Grapevine Viruses and Associated Vectors in Virginia: Survey, Vector Management, and Development of Efficient Grapevine Virus Testing Methods Taylor Jones Dissertation submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Plant Pathology, Physiology, and Weed Science Mizuho Nita, Chair Anton B. Baudoin Douglas G. Pfeiffer Xiaofeng Wang Naidu A. Rayapati May 3, 2016 Blacksburg, Virginia Keywords: Grapevine leafroll disease; Grapevine red blotch associated virus; Nitrocellulose Membranes; real-time PCR; Mealybugs; Grapevines; Virginia Grapevine Viruses and Associated Vectors in Virginia: Survey, Vector Management, and Development of Efficient Grapevine Virus Testing Methods Taylor Jones ABSTRACT In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels. Grapevine Viruses and Associated Vectors in Virginia: Survey, Vector Management, and Development of Efficient Grapevine Virus Testing Methods Taylor Jones GENERAL AUDIENCE ABSTRACT In order to aid the booming wine industry in the state of Virginia, U.S.A., a series of studies were conducted. A statewide survey between 2009 and 2014 uncovered 12 viruses (with nine being reported for the first time in the state) among Virginia vineyards. Three viruses, Grapevine leafroll-associated virus, Grapevine red blotch associated virus, and Grapevine rupestris stem pitting associated virus, were prevalent ones. More than 90% of our surveyed vineyards (out of 116 visited) contained at least one virus-infected grapevine. This widespread nature of grapevine viruses in Virginia led to the development of new virus testing methods: a gene-based diagnostic tool that allows rapid and specific detection of grapevine red blotch- associated virus and a special membrane-based sampling method, which allows growers to sample at their own convenience and allows storage of virus genetic materials for multiple years. Because of its robustness, we envision that this technology can be applicable beyond grapevine viruses. We also found insects that can transmit these viruses from vine to vine: grape mealybugs, Gill’s mealybugs, and obscure mealybugs. This is the first study to demonstrate the ability of Gill’s mealybug to transmit grapevine leafroll-associated virus. For the management of insect vectors, four insecticide trials were implemented over multiple seasons. Some insecticides significantly reduced the existing mealybug populations; however, even the best treatments were not able to completely stop the spread of the virus to a new vineyard. Lastly, a study on the effects of co-infection of the three most common viruses on grape berry chemistry showed sugar levels, color pigments, and acidity of grape berries were all negatively impacted. Acknowledgement I would like to acknowledge my advisor and mentor Dr. Mizuho Nita for his help and aid in project endeavors. I would like to thank former and current members of the Nita lab who worked along with me in data collection and experimentation: Shantal Hover, Amanda Bly, Sabrina Hartley, and Logan Howard. I would also like to thank members of my committee for their guidance and support on the projects outlined. I would like to acknowledge Dr. Naidu Rayapati and members of his lab at Washington State University for their continued help and support. I would also like to thank my source of funding: The Virginia Wine Board. iv Table of Contents List of Figures vi List of Tables viii List of Abbreviations xi Chapter 1: Introduction 1.1 Introduction to the topic 1 1.2 Conclusion and Objectives 27 1.3 References 29 Chapter 2: Occurrence of grapevine viruses in Virginia, U.S.A. vineyards and factors affecting virus infected vines. 2.1 Introduction 43 2.2 Materials and Methods 46 2.3 Results 52 2.4 Discussion 56 2.5 References 75 Chapter 3: Grapevine red blotch associated virus and the development of a real-time PCR assay. 3.1 Introduction 81 3.2 Materials and Methods 83 3.3 Results 89 3.4 Discussion 92 3.5 References 107 Chapter 4: Transmission of GLRaV-3 via Ferrisia gilli and evaluation of insecticidal treatments and spatio-temporal association of GLRaV-3 and mealybugs in Virginia Vineyards. 