The nucleotide sequence of the L RNA of Watermelon silver mottle virus (WSMoV) was determined. Combined with the previous work on M and S RNAs, the whole genomic sequence of this member of the genus Tospovirus was completed. The L RNA is 8,917 nucleotides in length, with one large open reading frame encoding a translation product of 2,878 amino acids (331.8 kDa) on the viral complementary strand. The L protein shares amino acid identities of only 44.3 and 46.5% with Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus, respectively; but an amino acid identity of 91.3% with Peanut bud necrosis virus. Among the sequenced tospoviruses, L protein was the most conserved gene product, whereas the nonstructural S protein was generally the most variable. Comparison of the deduced L protein of WSMoV with those of other members of the family Bunyaviridae revealed that its amino acid sequence includes the reported conserved motifs of RNA-dependent RNA polymerases. To develop a method for detecting tospo-viruses by reverse transcription-polymerase chain reaction (RT-PCR), two pairs of degenerate primers were designed from conserved regions of the L genes and used to amplify the corresponding regions of the L genes from total RNAs extracted from plant tissues infected with five serologically distinct tospoviruses. The DNA fragments obtained were identified as those of tospoviruses by restriction enzyme digestion and DNA sequencing. For field samples, watermelon and wax gourd infected with WSMoV, and lisianthus infected with TSWV were also successfully detected by these two pairs of degenerate primers, with a sensitivity similar to N-gene-specific primers. The results indicated that the RT-PCR with the degenerate primers is a fast and reliable method for detecting tospoviruses in different serogroups.

Calla lily chiorotic spot virus (CCSV), a tospovirus infecting Calla lily (Zantedeschia spp.) in Taiwan, with symptoms of chlorotic spots on leaves and stems, was previously reported serologically but distantly related to Watermelon silver mottle virus (WSM0V). To further characterize the virus, DNA fragments corresponding to the S RNA of the virus were amplified from total RNA extracted from CCSV-infected plants of Nicotiana benthwniana, cloned and sequenced. The full-length viral strand of the S RNA was determined as 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA was found identical to those of other tospoviruses, confirming that CCSV belongs to the genus Tospovirus. The N and the NSs proteins of CCSV share low amino acid identities, 20.9 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. The phylogenetic dendrogram of the N protein of CCSV compared with those of other tospoviruses in WSMoV serogroup indicates that CCSV is a distinct tospovirus. Thus, based on the results of molecular characterization of S RNA, we conclude that CCSV is a new tospovirus species belonging to WSMoV serogroup.