First finding of ‘Candidatus Phytoplasma trifolii’ (16SrVI group) associated with little leaf disease of Datura inoxia in India

Abstract

plants growingnearby brinjal (aubergine) fields in Ambedkar Nagar, U.P., India duringMarch 2008. The diseased plants exhibited proliferation of branches withshortened internodes and reduced-size leaves which give rise to the littleleaf appearance. To ascertain the association of a phytoplasma, total DNAwas extracted from ~100 mg of leaf tissue employing a phytoplasmaenrichment procedure (Ahrens & Seemuller, 1992). Direct polymerasechain reaction (PCR) was performed using P1/P6 universal primers specificto 16S rRNA gene of phytoplasmas (Deng & Hiruki, 1991), followed bya nested PCR from a 1:10 dilution of the P1/P6 PCR products usingR16F2n/R16R2 primers (Gundersen & Lee, 1996), which resulted inexpected size bands of ~1·5 kb and ~1·2 kb, respectively, for little leafaffected (2/2) but not symptomless (1/1) samples.Nested PCR amplicons from each sample were sequenced and theconsensus nucleotide sequence deposited in GenBank (Accession No.EU573925). BLAST analysis of the 16S rRNA partial sequence of thephytoplasma identified in little leaf-affected