First report of 16SrVI-A and 16SrXII-A phytoplasmas associated with alfalfa witches’ broom disease in Iran

In 2014-15 surveys, alfalfa witches’ broom (AWB) disease was observed in a number of Iranian provinces. The main symptoms were crown proliferation, witches' broom, little leaf, flower virescence, phyllody, sterility and stunting. To investigate the phytoplasma presence, total DNAs were extracted from 132 symptomatic and 19 asymptomatic alfalfa plants and were tested by direct PCR using P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) and nested PCR using R16F2n/R16R2 (Gundersen and Lee, 1996) primers. Amplicons were obtained only from all the symptomatic alfalfa plants and RFLP analyses with KpnI, AluI, HaeIII, HhaI, HpaII, MseI, RsaI, TaqI and HpaI enzymes showed the presence of two profiles. Mixed infections were never detected. From AWB samples collected in East Azarbayijan and Zanjan provinces, six amplicons were directly sequenced and showed 100% identity to each other (GenBank accession No. KU240021). The second profile was present in samples from Qom, Markazi, East Azarbayijan, Kordestan, Fars, Kermanshah, Kohgiluyeh and Boyer-Ahmad, Lorestan and Chaharamahal and Bakhtiari provinces; the 15 amplicons from Tabriz, Dehgolan, Arak, Qhorveh, and Shahrekian showed 100% identity in each area and formed 5 groups having 99% identity (GenBank accession Nos. KT763372, KT763371, KT781662, KT763373 and KT750060, respectively). The KU240021 strain sequence and its virtual RFLP indicated that it could be enclosed in the 16SrVI-A subgroup. The five-sequence group showed the highest homology with 16SrXII-A phytoplasma subgroup that was confirmed by virtual RFLP and phylogenetic analysis (MEGA 6.0). This is the first report of 16SrVI-A and 16SrXII-A phytoplasmas associated with AWB in Iran.