PLANT FUNGAL ENDOSYMBIONTS ALTER HOST-PARASITE RELATIONSHIPS BETWEEN GENERALIST HERBIVORES (LEPIDOPTERA: NOCTUIDAE) AND AN ENTOMOPATHOGENIC NEMATODE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian Albert Kunkel, B.S., M.S. ***** The Ohio State University 2003 Dissertation Committee: Approved by: Parwinder S. Grewal, Advisor A. Raymond Miller _______________________ Dave J. Horn Advisor Martin F. Quigley Department of Entomology Douglas J. Doohan ABSTRACT Perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreb.) contain symbiotic fungi (Neotyphodium spp.) that provide several ecological advantages to host plants: enhanced resistance to drought, disease, and insect herbivory. The resistance to insect herbivory is the result of fungus-produced alkaloids that are toxic to most herbivores. However, black cutworm, Agrotis ipsilon, is a generalist herbivore that is able to feed and develop on endophytic perennial ryegrass. As some insects can use plant secondary compounds to defend themselves against predators, I hypothesized that the cutworms fed on endophytic grasses would exhibit greater defense against a lethal endoparasitic nematode, Steinernema carpocapsae. To test this hypothesis, I developed a method to remove the fungal endophytes from the grass seed through a heat treatment. Laboratory experiments involving 4-5th instars support the hypothesis that A. ipsilon feeding on plants with high (>90%) incidence of endophyte are less susceptible to entomopathogenic nematodes than those feeding on plants with no or low incidence of endophyte. Field studies show decreased susceptibility to S. carpocapsae when larvae were confined to areas of endophytic grass (>75% infected). Early (2-3rd) instars were equally susceptible to nematode attack regardless of host plant. ii Endophytic grass fed to cutworm larvae did not influence nematode attachment behavior, or their ability to penetrate and successfully develop into adults. I examined the effects of ergot alkaloids that are produced by N. lolii such as, ergotamine, ergonovine, ergocryptine, ergocristine, and seed extract on nematode viability and infectivity. Ergonovine malate increased and ergocristine decreased the rates of nematode infectivity, whereas other treatments had no significant effect. I investigated the effects of ergocristine on Xenorhabdus nematophila, the symbiotic bacterium of S. carpocapsae. Bacterial growth and pathogenicity were significantly reduced when bacteria were grown in nutrient broth containing 200 µg/ml concentration of ergocristine. Further research demonstrated that several fungal alkaloids, including ergocristine, persist in or are sequestered by black cutworm and another noctuid, the fall armyworm. I conclude that A. ipsilon developing on endophytic grasses may acquire some level of resistance against entomopathogenic nematodes. This resistance is mediated through the effects of alkaloids; thus our results underscore the ability of N. lolii to affect trophic interactions through the production of alkaloids. iii I dedicate this work to my wife and parents. iv ACKNOWLEDGMENTS I would like to express my sincere gratitude to my major advisor Parwinder Grewal for his guidance, support, and patience, as being my mentor. I am grateful for the advice I received from my dissertation committee members, Drs. Ray Miller, Dave Horn, Martin Quigley, and Doug Doohan. I thank everyone in our laboratory, Kevin Power, Kevin McClure, Corrie Yoder, and Drs. Sukhbir Grewal, Gunpati Jagdale, Doug Richmond, Seppo Salminen, Somu Nethi, and Tim Miklasiewicz, for assistance and discussion about my various research projects. The use of Dr. Dan Herms’s freeze-dryer and Dr. Saskia Hogenhout’s capillary tube puller was appreciated. I would like to thank the department of entomology and Parwinder Grewal for providing financial support throughout my studies here at The Ohio State Unviversity. Additionally, I thank the The Ohio State University – OARDC competitive grants committee for awarding me a competitive grant. I am thankful for the support provided by my parents throughout my graduate education. I would like to thank my wife, Patti Kunkel, for all her assistance and support that she has provided throughout my graduate education. v VITA January 26, 1971………………………………….. Born – Covington, KY December 19, 1994……………………………….. B.S., Biological Sciences Northern Kentucky University Highland Heights, KY June 1995 - June 1998…………………………… Graduate Research Assistant University of Kentucky Lexington, KY August 1998……………………………………….. M.S., Entomology University of Kentucky Lexington, KY July 1998 – present ……………………………….. Graduate Research Associate The Ohio State University Wooster, OH PUBLICATIONS Refereed Journal Publications 1. Kunkel, B.A., D.A. Potter & D.W. Held. 2001. Lethal and sublethal effects of bendiocarb, halofenozide, and imidacloprid on Harpalus pennsylvanicus (Coleoptera: Carabidae) following different modes of exposure in turfgrass. J. Econ. Entomol. 