Identification of Potential Grapevine Red Blotch Virus Vector in Missouri Vineyards

Grapevine red blotch virus (GRBV), the causal agent of grapevine red blotch disease, has been detected in vineyards across the United States and throughout Missouri. Insect transmission of GRBV in cultivated vineyards of Missouri has not been investigated previously. The objectives of this study were to characterize the potential insect vectors present in four commercial vineyards that had previously been determined to be infected with GRBV, test potential vectors caught in vineyards and surrounding habitats for the presence of GRBV with the use of PCR, and investigate the ability of candidate vectors to acquire and transmit GRBV using controlled greenhouse experiments. Of the vineyard-collected insects tested over the course of this research, one species of treehopper, Entylia carinata, tested positive for GRBV. This species and one other treehopper, Enchenopa binotata, were selected for direct transmission assays. Both species successfully acquired GRBV from infected grapevines and transmitted GRBV to confirmed GRBV-free grapevines. E. carinata has been identified as a promising economic vector after insect samples from vineyards tested positive for GRBV, and monitoring data placed this species as the second-most abundant treehopper captured in traps. We do not consider E. binotata a likely economically significant vector because our monitoring data showed that this species was rare and only found along edge habitat surrounding vineyards, never inside vineyard rows. Samples of the most abundant treehopper, Micrutalis calva, have not tested positive, but its vector status remains unresolved. Further research on rates of secondary spread and transmission by M. calva are required, but these results provide evidence that insect transmission of GRBV is feasible in the region.

Grapevines face a host of abiotic and biotic stressors, ranging from soil nutrients to insect pests and disease pressure (Weaver 1976, Cox 2015. Viruses are of particular importance, with nearly 90 viruses and viroids identified from grapevines (Fuchs 2020). Many of these viral diseases shorten the lifespan of vineyards and reduce yield for growers (Maliogka et al. 2015). Seventy percent of viral plant diseases rely on an arthropod vector for transmission (Whitfeld et al. 2015); thus, understanding the role of vectors in virus transmission is critical to managing these diseases.
Grapevine red blotch disease (GRBD) is a recently-discovered disease caused by a single-stranded circular DNA virus called grapevine red blotch virus (GRBV) (Al Rwahnih et al. 2013, Yepes et al. 2018. GRBV is a geminivirus (family Geminiviridae) with isolates in two distinct clades placed in a unique genus called Grablovirus (Cieniewicz et al. 2017). Symptoms of this disease in European grape cultivars (Vitis vinifera) include the characteristic red blotches on leaves of red-berried cultivars or yellow blotches in white-berried cultivars. These foliar symptoms typically arise late in the growing season and first appear on older leaves (Cieniewicz et al. 2017). Alterations in berry chemistry like reduced sugar content (-1 to -4 Brix) and anthocyanin concentrations and delayed ripening have been documented (Blanco-Ulate et al. 2017, Girardello et al. 2019, Martínez-Lüscher et al. 2019, Bowen et al. 2020.
Viruses in the family Geminiviridae are transmitted by hemipteran insect vectors (Whitfeld et al. 2015). GRBV currently has one confirmed vector: the three-cornered alfalfa treehopper (Spissistilus festinus) successfully transmitted GRBV to uninfected Vitis vinifera grapevines under greenhouse conditions (Bahder et al. 2016a, Flasco et al. 2021). Other taxa have been implicated as vectors. The Virginia creeper leafhopper (Erythroneura ziczac) successfully transmitted the virus under greenhouse conditions, but subsequent assays did not replicate these results (Poojari et al. 2013, Bahder et al. 2016a. Several leafhopper and treehopper species collected from V. vinifera vineyards in New York and California tested positive for GRBV DNA, but their ability to transmit the virus directly has not been determined , 2019, Wilson et al. 2021.
