ABSTRACT FLOYD, LEAH EVELYN. Reservoir Hosts and Vectors of Xylella fastidiosa, Causal Agent of Pierce’s Disease of Grapevines, in North Carolina. (Under the direction of Turner Bond Sutton). Pierce’s disease (PD) of grapevines is caused by the xylem-limited bacterium Xylella fastidiosa (Xf) which is transmitted by leafhoppers and spittlebugs. Leafhopper populations were surveyed in six vineyards across North Carolina’s three geographic regions, in 2006 and 2007. Populations of two phloem-feeders, Agallia spp. and Paraphlepsius irroratus were compared with populations of known PD vectors, Graphocephala versuta and Oncometopia orbona as well as other leafhoppers and planthoppers. G. versuta was the dominant species in the Piedmont and Mountain regions in 2007 and in one Coastal Plain vineyard in 2006. Agallia spp. were most common in the Coastal Plain in 2007, and in the two Mountain and Piedmont vineyards in 2006. The possibility that a phloem-feeder may transmit Xf to grapevines was examined. The clover leafhopper, A. sanguinolenta, a phloem-feeder, did not transmit Xf to grapevines and the bacterium was not detected in its mouthparts using RT-PCR analysis. A reservoir host list of Xf was developed for North Carolina. This was developed using ground vegetation surveys conducted in three vineyards in the spring and fall of 2007-08 to identify and quantify plant species growing on the vineyard floor. Plant samples were collected and tested for the presence of Xf with ELISA and PCR. Fourteen of 40 plant species surveyed tested positive with ELISA and two were confirmed with PCR. Plant hosts of Xf identified in this study that have not been previously reported are: Chamaesyce maculata, Trifolium arvense, hop clover, Trifolium spp., Geranium carolinianum, Oxalis stricta, Festuca sp., Setaria sp., Hordeum pusillum, Poa trivialis and Ranunculus sp. Plant hosts of Xf identified that were previously reported to host PD-strains of the bacterium include: Trifolium repens, Plantago lanceolata, Digitaria sp., and Cynodon dactylon. To further examine the importance of potential groundcovers as reservoir hosts, experiments were conducted to find a groundcover that the known PD vector, Graphocephala spp. does not prefer for feeding. None of the plant species tested were found to be poor reservoir hosts, with regard to G. versuta feeding preference. Additionally, the effect of the presence of fungal endophytes on the survival of G. versuta was investigated. Those present in Festuca rubra spp. rubra and Festuca rubra ssp. commutata did not affect the survival of G. versuta. We recommend removing broadleaf weeds from the vineyard floor, and studies to find a suitable groundcover should be continued. Reservoir Hosts and Vectors of Xylella fastidiosa, Causal Agent of Pierce’s Disease of Grapevines, in North Carolina by Leah Evelyn Floyd A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science Plant Pathology Raleigh, North Carolina 2008 APPROVED BY: George G. Kennedy David F. Ritchie Turner B. Sutton Chair of Advisory Committee BIOGRAPHY Leah Evelyn Floyd was born July 16th 1984. She grew up in Oxford, North Carolina and attended J.F. Webb High School. Upon graduation, she began an undergraduate degree in the Department of Botany at North Carolina State University, where she became interested in plant pathology. Leah worked for the NCSU Plant Disease and Insect Clinic and the Department of Plant Pathology’s Fungal Genomics laboratory as an undergraduate. She began working for Dr. Turner Sutton during her senior year, and began her Master of Science degree in Plant Pathology at North Carolina State University in 2006 under his direction. ii ACKNOWLEDGMENTS I would like to thank Dr. Turner Sutton, chair of my advisory committee, for supporting this research, and for all the opportunities to practice extension and develop professionally. The learning experiences have certainly extended above and beyond scientific knowledge. Thanks to Dr. David Ritchie and Dr. George Kennedy, my committee members, for their guidance with this project. Thanks to all current and former members of the Sutton laboratory who have helped with the collection and processing of samples, and advised me along the way. I would also like to thank all of my family and friends for supporting me as I pursued my degree. Most of all, thanks to my parents for allowing me to focus on education throughout the years. iii TABLE OF CONTENTS LIST OF TABLES ………… ……………... ……………..……………………………... v LIST OF FIGURES ………................................................................................................ vi CHAPTER 1 ....................................................................................................................... 1 Introduction ............................................................................................................ 1 Materials and Methods ........................................................................................... 3 Leafhopper Population Surveys ................................................................. 3 Transmission Experiments ......................................................................... 4 RT-PCR Analyses ...................................................................................... 5 Results .................................................................................................................... 7 Leafhopper Population Surveys ................................................................. 7 Transmission Experiments ......................................................................... 9 RT-PCR Analyses ...................................................................................... 9 Discussion .............................................................................................................. 10 Literature Cited ...................................................................................................... 13 CHAPTER 2 ...................................................................................................................... 14 Introduction ............................................................................................................ 14 Materials and Methods ........................................................................................... 17 Ground Vegetation Surveys ....................................................................... 17 Testing Potential Reservoir Hosts for Xf ................................................... 17 Host Plant Suitability Studies .................................................................... 19 Fungal Endophyte Effects on Leafhopper Survival ……………………... 21 Results .................................................................................................................... 24 Ground Vegetation Surveys ....................................................................... 24 Testing Potential Reservoir Hosts for Xf ................................................... 25 Host Plant Suitability Studies …………………………............................. 25 Fungal Endophyte Effects on Leafhopper Survival ……………………… 25 Discussion .............................................................................................................. 26 Literature Cited ...................................................................................................... 32 APPENDIX ........................................................................................................................ 55 iv LIST OF TABLES CHAPTER 2 Table 1. Vineyard floor vegetative composition in 2007 in (A) Polk, (B) Yadkin and (C) Guilford counties, NC...........................................46 Table 2. Vineyard floor vegetative composition in 2008 in (A) Polk, (B) Yadkin and (C) Guilford counties, NC…………………...............48 Table 3. Plant species tested for Xf with ELISA, locations and dates for which samples were taken, and frequency with which species tested positive ....................................................………...........................50 v LIST OF FIGURES CHAPTER 1 Figure 1. Proportion of leafhoppers found in Polk Co. NC vineyard # 1 in (A) 2006 and (B) 2007…………………………………………….35 Figure 2. Proportion of leafhoppers found in Polk Co. NC vineyard # 2 in (A) 2006 and (B) 2007…………………………………………….36 Figure 3. Proportion of leafhoppers found in Alamance Co. NC vineyard in (A) 2006 and (B) 2007…………………………………...........................37 Figure 4. Proportion of leafhoppers found in Wake Co. NC vineyard in (A) 2006 and (B) 2007…………………………………………….38 Figure 5. Proportion of leafhoppers found in Currituck Co. NC vineyard # 1 in (A) 2006 and (B) 2007……………………………….........................39 Figure 6. Proportion of leafhoppers found in Currituck Co. NC vineyard # 2 in (A) 2006 and (B) 2007……………………………….........................40 Figure 7. Average number of Agallia spp. per trap across all vineyards in (A) 2006 and (B) 2007…………………………………………….41 Figure 8. Average number of Paraphlepsius irroratus per trap across all vineyards in (A) 2006 and (B) 2007…………………………………42 Figure 9. Average number of Graphocephala versuta per trap across all vineyards in (A) 2006 and (B) 2007…………………………………43 Figure 10. Average number of Oncometopia orbona per trap across all vineyards in (A) 2006 and (B) 2007…………………………………44 Figure 11. Average number of “other” leafhoppers per trap across all vineyards in (A) 2006 and (B) 2007…………………………………45 CHAPTER