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

Detection, Transmission, and Characterization of Grapevine Virus H in Croatia

A survey of recently discovered vitiviruses was performed on 113 Croatian autochthonous grapevine cultivars from the national collection “Jazbina” using one-step RT-PCR. The presence of grapevine virus H (GVH) was confirmed in nine (7.9%) cultivars and grapevine virus G in eight (7.1%), while the presence of grapevine viruses I and J were not detected. GVH was transmitted by the vine mealybug (Planococcus ficus) from a source plant to grapevine seedlings with a 10.5% transmission rate using a combination of 10 first and second instars per plant with 48 and 72 h of acquisition and inoculation access period, respectively. Transmission correlated with the presence of grapevine leafroll-associated virus 3 (GLRaV-3) in the GVH-source plant and recipient seedlings. No alternative GVH host was identified. A comparison of 356 nt fragments of the RdRP and CP coding regions showed nucleotide identity between the Croatian GVH isolates in the range of 95.5–99.2% and 97.5–99.4% and amino acid identity between 95.8 and 100% and between 98.3 and 100%, respectively. Comparison with foreign isolates revealed nucleotide sequence similarity in the RdRP and CP between 94 and 100% and between 97.7–100%, respectively. To the best of our knowledge, this is the first report of GVH in Croatia and the first identification of the vine mealybug as a vector of GVH.

Incidence of 14 grapevine viruses in Korean vineyards

The incidence of grapevine virus infections in Korean vineyards was investigated from July to October, 2020. A total of 177 petiole samples were collected from two or three different cultivars in each of four different regions; these were examined by reverse transcription-polymerase chain reaction assay for the presence of 14 major viruses. The overall occurrence of grapevine viruses was 91.0%, and the level of incidence was high irrespective of region or cultivar. The predominant viruses were grapevine leafroll-associated virus 3 (80.2%), grapevine fleck virus (70.6%), and grapevine rupestris stem pitting-associated virus (49.2%). Most grapevines were infected with multiple viruses, suggesting that Korean vineyards are likely to suffer economic losses resulting from viral diseases. This is the first extensive survey performed in Korea to observe the outbreak status of diverse grapevine viruses; surveys of this type can provide important information for the management of grapevine viruses in Korea.

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.

First report of grapevine rupestris vein feathering virus in Vitis vinifera L. from Japan

