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Article

Metagenomic Profiling of the Grapevine Virome in Canadian Vineyards

by
Bhadra Murthy Vemulapati
1,
Kankana Ghoshal
2,
Sylvain Lerat
3,
Wendy Mcfadden-Smith
4,
Mamadou L. Fall
5,
José Ramón Úrbez-Torres
6,
Peter Moffet
3,
Ian Boyes
2,
James Phelan
2,
Lucas Bennouna
2,
Debra L. Moreau
7,
Mike Rott
2 and
Sudarsana Poojari
1,*
1
Cool Climate Oenology and Viticulture Institute, Brock University, St. Catharines, ON L2S 3A1, Canada
2
Canadian Food Inspection Agency, Centre for Plant Health, Sidney Laboratory, 8801 East Saanich Rd, North Saanich, BC V8L 1H3, Canada
3
Département de Biologie, Université de Sherbrooke, 2500 Bd de l’Université, Sherbrooke, QC J1K 2R1, Canada
4
Ontario Ministry of Agriculture, Food and Rural Affairs, Vineland Station, ON L0R 2E0, Canada
5
Agriculture and Agri-Food Canada, 430 Gouin Boulevard, Saint-Jean-sur-Richelieu, QC J3B 3EB, Canada
6
Agriculture and Agri-Food Canada, Summerland Research and Development Centre, Summerland, BC V0H 1Z0, Canada
7
Kentville Research and Development Centre, Agriculture and Agri-Food Canada, Kentville, NS B4N 1J5, Canada
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(14), 1532; https://doi.org/10.3390/agriculture15141532
Submission received: 22 May 2025 / Revised: 5 July 2025 / Accepted: 7 July 2025 / Published: 16 July 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
Browse Figure
Versions Notes

Abstract

A high-throughput sequencing-based grapevine metagenomic survey was conducted across all grape-growing Canadian provinces (British Columbia, Ontario, Nova Scotia, and Québec) with the objective of better understanding the grapevine virome composition. In total, 310 composite grapevine samples representing nine Vitis vinifera red; five V. vinifera white; seven American–French red; and five white hybrid cultivars were analyzed. dsRNA, enriched using two different methods, was used as the starting material and source of viral nucleic acids in HTS. The virome status on the distribution and incidence in different regions and grapevine cultivars is addressed. Results from this study revealed the presence of 20 viruses and 3 viroids in the samples tested. Twelve viruses, which are in the regulated viruses list under grapevine certification, were identified in this survey. The major viruses detected in this survey and their incidence rates are GRSPaV (26% to 100%), GLRaV-2 (1% to 18%), GLRaV-3 (15% to 63%), GRVFV (0% to 52%), GRGV (0% to 52%), GPGV (3.3% to 77%), GFkV (1.5% to 31.6%), and GRBV (0% to 19.4%). This survey is the first comprehensive virome study using viral dsRNA and a metagenomics approach on grapevine samples from the British Columbia, Ontario, Nova Scotia, and Quebec provinces in Canada. Results from this survey highlight the grapevine virome distribution across four major grapevine-growing regions and their cultivars. The outcome of this survey underlines the need for strengthening current management options to mitigate the impact of virus spread, and the implementation of a domestic grapevine clean plant program to improve the sanitary status of the grapevine ecosystem.

