First Report of Grapevine Red Globe Virus, Grapevine Rupestris Vein Feathering Virus, and Grapevine Syrah Virus-1 Infecting Grapevine in Slovenia.

Abstract

To investigate the virome status of preclonal grapevine candidates, 82 dormant cuttings of six grape cultivars ('Laški rizling', 'Refošk', 'Rebula', 'Malvazija', 'Zeleni Sauvignon' and 'Pokalca') were taken in 2019 from vines in second cycle of clonal selection in Vipavska dolina (Primorska wine-growing region). All samples were pooled in twelve groups (1x 'Laški rizling', 1x 'Pokalca', 2x 'Rebula', 2x 'Zeleni Sauvignon', 3x 'Malvazija', and 3x 'Refošk') from which small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Life Technolgies), twelve cDNA libraries were constructed and sequenced on an Ion Proton Sequencer (Ion Torrent, Life Technologies, Waltham, MA, USA). The VirusDetect pipeline (Zheng et al. 2017) was used for bioinformatics analysis and 3.6 - 13.9 million reads per library were processed. According to the VirusDetect pipeline, grapevine red globe virus (GRGV; genus Maculavirus, family Tymoviridae) was detected in two libraries of cv. 'Refošk'. In one library, GRGV showed the highest coverage of 63.6% and nt identity of 92.71% with GRGV isolate Graciano-T101 (accession no. KX171166) from Spain, and was represented by 40 contigs with sequence depth of 53.0X. In the second library, GRGV showed the highest coverage of 45.8% and nt identity of 94.49% with GRGV isolate Graciano-T90 (accession no. KX109927) from Spain, and was represented by 36 contigs with sequence depth of 22.9X. Grapevine rupestris vein feathering virus (GRVFV; genus Marafivirus, family Tymoviridae) was found in eleven libraries representing six different cultivars. In six libraries, GRVFV showed the highest coverage of 37%-85% and nt identity of 88.53%-94.05% with GRVFV isolate CHASS (accession no. KY513702) from Switzerland, and was represented by 34-90 contigs with sequence depth of 11.0X-93.3X. In the other four libraries, GRVFV showed the highest coverage of 27.5%-64.8% and nt identity of 92.36%-93.87% with GRVFV isolate Mauzac (accession no. KY513701) from France, and was represented by 30-94 contigs with sequence depth of 5.4X-20.7X, while in one library, GRVFV showed the highest coverage of 30.2% and nt identity of 95.30% with GRVFV isolate SK925 (accession no. MH544692) from Slovakia, and was represented by three contigs with sequence depth of 5.6X. Grapevine Syrah virus-1 (GSyV-1; genus Marafivirus, family Tymoviridae) was detected in two libraries of cvs 'Laški rizling' and 'Malvazija'. In the library of cv. 'Laški rizling', GSyV-1 showed the highest coverage of 44.9% and nt identity of 95.36% with GSyV-1 isolate SK351 (accession no. KP221269) from Slovakia, and was represented by three contigs with sequence depth of 20.9X. In the library of cv. 'Malvazija', GSyV-1 showed the highest coverage of 48.8% and nt identity of 92.06% with GSyV-1 isolate SK30 (accession no. KP221256) from Slovakia, and was represented by 25 contigs with sequence depth of 29.4X. Results obtained by HTS were confirmed in individual samples using reverse transcription-polymerase chain reaction (RT-PCR). GRGV was amplified using specific primer pair RG6061F: 5'-CCGAGCTTCTCTCCAAGATCA-3' and RG6801R: 5'-ACTTAACGTAGGCCACTGGGT-3' (Cretazzo and Velasco 2017). GRVFV was amplified using specific primer pair GRVFV_6090F: 5'-CATCGTTCTGATCCTCAGCC-3' and GRVFV_6605R: 5'-AGAGACGCTGACCATGCCAC-3' (Glasa et al. 2019). GSyV-1 was amplified using specific primer pair SY5922F: 5'-CCAATGGGTCGCACTTGTTG-3' and SY6295R: 5'- ACTTCATGGTGGTGCCGGTG -3' (Glasa et al. 2015). Three out of 13 samples of 'Refošk' tested for GRGV, 44 out of 78 samples tested for GRVFV (2 'Laški rizling', 12 'Refošk', 9 'Rebula', 7 'Malvazija', 8 'Zeleni Sauvignon' and 6 'Pokalca') and 3 out of 8 samples tested for GSyV-1 (1 'Laški rizling' and 2 'Malvazija') were positive. Further verification of the amplicons was performed by bidirectional Sanger sequencing and the generated sequences were submitted to GenBank: GRGV (MW446914-MW446916), GRVFV (MW446917-MW446938) and GSyV-1 (MW446939-MW446941). Phylogenetic analysis showed that Slovenian GRGV isolates clustered separately from other GRGV isolates retrieved from the NCBI. Phylogenetic analysis showed that some Slovenian GRVFV isolates clustered with those from France or Slovakia, while some of them clustered separately. Phylogenetic analysis showed that Slovenian GSyV-1 isolates clustered with GSyV-1 isolates from Hungary and Slovakia. To our knowledge, this is the first report of GRGV, GRVFV and GSyV-1 infecting grapevine in Slovenia. Further analysis will be necessary to assess the prevalence of these viruses in Slovenian vineyards and to evaluate their impact on grapevine production.