Developing a GMO-Free RNA Interference Approach to Mitigate Red Blotch Negative Impacts on Grape Berry Ripening

RNA interference (RNAi) has been extensively used in crop protection platforms. Currently
most approaches are based on the conventional use of transgenic plants expressing doublestranded RNAs (dsRNAs) against selected targets (endogenous plant genes, virus, fungi,
bacteria). However, the use of Genetically Modified Organisms (GMOs) has raised scientific and
public concerns. Approaches alternative to the production of Genetically Engineered material
that could enable the direct exogenous application of RNA molecules to trigger RNAi has the
potential to address public and industry concerns with regards to RNAi field-based technology
(Dalakouras et al., 2020). There are several methods to deliver RNA in plants. Our project
proposes to develop dsRNAs application tools to mitigate the negative effects of Red Blotch
Virus on grape berry composition. As a first step of a long-term project, we propose to identify
through a genome wide study the genomic regions of the Red Blotch Virus that are targeted by
grapevine during the early stages of viral infection. Since the acceptance of the project in June
2020, the PI has recruited a graduate student from Italy who will be working on the project.
Through cooperation, the PL Deluc has been issued by the USDA-APHIS services a permit to
import two bitmer infectious clones containing two variant strains (NY175 and NY358) of the
Red Blotch virus (USDA -APHIS Permit #: 20-176-103m) These infectious clones will be
essential to study the early phases of grapevine GRBV infection through two main
methodologies that are describe below.

Layperson summary*
RNA interference (RNAi) is a conserved biological response across living organisms (animal or
plant cells) initiated by the presence of double-stranded RNA molecules from various pathogens,
including viruses. The RNA interference mechanism initiated in the plants will lead to a cascade
of molecular events that are meant to repress the activity of the virus and its propagation within
the plant. Once infected, the plants will recognize and produce specific nucleic regions of the
viral genome to activate the RNA silencing machinery. These regions are named “hot spots”
regions. The main goal of this project is to identify these “hot spots” for the Red Blotch Virus. In
the long-term, this knowledge could help developing innovative technology tools like ectopic
RNA molecules application in vineyards to mimic the presence of the virus and to make the
plants immune or “primed” to further infections like a vaccine will do. This might limit the
propagation of the Red Blotch Virus from plants to plants and could potentially mitigate its
negative effect on grape berry ripening of already infected plants. The current project will use
Next Generation Sequencing technologies to identify these “hot spots” during the early stages of
Red Blotch Virus infection in tissue culture material. Once the nucleic regions identified, the
continuum of the project will involve trials for systemic application, through spray, of these
RNA molecules to either infected plants to mitigate the negative effects of Red Blotch on
ripening, or to non-infected plants, to trigger their immune responses to viral infection. If
confirmed in a greenhouse setting, the next step will be to assess the ds-RNA formulation in
field-trails.

Investigation of the impact of grapevine red blotch-associated virus (GRBaV) on grapevine health and subsequent grape and wine composition and style

