First Report of Grapevine leafroll-associated virus 2 in Russian Grapevines (Vitis vinifera)

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Grapevine leafroll-associated virus 2 (GLRaV-2), a member of the genus Closterovirus within the family Closteroviridae, is associated with leafroll, young vine decline, graft union-incompatibility, and rootstock stem lesion disease (Poojari et al. 2013). The survey of a vineyard north of the Krasnodar region was conducted in September 2017. Twenty-six samples (one sample from each vine) with leaf curling, yellow or red leaves were collected from five table Vitis vinifera cultivars: Landish (five samples), Uybilei Novocherkasska (five samples), Kolobok (five samples), Livia (five samples), and Veles (six samples). Laboratory testing of the most important viruses (Grapevine virus A, Grapevine rupestris stem pitting-associated virus [GRSPaV], GLRaV-1, GLRaV-2, and GLRaV-3) was done by reverse transcription-polymerase chain reaction (RT-PCR) as described previously (Misbeh et al. 2007; Porotikova et al. 2016; Terlizzi et al. 2010). For GLRaV-2 detection, V2dCPf2 (5′-ACGGTGTGCTATAGTGCGTG-3′) and V2CPrl (5′-GCAGCTAAGTACGAATCTTC-3′) primers were used to amplify a 515-bp fragment of the major coat protein (CP) gene (Bertazzon et al. 2004). As a result, two samples tested positive for GLRaV-3, three samples tested positive for GRSPaV, and four samples (∼15% of total samples taken) of V. vinifera cultivar Livia tested positive for GLRaV-2 by PCR. No information was available on the presence of GLRaV-2 in Russia before we focused on GLRaV-2. RT-PCR amplicon of GLRaV-2 was sequenced in both directions with V2dCPf2 and V2CPrl primers (accession no. MH074870). BLAST analysis revealed 99% identity at the nucleotide level with GLRaV-2 isolate KP704586.1 from GenBank. Restriction fragment length polymorphism analysis of the sequenced isolate revealed it as belonging to GLRaV-2 group 3 (Bertazzon et al. 2004). Testing by enzyme-linked immunosorbent assay the 26 samples using antibodies specific to GLRaV-2 (AC Diagnostics, U.S.A.) confirmed the presence of GLRaV-2 in the three samples. Both positive and negative controls from AC Diagnostics were used (V208-P1 and V208-N1, respectively). The optical density range at 405 nm was 1.0 to 1.3 in the positive samples tested and 1.9 in the positive control. The plants that tested positive for GLRaV-2 had specific symptoms corresponding to red color of leaves started from the edge and chlorosis on the young leaves. This is the first report of GLRaV-2 in grapevines in Russia. This report contributes to increased awareness of the occurrence of the pathogen. Because GLRaV-2 can be transmitted through grafting, this report is important for GLRaV-2 containment by using of virus-free plants for propagation.