First report of grapevine virus L infecting grapevine in southeast France

Grapevine virus L (GVL) is a recently described vitivirus (family Betaflexiviridae) with a positive-sense single-stranded RNA genome. It has so far been reported from China, Croatia, New-Zealand, the United States and Tunisia (Debat et al. 2019; Diaz-Lara et al. 2019; Alabi et al. 2020; Ben Amar et al. 2020). It has significant genetic variability (up to 26% of nucleotide divergence between isolates) and the existence of four phylogroups has been proposed (Alabi et al. 2020). In the frame of a project investigating the possible links between grapevine trunk diseases and grapevine virome, viral high throughput sequencing (HTS)-based testing was performed on symptomatic and asymptomatic grapevines collected in July 2019 in vineyards of four areas in France (Bourgogne, Charentes, Gard, Gironde) corresponding to five cultivars of Vitis vinifera (Cabernet franc, Cabernet Sauvignon, Chardonnay, Sauvignon, Ugni blanc). Total RNAs were purified from powder of 105 trunk wood samples using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and RNA-seq libraries were prepared using Zymo-Seq RiboFree Total RNA Library Prep Kit (Ozyme, Saint Cyr l’Ecole, France). HTS was performed on a S4 lane of Illumina NovaSeq 6000 using a paired-end read length of 2x150 bp. The trimmed sequence reads obtained from Chardonnay plants CH30-75M (99.9 M) and CH37-19S (114 M) from a vineyard in Gard were analyzed using CLC Genomics Workbench v21 (Qiagen, Courtaboeuf, France) and revealed complex mixed infections. Besides contigs representing a complete GVL genome (average scaffold coverage: 6,197x and 2,970x, respectively), contigs from grapevine rupestris stem pitting virus (1,697x ; 1,124x), grapevine virus A (82x ; 95x), grapevine pinot gris virus (1,475x ; 866x), grapevine leafroll-associated virus 3 (5,122x ; 1,042x), hop stunt viroid (13,783x ; 29,514x) and grapevine yellow speckle viroid 1 (690x ; 1158x) were also identified. Plant CH37-19S was also co-infected by grapevine rupestris vein feathering virus (164x). The GVL contigs integrated respectively 320,000 and 152,000 reads (corresponding to 0.32% and 0.11% of filtered/trimmed reads, respectively). The GVL genomic sequences from each sample (7,616 nt) have been deposited in GenBank (Accession nos. OK042110 and OK042111, respectively). The two contigs are nearly identical (99.9% nt identity) and share respectively 97.5% and 95.9% with GVL-KA from the USA (MH643739) and GVL-RS from China (MH248020), the closest isolates present in GenBank. To confirm the presence of GVL, the original grapevines were resampled in the field and total RNAs were extracted as described above from cambial scrappings and leaves. Total RNAs were used for RT-PCR tests using primers targeting a 279-bp fragment corresponding to the 3’ end of the coat protein gene and part of the nucleic acid binding protein gene (Debat et al. 2019). The Sanger-derived sequences from the amplicons shared 100% nt identities with the corresponding sequences of the HTS assembled genomes, confirming the presence of GVL in both tissues of both grapevine samples. To our knowledge, this represents the first report of the occurrence of GVL in vineyards in France. Given the complex mixed infection present in the two analyzed grapevines, no conclusions can be drawn on the pathogenicity of GVL. Further efforts are needed to better understand GVL distribution and its potential pathogenicity to grapevine. References Alabi, O J., et al. 2020. Arch. of Virol. 165:1905-1909. Ben Amar, A., et al. 2020. Plant disease 104:3274. Debat, H., et al. 2019. Eur J Plant Pathol. 155:319. Diaz-Lara, A., et al. 2019. Arch. of Virol. 164:2573. Acknowledgments The authors are grateful to the “Plan National Dépérissement du Vignoble” (Mycovir project) for the financial support

Transmission of Grapevine leafroll-associated virus-1 (Ampelovirus) and Grapevine virus A (Vitivirus) by the Cottony Grape Scale, Pulvinaria vitis (Hemiptera: Coccidae)

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]−1, −3, and −4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV−1 and GVA. This is the first report of GLRaV−1 and GVA transmission from grapevine to grapevine by this species.

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 over the 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 and grafted grapevine cultivars, originating from different geographic regions that are representing important viticultural areas of Greece. Three new RT-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.

Distribution and Genetic Diversity of Grapevine Viruses in Russia

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.

