Biology and Spread of Grapevine Red Blotch-Associated Virus

Grapevine red blotch-associated virus (GRBaV) is associated with red blotch, a newlyrecognized emerging viral disease. We showed that GRBaV is the causal agent of the disease and microshoot tip culture can eliminate the virus from infected vines. Two distinct groups of GRBaV isolates exist in infected vines and currently available diagnostic tools are robust for a reliable detection of all isolates. Efforts to develop a serological detection assay for GRBaV are under way. Monitoring incidence of GRBaV in vineyards over time did not provide compelling evidence of spread. Research progress was extended to stakeholders through 15 presentations at conventions, field days and IPM conferences.

Grower Implemented Quantitative LAMP for Initiating and Adjusting Fungicide Program

The mission of this research is to increase the economic sustainability of grape production by providing decision support tools to aid in management of grape powdery mildew. In this project we propose to test the utility of a quantitative Loop mediated isothermal AMPlification assay (qLAMP) and handheld device for detection and quantification of airborne inoculum; thereby extending our  esearch on the use of inoculum detection as a decision support tool for managing grape powdery mildew. The specific objectives are:

  1. Test implementation of a grower preformed quantitative LAMP assay.
  2. Examine the effectiveness of adjusting fungicide interval based inoculum density.
  3. Assessment of quantitative LAMP (qLAMP) for estimating amount of fruit infection

Results: Due to reduced funding, Objective 1 had to be reduced and Objective 3 eliminated in the second year of this 3 year project. The extensive cooperation of several participating growers who shuttled samples from the upper Willamette Valley to Corvallis, allowed for us to complete Objective 2 using lab processed samples only.

Bio-Economic Analysis of Grape Leafroll Virus Epidemics in California

The work in this research project concerns three things. First, it is intended to improve understanding of what controls the spread of leafroll disease within and between vineyard blocks. Second, it aims to work out costs for finding and dealing with leafroll infections in California vineyards so that growers can make better-informed choices about disease management. Lastly, it is intended to look at some of the difficult issues concerning cooperation and shared costs and impacts in managing leafroll at a neighborhood level, and to act as a focus for outreach from UC Davis to support the grower community and UC Cooperative Extension in tackling leafroll disease.

Our analysis of leafroll disease progress data shows that the disease develops in a predictable way irrespective of grape variety. The disease is typically introduced to healthy vine blocks at random locations, consistent with dispersal of mealybug juveniles in wind gusts. Spread between infected and healthy blocks may cause these initial infections to edges of healthy blocks, but random infections, well away from the edges, are also possible. Random initial infections could also arise, in theory, from infected planting material, but cases where this happens would be expected to show up one to two years after block establishment or vine replacement and so should be identifiable by reference to block age when disease first appears. Once introduced to a block, disease intensifies around the initial infection in a way that is consistent with mostly plant-to-plant spread of mealybug crawlers.

The research on epidemic dynamics feeds into our second area of work. As part of the epidemiology studies we have characterized the degree of clumping of diseased vines around the initial infections. This statistical analysis of the pattern of diseased vines allows us to calculate the effect of clumping on sampling efficiency for detecting the disease. That is, we can work out how the tendency for diseased vines to occur in small focused patches initially affects the efficiency of time spent sampling for disease and also on the accuracy of estimates of the level of disease. In general, the level of patchiness we find for leafroll has significant impacts on both the efficiency of sampling and the certainty of estimates based on sampling. We provide some illustrative results from this analysis.Neighborhood groups for managing leafroll have now been established in the Napa region, partly in response to suggestions made in the early stages of this project. We have extended the work reported last year on attitudes among growers to include representatives of the grapevine nursery industry. The results show that individuals from nursery trade have a similar range of attitudes towards leafroll as growers. There was some evidence that different nursery companies may have a recognizable company-level collective attitude, but the sample size is small. Our modeling work of disease dynamics at the neighborhood scale has highlighted the importance of disease management within existing infected blocks. The contribution of new infections from infected planting material is relatively small when there is a high background level of disease from existing infections.

