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Research Report

Role of Cluster Thinning and Viral Load on the Effects of Grapevine Leafroll Disease in Merlot and Cabernet Sauvignon in British Columbia, Canada

April Roberts, Miranda Hart, Kevin Usher, José Ramón Úrbez-Torres
Am J Enol Vitic.  2025  76: 0760004  ; DOI: 10.5344/ajev.2024.24052

Abstract

Background and goals Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus that negatively affects grapevine health and fruit composition. This study investigated the use of crop thinning to mitigate the effects of GLRaV-3 in Merlot and Cabernet Sauvignon vines in the Okanagan Valley, and to understand if GLRaV-3 viral load correlates with decreased vine health and fruit composition.

Methods and key findings A commercial vineyard containing Merlot and Cabernet Sauvignon vines was studied over two years. GLRaV-3-positive (GLRaV-3(+)) and GLRaV-3-negative (GLRaV-3(−)) vines were paired and designated a cropping treatment, either 1.5 clusters per shoot (1.5 c/s) or one cluster per shoot (1.0 c/s). Vine health and fruit composition were measured during the growing season and at harvest, respectively. Viral load was measured at four stages during the growing season using Droplet Digital PCR (ddPCR). GLRaV-3(+) vines had increased crop load and titratable acidity (TA), and reduced total soluble solids. 1.0 c/s vines had lower TA and anthocyanins, and increased pH. Thinning GLRaV-3(+) vines significantly increased pH. GLRaV-3 viral load was negatively correlated with photosynthesis, stomatal conductance, SPAD values, cluster weight, berry weight, and yeast assimilable nitrogen, and positively correlated with skin and seed phenolics.

Conclusions and significance GLRaV-3 effects were mild and differed by cultivar and year. No consistent correlation was found between GLRaV-3 viral load and adverse vine health or fruit composition. Crop thinning did not improve vine health or fruit composition of GLRaV-3(+) or GLRaV-3(−) vines. This study shows no benefit to thinning vines lower than 1.5 c/s in the Okanagan Valley and thus, growers should keep their fruit to avoid diminishing returns.

Introduction

Grapevines host the most viruses of any cultivated crop (Fuchs 2023). Among them, those viruses responsible for grapevine leafroll disease (GLD) are the most widespread and significant. GLD symptoms encompass downward rolling of leaf margins, hence the name “leafroll”, as well as interveinal reddening in black-fruited cultivars (Naidu et al. 2015). The causative agents of GLD are grapevine leafroll-associated viruses (GLRaVs). To date, six positive-sense single-stranded RNA viruses of the family Closteroviridae have been associated with GLD. Among these, Grapevine leafroll-associated virus 3 (GLRaV-3) is the main etiological agent of GLD, due to its cosmopolitan distribution and strong negative influence on grapevine health and fruit composition (Maree et al. 2013, Burger et al. 2017, Song et al. 2021).

GLRaV-3 spreads primarily through infected clonal propagation material, and secondarily within and between vineyards via insect vectors, including several species of mealybug (Pseudococcidae) and soft scale (Coccidae) (Naidu et al. 2014). The financial impact of GLRaV-3 has been estimated at ~USD $29,902 to $226,405/ha in California (Ricketts et al. 2015), and USD $20,000 to $40,000 in New York (Atallah et al. 2012). A recent study conducted in the Okanagan Valley in British Columbia (BC) showed that GLRaV-3 was prevalent in the region, with a fast secondary spread as a result of the presence of grape mealybug (Pseudococcus maritimus) in the region (Poojari et al. 2017).

The effects of GLRaVs on grapevine health and fruit composition have been widely studied and highlighted in a few review articles which emphasize that GLRaV-3 is the most impactful GLRaV (Maree et al. 2013, Naidu et al. 2015, Burger et al. 2017, Song et al. 2021). GLRaV-3 is reported to decrease vine vigor, total yield, number of clusters, and berry weight, resulting in crop losses of up to 40% (Maree et al. 2013, Naidu et al. 2015, Burger et al. 2017). A reduction in photosynthetic capacity in the leaves of GLRaV-3-infected vines is also commonly reported, with metrics such as stomatal conductance and CO2 assimilation being the most affected (Maree et al. 2013, Burger et al. 2017, Song et al. 2021). The most consistently reported and significant effects in fruit from GLRaV-3-infected grapevines are decreased sugar accumulation and increased acidity (Maree et al. 2013, Naidu et al. 2015, Burger et al. 2017, Bowen et al. 2018, Song et al. 2021); the decrease in sugar accumulation is hypothesized to be due to altered source-sink relationships caused by impaired sugar translocation in virus-infected phloem tissue (Alabi et al. 2016).

There is no cure for a GLRaV-3 infection; once the virus is established in the vine, the plant is infected for life. Management of GLRaV-3 is limited to clean plant material, insect vector control, or vine removal (Fuchs 2020). Control of insect vectors may be accomplished using insecticides, mating disruption, or biological control with predators or parasitoids (Naidu et al. 2015, Cocco et al. 2021). These strategies are useful in combination if GLRaV-3 incidence is below 25%. Above that, vineyard replacement is recommended (Ricketts et al. 2015, Fuchs 2020, Hesler et al. 2022). Despite reported improvements in economic losses, vine removal and replant are costly and lengthy processes, and lead the virus to remain in vineyards, which increases the risk of spread within and between sites.

