Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2004 Identification of bacterial pathogens causing panicle blight of rice in Louisiana Xianglong Yuan Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Plant Sciences Commons Recommended Citation Yuan, Xianglong, "Identification of bacterial pathogens causing panicle blight of rice in Louisiana" (2004). LSU Master's Theses. 2883. https://digitalcommons.lsu.edu/gradschool_theses/2883 This Thesis 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 Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. IDENTIFICATION OF BACTERIAL PATHOGENS CAUSING PANICLE BLIGHT OF RICE IN LOUISIANA A Thesis 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 Master of Science In The Department of Plant Pathology and Crop Physiology By Xianglong Yuan B.S., University of Liaoning, 1985 M.S., Shenyang Agriculture University, 1990 May 2004 ACKNOWLEDGMENTS I wish to express my sincerest gratitude to Dr. M. C. Rush, my advisor, for giving me this opportunity to pursue my graduate studies in the Department of Plant Pathology and Crop Physiology at Louisiana State University. It was Dr. Rush that led me to enter the world of plant pathology, widening my field of vision in academia. I sincerely appreciate his guidance, advice, understanding, and patience throughout my time of study here, particularly in the research work and writing. His working attitude will necessarily be a beacon lighting my future voyage. I greatly appreciate Dr. A. K. M. Shahjahan’s help throughout my research efforts. His generous, patient guidance in my laboratory research, and his many excellent suggestions made during the past 2.5 years have helped my research efforts greatly. Special thank go to my committee members, Dr. D. E. Groth, Dr. S. D. Linscombe, and Dr. J. P. Jones for their suggestions on conducting my research and writing my thesis. It was their influence that greatly inspired me in developing my thesis. I acknowledge all of Chinese students that helped me at this university. Their great and generous support helped me to overcome many difficulties in my life and during my studies in Baton Rouge, LA. The most sincere appreciation belongs to my mom and dad. Their encouragement, love and care were the main source of power that prompted me to accomplish this thesis. I also express thankfulness to my other family members for their generous support. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................. ii ABSTRACT............................................................................................................v CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW .................1 1.1 Disease Distribution and Economic Importance....................................1 1.2 Symptoms and Epidemiology................................................................3 1.2.1 Symptoms ...............................................................................3 1.2.2 Epidemiology..........................................................................5 1.3 Detection and Identification Methods for Pathogenic Bacteria on Rice .........................................................................................................7 1.4 Classification, Nomenclature, and Pathological Characteristics of Pseudomonas and Related Genera .........................................................8 1.4.1 Classification and Nomenclature .............................................8 1.4.2 Characteristics of Burkholderia spp.........................................9 1.4.2.1 B. glumae .....................................................................9 1.4.2.2 B. Plantarii.................................................................10 1.4.2.3 The B. gladioli and B. cepacia Complex ...................10 1.4.2.4 P. fuscovaginae, P. syringae, Acidovorax avenae ( P. avenae) ...........................................................................11 1.5 Virulence..............................................................................................12 1.6 Disease Control....................................................................................13 1.6.1 Chemical Control..................................................................13 1.6.2 Biological Control.................................................................13 1.6.3 Developing Resistant Cultivars and Lines Using Genetics and Biotechnology Techniques.......................................13 CHAPTER 2. CLASSIFICATION, IDENTIFICATION, AND PATHOGENICITY OF BACTERIA ASSOCIATED WITH PANICLE BLIGHT IN RICE ............................................................................15 2.1 Materials and Methods.........................................................................15 2.1.1 Purification of Isolates .............................................................15 2.1.2 Identification of Isolates Using the BiologTM GN2 Microplate System ............................................................................16 2.1.3 Plant Materials .........................................................................17 2.1.4 Pathogenicity Tests……………………………....... ...............17 2.1.5 Functional Classification of the Identified Isolates .................21 2.2 Results and Discussion .......................................................................22 2.2.1 Purification and Identification of Isolates with the BiologTM GN2 System ....................................................................22 2.2.2 Pathogenicity Tests and Symptoms Produced .........................38 2.2.3 Colony and Culture Characteristics of the Bacterial Pathogens ..........................................................................................47 2.2.4 Clustering of Bacterial Strains Isolated from Rice Showing Panicle Blight into Functional Groups ..............................50 iii 2.3 Summary and Conclusions ..................................................................56 LITERATURE CITED ......................................................................................61 APPENDIX: BIBLIOGRAPHY OF RELATED LITERATURE...................80 VITA.....................................................................................................................97 iv ABSTRACT Four hundred and two bacterial isolates were isolated on the semi-specific S-PG medium from diseased rice tissues showing symptoms of panicle blighting. These isolates were purified using serial dilution in sterile water and replating on S-PG medium. A total of 420 single isolates were obtained. These isolates were subjected to pathogenicity tests on rice (Oryza sativa L. cv. Cypress). Based on these tests, 339 isolates were used in BiologTM tests and identified to the species level. Bacterial strains from 39 species in 16 genera were identified, including 52 isolates representing 15 Pseudomonas species and 261 isolates representing six Burkholderia species. The remaining 26 isolates included 14 other genera. Of 261 Burkholderia strains, 103 isolates were B. gladioli, 68 isolates were B. glumae, 60 isolates were B. multivorans, 25 isolates were B. plantarii, three isolates were B.cocovenenans and three isolates were B. vietnamiensis. The pathogenicity tests revealed that 69% or 234/339 isolates caused seedling infection, sheath rot, and/or panicle blighting. Most of the pathogenic strains were in the genera Burkholderia and Pseudomonas. The four most common species, B. glumae, B. gladioli, B. multivorans, and B. plantarii, comprised 90% of all of the pathogenic bacteria, suggesting that a complex of Burkholderia species were causing the panicle blight/sheath rot syndrome recently found in Louisiana. Five Pseudomonas species, with total of 16 isolates, were found to be associate with this disease, including two isolates of P. syringae pv zizanize, three isolates of P. fluorescens, three isolates of P. pyrrocinia, one isolate of P. spinosa and seven isolates of P. tolaasii. The symptoms of the disease on rice were only produced by the indicated bacterial strains. Symptoms included brown margined flag leaf sheath lesions, grain rot, sterile florets, grain discoloration, and leaf and sheath rot on inoculated plants. It was impossible to distinguish among Burkholderia species based on symptoms and colony morphology. Pathogenic Burkholderia strains produced a yellow pigment in King’s B medium. Avirulent strains did not produce this pigment. The isolates from rice were grouped by species and pathogenicity. It appeared that B. glumae and B. gladioli were the most common pathogenic species. v CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW 1.1 Disease Distribution and Economic Importance Panicle blighting has been an important sporadic problem in the southern rice production area of the United States for many years. A somewhat similar disease called “ear blight”, pecky rice, or grain discoloration has been attributed to fungal causal agents (Lee, 1992a; Lee, 1992b). This problem is characterized by discoloration of the grain and panicle branches, usually with distinct lesions, and many fungi have been described