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Biology and Control of Rice False Smut Caused by Ustilaginoidea Virens (Teleomorph Villosiclava Virens) Andrew Clayton Jecmen University of Arkansas, Fayetteville

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Biology and Control of Rice False Smut Caused by Ustilaginoidea Virens (Teleomorph Villosiclava Virens) Andrew Clayton Jecmen University of Arkansas, Fayetteville University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 5-2014 Biology and Control of Rice False Smut Caused by Ustilaginoidea virens (Teleomorph Villosiclava virens) Andrew Clayton Jecmen University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Molecular Biology Commons, and the Plant Pathology Commons Recommended Citation Jecmen, Andrew Clayton, "Biology and Control of Rice False Smut Caused by Ustilaginoidea virens (Teleomorph Villosiclava virens)" (2014). Theses and Dissertations. 2346. http://scholarworks.uark.edu/etd/2346 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Biology and Control of Rice False Smut Caused by Ustilaginoidea virens (Teleomorph Villosiclava virens) Biology and Control of Rice False Smut Caused by Ustilaginoidea virens (Teleomorph Villosiclava virens) A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Pathology by Andrew Clayton Jecmen Missouri Southern State University Bachelor of Science in Biology, 2010 May 2014 University of Arkansas This thesis is approved for recommendation to the Graduate Council __________________________________ Dr. David O. TeBeest Thesis Director __________________________________ __________________________________ Dr. Burt H. Bluhm Dr. Ken L. Korth Committee Member Committee Member __________________________________ __________________________________ Dr. Fred W. Spiegel Dr. A. Rick Bennett Committee Member Ex-Officio Member Abstract Rice false smut (FS), a disease caused by Ustilaginoidea virens (Cke.) Takahashi (1896), was first reported in northeastern Arkansas counties in 1997. The first objective of this research was to establish a collection of U. virens isolates from geographically diverse regions of Arkansas. Three U. virens isolates and chlamydospores from ‘Templeton’ and ‘Clearfield-151’ rice cultivars were used to determine the effects of temperature and pH on mycelial growth and germination. A nested-PCR protocol and histological methods were used to determine if U. virens infects and colonizes rice seedlings and spikelets on panicles. The sensitivity of three U. virens isolates was tested to analyze the inhibition of mycelial growth in vitro and to establish inhibitory concentrations to six technical and five analytical grade fungicides. Field and greenhouse tests were conducted to determine if fungicide seed treatments using technical grade fungicides could effectively reduce the incidence of U. virens rDNA in seedlings as measured by nested-PCR. Finally, field tests were conducted using fungicide seed treatments to control FS at two locations and disease was assessed by a visual disease assessment. We have an established collection of 190 isolates obtained from nine cultivars in seven counties of Arkansas, USA. Mycelial growth and germination of chlamydospores occurred between pH levels from 5.5 to 8.0. Mycelial growth and germination of chlamydospores occurred at temperatures from 18° to 34°C and from 18 to 26°C, respectively. Nested-PCR tests indicate the protocol is specific and sensitive for detecting U. virens in rice. Ribosomal DNA of U. virens was detected using nested-PCR from seedlings within three days after emergence from the soil and in 27.5 to 75% of spikelets in booted panicles before exsertion. Selected isolates of U. virens were sensitive to fungicides in-vitro but results from using nested-PCR in the greenhouse and field to screen seedlings for U. virens rDNA indicated a significant reduction in the incidence of U. virens in some seed treatments compared to controls. Seed treatments did not significantly reduce FS disease compared to controls in the field plots when measured by visual disease assessments. Acknowledgements I would like to take this opportunity to thank my committee members Drs. Burt Bluhm, Ken Korth, Fred Spiegel and the head of the Department of Plant Pathology, Rick Bennett for their contributions to initiating and ongoing support of this project. A special thanks to my supportive advisor Dr. David O. TeBeest for his tireless encouragement and careful guidance in preparation of this project and thesis. I also would like to honor my late father, Joseph Francis Jecmen DDS who constantly inspired me to pursue my dreams of studying plant sciences. He also passionately encouraged me to develop and contribute my talents to help others as he devoted his life to a ministry of healing and serving people in need. This project would not have been able to come to fruition without the relentless work in the persistent summer heat