Mealybug Pests and an Emerging Viral Disease: Vector Ecology and Their Role in Grape Leafroll Associated Virus Epidemiology

Grapevine leafroll-associated viruses (GLRaV) are a complex of viruses that cause leaf chlorosis and leaf margins to ?roll? downward. GLRaVs can reduce yields, delay fruit maturity, and impede fruit pigmentation. Our work concerns GLRaV field epidemiology with respect to its insect vectors. In field studies, we evaluated grape mealybug acquisition and transmission of GLRaV-3 from trunks, spurs, canes, and leaves. Two point three percent of mealybug crawlers acquired GLRaV-3 from vines in the field and transmitted leafroll virus to vines in the greenhouse. This is a lower transmission rate than previously reported from greenhouse studies. Surveys of grape mealybugs from five Napa County vineyards were taken monthly (May through October) and found 56%of grape mealybugs collected on leafroll-infected vines tested positive for GLRaV-3. The proportion of leafroll-infective mealybugs was related to the number of leafroll-positive vines in the vineyards surveyed. We continued a five-year field trial testing the impacts of ?zero tolerance? for mealybugs on GLRaV infection establishment and spread. A 20 acre vineyard planted in 2008 from certified virus-free scion, and bordered by older blocks that contained both GLRaV-infected vines and mealybugs, received pesticide treatments for mealybugs in 2009, 2010 and 2011. No mealybugs were found in visual inspections; however, pheromone traps showed the presence of male grape mealybugs. All vines were inspected annually for GLRaV symptoms. In 2009 and 2010, there was one new infected vine each year, while in 2011, six new vines tested positive for GLRaV-3. GLRaV-3 variants in the spray trial block (-3a, -3c, and -3d) were different than the GLRaV-3 variants in adjacent virus-infected blocks to the north and west (-3b). The leafroll-infected vines were randomly distributed inside the mapped plot, with similar numbers in insecticide and untreated vines. One hypothesis is that virus-carrying grape mealybug crawlers were ?blown? into the plot, and infecting previously-healthy vines during feeding. This trial will continue for two more years and to see if disease-free blocks can be established though the use of annual insecticide treatments to eliminate mealybug vectors. We investigated grape phylloxera as a possible vector of GLRaV. Previous studies in New Zealand excluded this insect as a vector and we consider this to be the standard guideline. Nevertheless, we are conducting trials to alleviate grower concerns and eliminate the possibility that phylloxera play a role in GLRaV transmission in California. After feeding on virus-positive vines in the greenhouse for six months, between 1 and 5%of phylloxera acquired GLRaV-3 or -5, while none acquired GLRaV-1 or -2. None of the 44 healthy vines planted in the same pots as virus-and phylloxera-infected plants developed GLRaV. Additionally, none of the 939 phylloxera collected from GLRaV-2 infected vineyards contained leafroll virus, leading to a conclusion that phylloxera is unlikely to be a vector of GLRaV.

