| Verticillium wilt that is caused by Verticillium dahliae Kleb is a vascular systemic soil transmission disease and reduce the yield and quality of potato. Collected potato plant that infected Verticillium wilt from Si zi wang qi, were isolated and identified, eventually identified as Verticillium dahliae. Transformation of plasmid mediated by Agrobacterium mediated genetic transformation was carried out with GFP labeled plasmid, and 47 positive transformants. We tested the biological characteristics of 8 transformants and select one stable transformants. Infection process of VDgfp3 in potato were studied. The results are as follows:1. In this study, we isolated Verticillium dahliae from potato yellow wilt plant, we verified that Verticillium dahliae by using of Koch’s postulation.2. The comparison of the Verticillium wilt resistance in each of the varieties, the disease index of the yun shu 301 is 53.33,and Zhi pi’s disease index is 26.67.3. A binary construct contained GFP gene was transferred via Agrobacterium tumefaciens-mediated system into V. dahliae isolate VD01.47 positive transformants were obtained by hygromycin B selection and molecular verification.4. Eight tansformants were randomly selected to characterize the colony morphology, growth rate, conidia production, toxin production and pathogenicity. The results showed that three transformants could produce more microsclerotia compared with the wild type strain; whereas, one transforment produced less microsclerotia. The significant difference on the growth rate of eight transformants was detected after cultured on medium for 10 days. The conidia production ability of the transformants were decreased to certain degree. The average germination rate of all the tested transformants was lower than that of the wild strain after 8h germination.Regarding to the toxin production, seven transformants showed increased tendency, while only one decreased compared with the wild type. The pathogencity results showed that, compared with wild strain, only one transformant showed increased tendency, five stayed at the same level as wild strain,the left two strains showed decreased pattern.5.Infection of susceptible cultivar Yunshu301 by VDgfp3. The results of microscopic observation indicated that the inoculation of 12h, the spores on the root surface, and the germination of the spores began to germinate at 24h. After inoculation with 7d, the hyphae expanded from the root to the cortex. 2w after inoculation, hyphae formation of root stem conduit, and no fluorescence signal was observed in the stem. After inoculation with 3w, the OFP of the pathogen hyphae was observed in the stem base, and the mycelium could be spread to the adjacent duct through the hole. In 4 weeks, the GFP was seen in the hyphae of the leaves of the stem upper and lower leaves. When 6-7w was inoculated,the upper leaves, the creeping stems and the vascular bundles of the tubers were able to detect the same fluorescence bands as the positive control.6w after inoculation, part of leaf Verticillium wilt symptoms, inoculation of 10 w of death.6. Using GFP primers to check after inoculation at different time points in different parts of the pathogen. The results showed that in root, stem, leaf and potato blocks were detected in the presence of GFP, indicating that these parts of Verticillium dahliae of colonization. |
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