Abstract
Background and goals This study uses trunk cambium sampling to improve detection of grapevine red blotch virus (GRBV) with the loop-mediated isothermal amplification (LAMP) assay, which can complement visual assessments and confirm GRBV infections. LAMP diagnostics of petiole tissues is optimal from harvest to dormancy and has shown poor detection of pre-symptomatic infections. Using Napa Valley vineyards, our goal was to validate a trunk sampling process to determine if LAMP assay diagnostics of cambium tissue could detect pre-symptomatic infections and detect GRBV on a year-round basis. We compared these results to LAMP assay testing of petiole tissue. We also surveyed LAMP assay users in the Napa Valley to assess practitioner experiences.
Methods and key findings This is the first study to demonstrate that trunk cambium is the most sensitive tissue for identifying pre-symptomatic infections, with an incidence of 33% compared to cane (16%) and petiole (2.7%) tissues. Optimal GRBV detection in petiole tissue was limited to veraison through early senescence; GRBV was detected in trunk cambium tissue year-round (budbreak through dormancy). Seventy percent of LAMP users were testing from 600 to more than 1000 vines per year, preferring trunk sampling with the primary purpose of identifying pre-symptomatic, infected vines.
Conclusions and significance Trunk cambium sampling is a new method for pre-symptomatic and year-round detection of GRBV with the LAMP assay. The grower community is adopting this strategy, and broader implementation of this approach will improve accurate estimates of GRBV incidence, leading to timely and informed management decisions to reduce the risk of spread.
- adoption of decision-support tools in agriculture
- grapevine red blotch disease (GRBD)
- grapevine red blotch virus (GRBV)
- loop-mediated isothermal amplification (LAMP)
- pre-symptomatic GRBV infection
- trunk sampling for GRBV detection
Introduction
Grapevine red blotch disease (GRBD), caused by grapevine red blotch virus (GRBV) (Yepes et al. 2018), is an ongoing threat to both the enological potential of winegrapes and to the economic viability of vineyards in regions where secondary spread has been documented (Cieniewicz et al. 2019, Dalton et al. 2019, KC et al. 2022). GRBV, the type member of genus Grablovirus in the family Geminiviridae, has a monopartite, circular DNA genome (Varsani et al. 2017), and is the first geminivirus reported for grapevines. GRBV can be transmitted by the three-cornered alfalfa hopper, Spissistilus festinus (Say) (Flasco et al. 2021, 2023b), and Vitis hosts include cultivated and free-living vines (Hoyle et al. 2022). GRBD extensively affects the metabolic pathways, biochemistry, and hormonal control of the host (Blanco-Ulate et al. 2017, Rumbaugh et al. 2022), resulting in delayed berry ripening (Martínez-Lüscher et al. 2019, Girardello et al. 2024), impaired sugar transport from leaves to berries (Martínez-Lüscher et al. 2019), and decreased synthesis of grape phenolics (Girardello et al. 2019, Pereira et al. 2021). GRBV also affects vineyard profitability, increasing the potential for vineyard removal at threshold values of disease incidence, while also subjecting infected vineyards to price penalties (Sudarshana et al. 2015, Ricketts et al. 2017), as GRBV-infected fruit decreases the quality of both white and red wine (Girardello et al. 2019, 2020, 2024).
The primary spread of GRBV across the United States (Krenz et al. 2014) and viticultural regions around the world (Krenz et al. 2023) is a consequence of its graft transmissibility (Al Rwahnih et al. 2013) and the unintended dissemination of infected plant material (Sudarshana et al. 2015). Secondary spread has been reported in southern Oregon (Dalton et al. 2019, KC et al. 2022) and Northern California (Cieniewicz et al. 2017a, 2019, Flasco et al. 2023a). The highest rates of in-field GRBV spread can be alarming, with an increase of up to 30% reported over three years (KC et al. 2022), or a two- to ten-fold increase over two years (Dalton et al. 2019), as observed in study vineyards in Oregon. In Northern California, GRBV increased from 9 to 14% incidence over the course of one year in a Cabernet franc vineyard (Cieniewicz et al. 2019), in which a spatial association between GRBV spread and the abundance of S. festinus populations was observed (Cieniewicz et al. 2018, 2019). Although vineyard managers employ strategies (e.g., vine removal, replanting) in an attempt to maintain GRBD incidence below 5% (KC et al. 2022), the underlying reasons for significant differences in secondary spread among vineyard sites remain unknown (Dalton et al. 2019).
