Biological and molecular characterization of phytoplasmas associated with GF-677 witches’-broom in Iran

GF-677 (prunus amygdalus × prunus persica) is one of the most suitable rootstocks for almond and peach trees. GF-677 cuttings were imported from Europe and planted in Bidzard and Estahban areas (Fars province) for further propagation and distribution to other Iranian stone fruit growing areas. During 2009 and 2010 surveys of GF-677 trees, witches’-broom disease (GFWB) was observed in both Bidzard and Estahban regions. The disease agents of GFWB from Bidzard (BGFWB) and Estahban (EGFWB) were transmitted from GF-677 to GF-677, bitter almond and peach seedlings by grafting and from experimentally inoculated bitter almond to seed-grown eggplant and periwinkle plants via dodder inoculation, causing phytoplasma-type symptoms. Total DNA extracted from naturally affected GF-677 trees in Bidzard and Estahban and experimentally inoculated plants reacted positively in direct PCR using P1/P7 and nested-PCR using P1/P7 (first round) and R16F2n/R16R2 (second round) primer pairs. On the basis of disease symptoms, positive reaction in PCR, transmission by dodder and grafting, BGFWB and EGFWB have phytoplasmal etiology. Blast searching showed that BGFWB and EGFWB phytoplasmas belong to the pigeon pea witches` broom (16SrIX) group. Using AluI, HpaII and HaeIII enzymes, RFLP patterns of BGFWB and EGFWB phytoplasmas were indistinguishable from each other and those of Neyriz (Fars, Iran) almond Witches’- broom (NAWB) phytoplasma. P1/P7 PCR ampliconsequences of BGFWB and EGFWB isolates were submitted to the GenBank database under the accession numbers JX445141 and JX445142, respectively. Virtual RFLP using 17 enzymes showed that BGFWB and EGFWB isolates are indistinguishable from NAWB phytoplasma (representative of 16SrIX-B subgroup) but different from Khafr (Fars, Iran) almond witches` broom (KAWB) phytoplasma which is classified with 16SrIX-C subgroup phytoplasmas. Phylogenetic analysis and homology percent, confirmed that among 16SrIX subgroups, BGFWB and EGFWB phytoplasmas were more similar to subgroup B of 16SrIX group than other subgroups.