First report of Candidatus Phytoplasma asteris associated with witches broom of Crotalaria tetragona in India

Crotalaria tetragona is mainly used as a green manure in Northeast India, since farmers in this region are averse to using chemicals. It is also used in contour hedgerow farming systems in the jhum fields (Ya and Tulachan, 2003). During field visits to Barapani, Meghalaya (Northeast India) in June-July, 2008, typical witches’ broomsymptoms were observed in almost 46% of C. tetragona plants (Fig. 1).

Total DNA was extracted from leaf samples of three randomly collected plants with symptoms and two symptomless plants. Nested PCR with the 16S rDNA phytoplasma universal primers R16F2m/R1-fU5/rU3 was carried out that yielded PCR amplicons in all tested plants with symptoms. No such products were obtained from symptomless plants. PCR products were purified (QIAquick gel extraction kit, USA), cloned (pGEM-T Easy vector, Promega) and sequenced in both directions using M13 forward and reverse sequencing primers (www.dnaseq.co.uk). The 16S rDNA sequences from the Crotalaria phytoplasma isolates showed 100% similarity with each other, and a representative was submitted to GenBank (Accession No. FJ185141). BLAST search analyses revealed that the Crotalaria phytoplasma sequence scored 99% similarity with members of group 16SrI, ‘Candidatus Phytoplasma asteris’. Phylogeny analyses (MEGA version 3.1) using reference phytoplasmas from GenBank confirmed sequencing results and placed the Indian Crotalaria phytoplasma in this group (Fig 2).

Crotalaria spp. have been recognized as a plant host for phytoplasmas from group 16SrII, ‘Ca. Phytoplasma aurantifolia’ in Asian countries, including India (IRPCM, 2004). The 16SrI ‘Ca. Phytoplasma asteris’ group has been reported from a number of plant hosts in India, including sesame (Khan et al., 2007), Cannabis sativa (Raj et al., 2008) and sandal (Khan et al., 2008). To our knowledge, this is the first record of a 16SrI ‘Ca. Phytoplasma asteris’ isolate in Crotalaria spp.