A potyvirus in Cymbidium spp. in northern India

Abstract

A potyvirus was isolated from diseased Cymbidium pendulum and C. tigrinum epiphytic orchids collected from Sikkim in northern India. ELISA, RT-PCR and Northern blot analysis revealed the association of potyvirus infection with disease symptoms. The sequencing of a RT-PCR-amplified amplicon using potyvirus group-specific primers showed that this virus is distinct but most closely related to Calanthe mild mosaic virus. This paper reports the occurrence of potyvirus in wild orchids especially in Cymbidium spp. Potyviridae are one of the largest and most economically important families of plant viruses. Orchids are the largest family of flowering plants with ∼30 000–35 000 species and they form a large part of the floriculture trade. Odontoglossum ringspot tobamovirus and Cymbidium mosaic potexvirus are the most frequently reported viruses in cultivated orchids, perhaps because both are highly contagious and spread by wounding when plants are pruned or divided for repotting. However, at least a dozen viruses, mostly potyviruses, have been recorded in orchids, including vanilla (Brunt et al. 1996). Most viruses are a serious threat to the orchid industry as they reduce the vigour of infected plants and decrease flower quality. The potyviruses previously isolated from orchids include Dendrobium mosaic (DeMV), Habenaria mosaic, Pecteilis mosaic, Vanilla mosaic and Vanilla necrosis potyviruses (Brunt et al. 1996). The polymerase chain reaction (PCR) has been shown to be very useful for detecting and characterising plant pathogens and is more sensitive than direct probing or serological techniques (Hadidi et al. 1995). There are now many published reports of the use of reverse transcription–PCR (RT-PCR) with degenerate primers, which produce specific DNA fragments from all species of a group. A survey was undertaken for screening of major viruses infecting orchids, which revealed widespread occurrence of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) in Indian orchids (Sherpa et al. 2006a, 2006b). However, some plants of Cymbidium pendulum and C. tigrinum with symptoms of mild mosaic and stunting gave negative results for CymMV and ORSV. These epiphytic orchids grow as wild plants in Sikkim, in the north-eastern region of India. These plants were further tested using Cucumber mosaic virus, Cymbidium ringspot virus and potyvirus group-specific antibodies. ELISA tests using potyvirus group-specific antibodies indicated the association of a potyvirus with diseased orchid samples. To further elucidate and confirm the identity of the virus, total RNA was extracted from the diseased leaves of C. pendulum and C. tigrinum using the RNAqueous TM Kit (Ambion, USA). RT-PCR was performed as described by Van der Vlugt et al. (1999) using the primer pair 'cp9502' (5 GCGGATCCTTTTTTTTTTTTTTTTT3), specific for 3 end of potyvirus genomes, and 'cpup' (5 TGAGGATCCT GGTGYATHGARAAYGG-3 , where Y = C or T, H = A, T or C, R = A or G), which hybridises with the coat protein region of potyvirus genomes. The cDNA was reverse transcribed in a 25-µL reaction mixture using 7 µL (1–2 µg) RNA, 0.2 µg reverse primer (cp9502), 2 µL of 40 mM dNTP mix, 10 units of human placental RNase inhibitor, 5 µL of 5× RT buffer and 200 units of M-MLV reverse transcriptase enzyme. The reaction mixture was incubated at 42 • C for 75 min and after that it was incubated at 70 • C for 5 min. PCR amplification was done in a GeneAmp PCR 9700 system (Applied Biosystems, USA) with a 50-µL reaction mixture containing 7 µL cDNA, 0.2 µg forward primer, 0.2 µg downstream primer, 5 µL of 10× PCR buffer, 3 µL of 10 mM dNTP mix and 1.5 units of Taq DNA polymerase. The DNA was amplified using 35 cycles of denaturation at 94 • C for 1 min, annealing at 54 • C for 2 min and elongation at 72 • C for 1 min, and a final elongation at 72 • C for 10 min. The amplified PCR-product was fractionated in a 1% agarose gel and then stained with ethidium bromide. The amplified fragment was cloned into a pGEM-T Easy vector (Promega, USA) and was then sequenced and digested with EcoRI and used for probe preparation. DNA was mixed properly with a random hexamer mixture (100 ng), 10× Klenow buffer, 10 mM dNTPs (-dATP), alpha 32 P dATP and Klenow enzyme (5U/µL) in a reaction mixture of 30 µL. The mixture was centrifuged briefly and kept at 37 • C for 1 h followed by incubation on ice for 5 min. Northern slot blot hybridisation (Fig. 1) was carried out with slight modification of the methods described in ExpressHyb Hybridization Kit (BD Bio Sciences, USA). ExpressHyb solution was warmed at 68 • C and stirred well to completely dissolve any precipitate. Membranes (10 × 10 cm) were prehybridised in a minimum total volume of 5–10 mL of ExpressHyb solution with continuous shaking at 68 • C for 30 min. An equal volume of buffer A (500 mM Tris-HCl, 500 mM NaCl, 5 mM EDTA, 0.5% SDS) was added to the