Fusarium Mycotoxins

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Preface The year 1984 saw the publication of the landmark Toxigenic Fusarium Species: Identity and Mycotoxicology by Walter F. O. Marasas, Paul E. Nelson, and T. A Toussoun. Their book brought order to a chaotic field and provided an important framework for the research of a generation of plant pathologists and mycotoxicologists. During the past two decades, great advances have been made in our knowledge of the genus Fusarium and its mycotoxicology; the time is ripe for a synthesis of this new information. Fusarium mycotoxicology stands at the interface of the scientific disciplines of chemistry, genetics, and biology, and aspects of all of these fields have been incorporated into this book. The book is divided into three parts. “Introduction to Fusarium Mycotoxicology� pro-

Fusariologists T. A. Toussoun, L. Burgess, P. E. Nelson, and W. F. O. Marasas in 1993. (Used with permission from the Fusarium Research Center, The Pennsylvania State University, and from Kathy Bechdel, photographer) iii

vides a brief, general introduction, including subjects as diverse as the discovery of mycotoxins, their toxicity and evolution, and mycotoxin risk assessment. Part One, entitled “Fusarium Mycotoxins”, comprises Chapters 1 through 5, which survey a range of Fusarium metabolites, with an emphasis on those whose toxicity and natural occurrence in foods and feeds worldwide are most relevant to mycotoxin risk assessment. Part One also includes the results of recent molecular biological studies of mycotoxin biosynthesis. Part Two, entitled “Mycotoxigenic Fusarium Species”, comprises Chapter 6, a survey of the genus Fusarium with an emphasis on those species whose plant pathogenicity and mycotoxin profiles are most relevant to mycotoxin risk assessment. The survey comprises 42 individual reports, each of which is focused on one mycotoxigenic species or group of similar species as defined by the most recent morphological, biological, and phylogenetic species concepts. I can only hope that this work will update the text of Marasas, Nelson, and Toussoun and inspire a new generation of scientists to explore the “endless forms …most wonderful” of Fusarium mycotoxins.

iv

Acknowledgments I gratefully acknowledge the contributions of many people to my 25 years of Fusarium research. First, I thank Hans VanEtten, then at Cornell University, and the late Paul Nelson at The Pennsylvania State University for introducing me to Fusarium. I am indebted to numerous individuals for their collaboration in the past and present and for their unpublished data, helpful discussions, and comments on drafts of this manuscript; any final errors are my responsibility. In particular, I thank Nancy Alexander, Guihua Bai, Marian Beremand, Gary Bergstrom, Deepak Bhatnagar, Robert Bowden, George Buechley, Robert Butchko, Lifeng Chen, Thomas Cleveland, Jon Duvick, Hal Gardner, David Geiser, Thomas Gordon, Linda Harris, Thomas Hohn, Andrew Jarosz, Jean Juba, Chagema Kedera, Gretchen Kuldau, John Leslie, Antonio Logrieco, Gyanu Manandhar, Hira Manandhar, Chris Maragos, Wally Marasas, Susan McCarthy, Susan McCormick, Antonio Moretti, Guisy Mulè, Gary Munkvold, Paul Nicholson, Ronald Plattner, Steven Poling, Robert Proctor, John Richards, Gary Samuels, Eric Schmidt, Keith Seifert, Gregory Shaner, Gayland Spencer, Jim Sweigard, Frances Trail, Gillian Turgeon, Klaus Weltring, Brenda Wingfield, Mike Wingfield, Jinrong Xu, Olen Yoder, and Kurt Zeller. I thank staff at the U.S. Department of Agriculture, National Center for Agricultural Utilization Research for their support, especially research assistant Stephanie Folmar, secretary Gail Bursott, and Steve Prather, who did the illustrations.

Lastly, I thank Randy Ploetz for suggesting this book project.

