Evaluating the Potential of Insect Vectors to Transmit Grapevine Red Blotch associated Virus (GRBaV)

At this time there is no accurate information on the epidemiology of grapevine red blotch-associated virus (GRBaV) – is it transmitted by insects or dispersed with the movement of infected planting material? Our goal is to screen possible vectors to determine if they can or cannot acquire GRBaV from infected vines and transmit GRBaV to clean vines. In 2013, replicated groupings of 30-50 western grape leafhopper, variegated leafhopper, Virginia creeper leafhopper, vine mealybug, blue-green sharpshooter, and grape whitefly were tested. Petiole samples from inoculated test plants were tested for the presence of GRBaV and, to date, none of the inoculated plants show symptoms of GRBaV and all petioles have tested negative. Subsamples of insects that were used in experiments have also tested negative. Based on the negative results of these studies, in 2014 trials we used more insects and allowed them to feed for longer acquisition and inoculation periods. Infected and uninfected vines were placed together in cages and adult insects were allowed to move between plants at will for 1-6 weeks, with replicated groupings of 600 western grape leafhopper and Virginia creeper leafhopper per cage, 30 blue-green sharpshooter per cage and 1500 grape whitefly per cage. The more sedentary vine mealybug crawlers were added to known infected plants at the rate of 1000 per plant for a 1 week acquisition period and were then moved to uninfected plants for a 1 week inoculation period.

To date, all recipient plants and exposed insects have tested negative for GRBaV. Plants from 2013 and 2014 will continue to be tested quarterly, for a period of 2 years, as it may take a year or longer for viral populations to reach detectable levels in inoculated vines. Field epidemiology was monitored at two sites. In a 20 ha block planted in 2008 the spread of grapevine leafroll-associated viruses (GLRaV) was mapped from 2009-2012, also recording ‘red leaf’ symptomatic vines’ that tested (PCR) negative for GLRaV. In both 2013 and 2014, there were about 150 ‘symptomatic vine’ that tested negative for GLRaV. In 2014 we surveyed and tested 156 of these suspect vines using new more complete primers for leafroll and primers for red blotch. Of these 156 vines, 136 tested positive for red blotch, 9 tested positive for leafroll and 11 tested positive for both red blotch and leafroll. The red blotch infected vines were randomly distributed within the plot, indicating that infection did not spread from previously infected vines, which is often indicating of vector movement. During field surveys we collected and tested western grape leafhoppers from red blotch infected vines Batches of leafhoppers from 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} (7 out of 14) of the GRBaV-positive vines tested positive for GRBaV. These results indicate that leafhoppers can acquire the virus by feeding on infected vines, but does not provide evidence that they can transmit GRBaV. We repeat for emphasis that acquisition does not imply transmission, basically the virus is in the bug after feeding on the vine.

Biology and Spread of Grapevine Red Blotch-Associated Virus

Grapevine red blotch-associated virus (GRBaV) is the causal agent of red blotch, a recently recognized viral disease of grapevines. We showed that GRBaV can infect rootstocks following agroinoculation, including 3309C that is asymptomatic while infected. This makes clear the potential for rootstocks to be involved in virus dissemination. Efforts to develop a serological detection assay for GRBaV have been challenging, suggesting a need to investigate the expression mechanism of the coat protein gene to facilitate developing approaches to produce antibodies that will recognize virions in infected grapevine tissue. The spatial distribution of GRBaV was mapped in selected vineyards in California and New York, and preliminary work designed to identify insect vectors has provided candidate leads among a few hemipteran insects. Survey efforts of alternative hosts revealed that free-living grapevines in riparian areas in close spatial proximity to diseased vineyards in California can be infected by GRBaV. Research progress was extended to stakeholders through presentations at grower’s conventions.

Investigation of the Impact of Grapevine Red Blotch-associated Virus on Grape and Wine Composition and Quality

Grapevine red blotch-associated virus (GRBaV) is the latest virus to be identified in grapevines. Since discovery in 2011, its widespread presence has been confirmed in several states and in different white and red wine grape varieties. At this stage very little is known about the effect of the red blotch virus on both grape development and composition at harvest as well as the long term effect on wine composition and quality. Several varieties should be studied to determine if the effects of the virus are variety specific. Preliminary data from the currently funded AVF investigation into the impact of GRBaV on grape and wine composition and quality show significant differences in basic chemical data for red blotch positive and negative grapes sampled at harvest. Berry samples were taken from vines at harvest from multiple sites investigated (three Chardonnay sites in Sonoma County, two Cabernet Sauvignon sites and one Merlot site in Napa County). A decrease in Brix was mostly obtained at harvest for grapes from red blotch infected grapevines irrespective of cultivar or clone compared to healthy grapevines. This decrease was up to 6{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} in Chardonnay grapes and respectively a maximum of 20 and 16{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} in Cabernet Sauvignon and Merlot grapes. Differences in pH were minor with TA values mostly higher in grapes from red blotch diseased grapevines.

