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.

 

Development of a Simulation Environment for Pathogen and Pest Spread in Vineyards

The overall goal of this research has been to develop an innovative modeling platform that can accurately simulate pathogen and pest spread in vineyards. The simulation tool will serve to help producers evaluate disease and pest management decisions using “virtual” crops. This system will allow producers to evaluate “what if” scenarios and to examine how to isolate individual management decisions that influence disease, pest, and plant development. The system can also be used to examine how row orientation, training system, etc. interact with climate and geography at new vineyard locations.

Completed research to date has focused on model development and integration. The project has successfully produced a model framework that integrates previously developed models for climate, plant growth, spore dispersion, pathogen infection, and colony growth. The system is currently able to simulate plant growth and disease progression throughout a growing season. An initial “vineyard builder” tool has been developed to rapidly build up the geometry of a particular vineyard of interest within the simulation system. Work is also underway to develop improved sub-models for meteorology and turbulence. The meteorological model will predict the three-dimensional turbulent wind field, which drives the airborne dispersion model. This work has involved comparing model outputs to field measurements, and making necessary modifications to the model to improve agreement between the two. Other work is developing improved models for airborne particle deposition to plant surfaces.

The overall modeling platform consists of a suite of coupled sub-models that represent the most important physical processes of disease spread such as plant growth, local climate, airborne dispersal by the turbulent wind, pathogen infection and colony growth. These state-of-the-art sub-models are among the most detailed simulation tools that have ever been developed for agricultural crops. Since they require substantial computational resources not provided by the processors of a standard desktop or laptop computer, we have overcome this limitation by using standard computers with a gaming graphics card. We have used the graphics card to accelerate many of the sub-models, meaning that very large simulations can be performed in a matter of minutes.

Using Post-Plant Nematicide Applications for Nematode Suppression

The spray applications with Movento were made on October 19, 2016. Watering scheduled as shown in the overview. On October 28 one of the grape plants was removed. Active leaves, and fibrous roots were excised, and provided after grinding to the Daane laboratory for analysis. Nematode population densities were determined at treatment time (October 18) and post-treatment (January 25). Soil samples were taken from 0-1 and 1-2 ft depth. After extraction, nematodes were identified and counted.

Nematode counts were analyzed before treatments (Fig. 1). Telone II fumigation reduced nematode numbers effectively. At treatment time, nematode numbers were similar among spirotetramat (Movento) treatments although some variability was measured. In parametric analysis, the “watering regiment Movento” treatment combinations analyzed as two-factor factorial design had no significant main effects or interactive effects (data not shown). Soil samples are currently processed to determine nematode numbers post-treatment.

The chemical detection of spirotetramat and its break-down products is initiated. It was possible to detect spirotetramat in the leaf tissues (Fig. 2). No spirotetramat was detected in the plants from the drench-treated plots. There were traces in the Telone-treated plots and in the non-treated controls. These appeared as false positives, and each case was only based on the detection of spirotetramat in one of the five replicate plants of the respective treatments. No spirotetramat was found in roots (data not shown).

The spirotetramat enol was found in leaves and roots from plants of all treatments (Fig. 3, 4). It is currently unclear how this can be explained. We are examining plants of the same source for the potential presence of some of these chemicals before treatment to assess if these could be carrying a back-ground amount of the chemicals. A careful review of our laboratory procedures did not provide any indication of experimental/technical errors that could have led to this find.

Conclusions and outlook

Progress has been made to establish the laboratory method to detect different forms of spirotetramat and its break-down products. A microplot experiment is in place that will be treated in the spring while allowing for additional destructive sampling of chemical analysis and nematode population density monitoring. Population densities of the nematode will provide additional clues on the efficacy of the treatments.

Evaluating the Effects of Grapevine Red Blotch-Associated Virus on Symptom Development and Fruit Maturity

A two year study in 2013 and 2014 evaluated the effect of grapevine red blotch disease on fruit produced on vines infected with Grapevine red blotch-associated virus (GRBaV). Vine growth was also monitored. In each of four sites, data vines were selected that tested positive or negative by qPCR for GRBaV and negative for several Grapevine leafroll associated viruses, vitiviruses and nepoviruses. Approximately 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the vines monitored were positive for Grapevine rupestris stem pitting-associated virus.  Data vines were located in four vineyards; one site each of Chardonnay, Cabernet Sauvignon, Merlot and Zinfandel.  Chardonnay and Cabernet Sauvignon vines were evaluated for two years and fruit was reduced in half of the GRBaV-positive vines to determine the effect of that practice on vine performance as compared to fruit from infected vines with a full crop load.

Foliar disease symptoms were evaluated in 2013 in Cabernet Sauvignon, Chardonnay and Merlot. Disease severity was an estimate of percent of leaf surface area expressing red color or chlorotic tissue associated with red blotch disease in red and white cultivars respectively. Across cultivars severity was greatest in the basal region of the canopies; however Chardonnay and Merlot canopies had a greater amount of leaf area affected by the disease compared to that of Cabernet Sauvignon. In Chardonnay severity was equally high in blades located in both the basal and middle regions of the canopy.

Red blotch disease consistently reduced sugar accumulation and increased malic acid in juice at harvest in Chardonnay, Cabernet Sauvignon and Merlot. Diseased vines had elevated titratable acidity in all cultivars and in Merlot differences were significant. Juice pH was increased in fruit from diseased Chardonnay and Zinfandel vines but not in Cabernet Sauvignon or Merlot. Crop reduction in diseased vines at early veraison in 2013 or just past fruit set on the same vines the following year did not significantly modify the effect of the red blotch disease on fruit composition.

Yield was reduced each year in diseased Chardonnay vines due to fewer clusters whereas Cabernet Sauvignon yield was not affected. In Zinfandel, GRBaV-positive vines produced fewer clusters with less mass than did GRBaV-negative vines although differences were not significant. Pruning weight was reduced in diseased Chardonnay in one of two years and not affected either year in Cabernet Sauvignon.

Biology and Spread of Grapevine Red Blotch-Associated Virus

Grapevine red blotch-associated virus (GRBaV) was isolated from table and wine grapes, as well as rootstocks, affected by red blotch, a recently recognized viral disease in grapevines.  Analysis of the genetic diversity among isolates of GRBaV indicates the existence of two groups (clades) of genetic variants (Krenz et al., 2014, Al Rwahnih et al., 2015).  Producing a full-length infectious clone of a representative isolate of each of the two clades showed systemic GRBaV infection of healthy grapevines following agroinoculation and the manifestation of typical disease symptoms, i.e. interveinal reddening on Vitis vinifera cvs. Cabernet franc, Cabernet Sauvignon, Syrah, Pinot noir and Pinot gris; and chlorotic and necrotic leaf areas on Vitis vinifera cv. Chardonnay, while infection was latent in rootstock genotype 3309C.  This work revealed that GRBaV isolates of both clades cause red blotch disease. Analysis of the spatio­temporal incidence of GRBaV in a selected vineyard of Cabernet franc in California and in New York was consistent with the occurrence of virus spread in the former but not in the latter vineyard.  GRBaV isolates spreading in California corresponded to phylogenetic clade 2.  A survey of alternate hosts in proximity to the diseased vineyard in California showed some free-living grapevines infected with GRBaV, suggesting the existence of a hemipteran vector. Insect sticky traps placed in the section of the California vineyard with extensive clustering of diseased vines in 2014 and 2015 revealed a diversity of insect families, genera and species that visited the vineyard, among which, the majority of specimens of three species consistently tested positive for GRBaV in PCR.  These species are investigated for their potential to transmit GRBaV in controlled conditions in the greenhouse.

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{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.