AN ABSTRACT OF THE DISSERTATION OF Christine Andrea Armer for the degree of Doctor of Philosophy in Entomology presented on August 28, 2002. Title: Entornopathogenic Nematodes for Biological Control of the Colorado Potato Beetle, Leptinotarsa decemlineata (Say) Redacted for privacy Abstract approved lalph E. Berry Suj7aRzto The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is the most devastating foliage-feeding pest of potatoes in the United States. Potential biological control agents include the nematodes Heterorhabditis marelatus Liu & Berry and Steinernema riobrave Cabanillas, Poinar & Raulston, which provided nearly 100% CPB control in previous laboratory trials, In the present study, laboratory assays tested survival and infection by the two species under the soil temperatures CPB are exposed to, from 4-37°C. H. marelatus survived from 4-31°C, and S. riobrave from 4-37°C. Both species infected and developed in waxworm hosts from 13-31°C, but H. marelatus rarely infected hosts above 25°C, and S. riobrave rarely infected hosts below 19°C. H. marelatus infected an average of 5.8% of hosts from 13- 31°C, whereas S. riobrave infected 1.4%. Although H. marelatus could not survive at temperatures as high as S. riobrave, H. marelatus infected more hosts so is preferable for use in CPB control. Heterorhabditis marelatus rarely reproduced in CPB. Preliminary laboratory trials suggested the addition of nitrogen to CPB host plants improved nematode reproduction. Field studies testing nitrogen fertilizer effects on nematode reproduction in CPB indicated that increasing nitrogen from 226 kg/ha to 678 kg/ha produced 25% higher foliar levels of the alkaloids solanine and chacomne. However, the increased alkaloids did not affect nematode infection of, nor reproduction in, CPB prepupae. Nematodes applied to field plot soil at 50 infectivejuveniles/cm2reduced adult CPB by 50%, and increased numbers of dead prepupae in soil samples up to five times more than in non-nematode plots. Laboratory studies of H. marelatus and its symbiotic bacteria in CPB hemolymph indicated that immune responses did not limit nematode reproduction. A 58kD CPB hemolymph protein apparently caused the symbiotic bacteria to switch to the secondary form, which does not produce antibiotics and enzymes necessary for nematode growth and reproduction. Despite heat denaturation of the protein, the nematodes did not reproduce unless lipids were added to the hemolymph. Therefore, while H. marelatus may provide high levels of CPB control, nutritional constraints on the nematode and its bacteria inhibit reproduction in CPB and limit long-term multi-generation control. ©Copyright by Christine Andrea Armer August 28, 2002 All Rights Reserved Entomopathogemc Nematodes for Biological Control of the Colorado Potato Beetle, Leptinotarsa decemlineata(Say) by Christine Andrea Armer A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented August 28, 2002 Commencement June 2003 Doctor of Philosophy dissertation of Christine Andrea Armer presented on August 28, 2002. Redacted for privacy or Redacted for privacy Co-Majo Prot'essor, representing Entomology Redacted for privacy of Redacted for privacy Dean of tYi aduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Redacted for privacy Christine Andrea Armer, Author ACKNOWLEDGEMENTS I would like to thank my parents for putting my education foremost, and always encouraging me and assisting me in choosing my way in the world. My husband, David Putzolu, has provided endless love and support through the research process. His patience and willingness to help have been invaluable, and his intelligence, logic, and understanding of human behavior have been an inspiration. No woman is an island, and I could not have completed this work without discussions that sparked new ideas and provided necessary information with students and faculty at OSU, as well as in other entomology departments around the country. Many thanks to all my friends (ayyyyyy!) at OSU and elsewhere who have provided interesting discussions, ideas, sympathy, understanding, and love. Innumerable thanks too to my committee members who provided support, knowledge, and friendship: Russell Ingham, Marcos Kogan, Alison Moldenke, Mary Powelson, and Gary Reed, and especially advisors Ralph Berry and Sujaya Rao. Funding was provided by EPA-STAR Graduate Fellowship #U9 15727, an Applied Insect Ecologists' Foundation Graduate Fellowship, a National Potato Council Auxiliary Graduate Scholarship, an Agriculture Research Foundation Grant from Oregon State University, and a Yerex Graduate Fellowship. As important as the funding to conduct the