Identification and characterization of a tospovirus causing chlorotic ringspots on Phalaenopsis orchids

Abstract

A putative virus-induced disease showing chlorotic ringspots on leaves of Phalaenopsis orchids has been observed in Taiwan for several years. A virus culture, 91-orchid-1, isolated from a Phalaenopsis orchid bearing chlorotic ringspot symptoms was established in Chenopodium quinoa and Nicotiana benthamiana, and characterized serologically and biologically. The virus reacted slightly with the antiserum of Watermelon silver mottle virus (WSMoV) but not with those of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Groundnut ringspot virus (GRSV). Isometric particles measuring about 70-100 nm were observed. Inoculation with isolated virus was conducted to confirm that 91-orchid-1 is the causal agent of chlorotic ringspot disease of Phalaenopsis orchids. To determine the taxonomic relationships of the virus, the conserved region of L RNA and the complete nucleocapsid gene (N gene) were cloned and sequenced. The sequence of conserved region of L RNA shares 83.8, 82.5, 64.4 and 64.9% nucleotide identities and 96.5, 97.7, 67.3 and 67.6% amino acid identities with those of Peanut bud necrosis virus (PBNV), WSMoV, TSWV and INSV, respectively, indicating that 91-orchid-1 is a tospovirus related to WSMoV. The complete nucleotide sequence of the N gene determined from a cDNA clone was found to be 828 nucleotides long encoding 275 amino acids. Sequence analyses of the N gene showed that 91-orchid-1 is an isolate of Capsicum chlorosis virus (CaCV) which has been reported to infect tomato and capsicum plants in Australia and Thailand. 91-orchid-1 is therefore designated as CaCV-Ph. To our knowledge, this is the first formal report of a tospovirus infecting Phalaenopsis orchids

Identification and characterization of a potyvirus causing chlorotic spots on Phalaenopsis orchids

“…Ultra-thin sections of symptomatic leaves of N. benthamiana plants infected with isolate 7-2 were prepared by the protocol described previously (Jan and Yeh 1995;Zheng et al 2008). Ultra-thin sections were cut with a diamond knife and collected on formvarcoated, carbon-stabilized copper grids (75 mesh).…”

“…The procedure of indirect enzyme-linked immunosorbent assay (indirect ELISA) followed the method described by Clark and Adams (1977) with some modifications (Zheng et al 2008). Rabbit antisera for ORSV, CymMV, CMV, Impatiens necrotic spot virus (INSV), Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), Watermelon silver mottle virus (WSMoV), Peanut chlorotic fan-spot virus (PCFV), Turnip mosaic virus (TuMV), Zantedeschia mild mosaic virus (ZaMMV), Zantedeschia mosaic virus (ZaMV), Dasheen mosaic virus (DsMV), Carnation mottle virus (CarMV), Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), CaCVPh or isolate 7-2 were individually prepared.…”

“…Immunoblotting was done as previously described (Jan and Yeh 1995;Zheng et al 2008). Leaf extracts of mock-inoculated and virus-infected C. quinoa were separated by SDS-PAGE and then transferred to a PVDF transfer membrane (Bio-Rad, California, USA).…”

“…The first-strand cDNA was synthesized with SuperScript™ III RNase H − Reverse Transcriptase (Invirtogen, California, USA) according to the instructions of the manufacturer and polymerase chain reaction (PCR) amplification was performed using TAKARA LA Taq DNA polymerase (TAKARA BIO Inc., Shiga, Japan). The procedures of reverse transcription-PCR (RT-PCR), DNA cloning and sequencing were described previously (Zheng et al 2008). Pot 2 and oligo d(T) primers were used to amplify the 3′ terminal region of the potyvirus genome.…”

“…In addition, we recently reported a tospovirus, Capsicum chlorosis virus (CaCV-Ph), causing chlorotic ringspots on Phalaenopsis orchids (Zheng et al 2008). CymMV, ORSV (Wey 1988), CMV (Ko 1988) and CaCV-Ph (Zheng et al 2008) are the only four viruses that have been reported to infect Phalaenopsis orchids in Taiwan.…”