Missouri grows ~688 ha of winegrapes with a wine industry that contributes 3.2 billion dollars annually to the state (Frank et al. 2015, Dunham et al. 2017. A comprehensive statewide virus survey conducted in 2017 revealed 35% of composite samples were infected with GRBV (Schoelz et al. 2021). Missouri commonly grows hybrid winegrape cultivars (crosses of V. vinifera and North American grape species [Vitis spp.]). Unlike V. vinifera cultivars, hybrid vines infected with GRBV are often completely asymptomatic (Atucha et al. 2018, Schoelz et al. 2021. It is unknown whether the negative fruit effects and overall decline in vine health documented in symptomatic V. vinifera cultivars occur in these asymptomatic hybrids. It is also unknown whether the confirmed vector, S. festinus, or other potential insect vectors contribute to GRBV transmission in Missouri vineyards. Our objectives were to (1) identify potential GRBV insect vectors in Missouri vineyards, focusing specifically on treehoppers and those leafhoppers that have previously tested positive for the virus, and (2) determine whether field-collected candidate vectors were carrying GRBV. We also sought to determine whether candidate vectors found in Missouri vineyards are capable of (3) acquiring GRBV from infected vines, and (4) transmitting GRBV to uninfected grapevines. An understanding of the potential role of insect transmission of GRBV in hybrid grape cultivars is essential to develop effective management strategies for this disease.

Materials and Methods
Candidate insect vector collection in vineyards. Potential insect vectors were collected from four commercial vineyards in central Missouri in 2018 and 2019. Sampled vines were hybrid cultivars commonly grown in Missouri, including the French-American hybrid cultivars Chardonel, Chambourcin, and Crimson Cabernet, and an American grape cultivar, Norton (Vitis aestivalis). Vineyard blocks used in this study were confirmed as infected with GRBV in a 2017 statewide virus survey (Schoelz et al. 2021).
In 2018, vineyards were sampled weekly for 19 consecutive weeks from budbreak in April to harvest in early October. In 2019, the sampling window was reduced to 12 consecutive weeks from budbreak in April to veraison in late July. The reduced sampling dates correspond with the peak insect abundance measured in 2018. Insects were collected using yellow sticky card traps (Pherocon,22 × 28 cm,Great Lakes IPM) secured to 1.8 m tall wooden 2.45 × 5.08 cm posts. Initially, three sticky cards were secured to each post at ground level, mid-canopy height, and within the fruit zone. The sticky card placement was reduced to a single mid-canopy level card, ~1 m from the ground, in 2019. In 2018, large amounts of grapevine leaves and vineyard debris were collected by the ground and fruit zone cards, reducing collection of insects. Fifteen posts were installed at each vineyard: five in the edge habitats surrounding the vineyards and 10 within the vineyard. Edge habitats consisted of tree lines with understory plants or weedy riparian areas and posts were located ~5 to 10 m from the nearest grapevine. The dominant plant species surrounding vineyards were recorded (Table 1) and free-living wild grape (Vitis sp.) was present in all habitats proximal to vineyards. Vineyard interior samples were located at various distances from the vineyard border, including samples near the end of rows and within the middle of the vineyard. All posts were at least 6 m apart. All vineyards were trained to high-wire bilateral cordon systems and had grassed alleyways with 2.43 m in-row spacing and 3.05 m between vines. Vineyard block size ranged from 0.98 ha to 1.57 ha, with edge habitat typically within 6 m of cultivated vines. Sticky cards were collected and replaced weekly, placed in a plastic bag, and stored in a -4°C freezer prior to processing.
Treehoppers and leafhoppers were identified to the lowest taxonomic level possible using dichotomous keys and voucher specimens (Delong 1948, Kopp andYonke 1974, Enns Entomological Museum, University of Missouri). Species-level determination of leafhoppers often requires dissection of male genitalia, which was not feasible with our sticky card sampling scheme, so some determinations were made to genus level. Additional insect specimens were collected using sweep nets and a D-Vac suction sampler (D-Vac Suction Sampler, Model 24), secured in plastic bags, and stored in a -4°C freezer for molecular testing of GRBV DNA presence. The insects collected using sweep nets and the D-Vac were not included in the statistical analyses.
