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.

Assessing Fungicide Resistance of grape powdery mildew in Wine, Table and Raisin Grapes

The 2018 assessment campaign processed >3000 samples from the Western US grape production
regions. Analysis of these ToughSpot samples for resistance to quinone outside inhibitor (QoI)
fungicides (FRAC Group 11) using the G143A qPCR assay indicated there is still widespread
resistance throughout Western grape production regions (71% samples with the G143A) but that
resistance is decreasing. Examination of samples from the Willamette Valley of Oregon indicates
that when growers rotate away from using QoI and DMI, the G143A allele occurrence significantly
decreases in the population (31% with G143A). These results were confirmed when isolate and
field sample DNA underwent genotyping by sequencing analysis of the cytb gene. These results
are similar to the QoI resistance observed throughout Oregon in 2015 and 2016. Analysis of these
samples using various molecular techniques and fungicide resistance bioassays to determine
resistance to demethylation inhibitor (DMI) fungicides (FRAC group 3) and succinate
dehydrogenase inhibitor (SDHI) fungicides (FRAC group 7) is ongoing. We have identified 7 E.
necator isolates that are resistant to at least one SDHI fungicide with only one isolate resistant to
more than one SDHI. Sequencing results indicate only one isolate has a known genetic mutation,
thus further sequencing is required. The Y136F qPCR assay for DMI resistance readily detects
the Y136F mutation associated with DMI resistance and can be used to determine DMI resistance
in single spore isolates. Its use on field samples is difficult to interpret because of the multiple
genotypes associated with DMI resistance. A leaf sample collection method was developed that
decreases sample collection and processing time by 40% and is cheaper to process. In addition, a
bark sampling method was developed that agrees with early spring G143A frequency, thus
allowing growers to assess their risk to QoI resistance during the dormant season. Both assays
will be made available to the industry in 2019. Using this project and Oregon Wine board funding
as a foundation for demonstrating grower support, we obtained funding from USDA-SCRI
program to significantly expand research efforts to develop regional networks for managing and
mitigating fungicide resistance.

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
2
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.

Understanding and managing the trunk disease Esca

Summary: Vine surgery (also known as trunk renewal) has been shown to be effective against
Eutypa dieback, but trunk pathogens cause mixed infections. Vineyards are thus likely to be
affected by more than one trunk disease. Furthermore, Eutypa dieback is not even the most
common trunk disease in California. It is important to also test the efficacy of vine surgery
against Esca because infections by two common Esca pathogens, Phaeomoniella chlamydospora
and Phaeoacremonium minimum, can occur at the base of the trunk, either during the
propagation process in the nursery or potentially by infections through roots after being planted
in the vineyard. To accomplish this objective, we established a replicated field trial in a
Sauvignon blanc vineyard in Lake County, CA. Vine surgery was done on vines with leaf
symptoms on shoots growing from only one cane, the idea being that the infection might be
restricted to that portion of the vasculature. In February 2018, we chain-sawed such vines above
the graft union and noted the presence of wood symptoms in 98 total data vines, which were
located among three experimental blocks in the vineyard. The vast majority of the 98 data vines
had wood symptoms, although they were restricted to less than 10% of the cross-section of the
trunk. This observation is important and is not so discouraging because a previous study of vine
surgery, combined with annual fungicide treatments of the cut trunk, showed that a higher
proportion of vines with wood symptoms on less than 20% of the trunk cross-section had normal
vigor and yields 8 years later, compared to vines with more advanced wood symptoms. In the 3rd
year after vine surgery (2020), the data vines will be fully trained and (assuming they are
healthy) are likely to be producing normal yields. After all, this is the goal of vine surgery, to
return vines to productivity and faster than would be possible with a replant. In January 2020,
we will submit a proposal to evaluate the progress of vine surgery, based on presence/absence of
leaf symptoms, vine growth, yields, and juice quality. The second objective of our study was to
identify the Basidiomycete wood-rotting fungi that cause Esca. For many years, there was an
untested assumption that these fungi infected vines after they were already infected by
Ascomycetes Phaeomoniella chlamydospora or Phaeoacremonium species. Our pathogenicity
tests show the wood-rotting fungi are pathogens when inoculated to vines alone. Because the
Basidiomycetes are assumed to be secondary pathogens, management practices have not been
evaluated against them. The most widespread Basidiomycete was Fomitiporia polymorpha.
This species was previously reported from one vineyard in California, so our finding expands its
range within the state. We described a new Basidiomycete species Inonotus vitis, which
represents the first report of Inonotus on grape in the Americas. Pathogenicity studies will be
established in January 2019 to evaluate whether I. vitis is pathogenic. If it is, then it might make
sense to include this species (along with F. polymorpha) in evaluations of pruning-wound
protectants.

Deep sequencing for trunk disease diagnostics

The aim of this multi-year project is 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 sequencingbased
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, metagenome
sequencing and meta-transcriptome 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 3rd and 4th year of 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 (Moralescruz
et al., 2018).
In summary, in these four 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).

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.

Assessing Fungicide Resistance of Grape Powdery Mildew in Wine, Table and Raisin Grapes

Throughout 2017, a field scouting campaign that covered Central and Northern California, Western Oregon, and Southern Washington yielded over 850 field samples and 64 isolates of Erysiphe necator. Analysis of these samples for resistance to quinone outside inhibitor (QoI) fungicides (FRAC Group 11) using the G143A qPCR assay indicated widespread resistance throughout all grape growing regions scouted (90% resistance among ToughSpot kit samples). These results were confirmed when isolate and field sample DNA underwent genotyping by sequencing analysis of the cytb gene. These results are similar to the QoI resistance observed throughout Oregon in 2015 and 2016. Analysis of these samples using various molecular techniques and fungicide resistance bioassays to determine resistance to demethylation inhibitor (DMI) fungicides (FRAC group 3) and succinate dehydrogenase inhibitor (SDHI) fungicides (FRAC group 7) is ongoing. A qPCR assay was developed to target a point mutation (Y136F) of the CYP51 gene that is a contributing factor to DMI resistance in E. necator; the mutation is present in 79.1% of tested 2017 samples (n=43). Analysis by sequencing of the SDHI gene complex has yielded 5 point mutations within two of the subunits in the SDH complex. Two of those mutations induce amino acid changes in their respective proteins and are being analyzed for potential contribution to SDHI resistance.

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.

Red Blotch – Associated Virus

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