First report of a 'Candidatus Phytoplasma asteris' related Phytoplasma associated with eucalyptus little leaf disease in Iran

During 2008-13 surveys, Eucalyptus camaldulensis little leaf (ELL) disease was observed in Firuzabad, Shiraz and Darab (Fars province, Iran), consisting of proliferation of auxiliary buds, formation of brooms on branches and trunk, yellowing, reddening and reduction of the leaf size, tree decline and death within 3-4 years from the onset of symptoms. Total DNA extracted from 15 ELL-affected and five symptomless plants was tested by direct and nested PCR using the phytoplasma primer pair P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen and Lee, 1996). Amplicons of ca. 1.25 kbp were obtained from 9 of 15 symptomatic but not from all symptomless and six symptomatic trees, probably due to the low titres and unevenly distribution of phytoplasma or the presence of PCR inhibitors in the latter. Nine R16F2n/R16R2 amplicons from infected samples (three from each sampled area) were directly sequenced in both directions, one of which from Firuzabad ELL (FELL) was deposited in GenBank (Accession No. KT992689). BLAST search showed that the FELL phytoplasma had 100% sequence identity with four 16SrI ('Candidatus Phytoplasma asteris') group-related phytoplasmas including mulberry dwarf, aster yellows, onion yellows, sasa witches' broom and epilobium phyllody phytoplasmas (AB693124, FJ824597, AP006628, AB293421, AY101386). Computer-simulated restriction analysis using iPhyClassifier revealed that the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment of FELL is identical (similarity coefficient 1.00) to onion yellows phytoplasma (NC_005303) representative of subgroup B of the 16SrI group. Phylogenetic analysis revealed that FELL phytoplasma clustered with 16SrI- B members closer to a phytoplasma associated with Eucalyptus yellows and witches' broom (AY685054). To our knowledge, this is the first report of the association of a 16SrI phytoplasma with ELL in Iran