Detection and characterization of GPGV and GYSVd-1 on Slovenian autochthonous and local grapevine varieties (Vitis vinifera L.)

Žlahtna vinska trta (Vitis vinifera L.) spada v družino vinikovk. V naravi jo okužuje okoli 70 virusov in sedem viroidov. Izmed sedmih viroidov sposobnih okužbe vinske trte, sta le dva, ki povzročita bolezensko stanje. V magistrskem delu smo preverili okužbo trt z virusom GPGV in viroidom GYSVd-1 pri sortah \u27Cipro\u27, \u27Rebula\u27, \u27Malvazija\u27, \u27Pokalca\u27, \u27Poljšakica\u27, \u27Volovnik\u27 in \u27Zeleni sauvignon\u27, in sicer z molekularno metodo RT-PCR in uporabo specifičnih začetnih oligonukleotidov. Na 15 vzorcih žlahtne vinske trte, od skupno 22, smo potrdili prisotnost virusa GPGV z dvema kompletoma začetnih oligonukleotidov. S parom začetnih oligonukleotidov Det-F1 in Det-R1 smo pomnožili lokus za proteinski plašč (CP), s parom Mer-F1 in Mer-R1 pa lokus za gibalni protein (MP). Pri določanju nukleotidnega zaporedja virusov vzorcev \u27Zeleni Sauvignon\u27 z bolezenskimi znaki in brez bolezenskih znakov smo določili razlike v posameznih bazah zaporedij. Na devetih vzorcih žlahtne vinske trte od skupno 13 smo potrdili prisotnost viroida GYSVd-1 s kompletom začetnih oligonukletidov (342P in 341Lj_M), od tega smo pri viroidih Rebule GPGV, Volovnika AV4 in Poljšakice AV dobili le delna zaporedja. Na osnovi dobljenih in urejenih zaporedij smo naredili filogenetsko analizo in ugotovili, s katerimi že identificiranimi in karakteriziranimi izolati v bazi podatkov (NCBI) se zaporedja naših vzorcev najbolj ujemajo. Pričakovano je večina zaporedij delila največje ujemanja z vzorci, ki so bili identificirani v Evropi. Izjema so bili trije vzorci, in sicer s trt Cipro AV1, Malvazija AV in Rebula 2TR. Cipro AV1 in Malvazija AV, ki smo jih pomnožili z začetnima oligonukletidoma Mer-F1 in Mer-R1, sta se v nukletidnem zaporedju najbolj ujemala z vzorcem, ki je bil identificiran na žlahtni vinski trti v ZDA. Vzorec Rebula 2TR, ki smo ga pomnožili z začetnima oligonukletidoma 342P in 341Lj_M, se je v zaporedju nukletidnega zaporedja najbolj ujemal z vzorcem s Kitajske.Grapevine (Vitis vinifera L.) is a species of a familyVitaceae. There are around 70 viruses and seven viroids infecting grapevine. In present study the infection of virus GPGV and viroid GYSVd-1 was verified on grapevine varieties \u27Cipro\u27, \u27Rebula\u27, \u27Malvazija\u27, \u27Pokalca\u27, \u27Poljšakica\u27, \u27Volovnik\u27 in \u27Zeleni sauvignon\u27 (Vitis vinifera L) using RT-PCR method and specific primers. 15 out of 22 samples of grapevine were positive on GPGV infection with two sets of specific primers. Resulting amplicons using specific primers Det-F1 and Det-R1 corresponded to a fragment of coat protein and Mer-F1 and Mer-R1 corresponded to a fragment of putative movement protein. We observed differences in the sequence of GPGV viruse obtained from symptomatic and nonsymptomatic grapevines of variety \u27Zeleni Sauvignon\u27. Nine out of 13 samples of grapevine were positive with GYSVd-1 infection, using speficific primers 342P and modified 341M designated as 341Lj_M. We got partial sequences on samples Rebula GPGV, Poljšakica AV and Volovnik AV4. Phylogenetic analysis was made with BLASTn sequence alignment against the non-redundant nucleotide NCBI collection and it revealed that most of sequences from our samples of grapevine are the most similiar to samples indentified and characterized in Europe. Exception were the samples Cipro AV1, Malvazija AV3 and Rebula 2TR. Samples Cipro AV1 and Malvazija AV3, amplified by using specific primers Mer-F1 and Mer-R1 showed the highest similarities with the isolate from USA. Rebula 2TR amplified by using primers 342P and 341Lj_M showed the highest similarity with an isolate from China