First Report of Grapevine virus A and Grapevine fleck virus in the Former Yugoslav Republic of Macedonia

Grapevine (Vitis vinifera L.) is an economically important crop and can host several different viruses, including those that have to be excluded from certified propagating material in Europe. Among these, the Vitivirus Grapevine virus A (GVA) and the Maculavirus Grapevine fleck virus (GFkV) are phloem-limited viruses that are associated with two different grapevine diseases, Kober stem grooving, belonging to the rugose wood complex, and fleck diseases, respectively. During a survey conducted in 2012 in the former Republic of Macedonia, symptomatic plants with reddening of leaves were collected for laboratory analyses. In this study, grapevine red varieties (Vranec, Francovka, and Pinot noir) from four different localities (Stip, Kavadarci, Valandovo, and Gevgelija) in Macedonia were examined. Thirty-four samples were analyzed by DAS-ELISA using commercially antibodies against Grapevine leafroll associated virus-3 (GLRaV-3). Ten selected samples were processed through DAS-ELISA and molecular assays also for the presence of GVA and GFkV. Total RNA was extracted as previously described (2) and retro-transcribed (RT) using random primers followed by PCR assay with primers GVA-MP (5'-GCCAGAGGTGTTTGAGACAAT-3') and GVA-CPdt (5'-TTTTGTCTTCGTGTGACAACCT-3') (1), which amplified a GVA-specific fragment of 986 bp, and with primers GFkV-U279 (5'-TGGTCCTCGGCCCAGTGAAAAAGTA-3') and GFkV-L630 (5'-GGCCAGGTTGTAGTCGGTGTTGTC-3') (3), which amplified a GFkV-specific region of 315 bp. Results from DAS-ELISA test showed the presence of GLRaV-3 in 21 tested samples and of GVA and GFkV in six and three out of 10 selected samples, respectively. GVA was found in Vranec and Francovka vines sampled in all the locations mentioned before, while GFkV was detected in Vranec and Pinot noir vines, in Stip, Kavadarci, and Gevgelija. These latter results were confirmed by RT-PCR assays; then, four GVA-specific and three GFkV-specific amplicons were sequenced from both directions to get a 3× coverage. For GVA fragment, a primer pair designed in the internal part of the sequence was also used. BLASTn analyses showed that (i) PCR products amplified with GVA-specific primers shared best nucleotide sequence identities, ranging from 91.7 to 93.7%, with GVA isolate at GenBank Accession No. X75433; (ii) PCR products amplified with GFkV-specific primers shared best nucleotide sequence identities, from 92.5 to 94.7%, with GFkV isolate at AJ309022. These evidences reinforced the serological and PCR resul