S.K. Raj*, S.K. Snehi, S. Kumar and M.S. Khan
Plant Molecular Virology, National Botanical Research Institute, Lucknow-226 001, U. P., India
Accepted: 23 Dec 2008
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.
Ahrens U, Seemuller E, 1992. Detection of DNA of plant pathogenic mycoplasma-like organism by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82, 828-832.
Deng S, Hiruki D, 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods 14, 53-61.
Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-151.
Schneider B, Cousin MT, Klinkong S, Seemüller E, 1995. Taxonomic relatedness and phylogenetic positions of phytoplasmas associated with diseases of faba bean, sunnhemp, sesame, soybean, and eggplant. Journal of Plant Disease and Protection 102, 225-232.
Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz KH, Seemuller E, Kirkpatrick BC, 1996. Phytoplasma-Specific PCR Primers Based on Sequences of the 16S-23S rRNA Spacer Region. Applied and Environmental Microbiology 62, 2988-2993.
©2008 The Authors