Survey of Vineyard Insects and Plants to Identify Potential Insect Vectors and Non-crop Reservoirs of Grapevine Red Blotch Virus

Grapevine red blotch virus (GRBV) is a DNA virus in the family Geminiviridae. This pathogen is the causal agent of grapevine red blotch disease, which affects cultivated grapevines and leads to negative effects on crop quality and yield. GRBV is present in commercial vineyards across North America, indicating spread may have been largely human mediated. That said, recent surveys have demonstrated that there appears to be secondary transmission, most likely by an insect vector. Here, vineyard insects and plants were surveyed to identify potential candidate vectors and non-crop plants that may act as reservoirs for this pathogen. Results reconfirm that GRBV is limited to Vitis spp., including both cultivated and wild grapevines. Eleven insect genera or species, field collected in vineyards, tested positive for GRBV using quantitative PCR. These insect taxa include unknown Aphididae, the Cicadellids Aceratagallia spp., Acinopterus angulatus, Caladonus coquilleti, Colladonus montanus reductus, Colladonus sp., Empoasca spp., Erythroneura elegantula, and Scaphytopius graneticus, along with the Membracid Spissistilus festinus and an unknown Delphacid. Of these organisms, S. festinus is already known to be capable of transmitting GRBV while Scaphytopius graneticus may merit closer evaluation as a candidate vector.

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 following 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 non-host of GRBV, the virus titer decreased over time in adult insects, as shown by qPCR. Snap bean proved to be a feeding host of S. festinus and a pseudo-systemic host of GRBV following 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 transstadially but not via seeds. The virus titer was significantly higher in guts and hemolymph relative to heads with salivary glands, and in 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) susceptibility to six generalist predators

Spissistilus festinus (Say) (Hemiptera: Membracidae) was shown to transmit Grapevine red blotch virus (GRBV) in a greenhouse study. Grapevines infected with GRBV exhibit reduced sugar accumulation, altered secondary metabolite production and delayed berry maturation that negatively impacts wine quality and economics. Augmentative biocontrol may be a useful integrated pest management (IPM) tool for suppressing S. festinus populations in vineyards, but minimal research has been conducted on testing potential predators against the different life stages of S. festinus. The susceptibility of S. festinus adults and nymphs (1st through 5th instar) to predation by six commercially available biocontrol agents in petri dish and bell bean plant arenas was determined under greenhouse conditions. No significant mortality of S. festinus nymphs or adults occurred when exposed to Cryptolaemus montrouzieri adults, C. montrouzieri larvae and Sympherobius barberi adults in petri dish or bell bean plant arenas. Significant mortality of 1st and 2nd instar nymphs of S. festinus in the presence of Zelus renardii nymphs was observed in petri dish but not in bell bean arenas. Hippodamia convergens adults and Chrysoperla rufilabris larvae both consumed a significant number of S. festinus nymphs in petri dish and bell bean arenas. No significant predation of S. festinus adults was documented in this experiment. Results of this study aid in identifying predators that may be suitable candidates for additional field testing to determine their potential efficacy as biocontrol agents of S. festinus in a vineyard setting.

Timing the Implementation of Cultural Practices for Spissistilus festinus (Hemiptera: Membracidae) in California Vineyards Using a Stage-Structured Degree-Day Model

The three-cornered alfalfa hopper, Spissistilus festinus (Say), was shown to transmit Grapevine red blotch virus (GRBV), the causative agent for Grapevine red blotch disease, in a greenhouse study on grapes. GRBV is a major concern of wine grape growers due to its economic impact on wine quality. Plants in the family Fabaceae are preferred hosts of S. festinus and are commonly planted as cover crops or present in a vineyard’s native vegetation. In late winter, during grapevine dormancy, S. festinus migrate into vineyards to feed and reproduce on these cover crop and weed hosts. Tilling vineyard floor vegetation provides growers an opportunity to disrupt the life cycle of early instars that are relatively immobile, reducing the S. festinus first-generation population. Nymphal presence is difficult to detect. First through third instars were not detected in sweep net samples in a 2-yr weekly sampling study, whereas fourth and fifth instars were first found on the same sample date as emerging adults. A degree-day model was developed and successfully predicted when early S. festinus instars are present in the vineyard to aid in exploiting the time period when S. festinus is most susceptible to cultural control measures.