Cluster thinning is a common viticultural practice in which fruit is removed from a grapevine at a selected stage during the growing season; this forces the vine to adjust resource allocation, resulting in altered berry composition (VanderWeide et al. 2024). As reduced sugar accumulation is a commonly reported finding in studies comparing fruit from GLRaV-3-infected vines to that from non-infected vines, we hypothesized that crop thinning could mitigate reduction of sugar accumulation, while concurrently improving overall vine health. Very few studies have been conducted to answer this question. Higher total soluble solids (TSS) and increased concentrations of proline and arginine were observed in GLRaV-3-infected vines in California, following a 50% reduction in cluster number 1 mo before veraison (Kliewer and Lider 1976). In Ontario, Canada, no significant improvement was noted when GLRaV-3-infected Cabernet franc vines were thinned 2 wk before veraison (Hébert-Haché 2015). Given the lack of research into the effects of crop thinning on GLRaV-3-infected vines, it is important to understand whether this cultural practice may be a suitable mitigation strategy in the Okanagan Valley, BC.

Though a model of GLRaV-3 infection was recently proposed by Song et al. (2021), it is unclear whether there exists a relationship between GLRaV-3 viral load and vine performance. Previous studies indicate the viral load of GLRaV-3 in black-fruited winegrapes to be highly variable throughout the growing season, with no consensus on a pattern. Increases in viral load of GLRaV-3-infected Pinot noir in Oregon occurred early in the season, followed by a leveling-off (Wright 2012). However, in a study on unspecified cultivars in California, viral load decreased in petiole tissue between May and August (Osman et al. 2018). In Ontario, viral load increased as the season progressed from bloom to harvest in Cabernet franc vines (Shabanian et al. 2020). A recent study on Crimson Seedless table grapes concluded that GLRaV viral load did not significantly correlate with fruit composition (Salo et al. 2024).

GLRaV-3 is an incurable threat to winegrape production in the Okanagan Valley; the presence of infection, insect vectors, and known negative effects to vine health and fruit composition demand action. Since GLRaV-3 is known to influence sugar accumulation, and cluster thinning is a common viticultural practice used to improve vine balance, the goal of our study was to understand if crop thinning can mitigate the negative effects of GLRaV-3, and if viral load influences vine performance and fruit composition.

Materials and Methods

Site characteristics and vine infection status

A field trial was conducted in both the 2021 and 2022 growing seasons at a commercial vineyard near Oliver, BC, Canada, that contained adjacent Merlot and Cabernet Sauvignon plantings. The entire vineyard was established in 2005. The vines were Cabernet Sauvignon clone 169 on 101-14 Mgt Vitis riparia × Vitis rupestris rootstock, and Merlot clone 412 on 3309C V. riparia × V. rupestris rootstock. Rows were 2.44 m wide with ~1.22 m vine spacing. All vines were double-cordon trained in north-south orientation and spur pruned. Shoots were vertically trained and held upright with three trellis catch wires in a vertical shoot-positioning system, and they were hedge trimmed regularly during the growing season. Water allocation, fertilization, and pest control were carried out according to standard production practices for the region. Temperature, relative humidity, and precipitation were recorded using portable HOBO (Onset Computer Corporation) data loggers at the field site from April to October of each season.

Both study blocks contained existing GLRaV-3 infections that were identified during a 2020 survey. A total of 408 leaf samples from GLD symptomatic and asymptomatic vines were collected from both Merlot and Cabernet Sauvignon blocks in September 2020. Five basal leaves per vine were collected evenly across the vine (one from the center near where the cordons join the trunk, and two from each cordon), bagged, and stored immediately on ice. Leaf samples were submitted to the Grapevine Virus Testing Laboratory in the Cool Climate Oenology and Viticulture Institute (CCOVI) at Brock University, St. Catharines, Ontario. Vines were tested for GLRaV-1, −2, −3, and −4 by endpoint reverse transcription PCR (RT-PCR), and for grapevine red blotch virus (GRBV) by endpoint PCR. The number of vines selected for study in each cultivar are described (Table 1). Infected (GLRaV-3(+)) vines were paired with adjacent non-infected (GLRaV-3(−)) vines 1:1, ensuring at least one guard vine in between. Each pair was assigned a treatment group based on industry-standard cropping levels of either 1.5 or 1.0 clusters/shoot (c/s). There were 80 Merlot (20 vines per infection status:treatment combination) and 72 Cabernet Sauvignon (18 vines per infection status:treatment combination) experimental vines. Thinning treatments were conducted at pea-sized berries stage each year to ~1.5 or 1.0 c/s. Infection status of all vines was confirmed by CCO-VI at the end of each growing season using petiole tissue, and in the winter using dormant wood. Vines for which infection status did not match the original survey were removed from analysis (Table 1).

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Table 1

Total sample number grouped by infection status of Merlot and Cabernet Sauvignon study vines, after confirmation by PCR testing in 2021 and 2022. GLRaV-3, grapevine leafroll-associated virus 3; (−), negative; (+), positive; c/s, clusters per shoot.

Vine health measurements

Leaf greenness and leaf gas exchange were measured at four measurement stages (bloom, pea-sized berries, veraison, and harvest). Leaf greenness was measured using a SPAD 502 Plus Chlorophyll Spectrophotometer (Spectrum Technologies Inc). Measurements were taken on 10 fully expanded mature leaves in the middle of the canopy. Leaf gas exchange (photosynthesis [Anet], stomatal conductance [gs], and transpiration rate [T]) was measured using a LI-COR LI-6400-XT Portable Leaf Gas Exchange System (LI-COR Biosciences). System specifications and measurement conditions were as described in Bowen et al. (2020). Forty vines (10 per infection status:treatment combination) were measured in each experiment. SPAD values and leaf gas exchange data were not collected for bloom in 2022, as higher-than-normal fungicide spray applications made the blocks inaccessible. Total yield per plant was measured at harvest using a field scale, and number of clusters per vine were recorded.

