Columnea latent viroid (CLVd): first report in tomato in France

Abstract

In summer 2007, leaves and fruits from tomato (Solanum lycopersicum) cv. Santa with symptoms of virus-like disease were received for diagnosis from glasshouse crops in Western France. Samples tested negative by ELISA for common tomato viral diseases: Cucumber mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, viruses in the Potyvirus group, Pepino mosaic virus and Tomato yellow leaf curl virus. Affected plants displayed viroid-like symptoms including severe leaf yellowing or reddening, in addition to distortion and stunting. To determine whether the symptoms were caused by a viroid infection, samples were first tested for Potato spindle tuber viroid (PSTVd) and the Pospiviroid group by reverse transcription-polymerase chain reaction (RT-PCR). The ‘semi-specific’ PSTVd-h/c primers (Shamloul et al., 1997), the universal Vir 1/2 (Mumford et al., 2000) and the Vid-FW/RE (Verhoeven et al., 2004) primer sets were used. All tests gave negative results, even with the last primer sets, initially designed to detect CLVd isolates. Since Columnea latent viroid (CLVd) is the only viroid not detected by the universal Vir 1/2 primers, a specific test based on primers pCLV4/pCLVR4 was conducted (Spieker, 1996) and a product of the predicted size (∼370 bp) was obtained. BLAST analysis of the sequenced amplicons (GenBank Accession Nos. FM995506 and FM995507) revealed that the highest identity (99·1% similarity) was with a CLVd isolate recently identified in tomato in England (Accession No. FJ394070, Nixon et al., 2010). Infection by CLVd was confirmed by two methods: mechanical inoculation on to a tomato plant cv. Marmande followed by RT-PCR using the specific pCLV4/pCLVR4 primers; or by a Scorpion real-time RT-PCR assay, followed by RT-PCR and sequencing using specific CLVd primers (J. Tomlinson & W. Monger, unpublished data, Accession Nos. FJ715462 and FJ715463). This is the first report of CLVd in tomato in France where the authorities have been duly informed. The origin of the infection is still unknown. The authors would like to thank E. Demonty and M. Collard for their excellent technical assistance.