First Report of Grapevine Rupestris Vein Feathering Virus in Grapevine in Slovakia

Abstract

Grapevine rupestris vein feathering virus (GRVFV), a tentative member of the genus Marafivirus (family Tymoviridae), was originally reported from a Greek source of Vitis vinifera ‘Sultanina’ displaying asteroid-like symptoms and eliciting vein feathering symptoms upon grafting on V. rupestris (El Beaino et al. 2001; Ghanem-Sabanadzovic et al. 2003). In August 2017, samples consisting of main veins of fully developed leaves of two grapevine plants were used for total RNA isolation using a Spectrum Plant Total RNA Kit (Sigma). Ribosome-depleted RNA preparations were used for cDNA library synthesis (Nextera XT, Illumina) followed by high-throughput sequencing (HTS) on an Illumina MiSeq platform (300-bp paired-end sequencing). BLAST analyses of contigs assembled from HTS reads using Geneious 8.1.9 (1.1 and 1.3 M) from these grapevine samples, referred to as SK809 and SK933, indicated the presence of multiple virus infections, including GRVFV (a total of 137 and 288 reads, respectively), mapped to the reference GRVFV genome (NC_034205; Reynard et al. 2017). The contigs from SK809 (530 bp) and SK933 (1,959 bp) showed 82 and 85% identity, respectively, to GRVFV. BLAST analyses revealed the additional presence of grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine virus T in SK809 and GLRaV-2, GLRaV-3, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus-1, and hop stunt viroid in SK933. Although sample SK809 was collected from a 12-year-old symptomless V. vinifera (cv. Welshriesling), the SK933 tissue originated from a ∼30-year-old grapevine, of unknown origin, showing pronounced chlorosis and leafroll. To confirm the presence of GRVFV, previously unreported in Slovakia, a pool of grapevine samples from four localities in western Slovakia was screened by reverse transcription polymerase chain reaction (RT-PCR) with newly designed primers GRVFV_6090F (5′-CATCGTTCTGATCCTCAGCC-3′) and GRVFV_6605R (5′-AGAGACGCTGACCATGCCAC-3′). To this purpose, total RNAs from phloem scrapings obtained in February 2018 from dormant canes or total RNAs from 2016 to 2017 were used as a template for RT-PCR. A 515-nt fragment spanning the end of the polyprotein gene was amplified from 12 of the 30 samples tested and, as expected, also from newly extracted preparations from SK809 and SK933 grapevines. The specificity of RT-PCR products was ascertained by Sanger sequencing (GenBank accession nos. MH544687 to MH544700). In the corresponding genome region (nucleotide positions 6,110 to 6,603 after primer removal), the Slovak isolates shared 85.8 to 90.1% nucleotide identity with the reference Mauzac isolate (NC_034205), whereas the average divergence among 14 partial sequences of Slovak isolates reached 11.9% (±0.9%). Phylogenetic analysis of all corresponding available partial sequences in GenBank and 14 Slovak isolates suggested the existence of two molecular groups. These results indicate multiple introductions of GRVFV in Slovakia. The GRVFV-infected grapevines of different cultivars (Veltliner, Chasselas, Dornfelder, and Welschriesling) exhibited a range of symptoms (vein clearings, chlorosis, mosaics, and leafroll) or were symptomless. However, it was not possible to assess the potential role, if any, of GRVFV in the observed symptomatology at this stage, because all sources were infected with at least one additional virus. To our knowledge, this is the first report of GRVFV infecting grapevines in Slovakia.