Grapevine Leafroll Disease: a Detailed, Broad-scope Study of Host and Pathogen Effects

Grapevine leafroll disease causes non-uniform maturation of fruit in Vitis vinifera, including poor color development in red grape varieties. The disease causes losses of as much as 20-40{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}, with delays of 3 weeks to a month in fruit maturation. To date 5 different viruses, namely Grapevine leafroll associated virus (GLRaV) types -1 through -4, and -7, have been conclusively shown to be associated with leafroll disease. In the case of GLRaV-4, several distinct leafroll disease-associated virus strains have been identified within the virus species. This project was planned as a detailed study of the effects of these viruses on variety Cabernet Franc grapevines. This grapevine produces a readily scored foliar response to leafroll virus infection. The analysis includes challenges with each agromonically significant GLRaV species, including types -1 and -2 (2 isolates each), -3 (3 isolates), -4, -5, -7 and -9 (one isolate each). Also, pairwise combinations of GLRaVs -1, -2, -3, -5 and -7 are being tested. The test vines are grafted onto a broad selection of different rootstock varieties. Nine different rootstocks are involved in the test, including AXR #1, Mgt 101-14, 110R, 3309C, 5BB, 420A, Freedom, St. George 15 and St. George 18. 15 replicates for each treatment are divided into three separate blocks each (5 replicate per treatment per block). The project has thus-far revealed a spectrum of differences in infection symptoms attributable to the different virus species, and to different combinations of these viruses and the grapevine varieties they infected. For example, it was observed that leaf symptoms produced by GLRaV-3 were more severe than those produced by GLRaV-4. In another example, it was found that GLRaV-2 induced more severe reactions on vines propagated specifically on rootstocks Freedom and 5BB. Those test vines exhibited red leaf symptoms, short internodes, and a near-lethal decline in vigor. Detailed analysis of these and other specific aspects of leafroll disease are on-going. Data collected from the experiment in 2011 revealed one particularly severe infective combination. Virus isolate LR132 (which contained both GLRaV-1 and Grapevine virus A) produced a severe infection in Cabernet Franc plants propagated on rootstocks 420A, Freedom, 3309C and 101-14. Many of these plants died a few months after inoculation. Whether the severity is due to a particular strain of GLRaV-1 found in the LR132 isolate, or to a synergy arising from the mixture of GVA with GLRaV-1 in the inoculums is under investigation.

Evaluating the Effects of Grapevine Red Blotch-Associated Virus on Symptom Development and Fruit Maturity

Red varieties of grapevines with leafroll-like symptoms that are not infected with leafroll-associated viruses have been found infected with grapevine red blotch-associated virus (GRBaV), a new virus first identified in 2011 and subsequently shown to be the causal agent of red blotch disease. Diseased vines have been identified in several counties in California and in other states. Effect of GRBaV infection on differences in berry composition over the ripening period have not been documented and foliar symptom development in red and white varieties has also not been characterized. A study was conducted in 2013 to clarify symptom development in foliage, fruit maturity and vine growth in Chardonnay, Cabernet Sauvignon, and Merlot. At each of the three sites, vines selected for the study were determined to be GRBaV positive or negative by qPCR assay as well as negative for all leafroll-associated viruses, vitiviruses and nepoviruses. To determine the effect of crop load on disease expression, crop was reduced at two sites by approximately 35{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} at the onset of veraison.

Foliar symptom expression in vine canopies increased with time and is greatest in older leaf tissue. The severity of foliar symptoms varied greatly across the three varieties; however, on all evaluation dates, vines positive for GRBaV had significantly greater percentage of symptomatic leaves in the basal and middle regions of canopies. In addition, symptomatic leaves in these regions had larger area (greater symptom severity) with red or chlorotic coloration in red and white varieties, respectively, than younger terminal leaves. At harvest, the severity of interveinal chlorotic blotch symptoms in Chardonnay was greater than the red blotch symptoms in Cabernet Sauvignon or Merlot. In Cabernet Sauvignon and Merlot, initial leaf symptoms in GRBaV positive vines were predominately leaves on which only red veins were present. In Merlot, the development of leaves with red interveinal tissue followed. In Cabernet Sauvignon, blades with only red veins remained the primary leaf symptom. In addition, reducing the crop at veraison in Cabernet Sauvignon may have resulted in an increase in virus symptom expression late in the growing season.

