Grapevine Virus A Research Articles

First reports of several viruses and a viroid including a novel vitivirus in Japan, found through virome analysis of bulk grape genetic resources.

Virome analysis was performed on 174 grape genetic resources from the National Agriculture and Food Research Organization, Japan. A total of 20 bulk samples was prepared by grouping the vines into batches of 6-10 plants. Each of the bulk samples was analyzed using high-throughput sequencing, which detected 27 viruses and 5 viroids, including six viruses and one viroid reported in Japan for the first time (grapevine viruses F, L, and T, grapevine Kizil Sapak virus, grapevine Syrah virus 1, grapevine satellite virus, and grapevine yellow speckle viroid 2). In addition, a novel vitivirus was detected with a maximum nucleotide sequence identity of only 58% to its closest relative, grapevine virus A (GVA). The genome of this novel virus was 7,461 nucleotides in length and encoded five open reading frames showing the typical genomic structure of vitiviruses. Phylogenetic trees of vitiviruses placed it in a distinct position nearest to GVA or grapevine virus F (GVF) in genomes and amino acids of deduced replication-associated protein (RAP) and coat protein (CP). The amino acid sequence identities of RAP and CP with GVA, GVF, and other vitiviruses were a maximum of 53% and 73%, respectively, which were significantly below the species demarcation threshold of 80% in the genus. The low identity and phylogenetic analyses indicate the discovery of a novel vitivirus species provisionally named grapevine virus P.

Understanding the dissemination of viruses and viroids identified through virome analysis of major grapevine rootstocks and RPA-based detection of prevalent grapevine virus B

Grapevine stocks are generally vegetatively propagated for the commercial cultivation. The propagation is predominantly done on rootstocks with >90 % of plant stocks propagated on Dogridge rootstock alone in India. Three additional rootstocks i.e. Paulsen, SO4 and Ramsey are also used for grapevine propagation on a limited scale. However, no information is available on the health status of these rootstocks being used by nurserymen and growers. Therefore, these rootstocks were collected from nurseries of five different locations and subjected to high-throughput sequencing (HTS) to determine the spectrum of viruses and viroids. The de novo assembly of unaligned reads revealed the presence of genome sequences of nine viruses viz. grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 (GLRaV-4), grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus D (GVD), grapevine virus F (GVF), grapevine virus L (GVL), grapevine fleck virus (GFkV) and five viroids viz. hopstunt viroid (HSVd), grapevine yellow speckle viroid 1 and 2 (GYSVd-1 and GYSVd-2), Australian grapevine viroid (AGVd) and grapevine latent viroid (GLVd). The identified viruses and viroids were further confirmed through RT-PCR and Sanger sequencing. Indexing for the presence of these graft-transmissible pathogens in 43 rootstocks and 23 scion samples collected through field surveys indicated that infected rootstocks are the probable source for dissemination of these viruses and viroids in grapevine scions. As GVB was the most prevalent virus in all the rootstocks and scions as observed in RT-PCR testing, a simplified crude sap-based recombinase polymerase amplification (RPA) assay was optimized for its rapid detection. The present study highlights that grapevine rootstocks are reservoir of viruses and viroids and source for their dissemination in grapevine vineyards. Findings of the study including the optimized simplified cost-effective isothermal nucleic acid-based detection technique for GVB will be highly useful for production of certified clean rootstocks of grapevine in India.

Simultaneous Detection of Grapevine Viruses via Multiplex Reverse Transcription Polymerase Chain Reaction: An Academic Approach

Grapevine viruses pose a significant threat to the viticulture industry, resulting in reduced grape yield, poor fruit quality, and economic loss. Accurate and timely detection of multiple viruses are essential for effective disease management and the implementation of appropriate control measures. The aim of this study was to assess the presence of grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine leafroll-associated virus‑1 (GLRaV-1), arabis mosaic virus (ArMV), and grapevine virus A (GVA) in 320 plant samples collected from the Aegean region of Turkey using the powerful technique of multiplex reverse transcription-polymerase chain reaction (RT-PCR). Through comprehensive analysis, 119 of the 320 infected samples were identified using PCR. The distribution of the detected viruses revealed the presence of 28 GFLV, 31 GLRaV‑1, 12 GFkV, 21 GVA, and 27 ArMV. By comprehensively analyzing the samples, the researchers were able to simultaneously detect and identify the target viruses using a single, cost-effective assay. These results reveal the prevalence and distribution of these viruses in the Aegean region, providing valuable insights into the viral landscape of grapevines in this important viticultural area. The high detection rates of these viruses highlight the urgent need for improved disease management strategies to safeguard vine health and productivity.

