Occurrence and identification of a phytoplasma associated with Pinus brutia witches’ broom disease in Isfahan, Iran

Between 2016 and 2017, symptoms of witches’ broom, proliferation, drying of twigs and ball-like structures formation were observed in Pinus brutia in urban green areas and parks in Isfahan (Iran). All samples from affected P. brutia trees resulted positive in nested-PCR using primer pairs targeting the phytoplasma 16S rRNA gene. Amplicons were obtained only from all the symptomatic pine tree samples and RFLP analyses of R16F2n/R16R2 amplicons with KpnI, AluI, HaeIII, HhaI, HpaII, MseI, RsaI,ThaI, BfaI and TaqI restriction enzymes showed the presence of phytoplasmas enclosed in the 16SrVI group. Four amplicons were sequenced and showed 100% identity to each other. One of these sequences was deposited in the GenBank database (accession number MK158098) and the virtual RFLP pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A confirming that the phytoplasma detected in pine is a member of 16SrVI-A subgroup. This is the first report of a 16SrVI-A phytoplasma strain associated with pine tree witches’ broom (P. brutia) disease.