1. Introduction

Grapevine (Vitis vinifera and Vitis spp.) is an important commodity in Canada, generating an annual economic impact of CAD 11.5 billion [1]. Grapevine cultivation in Canada occurs across four provinces, including Ontario (ON) (7168 ha, 58% of total); British Columbia (BC) (4848 ha, 36% of total); Québec (QC) (848 ha, 6% of total); and Nova Scotia (NS) (391 ha, 3% of total) [2]. In BC, ~102 wine grape cultivars are being cultivated, with the predominant V. vinifera being Cabernet franc, Cabernet Sauvignon, Chardonnay, Gewurztraminer, Merlot, Pinot Gris, Pinot Noir, Riesling, Sauvignon Blanc, and Syrah [3]. In ON, more than 50 cultivars are grown, with the main V. vinifera being Chardonnay, Cabernet Franc, Gamay, Merlot, Pinot Noir, and Riesling. The main hybrid cultivars in ON include Baco Noir, Seyval Blanc, and Vidal [4]. In QC, there are more than 80 cultivars planted and the predominant V. vinifera includes Merlot, Cabernet Sauvignon, Pinot Noir, Riesling, Chardonnay, and Pinot Gris, and the hybrids include Maréchal Foch, Frontenac Noir, Petite Perle, Seyval Noir, Seyval Blanc, St-Pepin, Frontenac Blanc, and Vidal [5]. Nova Scotia’s wine industry is largely based on the production of short-season, hardy French Hybrid cultivars including Baco noir, L’Acadie Blanc, Léon Millot, Lucy Kuhlman, Maréchal Foch, New York Muscat, Seyval, and Vidal [6].
Grapevines are known to host the largest number of viruses than any other plant crop, with some responsible for significant economic losses to grape and wine production worldwide. To date, a total of 102 viruses, belonging to 44 genera in 21 different virus families, have been identified in grapevines worldwide [7]. Based on disease symptomatology and severity, most viruses infecting grapevines were placed under four categories: rugose wood complex, leafroll, leaf degeneration or decline, and fleck complex [8,9]. More recently, two other important pathogenic viruses have been discovered on grapevines, including Grapevine red blotch virus (GRBV), a DNA virus causing grapevine red blotch disease [10,11], and Grapevine Pinot gris virus (GPGV), an ssRNA virus that may be causing chlorotic mottling and leaf deformation [12]. Viruses that are currently not placed under these four groups but are still important are Grapevine berry inner necrosis virus (GBNV), GPGV, and Grapevine vein-clearing virus (GVCV) [8]. GRBV, GPGV, and Grapevine fleck virus (GFkV) have also been reported to infect grapevines in Canada [13,14,15,16,17].
Extensive surveys carried out in major grapevine-growing regions in Canada focused on a (i) specific virus(es) [18,19]; (ii) a specific disease complex [20]; (iii) viruses of economic importance [21]; (iv) viral community and diversity in a specific province [22]; and (v) detection methods used for understanding the virus diversity [18,19,23]. Moreover, few reports are available on the status of viroid infections in grape-growing areas of ON [24,25] and QC [23]. Virus detection is key to the implementation of disease management strategies. Once a plant is infected with a virus, viroid, and/or phytoplasma, it remains infected throughout its lifetime and can serve as a virus source for vector transmission or via buds used for grafting. Therefore, it is important to develop and implement thorough surveillance programs to monitor the health status of vineyards. When establishing a new vineyard, the use of virus-free planting material is a first step to securing the sanitary status of the vineyards for years to come. Serological (ELISA) and molecular (PCR, qPCR) methods are available for the detection and identification of viruses in grapevines. High-throughput sequencing (HTS) has had a major impact on virus diagnostics and diversity studies in recent years. HTS is a valuable tool for the identification, genomic characterization and understanding of the disease etiology in agricultural crops [26]. During the past decade, the use of HTS methods facilitated the identification of more than 35 novel grapevine viruses worldwide, including 21 species with (+) ssRNA genomes [27,28,29,30]. Viral RNA/DNA, virus-derived small RNA (smRNA), total RNA (totRNA), and double-standard RNA (dsRNA) have all been effectively employed in HTS [31,32,33]. DsRNA could be advantageous as it represents the replicative form of most plant viruses and is more amenable to enrichment strategies. This reduces the amount of HTS data generation and simplifies bioinformatic analysis [34]. Most recently, advances have been made in grapevine virus research towards the development of a cost-effective and robust dsRNA enrichment method suitable for HTS. A comparison of three different dsRNA enrichment methods resulted in improved detection and diversity in the number of viruses and viroids using a ReliaPrep resin-based method and a B2-protein-based method [35].
In this study, a comprehensive survey was carried out for the detection and prevalence of viruses and viroids in Canadian vineyards using HTS. A total of 310 composite grapevine samples were collected from four provinces in Canada, representing the most important grapevine cultivation regions. The V. vinifera red, white, and American–French hybrid cultivars were collected, and dsRNA was extracted using the (i) ReliaPrep resin method and (ii) B2-protein methods. Here, we report the initial findings from this survey on the viruses and viroids detected and their distribution across Canada.

2. Materials and Methods

2.1. Plant Samples

Dormant cane cuttings were randomly collected between 2021 and 2023 from different vineyards in ON, BC, QC, and NS during dormancy (December to March) to not be biased by symptom expression. A total of 310 samples representing composite grapevine samples from 26 cultivars, including V. vinifera red, white, and American–French hybrids, were sampled in this study. Each composite comprised 5 to 10 cane samples. From BC, composite samples were collected from 9 cultivars; 67 composite samples from 14 cultivars from ON; 31 composite samples from 8 cultivars from NS; and 122 composite samples from 15 cultivars from QC were included in this study (Table 1). Cambium scrapings were collected from the canes and stored at −80 °C and ground in liquid nitrogen before storage at −80 °C until further use.

2.2. Viral Nucleic Acid Extraction

Double-stranded RNA (dsRNA) was used as the source of viral nucleic acid in this study. Isolation and enrichment of dsRNA from the samples were carried out by one of the following two methods: (a) the ReliaPrep resin-based method for the BC, ON, and NS samples and (b) the B2-Tag/Strep Tactin resin method for the QC samples. All the steps involved in the isolation and enrichment of dsRNA were carried out as described [35]. The isolated dsRNA was either processed immediately or stored at −80 °C until further use.

2.3. HTS Methodology

Quality checks of dsRNA extracts of ON and QC samples were performed with a Qubit 3.0 fluorometer (Invitrogen, Waltham, MA, USA) using the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The dsRNA extracts with purity of A260/A280 ratio of ~1.6 to 1.8 or higher were used in subsequent HTS assays. Library preparation was carried out using a TruSeq® Stranded mRNA Library Prep Plant (96 samples) (Illumina, San Diego, CA, USA) and Truseq RNA CD index plate (96 indexes) (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. The prepared libraries were sequenced to generate 150 bp paired-end reads using the TruSeq platform (Illumina, San Diego, CA, USA).