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Table of Contents Introduction to Fusarium Mycotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001 PART ONE. Fusarium Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 011 CHAPTER 1. Trichothecenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 013 Historical Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 013 Case Study 1: Alimentary Toxic Aleukia in Russia and Central Asia y Case Study 2: Akakabi-byo in Japan y Case Study 3: Swine Feed Refusal in the Central United States y Case Study 4: The Yellow Rain Controversy Trichothecene Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 019 Overview of Trichothecene-Producing Fusarium Species y Identification and Analysis of Trichothecenes y Natural Occurrence of Trichothecenes Trichothecene Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 033 Trichothecene Biosynthetic Pathway y Trichothecene Biosynthetic Enzymes y Trichothecene Biosynthetic Genes Trichothecene Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 053 Mechanism of Action of Trichothecenes y Biological Activity of Trichothecenes in Animal Systems y Biological Activity of Trichothecenes in Plant Systems y Applications of Trichothecenes to Plant Breeding CHAPTER 2. Zearalenones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 065 Historical Case Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 065 Case Study: Swine Estrogenic Syndrome in the Central United States Zearalenone Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 066 Overview of Zearalenone-Producing Fusarium Species y Identification and Analysis of Zearalenones y Natural Occurrence of Zearalenones Zearalenone Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 075 Zearalenone Biosynthesis vii

Zearalenone Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 076 Mechanism of Action of Zearalenones y Biological Activity of Zearalenones in Animal Systems y Biological Activity of Zearalenones in Plant Systems and Applications to Plant Breeding CHAPTER 3. Fumonisins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 079 Historical Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 079 Case Study 1: Equine Leukoencephalomalacia in the United States y Case Study 2: Swine Pulmonary Edema in the Central United States y Case Study 3: Esophageal Cancer in South Africa y Case Study 4: Neural Tube Defects Along the Texas–Mexico Border Fumonisin Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 084 Overview of Fumonisin-Producing Fusarium Species y Identification and Analysis of Fumonisins y Natural Occurrence of Fumonisins Fumonisin Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 095 Fumonisin Biosynthetic Pathway y Fumonisin Biosynthetic Genes Fumonisin Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Mechanism of Action of Fumonisins y Biological Activity of Fumonisins in Animal Systems y Biological Activity of Fumonisins in Plant Systems y Applications of Fumonisins to Plant Breeding CHAPTER 4. Other Selected Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Beauvericin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Chemistry y Genetics y Biology Enniatins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Chemistry y Genetics y Biology Fusaproliferin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Chemistry y Biology Fusaric Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Chemistry y Biology Fusarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Chemistry y Biology Moniliformin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Chemistry y Genetics y Biology CHAPTER 5. Other Selected Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Acuminatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Butenolide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chlamydosporol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Culmorin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cyclodepsipeptide HA23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cyclonerodiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Equisetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarochromanone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Gibberellins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Naphthoquinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sambutoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Wortmannin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

131 132 132 132 133 134 134 135 135 136 136 137

PART TWO. Mycotoxigenic Fusarium Species . . . . . . . . . . . . . . . . . . . . . . . . 139 CHAPTER 6. Selected Mycotoxigenic Fusarium Species . . . . . . . . . . . . . . . 145 Fusarium acuminatum and Fusarium armeniacum . . . . . . . . . . . . . . . . . .

Fusarium acutatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium andiyazi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium anthophilum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium avenaceum, Fusarium aywerte, and Fusarium nurragi . . . . . .

Fusarium begoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium beomiforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium camptoceras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium chlamydosporum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium circinatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium compactum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium concentricum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium crookwellense (syn. Fusarium cerealis) . . . . . . . . . . . . . . . . . . .

Fusarium culmorum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium denticulatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium dlamini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium equiseti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium fujikuroi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium globosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium graminearum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium guttiforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium konzum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium kyushuense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium lateritium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium moniliforme (see Fusarium verticillioides) . . . . . . . . . . . . . . . . .

Fusarium napiforme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium nygamai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium oxysporum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium phyllophilum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium poae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium proliferatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium pseudograminearum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium sacchari . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium sambucinum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium semitectum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium solani . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium sporotrichioides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium subglutinans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium thapsinum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium torulosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium tricinctum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium venenatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fusarium verticillioides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145 147 147 148 149 151 152 152 153 154 155 156 156 158 159 160 161 163 165 166 168 168 169 170 171 171 172 173 174 175 177 179 180 181 182 184 185 186 189 189 190 191 192

Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

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Introduction to Fusarium Mycotoxicology Whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved. —Charles Darwin (1859) In the famous last paragraph of On the Origin of Species, Charles Darwin asked the reader to contemplate the elaborately constructed forms of an entangled bank, clothed with plants of many kinds with their attendant birds, insects, and worms crawling through the damp earth. In this book, I ask the reader to examine the invisible world of microorganisms that live in intimate relationships with these plants and animals and to peer even more closely into the molecular world of metabolites that these microorganisms, especially the fungi, produce. Plant-associated fungi comprise a wide variety of groups, but few are as harmful to plant and animal health as the genus Fusarium, a large and ancient group that has evolved extraordinary chemical diversity. Fusarium metabolites are often polycyclic and highly complex and seem to have been designed by nature to be biologically active; some are mycotoxins, harmful to humans and animals.

