غلام خداکرمیان

Alfalfa witches' broom (AWB) is one of the most important diseases in Iran. During 2013 to 2015 AWB disease distributed areas were determined based on disease symptoms, ELISA test using polyclonal antibody and polymerase chain reaction (PCR). AWB phytoplasma strains from different areas were transferred to periwinkle mediated by dodder (Cuscuta campestris) and disease symptoms were observed. Phytoplasmas strains associated with AWB disease from collected diseased alfalfa samples were identified by biological and molecular assays. Propagation and maintenance of phytoplasma strains from different areas were achieved by graft inoculation to periwinkle. All symptomatic alfalfa plants collected from surveyed areas as well as inoculated periwinkle by dodder grafting in greenhouse showed presence of phytoplasma associated with AWB disease tested by PCR. The expected PCR product sized 1800, 1400 and 1200 bp were amplified in direct PCR using P1/P7 primers and nested PCR using P1/P7 (first step) and R16F2n/R16R2 and R16mF2/R16mR2 (second step) primer pairs. Real and virtual restriction fragment length polymorphism (RFLP) analysis of 1250 bp of R16F2nR16R2 amplicon in nested PCR showed that phytoplasmas associated with AWB disease in Iran were belongs to 16SrI, 16SrII, 16SrVI and 16SrXII ribosomal groups. The 16SrI group of AWB strains was observed only in West Azarbaijan which they failed to classify in any reported subgroups. The 16SrII-related AWB phytoplasmas detected in Fars, Yazd, Kerman, Esfahan, South Khorasan, Khuzestan, Hamedan and Bushehr provinces were members of D and C subgroups. Based on 16S rRNA gene and 16S-23S intergenic spacer sequences, two phytoplasma strains associated with AWB disease in Chahgeer (CAWB) and Juyom (JAWB), were grouped in 16SrII-C subgroup. These strains considered as distinct strains due to induction of different symptoms in common host plants, difference in natural insect vectors and no serological relationship. The 16SrVI subgroup A wa