New Host Plant Species of Grapevine Virus A Identified with Vector-Mediated Infections.

Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.

Genetic Diversity of Grapevine Virus A in Three Australian Vineyards Using Amplicon High Throughput Sequencing (Amplicon-HTS).

Shiraz disease (SD) is one of the most destructive viral diseases of grapevines in Australia and is known to cause significant economic loss to local growers. Grapevine virus A (GVA) was reported to be the key pathogen associated with this disease. This study aimed to better understand the diversity of GVA variants both within and between individual SD and grapevine leafroll disease (LRD) affected grapevines located at vineyards in South Australia. Amplicon high throughput sequencing (Amplicon-HTS) combined with median-joining networks (MJNs) was used to analyze the variability in specific gene regions of GVA variants. Several GVAII variant groups contain samples from both vineyards studied, suggesting that these GVAII variants were from a common origin. Variant groups analyzed by MJNs using the overall data set denote that there may be a possible relationship between variant groups of GVA and the geographical location of the grapevines.

CRISPR-based resistance to grapevine virus A.

Grapevine (Vitis vinifera) is an important fruit crop which contributes significantly to the agricultural sector worldwide. Grapevine viruses are widespread and cause serious diseases which impact the quality and quantity of crop yields. More than 80 viruses plague grapevine, with RNA viruses constituting the largest of these. A recent extension to the clustered regularly interspaced, short palindromic repeat (CRISPR) armory is the Cas13 effector, which exclusively targets single-strand RNA. CRISPR/Cas has been implemented as a defense mechanism in plants, against both DNA and RNA viruses, by being programmed to directly target and cleave the viral genomes. The efficacy of the CRISPR/Cas tool in plants is dependent on efficient delivery of its components into plant cells. To this end, the aim of this study was to use the recent Cas13d variant from Ruminococcus flavefaciens (CasRx) to target the RNA virus, grapevine virus A (GVA). GVA naturally infects grapevine, but can infect the model plant Nicotiana benthamiana, making it a helpful model to study virus infection in grapevine. gRNAs were designed against the coat protein (CP) gene of GVA. N. benthamiana plants expressing CasRx were co-infiltrated with GVA, and with a tobacco rattle virus (TRV)-gRNA expression vector, harbouring a CP gRNA. Results indicated more consistent GVA reductions, specifically gRNA CP-T2, which demonstrated a significant negative correlation with GVA accumulation, as well as multiple gRNA co-infiltrations which similarly showed reduced GVA titre. By establishing a virus-targeting defense system in plants, efficient virus interference mechanisms can be established and applied to major crops, such as grapevine.

Tokat İli Asma Alanında Bazı Asma Virüslerinin Moleküler Olarak Belirlenmesi ve Grapevine Virus A'nın Filogenetik Analizi

Grapevine (Vitis spp.) is one of the major fruit crop with high socioeconomic importance for Turkey. In vineyards, many harmful organism, especially virus infections, weaken the plant and lead to decreases in yield and quality, so it takes the lead in quarantine and certification. This study was carried out to determine some viral agents that cause yield loss in vines produced in Tokat, where viticulture is very important. Samples were collected from young leaves and one-year-old shoots of grapevines showing virus symptoms from some vineyard areas in Tokat Center and its districts. Collected 189 grapevine samples were subjected to the RT-PCR test, which is a molecular method using virus-specific primers, to detect the presence of Grapevine pinot gris virus (GPGV), Grapevine virus A (GVA), Strawberry latent ringspot virus (SLRSV). Out of a total of 189 plant samples, 80 (42.32%) of GVA, 3 (1.58%) of GPGV were detected and SLRSV (0%) was not detected. More than one virus was found in 2 (1.05%) of 189 tested samples. It was determined that the most common virus was GVA, the least detected virus was GPGV in plant samples collected from Tokat Center and its districts. Bidirectional sequence analysis of RT-PCR products of GVA-infected isolates were performed and phylogenetic analyzes were done by comparing them with reference isolates after they were aligned with the MEGAX computer program. Based on phylogenetic analysis studies, GVA showed differential branching with isolates registered in GenBank and isolates obtained in the study. GVA-infected isolates showed similarity with reference isolates at rates of 92-94%. In this study, molecular analysis of Turkish GVA isolates was performed. This molecular information is important as it will shed light on future studies.