This project has allowed us to associate the presence of GRBaV infection with a consistent delay in fruit maturity. For all three cultivars, fruit maturity was delayed in vines PCR positive for GRBaV. Brix was significantly lower on all sample dates and titratable acidity significantly greater on half the sample dates yet always elevated on other dates. At harvest, juice samples in all varieties had significantly higher malic acid in GRBaV positive vines as compared to GRBaV negative vines. Reducing crop load in GRBaV positive vines in Chardonnay and Cabernet Sauvignon did not significantly improve juice chemistry at harvest when compared to infected vines with full crop loads. Juice from fruit on Cabernet Sauvignon positive vines in which crop was reduced indicated a very slight improvement in ripening parameters although differences were not statistically significant. Red blotch disease did not affect vine yield. Berry weights in vines infected with GRBaV are at least as great as virus negative vines.

Development of Tools for Growers to Evaluate and Optimize Ecosystem Services of Birds in Vineyards

Establishment of songbird nestboxes in vineyards increases insectivorous bluebird populations. Our study investigated the diets of vineyard-nesting Western Bluebirds (Sialia mexicana) to document whether bluebirds consume insect pest species and offer growers ecosystem services in the form of pest control. To evaluate the impact of avian predation on arthropods in vineyards, we sampled both birds and arthropods across three vineyards and adjacent native forest patches. Over 4500 arthropods were collected, sorted, and identified. For bird sampling, we used non-invasive methods by gathering fecal samples from adult and nestling bluebirds to evaluate what prey were consumed. We tested several DNA extraction kits before developing a novel methodology that provided better quality and quantities of DNA from bird fecal samples. We applied next-generation sequencing to obtain a list of diet contents in the form of DNA sequences. We compared these sequences to a reference database that we constructed from DNA sequences of our collected arthropods. We found a rich and diverse abundance of arthropods in both the vineyard and adjacent woodland habitats. This signifies that insectivorous birds nesting in vineyards had access to plenty of food resources. Bluebirds were consuming a diverse diet comprised of many different arthropod orders, from millipedes to butterflies. We found that adult bluebirds regularly feed their nestlings isopods (also called roly-polys or pill bugs). These were abundant in the vineyards and are known to offer one of the few sources of calcium available to insectivorous animals. Calcium can be a limiting nutrient, and it is likely that high densities of isopods in vineyards offer nesting bluebirds high-quality prey items for their growing young.

No significant vineyard pests (including blue-green sharpshooters) were found in vineyard and woodland traps. This meant that avian populations did not have access to these pest insects, so it is not surprising that we did not find evidence of bluebirds consuming vineyard pests in this study. We did find evidence of bluebirds consuming treehoppers and caterpillars, so in vineyards where these pests are present, bluebird boxes may invite predators that successfully lower pest populations. We did not find evidence that bluebirds were consuming parasitic wasps (beneficial insects that lower pests populations). Consequently the presence of bluebirds did not harm growers. Nestboxes can bolster declining bird populations, and increasing vineyard nest box presence can be an important sustainability practice for growers. Consequently we connected with growers and presented our findings in numerous venues, distributing informational pamphlets and 100 nest boxes to eager growers. Our goal is to provide growers with the resources they need to maintain healthy populations of birds in their vineyard for year.

