Genetic diversity of flavescence dorée and closely related phytoplasma strains of the 16SrV taxonomic group in Europe.

In addition to the grapevine flavescence dorée phytoplasmas (ribosomal subgroups 16SrV-C and -D), other closely related members of group 16SrV infect Alnus spp. (16SrV-C), Clematis vitalba (16SrV-C), Spartium junceum (16SrV-C) and Rubus spp. (16SrV-E) in Europe. To investigate which phytoplasmas constitute discrete, species-level taxa, a high number of strains, mainly from grapevine and clematis, were analysed comparing their 16S rDNA and a set of two to five house-keeping genes. Whereas 16Sr DNA identities were well above 97.5% the threshold aimed to distinguish two ‘Candidatus Phytoplasma’ species, phylogenetic analysis of the combined sequences of tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genes showed that two discrete phylogenetic clusters can clearly be distinguished. The first cluster grouped flavescence dorée (FD), alder yellows (AldY), clematis and the Palatinate grapevine yellows (PGY) phytoplasmas, and the second cluster was constituted by Rubus stunt phytoplasmas. High genetic diversity was present among phytoplasma strains belonging to the first cluster and several subclusters could be recognized; whereas among Rubus stunt phytoplasmas all analyzed gene sequences resulted highly conserved. Studies based on sequencing, phylogenetic and RFLP analyses of 16S rDNA, rplV-rpsC and rpl15-secY gene sequences carried out mainly on flavescence dorée phytoplasma strains from Italy, France and Serbia confirmed the presence of genetic variability in phytoplasmas from both Flavescence dorée ribosomal subgroups. These subgroups were only differentiated for the presence of a SNP corresponding to a TaqI restriction site, however this difference enable to distinguish their geographical and epidemic distribution. Analyses on rplV-rpsC and rpl15-secY gene reinforced the clear strain differentiation between flavescence dorée phytoplasma strains of 16SrV-C and –D subgroups. Moreover it was proved the presence of about 10 different flavescence dorée phytoplasma strains in 16SrV-C subgroup, and of 3 different phytoplasma strains in 16SrV-D. In addition to their specificity of insect vector (Scaphoideus titanus and Oncopsis alni for phytoplasmas of the first cluster and Macropsis fuscula for Rubus stunt phytoplasma in the second cluster), the genomes of the phytoplasma strains belonging to the two distinct clusters above mentioned are differentiated enough to propose two novel putative taxa, ‘Candidatus Phytoplasma caudwellii’ for FD, AldY, PGY and clematis phytoplasmas, and ‘Candidatus Phytoplasma rubi’ for rubus stunt phytoplasmas. However, if the rules for the taxonomy of uncultured bacteria were to be strictly applied, this proposition would be not completely acceptable. Indeed, at the 16S rDNA level Rubus stunt phytoplasma can be described as a new species with several specific nucleotide polymorphisms in its 16S rDNA respect to 16SrV-C/D phytoplasmas, in addition to the clear speciation on the five non ribosomal genetic markers. On the contrary, the members of subgroup 16SrV-C and D (FD, AldY, Clematis vitalba and PGY phytoplasmas) could not be described as a new species because no specific oligonucleotide polymorphism can be found in their 16S rDNA. Therefore the entire rules as they stand could not be fulfilled for flavescence dorée and closely related phytoplasma isolate, except if a new rule more based on the genomo-species concept was to be established. This is quite relevant, especially considering that FD is a quarantine-regulated pathogen in Europe.