Interaction of Red Blotch Virus (GRBV) and Deficit Irrigation on Grapevine Water Relations, Disease Development, and Vine Productivity

A field experiment with two irrigation treatments – wet (W) and dry (D) – and two vine disease statuses – healthy (RB-) and infected (RB+) – was initiated in a commercial vineyard to understand the interaction between GRBV infection and deficit irrigation on disease development, vine productivity, and fruit quality. Irrigation treatments were imposed by varying the number of drip emitters per vine. W vines were irrigated at 100% of crop evapotranspiration (ETc), while D vines received water at 66% ETc. Within each irrigation treatment, RB- and RB+ vines (split-plot) were identified based on symptomology data from 2016. The identified vines were confirmed for GRBV positive and negative by PCR-based assays.

Measurements of vine water status (midday stem water potential; Ψstem) were made at regular intervals throughout the growing season beginning just after berry set until just before harvest. Disease severity was recorded every week after the first symptom appearance was observed on RB+ vines. At harvest, berry samples were collected for berry size and compositional analyses; and vine yield and yield components were determined.

There was no significant interaction between irrigation treatment and disease status on disease progression and severity. There was a significant interaction between irrigation treatment and vine disease status on Ψstem, but it depended on phenology. In other words, preveraison Ψstem was not affected by disease status but was significantly higher in RB+ vines postveraison. The higher Ψstem in RB+ vines resulted in larger berries and yield at harvest. Interestingly, RB+ also had greater clusters per vine and berries per vine in the W, but few of the differences in yield and yield components among treatments were significant.

Differences in juice composition among treatments were smaller than previously reported, but all treatment effects were more pronounced on berry secondary metabolites in skins and seeds. In juice, Brix was only slightly impacted by the experimental treatments, but there were no significant differences among treatments in juice pH or TA. In skins and seeds, significant differences among treatments were observed in both concentration and content of some secondary metabolites (anthocyanins and iron-reactive phenolics (IRPs)), but not others (tannins). Irrigation treatment had a greater effect than disease status on anthocyanins, while the converse was observed with respect to tannins and IRPs. Differences in secondary metabolism were most pronounced in seeds, which is likely related to the inhibition of ripening commonly observed in RB+ fruit.

Small differences among treatments in tannins coupled with the large differences in IRPs suggests that GRBV inhibits biosynthesis of flavonols (such as quercetin), which are the dominant class of non-iron-reactive phenolics, particularly in seeds. Since flavonols are implicated in wine mouthfeel, this may offer some explanation as to why GRBV-infected fruit produces lower quality wines. Taken together, these results suggest that keeping vines well-watered may mitigate some of the negative effects of GRBV infection, but ultimate changes in 2 secondary metabolism due to GRBV infection may necessitate using infected fruit for different wine programs (e.g. rosé and/or sparkling) or blending with lots from healthy vineyards.

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 patiotemporal 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.

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.

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

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.

Biology and Spread of Grapevine Red Blotch-Associated Virus

Grapevine red blotch-associated virus (GRBaV) is associated with red blotch, a newlyrecognized emerging viral disease. We showed that GRBaV is the causal agent of the disease and microshoot tip culture can eliminate the virus from infected vines. Two distinct groups of GRBaV isolates exist in infected vines and currently available diagnostic tools are robust for a reliable detection of all isolates. Efforts to develop a serological detection assay for GRBaV are under way. Monitoring incidence of GRBaV in vineyards over time did not provide compelling evidence of spread. Research progress was extended to stakeholders through 15 presentations at conventions, field days and IPM conferences.

Grower Implemented Quantitative LAMP for Initiating and Adjusting Fungicide Program

The mission of this research is to increase the economic sustainability of grape production by providing decision support tools to aid in management of grape powdery mildew. In this project we propose to test the utility of a quantitative Loop mediated isothermal AMPlification assay (qLAMP) and handheld device for detection and quantification of airborne inoculum; thereby extending our  esearch on the use of inoculum detection as a decision support tool for managing grape powdery mildew. The specific objectives are:

  1. Test implementation of a grower preformed quantitative LAMP assay.
  2. Examine the effectiveness of adjusting fungicide interval based inoculum density.
  3. Assessment of quantitative LAMP (qLAMP) for estimating amount of fruit infection

Results: Due to reduced funding, Objective 1 had to be reduced and Objective 3 eliminated in the second year of this 3 year project. The extensive cooperation of several participating growers who shuttled samples from the upper Willamette Valley to Corvallis, allowed for us to complete Objective 2 using lab processed samples only.