One challenge to understanding the variability in rates of virus spread across sites has been the ambiguity surrounding symptom onset for Vitis vinifera hosts infected with GRBV. For example, one study observed a 0, 4, or 12-mo latency period, defined as the time between GRBV detection and symptom development (Flasco et al. 2024). Similarly, an extended incubation period—the time between exposure to viruliferous S. festinus and symptom expression (Van der Plank 1963)—of up to 16 mo has been suggested (Flasco et al. 2023b). However, due to limited observations, this remains an open question (Krenz et al. 2023). A multiyear incubation period may explain why disease incidence across vineyards of multiple ages was generally higher in sites with mature vines (KC et al. 2022). Similar uncertainties exist in cases of primary spread via infected plant material. For instance, when a rootstock is the primary source of inoculum, it may take three to five years for symptoms to develop (Cieniewicz et al. 2017a), compared to one year if the scion material is the primary source of inoculum (Flasco et al. 2023a). Consequently, attributing GRBV infections to primary or secondary spread is challenging in the early years following vineyard establishment (Flasco et al. 2023a), and it may be difficult to determine whether disease symptoms are related to vector activity or if they are an artifact of a long incubation period. Scenarios such as this can result in uncertainty among growers regarding the factors that surround disease spread and the accurate estimation of disease incidence.
Another challenge to estimating GRBV incidence and spread is that symptom assessments and diagnostic testing typically focus on a discrete period surrounding harvest. There are two reasons for this. First, growers make GRBD management decisions based on visual symptom diagnosis (KC et al. 2022, Rohrs et al. 2023), but vines do not start expressing symptoms until later phenological stages (Blanco-Ulate et al. 2017, Rumbaugh et al. 2021), with symptom onset continuing over a 3 to 4-mo period from veraison to the start of senescence (Rohrs et al. 2023). Second, sampling during harvest or dormancy minimizes the impact of variability in GRBV detection resulting from the uneven distribution of the virus within the vine during early phenological stages (Setiono et al. 2018, DeShields and KC 2023). Consequently, growers typically focus on GRBD visual detection and diagnostic testing around harvest, even though epidemiologically-relevant events occur throughout the growing season. In Northern California, the first annual generation of S. festinus occurs between June and July (Cieniewicz et al. 2019, Preto et al. 2019), during the berry development period. Simultaneously, there are detectable GRBV levels in host leaf tissue and an availability of inoculum for virus transmission (Flasco et al. 2023c). A second generation of S. festinus is present from late August into fall (Preto et al. 2019, Sisterson et al. 2023), and by October, some cultivars have an increased GRBV load in leaf tissues (Flasco et al. 2023c).
Given the spatiotemporal variability of visual symptoms and the practical limitations of symptom assessment (Rohrs et al. 2023), diagnostic tools are required to complement visual assessments, accurately estimate disease incidence, and provide actionable information to limit secondary disease spread. This study aims to provide growers with improved strategies for GRBV detection by selecting the optimum tissue and timing for loop-mediated isothermal amplification (LAMP) assay diagnostics. The LAMP assay is a more economical and approachable point-of-use diagnostic tool than PCR, making it suitable for routine monitoring to inform vine removal decisions (DeShields and KC 2023). However, there is a limited window of harvest and dormancy during which the LAMP assay can optimally detect GRBV in petiole and cane tissues (DeShields and KC 2023). Furthermore, there has been limited detection of pre-symptomatic infections (vines infected with GRBV that have not expressed symptoms in previous years) in petiole and cane tissues tested with the LAMP assay (KC et al. 2022, Rohrs et al. 2023).
This study takes a new approach by using trunk cambium tissue for LAMP assay diagnostics. As a perennial tissue, the trunk cambium is a year-round reservoir for the virus, and sampling the cambium could avoid issues associated with detecting GRBV in annual tissues, such as seasonal fluctuations in the concentration of virus particles and non-uniform distribution within the grapevine canopy (Setiono et al. 2018, DeShields and KC 2023, Flasco et al. 2023c).
In Napa Valley, California vineyards with secondary GRBV spread, our first goal was to determine if LAMP assay diagnostics of trunk cambium tissue could be used to detect pre-symptomatic GRBV infections as well as GRBV in vines with known infection status across six phenological stages (budbreak, bloom, berry enlargement, veraison, harvest, dormancy). The second goal of this study was to determine the earliest phenological time point at which GRBV can be detected in basal petioles with the LAMP assay, and compare the temporal trends of GRBV detection in trunk cambium tissue to petiole tissues.
We also surveyed Napa Valley growers currently using the LAMP assay, with a goal to quantify metrics reflecting practitioner experience. As a part of ongoing efforts to manage a virus with an extended incubation period, uncertain timing of symptom onset, and secondary spread, Napa Valley growers have been employing trunk sampling methods and LAMP-GRBV assay diagnostics. Insights into practitioner experience may encourage broader use of the LAMP assay, equipping growers with a diagnostic tool and optimum sampling strategies that can improve the accuracy of GRBV incidence estimation and refine strategies for controlling spread.
