Infectious Clone Development of Zucchini Green Mottle Mosaic Virus Infecting Medicinal Plant <i>Trichosanthes kirilowii</i> and Establishment of a Serological Assay System

Trichosanthes kirilowii has long been cultivated for application in traditional Chinese medicine. In this study, we identified two isolates of zucchini green mottle mosaic virus (ZGMMV; species Tobamovirus cucurbitae) from T. kirilowii plants. We determined the complete genome sequences of ZGMMV isolates named ZGMMV-GL-1 and ZGMMV-GL-2. Each ZGMMV genome was 6517 nucleotides (nt) in length, with only a single nucleotide variation detected between two sequences. Sequence analysis revealed that the ZGMMV isolates from this study shared 88.07%-91.62% nucleotide identity with five other ZGMMV isolates deposited in GenBank. Phylogenetic analysis indicated that ZGMMV isolates can be clustered into two distinct groups; our two isolates shared the highest sequence similarity with the ZGMMV isolate from Nanning (GenBank accession no. MF066176) and clustered within Group Ⅱ. The coat protein (CP) gene was cloned from ZGMMV-infected T. kirilowii samples, and the CPZGMMV was expressed using the pET28(a) vector. Specific polyclonal antiserum CPZGMMV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPZGMMV antiserum, we developed a rapid, precise, and scalable diagnostic method for ZGMMV. Then, we constructed the full-length cDNA clones (ZGMMV-GL-1 and ZGMMV-GL-2). Additionally, the ZGMMV cDNA infectious clones from T. kirilowii were also able to infect Nicotiana benthamiana and Cucumis sativus systemically, inducing rough-textured and curled leaves in N.benthamiana, mosaic symptoms in C. sativus and T. kirilowii. In this study, we produced an antiserum against the ZGMMV coat protein and developed a sensitive, rapid and reliable diagnostic assay, which lays a technical foundation for the detection and monitoring of ZGMMV. Therefore, the establishment of the ZGMMV infectious clone facilitates further research on viral protein functions, plant-pathogen interactions, and the formulation of effective ZGMMV management strategies.