The Research On The Optimized Transformation System Of The Green Fluorescent Protein Gene To Fusarium Oxysporum From Solanaceae Crop And Its' Colonization Characteristics

Fusarium wilt of solanaceae crop caused by Fusarium oxysporum, is one of the most serious fungal diseases, and reported to be one of the major limiting factors for solanaceae crop production in China.The pathogens of Fusarium wilt were isolated from the tomato, eggplant and pepper, which were collected from the areas in Fujian province. One of F. oxysporum strains from the tomato was marked with the genes coding for GFP, stable transformants were chosen and used to reveal infected and location of the pathogen within the tomato plant by using the transformed strains as tools under CLSM. The major results are as follows:1. 40 Fusarium strains were isolated from the wilt plant of the tomato, eggplant and pepper which were collected from the areas of Zhangzhou, Fuzhou and Xiamen, in Fujian Province. 35 F. oxysporum strains were identified by morphological characteristics, molecular method and pathogenicity tests.2. To optimize the PEG-mediated transformation system of F. oxysporum, one of strain from the tomato was marked with the genes coding for gfp. The optimum method for the transformation of F. oxysporum from the tomato as follows: The tender mycelia were incubated at 28°C for 12-14 h with shaking at 120 r/min, then were collected and added 4 mL of an enzyme mixture containing 10 mg/mL Lywallzyme and 20 mg/mL Driselase. The suspension was incubated at 28°C with gentle shaking(70 r/min), protoplasts were filtered and transformed after 2.5-3 h. The morphology, growth rate and pH value of the gfp-tagged transformants are the same as the wild type strain. Stable transformants were chosen and saved.3. For the systemic infected and colonization of tomato plant by F. oxysporum, transformed strains were followed in whole roots and in longitudinal sections from 3 to 17 days after the inoculation by using CLSM. When inoculated for 3-6 days, the conidia or their germ-tubes of the wilt pathogen were observed to enter the plant through young roots. At inoculated for 10 days, wilt symptoms became visible and the roots and stems were necrosis, green fluorescence mycelia were visible in the root, and base of stem. At 17 days after pathogen inoculation, some of the plant branchs became dying and the stem were necrosis, the green-fluorescing mycelia were visible in the root, stem.4. For the colonization ability of F. oxysporum in the different type soils, gfp-transformaed strains were used to follow the changes of the amount of colonies for 32 days. The results shows F. oxysporum could keep at least one month in the different type soils, but the livability was descend with increasing the days. The best livability was in the soil from the rice field, but the least livability was in the sand.