Materials and Methods
Study sites
Four commercial vineyard blocks in Napa Valley were selected from four distinct American Viticultural Areas: St. Helena, Rutherford, Yountville, and Wooden Valley. The Yountville and Wooden Valley blocks were V. vinifera cv. Cabernet Sauvignon, the St. Helena block was V. vinifera cv. Malbec, and the Rutherford block was V. vinifera cv. Sauvignon blanc, representing the only white variety in this study (Table 1). Between 2010 and 2014, vineyard blocks were planted with various rootstocks. All blocks were cordon-trained and spur-pruned, except for the St. Helena block, which was cane-pruned only.
Descriptions of study blocks. AVA, American Viticultural Area.
These sites were selected based on their inclusion in a GRBV detection and disease ecology study that was conducted from 2021 to 2022. In that project, GRBD symptoms in the study blocks with black-fruited varieties were mapped annually, the presence of GRBV was confirmed with the LAMP assay, and spatial patterns reflected secondary spread (Rohrs et al. 2023). Because the Rutherford block did not have GRBD symptoms, we conducted LAMP testing of a subsample of vines to confirm the presence and spread of GRBV (unpublished). Symptomatic vines were removed annually at the Yountville site; insecticides targeting S. festinus were not applied in any of the blocks during the study period, and all other management decisions followed established practices for the Napa Valley region.
Sample collection
Detection of pre-symptomatic infections
To determine if LAMP assay diagnostics could detect GRBV in the trunk cambium tissue of pre-symptomatic vines, a total of 75 asymptomatic vines were selected from two sites, St. Helena and Yountville, where GRBD incidence in 2022 was 78% and 12%, respectively (Rohrs et al. 2023). Following GRBD symptom mapping in 2022 (Rohrs et al. 2023), asymptomatic vines were arbitrarily selected within and around areas of high disease aggregation. One trunk cambium sample was collected per vine on 10 Oct and 2 Nov 2022 (at St. Helena and Yountville, respectively). To compare GRBV detection in trunk versus petiole and cane tissues, one petiole sample (consisting of three basal petioles) and one cane sample (consisting of three, 10- to 15-cm sections from the basal portions of canes) were collected for each of the 75 target vines, from each half of the canopies. Petioles were collected on 5 or 7 Oct 2022 (St. Helena) and on 13 Oct 2022 (Yountville), and cane samples were collected after winegrapes were harvested for the season, on 4 Nov 2022 (St. Helena) and on 10 or 11 Nov 2022 (Yountville). All vines were visually assessed for GRBD symptoms in October 2023, according to methods in Rohrs et al. (2023).
Trunk sampling methodology
Four different study blocks (Rutherford, St. Helena, Wooden Valley, and Yountville) were sampled in 2023 to validate trunk sampling methodology and to compare the temporal trends of trunk and petiole tissue in GRBV detection. In previous work, a georeferenced grid of vines was created for each of the four blocks, and ArcGIS Pro (ver. 3.03, ESRI, Inc.) was used to randomly select vines for sampling and LAMP testing (Rohrs et al. 2023). From this set of vines, four GRBV-positive (GRBV+) and four GRBV-negative (GRBV−) vines were randomly selected at the Rutherford site and eight GRBV+ and four GRBV− vines were randomly selected at the Wooden Valley site. At the St. Helena site, 12 GRBV+ and eight GRBV-vines were selected, and at the Yountville site, 10 vines in each category were selected. Trunk cambium tissue was sampled from the 60 vines across the four sites at six phenological stages: budbreak (Eichhorn-Lorenz [E-L] stage 4 to 7), bloom (E-L 20 to 23), berry enlargement (E-L 29 to 31), veraison (E-L 35 to 36), harvest (E-L 38 to 39), and dormancy (Coombe 1995). At the Yountville site, five study vines were removed by the grower before the trunk cambium could be sampled at dormancy.
One petiole sample per vine was collected at each of three phenological stages (berry enlargement, veraison, and harvest) to compare GRBV detection in trunk cambium to petiole tissue. Each petiole sample consisted of three basal petioles collected across the length of the vine (cordons or canes, depending on pruning style).
Temporal trends of GRBV in petiole tissue
Samples were taken at two study blocks (Rutherford and Wooden Valley) over two growing seasons (2022 and 2023) to evaluate temporal trends of petiole tissue in GRBV detection. Target vines at the two study blocks were selected as previously described. Two petiole samples per vine were collected, each consisting of three basal petioles. As vines were trained to a quadrilateral trellis system, each sample represented either a northeast (NE) or southwest (SW) cordon position. Beginning in May (bloom; E-L 20 to 23) of each year (Coombe 1995), petioles were collected from target vines every two to three weeks, and collection was repeated until all samples tested consistently according to their known infection status. Due to climatic conditions, the calendar dates differed between the two study years and sample collection followed vine phenology.