Bio-economic Analysis of Grape Leafroll Virus Epidemics in California

Objectives of Proposed Research: 1. Analyze historical data maps of leafroll symptoms to elucidate quantitative parameters summarizing disease progress in time and space at an individual vineyard scale and at a landscape scale. 2. Develop a robust simulation model of disease spread based on the parameters elucidated in objective 1. 3. Validate the simulation analysis by comparing simulated disease dynamics against data collected from surveys of a range of vineyard types and an existing validation data set. 4.Use the simulation model in conjunction with financial data for grape production to explore a range of disease management tactics appropriate to different market sectors. 5.Analyze variation in levels of knowledge and opinion among growers concerning the impact of leafroll disease on grape/wine quality and vine economic life expectancy. 6.Explore the potential for developing area-wide disease management plans by facilitating grower focus groups for leafroll disease. Executive Summary: Progress has been made on six objectives of the project. The overall project is composed of two sub¬projects, the first concerning the basic epidemiology of the disease and its impacts at the individual planting block scale (Objectives 1 – 4), the second concerning the human-disease interaction (Objectives 5 and 6). Leafroll Epidemiology Objective 1: Analyses of historical data sets from the Napa Valley and other locations have revealed a consistency in the spatial and temporal statistical properties of leafroll epidemics that strongly suggests the same dispersal mechanism operates in all leafroll epidemics and that such epidemics will behave in a way that is predictable in general terms. Across four published studies (two from Spain, one from New Zealand, one from Napa) re-analysis of the original data suggest that leafroll disease incidence increased at an average rate of just under 12% of plants per year (mean percentage increase = 11.7%, minimum = 4.4%, maximum = 20.1%). The rate parameter of the logistic growth function estimated from the pooled disease incidence data was estimated to be 0.58. Starting from a completely healthy condition, with increase in disease incidence determined by these parameters it would take 8 years for disease incidence to reach 50% and after 15 years 99% of plants would be diseased. Analysis of the spatial pattern of disease incidence using a quadrat-based approach indicated that leafroll epidemics have an effective sample size of approximately 3 plants. This effective sample size is robust across studies involving epidemics covering different ranges of disease incidence, different sizes of vineyard, and different grape cultivars in different locations. The results indicate that leafroll has a highly patchy spatial pattern and a very short average dispersal distance within vine blocks. The consistency of the relationship is good news for efforts to develop robust simulations of the disease and for making predictions of disease impacts. However, the patchy nature of the disease has negative effects on sampling accuracy, and will make accurate estimates of true disease incidence relatively difficult to obtain at the outset of epidemics. Objective 2 The temporal and spatial statistics estimated from the historical data, together with simple generic rules about relationships between disease incidence measurements at different scales [1] provide the basic components of the simulation model for generating leafroll development patterns. The simulation model generates blocks of plants in which disease incidence increases according to a logistic growth curve and the pattern of diseased plants in the block is constrained in two ways. Following the analyses of published data the frequency distribution of diseased plants per quadrat in the simulated data is assumed to follow a time-dependent binomial distribution in which the sample size is 3 plants. Objective 3 ? Work is currently in progress to compare the simulation model with validation data collected in the Napa Valley. Objective 4 ? With the format of the simulation established we will be able to attach financial values to disease on a per plant basis and project revenue and costs for infected blocks according to using either generic information on costs or specific information for particular blocks of vines in specific locations. Human-disease interactions Objective 5 & 6 Research on objectives 5 and 6 was combined in a single study based on a Q-method approach. Three data generating workshops were held with key stakeholders in the Napa Valley grape growing and wine-making sectors. At each workshop participants were asked to write answers to a set of open-ended questions concerning the importance of leafroll and the different approaches that can be used to manage it, including cooperative pest management in neighborhood groups. We sorted the responses into subject area groups and generated a set of 47 response statements that summarized viewpoints expressed in the written responses. Email and telephone contacts were used to generate a second set of 37 respondents (with some overlap to the original workshop participants) who were invited to take part in the Q-sort exercise. In each Q-sort interview a respondent was asked to rank each of the statements on a scale from -5 (‘does not reflect my opinion’) to 5 (‘completely represents my opinion’). During the sorting the number of statements allowed in each category was constrained so the respondents were forced to rank the statements according to their own viewpoint. The analysis revealed a generally high level of awareness of the seriousness of the GLRaV among respondents, but a wide variety of views about the most important factors in the disease and how it should be managed. A small group of statements, focusing on the importance, availability and reliability of virus-tested planting material ranked highest of all statements (scoring about 75% of the maximum possible score achievable if all respondents had rated them in maximum accordance with their opinion). The lowest scoring statement concerned the relative importance of irrigation and leafroll to vine health (35% of maximum possible). Statements concerning the value of cooperation for leafroll management tended to score close to the average and did not have particularly high variances. These results indicate neither generally strongly favorable nor negative views about cooperation. Principal components analysis of the responses suggested that while each person had an essentially unique viewpoint there was some grouping among the respondents. We are currently analyzing the data in more detail, but the initial interpretation of the analysis is that the respondents can be classified by their viewpoints as: focused either on the financial aspects of the disease or its technical implications; either having an open-ended view of the problem or seeing it as an issue with a definite end-point; either being essentially strategic in approach or essentially tactical; either being optimistic or fatalistic.

