a most useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. Also, the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-Dglucuronic acid (X-Gluc) could be used to monitor the activity of b-glucuronidase in soil. Thus, cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil. As the biological strains of Trichoderma are difficult to distinguish from the indigenous strains of Trichoderma found in the field, Hermosa et al. (2001) developed a method to monitor these strains when applied to natural pathosystems. To this end they used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum, and T. longibrachiatum. Analysis of the respective RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identified one of their strains (T. atroviride 11), and clearly distinguished this strain from other closely related Trichoderma isolates, showing that SCAR (sequence-characterised amplified region) markers can be successfully used for identification purposes. An alternative approach, suitable to monitor the presence of several strains in one sample was presented by van Elsas et al. (2000) by selecting a nested PCR approach, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi including Trichoderma. Although only limited examples have so far been published, techniques like these and the fast current advance in PCR technology (such as real-time PCR to name only one) will stimulate further studies of the behavior of Trichoderma biocontrol agents in the field is now possible.

Molecular Biology of Biocontrol Trichoderma

GENOME ORGANIZATION AND REPRODUCTION

One of the major difficulties with Trichoderma biocontrol strains is their genetic instability, whose reason is only poorly understood at present. This is in part due to the fact that only little is known about the genome organization and its plasticity of Trichoderma. Not even the number of chromosomes is known with certainty: Fekete et al. (1996) separated six chromosomes in five Trichoderma biocontrol strains with sizes ranging from 3.7 to 7.7 Mb; estimated genome sizes were between 30.5 and 35.8 Mb. When fractionated chromosomes of the five species were probed with a fragment of the ech42 (endochitinase-encoding) gene, strong hybridization signals developed, but their physical position varied among species indicating a polymorphic chromosomal location. Herrera-Estrella et al. (1993) compared the molecular karyotype of T. reesei with that of T. atroviride (named erroneously T. harzianum in their study), and T. viride, and detected largely similar chromosomal organization of genes in different species, although T. viride seemed to lack the smallest chromosome. Similarly, Hayes et al. (1993), when karyotyping three biocontrol strains of T. harzianum (one parent and two mutants derived from it), found that the smallest chromosome was not present in the mutants. While all these studies revealed a low degree of chromosome polymorphism at the species level, the karyotypes were relatively constant. A report to the contrary (Gomez et al. 1997) is probably flawed by the use “T. harzianum” strains which in fact consisted of several different species (CP Kubicek, unpublished data). Thus, as expected for an asexual fungus, chromosome plasticity is unlikely responsible for the genetic instability of Trichoderma biocontrol strains. Molecular genetic work with Trichoderma spp. is still limited by the only rudimentary information about its genomic organization as is available for Aspergillus fumigatus (http://www.tigr.org/tdb/e2k1/afu1/) and Neurospora crassa (http://www-genome.wi.mit.edu/annotation/ fungi/neurospora). Genetic maps could so far not be constructed, because the teleomorphs of biocontrol species of Trichoderma (see Table 1) do not cross in axenic culture (CP Kubicek, unpublished data). Also, at the time of this writing, genome sequencing projects on selected species of Trichoderma have only just been initiated at a few places, and no results from these are yet available. However, a collection of 1151 ESTs of T. reesei grown on glucose and the sequence of the complete mitochondrial genome is already available in the Internet (http://trichoderma.iq.usp.br/TrEST.html), and can (because of the high similarity of nucleotide sequences of protein encoding genes within the genus (unpublished data) be used for picking genes from biocontrol strains as well). Interestingly, Seiboth and Hofmann (2002) found a similar genomic organization of several genes of galactose metabolism in T. reesei and N. crassa. This finding is highly

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interesting, as N. crassa has evolved about 200 million years ago (Berbee and Taylor 1993), whereas H. jecorina evolved only about 100 million years ago (KullnigGradinger et al. 2003), and thus the genomic organization of these genes has been maintained constant for about 100 million years. Hamer et al. (2001) have also recently reported that a 53-kb region of the genome of Magnaporthe grisea was also syntenic to a corresponding portion of the Neurospora genome. In a comprehensive study on hemiascomycetous yeasts, Llorente et al. (2000) demonstrated that even phylogenetic distant species such as S. cerevisiae and Yarrowia lipolytica exhibit 10.1 % of conserved synteny. If there is indeed a high degree of synteny between Neurospora and Trichoderma, this may be useful for studying the genomic organization of Trichoderma biocontrol strains. Probably due to reproduction, largely via asexual mechanisms, many species of Trichoderma reveal a high level of genetic stability (cf. Kubicek et al. 2002; Kullnig et al. 2000). T. harzianum, however, is a noteworthy exception, showing a remarkable intraspecific genetic and phenotypic variation, and this may also be related to the instability of the respective biocontrol species. The reason for this has not been explained yet. As the respective teleomorph (H. lixii) is known, the possibility of sexual recombination still needs rigorous testing. Transposons, which have been isolated from phylogenetically close fungal genera such as Tolypocladium or Fusarium, are another possibility. We have recently observed a very high noninduced mutation rate in one biocontrol strain of T. harzianum which would be compatible with the presence of a mobile element (C Gallhaup, RL Mach and CP Kubicek, unpublished). As far as nonchromosomal elements are concerned, plasmids have been detected in filamentous fungi almost exclusively in the mitochondrium (Bertrand 2002). They are generally stable genetic elements and vary between 1 –6 kb size. In accordance with this situation, Meyer (1991) detected mitochondrial plasmids in strains of T. viride and the biocontrol-relevant species T. asperellum (then named “T. viride 2”). A circular plasmid called pThr1, with a monomer size of 2.6 kb, was identified in the mitochondria of the biocontrol isolate T. harzianum T95 (Antal et al. 2002). It revealed no DNA sequence similarity with the mitochondrial genome of the isolate and contained a single 1818 bp open reading frame. The derived amino acid sequence exhibited similarity to the reverse transcriptases of the circular Mauriceville and Varkud retroplasmids of Neurospora spp. and the linear pFOXC2 and pFOXC3 retroplasmids of Fusarium oxysporum strains. In the regions of homology all of the seven conserved amino acid blocks characteristic of RTs could be found. In Fusarium oxysporum f. sp. conglutinans, these mitochondrial plasmids have been identified as factors determining the host specificity (Kistler and Leong 1986); unfortunately, corresponding investigations are still lacking for Trichoderma.

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MOLECULAR GENETIC BASIS OF BIOCONTROL

Arising from nectrotrophic ancestors, most of the currently known Trichoderma strains have developed highly effective antagonistic mechanisms to survive and colonize the basidiomycete-containing competitive environment of the rhizosphere, soil, and decaying wood. Active parasitism on host fungi, by penetration of host hyphae is probably the mechanism most studied (cf. Chet et al. 1998); it requires morphological changes of Trichoderma hyphae such as appressorium formation and coiling, and is further supported by the production of extracellular enzymes, and production of antifungal antibiotics. However this mechanism has mostly been observed in the laboratory, and application of Trichoderma in the field may involve additional mechanisms as well such as aggressive degradation of organic matter, thereby competing for nutrients which in saprobic phases may be a limiting factor. Also promotion of the growth and biological activities of saprobic bacteria and mycorrhizal fungi, and of plant-growth and induced resistance have been reported (for review see Herrera-Estrella and Chet, Chapter 57). Among these, mycoparasitism is the only process which has been studied on a molecular biological basis. HerreraEstrella and Chet (Chapter 57) give a detailed account on this, and I shall therefore treat this point here only very briefly: most attention has been paid to the enzymatic disruption of the cell wall of the fungus, thereby focusing on enzymes capable of hydrolysis its structural polymers (chitin, b-glucan, protein and others). Genes encoding endochitinases, N-acetyl-b-glucosaminidases, proteases, endo- and exo-glucan b-1,3-glucosidases, endoglucan-b1,6-glucosidases, lipases, xylanases, amylases, phospholipases, RNAses, and DNAses have been cloned from various biocontrol species of Trichoderma, and are listed in detail in the above-mentioned chapter (also see Benitez et al. 1998; Kubicek et al. 2001; Lorito 1998). Most of these enzymes showed very strong antifungal activity against a variety of plant pathogenic fungi in vitro. Several of these cell wall degrading enzymes, but most notably chitinases, have thus been demonstrated to have a great potential as active components in new fungicidal formulations or genetically modified plants. Interestingly, the endochitinases found in Trichoderma belong only to one (class V) of the several classes of the chitinases known from plants (Beintema 1994). The latter show a modular structure, and frequently contain protein domains capable of binding to chitin, which bear some resemblance to the cellulose-binding domains also found in Trichoderma cellulases. In contrast, none of the chitinases cloned from Trichoderma spp. so far has been shown to contain such a chitin-binding domain. To investigate the role of the latter, Limon et al. (2001) have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and

the cellulose-binding domain from cellobiohydrolase II of T. reesei. The chimeric chitinases had similar activities as the native chitinase towards soluble substrates, but higher hydrolytic activity on high molecular mass insoluble substrates (chitin or fungal cell walls). Unfortunately, no results from in vivo biocontrol tests were reported, and it remains thus unclear whether the presence of such a domain would improve the antagonistic abilities of Trichoderma biocontrol strains. The action of chitinases and glucanases is also strongly synergistic both with other chitinase components as well as with other components putatively involved in biocontrol, i.e., antibiotics (Jach et al. 1995; Lorito et al. 1994; 1996b; Schirmbo¨ck et al. 1994). In the case of the peptaibols, the mechanism of this enzyme – antibiotic synergism has been shown to be due to a synergistic effect of enzyme and the antibiotic on the maintenance of cell wall integrity (Lorito et al. 1996b). Peptaibols are linear oligopeptides of 12 –22 amino acids, which are rich in a-aminoisobutyric acid, N-acetylated at the N-terminus and containing an amino alcohol (Pheol or Trpol) at the C-terminus (Rebuffat et al. 1989), and known to form voltage-gated ion channels in black lipid membranes and modify the membrane permeability of liposomes in the absence of applied voltage (El Hadjji et al. 1989). Hence, while the chitinases reduce the barrier effect of the cell-wall, peptaibol antibiotics inhibit the membrane bound chitin- and b-glucan synthases and thereby impair the ability of the hyphae to repair the lytic effect of the enzymes on the cell walls polymers. The gene (tex1) encoding the enzyme synthesizing these peptaibols (peptaibol synthase) has recently been cloned from T. virens (Wiest et al. 2002). It comprises a 62.8 kb continuous open reading frame encoding a protein structure consisting of 18 peptide synthetase modules with additional modifying domains at the N- and C-terminii. Mutation of the gene eliminated production of all peptaibol isoforms, indicating that their formation is due to a relaxed substrate speciﬁcity of the individual synthase domains. Interestingly, the nucleotide sequence of tex1 is 100% identical to a 5,056-bp partial cDNA fragment of another gene ( psy1) isolated also from T. virens (Wilhite et al. 2001). These authors observed that psy1 disruptants grew poorly under low-iron conditions, and failed to produce the major T. virens siderophore, dimerum acid (a dipeptide of acylated N(a)-hydroxyornithine, thus suggesting that Psy1 plays a role in siderophore production. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the psy1disruption. The discrepancy between the results reported by Wiest et al. (2002); Wilhite et al. (2001) need to be explained before the importance of the tex1/psy1 gene in biocontrol can be estimated. Peptaibols, however, are certainly not the only secondary metabolites with synergistic action in host cell-wall degardation. Other components (e.g., gpentyl pyrone) was also found to be important for antagonism in vivo (Claydon et al. 1987; Howell 1998; Serrano-Carreon et al. 1993), and

Molecular Biology of Biocontrol Trichoderma

their mechanism of action thus awaits to be elucidated. 6-pentylapyrone is probably the most frequently studied of these metabolites, as it also exhibits a pronounced “coconutaroma” which can be used as a (for humans) nontoxic ﬂavoring agent. Its biosynthesis has been claimed to be derived from linolenic acid (Serrano-Carreon et al. 1993), but this conclusion was criticized by Sivasithamparam and Ghisalberti (1998), who consider it to be a product of polyketide biosynthesis. No other of the genes or proteins involved in Trichoderma secondary metabolism has as yet been characterized.

BIOCONTROL-SPECIFIC GENE EXPRESSION IN TRICHODERMA

In the laboratory, high-level induction of extracellular cellwall lytic enzymes is usually obtained by growing Trichoderma on purified chitin, fungal cell walls, or mycelia as sole carbon sources. No, or much less, induction is normally obtained when related compounds such as chitosan, cellulose, unpurified chitin, or laminarin are used. In addition, formation of most chitinolytic enzymes does not occur or is even inhibited by glucose, sucrose, and chitinolytic endproducts (Carsolio et al. 1994; Garcia et al. 1994; Lorito et al. 1996a; Margolles-Clark et al. 1996; Peterbauer et al. 1996), suggesting that direct induction and/or catabolic repression are major regulatory parameters for chitinase formation. Some researchers also found trace quantities of some chitinases (e.g. the 102-kDa N-acetyl-b-D -glucosaminidase, the 42-kDa endochitinase and the 33 kDa endochitinase) are produced constitutively (Carsolio et al. 1999; Garcia et al. 1994; Haran et al. 1995; Inbar and Chet 1995; MargollesClark et al. 1996). It should be noted that this does not rule out regulation of the respective promoters by induction only, due to the fact that the binding of DNA-binding proteins to their target sequences is an equilibrium, every promoter will partially be in transcriptionally active state, depending on the Kd and the concentrations of the respective proteins. Some of these findings have recently been supported by the analysis of gene expression. The expression of T. atroviride nag1 is triggered by fungal (B. cinerea) cell walls and the commercially available chitin monomer N-acetyl-glucosamine, and the oligomers di-N-acetylchitobiose and tri-N-acetylchitotriose (Mach et al. 1999). In contrast, ech42 expression in T. atroviride was also observed during growth on fungal cell walls, but could not be triggered by those chitin degradation products (Margolles-Clark et al. 1996; Corte´s et al. 1998; Mach et al. 1999), whereas in T. harzianum it is induced by N-acetyl-b-D -glucosamine (Garcia et al. 1994; Schickler et al. 1998). Digestion of the host cell walls with specific combinations of purified Trichoderma-secreted chitinases and glucanases (both endoand exo-acting) released products that strongly elicited ech42 and nag1 gene expression and consequent mycoparasitic activity. Lorito (2002) recently reported the purification of

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these low-molecular weight, biocontrol-inducing molecules released from the host cell walls, and showed that they were much more active in vitro than purified chitin or glucan monomers. The failure of T. atroviride to induce ech42 expression by chitin may be due to complex interactions within different regulatory circuits (Donzelli and Harman 2001): both ech42 and nag1 required both nitrogen starvation and the presence of chitin for induction, whereas gluc78 could be induced by nitrogen starvation alone. In the presence of low levels of ammonium (10 mM), both chito-oligomers and chitin triggered CHIT42 and CHIT40 (chitobiosidase) production. CHIT73 secretion occurred in the presence of N-acetylglucosamine and chito-oligomers, while chitin was less effective. These results indicate that the expression and secretion of cell wall-degrading enzymes by Trichoderma is nitrogen repressed, and that effects of carbon and nitrogen nutrition are interactive. The expression of ech42 from T. atroviride after prolonged carbon starvation is likely not due to a relieve from carbon catabolite repression, as it can be observed with glucose as well as with glycerol as a carbon source (Mach et al. 1999). In addition, ech42 gene transcription was triggered by some conditions of physiological stress (48C, high osmotic pressure, addition of ethanol; Mach et al. 1999), as well as during light-induced sporulation (Carsolio et al. 1994). Interestingly, T. harzianum chit33 expression, while being inducible by N-acetyl-b-D -glucosamine, was also triggered by carbon starvation, nitrogen starvation and physiological stress (de las Mercedes Dana et al. 2001), suggesting that stress-mediated regulation may be a general phenomenon involved in chitinase gene expression of Trichoderma spp. Some studies have so far been performed towards understanding how and in which order the chitinases are induced during mycoparasitic interaction. In their pioneering studies, Inbar and Chet (1992); (1995) demonstrated that formation of chitin-degrading enzymes in T. harzianum is elicited by a lectin-based physical interaction with the host, which was suggested to be the earliest event of interaction, and precede induction by possible chitooligomers (see chapter Herrera-Estrella and Chet). Inbar and Chet (1995) showed that a 102-kDa chitinase is specifically induced by contact with the host lectin, whereas formation of all the other chitinases requires the presence of the living host. They concluded that an N-acetyl-b-D -glucosaminidase with apparent denatured Mr of 102 kDa may be responsible for the first attack and induction for the other chitinases. However, Zeilinger et al. (1998), using the Aequorea victoria GFP as a nondisruptive reporter system, showed that ech42, but not nag1, was formed before any detectable contact of Trichoderma with its host. Similar studies with chit33:GFP in T. harzianum (de las Mercedes Dana et al. 2001) showed that this pre-contact gene expression did not occur with the 33-kDa endochitinase-encoding gene chit33, and therefore may be specific for ech42. Interestingly, ech42 gene expression was prevented when a dialysis membrane was placed between the two fungi (Zeilinger et al. 1999), but still

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occurred when a cellophane membrane was used for this purpose (Corte´s et al. 1998). This led to contradicting conclusions regarding the nature of the molecule triggering ech42 gene expression (Corte´s et al. 1998; Zeilinger et al. 1999); this issue was consequently solved by showing that the cellophane, but not the dialysis, membrane, was partially permeable to proteins of relatively large size (up to 100 kDa) (Kullnig et al. 2000). Thus the data from both studies (Corte´s et al. 1998; Zeilinger et al. 1999) were in perfect agreement and showed that ech42 is expressed before contact of Trichoderma with its host, probably representing one of the earliest event in mycoparasitism and biocontrol. By using two types of membranes (one permeable and one not permeable to proteins), which allowed the removal of either Trichoderma or Rhizoctonia colony from the plate and thus the performing of subsequent cultivations, Kullnig et al. (2000) also showed that a chitinase activity, secreted constitutively by Trichoderma, is essential for the triggering of ech42 gene expression. The nature of this enzyme is still unknown; and it could very well be either (constitutive amounts of) the 42 kDa endochitinase itself or the 102 kDa protein of Inbar and Chet (1995), or any other constitutively formed chitinase or chitinases. To this end, Brunner et al. (2002) deleted the nag1 (73-kDa N-acetyl-b-D -glucosaminidase encoding) gene of T. atroviride. These strains were unable to induce ech42 gene transcription under conditions of carbon starvation or in the presence of fungal cell-walls, and also lacked the formation of other enzyme activities capable of hydrolyzing PNP-NAcGlc, PNP-NAcGlc2, and PNP-NAcGlc3. Since the 102-kDa exochitinase does not occur in T. atroviride P1 (unpublished data), the 73-kDa enzyme may fulfil the role of the T. harzianum 102 kDa enzyme. Unfortunately, a characterization of the latter enzyme has not yet been published. The obvious antifungal activity of Trichoderma chitinases has consequently lead to attempts to improve or alter biocontrol properties of strains by chitinase gene manipulations. Somewhat conflicting data have been reported on the effect of overexpression and/or deletion of selected chitinase genes of Trichoderma. Carsolio et al. (1999) found no difference between an ech42- disrupted strain and its parent T. atroviride IMI 206040 in the biocontrol activity in glasshouse tests against Sclerotium rolfsii and R. solani on cotton, and therefore concluded that ech42 is not essential for biocontrol activity. In contrast, Woo et al. (1998); Baek et al. (1999) noted pronounced effects on the biocontrol efficacy of an ech42 gene disruption mutant of T. atroviride P1 or T. virens, respectively. The latter authors reported an increased and decreased biocontrol activity against R. solani on cotton in strains of T. virens containing two ech42 copies and a disrupted ech42 gene copy. Woo et al. (1998) also observed a significant reduction in antifungal activity for the ech42 disrupted strain and in vivo tests against B. cinerea by leaf inoculations of bean plants revealed a significant reduction of biocontrol ability of the disruptant strain. In contrast, a significant increase was noted for the biocontrol efficacy of soils heavily infested with R. solani. Macro- and

Kubicek

microscopic examinations of the attached seed coats suggested that the lack of the 42-kDa endochitinase may have stimulated the colonization of the spermo- and rhizosphere. Other cell wall hydrolases, whose effect on biocontrol has been studied, are the chitinases chit33 and nag1 and the proteinase prb1. b-Glucanases have also been tried but their overexpression appears to be counteracted by overexpression of acid proteases (Delgado-Jarana et al. 2000). Using a constitutively expressed pki1::chit33 fusion, Limoń et al. (1999) obtained recombinant strains with higher antagonizing activity against R. solani on agar plates. However, results from experiments with these mutants performed in glasshouse or soil have not been reported. T. harzianum transformants carrying two to ten copies of the prb1 gene significantly reduced the disease caused by R. solani in cotton plants under greenhouse conditions (Flores et al. 1997). Interestingly, culture filtrates of a T. atroviride nag1-delta strain, despite of their inability to induce chitinase gene expression (see earlier) exhibited a moderately reduced ability (40 –50%) to protect beans against infections by Rhizoctonia solani and S. sclerotiorum (Brunner et al. 2003). Therefore, while nag1 is essential for triggering chitinase gene expression in T. atroviride, the almost complete loss of chitinase activity only partially impairs biocontrol activity against R. solani and S. sclerotiorum. It is possible that this may be compensated by an increased formation of glucanolytic enzymes in this strain (unpublished data). A more general approach towards improvement of the biocontrol properties of T. harzianum CECT 2413 was presented by Rey et al. (2001); they selected improved biocontrol mutants by testing for the ability to produce wider haloes on pustulan, a polymer of beta-1,6-glucan, as a carbon source. Interestingly, the mutants exhibited two- to four times more chitinase, beta-1,3- and beta-1,6-glucanase activities than the wild type, and produced about three times more extracellular proteins. This mutant performed better than the wild type during in vitro experiments, overgrowing and sporulating on R. solani earlier, killing this pathogen faster and exerting better protection on grapes against B. cinerea.

6.1

Cis and Trans-Acting Genetic Factors Relevant to the Expression of Biocontrol Genes

Lorito et al. (1996a) ﬁrst used an in vitro approach to detect cis-acting motifs on the ch42 promoter being involved in mycoparasitism. They confronted Botrytis cinerea on agar plates with T. atroviride P1, prepared crude protein extracts from mycelia harvested at different phases during mycoparasitism, and used them in electrophoretic mobility shift assays. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1 (50 -SYGGRG-30 ; Kulmburg et al. 1993) provided evidence

Molecular Biology of Biocontrol Trichoderma

that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. These ﬁndings are consistent with the presence of two and three consensus sites, respectively, for binding of Cre1 in the two ech-42 promoter fragments used. In contrast, the protein-DNA complex from mycoparasitic mycelia does not involve Cre1, as its formation is unaffected by the addition of the competing oligonucleotides. Based on these ﬁndings, they offered a preliminary model for regulation of ech-42 expression in T. harzianum, which subsequently involves: (a) binding of Cre1 to two single sites in the ech-42 promoter; (b) binding of a “mycoparasitic” protein/protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (c) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the “mycoparasitic” protein–DNA complex (Lorito et al. 1996a). The cre1 gene from T. harzianum has been cloned (Ilmen et al. 1996), but no demonstration of its effect on biocontrol in vivo was as yet presented.

Figure 1 Scheme illustrating the hypothesis how chitinase gene expression could be triggered in T. atroviride, based on results from Brunner et al. (2002); Mach et al. (1999); Kullnig et al. (2000); Peterbauer et al. (2002a,b); Zeilinger et al. (1999). Circled plus and minus indicate activation and inactivation of a process, respectively, without implying the underlying mechanism. Proteins A, B and C refer to the Zn(6) cluster protein (Peterbauer et al. 2002a), the mycoparasitic regulator (Lorito et al. 1996a) and the BrlA-box binding starvation response repressor (see text), respectively. The black triangles indicate NAcGlc molecules, and symbolize NAcGlc, (NAcGlc)2, and (NAcGlc)3, respectively.

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Another cis-acting element was recently identified that may contribute to the regulation of ech42 gene expression: the ech42 promoter sequence contains two short nucleotide sequences which resemble the consensus for binding of the Aspergillus nidulans brlA (bristle) regulator (50 -MRAGGGR30 ; Chang and Timberlake 1992). The encoded BrlA protein is a general regulator of conidial development, which itself responds to carbon starvation (Skromne et al. 1995). Cell-free extracts of T. atroviride, prepared from mycelia subjected to carbon starvation, form a specific, consensus-dependent complex with BrlA site-containing oligonucleotide fragments of the ech42 promoter (K Brunner, CK Peterbauer, and CP Kubicek, unpublished data). Deletion of the promoter areas containing the BrlA sites in vivo resulted in a derepression of the starvation induced expression of ech42, but had no effect on the expression of ech42 during sporulation. This motif therefore likely binds a new repressor of Trichoderma rather than a sporulation specific regulator. The induction of nag1 by chitin oligomers has been studied in more detail, using a combination of promoter deletion, in vivo footprinting, and EMSA experiments, proteins binding to an AGGGG-element, to a CCAGN13CTGG motif and to a CCAAT-box were identified (Peterbauer et al. 2002a,b). Disruption of either of the two former binding sites in vivo resulted in an almost complete reduction of induction of nag1 expression by N-acetylglucosamine. The nature of the proteins binding to these three motifs is only partially understood: the spatial organization of the CCAGN13CTGG motif would be compatible with the binding of a Zn(II)2Cys6-type zinc finger protein (Todd and Andrianopoulos 1997), whereas the CCAAT-box binds a protein complex consisting of at least three proteins Hap2, Hap3, and Hap5, which were originally described in S. cerevisiae and more recently characterized from T. reesei (Zeilinger et al. 2001). According to Narendja et al. (1999); Zeilinger et al. (2003), their function is the establishment of an open chromatin structure at the promoter. The AGGGG-box is a motif which has been studied in detail in Saccharomyces cerevisiae and identified as a binding site for the Cys2His2 zinc finger proteins Msn2p and Msn4p, which are key regulators of the transcription of a number of genes coding for proteins with stress-protective functions (Ruis and Schu¨ller 1995). In Trichoderma, the occurrence of the AGGGG-box is not restricted to the nag1 promoter but also occurs in two other chitinase promoters, ech42 and chit33 (Lorito et al. 1996a; de las Mercedes Dana et al. 2001), consistent with a potential role in chitinase regulation. This would be compatible with a regulation of the expression of chitinase genes by metabolic stress, as shown both for ech42 and chit33 (see earlier). However, nag1 is not upregulated by stress (CK Peterbauer, unpublished data), and the presence of this motif must therefore serve another function. In this context it is interesting to note that an AGGGG-motif was also identified in the cutinase promotor of Haematonectria haematococca, where it appeared to be involved in maintaining the basal expression level (Ka¨mper et al. 1994). In Yarrowia lipolytica, the AGGGG motif is bound by

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the Mhy1p protein, whose transcription is dramatically increased during the yeast-to-hypha transition (Hurtado and Rachubinski 1999). In order to study whether a homologue of S. cerevisiae MSN2/4 and H. haematococca AGGGG-binding protein encoded by the open reading frame AAB04132 (which we call seb1, ¼stress element binding) has recently been cloned from T. atroviride (Peterbauer et al. 2002a,b). Its zinc finger domain has high amino acid sequence identity with S. cerevisiae Msn2/4 and the H. haematococca AGGGG-binding protein, and specifically recognizes the AGGGG sequence of the ech42 and nag1 promoter in band shift assays. However, a cDNA clone of seb1 was unable to complement a MSN2/4 delta mutant of S. cerevisiae. Despite the presence of AGGGG elements in the promoter of the chitinase gene nag1, no differences in its expression were found between the parent and a seb1-delta-strain. The EMSA analyses with cell-free extracts of the seb1-delta still showed the presence of proteins binding to the AGGGGelement in nag1 and ech42, and thus seb1 does not encode the protein binding to this sequence in the chitinase promoters. Rather, seb1 appears to be involved in osmotic stress response: seb1-mRNA accumulation was increased under conditions of osmotic stress (sorbitol, NaCl)—but not under other stress conditions (cadmium sulfate, pH, membrane perturbance), and growth of the delta-seb1 strain was significantly more inhibited by the presence of 1 M sorbitol and 1 M NaCl than that of the wild-type strain (Peterbauer et al. 2002a,b).

