Grapevine Red Blotch Virus Is Transmitted by the Three-Cornered Alfalfa Hopper in a Circulative, Nonpropagative Mode with Unique Attributes.

The transmission mode of grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) by Spissistilus festinus, the three-cornered alfalfa hopper, is unknown. By analogy with other members in the family Geminiviridae, we hypothesized circulative, nonpropagative transmission. Time-course experiments revealed GRBV in dissected guts, hemolymph, and heads with salivary glands after a 5-, 8-, and 10-day exposure to infected grapevines, respectively. After a 15-day acquisition on infected grapevines and subsequent transfer on alfalfa, a nonhost of GRBV, the virus titer decreased over time in adult insects, as shown by quantitative PCR. Snap bean proved to be a feeding host of S. festinus and a pseudosystemic host of GRBV after Agrobacterium tumefaciens-mediated delivery of an infectious clone. The virus was efficiently transmitted by S. festinus from infected snap bean plants to excised snap bean trifoliates (90%) or grapevine leaves (100%) but less efficiently from infected grapevine plants to excised grapevine leaves (10%) or snap bean trifoliates (67%). Transmission of GRBV also occurred trans-stadially but not via seeds. The virus titer was significantly higher in (i) guts and hemolymph relative to heads with salivary glands, and (ii) adults emanating from third compared with first instars that emerged on infected grapevine plants and developed on snap bean trifoliates. This study demonstrated circulative, nonpropagative transmission of GRBV by S. festinus with an extended acquisition access period compared with other viruses in the family Geminiviridae and marked differences in transmission efficiency between grapevine, the natural host, and snap bean, an alternative herbaceous host.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

Insights Into the Ecology of Grapevine red blotch virus in a Diseased Vineyard.

Limited information is available on the spread of Grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) in vineyards. To investigate ecological aspects of red blotch disease spread, sticky cards to catch flying insects were placed in 2015 (April to November) and 2016 (March to November) in a vineyard study site in California where disease incidence increased by nearly 20% between 2014 and 2016. Subsets of insect species or taxa were removed from sticky card traps and individual specimens were tested for the presence of GRBV by multiplex polymerase chain reaction. GRBV was consistently detected in Spissistilus festinus (Membracidae), Colladonus reductus (Cicadellidae), Osbornellus borealis (Cicadellidae), and a Melanoliarus sp. (Cixiidae). Populations of these four candidate vectors peaked from June to September, with viruliferous S. festinus peaking from late June to early July in both years. An assessment of co-occurrence and covariation between the spatial distribution of GRBV-infected vines and viruliferous insects identified a significant association only with viruliferous S. festinus. These findings revealed the epidemiological relevance of S. festinus as a vector of GRBV in a vineyard ecosystem. Sequencing coat protein and replicase-associated protein gene fragments of GRBV isolates from newly infected vines and viruliferous vector candidates further suggested secondary spread primarily from local sources and occasionally from background sources.

Distinct Red Blotch Disease Epidemiological Dynamics in Two Nearby Vineyards.

Grapevine red blotch virus (GRBV) causes red blotch disease and is transmitted by the three-cornered alfalfa hopper, Spissistilus festinus. GRBV isolates belong to a minor phylogenetic clade 1 and a predominant clade 2. Spatiotemporal disease dynamics were monitored in a 1-hectare 'Merlot' vineyard planted in California in 2015. Annual surveys first revealed disease onset in 2018 and a 1.6% disease incidence in 2022. Ordinary runs and phylogenetic analyses documented significant aggregation of vines infected with GRBV clade 1 isolates in one corner of the vineyard (Z = -4.99), despite being surrounded by clade 2 isolates. This aggregation of vines harboring isolates from a non-prevalent clade is likely due to infected rootstock material at planting. GRBV clade 1 isolates were predominant in 2018-2019 but displaced by clade 2 isolates in 2021-2022, suggesting an influx of the latter isolates from outside sources. This study is the first report of red blotch disease progress immediately after vineyard establishment. A nearby 1.5-hectare 'Cabernet Sauvignon' vineyard planted in 2008 with clone 4 (CS4) and 169 (CS169) vines was also surveyed. Most CS4 vines that exhibited disease symptoms one-year post-planting, likely due to infected scion material, were aggregated (Z = -1.73). GRBV isolates of both clades were found in the CS4 vines. Disease incidence was only 1.4% in non-infected CS169 vines in 2022 with sporadic infections of isolates from both clades occurring via secondary spread. Through disentangling GRBV infections due to the planting material and S. festinus-mediated transmission, this study illustrated how the primary virus source influences epidemiological dynamics of red blotch disease.