Grapevine Leafroll Disease, a General Detail Study and Evaluation

Grapevine leafroll disease causes poor color development in red grape varieties and non-uniform maturation of fruit in Vitis vinifera. It also has been reported to cause delay in fruit maturation from 3 weeks to a month and crop losses of as much as 20-40%. To date 11different viruses, named Grapevine leafroll associated virus (GLRaV) types -1 to -7, -9, -10, -11 and a newly discovered virus from a Carnelian clone which tentatively has been named GLRaV-Car are proven to be associated with leafroll disease. This project was planned to study the effects of GLRaV types -1 and -2 (2 isolates each), -3 (3 isolates), -4, -5, -7 and -9 (one isolate each) and a combination of two of types -1, -2, -3, -5 and -7 on Cabernet Franc grafted on 9 different rootstocks. Rootstocks used are AXR #1, Mgt 101-14, 110R, 3309C, 5BB, 420A, Freedom, St. George 15 and St. George 18. In this experiment 15 replicates for each treatment in three different blocks (5 replicate per treatment per block) were used. The data collected from the experiment in 2011 showed that the virus isolate LR132 (a mixed infection of GLRaV-1 and Grapevine virus A) had unfavorable reaction on Cabernet Franc plants propagated on 420A, Freedom, 3309C and 101-14 rootstocks and many of these plants died few months after inoculation. It is not clear whether a certain strain of GLRaV-1 is the cause of poor vine performance or the presence of GVA creates a synergistic effect and kills the plants. It was also found that the presence of GLRaV-2 had harmful reactions on vines propagated on Freedom and 5BB. These vines were week and exhibited red leaf symptoms, short internodes and almost dying. Furthermore, it was shown that different GLRaV types produce leaf symptoms with different severity, for example, it was observed that leaf symptoms produced by GLRaV-3 was more severe than by GLRaV-4.

Survey for Possible Reservoirs for Leafroll Viruses in North Coast

In out initial survey we detected Grapevine virus A (GVA) and /or Grapevine leafrollassociatedvirus 3 (GLRaV-3) in V. californica and V. californica x V. vinifera hybrids in the non-cultivated areas adjacent to three Napa Valley vineyards with leafroll disease symptoms. In addition, we identified Grapevine leafroll-associated virus 2 (GLRaV-2), GLRaV-3, GVA and/or Grapevine virus B (GVB) in V. californica and V. californica x V. vinifera hybrids in three riparian areas not near vineyards. This establishes that noncultivated Vitis spp. hosts for these leafroll and vitiviruses exist. Since our samples numbers were low, we returned to the three vineyards and two of the three riparian areas with positive V. californica and V. californica x V. vinifera hybrid samples and collected and additional 185 non-cultivated Vitis spp. and V. vinifera. We are currently testing these samples for GLRaV-2, GLRaV-3, GVA and GVB. When testing is complete, we will be able to estimate incidence at these sites. We also included two new vineyards with known GLRaV-2 and GLRaV-3 infections in order to extend our survey area to the more northern and southern regions of Napa County. All of the V. californica, V. californica x V. vinifera hybrids, and willow samples were negative. The material for biological assays has been collected.

Grapevine Leafroll Disease, a General Detail Study and Evaluation

Grapevine leafroll disease causes poor color development in red grape varieties and non-uniform maturation of fruit in Vitis vinifera. It also has been reported to cause delay in fruit maturation from 3 weeks to a month and crop losses of as much as 20-40%. To date 9 different viruses, named Grapevine leafroll associated virus (GLRaV) types 1 to 7, 9, and a newly discovered virus from a Carnelian clone which tentatively has been named GLRaV-Carnelian are proven to be associated with leafroll disease. This project was planned to study the effects of these GLRaV types 1 to 5, -7 and -9 and their combination of two on Cabernet Franc grafted on 9 different rootstocks including AXR #1, Mgt 101-14, 110R, 3309C, 5BB, 420A, Freedom, St. George 15 and St. George 18. In this experiment 20 replicates for each treatment will be used. Total of 6578 Cabernet Franc plants on 9 different rootstocks were produced by bench grafting. In 2009-2010, 3841 vines were inoculated with individual viruses or the combination of two per. Remaining 1186 plants will be inoculated in 2011. Seven acres of land in the Department of Plant Pathology Field Station at UC Davis has been prepared for planting by installing the drip irrigation pump and drip irrigation lines, leveling and setting up the trellis and planting 3841 virus-inoculated and healthy control plants. In 2010-2011, we continue the time course study choosing two months interval for testing the plants for virus titer. The viruses used in this experiment included GLRaV types 1-5 and GLRaV-2RG. The samples were tested by reverse transcriptase polymerase chain reaction (RT-PCR) and real time RT-PCR using TaqMan probe (qPCR) and the results were compared. The data showed that both methods were able to detect the viruses in the samples regardless of the time of collection. In general, the titer of the viruses selected in this investigation was higher in February and lowest in early growing season in the month of April.

