Establishment Of Agrobacterium Tumefacien– Mediated Transformation System Of Verticillium Dahliae And Construction Of T-DNA Insertional Mutagenesis Library

Verticillium dahliae is the causal agent of Cotton Verticillium Wilt, the most devastating diseases of cotton in the world. Agrobacterium tumefaciensis was used to transform Verticillium dahliae in research. Genetic transformation system in Verticillium dahliae mediatied by Agrobacterium tumefaciensis was established, and its transformation condition was studied. On the basis of above work, T-DNA insertional mutagenesis library of Verticillium dahliae mediated by Agrobacterium tumefaciensis was constructed. Construction of its ATMT library and screening of the library will establish the foundation for studying functional genomics of Verticillium dahliae and the molecular basis of pathogenicity, which will contribute to develop the new strategies for the control of cotton Verticillium wilt.The pathogenicity of different isolates was tested by wounded root method, and mean disease index of highly aggressive isolates HNZK-7 was 70.96. The results of wilting capability test of crude toxin indicated that crude toxin secreted by HNZK-7 has strong wilting capability, and its mean wilt index was 66.67. The result of hygromycin B sensitivity test showed that the concentration of hygromycin B which inhibited the growth of HNZK-7 was 50μg·mL^-1, the highly aggressive isolates HNZK-7 has a higher sensitivity to hygromycin B. Therefore, HNZK-7was selected to be the wild strain to constructed Verticillium dahliae T-DNA insertional mutagenesis library.The conditions for ATMT in Verticillium dahliae was further optimized based on the basic procedures established in Fusarium oxysporum by Mullins and co-workers,including length of co-cultivation period, the optimal concentration of hygromycin B in transformants selection, the optimal concentration of cefotaxime to inhibit Agrobacterium tumefaciensis, the concentration of AS, choice filters type in the co-cultivation, the concentration ratio between Agrobacterium tumefaciensis and Verticillium dahliae, etc. The results showed that co-cultivation time is 48 h , screening concentration of hygromycin B is 50μg·mL^-1, inhibiting concentration of cefotaxime is 200μg·mL^-1, the concentration of AS is 400μΜ, the filter type in the co-cultivation is nitrocellulose filter, the concentration ratio between Agrobacterium tumefaciensis and Verticillium dahliae is 250:1, and transformation efficiency is 200-600 transformants / 106. Transformation efficiency is 250 transformants / 106 using filter paper as the medium.By the means of ATMT, T-DNA insertional mutagenesis library of Verticillium dahliae mediated by Agrobacterium tumefaciensis was constructed, and 1019 transformants of HNZK-7 were obtained.To test the hygromycin B genetic stability of transformants, 25 transformants were randomly selected to culture 6 generations on PDA, then these transformants were put on PDA plate containing 70μg·mL^-1 hygromycin B. The results showed that all tested transformants could grow on PDA containing 70μg·mL^-1 hygromycin B, but HNZK-7 could not grow on PDA containing 70μg·mL^-1 hygromycin B.Morphology characteristic was observed with 30 randomly selected transformants and HNZK-7. The results showed that 4 transformants were found mutated in colony color, 2 transformants were brought down obviously in colony growth rate, 6 transformants had a weaker microsclerotia development capability, 2 transformants had a lower spores production capability. Moreover, 2 transformants were both found mutated obviously in colony growth rate and microsclerotia development capability, and 1 transformants was found mutated obviously in colony growth rate, microsclerotia development capability, spores production capability.