Mealybug transmission of Grapevine Leafroll-Associated Virus 3

The overarching goal of this research is to obtain information about the vector 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 little 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. In addition, 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. Lastly, there is no information on the consequences of insect-inoculated GLRaV-3 into plants in the field. This research addresses these significant gaps in knowledge.

We have completed all proposed experimental GLRaV-3 inoculations in greenhouse and field trials, using grape and vine mealybugs. Molecular diagnosis of test plants is ongoing. Though two GLRaV­3 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 co­infected source plant, indicating competition between variants. 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 experiments in Napa Valley in 2011 and 2012. Each vine was inoculated using 10 first instar mealybugs, and then treated with insecticide two days later. In 2011, we inoculated 60 mature vines cv Cabernet Franc, using grape mealybugs. Twenty vines tested positive for GLRaV­3 three months after inoculations. Symptoms appeared in June of the following year, and there were 29 symptomatic vines by July. The following year, the same 29 vines were symptomatic by May and tested positive for GLRaV-3. Berry quality was affected in symptomatic vines just one year after inoculations. 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.

Development and Implementation of Management Strategies for Grapevine Leafroll Disease and Mealybug Vectors

This project continues to develop management strategies for grape growers with grapevine leafroll disease. We recently completed a mapping project spanning 4 grape growing seasons (2010-2013) to determine the relationship between grape mealybug (GMB) populations and the incidence of grapevine leafroll disease (GLD) in Napa County vineyards. We surveyed 10  vineyards and conducted a preliminary analysis to demonstrate the link between the vector, GMB, and disease incidence. In vineyards with medium to high levels of GLD, rates of disease spread are influenced mainly by (1) the initial number of vines in a vineyard with GLD (disease pressure) and (2) GMB populations in the previous growing season. In vineyards with low levels of GLD, rates of disease spread are influenced by the initial disease incidence as well as the number of GMB found in the current season. These observations are consistent with similar studies in other grape growing regions around the world, and have clear implications for management: in order to successfully manage GLD incidence and spread, growers must evaluate disease pressure and GMB populations. Our preliminary analysis suggests that managing the vector alone may not be sufficient to decrease rates of disease spread, particularly in vineyards with medium to high GLD incidence.

Studies of other pests and diseases have demonstrated that a regional approach to disease management has more potential for success than individual efforts. This is especially true in Napa County, where a single or few growers rarely control large swaths of contiguous vineyard acreage. In Napa County, where neighboring vineyards share the burden of disease management, growers may jointly launch regional responses to GLD. We are developing a template for these regional efforts, with a pioneering group of 20 grape growers farming 1900 contiguous acres. The grower group is committed to implementation of coordinated, regional GLD management strategies that include monitoring of GLD and GMB, as well as lowering disease and vector pressure. Most importantly, the group shares information, communications and develops goals and activities at regular meetings (4-6 meetings per year) focusing on the implementation of GLD management strategies at a regional level.

This project is also developing the use of hyperspectral imaging to map the incidence of GLD in commercial vineyards and nurseries. Imaging spectroscopy provides a potentially valuable alternative to lab testing and field scouting in that it is efficient, non-destructive and relatively inexpensive. From an aircraft thousands of acres may be imaged in a single three-hour flight. Additionally, remote sensing provides continuous measurements to construct a map of the target vineyard. Our studies in Cabernet Sauvignon vineyards have shown a 94.7{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} correlation between leafroll-diseased vines identified in ground surveys with those identified through aerial imaging, suggesting a high level of confidence in the accuracy of our measurements for this variety, and potential to develop the use of this technology for other grape varieties. Hyperspectral airborne imaging could revolutionize the the collection of disease incidence data by increasing ease, accuracy and efficiency of measurements.

Management of Grapevine Leafroll Disease – What Level of Mealybug Control is Needed?

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 focused on control measures for the insect vectors of GLRaV. In 2013 we monitored changes in vine mealybug (VMB) populations in three areas that were formerly part of an areawide mating disruption program and showed that populations have increased in two out of the three of the areas since the removal of pheromone dispensers. Trap catches surrounding historical “hotspots” were also elevated, suggesting that mealybug populations in these areas are spreading. We continued a six-year field study mapping the establishment and spread of GLRaV in a 20 acre vineyard planted in 2008 from certified virus-free scion and bordered by blocks with GLRaV-infected vines and mealybugs. PCR tests revealed that 17 out of 25 vines that were visually identified as symptomatic for GLRaV in 2011 and 2012 were in fact infected by red blotch, which was present in the vineyard by at least 2011. Red blotch-infected vines were randomly distributed inside the mapped plot.

