Accepted for publication
First Report of Grapevine virus A (GVA) and Grapevine fleck virus (GFkV) in the Former Yugoslav Republic of Macedonia
Goce Delcev - University, Faculty of Agriculture, Department for Plant and Environmental Protection, Stip, Macedonia, The Former Yugoslav Republic of; firstname.lastname@example.org
Goce Delcev - University, Faculty of Agriculture, Department for Plant and Environmental Protection, Stip, Macedonia, The Former Yugoslav Republic of; email@example.com
Università degli Studi, Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territorio, Agroenergia, Milano, Italy; firstname.lastname@example.org
Università degli Studi, Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territorio, Agroenergia, Milano, Italy; email@example.com
University of Milan, Department of Agricultural and Environmental Sciences, via Celoria 2, Milan, MI, Italy, 20133; firstname.lastname@example.org
Grapevine (Vitis vinifera L.) is an economically important crop and can host several different viruses, including those ones that have to be excluded from the 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 the season 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 analysed 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 (1) and retro-transcribed (RT) using random primers followed by PCR assay with primers GVA-MP (5’-GCCAGAGGTGTTTGAGACAAT-3’) and GVA-CPdt (5’-TTTTGTCTTCGTGTGACAACCT-3’) (2) 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 twenty one 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 3X coverage. For GVA fragment also, a primer pair designed in the internal part of the sequence was 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 Accession No. AJ309022. These evidences reinforced the serological and PCR results indicating that GVA and GFkV were identified in examined grapevine plants in this study. Nucleotide sequences of GVA (accession numbers from KF594432 to KF594435) and GFkV (accession numbers from KF594429 to KF594431) were submitted to GenBank. To our knowledge, this is the first report of GVA and GFkV grapevine viruses in the Former Yugoslav Republic of Macedonia. References (1) MacKenzie D. J., McLean M.A., Mukerji S., and Green M. 1997: Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription – Polymerase Chain Reaction, Plant disease, Vol 81 No 2, 222–226. (2) De Meyer, J., Caudin, M., Bourquin, L., Jakab, G., Malnoe, P., Gugerli P., 2000: New primers for the molecular identification and detection of grapevine virus A (GVA), 138-140. Extended Abstracts, 13th Meeting of ICVG, Adelaide, 12-17 March 2000. (3) Shi B. J., Habili N. and Symons R. H. 2003. Nucleotide sequence variation in a small region of the Grapevine fleck virus replicase provides evidence for two sequence variants of the virus. Ann. Appl. Biol. 142:349-355.