Grapevine Virus Management in Lodi: A Collaborative Research & Integrated Outreach Effort to Help Solve a Statewide Challenge

Principal Investigator Stephanie Bolton successfully led an outreach project entitled “Grapevine Virus Management in Lodi: A Collaborative Research & Integrated Outreach Effort to Help Solve a Statewide Challenge” during the 2018-2019 grant funding cycle. The overall objective of this continuing project is to learn how to best manage and prevent grapevine virus disease in the 110,000 acres of Crush District 11, providing outreach tools and strategies to be shared with other regions across California. Grapevine viruses pose a severe threat to the sustainability of California viticulture by decreasing yields, lowering fruit quality, and decreasing vineyard lifespans. Traditional extension is unable to meet the outreach needs of this crisis alone and decades of scientific research are still needed. The good news is that there are virus management strategies that growers can implement in the short-term while we wait for science to catch up, which can be taught through real-world, hands-on integrated outreach from a team of growers, extension personnel, pest control advisors, and scientists established as the Lodi Grapevine Virus Research Focus Group. Our team meets monthly to conduct a thorough review of regional perceptions of viruses, virus management in the literature, current virus research projects, and management of viruses locally and internationally, which we use to produce practical advice for growers.
We hosted a Mealybug & Virus Outreach Meeting (April 2018), a Mealybug ID Workshop (May 2018), a Cryptolaemus Beetle Drone Demo (July 2018), a Leafroll Virus Tailgate Talk (October 2018), and several breakfast meetings where viruses and their vectors were discussed. We produced a “Nursery Ordering 101: Viruses” booklet, draft versions of two additional virus management booklets, a red leaf handout, a mealybug poster, lists of grapevine virus resources, a threecornered alfalfa hopper handout, and a virus comparison chart. Presentations were given at the Tree & Vine Expo (Turlock) and the Pierce’s Disease Research Symposium, with more talks scheduled for the near future. Outreach articles were written for the Lodi Grower Newsletter, the Pierce’s Disease Board Newsletter, online as blogs, and by the press. The first blind ring test for all virus testing labs in California was orchestrated by the Virus Focus Group during winter 2018-2019. Two Demonstration Vineyards were established – one managing leafroll virus through rogueing and another which is a full replant situation with rootstock trials in place. Virus testing across the region is documenting case studies and has elucidated the role of leafroll virus and Freedom rootstock in a mystery vine collapse. On April 4th, 2019, we will host a second Mealybug & Virus Outreach Meeting with invited speakers Gerhard Pietersen (South Africa), Marc Fuchs (Cornell University), Kent Daane (UCCE), and James Stamp (Consultant). This event will include outreach presentations, leadership sessions, and a grapevine virus influencer dinner for long-term strategizing. Outreach materials created, workshops and meetings hosted, and the communication channels which are opening between industry sectors are of utmost importance for the winegrape industry across the state of California, as we collectively lower virus inoculum and vector populations.

