Genomic characterization of Calla lily chlorotic spot virus and design of broad-spectrum primers for detection of tospoviruses

[[abstract]]Calla lily chlorotic spot virus (CCSV) collected from calla lily plants showing symptoms in Taiwan has been identified as a tentative species of the genus Tospovirus based on the comparison of S RNA sequences. In this investigation, the complete sequences of its L and M RNAs were determined. The L RNA contains 8911 nucleotides (nt) and encodes a putative RNA-dependent RNA polymerase of 2882 amino acids (aa) (332 kDa) in the viral complementary (vc) sense. The M RNA was shown to be 4704 nt in length, encoding a nonstructural NSm protein of 309 aa (35 kDa) in the viral sense and a Gn/Gc glycoprotein precursor (GP) of 1123 aa (128 kDa) in the vc sense. Phylogenetic analysis of the individual tospoviral proteins indicated that CCSV is closely related to Tomato zonate spot virus, a new tentative tospovirus isolated from tomato in southern China. Two degenerate primer pairs gL2740/gL3920c and gM410/gM870c, designed, after sequence comparison, from the highly conserved regions of the L and NSm genes, respectively, were successfully used to detect 12 greenhouse-cultured tospoviruses, including six formal species and six tentative species, by reverse transcription-polymerase chain reaction. They were also used to detect Melon yellow spot virus and Watermelon silver mottle virus from field cucurbit samples in Taiwan. The results indicate that the two degenerate primer pairs can be used for prompt detection of most tospoviruses and exploration of unknown tospovirus species