GENOMICS AND MANAGEMENT OF FUSARIUM ROOT ROT OF FIELD PEAS A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Kishore Chittem In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department: Plant Pathology December 2011 Fargo, North Dakota North Dakota State University Graduate School Title Genomics and Management of Fusarium Root Rot of Field Peas By Kishore Chittem The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Rubella S. Goswami Co-Chair Mohamed F. R. Khan Co-Chair Timothy L. Friesen Kevin McPhee Approved: 11-9-12 Jack B. Rasmussen Date Department Chair ii ABSTRACT Dry Pea or field pea (Pisum sativum L.) is an important cool season legume crop grown in the United States. Field peas are vulnerable to many diseases of which, soil borne diseases including wilt and root rot are of major economic importance and can cause significant reduction in yield. There is a dearth of satisfactory methods for control of root rot and no varieties with complete resistance to Fusarium root rot are currently available. Root rot disease was found to be prevalent in all the major pea growing counties of North Dakota surveyed in 2004, 2005, 2010 and 2011. Fusarium species were the most frequently isolated fungal species from the infected pea roots of which, F. oxysporum and F. avenaceum were the most common. 21 Field pea varieties were screened for resistance against F. avenaceum and F. solani f. sp. pisi, the Fusarium species traditionally associated with root rots of field pea in growth chamber experiments and field trials. Low levels of resistance were detected in a few cultivars but no variety was found to be completely resistant to any of the pathogens tested. Efficiency of precipitated calcium carbonate (PCC) in controlling Fusarium species most commonly associated with root rots was evaluated under in vitro and field conditions. Significant reduction in spore production, spore germination, and dry mycelial weight of Fusarium spp. were detected on PCC amended media in laboratory studies. In greenhouse and field experiments significant reduction in root rot disease severity was observed with PCC application compared to control. Fungal gene expression in artificially infected field pea roots and F. graminearum grown in culture was assessed using the Illumina mRNA-Seq technology. A total of 613 F. graminearum genes were found to be differentially expressed in planta on pea. Functional classes associated with amino acid metabolism, nitrogen metabolism, extracellular polysaccharide degradation, detoxification by degradation and defense related proteins were found to be significantly enriched in the up-regulated gene set as determined using FunCatDB. Expression of four up- iii regulated genes was confirmed by RT-PCR to validate the inferences from the sequencing results. iv ACKNOWLEDGEMENTS I would like to thank God, the almighty for his blessings and for giving me the strength and wisdom to accomplish this work. I express my deepest gratitude to my advisor, Dr. Rubella S. Goswami, for her persistent help, encouragement and guidance during my PhD program. It gives me great pleasure in acknowledging the support and help of my co-advisor Dr. Mohamed Khan, and my committee members Dr. Kevin McPhee and Dr. Timothy Friesen for their helpful comments, critiques and suggestions to improve my research. I would like to extend my gratitude to Dr. Samuel Markell, Robin Lamppa, Javier Delgado, Steven Chang, Travis Rath, Namratha Hegde, Debra Baer, William Yajima, and Aaron Carlson, for their help in my lab and field research. I would like to thank NCR-SARE and other funding sources for supporting my research. I would like to thank Dr. Jack B. Rasmussen and the entire faculty, staff and graduate students in Plant Pathology for continuous support and encouragements. My deepest gratitude is extended to my parents, my brothers Kiran Chittem, Praveen Chittem and friends for their prayers, love and sacrifices. v TABLE OF CONTENTS ABSTRACT............................................................................................................................... .iii ACKNOWLEDGMENTS.......................................................................................................... ..v LIST OF TABLES..................................................................................................................... .ix LIST OF FIGURES.................................................................................................................... ..x LIST OF APPENDIX TABLES................................................................................................ .xi LITERATURE REVIEW........................................................................................................... ..1 Introduction.................................................................................................................... ..1 Fusarium root rot of field pea: Importance and causal agents....................................... ..2 F. avenaceum: History, distribution and importance..................................................... ..3 Taxonomy....................................................................................................................... ..5 Morphology and colony characteristics......................................................................... ..6 Habit and habitat............................................................................................................ ..7 F. avenaceum toxins....................................................................................................... ..8 Phylogeny of F. avenaceum........................................................................................... .10 Disease screening methods and available sources of resistance.................................... .11 Current methods of root rot management in field peas and precipitated calcium carbonate as a soil amendment......................................................................... .13 Virulence factors identified in Fusarium – pea interaction............................................ .14 Literature cited............................................................................................................... .14 CHAPTER ONE. IDENTIFICATION AND CHARACTERIZATION OF FUSARIUM SPP. ASSOCIATED WITH ROOT ROTS OF FIELD PEA IN NORTH DAKOTA... .18 Abstract.......................................................................................................................... .18 Introduction.................................................................................................................... .19 vi Materials and methods................................................................................................... .20 Results............................................................................................................................ .23 Discussion...................................................................................................................... .25 Acknowledgements........................................................................................................ .28 Literature cited............................................................................................................... .32 CHAPTER TWO. IDENTIFICATION OF SOURCES OF RESISTANCE TO FUSARIUM AVENACEUM IN COMMERCIAL FIELD PEA VARIETIES.................................... .35 Abstract.......................................................................................................................... .35 Introduction.................................................................................................................... .35 Materials and methods.................................................................................................... .36 Results............................................................................................................................ .38 Discussion...................................................................................................................... .43 Literature cited............................................................................................................... .44 CHAPTER THREE. DETERMINING EFFICACY OF PRECIPITATED CALCIUM CARBONATE IN MANAGING FUSARIUM ROOT ROT OF FIELD PEAS........... .46 Abstract.......................................................................................................................... .46 Introduction.................................................................................................................... .46 Materials and methods................................................................................................... .48 Results............................................................................................................................ .53 Discussion...................................................................................................................... .59 Literature cited............................................................................................................... .63 CHAPTER FOUR. ASSESSMENT OF FUNGAL GENE EXPRESSION PATTERN ASSOCIATED WITH ROOT INFECTION OF FIELD PEA BY F. GRAMINEARUM......................................................................................................