The main and interactive effects of location (vineyard interior versus outside of the vineyard) and sample week on (1) total treehopper (Membracidae) abundance, (2) total leafhopper (Cicadellidae) abundance, (3) abundance of Micrutalis calva treehoppers, and (4) abundance of Entylia carinata treehoppers were determined by repeated measures analysis of variance (ANOVA), with vineyard included as a random blocking factor (PROC MIXED, SAS version 9.3; SAS Institute, Inc.). For all analyses, data were logarithmically transformed to fit the assumptions of ANOVA. In all cases, compound symmetry covariance structure was determined to be the best-fit using the Bayesian information criterion. Acquisition assay of GRBV by candidate vectors. Eight species of treehoppers and one species of leafhopper were collected at the University of Missouri Baskett Wildlife Research and Education Center (Boone Co., MO), a 917 ha research area. The nearest cultivated vineyard is ~8.28 km from our sampling site. Insects were collected in a weedy, riparian area. Some of the foliage identified was giant ragweed (Ambrosia trifida), common ragweed (Ambrosia artemisiifolia), sunflower (Helianthus sp.), and other herbaceous plants at least 2 m from a tree line. The insects were transported live in a cooler to the Curtis Hall Greenhouse on the University of Missouri campus for acquisition studies (photoperiod of 16:8, 24 to 32°C, and 35% RH; University of Missouri, Columbia). Treehoppers and leafhoppers were placed in mesh sleeve cages (sock enclosure, dimensions D25.4 × L50.8 cm, BioQuip Products, Inc.) on a GRBV-infected Crimson Cabernet grapevine, all contained inside a larger observation cage (model BugDorm-2120, dimensions W60 × D60 × H60 cm, MegaView Science Co. Ltd.). The grapevines used in the acquisition and transmission assays were one-year-old and were propagated from cane wood collected from one commercial vineyard identified as GRBV positive in a virus survey in 2016 (Schoelz et al. 2021). Grapevines were tested for GRBV before assays took place to confirm infection. Insects were placed on new tender growth to ensure successful feeding. Insects were allowed to feed on the grapevine for a 72 hr acquisition access period. They were then removed from the grapevine, placed in 1.5-mL microcentrifuge vials, and stored at -80°C prior to testing for the presence of GRBV DNA.
Individuals of the same insect species were combined into an aggregate sample of up to 50 mg tissue. Tissue was homogenized with disposable microtube pestles in 1.5-mL microcentrifuge tubes with 180 μL phosphate buffered saline, pH 7.2 (1×). Total DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's insect specimen To confirm successful DNA extraction from insect specimens, isolated DNA was also tested for the presence of a gene found in the mitochondria of insects, COI, by PCR using GoTaq Polymerase (Promega) (Lunt et al. 1996). The PCR primers COI-F (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and COI-R (5'-TAA ACT TCA GGG TGA CCA AAA AAT-3') amplified a 686-bp DNA segment of the COI genome. PCR primers were synthesized by Integrated DNA Technologies. PCR conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 40 sec, 47.9°C for 40 sec, and 72°C for 40 sec. At the conclusion of the final cycle, the temperature was held at 72°C for 5 min and then held at 4°C. PCR products were analyzed by gel electrophoresis in a 1.2% agarose gel run in TBE (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA).
Whole insect bodies were homogenized. Therefore, this assay does not distinguish insects that test positive due to the presence of GRBV in their gut versus acquisition of the virus in the salivary glands.
Assay for transmission of GRBV by candidate vectors. Two species of treehoppers, E. carinata and Enchenopa binotata, that tested positive for GRBV in the acquisition assays and for which there were sufficient numbers of wild individuals available, were selected for further transmission studies. Treehoppers were collected at the University of Missouri Baskett Wildlife Research and Education Center and the City of Columbia Capen Park (Boone Co., MO), a 12.9 ha municipal park with no cultivated vineyards nearby. Collected insects were transported to the University Curtis Greenhouse in a cooler.
Fifteen E. binotata and 15 E. carinata were used in the direct transmission assays. For both species, three groups of five insects were placed inside mesh sleeve bags secured to a Crimson Cabernet grapevine that was previously confirmed positive for GRBV. The insects were allowed to feed on the GRBV-positive vine for a 48 hr acquisition access period. Two E. binotata individuals died during the acquisition feeding period, resulting in two groups of four E. binotata and one group of five. There was no mortality of E. carinata in the acquisition assay. Insects were then transferred to six different Crimson Cabernet grapevines that were confirmed GRBV-free by PCR testing. Treehoppers were secured in mesh sleeve bags on vines with young, tender growth to facilitate successful insect feeding. Insects were allowed to feed on the virus-free vines for a 48 hr inoculation access period. There was no mortality of E. binotata or E. carinata during inoculation. Insects were then removed, placed into 1.5-mL microcentrifuge tubes, and stored in a -80°C freezer. As with the acquisition assay, all individual vines were contained inside of a larger observation cage.
Recipient plants were maintained within the greenhouse (photoperiod of 16:8, 24 to 32°C, and 35% RH; University of Missouri, Columbia) for four months to allow GRBV titer to build up before testing. Phloem scrapings from green cambium on canes were collected from each recipient vine. Additionally, tissue from leaf petioles or, if leaves had abscised, dormant buds, were collected. Up to 100 mg of each type of plant tissue were processed separately and homogenized for 2 min using 5 mm tungsten carbide beads in 2 mL microcentrifuge tubes in a TissueLyser II (Qiagen). Total DNA was extracted using a DNeasy Plant Mini Kit (Qiagen) following the manufacturer's protocol. DNA extracted from the different plant tissues was then tested for the presence of GRBV as described in the acquisition assay, with the same PCR primers and conditions used for detection of GRBV in insects.
Viral genome sequencing of infected vines used in transmission assay. GRBV viral DNA was isolated from a GRBVpositive donor vine and one of the recipient vines used for the transmission assay using a DNeasy Plant Mini Kit (Qiagen). PCR products were amplified using primers synthesized by Integrated DNA Technologies and PCR conditions described in the previous section, then purified for DNA sequencing using a QIAQuick PCR Purification Kit (Qiagen) and submitted for DNA sequencing at the University of Missouri Genomics Technology Core.

Candidate insect vectors collected in vineyards.
Over the two-year monitoring period, 1787 yellow sticky card traps were deployed and 65,870 individual leafhoppers and treehoppers were collected ( Table 2). The samples yielded 12 species of treehoppers and two leafhopper species that have been identified as candidate vectors of GRBV, Colladonus reductus and Osbornellus sp. (Cieniewicz et al. 2019). The previously confirmed insect vector of GRBV, the threecornered alfalfa treehopper (S. festinus), was not found over two years of monitoring.
Treehopper abundance peaked both inside and outside of the vineyard in June 2018 (F = 2.98, p < 0.0001) and 2019 (F = 451.41, p < 0.0001) (Figures 1A and 2A). There were significantly more treehoppers in the vineyard interiors than vineyard edges in June of both years. Peaks in total treehopper abundance are attributed largely to the most abundant species in our survey, M. calva. M. calva was significantly more abundant inside vineyards than in the edge habitats outside of the vineyards in the month of June (Figures 1B and 2B). The second most abundant species, E. carinata, was primarily found outside of the vineyards (Figures 1C and 2C).
Leafhoppers were more abundant than treehoppers, but leafhoppers overall shared a similar population dynamic trend as treehoppers. There was a population peak in June 2018 and 2019, followed by a gradual decrease throughout the season (Figure 3). There was no significant difference between the number of leafhoppers outside the vineyards and in the vineyard interiors in 2018. In 2019 there were significantly more leafhoppers outside of the vineyards. Two leafhopper taxa that are GRBV candidate vectors, C. reductus and Osbornellus sp., were also present (Cieniewicz et al. 2019). They were found in vineyard interiors and in edge habitats outside of vineyards.
