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 RNAdependent 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.

Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus

“…On the other hand, several consensus sequences can be found among tospoviral genomic sequences. Degenerate primers designed from the consensus nucleotide sequences of RdRp and NSm genes have been used to detect tospoviruses at the genus level [30, 31]. The virus strains with different genotypes will not be excluded when degenerate primers are used in amplification.…”

“…However, a single species-specific primer pair for diagnosis of tospoviruses in a farm is insufficient due to the complex virus categories which occurred in a crop or area. Degenerate primers designed from the conserved regions of genomic sequences simplify detection of tospoviruses in one tube-based RT-PCR, but they are inconvenient to identify virus species [30, 31]. The microarray is an efficient method for simultaneous detection and identification of various plant viruses in one assay to solve these problems [32].…”

Distribution and genetic diversity of Tomato spotted wilt virus following an incursion into Kenya

“…The three primer sets were used for comparison in case a virus isolate was not amplified with one of the sets. The degenerate primer pair gM410 and gM870c (Chen et al , ) and primers for INSV (Mumford et al , ) and TYRV (Birithia et al , ) were used to detect tospoviruses in symptomatic plants that were negative for TSWV, particularly from Bungoma and Loitokitok. GAPDH primers designed from Solamun lycopersicum glyceraldehyde‐3–phospate dehydrogenase (GAPDH) from NM_001279325 using primer3 were used as internal controls to check the quality of RNA used in the analysis.…”

PM 7/139 (1) Tospoviruses (GenusOrthotospovirus)

“…Other tests for broad detection of tospoviruses have been described by Chen et al . (2012) and Mumford et al . (1996b) (Appendix A).…”