Quantifying Risk for the Northern Root-Knot Nematode, Meloidogyne Hapla, in Potato in New York State
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QUANTIFYING RISK FOR THE NORTHERN ROOT-KNOT NEMATODE, MELOIDOGYNE HAPLA, IN POTATO IN NEW YORK STATE 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 Adrienne Marie Gorny August 2019 © 2019 Adrienne Marie Gorny QUANTIFYING RISK FOR THE NORTHERN ROOT-KNOT NEMATODE, MELOIDOGYNE HAPLA, IN POTATO IN NEW YORK STATE Adrienne Marie Gorny, Ph. D. Cornell University 2019 The Northern root-knot nematode, Meloidogyne hapla, is an important soilborne pathogen of potato in New York State. Initial pathogen populations (Pi) are often inversely correlated with crop loss and damage, with the economic threshold representing the Pi likely to result in significant crop loss, thus justifying use of control tactics. However, the economic threshold for M. hapla in potato in New York State is not well understood. Furthermore, standard methods of enumerating populations of nematodes from soil are time consuming and error prone. This research sought to quantify the risk of crop loss associated with M. hapla Pi, and develop a DNA-based soil test for prediction of risk. A method for isolating nematode DNA from 100 g of soil using super paramagnetic iron oxide nanoparticles was developed and tested using artificially inoculated soil and field collected samples. A species-specific, real-time quantitative PCR (qPCR) assay targeting the root-knot nematode effector gene 16D10 was developed for quantifying M. hapla. Spatial and spatiotemporal analysis of M. hapla and root-lesion nematodes, Pratylenchus spp., was conducted by intensively sampling three commercial potato fields in New York State. This informed enhanced sampling strategies and evaluated the potential for site-specific nematicide application. The influence of M. hapla Pi on crop loss was assessed by quantifying M. hapla Pi in commercial potato fields through manual extraction and a nested qPCR assay, and investigating associations with yield components. M. hapla Pi did not have a significant effect on total yield, number of tubers, tuber diameter, or tuber galling severity, suggesting the economic threshold for M. hapla in potato is higher than previously regarded (approximately 100 M. hapla second-stage juveniles (J2s) per 100 g soil), and/or tolerant cultivars were used. The response of the commercial potato cvs. Eva and Lamoka to differing Pi of M. hapla and P. penetrans was assessed through replicated field trials. Yield was not impacted even at the highest Pi tested (500 M. hapla J2s or 1,000 P. penetrans nematodes). These results will be used to guide future research to refine economic thresholds, explore advanced nematode management options, and assess potato cultivar susceptibility to plant-parasitic nematodes. BIOGRAPHICAL SKETCH Adrienne Marie Gorny was born in Cleveland, Ohio on June 25, 1992. She grew up in Canton, Michigan, where she attend the fledging All Saints Catholic School, and later attended Ladywood High School in Livonia, Michigan. From her Advanced Placement Biology class at Ladywood, she was drawn to studying plant science, and in 2010, began her undergraduate studies at Purdue University in West Lafayette, Indiana, where she majored in Plant Biology. From her mentors and classes at Purdue, she developed a focused interest in plant pathology, and participated in the Cornell University Summer Scholars program at the New York State Agricultural Experiment Station in 2013, which reaffirmed this interest. After graduating with college honors from Purdue in 2014, she returned to Cornell University to begin as an M.S./Ph.D. student in the laboratory of Dr. Sarah Pethybridge at Cornell AgriTech in Geneva, New York State, within the Section of Plant Pathology and Plant-Microbe Biology. At Cornell, her research centered on the quantitative epidemiology of the Northern root-knot nematode, Meloidogyne hapla, in potato. iii To my parents iv ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. Sarah Pethybridge for her guidance during my Ph.D. graduate program at Cornell University. Her mentoring throughout my time at Cornell has shown me the type of mentor that I strive to become. Our shared enjoyment of inspirational quotes always helped to keep our goals in front of us and perspective to our work. I would like to thank my committee members, Drs. Xiaohong Wang, Stephen Reiners, and Paul Esker for their helpful conversations, sage advice, and kind support. Thank you to Dr. Frank Hay for sharing his extensive knowledge of plant nematology and teaching many of the techniques and methods I found invaluable during my project. His insights were always helpful and discussions always worthwhile. Thank you to the New York State producers, especially Karl Hofmann and Kurt Brehm for kindly offering their potato fields for my sampling work and their enthusiastic collaboration. I have gained so much in talking with them and understanding their production systems. I would like to thank my parents for fostering my love of science and plants. Thank you to my Mom for teaching me to “think about it,” and to my Dad for encouraging me to dream big and “point at any problem and go solve it”. Thank you to Dr. Philip Osborne for his steadfast love, support, and encouragement throughout this degree. Thank you to my many wonderful friends, including Angela Kruse, Morgan Carter, Elizabeth Ceinewicz, and Alyssa Blachez. Thank you to “The Beetniks”: Lori Koenik, Eric Branch, Carol Bowden, Sean Murphy, Alex Silva, Audrey Klein, Dr. Noel Knight, and Dr. Sandeep Sharma for making a great team. Thank you to Larissa Osterbaan, Martha Sudermann, Anna Wallis, and Ali Cala for being fantastic office mates and always keeping the candy basket full. v Finally, this work would not have been possible without funding from the United States Department of Agriculture National Institute of Food and Agriculture (USDA- NIFA) Education and Literacy Initiative Pre-Doctoral Fellowship 2018-67011-28072, the USDA-NIFA Sustainable Agriculture Research & Education graduate student grant GNE15-103, the USDA-NIFA Crop Protection and Pest Management grant 2015- 70006-24160, and Extension and Teaching Assistantships from the Section of Plant Pathology and Plant-Microbe Biology at Cornell University. vi TABLE OF CONTENTS Page Biographical Sketch iii Dedication iv Acknowledgements v List of Figures x List of Tables xii Chapter 1: Introduction 1 References 20 Chapter 2: Isolation of Nematode DNA from 100 g of Soil Using Fe3O4 Super Paramagnetic Iron Oxide Nanoparticles 31 Abstract 31 Introduction 32 Materials and Methods Meloidogyne hapla Inoculum and Inoculation of Soil Samples 36 Comparison of Extraction Methods 36 Binding Capacity of the SPION Capture Method on 100 g of Soil 39 Sensitivity of the SPION Capture Method Using 100 g of Soil 40 Robustness of DNA Extraction with SPION from 100 g of Soil across Soil Type 40 Assessment of DNA Quantity and Quality 41 Statistical Analysis 42 Results Comparison of Extraction Methods 42 Binding Capacity of the SPION Capture Method on 100 g of Soil 43 Sensitivity of the SPION Capture Method Using 100 g of Soil 48 Robustness of DNA Extraction with SPION from 100 g of Soil across Soil Type 51 Discussion 53 References 58 vii Chapter 3: Development of a Species-Specific PCR for Detection and Quantification of Meloidogyne hapla in Soil Using the 16D10 Root- Knot Nematode Effector Gene 62 Abstract 62 Introduction 64 Materials and Methods Preparation of Nematodes and DNA Isolation from Pure Cultures and Soil 69 Development of Species-Specific Primers 70 Primer Specificity 71 Quantitative PCR 74 Generation and Validation of Population Standard Curve 74 Assay Sensitivity and Influence of Inhibitory Compounds 75 Assessment of Field Samples 76 Statistical Analysis 76 Results Development of Species-Specific Primers 77 Primer Specificity 77 Quantitative PCR 78 Generation and Validation of Population Standard Curve 78 Assay Sensitivity and Limit of Detection 80 Assessment of Field Samples 81 Discussion 82 References 87 Chapter 4: Spatial and Spatiotemporal Analysis of Meloidogyne hapla and Pratylenchus spp. Populations in Commercial Potato Fields in New York State 96 Abstract 96 Introduction 97 Materials and Methods Field Study Sites and Data Collection 100 Semivariogram Estimation and Modeling 103 Ordinary Kriging 104 Spatial Analysis by Distance IndicEs 105 Results Semivariogram Estimation and Modeling 106 Ordinary Kriging 109 Spatial Analysis by Distance IndicEs 109 Discussion 113 References 118 viii Chapter 5: Evaluating the Economic Threshold for the Northern Root-Knot Nematode (Meloidogyne hapla) in New York State Potato Production 123 Abstract 123 Introduction 125 Materials and Methods Soil Sampling and Yield Data Collection 128 Manual M. hapla Extraction and Quantification 131 DNA Extraction 131 Detection and Quantification of M. hapla 133 Statistical Analysis 135 Results Detection and Quantification of M. hapla 136 Relationship Between M. hapla Populations and Potato Yield Components 140 Discussion 144 References 149 Chapter 6: Responses of Two Potato Cultivars to the Northern Root-Knot Nematode, Meloidogyne hapla, and Root-Lesion Nematode, Pratylenchus penetrans, in New York State 155 Abstract 155 Introduction 156 Materials and Methods Trial Design 161 Data Collection 163 Statistical Analyses 163 Results Meloidogyne hapla 164 Pratylenchus penetrans 170 Discussion 171 References 174 Chapter 7: Conclusions 178 Appendix A: Establishment and Maintenance of a Hydroponics System for Rearing of Northern Root-Knot Nematode (Meloidogyne hapla) Second-Stage Juveniles 185 Summary 185 Introduction 186 Hydroponics System Establishment 186 Maintenance 189 Preparing Inoculum 190 References 192 ix LIST OF FIGURES Page Fig. 1.1. Life cycle of the root-knot nematode, Meloidogyne hapla on potato. 4 Fig. 1.2. Signs and symptoms of the Northern root-knot nematode (Meloidogyne hapla) on potato. 5 Fig. 2.1. Genomic DNA isolated using each extraction method and PCR amplification products were visualized on an agarose gel stained with GelRed. 44 Fig. 2.2. A SPION-based DNA extraction method was performed on 0.5 g of soil and compared to a standard phenol method and a commercial kit.