‘Bois noir’ (BN) - associated phytoplasmas were detected in Hungary since long time (Kolber et al., 1997), recently the presence of polymorphisms in some of the genes used as markers for strain differentiation was also reported (Contaldo et al., 2009). To further verify the consistence of molecular variability BN infected samples were collected in 2008-2010 from symptomatic grapevine plants from vineyards of variety Zweigelt located in Sopron area (near to the Austrian border), varieties Riesling-Sylvaner and Chardonnay from Etyek (near Budapest), unknown variety from Southeast Hungary, and from a variety Chardonnay from Eger. PCR/RFLP characterization was carried out on tuf gene (Langer and Maixner, 2004) using nested-PCR procedures, and restriction with HpaII enzyme showed that all the strains belong to tuf type-b (VK-II). Further molecular characterization on 16S rDNA gene, spacer region and beginning of 23S gene were carried out on 12 selected samples by RFLP analyses with TruI, BstUI, Hpy188I, and MboII restriction enzymes on diverse length amplicons. RFLP analyses on R16F2/R2 by BstUI and Hpy188I did not show differences among BN samples. The same amplicons digested with MboII show variability among BN strains: in particular three different profiles (a, b, and d) were observed. While the a profile was present in 4 samples, profile d was observed in 2 samples, and the c profile was observed in all the others. This last profile is very likely derived from a presence of a mixed infection by two BN strains recently differentiated on reference strains STOL and STOL-C (Contaldo et al., 2009, 2011). Selected grapevine samples were sequenced on 16S ribosomal region (about 1,500 bp), and virtual RFLP analyses carried out on R16F2/R2 amplicons showed in some cases differences between real and virtual RFLP profiles confirming the presence of mixed infection in samples with profile c. After Genbank search similar analyses on deposited BN strains showed identical Hpy188I profiles among a sample from Hungary and BN samples from Canada and Spain (EU086529 and AJ964960). These results clearly indicate that among BN strains belonging to tuf type-b there is a strain differentiation on 16S gene achievable using selected restriction enzymes. Further studies will clarify the epidemiological relevance of these strains in BN epidemics.
Acs Z., N. Contaldo, I. Ember, S. Paltrinieri, M. Kolber, B. Duduk, et al. (2011). ‘Bois noir’ in Hungary: tuf type b strain variability on 16S ribosomal gene. Roma : CRA, Centro di Ricerca Per la Patologia Vegetale.
‘Bois noir’ in Hungary: tuf type b strain variability on 16S ribosomal gene
CONTALDO, NICOLETTA;PALTRINIERI, SAMANTA;DUDUK, BOJAN;BERTACCINI, ASSUNTA
2011
Abstract
‘Bois noir’ (BN) - associated phytoplasmas were detected in Hungary since long time (Kolber et al., 1997), recently the presence of polymorphisms in some of the genes used as markers for strain differentiation was also reported (Contaldo et al., 2009). To further verify the consistence of molecular variability BN infected samples were collected in 2008-2010 from symptomatic grapevine plants from vineyards of variety Zweigelt located in Sopron area (near to the Austrian border), varieties Riesling-Sylvaner and Chardonnay from Etyek (near Budapest), unknown variety from Southeast Hungary, and from a variety Chardonnay from Eger. PCR/RFLP characterization was carried out on tuf gene (Langer and Maixner, 2004) using nested-PCR procedures, and restriction with HpaII enzyme showed that all the strains belong to tuf type-b (VK-II). Further molecular characterization on 16S rDNA gene, spacer region and beginning of 23S gene were carried out on 12 selected samples by RFLP analyses with TruI, BstUI, Hpy188I, and MboII restriction enzymes on diverse length amplicons. RFLP analyses on R16F2/R2 by BstUI and Hpy188I did not show differences among BN samples. The same amplicons digested with MboII show variability among BN strains: in particular three different profiles (a, b, and d) were observed. While the a profile was present in 4 samples, profile d was observed in 2 samples, and the c profile was observed in all the others. This last profile is very likely derived from a presence of a mixed infection by two BN strains recently differentiated on reference strains STOL and STOL-C (Contaldo et al., 2009, 2011). Selected grapevine samples were sequenced on 16S ribosomal region (about 1,500 bp), and virtual RFLP analyses carried out on R16F2/R2 amplicons showed in some cases differences between real and virtual RFLP profiles confirming the presence of mixed infection in samples with profile c. After Genbank search similar analyses on deposited BN strains showed identical Hpy188I profiles among a sample from Hungary and BN samples from Canada and Spain (EU086529 and AJ964960). These results clearly indicate that among BN strains belonging to tuf type-b there is a strain differentiation on 16S gene achievable using selected restriction enzymes. Further studies will clarify the epidemiological relevance of these strains in BN epidemics.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