Bud cold hardiness was determined at three time points each winter in both Merlot and Cabernet Sauvignon. One dormant cane was pruned from each vine and brought back to the Summerland Research and Development Centre for analysis. Canes were pruned to seven bud sections, and five buds per cane were placed in freezer trays. Differential thermal analysis was carried out as described in Bowen et al. (2020). Vines were spur pruned in early spring of the year following the growing season, and pruning weights were recorded in the field. Crop load (Ravaz index) was determined as the ratio of yield-to-pruning weight.

Fruit composition measurements

Two subsamples of berries were collected at harvest, one 50-berry subsample that was immediately frozen at −20°C for phenolic analysis, and a 70-berry subsample for immediate fruit composition analysis. Clusters were selected evenly across the vine, and the top, bottom, and each side of cluster were sampled for a total of 5 to 10 berries per cluster. After weighing, berries from the 70-berry subsample were crushed with a mortar and pestle and strained through cheesecloth to release their juice. TSS were measured using a Palette digital refractometer (ATAGO). Titratable acidity (TA) of juice was measured using an automated titrator (Metrohm). Five mL of juice were titrated with 0.1N sodium hydroxide to a pH endpoint of 8.1. Juice pH was measured using a pH meter with electrode (London Scientific). Yeast assimilable nitrogen (YAN) was measured using the formol method on an automated titrator (Metrohm). Berries from the 50-berry subsample were peeled to separate skins and seeds. Skins and seeds were lyophilized using a freeze drier (VirTual, SP Scientific). Seed samples were ground in liquid nitrogen using a freezer mill (SPEX SamplePrep LLC) and skins were ground in liquid nitrogen using a mortar and pestle. Anthocyanins were determined using the Glories method (1984) and tannins were determined using a methyl cellulose precipitation assay (adapted from Mercurio et al. 2007).

Quantification of GLRaV-3 viral load

GLRaV-3(+) vines were sampled at four measurement stages (bloom, pea-sized berries, veraison, and harvest) to determine viral load by Droplet Digital PCR (ddPCR). Five mature basal leaves were collected from each vine, placed in plastic zip-top bags, and stored in coolers on ice for transport. Immediately after arriving at the lab, petioles were separated from the leaf tissue, hand-torn into smaller pieces, and ground in liquid nitrogen using a mortar and pestle. RNA extraction was performed on ~100 mg of tissue using the Spectrum total plant RNA kit (MilliporeSigma), modified by an addition of 2.5% w/v PVP-40 to the lysis solution, as described in Xiao et al. (2018). After adding the lysis buffer, samples were incubated in a dry block for 5 min at 56°C. As specified in “Protocol A” of the Spectrum total plant RNA kit, 750 μL of binding buffer was added to the samples, followed by a DNase digest step, using the On-Column DNase 1 digestion kit (MilliporeSigma) according to manufacturer specifications. RNA concentrations were quantified using a Qubit fluorometer and the RNA BR (Broad-Range) assay kit (Thermo Fisher Scientific), following kit instructions. RNA quality was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Select RNA extracts were also run on a 1% agarose gel to ensure integrity.

Amplification and quantification were carried out using ddPCR with the Bio-Rad One-Step RT-ddPCR Kit for probes with target and reference in a duplex assay (Bio-Rad Laboratories, Inc.). Quantitative primers targeting GLRaV-3 ORF1a were used with forward sequence 5′–TGT-GTAGGGCTCAGAAGTC-3′ and reverse sequence 5′–GCGCGTAGGAATAGATCCT-3′, and a FAM labeled TaqMan hydrolysis probe with the sequence 5′-CCTGTTGTAGCTGGTTCTAGT-3′. Primers were previously developed by Dieter Kahl and Sudarsana Poojari (personal communication) using Primer-BLAST (National Centre for Biotechnology Information), and stability was confirmed by Net Primer (Premier Biosoft). The probe was developed based on the existing primer set using PrimerQuest software (Integrated DNA Technologies). The SAND family protein was used as a reference gene, with forward sequence 5′-CAACATCCTTTACCCATTGACAGA-3′ and reverse sequence 5′–GCATTTGATCCACTTGCAGATAAG-3′, as described in Reid et al. (2006) and Tashiro et al. (2016). A HEX labeled TaqMan hydrolysis probe with sequence 5′-TTGCACGTCCGTATCGCCAAGG-3′ was developed for SAND, with existing primer set using PrimerQuest software (Integrated DNA Technologies).

Each sample was serially diluted 1:1000 in molecular-grade water (ThermoFisher Scientific) (for optimized droplet resolution during analysis) before analysis in a 96-well plate. PCR master mix was prepared (per reaction) using 5.5 μL of supermix, 2.2 μL of RT enzyme, 1.1 μL of dithiothreitol, 0.99 μL of each 20 μM primer (3.96 μL total for all four primers), 0.275 μL of each 20 μM probe (0.55 μL total for both probes), and 3.19 μL of molecular-grade water. As recommended by the manufacturer due to viscosity of the solutions, all ddPCR reagents were vortexed for 30 sec and centrifuged before preparation of the mix, and the final master mix was also vortexed for 30 sec and centrifuged before aliquoting into a 96-well plate. Each plate was prepared with at least two positive controls, with RNA extracted as described above from confirmed GLRaV-3(+) greenhouse material, and at least two non-template controls containing confirmed GLRaV-3(−) greenhouse material. After the addition of samples and controls, the final 96-well plate was sealed using sealing foil and a PCR plate sealer (Bio-Rad), then vortexed for 10 sec, centrifuged for 1 min, then let stand for 10 min before droplet generation. Droplets were generated using a QX200 Automatic Droplet Generator (Bio-Rad) and PCR was carried out on a C1000 Touch Thermal Cycler (Bio-Rad) using the following thermocycling parameters set at a ramp rate of 2°C/sec: one cycle of reverse transcription at 50°C for 60 min, one cycle at 90°C for 10 min, 40 cycles at 95°C for 30 sec followed by 58°C for 1 min, 1 cycle of 98°C for 10 min, then held at 4°C.

To analyze the droplets, the plate was placed on a QX200 Droplet Reader (Bio-Rad) and analyzed using QX Manager Software with the “Direct Quantification (DQ)” experiment type in channel 1:FAM and channel 2:HEX. The average number of accepted droplets for all samples was 14,043 (range of 7648 to 17,787), however, samples containing under 10,000 droplets were re-run. The range of amplitude for positive GLRaV-3 droplets was generally between 1500 to 8000 and for negatives, between 700 to 1200. For SAND, positive droplets ranged between 5000 to 6000, and for negatives, from 1000 to 2000. Threshold values varied between plates but were always set just above the negative cluster. Equation 1 was used to determine viral load (Kahl et al. 2021).

virus titer(copiesng)=ddPCR conc.(copiesμL)*ddPCR rxn volume(μL)*DFNA volume in rxn(μL)*Qubit conc.(ngμL) Eq. 1

The Qubit concentration and ddPCR concentration were specific to each sample analyzed, with the ratio of reference gene-to-target gene used as the ddPCR concentration to normalize samples. The dilution factor was set to 1000, the ddPCR reaction volume 22 μL, and the nucleic acid (RNA) volume in the ddPCR reaction was 5.5 μL.

Statistical analyses

All statistical analyses were performed using the R programming language (ver. 4.2.1; R Core Team 2020) in R Studio (ver. 2022.12.0.353; RStudio Team 2020). An alpha value of 0.05 was used for all tests. Berry and cluster weight, yield, pruning weight, crop load, and all fruit composition data were analyzed using analysis of variance (ANOVA) with a Type III sum of squares, with the Anova function in the ‘car’ package (ver. 3.1-1; Fox and Weisberg 2019). Extreme outliers identified by boxplot methods were removed, and if any assumptions were violated, dependent variables were transformed. The best transformation for the data was determined using the bestNormalize function from the ‘bestNormalize’ package (ver. 1.9.0; Peterson and Cavanaugh 2020, Peterson 2021). If the assumption of heteroscedasticity could not be satisfied through variable transformation, White-adjusted p values were calculated using the white.adjust argument within the Anova function. Bonferroni tests were used as post-hoc pairwise comparisons with the emmeans function from the ‘emmeans’ package (ver. 1.8.4-1; Lenth 2023).

Repeated measurements (leaf gas exchange, SPAD values, bud hardiness, viral load) were assessed using a linear-mixed effects model structure with the lmer function from the ‘lme4’ package (ver. 1.1-31; Bates et al. 2015). Each parameter was assessed for effects of infection status, treatment, measurement stage, and their interaction terms, with a random intercept (repeated measure) for each vine. For the leaf gas exchange data from 2021, there was an additional random intercept term fitted for the measurement unit, to remove any potential variation due to machine measurement issues. The models were then analyzed using the Anova function (‘car’ package) with Type III sum of squares, Wald’s F test, and Kenward-Roger approximation for degrees of freedom. Models that did not meet assumptions had dependent variables transformed. To assess the effects of increased viral load on vine health and fruit composition, each individual parameter was modeled against the temporally-closest viral load measurement using Spearman’s rank-order correlation from the native cor.test function in base R ‘stats’ package (R Core Team 2020). Compact letter displays in tables and figures were calculated using the cld function from the ‘multcomp’ package (ver. 1.4.25; Hothorn et al. 2008). All figures were prepared using the ‘ggplot2’ package (ver. 3.4.1; Wickham 2016) and were stitched together using the ggarrange function from the ‘ggpubr’ package (ver. 0.6.0; Kassambara 2023).

Results

Site characteristics and vine infection status

The two years of this study were hotter than normal for the region. The average temperature for the 2021 growing season (12 May to 4 Oct) was 21.52°C, with a maximum of 43.98°C recorded on 30 June. The average temperature for the 2022 growing season (15 May to 4 Oct) was 20.23°C, with a maximum of 43.71°C recorded on 28 July. There was an average of 1682.67 and 1630.71 growing degree days (base 10°C) in 2021 and 2022, respectively.

During the initial assessment in 2021, 80 Merlot vines and 72 Cabernet Sauvignon vines were chosen for the study. GLRaV-3(−) vines did not test positive for any viruses included in the diagnostic PCR, and all non-infected vines remained negative throughout both years of study. In 2021, all vines that tested positive for GLRaV-3 were negative for all other GLRaVs tested, as well as negative for GRBV. However, in 2022, several vines that were GLRaV-3(+) in 2021 tested negative in 2022, leading to those vines being removed from the study, as well as additional testing from dormant canes to confirm all other results and validate the testing method. The final number of vines used for analysis was 74 Merlot, and 68 Cabernet Sauvignon. A breakdown of sample number by infection status and treatment is provided (Table 1).

