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INNOVATIVE TOOLS FOR STUDYING PLANT-RELATED OOMYCETE POPULATIONS OF POTENTIAL AND CURRENT THREAT IN AGRICULTURE IN ECUADOR By MARIA FERNANDA RATTI TORRES 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 2018 © 2018 Maria Fernanda Ratti Torres To my Obi-Wan Roberto, Yoda one for me. You have been nothing but supportive during these years, you make me proud of being your wife, but above all, you make me immensely happy. This is for you and for a marriage that has been put to rest for so long and it is ready to resume. ACKNOWLEDGMENTS I wish to thank my parents for all their effort and the patience, they are my pillar without whom I could not have pursued my Ph.D. studies. Also to my siblings Andrea and Pablo, my brother in law Carlos and my nephews, not only for their support, but for joking around all the time and cheering me up during this journey. Erica M. Goss deserves a special section only for her, but the formatting will not allow it. I cannot imagine having spent these years under anybody else’s guidance. She always challenged me to be better, to be calmed during stressful situations and to trust in myself. Her advices will be forever in my mind. Doing this research would have been impossible without helping hands around the world: Thanks to Esther Lilia P. for all her support, to Juan C., Carlos A., Jerry L. and Daynet S, for assisting with logistic and lab space in Ecuador. Also to Ricardo O., Frank M., Leonardo S., Janna B, Roberto Faedda, Natalia Peres, Shad Ali, and Edzard v.S., for providing isolates/DNA, advice and assistance that were required for this research. Also, to my Committee members: Randy P., Karen G. and Jiri H. that were very helpful with advices, suggestions, and corrections, they were a wonderful team and I learnt a lot from them. I want to thank my lab members: Marina A., who was always kind and whose help was extremely valuable; Jonelle J., Parker N., and Kelcey H., the smartest undergraduates in our Department. To my lab mates throughout my stay: Jianan W., Jackson P., Kola F., Fernanda I., Jeannie K., Brett L. and Ashish A. who were always willing to help, talk, or just listen. Thanks to all of them for making our lab such a nice place to work. 4 There are many people who were there pushing me giving words of courage when I needed it the most. I guess they call themselves friends, but they might as well be angels. Ellen D., Shankar K., Mpoki S., were my first lab and office mates, I wish the best for them. Also, to Ariena v.B. for giving me the opportunity to come here, she was the first person opening doors for me and I will be eternally grateful with her. A special thank for my chocolate club Ying and James, who will always be in my heart and in my mind. Thanks to my bestie Ana G., whose words of support always kept me up when I thought I could not have the strength to continue. This section would not be complete without my Ecuadorian fellas Lisbeth E., Daniel M., and Andres O. for making Gainesville an even more interesting place to live. But especially to my “Carachitas” Jorge and Miguel (honorary Ecuadorian), I do not know what I would have done without them, hope we can get together soon. I want to thank to my natural science teacher at Elementary school, Miss Betty V. and all the women around the world who are involved in science, they are an everyday inspiration. Finally, I cannot thank enough to my ultimate inspiration Roberto J., he is my Jedi master, partner, friend and my love. The Force will be with you, always. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 13 CHAPTER 1 OOMYCETE PATHOGENS IN TROPICAL AGRO-SYSTEMS ............................... 15 Emerging Pathogens in Crops and Agro-Ecological Interfaces .............................. 15 Overview of Oomycetes .......................................................................................... 17 Oomycete Communities in Tropical Soil: Cacao Farms .......................................... 19 Phytophthora nicotianae, Another Versatile Pathogen of Tropical and Subtropical Crops ................................................................................................ 20 Hybrid Phytophthora Species ................................................................................. 22 Objectives ............................................................................................................... 25 2 EVALUATION OF TWO BARCODING MARKERS FOR HIGH THROUGHPUT SEQUENCING OF OOMYCETES AS APPLIED TO CACAO AGRO- ECOSYSTEMS IN ECUADOR ............................................................................... 26 Introduction ............................................................................................................. 26 Materials and Methods............................................................................................ 30 Sampling Sites and Strategies ......................................................................... 30 Library Preparation and Sequencing ................................................................ 30 Database Assemblage and Taxonomy Assignment of Sequence Reads ......... 33 Statistical Analyses .......................................................................................... 34 Results .................................................................................................................... 36 Quality of Reads and General Statistics of Runs .............................................. 36 Mock Communities ........................................................................................... 36 Species Composition of Soil Samples .............................................................. 37 Taxa in Relationship with Land Use ................................................................. 40 Discussion .............................................................................................................. 41 3 PINEAPPLE HEART ROT ISOLATES FROM ECUADOR REAVEAL A NEW GENOTYPE OF PHYTOPHTHORA NICOTIANAE ................................................ 65 Introduction ............................................................................................................. 65 Materials and Methods............................................................................................ 68 6 Sampling and Morphological Identification of Isolates ...................................... 68 DNA Extraction and PCR Conditions for Microsatellites and Mitochondrial Markers ......................................................................................................... 69 SSR Genotyping and Analysis ......................................................................... 71 Multi-Locus Sequencing Analysis ..................................................................... 72 Results .................................................................................................................... 73 Identity of Pineapple Isolates and Morphological Description........................... 73 SSR Diversity and Host-Population Genetic Distance ...................................... 74 Population Structure Across Host-Populations ................................................. 75 Multi-Locus Sequencing ................................................................................... 76 Discussion .............................................................................................................. 76 4 EVALUATION OF HIGH RESOLUTION MELTING ANALYSIS AS A METHOD TO DIFFERENTIATE PHYTOPHTHORA HYBRIDS FROM THEIR PARENTAL SPECIES ................................................................................................................ 88 Introduction ............................................................................................................. 88 Materials and Methods............................................................................................ 92 Isolates Used in this Study ............................................................................... 92 Polymorphic Sites Mining and Primer Design ................................................... 93 PCR and High Resolution Melting .................................................................... 94 Results .................................................................................................................... 95 Primer Design and Evaluation .......................................................................... 95 P. infestans and P. andina Melting Curves ....................................................... 95 P. nicotianae, P. cactorum and P. x pelgrandis Melting Curves ....................... 96 Discussion .............................................................................................................. 97 5 GENERAL DISCUSSION AND CONCLUDING REMARKS ................................. 120 APPENDIX
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