The production of wine grapes is gaining widespread popularity and being carried out on approximately 2,200 hectares of land in Japan. Scions grafted onto rootstocks generally have been imported from the EU, USA, New Zealand, and Australia into Japan. Unfortunately, viruses have spread in Japanese vineyards by slipping through the net of plant quarantine. Grapevine rupestris vein feathering virus (GRVFV), which was detected in a Greek grapevine accessions, is a member of genus Marafivirus in family Tymoviridae (El Beaino et al. 2001). GRVFV has been detected in many countries such as USA, Canada, Australia, New Zealand, Italy, Spain, Switzerland, Czech Republic, Uruguay, and Pakistan (Jo et al. 2015; Eichmeier et al. 2016; Xiao and Meng 2016; Blouin and MacDiarmid 2017; Reynard et al. 2017; Cho et al. 2018; Mahmood et al. 2019; Wu et al. 2020). Herein we report GRVFV infection in Vitis vinifera L. grapevines from Japan. In February 2021, dormant canes from 18 V. vinifera cv. Cabernet Sauvignon with leafroll-like disease symptoms, growing in a vineyard located in Kanagawa Prefecture, were collected. No typical vein banding symptom by GRVFV were observed in the grapevines during the growing season. Total RNA was isolated from the canes using an RNeasy Plant Mini Kit and QIAshredder (Qiagen, Valencia, CA), and subjected to cDNA synthesis using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). RT-PCR was performed with GRVFV_6156F and GRVFV_6600R primers for GRVFV detection (Reynard et al. 2017). The expected 445 nucleotides (nt) amplification product was obtained from four of 18 grapevines. Sequence analysis of the products revealed 91% identities to corresponding sequences of GRVFV isolates CHASS (KY513702) and Mauzac (KY513701) from Switzerland. Genome walking to determine the whole-genome sequence of the GRVFV isolates from the four grapevines was performed. Briefly, the upstream and downstream of the 445 nt amplification product were amplified from first-strand cDNA using gene-specific primers designed from the product and CHASS-specific primers. Each amplified fragment was Sanger sequenced. Next, gene-specific primers were designed to obtain the complete genome of GRVFV as 13 overlapping DNA fragments from each of the four grapevine samples. An identical complete genome of 6,704 bp was assembled from the overlapping DNA fragments using MEGA 10 software and named as NA1 isolate (DDBJ accession no. LC619667). Phylogenetic analysis of the NA1 genome and corresponding sequences of GRVFV from other countries showed that NA1 formed a cluster with isolate NZ ChTK0004 from New Zealand (MF000326; Supplementary Figure 1). In pairwise comparisons, the complete NA1 genome was most identical at 88% and 87%, respectively to isolates NZ ChTK0004 and Mauzac. The predicted amino acid sequences of NA1 polyprotein shared high homologies (96%) to the corresponding polyprotein sequences of NZ ChTK0004 and Mauzac, suggesting that NA1 is genetically similar to GRVFV isolates from New Zealand and Switzerland. The NA1-infected Cabernet Sauvignon was co-infected with Grapevine leafroll-associated virus 3, Grapevine virus A, and Grapevine rupestris stem pitting-associated virus according to RT-PCR assay for grapevine virus detection (Nakaune and Nakano 2006). The results underscore the importance of intensifying quarantine measures to prevent introduction of exotic viruses via contaminated wine grape vegetative cuttings.

Variable Populations of Grapevine Virus T Are Present in Vineyards of Hungary

Grapevine virus T (GVT) is a recently described foveavirus, which was identified from a transcriptome of a Teroldego grapevine cultivar in 2017. Recently, we surveyed vineyards and rootstock plantations in Hungary using small RNA (sRNA) high-throughput sequencing (HTS), at a time when GVT had not yet been described. A re-analysis of our sRNA HTS datasets and a survey of grapevines by RT-PCR revealed the presence of GVT in most of the vineyards tested, while at rootstock fields its presence was very rare. The presence and high variability of the virus in the country was confirmed by sequence analysis of strains originating from different vineyards. In this study, we demonstrate the presence of GVT in Hungary and show its high diversity, suggesting that GVT presence may not seriously affect grapevine health and that it could have been present in European vineyards for a long time as a latent infection.

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.

Biological evidence and molecular modeling of a grapevine Pinot gris virus outbreak in a vineyard

Since its identification in 2003, little has been revealed about the spread of grapevine Pinot gris virus (GPGV), an emerging grapevine virus. According to studies from Italy, GPGV transmission in the vineyard can be fast but progressive over the years. To gain new insights into the spread of GPGV infections, we tested 67 grapevines in a single vineyard parcel in southern France. These vines were sampled over eight years (2013-2020) and tested for GPGV by RT-PCR using a new primer pair designed from the recently described genetic diversity of GPGV worldwide. While focusing on a portion of the samples (20), we observed a drastic increase in newly GPGV-infected vines from 2014 (5%, 1 of 20) to 2015 (80%, 16 of 20) and 2016 (90%, 18 of 20). Infected vines were scattered throughout the vineyard with no distinct pattern of distribution and some rare vines remained negative through 2020. Using all available genomic information, we performed Bayesian-based phylogeographic analyses that identified a major intra-vineyard transmission in 2014-2015. To test our model, we analyzed 47 additional grapevines and confirmed the outbreak of GPGV in 2015, validating our in-silico projection. Interestingly, some grapevines remained negative throughout the study, in spite of their close proximity to infected plants. These results raise questions on the dynamic of vector populations and environmental conditions that may be required for virus spread to occur in the vineyard.