Vine health measurements

SPAD values in Merlot and Cabernet Sauvignon vines were not significantly affected by GLRaV-3 infection or crop thinning treatment in either year of study (Figure 1). Leaf gas exchange parameters differed throughout the growing season based on the stage they were measured; neither GLRaV-3 infection nor crop thinning treatment significantly affected Anet, gs, or T at any of the four stages measured for Merlot or Cabernet Sauvignon in either year of study (Figure 1).

Figure 1
Figure 1

Effects of grapevine leafroll-associated virus 3 (GLRaV-3) ((−), negative; (+), positive) and crop thinning treatment (Infection:Thinning) in Merlot (A to D) and Cabernet Sauvignon (E to H) on photosynthesis (Anet), stomatal conductance (gs), transpiration rate (T), and SPAD values of study vines at four measurement stages in 2021 and 2022. Data points are means, with error bars indicating standard error of the mean. Different letters indicate statistically significant differences between means of Infection:Thinning for each measurement stage, as measured by analysis of variance (α = 0.05). Values were not available for Cabernet Sauvignon at harvest in 2021 or for either cultivar at bloom in 2022. c/s, clusters/shoot.

Cluster thinning was performed at pea-sized berries each season; final shoot and cluster counts at harvest are shown (Table 2). Average berry weight and average cluster weight were not significantly different with respect to vine infection status or crop thinning treatment for either cultivar in both years (Table 3). Yields consistently differed by treatment group for both cultivars across both years of study. Pruning weight for both cultivars was not significantly affected by GLRaV-3 infection or cluster thinning in either year. For Cabernet Sauvignon, GLRaV-3(+) vines had an average crop load that was 13% higher than GLRaV-3(−) vines in the 2021 season (Table 3); GLRaV-3 infection did not significantly affect Merlot crop load in either year. Bud hardiness was also not significantly affected by GLRaV-3 infection or crop thinning (Figure 2). However, there were significantly fewer viable buds in 2022 due to sustained freezing damage from evening temperatures below −22°C (with a low of −23.5°C) for several hours on 22 Dec, which caused bud mortality. This freezing event led to more significant bud death in Cabernet Sauvignon, with only 10% of vines having viable buds after the freeze, versus in Merlot, in which 29% of vines still had viable buds.

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Table 2

Average clusters at harvest, shoots at pruning, and clusters per shoot (c/s) of Merlot and Cabernet Sauvignon study vines in 1.5 and 1.0 c/s treatment groups in 2021 and 2022.

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Table 3

Effects of grapevine leafroll-associated virus 3 (GLRaV-3) infection and crop thinning, and their interaction on average berry and cluster weight, yield, pruning weight, and crop load of Merlot and Cabernet Sauvignon vines in 2021 and 2022. c/s, clusters/shoot; (−), negative; (+), positive; ANOVA, analysis of variance.

Figure 2
Figure 2

Effects of grapevine leafroll-associated virus 3 (GLRaV-3) ((−), negative; (+), positive) and crop thinning treatment (Infection:Thinning) on bud hardiness (LTE50) of dormant wood in Merlot (A) and Cabernet Sauvignon (B) vines at three measurement times in the 2021 (Nov 2021, Feb 2022, and March 2022) and 2022 growing seasons (Nov 2022, Feb 2023, and March 2023). Data points are means, with error bars indicating standard error of the mean. Different letters indicate statistically significant differences between means of Infection:Thinning for each growth stage, as determined by linear mixed-effects analysis for repeated measures (α = 0.05). c/s, clusters/shoot.

Fruit composition measurements

Across both growing seasons, GLRaV-3 infection was associated with decreased sugar accumulation for Merlot: an average of 1 Brix decrease in 2021, and 0.5 Brix decrease in 2022, when comparing GLRaV-3(−) vines to GLRaV-3(+) vines (Table 4). Cluster thinning treatment had no effect on juice TSS in Merlot berries for either year. Lastly, neither GLRaV-3 infection nor crop thinning treatment caused a significant change in TSS for Cabernet Sauvignon in either year of study (Table 4).

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Table 4

Effects of grapevine leafroll-associated virus 3 (GLRaV-3) infection and crop thinning, and their interaction on fruit composition of Merlot and Cabernet Sauvignon grape must in 2021 and 2022. c/s, clusters/shoot; TSS, total soluble solids; TA, titratable acidity; YAN, yeast assimilable nitrogen; ANOVA, analysis of variance; (−), negative; (+), positive.

The effects of GLRaV-3 infection and crop thinning on TA were cultivar- and year-dependent. In the 2021 growing season, fruit from Merlot 1.0 c/s vines had on average 5% lower TA than 1.5 c/s vines (Table 4). In 2022, GLRaV-3 infection caused a significant increase in TA, however cluster thinning had no effect. TA of Cabernet Sauvignon was not affected by crop thinning in either year, but it did increase by 4% and 6% in 2021 and 2022, respectively, as a result of GLRaV-3 infection.

Cluster thinning significantly increased Merlot juice pH across both years of study (Table 4), whereas GLRaV-3 infection had no effect. A significant interaction between crop thinning and GLRaV-3 infection in 2022 Cabernet Sauvignon resulted in increased pH in fruit from 1.0 c/s GLRaV-3 vines, compared to their 1.5 c/s counterparts.

There was no significant effect of GLRaV-3 infection on YAN in Cabernet Sauvignon, however, crop thinning increased Cabernet Sauvignon YAN by an average of 11% in the 2022 study season (Table 4). YAN in Merlot vines was not significantly affected by GLRaV-3 infection or crop thinning in either 2021 or 2022.