CONCLUSIONS

This and the accompanying review (see chapter 57) show that the molecular biology of Trichoderma has made tremendous progress during the last decade, both from a methodical as well as theoretical perspective. Gene manipulation in Trichoderma is now routine, as are more sophisticated approaches to study gene expression and its regulation. In addition, working models such as the one developed in our laboratory on the induction of chitinases during mycoparasitism (Figure 1), can now be investigated and critically tested in more detail. Yet a drawback of the current situation is that so far mainly genes encoding extracellular enzymes have been studied, and even these mostly under laboratory conditions. Because of the redundancy of the genes encoding extracellular enzymes such as chitinases (cf. Baek et al. 1999), the role of the individual enzymes in vivo still needs to be critically assessed. Other factors may be more relevant in the ﬁeld such as colonization (rhizosphere competence) and competition with the respective hosts. The development of strategies to clone the respective genes and their functional analysis will be the challenge of the future decade.

ACKNOWLEDGEMENTS Research by the author has been performed in collaboration with RL Mach, S Zeilinger, CK Peterbauer, and K Brunner. The projects are funded to CPK by the Jubila¨umsstuftung der ¨ sterreichischen Nationalbank (7817) and Austrian Science O Foundation (P13170-MOB).

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144 Kubicek CP, Bissett J, Kullnig-Gradinger CM, and Szakacs G (2002). Genetic and metabolic diversity of Trichoderma: a case study on South East Asian isolates. Manuscript submitted. Kullnig CM, Szakacs G, and Kubicek CP (2000). Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya. Mycological Res 104:1117 – 1125. Kullnig CM, Mach RL, Lorito M, and Kubicek CP (2000). Enzyme diffusion from Trichoderma atroviride (¼ T. harzianum P1) to Rhizoctonia solani is a prerequisite for triggering of Trichoderma ech42 gene expression before mycoparasitic contact. Appl Environ Microbiol 66:2232 –2234. Kullnig CM, Krupica T, Woo SL, Mach RL, Rey M, Benitez T, Lorito M, and Kubicek CP (2001). Confusion abounds over identities of Trichoderma biocontrol isolates. Mycol Res 105:700 –702. Kullnig-Gradinger CM, Szakacs G, and Kubicek CP (2003). Phylogeny and evolution of the genus Trichoderma: a multigene approach. Mycol Res. in press. Kulmburg P, Mathieu M, Dowzer C, Kelly JM, and Felenbok B (1993). Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CreA repressor mediating carbon catabolite represion in Aspergillus nidulans. Mol Microbiol 7:847 – 857. Limoń MC, Pintor-Toro JA, and Benı´tez T (1999). Increased antifungal activity of Trichoderma harzianum transformants that overexpress a 33-kDa chitinase. Phytopathology 89:254 – 261. Limon MC, Margolles-Clark E, Benitez T, and Penttila M (2001). Addition of substrate-binding domains increases substratebinding capacity and specific activity of a chitinase from Trichoderma harzianum. FEMS Microbiol Lett 198:57– 63. Llorente B, Malpertuy A, Neuveglise C, de Montigny J, Aigle M, Artiguenave F, Blandin G, Bolotin-Fukuhara M, Bon E, Brottier P, Casaregola S, Durrens P, Gaillardin C, Lepingle A, OzierKalogeropoulos O, Potier S, Saurin W, Tekaia F, ToffanoNioche C, Wesolowski-Louvel M, Wincker P, Weissenbach J, Souciet J, and Dujon B (2000). Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae. FEBS Letts 487:101 – 112. Lorito M (1998). Chitinolytic enzymes ands their genes. In: Harman GE, Kubicek CP eds. Trichoderma and Gliocladium. Vol. 2. London, UK: Taylor and Francis. pp 73 –99. Lorito M (2002). Mycoparasitism: what’s the plan of attack? Lecture at the 6th European Conference on Fungal Genetics (ECFG), Pisa, Italy, April 2002. Lorito M, Mach RL, Sposato P, Strauss J, Peterbauer CK, and Kubicek CP (1996a). Mycoparasitic interaction relieves binding of Cre1 carbon catabolite repressor protein to promoter sequence of ech-42 (endochitinase-encoding) gene of Trichoderma harzianum. Proc Natl Acad Sci USA 93:14868 – 14872. Lorito M, Farkas V, Rebuffat S, Bodo B, and Kubicek CP (1996b). Cell-wall synthesis is a major target of mycoparasitic antagonism by Trichoderma harzianum. J Bacteriol 178:6382 – 6385. Lorito M, Peterbauer CK, Hayes CK, Woo SL, and Harman GE (1994). Synergistic combination of cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiol UK 140:623 – 629. Mach RL, Peterbauer CK, Payer K, Jaksits S, Woo SL, Zeilinger S, Kullnig CM, Lorito M, and Kubicek CP (1999). Expression of

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Molecular Biology of Biocontrol Trichoderma the identiﬁcation of Trichoderma harzianum strains. Mycol Res 103:373 – 377. Schirmbo¨ck M, Lorito M, Wang YL, Hayes CK, Arisan-Atac I, Scala F, Harman GE, and Kubicek CP (1994). Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics: molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl Environ Microbiol 60:4364 – 4370. Seiboth B, Hofmann G, and Kubicek CP (2002). Lactose metabolism and cellulase production in Hypocrea jecorina: the gal7 (galactose-1-phosphate uridylyltransferase) gene is essential for growth on galactose but not for cellulase induction on lactose. Mol Gen Genomics 267:124 – 132. Serrano-Carreon L, Hathout Y, Bensoussan M, and Belin JM (1993). Metabolism of linoleic acid and 6-pentyl a-pyronebiosynthesis by Trichoderma species. Appl Environ Microbiol 59:2945 – 2950. Sivasithamparam K and Ghisalberti EL (1998). Secondary metabolism in Trichoderma and Gliocladium. In: Harman GE, Kubicek CP eds. Trichoderma and Gliocladium, Vol. 2. Enzymes, Biological Control and Commercial Application. London, UK: Taylor and Francis Ltd. pp 139 –191. Skromne I, Sanchez O, and Aguirre J (1995). Starvation stress modulates the expression of the Aspergillus nidulans brlA regulatory gene. Microbiol UK 141:21– 28. Stasz TE, Nixon K, Harman GE, Weeden NF, and Kuter GA (1989). Evaluation of phenetic species and phylogenetic relationships in the genus Trichoderma by cladistic analysis of isozyme polymorphism. Mycologia 81:391– 403. Todd RB and Andrianopoulos A (1997). Evolution of a fungal regulatory gene family: the Zn(II)2Cys6 binuclear cluster DNA binding motif. Fungal Genet Biol 21:388– 405. Turoczi G, Fekete C, Kerenyi Z, Nagy R, Pomazi A, and Hornok L (1996). Biological and molecular characterization of

145 potential biocontrol strains of Trichoderma. J Basic Microbiol 36:63– 72. Wiest A, Grzegroski D, Xu BW, Goulard C, Rebouffat S, Ebbole DJ, Bodo B, and Kenerley CM (2003). Identiﬁcation of peptaibols from Trichoderma virens and cloning of a peptaibol synthetase. J Biol Chem, (released electronically April 2002). in press. Wilhite SE, Lumsden RD, and Straney DC (2001). Peptide synthetase gene in Trichoderma virens. Appl Environ Microbiol 67:5055 –5062. Woo SL, Donzelli B, Scala F, Mach RL, Harman GE, Kubicek CP, Del Sorbo G, and Lorito M (1998). Disruption of ech42 (endochitinase-encoding) gene affects biocontrol activity in Trichoderma harzianum strain P1. Mol Plant-Microbe Interact 12:419– 429. Zeilinger S, Mach RL, and Kubicek CP (1998). Two adjacent protein binding motifs in the cbh2 (Cellobiohydrolase II-encoding) strain. Pl Mol Plant-Interact 12:419– 429. Zeilinger S, Galhaup C, Payer K, Woo SL, Mach RL, Fekete C, Lorito M, and Kubicek CP (1999). Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host. Fungal Genet Biol 26:131 –140. Zeilinger S, Ebner A, Marosits T, Mach R, and Kubicek CP (2001). The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element. Mol Genet Genomics 266:56– 63. Zeilinger S, Schmoll M, Pail M, Mach RL, and Kubicek CP (2003). Nucleosome transactions on the Hypocrea jecorina (Trichoderma reesei) cellulase promoter cbh2 reveal a novel role for the carbon catabolite repressor protein Cre1 in chromatin rearrangement. Manuscript submitted. Zimand G, Valinsky L, Elad Y, Cheb I, and Manulis S (1994). Use of RAPD procedure for the identiﬁcation of Trichoderma Strains. Mycol Res 98:531– 534.

13 The Biological Control Agent Trichoderma from Fundamentals to Applications A. Herrera-Estrella Centro de Investigacio´n y Estudios Avanzados, Unidad Irapuato, Irapuato, Me´xico I. Chet The Weizmann Institute of Science, Rehovot, Israel

Biocontrol must be effective, reliable, consistent, and economical before it becomes an important component of plant disease management. To meet these criteria, we must increase our understanding of the biology of the biocontrol agent in question, which in most cases is extremely limited. Furthermore, superior strains, together with delivery systems that enhance biocontrol activity, must be developed (Harman et al. 1989). In this context, many biological control agents can be modiﬁed genetically to enhance their attributes. In addition, we can now think of microorganisms with inhibitory activity against plant pathogens as potential sources of genes for disease resistance.

INTRODUCTION

The practice of monoculture in modern agriculture enables us to continue to provide foodstuffs for the world’s ever increasing population. Monoculture is, however, an ecologically unnatural situation, that is inherently unstable and offers considerable opportunity for the development of diseases. Plant disease control has now therefore become heavily dependent on fungicides to combat the wide variety of fungal diseases that threaten agricultural crops. Even with intensive fungicide use, the destruction of crop plants by fungal pathogens is a serious problem worldwide that annually leads to losses of about 15% (Logemann and Schell 1993). The use of pesticides in general, has also resulted in significant costs to public health and the environment. Studies aimed at replacing pesticides with environmentally safer methods are currently being conducted at many research centers. In this context, control of plant pests by the application of biological agents holds great promise as an alternative to the use of chemicals. It is generally recognized that biological control agents are safer and sounder environmentally than is reliance on the use of high volumes of fungicides and other antimicrobial treatments. The heightened scientific interest in biological control of plant pathogens is a response, in part, to growing public concerns over chemical pesticides. However, there is an equally greater need for biological control of pathogens that presently go uncontrolled or only partially controlled by these “traditional” means (Cook 1993). Thus, biological control should and can be justified on its own merits, without giving it importance at the expense of chemical controls.

TRICHODERMA AS A BIOLOGICAL CONTROL AGENT

The potential for the use of Trichoderma species as biocontrol agents was suggested 70 years ago by Weindling (1932) who was the ﬁrst to demonstrate the parasitic activity of members of this genus towards pathogens such as Rhizoctonia solani (Chet 1990; Weindling 1932). Since then, several species of Trichoderma have been tested as biocontrol agents; and have shown to attack a range of economically important aerial and soilborne plant pathogens (Chet 1987). In many experiments, showing successful biological control, the antagonistic Trichoderma was found to be a necrotrophic mycoparasite (Boosalis 1964; Chet and Elad 1982; Elad et al. 1983b). Mycoparasitism is deﬁned as a direct attack on a fungal thallus, followed by nutrient utilization by the parasite (Chet et al. 1997). Necrotrophic mycoparasites, such as 147

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Trichoderma, are those that kill the host cells before, or just after, invasion and use the nutrients released. These mycoparasites tend to be highly aggressive and destructive. They have a broad host range extending to wide taxonomic groups and are relatively unspecialized in their mode of parasitism. The antagonistic activity of necrotrophs is due to the production of antibiotics, toxins, or hydrolytic enzymes in such proportions as to cause death and destruction of their host (Manocha and Sahai 1993). In our view biocontrol by Trichoderma includes: (a) competition, (b) parasitism, (c) antibiosis, and (d) induction of defense responses in host plants, or the combination of some of them. Parasitism is a complex process including: (a) host recognition, (b) secretion of hydrolytic enzymes, (c) hyphae penetration and invasion (Figure 1), and (d) lysis of the host.

2.1

Host Recognition

In vitro, the ﬁrst detectable interaction shows that the hyphae of the mycoparasite grow directly towards its host (Chet et al. 1981). This phenomenon appears as a chemotropic growth of Trichoderma in response to some stimuli produced by the host (Chet and Elad 1983). When the mycoparasite reaches the host, its hyphae often coil around it or attach to it by forming hooklike structures. Although not a frequent event production of appressoria at the tips of short branches has been observed. Coiling appears to be controlled by lectins present on the host hyphae. A R. solani lectin that binds to galactose and fucose residues was shown to agglutinate conidia of a mycoparasitic strain of Trichoderma harzianum but did not agglutinate two nonparasitic strains (Barak et al. 1985; Elad et al. 1983a).

D -glucose

or D -mannose residues, apparently present on the cell walls of T. harzianum, inhibited the activity of a second lectin isolated from Sclerotium rolfsii. Inbar and Chet (1992; 1994) were able to mimic the fungus –fungus interaction in vitro using nylon fibers coated with either concanavalina A or the purified S. rolfsii lectin. During the interaction Trichoderma recognized and attached to the coated fibers, coiling around them and forming other mycoparasitismrelated structures, such as appresoriumlike bodies and hyphal loops (Inbar and Chet 1992; 1994). Recently, using the biomimetic system we showed that different lectins induce coiling. Furthermore, coiling of Trichoderma around the fibers in the absence of lectins can be induced by applying cAMP or the heterotrimeric G protein activator mastoparan (Rocha-Ramı´rez et al. 2002). Transgenic lines that overexpress the Ga subunit coil at higher frequency than untransformed controls. Furthermore, transgenic lines that express an activated mutant protein with no GTPase activity coil at an even higher frequency. In addition, lines that express an antisense version of the gene do not appear to coil in the biomimetic assay (Rocha-Ramı´rez et al. 2002).

2.2

Host Invasion

It has been proposed that penetration of the host mycelium takes place by partial degradation of its cell wall (Elad et al. 1983b,c). Interaction sites have been stained by fluoresceinisothiocyanate-conjugated lectins or calcofluor. The appearance of fluorescence indicated the presence of localized cell wall lysis at points of interaction between the antagonist and its host (Elad et al. 1983c). Furthermore,

Figure 1 Transmission electron micrographs of Trichoderma atroviride parasiting Rhizoctonia solani. A) T. atroviride (T) penetrates R. solani (R). B) Trichoderma (T) grows inside an R. solani (R) hyphae.

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analysis by electron microscopy has shown that during the interaction of Trichoderma spp. with either S. rolfsii or R. solani the parasite hyphae contacted their host and perforated their cell walls. These observations led to the suggestion that Trichoderma produced and secreted mycolytic enzymes responsible for the partial degradation of the host’s cell wall. Indeed, Trichoderma produces a complex set of glucanases, chitinases, lipases, and proteases extracellularly when grown on cell walls of R. solani (Geremia et al. 1991; Va´zquez-Garciduenãs et al. 1998). Table 1 summarizes the currently available information on this complex set of lytic enzymes produced by Trichoderma. Most attention has been paid to chitinases and several have been studied to some extent in different isolates or even species of the genus. The purification and characterization of three endochitinases secreted by T. harzianum was first reported by De la Cruz et al. (1992). They reported the isozymes to be 37, 33, and 42 kDa, respectively. Only the purified 42 kDa chitinase hydrolyzed Botrytis cinerea purified cell walls in vitro, but this effect was heightened in the presence of either of the other two isoenzymes (De la Cruz et al. 1992). The 42 kDa endochitinase has been found in most isolates. Recently, this enzyme has been proposed to play a major role in the regulatory circuits governing the expression of chitinases upon contact of Trichoderma with its host (Kubicek et al. 2001). Cloning of the genes coding for the 42 kDa secreted chitinases has allowed the construction of a phylogenetic tree,

which showed that they belong to family 18, class V of the glycosyl hydrolases. Interestingly, of the eight fungal species within this clade of the phylogenetic tree, all of them are either fungal or insect parasites and many of the corresponding genes have been implicated in their parasitic activity. However, the chitinolytic system of Trichoderma was recently found to be more complex. Two genes showing similarity to the one encoding the 33 kDa endochitinase described by De la Cruz et al. (1992) have been cloned from T. virens (Kim et al. 2002). These two genes are closely related, according to phylogenetic analysis, and belong to family 18, class III of the glycosyl hydrolases (Kim et al. 2002). Further, at least two types of N-acetyl-b- D glucosaminidases belonging to family 20 of the glycosyl hydrolases have been identified in T. harzianum and T. virens (Draborg et al. 1995; Kim et al. 2002). Chit 36 is another antifungal chitinase recently isolated from T. harzianum TM. This 36 kDa protein shares no significant homology to either Chit33 or 42 (Viterbo et al. 2001). In addition, a 40 kDa chitobiosidase and a 28 kDa exochitinase have been purified (Deane et al. 1998; Harman et al. 1993). In 1995, Haran and co-workers identified six distinct intracellular chitinases by activity on gels. This intracellular set of chitinases is apparently composed of two b-1,4-Nacetylglucosaminidases of 102 and 73 kDa, respectively, and four endochitinases of 52, 42, 33, and 31 kDa, respectively. From this set, the 102 kDa and the 73 kDa N-acetyl glucosaminidases and the 42 kDa endochitinase, were

Table 1 Trichoderma genes encoding cell-wall degrading enzymes Gene Th-En42 ech42 chit42 ech1 th-ch ENC1 ech2 ech3 chit33 cht1 cht2 chit36 nag1 exc1 nag1 exc2 nag2 bgn13.1 bgn1 bgn2 gluc78 bgn3 prb1

Trichoderma spp. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

atroviride atroviride harzianum virens harzianum harzianum virens virens harzianum virens virens harzianum atroviride harzianum virens harzianum virens harzianum virens virens atroviride virens atroviride

Strain

Encoded protein

References

P1 IMI206040 CECT2413 Tv29-8 Tam-61 T25-1 Tv29-8 Tv29-8 CECT2413 Tv29-8 Tv29-8 TM P1 T25-1 Tv29-8 T25-1 Tv29-8 CECT 2413 Tv29-8 Tv29-8 P1 Tv29-8 IMI206040

42-kDa endochitinase 42-kDa endochitinase 42-kDa endochitinase 42-kDa endochitinase 42-kDa endochitinase 42-kDa endochitinase 42-kDa endochitinase Endochitinase 33-kDa endochitinase 33-kDa endochitinase 33-kDa endochitinase 36-kDa endochitinase 73-kDa N-acetyl-b-D -glucosaminidase 73-kDa N-acetyl-b-D -glucosaminidase N-acetyl-b-D -glucosaminidase N-acetyl-b-D -glucosaminidase ?? N-acetyl-b-D -glucosaminidase 78 kDa b-1,3-endoglucanase 78 kDa b-1,3-endoglucanase 78 kDa b-1,3-endoglucanase 78 kDa exo-b-1,3-glucosidase b-1,6-endoglucanase 31-kDa subtilisinlike protease

Hayes et al. (1994) Carsolio et al. (1994) Garcı´a et al. (1994) Kim et al. (2002) Fekete et al. (1996) Draborg et al. (1996) Kim et al. (2002) Kim et al. (2002) Limo´n et al. (1995) Kim et al. (2002) Kim et al. (2002) Viterbo et al. (2001) Peterbauer et al. (1996) Draborg et al. (1995) Kim et al. (2002) Draborg et al. (1995) Kim et al. (2002) De la Cruz et al. (1995) Kim et al. (2002) Kim et al. (2002) Donzelli et al. (2001) Kim et al. (2002) Geremia et al. (1993)

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expressed differentially when Trichoderma was confronted with different hosts on plates (Haran et al. 1996; Inbar and Chet 1995). In conclusion, the complexity and diversity of the chitinolytic system of T. harzianum involves the complementary modes of action of a diversity of enzymes, all of which might be required for maximum efficiency against a broad spectrum of chitin containing plant pathogenic fungi. Trichoderma atroviride also secretes b-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of b-1,3-glucanase activity secreted by T. atroviride was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular b-1,3-glucanases upon induction with laminarin, a soluble b-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60 to 80 kDa, and their pIs are 5.0– 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted (Va´zquezGarciduenãs et al. 1998). bgn13, which encodes a 78 kDa protein from T. harzianum was the first endoglucanase gene identified (De la Cruz et al. 1995). Recently, two genes showing high homology to bgn13.1 have been identified in T. virens. These endoglucanases belong to family 55 of the glycosyl hydrolases (Kim et al. 2002). In addition, a 78 kDa exo-b-1,3-glucanase from T. harzianum has been characterized (Donzelli and Harman 2001). From the set of glucanases produced by Trichoderma two b-1,6-endoglucanase genes have been identified, one in T. harzianum and one in T. virens (Kim et al. 2002; Lora et al. 1995). These two genes encode nearly identical proteins belonging to family 5 of the glycosyl hydrolases (Kim et al. 2002). In 1993, Geremia and co-workers reported the isolation of a 31 kDa basic proteinase, which is secreted by T. harzianum during simulated mycoparasitism. The corresponding gene ( prb1) was cloned and characterized (Geremia et al. 1993) and was the first report of the cloning of a mycoparasitismrelated gene.

EXPRESSION OF MYCOPARASITISM RELATED GENES (MRGs)

Expression of extracellular chitinolytic enzymes is highly induced by growing Trichoderma on puriﬁed chitin, fungal cell walls, or mycelia as sole carbon source. It has been proposed that chitinolytic enzymes could be induced by soluble chito-oligomers (Reyes et al. 1989; St. Leger et al. 1986). This appears to be the case for the 73 kDa N-acetyl-bD -glucosaminidase of T. harzianum and T. atroviride, which are induced not only by chitooligomers but also by N-acetylb-D -glucosamine. The 42 kDa endochitinase of T. harzianum responds similarly to these compounds but ech42 expression in T. atroviride is not induced by the products of chitin degradation (Carsolio et al. 1999; De la Cruz et al. 1993;

Mach et al. 1999; Schikler et al. 1998; Ulhoa and Peberdy 1991; Ulhoa and Peberdy 1993). Expression of ech42 in T. atroviride is strongly induced during fungus –fungus interaction. Its expression is repressed by glucose, may be affected by other environmental factors, such as light and may even be developmentally regulated (Carsolio et al. 1994). In general, formation of most chitinolytic enzymes does not occur or is inhibited in the presence of glucose, sucrose, and chitinolytic end products (Carsolio et al. 1994; 1999; De la Cruz et al. 1993; Garcıá et al. 1994; Peterbauer et al. 1996; Ulhoa and Peberdy 1991). In addition, there is evidence suggesting that the expression of at least ech42 of T. atroviride and chit33 of T. harzianum is repressed by high levels of ammonium (Donzelli and Harman 2001; Mercedes de las et al. 2001). In this sense, the proteinase encoding gene prb1 responds to carbon and nitrogen limitation. It has also been suggested that the MRGs chit33, ech42, and prb1, respond to other types of physiological stress (Mach et al. 1999; de las Mercedes et al. 2001; Olmedo-Monfil et al. 2002). Recently, we found that the response of ech42 and prb1 to nutrient limitation depends on the activation of conserved mitogen activated protein kinase (MAPK) pathways (Olmedo-Monfil et al. 2002). The level of production of b-1,3-glucanases by T. atroviride is induced by the presence of cell walls of M. rouxii, N. crassa, S. cerevisiae, and R. solani (in ascending order of efficiency) and appears to be dependent on the amount of b-1,3-glucan present in the cell walls of these organisms (Va´zquez-Garciduenãs et al. 1998). Additional results obtained with a filtrate of autoclaved S. cerevisiae cell walls suggest that the induction observed with cell walls may be triggered by two components, one extractable and one that remains cell-wall bound (Va´zquez-Garciduenãs et al. 1998). In general, glucanase expression is repressed by glucose and in some cases, might be repressed by primary nitrogen sources (Donzelli and Harman 2001). In summary, expression of all enzymes from the cell-wall degrading system of Trichoderma appears to be coordinated. Suggesting a regulatory mechanism involving substrate induction and catabolite repression. The expression of the system is controlled at the level of transcription as indicated by Northern analysis of the available genes (Carsolio et al. 1994; De la Cruz et al. 1995; Donzelli and Harman 2001; Flores et al. 1997; Geremia et al. 1993; Kim et al. 2002; Limoń et al. 1995; Mercedes de las et al. 2001). An exciting finding in terms of signaling is that the induction of at least two MRGs, namely prb1 and ech42, is triggered by a diffusible factor produced by the host (Corte´s et al. 1998). Recently, it has been suggested that the activation of MRGs in response to the presence of the host, through such a molecule depends on the basal expression of ech42 in T. atroviride (Kubicek et al. 2001). However, in T. virens, induction of four MRGs in response to cell walls in ech1 (the homologue of ech42) knockout mutants is still observed. Whether a key molecule produced by the host in vivo switches on the expression of MRGs remains to be proven, as well as the role of Ech42 in the production of such a molecule.

Trichoderma in Plant Protection

ANTIBIOSIS

The involvement of volatile and nonvolatile antibiotics in the antagonism by Trichoderma has been proposed (Dennis and Webster 1971a,b). Indeed some isolates of Trichoderma excrete growth inhibitory substances (Claydon et al. 1987; Ghisalberti and Sivasithamparam 1991; Sivan et al. 1984). Claydon et al. (1987) identiﬁed volatile alkyl pyrons produced by T. harzianum that were inhibitory to a number of fungi in vitro. When these metabolites were added to a peat-soil mixture, they reduced the incidence of R. solani-induced damping-off on lettuce. However, there is insufﬁcient evidence to be conclusive about their contribution to pathogen suppression and disease reduction in situ. Trichoderma also produces linear oligopeptides of 12–22 aminoacids (peptaibols), which are rich in -aminoisobutyric acid, N-acetylated at the N-terminus and containing an amino alcohol at the C-terminus (Rebuffat et al. 1989; 1991). These oligopeptides are known to form voltage-gated ion channels in black lipid membranes and modify the membrane permeability of liposomes (El Hadjji et al. 1989). This suggested a scenario where cell-wall degrading enzymes weaken the cell wall and peptaibol antibiotics inhibit synthesis of cell-wall components, impairing the capacity of the hyphae to repair the effect of cell-wall degrading enzymes (Lorito et al. 1996a,b). This hypothesis is supported by the fact that the action of cellwall degrading enzymes is synergistic with that of antibiotics (Lorito et al. 1996a,b).

ROLE OF MRGs IN BIOCONTROL AND STRAIN IMPROVEMENT

A major challenge for researchers investigating the mechanisms involved in the parasitic activity of Trichoderma has been to establish the role of cell-wall degrading enzymes in the process. In fact, we have proposed to call all genes encoding cell-wall degrading enzymes MRGs, because of their apparent relation to the process, until their role is fully determined. Intensive efforts using genetic engineering are currently being directed at this goal. In 1997, Flores and coworkers generated transgenic T. atroviride lines carrying multiple copies of prb1. The resulting strains produced up to 20 times more proteinase and all strains tested were more effective in the control of R. solani. One strain reduced the disease incidence caused by R. solani on cotton plants to only 6% whereas the disease incidence for the nontransformed strain was 30% (Flores et al. 1997), demonstrating that prb1 plays an important role in biocontrol and the feasibility of strain improvement through genetic engineering. The role of the Trichoderma 42 kDa endochitinases in mycoparasitism has been addressed by genetic manipulation of the corresponding gene (ech42 and ech1) in T. atroviride and T. virens (Baek et al. 1999; Carsolio et al. 1999; Woo et al. 1999). In T. atroviride, several transgenic strains carrying multiple copies of ech42 were generated (Carsolio et al.

