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, -7 and -4 strains 5 and 9 (one isolate each). Also, pairwise combinations of GLRaVs -1, -2, -3, -4 strain 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.

In 2015, the vine performances were evaluated by measuring the trunk diameter, cane length, pruning weight, yield and fruit composition. Trunk diameter analysis showed that the plants propagated on rootstocks Freedom and 3309C were the most affected ones. For cane length measurements, the most affected rootstocks were 5BB and 3309C and the least affected ones were AXR and St. George 18. However, the two different isolates of GLRaV-2 (LR 103 and LR 119) had significant impact on cane length of plants propagated on rootstocks 101-14, 3309C, 5BB and Freedom. For pruning weight, the most affected rootstocks were 5BB, Freedom and 101-14 and the least affected were 110R, St. George 15 and St. George 18. For the yield the most affected rootstocks were 3309C and Freedom and no significant yield reduction were found on plant propagated on 110R, St. George 15 and St. George 18. Cluster count analysis was similar to total yield. Average berry weight analysis showed that the most affected rootstocks were 420A, Freedom and 5BB and no significant differences were found on rootstocks 110R, AXR, St. George 15 and St. George 18. The analysis also showed that both GLRaV-2 isolates (LR103 and LR119) in general have been more severely affecting the plants on panel of rootstocks.

 

Discerning Mechanisms of GRBaV Virus Disease (Red Blotch) Using Leaf Nutrient Transport and Photosynthesis Analyses

In 2015, considerable time was invested in establishing a relationship with the Stanford Synchrotron Radiation Lightsource Facility (SSRL) and working with their beam line scientist to develop a micro X-ray fluorescence method to image micronutrient distributions in grapevine tissue sections. Images of micronutrient distributions, concentrations and chemical states on a cellular level were not obtained this year due to a malfunction in the beam line equipment, but will be obtained in 2016. Nutrient levels in leaves in petioles at veraison were measured. Boron (B), an element essential for sugar transport across membranes, was observed to be accumulating in vines that tested positive for GRBaV virus (infected,[RB(+sanitize_seed_3md20fhorfc4wwg4w0ssogg8k)sanitize_seed_3md20fhorfc4wwg4w0ssogg8k]sanitize_seed_3md20fhorfc4wwg4w0ssogg8k). Iron (Fe) levels in RB(+) leaves were observed to be diminished compared to non-infected controls [RB (-sanitize_seed_3md20fhorfc4wwg4w0ssogg8k)sanitize_seed_3md20fhorfc4wwg4w0ssogg8k]sanitize_seed_3md20fhorfc4wwg4w0ssogg8k, a situation that may affect the photosynthetic metabolism since Fe is a major catalyst in the production of chlorophyll. These results suggest the need for SSRL analysis to better understand the changes of these elements, as well as other macro- and micronutrients, on a cellular level between RB(+) and RB(-) vines. Photosynthetic assimilation analysis showed decreased CO2 assimilation in the RB(+) mature leaves closest to fruit clusters yet at the same time starch accumulated in these leaves, suggesting phloem loading (transport) as being disrupted. Sucrose levels in RB(+) mature leaves were higher than RB(-) mature leaves. Sugar (brix) levels in the RB(+) fruit were 14{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} lower than RB(-) fruit. However in both treatments, sucrose levels between treatments were equally higher in younger RB(-) asymptomatic leaves compared to mature symptomatic leaves. These results, along with the results from Oberholster’s group, suggest that source sink dynamics are being altered by the virus

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

This project continues to inform and support the adoption of management strategies to minimize the incidence of leafroll disease spread in California vineyards. During the 2014-15 project year, we completed several objectives: (1) we finalized our analyses of the factors impacting leafroll disease spread, (2) developed methods for hyperspectral airborne imaging to identify diseased vines, and (3) supported the efforts of a group of growers working on regional leafroll disease management strategies. Our analyses of factors affecting disease spread suggest that the most important determinant of new disease is “block”—that is, there is great variability in the incidence of new disease explained by factors that are specific to the block and vintage (management practices, locationparticularly in relation to other diseased blocks, variety, vine age, vine health, etc.) Disease pressure, defined as the number of diseased vines present in the vineyard in the prior year, also impacts the incidence of new disease. And, in vineyards with medium to high disease incidence, mealybug populations affect disease spread. This suggests that in vineyards with low levels of disease, management practices that support vine health and decrease the number of diseased vines are critical to minimize disease spread.

