Identification and characterisation of watermelon green mottle mosaic virus as a new cucurbit-infecting tobamovirus

Abstract

A new virus tentatively named watermelon green mottle mosaic virus (WGMMV) was isolated from watermelon leaves showing mottle, mosaic and leaf crinkling symptoms in Taiwan. Electron microscopy identified tobamovirus-like, rod-shaped particles measuring about 18 nm x 300 nm in the crude saps. The enzyme-linked immunosorbent assay (ELISA) tests of symptomatic watermelon leaves using an antiserum against Cucumber green mottle mosaic virus (CGMMV) resulted in negative reactions. Genomic fragments of WGMMV were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers of cucurbit-infecting tobamovirus. Sequence analysis revealed that WGMMV genome contains 6,482 nucleotides (Gen Bank accession no. MH837097). WGMMV coat protein (CP) gene shares 55.9-82.2% nucleotide identities and 42.6-87.7% amino acid sequence identities and its complete genome shares 62.0-80.9% nucleotide sequence identities with other cucurbit-infecting tobamoviruses. Thus, WGMMV is a new tobamovirus. The virion origin of assembly sequence (OAS) containing a conserved GAGGUUG was found in the CP-coding region of CGMMV, WGMMV and Cucumber mottle virus (CuMoV). A comparison of genome organisation has revealed that both WGMMV and CuMoV, belonging to the sub-subgroup III, have a 29-nt overlap between the movement protein and CP coding genes. Full-length nucleotide sequence of WGMMV shares 80.9% identity with that of CuMoV. Watermelon seedlings inoculated with WGMMV showed similar symptoms with the diseased plants observed in the field. WGMMV proliferated in the inoculated seedlings as evidenced by the ELISA and western blotting analyses. Mechanical inoculation assays revealed that WGMMV caused mosaic symptoms in a wide array of cucurbitaceous plants. A primer pair specifically complementary to the WGMMV CP-coding gene was designed and used to successfully detect WGMMV using RT-PCR. The primers and WGMMV-specific antiserum will be useful for rapid detection of WGMMV.