Red Blotch – Associated Virus

Grapevine red blotch virus (GRBV), the causative agent of red blotch disease, is a member of the genus Grablovirus in the family Geminiviridae, and the first known geminivirus of Vitis spp.
(Cieniewicz et al. 2017a; Sudarshana et al., 2015; Varsani et al., 2017). Limited information is available on disease biology and epidemiology. Analysis of the spatiotemporal incidence of
GRBaV over a three-year period (2014–2016) in a selected 5-acre vineyard of Cabernet franc in California was consistent with the occurrence of virus spread. The incidence of diseased plants
increased by 1-2% annually. Spatial analysis of diseased plants in each year using ordinary runs analysis within rows and Spatial Analysis by Distance IndicEs (SADIE) demonstrated
aggregation, particular at the edge of the vineyard proximal to a riparian area, and isolated diseased vines. Analysis of epidemic spread fitting a stochastic spatiotemporal model using the
Monte Carlo Markov Chain method identified strong evidence for localized (within vineyard) spread. A spatial pattern consisting of a combination of strongly aggregated and randomly isolated symptomatic vines within 8-years post-planting suggested unique epidemic attributes compared to those of other grapevine viruses vectored by mealybugs and soft scales or by dagger nematodes for which typical within-row spread and small-scale autocorrelation are well documented (Cieniewicz et al., 2017b). These findings are consistent with the existence of a new type of vector for a grapevine virus. In contrast, similar spatiotemporal analyses of virus incidence in a diseased Merlot vineyard New York from 2014 to 2016 did not provide any evidence of GRBV spread. An insect vector of epidemiological relevance remains elusive for GRBV although the three cornered alfalfa treehopper (Spissistilus festinus [Say]) was recently shown to transmit GRBV in the greenhouse. To determine the diversity and distribution of potential vector candidates in the Cabernet franc vineyard in California, sticky cards were placed
from March to November in 2015 and 2016 in the area where disease incidence increased by nearly 20% between 2014 and 2016. Insects on sticky card traps were identified to species when possible by morphological characteristics and sequencing of the mitochondrial COI barcode region. Among the subsets of insect species/taxa that were removed from sticky cards and tested
by multiplex polymerase chain reaction, GRBV was consistently detected in Spissistilus festinus (Membracidae), Colladonus reductus (Cicadellidae), Osbornellus borealis (Cicadellidae) and a
Melanoliarus species (Cixiidae). Populations of these four candidate vectors peaked from June to September with viruliferous S. festinus culminating from late June to early July in both years. An assessment of co-occurrence and co-variation between GRBV-infected vines and viruliferous insects using the association function of SADIE identified a significant association between the
spatial distribution of infected vines and viruliferous S. festinus. These findings revealed the epidemiological significance of S. festinus as a vector of GRBV and the need for testing the
transmission capability of C. reductus, O. borealis, and the Melanoliarus species (Cieniewicz et al., 2017c). Altogether, our insights into the spread of GRBV and into population dynamics of S.
festinus and three other candidate vectors are important to inform epidemiological features of red blotch disease and devise disease management strategies.

Molecular Characterization and Improved Detection of Californian Isolates of Grapevine Pinot Gris Virus

Grapevine Pinot gris virus (GPGV) was recently detected in California vineyards. To gain a better understanding of the incidence and distribution of GPGV, we conducted field surveys throughout grape-growing regions in California and found that while the virus infects many varieties of grape, it has only been detected in the US in Napa County to date. The relationship between GPGV infection and symptoms remains complex. All California GPGV isolates share close homology with the asymptomatic reference isolates and when symptoms are observed in GPGV-positive vines, those vines were also infected with Grapevine fanleaf virus. Developing a serological detection method has been challenging, but we will continue this effort in collaboration with our Italian colleagues. Our molecular characterization of the virus has enabled us to develop an improved molecular detection assay which will facilitate monitoring the prevalence and natural spread of the virus. We have shared our research to date with stakeholders throughout the year.


Interpreting a Multi-Virus Survey and Designing and Delivering Virus Sampling Protocol for Industry-Wide Benefits

A. Analyze incidence of multiple virus diseases in a 2014 survey of grape blocks in the California north coast region and relate virus incidence to block planting date.

B. Interpret patterns of virus incidence in the 2014 survey in relation to entry of different virus diseases into the California grape certification system.

C. Develop a grower information pack and slide presentation to summarize survey information on long term changes in vine health and impact of clean plant strategies on virus incidence.

D. Adapt information from previous epidemiology studies on leafroll and Red Blotch to develop sample size calculations and sampling schemes for virus assessment in grape blocks.

E. Work with grower participation groups in Oakville and Lodi to demonstrate and evaluate virus sampling protocols

F. Develop grower information pack and slide presentation to summarize sampling approaches for virus management in different production situations

G. Make sample size calculations available online via a simple, free web page linked to supporting information on virus diagnostics and epidemiology.

