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M 06 E TY LIBRA L ll l“ll! lWIlTIWHNl.HHIHQIW flOQOd-llQ This is to certify that the dissertation entitled BIOECOLOGY OF Paraphlepsius irroratus (Say) (HOMOPTERA: CICADELLIDAE) : THE EFFECT OF THE X-DISEASE MYCOPLASMALIKE ORGANISM ON PHYSIOLOGICAL DEVELOPMENT presented by Carlos Garcia Salazar has been accepted towards fulfillment of the requirements for Ph.D. -degreein Entomology WM£7 Major professor DatejgaLrn—L /7Z/ MS U is an Affirmative Action/Equal Opportunity limitation 0-12771 LlE‘sRAR‘! Michigan Stale I University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or betore date due. r DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution chS-o.l —-—-—' BIOECOLOGY 0F Paraphlepsius irroratus (Say) (HOHOPTERA: CICADELLIDAE): THE EFFECT OF THE X-DISEASE HYOOPLASHALIKE ORGANISM OH PHYSIOLOGICAL DEVELOPMENT BY Carlos Garcia Salazar A DISSERTATION Sublitted to Michigan State university in partial fulfill-ant of the requirements for the degree of DOCTOR OF PHILOSOPHY Departnent of Entomology 1991 [1 ABSTRACT BIOECOLOGY OF Paraphlepsius irroratus (Say) (HOHOPTERA: CICADELLIDAE): THE EFFECT OF THE X-DISEASE HYCOPLASHALIKE ORGANISM OH PHYSIOLOGICAL DEVELOPMENT BY Carlos Garcia Salazar The vector-pathogen relationship between Paraphlepsius irroratus (Say) and the Eastern strain of the X-disease mycoplasmalike organism was studied, and the effect of temperature on physiological development of both, leafhopper vector and x-disease determined. Assessment of X-disease MLO acquisition and multiplication in MLO injected P. irroratus was done utilizing a DNA probe that showed specific hybridization with the Eastern strain of X-disease. Growth and development of P. irroratus, as well as the effect of the x-disease MLO on leafhopper longevity and survival was studied at constant and fluctuating temperatures regimens. Research showed that.P. irroratus required 602 i 31 and 518 i 66 DD (degree days base 8 °C) to complete the nymphal stage at constant and fluctuating temperature, respectively. At constant temperature, the mean generation time required 939 i 31 DD. The lower developmental threshold for P. irroratus was observed at 8 °C, and the upper threshold temperature was around 28 °C. Concerning the vector-pathogen association, there was a temperature-dependent pathogenicity of the X-disease MLO to its vector P. irroratus. Below 20 °C, the MLO accounted for 32% reduction in leafhopper longevity, compared with less than 6% above 25 °C. The X-disease MLO also affected leafhopper fecundity and a 48.8% fecundity reduction with respect to healthy females was observed in X-diseased P. irroratus. Compared with the X-disease MLO, the effect of the injection solution on leafhopper longevity and survival was negligible, and no statistical difference (P S 0.05) was observed between buffer or heat inactivated MLO injected leafhoppers and healthy non injected individuals. There was a temperature effect on MLO growth and multiplication in X-disease injected P. irroratus. The optimum multiplication of the Eastern strain of x-disease MLO in its leafhopper vector occurred in the 30-35 °C range. No multiplication was observed at 15 °C, and only a small titer was observed at 20 °C. Beyond 35 °C, the MLO titer declined. I dedicate this work to my parents Jose Cruz Garcia and Micaela Salazar de Garcia. Their love and moral support gave me the strength to succeed. To my wife, Maria Luisa Kiwen whose love, understanding and support in the most difficult moments of my career helped me not to yield to failure. To my daughters, Rocio and Isis who have always been the joy of my life. iv ACKNOWLEDGMENT I like to thank Dr. Mark E. Whalon, my Graduate Advisor and mentor, for the friendship and kindness always demonstrated to myself and my family during all of my years at Michigan State University. His advice and the trust he deposited in me gave direction to my dreams and made it possible for me to succeed where others met failure. I want to thank the other members of my guidance committee Drs. George S. Ayers, Stuart H. Gage, David R. Smitlly and Amy F. Iezzoni for their inputs and insightful advice throughout the planning, conduction and interpretation of my research. I am grateful to my good friend Dr. Thomas M. Mowry for his advice and encouragement in the early part of my research. His moral values and standards were always an example for me and the people who had the fortune to interact with him. Dr. L. N. Chiykowski deserves a special gratitude, the advice and biological material be supplied were critical for the success of my research. Likewise, special thanks to Dr. B. C. Kirkpatrick for his DNA probe from which the x-disease probe utilized in my research was derived. I also, want to thank my fellow graduate student Miss. Utami Rahardja and Mrs. Yang Tang Yong, our technician, for their invaluable contribution to the success of my research. Finally, I gratefully acknowledge the Mexican Government, who through the National Council of Sciences and Technology (CONACyT) and the National Institute of Forestry, Agricultural and Livestock Researches (INIFAP) financed my studies at Michigan State University's Department of Entomology. vi TABLE OF CONTENTS LIST OF TABLE ............................................ xii LIST OF FIGURES .......................................... xiv GENERAL INTRODUCTION..... ...... ............ .... ........... l The x-disease Importance............. ....... ...............1 X-disease Leafhopper Vectors ............................... 3 Biology of Paraphlepsius irroratus (Say) ................... 3 vector-Pathogen RelationShip O I O O O O O O O O O I O O O O O O O O O O O O O O O O O O O 5 Vector-Pathogen-Temperature Relationship...................7 ObjectiveSOOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0000000000000000011 Bibliography ............ ................................. 13 CHAPTER I. Temperature Related Development for the Speckled Leafhopper (Paraphlepsius irroratus (Say)) (Homoptera: Cicadellidae)............21 IntrOductionOOOOOOOOOOOO0....OOOOOOOOOOOOOOOOOOOO000......22 Materials and Methods.....................................24 1 O Leafhopper cu1ture O 0 ...... O O O O O 0 O O O O O O O O O O O O O O O O 0 O O O O O O 2 4 2. Treatments-0.0.0.000... ...... 0. ...... 00.... ......... 0.024 3. Egg Incubation Period...................... ....... .....25 4. Development Under Fluctuating Temperature..............26 vii 5. Statistical Analysis...................................28 6. Degree Days Estimate........................... ...... ..29 7. Test of Significance...................................30 Results...................................................31 1. Leafhopper Development at Constant Temperature.........31 2. Development at Fluctuating Temperature.................33 3. Development at Constant vs. Fluctuating Temperature....33 4. Lower Developmental Threshold..........................33 5. Upper Developmental Threshold..........................35 6. Physiological Time (DD) and Development................35 Discussion................................................38 Bibliography..................... ..... ....................43 CHAPTER II Temperature Dependent Pathogenicity of the X- disease Mycoplasmalike Organism (MLO) to its vector, Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae)...................46 Introduction..............................................47 Materials and Methods.....................................49 1. Leafhopper Culture.....................................49 2. Temperature and Injection Treatments...................50 3. Injection Protocol.....................................52 4. MLO Acquisition Determination..........................53 5. Dot Blot Hybridization.................................53 Results...................................................56 1. MLO Effect on Leafhopper