First Report of a New Subgroup 16Sr II-M 'Candidatus Phytoplasma aurantifolia' Associated with Witches'-Broom Disease of Tephrosia purpurea in India.

Wild indigo (Tephrosia purpurea (L.) Pers.) grows as a common weed throughout the Indian subcontinent. The plant has pinnate leaves, white or purplish flowers, and flat hairy pods, and is cultivated as a green manure crop. The plant extracts contain compounds such as tephrosin, an aromatic ester, prenylated flavonoid, and sesquiterpene (2) that have medicinal properties. The newly recognized disease, Tephrosia purpurea witches' broom (TPWB), was characterized by chlorosis, stunting, and proliferative branching, which were suggestive of phytoplasma infection during a field survey conducted in November 2013. To determine the presence of phytoplasma, 2 g of compound leaves from three symptomatic and asymptomatic plants were used for total DNA extraction using the CTAB method. The phytoplasma 16S rRNA gene was detected in all three symptomatic plants using nested PCR with universal phytoplasma primer pairs, P1/P7 followed by R16F2n/R16R2 (4). No amplification was observed in DNA isolated from asymptomatic plants. PCR fragments (1,246 bp in length) generated from symptomatic T. purpurea plants were sequenced directly using five different primers viz. 343R, 536F, 704F, 907R, and 1103F. TPWB phytoplasma 16S rRNA gene sequence (GenBank Accession No. HG792252) showed 99.12% homology with a 'Candidatus Phytoplasma aurantifolia' strain WBDL (U15442) when compared using the EzTaxon 16S rRNA database (3). Virtual restriction fragment length polymorphism (RFLP) analysis was carried out on the obtained sequence using iPhyClassifier (5). The virtual RFLP pattern derived from the HG792252 sequence was different to the reference patterns of previously established 16Sr groups and subgroups. The reference pattern of the 16Sr group II, subgroup C (AJ293216) was most similar with a similarity coefficient of 0.92, which placed it in a new subgroup, 16Sr II-M (1). Furthermore, virtual RFLP results were confirmed by digesting R16F2n/R16R2 amplicon with BstUI, DraI, HinfI, HpaI, and MseI restriction enzymes according to manufacturer's instructions. To our knowledge, this is the first report of a 'Ca. P. aurantifolia'-related strain associated with witches'-broom disease of T. purpurea in India. References: (1) H. Cai et al. Int. J. Syst. Evol. Microbiol. 58:1448, 2008. (2) A. K. Khalafalah et al. Pharmacognosy Res. 2:72, 2010. (3) O.-S. Kim et al. Int. J. Syst. Evol. Microbiol. 62:716, 2012. (4) C. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.