Molecular identification of chrysanthemum chlorotic mottle viroid infecting chrysanthemum in Karnataka, India

Chrysanthemum chlorotic mottle viroid (CChMVd, genus Pelamoviroid, family Avsunviroidae) has been reported to infect chrysanthemum (Dendranthema x grandiflorum). CChMVd was first reported in 1967 in the cultivar Yellow Delaware in New York State, USA (Dimock et al. 1971). Presence of CChMVd has previously been recorded, based on the symptoms and bioassay, in India (Singh et al. 1978). Karnataka State of India is the prominent chrysanthemum-growing region, with 19,161 ha cultivated under this crop and accounting for more than 40% of country’s flower production. During late summer 2010 (May and June), chrysanthemum leaves with yellow-green mottling and chlorotic spots were noted, indicating the possible association with CChMVd in Karnataka. Total RNA was extracted using a 2% CTAB buffer from leaf samples of symptomatic plants (Adkar-Purushothama et al. 2013). For the detection of CChMVd, reverse transcription PCR (RT-PCR) was performed using PIII/PIV as described previously (De la Peña et al. 1999). PCR products of the expected sizes (∼398 to 401 bp) were cloned and sequenced. BLAST analysis of the obtained sequences confirmed the presence of CChMVd in Karnataka. In order to estimate the incidence of CChMVd, the same RT-PCR assay was performed on 80 randomly selected chrysanthemum leaf samples of four different cultivars (Redgold, Bangalore, Sarval, and CO-1) collected from four chrysanthemum-growing districts of Karnataka. A PCR product of the size expected for CChMVd was detected in 8 of 80 samples analyzed, accounting for the infection incidence of 10%. To identify the prominent CChMVd variants, 10 clones obtained from the four representative samples were sequenced. To confirm the sequence of the primer regions, an additional set of primers CCh1/CCh2 were used for RT-PCR (Hosokawa et al. 2005) with high-fidelity LA-Taq polymerase (Takara-Bio, Japan). Alignment of all 40 sequences revealed the presence of at least two major sequence variants of CChMVd: CChMVd-Ind-1 (GenBank Accession No. KP262531) and CChMVd-Ind-3 (KP262533). Both the sequence variants had UUUC sequences (nucleotide position 82 to 85) in the tetraloop of the CChMVd, which is often associated with induction of symptoms in susceptible chrysanthemum cultivars (De la Peña et al. 1999). CChMVd-Ind-1was found to differ from CChMVd-Ind-3 by 17 nucleotide substitutions. BLAST analysis of CChMVd-Ind-1 showed 98% sequence identity with the CChMVd strain reported from Spain (GenBank Accession No. FJ647515) and from Himachal Pradesh state of India (FN646404), whereas CChMVd-Ind-3 showed 96% sequence identity with CChMVd strain W2-4, isolated from China (HQ891014). To our knowledge, this is the first molecular evidence for the presence of CChMVd infecting chrysanthemum in Karnataka State, India. The high degree of sequence diversity reported in the Karnataka isolates of CChMVd will help to clarify the evolution of this viroid and its possible threat to important crops