Characterization of 16SrII group phytoplasmas associated with alfalfa (Medicago sativa) witches’ broom disease in diverse areas of Iran.

Alfalfa witches’ broom (AWB) is one of the most important alfalfa diseases in Iran. To characterize 16SrII group phytoplasmas associated with this disease, symptomatic and asymptomatic plants were collected during 2013-2015 and subjected to direct and nested polymerase chain reaction (PCR) using P1/P7, R16mF2/R16mR2 and R16F2n/R16R2. PCR amplicons of ~1.8, ~1.4 and ~1.25 kb respectively, were obtained only from all symptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons showed that the phytoplasma associated with AWB disease were members of 16SrII group subgroups 16SrII-D and -C. Blast analysis of these amplicon sequences and sequence homology of collected strains and strain sequences retrived from GenBank (AWB strains Chahgeer, Juyom and Bushehr) confirmed that AWB phytoplasmas collected from Bafg, Ardakan, Bahabad and Herat (Yazd province), Nikshahr (Sistan-Baluchestan), Bam, Zarand, Jiroft (Kerman province), Bushehr (Bushehr province), Tabas (South Khorasan province), Jowkar (Hamedan province) and Zardenjan (Esfahan province) cluster with phytoplasma strains enclosed in the 16SrII-D subgroup, while AWB strains from Chahgeer (Yazd province) and Juyom (Fars province) cluster with phytoplasma strains in the 16SrII-C subgroup. Based on these results the predominant strains of 16SrII phytoplasmas associated with AWB disease in Iran were classified in the 16SrII-D subgroup. In Ashkezar and Abarkouh in Yazd province entire alfalfa farm was infected with witches’ broom disease. In 3 year alfalfa stands in Ashkezar alfalfa farms were plowed due to high incidence of the disease