Detection of Prunus necrotic ringspot virus in begonia by RT–PCR

Abstract

Begonia (family Begoniaceae), a pot plant, vegetatively propagated through cuttings, has been reported to be susceptible to four main viruses: Cucumber mosaic virus (CMV), Impatiens necrotic spot virus (INSV), Tobacco necrosis virus (TNV), and Tomato spotted wilt virus (TSWV). In addition, Arabis mosaic virus (AMV), Broad bean wilt virus 1 (BBWV 1) and Carnation mottle virus (CarMV) have also been reported from begonia (Albouy, 1995). During a survey in and around Palampur, India in 2001, begonia ( Begonia semperflorens ) plants were observed showing characteristic ring symptoms on leaves. A total of 30 plants were collected, both with and without symptoms, in order to identify the causal agent. Mechanical inoculation from infected leaves to healthy begonia plants produced characteristic rings, and dark coloured local lesions on inoculated Cyamopsis tetragonoloba , both typical of Prunus necrotic ringspot virus (PNRSV) (Brunt et al ., 1996). PNRSV, an ilarvirus, occurs worldwide in cultivated stone fruits ( Prunus spp.), and although its host range is wide, it has not previously been reported in begonia. Total RNA was isolated from 100 mg fresh affected leaf tissue using Qiagen RNeasy plant mini-kit (Qiagen, Crawley, UK), according to the manufacturer’s instructions. Reverse transcription (RT)–PCR was performed using PNRSV-specific primers as described by Spiegel et al. (1996), and RT–PCR amplified a c. 785 bp fragment indicating the presence of PNRSV in affected begonia. No product was amplified from symptomless begonia samples. As it was difficult to obtain virus-free begonia, RNA isolated from healthy Nicotiana tabacum was used as a negative control, and no PCR products were amplified.