CHARACTERIZATION OF FUSARIUM OXYSPORUM AND PHOMA SCLEROTIOIDES, PATHOGENS OF BIRDSFOOT TREFOIL AND ALFALFA A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Michael Johannes Wunsch May 2010 CHARACTERIZATION OF FUSARIUM OXYSPORUM AND PHOMA SCLEROTIOIDES, PATHOGENS OF BIRDSFOOT TREFOIL AND ALFALFA Michael Johannes Wunsch, Ph. D. Cornell University 2010 Soil-borne pathogens causing root and crown rots and vascular wilts are important causes of stand decline of perennial forage legumes. Fusarium wilt of birdsfoot trefoil (Lotus corniculatus), caused by an aggressive strain of Fusarium oxysporum, is associated with severe vascular discoloration, wilting and chlorosis, and rapid plant death of birdsfoot trefoil. Phoma sclerotioides, causal agent of brown root rot (BRR), causes root and crown lesions as alfalfa (Medicago sativa) and other perennial legumes emerge from winter dormancy, leading to increased plant mortality, reduced spring regrowth, and diminished yields. Fusarium wilt of birdsfoot trefoil has been a serious problem for trefoil production in parts of New York and Vermont since the 1970s. Analysis of F. oxysporum isolates causing this disease indicated that the pathogen has a unique host range relative to previously designated F. oxysporum formae speciales. Vegetative compatibility analysis and phylogenetic analysis of multilocus sequence data suggest that the pathogen is monophyletic. We propose designating the fungus F. oxysporum f. sp. loti. P. sclerotioides has long been known to be an important constraint to alfalfa production in Alaska and central and western Canada. In eastern North America, it has been reported only in Nova Scotia. Surveys of alfalfa production fields in Maine, New Hampshire, New York, and Ontario in 2004, 2005, and 2007 indicate that the pathogen is widespread in eastern North America, often occurring at high incidence levels. Patterns of infected alfalfa within fields suggest that the pathogen was not recently introduced. Surveys of alfalfa production fields in Colorado, New Mexico, and Pennsylvania conducted in 2006 and 2007 suggest that central Pennsylvania and high mountain valleys at the latitude of northern New Mexico may delineate the southern extent of the distribution of P. sclerotioides within alfalfa production regions of eastern and western North America, respectively. Phylogenetic analyses of multilocus sequence data places North American isolates of P. sclerotioides into multiple strongly supported clades, and morphological differences among isolates correspond to genetic differences. On the basis of genetic and morphological differences, we established seven infraspecific varieties within P. sclerotioides: P. sclerotioides var. sclerotioides, champlainii, viridis, obscurus, steubenii, macrospora, and saskatchewanii. BIOGRAPHICAL SKETCH Michael was born in Erie, Pennsylvania, and he grew up in Pennsylvania, Arizona, and Montana. He received a B.S. in biology and a B.A. in economics from the University of Missouri, Columbia in August 2000. Subsequent to his undergraduate studies, Michael served with the U.S. Peace Corps in the small community of El Cantoral, D.C., F.M. in central Honduras, where he facilitated efforts by vegetable crop growers to diversify their production and sell directly to supermarkets. In August 2003, Michael began his graduate work in plant pathology at Cornell University, where his research has focused on soil-borne fungal pathogens. At Cornell, Michael was an active member of the plant pathology graduate student association, helping with graduate student recruitment and other activities, and served as a teaching assistant in university’s introductory biology program for multiple years, instructing the laboratory component of introductory biology courses for both biology majors and non-majors. He is a member of the American Phytopathological Society and has presented his doctoral research at annual meetings of the American Phytopathological Society and the North American Alfalfa Improvement Conference. iii ACKNOWLEDGMENTS I would like to express my gratitude to my major advisor, Gary Bergstrom, for his support and guidance through all aspects of my graduate work. I am deeply grateful to have had the opportunity to work in his research program, and I am particularly grateful for the flexibility and intellectual freedom that I was granted in the development of my dissertation research. I extend my appreciation to members of my committee, Michael Milgroom, Loren Tauer, and Donald Viands, for their suggestions and support. Special thanks to Alexandra Baker, Hannah Baughman, Elizabeth Burrichter, Ryan O’Halloran, and Lelde Tamina for their assistance in the lab. I also extend a very deep