Association of 16SrII-D Phytoplasma With Phyllody Disease in Sesame (Sesamum indicum) in Telangana From Southern India

Sesame (Sesamum indicum L.) is an important and ancient oil-seed crop, domesticated over 3,000 years ago. Leaf-like flower (phyllody), virescence, and proliferation, which were typical symptoms of phytoplasma infection, were observed in sesame breeding lines in Hyderabad, Telangana, India during the cropping period between July and October 2012-14. The sesame plants were monitored for symptom development under natural infection in field conditions and leaf samples from three symptomatic and three asymptomatic plants were collected. Though leaf samples from asymptomatic and symptomatic plants were analyzed from 2012-14, the experimental figures presented correspond to the samples collected in September 2014. Total genomic DNA was isolated from the samples following a CTAB DNA extraction method (Smart et al. 1996). Genomic PCR with phytoplasma-specific P1/Tint primer pair (Smart et al. 1996) followed by nested PCR primer R16F2n/R16R2 (Lee et al. 1998) resulted in a PCR product of 1.6 and 1.25 kb, respectively, for all the three symptomatic samples whereas amplification of 16S rRNA gene was not detected in asymptomatic samples used as negative controls. The 1.25 kb DNA fragment obtained from three symptomatic samples was purified using the Nucleospin Gel and PCR Clean-up kit (Macherey Nagel), cloned into pGEMT Easy vector I (Promega) as per manufacturer’s instructions, and sequenced using M13 universal primer on an ABI 3730Xl automated sequencer at SciGenom sequencing services (SciGenom Labs, Cochin, India). The DNA sequences obtained from these samples share >99% similarity with each other and one of these sequences is deposited in GenBank with accession number KP297862. NCBI BLASTn analysis of KP297862 revealed 100% identity with Echinacea witches’-broom phytoplasma strain EWB6 (JF340080), papaya yellow crinkle disease phytoplasma (Y10097), and sesame phyllody phytoplasma strain Delhi (KC920750). Though phylogenetic analysis of KP297862 with representative sequences retrieved from GenBank revealed close proximity to Candidatus Phytoplasma aurantifolia (U15442), which belongs to 16SrII group (unpublished data); however, using iPhyClassifier (Zhao et al. 2009) for subgroup identification revealed sequence relatedness with similarity coefficient of 1.00 to Ca. P. australasia (Y10097), which represents 16SrII-D. An in silico RFLP digestion using 17 restriction enzymes followed by a real-RFLP digestion using six key restriction enzymes, viz., AluI, BamHI, EcoRI, KpnI, RsaI, and TaqI confirmed the phytoplasma identified in our study as 16SrII-D subgroup. Though phytoplasma association with sesame was identified in different regions of the country, like the subgroups 16SrI-B and 16SrII-C from Uttar Pradesh, 16SrI-B subgroup from Delhi and Bihar in northern India (Nabi et al. 2015), and subgroup 16SrI in Karnataka (Manjunatha et al. 2012) from southern India, the phytoplasma identified in our study belongs to 16SrII-D subgroup, which is being reported for the first time in sesame from Telangana in India. Early infection of sesame plants leads to total loss of seed production. Detection of the phytoplasma during early stages of plant growth would help in reducing the cultivation costs and for effective crop breeding program.