Survey for Possible Reservoirs for Leafroll Viruses in North CoastSurvey for Possible Reservoirs for Leafroll Viruses in North Coast

This project addresses one specific concern: are there reservoirs of virus in plants other than cultivated grapevines that might be an important factor in the epidemiology of grapevine leafroll disease? To answer this question, we collected samples of 83 wild grapes (Vitis spp.) and 85 non-Vitis weed, cover crop and native species found in and near nine vineyards symptomatic for leafroll disease, and tested them for grapevine leafroll associated viruses (GLRaV) 1-5, 7, and 9, and vitiviruses GVA, GVB, and GVD. We also collected wild Vitis samples from two habitats distant from vineyards to assess virus incidence in a natural environment. The wild Vitis spp. genotypes were determined by DNA fingerprinting, and the genotypes were then analyzed and grouped into three categories: V californica, V. vinifera hybrids, and wild V. vinifera plus rootstock cuttings. We detected GLRaV-2, GLRaV-3, GVA, and GVB in all three groups of wild Vitis. Overall virus incidence was 27 percent, but percent infection differed depending on the genotype. At least one of the four viruses was detected in: 18%of the V. californica samples, 48 percent of the V. vinifera hybrids; and 100 percent of the wild V. vinifera plus rootstock cuttings. We also detected GLRaV- 3 in one out of six willows (Salix spp.). No viruses were detected in any of the other 84 host species we sampled. Viruses were detected in wild Vitis spp. in habitats both near to and distant from vineyards. Preliminary evidence indicates that there is no correlation between virus infection in vineyards and in nearby wild Vitis spp. Our sample size is small, however, and further sampling is needed. An ornamental grapevine, ?Roger?s Red?, was included in our samples. It is grown for its red fall foliage and was previously thought to be V. californica. Roger?s Red tested positive for GLRaV-2 and GVB. DNA fingerprinting indicated that it is a hybrid of V. californica x V. vinifera ?Alicante bouschet?. Alicante bouschet is a tintureiro variety known to have red leaves in the fall. Both the virus infection and genetic background could account for its red leaves in the fall. This survey establishes that wild Vitis spp. are hosts for two of the most important leafroll-associated viruses?GLRaV-2 and -3? in addition to GVA and GVB. This is the first evidence of a leafroll virus in a woody host besides Vitis spp. and has important implications for leafroll disease management.

Etiology and Detection of the Cause of Syrah Decline in Syrah grapevine

Syrah decline disease is a well documented problem in California and France. It is characterized by swelling and cracking of the graft union, stem pitting and grooving, and premature leaf reddening. French scientist have been studying this problem since 1999 and have failed to find any correlation with genetic incompatibility, known pathogens (viruses, bacteria, fungi, viroids), or environmental conditions. The potential of the Syrah decline disease to be associated with a virus is suspicious and the symptoms observed support the hypothesis. In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using dsRNA as template and the Life Sciences 454 high-throughput sequencing. In phase 1 (year 1) of this project we selected FPS Syrah clone 6 which was showing sever pitting, grooving and wood necrosis on the woody cylinder including die back and declining. For control a healthy looking Syrah clone 8 was selected. The Life Science 454 sequencer produced 67.5 megabases of quality sequence data, and screened for sequences of viral or viroid origin. The data revealed that the Syrah clone 6 supported a mixed infection that included seven different RNA genomes including 4 viruses and 3 viroids. In the second phase (year 2) of the project, 5 more syrah clones were selected and dsRNAs were produced and sequenced by the high-throughput sequencing facilities. The clones selected included three which are reported to produce high incidence of Syrah decline syndrome (clons B0 and B1 obtained from France and clone 99) and 2 with moderate incidence (clones 525 and 877). Total of 76.6 megabases of sequence information, from 371,906 fragment reads (each approximately 200 bases long) were initially produced from these five source vines in which 354,441 were high quality reads. Among high quality reads, 253,867 showed sequences which identified mostly as viral nucleic acids, but plant, bacterial and fungal sequences (and unassigned sequences) were also detected. Further analysis of the remaining unassigned reads (100,574) is in progress. Up to date segments of RNA sequences have been identified which belongs to members of two virus family, Betaflexiviridae and Tymoviridae, each includes number of viruses infecting grapevines. For example, Grapevine rupestris stem pitting associated virus, Grapevine virus A, -B, -D and -E are members of Betaflexiviridae and Grapevine fleck virus, Grapevine syrah virus-1 and Grapevine rupestris vein feathering virus are members of Tymoviridae. Research is in progress to identify the viruses present in each of these 5 Syrah clones by further analysis of the available sequences; to investigate the correlation between each virus found and the disease syndrome; and finally to develop molecular methodologies for their detection.

