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Genetic Diversity of the Fungal Pathogen Corynespora Cassiicola and Its Fungicide Resistance

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Genetic Diversity of the Fungal Pathogen Corynespora Cassiicola and Its Fungicide Resistance Genetic diversity of the fungal pathogen Corynespora cassiicola and its fungicide resistance by Marina Nunes Rondon A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 12, 2020 Keywords: cassiicolin, cotton, G143A mutation, phylogeny, soybean, target spot Copyright 2020 by Marina Nunes Rondon Approved by Kathy S. Lawrence, Chair, Professor of Entomology and Plant Pathology Dennis P. Delaney, Professor Emeritus of Crop, Soil and Environmental Sciences Edward J. Sikora, Extension Specialist Professor of Entomology and Plant Pathology Jeffrey J. Coleman, Assistant Professor of Entomology and Plant Pathology Abstract Corynespora cassiicola C.T. Wei is a widespread phytopathogenic fungus that causes disease in tropical and subtropical regions in up to 400 plant species, including fruits, vegetables, ornamentals, forestry, and row crops. Disease symptoms can be observed on plant leaves, stems, roots, flowers, and fruits. Incidence of the disease, target spot, as well as severity has become more frequent across cotton and soybean producing countries, and significant yield losses can occur if it is not properly controlled. The fungus has a parasitic lifestyle coupled with a saprophytic and endophytic lifestyle which complicates disease management. The overall objective of this dissertation was to better understand the pathogen, its interaction with soybean plants, and risks associated with chemical control. The first chapter reviewed the literature on C. cassiicola focused on cotton and soybean, including pathogen biology and genetic diversity, and a detailed discussion on two essential IPM strategies emphasizing their importance to overcome the disease. The second chapter investigated the genetic diversity of C. cassiicola isolates from symptomatic leaves of cotton and soybean by studying the morphology, pathogenicity, and molecular phylogeny based on cassiicolin-encoding genes and four loci. The third chapter revealed the impact of C. cassiicola diversity on screening for resistance to target spot on soybean by comparing two screening methods: leaf wilting bioassay and plant inoculation. The fourth chapter determined the sensitivity profile of C. cassiicola isolates from cotton and soybean to five commercial fungicides, and if there is a fitness loss on C. cassiicola QoI-resistant isolates. Finally, the fifth chapter first reported C. cassiicola isolates from soybean in the United States with the G143A mutation in the cytochrome b gene that confers resistance to QoI fungicides. Altogether, the results of this dissertation provide useful insights for research on the management of target spot disease to reduce the risk of epidemics and yield losses. ii Acknowledgements I would like to extend my gratitude and appreciation to everyone that I had the privilege of meeting and working during my Ph.D. journey in the United States. I would like to express my deep gratitude to Dr. Kathy Lawrence for providing me the opportunity to pursue my research objectives with assistance, advice, encouragement, and financial support. I am very appreciative to my committee members Drs. Dennis Delaney, Edward Sikora, and Jeffrey Coleman, and the outside reader Dr. Wheeler Foshee for their time and contribution to my dissertation and completion of requirements for my Ph.D. I am especially thankful to Dr. Patricia Donald who carefully reviewed all of my publications since day one of my battle against writing in a second language. I would like to thank Drs. Ni Xiang, Bisho R. Lawaju, and Claudio Vrisman who provided assistance to conduct my research. I am thankful to all professors of the Entomology and Plant Pathology Department and other Departments who have broadened my knowledge on Plant Pathology and Statistics that will be very useful in my professional career. Thank you to the fellow graduate students Will Groover, David Dyer, Stephen Till, Meredith Hall, Katie Gattoni, WinDi Sanchez, Kara Gordon, Sloane McPeak, and Kate Armstrong and all undergraduate students for the countless hours in the conferences, field, laboratory, greenhouse; always showered with birthday cakes and hard work. Thank you to colleagues from other laboratories who have always helped with exchange of knowledge and lending lab materials when necessary. Thank you to my friends who have supported me throughout these years at Auburn, which I always considered as my Brazilian family in Auburn. We had a lot of fun, learning and experiencing a new culture outside our beloved country. Lastly but most importantly, I am very grateful to God, my parents, Marcos and Laura, sister Flavia, and family. They gave me support, love, encouragement, and prayers during this journey away from home. I am grateful and really appreciate all of you! iii Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii Chapter 1. Review of Literature ..............................................................................................11 Abstract ..........................................................................................................................................11 1. Introduction ........................................................................................................................ 12 1.1 Overview ......................................................................................................................... 12 1.2 Pathogen description........................................................................................................ 15 1.3 Disease symptoms ........................................................................................................... 17 1.4 Diversity of C. cassiicola isolates ................................................................................... 18 1.5 The use of fungicides to control target spot .................................................................... 20 1.6 Genetic control for disease resistance ............................................................................. 25 2. Conclusions and future prospects ...................................................................................... 28 3. Literature cited ................................................................................................................... 31 Chapter 2. Genetic diversity of Corynespora cassiicola isolated from cotton and soybean and relationships with morphology and pathogenicity ............................................................43 Abstract ..........................................................................................................................................43 1. Introduction ........................................................................................................................ 44 2. Materials and methods ....................................................................................................... 47 2.1 Isolates ............................................................................................................................. 47 2.2 Morphology ..................................................................................................................... 48 2.3 Pathogenicity ................................................................................................................... 50 2.4 DNA extraction, PCR amplification and sequencing ...................................................... 51 2.5 Phylogenetic analyses ...................................................................................................... 53 3. Results ................................................................................................................................ 54 3.1 Isolates ............................................................................................................................. 54 3.2 Morphology ..................................................................................................................... 54 3.3 Pathogenicity ................................................................................................................... 58 iv 3.4 PCR amplification of cassiicolin-encoding genes ........................................................... 58 3.5 Phylogenetic analysis ...................................................................................................... 59 4. Discussion .......................................................................................................................... 62 5. Literature cited ................................................................................................................... 66 Chapter 3. The impact of Corynespora cassiicola variability on two screening methods for target spot resistance on soybean ...............................................................................................83
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