Evaluating the Potential of High-Resolution Visible Remote Sensing to Detect Shiraz Disease in Grapevines

Background and Aims. Shiraz disease (SD) is a viral disease associated with Grapevine virus A that causes significant yield loss in economically important grape cultivars in Australia such as Shiraz and Merlot. Current diagnostic methods are time-consuming and costly. This study evaluates an alternative methodology using visible remote sensing imagery to detect SD in Shiraz grapevines. Methods and Results. High-resolution visible remote sensing images were captured of Shiraz grapevines in two South Australian viticultural regions over two seasons. The projected leaf area (PLA) of individual grapevines was estimated from the images. Virus-infected vines had significantly lower PLA than healthy vines in the early season but fewer difference after veraison. The lower PLA was only observed in grapevines coinfected with grapevine leafroll-associated viruses (GLRaVs) and Grapevine virus A (GVA). Shiraz vines infected with either GLRaVs or GVA had similar PLA to healthy vines. Conclusions. High-resolution RGB remote sensing technology has the potential to rapidly estimate SD infection in Shiraz grapevines. Our observations of shoot devigouration only in coinfected vines calls into question the etiology of SD. Further validation of the PLA technique incorporating different regions, seasons, cultivars, and combinations of viruses is needed for improving the robustness of the method. Significance of the Study. This preliminary study presents a new rapid and low-cost surveillance method to estimate SD infections in Shiraz vineyards, which could significantly lower the cost for growers who conduct on-ground SD visual assessments or lab-based tissue testing at the vineyard scale.

A Metagenomic Investigation of the Viruses Associated with Shiraz Disease in Australia.

Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD.

Potential Implications and Management of Grapevine Viruses in Mexico: A Review

Worldwide, virus infections in grapevines are of concern due to the potential for economic loss. Although the grape industry in Mexico is relatively small and focused mainly on the local market, production dates back to the time of the Spanish colonization. This manuscript discusses the findings on grapevine viruses in Mexico. Nine viruses have been identified in the last fifty years, including grapevine red blotch virus (GRBV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fanleaf virus (GFLV), and grapevine virus A (GVA). Important information is provided about these viruses and viral pathogens that have not yet been reported in Mexico, but represent an ongoing threat to plant health and grapevine production in other viticultural regions of the world. Strategies for virus control in vineyards are described. The information discussed here should be shared with growers and stakeholders to prevent future negative impacts on the Mexican grapevine industry and to save ancient grapevine accessions.

Identification of grapevine virus G and grapevine virus H in Sardinia, Italy

Grapevine virus G (GVG) and grapevine virus H (GVH) (genus Vitivirus) are recently discovered viruses. Analysis of 38 samples from Sardinian grapevine cultivars for the presence of GVG and GVH was carried out using RT-PCR. All samples were also tested for grapevine Pinot gris virus (GPGV) using RT-PCR, and for grapevine leafroll virus -1, -2 and -3, grapevine virus A (GVA) and B (GVB), arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and grapevine fleck virus (GFkV) using multiplex RT-PCR. GVG was confirmed in four vines, and GVH was detected in only one sample. In phylogenetic analyses of the coat protein (CP) region, the Sardinian GVG isolates clustered separately from isolates from Croatia and New Zealand. The Sardinian GVH isolate clustered with most sequences from other countries, but with greater affinity to isolates from California (USA) for the CP region, whereas it clustered with isolates from Croatia in the RNA-dependent RNA polymerase (RdRp) region. In addition to GVG and GVH, many samples were coinfected with GVA, viruses from the leafroll complex, and GPGV. This is the first record of GVG and GVH occurring in Italy.

Occurrence of Nine Grapevine Viruses in Commercial Vineyards of Mendoza, Argentina

Grapevine is a widely grown fruit crop that is seriously affected by different viruses, reducing grape yield and quality, as well as threatening profitability. Vineyard disease management requires accurate identification of viral infections. This study aimed to survey the presence of ten grapevine viruses in four geographic sites in the Mendoza province of Argentina. Two hundred twenty-three composite cane samples from 1060 plants of six cultivars were collected from 26 blocks distributed across 11 vineyards. The cane samples were screened by RT-PCR for the following viruses: grapevine leafroll-associated viruses 1-4 (GLRaV 1, 2, 3, and 4), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine virus A (GVA) and B (GVB), grapevine rupestris stem pitting associated virus (GRSPaV), and arabis mosaic virus (ArMV). The results showed an uneven occurrence of viruses through the sampled regions, with GRSPaV being prevalent (71.1%), followed by GFLV (28.9%), GFkV (20.6%), and GLRaV-2 (14.7%). GVB was not detected. This study revealed a moderate prevalence of viruses associated with economically impactful diseases in the vineyards surveyed.