Tissue handling and preparation for the LAMP assay
Trunk tissue
A variation of the “pin-prick” method of tissue acquisition for the LAMP assay, previously developed for petioles and canes (Romero Romero et al. 2019), was used to sample trunk cambium in the vineyard. The outer bark was first peeled away just below the head of the selected vine, and a sterilized razor was used to scrape away the remaining periderm to expose a small section (1 to 2 cm2) of cambium tissue (Figure 1A and 1B). To avoid permanent damage to the vine, care was taken to expose the periderm without causing extensive damage to the underlying vascular tissues. With a 10-μL sterile pipette tip, the exposed tissue was lightly scraped and a small segment (1 mm) of tissue was collected at the end of the pipette tip. For DNA elution, the pipette tip was placed in a prepared 5-mL centrifuge tube containing 10 μL of distilled water for at least 10 min. The centrifuge tube with the pipette tip was secured in a 25-place rack, transported in a cooler, stored at −20°C, and analyzed within one week of collection. To limit sample cross-contamination, nitrile gloves were used and changed between vine preparation and tissue collection, as well as between each sample vine, and a new sterilized razor blade was used to expose the cambium of each vine.
Sampling trunk cambium tissue for loop-mediated isothermal amplification (LAMP) assay diagnostics. Bark has been peeled away near the head of the vine and a sterilized razor is used to scrape away the periderm and expose the cambium tissue underneath (A). With a 10-μL sterile pipette tip, the exposed tissue is lightly scraped and a small segment of tissue (1 mm) is collected at the end of the pipette tip (B).
Petiole and cane tissue
Following field collection, cane and petiole samples were transported in a cooler and stored in a refrigerator (4°C) at the University of California Cooperative Extension, Napa. DNA templates for the LAMP assay were prepared via the “pin-prick” method within 24 hr of field collection (Romero Romero et al. 2019). For petiole samples, a sterile razor blade was used to expose the cross section of each petiole, which was lightly pricked three times with a sterile pipette tip (10 μL). To “prick” the cambium layer of cane tissue, the periderm of canes was removed with a sterile razor to expose the underlying cambium (DeShields and KC 2023). A sterile pipette tip (10 μL) was used to lightly scrape the exposed tissue. DNA was eluted as previously described, and DNA templates were frozen (−20°C) until analyzed with the LAMP assay, within one week of sample collection.
LAMP assay
Methods for the LAMP assay followed the standard protocols in Romero Romero et al. (2019). Primers were custom synthesized by IDT DNA Technologies and the Warm Start Colorimetric LAMP 2X was sourced from New England BioLabs. Positive controls consisted of diluted GRBV DNA obtained from AL&L Crop Solutions and negative controls consisted of leaf petiole material that had previously tested negative for GRBV. LAMP reactions were completed at 65°C for 35 min, and GRBV+ results were indicated by a pink-to-yellow color change in the colorimetric LAMP-reaction mix.
Surveying users of the LAMP assay for GRBV detection in Napa County
A survey was developed to understand how and why members of the winegrape industry are adopting the LAMP-GRBV assay as a decision-support tool. Demographic information about job roles and number of people involved in LAMP assay implementation was collected, as were user opinions on the importance of the assay, from a disease management perspective. The survey also addressed logistical topics such as time to train users to proficiency, number of samples processed per year, tissue types tested (trunk cambium, petiole, cane), and approximate cost to process samples (excluding equipment required to carry out the assay). The survey was distributed to a total of 20 organizations known to be using the LAMP assay between 2021 to 2024 and was limited to one response per organization, to be completed by someone responsible for conducting or overseeing the assay. A total of 10 organizations responded, using the Survey123 tool (Esri).
Results
Detecting pre-symptomatic GRBV infections using trunk, petiole, and cane tissue
Comparing trunk, cane, and petiole samples from vines that had not expressed symptoms in previous years, pre-symptomatic infections were detected for 33% (25/75) of vines tested using trunk cambium, 16% (12/75) using cane tissue, and 2.7% (2/75) using petiole tissue (Table 2A). In total, 52% (39/75) of the vines had visual symptoms in 2023 (Table 2B). In the 2022 LAMP assay results for symptomatic vines, GRBV was not detected in 36% (14/39) of vines using trunk tissue, 72% (28/39) using cane tissue, and 95% (37/39) using petiole tissue. Additionally, there was one vine that did not have symptoms during visual assessments in 2023, despite a positive GRBV result from a cane sample.
Trunk, petiole, and cane sampling study for pre-symptomatic detection of grapevine red blotch virus (GRBV)-infected vines at the St. Helena and Yountville study sites in 2022. A) GRBV-positive (GRBV+; n = 25) and GRBV-negative (GRBV−; n = 50) results for the petiole and cane tissue samples, reported according to whether GRBV was detected in trunk cambium tissue. All detection was conducted following established protocols for the loop-mediated isothermal amplification (LAMP)-GRBV assay. B) Presence (n = 39) or absence (n = 36) of grapevine red blotch disease (GRBD) visual symptoms, for all vines assayed for pre-symptomatic infections in 2023.
