Multigene analysis of an aster yellows phytoplasma strain showing interoperon heterogeneity

Identification of phytoplasmas associated with carrot yellows in Serbia allow to identify 16SrI-A and 16SrI-B subgroups (Duduk et al., Annals of Applied Biology, 154, 219-229. 2009). However, PCR amplification of 16SrDNA followed by RFLP analysis, cloning and sequencing showed clearly presence of interoperon heterogeneity in one of the samples: two different sequences were obtained associated with two different RFLP profiles. Homology comparison among the sequences clustered in 16SrI-B clade showed that cloned sequences of the strain are closer to some other 16SrI-B strain sequences than to each other. Moreover phylogenetic analyses of the two operons showed that, while one operon is clustering in the 16SrI-B clade, the other operon is clustering out of it. The two operons of the same phytoplasma can be affiliated to different 16SrI subgroups according to RFLP analyses and this is supported also by phylogenetic analyses. Therefore, additional genes such as the l22 and s3 ribosomal protein genes, the tuf gene coding the elongation factor Ef-Tu, the putative aa kinase gene and ribosomal recycling factor gene, and a phytoplasma DNA helicase gene were studied to molecularly characterize this aster yellows strain from carrot. The RFLP and sequence analyses of PCR amplified ribosomal protein genes clearly showed that the strain is different from those affiliated with rpI-B and from all other strains in rpI subgroup tested. This strain was also differentiable from all other strains by RFLP analyses of putative aa kinase gene and ribosomal recycling factor gene, while analyses of tuf gene and of DNA helicase gene did not supported the difference and did not show any polymorphism, respectively. The presence of 16S rRNA interoperon sequence heterogeneity is not uncommon in phytoplasmas, and although the difference in homology between two operons is relatively small, when differences occur in restriction sites, misidentification or assignment of the same phytoplasma to two different 16S rRNA subgroups is possible. However, the use of other genes present as single copy in the phytoplasma genome can be helpful in discriminating when different phytoplasma populations are present in mixed infection from the presence of interoperon sequence heterogeneity.