Transmission of Grapevine Leafroll-Associated Viruses and Grapevine Virus A by Vineyard-Sampled Soft Scales (Parthenolecanium corni, Hemiptera: Coccidae).

Grapevine-infecting ampelo- and vitiviruses are transmitted by scale insects belonging to several species, among which is the European fruit lecanium, Parthenolecanium corni (Bouché) (Hemiptera Coccidae). Our objective was to characterize the transmission biology of grapevine leafroll-associated viruses (GLRaV) and grapevine virus A (GVA) by this soft scale species in order to evaluate its ability to spread these viruses. In transmission experiments with nymphs sampled from different vineyards infected with GLRaV 1, 2, 3 and GVA, P. corni transmitted only GLRaV 1 and GVA to healthy vines. GVA was predominantly transmitted along with GLRaV 1, whereas the latter could be transmitted alone from single or co-infected vines. Vineyard-sampled second instar nymphs were more efficient than first instars at transmitting GLRaV 1, whereas both instars displayed similar transmission rates for GVA. Short virus inoculation access periods and the absence of virus in eggs of females living on infected grapevines fulfilled the criteria of non-circulative semi-persistent transmission mode.

Transmission of Grapevine Ampelo- and Vitiviruses by the Bohemian Mealybug Heliococcus bohemicus Šulc (Hemiptera: Pseudococcidae).

Grapevine-infecting ampelo- and vitiviruses are transmitted by several scale insect species, including the Bohemian mealybug, Heliococcus bohemicus Šulc. Virus infectivity experiments were performed with this species to study the transmission ability of natural populations living in infected vineyards in Alsace, France. Mealybugs were sampled on vines infected by grapevine leafroll-associated viruses (GLRaV-1, -2, and -3) and by grapevine virus A (GVA), either alone or in combinations. Out of six natural populations tested, only one, located at Bennwihr, was able to transmit GLRaV-1 and -3 to healthy vines, though with low transmission rates (1.6 and 11.8%, respectively). Mealybugs from Bennwihr were also able to transmit GLRaV-3 from grapevines of another location where H. bohemicus was not a vector. Conversely, mealybugs from two other locations did not transmit any virus acquired from infected grapevines at Bennwihr. These results suggest differences in vector ability between H. bohemicus populations. Moreover, laboratory experiments were developed to estimate the minimal acquisition and inoculation access periods (AAP and IAP, respectively) for virus transmission of GLRaV-1 and -3, and GVA. First instar nymphs transmitted GLRaV-1 after 6 h AAP, GLRaV-3 and GVA together after 1 h AAP, and the three viruses after only 1 h IAP, supporting a semi-persistent mode of transmission. Second instar nymphs fed on multi-infected grapevine for 72 h then starved or fed on potatoes tested positive by RT-PCR for GLRaV-1 and -3 after up to 35 and 40 days, respectively, contrasting with the short retention times generally observed for mealybugs. These findings provide new knowledge of the vector ability of H. bohemicus.

Survey for Major Grapevine Viruses in Commercial Vineyards of Northwestern Argentina.

This study aimed to survey the occurrence of eight grapevine viruses in commercial vineyards located in the Calchaquíes Valleys in the northwest region of Argentina. A total of 103 samples of mature canes of vines showing either none or some viral-like symptoms were randomly collected. The samples were tested by RT-PCR/PCR-based assays for the screening of the following viruses: Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4), Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated viruses (GRSPaV), and Grapevine red blotch virus (GRBV). Sixty percent of the analyzed samples showed infection with some of the analyzed viruses, except GRBV. GLRaV-3 and GFLV were the most frequent viruses, present in 34% and 21% of the positive samples, respectively. This study represents the first survey report of the presence of grapevine viruses in the region of the Calchaquíes Valleys and contributes to the knowledge to maintain the sanitary status of commercial vineyards in Argentina.

