During 2014–2015 phytoplasmas associated with alfalfa witches’ broom (AWB) in the western areas of Iran were characterized by molecular analyses studies. Polymerase chain reaction was carried out using direct and nested as says to amplify the 16S ribosomal gene for phytoplasma identification. RFLP analysis of R16F2n/R16R2 amplicons with selected restriction enzymes and phylogenetic studies showed that AWB associated phytoplasma strains from Salafchegan (Qom province), Arak (Markazi province), Tabriz (East Azerbaijan province), Dehgolan and Qhorveh (Kordestan province), Eghlid (Fars province), Mahidasht (Kermanshah province), Dehdasht (Kohgiluyeh and Boyer-Ahmad province), Borujerd (Lorestan province), Chenar-e Mahmudi and Shahrekian (Chaharmahal and Bakhtiari province) belong to 16SrXII-A subgroup (“stolbur”), while AWB associated phytoplasmas from Jowkar (Hamedan province) belong to 16SrII-D subgroup. Disease incidence was variable in the different areas and the highest infection rate (14.7%) was observed in Chenar-e Mahmudi (Chaharmahal and Bakhtiari province). The highly AWB affected areas were generally located in the warmer parts of each region in sparse fields. This is the first report and characterization of 16SrXII-A phytoplasmas associated with alfalfa witches’ broom diseases in Iran and occurrence of 16SrII-D in western areas of Iran.