151

1999), as well as the corresponding gene disruptants (Carsolio et al. 1999; Woo et al. 1999). The level of extracellular endochitinase activity when T. atroviride was grown under inducing conditions increased up to 42 fold in multicopy strains as compared to the nontransformed strain. Multicopy transformants reduced disease incidence by about 10%. Furthermore, a 30% higher degradation of the chitin content in R. solani cell walls was observed during interaction with the overexpressing Trichoderma than with the wild type, when quantified by transmission electron microscopy (Carsolio et al. 1999). In the case of the gene disruptants no differences in their efficiency to control R. solani or S. rolfsii were observed in greenhouse experiments, as compared to the nontransformed control strains (Carsolio et al. 1999). In a second study (Woo et al. 1999), a reduction of the antifungal activity in vitro of the ech42 disrupted strains towards B. cinerea was observed. However, in vivo tests against B. cinerea by leaf inoculation of bean plants revealed a significant reduction of their biocontrol capacity. Contrasting with these results, ech42 gene disruptants showed increased efficiency to control R. solani in soil. Similar experiments in T. virens showed increased and decreased biocontrol activity against R. solani on cotton using ech1 overexpressing lines and gene disruptants, respectively (Baek et al. 1999). The role of Chit33 and Chit36 from T. harzianum in biocontrol has also been tested by expressing the corresponding gene at high levels using the strong constitutive pki promoter (Limoń et al. 1999; Viterbo et al. 2001). Test of R. solani control under greenhouse conditions suggested higher efficiency of Trichoderma transformants bearing the chit36 gene under the pki promoter, but this did not reach statistical significance (Viterbo et al. 2001). The transgenic lines generated overexpressing Chit 33 showed higher antagonistic activity against R. solani on agar plates. However, in vivo experiments with these transgenic lines have not been reported. In another attempt to increase the effectiveness of T. harzianum, it was transformed with a bacterial chitinase gene from Serratia marcescens under the control of the CaMV35S promoter. Two transformants showed increased constitutive chitinase activity and expressed a protein of the expected size (58 kDa). When evaluated in dual cultures against the phytopathogenic fungus S. rolfsii both showed higher antagonistic activity, as compared to the nontransformed control (Haran et al. 1993). Unfortunately, no in vivo experiments were reported using these strains. Recently, the role of glucanases in the interaction between T. harzianum and Pythium ultimum was studied (Benhamou and Chet 1997). Contact between the two fungi was accompanied by the deposition of a cellulose-enriched material at potential penetration sites. Trichoderma was able to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, cellulase production was not the only critical trait involved in the process. A marked alteration of the b-1,3-glucan component of the Pythium cell wall was also observed, suggesting that b-1,3-glucanases played a key role in the antagonism. In yet another study,

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T. longibrachiatum transformants carrying extra copies of the egl1 gene (a cellulase encoding gene) were evaluated for their biocontrol activity against P. ultimum on cucumber seedlings (Migheli et al. 1998). The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under nonselective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain. Biocontrol agents tolerant to specific pesticides could be constructed using molecular techniques. Resistance to the fungicide benomyl is conferred by a single amino-acid substitution in one of the b-tubulins of T. viride; the corresponding gene has been cloned and proven to work in other Trichoderma species (Goldman et al. 1993), thereby producing a biological control agent that could be applied simultaneously or in alternation with the fungicide. However, these strains have not been tested in biocontrol experiments. Molecular techniques may eventually be used to transfer several beneficial traits, such as the production of one or more antibiotics and pesticide tolerance, to an aggressive phyllosphere colonizer.

COMPETITION

Competition occurs between microorganisms when space or nutrients (i.e., carbon, nitrogen, and iron) are limiting and its role in the biocontrol of plant pathogens has been studied for many years, with special emphasis on bacterial biocontrol agents (Weller 1988). Implicit in this deﬁnition is the understanding that combative interactions such as antibiotic production, mycoparasitism, or the occurrence of induced resistance in the host are excluded even though these mechanisms may form an important part of the overall processes occurring in the interaction. In the rhizosphere, competition for space as well as nutrients is of major importance. Thus, an important attribute of a successful rhizosphere biocontrol agent would be the ability to remain at high population density on the root surface providing protection of the whole root for the duration of its life. Recently, it was found that a strain of T. harzianum (T-35) that controls Fusarium spp. on various crops might take advantage of competition for nutrients and rhizosphere colonization (Sivan and Chet 1989).

INDUCED RESISTANCE

Induced resistance is a plant response to challenge by microorganisms or abiotic agents such that following the inducing challenge de novo resistance to pathogens is shown

in normally susceptible plants (de Wit 1985) Induced resistance can be localized, when it is detected only in the area immediately adjacent to the inducing factor or systemic, where resistance occurs subsequently at sites throughout the plant. Both localized and systemically induced resistances are nonspeciﬁc. Recently, the potential of T. harzianum T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with Trichoderma in an aseptic, hydroponic system (Yedidia et al. 1999). Trichoderma-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from Trichoderma-treated roots revealed penetration of T. harzianum, mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as well as deposition of newly formed barriers. These typical host reactions were found beyond the sites of potential fungal penetration. Wall appositions contained large amounts of callose and inﬁltrations of cellulose. Further biochemical analyses revealed that inoculation with the fungus resulted in increased peroxidase and chitinase activities in roots and leaves of treated seedlings, providing evidence that T. harzianum may induce systemic resistance mechanisms in cucumber plants (Yedidia et al. 1999).

PLANT GROWTH PROMOTION

Microbial interactions with plant roots are known to affect profoundly plant nutrient status and, for manganese at least, to affect plant resistance to pathogens (Huber and McCay-Buis 1993). In addition to their biocontrol characteristics, Trichoderma species also exhibit plant-growth-promoting activity (Baker 1989; Chet 1987; Harman and Bjorkman 1998; Inbar et al. 1994; Kleifeld and Chet 1992; Naseby et al. 2000). In spite of their theoretical and practical importance, the mechanisms responsible for the growth response due to Trichoderma have not been investigated extensively. Since growth enhancement has been observed in the absence of any detectable disease (Chang et al. 1986; Harman and Bjorkman 1998; Naseby et al. 2000) and in sterile soil (Windham et al. 1986), it is not thought to be a side effect of suppression of disease or minor plant pathogens. Other mechanisms, including production of hormonelike metabolites and release of nutrients from soil or organic matter, have been proposed (Kleifeld and Chet 1992; Windham et al. 1986). The plant-growth-promoting capacity of T. harzianum to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity was recently investigated (Altamore et al. 1999). T. harzianum was able to solubilize MnO2, metallic zinc, and rock phosphate (mostly calcium phosphate). Fe2O3, MnO2, Zn, and rock phosphate were also solubilized by cell-free culture filtrates. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn.

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In liquid culture, T. harzianum also produced diffusible metabolites capable of reducing Fe(III) and Cu(II). Solubilization of metal oxides by Trichoderma involves both chelation and reduction (Altamore et al. 1999).

TRICHODERMA AS A SOURCE OF GENES FOR CROP IMPROVEMENT

One of the major goals of plant genetic engineering is to protect plants from diseases. There are many examples of the introduction of chitinase genes into plants resulting in an enhancement of resistance of the host plant to fungal pathogens (Broglie et al. 1993; Lin et al. 1995; Vierheilig et al. 1993). However, the desired levels of resistance for successful commercial application have not yet been reached. The use of potent chitinases with proven antifungal activity is thus an attractive alternative. Because Ech42 from T. atroviride fulﬁlls these criteria, the corresponding gene was introduced into tobacco and potato (Lorito et al. 1998). High expression levels of the fungal gene were obtained in different plant tissues, with no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among different transformed lines. Transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, Alternaria solani, B. cinerea, and the soilborne pathogen R. solani. Interestingly, the levels of tolerance reached in these experiments were higher than those previously achieved by expression of bacterial or plant chitinases (Lorito et al. 1998). A similar strategy was used to improve scab resistance of apple (Bolar et al. 2000). The endochitinase gene (ech42), as cDNA and genomic clones, was transferred into apple cv. Marshall McIntosh. Eight lines propagated as grafted and self-rooted plants were inoculated with Venturia inaequalis. Six transgenic lines expressing the endochitinase were more resistant than controls. Disease severity in the transgenic lines tested compared with nontransformed controls was reduced, as well as the number of lesions and the leaf area infected. However, in contrast with the results previously reported (Lorito et al. 1998), expression of the endochitinase also had negative effects on the growth of both inoculated and uninoculated plants (Bolar et al. 2000). In a more recent investigation the same group introduced either an endochitinase or an exochitinase, both from Trichoderma, into apple plants (Bolar et al. 2001). In agreement with their previous results resistance to V. inaequalis correlated with the level of expression of either enzyme. Plants expressing both enzymes simultaneously were more tolerant that plants expressing either enzyme alone. Their results indicate that the two enzymes acted synergistically to limit disease development.

CONCLUSIONS

Although Trichoderma is widely used in the ﬁeld to control plant diseases and its commercialization has signiﬁcantly

increased, our understanding of the mechanisms used by Trichoderma to antagonize phytopathogenic fungi is still very limited. Little is known on the signaling pathways that determine host recognition, although there is evidence of the involvement of conserved signaling pathways such as heterotrimeric G proteins in hyphal coiling. At later stages of the interaction, MAPK pathways appear to participate in the regulation of the expression of MRGs. However, we are still just beginning to untangle the networks determining host recognition. An important number of genes encoding cell-wall degrading enzymes have been cloned. In most cases, their expression correlates with conditions that simulate the actual interaction with the host and some of them have even been used to generate improved strains. However, the fact that several of the cloned MRGs respond to multiple environmental signals that they are subjected to catabolite repression, and that none of them is expressed specifically at the sites of interaction, suggests that these genes maybe part of a specialized saprophytic response. Thus, the corresponding enzymes are more likely to participate in the utilization of the host’s cellular components as a food supply at the end of the parasitic process. An alternative explanation is that the interaction of Trichoderma with a host is interpreted by the parasite as a stress signal and that MRGs are in fact stress responsive genes. It is likely that genes coding for key enzymes such as those expressed specifically at the site of interaction where penetration or cell wall perforations are observed, have not been yet identified. The use of functional genomics strategies will certainly be a major step towards the identification of genes playing key roles in mycoparasitism by Trichoderma. Trichoderma has already proven to be an important source of genes for engineering plants for pathogen resistance. Yet, there is still a complete battery of genes that should be tested for this purpose, as well as combinatory strategies using several Trichoderma genes. Induction of defense responses in host plants and plant growth promotion are important attributes of Trichoderma, whose study was neglected for a long time. The recent evidence on these two aspects makes Trichoderma an even more attractive organism for large-scale application as a biological control agent. In spite of our limited knowledge on the mechanisms underlying the mycoparasitic activity of Trichoderma, it is clear that it is an excellent model system for the study of interfungal parasitic relationships and that it has an enormous potential for a variety of biotechnological applications.

ACKNOWLEDGEMENT The authors wish to thank Dr. June Simpson for critical reading of the manuscript.

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Herrera-Estrella and Chet Cook RJ (1993). Making greater use of introduced microorganisms for biological control of plant pathogens. Annu Rev Phytopathol 31:53 –80. Corte´s C, Gutie´rrez A, Olmedo V, Inbar J, Chet I, and HerreraEstrella A (1998). The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor. Mol Gen Genet 260:218 – 225. De la Cruz J, Hidalgo-Gallego A, Lora JM, Benı´tez T, Pintor-Toro JA, and Llobell A (1992). Isolation and characterization of three chitinases from Trichoderma harzianum. Eur J Biochem 206:859 – 867. De la Cruz J, Rey M, Lora JM, Hidalgo-Gallego A, Domıńguez F, Pintor-Toro JA, Llobell A, and Benı´tez T (1993). Carbon source control on b-glucanases, chitobiase and chitinase from Trichoderma harzianum. Arch Microbiol 159:1– 7. De la Cruz J, Pintor-Toro JA, Benı´tez T, Llobell A, and Romero LC (1995). A novel endo-b-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 177:6937 – 6945. Deane EE, Whipps JM, Lynch JM, and Peberdy JF (1998). The purification and characterization of a Trichoderma harzianum exochitinase. Biochim Biophys Acta 1383:101 – 110. Dennis C and Webster J (1971a). Antagonistic properties of species-groups of Trichoderma. I. Production of non-volatile antibiotics. Trans Brit Mycol Soc 57:25 – 39. Dennis C and Webster J (1971b). Antagonistic properties of speciesgroups of Trichoderma. II. Production of volatile antibiotics. Trans Brit Mycol Soc 57:41 – 48. Donzelli BGG and Harman GE (2001). Interaction of ammonium, glucose, and chitin regulates the expression of cell wall degrading enzymes in Trichoderma atroviride strain P1. Appl Environ Microbiol 67:5643 –5647. Donzelli BG, Lorito M, Scala F, and Harman GE (2001). Cloning, sequence and structure of a gene encoding an antifungal glucan 1,3-beta-glucosidase from Trichoderma atroviride (T. harzianum). Gene 277:199 – 208. Draborg H, Kaupinnen S, Halkier T, Dalboge H, and Christgau S (1995). Molecular cloning and expression in Sacharomyces cerevisiae of two exochitinases from Trichoderma harzianum. Biochem Mol Biol Interact 36:781– 791. Draborg H, Christgau S, Halkier T, Ramussen G, Dalboge H, and Kaupinnen S (1996). Secretion of an enzimatically Trichoderma harzianum endochitinase by Sacharomyces cerevisiae. Curr Genet 29:404– 409. El Hadjji M, Rebuffat S, Le Doan T, Klein G, Satre M, and Bodo B (1989). Interaction of trichorzianines A and B with model membranes and with amoeba Dictyostelium. Biochim Biophys Acta 978:91 –104. Elad Y, Barak R, and Chet I (1983a). Possible role of lectins in mycoparasitism. J Bacteriol 154:1431 –1435. Elad Y, Barak R, Chet I, and Henis Y (1983b). Ultraestructural studies of the interaction between Trichoderma spp. and plant pathogenic fungi. Phytopathol Z 107:168 –175. Elad Y, Chet I, Boyle P, and Henis Y (1983c). Parasitism of Trichoderma spp. on Rhizoctonia solani and Sclerotium rolfsii. Scanning electron microscopy and fluorescens microscopy. Phytopathology 73:85 –88. Fekete C, Weszely T, and Hornok L (1996). Assignment of a PCRamplified chitinase sequence cloned from Trichoderma hamatum to resolved chromasomes of potential biocontrol species of Trichoderma. FEMS Microbiol Lett 145:385 – 391.

Trichoderma in Plant Protection Flores A, Chet I, and Herrera-Estrella A (1997). Improved biocontrol activity of Trichoderma harzianum strains by overexpression of the proteinase encoding gene prb1. Curr Genet 31:30 – 37. Garcıá I, Lora JM, De la Cruz J, Benı´tez T, Llobell A, and Pintor-Toro JA (1994). Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum. Curr Genet 27:83 – 89. Geremia R, Jacobs D, Goldman GH, Van Montagu M, and Herrera-Estrella A (1991). Induction and secretion of hydrolytic enzymes by the biocontrol agent Trichoderma harzianum. In: Beemster ABR, Bollen GJ, Gerlagh M, Ruissen MA, Schippers B, Tempel A eds. Biotic Interactions and Soil-Borne Diseases. Amsterdam: Elsevier. pp 181 – 186. Geremia RA, Goldman GH, Jacobs D, Ardiles W, Vila SB, Van Montagu M, and Herrera-Estrella A (1993). Molecular characterization of the proteinase-encoding gene prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 8:603– 613. Ghisalberti EL and Sivasithamparam K (1991). Antifungal antibiotics produced by Trichoderma spp. Soil Biol Biochem 23:1011 – 1020. Goldman GH, Temmerman W, Jacobs D, Contreras R, Van Montagu M, and Herrera-Estrella A (1993). A nucleotide substitution in one of the b-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-ylcarbamate. Mol Gen Genet 240:73– 80. Haran S, Schickler H, Pe’er S, Logemann S, Oppenheim A, and Chet I (1993). Increased constitutive chitinase activity in transformed Trichoderma harzianum. Biological Control 3:101– 103. Haran S, Schickler H, Oppenheim A, and Chet I (1996). Differential expression of Trichoderma harzianum chitinases during mycoparasitism. Phytopathology 86:980– 985. Harman GE and Bjorkman T (1998). Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: Kubicek CK, Harman GE eds. Trichoderma and Gliocladium. London, England: Taylor and Francis. pp 229 – 265. Harman GE, Taylor AG, and Stasz TE (1989). Combining effective strains of Trichoderma harzianum and solid matrix priming to provide improved biological seed treatment systems. Plant Dis 73:631– 637. Harman GE, Hayes CK, Lorito M, Broadway RM, Di Pietro A, and Tronsmo A (1993). Chitinolytic enzymes of Trichoderma harzianum, purification of chitibiosidase and endochitinase. Phytopathology 83:313 –318. Hayes CK, Klemsdal S, Lorito M, Di Pietro A, Peterbauer C, Nakas JP, Tronsmo A, and Harman GE (1994). Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum. Gene 138:143 – 148. Huber DM and McCay-Buis TS (1993). A multiple component analysis of the take-all disease of cereals. Plant Dis 77:437– 447. Inbar J and Chet I (1992). Biomimics of fungal cell – cell recognition by use of lectin-coated nylon fibers. J Bacteriol 174:1055 –1059. Inbar J and Chet I (1994). A newly isolated lectin from the plant pathogenic fungus Sclerotium rolfsii: purification, characterization and role in mycoparasitism. Microbiology 140:651 – 657.

155 Inbar J and Chet I (1995). The role of recognition in the induction of specific chitinases during mycoparasitism by Trichoderma harzianum. Microbiology 141:2823 –2829. Inbar J, Abramsky M, Cohen D, and Chet I (1994). Plant growth enhancement and disease control by Trichoderma harzianum in vegetable seedlings grown under commercial conditions. Eur J Plant Pathol 100:337 – 346. Kim D-J, Baek J-M, Uribe P, Kenerley CM, and Cook DR (2002). Cloning and characterization of multiple glycosyl hydrolase genes from Trichoderma virens. Curr Genet 40:374– 384. Kleifeld O and Chet I (1992). Trichoderma harzianum—-interaction with plants and effect on growth response. Plant Soil 144:267 –272. Kubicek CP, Mach RL, Peterbauer CK, and Lorito M (2001). Trichoderma: from genes to biocontrol. J Plant Pathology 83:11– 23. Limoń MC, Lora JM, Garcia I, De la Cruz J, Llobell A, Benitez T, and Pintor-Toro JA (1995). Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum. Curr Genet 28:478– 483. Limoń MC, Pintor-Toro JA, and Benı´tez T (1999). Increased antifungal activity of Trichoderma harzianum transformants that overexpress a. 33-kDa chitinase. Phytopathology 89:254– 261. Lin W, Anuratha CS, Datta K, Potrykus I, Muthukrishnan S, and Datta SK (1995). Genetic engineering of rice for resistance to sheath blight. Biotechnology 13:686– 691. Logemann J and Schell J (1993). The impact of biotechnology on plant breeding, or how to combine increases in agricultural productivity with an improved protection of the environment. In: Chet I ed. Biotechnology in Plant Disease Control. New York: Wiley-Liss, Inc. p 14. Lora JM, De la Cruz J, Llobell A, Benı´tez T, and Pintor-Toro JA (1995). Molecular characterization and heterologous expression of an endo-b-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum. Mol Gen Genet 247:630 – 645. Lorito M, Farkas V, Rebuffat S, Bodo B, and Kubicek CP (1996a). Cell wall synthesis is a major target of mycoparasitic antagonism by Trichoderma harzianum. J Bacteriol 178:6382– 6385. Lorito M, Woo SL, D’Ambrosio M, Harman GE, Hayes CK, Kubicek CP, and Scala F (1996b). Synergistic interaction between cell wall degrading enzymes and membrane affecting compounds. Mol Plant-Microb Interact 9:206 –213. Lorito M, Woo S, Garcıá-Fernańdez I, Colucci G, Harman GE, Pintor-Toro JA, Filippone E, Muccifora S, Lawrence CB, Zoina A, Tuzun S, and Scala F (1998). Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 95:7860 –7865. Mach RL, Peterbauer CK, Payer K, Jaksits S, Woo SL, Zeilinger S, Kullnig C, Lorito M, and Kubicek CP (1999). Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals. Appl Environ Microbiol 65:1858 –1863. Manocha MS and Sahai AS (1993). Mechanisms of recognition in necrotrophic and biotrophic mycoparasites. Can J Microbiol 39:269– 275. Mercedes de las D, Limoń MC, Mejıás R, Mach RL, Benı´tez T, Pintor-Toro JA, and Kubicek CP (2001). Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum. Curr Genet 38:335 –342.

156 Migheli Q, Gonza´lez-Candelas L, Dealessi L, Camponogara A, and Ramoń-Vidal D (1998). Transformants of Trichoderma longibrachiatum overexpressing the beta-1,4-endoglucanase gene egl1 show enhanced biocontrol of Pythium ultimum on cucumber. Phytopathology 88:673– 677. Naseby DC, Pascual JA, and Lynch JM (2000). Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum populations, soil microbial communities and soil enzyme activities. J Appl Microbiol 88:161– 169. Olmedo-Monfil V, Mendoza-Mendoza A, Go´mez I, Corte´s C, and Herrera-Estrella A (2002). Multiple environmental signals determining the transcriptional activation of the mycoparasitism related gene prb1 in Trichoderma atroviride. Mol Gen Genom 267:703 –712. Peterbauer CK, Lorito M, Hayes CK, Harman GE, and Kubicek CP (1996). Molecular cloning and expression of the nag1 gene gene) from (N-acetyl-b-D -glucosaminidase-encoding Trichoderma harziaunm P1. Curr Genet 30:325– 331. Rebuffat S, El Hajji M, Hennig P, Devoust D, and Bodo B (1989). Isolation, sequence and conformation of seven trichorzianines B from Trichoderma harzianum. Int J Peptide Prot Res 34:200 – 210. Rebuffat S, Prigent Y, Auvin-Guette C, and Bodo B (1991). Tricholongins BI and BII 19-residue peptaibols from Trichoderma longibrachiatum. Solution structure from twodimensional nuclear magnetic resonance spectrometry. Eur J Biochem 201:661 – 674. Reyes F, Calatayud J, and Martıńez MJ (1989). Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall. FEMS Microbiol Lett 60:119– 124. Rocha-Ramı´rez V, Omero C, Chet I, Horwitz BA, and HerreraEstrella A (2002). A Trichoderma atroviride G protein a-subunit gene, tga1, involved in mycoparasitic coiling and conidiation. Eukaryotic Cell: 0. in press. Schikler H, Haran S, Oppenheim A, and Chet I (1998). Induction of the Trichoderma harzianum chitinolytic system is triggered by the chitin monomer N-acetylglucosamine. Mycol Res 102:1224 – 1226. Sivan A and Chet I (1989). The possible role of competition between Trichoderma harzianum and Fusarium oxysporum on rhizosphere colonization. Phytopathology 79:198 – 203. Sivan A, Elad Y, and Chet I (1984). Biological control effects of a new isolate of Trichoderma harzianum on Pythium aphanidermatum. Phytopathology 74:498– 501.

Herrera-Estrella and Chet St. Leger R, Cooper RM, and Charnley AK (1986). Cuticle degrading enzymes of entomopathogenic fungi: regulation of production of chitinolytic enzymes. J Gen Microbiol 132:1509 – 1517. Ulhoa CJ and Peberdy JF (1991). Regulation of chitinase synthesis in Trichoderma harzianum. J Gen Microbiol 137:2163 – 2169. Ulhoa CJ and Peberdy JF (1993). Effect of carbon sources on chitobiase production by Trichoderma harzianum. Mycol Res 97:45 – 48. Va´zquez-Garciduen˜as S, Leal-Morales CA, and Herrera-Estrella A (1998). Analysis of the b-1,3-glucanolytic system of the biocontrol agent Trichoderma harzianum. Appl Environ Microbiol 64:1442 – 1446. Vierheilig H, Alt M, Neuhaus J-M, Boller T, and Wiemken A (1993). Colonization of transgenic N. sylvestris plants, expressing different forms of N. tabacum chitinase, by the root pathogen, Rhizoctonia solani, and by the mycorrhizal symbiont, Glomus mosseau. Mol Plant-Microbe Interact 6:261 –264. Viterbo A, Haran S, Friesem D, Ramot O, and Chet I (2001). Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiol Lett 200:169 – 174. Weindling R (1932). Trichoderma lignorum as a parasite of other fungi. Phytopathology 22:837– 845. Weller DM (1988). Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev Phytopathol 26:379 –407. Windham MT, Elad Y, and Baker R (1986). A mechanism for increased plant growth induced by Trichoderma spp. Phytopathology 76:518– 521. Wit de PJGM (1985). Induced resistance to fungal and bacterial diseases. In: Fraser RSS ed. Mechanisms of Resistance to Plant Diseases. Dordrecht: Nijhoff/Junk. pp 405 –424. Woo SL, Donzelli B, Scala F, Harman GE, Kubicek CP, Del Sorbo G, and Lorito M (1999). Disruption of the ech42 (endochitinase-encoding) gene affects biocontrol activity in Trichoderma harzianum P1. Mol Plant-Microb Interact 12:419 –429. Yedidia I, Benhamou N, and Chet I (1999). Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl Environ Microbiol 65:1061 – 1070.

14 Biological Control of Fungal Diseases on Vegetable Crops with Fungi and Yeasts Zamir K. Punja Simon Fraser University, Burnaby, British Columbia, Canada Raj S. Utkhede Paciﬁc Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada

stages of seedling growth, causing seed decay and damping-off. Examples of these fungi are Rhizoctonia solani Ku¨hn, various species of Fusarium and Pythium, and Sclerotium rolfsii Sacc. Fungal pathogens that infect the roots and crown of developing plants, causing root and crown rots and vascular wilts, have also been researched for biological control strategies. These include fungi such as Pythium spp., Fusarium spp., and Sclerotinia sclerotiorum (Lib.) de Bary. A third group of foliar-infecting fungi of vegetable crops that cause leaf spots and blights and stem infection, also have biological control strategies developed against them. These include Botrytis cinerea Pers. ex Fr. (gray mold), Didymella bryoniae (Auersw.) Rhem (gummy stem blight), S. sclerotiorum (white mold), and Sphaerotheca and Erysiphe spp. (powdery mildews). Many different fungal and yeast biological control agents have been identified and evaluated for disease control potential against the above-mentioned pathogens, and some have been formulated and brought to market to provide disease control options for producers of vegetable crops. The use of biological control agents may be particularly attractive for vegetable crops grown in glasshouses, due to the high market value of these crops and the possibility for control of environmental parameters, particularly temperature and relative humidity (Paulitz and Be´langer 2001). These are important variables that can significantly influence the efficacy of biological control agents under natural field conditions (Paulitz 1997). The rationale for development of biological control agents against fungal diseases on vegetable crops was to provide an additional/alternative approach to augment/replace the use of

INTRODUCTION

Vegetable crops may be produced as both fresh market and processed commodities and can be grown under ﬁeld conditions or in controlled environments, such as glasshouses or other similar structures. There are numerous fungal diseases that attack a wide range of these vegetable crops (Howard et al. 1994), thereby reducing crop yield and quality. Methods for disease control have included the use of cultural practices to reduce pathogen inoculum and disease incidence, development of resistant cultivars, as well as the application of chemical fungicides to inhibit pathogen development. The use of biological control strategies has also demonstrated the potential of fungi and yeasts in reducing a range of fungal pathogens that cause various diseases on vegetable crops. In this chapter, some examples of recent successes in biological control of fungal diseases of vegetable crops using fungi and yeasts, and the mechanisms by which pathogen control was achieved will be reviewed. In addition, the utilization of techniques in biotechnology to aid in the implementation of biological control strategies for disease control will be reviewed. These include techniques to investigate mechanism(s) of action of the biological control agent, development of strains with enhanced efﬁcacy through genetic manipulation, monitoring the growth and spread of biocontrol agents using molecular techniques, and characterization of strains using genetic markers and biochemical methods. The diseases to be considered in this chapter for which biocontrol strategies have been described include those caused by pathogenic fungi that infect the seed and early 157

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chemical fungicides, to provide a level of disease control in the absence of crop genetic resistance, and to augment cultural control practices to further minimize the impact of these diseases and reduce chemical residues in food. For example, chemical fungicides typically have provided adequate control of many fungal pathogens. However, fungicide resistance problems, concerns regarding pesticide residues, and revocation of registration of certain widely-used fungicides, have led to increased activity in the development of biocontrol agents against foliar fungal pathogens. For a potential biological control agent to reach the stage of commercial deployment, numerous criteria have to be satisﬁed and considerable data need to be obtained to demonstrate aspects of efﬁcacy, survival, adaptability, and scale-up. These aspects are reviewed elsewhere (Avis et al. 2001a; Cook 1993; Harman 2000; Lumsden et al. 1996) and will not be discussed in this chapter. Several agricultural chemical companies and a number of companies with specialized agricultural products have invested in the discovery and development of biological control agents to complement synthetic pesticides for the control of diseases on horticultural crops. These products are targeted to markets where they have the best chance of performing and where there is the most need, e.g., for control of seed and rootinfecting pathogens on seedlings (Whipps 2001). A range of commercially available biological control products for plant disease control is now available (Fravel 2000) and are more likely to be brought to market in the future. Molecular methods have been described that can be adapted for use to ensure quality control and monitoring of the biocontrol agents (Avis et al. 2001a).