Because many mycotoxigenic Fusarium species are aggressive pathogens of agricultural plants, they can cause mycotoxin contamination of cereal grains and other plant-based foods, thereby impairing human and animal health. The ultimate goal of Fusarium mycotoxicology is to provide a secure and safe food supply by identifying mycotoxins and mycotoxigenic species, by assessing risks of mycotoxins to human and animal health, and by reducing mycotoxin contamination of human foods and animal feeds.

DISCOVERING FUSARIUM MYCOTOXINS Fusarium mycotoxicology began in 1809 with the identification of the genus Fusarium by Johann H. F. Link, director of the Botanic Garden in Berlin (Table 1). Link characterized the genus by the fusiform or 1

2

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INTRODUCTION Table 1. Landmarks in Fusarium Mycotoxicology (1809–2004)

Date 1809 1822 1834 1853 1877 1882 1886 1901

Event

Link describes the genus Fusarium.

Fries describes the sexual stage Gibberella genus (Fries 1822).

Schweinitz describes Gibberella zeae on maize in the United States.

Tulasne writes the first description of a mycotoxicosis: ergotism caused by Claviceps species.

Saccardo describes Oospora verticillioides from maize associated with pellagra in Italy.

Palchevski reports toxic bread in Siberia.

De Bary proposes that toxins play a role in plant disease.

Buckley and MacCallum associate acute encephalitis of horses with moldy feed in Maryland, United States (Buckley and MacCallum 1901).

1904 Peters and Sheldon correlate consumption of maize contaminated with Fusarium moniliforme with animal toxicoses in Nebraska, United States (Peters 1904; Sheldon 1904).

1918 The first mycotoxin, ergotamine from Claviceps species, is structurally characterized.

1923 Dounin reports Fusarium head blight and toxic bread in western Russia (Dounin 1926).

1928 Mains and colleagues report swine feed refusal of barley in Indiana, United States (Mains et al. 1930).

1934 Yabuta and colleagues report the structure of fusaric acid from Fusarium species.

1935 Wollenweber and Reinking publish the first great atlas, Die Fusarien, in Berlin, describing 142 species of the genus Fusarium (Wollenweber and Reinking 1935).

1936 Christensen and Kernkamp report toxicity of blighted barley to swine in Minnesota, United States (Christensen and Kernkamp 1936).

1940 Snyder and Hansen reduce all Fusarium strains to nine species.

1940s Outbreaks of alimentary toxic aleukia in humans in Russia follow consumption of overwintered grains contaminated with Fusarium species.

1947 Gäumann isolates a phytotoxic enniatin mixture from Fusarium species. Structures are reported 20 years later.

1957 Gäumann reports that fusaric acid is a plant wilt toxin (Gäumann 1957).

1960 Consumption of feed contaminated with aflatoxin is correlated with outbreaks of Turkey X disease in England.

1960s Consumption of feed contaminated with Fusarium graminearum is correlated with outbreaks of estrogenic syndrome in swine in the central United States.

1960s– Structures of the Fusarium trichothecenes, including diacetoxyscirpenol, deoxynivalenol, 1970s T-2 toxin, and nivalenol, are reported in Europe, Japan, and the United States.

1966 Urry and colleagues report the structure of zearalenone from Fusarium species (Urry et al. 1966).

1969 Hamill and colleagues report the structure of beauvericin from Beauveria species. In 1991, this mycotoxin is reported from Fusarium species (Hamill et al. 1969).

1970s Consumption of grain contaminated with Fusarium graminearum is correlated with outbreaks of akakabi-byo in humans in Japan.

1974 Springer and colleagues report the structure of moniliformin from Fusarium species (Springer et al. 1974).

1977 McLaughlin and colleagues and Ueno report that trichothecenes inhibit protein synthesis (McLaughlin et al. 1977; Ueno 1977).