Objective A: As a reminder, we surveyed approximately 100 blocks of wine grape in the North Coast region in the fall of 2014 for the incidence of nine viruses, including Grapevine Red Blotch associated Virus (GRBaV) and Grapevine Leafroll associated Virus (type 3) (GLRaV-3). Ten randomly selected vines in each block were sampled by collecting two petioles per vine.   Samples were tested using a set of species-specific PCR primers after DNA/RNA extraction.

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%, 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.

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%per individual mealybug.

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 General Detail Study and Evaluation

Grapevine leafroll disease causes poor color development in red grape varieties and non-uniform maturation of fruits in Vitis vinifera. It also has been reported to cause delay in fruit maturation from 3 weeks to a month and crop losses of as much as 20-40%.  This project was planned to study the effects of GLRaV types -1 and -2 (2 isolates each), -3 (3 isolates), -4, -5, -7 and -9 (one isolate each) and a combination of two of types -1, -2 and -3 each and with -5 and -7 on Cabernet Franc grafted on 9 different rootstocks.  Rootstocks used are AXR #1, Mgt 101-14, 110R, 3309C, 5BB, 420A, Freedom, St. George 15 and St. George 18.

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

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

Mealybug Pests and an Emerging Viral Disease: Vector Ecology and Their Role in Grape Leafroll Associated Virus Epidemiology

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 concerns GLRaV field epidemiology with respect to its insect vectors. In field studies, we evaluated grape mealybug acquisition and transmission of GLRaV-3 from trunks, spurs, canes, and leaves. Two point three percent of mealybug crawlers acquired GLRaV-3 from vines in the field and transmitted leafroll virus to vines in the greenhouse. This is a lower transmission rate than previously reported from greenhouse studies. Surveys of grape mealybugs from five Napa County vineyards were taken monthly (May through October) and found 56%of grape mealybugs collected on leafroll-infected vines tested positive for GLRaV-3. The proportion of leafroll-infective mealybugs was related to the number of leafroll-positive vines in the vineyards surveyed. We continued a five-year field trial testing the impacts of ?zero tolerance? for mealybugs on GLRaV infection establishment and spread. A 20 acre vineyard planted in 2008 from certified virus-free scion, and bordered by older blocks that contained both GLRaV-infected vines and mealybugs, received pesticide treatments for mealybugs in 2009, 2010 and 2011. No mealybugs were found in visual inspections; however, pheromone traps showed the presence of male grape mealybugs. All vines were inspected annually for GLRaV symptoms. In 2009 and 2010, there was one new infected vine each year, while in 2011, six new vines tested positive for GLRaV-3. GLRaV-3 variants in the spray trial block (-3a, -3c, and -3d) were different than the GLRaV-3 variants in adjacent virus-infected blocks to the north and west (-3b). The leafroll-infected vines were randomly distributed inside the mapped plot, with similar numbers in insecticide and untreated vines. One hypothesis is that virus-carrying grape mealybug crawlers were ?blown? into the plot, and infecting previously-healthy vines during feeding. This trial will continue for two more years and to see if disease-free blocks can be established though the use of annual insecticide treatments to eliminate mealybug vectors. We investigated grape phylloxera as a possible vector of GLRaV. Previous studies in New Zealand excluded this insect as a vector and we consider this to be the standard guideline. Nevertheless, we are conducting trials to alleviate grower concerns and eliminate the possibility that phylloxera play a role in GLRaV transmission in California. After feeding on virus-positive vines in the greenhouse for six months, between 1 and 5%of phylloxera acquired GLRaV-3 or -5, while none acquired GLRaV-1 or -2. None of the 44 healthy vines planted in the same pots as virus-and phylloxera-infected plants developed GLRaV. Additionally, none of the 939 phylloxera collected from GLRaV-2 infected vineyards contained leafroll virus, leading to a conclusion that phylloxera is unlikely to be a vector of GLRaV.