We report the first identification of novel viruses, and sequence of an entire viral genome, by a single step of high-throughput parallel sequencing of small RNAs from diseased, as well as symptomless plants. Contigs were assembled from sequenced total siRNA from plants using small sequence assembly software and could positively identify RNA, ssDNA and dsDNA reverse transcribing viruses and in one case spanned the entire genome. The results present a novel approach which cannot only identify known viral pathogens, occurring at extremely low titers, but also novel viruses, without the necessity of any prior knowledge.
At the beginning of 2000, a damaging disease developed on protected tomato (Lycopersicon esculentum) crops grown in polyethylene greenhouses in different regions of Spain. Production losses were estimated at 15 to 80%. The tomato plants showed a variety of symptoms. The most common symptoms were leaf distortion, chlorosis, and mosaic. Some plants showed a dark green mosaic and bubbling of the leaf surface. Green striations were also observed on the stem and sepals. Most of the diseased plants had discolored fruits. Symptoms decreased as environmental temperature increased. The involvement of Pepino mosaic virus (PepMV) was suspected. To identify the etiological agent, ≈500 symptomatic tomato plants were collected from several locations in Alicante, Murcia, Almeria and the Canary Islands. Flexuous viral particles 510 nm long were observed by transmission electron microscopy, suggesting the presence of a potexvirus in the tissue extracts analyzed. All samples were tested by ELISA (enzyme-linked immunosorbent assay), using polyclonal antibodies to Narcissus mosaic virus (Adgen, Auchincriuve, Scotland), a virus serologically related to PepMV, and two antisera specific to PepMV (Adgen, Scotland and DMSZ, Braunschweig, Germany). PepMV was detected in 35% of the samples. Like PepMV, the virus infected (as confirmed by ELISA) greenhouse-grown Datura stramonium, Nicandra physalodes, Nicotiana benthamiana, N. clevelandii, Solanum tuberosum, and Vigna sinensis and did not infect Capsicum anuum, Cucumis sativus, Chenopodium amaranticolor, C. quinoa, Petunia × hybrida, Phaseolus vulgaris, Physalis floridana, N. glutinosa, N. rustica, or N. tabacum. The virus did infect Gomphrena globosa, which normally is not infected by PepMV. The first report of PepMV was on pepino (Solanum muricatum) in Peru in 1974 (1), but this virus has been recently reported in the Netherlands, England, Germany, and France on protected tomato crops (2). To our knowledge, this is the first report of PepMV in Spain, including the Canary Islands. References: (1) R. A. C. Jones et al. Ann. Appl. Biol. 94:61, 1980. (2) European and Mediterranean Plant Protection Organisation (EPPO). Alert List Viruses. On-line publication/2000/003.
Pepino mosaic virus (PepMV) is a potexvirus recently identified as the causal agent of a new disease occurring in protected tomato (Lycopersicon esculentum Mill.) crops in the Netherlands (2). PepMV has been subsequently identified in England, Germany, Italy, Morocco, Portugal, and Spain. The new disease has become a serious problem for tomato production in Europe. Most infected tomato plants expressed leaf distortion, chlorosis, and yellow mosaic. Other plants expressed mosaic and bubbling of the leaf surface. Tomato fruits showing severe discoloration and mosaic were observed in protected tomato crops. Symptoms attenuated in tomato plants as the ambient temperature increased. At present, only Solanum muricatum Ait. (Peruvian pepino) and L. esculentum are affected by PepMV.To determine possible reservoir hosts for this virus, 70 samples from Amaranthus sp., A. viridis (L.) Britton et al., Chenopodium murale L., Convolvulus arvensis L., Malva parviflora L., Nicotiana glauca Grah., Polypogon monspeliensis (L.) Desf., Senecio vulgaris L., Sisybrium sp., Solanum nigrum L., and Sonchus oleraceus L. were analyzed. The plants were collected around greenhouses affected by PepMV from different regions in Spain (Murcia and Canary Islands). The samples were analyzed for PepMV by double-antibody sandwich enzyme-linked immunosorbent assay with a commercial antiserum (DSMZ AS-0554, Biologische Bundesantstal, Braunschweig, Germany). Only Amaranthus sp., M. parviflora, N. glauca, Solanum nigrum, and Sonchus oleraceus tested postive. The presence of PepMV in these weed species was confirmed by electron microscopy and reverse transcription-polymerase chain reaction using degenerate primers for potexvirus (1). All the hosts analyzed were asymptomatic. However, symptoms were reproduced by mechanically inoculating tomato plants with sap from naturally infected weeds. To our knowledge, this is the first report of natural infection of weeds by PepMV. References: (1) A. Gibbs et al. J. Virol. Methods 74:67, 1998. (2) R. A. A. Van der Vlugt et al. Plant Dis. 84:103, 2000.
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