and droughts by my advisor Dr. David O. TeBeest and the field staff at the University of Arkansas Research Stations at Newport and Pine Tree. They all deserve big thanks! Other big thanks are due to the Agriculture Diagnostic Laboratory for Research Soil Analyses, Jeff Velie in the Agricultural Statistics lab for statistical data analysis and Alma Laney for advice in sequence analysis and manipulations. A special thank you is warranted for the ongoing support and intellectual challenge of my peers. I am very thankful to have enjoyed the plentiful learning resources of very helpful staff and outstanding Professors here in the Department of Plant Pathology at the University of Arkansas! Dedication I would like to dedicate this thesis to the future generations of researchers, for we all stand on the shoulders of those who precede us. No combination of words may ever fully express what a very wonderful opportunity it is to have the privilege of studying what is known and exploring the unknown. I wish you the best in future endeavors! Table of Contents Abstract Acknowledgments Dedication Chapter I. Introduction and review of relevant literature on infection of rice by Ustilaginoidea virens A. Rice and rice production 1 1. Brief history of rice production 1 2. Amount of production/top rice growing areas 1 3. Rice production in the United States 1 4. Importance of managing diseases 1 B. Ustilaginoidea virens (etiological agent of rice false smut) 1 1. Taxonomy of Ustilaginoidea virens 2 2. Distribution of Ustilaginoidea virens 2 3. Mycotoxins produced by Ustilaginoidea virens 2 C. Features of Ustilaginoidea virens and the FS-Rice pathosystem 3 1. Host specificity 3 2. Morphology and culturing Ustilaginoidea virens 3 3. Morphology of Villosiclava virens 4 4. Characteristics of false smut disease 4 5. Disease cycle and epiphytology 4 6. Factors related to pathogenicity 7 D. Molecular detection methods for Ustilaginoidea virens 7 1. Histopathology 7 2. Nested-PCR for detection of U. virens in rice 7 E. Methods for controlling false smut in rice 8 E. Goals 9 F. Hypothesis 9 G. Scientific objectives 10 II. Isolation, culture, preservation and the effects of pH and temperature on germination of chlamydospores and mycelial growth A. Introduction 19 B. Materials and Methods 20 1. Collection of samples to prepare U. virens isolates 20 2. Isolation of U. virens from spore balls 20 3. Cryogenic storage of the isolate collection 21 4. Identification of single chlamydospore isolates as U. virens 21 5. Identification of isolates with selective nested-PCR primers 22 6. Description of the white false smut found in this study 22 7. Effect of pH on germination of one-, three- and five-month-old U. virens chlamydospores in agar adjusted to pH ranging from 5.5 to 8.0 25 8. Growth of U. virens isolates I-6E, I-7E and I-8E on agar adjusted to pH ranging from 5.5 to 8.0 26 9. Germination of U. virens chlamydospores at temperatures 18°C, 22°C and 26°C 27 10. Growth of U. virens isolates I-6E, I-7E and I-8E at temperatures 18°C, 22°C, 26°C, 30°C, 34°C, 36°C and 38°C 27 11. Data analysis 28 C. Results 28 1. Collection of U. virens isolates: 2010 and 2011 surveys 28 2. Identification and verification of U. virens 29 3. Description of the white false smut found in this study 30 4. Effect of pH on germination of one-, three- and five-month-old U. virens chlamydospores in agar adjusted to pH ranging from 5.5 to 8.0 34 5. Effect of pH on growth of U. virens isolates I-6E, I-7E and I-8E in buffered agar 35 6. Effect of temperatures 18°C, 22°C and 26°C on germination of U. 35 virens chlamydospores 7. Growth of U. virens isolates I-6E, I-7E and I-8E at temperatures 18°C, 22°C, 26°C, 30°C, 34°C, 36°C and 38°C 35 D. Discussion 36 III. Investigating nested-PCR as a strategy for detection of Ustilaginoidea virens during asymptomatic colonization of rice tissues. A. Abstract 60 B. Introduction 60 C. Materials and Methods 65 1. Source of isolates and culture preparation 65 2. Plant material 65 3. Isolation of fungi from rice germlings, seedlings and spikelets from 66 booted panicles 4. DNA extraction from cultured mycelium and rice tissues 67 5. Conditions for detection of U. virens by simple and nested-PCR 69 6. The specificity of nested-PCR primers and conditions used for detection of U. virens isolates and one Ustilaginoidea isolate I-9E 71 a. Part 1: The specificity of nested-PCR to U. virens isolates from Arkansas 72 b. Part 2: The specificity of nested-PCR to other rice Pathogens 72 c. Part 3: The specificity of nested-PCR to pathogens of other crops 73 d. Part 4: The specificity of nested-PCR to fungi isolated from crowns of rice germlings, seedlings and spikelets from booted panicles 73 7. Sequence analysis and multiple sequence alignment of the amplified products from nested-PCR 74 8. Sensitivity in U. virens detection in the presence of rice DNA 76 9.
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