1977 Hidy and colleagues report estrogenic activity of zearalenone (Hidy et al. 1977).

1981 Consumption of maize contaminated with Fusarium verticillioides is correlated with a high rate of human esophageal cancer in South Africa.

1981 Officials of the U.S. government allege the use of trichothecenes as biological warfare agents in Southeast Asia.

1984 Gelderblom and colleagues report the mutagenic activity and structure of fusarin C from Fusarium species (Gelderblom et al. 1984a).

1984 Marasas, Nelson, and Toussoun publish the landmark text Toxigenic Fusarium Species: Identity and Mycotoxicology (Marasas et al. 1984). (continued on next page)

FUSARIUM MYCOTOXICOLOGY

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Table 1. (continued from previous page) Date 1985– 1991 1988

1988

1989

1989

1990s

1990

1991

1991 1993

1993 1996 1995 1996 1996

1997 1998

1999 2000 2000 2002 2003

2004

Event According to officials with the United Nations Special Commission, Iraq engages in largescale production of trichothecenes and other mycotoxins for potential weapons development.

Bezuidenhout and colleagues report the structure of fumonisins from Fusarium species (Bezuidenhout et al. 1988). They are also reported independently as macrofusine by Laurent and colleagues in 1989 (Laurent et al. 1989a,b).

Marasas and Kellerman and colleagues cause leukoencephalomalacia in horses with pure fumonisin by intravenous injection and, in 1990, by oral dosing (Marasas et al. 1988a; Kellerman et al. 1990).

Hohn and colleagues clone trichodiene synthase, the first trichothecene biosynthetic gene and, in 1992, discover the trichothecene gene cluster (Hohn and Beremand 1989b; Hohn et al. 1995).

Desjardins and colleagues report that trichothecenes are required for Fusarium sporotrichioides and (in 1992) Fusarium sambucinum to cause parsnip root rot but not potato tuber dry rot (Desjardins et al. 1989, 1992).

Governments of countries in Europe, North America, South America, and elsewhere publish regulations for deoxynivalenol, fumonisins, and zearalenones in human foods and animals feeds.

Harrison and colleagues show that pulmonary edema in swine can be caused by intravenous injection of fumonisin (Harrison et al. 1990). In 1998, Gumprecht and colleagues cause the disease by oral dosage (Gumprecht et al. 1998).

Gelderblom and colleagues show that liver cancer in experimental rats can be caused by ingestion of dietary fumonisin (Gelderblom et al. 1991). In 2001, the U.S. Food and Drug Administration confirms that dietary fumonisin causes liver and kidney cancer in rodents.

Wang and colleagues discover that fumonisins inhibit sphingolipid biosynthesis (Wang et al. 1991).

The International Agency for Research on Cancer rates the toxins produced by Fusarium moniliforme (now Fusarium verticillioides) as group 2B carcinogens (possibly carcinogenic to humans) (IARC 1993).

Haese and colleagues discover the enniatin synthetase gene (Haese et al. 1993).

Xu and Leslie publish a genetic map of Fusarium verticillioides (Xu and Leslie 1996).

Ritieni and colleagues report the structure of fusaproliferin from Fusarium species (Ritieni et al. 1995).

Herrmann and colleagues report that production of enniatins enhances the ability of Fusarium avenaceum to cause potato tuber dry rot (Herrmann et al. 1996a,b).

Desjardins, Proctor, and colleagues report that production of trichothecenes enhances the ability of Fusarium graminearum to cause wheat head blight in field tests (Desjardins et al. 1996b).

Stevens and Tang discover that fumonisin inhibits the folate receptor, a possible mechanism for neural tube defects (Stevens and Tang 1997).

Kimura and colleagues report the Fusarium gene TRI101, which confers resistance to trichothecenes (Kimura et al. 1998a). This is also reported independently by McCormick and colleagues in 1999 (McCormick et al. 1999).

Proctor and colleagues discover the fumonisin biosynthetic gene cluster (Proctor et al. 1999).

Beremand and colleagues publish a gene expression library of Fusarium sporotrichioides.

Brandwagt and colleagues discover the tomato gene ASC-1, which confers resistance to fumonisins (Brandwagt et al. 2000).

Jurgenson and colleagues publish a genetic map of Fusarium graminearum (Jurgenson et al. 2002a).