2.4. HTS Data and Virtool Analysis

Virtool, a cloud-based web application [36], was used for HTS raw data management, quality control, and analysis. HTS reads that passed quality control were mapped against a database of plant virus and viroid genomic sequences using a modified version of Pathoscope 2.0 [31]. Virtool analyzes the weight (calculated proportion of reads mapping to a virus); read depth (a measure of number of times a genome is covered by mapped reads); and read coverage (a measure of how well the mapped reads cover the viral genome) parameters from Pathoscope workflow for analysis. Viruses or viroids with sequences (a) >15% coverage or (b) >0.001 weight in Virtool were considered a strong indicator of positive detection [31,36] (Supplementary Figure S1). The NuVs workflow in Virtool was selected for screening novel viruses. The NuVs workflow in Virtool was also used for the discovery of novel viral sequences from the library of samples tested from all the four provinces.

3. Results

HTS data analysis of 310 grapevine composite samples collected from four provinces in Canada resulted in the identification of 20 viruses and 3 viroids. The majority of the composite samples were infected with at least one virus. The viruses identified by HTS in this study were consolidated based on (i) the disease complex group and (ii) the regional and varietal distribution of viruses in the cultivars.
Based on the disease complex, the viruses and viroids identified were sorted into five groups: (A) rugose wood complex, (B) leafroll complex, (C) leaf degeneration or decline complex, (D) fleck complex, and (E) other viruses. The viroids and viroid-like particles were included in group-F.

3.1. Viruses Associated with Disease Complex

  • Group-A (rugose wood complex-associated viruses)
Five viruses were associated with the “rugose wood complex”. All five major viruses in group-A were identified in samples from BC, four from NS, two from ON, and one from QC. The incidence of Grapevine rupestris stem pitting-associated virus (GRSPaV) was the highest in all the four provinces. In BC and NS, GRSPaV was detected in 100% of the samples while in QC and ON, GRSPaV was identified in 51.6% and 41.8% of the samples. Grapevine virus A (GVA) was detected in 26.6% and 3.2% of the samples from BC and NS and was not detected in ON and QC samples. The occurrence of Grapevine virus B (GVB) was confirmed in samples from BC, ON, and NS at 6%, 1.5%, and 13% while samples from QC were negative for GVB. Grapevine virus D (GVD) was only detected in BC samples at 1.3% while Grapevine virus E (GVE) was detected in samples from NS and BC at 22.6% and 1.3% incidence. GVE was not detected in ON and QC samples (Figure 1A).
  • Group-B (leafroll complex-associated viruses)
Of the six leafroll complex-associated viruses, Grapevine leafroll-associated virus -1, -2, and -3 (GLRaV-1, -2, -3) were detected in the samples tested. The percentage of infection due to GLRaV-3 was the highest in all the provinces for this group of viruses. The incidence of GLRaV-3 was 63%, 42% 18%, and 15% in BC, NS, ON, and QC while GLRaV-2 was detected in 13%, 12.7%, 6%, and 1% of samples in NS, BC, ON, and QC. GLRaV-1 incidence was 6%, 4%, 3.2%, and 0% in ON, BC, NS, and QC, respectively (Figure 1B).
  • Group-C (leaf degeneration or decline complex-associated viruses)
This group comprises about 12 viruses of which a limited detection of only one virus was confirmed in this survey. No leaf degeneration-associated viruses were detected in ON and QC. Arabis mosaic virus (ArMV) was detected in 1.3% of the samples from BC and 3.2% of the samples in NS (Figure 1C).
  • Group-D (fleck complex-associated viruses)
Four viruses, including Grapevine redglobe virus (GRGV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine asteroid mosaic-associated virus (GAMaV), and Grapevine fleck virus (GFkV), associated with the fleck disease complex, were detected in samples. Incidence of GRGV was highest at 52% and 51.6% in BC and NS compared to QC at 1.6% and not detected in samples from ON. GRVFV was confirmed in samples from BC and NS at 52% and 32.3% infection. GAMaV was detected in samples from BC and NS at 7.6% and 16% rate of infection. GFkV infection was detected in all the four provinces at 31.6%, 19.4%, 11.5%, and 1.5% in BC, NS, QC, and ON (Figure 1D).
  • Group-E (other viruses)
GPGV and GRBV are the two viruses that were detected in this group. A significant rate of GPGV infection was observed in all four provinces. GPGV was detected in 77.2% of samples in BC, 71% in NS, 24% in ON, and 3.3% in QC. The presence of GRBV was confirmed in 69% of samples in QC, 19.4% in ON, and 10% in NS. GRBV was not detected in BC samples (Figure 1E).
  • Group-F (viroid and viroid-like particles)
In this survey, three viroid and(or) viroid-like infections were identified, which includes Hop stunt viroid (HSVd), Grapevine yellow speckle viroid (GYSVd), and Grapevine hammerhead viroid-like RNA (GHVd). In this study, the exact identification of GYSVd-1 and -2 by Virtool was unreliable because of the inclusion of a single GYSVd reference isolate genome for analysis. Hence, GYSVd-1 or -2 positive samples were considered as GYSVd positive. All three were found in samples in BC. HSVd was detected in samples from all the four provinces tested. HSVd was detected in 100% of samples in BC followed by 87% in NS, 47.8% in ON, and 30% in QC. The prevalence of GYSVd was 85% in BC, 41.9% in NS, 2.9% in ON, and 0% in QC. The prevalence of GHVd was observed in 13.9% in BC, 4.48% in ON, 3.23% in NS, and not detected in QC samples (Figure 1F).