Effects of Pre- and Post-Harvest Practices on the Replenishment of the Nitrogen Reserve Pool in the Permanent Structures of Grapevines

The objective of this study was to quantify the amount of N remobilized and/or taken up from the soil or N fertilizer and put into the N reserve pool (within the trunk and root system) of field-grown grapevines.  This study also determined the effectiveness of a post-harvest N fertilization application on the dynamics of the reserve N pool.  Nitrogen within whole vines was quantified using destructive harvests.  Several treatments were imposed to assess their effect on the replenishment of N reserves independent of remobilization.  They included: 1.) The application of N during the growing season, 2.) The application of N post-harvest and  3.) Fifty percent of the leaves in the canopy removed after harvest to mimic the effects of mechanically harvesting a vineyard.

Petiole NO3-N at bloom for the non-fertilized treatment averaged less than 100 ppm (dry weight), a value many consider to indicate a N deficiency.  The application of a nitrogen fertilizer one month after budbreak (albeit at only ½ the total amount applied) significantly increased NO3-N, NH4-N and total N of the petioles compared to the no N treatment.  The NO3-N and NH4– petiole values measured at bloom were in excess of 2500 and 1800 ppm, respectively.  These values would be considered excessive by some.

A N budget for vines in the fertilized (‘+N’) and non-fertilized (‘–N’) treatments was determined at fruit maturity and at the end of the season (after leaf fall).  While it can be surmised from the previous paragraph that the vines in this vineyard may have been N deficient, vines from the ‘–N’ treatment still accumulated 58.9 g N/vine (78 kg N/ha; 69 lbs N/acre) in the leaves, stems, fruiting canes and clusters at fruit maturity with 82.2 g N/vine (109 kg/ha; 97 lbs N/acre) in the trunk and roots.  The amount of N in the leaves, stems, fruiting canes and clusters at fruit maturity for the ‘+N’ treatment was 72.9 g N/vine (96.5 kg/ha; 86 lbs/acre) while that in the trunk and roots was 103 g N/vine (136 kg/ha; 121 lbs/acre). N fertilizer recovery efficiency (REN) can be determined by comparing the uptake of N in plants with that of a non-fertilized treatment.  Vines in the ‘+N’ treatment accumulated 35.1 g N/vine more than that of the non-fertilized control at fruit maturity.  Since 34 g N/vine was applied to the fertilized vines the REN would be ~100{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}.

The REN was also calculated for data collected at the end of the season, subsequent to leaf fall.  The non-fertilized control would be another ‘–N’ treatment cohort of vines while the fertilized treatment would be the ‘PH +N’ treatment.  The amount of N taken up from the soil by the ‘PH +N’ treatment was 7.1 g/vine greater than that of the ‘–N’ treatment.  Since the ‘PH +N’ treatment was fertilized with 25.5 g N/vine, the REN would be ~ 28{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} ((7.1/25.5) * 100).  Therefore, under the conditions of this study, a fertilizer application one month after budbreak and at berry set was more efficient than the post-harvest application of N.

Mealybug Transmission of Grapevine Leafroll-Associated Virus 3

The overarching goal of this research is to obtain information about transmission of Grapevine leafroll-associated virus 3 (GLRaV-3), the primary virus species associated with spread of the economically damaging Grapevine Leafroll Disease (GLD) in Napa Valley. Such information is necessary to inform control strategies, it is clear that knowledge-based management of vector-borne diseases requires a robust understanding of how the pathogen spreads in vineyards. Mealybugs are the vectors associated with spread of GLD, but nothing is known about differences in transmission efficiency among mealybug species inhabiting vines in California. Furthermore, genetically distinct variants of GLRaV-3 exist but nothing is known about differences among these variants in terms of their ability to spread, or what the relevance of that variation is to GLD epidemiology. Lastly, all previous GLRaV-3 transmission studies were done under greenhouse conditions, and it is not known how well the results of such studies predict transmission in vineyards. This research addresses these significant gaps in knowledge.

We have completed proposed single and simultaneous mixed GLRaV-3 variant inoculations in greenhouse trials, using grape and vine mealybugs. Though two GLRaV-3 variants from singly infected source plants did not differ in transmission efficiency, the transmission efficiency of one variant was substantially lower when acquisition occurred from a co-infected source plant, indicating inhibition of transmission by the other variant. This may mean that one variant can be transmitted more efficiently than another and increase its incidence in the landscape (e.g. Napa Valley). It is not known whether some GLRaV-3 variants are more pathogenic than others.

