A Survey of Grapevine Leafroll-Associated Virus 1 and 3 in the South of Ukraine and Development of Primers for GLRaV-3 Identification

Abstract

Viticulture is one of the most intensive and complex branches of Ukraine agriculture. Grapevine virus diseases are responsible for considerable economic losses to grape productivity and wine industries. One of the most notable and widespread viruses associated with vine leafroll disease is grapevine leafroll-associated viruses (GLRaV), belonging to the genus Ampelovirus, family Closteroviridae. Thus, the aim of this study was to conduct a survey targeting two viruses involved in the grapevine leafroll, namely grapevine leafroll-associated virus 1 (GLRaV-1) and virus 3(GLRaV-3) distributed in commercial wine grapes growing in the vineyards of the Ovidiopol and Bolhrad districts of the Odesa region. For efficient and accurate virus detection, we aimed to design universal primers based on conserved nucleotide sequences. Methods. Virus surveys of vineyards, visual diagnosis, immunoassay (ELISA), polymerase chain reaction with reverse transcription (RT-PCR), and Sanger sequencing of partial genome sequences of GLRaV. Results. The results obtained indicate that grapevine leafroll disease symptoms in field-grown grapevines in the south of Ukraine are caused by GLRaV-3. GLRaV-1 was not detected in any of the samples tested. To confirm the presence of GLRaV-3 in the samples, specific primers were designed targeting the coat protein region (GLRaV3-7f 5’-AGTAGGGGATGCAGCACAAG-3’; GLRaV3-7r 5’-ATCCAAAGCTATTCCCTTGC-3’) of the virus. A new set of primers (GLRaV3-7f / GLRaV3-7r) has been validated for sensitive detection of GLRaV-3 by RT-PCR and may be useful for routine virus detection in the laboratory as well as for large-scale testing. The partial coat protein gene of the isolate, GLRaV-3 ukr, distributed in the south of Ukraine, was sequenced, and the obtained sequence was deposited in GenBank under Acc. No. ON015835. The phylogenetic study demonstrated that GLRaV-3 ukr was closely related to isolates from Russia (MZ065372 and MZ065370).