| Cucumber green mottle mosaic virus(CGMMV) belongs to the genus Tobamovirus,which leads to devastating losses on cucurbitaceous crops. The virus occurs all over the world and is listed as the plant importing quarantine pest in our country. It has been reported in Shandong, Guangdong, Hubei, Liaoning province of China. According to the reports CGMMV had a tendency to expand year by year on cucurbitaceae crops in Hebei province. In order to find out effective control methods, the isolates collected from Hebei were identified and the dominant strain of Hebei was analyzed. The dominant strain was used as material to screen attenuated isolate. At last the infectious c DNA clone of CGMMV was constructed. The main results are as follows:1. The classification status of CGMMV from Hebei was clarified and the dominant strain was analyzed. The complete genome sequence, classification status and phylogenetic characteristics of the dominant strain was revealed. Five isolates of CGMMV was isolated and purified from the samples collected from the cucurbitaceae crops in Hebei province. Coat protein(CP) was sequenced and alignment and phylogenetic analysis of CP were conducted to Hebei isolates with other seventeen isolates. The dominant strain CGMMV-chb was confirmed by analysis of the CP sequence of five isolates. The complete genome of CGMMV-chb was determined. Alignment and phylogenetic analysis of CGMMV-chb with twelve other isolates was conducted. The results showed that isolates from Hebei, other region of China, Japan and Korea were classified into the same group and shared relative closer relationship. Hebei isolates shared more distant relationship with CGMMV-GR5 and CGMMV-GR3 from Greece. The CP sequence analysis showed that the identities were 100% for the three isolates of CGMMV-chbz, CGMMV-chbx and CGMMV-chbl among five Hebei isolates, so the three isolates maybe the dominant isolate. The complete genome of the dominant isolate, CGMMV-chb, contained 6422 nucleotides and four open reading frames(ORFs), coding four proteins including 128.7 k Da protein, 186.4 k Da protein, 28.9 k Da movement protein and 17.4 k Da coat protein. The relationship of CGMMV-chb was closest to CGMMV-KW from Korea and farthest to CGMMV isolate Ec from Israel.2. The two CGMMV-chb attenuated mutants were obtained by artificial methods. The wild isolate of CGMMV was mutated by three artificial mutation methods, including heat treatment, nitrous acid treatment and combination treatment. The small local lesion was isolated from chenopodium amaranticolour after being attenuated by three artificial methods. All of the single small local lesions were inoculated to systematic host plants cucumber and the symptomless isolates were acquired. The virulence of the attenuated isolates of CGMMV-chb was determined and the multiplication rates and host range were also tested. The results showed that the 127 small local lesions were obtained by the three artificial methods and inoculated to systematic host plants. Two attenuated isolates showed symptomless and positive in RT-PCR, numbered as CGMMV-066 and CGMMV-113. The host plants chenopodium amaranticolour and cucumber were continuous and repeated inoculated 4 times to identify the virulence of CGMMV-066 and CGMMV-113. The proliferation rate of CGMMV-066 and CGMMV-113 showed slower and lower than wild strains by semi-quantitative RT-PCR. The host range of the two attenuated isolates was no significant difference with the wild strain. So CGMMV-066 and CGMMV-113 could be used as the material for attenuated vaccine.3. The infectious c DNA clone of CGMMV-chb was constructed in this study. Full-length CGMMV-chb c DNA was cloned into the vector p UC19 at first. The lineared recombinant vector was used as template for in vitro transcription, and the transcripts were inoculated to host plants by mechanical inoculation. Inoculated plants showed similar symptoms with the typical symptom of wild strains, which was confirmed by mechanical transmission to new plants, RT-PCR and western blot. |
