Molecular characterisation of bois noir phytoplasmas from Bosnia and Herzegovina

Bois noir (BN) phytoplasmas were associated with grapevine yellows diseases in Bosnia and Herzegovina (Delic and Lolic, 2010). The disease is widely to moderate distributed in vineyards with both domestic and imported grapevine cultivars. Molecular characterisation of selected strains of BN phytoplasmas detected in heavily BN-infected vineyards in was done to verify the presence of genetic variability. Thirty-five DNA samples of grapevines that were shown to be positive by PCR assays on the 16S rDNA were selected for the study. Polymorphisms were studied in the 16S rDNA, Tuf and ribosomal protein genes of the selected samples. Tuf1f/r, TufAYf/r, and TufINT1f/TufINT4r primer pairs were used in nested PCR for amplification of Tuf genes; RFLP analyses with HpaII showed the presence of the tuf type-b (VK-II) in all the tested samples. The BN infected samples were further amplified with rp/STOL primers and PCR products were digested with TaiI and SspI restriction enzymes. Finally 16S rDNA genes were amplified with P1/P7 primers in direct PCR and with R16F2n/R16R2 primers in nested PCR assays: both types of amplicons obtained were restricted with MboII and RsaI, MboII, Hpy188I enzymes, respectively. Restriction profiles obtained after RFLP analyses on 16S rDNA and ribosomal protein amplicons weren't uniform. Some samples showed profiles comparable with some of those recently published (Contaldo et al., 2009), while some of the profiles of other samples were totally different from any of those reported in literature. Therefore, polymorphism of these regions should be further studied to better understand BN epidemiology in Bosnia and Herzegovina.