Identification of Potential Grapevine Red Blotch Virus Vector in Missouri Vineyards

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, No-Bait Traps, 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 and Yonke 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 protocol. DNA extracted from insect specimens was tested for the presence of GRBV by PCR using GoTaq Polymerase (Promega). The PCR primers (GRBV-For621 5’-TCA ACT GAG TAG ACG CGT TGC-3’ and GRBV-Rev1261 5’-TCA ACA TCA TTC CGT CCT CCA-3’) amplified a 640-bp DNA segment of the GRBV genome from nucleotide 621 to 1261. PCR primers were synthesized by Integrated DNA Technologies. PCR conditions were 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 60 sec. At the conclusion of the final cycle, the temperature was held at 72°C for 10 min and then held at 4°C. PCR products were analyzed by gel electrophoresis in a 1.5% agarose gel run in TBE (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA).

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 GRBV-positive 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.