BIOCONTROL OF SEED ROTS AND DAMPING-OFF DISEASES

Germination of plant seeds is accompanied by the exudation of host nutrients into the soil environment, which frequently attract potentially damaging fungi such as Pythium spp., R. solani, and Fusarium spp. These fungi utilize the seed and root exudates as an energy source for germination and growth, and subsequently penetrate and colonize the seed and root hairs, causing rot and damping-off of emerging seedlings. These fungi are favored by cool (15 –208C) and moist conditions. Fungal biological control agents have been described which when applied to the surface of seed, to the planting substrate, or when applied shortly after seed germination, can utilize the host nutrient exudates and colonize the seed and developing roots to compete with and exclude the pathogenic fungi. In addition, many of these biocontrol agents secrete hydrolytic enzymes and antibiotics that inhibit the development of the pathogenic fungi. The most widely-researched of these biocontrol agents are species of Trichoderma and Gliocladium and to a lesser extent Penicillium spp. (Table 1).

BIOCONTROL OF ROOT AND CROWN ROTS AND VASCULAR WILT DISEASES

Pathogenic fungi which infect the root system and crown tissues through root hairs, natural openings, or wounds, can rapidly colonize these tissues and enter the vascular tissues, causing decay and death of the plants. Most of these pathogens infect during the early stages of plant development, although disease symptoms may only be manifested later. Fungal biological control agents have been described which when applied to the seed, planting medium, or roots of plants, can colonize the root system, occupy potential infection sites, and compete with the pathogen. In addition, these agents may enhance resistance in the plants through induction of various defense responses, and secrete hydrolytic enzymes and antibiotics that inhibit pathogen growth and development. The most widely-researched of these biocontrol agents are antagonistic fungi (Trichoderma and Gliocladium species) and nonpathogenic, closely-related fungi (Pythium, Fusarium, and Rhizoctonia species), as well as mycoparasitic distantly related fungi such as Talaromyces, Coniothyrium, Sporidesmium, Stachybotrys, and Verticillium (Table 1).

3.1 3.1.1

Trichoderma As a Biocontrol Agent of Seedling Root, Crown, and Vascular Diseases Trichoderma Species Identiﬁcation

The genus Trichoderma contains species that occur in soils throughout the world. Most species are fast-growing saprophytes with the ability to survive under a range of environmental conditions by utilizing different substrates for growth (Hjeljord and Tronsmo 1998; Samuels 1996). The most common biological control agents in the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride (Hermosa et al. 2000). Characterization of 16 biocontrol strains, identiﬁed previously as Trichoderma harzianum Rifai and one biocontrol strain recognized as T. viride, has been carried out using several molecular techniques. A certain degree of polymorphism was detected among isolates in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) of ribosomal DNA revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum –T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride –T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride –T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum that cause mushroom green mold. A similar study by Dodd et al. (2000) utilized ITS1 and ITS2 sequence data to group 50 isolates of Trichoderma species with biocontrol potential, while ITS1

Nonpathogenic Rhizoctonia P. oligandrum P. oligandrum T. flavus T. flavus T. flavus C. minitans S. sclerotivorum C. foecundissimum

G. virens GL-3, GL-21 G. catenulatum J1446 P. oxalicum Nonpathogenic F. oxysporum Nonpathogenic F. oxysporum, F. solani Nonpathogenic F. oxysporum Nonpathogenic R. solani

T. harzianum T. harzianum, T. hamatum T. harzianum T. harzianum T. hamatum, T. harzianum, T. viride, T. virens T. harzianum T. longibrachiatum G. virens G. virens G. virens GL-3

Biocontrol agent

Knudsen and Eschen (1991) Migheli et al. (1998) Larkin and Fravel (1998) Koch (1999) Mao et al. (1998) Ristaino et al. (1994) Niemi and Lahdenpera¨ (2000) and Punja and Yip (unpublished) De Cal et al. (1999) Alabouvette et al. (1993), Duijff et al. (1998), and Fuchs et al. (1999) Larkin and Fravel (1998)

Mandeel and Baker (1991) Cubeta and Echandi (1991), Harris and Adkins (1999), and Villajuan-Abgona et al. (1996) Jabaji-Hare et al. (1999) and Ross et al. (1998) McQuilken et al. (1992) Al-Rawahi and Hancock (1998) Madi et al. (1997) Engelkes et al. (1977) and Stosz et al. (1996) Madi et al. (1997) Budge and Whipps (2001) Adams and Fravel (1990) Lewis and Larkin (1998)

S. sclerotiorum on pea P. ultimum on cucumber F. oxysporum f. sp. lycopersici on tomato P. ultimum on cucumber; R. solani on peas R. solani, P. ultimum, S. rolfsii, and F. oxysporum on tomato and pepper S. rolfsii on carrot Pythium on cucumber F. oxysporum f. sp. lycopersici on tomato F. oxysporum f. sp. lycopersici on tomato F. oxysporum f. sp. lycopersici on tomato

F. oxysporum f. sp. cucumerinum on cucumber R. solani and Pythium on cucumber and pepper

Pythium spp. on cress V. dahliae on pepper S. rolfsii on bean V. dahliae on eggplant S. rolfsii on bean S. sclerotiorum on lettuce S. minor on lettuce P. ultimum and R. solani on eggplant and pepper

R. solani on bean, cabbage

Datnoff et al. (1995), Nemec et al. (1996), and Sivan and Chet (1993) Larkin and Fravel (1998) Ahmed et al. (1999) Woo et al. (1999) Lewis et al. (1998)

References

F. oxysporum f. sp. radicis-lycopersici on tomato F. oxysporum f. sp. lycopersici on tomato P. capsici on pepper P. ultimum and R. solani on bean R. solani on eggplant

Target pathogen and host

Table 1 Examples of fungal biological control agents that prevent seed rots, damping-off, and root, crown, and vascular diseases caused by pathogenic fungi on vegetable crops

Biological Control of Vegetable Diseases 159

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sequence data and RFLP analysis were used to distinguish amongst isolates of T. harzianum (Gams and Meyer 1998). These studies demonstrated the utility of molecular methods to resolve the identity of strains of Trichoderma with potential biocontrol activity that were overlapping in morphological features. This approach could also be used to develop strainspeciﬁc markers for a desired biocontrol strain. Molecular markers were developed and used to detect and trace a strain of T. hamatum in potting mix (Abbasi et al. 1999).

3.1.2

Trichoderma Biocontrol Activity

Several studies have shown that T. harzianum can control diseases caused by many root-infecting pathogens, including Fusarium, Rhizoctonia, and Pythium (Table 1). T. harzianum strain KRL-AG2, commercially formulated as F-Stop, when added to a potting soil mix prior to seeding with tomatoes, reduced the incidence and severity of Fusarium root and crown rot caused by Fusarium oxysporum f. sp. radicislycopersici (Datnoff et al. 1995). T. harzianum strain T-22 also provided control of Fusarium crown rot of tomato (Nemec et al. 1996), and the fungus could be recovered from the roots of treated plants 26 days after application, suggesting it had colonized the roots. T. hamatum reduced the incidence of Fusarium wilt of tomato, caused by F. oxysporum f. sp. lycopersici, when added to potting medium prior to seeding (Larkin and Fravel 1998). A commercial formulation of T. harzianum (Rootshield strain T-22) was also evaluated against this disease and was found to signiﬁcantly reduce it when incorporated into potting mix at 0.2% (Larkin and Fravel 1998). Strain T-22 of T. harzianum was generated by fusing a mutant strain capable of colonizing plant roots with a strain able to compete with bacteria under iron-limiting conditions using protoplast fusion techniques (Harman 2000). This new strain had the enhanced ability to colonize the root system of host plants, resulting in greater efﬁcacy as a biological control agent for long-term root protection (Harman 2000; Harman and Bjo¨rkman 1998; Sivan and Harman 1991). A number of commercial formulations are available that contain strains of T. harzianum for use against different diseases on a wide range of crops. These products are registered for use against a number of soilborne pathogens. Among these products, RootShielde, T-22G, and T-22 Planter Boxe contain T. harzianum strain T-22 that is able to survive well in the rhizosphere of plants (Fravel 2000).

3.1.3

Trichoderma Mechanism of Action

Trichoderma species can confer biological control against soilborne diseases through a number of mechanisms, including antibiosis, parasitism, competition, and the induction of host plant resistance (Hjeljord and Tronsmo 1998). Trichoderma species are known to produce a range of volatile and nonvolatile secondary metabolites, some of which inhibit other microorganisms and are considered to be antibiotics. These fungi can also penetrate and infect

pathogen structures, such as hyphae, causing them to be degraded through the production of cell wall degrading enzymes, such as chitinases, glucanases, cellulases, and proteinases (Geremia et al. 1993; Schirmbock et al. 1994; Thrane et al. 1997; Zeilinger et al. 1999). Trichoderma species can compete with pathogens for nutrients, rapidly colonize a substrate and exclude pathogens from infection sites, and colonize senescing tissues and wounds to reduce pathogen colonization (Hjeljord and Tronsmo 1998). Some strains of Trichoderma are good root colonizers (rhizosphere competent). It has also been reported that T. harzianum (strain T-22) has the ability to directly enhance root growth and plant development in the absence of pathogens (Harman 2000), and it has been suggested that this was due to the production of a growth-regulating factor by the fungus (Windham et al. 1986). Altomare et al. (1999) proposed that the ability of T. harzianum to increase plant growth was partially due to the organism’s ability to solubilize nutrients, thus making them more available to host plants. These observations indicate the versatility through which Trichoderma species can manifest biological control activity. Finally, it has been reported that a strain of T. harzianum was able to trigger host defense mechanisms in cucumber plants through enhanced cell wall depositions and induction of defense enzymes, suggesting an indirect effect in the host plant by the biocontrol agent (Yedidia et al. 1999).

3.1.4

Biotechnological Manipulations of Trichoderma

Techniques in biotechnology have been applied to elucidate the role of hydrolytic enzymes, such as chitinases and glucanases, in mycoparasitism by Trichoderma that could lead to biological control activity. Transformants of T. longibrachiatum expressing extra copies of the b-1,4-endoglucanase gene egl1 were found to be better at suppressing Pythium development on cucumber compared to wild-type strains (Migheli et al. 1998). In addition, transformants of T. harzianum overproducing the proteinase gene prb1 had up to a five-fold increase in ability to protect cotton seedlings from R. solani (Flores et al. 1996). Transformants of T. harzianum overexpressing an endochitinase gene chit33 were more effective in inhibiting growth of the pathogen R. solani in vitro compared with wild-type strains (Limoń et al. 1999). A mutant of T. harzianum that was selected for its enhanced ability to hydrolyze pustulan, a polymer of b-1,6-glucan, had 2 –4 times more chitinase, b-1,3 and b-1,6 glucanase activity compared to the wild-type, produced three times more extracellular proteins and other compounds, and showed greater inhibition of B. cinerea in vitro (Rey et al. 2001).These studies reaffirm the roles played by fungal enzymes in biocontrol of plant pathogens and also highlight the successes in manipulating biocontrol strains to genetically engineer them to enhance efficacy. Specificity in the activity of the hydrolytic enzymes was suggested in a study by Woo et al. (1999), in which the endochitinase ech42 gene encoding for the secreted 42 Kda endochitinase

Biological Control of Vegetable Diseases

(CHIT 42) was silenced by targeted disruption; it was found that the endochitinase-deﬁcient mutant had similar activity as the wild-type strain against Pythium ultimum, but had enhanced activity against R. solani, and reduced activity against B. cinerea. A genetically marked strain of T. harzianum was developed by transformation with the b-glucuronidase (uid A) gene and the hygromycin B (hygB) gene for use in population dynamics studies (Thrane et al. 1995). Techniques utilizing protoplast transformation as well as particle bombardment of conidia have been described for Trichoderma (Lorito et al. 1993; Thrane et al. 1995). Population densities of the transformed strain could be monitored in a potting mix (Green and Jensen 1995), and the presence of the biocontrol agent around wounded tissues was reported.

3.2

Gliocladium As a Biocontrol Agent of Seedling, Root, Crown, and Vascular Wilt Diseases

Gliocladium virens Miller, Giddens and Foster, a biocontrol agent of a wide range of fungal pathogens, is now classiﬁed in the genus Trichoderma due to similar morphological characteristics that are shared with members of this genus and DNA analysis supported the inclusion of G. virens with the Trichoderma genus (Rehner and Samuels 1994). One characteristic of G. virens is the ability to produce the antibiotic metabolites gliotoxin and viridin, a characteristic not generally shared with other species of Trichoderma (Papavizas 1985). G. virens (T. virens) strain GL-3 was evaluated as a seed treatment on tomato against several pathogens, including R. solani, P. ultimum, S. rolfsii, and F. oxysporum f. sp. lycopersici. The treatment resulted in signiﬁcantly higher seedling establishment (Mao et al. 1998). A commercial formulation of strain GL-21 of G. virens is registered for use in the United States, under the trade name SoilGarde, and can control P. ultimum and R. solani on vegetables and ornamental seedlings (Koch 1999; Lumsden et al. 1996). Like T. harzianum strain T-22, certain strains of G. virens have the ability to colonize the rhizosphere of plant roots (Harman 2000). In addition, the production of gliotoxin occurs rapidly (within a few hours) and can persist for several days to provide high levels of pathogen suppression (Lumsden et al. 1992; Wilhite and Straney 1996). Gliotoxin has also been shown to act synergistically with endochitinase in G. virens (Di Pietro et al. 1993). A related species, G. catenulatum Gilman and E. Abbot, has been reported to be effective in reducing the incidence of damping-off diseases, caused by P. ultimum and R. solani (McQuilken et al. 2001). Incorporation of a wettable powder formulation of G. catenulatum strain JI446 into peat-based growing media or application as a drench reduced dampingoff due to P. ultimum and R. solani. Two commercial formulations of G. catenulatum strain J1446 (Prestop and Primastop) have been recently developed (Fravel 2000; Niemi

161

and Lahdenpera¨ 2000). Primastop is currently registered in Europe and in a number of regions of the United States, and Prestop is expected to be registered in the near future (Niemi and Lahdenpera¨ 2000). G. catenulatum strain J1446 was able to colonize cucumber roots extensively 5 weeks following its application, indicating that the fungus has the ability to survive and proliferate in the rhizosphere of plants. This rhizosphere competence, coupled with its reported ability to act as a mycoparasite (McQuilken et al. 2001) makes G. catenulatum a strong candidate as a biological control agent against a number of vegetable diseases. We have evaluated this biocontrol agent against Pythium root and crown rot of cucumber caused by P. aphanidermatum. Application at the time of seeding signiﬁcantly reduced plant mortality and enhanced seedling growth (Figure 1).

3.3

Nonpathogenic Fungi As Biocontrol Agents of Seedling, Root, Crown, and Vascular Wilt Diseases

Nonpathogenic and hypovirulent strains of fungi that are closely related taxonomically to plant pathogenic species have been reported to provide biological control of a number of pathogens, including species of Fusarium, Rhizoctonia, and Pythium. Competition by nonpathogenic strains for host plant nutrients, colonization of roots and infection sites to preclude the pathogen, parasitism of pathogen hyphae, and induction of host plant resistance are mechanisms through which these nonpathogenic strains achieved biological control (Sneh 1998). Nonpathogenic Fusarium species (F. oxysporum and F. solani) provided control of Fusarium wilt of tomato

Figure 1 Effect of G. catenulatum, formulated as Prestop, on reducing root rot and damping-off caused by P. aphanidermatum on cucumber seedlings. The plant on the left received the biocontrol agent at seeding time followed by the pathogen 10 days later; the plant in the center received the pathogen only 10 days after seeding; the plant on the right is the uninoculated control. Photograph was taken at 28 days after seeding.

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(Alabouvette et al. 1993; Fuchs et al. 1999; Larkin and Fravel 1998) through systemic induction of host resistance, and an increase in hydrolytic enzyme activity was reported in treated plants (Duijff et al. 1998; Fuchs et al. 1999). Induction of resistance by nonpathogenic fungi has also been reported to occur for binucleate Rhizoctonia species (Jabaji-Hare et al. 1999; Xue et al. 1998), P. oligandrum (Benhamou et al. 1997), and Penicillium oxalicum (DeCal et al. 1999). In plants treated with these biocontrol agents, induction of host defense responses, alterations of the plant cell wall, and enhanced expression of antifungal enzymes were reported (Benhamou et al. 1997; Jabaji-Hare et al. 1999; Xue et al. 1998). Further studies on the mechanisms by which these fungi elicit the host defense responses should provide interesting information on this group of biocontrol fungi. Formulations for biocontrol fungi such as binucleate Rhizoctonia species have been described (Honeycutt and Benson 2001).

3.3.1

Biotechnological Manipulations of Nonpathogenic Biocontrol Fungi

Techniques in biotechnology have been applied primarily to the nonpathogenic F. oxysporum strains that provide biocontrol of Fusarium wilt on a number of plant species. By creating strains expressing the marker gene b-glucuronidase (GUS) through genetic transformation, the role of competition for root colonization between pathogenic and nonpathogenic strains could be elucidated (Eparvier and Alabouvette 1994). Nonpathogenic strains differed in their ability to colonize roots and to preclude the pathogenic strains, and the use of GUS-marked strains utilizing the glyceraldehyde-3-phosphate dehydrogenase promoter provided an estimation of fungal metabolic activity on the roots (Eparvier and Alabouvette 1994). A strain of nonpathogenic F. oxysporum transformed with the b-glucuronidase (GusA) and hygromycin B resistance (Hph) genes could be detected at levels as low as 1 ng of mycelia and estimates of fungal biomass on tomato roots were shown to be considerably higher compared to a plating assay method (Bao et al. 2000), indicating this was a sensitive and rapid assay for this biocontrol agent in planta. The colonization by the transformed strain of plant roots could be assessed in relation to the extent of colonization of a pathogenic strain of F. oxysporum (Bao and Lazarovits 2001).

3.4

Mycoparasites As Biocontrol Agents of Seedling, Root, Crown, and Vascular Wilt Diseases

Nonpathogenic fungi can act as mycoparasites, as exempliﬁed by P. oligandrum, P. nunn, and P. periplocum, which are mycoparasitic on other species of Pythium and can reduce pathogen infection levels and reduce disease (Berry et al. 1993; McQuilken et al. 1992; Paulitz and Baker 1987). Other mycoparasitic fungi include Stachybotrys elegans and

Verticillium biguttatum affecting R. solani (van den Boogert and Velvis 1994; Tweddell et al. 1995), Coniothyrium minitans on Sporidesmium sclerotiorum (Budge and Whipps 2001), S. sclerotivorum on a number of sclerotial-forming soilborne fungi (Mischke 1998), and Talaromyces ﬂavus on S. rolfsii (Madi et al. 1997). In addition, T. ﬂavus was reported to produce glucose oxidase and potentially peroxide, which was lethal to sclerotia of V. dahliae (Stosz et al. 1996). Species of Trichoderma and Gliocladium are also known to be mycoparasitic, as discussed in previous sections of this chapter. All of these mycoparasitic fungi have been demonstrated to reduce diseases caused by a number of different pathogens on a range of vegetable crop species (Table 1).

BIOCONTROL OF FOLIAR-INFECTING FUNGI

Pathogenic fungi which infect the leaves and stems of developing plants may enter through senescing tissues, wounded regions, or natural openings, or may penetrate host tissues directly. These fungi can infect plants at all stages of development, and are favored by warm (20 –258C) and humid conditions. Infection results in blighting of the foliage, premature leaf senescence, and compromised plant growth and yield. Biological control agents have been described which when applied to the foliage, can reduce primary infection as well as reduce pathogen development and sporulation, and can colonize wounds and other tissues to preclude pathogen establishment or development. Some of the biological control agents can act as mycoparasites and reduce pathogen growth directly, while others may secrete hydrolytic enzymes and antifungal compounds to reduce pathogen development, or alter pathogen physiology to reduce disease-causing potential. The most widely-researched of these biocontrol agents are fungi (Trichoderma, Ulocladium, Ampelomyces, and Verticillium) and yeasts (Aureobasidium, Cryptococcus, Rhodosporidium, and Rhodotorula).

4.1

Biocontrol of Gray Mold

Botrytis cinerea Pers:Fr. is an important pathogen on many vegetable crops grown under greenhouse conditions as well as under field conditions. Under high humidity conditions or when free moisture is present on the plant surface, the pathogen infects fruits, flowers, leaves, and stems causing tissue decay. This is followed by prolific sporulation of the pathogen, producing a gray mold appearance. Wounded tissues are especially susceptible to this pathogen. Much of the research activity to achieve biological control of B. cinerea on vegetable crops has centered around the use of T. harzianum, followed by Ulocladium spp. and a number of yeasts, as described later.

Biological Control of Vegetable Diseases

4.1.1

Trichoderma As a Biocontrol Agent of Botrytis Cinerea

Isolate T-39 of T. harzianum (marketed as Trichodexe) provided control of gray mold as well as a number of other fungal diseases of cucumber under commercial greenhouse conditions (Elad 2000a). T. harzianum T-39 was applied as part of a gray mold management program in alternation with chemical fungicides. The biocontrol agent was effective when applied in formulations containing two concentrations of the active ingredient (0.2 and 0.4 g/l), at around 1010 cfu/g of T. harzianum (Elad et al. 1993). A number of other research studies have conﬁrmed the efﬁcacy of T. harzianum strains in reducing development of B. cinerea on crops such as cucumber and tomato under laboratory conditions and on greenhouse-grown plants (Dik and Elad 1999; Dik et al. 1999; O’Neill et al. 1996; Utkhede et al. 2000). Mechanisms involved in the biological suppression of infection and inoculum potential of B. cinerea by Trichoderma are numerous and variable and the involvement of two or more mechanisms has been demonstrated in several studies. Reported combinations include antibiosis with enzyme degradation of B. cinerea cell walls and parasitism (Be´langer et al. 1995); competition for nutrients followed by interference with pathogenicity enzymes of the pathogen or with induced resistance; and alteration of plant surface wettability combined with antibiosis (Elad 1996). Since, germinating B. cinerea conidia are dependent on the presence of nutrients to initiate pathogenesis, competition for nutrients is important in biocontrol. Pathogen conidial viability and germination capacity are also potentially affected by the presence of antibiotics produced by Trichoderma and present in the phyllosphere. Slower in action are mechanisms involving induced resistance in the host plant and production of hydrolytic enzymes that degrade B. cinerea cell walls. The latter has been demonstrated much more convincingly in vitro than in the phyllosphere. Biocontrol in established lesions and reduction of sporulation of Botrytis on necrotic plant tissues is a means to minimize secondary spread of pathogen inoculum. Zimand et al. (1996) also demonstrated that the presence of T. harzianum at the site where B. cinerea infects can have an adverse effect upon activity of pathogen enzymes involved in pectin degradation and host cell wall destruction, e.g., pectinase, cutinase, and pectate lyase. Since such enzymes are intimately involved in the infection process by B. cinerea, the effect of the biocontrol agent in reducing their activity in vitro and on the surface of plant leaves could also limit disease development by the pathogen (Kapat et al. 1998). The inhibition of pathogen enzymes was proposed to be due to the secretion of serine proteases by T. harzianum (Elad and Kapat 1999), which could also inhibit pathogen spore germination. The presence of protease inhibitors was found to reduce the biocontrol activity. The potential role of induced plant resistance by T. harzianum for control of B. cinerea was demonstrated by De Meyer et al. (1998), wherein application of the biocontrol agent to roots or leaves of a number of different plant species was observed to provide protection

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against the pathogen on leaves that were spatially separated from the site of application of T. harzianum. This was attributed to induction of systemic resistance that delayed or suppressed spreading lesion formation (De Meyer et al. 1998).

4.1.2

Saprophytic Fungi and Yeasts As Biocontrol Agents of Botrytis Cinerea

The leaf surface of plants (phylloplane) is frequently colonized by a range of saprophytic fungi and yeasts, which rely on plant nutrient exudates and a range of other carbon/ nitrogen sources for their survival, e.g., damaged or senescing tissues, pollen grains, insect honeydew. If present in the same niche as plant pathogenic fungi, these saprophytes may compete with pathogens for nutrients, infection sites, or reduce growth and sporulation of the pathogen on host tissues through competition or antagonism (Fokkema 1993). Recovery of selected fungi and yeasts and reapplication to the leaf or stem surface has identified a number of potential biological control agents that can reduce diseases caused by B. cinerea. On onion leaf tissues, the saprophytic fungi Alternaria alternata, Chaetomium globosum, Ulocladium atrum, and U. chartarum suppressed sporulation of the pathogen significantly when applied after pathogen inoculation (Ko¨hl et al. 1995; 1999). A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been described to detect and quantify U. atrum in colonized plant tissues (Karpovich-Tate and Dewey 2001) and could be useful in monitoring of this biocontrol agent. Application of the saprophytic fungus Cladosporium cladosporioides to wounds on tomato stems was reported to reduce infection by B. cinerea in laboratory and greenhouse experiments (Eden et al. 1996). The yeast-like fungi Aureobasidium pullulans and Cryptococcus albidis significantly reduced sporulation of B. cinerea on pruning wounds and stems of cucumber and tomato under laboratory and greenhouse conditions (Dik and Elad 1999; Dik et al. 1999). Another yeast, Rhodosporidium diobovatum, when applied to tomato stems, reduced lesion size due to B. cinerea and the treated plants yielded higher fruit when compared to the untreated controls (Utkhede et al. 2000). Both C. albidus and Rhodotorula glutinis reduced sporulation of B. cinerea on bean and tomato leaves and reduced disease levels (Elad et al. 1994).

4.1.3

Mechanisms of Action of Yeasts Against Botrytis Cinerea

Yeasts can compete effectively against B. cinerea for nutrients, such as glucose and fructose (Filonow 1998; Filonow et al. 1996), thereby reducing pathogen colonization of plant tissues and sporulation (Elad et al. 1994). Yeasts such as Aureobasidium have also been reported to produce mycotoxins in culture (Schrattenholz and Flesch 1993). Yeast cells may attach to pathogen hyphae, as demonstrated for B. cinerea, and cause them to degrade (Cook et al. 1997) through secretion of cell wall degrading enzymes

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(Wisniewski et al. 1991). The production of cell wall degrading enzymes, such as b-1,3-glucanases has also been documented in yeasts such as Pichia anomola that are effective biocontrol agents against B. cinerea as a postharvest treatment (Jijakli and Lepoivre 1998). In vivo studies with Candida saitoana in apple demonstrated that B. cinerea hyphae had degenerated (El-Ghaouth et al. 1998), implicating the possible role of toxins and/or enzymes. In addition, plant cells in the vicinity of the yeasts appeared to have enhanced structural defense responses, suggesting an induction of defense in the host plant may have occurred. Stimulation of host cell defenses by the yeast C. oleophila was recently described (Droby et al. 2002).

G. catenulatum J1446 as preventative treatments to wounded stem tissues of cucumber followed by inoculation with the pathogen. They demonstrated that both microbial agents signiﬁcantly reduced disease development when compared to plants treated with water. Anthracnose disease of cucumber, caused by Colletotrichum magna, was reduced when a nonpathogenic mutant was applied to seedlings to achieve colonization and induction of defense responses that subsequently protected treated plants against the pathogen (Redman et al. 1999).

4.1.4

Powdery mildew fungi are obligate parasites of plants that derive nutrients and water from their host, thereby reducing growth and yield through the acquisition of photosynthates. The fungi penetrate into the epidermis directly and establish a parasitic relationship with the plant host through the formation of haustoria, the nutrient-absorbing structures. Mycelial growth and sporulation occur on the surface of leaves and stems, resulting in a white fuzzy mildew appearance. Over the years, powdery mildew diseases have been managed through the use of chemical fungicides and genetic resistance, but recent reports have highlighted the potential of biological control methods. Fungal and yeast biological control agents have been described which can reduce sporulation and growth of mildew pathogens, thereby minimizing their damaging effects to host plants. The fungal biocontrol agents are mostly mycoparasites, while the yeasts produce antibiotics and hydrolytic enzymes that cause the mildew hyphae and conidia to collapse and be rendered nonviable.

Biotechnological Techniques Applied to Yeasts

Yeasts with biocontrol potential against gray mold have been characterized using molecular techniques to provide a method to distinguish between closely-related strains and to identify and monitor survival of strains after application (Schena et al. 2000; 2002). These techniques include arbitrarily primed polymerase chain reaction (AP-PCR), random amplified polymorphic DNA (RAPD-PCR) analysis, and sequencecharacterized amplification region (SCAR) analysis. In addition, transformation of the yeast Metschnikowia with green fluorescent protein (GFP) was achieved and colonies could be visualized under epifluorescence (Nigro et al. 1999). The transformed strains behaved similarly to the wild-type strains in biocontrol activity against B. cinerea and in growth rates. Another yeast, A. pullulans, was also transformed with GFP and colonies were readily visible on apple leaf surfaces when subjected to fluorescence and could be quantified (Wymelenberg et al. 1997). Genetic transformation of the yeast Saccharomyces to express a cecropin A-based peptide with antifungal activity was recently described (Jones and Prusky 2002). The transformants inhibited the growth of Colletotrichum and reduced fungal decay of tomato fruits when applied prior to pathogen inoculation. The expression of the antifungal peptide in the biological control agent suggests a new approach for disease control.