The complete genome sequence of Fusarium graminearum is published by the Broad Institute at the Massachusetts Institute of Technology, with support from the United States Department of Agriculture and the National Science Foundation.

Schmidt, Trail, and Song and colleagues discover biosynthetic genes for equisetin, fusarins, and zearalenones in Fusarium species (Song et al. 2004, Sims et al. 2005).

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spindlelike shape of its macroconidia, which remains a characteristic morphological trait of the asexual stage (anamorph) of this fungus. In 1821, the Swedish botanist Elias M. Fries described the genus Gibberella, which is the sexual stage (teleomorph) of all major mycotoxigenic Fusarium species. During the first 100 years of Fusarium history, the important mycotoxigenic species F. graminearum and F. verticillioides were described; by 1930, both of these species were associated with moldy grain toxicoses in farm animals. Numerous studies have now proven that ingestion of feed or culture material contaminated with F. graminearum and related species causes hemorrhagic syndrome, estrogenic syndrome, and feed refusal in farm animals, especially in swine.

Ingestion of materials contaminated with F. verticillioides and related species causes leukoencephalomalacia in horses, pulmonary edema in swine, and cancers of the liver and kidney in rodents.

The golden age of discovery of Fusarium mycotoxicology began in 1961 with the determination of the structure of the trichothecene diacetoxyscirpenol and ended in 1991 with proof of the carcinogenicity of fumonisins in experimental rodents (Table 1). During this 30-year period of intense effort by an international community of scientists, chemists discovered the three major classes of Fusarium mycotoxins, the fumonisins, trichothecenes, and zearalenones, as well as minor mycotoxins, such as beauvericin, fusaproliferin, fusarins, and moniliformin.

Toxicologists proved the roles of fumonisins in equine leukoencephalomalacia, swine pulmonary edema, and carcinogenesis in rodents; of trichothecenes in hemorrhagic syndrome and feed refusal in several animal species; and of zearalenones in swine estrogenic syndrome. Furthermore, during this period, trichothecenes and fumonisins were associated epidemiologically with human disease syndromes in Europe, Africa, and Asia. By 1991, biochemists had identified mechanisms of toxicity of all three major classes of mycotoxins: inhibition of sphingolipid biosynthesis by fumonisins, inhibition of ribosomal protein synthesis by trichothecenes, and binding of zearalenones to the estrogen receptor.

As the twentieth century ended, Fusarium mycotoxicology entered the age of genomics (Table 1). Our research group at the U.S. Department of Agriculture discovered the trichothecene biosynthetic gene cluster in F. sporotrichioides in 1993 and the fumonisin biosynthetic gene cluster in F. verticillioides in 1999. During the 1990s, research groups in Germany cloned Fusarium genes for enniatin synthetase and bikaverin polyketide synthase and discovered a gibberellin biosynthetic gene cluster in F. fujikuroi. During this decade, gene expression libraries of various mycotoxigenic Fusarium species also became available.

The field of Fusarium genomics was accelerated in 2003 when the U.S. Department of Agriculture and National Science Foundation jointly supported the sequencing and public release of the complete genome of F. graminearum. Access to this first-published Fusarium genome revealed

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the presence of dozens of candidate genes for polyketide synthetases, nonribosomal peptide synthetases, sesquiterpene cyclases, and other types of enzymes that synthesize mycotoxins and other biologically active metabolites. Comparison of DNA sequences per se cannot supply details of mycotoxin biosynthetic pathways; this information must be obtained by appropriate experimentation. Fortunately, Fusarium species are highly amenable to the techniques of biochemistry, classical genetics, and molecular genetics necessary to validate the function of candidate genes. With the first complete Fusarium genome and several Fusarium gene expression libraries on hand, researchers worldwide are working at a rapid pace to identify biosynthetic and regulatory genes for individual mycotoxins and other biologically active metabolites.

