ASSOCIATION OF MEMBERS OF CLOVER PROLIFERATION (16SrVI) AND PIGEON PEA WITCHES BROOM (16SrIX) PHYTOPLASMA GROUPS WITH TOMATO BIG BUD DISEASE IN IRAN

Tomato plants with symptoms of big bud were observed in Khorasan Razavi, Khorasan Shomali, Azerbaijan Gharbi, Azerbaijan Sharghi, Kermanshah, Kurdistan and Fars provinces of Iran. Affected tomato plants showed proliferation of lateral shoots, small thickened and chlorotic leaves, purple coloration, hypertrophic calyxes, floral virescence, phyllody and failure of fruit setting. Agents of tomato big bud (TBB) disease from these provinces were transmitted from naturally symptomatic tomato plants to tomato by graft and from tomato to periwinkle via dodder inoculation. The presence of phytoplasma in affected plants was shown by direct and nested polymerase chain reaction (PCR) assays using the phytoplasma-specific primer pairs, P1/P7 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of nested R16F2n/R2 primed PCR product (1.2 kbp) identified TBB phytoplasmas from Kurdistan and Khorasan Shomali provinces as members of the pigeon pea witches’broom (16SrIX) group, and TBB phytoplasmas from Khorasan Razavi, Azerbaijan Gharbi, Azerbaijan Sharghi, Kermanshah and Fars provinces as members of the clover proliferation (16SrVI) group. Phylogenetic analysis of 16S-23S spacer region and putative restriction site analysis of the R16F2n/R2 primed sequence also classified TBB phytoplasmas in the clover proliferation (16SrVI) and pigeon pea witches’broom (16SrIX) phytoplasma groups. The same analyses showed that TBB phytoplasmas related to clover proliferation group belong to subgroup 16SrVI-A and those related to pigeon pea witches’broom group belong to subgroup 16SrIX-E. To our knowledge this is the first report of tomato big bud disease in Khorasan Razavi, Khorasan Shomali, Azerbaijan Sharghi, Kermanshah and Kurdistan provinces and the first report of subgroup classification of associated phytoplasmas.