Distinct responses of Vitis aestivalis ‘Norton’ and Vitis vinifera ‘Kishmish Vatkana’ to seven viruses revealed by small RNA sequencing

Grapevines are frequently infected by multiple viruses. Our previous study showed that ‘Norton’ grapevine (Vitis aestivalis) is resistant to grapevine vein clearing virus, a DNA virus in the family Caulimoviridae. To study the reaction of ‘Norton’ to RNA viruses, we transferred seven RNA viruses to ‘Norton’ from ‘Kishmish Vatkana’ (‘KV’) (Vitis vinifera) via graft-transmission. We profiled viral small RNAs (vsRNAs) of the seven viruses and compared viral titers in ‘Norton’ and ‘KV’. Total vsRNAs of grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, grapevine virus A (GVA) and grapevine Pinot gris virus (GPGV) were significantly less abundant in ‘Norton’ than in ‘KV’, but total vsRNAs of grapevine fleck virus (GFkV) were more abundant in ‘Norton’ than in ‘KV’. Total vsRNAs of grapevine rupestris stem pitting-associated virus (GRSPaV) were not different between ‘Norton’ and ‘KV’. Grafting direction of ‘Norton’ to ‘KV’ or ‘KV’ to ‘Norton’ did not affect the quantity of vsRNAs. The genome coverage of GLRaV-1, GLRaV-2, GLRaV-3 and GVA vsRNAs was lower in ‘Norton’ than ‘KV’. The 21-nt and 22-nt classes of vsRNAs were predominant for all seven viruses. Virus quantification by qPCR indicated that GLRaV-1 was undetectable in ‘Norton’, GLRaV-2, GLRaV-3, and GVA were less abundant in ‘Norton’, but GFkV was more abundant in ‘Norton’ than in ‘KV’. These results demonstrated that ‘Norton’ grapevine suppresses GLRaV-1, GLRaV-2, GLRaV-3, and GVA, but supports GFkV in comparison with ‘KV’. This study revealed new facets of complex molecular interactions between grapevines and multiple viruses.

Predominance and Diversity of GLRaV-3 in Native Vines of Mediterranean Croatia

Sixteen grapevine cultivars from Mediterranean Croatia were surveyed for the presence of 10 of the most economically important grapevine viruses. The presence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine leafroll associated virus-1, -2, and -3 (GLRaV-1; GLRaV-2 and GLRaV-3), Grapevine virus A (GVA) and B (GVB), Grapevine fleck virus (GFkV), Grapevine rupestris stem pitting associated virus (GRSPaV), and Grapevine Pinot gris virus (GPGV) were tested by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). All 71 analyzed clones were positive for the presence of one or more viruses. The most abundant one, detected in almost 95% of samples was GLRaV-3. In most of cases it was reported in mixed infections with GVA, GRSPaV, and GPGV. Virus genomes of GLRaV-3 infected vines were further characterized molecularly in order to determine their genetic diversity. Different genomic variants of heat shock 70 protein homologue (HSP70h) were identified by single-strand conformation polymorphism (SSCP) and sequenced. Sequence analysis confirmed their clustering into phylogenetic group I and/or phylogenetic group II. This study emphasizes the wide virus heterogenicity in Mediterranean vines and the predominant presence of GLRaV-3 phylogenetic groups I and II, either individually or in combination.

Spatial Distribution Patterns of Parthenolecanium corni (Hemiptera, Coccidae) and of the Ampelovirus GLRaV-1 and the Vitivirus GVA in a Commercial Vineyard

Distribution patterns of the European fruit lecanium Parthenolecanium corni (Bouché) and of grapevine leafroll-associated virus-1 (GLRaV-1) and grapevine virus A (GVA) were monitored from 2003 to 2015 in a Riesling vine plot in the northeast of France. Virus spread was compared between two periods: 2003–2008 and 2009–2014. The percentage of infected vines increased from 54 to 78% for GLRaV-1 and from 14 to 26% for GVA. The spatial distribution of viruses and of P. corni was analysed using permutation tests and revealed an aggregative pattern. Virus distribution was not associated with the density of P. corni population on grapevines. However, GLRaV-1 and GVA spread mainly from initially infected vines. New GLRaV-1 and GVA infections were more frequent on vines near primarily infected vines, first anisotropically along the row, then between neighbouring rows. Virus spread was similar to those described in literature with grapevine mealybug species. This slow vine-to-vine progression suggests that P. corni was responsible for the virus spread, in accordance with the low mobility and low transmission capacities of its local population.

Virus Pathogens in Australian Vineyards with an Emphasis on Shiraz Disease

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on “Shiraz Disease” (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.