4.2

Biocontrol of Leaf and Stem Blights

Didymella bryoniae (Auersw.) Rehm (anamorph Phoma cucurbitacearum Fr.:Fr.) Sacc. is an important pathogen on greenhouse- and ﬁeld-grown cucumbers and other cucurbits, and causes the disease gummy stem blight. The disease is favored by warm, humid conditions and the pathogen infects stems, fruit, leaves, and ﬂowers of susceptible plants, especially through wounded or senescing tissues, and natural openings such as stomata and hydathodes. There are few reports on the potential of using biological control agents to control this disease. Utkhede and Coch (unpublished) applied the yeast R. diobovatum and the biocontrol agent

4.3

4.3.1

Biocontrol of Powdery Mildews

Fungi As Biological Control Agents of Powdery Mildews

Verticillium lecanii has been described as a mycoparasite of powdery mildew fungi as well as a pathogen of insects and it has been developed as a biocontrol agent of insects on greenhouse crops. Strains of V. lecanii differed in their level of antagonism against the powdery mildew pathogen of cucumber, Sphaerotheca fuliginea, under laboratory conditions (Askary et al. 1998). Application to cucumber leaves prior to mildew infection and incubation under high (. 95%) relative humidity conditions reduced mildew development (Verhaar et al. 1997). The high humidity requirement for growth of this mycoparasite was reduced by the addition of an oil formulation (Verhaar et al. 1999). Infection of S. fuliginea by V. lecanii resulted in disorganized cytoplasm and plasmalemma disruption, possibly due to chitinase enzyme activity (Askary et al. 1997). Another mycoparasite, Ampelomyces quisqualis, has been extensively studied as a biocontrol agent of powdery mildew of cucumber. The mycoparasite infects the mildew pathogen and forms pycnidia in association with colonized mycelium,

Biological Control of Vegetable Diseases

reducing growth and sporulation of the pathogen. Cells of the mycoparasite grow inside the mildew hyphae, gradually causing them to degenerate. High levels of b-1,3-glucanase activity were reported in A. quisqualis (Rotem et al. 1999) and exposure of mildew hyphae to the enzymes caused them to degrade. A commercially available formulation of A. quisqualis AQ10 has been extensively evaluated against powdery mildew development. On cucumbers grown in the greenhouse, AQ10e was very effective in reducing mildew development (Elad et al. 1998). On ﬁeld-grown cucurbits, AQ10e also suppressed mildew development and increased yield when compared to the nontreated plants (McGrath and Shishkoff 1999).

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Molecular techniques have been used to characterize strains of A. quisqualis. The RFLP analysis of the nuclear rDNA ITS region and sequence analysis among a worldwide collection of isolates revealed considerable intraspeciﬁc variation (Kiss 1997; Kiss and Nakasone 1998). Isolates of the same genetic background were found in widely different areas and genetically different isolates could be found in a given area.

4.3.2

Yeasts As Biological Control Agents of Powdery Mildew

Pseudozyma (Sporothrix) ﬂocculosa is a yeast-like fungus with demonstrated biocontrol activity against powdery

Figure 2 Effect of Tilletiopsis pallescens on development of powdery mildew (S. fuliginea) on cucumber leaves. (A) A mildew-infected leaf showing chains of conidia and mycelium. (B) A mildew colony treated with a 3-day-old liquid culture of T. pallescens. Note collapsed conidia and mycelium. Photograph was taken 2 days following treatment. (C) Spore masses of Tilletiopsis adjacent to mildew conidia. Note intact mildew conidia on left and collapsed condia on the right.

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mildew fungi, especially on cucumber and rose (Be´langer and Benyagoub 1997; Belanger et al. 1994). Cytochemical investigations have shown that the yeast induces a rapid collapse of mildew spores and hyphal cells (Hajlaoui et al. 1992). Extracellular fatty acids with antifungal properties were produced by P. flocculosa and reported to be the principle mode of action in biological activity against powdery mildew (Benyagoub et al. 1996), by disrupting the cytoplasmic membrane in a range of fungi (Avis and Be´langer 2001). A fungicide-tolerant strain of the yeast was selected which could be used in conjunction with chemical control methods to reduce powdery mildew development (Benyagoub and Be´langer 1995). In a comparative study of three biological control agents against powdery mildew of cucumber, i.e., V. lecanii, A. quisqualis, and P. flocculosa, it was shown that P. flocculosa gave the best disease control (Dik et al. 1998). Molecular techniques have been used to characterize strains of Pseudozyma flocculosa (Avis et al. 2001b). Ribosomal DNA sequences and random amplified microsatellites were used to distinguish among different strains of this species and to develop isolate-specific markers to monitor spread and confirm genetic fidelity of the strains. A strain of P. flocculosa has been formulated and produced commercially under the name Sporodexe for use in control of powdery mildew on a number of crops grown under greenhouse conditions. Species of Tilletiopsis are saprophytic yeast-like fungi that occur as epiphytes on the leaf surface of various plant species and which have been demonstrated to have biological control activity against powdery mildew diseases (Hijwegen 1992; Knudsen and Skou 1993; Urquhart et al. 1994). Scanning

electron microscopic studies have revealed that mildew hyphae and spores appeared collapsed after treatment with Tilletiopsis (Figure 2) (Urquhart and Punja 1997). It was postulated that extracellular antifungal compounds were involved in biocontrol activity that included fatty acid esters and hydrolytic enzymes (Urquhart and Punja 2002). Various species of Tilletiopsis have demonstrated biological control activity, including T. albescens, T. minor, T. pallescens, and T. washingtonensis (Table 2). These species could be distinguished using RAPD analysis of PCR-generated DNA with random primers (Urquhart et al. 1997). Intraspeciﬁc variation was also noted and DNA ﬁngerprints were generated for some isolates that could be useful for monitoring the distribution and spread of certain isolates.

CONCLUSIONS

The numerous reports of success in the utility of fungal and yeast biological control agents to reduce fungal diseases on vegetable corps illustrate the potential of this approach for disease management. In addition, the applications of techniques in biotechnology are providing numerous examples of how these biocontrol agents can be characterized, monitored, and investigated in more depth. However, there are unique requirements in working with microbial biocontrol agents that must be recognized if this approach to disease control is to be successful. Environmental conditions, particularly temperature and moisture, can greatly inﬂuence the degree to which fungal and yeast biological control agents can affect fungal diseases on vegetable crops, even in greenhouse environments. Therefore,

Table 2 Examples of fungal and yeast biological control agents that reduce foliar diseases on vegetable crops caused by pathogenic fungi Biocontrol agent

Target pathogen and host

T. harzianum

B. cinerea on cucumber

T. harzianum

B. cinerea on tomato

T. harzianum T. harzianum G. catenulatum A. quisqualis AQ10 A. pullulans C. albidus

C. fulvum on tomato S. fuliginea and S. fusca on cucumber D. bryoniae on cucumber S. fusca on cucumber B. cinerea on tomato and cucumber B. cinerea on bean, tomato, and cucumber

R. glutinis R. diobovatum C. cladosporioides S. ﬂocculosa T. albescens, T. minor, T. pallescens, and T. washingtonensis

B. cinerea on bean and tomato B. cinerea on tomato B. cinerea on tomato S. fuliginea on cucumber S. fuliginea on cucumber

References Dik and Elad (1999) and Elad et al. (1993; 1998) Dik and Elad (1999), Migheli et al. (1994), and Utkhede et al. (2000) Elad et al. (2000a) Elad et al. (1998; 2000b) Utkhede and Coch (unpublished) Elad et al. (1998) Dik and Elad (1999) Dik and Elad (1999) and Elad et al. (1994) Elad et al. (1994) Utkhede et al. (2000) Eden et al. (1996) Dik et al. (1998) Hijwegen (1992), Knudsen and Skou (1993), Urquhart and Punja (1997), and Urquhart et al. (1994)

Biological Control of Vegetable Diseases

careful monitoring and recording of environmental variables is a requisite. The biological control agents are generally most effective when applied as a preventative treatment, prior to or at the onset of disease, and multiple applications may be needed to provide longer-term disease suppression. At high levels of disease pressure, biological control agents can be anticipated to perform less well. Some of the agents may be used in combination with, or in alternation with, chemical fungicides if it can be demonstrated that their survival is not adversely affected. Similarly, it may be possible that combinations of biocontrol agents may be more effective than single organisms although little research has been done in this area. Biological control agents that affect more than one disease should have greater market potential than those that specifically target a particular disease. It is not clear whether different plant hosts may have an influence on the efficacy of these biocontrol agents. Notwithstanding these conditions, the use of fungal and yeast biological control agents has generated significant interest in both the scientific research and product development arenas to ensure that commercially viable products will continue to be brought to market.

ACKNOWLEDGEMENTS Funding for aspects of this work was provided by the British Columbia Greenhouse Growers’ Association, the National Research Council of Canada IRAP Program, the Natural Sciences and Engineering Research Council of Canada Biocontrol Network Program, and Agriculture and Agri-Food Canada Matching Investments Initiative Program.

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Biological Control of Vegetable Diseases Wilhite SE and Straney DC (1996). Timing of gliotoxin biosynthesis in the fungal biological control agent Gliocladium virens (Trichoderma virens). Appl Microbiol Biotechnol 45:513 – 518. Windham MT, Elad Y, and Baker R (1986). A mechanism for increased plant growth induced by Trichoderma spp. Phytopathology 76:518 –521. Wisniewski M, Biles C, Droby S, McLaughlin R, Wilson C, and Chalutz E (1991). Mode of action of the postharvest biocontrol yeast, Pichia guilliermondii. I. Characterization of attachment to Botrytis cinerea. Physiol Mol Plant Pathol 39:245– 258. Woo SL, Donzelli B, Scala F, Mach R, Harman GE, Kubicek CP, Del Sorbe G, and Lorito M (1999). Disruption of the ech42 (endochitinase-encoding) gene affects biocontrol activity in Trichoderma harzianum P1. Mol Plant – Microbe Interact 12:419– 429. Wymelenberg AJV, Cullen D, Spear RN, Schoenike B, and Andrews JH (1997). Expression of green ﬂuorescent protein in

171 Aureobasidium pullulans and quantiﬁcation of the fungus on leaf surfaces. Biotechniques 23:686– 690. Xue L, Charest PM, and Jabaji-Hare SH (1998). Systemic induction of peroxidases, 1,3-b-glucanases, chitinases and resistance in bean plants by binucleate Rhizoctonia species. Phytopathology 88:359– 365. Yedidia I, Benhamou N, and Chet I (1999). Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl Environ Microbiol 65:1061 –1070. Zeilinger S, Galhaup C, Payer KL, Woo S, Mach R, Fekete-Csaba L, Lorito M, and Kubicek CP (1999). Chitinase gene expression during mycoparasitic interactions of Trichoderma harzianum with its host. Fungal Genet Biol 26:131 –140. Zimand G, Elad Y, and Chet I (1996). Effect of Trichoderma harzianum on Botrytis cinerea pathogenicity. Phytopathology 86:1255 –1260.

15 Control of Postharvest Diseases of Fruits Using Microbes Wojciech J. Janisiewicz Appalachian Fruit Research Station, U.S. Department of Agriculture – Agricultural Research Service, Kearneysville, West Virginia, USA

has been increasingly curtailed by the perceived hazard to humans and the environment. This has resulted in new regulations restricting or eliminating their use in this country and abroad. It has become increasing difficult to find and register new fungicides to replace those to which postharvest pathogens have developed resistance (Gullino and Kuijpers 1994; Ragsdale and Sisler 1994). Thus, there has been a need to find effective alternatives to synthetic fungicides. None of the alternative methods developed during the past two decades have had the broad spectrum of activity as synthetic fungicides. Recently, biological control has emerged as an alternative (Janisiewicz and Korsten 2002). The full potential of biocontrol has not yet been realized because the mechanisms of biocontrol have not been explained. A fuller understanding of the antagonistic mechanisms will eventually help manipulate and improve the biocontrol system. Although this method has some limitations, these limitations can be addressed by combining biological control with other alternative methods (Conway et al. 1999; El-Ghaouth et al. 2000b; Janisiewicz et al. 1998; Smilanick et al. 1999). In this chapter, the key elements in the development of biological control of postharvest diseases (BCPD) of fruits, and the current status and future prospects of BCPD of fruits using examples of fungal and bacterial antagonists are discussed.

INTRODUCTION

Losses from postharvest diseases of fruits have been substantial at the storage, wholesale, retail, and consumers levels. The total losses are very difficult to establish because research has generally considered only one or two levels, and little work has been done to determine losses at the consumer level. Nevertheless, in the United States, losses are estimated to range from 5% for citrus to as much as 20% for strawberries (Cappellini and Ceponis 1984; Eckert and Ogawa 1985). Most of the fruit decay results from infection through wounds made during harvest and postharvest handling, but for some fruits, infection takes place in the orchard during the growing season, and remains latent. As fruit mature in storage the pathogens become active again and invade fruit tissue. A variety of approaches have been used to reduce postharvest fruit decays, including sanitation to reduce pathogen inoculum, gentler handling of fruit to reduce wounding (Sommer 1982), physical treatments such as hot water dips and hot air treatments that kill pathogens (Falik et al. 1995; Lurie et al. 1998), storing produce at low temperatures or in modified atmosphere which stop or reduce growth of the pathogens (Sommer 1982), treating fruit with chemicals that enhance natural resistance (El-Ghaouth 1998), with synthetic fungicides (Eckert and Ogawa 1985; Eckert and Ogawa 1988), and, more recently, with biocontrol agents (Droby et al. 1998; Janisiewicz and Jeffers 1997; Janisiewicz and Korsten 2002; Korsten et al. 1995; Usall et al. 2001). Fungicides have been, by far, the most widely used remedy against fruit decay because they are easy to apply and generally, one fungicide is effective against most of the pathogens on a specific crop. Storage of some fruits for extended periods, e.g., citrus fruits, is totally dependent on the use of fungicides. But postharvest use of synthetic fungicides

PATHOGENS TARGETED FOR BCPD OF FRUITS

The BCPD of fruits can be approached from the perspective of the host plant (different fruits), habitat for the microorganisms (wound, intact surface), and the pathogen’s strategy used to infect fruit. The pathogen’s strategy has been emphasized most frequently because many economically 173

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important postharvest diseases of fruits are caused by necrotrophic pathogens (Dennis 1983). These pathogens invade mainly through wounds, and require nutrients for spore germination and initiation of the pathogenic process, which makes them vulnerable to competition for nutrients from surrounding microorganisms. Other mechanisms of biocontrol described later in this chapter may also be involved, but the prevailing evidence suggests that their role is secondary (Janisiewicz and Korsten 2002). Incipient or latent infections that generally occur in the field are less prone to biological control because the pathogen has already established a parasitic relation with the host. However, these pathogens can be controlled by antagonists that prevent infection in the field, perpetuate latency by removal of nutrients from areas surrounding the appressoria, or perhaps those that can inhibit pathogen development by the production of antifungal substances or by direct parasitism (Koomen and Jeffries 1993; Korsten and Jeffries 2000; Leibinger et al. 1997). The greatest progress in BCPD of fruits has been made against typical wound-invading necrotrophic postharvest pathogens such as Penicillium expansum which causes blue mold of apple, pear, and cherries, Botrytis cinerea which causes gray mold of pome fruits (Janisiewicz and Jeffers 1997), P. italicum and P. digitatum which causes blue and green-mold of citrus fruits, respectively (Droby et al. 1998), and against the wound invading phase of brown rot decay of stone fruits caused by Monilinia fructicola (Pusey et al. 1988). Although the likelihood of infection is dependent on the concentration of fungal spores, the biocontrol strategy has always focused on the preemptive colonization of the wounds by the antagonist to prevent infection, and not on reduction of the pathogen inoculum. Thus, the control of these decays was achieved by the application of antagonists to wounds, simultaneously with the pathogen or shortly after the infection took place. Other potential candidates for this type of biological control are pathogens invading through cut stem of bananas, mangos, and papayas (Eckert 1991). Since the development of the pathogen depends on fruit maturity and the environment, these factors have been critical in the pathogen-antagonist interaction and resulting biological control. Significant successes were achieved with biocontrol of latent infections caused by Colletotrichum spp. on mango and avocado (Korsten and Jeffries 2000), and to a lesser extent by B. cinerea on strawberries (Helbig 2002; Ippolito et al. 1998; Peng and Sutton 1990; Takeda and Janisiewicz, unpublished results). Biological control of these diseases must start in the field, relies on multiple application of the antagonist, and is generally more difficult to achieve.

ANTAGONIST SELECTION

The ﬁrst successful attempts in BCPD of fruits, which stimulated research in postharvest biological control, used soil isolated bacterium, Bacillus subtilis, to control brown rot

of stone fruits caused by M. fructigena (Pusey and Wilson 1984). Subsequent works focused on screening natural microflora from the aerial surfaces of apple and pear trees for antagonistic activity against decays caused by P. expansum and B. cinerea (Janisiewicz 1987). This resulted in the isolation of many bacteria and yeasts that were effective in controlling fruit decays caused by these pathogens. Isolation from the fruit surfaces has become a standard practice and is the most efficient source of antagonists against postharvest fruit pathogens of temperate, subtropical, and tropical fruits (Adikaram and Karunaratne 1998; Arras 1993; Chalutz et al. 1988; Chand-Goyal and Spotts 1996; Droby et al. 1999; Guinebretiere et al. 2000; Huang et al. 1992; Kanapathipillai and bte Jantan 1985; Lima et al. 1998; Qing and Shiping 2000; Teixido´ et al. 1998a; Testoni et al. 1993; Zahavi et al. 2000). A variety of enrichment procedures, employing either fruit juice or tissue, have been used to isolate microorganisms best suited to colonize wounded fruit tissue (Janisiewicz 1991; 1996; Wilson et al. 1993). The enrichment procedures appear to favor isolation of the resident fruit microflora, with the yeasts being isolated most frequently, followed by bacteria. Filamentous fungi have been isolated only sporadically (Janisiewicz 1996; Wilson et al. 1993). The rapid colonization of wounds by yeasts is necessary for preemptive exclusion of the wound-invading pathogens. The number of species that are residents on a specific kind of fruit is limited, and reports from various laboratories worldwide increasingly describe biocontrol potential of the same antagonist species isolated at different locations (ChandGoyal and Spotts 1996; 1997; Falconi and Mendgen 1994; Ippolito et al. 2000; Janisiewicz et al. 1994; 2001; Leibinger et al. 1997; Lima et al. 1998; McLaughlin et al. 1992; Roberts 1990; Wisniewski et al. 1988). Recent studies; however, indicate great diversity within an antagonist species, even at a single geographical location, with regard to effectiveness in controlling fruit decays and other factors important in commercializing a biocontrol agent (Janisiewicz et al. 2001; Schena et al. 1999). Thus, investigating the same species of the antagonist at various locations may lead to finding an antagonist with superior attributes. An effective antagonist may also be found by screening starter cultures used for food products (Pusey 1991), various culture collections (Filonow et al. 1996), and even by exploring an aquatic environment, as is the case with bacteriophages used against soft rotting bacteria (Eayre et al. 1995). In addition to being effective in controlling fruit decays, antagonists should have certain attributes to make them good candidates for commercialization. These include: compatibility with postharvest practices (storage temperatures, relative humidity-RH, storage atmosphere with elevated CO2 and reduced O2, handling in water, heat drying tunnels, etc.), treatments and additives (waxes, antioxidants, flotation salts), ability to grow efficiently in a commercially used media for mass production, ease of formulation, and the lack of potentially deleterious effects on human health that would disqualify them from being approved by regulatory agencies. Human safety, in particular, necessitates a thorough

Biological Control of Postharvest Diseases of Fruits

approach in identifying an antagonist. Misidentiﬁcation may result in abandonment of commercial development of the antagonist, and, if not detected early, may be very costly.

MECHANISMS OF BIOCONTROL

Various mechanisms of biocontrol have been suggested for antagonists effective in BCPD of fruits and often more than one mechanism was implicated for a single antagonist. In no case, however, was the biocontrol mechanism fully explained. The putative mechanisms included competition for limiting nutrients and space, lysis, induced resistance, direct parasitism, and production of inhibitory substances. Attachment of antagonists to a fungal hyphae was observed in some antagonist-pathogen interactions, but its role remains largely speculative (Arras et al. 1998; Wisniewski et al. 1989; 1991). The main reasons for the limited knowledge in mechanism of biocontrol have been a lack of appropriate methods to study microbial interactions in wounds of fruit, and the fact that progress in BCPD was driven by advances in microbial ecology of the antagonists. However, recent advances in microbial sensing of nutrients on plants (Lindow et al. 2002), molecular approaches (Bassett and Janisiewicz 2001; Jijakli et al. 2001; Jones and Prusky 2002; Yehuda et al. 2001), and a method allowing separation of competition for nutrient and space using natural substrates (Janisiewicz et al. 2000) may lead to better explanation of the signiﬁcance of various biocontrol mechanisms. Progress in microbial ecology of the antagonists led the commercialization of BCPD of fruits in a relatively short period of time, but further expansion will greatly depend on achieving the full potential of BCPD, for which knowledge of the mechanisms of biocontrol will be essential. Bacterial antagonists such as Bacillus spp. (Gueldner et al. 1988) or Pseudomonas spp. (Bull et al. 1998; Janisiewicz and Roitman 1988) produce a variety of antifungal compounds in artiﬁcial media, which by themselves can provide effective control of postharvest decays of fruits (Bull et al. 1998; Janisiewicz et al. 1991; Takeda and Janisiewicz 1991). But the role of these antifungal compounds as the biological control mechanism is uncertain, because they either can not be detected in fruit wounds after inoculation with the antagonist (Bull, personal communication), or pathogen mutants resistant to these inhibitory substances are still controlled by the antagonists (Smilanick, personal communication). Yeast antagonist such as Pichia anomala strain K (Gravesse et al. 1998; Jijakli and Lepoivre 1998), P. guilliermondii (Arras et al. 1998; Wisniewski et al. 1991), or yeast like Aureobasidum pullulans (Castoria et al. 2001) effective in controlling gray mold of apples, produce b-1,3-glucanase, which caused lysis of the B. cinerea hyphae. Production of this enzyme by P. anomala strain K was stimulated in the presence of cell wall preparations of B. cinerea in apple wounds resulting in improved biocontrol (Gravesse et al. 1998; Jijakli and Lepoivre 1998). This

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enzyme also increased in apple wounds treated with the A. pullulans cells, but how much of this increase could be attributed to production by the antagonist, on the fruit itself, was not resolved (Castoria et al. 2001). This antagonist can also produce aurebasidins, antibiotics whose role has not been determined. The antagonistic yeast, Candida saitoana, effective in reducing decays of citrus and apple, induced chitinase activity in apple (Wilson and El Ghaouth 1993). C. oleophila, used in the commercial product Aspire, induced resistance responses such as production of chitinase, b-1,3-endoglucanases, PAL, phytoalexins scoparone and scopoletin and ethylene in flavedo tissue of grapefruit (Droby et al. 2002). The contribution of these induced resistance responses to biocontrol was not determined. The yeast C. famata, effective in reducing green mold caused by P. digitatum on oranges, increased the phytoalexins scopoletin and scoparone 12-fold in fruit wounds after four days, but the role scoparone in biocontrol is uncertain due to its relatively slow production (Arras 1996). The antagonists Cryptococcus laurentii and Sporobolomyces roseus, effective against gray mold of apple, utilized the apple volatile, butyl acetate, which stimulated germination and adhesion to membranes of B. cinera conidia (Filonow 1999; 2001). The significance of these phenomena in the biological control was not established due to the technical difficulties in conducting this type of experiment in fruit wounds. When these antagonists were applied to harvested fruits, they colonized fruit wounds rapidly, and competition for limiting nutrients and space was suggested as an important biocontrol mechanism. Removal of limiting nutrients may also be responsible for maintaining the dormancy of Colletotrichum spp. appresoria on fruit treated with antagonistic Bacillus spp. (Korsten and Jeffries 2000).

IMPROVING BCPD

The goal of the biocontrol improvement program is to realize the full potential of biological control. This may be accomplished by more extensive strain selection of the same antagonist species, as indicated earlier in the case of M. pulcherrima, by manipulating antagonists and/or their environment, and by applying the antagonists before harvest in addition to one after harvest. Postharvest application of the antagonists mixture of P. syringae and S. roseus (Janisiewicz and Bors 1995), and C. sake and Pantoea agglomerans (Nunes et al. 2002) improved efﬁcacy of biocontrol of blue mold of apples compared to the individual antagonist. An orchard application of a mixture of A. pullulans with Rhodotorula glutinis was more effective than the individual antagonists, and suppressed apple decays caused by Penicillium spp., B. cinerea, and Pezicula malicorticis after harvest to the same level as the commonly used fungicide Euparen (Leibinger et al. 1997). Nutrient utilization proﬁle of individual antagonists was successfully used to develop antagonist mixtures with a minimum of nutrient overlap between the antagonists and

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resulted in biocontrol of blue mold of apples that was superior to the individual antagonists (Janisiewicz 1996). Physiological manipulation has been focused on improving antagonist fitness by growing them under various conditions that improved resistance to desiccation and survival on the fruit. This is of particular importance to antagonists that are applied in the orchard for control of postharvest decays. C. sake cells grown under water stress caused by addition of glucose or glycerol increased after application to apple trees, while those grown on unmodified media did not (Teixido´ et al. 1998b). This yeast was more water-stress tolerant when grown on a molasses-based medium than on a medium where water activity (aw) was modified by the addition of NaCl (Abadias et al. 2001b). In preparing a freeze-dried formulation, viability of the C. sake cells was best maintained when 10% skim milk was combined with other protectants such as lactose, glucose, fructose, or sucrose (Abadias et al. 2001a). In general, the highest viability of the C. sake cells occurred when the protection and rehydration media were the same. Control of blue mold of apples was improved by the addition of the amino acids L-asparagine or L-proline to the P. syringae antagonist treatment suspensions (Janisiewicz et al. 1992). These amino acids were selected after screening various C and N sources for their effect on the antagonist and pathogen growth. Both amino acids increased population of the antagonist more than 10-fold in the fruit wounds. The addition of the glucose analog, 2-deoxy-D -glucose, to the antagonistic yeasts S. roseus and C. saitoana significantly improved decay control on apple and citrus, respectively (ElGhaouth et al. 2000c; Janisiewicz 1994). 2-deoxy-D -glucose can be up taken by the pathogens but it cannot be metabolized as energy source, resulting in reduced pathogen growth, which gives the advantage to the antagonists, and improves biocontrol. Ammonium molybdate stimulated population of the antagonistic yeast C. sake (CPA-1) and improved control of blue mold of apple and pear after harvest (Nunes et al. 2001a). This nutrient also has fungicidal activity and inhibited germination of P. expansum and B. cinerea spores in vitro, and reduced blue mold, gray mold, and Rhizopus stolonifer decay of apple in pre- and postharvest applications (Nunes et al. 2001b). Use of genetic manipulation to improve BCPD has great potential, but little research has been done in this area. The appearance of decay symptoms on avocado fruit was delayed when the fruit were dipped in a suspension containing a reduced-pathogenicity mutant of the avocado pathogen Colletotrichum gleosporioides (Yakoby et al. 2001). This mutant was generated by restriction enzyme-mediated integration (REMI) transformation, and induced natural resistance of avocado by increasing production of the antifungal diene from 700 to 1,200 mg/g fresh weight 9 days after inoculation. Saccharomyces cerevisiae transformed with a cercopin A-based peptide, that inhibits germination of C. cocodes at 50 mM, was able to inhibit the growth of germinated spores, and inhibited decay development caused by C. cocodes on wounded tomato (Jones and Prusky 2002).

Janisiewicz

This work demonstrated that microorganisms that colonize fruits can be used as vehicles for providing decay control which may include various biocontrol traits.

