A minimum of five buffer vines were left untreated at each end of the rows

There is no evidence of GLRaV-3 transovarial passage . While laboratory-based transmission studies have been informative, there is still a need for field-based transmission experiments to accurately estimate expected vector transmission efficiency and disease incubation time within the host. Information is lacking regarding how soon disease symptoms will appear after vector-borne infections are initiated, or how soon crop quality will be affected. The goal of our study was to obtain information about vector-borne transmission of GLRaV-3 and subsequent disease progression under commercial vineyard conditions. We performed a controlled GLRaV-3 transmission study into mature V. vinifera cv. Cabernet franc vines in Napa Valley, CA USA. We used first instars of the vector Pseudococcus maritimus , a mealybug species that is a common vineyard pest and native to North America . We performed a concurrent laboratory study, with identical experimental design, to compare pathogen transmission efficiency under controlled laboratory and field conditions. In the field study, dutch buckets system we estimated transmission efficiency and monitored time to GLRaV-3 detection via molecular diagnostics, appearance of symptoms, and effects of disease on berry quality.

Our findings are key to informing sound management practices with respect to understanding spread and progression of disease in a commercial vineyard. Furthermore, we provide a previously missing link between controlled laboratory studies and realistic vineyard conditions.Virus-infected dormant cuttings of V. vinifera cv Cabernet Sauvignon were used as source of GLRaV-3 in our transmission experiments. Foundation Plant Services at the University of Califtornia, Davis provided accession LV89-01 from their Virus Source Vineyard, which is known to be infected with genetic variant group III of GLRaV-3, Grapevine virus B , and Grapevine fleck virus . This accession was chosen because genetic variant group III of GLRaV-3 is common in Napa Valley . Plant cuttings were cut to three buds each, treated with RootBoost rooting hormone, planted in 1:1 vermiculite: perlite, and kept on a mist bench for 6 weeks, until a few leaves were produced and roots were approximately 2.5 cm long. Cuttings were then removed from the mist bench and transplanted to 10 cm pots with a growth medium consisting of 2:1:1 SuperSoil: perlite: sand , and kept in the greenhouse until used as virus sources in transmission experiments. GLRaV-3 infection was confirmed by molecular diagnostics prior to use for source material in transmission studies. Virus-free dormant V. vinifera cvPinot noir cuttings were also provided by Foundation Plant Services, collected during winter dormancy 2011, and propagated in the same manner as the virus-infected source cuttings. We used Ps. maritimus as the mealybug vector, which is difficult to maintain in insect colonies; therefore we relied on field collections for experimental inoculations. To obtain virus-free first instar Ps. maritimus mealybugs, third instar females were collected from a vineyard in Pope Valley, CA in May 2011, and allowed to mature and oviposit in the laboratory.

The third instar females were collected from underneath the bark of the trunks and cordons of mature grapevines and placed into gel capsules for transport to the laboratory. The mealybugs were immediately transferred to 100 mm petri dishes, each containing one piece of 70 cm Whatman filter paper. The mealybugs were kept in darkened conditions at 25 °C: 20 °C, 16: 8 h day: night temperatures- females were removed and discarded after oviposition. The filter papers containing ovisacs were pinned to the underside of the aforementioned infected source plants, which were then kept in a growth chamber until the first instar mealybug crawlers hatched. Approximately 72 h after hatching on the infected source plants, mealybugs were transferred to mature vines in the vineyard and to uninfected vines in the laboratory, for a 48 h inoculation access period. The timing of hatching led us to perform field inoculations on 18 July 2011, which coincided with the emergence of the new Ps. maritimus generation in Napa Valley. Twenty replicate source vines were propagated and used, with one to five recipient test vines inoculated per source plant in each inoculation experiment . All recipient test vines were treated with an insecticide upon completion of the inoculation access period.The experimental field inoculations were located in three rows of a vineyard block of V. vinifera cv. Cabernet Franc clone 01 grafted to 110R rootstock, obtained from Duarte Nursery and planted in Oakville, Napa Valley, CA in 1994. No vines in the experimental area were symptomatic for grapevine leaf roll disease prior to our experimental inoculations.

To confirm initial GLRaV-3-free status prior to inoculations, three petioles were collected from each experimental vine in July 2011 before inoculations were performed, for diagnostic testing . The block consisted of 8315 vines planted at 588 vines per hectare. Row spacing was 1.8 m, and vine spacing was 1.5 m, with a vertical shoot positioning trellis system and bilateral pruning. Row direction was northwest-southeast. Drip irrigation was provided using one 3.8 – L·h–1 emitter every 1.5 m. Experimental vines were spaced every third vine, and treatments were fully randomized. The three treatments included inoculations with no leaf cages, inoculations using mesh leaf cages, and negative controls for which no experimental manipulation was performed. Each treatment included 30 replicate vines, for a total of 90 experimental vines. The experiment comprised an area including 360 total vines, including the 90 experimental vines plus the spacer vines. The spacer vines were monitored periodically throughout the study for symptoms of grapevine leaf roll disease. A survey for any signs of mealybugs was performed in October 2012. On 11 October 2012, 15 months post inoculation, a commercial testing service collected and analyzed material from some vines that were symptomatic for grapevine leaf roll disease in the experiment and tested for a broad panel of known grape pathogens: GLRaV-1, GLRaV-2, GLRaV-2 strain Red Globe, GLRaV-3, GLRaV-4, GLRaV-4 strain 5, GLRaV-4 strain 6, GLRaV-4 strain 9, GLRaV-7, Syrah virus 1, Grapevine virus A, GVB, Grapevine virus D, Grapevine fanleaf virus, Xylella fastidiosa, GFkV, Rupestris stem pitting-associated virus, Rupestris stem pitting-associated virus strain Syrah, and Grapevine red blotch-associated virus. The vineyard block was not treated with insecticide prior to inoculations during the 2011 growing season. For inoculations, ten Ps. maritimus first instar insects were gently moved with a paintbrush from leaves of infected source plants onto the underside of one fully expanded mid-height leaf, located on a vertical cane growing from a middle spur on the south cordon of each grapevine. For the caged treatment, a cloth mesh cage was placed over the inoculated leaf and secured at the petiole using a twist tie. For the uncaged treatment, no covering was used on the inoculated vine. The experimental area was commercially treated with spirotetramat insecticide on 20 July 2011, after a 48 h inoculation access period. After inoculations the experimental area was managed following standard commercial practices.

