ABSTRACT GALLUP, COURTNEY ANNE. The Impacts of Black Shank Resistance Management on the Population Biology of Phytophthora nicotianae in Tobacco. (Under the direction of Howard David Shew.) Black shank of tobacco, caused by the oomycete Phytophthora nicotianae, is an important disease of tobacco. Emergence of race 1 has led to loss of major gene resistance and to questions on the genetic diversity in the pathogen in North Carolina. Race 3 is reported for the first time in NC. Race 3 is virulent on plants with the Phl gene and not the Php gene and causes severe disease symptoms on mature plants. Isolates of race 3 were recovered from locations where the Phl gene was deployed and in fields characterized as the wild-type race, race 0, with no history of single-gene resistance. In order to determine whether races 1 and 3 can develop as natural variants from race 0, and to track loss of Php and Phl virulence in races 1 and race 3, soil was infested with one race of P. nicotianae and planted with tobacco varieties with multigenic resistance. Isolates were recovered after five months and screened for race. Additionally, zoospore isolates were derived from progenitor zoospore isolates representing the three races. Zoospore progeny were screened to identify changes in virulence during asexual sporulation. A subset of zoospore progeny was subjected to Fluorescent Amplified Fragment Length Polymorphism analysis to investigate genetic diversity generated through clonal sporulation. Results showed a gain and/or loss of virulence within all race progeny in soil and single-zoospore isolates. Race 1 was the most stable phenotype, with 91% in infested soil and 99.7% of the zoospore progeny retaining the virulence phenotype. The race structure in soil infested with races 0 and 3 were similar after five months. Races were recovered in a 2:1 ratio (race 0: race 3) with a small percentage of race 1. Races 0 and 3 zoospore progeny also segregated. Race 0 progeny were 67% race 0 and 33% gained virulence to the Phl gene (race 3). Similarly, 68% of the race 3-derived progeny retained the parental virulent phenotype, 31% lost the virulent phenotype (race 0), and 1% gained virulence to the Php gene (race 1). Estimates of genetic diversity within each group of related zoospores ranged from 0.17013 to 0.44196. Phenotypic and genotypic investigations revealed that asexual sporulation may be a major source of variation in natural populations. A state-wide survey of P. nicotianae populations was conducted in NC tobacco-producing regions. Isolates were obtained from 76 tobacco fields in 23 counties and screened for race and mating type. Race 1 was predominant in most regions, with 59% of fields consisting of 90 to 100% race 1. The occurrence of race 1 within fields was positively correlated with the history of monogenic resistance deployment. Race 3 was identified in low frequency throughout the state, primarily in wild-type populations where no monogenic resistance was deployed. The A1 and A2 mating types were found throughout NC and were recovered concurrently from multiple fields. Pairings of isolates from within fields yielded viable oospores, indicating for the first time, the potential for sexual reproduction by P. nicotianae. A subset of the survey isolates were screened for sensitivity to the fungicide mefenoxam. All isolates were sensitive, with a mean EC50 value of 0.4 μg/ml mefenoxam, indicating fungicide applications are still a reliable method of black shank management. Results reveal a rapid state-wide shift toward race 1, correlating with the deployment of monogenic resistance and indicate that sexual recombination may be important in generating variation within the pathogen population. The Impacts of Black Shank Resistance Management on the Population Biology of Phytophthora nicotianae in Tobacco by Courtney Anne Gallup A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Plant Pathology Raleigh, North Carolina 2009 APPROVED BY: _________________________________ _________________________________ Dr. H. David Shew Dr. Ignazio Carbone Chair of Advisory Committee _________________________________ _________________________________ Dr. Marc Cubeta Dr. Kelly Ivors _________________________________ Dr. Jennifer Nicholson DEDICATION I dedicate my dissertation to my loving husband, Bill. I couldn’t have made it through graduate school so happily without all of the love and support you have given me through these years in North Carolina. You’re my favorite! ii BIOGRAPHY Courtney Gallup was born into a Marine Corps family on January 5, 1980 in Honolulu, Hawaii. She moved nationally and internationally throughout her childhood. She graduated from North Nash Senior High School in Rocky Mount, NC in 1998. As an undergraduate, Courtney attended North Carolina State University and received B.S. degrees in Horticultural Science and Botany. During these years, she interned at the J.C. Raulston Arboretum, assisted Dr. C. Lee Campbell in the NCSU Plant Pathology Department, and completed teaching assistantships for the laboratory sections of Introductory Botany, and later Introductory Plant Pathology. In her third year, she studied agroecology and ecology in Monteverde, Costa Rica. In her final year, she moved under the guidance of Dr. H. D. Shew as a student research assistant working with soilborne pathogens and assisting in the preparation of PP315 for distance education. In January of 2004, she followed her interests by pursuing graduate degree in the NCSU Plant Pathology Department under the direction of Dr. David Shew. Her research focused on clarifying the race structure and characterizing populations of Phytophthora nicotianae across North Carolina in order to enhance our understanding of pathogen races and how they may impact future management of the disease. The second major focus of her research aimed to describe pathogen variability arising from sexual and asexual recombination. Characterizing the level of genetic variability will help elucidate how this variability influences the development of pathogen races and how human activities drive pathogen evolution. iii ACKNOWLEDGEMENTS I would like to thank the members of my advisory committee, Drs. David Shew, Kelly Ivors, Marc Cubeta, Ignazio Carbone, and Jennifer Nicholson for the unfailing support and guidance. I would especially like to thank Dr. David Shew for his mentorship and encouragement both professionally and personally. I am deeply grateful to all the members of the labs of David and Barbara Shew not only for their contribution to this work, but especially for their friendship and support: Arlene Mendoza-Moran, Joyce Hollowell, Damon Smith, Virginia Curtis, Elizabeth Schoenbaum, Heather Bartone, Anna Kerns, Ed Hassell, Cameron Ward, Sarah Ruark, Kestrel Lannon, Sarah Amend, Mary-Claire Garrison, and others. I would like to specifically recognize Kestrel Lannon for her Herculean feats in the lab. Her contribution to this work is expansive. It was a comfort being able to rely on her help and her humor throughout this process. I also acknowledge my sources of funding: the Tobacco Research Commission, the North Carolina Tobacco Foundation, Inc., and the Tobacco Trust Fund of NC. Most importantly, I thank my husband, Bill, my parents, Jim and Andrea Reuter, my brother, Christian Reuter, my parents-in-law, Fred and Beverly Gallup, and my brothers- and sisters-in-law, Jeff and Andrea Gallup, and Dan and Kendra Gallup, for their love and encouragement. iv TABLE OF CONENTS List of Tables .......................................................................................................................... vii List of Figures............................................................................................................................x CHAPTER 1: RACE 3 OF PHYTOPHTHORA NICOTIANAE, THE CAUSAL AGENT OF BLACK SHANK OF TOBACCO IN NC .............................1 Abstract ..................................................................................................................................2 Introduction ............................................................................................................................3 Materials and Methods...........................................................................................................6 Results ..................................................................................................................................10 Discussion ............................................................................................................................13 Acknowledgements ..............................................................................................................16 Literature Cited ....................................................................................................................16 CHAPTER 2: DEVELOPMENT, STABILITY, AND GENETIC VARIABILITY OF PHYTOPHTHORA NICOTIANAE RACES IN NC TOBACCO FIELDS........................28 Abstract ................................................................................................................................29 Introduction ..........................................................................................................................29 Materials and Methods.........................................................................................................34 Results ..................................................................................................................................43