The cpn60 gene as an additional marker for 'Candidatus Phytoplasma asteris' strain differentiation.

The phytoplasma classification was firstly established using RFLP analysis with a number of restriction enzymes on 1,200 bp amplicons in their 16S rDNA. Obtained phytoplasma 16Sr groups have been shown to be consistent with the groups (clades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, confirming phylogenetical validity of this grouping. However the recent introduction of ‘Candidatus’ status, which is based on 16S sequences, does not always provides the molecular distinction necessary for epidemiological studies towards disease control. For ‘Candidatus Phytoplasma asteris’-related phytoplasmas numerous different diseases, plant species and insect vectors were described and attributed to different 16SrI subgroups. However because of the highly conserved nature of the 16S rRNA gene, and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, more variable single copy genes were often shown to be more suitable for finer phytoplasma differentiation. Additional genes such as ribosomal protein (rp), secY, tuf, have been used for ‘Ca. P. asteris’-related strains differentiation corroborating the 16S rDNA designed subgroups. Several publically available sequences of the 3.6 kb phytoplasma fragments, containing cpn10, cpn60, amp and nadE genes allowed design of primers for specific amplification of 1,397 bp fragments containing phytoplasma cpn60 gene. Variability of amplified sequences was then studied in 27 ‘Ca. P. asteris’-related strains belonging to different 16SrI subgroups. The RFLP analyses of the amplified fragment with TruI and AluI restriction enzymes confirmed the reported differentiation among 16SrI-A, I-B, I-C, I-F and I-P subgroups, and showed further differentiation in strains assigned to 16SrI-A, 16SrI-B and 16SrI-C subgroups. However, analyses of cpn60 gene failed to discriminate strains in subgroups 16SrI-L and 16SrI-M (described on the basis of 16S rDNA interoperon heterogeneity) from strains in subgroup 16SrI-B, as it was also shown on tuf and rp genes. On the contrary, the 16SrI unclassified strain ca2006/5 from carrot (showing interoperon heterogeneity) was differentiable on both rp and cpn60 genes from the strains in subgroup 16SrI-B. These results indicate that interoperon sequence heterogeneity of strains AY2192, and PRIVA (16SrI-L), AVUT (16SrI-M), and ca2006/5 resulted in multigenic changes - one evolutionary step further - only in the latter case. Phylogenetic analyses carried out on full cpn60 gene (1,610 bp) are in agreement with 16Sr-based phylogeny, and confirmed also the further differentiation detected by RFLP analyses on specifically amplified and sequenced 1,397 bp fragments. Maximum sequence variability among the 16SrI group examined strains was 6.2% mismatches in cpn60 gene, while maximum reported sequence variability among the same phytoplasmas was 2.6% for 16S rDNA, 3.6% for tuf and 3.2% for rp genes.