In vineyards with greater incidence of disease, management practices may also include strategies to minimize mealybug populations. These observations are consistent with similar studies in other grape growing regions around the world, and have clear implications for management: in order to determine which management strategies should be adopted, growers must evaluate disease pressure and GMB populations. In Napa County, where neighboring vineyards share the burden of disease management, growers may jointly launch regional responses to GLD. We have developed a template for these regional efforts, with a pioneering group of 20 grape growers farming 1900 contiguous acres. The grower group (LAMBA) 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. The group shares information and develops goals and activities at regular meetings and focuses on the implementation of GLD management strategies at a regional level.

We have also developed 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. Our preliminary analysis of hyperspectral images of Cabernet Sauvignon vineyards have shown 75 to 90{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} correlation between leafroll-diseased vines identified in ground surveys with those identified through aerial imaging. Further analyses planned for 2015 will refine these figures and help develop the use of this technology for this and other grape varieties.

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

Organic grape growers in Mendocino and Lake County have been experiencing severe outbreaks of the Virginia creeper leafhopper (Erythroneura ziczac) for the past 3 years. Feeding by E. ziczac causes leaf stippling and reduced photosynthesis which can impact crop yield and quality. The primary natural enemies of E. ziczac are the small ‘mymarid’ egg parasitoids Anagrus daanei and Anagrus tretiakovae. A related pest, the Western grape leafhopper (Erythroneura elegantula) is also parasitized by A. daanei as well as Anagrus erythroneurae. Erythroneura ziczac and E. elegantula are commonly found together in North Coast vineyards. Anagrus daanei is the parasitoid species of most importance for E. ziczac control, whereas A. tretiakovae is rarely found in California.

Our approach to improving E. ziczac control involves a combination of short- and long-term strategies. Short-term work focuses on the evaluation of Organic Materials Review Institute (OMRI) approved pesticides. In 2014, we tested Stylet oil and DeBug® Turbo (applied twice) and Pyganic® (applied once) on the development of the first leafhopper brood. All of these products significantly reduced E. ziczac nymph populations relative to an untreated control.

Long-term strategies are focused on the identification and evaluation of Anagrus parasitoids to improve biological control. A survey in Mendocino, Lake, Napa (Pope Valley), Yolo and El Dorado County vineyards found that E. ziczac parasitism was consistently low (0-2{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}) with the exception of Yolo County, where rates reached 10-15{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}. Surprisingly, A. daanei was attacking E. elegantula in all of the surveyed vineyards, but only in Yolo County was it attacking both E. elegantula and E. ziczac. Therefore, we questioned whether or not the A. daanei in Yolo County are the same species as the A. daanei that don’t attack E. ziczac in other regions. Molecular comparison of the A. daanei from different Californai regions is still in progress, but to date morphological evaluations have not shown any differences among the A. daanei populations tested. We conducted a trial in which we forced A. daanei from Mendocino County onto E. ziczac eggs in order to see whether or not, in the absence of their preferred E. elegantula host, they would attack the E. ziczac eggs. Findings from this study indicated they would not.

In another trial, we separately inoculated potted grape vines with E. ziczac eggs from Mendocino, Lake and Yolo County and then exposed sets of these vines to the A. daanei in each of these regions. Results showed fairly consistent parasitism of all three E. ziczac populations by the A. daanei in Yolo County. Having verified that the A. daanei in Yolo County will readily attack the E. ziczac population in Mendocino and Lake County, we now feel that there is adequate evidence to support a collection and re-release program in which A. daanei from Yolo County are introduced into Mendocino and Lake County vineyards. This redistribution of California parasitoid material would be carried out in conjunction with an area-wide IPM program to promote additional best management practices to further reduce E. ziczac outbreaks.

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).