First Report of a Subgroup 16SrIX-E (‘Candidatus Phytoplasma phoenicium’-Related) Phytoplasma Associated with Phyllody and Stem Fasciation of Sunn Hemp (Crotalaria juncea L.) in India

Abstract

Sunn hemp (Crotalaria juncea) is widely grown in tropical and subtropical regions for fiber and green manure. During the summer of 2016, sunn hemp plants in different experimental fields of the CRIJAF research farm, Barrackpore, India, exhibited symptoms characteristic of phytoplasmal disease. Symptoms included phyllody, bunchy top with bending, little leaf, leaf vein discoloration, and stem fasciation (flattened stem). Approximately the incidence of the disease was less than 2%. Leaf samples were collected from different plants with characteristic symptoms as well as without symptoms. Total genomic DNA was extracted from three symptomatic and three asymptomatic leaf samples using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was carried out with phytoplasma 16S rRNA universal primer set P1/P7 followed by nested primer pair R16F2n/R16R2, resulting in DNA amplicons that were 1.8 and 1.2 kb, respectively, in all symptomatic samples tested (Deng and Hiruki 1991; Lee et al. 2004). No amplification was found in the case of symptomless samples, suggesting a possible association of a phytoplasma with the disease. Six PCR products were purified and cloned in a pGEM-T Easy vector (Promega) and sequenced. One of the sequences of 1,844 bp, which proved to be identical to the others, was deposited in GenBank (accession no. KY973967). BLASTn similarity search revealed that the sequence shared 100% identity to rDNA of ‘Candidatus Phytoplasma phoenicium’ (GQ925919) and 98.5% similarity with ‘Ca. P. phoenicium’ (AF515636). Additionally, analysis of the 16Sr group/subgroup classification, based on in silico restriction fragment length polymorphism analyses using iPhyClassifier, indicated that the sunn hemp phytoplasma is a member of the 16SrIX-E ‘Ca. P. phoenicium’ subgroup (Zhao et al. 2009). In a phylogenic tree deduced by using the neighbor-joining algorithm, the present sunn hemp phytoplasma clustered with other 16SrIX-E (‘Ca. P. phoenicium’) phytoplasma strains. Amrasca biguttula biguttula noticed in the sunn hemp field is the probable vector species transmitting the ‘Ca. P. phoenicium’. Although ‘Ca. P. phoenicium’ 16SrIX-E subgroup has been previously reported in different hosts (Bayat et al. 2013; Davis et al. 2010), to our knowledge, this is the first report of ‘Ca. P. phoenicium’ infecting sunn hemp in India.