Surveys carried out to identify ‘bois noir’ (BN) phytoplasmas in grapevine yellows outbreaks in vineyards located in Serbia and in several Italian regions were carried out from 2008 to 2010. The presence of BN phytoplasmas was preliminarily demonstrated by RFLP analyses with TruI restriction enzyme on R16F2/R2 amplicons. About 60 samples were selected for further molecular characterization. Reference strains maintained in periwinkle were STOL (from Serbia), STOLC and STOL-PO (from France). RFLP analyses on R16F2/R2 amplicons with Hpy188I, Hpy8I, MboI, MboII, TruI, RsaI, BstUI, AluI, and Tsp509I restriction enzymes were carried out. Tsp509I was only differentiating one samples from Veneto region in Italy, while different profiles were observed in several of the examined samples when MboII, Hpy188I, and AluI were used. In particular, three different profiles were observed with AluI in samples from Veneto and Tuscany (Italy), and Serbia. Using MboII four different profiles (a-b-c-d) were observed in samples from Veneto, Tuscany and Emilia, while only one of them (profile a) was observed in the samples from Serbia. RFLP profiles of reference strains were a for STOL, b for STOL-PO and c for STOLC. Considering that the profile c is referring to an amplicons longer than the expected one, the presence of interoperon heterogeneity and/or of mixed BN strain infection could be hypnotized for this profile. Some of the samples having profile c when digested with Hpy188I showed further polymorphism. The reference strains plus 7 field collected BN strains chosen among those showing the above described polymorphisms were sequenced on the full 16S gene. The sequences were assembled using DNA STAR software, and compared with selected sequences of phytoplasmas in GenBank database using Blast N 2.2.18. Obtained aligned sequences ranged from 1,300 to 1,500 bp, all showing 99% homology among them self and with several of the 16SrXII-related strains deposited in Genbank. Virtual RFLP analyses on R16F2/R2 amplicons were carried out, using pDRAW32 program (AcaClone Software) and it was possible to confirm the variability detected in real RFLP analyses in the majority of BN sequenced strains. The comparison between real and virtual RFLP analyses showed in some cases different profiles when digested with MboII. Strain STOLC showed in this case a b profile confirming that profile c is formed by mixed infection or interoperon heterogeneity. On the other hand the presence of a d profile was detected in one of the sequenced strains from Serbia showing an a profile in RFLP analyses. BN strains differentiation on 16S rDNA on field collected uncloned amplicons indicates that the detected variability could be related with different epidemiological behaviours.
Molecular variability on 16S rDNA of ‘bois noir’ phytoplasmas in grapevine from Italy and Serbia
CONTALDO, NICOLETTA;DUDUK, BOJAN;PALTRINIERI, SAMANTA;BERTACCINI, ASSUNTA
2011
Abstract
Surveys carried out to identify ‘bois noir’ (BN) phytoplasmas in grapevine yellows outbreaks in vineyards located in Serbia and in several Italian regions were carried out from 2008 to 2010. The presence of BN phytoplasmas was preliminarily demonstrated by RFLP analyses with TruI restriction enzyme on R16F2/R2 amplicons. About 60 samples were selected for further molecular characterization. Reference strains maintained in periwinkle were STOL (from Serbia), STOLC and STOL-PO (from France). RFLP analyses on R16F2/R2 amplicons with Hpy188I, Hpy8I, MboI, MboII, TruI, RsaI, BstUI, AluI, and Tsp509I restriction enzymes were carried out. Tsp509I was only differentiating one samples from Veneto region in Italy, while different profiles were observed in several of the examined samples when MboII, Hpy188I, and AluI were used. In particular, three different profiles were observed with AluI in samples from Veneto and Tuscany (Italy), and Serbia. Using MboII four different profiles (a-b-c-d) were observed in samples from Veneto, Tuscany and Emilia, while only one of them (profile a) was observed in the samples from Serbia. RFLP profiles of reference strains were a for STOL, b for STOL-PO and c for STOLC. Considering that the profile c is referring to an amplicons longer than the expected one, the presence of interoperon heterogeneity and/or of mixed BN strain infection could be hypnotized for this profile. Some of the samples having profile c when digested with Hpy188I showed further polymorphism. The reference strains plus 7 field collected BN strains chosen among those showing the above described polymorphisms were sequenced on the full 16S gene. The sequences were assembled using DNA STAR software, and compared with selected sequences of phytoplasmas in GenBank database using Blast N 2.2.18. Obtained aligned sequences ranged from 1,300 to 1,500 bp, all showing 99% homology among them self and with several of the 16SrXII-related strains deposited in Genbank. Virtual RFLP analyses on R16F2/R2 amplicons were carried out, using pDRAW32 program (AcaClone Software) and it was possible to confirm the variability detected in real RFLP analyses in the majority of BN sequenced strains. The comparison between real and virtual RFLP analyses showed in some cases different profiles when digested with MboII. Strain STOLC showed in this case a b profile confirming that profile c is formed by mixed infection or interoperon heterogeneity. On the other hand the presence of a d profile was detected in one of the sequenced strains from Serbia showing an a profile in RFLP analyses. BN strains differentiation on 16S rDNA on field collected uncloned amplicons indicates that the detected variability could be related with different epidemiological behaviours.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