Temporal trends in GRBV detection for trunk cambium compared to petiole tissue
At all study sites, trunk samples collected from GRBV+ vines consistently tested positive during all six phenological stages (Table 3). Exceptions include one vine at the Wooden Valley site that tested negative at bloom (Table 3B) and three vines at the Yountville site, two of which tested negative at budbreak and one of which tested negative at bloom and berry enlargement, but not at budbreak (Table 3D). Therefore, out of the 207 cambium samples tested from known GRBV+ vines, false negative results (2.4%; 5/207) only occurred between budbreak and berry enlargement. There was also one apparent false positive for a vine at the Wooden Valley site at bloom (Table 3B).
Loop-mediated isothermal amplification (LAMP) assay results for grapevine red blotch virus (GRBV) detection, using trunk cambium and petiole tissue, at four study sites in 2023. GRBV positive (+) and negative (−) results for previously GRBV+ (red print) and GRBV− (black print) vines are reported according to phenological stage: budbreak (BB), bloom (BL), berry enlargement (BE), veraison (VE), harvest (HA), and dormancy (DO). Petioles were collected only during the BE, VE, and HA periods.a
In conjunction with cambium samples, petioles were also collected at berry enlargement, veraison, and harvest. Petiole samples generally tested positive for GRBV+ vines, but we observed greater site-specific variability and achieved more consistent results from veraison through early senescence. Specifically, there were eight vines with false negative petiole results at berry enlargement; only one of those vines still tested negative at veraison. Most false negative results were from vines at the Yountville site (six vines; Table 3D), with one vine each at the Rutherford and St. Helena sites (Table 3A and 3C). No false negative results for petiole samples at the Wooden Valley site (Table 3B) were observed.
Although not the focus of this part of the study, pre-symptomatic infections in five vines were also detected, two at the Yountville site and three at the St. Helena site. Trunk cambium was more sensitive than petiole tissue: both pre-symptomatic infections in the Yountville site were detected at berry enlargement with trunk cambium tissue, and one of these vines was detected via petiole tissue at veraison. At the St. Helena site, two pre-symptomatic vines tested positive from trunk cambium at bloom and the third was GRBV+ at veraison. Petiole tissue did not test positive for all three of these vines until veraison.
Temporal trends in GRBV detection for petiole samples
At the Rutherford site, consistent GRBV testing results with petiole tissue from GRBV+ vines were achieved by late June (2022) or mid-July (2023), corresponding to the berry enlargement period (Table 4A). Similarly, at the Wooden Valley site, consistent results were achieved by mid-June (2022) and early July (2023) (Table 4B). The highest incidence of false negative results in the known GRBV+ vines, and of inconsistent results between samples originating from the two cordon positions (NE, SW), were observed during the prebloom (May) to berry enlargement period (June) (Table 5). Specifically, for this period in 2022 and 2023, there was a total of 24 (Rutherford) or 48 (Wooden Valley) observations (vine × date). For both cordon positions, 25% (6/24) or 52% (25/48) had positive results, 50% (12/24) or 19% (9/48) had false negative results, and 25% (6/24) or 29% (14/48) had mixed results (one positive and one negative). Overall, there were fewer consistent positive results (43%) prior to berry enlargement and a higher incidence of false negative (29%) and mixed results (28%).
Temporal trends in grapevine red blotch virus (GRBV) detection from petiole tissue at two sites in 2022 and 2023.a
Comparison of loop-mediated isothermal amplification (LAMP) assay results in 2022 and 2023 for known grapevine red blotch virus-infected (red print) vines during the prebloom through berry enlargement period at the Rutherford (RD) and Wooden Valley (WV) study sites.
After veraison, the likelihood of a false negative result with the LAMP assay for a known GRBV+ vine decreased substantially. In total, we had three false negative results surrounding or following veraison. Specifically, one petiole sample tested negative at the Rutherford site on 21 Aug 2023, and there were two negative test results at the Wooden Valley site on 8 July 2022, which was just prior to veraison (18 July) (Table 4B). In total, 272 petiole samples from known GRBV+ vines were tested, and the incidence of false negative results after veraison was 1.1% (3/272).
Trends in adopting the LAMP assay as a decision-support tool in Napa Valley
Respondents to the LAMP user survey represented a range of viticultural positions, including viticulturist (n = 3), director (n = 2), vineyard manager (n = 1), winemaker (n = 1), laboratory manager (n = 1), intern (n = 1), and production assistant (n = 1). Three participants managed 20 to 40 ha, five managed 40 to 200 ha, and two managed more than 400 ha. Half of respondents felt sufficiently proficient to conduct LAMP by themselves after processing fewer than 50 samples, whereas four felt they needed considerably more practice (Table 6). Users’ estimated cost to process each sample, including supplies and labor, was $5.00 to $10.00, although two participants estimated less than $5.00. Although the cost implications were not explored, two companies reported hiring employees (technician; intern) for the sole purpose of conducting LAMP testing. Respondents preferred assaying trunk tissue over cane or petiole tissue and reported sampling trunk tissue more frequently (100%) than canes (30%) or petioles (20%). Six respondents used trunk tissue exclusively, explaining that it was the most readily available tissue for the period they sampled (winter, during or after pruning), was easy to collect the sample, and they perceived it to be more reliable for their specific purposes.
