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A survey of viruses affecting grapevine in the wine regions of the Western Cape Province in South Africa was conducted. The survey determined the relative abundance of five different Grapevine leafroll-associated virus 3 (GLRaV-3) variants. Virus profiles were also determined for individual vines. A total of 315 plants were sampled and analysed over two growing seasons. Five GLRaV-3 variants were detected as either single or mixed infections, with GLRaV-3 variant groups II and VI being the most prominent as single infections and in combinations with each other and other variants. An analysis of the distribution of variants per region showed that single infections of variant groups II and VI occurred predominantly in certain regions, and were equally distributed in the red and white cultivars studied. The distribution of a recently identified, unclassified variant of GLRaV-3 (represented by isolates GH24 and GTG10) was included in the study. The overall analysis showed that infection with variant groups II and VI were the most abundant among the samples with 49.8 and 47.6 %, respectively, followed by variant group I, variants similar to isolate GH24 and variant group III with 16.2, 13.3 and 2.5 % infection, respectively. Mixed infections, representing 36 virus combinations, were found in 251 plants. The most abundant virus combination was GLRaV-3 with Grapevine virus E (GVE), found in 28 % of the plants. GLRaV-3 was the predominant virus detected in the samples with a frequency of 80 % detection, followed by GVE (57.4 %), Grapevine rupestris stem pitting-associated virus (GRSPaV) (36.8 %), Grapevine virus A (GVA) (19.3 %), Grapevine virus F (GVF) (16.25 %), Grapevine leafroll-associated virus 2 (GLRaV-2) (8.25 %), Grapevine leafroll-associated virus 1 (GLRaV-1) (1.58 % infection) and Grapevine leafroll-associated virus 4 (GLRaV-4 like) (0.6 %). Most of the plants tested were infected with multiple viruses. The complexity of virus populations detected in this study, highlights the need for detection methods able to identify all viruses and their variants in vineyards. The information generated in this study will assist in the development of reliable detection assays that will benefit the monitoring of disease spread and aid in the efficient management of Grapevine leafroll disease (GLD).

Viral diseases can seriously damage the vineyard productivity and the quality of grape and wine products. Therefore, the study of the species composition and range of grapevine viruses is important for the development and implementation of strategies and tactics to limit their spread and increase the economic benefits of viticulture. In 2014–2019, we carried out a large-scale phytosanitary monitoring of Russian commercial vineyards in the Krasnodar region, Stavropol region and Republic of Crimea. A total of 1857 samples were collected and tested for the presence of Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine leafroll-associated virus-1 (GLRaV-1), Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine fanleaf virus (GFLV), and Grapevine fleck virus (GFkV) using RT-PCR. Out of all samples tested, 54.5% were positive for at least one of the viruses (GRSPaV, GVA, GLRaV-1, GLRaV-2, GLRaV-3, GFLV, GFkV) in the Stavropol region, 49.8% in the Krasnodar region and 49.5% in the Republic of Crimea. Some plants were found to be infected with several viruses simultaneously. In the Republic of Crimea, for instance, a number of plants were infected with five viruses. In the Krasnodar region and the Republic of Crimea, 4.7% and 3.3% of the samples were predominantly infected with both GFkV and GRSPaV, whereas in the Stavropol region, 6% of the selected samples had both GLRaV-1 and GVA infections. We carried out a phylogenetic analysis of the coat protein genes of the detected viruses and identified the presence of GVA of groups I and IV, GRSPaV of groups BS and SG1, GLRaV-1 of group III, GLRaV-2 of groups PN and H4, GLRaV-3 of groups I and III. The results obtained make it possible to assess the viral load and the distribution of the main grapevine viruses on plantations in the viticultural zones of Russia, emphasizing the urgent need to develop and implement long-term strategies for the control of viral diseases of grapes.