INTEGRATING BCPD WITH OTHER ALTERNATIVES

Although BCPD can provide levels of control that are commercially acceptable, the performance margin of biocontrol is generally lower than for fungicides. For example, higher concentrations of the antagonist must be used to achieve the same control of decay as fruit mature. To increase the performance margin of biocontrol, attempts have been made to integrate biocontrol with other alternatives to synthetic fungicide methods that were developed mainly during the past two decades (Conway and Sams 1983; Falik et al. 1995; Smilanick et al. 1995; 1997; 1999; Smoot and Melvin 1965; Spotts 1984; Spotts and Chen 1987; Tukey 1993). These methods alone did not provide commercially acceptable control of fruit decay, but in combination with biocontrol increased its performance margin. Infiltration of apples with calcium chloride alone reduced blue mold decay by approximately half (Conway and Sams 1983), but in combination with the antagonist, P. syringae, resulted in greater reduction of fruit decay than either treatment alone (Janisiewicz et al. 1998). The effects of calcium treatment were greatest on more mature fruit, inoculated after 3 or 6 months in storage, when the effectiveness of biocontrol declines (Conway et al. 1999). Combining biocontrol with a calcium treatment complements each other to overcome the shortcomings of each, and may allow for reduced amounts of both products to be used without compromising decay control. In addition, applying lower calcium concentrations would reduce potential calcium injury, while maintaining other benefits, including alleviating storage maladies, such as bitter pit. The addition of calcium chloride to the yeast antagonist Candida sp. also improved control of blue mold and gray mold on apples (McLaughlin et al. 1990; Wisniewski et al. 1995). Treating apples with hot air (4 d at 388C) may virtually eliminate blue mold of apple but it has no residual effect, and any inoculation with pathogens following heat treatment results in decay (Falik et al. 1995; Lurie et al. 1998). Combining antagonistic yeasts or bacteria with heat treatment improved control of blue mold on apples (Conway et al. 1999). The heat treatment eradicated P. expansum infections up to 12 h after inoculation, and yeast and bacterial antagonists provided the residual effect. The heat treatment complemented the lack of eradicative activity of the antagonists, the major shortcoming of BCPD. It may have additional benefits of eradicating pathogens from fruit bins and storage rooms (Douglas 1998). Substances generally regarded as safe (GRAS), such as sodium carbonate, sodium bicarbonate, ethanol, acetic acid, hydrogen peroxide, chitosan, or some edible coatings can

Biological Control of Postharvest Diseases of Fruits

reduce pathogen germination and growth. They are acceptable to the consumers, and in contrast to synthetic fungicides, do not have the prospect of a lengthy and costly approval process from regulatory agencies. For example, sodium carbonate has been used to treat lemons in commercial packinghouses (Smilanick et al. 1999). Combining 3% sodium carbonate with the antagonist P. syringae ESC-10 was superior to the individual treatments in controlling green mold on citrus (Smilanick et al. 1999). This compound also improved control of blue mold and gray mold of oranges in combination with antagonist P. agglomerans (CPA-2) (Teixido et al. 2001). Sodium carbonate has up to 24 h of eradicative activity, but little residual activity, which, like heat treatment, complements biocontrol. Treatment of lemons with 10% ethanol reduced green mold to less than 5% (Smilanick et al. 1995). The addition of 10% ethanol to suspensions of S. cerevisiae strains 1440 and 1749, which had little biocontrol activity, reduced gray mold decay of apple from more than 90% to close to 0% (Mari and Carati 1998). Chitosan and its derivatives can reduce fungal growth and induce resistance responses in harvested fruits and vegetables (Allan and Hadwiger 1979; El-Ghaouth 1998). The addition of 0.2% glycolchitosan to a suspension of the antagonist C. saitoana increased control of green mold of oranges and lemons, and gray and blue mold of apples over that of the antagonist alone (El-Ghaouth et al. 2000a, b). There are many more possibilities for combining biocontrol agents with GRAS substances or other nonfungicidal treatments. The above examples of improving biocontrol and integrating biocontrol with other nonfungicidal treatments demonstrate that biocontrol is amenable to manipulation and can be easily integrated with various decay control measures resulting in additive or synergistic effects. Strategies must be developed for the integration of various treatments in order to maximize decay control (Conway et al. 1999). Various control measures may be applied in succession and these applications may be customized to ﬁt different postharvest practices.

COMMERCIALIZATION OF BCPD

Finding an industry partner is essential for the commercialization of any antagonist for postharvest biocontrol. Formulation, pilot tests, toxicology tests, and registration of the product are expensive and entail more than most research programs in government laboratories or academia are equipped to handle. Although there are examples of commercialization of biocontrol products for plant diseases control by individual scientists, especially from academia, they generally involve creation of a private company that can generate venture capital. USDA/ARS has developed a number of useful vehicles that allow private industry to commercialize inventions created in government laboratories by working jointly with the government scientists. These include simple Material Transfer Agreements, or

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Memorandums of Understanding, which allow private industry the initial exploration of the commercial potential of an invention, or more definitive, Cooperative Research and Development Agreements (CRADA) that specify the role of each party in the commercial development and ownership of the final product. Having a private industry partner warrants closer scrutiny of the economic impact of the disease, importance of the disease to be controlled, and the potential for biocontrol to be competitive with the other control measures in terms of efficacy and cost. A CRADA was essential in the commercialization of BioSavee by EcoScience Corp., which is based on P. syringae, and Aspiree by Ecogen-Israel Partnership, Ltd., which is based on C. oleophila. For example, under the CRADA, mass production by fermentation and biomass yield were determined for the P. syringae antagonist first, and EcoSciene Corp. investigated the potential for registration and formulation of the antagonist. This was followed by joint biocontrol feasibility and up-scale tests under simulated commercial conditions, biocontrol tests with various formulations developed by EcoScience Corp., and the pilot test with the final formulation. EcoScience Corp. developed safety data and registered the product. Production, marketing, and quality control were conducted by EcoScience Corp. Also, essential for the success of BioSave was the well-developed distribution of the product, skillful technical assistance, and rigorous quality control. This approach could be used as a model for successful public/private sector cooperation in the commercialization of other antagonists for BCPD (Stack 1998). The commercial development of Avogreen in South Africa, which contains B. subtillis and is applied in the field for the control of postharvest diseases of avocado, followed a slightly different path (L. Korsten, personal communication). Once registered, the approach was “to make the system work in the hands of the farmer.” First, growers tested the product on a limited scale and were encouraged to integrate the product with existing copper sprays. By slowly phasing in biocontrol, growers gained confidence in the product. They were provided with technical support to calculate dosages, and develop suitable spray schedules adjusted for their spray equipment, cultivars used, age, and history of their orchards and disease profiles. Different formulations were developed to adapt biocontrol to different mixing systems, and for integration with existing chemicals and application methods. Application guidelines were subsequently developed for all possible application systems and for different customer needs. The wettable carrier was found to be more desirable from a production point of view because it sustained excellent cell and biomass densities, had acceptable shelf life, and was more economical. Rigorous quality control has been an integral part throughout the development of this product. Marketing biological control products requires extensive knowledge in the fields of biological- and integrated control, production systems, and microbial ecosystems. The effectiveness of implementing biocontrol alone or in integrated systems will largely depend on knowledge of the product,

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thorough understanding of its complexity, and transferring this knowledge to the market place. These aspects are often neglected in the commercialization of biocontrol.

CONCLUSIONS

Work conducted on BCPD demonstrates that in some cases biological control alone can provide adequate decay control, but in others it must be integrated with additional control measures. BCPD is compatible with many alternatives to fungicide treatment, can be easily adapted to current postharvest practices, and be used in a cascade system, where each additional control measure further reduces fruit decay. Recent advances in physiological and genetic manipulation of the yeast biocontrol agents and the development of superior antagonist mixtures that led to significant increases in the efficacy of BCPD are only the tip of the iceberg in showing what can be accomplished with the postharvest biocontrol system. They are also indicative that, in the future, there will be additional situations where biocontrol treatment alone will be adequate for the control of postharvest fruit decays. Greater effort is needed to identify circumstances where currently available biocontrol can be used alone. This includes not only replacing a fungicide treatment but also instances where no fungicides are registered for postharvest use and losses due to decay are significant, e.g., Botrytis rot on pomegranates designated for processing. There are an increasing number of situations where a fungicide is registered for postharvest use in the United States but not in other countries, particularly in Europe. This may restrict export markets or increase losses if major decay control measures are not implemented for fruit designated for export. In many of these instances, biological control can be an acceptable treatment. Currently, the European Community is sponsoring a large international project on biological control of postharvest diseases of fruits as an alternative to synthetic fungicide treatment after harvest. Experience with the commercial products for BCPD such as Aspiree, Avogreene, and BioSavee, indicate that the fruit industry is receptive to the new control measures and will implement them if they provide adequate control, are cost effective and compatible with current postharvest practices. Continuous expansion of postharvest biocontrol research worldwide and the related successes, even in the most challenging areas such as control of latent infections creates an optimistic picture for the future of BCPD of fruits.

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179 Gueldner RC, Reillly CC, Pusey PL, Costello CE, Arrendale RF, Cox RH, Himmelsbach DS, Crumley FG, and Cutler HG (1988). Isolation and identiﬁcation of iturins as antifungal peptides in biological control of peach brown rot with Bacillus subtilis. J Agric Food Chem 36:366– 370. Guinebretiere MH, Nguyen-The C, Morrison N, Reich M, and Nicot P (2000). Isolation and characterization of antagonists for the biocontrol of the postharvest wound pathogen Botrytis cinerea on strawberry fruits. J Food Prot 63:386– 394. Gullino ML and Kuijpers LAM (1994). Social and political implications of managing plant diseases with restricted fungicides in Europe. Annu Rev Phytopathol 32:559 – 579. Helbig J (2002). Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry. BioControl 47:85– 99. Huang Y, Wild BL, and Morris C (1992). Postharvest biological control of Penicillium digitatum decay on citrus fruit by Bacillus pumilus. Ann Appl Biol 120:367 – 372. Ippolito A, Nigro F, Romanazzi G, and Campanella V (1998). Field application of Aureobasidium pullulans against Botrytis storage rot of strawberry. Proceedings of COST 914-COST 915 Joint Workshop on Non Conventional Methods for the Control of Postharvest Disease and Microbial Spoilage, Bologna, Italy, pp 127 –133. Ippolito A, El-Ghaouth A, Wilson CL, and Wisniewski M (2000). Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses. Postharvest Biol Technol 19:265 –272. Janisiewicz WJ (1987). Postharvest biological control of blue mold on apples. Phytopathology 77:481 –485. Janisiewicz WJ (1991). Control of postharvest diseases of fruits with biocontrol agents. Proceedings of the International Seminar on Biological Control of Plant Diseases and Virus Vectors, Tsukuba, Japan, pp 56– 68. Janisiewicz WJ (1994). Enhancement of biocontrol of blue mold with the nutrient analog 2-deoxy-D -glucose on apples and pears. Appl Environ Microbiol 68:2671 –2676. Janisiewicz WJ (1996). Ecological diversity, niche overlap, and coexistence of antagonists used in developing mixtures for biocontrol of postharvest diseases of apples. Phytopathology 86:473– 479. Janisiewicz WJ and Bors B (1995). Development of a microbial community of bacterial and yeast antagonists to control woundinvading postharvest pathogens of fruits. Appl Environ Microbiol 61:3261 –3267. Janisiewicz WJ and Jeffers SN (1997). Efﬁcacy of commercial formulation of two biofungicides for control of blue mold and gray mold of apples in cold storage. Crop Prot 16:629– 633. Janisiewicz WJ and Korsten L (2002). Biological control of postharvest diseases of fruits. Annu Rev Phytopathol 40:411– 441. Janisiewicz WJ and Roitman J (1988). Biological control of blue mold and gray mold on apple and pear with Pseudomonas cepacia. Phytopathology 78:1697 – 1700. Janisiewicz W, Yourman L, Roitman J, and Mahoney N (1991). Postharvest control of blue mold and gray mold of apples and pears by dip treatment with pyrrolnitrin, a metabolite of Pseudomonas cepacia. Plant Dis 75:490– 494. Janisiewicz WJ, Usall J, and Bors B (1992). Nutritional enhancement of biocontrol of blue mold on apples. Phytopathology 82:1364 –1370.

180 Janisiewicz WJ, Peterson DL, and Bors R (1994). Control of storage decay of apples with Sporobolomyces roseus. Plant Dis 78:466 – 470. Janisiewicz WJ, Conway WS, Glenn DM, and Sams CE (1998). Integrating biological control and calcium treatment for controlling postharvest decay of apples. HortScience 33:105 – 109. Janisiewicz WJ, Tworkoski TJ, and Sharer C (2000). Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients. Phytopathology 90:1196 – 1200. Janisiewicz WJ, Tworkoski TJ, and Kurtzman CP (2001). Biocontrol potential of Metchnikowia pulcherrima strains against blue mold of apple. Phytopathology 91:1098 –1108. Jijakli MH and Lepoivre P (1998). Characterization of an exo-b-1,3glucanase produced by Pichia anomala strain K, antagonist of Botrytis cinerea on apples. Phytopathology 88:335 –343. Jijakli MH, Declerq D, Cognet S, Massart S, Grevesse C, and Lepoivre P (2001). Use of molecular tools to enhance antagonistic activity of yeasts against postharvest diseases of apples. Phytopathology 91:S154. Jones RW and Prusky D (2002). Expression of an antifungal peptide in Saccharomyces: A new approach for biological control of the postharvest disease caused by Colletotrichum coccodes. Phytopathology 92:33 –37. Kanapathipillai VS and bte Jantan R (1986). Approach to biological control of anthracnose fruit rot of bananas. Proceedings of the First Regional Symposium on Biological Control, Pertanian, pp 387 –398. Koomen I and Jeffries P (1993). Effects of antagonistic microorganisms on the post-harvest development of Colletotrichum gloeosporioides on mango. Plant Pathol 42:230 – 237. Korsten L and Jeffries P (2000). Potential for biological control of diseases caused by Colletotichum. In: Prusky D, Freeman S, Dickman MB eds. Colletotrichum Host Specificity, Pathology and Host-Pathogen Interaction. St. Paul, MN: APS Press. pp 266 –295. Korsten L, De Jager ES, De Villiers EE, Lourens A, Kotze JM, and Wehner FC (1995). Evaluation of bacterial epiphytes isolated from avocado leaf and fruit surfaces for biological control of avocado postharvest diseases. Plant Dis 79:1149 –1156. Leibinger W, Breuker B, Hahn M, and Mendgen K (1997). Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field. Phytopathology 87:1103 –1110. Lima G, De Curtis F, Castoria R, and De Cicco V (1998). Activity of the yeasts Cryptococcus laurentii and Rhodotorula glutinis against post-harvest rots on different fruits. Biocontrol Sci Technol 8:257 –267. Lindow SE, Monier J-M, and Leveau JHJ (2002). Characterizing the microhabitats of bacteria on leaves. Proceedings of the 10th International Congress on Molecular Plant-Microbe Interaction, Madison, Wisconsin, pp 241 –249. Lurie S, Falik E, Klein JD, Kozar F, and Kovacs K (1998). Postharvest heat treatment of apples to control San Jose (Quadraspidiotus perniciosus Comstock) and blue mold (Penicllium expansum Link) and maintain fruit firmness. J Am Soc Hortic Sci 123:110 – 114. Mari M and Carati A (1998). Use of Saccharomyces cerevisiae with ethanol in the biological control of grey mould on pome fruits. Proceedings of COST 914 –COST 915 Joint Workshop on Non

Janisiewicz Conventional Methods for the Control of Postharvest Disease and Microbial Spoilage, Bologna, Italy, pp 85 –91. McLaughlin RJ, Wisniewski ME, Wilson CL, and Chalutz E (1990). Effect of inoculum concentration and salt solutions on biological control of postharvest diseases of apple with Candida sp. Phytopathology 80:456 –461. McLaughlin RJ, Wilson CL, Droby S, Ben-Arie R, and Chalutz E (1992). Biological control of postharvest diseases of grape, peach, and apple with the yeasts Kloeckera apiculata and Candida guilliermondii. Plant Dis 76:470– 473. Nunes C, Usall J, Teixido´ N, Miro´ M, and Vinãs I (2001a). Nutritional enhancement of biocontrol activity of Candida sake (CPA-1) against Penicilliium expansum on apples and pears. Eur J Plant Pathol 107:543 –551. Nunes C, Usall J, Teixido N, Ochoa de Eribe X, and Vinas I (2001b). Control of post-harvest decay of apples by pre-harvest and postharvest application of ammonium molybdate. Pest Manag Sci 52:1093 – 1099. Nunes C, Usall J, Teixido N, Torres R, and Vinas I (2002). Control of Penicillium expansum and Botrytis cinerea on apples and pears with the combination of Candida sake and Pantoea agglomerans. J Food Prot 65:178– 184. Peng G and Sutton JC (1990). Biological methods to control grey mould of strawberry. Brighton Crop Prot Conf 1:233 –240. Pusey PL (1991). Antibiosis as mode of action in postharvest biological control. Proceedings of Workshop on Biological Control of Postharvest Diseases of Fruits and Vegetables, Shepherdstown, West Virginia, pp 127 –141. Pusey PL and Wilson CL (1984). Postharvest biological control of stone fruit brown rot by Bacillus subtilis. Plant Dis 68:753 –756. Pusey PL, Hotchkiss MW, Dulmage HT, Baumgardner RA, Zehr EI, Reilly CC, and Wilson CL (1988). Pilot test for commercial production and application of Bacillus subtilis (B-3) for postharvest control of peach brown rot. Plant Dis 72:622 –626. Qing F and Shiping T (2000). Postharvest biological control of rhizopus rot of nectarine fruits by Pichia membranefaciens. Plant Dis 84:1212 – 1216. Ragsdale NN and Sisler HD (1994). Social and political implications of managing plant diseases with decreased availability of fungicides in the United States. Annu Rev Phytopathol 32:545 –557. Roberts RG (1990). Postharvest biological control of gray mold of apple by Cryptococcus laurentii. Phytopathology 80:526 –530. Schena L, Ippolito A, Zahavi T, Cohen L, Nigro F, and Droby S (1999). Genetic diversity and biocontrol activity of Aureobasidium pullulans isolates against postharvest rots. Postharvest Biol Technol 17:189– 199. Smilanick JL, Margosan DA, and Henson DJ (1995). Evaluation of heated solutions of sulfur dioxide, ethanol, and hydrogen peroxide to control postharvest green mold of lemons. Plant Dis 79:742 –747. Smilanick JL, Mackey BE, Reese R, Usall J, and Margosan DA (1997). Influence of concentration of soda ash, temperature, and immersion period on the control of postharvest green mold of oranges. Plant Dis 81:379– 382. Smilanick JL, Margosan DA, Milkota F, Usall J, and Michael I (1999). Control of citrus green mold by carbonate and bicarbonate salts and influence of commercial postharvest practices on their efficacy. Plant Dis 83:139– 145. Smoot JJ and Melvin CF (1965). Reduction of citrus decay by hot-water treatment. Plant Dis Rep 49:463 –467.

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16 Arbuscular Mycorrhizal Fungi in Plant Disease Control Lisette J.C. Xavier / Susan M. Boyetchko Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada

and microbiological activities occur (Lynch 1990). The mycorrhizosphere is the region of the rhizosphere that is subjected to modifications following AMF colonization of the host plant (Linderman 1988). Induced biochemical changes in the plant as a result of AMF root colonization is collectively termed the “mycorrhizosphere effect.” The mycorrhizosphere effect typically results in a transient or permanent shift in the resident microbial community that may favor the elimination or proliferation of pathogens (Edwards et al. 1998; Meyer and Linderman 1986; Nemec 1994; Paulitz and Linderman 1989). In general, these changes are mediated by modifications in host root membrane permeability that subsequently leads to modifications in root exudate composition (Graham et al. 1981; Ratnayake et al. 1978). Meticulous management of the mycorrhizosphere may serve as an effective, safe, and environmentally friendly alternative to conventional methods of plant disease control.

INTRODUCTION

Biological control of plant pathogens presents a compelling method of increasing plant yields by suppressing or destroying pathogens, enhancing the ability of plants to resist pathogens, and/or protecting plants against pathogens. Micro-organisms antagonistic to plant pathogens may be derived from the resident microbial community or may be of foreign origin. Although there are concerns towards the release of an organism of foreign extraction, in general, biological control presents a myriad of benefits such as being a component of the environment, resistant to development of chemical pesticide resistance, being relatively safe and risk free, and by being compatible with sustainable agriculture. Arbuscular mycorrhizal fungi (AMF) form one such group of organisms that can act as bioprotectors of plants. These zygomycetous fungi that form specialized structures such as arbuscules and/or vesicles are obligate biotrophs and utilize host photosynthates for their growth. They are ubiquitous and co-exist with over 80% of terrestrial plants including agricultural or horticultural crops. Their interactions with rhizosphere flora and fauna influence the growth and fitness of the associated plants (Azcon-Aguilar and Barea 1992; Fitter and Sanders 1992). An incompatible association between the host plant and the indigenous AMF community can lead to serious losses in crop yields, indicating the significance of AMF in crop production. In contrast, a compatible association can result in enhanced plant productivity, through enhanced host P nutrition (Ravnskov and Jakobsen 1995), prevention or control of plant diseases caused by soil-borne pathogens (Caron 1989a; St-Arnaud et al. 1995), and/or enhancement of plant hormonal activity (Frankenberger and Arshad 1995). The rhizosphere, a zone of soil loosely surrounding the roots, is a dynamic environment wherein complex chemical

2 2.1

EXAMPLES OF AMF-MEDIATED PLANT DISEASE CONTROL Phytopathogenic Fungi

Plant pathogenic fungi contribute signiﬁcantly to crop damage and yield loss, followed by plant pathogenic bacteria and viruses. The potential of AMF to control various plant pathogenic fungi has been clearly demonstrated (Becker et al. 1999; Bodker et al. 2002; Boyetchko and Tewari 1996; Duchesne et al. 1989; Kapoor et al. 1998; Kasiamdari et al. 2002; Kegler and Gottwald 1998; Krishna and Bagyaraj 1983). In contrast, there are reports wherein AMF inoculation did not have any effect on disease severity (Guillon et al. 2002; Larsen and Bodker 2001; Wyss et al. 1991; Zambolin and Schenck 1983). In order for practical and routine use of 183

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AMF as protectors of plants against plant pathogenic fungi, AMF performance must be consistent, specific, and effective. Specificity of AMF for the control of crop diseases is crucial in order to mitigate any nontarget effects to beneficial micro-organisms. However, there are conflicting reports on the specificity of AMF. For example, inoculation of micropropagated banana with Glomus intraradices and a Glomus spp. isolate reduced rhizome necrosis and external disease symptoms caused by Fusarium oxysporum f. sp. cubense, but differences between the two AMF isolates were not noted, indicating that either both AMF species were equally effective against the pathogen or that they lacked specificity (Jaizme-Vega et al. 1998). In contrast, eggplant and cucumber seedlings transplanted into soils inoculated with G. versiforme and subsequently challenged with Verticillium dahliae and Pseudomonas lacrymans alleviated wilt symptoms caused by V. dahliae, but not G. mosseae, Glomus spp.-1, or Glomus spp.-2, indicating species-specific antagonistic symbiont – pathogen interactions (Li et al. 1997). Pozo et al. (1999) demonstrated the expression of two new basic glucanase isoforms, a phytoalexin elicitor-releasing factor between G. mosseae and G. intraradices used for the control of Phytophothora parasitica var. nicotianae. Because of the potential of AMF as bioprotectors against phytopathogens, this is an area that needs further study. In order to enhance AMF efficacy, some researchers have used an AMF species mixture or a combination of microorganisms including AMF that act in concert to eliminate pathogens. For example, co-inoculation of groundnut with G. fasciculatum, Gigaspora margarita, Acaulospora laevis, and Sclerocystis dussii eliminated the damaging effects of Sclerotium rolfsii (Kulkarni et al. 1997). Also, tobacco inoculated with a mixture containing G. fasciculatum and Trichoderma harzianum effectively controlled damping-off caused by Pythium aphanidermatum and black shank disease caused by P. parasitica var. nicotianae (Sreeramulu et al. 1998). In some cases, microbial mixtures act synergistically with pesticides to result in effective control of plant diseases. A combination of wheat straw, carbendazim, G. fasciculatum, and T. viride protected safflower seedlings from the root rot pathogen Macrophomina phaseolina, resulting in 100% seedling survival (Prashanthi et al. 1997). Sharma et al. (1997) effectively managed ginger yellows disease caused by F. oxysporum f. sp. zingiberi using a combination of Gi. margarita, pine needles, and T. harzianum. The requirement of a fully established AM symbiosis for elicitation of bioprotective activity by AMF has been disputed. The invasion of phytopathogenic fungi is said to be prevented by an aggressively root colonizing AMF species, indicating that AMF root colonization was satisfactory for control of disease. For example, Feldmann and Boyle (1998) found an inverse correlation between G. etunicatum root colonization of begonia cultivars and susceptibility to the foliar pathogen caused by the powdery mildew fungus Erysiphe cichoracearum. However, it was not clear whether G. etunicatum colonization preceded infection by E. cichoracearum or whether pathogen suppression was

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accompanied by other mechanisms of biocontrol. Using an in vitro system, Filion et al. (1999) demonstrated that extracts from the extraradical mycelium of G. intraradices reduced the conidial germination of F. oxysporum f. sp. chrysanthemi. Alternatively, alterations in the chemical equilibrium of the mycorrhizosphere may have resulted in pathogen control. In another study, pea mutants defective for mycorrhization and nodulation challenged with Aphanomyces euteiches required a fully established AMF symbiosis for protection against the pathogen (Slezack et al. 2000). Several researchers have also demonstrated AMFmediated reduction of root rot disease in cereal crops (Boyetchko and Tewari 1988; Grey et al. 1989; Rempel and Bernier 1990; Thompson and Wildermuth 1989) and take-all disease of wheat (Graham and Menge 1982). Phytophthora spp., which cause diseases in a variety of plants have been model systems for AMF-mediated plant disease control (Cordier et al. 1996; Guillemin et al. 1994; Mark and Cassells 1999; Norman and Hooker 2000; Pozo et al. 1996; Trotta et al. 1996). Using the AMF species G. intraradices and pathogen F. oxysporum f. sp. lycopersici on tomato, Caron and co-workers have shown that the growth medium used (Caron et al. 1985), the application of P (Caron et al. 1986a), and pretreatment of the growth medium with AMF (Caron et al. 1986c) can influence disease severity. Despite proof of AMF potential in controlling plant diseases, few published reports have successfully demonstrated biological control of plant pathogens by AMF in the field (Bodker et al. 2002; Newsham et al. 1995; Torres-Barragan et al. 1996). Newsham et al. (1995) showed that pre-inoculating the annual grass Vulpia ciliata var. ambigua with an indigenous Glomus sp. and re-introducing the grass into a natural grass population extended a favorable effect against an indigenous F. oxysporum. Onion pretreated with Glomus sp. Zac-19 delayed the development of onion white rot caused by S. cepivorum by two weeks in the field and protected onion plants for 11 weeks after transplanting in the field and resulted in a yield increase of 22% (Torres-Barragan et al. 1996). One of the first reports on the effect of indigenous AMF on the development of introduced A. euteiches infection and disease development on field-grown pea (Bodker et al. 2002) showed that there was no correlation between AMF root colonization and disease incidence or severity, and emphasized the importance of field evaluations for authenticating the use of AMF as biocontrol agents. Although the indigenous AMF community composition was not described, this study underscores the importance of a richly diverse indigenous AMF community to defend plants from plant pathogens. Thus, there appears to be tremendous potential for AMF control of plant pathogens and the need for more detailed and well-planned and executed studies that will address problems of inconsistent and unreliable results.

2.2

Plant Pathogenic Bacteria

The AMF interact with functionally diverse bacteria such as diazotrophs, biological control agents, and other common

Plant Disease Control Using AMF

rhizosphere inhabitants (Nemec 1994) that often result in significant alterations to plant growth, yield, and nutrition. Interactions between mycorrhizal fungi and bacteria may have detrimental (Filion et al. 1999; Shalaby and Hanna 1998; 2001) or beneficial effects (Edwards et al. 1998; Gryndler and Hrselova 1998; Li et al. 1997; Ravnskov and Jakobsen 1999), or have no effect at all on the plant pathogenic bacterium (Otto and Winkler 1995). Glomus mosseae prevented the infection of soybean plants by P. syringae (Shalaby and Hanna 1998), by suppressing the population density of the pathogen in soybean rhizosphere. Li et al. (1997) also found that G. macrocarpum reduced the infection caused by P. lacrymans in eggplant and cucumber, although no positive growth or yield effect was noted, indicating tolerance to the pathogen as a possible mode of action. Inoculation of mulberry with G. fasciculatum or G. mosseae in combination with 60–90 kg of P per hectare per year reduced the incidence of bacterial blight caused by P. syringae pv. mori (Sharma 1995). Inoculation of grapevines with AMF reduced the number of fluorescent pseudomonads on the rhizoplane thereby reducing the incidence of grapevine replant disease (Waschkies et al. 1994). Similarly, a reduction in the colonization of apple seedling rootlets by actinomycetes causing replant disease was reported, while a proportionate increase in root colonization by AMF was noted (Otto and Winkler 1995).