We also set up an experiment in Napa Valley in summer 2011, inoculating 60 mature grapevines with GLRaV-3 using grape mealybugs as vectors. Each vine was inoculated using 10 first instar mealybugs, and then treated with insecticide two days later. Three months after inoculation, 20 of 60 plants tested positive for GLRaV-3 from our inoculations. No symptoms appeared in 2011. During the following growing season, GLD symptoms first began to appear in our experimental vines in June. By July, symptoms appeared in 29 of 60 experimental vines, and no other vines became symptomatic in 2012. Berry quality was affected in symptomatic vines compared to asymptomatic vines in the experiment.

Transmission in a parallel greenhouse experiment was higher than in the vineyard. In 2012 we set up a second field inoculation experiment in Napa to compare transmission of two different GLRaV-3 variants, by grape and vine mealybugs, in both Chardonnay and Pinot Noir. Inoculations were completed in July. Results from the 2012 inoculations are pending. This is the first time it has been shown that GLD symptoms due to mealybug inoculation of GLRaV-3 into established mature vines (~15 years old) in commercial vineyards are expressed in the following growing season. Results also showed that the entire vines were symptomatic in 2012, instead of just the inoculation site. Lastly, transmission success in the field was about 6{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} per individual mealybug.

Maintaining UC IPM Pest Management Guidelines for Grape – 2012

UC IPM Pest Management Guidelines: Grape, the University of California’s official guidelines for managing pests in grapes, is being revised. The version being revised includes two year-round IPM programs (table grapes and wine and raisin grapes), 11 general information sections, 11 diseases, 7 weed sections; 24 insect and mites, and 1 nematode section. Revisions to be made include adding new pesticides and removing unregistered ones; improved management information such as discussions about herbicide drift reduction and the use of mating disruption for vine mealybug; and new pests: Virginia creeper leafhopper, spotted wing drosophila, and vertebrates (birds, deer, ground squirrel, meadow vole, pocket gopher, and rabbits)

Mealybug vectors and Grapevine Mealybug Vectors and Grapevine Leafroll Dsease: Temporal and Spatial Studies

Grapevine leafroll disease, caused by a complex known as grapevine leafroll-associated viruses, is a worldwide threat to vineyard health and sustainability. Mealybugs are the main agents (vectors) responsible for virus movement between vines in the field. This project continues to study the relationship between mealybug populations and the incidence of leafroll disease in vineyards. The ultimate goal is to develop and deploy best management practices for leafroll disease and vineyard mealybugs as vectors of leafroll viruses. The project not only focuses on development of these practices, but also works with grape growers to use these tools in a disease management program. For example, we have successfully developed the use of male GMB traps as a monitoring and decision-making tool and provided significant formal and informal educational efforts on the use of these traps. Traps are more sensitive detection tools that can supplement labor-intensive ground surveys. We provided training sessions on trap deployment and male mealybug identification to ensure that the traps are being used correctly, at the appropriate time of year, and that the trap data are useful to growers. Grape growers are increasingly adopting the use of GMB traps as a management tool. We have also worked with growers to explore and implement other effective and timely management practices, such as insecticide sprays and vine removal.

Our preliminary analysis has shown that prior infection rates of leafroll disease and mealybug populations in the current year will affect the number of vines that develop leafroll disease symptoms in the following year. This has implications for vine removal programs: in a given year, there may be a percentage of vines that contain virus particles but are not showing disease symptoms. This and other complementary projects worldwide are developing best management practices for leafroll disease management that include monitoring and management of vector populations, identification and removal of diseased vines or vineyards, planting of material free of known grapevine pathogens, and regional approaches to management. In future studies we propose to continue to develop new practices, fine-tune current practices to determine when, where and how they can be used most effectively, develop a regional approach to management, and strengthen our educational efforts.