With the exceptions of skin anythocyanins and skin flavonols, GLRaV-3 infection did not significantly affect the phenolic profiles of skins and seeds in either Merlot or Cabernet Sauvignon. In 2021, skin flavonols increased in GLRaV-3(+) Merlot (Table 5), and in Cabernet Sauvignon, both skin anthocyanins and skin flavonols were 16% lower for 1.0 c/s vines than their 1.5 c/s counterparts. Skin flavonols were also reduced by 11% in the 2022 Cabernet Sauvignon 1.0 c/s vines, compared to the 1.5 c/s vines. Additionally, the interaction between crop thinning and GLRaV-3 infection significantly reduced the seed tannins of Cabernet Sauvignon vines, but only in 2022 (Table 5).

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Table 5

Effects of grapevine leafroll-associated virus 3 (GLRaV-3) infection and crop thinning, and their interaction on skin and seed phenolic profiles of Merlot and Cabernet Sauvignon in 2021 and 2022. c/s, clusters/shoot; (−), negative; (+), positive; ANOVA, analysis of variance.

Quantification of GLRaV-3 viral load

Across both years of study, viral load followed a seasonal pattern in both cultivars (Figure 3). For Merlot, the lowest viral load was observed at bloom, with an average of 68,221 copies/ng for 2021 (data not available for 2022), and the highest viral load was observed at pea-sized berries (258,713 copies/ng in 2021 and 158,236 copies/ng in 2022). The average GLRaV-3 viral load at veraison was 79,423 copies/ng in 2021 and 32,418 copies/ng in 2022, and at harvest, 222,119 copies/ng in 2021 and 99,581 copies/ng in 2022. Trends in viral load for Cabernet Sauvignon in 2022 (data for bloom not available) were similar to those observed for Merlot, however, in 2021, GLRaV-3(+) Cabernet Sauvignon vines did not show a significant change in viral load until harvest (Figure 3). In addition, viral loads for Cabernet Sauvignon were generally lower than for Merlot. GLRaV-3 viral load in Cabernet Sauvignon at bloom was 74,850 copies/ng in 2021 (data not available for 2022), at pea-sized berries, 60,078 copies/ng in 2021 and 57,736 copies/ng in 2022, at veraison, 42,851 copies/ng in 2021 and 34,591 copies/ng in 2022, and at harvest, 101,693 copies/ng in 2021 and 56,728 copies/ng in 2022.

Figure 3
Figure 3

Changes in grapevine leafroll-associated virus 3 (GLRaV-3) viral load in infected Merlot (open circles, A and B) and Cabernet Sauvignon (filled circles, C and D) vines across four measurement stages in 2021 (A, C) and 2022 (B, D). Boxes enclose the 25th (bottom) and 75th (top) percentiles of the data, with a bold line at the median value. Whiskers indicate standard deviation. Different letters indicate statistically significant differences between growth stages, as determined by linear mixed-effects analysis for repeated measures (α = 0.05). Data is not available for bloom in 2022. Y-axis values: Ne+05 = N × 105.

In 2021, there was a negative correlation between viral load and Anet and gs in leaves of Merlot vines at veraison, and in 2022, there was a negative correlation between viral load and T at pea-sized berries (Table 6). SPAD values of Cabernet Sauvignon leaves showed significant negative correlations to viral load at bloom, pea-sized berries, and harvest in 2021, as well as at veraison and harvest in 2022 (Table 6). There were no significant correlations between GLRaV-3 viral load and fruit composition of Merlot in either year of study; however, seed flavonols and seed tannins had positive correlations with viral load at harvest (Table 7). There was a significant negative correlation between GLRaV-3 viral load and YAN of Cabernet Sauvignon in 2021 (Table 7), and in 2022, negative correlations were also present with average cluster weight and average berry weight in Cabernet Sauvignon (Table 7). In 2022, skin phenolics were positively correlated with viral load in Cabernet Sauvignon for both anthocyanins and tannins.

View this table:
Table 6

Correlation (Spearman’s ρ) between grapevine leafroll-associated virus 3 (GLRaV-3) viral load, leaf greenness, and leaf gas exchange for Merlot and Cabernet Sauvignon vines in 2021 and 2022. Anet, photosynthesis; gs, stomatal conductance; T, transpiration rate.

View this table:
Table 7

Correlation (Spearman’s ρ) between grapevine leafroll-associated virus 3 (GLRaV-3) viral load measured at harvest on yield-related vine health parameters and fruit quality parameters in Merlot and Cabernet Sauvignon in 2021 and 2022.

Discussion

This is the first study documenting the use of crop thinning to mitigate the effects of GLRaV-3 infection in Merlot and Cabernet Sauvignon in the Okanagan Valley. The utility of cluster thinning to improve fruit composition in wine-grapes has been studied (VanderWeide et al. 2024), but very few studies have applied this technique as a tool to counteract the effects of GLRaV-3 (Kliewer and Lider 1976, Hébert-Haché 2015). This is also the first study to measure GLRaV-3 viral load using ddPCR, and one of a few attempting to correlate viral load to vine health and fruit composition of infected vines (Salo et al. 2024). Our data demonstrates minimal effects of thinning from 1.5 to 1.0 c/s in GLRaV-3(+) or GLRaV-3(−) vines. In addition, no strong correlations were observed between vine health or fruit composition and GLRaV-3 viral load, suggesting that further research is needed into how this complex pathosystem influences GLD. We argue that crop thinning has no utility to combat GLRaV-3, and offers little benefit to improve the fruit composition of non-infected Merlot and Cabernet Sauvignon vines when thinning from 1.5 to 1.0 c/s.