2.3

Phytopathogenic Viruses

Viruses remain the least studied amongst all the plant diseasecausing target organisms listed for mycorrhizae-mediated biocontrol. The general response of mycorrhizal plants to the presence of viral pathogens is as follows: (a) mycorrhizal plants apparently enhanced the rate of multiplication of viruses in some plants (Daft and Okusanya 1973; Nemec and Myhre 1984), (b) more leaf lesions were found on mycorrhizal plants than on nonmycorrhizal plants (Scho¨nbeck 1978; Scho¨nbeck and Dehne 1979), and (c) the number of AMF spores in the rhizosphere was reduced considerably (Jayaraman et al. 1995; Nemec and Myhre 1984). Enhanced viral multiplication and activity in mycorrhizal plants is speculated to be attributed to higher P levels compared to nonmycorrhizal plants. A similar effect was noted in nonmycorrhizal plants fertilized with P (Daft and Okasanya 1973; Shaul et al. 1999). Some workers found that host plants were more susceptible to AMF colonization following infection by a virus. For example, Scho¨nbeck and Spengler (1979) reported that following the inoculation of mycorrhizal and nonmycorrhizal tobacco (Nicotiana glutinosa L.) with tobacco mosaic virus (TMV), mycorrhizal plants exhibited higher levels of AMF colonization. In contrast, mung bean yellow mosaic bigeminivirus reduced the AMF colonization and yield of mycorrhizal plants (Jayaraman et al. 1995), while lack of response to viral infection by a mycorrhizal host was also demonstrated (Takahashi et al. 1994). Early studies using electron

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microscopy revealed that mycorrhizae were not viral vectors because virus particles were absent in the AMF hyphae and around arbuscules, suggesting that AMF did not interact with viruses (Jabaji-Hare and Stobbs 1984). Thus, potential for the biocontrol of plant pathogenic viruses using mycorrhizae does not appear to be promising. However, it may be worthwhile to investigate the role of viruses in the reduction of mycorrhizal colonization and related host plant effects.

MODES OF MYCORRHIZAE-MEDIATED DISEASE CONTROL

3.1 3.1.1

Host Nutritional Effects Improved Plant Nutrition

Mycorrhizal plants are generally able to tolerate pathogens and compensate for root damage and photosynthate drain by pathogens (Azcon-Aguilar and Barea 1992; Declerck et al. 2002), because AMF enhance host nutrition and overall plant growth. For example, Declerck et al. (2002) found that G. proliferum and a Glomus sp. isolate not only stimulated growth and increased shoot P content of banana in the presence and absence of the root rot fungus Cylindrocladium spathiphylli, but also reduced root damage by the pathogen, indicating direct interactions between the AMF and the pathogen. In contrast, some reports indicate that AMF are capable of biological control activity (Boyetchko and Tewari 1988; Grey et al. 1989; Rempel and Bernier 1990). It is believed that AMF interact equally with host plants, but in fact AMF prefer one host or host cultivar over another, as shown by Grey et al. (1989) who reported that mycorrhizal barley cultivar WI2291 not only exhibited greater control of the barley common root rot pathogen Bipolaris sorokiniana than a mycorrhizal cultivar Harmal, but also produced significantly higher yields. On the other hand, biological control activity is dependent on the AMF species as demonstrated for common root rot of barley by Boyetchko and Tewari (1992). There are suggestions that root colonization by natural AMF communities occurring in field soils has an inverse relationship with B. sorokiniana infection, indicating not only a direct interaction between the AMF and the pathogen, but also an AMF-mediated improvement in host nutrition (Thompson and Wildermuth 1989). In contrast, there are also reports suggesting a lack of interaction between AMF and B. sorokiniana under field conditions (Wani et al. 1991). Interaction between naturally occurring AMF and pathogens or the lack thereof in the field likely depends on the distribution of the organisms particularly under the different crop rotations. Significant reductions in disease severity as a result of AMF colonization and enhanced P uptake followed by modifications in root exudation patterns has also been reported for take-all disease of wheat (Graham and Menge 1982). Improvement in host P nutrition is one of the earliest proposed mechanisms of AMF-mediated pathogen or disease tolerance that is still very pertinent.

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3.1.2

Xavier and Boyetchko

Tolerance to Pathogen

Arbuscular fungi are known to enhance plant tolerance to pathogens without excessive yield losses, and in some cases, enhance pathogen inoculum density. This compensation is apparently related to enhanced photosynthetic capacity (Abdalla and Abdel-Fattah 2000; Heike et al. 2001; Karajeh and Al-Raddad 1999) and a delay in senescence caused by the pathogen, which cancels the positive relationship between disease severity and yield loss (Heike et al. 2001). For example, soybean plants grown in the soil infested with M. phaseolina, Rhizoctonia solani, or F. solani exhibited lower shoot and root weight and plant height compared to control plants in soil not infested with the pathogens or with G. mosseae (Zambolin and Schenck 1983). The incidence of infection by the pathogens was not affected by G. mosseae colonization but the mycorrhizal plants were able to tolerate infection of pathogens better than nonmycorrhizal plants. The efficacy and efficiency of AMF in promoting plant growth enables mycorrhizal plants to tolerate pathogens, as demonstrated by Hwang (1988) using alfalfa challenged with P. paroecandrum and Karajeh and Al-Raddad (1999) using olive seedlings. It is unclear whether mycorrhizal alfalfa tolerated P. paroecandrum or if other additional mechanisms were involved. Despite the presence of a pathogen benefits of AMF to susceptible hosts can occur until a pathogen inoculum threshold level, beyond which no AMF-mediated benefits can be realized (Stewart and Pfleger 1977). On the other hand, high tissue P levels in mycorrhizal plants may not only improve vigor and fitness of the plant but also modify pathogen dynamics in the mycorrhizosphere by modifying root exudation (Davis and Menge 1980; 1981; Kaye et al. 1984). Tolerance of the plant to a pathogen can vary depending on the AMF species and their ability for enhancing host nutrition and growth, although some ineffective AMF species reduce pathogen entry by triggering a defense reaction in plants (Davis and Menge 1981). For example, Matsubara et al. (2000) noted that there were significant differences in the ability of Gi. margarita, G. fasciculatum, G. mosseae, and Glomus sp. R10 to not only enhance asparagus growth but also in their ability to tolerate the severity of violet root rot caused by Helicobasidium mompa. Asparagus seedlings inoculated with Glomus sp. R10 had the lowest incidence of violet root rot. This important fact highlights the care that needs to be exercised in the selection of AMF species for biological control of diseases.

3.1.3

Qualitative and Quantitative Alterations in Pathogen Biomass

Modiﬁcations in root exudate composition following changes in host root membrane permeability as a result of AMF colonization (Graham et al. 1981) can enforce changes in the rhizosphere microbial equilibrium (Brejda et al. 1998; Edwards et al. 1998; Kaye et al. 1984; Meyer and Linderman 1986). Changes in the rhizosphere microﬂoral community can

collectively benefit host plants by creating favorable conditions for the proliferation of microflora antagonistic to pathogens such as Phytophthora and Pythium spp. as shown for eucalyptus seedlings by Malajczuk and McComb (1979). Unfavorable conditions induced by AMF colonization resulted in qualitative changes in the mycorrhizosphere that prevented P. cinnamoni sporangial induction in tomato plants (Meyer and Linderman 1986). Proliferation of G. mosseae inside grapevine roots was associated with a significant reduction in replant disease-causing fluorescent pseudomonad inoculum in soil (Waschkies et al. 1994). Promoting AMF diversity that will ensure that at least a component of the AMF community may be active against pathogens can further enhance the benefits of this mechanism.

3.2

Competition

The AMF spores in soil are not known to compete for nutrients as spore reserves are utilized for survival until root contact is achieved. Following root entry, competition can occur for infection sites, host photosynthates, and root space (Smith and Read 1997). Competition between AMF and pathogens can be used for physical exclusion of pathogen (Davis and Menge 1980; Hussey and Roncadori 1982; Smith 1988), if the host is preinoculated with AMF. Simultaneous colonization of AMF and the pathogen may not provide a competitive edge for AMF for inoculum build-up (Daniels and Menge 1980) because of its relatively slow growth rate compared with the pathogen. In contrast, some others have noted that competition may not occur between AMF and other organisms (Sempavalan et al. 1995). Competition, as a mechanism of suppressing pathogens by AMF did not receive much consideration, because in some cases pathogens were suppressed even in noncolonized root portions that was later described as induced resistance by AMF (Pozo et al. 1999). In addition, inconsistencies with regard to prerequisites and AMF effects on pathogens have contributed to a lack of interest.

3.3

Physiological and Biochemical Alterations of the Host

Following AMF colonization, host root tissue P levels are typically enhanced which modify the phospholipid composition and therefore the root membrane permeability resulting in a reduction in the leakage of net amount of sugars, carboxylic acids, and aminoacids into the rhizosphere (Graham and Menge 1982; Ratnayake et al. 1978; Schwab et al. 1983). These alterations arrest the chemotactic effect of pathogens to plant roots and discourage pathogen entry. Prior inoculation of maize plants with G. mosseae decreased the number of Alternaria alternata colony forming units, but when both organisms were inoculated at the same time, there was no effect on pathogen inoculum density in soil (McAllister et al. 1996). It is possible that the G. mosseae

Plant Disease Control Using AMF

symbiont altered membrane permeability of the host roots, thereby reducing the quality and quantity of substances exuded by the roots (Graham et al. 1981), restricting pathogen propagule germination, indicating that the timing of inoculation can enhance biocontrol activity.

3.3.1

Systemic-induced Resistance

Systemic-induced resistance (SIR) is typically the sustained induction of resistance or tolerance to disease in plants by previously inoculating with a pathogen, exposing to an environmental influence or treating with a chemical, which may or may not have antimicrobial activity (Handelsman and Stabb 1996; Kuc 1995). Researchers have suggested that AMF-inoculated plants may employ SIR as a mechanism of biocontrol (Benhamou et al. 1994; Brendan et al. 1996; Trotta et al. 1996). The SIR phenomenon in mycorrhizal plants is demonstrated as localized and systemic resistance to the pathogen (Cordier et al. 1998). An increase in the lignin deposition in plant cell walls following AMF colonization can restrict the spread of pathogens (Dehne and Scho¨nbeck 1979). Using a split root system, Cordier et al. (1998) demonstrated that G. mosseae protected tomato plants against P. parasitica by reducing pathogen development and spread by increasing cell wall appositions containing callose close to the intercellular hyphae and accumulation of phenolic compounds and plant cell defense responses. Root damage was observed in portions of mycorrhizal root systems not containing mycorrhizal structures. The SIR reaction to the pathogen in mycorrhizal plants was further illustrated by host wall thickenings containing nonesterified pectins and pathogenesis related (PR)-1 protein in the nonmycorrhizal areas of the roots. They also noted that the PR-1 protein was found only in the pathogen-invaded tissues of pea. These responses were observed in the nonmycorrhizal pathogen-infected root tissues that ultimately led to cell death. Bodker et al. (1998) reported that the observed increased resistance to A. euteiches in G. intraradices-inoculated pea was probably due to an “induced systemic factor,” induced by G. intraradices. The AMF-mediated SIR phenomenon is speculated to play a role in the protection of potatoes against post-harvest suppression of potato dry rot, wherein dry rot in G. intraradix-inoculated potato was reduced by up to 90% compared to uninoculated control (Brendan et al. 1996). This finding suggests that the benefits of AMF inoculation for disease control surpasses the growth and reproduction phase of the host and extends to the storage phase of the product. The area of SIR response in mycorrhizal plants is still developing and several aspects including whether all AMF species can equally elicit a SIR response in the host are not known. Some researchers have examined the role of PR proteins in the disease control process mediated by AMF (Liu et al. 1995). Enhanced levels of 10 different PR proteins were detected in cotton plants inoculated with G. mosseae, G. versiforme, or Scl. sinuosa challenged with V. dahliae compared with plants not challenged by the pathogen. The PR proteins retarded the hyphal growth of V. dahliae and killed

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their conidia. This appears to be a promising ﬁeld that can be used for the effective control of plant diseases.

3.3.2

Phytoalexins and Phytoanticipins

Phytoalexins are produced in response to microbial infection (Paxton 1981), whereas phytoanticipins are stored in plant cells in anticipation of or prior to pathogen attack (VanEtten et al. 1995). The level of phytoalexins elicited by pathogens has been shown to be much higher than those elicited by symbiotic organisms (Wyss et al. 1991). The function of an isoflavonoid molecule as a phytoalexin or phytoanticipin can be predicted based on the cellular location of the molecule (Stafford 1992). An increase in the level of total soluble plant phenolics such as isoflavonoids or flavonoids, lignin, syringic, ferulic or coumaric acids, etc. have been reported as synthesis of phytoalexins following AMF colonization of roots (Harrison and Dixon 1993; Morandi 1989; 1996). Some flavonoids that are not true phytoalexins may also respond to AMF colonization of roots (Harrison and Dixon 1993; Morandi and Le-Quere 1991; Volpin et al. 1995). The production of phytoalexins as a result of pathogen invasion in mycorrhizal plants has been explored. Tomato plants inoculated with G. mosseae posed greater resistance to the pathogen F. oxysporum and were found to have increased phenylalanine and b-glucosidase activity and total phenol content in their roots compared to plants inoculated with either organism alone (Dehne and Schonbeck 1979). Sundaresan et al. (1993) reported that a purified ethanol fraction of mycorrhizal cowpea root extract inhibited F. oxysporum in vitro. However, the isoflavonoid was not identified. Production of phytoalexins in mycorrhizal plants appears to be independent of the effect of fertilizer addition (Caron et al. 1986b). In general, in the presence or absence of pathogens in plant roots, phytoalexins are induced in mycorrhizal plants that neutralize the negative effects of pathogens.

3.3.3

Hydrolases

Differential expression of defense-related genes in mycorrhizal plants has been the recent focus of AMFmediated biocontrol (Blee and Anderson 1996; DumasGaudot et al. 1996; Lambais and Mehdy 1995; Pozo et al. 2002). Researchers have shown that AMF enter into host (e.g., tomato) roots and induce a local, weak, and transient activation of the host defence mechanism against pathogens such as P. parasitica, which involves the induction of hydrolytic enzymes such as chitinase, chitosanase, b-glucanase, and superoxide dismutase (Pozo et al. 2002). In addition, portions of the mycorrhizal root system not containing mycorrhizal structures appear to have alterations in the constitutive isoforms of the enzymes indicating systemic changes following AMF colonization (Pozo et al. 2002). A high positive correlation between the level of glucanase activity in host tissues and pathogen resistance has

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been established (Graham and Graham 1991). Further studies examining the role of these glucanases will help in the development of strategies for control of pathogens using AMF.

3.4

Antibiosis

Reports on the production of antimicrobial substances by AMF are not common. However, recently, it was shown that antimicrobial substances (unidentified) produced by the extraradical mycelium of the AMF species G. intraradices reduced conidial germination of F. oxysporum f. sp. chrysanthemi, which was independent of changes in pH (Filion et al. 1999). Budi et al. (1999) isolated a Paenibacillus sp. strain from the mycorrhizosphere of Sorghum bicolor plants inoculated with G. mosseae that exhibited significant antagonism against P. parasitica. Regardless of the source of these biocontrol activities, it is important to realize and utilize the significance of AMF in plant disease control. Additional research in this are may prove to be fruitful in the control of pathogenic bacteria and fungi.

4.1

CHALLENGES AND STRATEGIES TO ENHANCING ARBUSCULAR FUNGAL EFFICACY IN DISEASE CONTROL Challenges

Although the efficacy of an AMF species on plant pathogens has been assessed under controlled environments and usually in the absence of other AMF or other organisms (Budi et al. 1999; Kasuya et al. 1996; Li et al. 1997), research indicates that the potential of AMF for control of plant pathogens is high. Limitations in AMF research pertaining to biological control of plant diseases under field conditions are two fold, (a) production of large quantities of AMF inoculum is not feasible because of the obligate biotrophicity status of AMF, and (b) negative interactions between the introduced AMF and the indigenous AMF and microbial community after introduction into field. Challenges posed by interactions between AMF and indigenous microbial community and soil and environmental conditions often determine the success of AMF inoculation in disease control under field conditions. An appreciation of factors that influence AMF efficacy as biological control agents can further enhance their survival, competitiveness and efficacy. For example, it is known that intraradical proliferation of AMF within roots is a hostregulated event (Bever et al. 1996). Therefore, a highly mycotrophic host or host cultivar may be more favorable for AMF proliferation and reproduction than one that is not highly mycotrophic (Feldmann and Boyle 1998; Xavier 1999). In addition, nonconducive soil-environment combinations such as high soil P levels and soil disturbance can affect AMF colonization (Bever et al. 1996; Gazey et al. 1992; Stahl et al. 1988; Stutz and Morton 1996) and efficacy

(Graham et al. 1981; Menge et al. 1978; Ratnayake et al. 1978). Colonization of host roots by AMF is a crucial component in the AMF-mediated SIR response of host plants to plant pathogens as the expression of an SIR response requires a threshold level AMF presence within host roots (Cordier et al. 1996; 1998). The effect of phosphorus on AMF efficacy may be direct, wherein inoptimum P levels impair AMF activity and therefore its ability to effectively control pathogens. In contrast, soil disturbance has an indirect effect where AMF efficacy may be altered by a delay in mycelial network initiation and diversion of carbon for the synthesis or repair of the external mycelial network and not nutrient uptake. Zak et al. (1982) suggested that the ability of AMF to effectively re-establish their mycorrhizal association after disturbance might partially determine their success in a disturbed site. It is not known to what extent soil disturbance affects the biocontrol activities of AMF, but it disrupts the external mycelial network resulting in a severe reduction in mycorrhizal efficacy (Evans and Miller 1990; Stahl et al. 1988). A richly diverse AMF community ensures qualitatively and quantitatively the presence of AMF species desired for specific activities such as biological control of plant pathogens. However, choice of host genotype and rotation (Bever et al. 1996; Johnson et al. 1992; Talukdar and Germida 1993), levels of fertilizer application (Baltruschat and Dehne 1982; Jasper et al. 1979; McGonigle and Miller 1993; 1996; Vivekanandan and Fixen 1991), tillage (Evans and Miller 1990; McGonigle and Miller 1993; Vivekanandan and Fixen 1991), pesticide application (Manjunath and Bagyaraj 1984; Schreiner and Bethlenfalvay 1997), and the effect of associated micro-organisms (Andrade et al. 1995; Xavier and Germida 2002) are some critical factors that can indirectly alter AMF diversity in soils. For example, continuous cropping selectively enhances the proliferation of parasitic AMF, which are relatively fast growing compared to beneficial AMF, leading to alterations in mycorrhizal biodiversity in the rhizosphere (Johnson et al. 1992). Similarly, one particular AMF host selects from an indigenous AMF pool resulting in the selective enrichment of certain AMF species over others (Xavier 1999). Studies assessing the significance of AMF biodiversity in AMF-mediated biocontrol are rare but critical. Given the importance of AMF to plant health and the complexity of the various microbial interactions, all the relevant factors have to be considered before AMF selected as biocontrol agents can effectively function in the field.

4.2

Strategies

Biological control of plant diseases by AMF under ﬁeld conditions is the effect of interactions between AMF and various groups of organisms in the rhizosphere. Sikora (1997) proposed “biological system management,” a holistic approach for improving plant root systems that adopts

Plant Disease Control Using AMF

speciﬁc cultural practices that promote plant defense mechanisms such as tolerance and/or resistance to pathogens, and the use of organisms that are antagonistic towards pathogens and that target sensitive developmental stages of pathogens. This approach offers a viable alternative to integrated pest management and inundative approaches such as the application of high levels of microbial inocula to the nonrhizosphere soil for biological control purposes, and underscores the signiﬁcance of mycorrhizae in root health (Sikora 1997).

4.2.1

Improved Understanding of Microbial (AMF) Ecology and Ecosystem Functioning

It is common knowledge that AMF functioning in natural ecosystems can be altered by various factors including interactions with other organisms. However, speciﬁcs on the topic are lacking. Knowledge generated from studies addressing AMF efﬁcacy in a typical rhizosphere community, under moisture and salinity stress and soil disturbance, in soils containing extreme indigenous AMF levels, and in the presence of antagonizers is required for the development of effective AMF biocontrol agents.

4.2.3

(Reena and Bagyaraj 1990a,b; Talukdar and Germida 1994; Vinayak and Bagyaraj 1990). Screening procedures must include selection pressure similar to that in which the AMF will be applied. Research shows that plant pathogens can be controlled not only by the use of biocontrol agents, but also by the induction of resistance responses in plants. Inoculating plants with AMF has been shown to induce resistance in plants. Such plant immunizations are a viable approach for transplant crops because of the ease of AMF inoculation, while more innovative methods are required with directsown crops.

Enhanced AMF Biodiversity

A diverse AMF community contains a mycorrhizal assemblage and species abundance that naturally aid the host to endure adverse conditions to ultimately enhance plant growth. Research shows that inclusion of host crops (Bever et al. 1996; Johnson et al. 1992) and/or cultivars that exhibit high mycorrhizal responsiveness can signiﬁcantly improve AMF functioning (Boyetchko and Tewari 1995; Xavier and Germida 1998). Therefore, rotation of crops that are dependent on mycorrhizae will ensure early AMF root colonization and high sporulation of even the most sensitive AMF species in soil. Minimal disturbance to the soil also guarantees early contact between an emerging seedling and the AMF hyphal network in soil that distributes nutrients and initiate early colonization of AMF propagules in soil. Excessive fertilizer and pesticide use can alter plant chemistry and cause changes in AMF assemblage and abundance, resulting in a poor AMF community that does not beneﬁt the host (Gazey et al. 1992; Jasper et al. 1979; Johnson et al. 1992). Caution in the choice of cultural practices that potentially alter AMF diversity would prove to be fruitful.

4.2.2

189

Selection of Effective AMF

Bagyaraj (1984) suggested that AMF species selection for a desired activity must be based on their ability for survival, aggressive colonization of host roots, and efﬁcacy. Use of AMF species originally isolated from test host roots has proven advantageous for many plant species including agricultural crops, forest tree species, and orchard crops

4.2.4

Superior Application Technology

Research shows that “priming” plants against pathogens using selective AMF inocula (or plant immunization) helps protect plants by inducing a SIR response (Cordier et al. 1998). The inoculum may be applied to seeds, transplanted crops, or plantlets produced through tissue culture before being transplanted into pathogen-infested ﬁelds. Application of the agent prior to transplanting eliminates the need for complex formulations and application techniques, guarantees “targeted placement,” and greater biocontrol activity, reduces costs associated with application and has a minimal impact on the environment (Boyetchko 1996; Glass 1993). Inoculum may include one or more AMF species or other organisms such as bacteria or fungi that exhibit sustained and coordinated biocontrol activity. The application of a multiple agent mixture may concurrently confer control for more than one plant disease by more than one mechanism rather than single inoculants targeted for control of only one plant disease or pathogen. Application of two or more biocontrol agents targeting different life stages of a pathogen may also be more effective than sequential application of the biocontrol agents. In some instances, augmenting soil with organic amendments such as forest humus and charcoal compost has enabled a signiﬁcant reduction in disease severity (Kobayashi 1993; Wei et al. 1987).

CONCLUSIONS

Literature presents a wealth of evidence to indicate potential for AMF-mediated control of plant diseases. Although there are challenges in the form of nonculturability of AMF and therefore mass multiplication for agricultural crops, there is promise for nondirect sown crops, which is currently undervalued and underexploited. The AMF, by increasing crop productivity using existing resources, avoiding resistance development to chemicals, maintaining pollution and risk-free disease control, and conforming to sustainable agricultural practices, offers more than mere plant disease control. In the future, mycorrhizosphere management must become one of the viable and ecosystem friendly solutions to managing plant diseases and reducing pathogen inoculum.

190

ACKNOWLEDGEMENTS The ﬁnancial support provided by Western Grains Research Foundation, Westco Fertilizers, and Agriculture and Agri-Food Canada Matching Investment Initiative is acknowledged.

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Xavier and Boyetchko Xavier LJC and Germida JJ (1998). Response of spring wheat cultivars to Glomus clarum NT4 in a P-deﬁcient soil containing arbuscular mycorrhizal fungi. Can J Soil Sci 78:481 –484. Xavier LJC and Germida JJ (2003). Bacteria associated with Glomus clarum spores inﬂuence mycorrhizal activity. Soil Biol Biochem 35:471– 478. Xavier LJC (L. Johnny) (1999). Effects of interactions between arbuscular mycorrhizal fungi and Rhizobium leguminosarum on pea and lentil PhD dissertation, Saskatoon, Saskatchewan, Canada: University of Saskatchewan. Zak JC, Danielson RM, and Parkinson D (1982). Mycorrhizal fungal spore numbers and species occurrence in two amended mine spoils in Alberta, Canada. Mycologia 74:785 – 792. Zambolin L and Schenck NC (1983). Reduction of the effects of pathogenic root-infecting fungi on soybean by the mycorrhizal fungus, Glomus mosseae. Phytopathology 73:1402 – 1405.

17 Commercialization of Arbuscular Mycorrhizal Biofertilizer Pragati Tiwari / Alok Adholeya The Energy and Resources Institute, New Delhi, India Anil Prakash Barkatullah University, Bhopal, Madhya Pradesh, India

technological application implies a direct relevance to commercialization for mass multiplication of the invented bio-product so as to reach the masses. It is encouraging that both, government and industry are becoming more responsive to natural approaches to growing environmental problems. Individuals and organizations worldwide are coming to realize that excessive use of chemical fertilizers can negatively impact water quality and the environment as a whole. The joint effort, if addressed properly, may lead to a healthy environment for future generations. Biofertilizers include environment-friendly fertilizers with organisms such as: (a) Rhizobium strains for legumes, (b) Azotobacter strains for nonlegume crops, (c) VAM strains for use in agriculture, horticulture, and plantation crops, and (d) phosphorus solubilizing bacteria (PSB) –phosphorus dissolving bacteria strains. Most common among these are symbiotic mycorrhiza, Rhizobium members and cyanophyceae group, which deliver plant nutrition, disease resistance, and tolerance to adverse soil and climatic conditions. Biofertilizers also known as microbial inoculants may be involved in symbiotic and associative microbial activities with higher plants. They are natural mini-fertilizer factories that are an economical and safer source of plant nutrition and can increase agricultural production and improve soil fertility. They have great potential as a supplementary, renewable, and environment-friendly source of plant nutrients and are important components of any integrated plant nutrient system. Research on biofertilizers has focussed on biological N2 ﬁxation, plant growth promoting bacteria (PGPR’ B) and phosphorus solubilizing microbes (Hegde et al. 1999). Research and development activities involved in this demanding but unexplored ﬁeld

INTRODUCTION

Human society today demands the production of high quality food in a most sustainable way causing least damage possible to the environment. Expected benefits include increase in the efficiency of crop production, reduction in agrochemical inputs, and an evaluation of the safety and bioethical aspects in relation to public acceptability. High productivity agriculture exacts a high cost in terms of energy and the environment. Typically, fertilizer and pesticides are used at high levels in the intensive production of plants. More than 150 years of over cultivation with synthetic fertilizers and pesticides has left our soils depleted of the natural biota needed to facilitate the growth of crops. A less costly and nondestructive means of achieving high productivity rests on a establishment of the viable low input farming system. However, to implement such a plan, we must develop plant systems that can efficiently scavenge and utilize soil nutrients present at low levels. The judicious use of nature’s own biofertilizers by their biotechnological applications appears to be a suitable answer to this problem. Role of biotechnology in sustainable agriculture can offer a great help towards modern agriculture improvement. In the present review, we have discussed the role of biotechnology, potential biofertilizers with special reference to mycorrhizal biofertilizers, their so far reported synergies, mycorrhizal potential and methodologies for its mass multiplication, different constraints in its commercialization and its future role in achieving sustainable agriculture. Biotechnology has been defined as the integrated use of biological, physical, and engineering sciences to achieve technological application of biological systems. The goal of 195

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include microbiological, biochemical, serological, molecular, and ultrastructural techniques, followed by extensive field trials for crop testing before releasing them for agricultural use. During the last decade the phenomenal increase in the production and promotion of biofertilizers in agriculture has been the result of special attention given by the government and interest by entrepreneurs in setting up biofertilizer production facilities. Farmers have also realized the benefits of biofertilizers. For ensuring the rapid growth of biofertilizer usage, constant research support is critical, as it will provide the latest information on improvements of their production technology, applications under different agro-climatic conditions and help in standardizing handling and storage norms. The role of biofertilizers like arbuscular mycorrhizal (AM) fungi in the growth and multiplication of crop plant can prove to be the most effective alternative to fertilizers for enhancing growth and biomass production. Its application has additional benefits like improved vigor and nutrient uptake, disease resistance, and drought tolerance. The economic surplus is used to assess the impact on the overall economic growth and its contribution to economic efficiency and environmental security.