3.2. Status of Virome Distribution in Different Regions and Grapevine Cultivars

In BC, 12 viruses were identified among the V. vinifera cultivars tested with an incidence ranging from 5.6% to 100%. The most infected cultivar was Chardonnay with eleven viruses and three viroids followed by Merlot with seven viruses and four viroids. The least infected cultivar(s) was Gamay Noir and Cabernet Sauvignon with three viruses and two viroids. A 100% infection rate of GRSPaV was observed in each of the nine cultivars tested. GLRaV-3 was detected in four V. vinifera cultivars (Cabernet franc, Cabernet Sauvignon, Sauvignon blanc, Pinot gris) with 100% incidence. GPGV was detected in four V. vinifera cultivars (Cabernet franc, Cabernet Sauvignon, Pinot Noir, and Pinot gris) with an incidence ranging from 25 to 100%. The percentage of infection of ArMV and GVE was the lowest at 5.6% each in Chardonnay. Three viroids were found to be infecting grapevines in BC with an incidence ranging from 12.5% to 100%. HSVd was detected in all the nine cultivars with 100% incidence; GYSVd incidence ranged from 25 to 100% in the nine cultivars while the incidence of GHVd was at 12.5–30.4%, limited to three cultivars (Table 2).
In ON, 10 viruses and 2 viroids were detected among 14 cultivars tested. Incidence of GRSPaV was detected in 12 cultivars and was higher compared to other virus infections. GVA and GFkV infections were limited to cultivar, viz., Cabernet Franc and GVB infections were detected only in Petit Verdot. Single virus infections were observed in Cabernet Sauvignon and Gamay Noir for GRSPaV and GLRaV-3, respectively. The number of virus and viroid infections was less in comparison to BC. One composite sample was positive for Grapevine Syrah Virus-1 (GSyV-1) with 20% overall incidence whereas 60% of the samples were identified to be infected with HSVd. Group-E viruses were not detected from ON samples in this study (Table 3).
In NS, 12 viruses and 3 viroids were confirmed in the cultivars tested with varying rates of incidence. GRSPaV was detected in all the eight cultivars at a 100% rate of infection. GVA, GVB, and GLRaV-1 were detected in one cultivar each while the remaining viruses were detected in more than one cultivar. Vidal Blanc was infected with nine viruses, the highest of all the cultivars tested in NS. HSVd was the most prevalent with a 100% incidence in five cultivars followed by GYSVd with 100% incidence in one cultivar (Chardonnay) (Table 4).
In QC, from the 15 cultivars tested, six viruses and one viroid infection were detected. All the cultivars tested were positive for at least one of the grapevine viruses. Group-C viruses were not detected from the samples from QC. GRSPaV infection was detected in 11 cultivars, and 100% incidence was noticed in 3 cultivars. GRBV was detected in seven cultivars with a 100% incidence observed in five cultivars whereas GPGV was detected in three cultivars with a 100% incidence in Vidal Blanc. GLRaV-3 was detected in six cultivars with 100% incidence in three cultivars. GLRaV-2 was detected only in Cabernet Sauvignon. Of the viroids, only HSVd was detected in nine cultivars (Table 5).

