Naslov
A comparison of stolbur phytoplasma isolates from Croatian grapevine by analyses of ribosomal and non-ribosomal gene regions

Autori
Šeruga, Martina ; Škorić, Dijana ; Kozina, Bernard ; Ćurković Perica, Mirna ; Krajačić, Mladen

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Extended abstracts of the 14th meeting of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine / Boscia, Donato - Locorotondo : Department of Plant Protection and Applied Microbiology, University of Bari, 2003, 96-96

Skup
14th meeting of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine

Mjesto i datum
Bari, Italija; Locorotondo, Italija, 12.09.2003. - 17.09.2003

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
stolbur; 16S rRNA gene; elongation factor EF-Tu gene; non-ribosomal regions; PCR; RFLP; SSCP; grapevine cultivars; phytoplasma relatedness

Sažetak
So far, the only detected etiological agent of phytoplasmoses in Croatian grapevines is a phytoplasma classified on the basis of the analysis of 16S rRNA gene region as a member of ribosomal subgroup 16SrXII-A (stolbur ; bois noir). Almost fifty stolbur phytoplasma isolates from various cultivars and different geographic regions of the country were used in this study in order to be compared and differentiated. Gene regions other than 16S rDNA were also analyzed: gene for the elongation factor EF-Tu and two stolbur-specific non-ribosomal regions amplified by primer pairs G35pm and STOL11f2/r1. Nucleic acids used for analyses were extracted by two procedures described in Prince et al. and Daire et al. with some modifications. Amplification of phytoplasma 16S rRNA gene was performed in a direct PCR by using R16F1/R0 universal phytoplasma primer pair. The nested PCRs were performed with R16F2/R2, 16R738f/R1232r and R16(I)F1/R1 primers, as already described. Major portion of the tuf gene coding for the elongation factor EF-Tu was amplified by employing the fTufu/rTufu primer pair. PCR conditions in all experiments were as in Schaff et al. with the exception of annealing temperature for the amplification of tuf gene that was 55°C in our experiments. A set of restriction enzymes (Tru9I, TspEI, AluI, KpnI) was used in routine RFLP analyses. The problem of finer differentiation of stolbur isolates was approached by using HMA (heteroduplex mobility assay, and SSCP (single-strand conformation polymorphism analysis. RFLP profiles of 16S rRNA amplicons for all of the isolates tested were the same regardless of the extraction method used. The tuf gene restriction patterns obtained by digestion of PCR product from samples extracted by modified Daire et al. revealed additional bands not resembling any other known phytoplasma profiles. The extra bands were not a result of an unspecific amplification visible in the PCR experiments and could be of non-phytoplasmal origin due to the less selective DNA extraction. Restriction analyses of other amplified regions were not informative enough to make a finer distinction. The results of HMA and SSCP analyses confirmed a very strong relatedness among our stolbur isolates from different grapevine cultivars. It would be interesting to determine their relationship with stolbur isolates from other, especially neighbouring countries.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA