Similarly, Flores et al. observed that temperatures above 22 ◦C attenuated infection symptoms and increased gene silencing. Thus, in 2017, the infected grapevines on both rootstocks could have experienced a reduction in GRBV impacts due to the high temperatures causing viral gene silencing and a decrease in viral DNA. However, the gene expression and regulation of transcriptional factors need to be investigated further to understand the correlation between extreme heat and disease expression in GRBD infected grapevines. At harvest, a three-way ANOVA indicated that seasonal differences play a large role in the extent of disease symptoms in terms of anthocyanin content at harvest and through ripening which was not observed for total tannin and total phenolic content. Past studies have indicated that anthocyanin accumulation in grapes are highly susceptible to variations in temperature, with high temperatures leading to anthocyanin degradation and inhibition of biosynthetic pathways, whereas tannin concentrations are less sensitive to environmental factors. Therefore, regarding anthocyanin content, the temperature differences between the two seasons may have had a compounding effect with GRBD infection in grapevines.Similar to previous results, large square planting pots the severity of GRBD symptoms depends not only on season, but also on rootstock. Anthocyanin levels through ripening and at harvest in 2017 for CS 110R infected grapevines were more impacted than in 2016 which was not observed for CS 420A .
Previous work described the impact GRBV has on grape metabolism and demonstrated that GRBV inhibits the phenylpropanoid pathway in grapes, which is responsible for the synthesis of flavonoids. As previously mentioned, temperature plays a large role in anthocyanin content in grapes, where higher temperatures lead to lower anthocyanin levels. Therefore, it is possible that the extreme heat in 2017 acted as a secondary stressor to infected grapevines, and potentially caused larger decreases in anthocyanin levels through ripening than in 2016. However, this was only observed for rootstock 110R, suggesting that infected grapevines on this rootstock are potentially more susceptible to temperature fluctuations. In addition, the difference in the rate of ripening between RB and RB data vines , was larger for CS 110R than for CS 420A. This indicates that the virus differentially impacted the rate of translocation of sugars from the leaves to the berries depending on the rootstock. Additionally, at harvest CS 110R RB grapevines consistently had higher levels of total tannins and phenolics than RB grapes, where the opposite was observed for CS 420A . The former has been seen in prior research by Girardello et al. which screened the impact of GRBD on three varieties across seven sites. One of the varieties which had significantly higher proanthocyanidin values in RB grapes compared to RB was CS on rootstock 110R. Flavonoid biosynthesis such as flavan-3-ols and tannins has been correlated to abiotic and biotic stress responses in the grape. It is possible that the higher content of tannin observed in CS 110R infected grapes is correlated to a plant induced defense response, which was less significant in CS 420A. Lastly, the volatile aroma profiles between RB and RB were more similar for grapes from rootstock CS 420A compared to rootstock CS 110R, indicating that choice of rootstock has an influence on disease expression and may have various effects on secondary metabolites.
Rootstock 110R has high drought tolerance and is a moderately high vigor rootstock; whereas 420A has less drought tolerant and induces lower vigor in the scion in comparison. Lower vigor can result in a change in microclimate by increasing sun exposure, overall changing berry ripening and composition. Previous research that investigated the impact of GRBV on vine physiological found similarly that CS110R grapes exhibited more symptoms than CS 420A. In this study, RB grapevines had higher sugar content in the leaves, lower sugar content in the grapes, and higher water potential than RB grapevines. These differences were more drastic for CS 110R than CS 420A grapevines. In addition, CS 110R had higher water potential than CS 420A across disease status, correlating to the high vigor of 110R. Overall, this study concluded that GRBV inhibited the translocation mechanisms of photosynthetic products from the source to the sink . Taken together, this suggests that there is a larger impairment to translocation mechanisms in the CS 110R grapevine than CS 420A grapevines.All water used during extractions and other analyses was 18MΩ·cm deionized water from a Milli-Q Element system . All ethanol was purchased from KOPTEC . ACS grade acetone was used during phenolic extractions, along with 37% HCl, which was purchased from Sigma Aldrich . Ascorbic acid, maleic acid, bovine serum albumin, glacial acetic acid, ferric chloride, triethanolamine, and NaCl were purchased from Sigma Aldrich .
