The Role of Rootstocks and Single and Mixed Infections of Grapevine Leafroll Associated Virus-3 and Grapevine Virus A in Sudden Vine Collapse

Eighteen blocks in 12 vineyards, located in five different counties, were identified as having clusters of vines characteristic of sudden vine collapse (SVC). The vines included eight scion varieties, two rootstock varieties, and one own-rooted. Leaf samples were collected from both symptomatic and asymptomatic vines within SVC clusters. In addition, asymptomatic vines outside of the SVC cluster were included as an indication of the overall infection status of the block. Samples were processed and tested by RT-qPCR for GLRaV-1, -2, -3, GVA and GVB. Eighty-seven percent of all vines were positive for GLRaV-3, 73% were positive for both GLRaV-3 and GVA, and 26% were positive for both GLRaV-3 and GVB. All GVA and GVB infections were co-infected with GLRaV-3. One vine was positive for GLRaV-1 but no GLRaV[1]2 positive vines were detected. A comparison of GLRaV-3 and GVA infection rates in symptomatic versus asymptomatic vines within SVC clusters indicated that overall, the rates were slightly but significantly higher in symptomatic vines. Analysis at the block level indicated that this positive correlation was only significant in two out of 16 blocks due to the constraints of small sample size. Therefore, GLRaV-3/GVA co-infection rates in symptomatic versus asymptomatic vines were not significantly different in most of the blocks we sampled. A comparison of GLRaV-3/GVA infection rates in asymptomatic vines within and outside SVC clusters indicated that only two blocks had significantly higher co-infection rates in asymptomatic vines within clusters. Conversely, one block had significantly higher infection rates in asymptomatic vines outside the SVC cluster. These observations, coupled with the overall high percentage of vines positive for GLRaV-3 and GVA indicates that the blocks we selected for this study had high GLRaV-3/GVA co-infection rates despite the lack of disease symptoms outside SVC clusters. Fewer GVB positive vines were detected, and these infections were almost evenly divided between symptomatic and asymptomatic vines within SVC clusters, and between asymptomatic vines within and outside the SVC cluster. To track the progression of SVC in these 18 blocks over the next two years, a group of 300 SVC symptomatic and asymptomatic vines within SVC clusters were mapped by row and vine location. For the rootstock field trial, approximately 80 vines each of nine different rootstocks were propagated from dormant cuttings and chip-bud grafted with one bud from Pinot gris 09. These vines were transplanted into 1-gallon pots and are being kept in a FPS screenhouse until spring 2022. In addition, vines positive for GLRaV-3, GLRaV-3 and GVA, GLRaV-1 and GVA, and GLRaV-2 and GVB have been identified and analyzed by high throughput sequencing to verify their infection status. A field site has been identified at the UCD Armstrong Field Station and prepared for planting vines in the spring 2022.

Controlling Grapevine Trunk Diseases in California

A total of 20 vineyards belonging to 10 counties in California were sampled during summer 2019. Cordon, trunk and root tissue were taken from vines using non-destructive methods in order to isolate, analyze and study endophytic bacterial communities between healthy and diseased vines exhibiting typical trunk disease symptoms. A collection of over 1,750 isolates was obtained, from which 27.43% has been screened for their potential antifungal effect against the main GTD-causing pathogens in vitro. A set of 80 bacterial isolates was selected after a first screening against Neofusicoccum parvum, exhibiting >40% of inhibition of the pathogen mycelium. Phylogenetic analyses showed that 57 isolates belong to the genus Bacillus, 8 isolates to Variovorax, 6 isolates to Pseudomonas, 3 isolates to Stenotrophomonas, 2 isolates to Pantoea, 2 isolates to Lysobacter, 1 isolate to Lysinibacillus and 1 to Klebsiella. These isolates are currently being tested using the same methodology against other important GTD pathogens (Diplodia seriata, Eutypa lata, Diaporthe ampelina, Phaeomoniella chlamydospora, Phaeoacremonium minimum and Ilyonectria liriodendri) and a second selection will be performed with bacterial isolates exerting the higher percentages of inhibition against the majority of pathogens in order to be tested in greenhouse and field experiments. Furthermore, a fungicide trial was set over the summer of 2019 in three different nurseries (Winters, Wasco and Bakersfield), including chemical and biological products, using a vacuum chamber to infiltrate the fungicides through the vessels of dormant cuttings prior the grafting process. Fourteen treatments were included, and vines were grafted. Callus formation was evaluated 18 days after the treatments and results indicate frequencies of callusing ratings were similar among treatments, being in average 18.1% of rootstocks showing 100% of callusing, 48.0% showing 80-99%, 22,4% showing 60-79%, 7.7% showing 40-59% and 4.9% showing below 40%. Plants were further planted in pots according to each nursery protocols and kept in the facilities for approximately 3 months. In October 2019, actively growing vines were transported to the UC Davis Department of Plant Pathology field station located in Davis, CA, to be planted in the field under a completely randomized block design. In one of the nurseries, treated vines were planted in the ground after the callusing. The rooting of these plants will be evaluated in March during their transplantation to the field. Trunk disease incidence and severity will be evaluated yearly during the summer season.

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project was to develop rapid and cost-effective diagnostic methods for detection, identification, and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. Healthy vines are essential for the successful establishment and sustainability of all grape production systems. Since wood pathogens may remain asymptomatic in young, non-stressed vines, propagation material may contain latent fungal infections and may become symptomatic after planting and serve as a source of inoculum for further infections of potentially clean plants. Methods of virus detection and eradication have been crucial in ensuring that the material in germplasm repositories and clean plant programs is free of known viruses. There remains much to be developed in terms of fungal pathogen detection. Our laboratories have developed comprehensive genomic information on several ascomycetes associated with the most common and aggressive trunk diseases, which provides the unprecedented opportunity for the implementation of new sequencing based diagnostic tools that take advantage of Next Generation Sequencing (NGS) technologies. By allowing the testing of mother plants in foundation blocks and propagation material in nurseries, we expect that the applications of deep sequencing diagnostics will help establish a certification program for trunk pathogen-free germplasm and reduce the amount of trunk pathogens introduced into vineyards at planting as well as the incidence of young vine decline. Deep-sequencing diagnostics will also help identify disease-causing organisms associated with diseased vines in older vineyards.

In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies. We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and metatranscriptome sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the relative abundances of the different infecting species. This work was published in Molecular Plant Pathology (Morales-cruz et al., 2017). Among all methods
tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization to improve sensitivity and specificity for diagnostics purposes. In the second year of the project (2016-2017), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c) developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of sample, reagents and waste. In the second phase on the project, our effort focused on the development and optimization of a new set of optimized primers for ITS-seq designed specifically to target the ITS of grapevine trunk pathogens. The primers as well as the method are publicly available and described in a peer-reviewed article published in December 2018 (Morales-cruz et al., 2018).

In summary, in these five years we have:
1. Applied NGS to trunk pathogen diagnostics and demonstrated that NGS provides qualitatively and quantitatively accurate simultaneous identification of multiple trunk pathogens directly from grapevine wood samples (Morales-Cruz et al., 2017 Mol Plant Pathol).
2. Developed a new protocol with optimized diagnostic markers for NGS ITS-seq diagnostics of trunk diseases, which is publicly available and described in detail in Morales-Cruz et al. (2018; BMC Microbiology).

We publish all protocols, which can be used freely by diagnostic and research labs. We are now seeking funds to survey propagation material and determine the association between pathogen contamination in propagation material and trunk disease incidence in young vineyards.

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.

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.