We conducted a study of aerial grape and vine mealybug dispersal at two 10-acre vineyard sites in the Napa Valley, using sticky traps that were deployed in a grid pattern throughout each site. We have not observed mealybugs on the sticky traps that have been processed thus far, suggesting that aerial dispersal by mealybugs is uncommon in Napa vineyards. Work examining the temperature development rates of grape, obscure and Gills mealybugs is ongoing; we began this work with grape mealybug but were unable to maintain sufficient numbers on vines, and will attempt the work again in 2014 with added improvements.

We conducted several transmission experiments to: 1) compare GLRaV-3 transmission efficiency of mealybugs of different ages; 2) detect GLRaV-3 in various grapevine tissues after first inoculation; and 3) determine latency period between first inoculation and successful transmission of GLRaV-3 by vine mealybug (VMB) crawlers. The results indicate that VMBs of all ages are capable of transmitting GLRaV-3 in grapevines, but that first instar VMBs (crawlers) are the most efficient stage. The detection experiments showed that GLRaV-3 can be detected in potted grapevines 3-4 weeks after inoculation and crawlers can acquire and transmit GLRaV-3 from newly inoculated vines 2 weeks after first inoculation. The findings indicate that the infected grapevines could serve as a virus source long before the appearance of GLD symptoms. The stage-specific transmission studies showed that VMB (crawlers) are the most efficient stage and based on biology of VMB crawlers are also the most dispersible stage. Therefore, the GLD management efforts should focus primarily on effective control of VMB crawlers. However, previous studies have shown that application of even some of the highly effective insecticides after VMB infestation may not prevent GLRAV-3 inoculation by VMB crawlers in newly infested blocks. In this situation, application of fast acting products in a proactive manner would be the best approach to manage GLD in vineyards.

Egg Parasitism of the Virginia Creeper (Erythroneura ziczac),A Newly Invasive Leafhopper Pest in California

Grape growers in Mendocino/Lake County are experiencing outbreaks of the Virginia creeper leafhopper (Erythroneura ziczac) [Hemiptera: Ciccadellidae]. Feeding by E. ziczac causes leaf stippling, loss of photosynthetic capacity and can ultimately reduce crop yield and quality. This leafhopper is also thought to transmit the newly discovered grapevine virus “RedBlotch Disease”. The primary egg parasitoids of the Virginia creeper leafhopper (VCLH) are Anagrus daanei and Anagrus tretiakovae [Hymenoptera: Mymaridae]. A related vineyard pest, the Western grape leafhopper (Erythroneura elegantula, WGLH) is also parasitized by A. daanei as well as Anagrus erythroneurae. VCLH and WGLH are commonly found together in many North Coast vineyards. In California, A. daanei is the parasitoid species of most importance for VCLH control, as A. tretiakovae has never been found in California.

Over the past year we focused on determining parasitism levels and parasitoid species present in vineyards infested with VCLH and WGLH. Mendocino County surveys found that VCLH parasitism was practically non-existent while parasitism of WGLH eggs occurred with relatively high frequency. We isolated and reared the Anagrus species attacking WGLH eggs in these vineyards and found 87{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} A. erythroneurae and 13{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} A. daanei. While A. daanei is known to attack both WGLH and VCLH eggs, they are only attacking WGLH in Mendocino County. We subsequently reared Anagrus specimens from parasitized VCLH eggs from a vineyard in Yolo County. These specimens were identified as A. daanei. This finding brings into question the A. daanei populations found in these two counties – why is A. daanei attacking VCLH in Yolo, but not in Mendocino County? We will address this with our work in 2014.

We sampled for Anagrus and leafhopper species in the natural and cultivated habitats surrounding North Coast vineyards. While A. erythroneurae could be found on many host plants, we found A. daanei was very restricted in host diversity and overall in low abundance, which could explain the lack of VCLH parasitism. While we did find small populations of VCLH and WGLH on a variety of non-crop plants during the growing season, both pests appeared to overwhelmingly prefer cultivated grapes during the growing season and in the winter reside in vineyard leaf litter. The most common non-crop host was wild grape and VCLH actually appears to be reproducing on it. Work in 2014 will further evaluate VCLH use of wild grapes as refugia and reproductive sites.

We conducted a spray trial to determine effectiveness of OMRI approved products for VCLH control. Three insecticides were tested: Pyganic®, Mycotrol® and Grandevo™. Applicationtiming was scheduled to target young leafhopper nymphs (mid-June). Pyganic® significantly reduced nymph populations compared to the control while Mycotrol® and Grandevo™ were not significantly different from the control after the first or the second application. Further trials are planned in 2014 to evaluate application timing and frequency for non-OMRI products.

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.