GLRaV-3 infection did not significantly affect any leaf exchange parameters (Anet, gs, or T) or SPAD values compared to GLRaV-3(−) vines in this study. Our findings are contradicted by the majority of studies which have reported GLRaV-3 infection decreases leaf gas exchange and leaf chlorophyll (Song et al. 2021). The lack of virus effects on these vine health parameters could be due to the mild symptom development that was observed in this study, or due to environmental and seasonal variation (neither variation was captured across the two years that were examined in this study). Across 2021 and 2022, crop thinning did not significantly affect leaf gas exchange or SPAD values in GLRaV-3(+) or GLRaV-3(−) vines of Merlot or Cabernet Sauvignon. These findings are unique to this work, as other studies on crop thinning in GLRaV-3 vines did not measure SPAD values or leaf gas exchange (Kliewer and Lider 1976, Hébert-Haché 2015). Our findings, specifically in non-infected vines, are supported by Bowen et al. (2011), who concluded that cluster thinning did not significantly influence photosynthesis in Okanagan Valley Merlot and Cabernet Sauvignon. Therefore, in a case where GLRaV-3 is a significant hinderance to leaf gas exchange, crop thinning may not be a suitable solution.

Additionally, yield components were minimally affected by GLRaV-3 infection or crop thinning in this study. This was not unexpected, as it has been recently summarized that the effects of GLRaV-3 on yield components like berry weight are inconsistent among studies, and geographic, cultivar, and genetic variation likely play a role in the observed variation (Song et al. 2021). The lack of effects from crop thinning in this study could be due to the timing at which thinning was performed. A meta-analysis on the effects of cultural practices on vine yield components summarized that timing of crop thinning plays a crucial role (Cameron et al. 2024). Early season cluster thinning (Eichhorn-Lorenz [E-L] ≤ 33, as performed in our study) led to lower yield reductions due to compensatory increases in the number of berries per cluster. The severity of thinning also affects yield reduction, such that yield was lower in vines where 50% or more of clusters were removed, though berry weight and number slightly increase to compensate for this (Cameron et al. 2024).

Our study showed that in Cabernet Sauvignon, crop loads in GLRaV-3(+) vines were on average 13% higher when compared to GLRaV-3(−) vines in 2021. However, there was no significant interaction between crop thinning and infection for either cultivar in either year. The lack of an effect from crop thinning could have been due to the low crop loads we observed in our study, specifically in Cabernet Sauvignon. Crop load assessment is an important tool in viticulture as it is a good indication of vine balance and how capable the vine is to properly ripen its fruit (Kliewer and Dokoozlian 2005). Hébert-Haché (2015) was cautious in drawing conclusions from their data regarding a lack of benefit from crop thinning in GLRaV-3-infected Cabernet Sauvignon, as crop load information was not available for their study. In our study, crop loads were on average 4.5 and 6.8 for Merlot in 2021 and 2022, respectively, while for Cabernet Sauvignon, the respective values were just 1.8 and 2.3. A vine in balance should have a crop load ~4, which is indicative of its ability to produce high-quality fruit (Kliewer and Dokoozlian 2005). Cabernet Sauvignon vines in this study are far from that value, which may have impeded vines from improving health and fruit composition following crop thinning.

GLRaV-3 infection has been consistently shown to decrease TSS (Song et al. 2021), including in Cabernet franc in the Okanagan Valley (Bowen et al. 2018). The increase in leaf area-to-fruit ratio that results from crop thinning could increase the availability of photosynthetic products to ripening berries. However, GLRaV-3 infection is thought to cause discontinuities in phloem tissue and disrupt the flow of nutrients from source to sink (Naidu et al. 2015, Song et al. 2021). Despite this, no significant effect of crop thinning on TSS levels in GLRaV-3(+) Merlot or Cabernet Sauvignon was evident in our study in either 2021 or 2022. This is supported by a similar study on Cabernet franc, in which crop thinning had no significant effect on TSS in an Ontario vineyard (Hébert-Haché 2015), but it contrasts with a study on Burger vines, where GLRaV-3-infected vines had higher TSS levels following a 50% reduction in clusters (Kliewer and Lider 1976). Our results could be different due to a less significant reduction in cluster number or due to the differences in climate and growing region, as the 1976 study was performed in California. However, Cabernet Sauvignon does not respond to source-sink manipulation or environmental conditions as readily as other cultivars, such as Merlot (VanderWeide et al. 2024). Additionally, Cabernet Sauvignon vines in this study may have been out of balance, as shown by the low Ravaz index values, which could have hampered the benefit of crop thinning. Interestingly, VanderWeide et al. (2024) also highlighted that the severity of cluster thinning is more influential on fruit composition than the timing of thinning; this contrasts with Cameron et al. (2024), who asserted that the timing of cluster thinning is more influential on yield components. Since the present study only performed crop thinning at pea-sized berries, we cannot determine the effect that timing has on fruit composition, however, our data do not suggest that more severe cluster thinning improves fruit composition. The effects of crop thinning on other fruit composition parameters in this study were largely year- and cultivar-dependent. This was not unexpected, as previous work on cluster thinning in the region reported year-to-year differences in fruit composition, and that growing season is likely a more influential pressure than cropping treatment (Bowen et al. 2011). It is crucial to highlight that results of this study could be significantly confounded by unseasonably high average temperatures during the growing season across both years of this study and would have benefitted from additional years of data with more typical growing conditions. Growing region and environmental conditions have been shown to occasionally have stronger influence on vine health and fruit composition than GLRaV-3 (Bowen et al. 2018, Song et al. 2021). To mitigate this confounding factor, longer-term studies should assess the true effects of GLRaV-3 and the utility of crop thinning on vines in the Okanagan Valley.