4.1 Introduction 110 4.2 Materials and Methods 112 4.3 Results 124 4.4 Discussion 129 4.5 References 153 Chapter 5: Nitro-pure nitrocellulose membranes as an effective solid storage and testing medium for grapevine viruses. 5.1 Introduction 158 5.2 Materials and Methods 162 5.3 Results 169 5.4 Discussion 173 5.5 References 184 Chapter 6: Changes in basic grapevine berry chemistry due to effects of single and co- infections of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus, and Grapevine red blotch associated virus. 6.1 Introduction 189 6.2 Materials and Methods 193 6.3 Results 197 6.4 Discussion 199 6.5 References 206 v List of Figures Chapter 2. Figure 2.1: The state of Virginia, U.S. and its five “wine regions” divided on 74 the map. Chapter 3: Figure 3.1: Symptoms and genome of GRBaV. 102 Figure 3.2: The evolutionary history was inferred for GRBaV V2 gene 103 isolates (rooted with Maize streak virus) from VA and GenBank using the Neighbor-Joining method. Figure 3.3: Determination of efficiency of real-time PCR assays using 105 standard curve analysis and Ct slope method with seven concentrations covering a 6-log dilution range for two extraction methods of the sample for Primer/Probe set 3 and the endogenous control. Figure 3.4: Non-significant general trend of 2 –ΔCt values of GRBaV positive 106 grapevines by month and region of sampling. Chapter 4: Figure 4.1: Spread of GLRaV-3 through 10 rows x 10 vine field of cultivar 142 Chardonnay. Figure 4.2: AHS AREC Cabernet Sauvignon trial diagram: Cabernet 143 Sauvignon inter-planted vineyard layout at the Winchester, AHS AREC, VA, USA. Figure 4.3: The mean and standard error of mealybug counts per treatment per 144 date from the Cabernet Sauvignon AHS AREC interplanting trial in Winchester, VA, USA in 2012 (A), 2013 (B), and 2014 (C). Figure 4.4: Spread of GLRaV-3 through the vineyard at the Cabernet 146 Sauvignon AHS AREC interplanting trial. Figure 4.5: The mean and standard error of mealybug count per treatment per 147 date from the Merlot AHS AREC trial in Winchester, VA, USA throughout time in 2012 (A), 2013 (B), and 2014 (C). Figure 4.6: The mean and standard error of mealybug count per treatment per 149 date from the Orange County, CA Chardonnay trial in 2013 and 2014 vi seasons. Figure 4.7: The mean and standard error of mealybugs per treatment per date 151 from the Orange County, VA Rkatsiteli trial in Orange, VA, USA in 2012 (A), 2013 (B), and 2014 (C). Chapter 5: Figure 5.1: PCR amplification of the V2 gene of GRBaV (~446bp) isolates 180 using varying extraction buffers and template combinations. Figure 5.2: Mean Ct values per sample based on three independent 181 amplifications per sample for the resveratrol synthase gene (endogenous control) (A) and V2 gene of GRBaV (B). Figure 5.3: Determination of efficiency of real-time PCR assays using 183 standard curve analysis and Ct slope method with seven concentrations covering a 6-log dilution range for the NPN membrane extraction method for GRBaV. vii List of Tables Chapter 2. Table 2.1: Primers used for conventional and RT-PCR detection of grapevine 64 viruses. Table 2.2: Grapevine virus survey samples by virus species tested and number 65 found. Table 2.3: Counts of the most common types of single and co-infections of all 66 grapevine viruses found in this study. Table 2.4: Kendall’s tau (upper right triangle) and Pearson’s correlation 68 coefficient (lower left triangle) values and P-values among six commonly found grapevine viruses, Virginia 2009-2014. Table 2.5: Differences in test statistics and estimated variances for the effect of 69 region (R), vineyard within the region (V), and cultivar within the farm (C) on virus detection in the Virginia grapevine virus survey sample 2009-2014. Table 2.6: Effect of vine age (planting time period) on the percentage of viruses 70 found in the Virginia grapevine virus survey sample 2009-2014.