MORPHOLOGICAL CHARACTERIZATION, VIRULENCE, AND FUNGICIDE SENSITIVITY EVALUATION OF PHYTOPHTHORA PALMIVORA By GABRIEL ANDRES TORRES-LONDONO A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Pathology - Doctor of Philosophy 2016 ABSTRACT MORPHOLOGICAL CHARACTERIZATION, AND VIRULENCE AND FUNGICIDE SENSITIVITY EVALUATION OF PHYTOPHTHORA PALMIVORA By Gabriel Andres Torres-Londono Phytophthora palmivora is restricting tropical plant pathogen for tropical and subtropical crops. Management of the pathogen has relied mostly on cultural practices and plant breeding; however, absence of these strategies in crops such as oil palm have resulted in catastrophic epidemics. During the present study a total of 150 isolates of P. palmivora obtained from 17 countries and 16 different hosts were studied. Differences in morphology, cultural growth, virulence and in-vitro and in-vivo response to nine fungicides, including the broadly used active ingredient mefenoxam, were elucidated. Differences in sporangia, chlamydospore and oospore measured parameters were observed among isolates; however, they ranged within those described for the species. Differences in sporangia length, sporangia breadth, and chlamydospore diameter seems to be influenced by the host family. This influence was not observed for the oospore characteristics. Virulence of isolates from Citrus spp. were also distinguishable from the rest of families when they were tested on apples; the lesion diameter of this isolates (31.9 mm) was about 10 millimeters shorter than the average of the rest of the families (41.7 mm). No significant differences were observed within the other families. Isolates from the Americas caused larger lesions than those from Asia and Oceania. Only one isolate of P. palmivora (13720) was intermediately sensitive to mefenoxam at 100 ppm. In order to identify possible active ingredients that could be used to manage diseases caused by P. palmivora, efficacy of the fungicides captan, cyazofamid, dimethomorph, fluopicolide, mandipropamid, mefenoxam, oxathiapiprolin, zoxamide, and potassium phosphite were tested on petunia bedding plants. Total control of the disease was observed at 21 days after inoculation with mefenoxam, captan and oxathiapiprolin. Success in disease control was followed by manidporpamid (83%), and fluopicolide and dimethomorph and zoxamide which presented an intermediate level of control (50%). The rest of the fungicides were not different from the inoculated control. With the exception of potassium phosphite, the in-vitro response to the selected fungicides was evaluated using the spiral plating technique. The effective concentration (EC50) for each isolate was elucidated by measuring the distance (mm) between the center of the plate and the point where 50% of lateral growth was observed. The data generated during the study were analyzed using the R-package ECX, developed during the present study. All products were effective in controlling most of the isolates at the selected ranges. Four isolates were not controlled with mefenoxam; however, the maximum dose of mefenoxam evaluated (0.289 mg/L) with the spiral plating technique does not implies resistance. The present study demonstrates the variability of the P. palmivora population, and provides some insight in its control using fungicides. To Maritza for her immeasurable love and support, To Emilia for coming to my life and inspiring my way to happiness To my parents Gabriel and María Teresa for their support To my brothers, sisters nephews and niece for be there for me To my friend Gerardo Martinez for encouraging me to take this journey iv ACKNOWLEDGMENTS I want to express my gratitude to Dr. Mary K. Hausbeck for accepting me into her program and supporting me to conduct the research on Phytophthora palmivora. I also want to thank my graduate committee members Drs. Ray Hammerschmidt, Annemiek Schilder, and Christina DiFonzo for their feedback and contribution to accomplish my doctoral studies. I also want to thank Dr. William Kirk for his feedback and support for the fungicide testing chapter and Dr. Mathieu Ngouajio as former committee member. I want to specially thank Dr. Jianjun Hao for supporting me to combine two different sciences (computer and plant pathology) into a useful tool for fungicide testing, and also for his critical review of this thesis. I also want to express my gratitude to my Colombian friends in the department and lab, to my lab friends and members, especially to Dr. Rachel Naegele, and Sheila Linderman for her critical review of parts of this thesis. Finally, but not least, I want to express my gratitude to my wife Maritza, for her unconditional support during this grateful experience. v TABLE OF CONTENTS LIST OF TABLES ................................................................................................................... VIII LIST OF FIGURES ..................................................................................................................... X CHAPTER I: LITERATURE REVIEW ....................................................................................... 1 General life cycle of plant pathogenic Phytophthora species:............................................ 1 Dispersal of Phytophthora .................................................................................................. 2 Phytophthora palmivora ..................................................................................................... 3 Fungicide control of Oomycetes ......................................................................................... 7 Fungicide resistance ............................................................................................................ 9 Bibliography ..................................................................................................................... 12 CHAPTER II: CHARACTERIZATION OF MORPHOLOGY, MATING TYPE AND VIRULENCE OF PHYTOPHTHORA PALMIVORA ISOLATES ................................... 18 Abstract ............................................................................................................................. 18 Introduction ....................................................................................................................... 19 Materials and methods ...................................................................................................... 21 Phytophthora palmivora isolates. ................................................................... 21 Morphological characterization ...................................................................... 21 Mefenoxam sensitivity .................................................................................... 24 Pathogenicity tests .......................................................................................... 25 Data analysis ................................................................................................... 26 Results ............................................................................................................................. 27 Morphology..................................................................................................... 27 Pathogenicity and virulence ............................................................................ 35 Discussion ......................................................................................................................... 39 Acknowledgments............................................................................................................. 43 Bibliography ..................................................................................................................... 44 CHAPTER III: SENSITIVITY OF PHYTOPHTHORA PALMIVORA TO NINE FUNGICIDES ................................................................................................................... 49 Abstract ............................................................................................................................. 49 Introduction ....................................................................................................................... 50 Materials and methods ...................................................................................................... 52 Isolates ............................................................................................................ 52 In-vitro assays on the sensitivity of P. palmivora to fungicides ..................... 52 In-vivo assays on the sensitivity of P. palmivora to fungicides ..................... 56 Data analysis ................................................................................................... 57 Results ............................................................................................................................. 59 In-vitro assay ................................................................................................... 59 In-vivo assay ................................................................................................... 60 Discussion ......................................................................................................................... 62 vi Acknowledgments............................................................................................................. 64 Bibliography ..................................................................................................................... 65 CHAPTER