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

Datura inoxia (family Solanaceae) is an annual invasive weed grown in India, and used as a medicinal plant for therapeutic purposes. A disease characterized by little leaf was observed on D. inoxia plants growing nearby brinjal (aubergine) fields in Ambedkar Nagar, U.P., India during March 2008. The diseased plants exhibited proliferation of branches with shortened internodes and reduced-size leaves which give rise to the little leaf appearance (Fig 1).

To ascertain the association of phytoplasma, total DNA was extracted from ~100 mg of leaf tissue employing a phytoplasma enrichment procedure (Ahrens and Seemuller, 1992). Direct polymerase chain reaction (PCR) was performed using P1/P6 universal primers specific to 16S rRNA gene of phytoplasmas (Deng & Hiruki, 1991), followed by a nested PCR from a 1:10 dilution of the P1/P6 PCR products using R16F2n/R16R2 primers (Gundersen & Lee, 1996), which resulted in expected size bands of ~1.5 kb and ~1.2 kb, respectively, for symptomatic (2/2) but not healthy (1/1) samples.

Nested PCR amplicons from each sample were sequenced and the consensus nucleotide sequence deposited in GenBank (Accession No. EU573925). BLAST analysis of the 16S rRNA partial sequence of the phytoplasma identified in little leaf-affected D. inoxia revealed its highest identity (97%) with those of members of group 16SrVI, ‘Candidatus Phytoplasma trifolii’. Phylogenetic analysis using MEGA 4.0 confirmed the Datura little leaf phytoplasma as a ‘Ca. Phytoplasma trifolii’-related strain (Fig 2). The group 16SrVI was previously associated with brinjal little leaf disease in India (Schneider et al., 1995; Smart et al., 1996). However, this is the first record of a 16SrVI phytoplasma affecting D. inoxia, which is suggested as a possible new ‘Candidatus Phytoplasma’ species based on sequence analysis.