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Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with Cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly-constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (Agroinfiltration and in vitro RNA transcription) and a modified Hepatitis delta virus (HDV) ribozyme sequence to precisely cleave RNA transcripts at the 3’ end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to Tobacco mosaic virus (TMV); 49.85% to Pepper mild mottle virus (PMMoV); 50.47% to Tomato mosaic virus (ToMV); 60.9% to Zucchini green mottle mosaic virus (ZGMMV); and 60.96% to Kyuri green mottle mosaic virus (KGMMV), confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as ‘Russian’, ‘Israeli’, and ‘Asian’ groups. All of our new Korean isolates fell within the ‘Asian’ clade.
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