Three months after inoculations, dutch buckets the petiole of the inoculated leaf was collected on 14 October 2011 for diagnostic testing. In the instance where that petiole had fallen off the vine or could not be found, a petiole near the inoculated leaf was collected; inoculated petioles were missing from 9 of 60 inoculated vines. Immediately following the first appearance of symptoms in 2012 and 2013, petioles were collected from each experimental vine and tested for presence of GLRaV-3. Petioles were collected from each experimental vine in September 2014, and tested for the presence of GLRaV-3, GVB, and GFkV. On each sampling date, three petioles were collected from each vine and pooled for diagnostic testing. If a vine had symptomatic leaves at the time of sample collection, symptomatic leaves were preferentially collected over asymptomatic leaves. During each growing season in 2011 through 2014 , experimental vines were surveyed regularly for visible leafroll disease symptoms, beginning immediately after inoculations. On each survey date vines were marked as either asymptomatic or symptomatic, with surveys beginning in May and continuing through October. Shortly after symptoms first emerged in 2012, a detailed symptom survey of each symptomatic vine was performed to determine possible variation in disease symptom severity among vines and if there was an association between location of inoculation and initial appearance of symptoms within vines. For this survey, the position of each spur and the number of symptomatic and asymptomatic leaves on each spur were recorded. In Year Two, berry quality of all vines was measured three times during the weeks immediately preceding commercial harvest. Degrees Brix , pH, and titratable acidity were measured on 31 August, 21 September, and 3 October 2012, and harvest was 4 October 2012. In Year Three, berry quality of a randomly selected subset of 30 vines was measured on 28 August and 14 September, and harvest was 14 September 2013. The 30 vines were evenly divided between uninfected negative controls, uninfected and infected vines from the caged inoculation treatment, and uninfected and infected vines from the uncaged inoculated treatment. For berry quality analysis, on each sampling date approximately 200 berries were collected from each vine to minimize variance in measurements . Within each grapevine, berries were collected from the top, middle, and bottom of each harvestable cluster of grapes and pooled for laboratory analysis. All samples were processed by Constellation Laboratories in Califtornia, USA. Total soluble solids as °Brix were measured using an Atago refractometer, and pH was measured using an Orion pH meter. Titratable acidity of the juice was measured via direct titration with 0.1 N NaOH, using phenolphthalein as an indicator.To compare field inoculations in an established commercial vineyard with controlled laboratory studies, a laboratory experiment including the same treatments was performed, using the same collection of Ps. maritimus and same known infected source plants. Ps. maritimus first instars were moved from known infected source plants to uninfected recipient test plants on 15 July 2011 and allowed a 48 h inoculation access period, after which plants were treated with a contact insecticide. Mealybugs on half of the recipient test plants were caged on the underside of one leaf using clip cages, as described by Tsai et al. , and mealybugs remained uncaged on the other half of the recipient test plants. Thirty replicate recipient test plants were used for each treatment, caged and uncaged, for a total of 60 recipient test plants. Additionally, a replicate of 30 uninoculated plants was kept in the greenhouse following inoculations to assure that all GLRaV-3 infections were due to our experimental inoculations. Following inoculations, plants were kept in a greenhouse with 16:8 light: dark using supplemental lighting as needed, and 25 °C: 22 °C day: night temperatures, with regular fertilizer and insecticide treatments. Because disease symptoms under greenhouse conditions are not reliable for diagnosis of GLRaV-3, we used molecular testing to confirm infection with GLRaV-3. Four months after inoculations, at which time new GLRaV-3 infections can be reliably detected using molecular diagnostics, three petioles per inoculated test plant were collected and stored at −80 °C until testing for GLRaV-3 .To test whether the newly infected field vines could be a source of GLRaV-3 one season after mealybug inoculations, a transmission experiment was performed in the laboratory from cuttings of these newly infected fieldvines. Ps. maritimus were not used because of the abovementioned difficulty in obtaining virus-free first instars for transmission experiments. Instead we used first instars of Planococcus ficus, which are easily maintained in colonies and therefore can be ready for use in transmission studies at any time. Furthermore, Pl. ficus is a known vector of GLRaV-3 . Field cuttings were collected on 4 October 2012 and the stem bases were placed in flasks of water. First instar Pl. ficus were allowed a 24 h acquisition access period on the field cuttings, then transferred to the underside of a leaf of virus- free V. vinifera cv. Pinot noir recipient test vines; ten insects per recipient test vine were confined using a leaf cage for a 24 h inoculation access period. Following inoculations, plants were treated with a contact insecticide and then kept in a greenhouse for four months until petiole sample collection for diagnostic detection of GLRaV-3. For this experiment, a randomly selected subset of experimental field vines of each treatment was tested as a potential GLRaV-3 source. In total, nine symptomatic vines were tested; five from the caged inoculation treatment and four from the open inoculation treatment, and seven recipient test vines were inoculated in the laboratory from each symptomatic field vine. One of these 63 recipient test vines died before petiole sample collection to test for infection with GLRaV-3.