First Report on the Occurrence of Virescence of Chrysanthemum Associated with 16SrII-A Group Phytoplasma in India

Chrysanthemum (Chrysanthemum indicum L.) is an important floriculture crop grown in different parts of Karnataka, India for its beautiful cut flowers. Recently, there has been a decline in flower quality due to major pests and diseases. A field survey conducted during 2012-2013 in chrysanthemum-growing areas revealed the occurrence of virescence with an incidence of 5 to 10% in nearly 6 to 8 ha of chrysanthemum fields in Mandya and Mysore districts. Symptoms including malformed flowers with greenish florets and reduced flower size and floral virescence with sepaloidy were observed. Diseased leaves and flower parts were collected and used for identification of the causative agent. Ten samples (6 symptomatic, 2 healthy, and 2 phytoplasma-associated with phyllody of Catheranthus roseus as positive control) were used for total genomic DNA isolation using GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) and the DNA was used as template to amplify the phytoplasma-specific 16SrDNA gene using P1/P7 primers followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected only with DNA from symptomatic and positive control samples. Nested PCR products of all six phytoplasma-associated samples were purified and sequenced from both the directions using a ABIx370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The 16SrDNA sequences were aligned using CodonCode Aligner (Centerville, MA) and analyzed by BLASTn. The analysis revealed that all six sequences shared 100% identity with Cassava witches broom (KM280680.1) and Peanut witches broom (JX403944.1) phytoplasma. The in-silico RFLP pattern of F2n/R2 primed region of 16SrDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 99.8% nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup A (Peanut witches broom phytoplasma; Accession No. L33765.1). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chrysanthemum virescence was identified as a member of 16SrII-A group. The representative sequence was deposited in GenBank with Accession Number KP027534.1. Phytoplasma associated with little leaf and witches broom of chrysanthemum have been reported earlier from India and were assigned to groups 16SrI-A and 16SrII, respectively (Kumar et al. 2012; Raj et al. 2007). Chrysanthemum virescence phytoplasma belonging to Candidatus phytoplasma aurantifolia has been reported from Japan and has not been assigned to any group (Naito et al. 2007). The occurrence of virescence is of great importance as it affects the overall productivity and quality of flowers, which reduces the market value of chrysanthemum. To our knowledge, this is the first report of a group 16SrII-A phytoplasma associated with chrysanthemum virescence in India.