Survey responses collected from organizations using the loop-mediated isothermal amplification (LAMP) assay for grapevine red blotch virus detection in Napa Valley (n = 10). Responses pertained to sampling logistics such as tissue type and preference, the estimated costs to process samples, the number of staff involved in sampling and processing, and the time to proficiency for new users. Users also reported the number of people external to their organization with whom they discussed the LAMP assay.
LAMP was adopted by two respondents in 2021, by four in 2022, and by nine in both 2023 and 2024 (Supplemental Figure 1). During this period, the overall number of assayed vines increased substantially, and by 2024, 600 to more than 1000 vines were annually assayed by 70% of users. This is consistent with respondent perception that the LAMP assay is a highly useful decision-support tool—with an average rating of 1.9 out of 2—that can be implemented for a variety of reasons (Supplemental Figure 2). Respondents are most commonly using the LAMP assay to identify pre-symptomatic infected vines and to delineate areas of a block to remove. The assay is also being used to confirm infections in asymptomatic cultivars and verify visual assessments, and less commonly, to confirm infections when mapping is not possible.
We estimate that knowledge of LAMP was spread by the 10 survey participants to more than 66 contacts in the viticulture industry (Table 6). Participating companies involved two to four staff members in sample collection, processing, and LAMP analysis (28 total), with an additional three staff members witnessing all required steps for the LAMP diagnostics (31 total). All participants had also communicated about LAMP to at least 38 industry contacts outside their company. Three participants knew other growers who were also using LAMP.
Discussion
Members of the winegrape industry typically concentrate on GRBD visual diagnostics in the period surrounding harvest, anchored by the timing of symptom expression in red wine cultivars. However, this strategy overlooks the fact that the virus is detectable in plant tissue earlier in the growing season (DeShields and KC 2023, Flasco et al. 2023b) and that infections in certain cultivars, such as Sauvignon blanc, must be diagnosed via molecular assay in the absence of visual symptoms. Furthermore, anchoring diagnostics to symptom expression may not be an appropriate strategy for detecting a virus with an extended incubation period (Flasco et al. 2023b) and uncertain timing of initial symptom onset. To expand the diagnostic period beyond symptom expression, growers must have confidence in the reliability of the LAMP assay at different times of the year and should be informed about which tissues can be sampled, and when.
This study took a new approach to pre-symptomatic and year-round detection of GRBV by focusing on LAMP assay diagnostics of trunk cambium tissue. This is the first study to demonstrate that trunk cambium is the most sensitive tissue for detection of pre-symptomatic infections in blocks experiencing secondary disease spread. This study also reports the highest detection of pre-symptomatic GRBV infections, compared to previous studies. For instance, prior studies reported 0.4% incidence of pre-symptomatic detections for petiole tissue using LAMP (Rohrs et al. 2023), 0.5% incidence using qPCR (KC et al. 2022), and 1.7% incidence with multiplex PCR (Cieniewicz et al. 2017a). Consistent with these studies, we also demonstrated that petiole tissue was a very poor indicator (2.7% incidence) of pre-symptomatic infection. Cane tissue detected half of the pre-symptomatic infections that were identified with trunk cambium, although cane tissue identified three GRBV-positive vines that were not detected from trunk cambium. We did not detect GRBV in all of the vines that ultimately developed symptoms during the 2023 growing season, however, the proportion of undetected infections was much less for trunk cambium (36%) than for cane (72%) or petiole (95%) samples. This suggests that there is still a need for visual symptom assessment as part of a comprehensive strategy to identify and catalog diseased vines. LAMP diagnostics of the trunk cambium will augment visual assessments by reinforcing symptom observations and identifying pre-symptomatic infections, contributing to more accurate estimates of disease incidence and spread.
The trunk sampling process was validated and we determined that GRBV can be consistently detected in trunk cambium with the LAMP assay year-round, from budbreak through dormancy. From veraison through dormancy, there were no false negative results produced. From budbreak through berry enlargement, the incidence of false negatives was 2.4%, or 5/207, and during this period, multiple samples from the same trunk could be taken to reduce the likelihood of obtaining a false negative result. Overall, the trunk sampling results demonstrate that growers can use the trunk sampling technique to expand the period of LAMP diagnostics throughout the year. This has practical advantages such as shifting viral disease-focused labor demands away from the harvest period (Hobbs et al. 2023), and also helps growers that are managing secondary spread to gain confidence in their management decisions. It may also align LAMP diagnostics with epidemiologically relevant periods, such as the emergence of the first generation of S. festinus in the vineyard.