Polymerase chain reaction (PCR) is routinely used for the detection of grapevine red blotch virus (GRBV), a virus with a circular single-stranded DNA genome, in grapevine tissue. Since the preparation of purified grapevine DNA is time consuming, a user-friendly AmplifyRP Acceler8 assay was developed to quickly, specifically, and sensitively identify GRBV-infected grapevine samples. The sensitivity of AmplifyRP Acceler8 for GRBV detection is approximately 100 times higher than that of PCR. GRBV is consistently detected by AmplifyRP Acceler8 up to a 10⁻⁸ dilution of infected grapevine leaf crude extracts diluted in healthy grapevine leaf crude extracts and nearly 10 copies of plasmid DNA containing a GRBV genomic fragment in a matrix of healthy grapevine leaf crude extracts. The test has no cross reactivity to grapevine tissue, nor to several grapevine-infecting pathogens, including arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leaf roll-associated virus 1, grapevine leafroll-associated virus 2, grapevine leafroll-associated virus 3, grapevine leafroll-associated virus 4 strain 5, tomato ringspot virus, tobacco ringspot virus, Xylella fastidiosa, and Botrytis cinerea. Dried GRBV reaction pellets provided in the AmplifyRP Acceler8 kit contain all the necessary reaction components and are stable for at least 5 weeks at –20°C, 4°C, 22°C, and 37°C, providing convenience for transportation and field application. The AmplifyRP Acceler8 assay generated results consistent with PCR and real-time PCR outputs.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020–2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.

Grapevine leafroll-associated virus-2 (GLRaV-2) and GLRaV-3 are both (+) ssRNA viruses of the family Closteroviridae and are involved in grapevine leafroll, the most destructive viral disease affecting the global grape/wine industry. Outbreaks of the disease were recently reported in Canada, causing serious concerns to the grape/wine industry. Reliable, sensitive and timely detection is key to the control of the disease. However, information on their seasonal dynamics and tissue distribution under cool climate conditions has been rather limited. We conducted a two-year comprehensive study to elucidate the temporal variation and spatial distribution of both viruses through symptom monitoring, Western blotting and RT-qPCR. Sampling was done monthly from commercial Cabernet Franc and Chardonnay vines from May to October in 2015 and 2016. Both viral RNA and capsid protein levels in leaves remained low during May and June, steadily increased from late July, and peaked in September or October. Interestingly, young berries collected in June contained high levels of viral RNA for both viruses. As expected, the viral RNA levels of GLRaV-2 detected by RT-qPCR using primers targeting the CP region were much higher than those by using primers targeting the genomic RNA. Surprisingly, virtually the same levels of viral RNA were detected for GLRaV-3 regardless of the targeted genomic regions. To ensure accurate detection of both viruses, we recommend using young berries early in the season and leaves and cambial scrapings from late July to harvest. To our knowledge, this is the first report on the seasonal dynamics and tissue distribution of two major grapevine viruses in Canada.

Grapevine leafroll disease affects the health status of grapevines worldwide. Most studies in Australia have focused on grapevine leafroll-associated viruses 1 and 3, while little attention has been given to other leafroll virus types, in particular, grapevine leafroll-associated virus 2 (GLRaV-2). A chronological record of the temporal occurrence of GLRaV-2 in Australia since 2001 is reported. From a total of 11,257 samples, 313 tested positive, with an overall incidence of 2.7%. This virus has been detected in 18 grapevine varieties and Vitis rootstocks in different regions of Australia. Most varieties were symptomless on their own roots, while Chardonnay showed a decline in virus-sensitive rootstocks. An isolate of GLRaV-2, on own-rooted Vitis vinifera cv. Grenache, clone SA137, was associated with severe leafroll symptoms after veraison with abnormal leaf necrosis. The metagenomic sequencing results of the virus in two plants of this variety confirmed the presence of GLRaV-2, as well as two inert viruses, grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine rupestris vein feathering virus (GRVFV). No other leafroll-associated viruses were detected. Among the viroids, hop stunt viroid and grapevine yellow speckle viroid 1 were detected. Of the six phylogenetic groups identified in GLRaV-2, we report the presence of four groups in Australia. Three of these groups were detected in two plants of cv. Grenache, without finding any recombination event. The hypersensitive reaction of certain American hybrid rootstocks to GLRaV-2 is discussed. Due to the association of GLRaV-2 with graft incompatibility and vine decline, the risk from this virus in regions where hybrid Vitis rootstocks are used cannot be overlooked.