94:60-67. vi 2. Kunkel, B.A., D.A. Potter, & D.W. Held. 1999. Impact of halofenozide, imidacloprid, and bendiocarb on beneficial invertebrates and predatory activity in turfgrass. J. Econ. Entomol. 92:922-930. 3. Kunkel, B.A. & J.M. Hastings. 1996. Intersexual difference in feeding frequency and prey size in the robber fly Promachus albifacies (Diptera:Asilidae): possible influence of male mating behavior. Trans. Kentucky Acad. Sci. 57:1-5. 4. Luken, J.O., T.C. Tholemeier, L.M. Kuddes, & B.A. Kunkel. 1995. Performance, plasticity, and acclimation of the nonindigenous shrub Lonicera maackii (Caprifoliaceae) in contrasting light environments. Can. J. Bot. 73:1953-1961. 5. Luken, J.O., T.C. Tholemeier, B.A. Kunkel, & L.M. Kuddes. 1995. Branch architecture plasticity of Amur honeyscukle (Lonicera maackii (Rupr.) Herder): initial response in extreme light environments. Bull. Tor. Bot. Club 122(3):190-195. FIELDS OF STUDY Major Field: Entomology vii TABLE OF CONTENTS PAGE ABSTRACT……………………………………………………………. ii DEDICATION…………………………………………………………. iv ACKNOWLEDGEMENTS……………………………………………. v VITA……………………………………………………………………. vi LIST OF TABLES……………………………………………………… x LIST OF FIGURES…………………………………………………….. xi CHAPTERS: Chapter 1 Introduction………………………………………………………. 1 Chapter 2 A simple method to remove Neotyphodium endophytes from perennial ryegrass and tall fescue seed 2.1 Abstract……………………………..……………………….……… 12 2.2 Introduction……………………………………………….………… 13 2.3 Materials and Methods…………………………………………..….. 14 2.4 Results………………………………………………………………. 18 2.5 Discussion……………………………………………………….….. 20 2.6 Acknowledgments……………………………………………….….. 23 Chapter 3 Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to an entomopathogenic nematode 3.1 Abstract……………………………………………………………… 37 3.2 Introduction……………………………………………………. …… 38 3.3 Materials and Methods………………………………………… …… 40 3.4 Results…………………………………………………………. …… 47 viii 3.5 Discussion…………………………………………………………… 50 3.6 Acknowledgments…………………………………………………… 54 Chapter 4 Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to an entomopathogenic nematode: the mechanism 4.1 Abstract…………………………………………………………….. 63 4.2 Introduction………………………………………………………… 64 4.3 Materials and Methods…………………………………………….. 66 4.4 Results…………………………………………………………. ….. 73 4.5 Discussion…………………………………………………………. 76 4.6 Acknowledgments…………………………………………………. 80 Chapter 5 Fate of Neotyphodium lolii - produced alkaloids in generalist herbivores, Agrotis ipsilon and Spodoptera frugiperda and possible ecological consequences 5.1 Abstract…………………………………………………………….. 92 5.2 Introduction…………………………………………………….….. 92 5.3 Materials and Methods……………………………………………... 95 5.4 Results……………………………………………………………… 100 5.5 Discussion………………………………………………………….. 101 5.6 Acknowledgments…………………………………………………. 106 Bibliography………………………………………………………………… 111 ix LIST OF TABLES Table Page 5.1 The amount of endophyte produced alkaloids (:g/g dry weight) and perloline methyl ether (:g/g dry weight) found in pupae or hemolymph S. frugiperda larvae fed on endophyte infected (HE) or endophyte free (NE) grass clippings…. 110 x LIST OF FIGURES Figure Page 2.1 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass infected with Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments…………..……………………. 24 2.2 Effect of seed treatments on ‘Palmer III’ perennial ryegrass infected with Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments……………………………….. 25 2.3 Effect of seed treatments on ‘Alamo’ tall fescue infected with Neotyphodium endophyte. Percentage (± SEM) of plants infected with endophyte grown in 1999 from seeds stored in woven polypropylene bags during treatment, • = 4°C seed treatment and ∆ = 37°C seed treatment. Weeks refers to the number of weeks seeds were exposed to treatments..………………………………………… 26 2.4 Effect of seed treatments on ‘Goalkeeper’ perennial ryegrass germination when infected with the Neotyphodium endophyte. Percent seeds (± SEM) germinated in 1999 that were stored