Since its identification in 2011, grapevine red blotch disease has been found to be wide spread in the United States. This disease is caused by grapevine red blotch virus (GRBV, in 2017 the name changed from grapevine red blotch-associated virus (GRBaV)) infection of grapevines. Over the past four years, we have investigated the impact of GRBV on grape composition and resulting wine quality across varieties, rootstocks, seasons and sites. This investigation completed a two-year study on Cabernet Sauvignon (CS) and Merlot (ME) grape varieties, examining the impact of GRBV on grapes at harvest and on the resulting wine composition. Additionally, the effect of rootstock on disease expression was also investigated as the CS grapevines were grafted onto two different rootstocks: 110R and 420A.
Grapes through development were analyzed for sugar and anthocyanin content, as well as pH and TA levels. The phenolic profiles of harvested grapes and final wines were analyzed by RP-HPLC and a modified protein precipitation (PP) assay. Volatile profiles of harvested grapes and final wines were analyzed using HS-SPME-GC-MS. Results from these analyses were compared to the sensory evaluation of the four wine treatments through a trained panel.
Consistently through the four years, symptomatic grapevines (RB (+)) resulted in lower sugar content, less anthocyanin accumulation, and higher titratable acidity (TA) when compared to asymptomatic grapevines (RB (-)). Due to these observations, chaptalization was used to adjust the soluble sugars (SS) (and therefore ethanol content of final wines) of the diseased grapes (RB (+) S) at crush to be the same as the healthy grapes. Chaptalization could potentially determine if ethanol concentration differences between wines made from RB (-) and RB (+) grapes were contributing to phenolic differences in the wines. In another treatment, diseased grapes were harvested a second time (RB (+) 2H) when the SS reached similar values as RB (-) grapes.
The results of this investigation confirm previous findings, that disease expression is dependent on genotypic and environmental factors. Anthocyanin and sugar accumulation were significantly lower in RB (+) grapes than RB (-), yet, the level of disease impact varied from rootstock, variety and location. Vine physiological measurements and phenolic profile analysis on harvested grapes indicated a potential larger impact on RB (+) vines on 420A rootstocks. In general a second harvest was successful at increasing anthocyanin, some phenolic, and volatile concentrations in infected grapes. The phenolic extraction during fermentation was followed and revealed that longer hang time (RB (+) 2H) increased extraction more than chaptalization (RB (+) S). Chemical analysis of the final wines indicated that both mitigation strategies (chaptalization and sequential harvesting) alleviated some of the differences due to disease status. This indicate that both ethanol content and cell wall integrity may play a role in phenolic extractability during winemaking.

Interaction of red blotch virus (GRBV) and deficit irrigation on grapevine water relations, disease development, and vine productivity

The second year of a field experiment with two irrigation treatments – wet (W) and dry (D) – and two vine disease statuses – healthy (GRBV-) and infected (GRBV+) – was continued in a commercial vineyard to understand the interaction between GRBV infection and deficit irrigation on disease development, vine productivity, and fruit quality. W vines were irrigated at 100% of crop evapotranspiration (ETc), while D vines received water at 50% ETc. Within each irrigation treatment, GRBV- and GRBV+ vines (split-plot) that were previously identified in early 2017 based on symptomology data from 2016. The identified vines were confirmed as GRBV+ and GRBV- by PCR-based assays. GRBV- vines from 2017 were re-tested in early 2018 to confirm disease status.
In both years, measurements of vine water status (midday stem water potential; stem) were made at regular intervals throughout the growing season beginning just after berry set until just before harvest. Similarly, disease severity was recorded every week after the first symptom appearance was observed on GRBV+ vines. At harvest, berry samples were collected for berry size and compositional analyses; and vine yield and yield components were determined.
With respect to vine water relations and gas exchange, there were no significant interactions among experimental treatments. Irrigation treatment and disease status both impacted these aspects of vine physiology, but they acted independently, with water deficits consistently reducing vine water status, and GRBV infection consistently increasing it. In other words, GRBV infection had the same effect on vine water status (stem water potential) under both well-watered and deficit conditions. However, the significant impact of GRBV infection on vine water status only arose post-veraison – at the same time that foliar symptoms became visible. The increase in post-veraison water status under GRBV+ conditions was likely a consequence of reduced stomatal conductance, which in turn reduced net photosynthesis.
Berry development was similarly impacted by the treatments independently, with consistently larger berries in W and GRBV+ vines. This was observed at nearly every sample date in each year, but differences between vines of different disease status only became significant post-veraison. TSS were also only significantly different between GRBV- and GRBV+ vines post-veraison, and there was a limited impact of irrigation treatment. In contrast, pH and TA were more variable among treatments and years, suggesting that GRBV has a limited effect on organic acid metabolism.
Irrigation treatment and disease status impacted skin and seed phenolic concentrations to varying degrees over two seasons. Whereas irrigation treatment and disease status impacted skin phenolic concentration together, disease status alone impacted seed phenolic concentration. In both years, skin anthocyanin concentration was increased with deficit irrigation – in both GRBV- and GRBV+ vines – but only increases in GRBV- vines were statistically significant. Conversely, skin tannin and iron-reactive phenolic concentrations were variably affected by treatments, and results were not consistent between years. In seeds, there were no effects of the irrigation treatments, but disease status significantly reduced both tannin and iron-reactive phenolic concentrations. This effect was consistent between years. All together, these results
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suggest that the genetic control of phenolic metabolism by GRBV infection is stronger than the environmental control due to vine water deficits. Furthermore, experimental results suggest that keeping vines well-watered may mitigate some of the negative effects of GRBV infection, but ultimate changes in 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.
In contrast to 2017 data on disease severity, significant differences on rate of disease progression as well as disease severity were observed in 2018 between the wet and dry treatments. The vines in wet irrigation treatment showed significantly low disease severity at harvest and two weeks prior to harvest. The differences were observed as significant increase in vine canopy in irrigation treatment compared to vines in dry treatment; as a result percent of symptomatic leaves in wet treatment vines were less compared to dry treatment vines. Even though, the virus expression remained same (symptomatic) within wet and dry treatments, it would be informative to assess the status of virus (quantity) within each treatment. Furthermore, the carry over effect of less severe vines on vine health as well as fruit qualities would be an additional information on long-term management of GRBV infected vines. On this regard, we wanted to continue the project for another year, where the focus will be on virus quantification, vine health, and fruit quality as a result of carry over effects within less severe vines while keeping all treatments as is.

