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DEVELOPING AND TESTING INTEGRATED PEST MANAGEMENT STRATEGIES: FROM COVER CROP TREATMENTS TO THE CHARACTERIZATION AND DETECTION OF PSEUDOPERONOSPORA CUBENSIS AND P. HUMULI 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 Carly Faye Summers August 2015 © 2015 Carly Faye Summers ABSTRACT DEVELOPING AND TESTING INTEGRATED PEST MANAGEMENT STRATEGIES: FROM COVER CROP TREATMENTS TO THE CHARACTERIZATION AND DETECTION OF PSEUDOPERONOSPORA CUBENSIS AND P. HUMULI Carly Summers, Ph.D. Cornell University 2015 The practice of integrated management of plant diseases incorporates a vast body of scientific information for effective pathogen control as well as environmental stewardship. Pathogen biology, environmental and epidemiological patterns, preventative cultural practices and judicious chemical applications are a few examples of the scientific areas of research used to support integrated disease management. This body of work focuses on research involved in the integrated disease management approach: cover cropping, early detection of plant pathogens and a pathogen population study. The first chapter explores the effects of mixed-species cover crops on organic tomato crop productivity and disease severity in the field. This research, taken over three field seasons (2010, 2011 and 2012) at sixteen field sites in three states, Maryland, New York and Ohio (MD, NY and OH), each with distinct soilborne disease pressure, was based on measurement of plant growth and vigor, crop yield and disease ratings of naturally occurring infection over time. Our research revealed no short-term effects of cover crop treatments on productivity or disease severity in tomato crops. The second chapter looked at the same tomato crops, turning attention to how the cover crops affect fungal and oomycete pathogen presence in the rhizosphere of tomato plants. For this, two years (2010 and 2011) of field samples from 260 plots were evaluated using two molecular tools: macroarray analysis, a diagnostic tool used to test for the presence of 31 solanaceous pathogens, and terminal restriction fragment length polymorphism (T-RFLP), used to evaluate the microbial fingerprint of the samples. Our findings showed that cover crops did not affect pathogen presence in the rhizosphere and that macroarray was able to detect certain fungi with much greater sensitivity than T-RFLP. Chapter 3 explores the development of techniques used for early detection of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildews, respectively. Two techniques were designed which allow for detection and differentiation of both pathogens from environmental samples: real-time PCR with locked-nucleic acid probes and high resolution melt curve analysis. These assays were then used to test for pathogen presence in air samples collected in New York using spore traps. Chapter 4 presents the results of a comparative analysis between RNA sequencing (RNA-seq) and genotyping by sequencing (GBS) conducted in order to determine which technique is most effective in single nucleotide polymorphism (SNP) identification for Pseudoperonospora cubensis and P. humuli, as well as to discover novel SNPs that determine population structure between and among these two genetically similar species. Finally, included in the appendix is a case study summarizing a collaboration with St. John’s University in Tanzania, facilitated by a Horticulture Collaborative Research Support Program grant. The project aims were to train a graduate student in extension talks for IPM of tomato, implement the talks with growers and a pesticide alternative in an experiment-based field study. BIOGRAPHICAL SKETCH Carly Faye Summers received a Bachelor of Arts in Biochemistry from New College, a small liberal arts college in Sarasota, Florida. After graduating in 2005, she served in the Peace Corps in Ahuachapán, El Salvador, for two years and three months. As part of the Agroforestry and Environmental Education field unit, she worked with farmers, families and schools in various areas from integrated pest management to recycling. Upon return to Florida, she worked as a research chemist at Archer Pharmaceuticals, a systematics botanist at Selby Gardens then a technician in a plant pathology laboratory at the University of Florida. In 2010 she began her PhD work at Cornell, working with Dr. Christine Smart. iii For my loving family~ Nancy Jo Summers, James Eric Summers and Rebecca Claire Summers. iv ACKNOWLEDGEMENTS A warm thank you is extended to my advisor, Dr. Christine Smart, for inviting me to be part of a program centered upon dedication to growers, students and community. I greatly appreciated the chance to mentor undergraduates each summer, explore my international agriculture interests, and take part in grower field trials as well as the local school garden program. Thank you to committee members Drs. William Fry, George Abawi and Marc Fuchs for your time, feedback and encouragement. Thank you to Holly Lange for her support in the lab and field– and of course the mountain biking! Working with lab members Amara Dunn, Lisa Jones, Matt Tancos, Zach Hansen, Rachel Kreiss and Maryn Carlson was definitely one of the highlights of my graduate work. Thank you to the undergraduate summer scholars who worked with me: Gussie Williams, Alexandra Curtis and Eva Beyen. Thank you to Don Lotter and Sophia Chami in Tanzania for our collaboration through the CRSP USAID grant work. I am also grateful for the graduate student garden in Geneva each summer, facilitated by Steve Reiners and Chris Smart. Finally, I’d like to thank my close friends Angela Beidler, Kaitlin Berger, Belinda Bonnen, Rachael McLean, Ellen Crocker, Tuyen Nguyen and Spencer Horchler. My partner Colwyn Gulliford, thank you for embarking on this journey with me. And to my loving family, Nancy Jo, Eric and Rebecca Claire– you are my sunshine. My research was supported by grants from Trellis HortCRSP USAID, the Einaudi Center International Research, the Toward Sustainability Foundation, the American Phytopathological Society Kenneth F. Baker and R. James Cook Student Travel Award, NSF pre-doctoral fellowship (DGE-1144153) and USDA-NIFA grant number 2009-55605-05184. v TABLE OF CONTENTS Biographical Sketch ......................................................................................................................... iii Dedication ......................................................................................................................................... iv Acknowledgements .......................................................................................................................... v Table of Contents ............................................................................................................................ vi List of Figures ................................................................................................................................... vii List of Tables .................................................................................................................................... viii Introduction ...................................................................................................................................... 1 Chapter 1. Single Season Effects of Mixed-Species Cover Crops on Tomato Health (cultivar Celebrity) in Multi-State Field Trials ............................................................................ 8 Chapter 2. Fungal and Oomycete Pathogen Detection in the Rhizosphere of Organic Tomatoes Grown in Cover Crop-Treated Soils .......................................................................... 51 Chapter 3. Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis ......................................................................................... 75 Chapter 4. Identification of genetic variation between obligate plant pathogens Pseudoperonospora cubensis and P. humuli using RNA sequencing (RNA-seq) and genotyping-by-sequencing (GBS) .................................................................................................. 113 Conclusions ....................................................................................................................................... 152 Appendix I: Tomato integrated pest management in Tanzania ................................................ 158 Appendix II: Neighbor-joining trees of concatenated SNPs .................................................... 163 vi LIST OF FIGURES 1.1 Productivity Graphs .................................................................................................................... 28 1.2 Disease Graphs ............................................................................................................................ 41 3.1 Linear regression of 10-fold dilutions of Pseudoperonospora cubensis and P. humuli DNA against corresponding mean quantification cycle values ....................................................... 96 3.2 Melt and difference curves from a high resolution melt curve assay for Pseudoperonospora cubensis and P. humuli samples .................................................................................................... 99 3.3 Melt and difference curves from a high resolution melt curve assay for Pseudoperonospora cubensis and P. humuli when conducted
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