Given the lack of improvement that cluster thinning provided to non-infected vines in this study, we calculated the price differences between 1.5 c/s and 1.0 c/s Merlot and Cabernet Sauvignon by calculating price per acre using our yield data, yearly crop pricing reports (https://bcwgc.org/annual-crop-assessment), and vines per acre data (following conversion to hectares) for the region (https://vinetech.ca/help-tips/planting-vines-per-acre/). It is estimated that by cropping from 1.5 to 1.0 c/s in non-infected Merlot, growers could lose CAD $9350.91/ha and $16,956.74/ha, based on yields from 2021 and 2022, respectively. Costs from thinning non-infected Cabernet Sauvignon were $6874.03/ha in 2021 and $11,523.43/ha in 2022. We believe our data demonstrate that the benefits of crop thinning do not outweigh these costs, and therefore, to avoid risking financial losses without improvements to fruit composition, Okanagan Valley grapegrowers should thin to no lower than 1.5 c/s.

This is the first report to quantify GLRaV-3 viral load using ddPCR. During the seasons studied, GLRaV-3 viral load in both Merlot and Cabernet Sauvignon followed similar seasonal trends, with respect to measurement stage for both cultivars. This bimodal trend of peaking viral load at peasized berries, and again at harvest, has not been reported before for GLRaV-3. Wright (2012) noted a polynomial pattern from June to October in the viral load of mature Pinot noir tissue infected with GLRaV-3. Work by Osman et al. (2018) did not find a significant difference in GLRaV-3 viral load measured using reverse transcription quantitative (real-time) PCR (RT-qPCR) throughout the season, though on average, viral loads were similar to those reported in this study. Similar to Wright (2012), the levels of GLRaV-3 in Cabernet franc and Chardonnay from Ontario showed a steady increase as the season progressed, with peaks near harvest, when measured using RT-qPCR (Shabanian et al. 2020). Given the current research, it may be speculated that changes in GLRaV-3 viral load may be influenced by cultivar and by specific climactic conditions in the growing region. This is the first report in Canada attempting to correlate virus effects to GLRaV-3 viral load measured by ddPCR, and to our knowledge, it is the first study correlating GLRaV-3 viral load to vine physiological parameters. Despite many vine health and fruit quality parameters being negatively correlated to GLRaV-3 viral load in this study, most of these parameters were not significantly affected by GLRaV-3, compared to non-infected vines. This suggests the possibility of a GLRaV-3 viral load threshold level, below which the magnitude of impact to vine health and fruit composition is not statistically significant. Recent work in Crimson Seedless grapes showed that GLRaV-3 viral load significantly affected the TSS and TA of GLRaV-3-infected vines, but a relationship between GLRaV-3 viral load and fruit composition was not found. The study concluded that viral load may be less important than the overall influence of GLRaV-3 infection to deregulate gene expression (Salo et al. 2024). To better understand what influences the pathology of GLD, we recommend that long-term studies track the influence of GLRaV-3 and GLRaV-3 viral load on vine health and fruit composition.

Conclusion

This study determined that cluster thinning from 1.5 to 1.0 c/s is not sufficient to mitigate the negative effects of GLRaV-3 in Merlot or Cabernet Sauvignon in the Okanagan Valley, although these effects were minimal and may have been confounded by higher-than-average temperatures across growing seasons. Additionally, viral load is not related to changes in vine health or fruit composition in these cultivars, and cluster thinning did not improve the fruit composition of GLRaV-3(−) vines. Therefore, the results from this study suggest that growers should not thin vines lower than 1.5 c/s, due to the risk of financial loss without an improvement in fruit composition.

Footnotes

  • This research was possible thanks to funds provided by Agriculture and Agri Food Canada and the British Columbia Wine Grape Council under the Canadian Agricultural Partnership Grape and Wine Cluster. We thank our commercial partner for permitting use of their vineyard blocks for this work. Technical support was provided by Jane Theilmann, Brad Estergaard, Steve Marsh, Julie Boulé, John Drover, Steve Orban, Dieter Kahl, Melanie Walker, and Emmanuelle Jean. Special thanks to students Jared Hrycan, Mia Alexander, Brett Pfliger, Cassandra Bruce, and Madeline MacIntosh. Virus testing was provided by Dr. Sudarsana Poojari at Brock University Cool Climate Oenology and Viticulture Institute.

  • Roberts A, Hart M, Usher K and Úrbez-Torres JR. 2025. Role of cluster thinning and viral load on the effects of grapevine leafroll disease in Merlot and Cabernet Sauvignon in British Columbia, Canada. Am J Enol Vitic 76:0760004. DOI: 10.5344/ajev.2024.24052

  • By downloading and/or receiving this article, you agree to the Disclaimer of Warranties and Liability. If you do not agree to the Disclaimers, do not download and/or accept this article.

  • All data underlying this study are included in the article and its supplemental information.

  • Received September 2024.
  • Accepted December 2024.
  • Published online February 2025

This is an open access article distributed under the CC BY 4.0 license.

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Role of Cluster Thinning and Viral Load on the Effects of Grapevine Leafroll Disease in Merlot and Cabernet Sauvignon in British Columbia, Canada
April Roberts, Miranda Hart, Kevin Usher, José Ramón Úrbez-Torres
Am J Enol Vitic.  2025  76: 0760004  ; DOI: 10.5344/ajev.2024.24052
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