Grapevine red blotch-associated virus is Present in Free-Living Vitis spp. Proximal to Cultivated Grapevines.

Red blotch is an emerging disease of grapevine associated with grapevine red blotch-associated virus (GRBaV). The virus spreads with infected planting stocks but no vector of epidemiological significance has been conclusively identified. A vineyard block of red-blotch-affected Vitis vinifera 'Cabernet franc' clone 214 was observed in California, with a clustering of infected, symptomatic vines focused along one edge of the field proximal to a riparian habitat with free-living Vitis spp. No genetic heterogeneity was observed in a 587-nucleotide region of the GRBaV genome in a population of 44 Cabernet franc clone 214 isolates. By contrast, genetic differences were observed in isolates from other cultivars and clones growing in adjacent blocks. GRBaV was confirmed infecting four free-living vines, two of which were shown to be V. californica × V. vinifera hybrids. The genomes of three free-living GRBaV vine isolates and seven from V. vinifera cultivars were compared; free-living vine isolates were shown to be more similar to each other and a 'Merlot' isolate than to the other cultivated vine isolates. The finding that GRBaV is present in free-living Vitis spp. indicates the virus can be spread by natural (nonhuman-mediated) means, and we hypothesize that in-field spread of GRBaV is occurring.

Grapevine Red Blotch-Associated Virus, an Emerging Threat to the Grapevine Industry.

Grapevine red blotch-associated virus (GRBaV) is a newly identified virus of grapevines and a putative member of a new genus within the family Geminiviridae. This virus is associated with red blotch disease that was first reported in California in 2008. It affects the profitability of vineyards by substantially reducing fruit quality and ripening. In red-berried grapevine cultivars, foliar disease symptoms consist of red blotches early in the season that can expand and coalesce across most of the leaf blade later in the season. In white-berried grapevine cultivars, foliar disease symptoms are less conspicuous and generally involve irregular chlorotic areas that may become necrotic late in the season. Determining the GRBaV genome sequence yielded critical information for the design of primers for polymerase chain reaction-based diagnostics. To date, GRBaV has been reported in the major grape-growing areas in North America and two distinct phylogenetic clades have been described. Spread of GRBaV is suspected in certain vineyards but a vector of epidemiological significance has yet to be identified. Future research will need to focus on virus spread, the production of clean planting stocks, and the development of management options that are effective, economical, and environmentally friendly.

Predictive Modeling of Proteins Encoded by a Plant Virus Sheds a New Light on Their Structure and Inherent Multifunctionality.

Plant virus genomes encode proteins that are involved in replication, encapsidation, cell-to-cell, and long-distance movement, avoidance of host detection, counter-defense, and transmission from host to host, among other functions. Even though the multifunctionality of plant viral proteins is well documented, contemporary functional repertoires of individual proteins are incomplete. However, these can be enhanced by modeling tools. Here, predictive modeling of proteins encoded by the two genomic RNAs, i.e., RNA1 and RNA2, of grapevine fanleaf virus (GFLV) and their satellite RNAs by a suite of protein prediction software confirmed not only previously validated functions (suppressor of RNA silencing [VSR], viral genome-linked protein [VPg], protease [Pro], symptom determinant [Sd], homing protein [HP], movement protein [MP], coat protein [CP], and transmission determinant [Td]) and previously identified putative functions (helicase [Hel] and RNA-dependent RNA polymerase [Pol]), but also predicted novel functions with varying levels of confidence. These include a T3/T7-like RNA polymerase domain for protein 1AVSR, a short-chain reductase for protein 1BHel/VSR, a parathyroid hormone family domain for protein 1EPol/Sd, overlapping domains of unknown function and an ABC transporter domain for protein 2BMP, and DNA topoisomerase domains, transcription factor FBXO25 domain, or DNA Pol subunit cdc27 domain for the satellite RNA protein. Structural predictions for proteins 2AHP/Sd, 2BMP, and 3A? had low confidence, while predictions for proteins 1AVSR, 1BHel*/VSR, 1CVPg, 1DPro, 1EPol*/Sd, and 2CCP/Td retained higher confidence in at least one prediction. This research provided new insights into the structure and functions of GFLV proteins and their satellite protein. Future work is needed to validate these findings.

Two Distinct Genotypes of Spissistilus festinus (Say, 1830) Reproduce and Differentially Transmit Grapevine Red Blotch Virus.

Two phenotypically similar but genetically distinct genotypes of Spissistilus festinus (Say, 1830) (Hemiptera: Membracidae), a pest of legume crops in Southern United States and a vector of grapevine red blotch virus (GRBV) in California vineyards, exist. No information is available on whether the two S. festinus genotypes, i.e., California (CA) and Southeastern (SE), are sexually compatible or whether the SE genotype can transmit GRBV. In this study, we established mixed mating S. festinus pairs for which the F1 offspring varied phenotypically compared with the offspring of same genotype pairs but acquired GRBV isolate NY175 at similar rates (p = 0.96) and with a similar viral genome copy number (p = 0.34). Likewise, rates of GRBV acquisition were alike for the two parental CA (58%, 61/105) and SE (61%, 65/106) genotypes (p = 0.74), though the GRBV copy number in the salivary glands was overall significantly higher for SE than CA individuals (p = 0.02). Furthermore, the GRBV transmission rate was significantly higher for the SE genotype (89%, 16/18) than the CA genotype (50%, 8/16) (p = 0.04). These results revealed the existence of two sexually compatible S. festinus genotypes with distinct GRBV transmission abilities, suggesting the need to study GRBV ecology in Southeastern United States and areas where the two genotypes might co-exist.

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.

Grapevine leafroll-associated virus 3.

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.