Identification of 16SrKSII-A and 16SrKSII-H phytoplasma subgroup in Russia, using 16S rDNA NGS analysis

The RFLP analysis of the 16S rDNA revealed two distinct phytoplasma groups, 16SrXII-A and 16SrXII-H, suggesting the presence of diverse phytoplasma strains within the analyzed isolates from the Russian State Collection of Pathogenic Microorganisms. Partial 16S rDNA sequences of four phytoplasma, 'Candidatus Phytoplasma', isolates belonging to the 16SrXII group from the three plant species: common hop (Humulus lupulus L.), Indian datura (Datura metel L.) and grapevine (Vitis vinifera L.) are presented. DNA of gene coding 16S rRNA was amplified, using nested PCR with primers P1/16Sr-SR and R16F2n/R16R2.Fragment of 16S rDNA of the 1.2 Kb was eluted from the corresponding band after agarose gel electrophoresis, purified and used to generate libraries. Sequencing of DNA libraries was performed on a MiSeq sequencer, while assembly of sequences was carried out using the QIAGEN CIC Genomics Workbench software. To compare 16S rDNA sequences with reference strain, the MEGA 5.2 program was used. Two phytoplasma isolates from grapevine (NCBI acc. nos. OQ120463 and OQ120464) and one from datura (OQ130742) have shown 100% and 99.9% similarity to the reference strain and to previously deposited phytoplasma isolates from Russia infecting potato (Solanum tuberosum L.) and alfalfa (Medicago sativa L.), as well as 99.9-99.7% similarity to 'Ca. P. solani' isolates from grapevine from Spain, Italy, Georgia, and from tobacco in Serbia. Phytoplasma isolate sequenced from common hop belonged to the 16SrXII-H subgroup, closely related to 'Ca. P. convolvuli'. This type of phytoplasma was discovered and sequenced for the first time on the territory of the Russian Federation. Its similarity to the species 'Ca. P. solani' did not exceed 97.2%, and it reached 99.8% relatively to the reference 'Ca. P. convolvuli' isolate (JN833705) from field bindweed (Convolvulus arvensis L.).