Development of Low Density PCR Array (LDA) for the Detection of Pathogens in Grapevines

The goal of this project was to develop sensitive and reliable molecular methods for the detection of viruses in grapevine. The method developed in this project is called “Low Density PCR Array” (LDA) and is a derivative of real time RT-PCR with TaqMan probe (TaqMan RT-PCR). LDA system uses a fluorescently tagged TaqMan probe and it fluoresces in the presence of target viral RNA in an incremental level following each PCR cycle. The released fluorescent signal then is analyzed by a laser-based thermocycler and evaluated.

In this project we also optimized the buffer for the 6700 automated Nucleic Acid Workstation for sample preparation and RNA extraction for use in LDA system. The results showed that the 2X ABI lysis buffer (from Applied Biosystems Inc.) worked better than 1X (the buffer routinely used for the extraction of total RNA from different biological tissues by the automated 6700 Workstation) for grapevine tissue and it was comparable to the expensive and time consuming RNeasy column kit produced by Qiagen Inc. The 6700 automated system is quite fast and inexpensive and uses a 96 well plate format and can prepare RNA from 96 samples in approximately 2 hours. Furthermore, the system capacity is approximately 400 samples per day and built into a BSL2 laminar flow hood to reduce cross contamination between samples. In an experiment, total RNA extraction methods were compared by using 29 vines infected with Grapevine leafroll associated virus (GLRaV) types 2, 3 or their combination. The RNA extraction methods used were Qiagene method and the 6700 automated workstation using 1X and 2X lysis extraction buffer. The TaqMan RT-PCR results showed that 15, 12 and 19 plants were tested positive for GLRaV-2 by using Qiagen, 1X and 2X buffer, respectively. For GLRaV-3 the numbers tested positive were 9, 7 and 9 respectively.

Primers and TaqMan probes were also designed for 16 different viruses in grapevine and all were evaluated for their efficiency in detecting the viruses. These viruses included GLRaV-1 to -7, -9, Car; GLRaV-2-RG; Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFKV), Rupestris stem pitting associated virus (RSPaV); Grapevine virus A (GVA), B (GVB) and D (GVD). The LDA system developed for these 16 viruses was compared with TaqMan RT-PCR and the standard RT-PCR using 29 different grapevine cultivars and selections with multiple virus infection from different grape growing regions in the world. The results showed that the LDA method was comparable to TaqMan RT-PCR in detecting viruses and both were superior to RT-PCR. The efficiency of RT-PCR, TaqMan RT-PCR and LDA in detecting viruses in different growing seasons were also compared using five vines each infected with GLRaV-1, -2, -3 and -5 (closteroviruses); GVA and RSPaV (rugose wood complex); GFLV (nepovirus); and GFkV (maculavirus). The results showed that TaqMan RT-PCR and LDA methods could consistently detect the viruses in different growing season. The developed LDA method has been incorporated into testing scheme at Foundation Plant Services at UC Davis.

Etiology and Detection of the Cause of Syrah Decline in Syrah grapevine

Syrah decline disease is a well documented problem in California and France. It is characterized by swelling and cracking of the graft union, stem pitting and grooving, and premature leaf reddening. French scientist have been studying this problem since 1999 and have failed to find any correlation with genetic incompatibility, known pathogens (viruses, bacteria, fungi, viroids), or environmental conditions. The potential of the Syrah decline disease to be associated with a virus is suspicious and the symptoms observed support the hypothesis.