A total of 1168 insects collected from the field were assayed for the presence of GRBV. Of the field-caught specimens, two pooled samples of E. carinata tested positive for GRBV (Table  2). One pooled sample contained eight individual insects with four removed from sticky cards in the edge habitat, and four removed from sticky cards in the vineyard interior. The other positive sample contained seven E. carinata, all found on one vineyard interior card. The percent of insects that tested positive for GRBV is listed as a range dependent on the number of individuals in one aggregate sample weighing 50 mg ( Table 2).

Acquisition of GRBV by candidate vectors.
Six treehopper species tested positive for GRBV after feeding on infected grapevines for a 72 hr acquisition period (Table 3). The leafhopper species Graphocephala coccinea and one species of treehopper, Archasia pallida, tested negative for GRBV. The molecular results for the treehopper M. calva were inconclusive because the DNA extracted was not usable, as it did not test positive for the control gene.
Transmission of GRBV by candidate vectors. Both species of treehoppers selected for direct transmission assays (E. binotata and E. carinata) successfully transmitted GRBV to virus-free grapevines ( Table 4). None of the recipient vines had symptoms of GRBV when plant material was collected for PCR assays, four months postinoculation. DNA extracted from grapevine tissue tested positive for GRBV. In some instances, phloem scrapings and petiole tissue tested positive for GRBV, while bud tissue did not (Table 4). These inconsistencies may be due to differences in virus titer among various plant tissues (Setiono et al. 2018), which should be further investigated, or may be due to incomplete disruption of the bud tissue during extraction.
GRBV DNA was isolated from donor and recipient vines and the nucleotide sequence determined for approximately two thirds of the virus genome of each (2050 bp). The nucleotide sequences were compared using the Global Align program of BLAST (Zhang et al. 2000), showing that they were 100% identical over the 2050 nucleotide stretch. A separate BLAST nucleotide search of the sequences showed that they were 99.90% identical to MO-CC7 (Schoelz et al. 2018), a GRBV isolate recovered from the same Crimson Cabernet vineyard as the donor vine. The comparison of virus sequences recovered from donor and recipient vines is consistent with the hypothesis that the transmitted virus originated from the source vine in our greenhouse and was not the result of contamination by prior insect acquisition of GRBV in the field. Table 2 Abundance of treehoppers (Membracidae) and leafhoppers (Cicadellidae) at monitoring sites in four commercial Missouri vineyards in 2018 and 2019. Samples were collected weekly from budbreak to harvest in 2018 and from budbreak to veraison in 2019.
"Inside" refers to insects trapped on sticky cards placed in interior vineyard rows. "Outside" refers to insects trapped on sticky cards placed along the edge habitats surrounding vineyards. Selected species of insects were tested using standard PCR for grapevine red blotch virus.

Discussion
The goal of this research was to identify the insect vectors of GRBV in Missouri vineyards. We sampled the candidate vector community in four vineyards throughout the growing season and tested field-collected individuals for GRBV. Of the 1168 individuals tested, only 0.06 to 4% were positive for GRBV. All individuals testing positive were of the treehopper species E. carinata. Given the low likelihood of finding GRBV-positive individuals in the field, we tested the ability of candidate vectors to acquire the virus directly by feeding on confirmed GRBV-infected grapevines and to transmit the virus to GRBV-free grapevines in the greenhouse. Six species of treehoppers tested positive for GRBV after an acquisition access period of 72 hrs on infected Crimson Cabernet grapevines (Table 3), indicating potential acquisition of the virus. Because whole insects were homogenized and the salivary glands were not dissected, this result is consistent with, but does not confirm, acquisition of the virus by the treehoppers. Further testing revealed that two species of treehoppers, E. carinata and E. binotata, successfully infected grapevines
Entylia sp. have also tested positive in vineyards in New York (Cieniewicz et al. 2019). Additionally, direct transmission of GRBV by E. carinata was confirmed in our greenhouse assays. E. carinata is commonly found feeding and reproducing on herbaceous weeds in the family Asteraceae like ragweed (Ambrosia sp.), horseweed (Conyza sp.), and fleabane (Erigeron sp.) (Kopp and Yonke 1974). E. carinata was abundant in edge habitats surrounding vineyards, but is also commonly found in vineyard interiors, especially at the end of vineyard rows near edge habitats. The abundance of E. carinata caught in traps in the vineyards was nearly 10% less in 2019. This may be due to the reduced sampling period and the reduced number of sticky card traps deployed. Further assays and monitoring should be conducted with this species.