Urea and NaOH were purchased from Thermo Fischer , and potassium bitartrate and potassium metabisulfite were purchased from ACROS organics-Thermo Fischer . For headspace solid-phase microextractiongas chromatography-mass spectrometry analysis, sodium citrate dehydrate was purchased from Thermo Fischer . Internal standards, 2-octanol and 2-undecanone were purchased from Sigma Aldrich .We used Cabernet Sauvignon grapevines grafted onto 110R and 420Vineyard . The grapevines were trained to a bilateral cordon, in a vertical shoot positioned system. Vineyard management followed standard commercial practices for the region. The grapevines were drip-irrigated at 50% of crop evapotranspiration as reported previously. For several years prior to the initiation of this study, GRBD symptoms had been monitored for each vine in this block. Petiole samples from a subset of vines from this block were tested by qPCR analysis at Agri-Analysis LLC laboratories in Davis, CA to confirm the healthy and GRBV status of the grapevines. In addition, the plant material was screened for the presence of the three most common grapevine leafroll associated virus as well as Rupestris stem pitting-associated virus.The field design of this project was a completely randomized design without blocking. Twenty and twenty-five data vines that tested positive and negative for GRBV were selected for each rootstock in 2016 and 2017, respectively. Data vines were further subdivided into four and five vines for each vineyard replicate in 2016 and 2017, respectively . Vines were sampled every two weeks pre-veraison and weekly two weeks after veraison until harvest. Fifteen berries were randomly collected from different parts of the cluster and canopy of each vine and used to determine ripening progression. At harvest, the sampling was wider to include the vines utilized for winemaking. The values from the data vines regarding ◦Brix, pH, and TA , were compared to the values of asymptomatic and symptomatic vines , which agreed, indicating that symptomology is a strong indicator of virus status. Primary metabolites and components of harvest yield were measured from each data vines replicate . For RB and RB , 500 berries were randomly collected from harvest lots and stored at −80 ◦C until phenolic analysis and volatile aroma compound analysis could be performed.For the phenolic extraction, five sets of 20 berries from the RB and RB grapevines at harvest were randomly selected from grapes stored at −80 ◦C and weighed. Phenolic compounds were extracted similar to that described for anthocyanins with the addition of a subsequent extraction with an acetone solution in the same ratio of 1:10 w/v. After an 18-h, overnight extraction at 4 ◦C, the solution was centrifuged, and the supernatant collected. The ethanol and acetone extractions were combined, plastic square planter pots concentrated under reduced pressure at 34 ◦C, quantitatively transferred to a 10 mL volumetric flask with acidified methanol, and stored at −20 ◦C for up to one month until analysis was performed. A modified protein precipitation assay was used to determine total phenolics, total anthocyanins, and total tannins. Samples were thawed and diluted to fit the limitations of the spectrophotometer . Using a Genesys10S UV-Vis Spectrophotometer, total phenolics and total tannins were measured at 510 nm absorbance and expressed as catechin equivalents ; whereas total anthocyanins were measured at 520 nm absorbance.Statistical analysis was conducted in the R language . All analyses used an α of 0.05 for statistical significances.
One-way analysis of variance and three-way ANOVA with three-way interactions were used to determine significant differences between samples. For a three-way ANOVA with three-way interactions, only the interactions of virus status to rootstock and virus status to year were considered to determine the influence genotypic or seasonal factors had on virus status. Virus status, rootstock, and year were all considered fixed effects for the purpose of determining the genotypic and temporal effects on disease status. A Tukey’s honestly significant difference test was used for post hoc analysis. Principal component analysis was used to display the variance in volatile analysis.While San Joaquin Valley vineyards are currently fertilized with boron through the soil and foliage , some growers have expressed interest in applying boron via drip irrigation or “fertigation.” Fertigation is a relatively simple, cost-effective and efficient way to apply nutrients. However, irrigation water with more than 1 part per million boron can lead to vine toxicity, so the safety of boron fertigation is also a concern. Our research evaluates the safety and efficacy of boron fertigation in grapevines using drip irrigation. Boron is unique among the micronutrients due to the narrow range between deficiency and toxicity in soil and plant tissues. For grapevines, this range is 0.15 ppm to 1 ppm in saturated soil extracts, and 30 ppm to 80 ppm in leaf tissue. The goal of boron fertilization of grapevines is to keep tissue levels within this narrow range, since both deficiency and toxicity can have serious negative effects on vine growth and production. Fertilization amounts must be precise to avoid toxicity while providing adequate boron to satisfy grapevine requirements . On the east side of the San Joaquin Valley, boron deficiency of grapevines occurs on soils formed from igneous rocks of the Sierra Nevada. This parent material is low in total boron, which is crystallized in borosilicate minerals that are highly resistant to weathering. Boron deficiency is often associated with sandy soils and vineyard areas with excessive leaching, such as in low spots or near leaky irrigation valves. Vine symptoms of boron deficiency are more widespread and pronounced following high rainfall years, when greater amounts of soluble boron are leached from the root zone. In addition, snowmelt water has very low levels of boron, and vineyards irrigated primarily with this water have a greater risk of deficiency. Boron is required for the germination and growth of pollen during flowering, and vines that are deficient in this micronutrient will have clusters that set numerous shot berries, small berries with a distinctive pumpkin shape. When boron deficiency is severe, vines produce almost no crop. Foliar symptoms appear in the spring: shoots have shortened, swollen internodes and their tips sometimes die, and leaves have irregular, yellowish mottling between the veins. Grapevines are also sensitive to too much boron. Toxicity is common on the west side of the San Joaquin Valley, where most soils are derived from marine sedimentary and metasedimentary parent material that is rich in easily weathered boron minerals. Symptoms of boron toxicity include leaves that are cupped downward in the spring and that develop brown spots adjacent to the leaf margin in middle or late summer, intensifying and leading to necrosis as boron accumulates. Yields are reduced, the result of diminished vine vigor and canopy development. When foliar boron sprays are applied in excess in the spring, juvenile leaves become cupped within 2 weeks; however, vines quickly recover and yields are usually unaffected. Toxicity also occurs when boron fertilizer is applied in excess, regardless of the soil type, and this can lead to yield loss. Over-fertilization is the sole reason for boron toxicity on the east side of the San Joaquin Valley, so it is critical to establish how much boron fertilizer can be applied safely and effectively. Our research investigated the uptake of boron by grapevines when fertilizer was applied with a drip-irrigation system.Research was conducted from 1998 to 2001 in a mature ‘Thompson Seedless’ raisin vineyard near Woodlake in Tulare County. The vineyard was planted in Cajon sandy loam on a recent alluvial fan associated with the Kaweah River. This soil is derived from granitic parent materials, and the surface soil is highly micaceous with a slight to moderate amount of lime. The underlying soil has a coarse, sandy texture. At the onset of this study, the vineyard’s boron status was in the questionable range for deficiency.