Intra and extragenomic variation between 16S rRNA genes found in 16SrI-B-related phytopathogenic phytoplasma strains

Phytoplasmas are bacterial pathogens that pose a threat to cereal and vegetable production worldwide. However, there is limited information regarding the occurrences of phytoplasmas in Central Europe. In this study we examined samples taken from crop and ornamental plants showing symptoms indicative of phytoplasma infection. Phytoplasmas related to 'Candidatus Phytoplasma asteris' were detected in eight plant species (carrot, lupine, pea, cauliflower, mustard, maize, juniper and marigold). Based on the restriction fragment length polymorphism analysis of the 16S rDNA, the phytoplasmas were classified as subgroup 16SrI-B, 16SrI-L or a complex strain, which harboured the I-B and I-L-type 16S rRNA genes. Individual 16S rDNA sequences were analysed from those heterogeneous strains and as many as five genetic variants were distinguished. By operon-specific amplification of the 16S rRNA genes with primers complementary to RP166 phytoplasma genome, we detected 16SrI-L/B phytoplasma strains in the multivariant samples. The I-L-type sequences were amplified from rrnA and the I-B-type sequences were amplified from rrnB. Molecular phylogeny based on the 16S rRNA showed that all the detected phytoplasma variants were clustered with a 'Ca. Phytoplasma asteris' subclade and were closely related to the 16SrI-B subgroup. Despite the variability of 16S rDNA, the strains from all the eight crops shared identical tuf gene sequences. We report, for the first time, 16SrI-B phytoplasma infecting lupine, field pea, cauliflower and mustard, 16SrI-L phytoplasma infecting maize and field pea and 16SrI-L/B phytoplasma infecting carrot, lupine and marigold. We further demonstrate variability in the 16S rRNA genes found in the 16SrI-B-related phytoplasma genomes worldwide. For clearer and more unambiguous nomenclature, we propose that in the future, the more intuitive subgroup 16SrI-L/B be used for heterogenic phytoplasma strains instead of 16SrI-(B/L)L.