E. binotata, the two-marked treehopper, successfully transmitted GRBV in transmission studies, but we do not consider it of economic importance because it was rare and only found in edge habitats outside vineyards, never inside vineyards. Among samples collected from edge habitats surrounding vineyards, no individuals tested positive for GRBV. While we do not consider this insect to be a likely economic vector, its ability to transmit GRBV is significant, as it demonstrates that the potential vector community may be broad.
Surveys for GRBV in vegetation adjacent to cultivated vineyards in California have found alternate hosts growing in riparian edge habitats (Bahder et al. 2016b, Wilson et al. 2021. Thirteen species of woody herbaceous plants growing around three vineyards were tested for the presence of GRBV. Two species, including wild grape (Vitis californica × V. vinifera), tested positive for the virus. The candidate insect vectors monitored in our study, including E. carinata, were present in the edge habitats surrounding cultivated vineyards in addition to vineyard interiors. Wild Vitis was present in the edge habitat of all four vineyards surveyed. A 2021 survey of the prevalence of GRBV in wild Vitis sp., Ampelopsis sp. (a vine in the Vitaceae family), and four species of Roundup-resistant weeds (Solanum carolinense, Conyza canadensis, Ambrosia artemisiifolia, and Ambrosia trifida) in 13 different Missouri vineyards found that 13.24% of 137 wild Vitis samples and one sample of Ampelopsis sp. tested positive for GRBV, but no samples of the four weed species tested positive for GRBV (Dean Volenberg, personal communication). The presence of GRBV in riparian edges could provide a reservoir of virus inoculum for a mobile insect vector which may move from these riparian areas into vineyards, indicating spread may be inevitable. Spatial data from this study demonstrates that E. carinata is abundant in habitats surrounding vineyards and common along vine-yard edge rows, potentially indicating movement from edge habitats to vineyard rows. These insects may feed on alternate GRBV hosts in surrounding habitats while moving between edge habitats and cultivated grapevines. Further research on GRBV reservoirs in Missouri vineyards is crucial to ensure vineyards are not reinfected after GRBV-positive vines are removed and replaced.

Conclusion
Insect transmission of GRBV in hybrid cultivars is possible in Missouri vineyards. The successful transmission of the virus by two species of treehoppers, E. binotata and E. carinata, under greenhouse conditions demonstrates that the molecular mechanisms of virus transmission exist. However, the spatiotemporal occurrence of the treehoppers and the presence of infected individuals in vineyards indicate that E. carinata is the species that is most likely to play an economic role. Continued monitoring for infected E. carinata individuals and a better understanding of phenology and host plants of these treehoppers is necessary. Monitoring individual vines for secondary spread is essential to understanding whether these insects are vectors of economic significance. Further research, including an economic impact study on common Missouri cultivars and the effect of GRBV on fruit and wine yield and quality among asymptomatic cultivars, is required to develop a management plan to prevent spread of this disease in Missouri vineyards. Additionally, understanding whether alternate sources of inoculum exist in the environment surrounding cultivated grapevines will play a crucial role in control of secondary spread of GRBV via insects. Table 4 Transmission studies of grapevine red blotch virus were conducted with two species of treehoppers, Entylia carinata and Enchenopa binotata. PCR assays were performed on grapevine tissue collected from recipient plants four months postinoculation.

Treehopper species feeding on vine
Vine number and tissue type Result