Detection and identification of Candidatus phytoplasma trifolii on apple trees in Iran

During spring and summer of 2018, suspected symptoms of the phytoplasma disease such as leaf shrinkage and yellowing as well as leaves with abnormally-enlarged stipules were observed in apple orchards in Khorasan Razavi province and 33 leaf samples were collected. Samples of symptomatic apple tree were assayed for infection with phytoplasma, using universal primer pairs P1 / P7 and nested PCR using primer pair R16F2n / R16R2, fU5 / rU3 and R16MF2 / R16MR2 in Polymerase Chain Reaction (PCR). The universal PCR products were 1800 bp fragments and the nested-PCR products were 1250, 876 and 1400 bp fragments, respectively, which were amplified by primer pairs. Of 33 leaf samples from apple, 18 samples were tested positive for phytoplasmas. The R16MF2/R16MR2 primed nested PCR products were cloned, sequenced and submitted into GenBank. Comparison of the obtained sequences with the sequences of NCBI using BLAST software and Restriction Fragment Length Polymorphism (RFLP) analysis of the nested PCR products using the enzymes AluI, RsaI and TruI (MseI) indicated that phytoplasma in apple trees in Razavi Khorasan province was most similar to Candidatus Phytoplasma trifolii related to Clover Proliferation (CP) 16SrVI group. Analysis of sequences using iPhyClassifier software, determination of nucleotide similarity, and phylogenetic analysis of full-length 16S rDNA showed that phytoplasma in apple tress of Razavi Khorasan province belongs to 16SrVI-A subgroup. This is the first report on Ca. Phytoplasma trifolii infections in apple trees in Iran and other regions across the world.