Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2012 Molecular Approaches to Detect and Control Cercospora kikuchii in Soybeans Ashok Kumar Chanda Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Plant Sciences Commons Recommended Citation Chanda, Ashok Kumar, "Molecular Approaches to Detect and Control Cercospora kikuchii in Soybeans" (2012). LSU Doctoral Dissertations. 3002. https://digitalcommons.lsu.edu/gradschool_dissertations/3002 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. MOLECULAR APPROACHES TO DETECT AND CONTROL CERCOSPORA KIKUCHII IN SOYBEANS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College In partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Plant Pathology and Crop Physiology by Ashok Kumar Chanda B.S., Acharya N. G. Ranga Agricultural University, 2001 M.S., Acharya N. G. Ranga Agricultural University, 2004 August 2012 DEDICATION This work is dedicated to my Dear Mother, PADMAVATHI Dear Father, MADHAVA RAO Sweet Wife, MALA Little Angel, HAMSINI ii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisors Dr. Zhi-Yuan Chen and Dr. Raymond Schneider, for giving me the opportunity to pursue this doctoral program, valuable guidance throughout my research as well as freedom to choose my work, kindness and constant encouragement, and teaching me how to become a molecular plant pathologist. I am very grateful to my committee members, Dr. Christopher Clark, Dr. Jeffrey Hoy, and Dr. Kenneth Damann, for their constructive criticism and valuable suggestions from the time I proposed until the completion of my research and especially for their critical review of my dissertation. My special thanks to our head, Dr. Lawrence Datnoff, for his constant encouragement and motivation. I would like to thank all the people who helped me in completion of this dissertation research. I owe special thanks to PPCP family for their love and affection to me and my family during the stay at LSU. I thank all my friends, especially Suman, Ramu, Dina, Freddy, Sunny, Nicole, Syam, Alessandro, Yi for their help, sharing knowledge, and for the memorable times we had together. I am very thankful to my family members, Amma, Nana, Vijay Anna, Malathi Vadina, Patricia mama for their unconditional love and support during my time away from home. Amma deserves special thanks for her love and staying with us to help with our baby girl during the final stage of writing this dissertation. If I can thank only one person, it will be my sweet wife, MALA, for always being with me in ups and downs in research and in life, for her unconditional love and support, taking care of everything during drafting of this dissertation. Thanks to our little angel HAMSINI for bringing us lots of joy in our life. iii TABLE OF CONTENTS DEDICATION ................................................................................................................................ ii ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix LIST OF ABBREVIATIONS ....................................................................................................... xv ABSTRACT .................................................................................................................................. xx CHAPTER: 1. INTRODUCTION .................................................................................................. 1 1.1 Justification ...................................................................................................................... 1 1.2 Objectives ......................................................................................................................... 4 CHAPTER 2: REVIEW OF LITERATURE .................................................................................. 5 2.1 Soybean ............................................................................................................................ 5 2.2 Soybean Growth and Development.................................................................................. 5 2.3 Yield Loss Caused by Cercospora kikuchii ..................................................................... 6 2.4 Cercospora kikuchii ......................................................................................................... 7 2.4.1 Cercospora kikuchii and Latent Infection of Soybeans ............................................ 7 2.4.2 Purple Seed Stain (PSS) ............................................................................................ 9 2.4.3 Cercospora Leaf Blight (CLB)................................................................................ 10 2.4.4 Resistance to PSS and CLB .................................................................................... 10 2.4.5 Management of Cercospora Leaf Blight ................................................................. 11 2.5 Cercosporin .................................................................................................................... 12 2.5.1 Cercosporin and Mode of Action ............................................................................ 12 2.5.2 Role of Cercosporin in Plant Diseases .................................................................... 13 2.5.3 Cercosporin Biosynthesis........................................................................................ 14 2.5.4 Resistance to Cercosporin ....................................................................................... 18 2.5.5 Factors Affecting Cercosporin Production ............................................................. 20 2.6 Real-Time Polymerase Chain Reaction (Real-time PCR) ............................................. 20 2.6.1 Available Real-Time PCR Technologies ................................................................ 20 2.6.2 Application of Real-Time PCR to Study Plant Pathogens...................................... 22 iv 2.7 Proteomics ...................................................................................................................... 24 2.7.1 Introduction and Advantages .................................................................................. 24 2.7.2 2-Dimensional Electrophoresis ............................................................................... 26 2.7.3 Different Staining Techniques ................................................................................ 27 2.7.4 Application of Proteomics to Study Plant Pathogenic Fungi ................................. 28 2.7.5 Application of Proteomics to Study Secreted Proteins from Plant Pathogenic Fungi ……………………………………………………………………………………..30 CHAPTER 3: LATENT INFECTION BY CERCOSPORA KIKUCHII, CAUSAL AGENT OF CERCOSPORA LEAF BLIGHT IN SOYBEAN ........................................................................ 36 3.1 Introduction ......................................................................................................................... 36 3.2 Materials and Methods ........................................................................................................ 38 3.2.1 Cercospora Species ................................................................................................ 38 3.2.2 Development of Specific Real-time PCR Primers and Probe for C. kikuchii ......... 39 3.2.3 Real-Time PCR Assay and Quantification of DNA of C. kikuchii in Field Samples ……………………………………………………………………………………..40 3.2.4 Plant Materials, Sampling Details, and Fungicide Sprays ...................................... 42 3.2.5 DNA Extraction ...................................................................................................... 42 3.2.6 Cercospora Leaf Blight Disease Severity Ratings .................................................. 44 3.2.7 Data Analysis .......................................................................................................... 44 3.3 Results ............................................................................................................................ 45 3.3.1 Developing Real-Time PCR Primers Specific to C. kikuchii ................................. 45 3.3.2 Standard Curves and Sensitivity of Real-Time PCR Assays .................................. 47 3.3.3 Visual Rating of Disease Severity and Quantification of C. kikuchii DNA in Soybean Leaf Samples Using Real-Time PCR ..................................................................... 47 3.4 Discussion ...................................................................................................................... 56 CHAPTER 4: IDENTIFICATION AND CHARACTERIZATION OF LIGHT-INDUCIBLE PROTEINS FROM CERCOSPORA KIKUCHII .........................................................................