An advantage of using trunk cambium for the LAMP assay is that the method of tissue acquisition, pricking the targeted tissue with a pipette tip, can be implemented in the vineyard. However, as this poses a higher risk for sample contamination, strict adherence to sanitary practices is required. Although we minimized this risk by changing gloves between preparing the trunk and collecting the samples, there was one false positive test result (0.8% incidence; 1/120) at bloom, likely resulting from cross-contamination. Treating and sealing trunk wounds should be considered when sampling cambium tissue during periods of high pressure from trunk pathogens.
GRBV can be detected in trunk cambium year-round, however, LAMP assay diagnostics of petiole tissue was reliable only from veraison through early senescence. From bloom through berry expansion, our results with petiole tissue were variable, with consistent detection of GRBV in less than half of the samples from known positive vines. This is likely explained by the uneven distribution of virus particles within the host that is characteristic of GRBV (Setiono et al. 2018, Flasco et al. 2023b) and other phloem-limited viruses of grapevine such as grapevine fanleaf virus (Krebelj et al. 2015), grapevine leafroll-associated virus 3 (Tsai et al. 2012), and Rupestris stem pitting-associated virus (Hu et al. 2018). Perennial tissues are the only persistent organs in cultivated grapevines where phloem-limited viruses reside year-round. During early spring and subsequent phenological stages, these viruses are translocated to newly expanding shoots, leaves, and inflorescences, and distribution varies as the pathogen gradually colonizes these tissues (Setiono et al. 2018). Thus, the optimal time to sample petiole tissue is from veraison through early senescence, when we saw a very low incidence (1.1%) of false negative results. Testing during earlier phenological stages (prior to berry enlargement) can yield a high proportion of false negative results. For greater reliability, assays conducted earlier in the season with petiole tissue could use a different diagnostic method such as Endpoint PCR or qPCR (DeShields and KC et al. 2023). Because local climate, seasonal weather, and site-specific factors affect growth and development of the grapevine, coordinating diagnostic testing with phenological development rather than with calendar date will provide more consistent results and make the guidelines applicable across the wide range of climatic regions.
LAMP survey on user challenges and experience
The 10 LAMP adoption survey respondents represent half of the total organizations of which we are aware that use the LAMP assay in Napa Valley, with an uptick in adoption between 2022 and 2023. Respondents perceive the LAMP assay as a highly useful decision-support tool for GRBD management. Trunk tissue is overwhelmingly preferred for diagnostics, consistent with the reasons given for assay adoption: to identify pre-symptomatic infected vines and to delineate areas of a block for removal. With an approximate sample processing cost of $5.00 to $10.00, and a reasonably short training time of 50 samples to reach proficiency, there is likely potential for greater adoption of the LAMP assay. Doing so may require additional staff, depending on the expertise and responsibilities of the existing team and the extent of intended sampling. To influence more widespread adoption, knowledge of the assay and perceptions of its usefulness can be shared by extension professionals and reinforced through established agricultural social networks (Lowder et al. 2024).
Management implications
Detecting and removing GRBV-infected vines is a consistent and widespread strategy to manage GRBV secondary spread (Cieniewicz et al. 2017b, KC et al. 2022). However, given an extended lag time between vector or graft-mediated inoculation and symptom development (Cieniewicz et al. 2017a, Flasco et al. 2023a), and the ability of pre-symptomatic vines to serve as a source of inoculum for secondary spread (Flasco et al. 2023b), the use of trunk cambium to improve detection of pre-symptomatic infection could transform the industry’s approach to GRBV interventions. Further studies are needed to focus on pre-symptomatic GRBV detection across multiple sites, as the duration between inoculation and symptom development may depend on factors such as viral isolate and host (species, cultivar, age). Exploring these factors could improve our understanding of the parameters around detection and refine removal efforts, as has been done previously for plum pox virus (Gottwald et al. 2013, Rimbaud et al. 2015a, 2015b) which, similarly, may have a long incubation period and variable symptom expression.