4. Discussion

In recent years, the incidence of grapevine viral diseases poses a serious concern to the profitability and sustainability of the grape and wine sector [37]. Several surveys were conducted in major grapevine-growing provinces of Canada over the last 25 years to understand virus incidence and severity [15,18,20,22,38]. These earlier surveys relied on available virus-specific ELISA and (or) PCR detection assays and focused on the presence of selected viruses of concern. ELISA and end-point PCR methods are impractical and laborious to detect all grapevine viruses since they rely either on the availability of virus-specific antibodies or primers for detection. For the first time, this study implemented a multi-provincial survey of Canadian grapevines using viral dsRNA enrichment in combination with HTS to detect viruses and viroids infecting grapevines. dsRNA-enriched extracts from plants are known to contain concentrated nucleic acids from viruses with either RNA and DNA genomes and are an ideal source of enriched viral nucleic acids for studies employing HTS. At present, HTS-based viral discovery has been the preferred technique over other methods due to the fact that it is independent of any virus bias as it provides the researcher with an integral snapshot of all viral and viroid species from a set of pooled test samples [39]. The outcome would be a comprehensive list of already reported novel or unknown viruses and viroids existing in the sample. Over the last decade, HTS methods assisted in the detection of more than 35 grapevine viruses. HTS-based surveys were conducted in several grapevine-growing regions, resulting in the detection of existing and new viruses. In Quebec, Canada, the incidence of GRSPaV, GLRaV-2, and GLRaV-3, HSVd, and other viruses was reported [23]. Grapevine-associated tymo-like virus (GaTLV) were detected in Canyon and Nez Perce counties in ID, USA [40], 10 viruses and 2 viroids were detected in counties in eastern, Western and middle Tennessee in TN, USA [41], Grapevine rupestris vein feathering virus (GRVFV) was detected in Canyon County in ID, USA [42], Grapevine red globe virus (GRGV) in grapevine nurseries [43] and Grapevine rupestris vein feathering virus (GRVFV) in eastern Washington State, USA [44]. The viruses identified in this survey were categorized based on their disease complex into five groups (group A to E) and the viroids were included under the sixth group (group-F).
In this HTS-based comprehensive grapevine virus survey from four provinces in Canada, group-B viruses (leafroll complex) were the most widely distributed in comparison to the other virus groups. Of the leafroll complex, GLRaV-3 was the most prevalent with the highest incidence in all four provinces followed by GLRaV-2 and -1. GLRaV-4, -7, and -13 were not detected from the samples tested. The higher incidence of GLRaV-3 could be attributed to the natural transmission of the virus by scale insects and mealybugs or due to planting of infected planting material. Several studies indicated the insect transmission of GLRaV-3, which included mealybugs and scale insects [45,46,47]. The lower incidence of GLRaV-2 could be due to the lack of natural vectors for the virus transmission, low virus retention, or low frequency of transmission. Epidemiological and virus diversity studies on leafroll disease in BC during 2014 and 2015 using DAS-ELISA and RT-PCR detected the prevalence of four leafroll viruses (GLRaV -1, -2, -3, -4). GLRaV-3 was found to be the most widespread with 16.7% incidence followed by GLRaV 1 (3.8%), -2 (3.0%), and -4 (3.9%), respectively. In this survey, there is an increase in the incidence of GLRaV-2 in BC at 12.7% compared to the previous BC survey [20]. The increase in the incidence of leafroll viruses in BC might be attributed to the higher transmission frequency of GLRaV-3 by natural vectors while higher GLRaV-2 detection in the absence of a natural vector could be due to the use of infected planting material in later years with some blocks spreading up to 25% a year and most of the blocks chosen to be pulled out due to the disease spread could have accounted for the significant increase in the virus incidence in the samples tested. A survey conducted in Nova Scotia from 2016 to 2018 for the detection of seven viruses (GLRaV-1, -2, -3, -4, GFLV, GRBV, and GPGV), employing PCR/RT-PCR, indicated that leafroll disease caused by GRLaV-3 was the most prevalent with a high incidence rate of 22.8% followed by 3.4% incidence of GLRaV-1. None of the samples were positive for GLRaV-2 and -4. Multiple viruses were noticed in 3% grapevine samples collected from NS. Observing the results from the current survey (2021–2023), there has been an increase in the incidence of GLRaV-3 in NS compared to an earlier survey [21]. This increase in the incidence rate indicates the possible role of natural vectors spreading GLRaV-3. Vector transmission of GLRaV-3 was reported to be carried out by mealybugs [48,49] and scale insects [48,50]. However, the role of natural vectors leading to an increase in the incidence of GLRaV-3 in NS has not been confirmed. Negligible incidence of GLRaV-7 and -13 in the current and earlier surveys could be due to the fact that these two viruses are less common and the absence of a natural vector for the transmission of GLRaV-7. In ON, incidences of GLRaV-1, -2, and -3 were detected at 6%, 6%, and 18%, respectively. Compared to the previous survey performed during 2015–2016 [22], there is an increase in the incidence of GLRaV-1 and -2. In this survey, the incidences of GLRaV-1 and -2 were 6% each compared to 2.1% and 4.4% from a recent previous survey conducted in ON. However, the incidence of GLRaV-3 in this survey was recorded as lower compared to a recent survey that recorded 47.9% incidence in ON. A possible explanation for the lesser incidence of leafroll viruses in ON could be due to the virus distribution at different locations and smaller sample size tested in comparison to the earlier provincial surveys in ON [22]. A similar scenario of a higher incidence of GLRaV-3 among the leafroll complex has been reported from other grapevine-growing regions outside Canada. Earlier, a survey for viruses in Oregon (OR) and Washington (WA), USA, indicated the highest incidence of GLRaV-3 in OR (4.4%) and WA (6.5%) followed by GLRaV-1 and -2, respectively [51]. A survey of vineyards in Missouri for 19 viruses tested using RT-qPCR indicated the presence of GLRaV-3 at 53% followed by GLRaV-2 at a 19% rate of infection among other grapevine viruses [52]. A survey conducted during 2018 to 2020 for seven grapevine viruses in New England vineyards in USA using ELISA and RT-PCR methods determined a higher incidence rate of GLRaV-3 (27.59%) followed by GLRaV-1 (13.52%) and GLRaV-2 (11.03%), respectively [53]. A recent survey of grapevines in