Evolutionary Analysis of Grapevine Virus A: Insights into the Dispersion in Sicily (Italy)

Grapevine virus A (GVA) is a phloem-restricted virus (genus Vitivirus, family Betaflexiviridae) that cause crop losses of 5–22% in grapevine cultivars, transmitted by different species of pseudococcid mealybugs, the mealybug Heliococcus bohemicus, and by the scale insect Neopulvinaria innumerabilis. In this work, we studied the genetic structure and molecular variability of GVA, ascertaining its presence and spread in different commercial vineyards of four Sicilian provinces (Italy). In total, 11 autochthonous grapevine cultivars in 20 commercial Sicilian vineyards were investigated, for a total of 617 grapevine samples. Preliminary screening by serological (DAS-ELISA) analysis for GVA detection were conducted and subsequently confirmed by molecular (RT-PCR) analysis. Results showed that 10 out of the 11 cultivars analyzed were positive to GVA, for a total of 49 out of 617 samples (8%). A higher incidence of infection was detected on ‘Nerello Mascalese’, ‘Carricante’, ‘Perricone’ and ‘Nero d’Avola’ cultivars, followed by ‘Alicante’, ‘Grecanico’, ‘Catarratto’, ‘Grillo’, ‘Nerello Cappuccio’ and ‘Zibibbo’, while in the ‘Moscato’ cultivar no infection was found. Phylogenetic analyses carried out on the coat protein (CP) gene of 16 GVA sequences selected in this study showed a low variability degree among the Sicilian isolates, closely related with other Italian isolates retrieved in GenBank, suggesting a common origin, probably due to the exchange of infected propagation material within the Italian territory.

Phylogenetic Marker Selection and Protein Sequence Analysis of the ORF5 Gene Product of Grapevine Virus A.

Grapevine virus A (GVA), the type species of the Vitivirus genus, is one of the causal agents of the Kober stem grooving disease of the rugose wood complex and one of the most frequently detected viruses in grapevine. There is little information on GVA gene(s) marker useful for phylogenetic analysis. To this aim, a total of 403 leaf samples were collected from vineyards of East and West Azarbaijan provinces in the Northwestern provinces of Iran during 2014–2016 and tested by DAS-ELISA and RT-PCR using ORF5-specific primers. GVA was detected in 56 symptomatic samples, corresponding to 14% of infection, while it was not detected in asymptomatic samples. The ORF5 (p10) protein sequence of eight Iranian isolates was compared to other vitiviruses, showing that the most conserved region resides in the N-terminus, carrying an arginine-rich motif followed by a zinc-finger motif. Next, to define a robust phylogenetic marker representative of the whole genome sequence suitable for phylogenetic and evolutionary studies, phylogenetic trees based on the full genome sequences of all the available GVA isolates and on individual genomic regions were constructed and compared. ORF1, which encodes the RNA-dependent RNA polymerase, was found to be the best phylogenetic marker for GVA classification and evolution studies. These results can be used for further research on phylogenetic analyses, evolution history, epidemiology, and etiology of rugose wood complex, and to identify control measures against GVA and other vitiviruses.

Prevalence of Virus Infections and GLRaV-3 Genetic Diversity in Selected Clones of Croatian Indigenous Grapevine Cultivar Plavac Mali.

The cultivar Plavac Mali (Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 (GLRaV-1, GLRaV-2 and GLRaV-3, respectively), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription–polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard.

DETECTION OF VIRUSES OF THE RUGOSE WOOD COMPLEX ON VINEYARDS OF THE ODESSA REGION

DETECTION OF VIRUSES OF THE RUGOSE WOOD COMPLEX ON VINEYARDS OF THE ODESSA REGION

Prevalence and Genetic Diversity of Viruses Associated with Rugose Wood Complex in Greek Vineyards.

Rugose wood is one of the most important disease syndromes of grapevine, and it has been associated with at least three viruses: grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A (GVA), and grapevine virus B (GVB). All three viruses show a worldwide distribution pattern, and their genetic composition has been the focus of extensive research in past years. Despite their first record in Greece almost 20 years ago, there is a lack of knowledge on the distribution and genetic variability of their populations in Greek vineyards. In this context, we investigated the distribution of GRSPaV, GVA, and GVB in rootstocks, self-rooted vines, and grafted grapevine cultivars originating from different geographic regions that represent important viticultural areas of Greece. Three new reverse transcription-PCR assays were developed for the reliable detection of GRSPaV, GVA, and GVB. Our results indicated that GVA is the most prevalent in Greek vineyards, followed by GRSPaV and GVB. However, virus incidence differed among self-rooted and grafted grapevine cultivars or rootstocks tested. Selected isolates from each virus were further molecularly characterized to determine their phylogenetic relationships. All three viruses exhibited high nucleotide diversity, which was depicted in the constructed phylogenetic trees. Isolates from Greece were placed in various phylogroups, reinforcing the scenario of multiple introductions of GVA, GVB, and GRSPaV in Greece and highlighting the effect of different transmission modes in the evolutionary course of the three viruses.