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The Pennsylvania State University The Graduate School College of Agricultural Sciences ELM YELLOWS PHYTOPLASMA DETECTION IN TREES AND INSECTS A Thesis in Plant Pathology by Padmini Herath © 2009 Padmini Herath Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2009 ii The thesis of Padmini Herath was reviewed and approved* by the following: Gary Moorman Professor of Plant Pathology Thesis Adviser Donald Davis Professor of Plant Pathology Frederick Gildow Professor of Plant Pathology Head of the Department of Plant Pathology Gregory Hoover Sr. Extension Associate *Signatures are on file in the Graduate School. iii ABSTRACT A rapid, sensitive and accurate method to detect Elm Yellows (EY) phytoplasma and to identify EY- positive elm trees and the possible vectors of EY phytoplasma was developed using a real– time PCR (RT-PCR) procedure based on the TaqMan MGB probe. Primers and the TaqMan probe were designed based on the EY specific protein translocation secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trnL gene of Ulmus americana. Using the RT-PCR assay it was possible to detect phytoplasma in samples that tested negative with nested PCR by a commercial company, thereby demonstrating the superior sensitivity of the novel method. The RT- PCR test developed did not cross react with either Illinois elm yellows phytoplasma or aster yellows phytoplasma DNA. A survey of the elms on the Pennsylvania State University, University Park campus (PSU-UP) was started in May 2008 and continued through June 2009. The RT-PCR detected 35 EY- positive trees. Threshold cycle (CT ) values obtained from the EY infected elms ranged from 15- 37. A survey for leafhoppers was carried out from May to July 2008, at six locations on and around the PSU campus. Based on leafhopper morphology, 17 different taxa belonging to 5 subfamilies of the family Cicadellidae were collected from insect traps. Scaphoideus sp. and Allygus sp. were most abundant. EY phytoplasma was detected in 14 leafhopper morphological groups. iv TABLE OF CONTENTS List of Tables…………………………………………………………………………………….. v List of Figures…………………………………………………………………………………….vi Acknowledgements……………………………………………………………………………....vii Chapter 1.LITERATURE REVIEW………………………………………………………………1 Introduction…………………………………………………………………………….1 Elm Yellows Disease…………………………………………………………………..2 Phytoplasma……………………………………………………………………………4 Real-Time PCR………………………………………………………………………...7 References……………………………………………………………………………...9 Chapter 2.DEVELOPMENT OF REAL-TIME PCR TO DETECT ELM YELLOWS PHYTOPLASMA………………………………………………………..13 Introduction…………………………………………………………………………...13 Material and Methods………………………………………………………………...14 Results………………………………………………………………………………...19 Discussion…………………………………………………………………………….22 References…………………………………………………………………………….24 Chapter 3. ELM YELLOWS AT THE PENNSYLVANIA STATE UNIVERSITY…………....26 Introduction…………………………………………………………………………...26 Material and Methods………………………………………………………………...27 Results………………………………………………………………………………...28 Discussion…………………………………………………………………………….33 References…………………………………………………………………………….35 Chapter 4. INSECT VECTORS OF ELM YELLOWS PHYTOPLASMA …………………….36 Introduction…………………………………………………………………………...36 Material and Methods………………………………………………………………...37 Results………………………………………………………………………………...38 Discussion…………………………………………………………………………….45 References…………………………………………………………………………….48 v LIST OF TABLES Table 2.1. Primers and probe sequences for detection of the secY EY phytoplasma gene and the U. americana trnL chloroplast gene………………………………....17 Table 3.1. EY phytoplasma detection in elm trees at PSU-UP……………………………….28 Table 3.2. CT values of EY phytoplasma DNA extracted from elms……………...…………29 Table 3.3. Distribution of EY phytoplasma in the tree 29-37. Twig, trunk bark and leaf samples were analyzed using the TaqMan real-time PCR and the nested PCR-RFLP …..………………………………………………….....30 Table 3.4. Distribution of EY phytoplasma in the tree C 4. Twig, trunk bark and leaf samples were analyzed using the TaqMan real-time PCR and the nested PCR- RFLP ……………………………………………………..31-32 Table 4.1. Family Cicadellidae leafhoppers captured from elm trees………………………....39 Table 4.2. Leafhoppers collected at six sites of EY occurrence near PSU in 2008 and detection of EY phytoplasma by TaqMan real-time PCR………….....40-44 vi LIST OF FIGURES Figure 1.1. The plot of fluorescence signal vs PCR cycle number…...........................................9 Figure 2.1. The sequence of the secY gene(GenBank no: AY 197690) used to design the primers/probe to detect EY phytoplasma by TaqMan real-time PCR………………………………………………………...16 Figure 2.2. RT-PCR on EY reference sample…………………………………………………20 Figure 2.3. Agarose gel (1.5 %) showing amplification products obtained from nested PCR………………………………………………………...21 Figure 3.1. Location of the EY infected trees at the PSU-UP campus……………………….29 Figure 3.2. Tree number 29-37 with subsample numbers………….