Palestine based on ELISA and RT-PCR confirmed a higher incidence of GLRaV-3 (23.8%), which indicates the severity of GLRaV-3 [54]. An extensive survey conducted during 2019 for five major grapevine viruses in Bosnia and Herzegovina using ELISA and RT-PCR confirmed that GLRaV-3 had the highest incidence of 84% followed by GLRaV-1 (14%) among other viruses tested [55]. HTS analysis of grapevine samples from the Don ampelographic collection in Russia revealed the highest incidence of GLRaV-3 (37%) followed by GLRaV-1 (16%) among the leafroll viruses tested [56]. Incidence of group-A viruses (rugose wood complex) was also distributed across the four provinces with BC and NS showing a higher incidence of viruses compared to ON and QC. A 100% incidence of GRSPaV was observed in BC and NS. The highest incidence of GRSPaV has been reported in Mendoza, Argentina (71.3%) [57], Missouri, USA (59%) [52], the Don ampelographic collection (98%), and Dagestan (62–98%) in Russia [56,58], respectively. In this survey, the prevalence of GVA, GVB, GVD, and GVE was noticed in BC samples. However, the incidence of GRSPaV, GVA, and GVB in ON samples in this survey was observed to be lower compared to an earlier survey that reported an 84%, 6.2%, and 3% incidence, respectively [22]. An important factor for the lower incidence of rugose wood complex viruses detected in this survey could be due to the CFIA regulations and quarantine measures that could have resulted in the lower incidence of GVA, GVB, and other viruses.
A close observation of the incidence of group-E viruses (other viruses) in this study shows an increase in the virus incidence in comparison to the previous surveys in Canada. The most notable rise is the infection rate of GRBV and GPGV in NS with GRBV at 10% and GPGV at 71% compared to the 4.6% and 3.2% from the 2016–2018 NS survey by Poojari et al. [21]. In ON, there has been a marginal increase in the incidence of GPGV and GRBV infection compared to an earlier survey [22]. The incidence of GRBV and GPGV in an earlier survey in ON was 18.3% and 21.6% whereas in this survey, GRBV and GPGV incidence was at 19.4% and 24%. In BC, the incidence of GRBV was not detected in any of the samples tested. This could be attributed to the management practices being implemented in BC to reduce the virus load and negative impacts of viral diseases in vineyards, including roguing. Roguing is a plant disease management method where undesirable plants are identified and removed to improve the crop. Roguing has been shown to successfully manage Grapevine leafroll disease (GLRD) in different countries [59,60] and was implemented in BC, resulting in a significant reduction in GRBV incidence [61].
The fleck disease complex viruses were predominantly seen distributed in BC and NS samples with GRGV and GRVFV being more prevalent in the samples tested. Compared to the previous survey on GFkV with 29.7% incidence, there has been a marginal decrease in the incidence of GFkV at 28% in this survey [17]. The least prevalent of the virus disease complexes was observed in group-C (infectious disease/decline complex) with ArMV infection in 1.3% samples in BC and 3.2% in NS. None of the group-C viruses were detected in ON and QC. Other notable viruses detected in this study with varying incidence rates were GFkV, GRGV, and GVE. The survey results highlight the fact that GRSPaV, GLRaV-3, and GRBV are the most prevalent grapevine viruses with a higher rate of infection.
Virtool analysis of the metagenomic data of the 310 samples in this study determined the incidence of viruses that were not reported earlier from these four provinces. Grapevine rupestris vein feathering virus (GRVFV) infection was earlier reported from Quebec [23] while in this survey, the incidence of GRVFV in BC and NS was 46% and 32.3%. This is the first report of GRVFV from these two provinces in Canada while GRVFV was not detected in the samples from ON and QC. Other viruses detected in this HTS analysis include Pepino mosiac virus (PepMV), Turnip crinkle virus (TCV), and Grapevine-associated tymo-like virus (GaTLV). One composite sample from QC was positive for PepMV and TCV while one composite sample from ON was positive for GaTLV. In earlier studies based on mechanical inoculation studies, the status of fungicide application season, and swipe tests, it was suggested that GaTLV could be a likely surface contaminant [39]. The presence of PepMV and TCV in QC, however, needs to be confirmed by PCR to ascertain whether the sample was PCR positive or if this could be due to sample contamination. Similarly, the presence of GaTLV in one composite from ON could be a result of surface contamination or a mycovirus associated with the grapevine phytobiome, which requires further investigation. This HTS-based grapevine virome survey emphasizes the importance of determining the virome status of grapevine viruses and viroids from four major grapevine-growing provinces in Canada. This survey highlights the advantages of employing HTS-based surveys for virus discovery in grapevines. Overall, in this survey, 20 viruses and 3 viroids were identified in different grape cultivars at different incidence rates. About 12 viruses that were detected in this survey are included in the list of 23 viruses in the Canadian Grapevine Certification Network (CGCN) certification program under approved virus-testing methods and viruses of concern [62]. These 12 viruses are ArMV, GLRaV-1, -2, -3, GFkV, GVA, GVB, GVD, GVE, GRBV, GPGV, and GRSPaV. The majority of the viruses identified in this survey are commercially important and cause serious economic losses to the grapevine industry. These viruses are critical in the decisions made by the CGCN to develop and implement suitable measures towards the implementation of clean plant programs. The outcomes of this survey indicate the importance of vineyard surveillance to adopt necessary disease management measures to mitigate viral diseases and improve vineyard ecosystem across Canada.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15141532/s1, Figure S1: Virtool analysis interface of HTS data analysis. The pathoscope workflow was used for detection and identification of viruses from Illumina sample library. Pathoscope analysis of a composite sample (DSN03_S22) resulted in the identification of three viruses. Based on the weight and coverage parameters, the sample is positive for (a) Grapevine red blotch virus (GRBV) and (b) Grapevine Pinot gris virus (GPGV). The sample was considered negative for Grapevine leafroll virus 3 (GLRaV-3) as the Virtool requirements were not met by sample (c); Table S1: List of grapevine cultivars and viruses referred to in this study. List of viruses and their acronyms (based on disease complex and other viruses) are summarized below. (The viruses identified in this study were italicized and bold).

