Alfalfa witches’ broom (AWB) is a devastating disease distributed mainly in tropical provinces of Iran and widespread all over the Sistan-Baluchestan province. The highest incidence was found in Rask (Sarbaz) (70%), Sefidaba (Zabol) (46.26%), Sarboog (Nikshahr) (45.4%), Mirjaveh (Zahedan) (40%), Shams Abad (Saravan) (32.2%), Namardian (Iranshahr) (27%), Kahir (Chabahar) (25.7%) and Najaf Abad (Khash) (17.8%). Dodder inoculation of periwinkle plants with AWB agents from different areas induced undistinguishable symptoms. Detection and identification of AWB associated phytoplasmas was based on characteristic disease symptoms and direct and nested polymerase chain reaction using P1/P7, R16mF2/ R16mR2 and R16F2n/ R16R2 primer pairs. RFLP and phylogenetic analyses of 16S rDNA showed that AWB associated phytoplasmas from Sistan-Baluchestan belong to peanut witches’ broom (16SrII) group. Virtual digestion of R16F2n/R16R2 sequence of a Nikshahr AWB strain exhibited RFLP profiles referable to those of members of the 16SrII phytoplasma group, subgroup D
