THE FIRST REPORT OF A 16SrXIIA PHYTOPLASMA ASSOCIATED WITH TOMATO BIG BUD DISEASE IN IRAN
M. Salehi, S.A. Esmailzadeh Hosseini
In 2013 surveys, up to 7.5% incidence of big bud disease was observed in tomato fields of Kazerun area (Fars province, Iran). The main disease symptoms were big bud, little leaf, flower virescence, phyllody, proliferation and sterility. To investigate the phytoplasma presence, total DNAs extracted from four symptomatic and four symptomless tomato plants were tested by nested PCR using P1/P7 primer pair followed by R16F2n/R16R2 primers (Lee et al., 1998). Amplicons of ca. 1.8 and 1.2 kb, respectively were amplified in samples of symptomatic plants but not of symptomless ones. Four P1/P7 amplicons from symptomatic tomato plants were cloned and sequenced. The obtained 16S rDNA sequences showed 100% sequence identity with each other and a representative of these sequences deposited in GenBank (Accession No. KX098490). BLAST search using full length 16S rRNA gene sequence revealed that Kazerun tomato big bud (KTBB) sequence showed 100% identity with a 'Candidatus Phytoplasma solani' strain (AF248959), representative of 16SrXII-A subgroup. Computer-simulated restriction analysis using iPhyClassifier showed that the RFLP profile of KTBB 16S rDNA F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16SrXII-A (AF248959). Phylogenetic analysis revealed that KTBB phytoplasma clustered with16SrXII group phytoplasmas closest to 16SrXII-A subgroup reference strain (AF248959). To our knowledge this is the first report of a 16SrXII-A phytoplasma associated with TBB disease in Iran. 16SrXII- related phytoplasmas have been previously reported from grapevine (Salehi et al., 2014) and Cannabis sativa (Vali Sichani et al., 2011) in Iran.