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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9427775 Possible biochemical mechanisms of pathogenicity in Phytophthora sojae Rivera-Vargas, Lydia Ivette, Ph.D. The Ohio State University, 1994 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 POSSIBLE BIOCHEMICAL MECHANISMS OF PATHOGENICITY IN Phytophthora aojae DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Lydia I. Rivera Vargas, B.S., M.S. The Ohio State University 1994 Dissertation Committee: Approved by Dr. T .L. Graham Dr. I. Deep J JL Adviser Dr. A.F. Schmitthenner Dr. S. St. Martin Department of Plant Pathology To my niece, Adriana ii ACKNOWLEDGMENTS I would like to express my gratitude to my adviser. Dr. T.L. Graham for his guidance throughout the research. X am also very thankful to the other members of my committee. Dr. I. Deep, Dr. A.F. Schmitthenner and Dr. S. St. Martin for their suggestions and comments. Specials thanks to Dr. M.Y. Graham for her guidance with the electrophoretic work. Also, X would like to express my gratitude to The Ohio State University, specially the Fellowship Office and the Dept, of Plant Pathology for their support throughout my studieB. X extend my thanks to Mr. J. Diaz, technician at the electron microscope lab for his advice during that part of the research. X express my sincere appreciation to my friends for their encouragement and support throughout my studies. To my family, all my love for their continuous faith in me. Columbus, Ohio Lydia I. Rivera-Vargas May, 1994 iii VITA May 12, 1959 ............. Born - May ague z, Puerto Rico EDUCATION 1977 - 1981 .............. B.S., University of Puerto Rico Mayaguez Campus 1981 - 1985 .............. M.S., University of Puerto Rico Mayaguez Campus 1985 -.1989 .............. Research Assistant, Plant Pathology Lab., Department of Crop Protection, University of Puerto Rico, Mayaguez Campus 1989 -.1992 .............. Patricia Robert Harris Fellow, Fellowship Office, The Ohio State University, Columbus, Ohio 1992 - 1994 .............. Teaching Assistant, Department of Plant Pathology, The Ohio State University, Columbus, Ohio PUBLICATIONS 1. Rivera-Vargas, L.I. and Hepperly, P.R. 1987. Assessment of Chinese straw mushroom (Volvariella volvacea) fungal competitors on sugarcane bagasse. In: Cultivating Edible Fungi. Elsevier Publishing Co., The Netherlands, pp. 341 - 349. 2. Rivera-Vargas, L.I. and Hepperly, P.R. 1987. Internal mycoflora of Chinese straw mushroom basidiocarps - "in vitro" effects on mushroom growth. J. Agric. Univ. P.R. 71(2): 159-164. 3. Rivera-Vargas, L.I. and Hepperly, P.R. 1987. Fungicides to control fungal competitors in Chinese straw mushroom. J. Agric. Univ. P.R. 71(2): 165-175. 4. Rivera-Vargas, L.I., Schmitthenner, A.F. and Graham, T.L. 1993. Soybean flavonoid effects on and metabolism by Phytophthora sojae. Phytochemistry. 32(4): 851-857. 5. Rivera-Vargas, L.I. and Graham, T.L. 1993. A Possible Role for Pectin Lyase in Phytophthora aojae Pathogenicity. (Abstr.) Phytopathology iv FIELD OF STUDY Major Field: Plant Pathology Biochemical studies of host-pathogen interactions under the guidance of Dr. Terrence L. Graham. v TABLE OF CONTENTS DEDICATION ........................................... ii ACKNOWLEDGEMENTS ...................................... iii VITA ................................................. iv LIST OF TABLES ....................................... viii LIST OF FIGURES ...................................... xiii LIST OF PLATES ....................................... xvii GENERAL INTRODUCTION ................................... 1 CHAPTER PAGE I. SOYBEAN FLAVONOID EFFECTS ON AND METABOLISM BY Phytophthora sojae .......................... 10 Introduction..... ...................... 10 Materials and Methods .................. 18 Results and Discussion ................ 25 Conclusions ....................... 74 II. Phytophthora sojae CATALASE ................ 76 Introduction..... ...................... 76 Materials and Methods .................. 86 Results . .......................... 91 Discussion ............................ 96 Conclusions ....................... 102 III. Phytophthora sojae PECTOLYTIC ENZYMES ...... 104 Introduction..... ..................... 104 Materials and Methods .............. 114 Results .............................. 129 Discussion ........................... 176 Conclusions .......................... 183 GENERAL CONCLUSION .................................... 184 LIST OF REFERENCES .................................... 185 vi APPENDICES .......................................... 198 A. Data Relative to Chapter I .................. 198 B. Data Relative to Chapter III ............... 231 vii LIST OF TABLES TABLE PAGE 1. Effects of different plant natural compounds on P. sojae growth "in culture .................... 46 2. Microscopic affects of different plant natural compounds on P. sojae .............................. 52 3. Possible mechanisms suggested in the chemical conversion of plant natural compounds by P. sojae .......................................... 73 4. Major functions of catalase ........................ 79 5. Effect of different peroxide concentrations on P. sojae catalase activity ....................... 91 6. Constitutive levels of catalase in different P. sojae isolates .................................. 92 7. Constitutive levels of catalase in P. sojae isolate of race 3 over a period of 9 days of growth ........... 93 8. Induced catalase activity in P. sojae isolate of race 3 using peroxide and plant phenolic compounds ......... 95 9. Detection of different phenolic compounds used to induced catalase activity in 3 different P. sojae fractions using HPLC analysis ............. 95 10. Cotyledon lesion Bize of soybean cultivars W and W79 infected with P. sojae races 3 and 4 ............... 131 11. Histological data from soybean cultivar W7S infected with P. sojae races 3 (Incompatible) and 4 (Compatible) at different time intervals ........................ 132 12. PG specific activity (U/mg of protein) in P. sojae race 4 mycelial portions ............. 151 13. PG specific activity (U/mg of protein) in P. sojae race 4 filtrate portions ........................... 151 14. PNL specific activity in P. sojae race 3 induced using different soybean fractions .................. 159 viii TABLE PAGE 15. PL specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4) .................................... 169 16. PL specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) .................................... 169 17. PG specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4) ................................... 170 18. PG specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ................................... 170 19. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4} ................................... 172 20. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ................................... 172 21. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4) .... 173 22. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ................................... 173 23. P. sojae (race 1) growth on lima bean medium amended with various concentrations of apigenin............... 199 24. P. sojae (race 3) growth on lima bean medium amended with various concentrations of apigenin................ 199 25. P. sojae (race 12) growth on lima bean medium amended with various concentrations of apigenin................ 199 26. P. sojae (race 1) growth on lima bean medium
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