Molecular differentiation of 16SrIX-I phytoplasmas detected in Onobrychis viciifolia leaf yellowing in Iran from phytoplasmas in 16SrIX-J subgroup

From 2014 in Chaharmahal and Bakhtiari province, Iran, Onobrychis viciifolia plants were observed with symptoms of yellowing, little leaf and reddening of leaflet margins and dwarfing and up to 32% of disease incidence was observed. Total DNA extracted from symptomatic and asymptomatic plants was tested by direct and nested PCR using P1/P7 followed by R16mF2/ R16mR2 and R16F2n/R2 primer pairs. Amplicons of about 1.8, 1.4 and 1.25 kbp were obtained from all symptomatic plants, but not from the asymptomatic ones. Restriction fragment length polymorphism analysis on R16F2n/R16R2 amplicons showed identical restriction profiles in all samples, referable to those of phytoplasma strains in the 16SrIX group. One sample from Kiar (KY) was directly sequenced and the fragment corresponding to the R16F2n/R16R2 amplicon was submitted to GenBank (Accession Number KX461906). Sequence comparison by BLAST analysis showed the highest sequence identity with pigeon pea witches\u2019 broom phytoplasmas. Phylogenetic analysis confirmed that KY phytoplasma clustered with \u2018Candidatus Phytoplasma phoenicium\u2019 with 99.4% sequence identity to the reference strain for this phytoplasma (GeneBank Accession number AF515636), and is therefore, a \u2018Ca. P. phoenicium\u2019-related strain. Computer-simulated analysis using iPhyClassifier showed that the RFLP pattern of KY phytoplasma was different from those of all phytoplasma subgroups in 16SrIX group and the highest similarity was 0.95 with 16SrIX-E (GeneBank Accession number GQ925918) with differences in AluI and TaqI restriction enzymes patterns; the strain was enclosed in the 16SrIX-I subgroup. However a chicory bushy stunt phytoplasma showing identical profile with KY phytoplasma was proposed later as a new subgroup as 16SrIX-J (GeneBank Accession number KY986922). Molecular comparison of the two phytoplasma sequences allow to verify that the two subgroups are differentiated by four single nucleotide polymorphisms at the 3\u2019 of the 16S rDNA sequence not enclosing restriction sites. Since the phytoplasma in O. viciifolia from Iran is well distinguished by RFLP analyses from all reported subgroup but the 16SrIXJ, it could be confused with it in epidemiological studies if the sequences are not compared. Phytoplasmas in the 16SrIX-I subgroup are quite represented in Iran where they were reported in Lactuca serriola phyllody and Bushehr eggplant big bud, however for their differentiation from the 16SrIX-J phytoplasma reported in Saudi Arabia in chicory it will be necessary the sequence comparison or the development of new specific primers on other phytoplasma genes