Grapevine leafroll disease causes poor color development in red grape varieties and non-uniform maturation of fruits 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%.  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 and -3 each and with -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.

The data collected from the experiment in 2012 showed that the mild leaf symptoms started in the month of July for GLRaVs-1, -2 and -3 and become very severe when recorded in November.   Plants inoculated with GLRaVs-4, -5,-7 and 9 were asymptomatic in July and showed mild leaf symptoms in November except for GLRaV-7 that stayed asymptomatic.  Leaf symptoms differences were not observed between different isolates of the same virus except for the isolate LR132 (GLRaV-1+GVA) which had a lethal reaction on Cabernet Franc plants propagated on 420A, Freedom, 3309C and 101-14 rootstocks.

In treatments with two different virus isolates, more severe symptoms were noticeable even in cases that one of the isolates in the treatment belonged to the group with mild symptoms (GLRaV-4, -5 and -9).  When the effects of viruses on different rootstocks were compared, no significant differences were observed except for Cabernet Franc plants propagated on AXR-1 which were showing slightly milder symptoms.  Both isolates of GLRaV-2 showed solid red leaf symptoms on plants propagated on 3309C, Freedom, 101-14, 5BB and 110R and in many cases the plants were observed to be weaker and many having short internode and were stunted. Both of these GLRaV-2 isolates killed the plants propagated on 5BB rootstock.

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.

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.

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.

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.