Completion of the genome sequence of Watermelon silver mottle virus and utilization of degenerate primers for detecting tospoviruses in five serogroups.

The nucleotide sequence of the L RNA of Watermelon silver mottle virus (WSM oV) was determined. Combined with the previous work on M and S RNAs, the wh ole 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 vir al complementary strand. The L protein shares amino acid identities of only 44.3 and 46.5% with Tomato sparred wilt virus (TSWV) and Impatiens necroti c spot virus, respectively; but an amino acid identity of 91.3% with Peanut bud necrosis virus. Among the sequenced tospoviruses, L protein was the mo st conserved gene product, whereas the nonstructural S protein was generall y the most variable. Comparison of the deduced L protein of WSMoV with thos e of other members of the family Bunyaviridae revealed that its amino acid sequence includes the reported conserved motifs of RNA-dependent RNA polyme rases. To develop a method for detecting tospoviruses by reverse transcript ion-polymerase chain reaction (RT-PCR), two pairs of degenerate primers wer e designed from conserved regions of the L genes and used to amplify the co rresponding regions of the L genes from total RNAs extracted from plant tis sues infected with five serologically distinct tospoviruses. The DNA fragme nts 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 successful ly detected by these two pairs of degenerate primers, with a sensitivity si milar to N-gene-specific primers. The results indicated that the RT-PCR wit h the degenerate primers is a fast and reliable method for detecting tospov iruses in different serogroups.