First report of mild mosaic in ground orchid, Phaius tankervilleae, in India associated with infection of Calanthe mild mosaic virus

Phaius tankervilleae is a species of ground orchid widespread in the Indian subcontinent, China, Japan, Malaysia, Philippines, the U.S.A. (Florida and Hawaii), and Australia, and it is grown commercially in India. During a December 2016 survey in Sikkim and Darjeeling hills, several plants of P. tankervilleae were observed with mild mosaic and streak symptoms in the germplasm collection of National Research Centre for Orchids, Pakyong, Sikkim. Negatively stained preparations of symptomatic leaf samples showed flexuous filamentous particles measuring about 800 nm long and 12 nm wide under the electron microscope. To further characterize this virus, total RNA was extracted from symptomatic leaf tissues of P. tankervilleae, using an RNeasy plant mini kit (Qiagen, Valencia, CA). RT-PCR results showed the absence of Cymbidium mosaic virus and Odontoglossum ringspot virus in the sample. RT-PCR was further performed using potyvirus generic primers (van der Vlugt et al. 1999) that target the partial coat protein (CP) region and complete 3′-nontranslated region (NTR). The resulting PCR amplicon (647 bp) was sequenced using the potyvirus generic primers. BLASTN sequence analysis revealed 95% identity with the CP region of Calanthe mild mosaic potyvirus (CalMMV, AB011404). A potyvirus closely related to CalMMV from Cymbidium species has been previously reported in Sikkim by Singh et al. (2007). To characterize the complete coat protein gene, specific primers Cal-forward (5′-GGGGACAAGAGTGAGTTGGAT-3′) and Cal-reverse (5′-CATATAGCGGGCACCATTGAG-3′) were designed using the sequence of CalMMV (AB011404) CP gene. Reverse transcription was performed using the tagged oligo(dT) reverse primer (5′-CACGGATCCCGGG(T)17VGC-3′) (Gibbs and Mackenzie 1997). PCR was carried out using CalMMV CP gene specific Cal forward and Cal reverse primers or using CalMMV-CP specific forward and tagged oligo(dT) reverse primer. The resulting amplicons (∼800 bp and ∼1 kb) were cloned separately into the pGEM-T Easy vector (Promega, Madison, WI) and two clones of each amplicon were sequenced. Sequence analysis of both the clones (∼800 bp and ∼1 kb) revealed the length of the coat protein to be 807 nt and the sequence was submitted to NCBI GenBank as accession no. MF385014. Pair-wise sequence alignment of both the amplicons revealed 94 and 87% nt sequence identity as well as 95 and 98% identity at the amino acid level with the corresponding CP gene sequence of CalMMV Japan isolate (804 bp) and partial CP gene sequence of Cymbidium potyvirus isolate (453 bp) (AJ871476), respectively. The amino acid sequences (DAG and WCIEN) conserved among the majority of potyviruses were also present. This is the first report of occurrence of CalMMV in ground orchid, P. tankervilleae from India, and this study will help in developing diagnostics for the virus-free production of ground orchid.