FIRST REPORT OF GRAPEVINE YELLOW SPECKLE VIROID1 INFECTING GRAPEVINES IN SLOVAKIA
Z. Vozárová, N. Sihelská, L. Predajňa, K. Šoltys, M. Glasa
Grapevine (Vitis spp.) has been shown to be a natural host for at least five viroid species belonging to the family Pospiviroidae (Di Serio et al., 2014). During a survey in 2016, samples collected from grapevines growing in the western part of Slovakia were individually analysed by Illumina next-generation sequencing of cDNA libraries prepared from total RNAs isolated from leaf petioles. Analysis of the sequence data from two grapevines (labelled as SK02 and SK503 and originated from cvs. Pinot gris and Dornfelder, respectively) revealed contigs covering the whole genome of Grapevine yellow speckle viroid-1 (GYSVd-1). To confirm the presence of GYSVd-1 in the original grapevine plants, two sets of GYSVd-1-specific primers were used for RT-PCR, i.e. GYSVd-1-mF/GYSVd-1-mR2 (Hajizadeh et al., 2012) and primers GYSVd-F (5'CCGGATCTTCTTGCTTGTGG3', sense)/GYSVd-R (5'GATTGCACTCGCGGGGCG3', anti- sense), newly designed from GYSVd1 sequences retrieved from GenBank (accessed in May, 2016). RT-PCR with both primer sets confirmed the presence of GYSVd-1 in the two grapevines and sequences identical to the initial Illumina contigs were obtained upon direct sequencing of PCR am- plicons (GenBank accession Nos. KY039378 and KY039380). Subsequently, 35 grapevines grown in different locations of western Slovakia were tested by RT-PCR for GYSVd-1 infec- tion, allowing the identification of three additional GYS- Vd-1 positive samples from unrelated vineyards (samples SK610, SK612 and SK657 from local white-berry varieties, KY039376-77 and KY039379). Comparison of the complete genomes of these five isolates, 366-368 nucleotides in size, showed pairwise nt identities of 94.2-99.5%. All five grape- vines were also tested for the presence of Hop stunt viroid (HSVd), Grapevine yellow speckle viroid-2 (GYSVd-2) and Citrus exocortis viroid (CEVd) by RT-PCR using primers described previously (Hajizadeh et al., 2012). These tests confirmed only the presence of HSVd in all samples. In addition, all the GYSVd-1-positive grapevines were shown to be simultaneously infected by two or more virus agents, complicating the determination of disease etiology. The phy- logenetic analysis of 154 complete sequences, including those determined in this study and those retrieved from databases, showed that GYSVd-1 clustered in four main groups, as previously suggested (Fajardo et al., 2016). The phyloge- netically-related Slovak isolates SK503, SK610, SK612 and SK657 were most closely related to GYSVd-1 isolates from Germany (X87910 and X89715-19) and the Czech Republic (KT000350). On the contrary, isolate SK02 clustered with GYSVd-1 isolates of Asian origin (KP993474 and JQ686712). To the best of our knowledge, this is the first report of GYS- Vd-1 presence in Slovakia, extending the information on its geographical distribution.