Recent genome comparisons among filamentous members of the class Ascomycetes (Euascomycetes) indicate that F. graminearum and other plant pathogens contain far more genes for polyketides, peptides, terpenes, and other metabolites than do nonpathogens such as Neurospora crassa. The phylogenetic distribution and the toxicity of many of these metabolites to plants raise the possibility that these metabolites play a role in plant pathogenesis. However, gene disruption analyses have already shown that a universal role in the virulence of Fusarium species should not be assumed for any metabolite. For example, studies with gene disruption mutants have shown that trichothecenes enhance the ability of F. graminearum to cause wheat head blight but have no detectable effect on the ability of F. sambucinum to cause potato tuber dry rot. Likewise, fumonisins have no detectable effect on the ability of F. verticillioides to cause maize ear rot, although the structurally similar Alternaria alternata f. sp. lycopersici (AAL) toxin is a major virulence factor of the euascomycete Alternaria alternata on tomato. As biosynthetic genes for additional metabolites are identified, it should be possible to use gene disruption mutants to test their roles in the plant pathology of Fusarium species. But it always should be kept in mind that the role of any metabolite in plant pathogenesis may vary dramatically with each metabolite, Fusarium species, and plant host.

Phylogenetic comparisons among Fusarium species have only just begun, but they already indicate that patterns of distribution and diversity of mycotoxins within the genus are complex and that associations with plant host and pathogenicity are not obvious. Although surveys have been fragmentary, some metabolites, such as beauvericin, fusarins, and moniliformin, appear to be distributed more or less throughout the genus. In contrast, trichothecene and fumonisin gene clusters have not been found to co-occur in any well-documented Fusarium species, although both of these classes of mycotoxins have been found in other plant-pathogenic euascomycetes. The fumonisin gene cluster is present but the trichothecene gene cluster is absent from the monophyletic G. fujikuroi clade and its sister clade F. oxysporum. Conversely, the

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INTRODUCTION

trichothecene gene cluster is present but the fumonisin gene cluster is absent from the F. graminearum (sexual stage G. zeae) and F. sambucinum (sexual stage G. pulicaris) clades. All of these Gibberellalinked clades are monophyletic, thus the Gibberella common ancestor may have contained both fumonisin and trichothecene genes. Current discontinuous patterns of distribution could have arisen by loss of the fumonisin gene cluster in the lineage leading to the F. graminearum and F. sambucinum clades and by loss of the trichothecene gene cluster in the lineage leading to the G. fujikuroi clade. Gene loss hypotheses are supported by the discontinuous distribution of the fumonisin gene cluster within the G. fujikuroi clade itself. On the other hand, horizontal gene transfer from other euascomycetes cannot be ruled out, especially because many of the fumonisin and trichothecene biosynthetic genes are organized in tight clusters. However, there is no evidence, in the vicinity of the fumonisin and trichothecene gene clusters, of the transposable elements or repetitive DNA that have been associated with horizontal gene transfer in other microorganisms.

Historically, mycotoxins have been called secondary metabolites because their biosynthesis is not required for the primary functions of growth and reproduction. Some early authors even proposed that fungal secondary metabolites were not heritable, adaptive Darwinian traits at all, but rather were simply waste products whose structures were determined solely by chemical constraints. This now-discredited theory has obvious parallels with the views on morphology of D’Arcy Thompson in his classic On Growth and Form published first in 1917 (Thompson 1948). Thompson argued that the forms of the living world result directly from physical forces analogous to the forces of gravity and surface tension that shape a drop of water. Like many biologists of the Victorian era, Thompson was unable to accept the primary role of heredity and adaptation in determining form and function. As we approach the year 2009, the bicentennial of Charles Darwin’s birth, we recognize that the “elaborately constructed forms” of living organisms and of their metabolites result from Darwinian inheritance and selection. For example, the mycotoxin enniatin, a complex cyclic depsipeptide, is not a means for disposing of waste isoleucine but is an inherited trait that enhances the virulence of F. avenaceum on potato tubers. It is necessary to be cautious, however, and not to err in the opposite direction by assuming that the current utility of a trait is evidence of the historical origin of that trait. Even in 1859, Darwin recognized that some traits were likely to have undergone a functional shift from one use to another during evolution, a concept termed “exaptation” by the modern evolutionary biologist Stephen Jay Gould (Gould 2002). In the genus Fusarium, for example, trichothecene mycotoxins now enhance the virulence of F. graminearum on wheat but may have evolved for quite a different purpose in the ancestral Fusarium species. Since trichothecenes are toxic to

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a wide range of eukaryotes, including fungi, they may have originally functioned to enhance the competitiveness of Fusarium species with other fungi in soil environments.