Author Contributions

Conceptualization, J.R.Ú.-T., D.L.M., M.R., and S.P.; methodology, B.M.V., K.G., W.M.-S., M.L.F., J.R.Ú.-T., P.M., I.B., J.P., L.B., D.L.M., M.R., and S.P.; software, K.G., J.R.Ú.-T., P.M., I.B., and J.P.; validation, B.M.V., K.G., M.L.F., J.R.Ú.-T., P.M., S.L., L.B., M.R., and S.P.; formal analysis, B.M.V., K.G., J.R.Ú.-T., J.P., and S.P.; investigation, B.M.V., M.L.F., J.R.Ú.-T., P.M., M.R., and S.P.; resources, B.M.V., W.M.-S., M.L.F., P.M.,S.L., D.L.M., M.R., and S.P.; data curation, B.M.V., K.G., M.L.F., J.R.Ú.-T., and I.B.; writing—original draft, B.M.V., J.R.Ú.-T., and S.P.; writing—review and editing, B.M.V., J.R.Ú.-T., M.R., and S.P.; visualization, B.M.V., K.G., S.L., and J.R.Ú.-T.; supervision, S.P., and M.R.; project administration, S.P.; funding acquisition, W.M.-S., D.L.M., M.R., and S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Genome British Columbia and Genome Canada through the project (code 189GRP), entitled “CLEan plAnt extractioN sEquencing Diagnostics (CLEANSED) for Clean Grapevines in Canada”.

Institutional Review Board Statement

Not Applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author due to restrictions on data sharing policies from different institutions and the large data size.