The long-tailed mealybug,  Pseudococcus longispinus , is an important insect pest in grapevine growing areas in several countries, including Brazil. Metagenomic analysis of nucleic acids extracted from insect vectors makes it possible to study the diversity of insect viruses in addition to plant pathogenic viruses. In this study, insects ( Ps. longispinus ) were collected, and pooled throughout a plot of virus disease symptomatic vines, cultivated in growing beds, and analyzed by high throughput sequencing (HTS). The complete genome of grapevine leafroll-associated virus 2 and 3 (GLRaV-2 and -3) and a partial sequence of grapevine virus A (GVA) with two complete ORFs (coat protein and RNA-binding protein) were assembled from mealybug extracts and exhibited high nucleotide identities, up to 99%, with previously characterized homologous Brazilian isolates from grapevines. This information was validated by the detection of these viruses in the original symptomatic vines (N=76), from where mealybugs were collected, equivalent to an incidence of 34.2%, 89.5% and 36.8% for GLRaV-2, GLRaV-3 and GVA, respectively. Although one of these viruses is not transmitted by mealybugs (GLRaV-2), prospection of plant viruses infecting grapevine plants by analyzing the metagenome of insects could represent a relevant alternative to improve monitoring of viral diseases aiming at the management and control of viral diseases in vineyards or cultivation fields. This work is the first analysis of the  Ps. longispinus virome in Brazil focusing on grapevine viruses.

A statewide survey of commercial vineyards in Virginia (VA), USA was conducted in 2009–2011 seasons to examine the occurences of Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine fleck virus (GFkV), and mealybugs. Out of 415 samples (comprising 41 wine grape cultivars) from 77 locations (vineyards), GLRaV-2, GLRaV-3 and GFkV were detected by RT-PCR in 8, 25, and 1 %, respectively, and 64 % of vineyards were positive for at least one of the three viruses. Samples from 100 wild Vitis sp. tested negative for the three viruses. Both the grape (Pseudococcus maritimus) and Gill’s (Ferrisia gilli) mealybug were found in VA vineyards. Although regional effect was not significant (P > 0.05), examination of variance showed higher variability of GLRaV-3 incidence at cultivar scale within a vineyard than at a smaller spatial scale (i.e., at sampling site). In addition, the probability of finding a GLRaV-3-infected vine was higher in the presence of mealybugs (P < 0.001) and with increased vine age (P < 0.001). These results also suggest movement of GLRaV-3 by mealybugs in VA vineyards. The high frequency of virus-infected vines emphasizes the importance of clean plant materials as well as management of mealybugs. This is the first study reporting the presence of Ferrisia gilli, GLRaV-2, GLRaV-3, and GFkV in VA vineyards.

The ribosomal-depleted total RNA from white-berry grapevine (Vitis vinifera, SK933) plant showing severe chlorosis and downrolling of leaves was used for the high-throughput sequencing (HTS) analysis in order to unravel the potential contribution of the viral pathogens to the symptomatology observed. The combination of de novo assembly and mapping of ca. 1.1 millions of HTS reads enabled to identify and characterise a complex viral/viroid infection involving Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Syrah virus-1 (GSyV-1) and Hop stunt viroid (HSVd). The determined nearly complete genomes of GLRaV-2 SK933 showed its high genetic divergence from previously characterised isolates. In case of GRSPaV, two variants representing different evolutionary lineages have been identified in the plant. The results further pinpoint the complexity of grapevine viral diseases and show that mixed virus infection of grapevine is rather a rule than an exception.

Evaluation of the prevalence of virus and viroid infections was conducted in a grapevine field collection in Valencia, Spain. Samples of autochthonous and traditional grapevine cultivars were collected during November 2011 and tested for the presence of fourteen viruses and five viroids, using RT-PCR. The prevalent viruses were Grapevine rupestris stem pitting-associated virus (GRSPaV: 49% infected samples) and Grapevine leafroll-associated virus 2 (GLRaV-2:15% of samples). GLRaV-1, GLRaV-3, GLRaV-4 (variants 4 and 5), Grapevine fanleaf virus, Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) and Grapevine virus A were also detected. Hop stunt viroid (HSVd: 92% of plants infected) and Grapevine yellow speckle viroid 1 (6% of plants) were also detected. Mixed infections with two, and up to six different viruses and/or viroids were common. Only five samples (4%) were free from 19 pathogens tested. This is the first report of GLRaV-4 (variants 4 and 5) in the Valencia region of Spain, and the first record of GRSPaV and GRVFV in this country. Phylogenetic analyses performed with the sequences of these viruses showed that the Spanish isolates of GLRaV-4, GFkV and HSVd belong to new phylogenetic groups.