MEASURING AND MANAGING RISK With the domestication of cereal grains, the rise of agriculture, and the development of food storage systems several thousand years ago, human populations in Eurasia and Mesoamerica increased their food security and their survival. Wheat and rice became staple grains of agricultural communities of Eurasia, while maize became a staple grain of the Americas. After Columbus reached the Americas in 1492, sixteenth-century ships transported wheat and other Eurasian grains to the Americas and maize to Europe. During the 500 years since Columbus, wheat has become a new food crop in the Americas, while maize has spread throughout the world as a human food and as feed for livestock. The domestication and spread of grain crops increased food security but, at the same time, created opportunities for disease outbreaks in humans and animals because of consumption of moldy grain that was contaminated with mycotoxins. Historically, mycotoxicoses were often thought to be caused by poisonous air, water, or soil, or even by witchcraft. By the end of the twentieth century, however, careful documentation of numerous disease outbreaks proved that they were caused by the consumption of mycotoxins in moldy grain.

Unlike insecticides and other man-made chemicals, mycotoxins are natural toxins and thus cannot be completely eliminated from the food supply. Therefore, the goal of mycotoxicology is to determine sciencebased levels of acceptable risk and to reduce mycotoxins to acceptable levels. Risk assessment of each mycotoxin begins with the determination of its mechanism and level of toxicity in a variety of human and animal cell and tissue systems. Because of ethical issues surrounding studies of toxicity in human subjects, mycotoxins are usually tested on animal subjects and the results are extrapolated to humans. Doseresponse studies in animals are used to determine the mycotoxin level at which no adverse effect is observed. Because of uncertainties in extrapolating data from animals to humans, the no-adverse-effect level is divided by a safety factor to determine an acceptable daily intake.

Using this approach, the Joint Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) has determined no-adverse-effect levels for a number of mycotoxins. For fumonisins, for example, JECFA used data from dose-response studies of kidney toxicity in the rat to determine a no-adverse-effect level of 200 Âľg per kilogram of body weight per day for humans (JECFA 2001). With a safety factor of

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INTRODUCTION

100, the acceptable daily intake of fumonisins would become 2 µg per kilogram of body weight per day.

Risk assessment also requires a determination of the total dietary exposure to the mycotoxin by the target population. Dietary exposure to a mycotoxin can be estimated by surveys of the mycotoxin contamination of a commodity and of the average consumption of a commodity. Cereal grains are not all equally susceptible to mycotoxin contamination; for example, fumonisin contamination is frequent in maize but rare in rice and wheat. In developed countries, populations are likely to consume a more varied diet with a lower average consumption of cereal grains; thus, their total dietary exposure to mycotoxins from grains tends to be lower. For example, for a maize sample contaminated with fumonisins at the relatively low level of 1.0 µg/g dry weight, consumption of less than 50 g per day by a 70-kg person in a typical varied diet would yield an acceptable daily fumonisin intake of less than 0.7 µg per kilogram of body weight per day. In developed countries, improved standards of crop management and storage, along with public education, extensive surveillance, and stringent regulations, also function to limit the exposure of humans and animals to mycotoxins.

In many less-developed countries, tropical climates and poor storage conditions allow fungal growth and mycotoxin contamination in cereal grains and other commodities. Because of limited resources, mycotoxin surveillance programs in less-developed countries often are lacking or focused on export commodities. Furthermore, cereal grains can constitute the overwhelming majority of the diet, particularly among rural populations. Thus, a 70-kg person who each day consumes 500 g of maize with 1.0 µg of fumonisin per gram would consume 7 µg of fumonisins per kilogram of body weight, which is higher than the acceptable level of 2 µg per kilogram of body weight per day. Surveys in Africa, Asia, and South America indicate that the level of fumonisins or trichothecenes often exceeds 5 µg/g in grain being used for human consumption, thus creating an even greater health risk. Estimates of high dietary intakes of fumonisins and trichothecenes by some populations in less-developed countries have been confirmed by the analysis of blood and urine for mycotoxin metabolites and other biological markers that indicate mycotoxin exposure.

