Detection and partial molecural characterization of grapevine virus H from autochthonous vine varieties

Vinova loza (Vitis vinifera L.) poznata je kao kultura s najvećim brojem poznatih viroza, a samo manji broj virusa ima ekonomsku važnost te se oni povezuju s najvažnijim skupinama bolesti vinove loze, a jedna od njih je i naboranost drveta vinove loze. Kompleks naboranosti drveta vinove loze povezan je s vrstama roda Vitivirus u koje se ubraja i H-virus vinove loze (grapevine virus H, GVH). Istraživanje u kojem je testirano 113 trsova vinove loze, provedeno je na autohtonim sortama iz kolekcijskog nasada smještenog na vinogradarsko-vinarskom pokušalištu Jazbina. Detekcija virusa provedena je uporabom PCR metode u stvarnom vremenu te je potvrđena konvencionalnom RT-PCR metodom čiji su produkti vizualizirani nakon provedbe horizontalne gel elektroforeze. U ovom radu eksperimentalno je po prvi puta u Hrvatskoj dokazana prisutnost GVH u postotku zaraze od ~8% na sortama Gustopupica, Malvazija istarska, Muškatel, Babica plosnata, Brajdica bijela, Plavčina, Bljuzgavac, Svrdlovina crna i Kozak. Izolati su sekvencirani u oba smjera i to u dva područja genoma (RNA ovisna RNA polimeraza (RdRp) i protein omotača (CP)) te je njihova međusobna usporedba, kao i usporedba sa stranim izolatima, pokazala razlikovanja na nukleotidnoj i aminokiselinskoj razini. Unutar sekvenci hrvatskih izolata RdRp područja pronađena je sličnost između 95.5 do 99.15% na nukleotidnoj te 95.76 do 100% na aminokiselinskoj razini, dok je unutar CP područja sličnost varirala od 97.47 do 99.43% na nt i od 98.3 do 100% na ak razini. Filogenetskom analizom utvrđeno je grupiranje izolata prema geografskom podrijetlu samo kod sekvenci RdRp područja genoma.Grapevine (Vitis spp.) is known as the cultivated crop with the highest number of known viruses. Only a small number of them are of economic importance and they are associated with the most important groups of grapevine diseases, one of them is rugose wood complex. The rugose wood complex is associated with species of the genus Vitivirus, which also includes the grapevine virus H (GVH). The research, in which 113 grapevine plants were tested, was carried out on autochthonous varieties from a collection plantation located on the Jazbina viticultural-winemaking trial ground. Detection of the virus was performed by conducting real-time PCR method and subsequently confirmed by horizontal agarose gel electrophoresis of conventional RT-PCR amplified products. In this paper, the presence of GVH in the percentage of infection of ~8% was experimentally proven for the first time in Croatia in grapevine varieties Gustopupica, Malvazija istarska, Muškatel, Babica plosnata, Brajdica bijela, Plavčina, Bljuzgavac, Svrdlovina crna and Kozak variety. Two regions of the genome (RNA-dependent RNA polymerase (RdRp) and coat protein (CP)) of isolates were sequenced in both directions and by comparing them mutually and with foreign isolates, differences at the nucleotide and amino acid levels were shown. Sequences of 9 Croatian isolates of GVH in RdRp region showed a similarity between 95.5 and 99.15% at the nucleotide level and 95.76 to 100% at the amino acid level, while the sequence identity within the CP region ranged between 97.47 and 99.43% nt and 98.3 to 100% aa. Phylogenetic analysis revealed grouping of isolates by geographical origin only in the sequences of the RdRp region of the genome