Evaluation of PCR-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium Dahliae in Woody Hosts by Real- Time Polymerase Chain Reaction
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University of Kentucky UKnowledge Theses and Dissertations--Plant Pathology Plant Pathology 2014 Evaluation of PCR-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium Dahliae in Woody Hosts by Real- Time Polymerase Chain Reaction Baker Diwan Getheeth Aljawasim University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Aljawasim, Baker Diwan Getheeth, "Evaluation of PCR-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium Dahliae in Woody Hosts by Real-Time Polymerase Chain Reaction" (2014). Theses and Dissertations--Plant Pathology. 13. https://uknowledge.uky.edu/plantpath_etds/13 This Master's Thesis is brought to you for free and open access by the Plant Pathology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant Pathology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Baker Diwan Getheeth Aljawasim, Student Dr. Paul Vincelli, Major Professor Dr. Lisa J. Vaillancourt, Director of Graduate Studies EVALUATION OF PCR-BASED METHODS FOR RAPID, ACCURATE DETECTION AND MONITORING OF VERTICILLIUM DAHLIAE IN WOODY HOSTS BY REAL-TIME POLYMERASE CHAIN REACTION THESIS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Agriculture, Food, and Environment at the University of Kentucky By Baker Diwan Getheeth Aljawasim Lexington, Kentucky Director: Dr. Paul Vincelli, Professor of Plant Pathology Lexington, Kentucky 2014 Copyright© Baker Aljawasim 2014 ABSTRACT OF THESIS EVALUATION OF PCR-BASED METHODS FOR RAPID, ACCURATE DETECTION AND MONITORING OF VERTICILLIUM DAHLIAE IN WOODY HOSTS BY REAL-TIME POLYMERASE CHAIN REACTION Verticillium wilt, caused by Verticillium dahliae Kleb, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration symptoms, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time PCR-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead-beating, and extracting using a CTAB method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species- specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detecting and monitoring, thus informing plant pathogen management decisions. KEYWORDS: Verticillium dahliae, Verticillium wilt, real-time PCR, woody plants. Baker Aljawasim Apr 8th 2014 EVALUATION OF PCR-BASED METHODS FOR RAPID, ACCURATE DETECTION AND MONITORING OF VERTICILLIUM DAHLIAE IN WOODY HOSTS BY REAL-TIME POLYMERASE CHAIN REACTION BY Baker Diwan Getheeth Aljawasim Dr. Paul Vincelli Director of Thesis Dr. Lisa J. Vaillancourt Director of Graduate Studies Apr 8th 2014 To my family, my father, my mother, my friends, and especially Higher Committee for Education Development in Iraq (HCED) ACKNOWLEDGEMENTS First of all, I would thank my advisor, Paul Vincelli, for his support, guidance, and patience with me during my work in University of Kentucky. I think any student will be grateful to work with a great person like him. Dr. Paul taught me the fundamentals of science, and how can I think as a scientist to overcome any problem during my career. I would like to extend my gratitude to my committee members Dr. Nicole Ward Gauthier and Dr. A Bruce Downie for their helpful advice and instructions. I greatly appreciate the extensive support offered by Extension Plant Pathology staff members (Bernadette, Ed, Julie, Sara, and Doug). Especially, I really want to express my deep gratitude to Bernadette for her extensive support, assistance, and help with my experiments in the laboratory. Also, I want to thank Dr. K. Subbarao from University of California, Davis for providing us isolates of V. dahliae. I would like to thank all the graduate students in the Plant Pathology Department at the University of Kentucky for their help and support. Also, I would like to thank all faculty and staff of the Department of Plant Pathology, especially Dr. Christopher L. Schardl, Dr. Lisa Vaillancourt, Dr. Mark Farman, Dr. Pradeep Kachroo, Dr. Aardra Kachroo and, Dr. Kenneth W. Seebold, Shirley Harris, and Alicia Landon for their support and help. Lastly, I sincerely thank the Higher Committee for Education Development (HCED) in Iraq for their great support and confidence in me, and I hope to pay my country back by participating in Iraq‟s development, especially in iii agricultural disciplines. I will not forget my father, my mother, my brothers, and my sister for supporting and praying for me to achieve all my dreams. I will never forget that my father said “Go to United States and get your degree, and all people in your neighborhood will support you,” and my mother‟s tears when I left my country. I hope that I can achieve my big dream by earning my PhD degree from United States as well. Actually, I am grateful for being at the University of Kentucky not only for high level of education but also for great culture and atmosphere for studying. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................. iii LIST OF TABLES .......................................................................................................... vii LIST OF FIGURES ........................................................................................................ viii CHAPTER ONE: LITERATURE REVIEW .................................................................... 1 I. Verticillium Wilt ............................................................................................... 1 History of Verticillium Wilt.................................................................. 2 Classification of V. dahliae ................................................................... 4 Morphological and Anatomical Characters .......................................... 6 Disease Cycle ........................................................................................ 8 Symptoms ........................................................................................... 11 II. Management of Verticillium Wilt ................................................................... 14 Physical Methods ................................................................................ 14 Chemical Methods .............................................................................. 17 Fertilization Methods .......................................................................... 21 Biological Methods ............................................................................. 23 Resistant Cultivars .............................................................................. 26 III. Traditional Detection Methods for V. dahliae ................................................ 27 Detection of Microsclerotia from the Soil .........................................