The vine’s leaf petioles and blades contained about 30 ppm boron. While the foliage had no symptoms of boron deficiency, in the past the grower had observed sticking caps and pumpkin-shaped shot berries, which are indicative of boron deficiency. During the course of the research, the vineyard was drip-irrigated from April through October. The vineyard canopy covered 60% of the land surface during summer months and about 20 inches of water was applied during the season. Boron treatments consisted of applying fertilizer in varying amounts 3 weeks prior to bloom on May 18, 1998, and then again 3 weeks prior to bloom the following year, on May 3, 1999. Growers who fertigate grapevines with a drip system generally inject material into the irrigation water over a 45-to- 60-minute period at the beginning of an irrigation set. We simulated fertigation by applying Solubor, a soluble boron product , to a shovel-sized hole beneath drippers during the first hour of the irrigation set. By doing this, precise amounts of boron could be applied to each plot and plot size could be reduced. The experiment was designed as a randomized complete block with five treatments, five blocks and five vine plots . To evaluate the rate of boron uptake and accumulation in tissue with consecutive years of fertilization, square pots for planting grape tissue samples were collected in 1998 and 1999 at bloom and then again about 6 weeks later during veraison.

Veraison is the stage of development where berries begin to soften and/or color. To evaluate carryover, leaf tissue samples were also collected at this Tulare County site at both bloom and veraison in 2001, 2 years after the fertilization was discontinued. In each case, 100 petioles and 50 blades were sampled per plot from the center three vines. Petioles and blades were taken opposite inflorescences during bloom, and recently matured leaves were sampled at veraison. Samples were oven-dried, ground in a Wiley mill and sent to the UC Davis DANR Analytical Laboratory for analysis of total boron. Statistical analysis was by ANOVA using least significance difference to separate treatment means. A second experiment was conducted in 1998 in Fresno County near Selma, in a mature Thompson Seedless raisin vineyard planted on Pollaski sandy loam and drip-irrigated. The soil was formed in place from the weathering of softly to moderately consolidated granitic sediments. The particle size distribution of the surface soil is 63% sand, 25% silt and 12% clay. At the onset of the experiment, boron tissue levels were in the adequate range, 40 ppm. In both experiments, drip irrigations during the season were based on a schedule using historical evapotranspiration and developed for raisin vineyards in the San Joaquin Valley . The irrigation source was high-quality pump water with a boron concentration less than 0.1 ppm. The experimental design and methods were identical in both vineyards, except that the 1/16-poundper-acre boron treatment was omitted in the second vineyard. The Fresno County trial was discontinued after tissue samples were taken at bloom and veraison in 1998.

At both the Tulare and Fresno county sites, boron uptake was rapid when fertilizer was applied in the spring. In both vineyards, applying boron at 2/3 or 1 pound per acre increased the boron concentration in blades by bloom, 3 weeks after application. Boron increased further in blades by veraison . In the Tulare County vineyard, boron in bloom tissue increased from a questionable deficiency range to adequate; at the Fresno location, boron in bloom tissue increased from 40 ppm to 54 ppm, a dramatic increase considering boron fertilizer was applied just 3 weeks prior. This indicates that boron uptake is rapid. None of the fertigation treatments resulted in either symptoms of boron toxicity or deficiency. Applying boron at 1/3 pound per acre or less did not significantly increase boron tissue levels by bloom or veraison at either site the first year. Fertigation over consecutive years was evaluated at the Tulare County location. Boron in grapevine tissue continued to increase with consecutive years of application. At the higher fertilizer rate , boron levels in blades increased from 35 ppm in control vines to about 60 ppm. We speculate that continuing with annual applications of 1 pound boron per acre would result in toxicity within 4 to 5 years. The 1/3-pound-per-acre rate significantly elevated boron in blades by veraison of the second year to adequate levels . There were no visual signs of toxicity in any of the fertilizer treatments, even when boron was applied at 2 pounds per acre in a single application. Boron levels in tissue remained unchanged 2 years after fertilization was discontinued at the Tulare County location . This indicates substantial treatment longevity with fertigation of a drip-irrigated vineyard. Rainfall during this experiment was below normal, which helped minimize leaching. Also, well-managed drip irrigation minimizes leaching.