Influence of Riparian Habitat and Ground Covers on Threecornered Alfalfa Hopper (Hemiptera: Membracidae) Populations in Vineyards

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch disease, which affects wine grapes and leads to reduced crop yield and quality. While some virus spread can be attributed to the propagation of infected plant material, a greenhouse assay recently demonstrated that the threecornered alfalfa hopper (Membracidae: Spissistilus festinus Say) can transmit GRBV between grapevines. While S. festinus is not considered an economic pest of wine grapes, this species is present in California vineyards and their feeding can cause petiole girdling. Recent surveys have noted a correlation between S. festinus populations and GRBV-positive vines in vineyard areas adjacent to riparian habitat. Here, S. festinus populations were monitored over a 2-yr period at multiple vineyard sites adjacent to riparian habitats. At each site, insects were sampled from ground covers and the vine canopy at the vineyard edge and interior, and vines in both locations were evaluated for petiole girdling. Results indicate that there was no difference in abundance of S. festinus at the vineyard edge and interior. Populations in the vine canopy were highest in the late spring and early summer, and this was followed by the appearance of petiole girdling, indicating a key period of potential GRBV transmission. Furthermore, activity in the vine canopy appears to be amplified when the quality of ground covers is reduced as the season progresses. That said, overall populations of S. festinus were relatively low and additional work is needed to characterize the timing and efficiency of transmission under field conditions.

Treehopper voucher specimens collected in from Benton, Josephine, and Yamhill Counties, Oregon

Treehopper insect populations (Hemiptera: Membracidae) were surveyed in 2018 in Benton, Josephine, and Yamhill Counties, Oregon to determine their potential roles in the epidemiology of Grapevine red blotch virus. Stictocephala basalis and Tortistilus albidosparsus were identified through a taxonomic assessment of samples collected by hand near vineyards and in a natural area. Historical presence of Spissistilus festinus in the Willamette Valley is discussed. Voucher specimens were deposited in the Oregon State Arthropod Collection and at the United States National Museum of Natural History.

Two Distinct Genotypes of Spissistilus festinus (Say, 1830) (Hemiptera, Membracidae) in the United States Revealed by Phylogenetic and Morphological Analyses

Spissistilus festinus (Say, 1830) (Hemiptera: Membracidae) is a frequent pest of leguminous crops in the Southern United States, and a vector of grapevine red blotch virus. There is currently no information on the genetic diversity of S. festinus. In this study, populations of S. festinus were collected in 2015–2017 from various crops and geographic locations in the United States, and fragments of the mitochondrial cytochrome C oxidase 1 (mt-COI) gene and the nuclear internal transcribed spacer 2 (ITS2) region were characterized by polymerase chain reaction and sequencing. Maximum-likelihood and Bayesian analyses of the mt-COI and ITS2 sequences yielded similar phylogenetic tree topologies, revealing two distinct genetic S. festinus lineages with all of the specimens from California comprising one phylogenetic clade, alongside a single GenBank entry from Arizona, and all specimens from the Southeastern United States comprising a statistically-supported distinct clade, regardless of host and year of collection. The mt-COI gene fragment showed up to 10.8% genetic distance between the two phylogenetic clades. These results suggest the existence of two genotypes within S. festinus in the United States. The only distinct morphological trait between the two genotypes was a less elevated pronotum in the representative specimens from California, compared to the representative specimens from the Southeastern United States. Since this phenotypic feature is inconspicuous, a diagnostic polymerase chain reaction targeting a variable region of the mt-COI fragment was developed to reliably distinguish between the specimens of the two genotypes of S. festinus and to facilitate their specific identification.