The Three-Cornered Alfalfa Hopper, Spissistilus festinus, Is a Vector of Grapevine Red Blotch Virus in Vineyards.

Spissistilus festinus (Hemiptera: Membracidae) transmit grapevine red blotch virus (GRBV, Grablovirus, Geminiviridae) in greenhouse settings; however, their role as a vector of GRBV in vineyards is unknown. Following controlled exposures of aviruliferous S. festinus for two weeks on infected, asymptomatic vines in a California vineyard in June and a 48 h gut clearing on alfalfa, a nonhost of GRBV, approximately half of the released insects tested positive for GRBV (45%, 46 of 102), including in the salivary glands of dissected individuals (11%, 3 of 27), indicating acquisition. Following controlled exposures of viruliferous S. festinus for two to six weeks on GRBV-negative vines in vineyards in California and New York in June, transmission of GRBV was detected when two S. festinus were restricted to a single leaf (3%, 2 of 62 in California; 10%, 5 of 50 in New York) but not with cohorts of 10-20 specimens on entire or half shoots. This work was consistent with greenhouse assays in which transmission was most successful with S. festinus exposed to a single leaf (42%, 5 of 12), but rarely occurred on half shoots (8%, 1 of 13), and never on entire shoots (0%, 0 of 18), documenting that the transmission of GRBV is facilitated through the feeding of fewer S. festinus on a restricted area of grapevine tissue. This work demonstrates S. festinus is a GRBV vector of epidemiological importance in vineyards.

Transmission of Grapevine Red Blotch Virus by Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) between Free-Living Vines and Vitis vinifera 'Cabernet Franc'.

Grapevine red blotch disease emerged within the past decade, disrupting North American vine stock production and vineyard profitability. Our understanding of how grapevine red blotch virus (GRBV), the causal agent of the disease, interacts with its Vitis hosts and insect vector, Spissistilus festinus, is limited. Here, we studied the capabilities of S. festinus to transmit GRBV from and to free-living vines, identified as first-generation hybrids of V. californica and V. vinifera 'Sauvignon blanc' (Vcal hybrids), and to and from V. vinifera 'Cabernet franc' (Vvin Cf) vines. The transmission rate of GRBV was high from infected Vcal hybrid vines to healthy Vcal hybrid vines (77%, 10 of 13) and from infected Vvin Cf vines to healthy Vcal hybrid vines (100%, 3 of 3). In contrast, the transmission rate of GRBV was low from infected Vcal hybrid vines to healthy Vvin Cf vines (15%, 2 of 13), and from infected Vvin Cf vines to healthy Vvin Cf vines (19%, 5 of 27). No association was found between transmission rates and GRBV titer in donor vines used in transmission assays, but the virus titer was higher in the recipient leaves of Vcal hybrid vines compared with recipient leaves of Vvin Cf vines. The transmission of GRBV from infected Vcal hybrid vines was also determined to be trans-stadial. Altogether, our findings revealed that free-living vines can be a source for the GRBV inoculum that is transmissible by S. festinus to other free-living vines and a wine grape cultivar, illustrating the interconnected roles of the two virus hosts in riparian areas and commercial vineyards, respectively, for virus spread. These new insights into red blotch disease epidemiology will inform the implementation of disease management strategies.

Spatiotemporal spread of grapevine red blotch-associated virus in a California vineyard.

Grapevine red blotch-associated virus (GRBaV), 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. Limited information is available on the epidemiology of red blotch disease. A 2-hectare Vitis vinifera cv. 'Cabernet franc' vineyard in Napa County, California, USA was selected for monitoring GRBaV spread over a three-year period (2014-2016) based on an initially low disease incidence and an aggregation of symptomatic vines at the edge of the vineyard proximal to a wooded riparian area. 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. Spatiotemporal analysis between consecutive years within the association function of SADIE revealed a strong overall association among all three years (X=0.874-0.945). 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. These findings are consistent with the existence of a new type of vector for a grapevine virus.