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Molecular Characterization of Southeastern Armillaria Isolates

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Molecular Characterization of Southeastern Armillaria Isolates MOLECULAR CHARACTERIZATION OF SOUTHEASTERN ARMILLARIA ISOLATES by KAROL LEIGH KELLY (Under the Direction of Kathryn C. Taylor) ABSTRACT The genus Armillaria contains white-rot basidiomycetes pathogenic to woody plant hosts worldwide. Of particular interest are the species found in southeastern peach (Prunus persica) orchards causing Armillaria root rot. Isolates collected during the past four years from orchards in Georgia, South Carolina, and Alabama were identified to species and grouped within species based on molecular analysis of the internal transcribed spacer (ITS) regions and the intergenic spacer (IGS) regions of the ribosomal DNA (rDNA). Thirty isolates of A. tabescens and nine isolates of A. mellea were identified from these orchards. Restriction fragment length polymorphism (RFLP) analysis of the IGS region with Alu I revealed two groups of A. tabescens and one group of A. mellea. Similarly, the ITS region was amplified with primer sets At- ITS1/Am-ITS1/ITS-2 and ITS-1/ITS-4 and subsequently restricted with Mbo II and Hha I. These restrictions indicated two groups of A. tabescens and four groups of A. mellea. INDEX WORDS: Armillaria mellea, Armillaria tabescens, Armillaria root rot, Basidiomycetes, intergenic spacer region, internal transcribed spacer region, peach, polymorphism, Prunus persica, rDNA, restriction analysis MOLECULAR CHARACTERIZATION OF SOUTHEASTERN ARMILLARIA ISOLATES by KAROL LEIGH KELLY B.S., Georgia College and State University, 1996 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2004 © 2004 Karol Leigh Kelly All Rights Reserved MOLECULAR CHARACTERIZATION OF SOUTHEASTERN ARMILLARIA ISOLATES by KAROL LEIGH KELLY Major Professor: Kathryn Taylor Committee: Mark Rieger Harald Scherm Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2004 DEDICATION I would like to dedicate this thesis to my wonderful family. My parents, Sam and Judy Kelly, have believed in me since the very beginning. They are the most humble, honest, and wonderful people that I know. I am so lucky to have them in me life. They have taught me that I can do anything that I put my mind to and that I should always look for the best in people. I don’t think there are better parents anywhere. I love y’all with all of my heart. I would also like to dedicate this to my sister Lisa. She is good at keeping me honest in everything that I do. She also has the best heart of anyone I know and will make a positive difference in the world. I love you. Finally, I would like to dedicate this to my split-apart, Kristen. There is not a doubt in my mind that I would not have made it through this research without your help. You have been my cheerleader, my shoulder to cry on, and my best friend through it all. I look forward to spending the rest of my life with you… I love you. iv ACKNOWLEDGEMENTS Not only do I have the most wonderful family in the world, I think I also have the best friends. Everyone was such a help to me whether it was as a mentor, proof-reader, or the most important of all moral support – most involved had multiple rolls. I couldn’t have undertaken this work at all without the support of Kathy Taylor. I’ve been fortunate to have you as a boss, mentor, professor, and pain in my butt (I meant to say friend). I never would have had the opportunity for this degree had it not been for you. Stacy, maybe one day you will be able to see the river despite the forest (that you tried to drive through). Thanks for all that you have done for me. You are a wonderful friend and huge pain in the butt. Janice and Merry (and Charlotte and Laura), I couldn’t have made it without all of your therapy sessions and proofing. Y’all have been good friends and support to me. Carol and Henry, you were a continuous source of wisdom, inspiration, and laughs. I am so thankful that those random chance meetings have made us friends. I am continually awed by your intellect and kindness – not many people have such a wonderful combination. Dr. Scherm, Dr. Rieger, and Dr. Beckman, I know that I could not have made it without your persistent, driving forces and advice. I am so lucky to have had y’all as mentors and instructors during this time. I have learned so much from you. I count myself lucky to have been your student. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES........................................................................................................................ vii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION .........................................................................................................1 2 MATERIALS AND METHODS.................................................................................20 3 RESULTS ....................................................................................................................31 4 DISCUSSION..............................................................................................................43 REFERENCES ..............................................................................................................................55 vi LIST OF TABLES Page Table 1: Armillaria isolates collected from southeastern peach orchards and used in the current study................................................................................................................................28 Table 2: Polymerase chain reaction (PCR) primers used to amplify regions of Armillaria DNA…............................................................................................................................29 Table 3: Summary of the molecular analysis of known and unknown Armillaria isolates..... 35-36 Table 4: Identification and classification of southeastern Armillaria tabescens isolates. .............52 Table 5: Identification and classification of southeastern Armillaria mellea isolates...................53 vii LIST OF FIGURES Page Figure 1: Schematic illustration of the typical arrangement of rDNA of basidiomycetes, including primer locations.............................................................................................19 Figure 2: Geographical distribution of Armillaria isolates collected from southeastern peach orchards .........................................................................................................................30 Figure 3: Analysis of the IGS and ITS of known A. tabescens isolate TA1 and known A. mellea isolate ST5 ....................................................................................................................37 Figure 4: IGS region of Armillaria isolates amplified with primers LR12R/O-1 and restricted with Alu I.......................................................................................................................38 Figure 5: IGS region of Armillaria isolates amplified with primers based on the sequences of European isolates (Sicoli et al., 2003) to identify A. mellea and A. tabescens….. .......39 Figure 6: ITS region of Armillaria isolates amplified with primer set ITS-1/ITS-4 and the primer set developed by Bryson et al. (2003) to discriminate among southeastern Armillaria spp. ................................................................................................................................40 Figure 7: ITS regions of putative A. tabescens isolates amplified with primer set ITS-1/ITS-4 and restricted with Hha I and Mbo II to visualize polymorphisms among isolates.............41 Figure 8: ITS regions of putative A. mellea isolates amplified with primer set ITS-1/ITS-4 and restricted with Hha I......................................................................................................42 Figure 9: Geographical distribution of Armillaria “groups” as established by molecular characterization of IGS and ITS region.........................................................................54 viii CHAPTER 1 INTRODUCTION Purpose of the Study The southeastern peach industry. According to the National Agricultural Statistics Service (NASS-USDA, 2004), 45,350 metric tons of peaches (Prunus persica L. Batsch) were produced for shipping from Georgia during the 2002 season. The farm-gate value for the Georgia and South Carolina peach industries combine to exceed $70 million annually. California is the only state with a higher production level. Approximately 5670 of Georgia’s hectares are planted in bearing-age commercial peaches (NASS-USDA, 2004). Because of the warm and humid climate in the southeastern United States, it is a constant challenge to maintain tree health during and after orchard establishment. The problems that affect peach production and orchard longevity are both biotic and abiotic including fungi, bacteria, arthropods, nematodes, sunscald, frost damage, hail, drought, and even occasionally flooding. Many of the biotic problems tend to be heightened when replanting on old peach sites (Hine, 1961; Koch, 1955; Savage et al., 1953). Good management practices (such as proper planting, fertilization, and weed control), advances in chemical treatments (e.g., fumigants, fungicides, herbicides, and insecticides), and increased plant vigor (resistant rootstocks and improved scion
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