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 Effect of Grapevine Red Blotch Associated Virus Infection on Vine Nutrition

 

 

Principal Investigators:

Rhonda J. Smith, Monica L. Cooper, Mysore R. Sudarshana

 

  1. Summary:

A one year study in 2015 evaluated the differences in vine mineral nutrient levels as influenced by infection with grapevine red blotch-associated virus (GRBaV) in vines that were not co-infected with several other grapevine viruses. Nutrients were assessed in a Chardonnay and a Cabernet Sauvignon vineyard located in Sonoma and Napa Counties respectively. The two sites were selected based on extensive virus testing that had occurred in 2013 which identified 10 vines at each site that were GRBaV-positive and 10 vines that were GRBaV-negative.

The 20 vines selected at each site all tested negative for 10 grapevine leafroll-associated viruses, four nepoviruses [Arabis mosaic virus, Grapevine fanleaf virus (GFLV), Tobacco ringspot virus, and Tomato ringspot virus]; and four vitiviruses [Grapevine virus A, Grapevine virus B, Grapevine virus D, and Grapevine virus E]. Of the 40 vines selected for the study, thirty-one tested positive for grapevine rupestris-associated virus. Virus testing conducted on samples collected in December 2015 confirmed that vines had remained negative for viruses known to be naturally spread by insects, as well as for GFLV. In addition, all vines previously determined to be GRBaV-negative remained uninfected.

Petioles were collected at bloom and veraison from each vine and tissue collected from an individual vine represented a single sample, thus for each cultivar on each sample date 10 samples were collected from vines that tested negative for GRBaV and 10 samples were collected from vines that tested positive for GRBaV. Petiole analyses on samples collected at bloom included total N, nitrate-N, total P, phosphate-P, extractable and total K, Ca, Mg, S, Zn, B, Fe, Mn and Cu. At veraison, petiole tissue was analyzed for total N and extractable K.

Chardonnay vines infected with GRBaV had significantly greater total N and total K at bloom and total N at veraison when compared to vines that were not infected. Extractable K was greater at bloom in infected vines but not at veraison. Nitrate-N and phosphorous were not affected by virus status in Chardonnay. Vines that tested GRBaV-negative had significantly greater Ca, Mg, Zn, Mn and Cu at bloom when compared to infected vines; however these minerals were at adequate levels regardless of virus status. Boron and iron were not affected by virus status in Chardonnay.

Cabernet Sauvignon vines infected with GRBaV had significantly greater phosphorous at bloom than did vines that were not infected. Nitrate-N was reduced in infected vines. Total N and potassium at both bloom and veraison were not affected by virus status. Vines that tested GRBaV-negative had significantly greater Ca and elevated Mg at bloom when compared to infected vines. Zinc, Mn and Cu were not affected; however in infected vines, boron was increased and iron levels decreased.

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%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%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%(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.