Under drip irrigation, salts tend to accumulate near the soil surface and 2 to 3 feet away from the drip line, with minimal water and salt movement below the root zone when irrigations are accurately scheduled . Boron concentrated more in the blades than in the petioles in response to fertilization. At the onset of the Tulare County experiment, boron concentrations in petioles and blades were similar at 31 ppm and 34 ppm, respectively. Fertilizing with 1 pound boron per acre for 2 consecutive years resulted in a 25% increase of boron in petioles but a 76% increase in blades . Allfertilizer treatments increased boron in blades more than in petioles, indicating that blades should be sampled when monitoring the vines’ boron status following fertilization. Potential boron toxicity values at the time of sampling during the bloom period are 80 ppm for petioles and 120 ppm for blades, and in mid- to late summer are 100 ppm for petioles and 300 ppm for blades.Annual boron fertigation at 1/3 pound per acre elevated grapevine tissue levels from questionable to the adequate range within 2 years . In addition, tissue boron levels remained unchanged 2 years after fertilization was discontinued. This is probably because leaching was reduced by two factors: below-normal rainfall and accurately scheduled drip irrigations. After fertilization, boron was concentrated more in blades than in petioles, indicating that blades are the best choice for monitoring toxicity. Blade samples should be monitored on a routine basis and fertilizer amounts should be adjusted accordingly to avoid boron toxicity or deficiency. The results of this research can be applied to other drip-irrigated vineyards in the San Joaquin Valley under similar conditions: rapidly drained soils, high quality irrigation water, and low boron content in soil, water and vine tissue. In other regions of the state where winter rainfall is much higher, there would presumably be more leaching of boron fertilizer during winter months and less carryover time after fertilization is discontinued. In contrast, less leaching and greater carryover of boron would be expected in areas of less rainfall or on soils with finer texture and higher water-holding capacity. The amount of boron fertigation used in a maintenance program will vary with leaching potential. These variables underscore the importance of monitoring boron in tissue when developing a long-term fertilization program. More than three quarters of all plant viruses are transmitted by insects , and information regarding key biological traits of vector-borne pathogens is needed to inform effective control strategies. For example, knowledge of transmission efficiency can aid in predicting rates of pathogen spread . Another key parameter in estimating the rate of appearance of newly diseased hosts is the pathogen incubation period, the time between initial infection and when symptoms become evident. Despite the importance of transmission efficiency and incubation period with respect to the development of disease management strategies, data are often not available and, when available, square pots plastic are usually derived from research performed under artificial conditions such as greenhouse environments. Grapevine leafroll-associated virus 3 , in the genus Ampelovirus, family Closteroviridae, is the primary virus species associated with grapevine leafroll disease in vineyards of wine growing regions worldwide . GLRaV-3 can cause interveinal reddening and downward rolling in red berried grape varieties , inhibits photosynthesis, decreases vine lifespan, and reduces fruit yield and quality . GLRaV-3 is one of the most common and detrimental viruses of grapevines, and has led to economic losses of 25 % or more . Spread of GLRaV-3 in vineyards and vector-borne transmission in controlled laboratory studies were first documented in South Africa , and since then GLRaV-3 spread in vineyards and transmission by several mealybug species have been documented in wine growing regions worldwide .

Although multiple grape-colonizing mealybug species transmit GLRaV-3, estimates of vector transmission efficiency vary both among and within mealybug species . GLRaV-3 is transmitted in a semi-persistent manner with no latent period required between acquisition and inoculation by vectors; transmission can occur after access periods of as little as one hour, and reaches a maximum after access periods of 24 hours . First instar mealybugs are the most efficient vectors, and mealybugs lose the ability to transmit GLRaV-3 four days after being removed from an infected source . 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, 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 California, 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.