In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using Life Sciences 454 high-throughput sequencing. 67.5 megabases of quality sequence data were derived from reverse-transcribed cDNA fragments, and screened for sequences of viral or viroid origin. The data revealed that a Syrah vine, FPS clone 6, showing decline symptoms supported a mixed infection that included seven different RNA genomes. Four viruses and three viroids were identified. Three viruses were found to be the predominant agents in a declining Syrah plant. Two of these were known viruses, each of which has been observed to cause a mild or asymptomatic infection when present singly. Grapevine rupestris stem-pitting associated virus (GRSPaV, a foviavirus) was the most prevalent virus in the census, as represented by fragment count. Another predominant virus found was a marafivirus, Grapevine rupestris vein-feathering virus (GRVFV) and The third one

was a new virus tentatively named Grapevine Syrah virus 1 (GSyV-1) and was most closely homologous to GRVFV. GSyV-1 differed from other marafivirus species by 40% or more and had not been identified in multiple analyses done using previously available technologies but the high number of fragments generated by the 454 analysis allowed for its identification against the background of the related and more prevalent GRVFV. The fourth virus found was a known virus, Grapevine leafroll associated virus 9, with low reads indicating to be present in the plant at low titer. Diagnostic primers were designed from unique sequence regions on the genome of GSyV-1. A field survey was conducted on samples collected from Napa, Sonoma and Yolo Co. using the specific detection primers for the virus. A preliminary analysis of 154 plants in vineyards where decline was evident showed only thirty vines (19%) positive for the virus. Further surveys are underway.

In the second experiment, samples were prepared from five Syrah clones and submitted to 454 sequencing facilities on November 2008 and the sequences were ready in early December. Total reads of 371,906 reads with approximately 200 nucleotides per read were produced and the sequence analysis is in progress. The Syrah clones included in this experiment were, clones 99 (high incidence of decline according to French scientists), 877 and 525 (both with moderate incidence of decline). Two other clones are designated as B0 and B1 which were obtained from France for this research project. B0 clone showed cracks at the union with no red leaf canopy and B1 showed cracks with red leaf canopy.

Grapevine Leafroll Associated Virus (GLRaV) types 4 & 6, Molecular Charachterization, Biological Study and RT-PCR Detection

Sequencing of the genomic RNA of Grapevine leafroll associated virus (GLRaV) -4 and -6 was completed. In this effort 13250 and 13811 nucleotides (nt), respectively, for GLRaV-4 and -6 were sequenced. These sequences included all open reading frames (ORF, or functional genes) of the viruses. From the organizational point of view, genomes of these two viruses appeared virtually identical and encoding for 6 viral proteins: replicase-associated protein (encoded by ORF1a and ORF1b by ribosomal frame shift), small hydrophobic protein (p5), HSP-70 homologue (HSP70h), 60K protein (p60), viral coat protein (CP) and protein with a molecular mass of 23K (p23) at the very extreme 3?end. The genome organization of GLRaV-4 and -6 suggests that both viruses belong to the genus Ampelovirus in the Family of Closteroviridae of plant viruses. Amino acid (aa) comparisons of the ORFs showed that the ORF2 (P5) was the most conserved gene among GLRaVs-4, -6 and -9 and showed 89-91%identity. In comparing the aa sequences of the coat protein (CP) of GLRaVs-4, -5, -6 and -9, it showed that the size of the viral coat proteins were slightly different in length and ranged from 268 aa for GLRaV-9 to 272 aa in GLRaV-4. GLRaV-4 and GLRaV-6 CPs are slightly closer to GLRaV-5 and GLRaV-9 (83-85%) than to each other (81%). However, in all cases identity among the viruses was lower than the threshold for the species demarcation established by the International Committee on the Taxonomy of Viruses (ICTV). The phylogenetic analysis also clearly demonstrated that these two viruses clustered with others in the genus Ampelovirus. Furthermore, the data showed that GLRaVs-4, -5, -6 and -9 are clustered together and have closer relationship compared to GLRaV-1 and -3 the other two grapevine viruses in this genus.