Decades of research on the toxicity of Fusarium metabolites and their natural occurrence in human foods and animal feeds worldwide have identified fumonisins, trichothecenes, and zearalenones as key targets for risk management. However, most strategies that decrease the levels of these major classes of mycotoxins are likely to reduce levels of other mycotoxins as well. Mycotoxin risk management begins in the field with improved farming practices and improved crop plants with increased resistance to insect damage, fungal infection, and mycotoxin production. Risk management continues with improved harvesting prac-

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tices and storage conditions. Mycotoxin testing by grain inspection services, food and feed industries, and other agencies ensures that the commodity meets regulatory requirements. Depending on mycotoxin levels, the commodity may be diverted from human food uses to animal feed or to nonfood uses, such as ethanol production, often at a significant monetary loss to the producer. With both science and common sense as guides, the benefits of a food supply with reduced mycotoxins must be weighed against the monetary costs of mycotoxin management to farmers, agricultural industries, governments, and consumers, especially in less-developed countries.

For mycotoxicologists and plant pathologists in the twenty-first century, the challenges of Fusarium mycotoxicology are diverse. We need a better understanding of how climate change, conservation tillage, low genetic diversity of crop plants, pesticide and herbicide use, and other factors impact the interactions of mycotoxigenic Fusarium species with crop plants in modern, large-scale, production agriculture. But we also need to increase the food security and food safety of maize and other grains in the complex, indigenous agricultural systems of Africa, Asia, and the Americas that feed many of the world’s poorest human populations. We need more information on populations of mycotoxigenic Fusarium species worldwide to better understand how these species have coevolved with crop plants, particularly as new crops have spread throughout the world during the 500 years since Columbus. Understanding the “elaborately constructed forms” of Fusarium species and their mycotoxins is critical to ensuring a secure and safe food supply.

But the extraordinarily adaptive genus Fusarium also offers unique opportunities to study the coevolution of fungi and plants in space and time—“how the innumerable species inhabiting this world have been modified, so as to acquire that perfection of structure and coadaptation which most justly excites our admiration” (Darwin 1859).

PART ONE

Fusarium Mycotoxins Part One of this work surveys a range of Fusarium metabolites, with an emphasis on those whose toxicity and natural occurrence are most relevant to mycotoxicology. The survey begins with individual chapters on three major classes of mycotoxins that have been proven to cause animal disease outbreaks: Chapter 1 on trichothecenes, Chapter 2 on zearalenones, and Chapter 3 on fumonisins. Trichothecenes and fumonisins also have been implicated in human disease outbreaks. Each of these three chapters begins with a presentation of historical case studies of suspected mycotoxicoses in humans and animals. These stories range from the trichothecene yellow rain controversy and the zearalenone estrogenic syndrome to the possible role of fumonisins in neural tube defects. Each chapter continues with a survey of individual mycotoxin production among species of the genus Fusarium, a brief overview of analytical methods for mycotoxin detection, and a summary of the natural occurrence of the mycotoxin worldwide. The chapters on trichothecenes and fumonisins also contain detailed updates on toxin biosynthesis and genetics. Each chapter finishes with a discussion of toxin biological activity, with a focus on the information relevant to food and feed safety risk assessment and to practical approaches to plant disease control.

The review of Fusarium mycotoxins continues in Chapter 4 with similar but shorter presentations of six other mycotoxins of emerging interest: beauvericin, enniatins, fusaproliferin, fusaric acids, fusarins, and moniliformin. This group of minor mycotoxins includes some metabolites that are carcinogenic or toxic to experimental animals and some that have been implicated in animal mycotoxicoses. In general, only limited data are available on the natural occurrence and risk assessment of these six mycotoxins and on their biosynthesis. The review concludes in Chapter 5 with brief updates on 12 additional Fusarium metabolites that were selected for their novel chemistry or biological activity. Comprehensive surveys of all biologically active metabolites of 11

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the genus Fusarium are beyond the scope of this book but can be found in a number of sources (Betina 1989; Cole et al. 2003; De Nijs et al.

1996; Stoessl 1981; Thrane 1989, 2001; Turner and Aldridge 1983; Vesonder and Golinski 1989). Assigning many of these minor metabolites to specific Fusarium species is often difficult because of incomplete or incorrect identification of the fungal strains from which they were isolated.

For comprehension and cross-comparisons between studies, mycotoxin concentrations have been standardized as µg/g dry weight for solid samples and as µM for liquids. For mycotoxin survey data, the number of samples tested and the detection limit are given, if available. Genetic nomenclature also has been standardized. Genes are indicated by three italicized uppercase letters followed by a number. Proteins are indicated by the same combination of uppercase letters and number, but are not italicized. For further clarity, the nomenclature for genes and proteins usually specifies the “TRI3 gene” and the “TRI3 protein”.