MYCORRHIZAL ASSOCIATIONS

Mycorrhizal associations include many taxas of fungi belonging to members of Zygomycetes, Ascomycetes and Basidiomycetes and Deutromycetes. A characteristic feature of these fungi is that they are generally widespread in soils, exhibit a strong biotrophic dependence on their host plants, and are rarely free living saprophytes. Different types of mycorrhiza are classified into seven different categories on the basis of the extent of root penetration. Among them arbuscular mycorrhizas are the most common and have gained tremendous importance in present day agriculture. The AM symbiosis is the association between fungi of the order Glomales (Zygomycetes) and the roots of terrestrial plants (Harley and Smith 1983). Conservative estimates predict that this ancient symbiosis, dating back to the early Devonian age (398 million years ago), occurs in approximately 90% of the Earth’s land plants (Remy et al. 1994). The AM symbiosis is increasingly gaining recognition as an important integral component of natural ecosystems throughout the world. The AM fungus –plant association is a mutually beneficial event: the plant supplies the fungus with carbon (from its fixed photosynthates) while the fungal symbiont assists the plant in phosphate uptake and also converts some unavailable mineral nutrients to available forms for the plant. This bidirectional exchange of nutrients takes place through extensively branched haustoria, commonly called arbuscules. In addition to the nutrient uptake, mycorrhizal fungi improve the performance also of other beneficial microbes, help in resisting root pathogens, and increase the tolerance to extremes of environmental and biological stress. With increased nutritional consumption and a higher uptake of desirable nutrients due to mycorrhization, the biomass, both

above and below the ground, increases. Decomposed biomass, when recycled, improves the soil fertility manifold and this is how mycorrhiza helps in restoring ecosystems. Above-ground plant development is influenced by belowground microbial activity. In the presence of mycorrhizal fungi, other micro-organisms such as PSB, many free-living nitrogen-fixing organisms and Rhizobium work more efficiently, improving soil fertility and plant growth. Mycorrhizas remain in the soil and form active links with growing plants and mutually benefit each other. Papers advocating the valuable potential of mycorrhizal inoculations in plant establishments have been published since the 1960s but comprehensive information on their practical exploitation by multiple field trials has not been presented so far (Findlay and Kendle 2001). Immense potential of mycorrhiza has not been so far exploited due to its uncultivable nature unlike other biofertilizers. Mycorrhizas are conventionally propagated using pot-based methods with host trap plants. The disadvantage of this mode is the low recovery of mycorrhizal propagules, contamination by saprobes, pathogens and other mycorrhizal fungi because of improper management techniques and long gaps duration between setup and harvest. Several alternatives to this mode have been designed, but in all current methodologies of cultivating AM fungi, host plant is indispensable. Many variants of these methods have been proposed by various workers to culture glomalean endomycorrhizal fungi, with a bewildering array of claims and counterclaims. These will be described in detail in the present review. All of these involve a plant host, either intact or as root explants. Methodological differences focus mainly on differences in the cultural environment, the most dramatic being the interface between fungus, plant root, and external matrix. The various modes include pot base techniques, hydroponic films (Elmes et al. 1984) or in aeroponic mist chambers (Hung and Sylvia 1988) and the recent in vitro root organ culture (ROC) system (Becard and Fortin 1988).

2.1

Pot-Based Techniques

The traditional and most widely used approach has been to grow fungus with a host plant in a solid growth medium consisting of one or a combination of the solid growth media such as soil, sand, peat, vermiculite, perlite, clay, or various types of composted barks. The mycorrhizal inoculum has not been conveniently mass-produced by traditional sand-based pot culture techniques and different micro-organisms frequently contaminate it. In addition, the volume and weight of the inoculum produced by solid growth cultures was sometimes too large and bulky to carry and utilize (Wang and Tschen 1994). Alternative particle size distributions of substrates vary the inoculum production, the ideal substrate proposed for optimum production is proposed to be low in nutrients and carbon (Gaur and Adholeya 2000).

Commercialization of AM Biofertilizer

2.2

Aeroponic Culture Techniques

It is a soil-less plant culture system in which nutrient solutions are intermittently or continuously misted onto plant roots. This system allows efﬁcient production of AM fungi, free of a physical substrate. The colonized root material can be sheared, resulting in inocula with very high propagule densities. Furthermore, large quantities of spores can be obtained from the culture system. Aeroponic culture has worked well for several species of Glomus. It typically takes 12– 15 weeks to obtain an inoculum. There is a 3-week “inoculation phase,” followed by 9 weeks of aeroponic culture for colonized roots or 12 weeks for spore formation. This also has many disadvantages, because the system is also open to other undesirable microbes, which may harbor and propagate along side. Also, the assembly is huge and requires a lot of space; and regular monitoring of the nutrient solution and its ﬂow is required.

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Isolation and inoculum production of ectomycorrhiza (EM) and AM fungi present very different problems. Many EM fungi can be cultured on artificial media. Therefore, isolates of EM can be obtained by placing surface-disinfected portions of sporocarps or mycorrhizal short roots on growth medium. The resulting fungal biomass can be used directly as inoculum but, for ease of use, inoculum often consists of the fungal material mixed with a carrier or bulking material such as peat. They can now be produced in a fermentor and by entrapping the mycelium in alginate beads (Le Tacon et al. 1985; Mauperin et al. 1987). Obtaining isolates of AM fungi is more difficult because they will not grow apart from their hosts. Spores can be sieved from soil, surface disinfected, and used to initiate “pot cultures” on a susceptible host plant in sterile soil or an artificial plant-growth medium. Inoculum is typically produced in scaled-up pot cultures. Alternatively, hydroponic or aeroponic culture systems are possible; a benefit of these systems is that plants can be grown without a supporting substrate, allowing colonized roots to be sheared into an inoculum of high propagule number.

Root Organ Culture Technique

The ROC system is the most attractive cultivation methodology for research; it uses root-inducing transferDNA-transformed roots of a host plant to develop the symbiosis on a specific medium in vitro (Becard and Fortin 1988). The pathogenic condition known as “hairy root” occurs due to the transfer of root inducing Ri-plasmid from the bacterium Agrobacterium rhizogenes (Schenck 1992). These techniques, though challenging have proved useful additions to our knowledge for various aspects of the AM fungal-host symbiosis (Douds 1997). The problem of producing inoculum in bulk is addressed by the ROC of AM isolates, which provides pure, viable, contamination-free inoculum using less space and thus has an edge over the conventional mode of pot-culture multiplication. Pioneering work on in vitro cultures was initiated in the early 1960s (Chabot et al. 1992). The use of Ri T-DNA transformed roots of Daucus carrota as host by A. rhizogenes has permitted an increase in spore production of Glomus mosseae (Mugnier and Mosse 1987). Only few fungal species have being successfully grown using ROC mode such as Gigaspora margarita, Glomus fasciculatum, and G. macrocarpum, G. intraradices, Gigaspora gigantia, G. versiforme, G. caledonium, G. clarum, G. fistulosum, and G. etunicatum (Chabot et al. 1992; Declerck et al. 1996a,b; Declerck et al. 1998; Diop et al. 1992; Douds and Becard 1993; Gryndler et al. 1998; Karandashov et al. 2000; Pawlowska et al. 1999; Souza and Barbara 1999). Though process of bringing them is very difficult, but the list of the new species under in vitro is increasing every day. Mass production of spores is prerequisite and mathematical models may be useful as descriptive and predictive tools of sporulation dynamics (Declerck et al. 2001). This mode of culturing AM fungi also provides an opportunity for biochemical and molecular investigations of AM symbiosis, which are otherwise unclear.

BENEFITS OF MYCORRHIZAL INOCULATION

The practical application of mycorrhiza in agriculture is relatively new, though its importance has been evident for some 400 million years. The unique advantage of mycorrhizal organisms is that they not only survive in the most stressful environments but also make the plant to do so. The role of mycorrhiza in land reclamation is most recognized these days. Application of mycorrhizal biofertilizer provides a most desirable solution to many such environmental problems. These phosphate-solubilizing biofertilizers are suggested as an alternative or supplements to chemical fertilizers. Some of the beneﬁts offered by mycorrhizal fungi to plants and general soil health improvement are listed below. However, these are not discussed in detail in the present review: (a) Alleviation of nutrient stress. Under deﬁciency conditions, mycorrhizal fungi can increase nutrient uptake. They facilitate the uptake of nutrients such as phosphorus. Difference among arbuscular mycorrhizal fungi (AMF) for plant P acquisition has also been associated with differences in development and function of hyphae like intraradical and extraradical mycelia (Boddington and Dodd 1998; 1999). Many immobile trace elements such as N, S, Ca, Mg, K, Zn, Cu, etc, are also known thus providing better nutrition to the host plant (Clark 1997; Persad-Chinnery and Chinnery 1996). Several reviews are available on the enhanced acquisition of mineral nutrients in plants with mycorrhization (Clark 1997; George et al. 1994; Smith and Read 1997). Turnau et al. (1993) proposed that polyphosphates in the fungal hyphae could sequester metals and minimize transfer to roots of the mycorrhizal plants in stressed conditions, (b) Enhancement of rooting and reduction of transplant shock. Mycorrhizal fungi stimulate root production and dramatically increase the volume of soil

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the plant can explore. This is especially important on disturbed sites, where nutrients and water availability might otherwise inhibit plant growth, (c) Alleviation of drought stress. The increased root volume allows more water to be taken up. Mycorrhizal fungi also enhance the host’s osmotic adjusting capabilities, allowing some plants to continue extracting water from soils as they become drier (Ellis et al. 1985; Morte et al. 2000), (d) Stabilization and aggregation of soil. Mycorrhizal fungi encircle soil particles and glue larger soil particles together into aggregates. This increases the surface absorbing area of roots 10 to 100 times. They release powerful chemicals in the form of exudates into the soil that dissolve the hard to capture tightly bound soil nutrients. This improves soil structure, producing humic compounds and organic “glues” that bind soils into aggregates and improve soil porosity, increasing air and water movement though the soil while reducing erodibility. Reports are also available suggesting the presence of a protein called Glomalin, which seems to be involved in a very important hypha-mediated mechanism of soil aggregate stabilization (Borie et al. 2000; Degens et al. 1996; Rillig et al. 2002), (e) Suppression of disease. Mycorrhizae directly and/or indirectly antagonize disease organisms, increase the number of biocontrol agents around the roots, occupy potential infection sites on the root, and increase host plant vigor to the extent that it can survive disease (Datnoff et al. 1995; St Arnaud et al. 1997; Thomas et al. 1994). They act as biocontrol agent, (f) Enhancement of nutrient transfer between plants. This is especially important when nitrogen fixing and non-nitrogen fixing species are planted together. Mycorrhizal roots exploit the soil profile beyond the depletion zone surrounding the absorbing root and its root hairs. Mycorrhizal modifications of the nutrient uptake properties are dependent on the development of extramatrical hyphae in soil, hyphal absorption of phosphate and other micronutrients, their translocation through hyphae over considerable distances and subsequent transfer from fungus to root cells, (g) Enhancement of beneficial interactions with other microbes. Mycorrhizal fungi increase the nitrogen made available to the plant by both symbiotic and free-living bacteria. They also increase phosphate uptake to the plant by PSB and support biocontrol agents that are antagonistic to pathogenic organisms, (h) Salt stress. The enhancement of mineral acquisition especially that of P, K, Zn, Cu, and Fe due to AMF inoculation is more pronounced under salt-stressed conditions. Studies indicate that AMF-inoculated plants have a greater tolerance to salt stress than un-inoculated plants (Al-Karaki and Hammad 2001; Cantrell and Linderman 2001).

AM BIOFERTILIZERS HAVE AN EDGE OVER OTHER BIOFERTILIZERS

This group of biofertilizers is the only among others having fungal system involved. Other biofertilizers exploit bacteria

most commonly. Also this offers wide applicability with a wide range of plants having little selectivity, which is commonly reported in other biofertilizers. Though some exceptions exist with certain nonmycorrhizal families like chenopodiaceae, brassicaceae, and few nonhost plants of nyctaginaceae etc. The storage conditions also are very simple with no extra infrastructural requirements like low temperature and moisture content. Shelf-life is comparatively long. Bacterial systems have short life and cause cell death easily. The hyphae of fungal system can extend much beyond (a few meters away) the depletion zone and thus can acquire nutrients from a much wider area. The fungal system also produces vegetative structures like chlamydospores and zygospores, which become dormant during periods of environmental stress and germinate with the return of favorable conditions. Thus, they are better equipped for combating unfavorable conditions and have longer shelflives compared to bacterial systems. These biofertilizer organisms are broad-spectrum and nonspeciﬁc. A single species is known to colonize approximately 90% of land plants. These biofertilizers have broad ecological adaptability and are known to occur in deserts as well as arctic, temperate, tropical, and other habitats. They offer a 25–50% reduction in phosphorus fertilizer application depending on the plant.

4.1

Interaction of Natural Biofertilizers

Different biofertilizers have shown nitrogen-fixing, phosphorus-solubilizing, and phytohormone-producing abilities and are used as for increasing agricultural productivity, for e.g., (Brady)rhizobium for legumes (grain, fodder), plant growth promoting bacteria (PGPR) for cereals (wheat, rice, grasses, etc.), Azolla for the rice ecosystem, and actinomycetes (Frankia spp.) for forest trees. The AM biofertilizer is known to increase the nitrogen-fixing potential of the legumes when given together with Rhizobium (Chaturvedi and Kumar 1991) and Bradyrhizobium (Werner et al. 1994; Xie et al. 1995). The mycorrhiza first stimulates the nodule bacteria in a sequential process by increasing the tissue phosphorus content; this results in improved nodulation. There are also reports of positive interaction between Azotobacter/Azospirillum, and AM fungi (Alnahidh and Gomah 1991). The AM colonization favorably affects the population of these free-living N-fixing bacteria and thus stimulates better growth of plants. The AM colonization also has a stimulatory effect with different nonlegume nitrogenfixing plant species. In Casuarina sp., the double inoculation of AM and Frankia improves plant growth and nodulation (Sempavalan et al. 1995). Two groups of bacteria, chemo-organotrophs like some Pseudomonas and Bacillus sp., and chemo-lithotrophs, such as Thiobacillus sp., are able to solublize insoluble phosphates.

Commercialization of AM Biofertilizer

The AM when given in addition to these bacteria, improve the plant performance. The AM favors the early establishment and efficacy of these bacteria. The synergistic effect of these fungi should thus be exploited on a large scale in the form of biofertilizers to increase the nitrogen-fixing potential of legumes and nonlegume plant species as well as with different phosphate solubilizers. Mycorrhizal fungi interact with a wide assortment of organisms in the rhizosphere. The result can be positive, neutral, or negative on the mycorrhizal association or a particular component of the rhizosphere. For example, specific bacteria stimulate EM formation in conifer nurseries and are called mycorrhization helper bacteria. In certain cases these bacteria eliminate the need for soil fumigation (Garbaye 1994).

ENVIRONMENTAL CONCERN ON CHEMICAL FERTILIZER USAGE

Fertilizer production is also an environmental concern. For every ton of phosphoric acid produced, five tons of phosphogypsum are generated. Phosphogypsum is a solid material that results from the reaction of phosphate rock with sulfuric acid. Although it is nearly identical to natural gypsum, it may contain small amounts of sand, phosphate, fluorine, radium, and other elements present in phosphate ore. Federal regulations restrict both use and research involving phosphogypsum because of its radium content and require phosphogypsum to be stacked on the ground. A limited amount of phosphogypsum, with a minimal radium content, is used as an agricultural soil amendment. During the past 50 years, more than 700 MT have accumulated in Florida alone. These enormous stacks, some covering an area of more than 300 hectares and up to 60 m high, have settling ponds on top that contain highly acidic water that can overflow into waterways. New regulations have been enacted to guard against potential spills (Johnson and Traub 1996). Mycorrhiza offers an alternative to many problems in an ecofriendly, sustainable, and economical way besides creating employment and facilitating poverty reduction. In situations where the native mycorrhizal inoculum potential is low or ineffective, providing appropriate fungi for the plant production system is worth considering. With the current state of technology, inoculation is best for transplanted crops and in areas where soil disturbance has reduced the native inoculum potential. The first step in any inoculation program will be to obtain an isolate that is both infective, and able to penetrate and spread in the root, and effective, or able to enhance the growth and stress tolerance of the host. Individual isolates of mycorrhizal fungi vary widely in these properties, so screening trials are important to select isolates that will perform successfully. Screening under actual cropping conditions is best because indigenous mycorrhizal fungi, pathogens, and soil chemical and physical properties will influence the result.

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MAJOR CONSTRAINTS AND SOLUTIONS IN COMMERCIALIZATION OF AM BIOFERTILIZER

Perhaps the most important deterrent to the commercial use of mycorrhizal fungi globally is the lack of large-scale multilocation field trials in a variety of agricultural soils and an absence of general lack of awareness among the users. Without such an activity it will be difficult to establish a market for mycorrhizal inoculum. Without a market there is little incentive for a commercial setup to initiate the production of inoculum on a commercial scale, and only large-scale production will make large-scale field trials possible. Other important issues responsible for the general lack of trust amongst the users in its potential are: (a) The lack of cost-benefit analysis to determine the economics of mycorrhizal applications, and (b) The general trend towards excessive fertilization to substitute for the lack of mycorrhizal fungi. Once large-scale applications of the potential of mycorrhizal inocula are proven to common masses on multi-location fields, lightweight commercial mycorrhizal formulations will need to be developed and new application methods will be devised. Most importantly, from large-scale field tests, cost-benefit analysis will be done accurately to determine the economic benefit derived from the use of mycorrhizal fungi. In the end, this will be the determining factor in the commercial application of mycorrhizal fungi. Biological scientists are rarely able to critically assess the economic factors involved in the application of a new technology. It is important to design the total economic and infrastructure requirement for the setting up a production facility and strict regulatory norms for the quality assessment of the finished product before the release of the product in the market. The involvement of the scientific community is important to define such norms. The field of applied AM research has suffered for many years from the “chicken-and-egg” syndrome. The inoculum was not widely used because it was not readily available, and it was not available because it was not used. The recent boom in commercial AM inoculants will help break out of the cycle. There have been numerous inquiries about the quality of available inoculum. Unfortunately, only little data on which to base recommendations are available. To remedy this situation, the initiation of a quality control assay (QCA) for commercial AM products is essential which will involve conducting a standard mycorrhizal colonization percentage (MCP) assay on commercial AM inoculum received under before making them available in the market. This will need regular supervision and knowledge of the correct mode of production, formulation, and delivery. Biofertilizers represent an affordable industry for many developing countries. In many African countries, the use of inorganic fertilizer has increased soil acidity, reducing the yield per ton of fertilizer. Biofertilizers are cheap to manufacture, suitable for small-scale farmers if produced locally (eliminating distribution costs), and the investment in

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technology is far lower than that of inorganic fertilizers. Biofertilizers have been produced, packaged, and sold commercially in India, while in a number of African and Latin American countries, biofertilizers have been produced at national research centers. Most importantly, the demand for biofertilizers has outstripped production in almost all these countries. It is estimated that about $40,000 –$50,000 is required to build a 100 – 150 MT biofertilizer plant. Alternatively, $500,000 for 10 plants in different locations could produce up to 1000 –1500 MT to meet the demand by rural farmers. With increased production capacity, biofertilizers have a market locally and possibly internationally. Biofertilizers present developing countries with a unique opportunity to enhance their crop yields. Countries like Bangladesh, Brazil, Kenya, Tanzania, Zimbabwe, and Zambia have had successful pilot plants for the production of biofertilizers, and demand has often exceeded production. If any of these countries built a production plant with local and regional markets in mind, they could be exporting their products. India has developed many biofertilizers that are currently on the market for gardeners and farmers. If these products are coupled with crop rotation and irrigation, it is possible to increase crop yields of legumes and cereals. Biopesticides, too, could help increase crop yield, reduce import bills, and increase export earnings. Taken together, they could provide an affordable source of agricultural inputs that would challenge chemical use in rural areas. Chemical fertilizer and pesticide imports and exports from developing countries are low, and production yields are very poor, especially in Africa. Biotechnology will depend on renewable raw materials, and agriculture should play a big role in developing countries’ exports.

6.1

and its long-term viability in the commercial sector are listed here. (b) Inoculum registration. Concerns on ecological, biosafety, and bio-ethics demand the requirement for microbial inoculants to be approved and registered. There is need for a centralized government-regulated agency to provide the guidelines for AM fungi-specific standards of inoculum use. (c) Quality control. Specific protocols for quality control of AM fungal inoculum need to be developed and standardized for application. This is essential not only as a guarantee for producers and users but also for the protection of ecosystems. This would help in quality management and assessment of inoculum potential with every batch of inocula produced. Quality control of commercial AM inoculum is extremely important for developing faith in the user community for its effectively potential. Unless this is achieved, the potential will remain unexplored among the other biofertilizers. (d) Technology transfer. The product concept for AM fungal inoculum is particularly suitable for industries. Scaling up of production and use of AM fungal inoculum is only economically feasible for them if structures to run concerted field experiments is available. This needs to be offered by researchers working in the area through case studies in the areas of horticulture, fruit production, and revegetation of desertified ecosystems. In India, The Energy and Resources Institute, New Delhi has developed AM mass production technology, which was transferred to two leading industries, however, this is a small move for an agricultural country where economy is important and based on the yield production.

Mycorrhizal Commercialization Techniques and Their Formulations

The use of AM fungi in plant biotechnology differs from that of other beneficial soil micro-organisms because the fungi involved are obligate symbionts and therefore recalcitrant to pure culture. Thus specific procedures are required to culture and handle them; specific tools have to be developed and provided to biotechnological producers. (a) Inoculum technology. Plant inoculation with AM fungi results in the formation of a mycorrhizosphere with selective consequences on other important soil micro-organisms. Therefore the use of AM fungi in plant production needs an appropriate inoculum technology compatible with that used for other beneficial soil micro-organisms. Development of second generation inocula, derived from mixing AM fungi with other inocula, is one such major activity. The use of such inocula will improve plant fitness, and soil aggregation and stability, so increasing yield by biological means. Some of the important issues related to AM biofertilizer commercialization

6.2

List of Producers and Formulators of Commercial AM Inoculum

These bio-inoculants are now formulated in different formulations. These are designed on the basis of their application to different crops and locations. They are available in the form of powders, tablets/pellets, gel beads, and balls. Intraradical forms of Glomus sp. (vesicles and mycelium fragments) were entrapped in alginate and used as inocula. Isolated intraradical material was found to regenerate in alginate beads and the regenerated mycelium infected roots under controlled conditions (Declerck et al. 1996a,b; Strullu and Plenchette 1991). Glass beads have also been suggested an inoculum type with spores and mycelia inside (Redecker et al. 1995). The application in nursery plantations is normally done using pellets or tablets placed just below the seeds or small plantlets initiating mycorrhization in the hardening phase. Alternative approaches for inoculum disbursement include broadcasting in the ﬁeld or mycorrhizal products often contain other ingredients designed to increase

Commercialization of AM Biofertilizer

Company AgBio Inc., Westminster Accelerator Horticultural Products AgBio Inc., Westminster Bio-Organics Supply, Camarillo Becker-Underwood, Ames BioScientiﬁc, Inc., Avondale EcoLife Corporation, Moorpark First Fruits Horticultural Alliance, Inc. J.H. Biotech, Inc. Mikro-Tek Inc., Timmins Mycorrhizal Applications. Grants Pass BioGrow TM Plant Health Care, Inc. MycorTM VAM MiniPlug TM Premier Horticulture, Red Hill Premier Tech Reforestation Technologies, Salinas Roots Inc., Independence T & J Enterprises, Spokane TIPCO, Inc., Knoxville Tree of Life Nursery, San Juan Capistrano Tree Pro, West Lafayette Biological Crop Protection Ltd Bio-Organics Biorize Central Glass Co., Chemicals Section Global Horticare, Idemitsu Kosan Co. MicroBio, Ltd N-Viron Sdn. Bhd PlantWorks Ltd., Sittingbourne Triton Umweltschutz GmbH KCP Sugar and Industries Corporation Ltd Cadila Pharmaceuticals Ltd

the effectiveness of the mycorrhizal spores. For example, organic matter is often added to encourage microbial activity, soil structure, and root growth. Stress vitamins improve nutrient uptake and build root biomass. Water absorbing gels help “plaster” beneficial mycorrhizal spores in close proximity to feeder roots and encourage favorable soil moisture conditions for mycorrhizae to form and grow. Organic biostimulants, in general, are effective ingredients in mycorrhizal products. By promoting field competitiveness, stress resistance, and nutrient efficiency, biostimulants reduce barriers to rapid mycorrhizal formation especially during the critical period following root initiation or transplanting. A list of commercially available mycorrhizal inocula is provided in the table above. Recent advances in the in vitro mode of mass multiplication like optimizing various growth parameters

201

Country Colorado, USA Ohio, USA Colorado, USA California, USA Iowa, USA Arizona, USA California, USA Triadelphia, West Verginia Sarasota, Florida, USA Ventura, California, USA Ontario, Canada Oregon, USA North America Pennsylvania, USA North America Pennsylvania, USA Que´bec, Canada California, USA Montana, USA Washington, USA Tennessee, USA California, USA Indiana, USA Kent, UK Medillin, Columbia Dijon, France Tokyo, Japan Lelystad, Netherlands Sodegaura, Chile Royston, Herts, UK Malaysia UK Bitterfeld, Germany Andhra Pradesh, India Ahmedabad, India

like pH, media manipulations (Douds 2002) can further increase the recovery of propagules. Recent report on the success of co-culturing two different genera together with single host under in vitro as it occurs in nature, opens a new scope of an in vitro consortium package as inoculum, which may prove more superior in varied edapho-climatic regions where multiple mycorrhizal isolates may function better than single isolate inoculation for future (Tiwari and Adholeya 2002). Industry-based research documentation’s as such are not available to the end users but a recent brief insight into some of the potential techniques by Moutoglis and Beland (2001) along with other alternative production techniques such as bioreactor-based production techniques proposed by Jolicoeur et al. (1999); Jolicoeur and Pirrier (2001) making use of ROC proposes a bright future for AM biofertilizer.

202

Tiwari et al.

CONCLUSIONS

A lot of research done in the past few decades has enabled these fungi to emerge as a potential biofertilizer, a cheap and environment friendly alternative to expensive, harmful chemical fertilizers. This aspect of an alternative to conventional route to more food grain production in a sustainable manner especially gains significance for a developing countries where judicious and large scale utilization of this technology can prove very useful for getting maximum and long-term gains in various wasteland reclamation, reforestation, and afforestation programs apart from giving a much desirable thrust in the production of important agricultural crops. The AM biofertilizer technology can be called poor man’s technology. Taking into account the amount of nutrient supplied, biofertilizers are many times cheaper than chemical fertilizers. Biofertilizers improve the quality of produce. They are cheap and economical, the cost benefit ratio is more than 1: 10. It is an ecofriendly practice, improves natural characters of the soil. Uses of biofertilizers maximize ecological benefits and minimize environmental hazards. The demand of biofertilizers is increasing at a tremendous pace, which necessitates the inculcation of the more units to be established in the field to rope of the outgrowing demand potential and the challenges of fabulous future scope. Despite many lacunas in its commercialization and delivery to farmers for exploitation of its potential in agriculture, there is little doubt that AM fungi will emerge as a potential tool for improving crop plants as a promising biofertilizer. Future upgradations in the mode of the AM biofertilizer technology development, redefining the ratelimiting factors and exploration of possible AM combinations along with other potential biofertilizers together as a single package for end users might bring a major boon to agriculture sector using nature’s biofertilizers.

ACKNOWLEDGEMENTS The authors wish to thank the Director General, Tata Energy Research Institute, New Delhi, India for infrastructural and the Department of Biotechnology, Ministry of Science and Technology, India for ﬁnancial support for development of TERI’ s Mycorrhizal Mass Production Technology and its transfer to industry for commercialization.

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