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Corynespora Cassiicola) Resistance in Cotton (Gossypium Hirsutum L.

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Corynespora Cassiicola) Resistance in Cotton (Gossypium Hirsutum L. Screening for target spot (Corynespora cassiicola) resistance in cotton (Gossypium hirsutum L.) by Carly Moore A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama May 4, 2019 Keywords: cotton, target spot, Corynespora cassiicola, inoculation Copyright 2019 by Carly Moore Approved by Jenny Koebernick, Chair, Assistant Professor of Crop, Soil and Environmental Sciences Kira Bowen, Professor of Plant Pathology Charles Chen, Professor of Crop, Soil and Environmental Sciences Abstract A critical problem for Alabama cotton growers is the presence of target spot (Corynespora cassicola), a foliar disease which is responsible for combined annual yield losses of >$70 million in AL, GA and the FL Panhandle. Currently, there are no resistant commercial cultivars and the only management defense are fungicide applications. Timing of application and implications for resistance to the fungicides are high. A resistant variety is the only long term solution, therefore this study focuses on the inoculation method and growing conditions of C. cassiicola in order to develop a greenhouse protocol to screen for the variety response to target spot. Preliminary experiments focused on inoculation techniques and environmental conditions favoring pathogen reproduction. It was determined that a spore suspension was preferred over a mycelium broth, the temperature required for growth was 24-32°C and that the plants had to be under consistent moisture for at least 12 hours. Future experiments will compare cultivars known to be different in a field setting to validate this protocol. Key words; cotton, target spot, Corynespora cassiicola, inoculation ii Acknowledgments I would like to thank my mother and my father for their endless support in whichever path I choose. I would also like to thank my paternal grandfather, Dr. Wellington Moore, for inspiring me to run towards challenge and always seek wisdom and my maternal grandmother, Phyllis Rothert, for being a remarkable example of the kind of person I should aspire to be. iii Table of Contents Abstract ................................................................................................................................... ii Acknowledgments ................................................................................................................. iii List of Tables ........................................................................................................................ vii List of Figures ...................................................................................................................... viii List of Abbreviations .............................................................................................................. ix Literature Review .................................................................................................................. 1 Cotton Growth and Development ................................................................................ 1 Common Foliar Diseases ............................................................................................ 4 Bacterial ................................................................................................................. 4 Fungal .................................................................................................................... 5 Target Spot ................................................................................................................. 9 Target Spot as a Threat ........................................................................................... 9 Corynespora cassiicola ..............................................................................................10 Target Spot of Cotton ............................................................................................10 Corynespora cassiicola – The Fungus ...................................................................10 Isolation and Culture Methods ...............................................................................11 Inoculum ....................................................................................................................12 Common Laboratory Inoculum Sources ................................................................12 Resistance in Soybean ................................................................................................14 Fungicide Control ......................................................................................................14 Objectives ..................................................................................................................15 iv References ..................................................................................................................17 Chapter 1 ..............................................................................................................................21 Introduction ..................................................................................................................21 Materials and Methods .................................................................................................24 Inoculum ................................................................................................................24 Isolate Collection ...............................................................................................24 Mycelium Fragment V8 broth ............................................................................24 Spore Suspension ...............................................................................................25 Genetic Confirmation .............................................................................................26 Gel Electrophoresis ............................................................................................27 BLAST ..............................................................................................................28 Preliminary Inoculation ..........................................................................................28 Experiment – Trial 1 ..............................................................................................29 Experiment – Trial 2 ..............................................................................................30 Experiment – Trial 3 and 4 ......................................................................................30 Data Analysis................................................................................................................31 Result and Discussion ...................................................................................................32 Mist Chamber Analysis ...........................................................................................32 V8 Corynespora cassiicola Broth Inoculum ......................................................32 Leaf Wetness Duration, Temperature, and Environment ....................................33 Concentration and Replication Adjustments ......................................................34 Conclusion ...................................................................................................................35 References ....................................................................................................................36 v Chapter 2 ................................................................................................................................40 Introduction ..................................................................................................................40 Materials and Methods ..................................................................................................42 2017 Field Information ............................................................................................42 Rating Scale ........................................................................................................42 2018 Field Information ............................................................................................43 Data Analysis .........................................................................................................44 Result and Discussion ...................................................................................................45 Conclusion ....................................................................................................................47 References ....................................................................................................................48 Appendix 1. Tables ................................................................................................................50 Appendix 2. Figures ..............................................................................................................58 Appendix 3. Protocol Development Troubleshooting .............................................................67 vi List of Tables Table 1. ANOVA table for mean target spot ratings comparing 1.5x10^4 spores/ml with 4.0x10^4 spores/ml. ................................................................................................................50 Table 2. Mean values for target spot ratings for 1.5x10^4 spores/ml. ......................................51 Table 3. Mean values for target spot ratings for 4.0x10^4 spores/ml .......................................52 Table 4. ANOVA of
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