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.

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.

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.

Background and goals Grapevine red blotch virus (GRBV) is the causal agent of red blotch disease, a major threat to grape production and vineyard profitability. Several species of Cicadellidae and Membracidae, including the three-cornered alfalfa hopper (Spissistilus festinus), have been reported to transmit GRBV in the greenhouse, but their capacity to act as vectors in the vineyard remains a source of confusion. This article examines the literature on GRBV transmission and provides recommendations to convincingly identify arthropod vectors. Methods and key findings We thoroughly evaluated transmission assays of GRBV that have been reported thus far, and critically analyzed their strengths and limitations. We identified experimental ambiguities and discussed how these have prevented the recognition of S. festinus as a GRBV vector of epidemiological relevance in the vineyard. Based on our assessments and given the circulative transmission mode of GRBV, we formulated a few recommendations to accurately determine how GRBV spreads, and by which insect vectors. These include the testing of the salivary glands of vector candidates for the virus, the determination of an optimal acquisition access period, the use of alfalfa plants for insect gut clearing following the access acquisition period and preceding the inoculation access period, and the inclusion of excised grapevine leaves as virus recipient tissue in transmission assays. Conclusions and significance There is a great deal of confusion about how GRBV spreads, and which insect transmits the virus in the vineyard. Our recommendations pertain to the accurate identification of arthropod vectors, which in turn provide an opportunity to address uncertainties relating to GRBV transmission, and will aid in the development of red blotch disease management strategies.

The epidemiological relationship between arthropod dispersal and plant communities is poorly understood at the landscape level. We investigated the connectivity of natural and cultivated plants in vineyard ecosystems for red blotch disease dynamics by characterizing the dietary history of Spissistilus festinus, a treehopper vector of grapevine red blotch virus. Molecular analysis of the gut content of 205 S. festinus caught in 71 vineyard sites and the flora proximal to vineyards in Napa Valley, California, revealed a total of 171 genera from 60 plant families with a preeminence of Asteraceae, Fabaceae, and Vitaceae over two growing seasons, illustrating dietary profiles composed primarily of natural vegetation. An indicator species analysis identified the strength of ecological associations by showing distinct seasonal feeding trends related to weather patterns and host repertoires unique to some vineyard sites with estimated S. festinus travel distances of up to 2 km. Potential reproductive and overwintering hosts of S. festinus were identified in natural habitats, and an ecological relatedness between free-living vines in riparian corridors and vineyards for grapevine red blotch virus transmission was documented. Together, our findings on landscape vegetation connectivity and S. festinus dispersal transformed our understanding of red blotch disease ecology and informed disease management options. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .