BIOCHEMICAL MODE OF RESISTANCE TO MULTIPLE INSECT PESTS IN A ROMAINE LETTUCE CULTIVAR By AMIT SETHI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1 © 2007 Amit Sethi 2 To my beloved father, Amar L. Sethi who has been my role-model for hard work, persistence and personal sacrifices, and who instilled in me the inspiration to set high goals and the confidence to achieve them, and his words of encouragement and push for tenacity ring in my ears. 3 ACKNOWLEDGMENTS It gives me immense pleasure to record my thanks and sense of profound gratitude to my major advisor, Dr. Heather J. McAuslane for her kind inspiration, constant supervision, constructive criticism and encouragement throughout the period of my Ph.D, especially time spent in informal discussions and training that have all been a valuable part of my learning experience. Expressing sense of gratitude and admiration for the kind help extended by Dr. Hans T. Alborn (CMAVE, USDA) and Dr. Bala Rathinasabapathi (Departemnt of Horticultural Sciences) is not mere obedience of convention, but a real appreciation. I am also highly obliged to Dr. Gregg S. Nuessly and Dr. Russell T. Nagata (Everglades Research and Education Center), the members of my committee for their guidance and valuable suggestions for the improvement of this dissertation project. I owe my sincere thanks to Jennifer Hogsette, Jennifer Meyer, and Debbie Boyd for their timely help in the insect colony maintenance when I was away for the conferences. I am thankful to Dr. Peter Teal (CMAVE, USDA) for providing greenhouse space for growing lettuce plants and also Julia Meredith (CMAVE, USDA) for taking care of plants when I was away for scientific conferences. I am also thankful to Dr. Marty Marshall (Departement of Food Science and Food Nutrition) for use of his spectrophotometer. Words fail me to convey the depth of my feelings and gratitude to my lab mates Jennifer Meyer, Karla Addesso, Jennifer Hogsette and Murugesan Rangasamy for their encouragement, generosity and memorable association. I seize the opportunity to express my moral obligations to my brothers and their families, and in-laws for their encouragement and moral support. My father deserves my heartiest thanks 4 for his magnanimity, inspiration and encouragement at times of despair that helped me in innumerable ways in making this effort a success. No appropriate words could be traced in the presently available lexicon to acknowledge the sacrifices, selfless devotion, love and unflinching support extended by my beloved wife Dr. Ramandeep Kaur to complete this study. Putting it last, but feeling it first, I owe God who has given me courage, patience and motivation from time to time in completing my degree successfully. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................10 ABSTRACT...................................................................................................................................14 CHAPTER 1 REVIEW OF LITERATURE.................................................................................................16 Introduction.............................................................................................................................16 Origin and History of Lettuce.................................................................................................17 Types of Lettuce .....................................................................................................................18 Insect Pests and Lettuce..........................................................................................................18 Host Plant Resistance .............................................................................................................21 Biochemical Basis of Host Plant Resistance ..........................................................................24 Host Plant Resistance Due To Proteins...........................................................................24 Protease inhibitors....................................................................................................25 Cysteine protease......................................................................................................25 Oxidative enzymes ...................................................................................................26 Proteins of the cell wall............................................................................................28 Secondary metabolism pathways .............................................................................29 Enzymes involved in secondary metabolism ...........................................................29 Host Plant Resistance Due To Secondary Plant Compounds..........................................30 Phenolics ..................................................................................................................31 Flavonoids ................................................................................................................33 Terpenoids................................................................................................................35 Host Plant Resistance in Lettuce to Insect Pests ....................................................................37 Aphids..............................................................................................................................37 Cabbage Looper...............................................................................................................39 Banded Cucumber Beetle................................................................................................40 Leafminer ........................................................................................................................41 Helicoverpa species.........................................................................................................42 Spodoptera species ..........................................................................................................42 Bemisia species or strains................................................................................................43 Thrips...............................................................................................................................43 Research Goals .......................................................................................................................44 6 2 HOST PLANT RESISTANCE IN ROMAINE LETTUCE AFFECTS LARVAL FEEDING BEHAVIOR AND BIOLOGY OF TRICHOPLUSIA NI AND SPODOPTERA EXIGUA (LEPIDOPTERA: NOCTUIDAE) ..............................................46 Introduction.............................................................................................................................46 Materials and Methods ...........................................................................................................48 Plants ...............................................................................................................................48 Insects..............................................................................................................................48 Neonate Survival and Development to Third Instar........................................................49 Survival and Development from Neonate to Adult Emergence......................................50 Fecundity and Longevity of Subsequent Generation ......................................................51 Results.....................................................................................................................................51 Neonate Survival and Development to Third Instar........................................................51 Larval Feeding Behavior .................................................................................................52 Survival and Development from Neonate to Adult Emergence......................................53 Fecundity and Longevity of Subsequent Generation ......................................................54 Discussion...............................................................................................................................54 3 ROMAINE LETTUCE LATEX DETERS FEEDING OF BANDED CUCUMBER BEETLE (COLEOPTERA: CHRYSOMELIDAE) ...............................................................68 Introduction.............................................................................................................................68 Materials and Methods ...........................................................................................................71 Plants and Insects ............................................................................................................71 Artificial Diet Preparation ...............................................................................................73 Latex Collection and Solvent Extraction.........................................................................74 Bioassay Conditions ........................................................................................................75