research was the support of knowing others believed in the value of my research. CONTRIBUTION OF AUTHORS Dr. Gary Reed provided vital guidance for field plot set-up, maintenance, and data collection for the field study presented in Chapter 3. He presented background information on agronomic techniques, grower concerns, and costs involved in agribusiness. He also provided valuable discussions throughout the sampling process and offered ideas on new topics to study. Sarina Jepsen assisted with data collection and helped develop field monitoring techniques for Chapter 3. Conversations with her, based on her questions and ideas, helped clarify the direction and goals of the field study. TABLE OF CONTENTS Page CHAPTER 1: INTRODUCTION..................................................... 1 COLORADO POTATO BEETLE (CPB): ECONOMIC IMPORTANCE 1 CPB BASIC BIOLOGY AND BIOGEOGRAPHY............................. 1 CPB HOST PLANTS................................................................. 3 CPB RESISTANCE TO TOXINS................................................. 6 PLANT BREEDING FOR RESISTANCE TO CPB........................... 7 CPB CHEMICAL CONTROL..................................................... 10 CPB CONTROL BY PREDATORS, PARASITES, AND PATHOGENS 11 CPB CONTROL BY ENTOMOPATHOGENIC NEMATODES............. 12 ENTOMOPATHOGENTC NEMATODE LIFE CYCLE....................... 12 INSECT HOST RESISTANCE TO ENTOMOPATHOGENIC NEMATODES...................................................................... 15 SEQUESTRATION OF HOST-PLANT CHEMICALS........................ 17 RESEARCH OBJECTIVES........................................................ 18 REFERENCES........................................................................ 22 CHAPTER 2: LONGEVITY AND INFECTIVITY OF ENTOMOPATHOGENIC NEMATODES AT TEMPERATURE EXTREMES............................................................................ 33 ABSTRACT........................................................................... 33 INTRODUCTION................................................................... 35 MATERIALS AND METHODS.................................................. 37 TABLE OF CONTENTS (CONT1MJED) Page RESULTS.............................................................................. 44 DISCUSSION........................................................................ 51 ACKNOWLEDGEMENTS........................................................ 57 REFERENCES........................................................................ 58 CHAPTER 3: FIELD EFFICACY OF THE ENTOMOPATHOGENIC NEMATODE HETERORJIABDJTJS MARELA TUS LJU & BERRY FOR COLORADO POTATO BEETLE CONTROL....................................60 ABSTRACT.......................................................................... 60 INTRODUCTION................................................................... 62 MATERIALS AND METHODS.................................................. 65 RESULTS............................................................................. 74 DISCUSSION........................................................................ 81 ACKNOWLEDGEMENTS........................................................ 88 REFERENCES....................................................................... 88 CHAPTER 4: ANTI-PATHOGENIC DEFENSES IN COLORADO POTATO BEETLE }IEMOLYMPH................................................ 92 ABSTRACT...........................................................................92 INTRODUCTION................................................................... 94 MATERIALS AND METHODS.................................................. 97 RESULTS.............................................................................109 TABLE OF CONTENTS (CONTINUED) Page DISCUSSION........................................................................121 ACKNOWLEDGEMENTS.........................................................129 REFERENCES.......................................................................129 CHAPTER 5: CONCLUSIONS.......................................................133 BIBLIOGRAPHY........................................................................ 140 LIST OF FIGURES Figure 2.1. Four-year (1998-200 1, inclusive) average maximum and minimum soil temperatures at a depth of 7.5 cm in unirrigated soil......................... 38 2.2. Average Heterorhabditis marelatus infective period vs. percent of waxworms infected, as mediated by temperature..............................48 2.3. Length of Steinernema riobrave infective period compared to percent of waxworms infected over a broad range of temperatures...................... 49