Acknowledgments

Our sincere thanks to the grape growers of British Columbia, Ontario, Nova Scotia, and Quebec for allowing us to access their vineyards and collect the vine samples for this project.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 15 01532 g001
Figure 1. High-throughput sequencing (HTS) analysis of grapevines from British Columbia (BC), Ontario (ON), Nova Scotia (NS), and Quebec (QC) provinces of Canada. The graph summarizes the percentage of virus incidence based on the disease complex in grapevines. (A) Rugose wood complex, (B) leafroll disease complex, (C) infectious degeneration/decline, (D) fleck disease complex, (E) other viruses, (F) viroids. The acronyms of viruses and cultivars are given in Supplementary Table S1.
Figure 1. High-throughput sequencing (HTS) analysis of grapevines from British Columbia (BC), Ontario (ON), Nova Scotia (NS), and Quebec (QC) provinces of Canada. The graph summarizes the percentage of virus incidence based on the disease complex in grapevines. (A) Rugose wood complex, (B) leafroll disease complex, (C) infectious degeneration/decline, (D) fleck disease complex, (E) other viruses, (F) viroids. The acronyms of viruses and cultivars are given in Supplementary Table S1.
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Table 1. List of cultivars from each province included for metagenomic analysis.
Table 1. List of cultivars from each province included for metagenomic analysis.
British Columbia (BC)Nova Scotia (NS)Ontario (ON)Quebec (QC)
V. vinifera (Red)V. vinifera (White)V. vinifera (Red)V. vinifera (Red)
MerlotChardonnayCabernet FrancMerlot
Pinot noirRieslingMerlotCabernet Sauvignon
Gamay noirSauvignon BlancCabernet SauvignonPinot Noir
Cabernet francPinot GrisGamay noirVitis Riparia
Cabernet SauvignonHybrid RedPinot noirV. vinifera (White)
V. vinifera (White)MarquetteMalbecRiesling
ChardonnayNew York MuscatPetit VerdotChardonnay
RieslingHybrid WhiteShirazPinot Gris
Sauvignon BlancL’Acadie BlancV. vinifera (White)Hybrid Red
Pinot GrisVidal BlancChardonnayMaréchal Foch
GewurztraminerFrontenac Noir
RieslingPetite Perle
Hybrid RedSeyval Noir
Baco noirHybrid White
MarquetteSeyval Blanc
Hybrid WhiteSt-Pepin
VidalFrontenac Blanc
Vidal
Table 2. Details of viruses and viroids and their incidence in grapevine cultivars from British Columbia (BC). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
Table 2. Details of viruses and viroids and their incidence in grapevine cultivars from British Columbia (BC). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
CultivarGROUP-AGROUP-BGROUP-CGROUP-DGROUP-EGROUP-F
GRSPaVGVAGVBGVEGLRAV-1GLRAV-2GLRAV-3ArMVGFkVGAMAVGRVFVGRGVGPGVGRBVHSVdGYSVdGHVd
V. vinifera (Red)
Merlot100260003969.6043.5069.61378010010030.4
Pinot Noir10001000105007010107010001001000
Gamay Noir100000000000251002501001000
Cabernet franc1005050000100025012.550100010010012.5
Cabernet Sauvignon100000001000000010001001000
V. vinifera (White)
Chardonnay10022115.611044.45.622.227.894.48166.7010010016.5
Riesling10000000250250100872501001000
Sauvignon blanc10075000010002500757501001000
Pinot gris1001000025010000025501000100250
Table 3. Details of viruses and viroids and their incidence in grapevine cultivars from Ontario (ON). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
Table 3. Details of viruses and viroids and their incidence in grapevine cultivars from Ontario (ON). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
CultivarGROUP-AGROUP-BGROUP-DGROUP-EGROUP-F
GRSPaVGVAGVBGLRAV-1GLRAV-2GLRAV-3GFkVGPGVGRBVHSVdGYSVd
V. vinifera (Red)
Cabernet franc2040020002020802020
Merlot54.50000100954.5360
Cabernet Sauvignon250000000000
Gamay noir000005000000
Pinot noir16.700016.766.700000
Malbec16.70033.3016.7016.7066.716.7
Petit Verdot83.3016.7016.750066.7083.30
Shiraz000000000600
V. vinifera (White)
Riesling80000020060201000
Chardonnay33.300000066.7033.30
Gewurztraminer33.300000066.7033.30
Hybrid red
Baco noir1000000005010000
Marquette83.30033.300016.7083.30
Hybrid white
Vidal Blanc66.700033.300001000
Table 4. Details of viruses and viroids and their incidence in grapevine cultivars from Nova Scotia (NS). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
Table 4. Details of viruses and viroids and their incidence in grapevine cultivars from Nova Scotia (NS). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
CultivarGROUP-AGROUP-BGROUP-CGROUP-DGROUP-EGROUP-F
GRSPaVGVAGVBGLRAV-1GLRAV-2GLRAV-3ArMVGFkVGAMAVGRVFVGPGVGRBVHSVdGYSVdGHVd
V. vinifera (White)
Chardonnay10000033.333.30010010066.701001000
Riesling100002500000251000100750
Sauvignon Blanc1000000000050100010000
Pinot gris10016.700066.7033.30083.3083016
Hybrid red
Marquette10000000000066.71006600
New York Muscat10002500100025250250100500
Hybrid white
Vidal Blanc1000750751002575251001000100750
L’Acadie Blanc10000000000010003300
Table 5. Details of viruses and viroids and their incidence in grapevine cultivars from Quebec (QC). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
Table 5. Details of viruses and viroids and their incidence in grapevine cultivars from Quebec (QC). The highest virus incidence is represented in bold font. Details of the acronyms of viruses and grapevine cultivars are given under Supplementary Table S1.
CultivarGROUP-AGROUP-BGROUP-DGROUP-EGROUP-F
GRSPaVGLRAV-2GLRAV-3GFkVGPGVGRBVHSVd
V. vinifera (Red)
Merlot5000000100
Cabernet Sauvignon502500000
Pinot Noir750305015045
Vitis Riparia0000000
V. vinifera (White)
Riesling0010000050
Chardonnay66.6022.25.5010044.4
Pinot Gris66.60016.6010083.3
Hybrid red
Maréchal Foch55.5022.21108933.3
Frontenac Noir001000000
Petit Perle100000000
Seyval Noir66.600001000
Hybrid white
St-Pepin10000000100
Frontenac Blanc000001000
Vidal Blanc1000016.610010016
Seyval Blanc36.601003.36016.6
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MDPI and ACS Style

Vemulapati, B.M.; Ghoshal, K.; Lerat, S.; Mcfadden-Smith, W.; Fall, M.L.; Úrbez-Torres, J.R.; Moffet, P.; Boyes, I.; Phelan, J.; Bennouna, L.; et al. Metagenomic Profiling of the Grapevine Virome in Canadian Vineyards. Agriculture 2025, 15, 1532. https://doi.org/10.3390/agriculture15141532

AMA Style

Vemulapati BM, Ghoshal K, Lerat S, Mcfadden-Smith W, Fall ML, Úrbez-Torres JR, Moffet P, Boyes I, Phelan J, Bennouna L, et al. Metagenomic Profiling of the Grapevine Virome in Canadian Vineyards. Agriculture. 2025; 15(14):1532. https://doi.org/10.3390/agriculture15141532

Chicago/Turabian Style

Vemulapati, Bhadra Murthy, Kankana Ghoshal, Sylvain Lerat, Wendy Mcfadden-Smith, Mamadou L. Fall, José Ramón Úrbez-Torres, Peter Moffet, Ian Boyes, James Phelan, Lucas Bennouna, and et al. 2025. "Metagenomic Profiling of the Grapevine Virome in Canadian Vineyards" Agriculture 15, no. 14: 1532. https://doi.org/10.3390/agriculture15141532

APA Style

Vemulapati, B. M., Ghoshal, K., Lerat, S., Mcfadden-Smith, W., Fall, M. L., Úrbez-Torres, J. R., Moffet, P., Boyes, I., Phelan, J., Bennouna, L., Moreau, D. L., Rott, M., & Poojari, S. (2025). Metagenomic Profiling of the Grapevine Virome in Canadian Vineyards. Agriculture, 15(14), 1532. https://doi.org/10.3390/agriculture15141532

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