………………………….31 Figure 3.3. Tree number C4 with subsample numbers………………………………………...33 vii ACKNOWLEDGEMENTS I am deeply indebted to Prof. Gary Moorman, my advisor, for his advice and constant encouragement throughout the entire period of study. Without his support it would not have been possible to complete this research. I sincerely appreciate him giving me the opportunity to undertake this research project. I express my deep gratitude to Greg Hoover, for identifying the leafhoppers and sharing his “hands on experience” with me. I am very grateful to Prof. Fred Gildow and Prof. Don Davis for their thoughtful advice and constant encouragement. I express my sincere thanks to Drs. Sarah Melissa Waitiak, Deborah Grove, Irmgard Siedle- Adams and Xinshun Qu who kindly advised me and assisted me in the real-time PCR experiments. Discussions with them are gratefully appreciated and they all were wonderful sources of knowledge. My sincere thanks go to The Pennsylvania State University for financing this research project. I am very grateful to Jeff Dice, Kris Edson and other Penn State arborists for their tireless help with the field experiments. I gratefully acknowledge Dr. Ing-Ming Lee for providing Elm Yellows phytoplasma and Illinois Elm Yellows phytoplasma DNA samples. Special thanks to Prof. David Geiser, Prof. Scott Isard and Prof. Beth Gugino for allowing the use of their laboratory facilities. In addition, I am deeply indebted to Prof. David Geiser for his help in getting started with my M.S. program and serving as my advisor during the first year. I am also very grateful to Rachel Leonard, Emily Dice and Camille Francl for their help in laboratory experiments. Their dedication is really appreciated. I also thank my fellow lab mate Maria Burgos for her friendship and encouragement. Finally I am very grateful to my husband Nuwan and daughter Malithi for their support and patience throughout this period. 1 CHAPTER 1 LITERATURE REVIEW INTRODUCTION American elm (Ulmus americana L.) trees are well known for their great aesthetic value as shade trees because of the graceful, arching, vase-like architecture and tolerance to stressful conditions. Dutch elm disease (DED), caused by an ascomycete fungus Ophiostoma ulmi (Buisman) Nannf., has all but eliminated this tree species from urban forests in the eastern U.S. Numerous cultivars of DED resistant elms have been developed (25,30). Yet another widespread and serious disease of elms for which there is no resistance is the Elm Yellows (EY; previously called elm phloem necrosis). This disease is caused by the phytoplasma, „Candidatus Phytoplasma ulmi‟, which is classified as a member of the “Elm Yellows” group. At present, elms on The Pennsylvania State University campus are affected by EY, causing serious concerns among the Penn State community. Most of the research on EY has focused on mapping disease prevalence in different parts of the world and the identification of insect vectors using the Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) methodology. Nevertheless, a rapid and sensitive diagnostic method for high throughput large scale analyses is still lacking. Therefore my research goals were to develop a rapid and accurate diagnostic method to detect EY phytoplasma, to detect the presence of EY phytoplasma in elm trees on the Penn State campus and to identify possible vectors of the EY phytoplasma. The successful completion of the research program will advance our understanding of EY and facilitate its early detection, vector identification and disease control. 2 1) Elm Yellows Disease A. History and Epidemics EY was first described in detail by Swingle in 1938 (31). However, the presence of EY had been reported as early as 1882 in the Ohio River valley and was thought to be caused by a virus (3, 31). Following its first description, EY was reported in the eastern USA and in central and southern Europe. In the USA, EY is known for epidemics that kill nearly all native elms in affected localities. Several outbreaks of EY have been reported from Ohio (1940), Illinois (1960), Pennsylvania and New York (1970) (14). According to Lanier et al. (14) approximately 58% of the elms were infected and lost from 1981 through 1984 in Syracuse, New York. However, EY does not spread rapidly. An outbreak in New York observed over 15 years advanced only at a rate of 6 km /year on average (14). In contrast the disease in Europe is of little importance and usually not lethal. For example in northern Italy, EY has appeared in scattered