thank you to all of my collaborators: Ken Bassendowski and Bruce Gossen (Agriculture and Agri-Food Canada), Merlin Dillon (San Luis Valley Research Center, Colorado State University), Craig Grau (University of Wisconsin), Marvin Hall (The Pennsylvania State University), Julie Hansen (Department of Plant Breeding and Genetics, Cornell University), Richard Kersbergen (University of Maine Extension), Deborah Samac (USDA-ARS), Robert Schindelbeck and Harold van Es (Department of Crop and Soil Sciences, Cornell University), Howard Schwartz (Colorado State University), Albert Tenuta (Ontario Ministry of Agriculture, Food and Rural Affairs), Rey Torres (New Mexico State University Extension), and extension educators from Cornell Cooperative Extension, Colorado State University Extension, Montana State University Extension, University of New Hampshire Extension, New Mexico State University Extension, The Pennsylvania State University Extension, and University of Wisconsin Extension. Finally, I thank my parents and Marie Lepeltier for their love and support; without them, none of this would have been possible. iv TABLE OF CONTENTS Biographical sketch ……………………………………………………………….. iii Acknowledgments………………………………………………………………... iv Table of contents…………………………………………………………………... v List of Figures…………………………………………………………………........ viii List of Tables………………………………………………………………………. ix CHAPTER 1: FUSARIUM WILT OF BIRDSFOOT TREFOIL AND BROWN ROOT ROT OF ALFALFA AND OTHER PERENNIAL LEGUMES: BACKGROUND AND RESEARCH OBJECTIVES……………………………. 1 1.1 Perennial forage legumes…………………………………………….... 1 1.2 Fusarium wilt of birdsfoot trefoil……………………………………… 2 1.3 Brown root rot of perennial forage legumes…………………………... 4 1.4 Disease management…………………………………………………... 5 1.5 Objectives of dissertation research……………………………………. 5 CHAPTER 2: CHARACTERIZATION OF FUSARIUM OXYSPORUM F. SP. LOTI FORMA SPECIALIS NOV., A MONOPHYLETIC PATHOGEN CAUSING VASCULAR WILT OF BIRDSFOOT TREFOIL 2.1 Abstract……………………………………………………………….. 9 2.2 Introduction…………………………………………………………… 10 2.3 Materials and methods………………………………………………… 14 2.4 Results…………………………………………………………………. 26 v 2.5 Discussion……………………………………………………………... 29 CHAPTER 3: DISTRIBUTION, IMPACT, AND SOIL ENVIRONMENT OF PHOMA SCLEROTIOIDES IN NORTHEASTERN U.S. ALFALFA FIELDS…. 39 3.1 Abstract……………………………………………………………….. 39 3.2 Introduction…………………………………………………………… 40 3.3 Materials and methods………………………………………………… 43 3.4 Results…………………………………………………………………. 62 3.5 Discussion…...………………………………………………………… 71 CHAPTER 4: FIRST REPORT OF BROWN ROOT ROT OF ALFALFA, CAUSED BY PHOMA SCLEROTIOIDES, IN COLORADO, MAINE, NEW MEXICO, ONTARIO, AND PENNSYLVANIA……………………………………………. 80 4.1 Abstract……………………………………………………………….. 80 4.2 Introduction…………………………………………………………… 81 4.3 Materials and methods………………………………………………... 83 4.4 Results……………………………………………………………….... 86 4.5 Discussion…………………………………………………………….. 91 CHAPTER 5: GENETIC AND MORPHOLOGICAL EVIDENCE THAT PHOMA SCLEROTIOIDES, CAUSAL AGENT OF BROWN ROOT ROT OF ALFALFA, IS COMPOSED OF A SPECIES COMPLEX……………………………………….. 96 5.1 Abstract……………………………………………………………….. 96 5.2 Introduction……………………………………………………………. 97 vi 5.3 Materials and methods………………………………………………… 100 5.4 Results…………………………………………………………………. 113 5.5 Discussion……………………………………………………………... 128 CHAPTER 6: FUSARIUM WILT OF BIRDSFOOT TREFOIL AND BROWN ROOT ROT OF ALFALFA: DIRECTIONS FOR FUTURE RESEARCH……… 142 6.1 Fusarium wilt of birdsfoot trefoil……………………………………… 143 6.2 Brown root rot of alfalfa………………………………………………. 145 vii LIST OF FIGURES FIGURE 1.1 Foliar symptoms of Fusarium wilt of birdsfoot trefoil……………… 3 FIGURE 1.2 Root necrosis associated with Fusarium wilt of birdsfoot trefoil…... 3 FIGURE 1.3 Characteristic symptoms of brown root rot of alfalfa……………..... 4 FIGURE 2.1 Maximum parsimony analysis of multilocus sequence data of Fusarium oxysporum isolates, including F. oxysporum pathogenic to birdsfoot trefoil……… 30 FIGURE 3.1 Distribution and incidence of brown root rot (BRR) in New York, Vermont and New Hampshire alfalfa production fields sampled in spring 2005….. 62 FIGURE 3.2 In-vivo pycnidium and conidium sizes of single-conidium Phoma sclerotioides isolates from New York, Vermont and New Hampshire……………. 63 FIGURE 3.3 Scatter plots of spring regrowth of individual alfalfa plants relative to brown root rot severity……………………………………………………………... 65 FIGURE 3.4 Contour maps of the spatial patterns of brown root rot incidence, stand density, and soil characteristics in two New York alfalfa production fields………. 67 FIGURE 5.1 Maximum parsimony and maximum likelihood analyses of concatenated sequence data of P. sclerotioides isolates………………………………………….. 115 FIGURE 5.2 Congruence of clades as determined from maximum parsimony