The first year’s data indicate that red blotch disease resulted in a decrease in total phenols and tannins in Chardonnay grapes of up to 25{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}. Phenolic results for the different Cabernet Sauvignon and Merlot sites were more variable. There was only a clear decrease in tannin concentration for one of the Cabernet Sauvignon sites (18{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}). However, the total anthocyanin concentration was 20 to 38{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} lower in all red blotch infected red grapes. This indicates variability in response to red blotch infection within a variety. Future analyses to determine the individual phenolic profiles and tannin compositions of red blotch infected and non-infected grapes may shed more light on these findings. Wines could only be made from one of the Chardonnay sites in addition to the two Cabernet Sauvignon sites described due to the fact that we were unable to obtain enough GRBaV negative grapevines for winemaking. All analyses will be repeated on the completed wines to coincide with descriptive sensory analysis. Final results will enable us to relate grape and wine composition as well as potentially find a correlation between differences in mouthfeel properties of the wines and changes in phenol and tannin compositions due to red blotch disease. At least one more year’s data is needed to confirm results and determine the potential impact of seasonal variability on the expression of GRBaV. It is important to determine the impact of GRBaV on grape and wine composition so that recommendations can be made to the industry regarding the future of infected vines.

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 cultivar 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. In 2014, the vine performances were evaluated by measuring the trunk diameter, cane length, pruning weight, yield and fruit composition. Trunk diameter analysis did not show much differences on each rootstock treated with different GLRaVs and virus isolates. For cane length measurements, the data showed that St. George 15 and St. George 18 rootstocks were not affected by different treatments. However, the two different isolates of GLRaV-2 (2B and 2C) had significant impact on cane length of plants propagated on rootstocks 101-14, 3309C, 5BB and Freedom. The yield did not show any significant difference between different treatments on rootstocks 110R, 420A, 5BB, AXR, Freedom, St. George 15 and -18. Pruning weight analysis did not show any differences between different treatments and rootstocks 110R, 420A, St. George 15 and -18. However, significant differences were observed between different treatments and the rootstocks 101-14, 3309C, 5BB and Freedom. Rootstock AXR was less affected. The analysis also showed that both GLRaV-2 isolates (2B and 2C) in general have been more severely affected the plants on panel of rootstocks.

Development and Application of Next Generation Sequencing to Facilitate the Release of New Grapevine Accessions in Quarantine and Certification Programs

This project is involved with the characterization of the technical tool “NGS” as a method of detection of grapevine viral pathogens. The objective is to demonstrate that, by every measure, NGS is superior to the current biological indexing screen for the certification of grapevine accessions for release into the field. The project is proceeding with the side-by-side comparison of the two diagnostic procedures. Both procedures focus on the identification of viral pathogens. The bioassay identifies virus infections through the symptoms caused in indicator plants in the field. NGS identifies viruses through the sequences of their genomes, found in the laboratory by total genomic deep sequencing analysis of extracts of DNA and RNA from infected vines. We have not yet described the biological index screening results, since it will take two years to get those results from field trials begun during the course of this project. In the past year, dsRNA samples were prepared from54 grapevine cultivars and accessions and sequenced by an Illumina platform. Sequences from 15 of these accessions have been analyzed and work on remaining 39 samples is in progress. For a comparison and optimization of the NGS analysis, small interfering RNA and total RNA were also prepared from selected grapevine accessions in our list and sequenced and the analysis is pending. The initial NGS data analysis of the subset of the samples (15 samples) from this project already suggests that many more virus species will be revealed by deep sequencing than can be identified by the biological field assay on indicator plants. We will have to wait for the bioassay results to be scored before we can make quantitative deductions about the comparative sensitivity, accuracy, and comprehensiveness of the two methods.

Development of the Vineyard Advisor – A Mobile Application for Grape Disease and Pest Management Recommendations

The Vineyard Advisor mobile application was designed to provide up-to-date recommendations on disease and pest management for commercial grape production, including the most current federal pesticide labels. For a given grape disease, pest, or weed problem, the Vineyard Advisor delivers management recommendations following a standardized format with the following sections: Problem name; When to take action; Cultural management practices; Organic materials; Registered pesticides; and References. Recommendations are derived from published resources including viticulture books and Extension pest management guides from several states. Current federal pesticide labels are retrieved from the U.S. Environmental Protection Agency (EPA) using a modification of the system employed by the Mobile Access to Pesticides and Labels tool produced by the National Pesticide Information Center.

The Vineyard Advisor can be used by a vineyard manager who has observed a disease problem, for example, to look up the recommended cultural practices to manage the disease as well as a list of pesticide products labeled for control of that specific disease on grapes. The pesticide product is linked to PDF files of the most current national product labels approved by EPA. Alternatively, the vineyard manager could use the app to search directly for a specific pesticide product labelled for grapes. This feature is particularly valuable as changes occur in pesticide product availability or directions-for-use. The Vineyard Advisor pesticide label database is updated in concert with EPA updates to their database, which typically occur on a weekly schedule.

The Vineyard Advisor also seamlessly integrates with the Vineyard Doctor diagnostic system, through the Problem Profile pages of the Vineyard Doctor. An example of this use would be a vineyard manager using the Vineyard Doctor to identify an insect problem in the vineyard, and following the management recommendations link on the Problem Profile to the corresponding insect management recommendations page on the Vineyard Advisor. The scope of vineyard problems for which the Vineyard Advisor provides management recommendations fully coincides with the 150+ problems within the Vineyard Doctor system, and additionally provides weed management recommendations including links to herbicide labels. The Vineyard Advisor can be accessed by any computer with internet access as a web service at <http://vineyardadvisor.tamu.edu> or via mobile applications downloadable from the Apple Store (iOS) or Google Play (Android operating system).

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.