While the impact of pre-symptomatic infections in GRBV epidemiology has not been characterized, the proportion of pre-symptomatic infections present in a vineyard may have complex implications for disease management. When this question arose previously, poor pre-symptomatic detection using petiole tissue led the authors to conclude that symptom-based management practices are reliable for identifying asymptomatic vines, and that there is no evidence for the need to remove nearby asymptomatic vines within the same row as diseased vines (KC et al. 2022). However, our study suggests greater incidence of pre-symptomatic infections than previously described, which may have important epidemiological significance. Pre-symptomatic infections may necessitate the adjustment of vine removal practices to account for the incidence and spatial distribution of these vines. For example, spatial roguing strategies for grapevine leafroll-associated viruses were developed from predictive modeling of disease spread and bioeconomic factors. Spatial roguing eliminates symptomatic vines and the two immediate, within-row, neighboring vines on each side of the infected vine, regardless of infection status (Atallah et al. 2015). This strategy was validated in a Cabernet franc vineyard in New York, where spatial roguing was the dominant factor in reducing the incidence of leafroll disease to less than 1% over the course of six years (Hesler et al. 2022). Adapting a spatial or zonal roguing approach to GRBD management could integrate the newly developed practice of trunk cambium sampling to justify removing either multiple neighbors of a symptomatic vine or larger zones of vines in areas of aggregated disease incidence, resulting in improved disease management outcomes for the viticulture community.
Conclusion
This study provides the winegrape industry with guidelines for identifying the optimal tissue to sample and the appropriate timing for LAMP assay diagnostics of GRBV throughout the growing season. Petiole tissue is easily accessible, can be sampled multiple times throughout the season, and is the quickest to process with the “pin-prick” tissue acquisition step of the LAMP assay. Petiole tissue is useful to diagnose GRBD infections across the 3- to 4-mo period of symptom expression, from veraison to the beginning of senescence, and can also be used to detect GRBV infections in Sauvignon blanc, a cultivar that does not express GRBD visual symptoms.
Cane sampling has been an industry standard for postharvest GRBV diagnostics, but because of the destructive nature of sample collection, its use is limited to postharvest through pruning. Furthermore, the proportion of pre-symptomatic infections is underestimated when using cane tissue for LAMP diagnostics (and likewise, diagnostic testing of petiole tissue does not reliably detect such infections). Trunk cambium is therefore the optimal tissue to detect pre-symptomatic GRBV infections, as it can be used at least one year prior to symptom development and can detect GRBV throughout the growing season.
Trunk sampling is more time intensive and requires sanitary measures during field sampling, however, efficiency can be improved by targeting sampling to areas where there is a higher likelihood of pre-symptomatic infections. Trunk sampling may also be used in a spatial roguing scenario: the presence of pre-symptomatic vines can be used to justify the removal of a section of the vineyard and to delineate the removal boundaries. In fact, LAMP users indicated that the assay is important for these purposes, and that they preferentially sample trunk cambium over cane or petiole tissue. Using trunk cambium tissue extends GRBV diagnostics over a longer period during the growing season; allows for the detection of pre-symptomatic GRBV infections; and supports timely, informed management decisions to reduce the risk of secondary spread and limit the economic damage resulting from GRBD.
Supplemental Data
The following supplemental materials are available for this article in the Supplemental tab above:
Supplemental Figure 1 Index of total vines tested and the trend in adoption of the grapevine red blotch virus loop-mediated isothermal amplification (LAMP) assay in Napa Valley from 2021 to 2024, among 10 reporting organizations. The sum of the ratings for total vines tested is an index representing how LAMP assay adoption increased, not an exact count of tested vines. Participants used a rating scale to report how many vines they tested each year, on a scale of 1 to 6 (1 = <50; 2 = 50 to 100; 3 = 100 to 300; 4 = 300 to 600; 5 = 600 to 1000; 6 = 1000+). Total ratings across respondents were summed, with a maximum possible score of 60 if all 10 reporting organizations had all tested 1000+ vines each.
Supplemental Figure 2 Average (mean) overall usefulness rating and importance of reasons for adopting the loop-mediated isothermal amplification (LAMP) assay as a decision-support tool for grapevine red blotch disease management, as perceived by 10 survey respondents from the winegrape industry in Napa Valley. Participants used a Likert scale to rank their responses, where 0 = Not Important, 1 = Somewhat Important, and 2 = Very Important. GRBV, grapevine red blotch virus.
Footnotes
This work was funded by a grant from the California Department of Food and Agriculture, Pierce’s disease and glassy-winged sharpshooter program, Agreement Number 21-0273-000-SA. We gratefully acknowledge the technical support received from Dr. Keith Perry, Cornell University, who helped us learn and troubleshoot the LAMP assay. We are continually grateful to the grapegrowing community in Napa Valley, whose insights and collaboration make our work possible and relevant. We would particularly like to recognize the contributions of Ashton Leutner: early adopter, innovator, and enthusiastic supporter of the LAMP assay, as well as the inspiration for trunk cambium sampling.
Rohrs JK, Fendell-Hummel HG, MacDonald SL, Hobbs MB and Cooper ML. 2024. Trunk cambium facilitates pre-symptomatic and year-round detection of grapevine red blotch virus using the LAMP assay. Am J Enol Vitic 75:0750023. DOI: 10.5344/ajev.2024.24034
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All data underlying this study are included in the article and its supplemental information.
- Received June 2024.
- Accepted September 2024.
- Published online December 2024
This is an open access article distributed under the CC BY 4.0 license.
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