Construction Of Cotton Fusarium Wilt Mutant Library And Its Infection In Gossypium Barbadense

Cotton Fusarium wilt is one of the main diseases that hinder the growth and development of cotton.Gossypium barbadense is more sensitive to Fusarium wilt than upland cotton,and there is a lack of research on Fusarium wilt related genes and their infection in Gossypium barbadense.Agrobacterium-mediated T-DNA random insertion mutation is a common and effective method for gene function research in plants and fungi.In this paper,the GFP gene was integrated into the cotton Fusarium wilt by Agrobacterium-mediated method and the genetic transformation system was optimized.The mutant library of cotton Fusarium wilt was preliminarily constructed,and the phenotypes and pathogenicity of some mutants were analyzed to obtain some strains with significant differences in traits.At the same time,a mutant similar to the wild type was selected to infect sea Gossypium barbadense,and its infection in different parts of sea Gossypium barbadense at different times was observed.The main research results are as follows:（1）ATMT-mediated transformation of Fusarium oxysporum f.sp.tritici was successfully carried out,and it was found that the Trp C promoter in fungal expression vector could more effectively activate the expression of foreign genes in Fusarium oxysporum f.sp.tritici,while the plant expression vector Ca MV35S could not.（2）The Agrobacterium-mediated transformation system of Verticillium dahliae was constructed and the transformation program was optimized.The optimum conditions for ATMT transformation of Verticillium dahliae were as follows:150μg/m L hygromycin could completely inhibit the growth of Verticillium dahliae,200μg/m L cefotaxime sodium could completely inhibit the growth of Agrobacterium LBA4404,the initial concentration of Agrobacterium was OD₆₀₀=0.2,the pre-culture time of Agrobacterium was 8 h,the conidia concentration of Verticillium dahliae was 10⁵/m L,the ratio of spore suspension and Agrobacterium LBA4404 was 1:1,the AS concentration was 200μmol/m L,the co-culture time of Agrobacterium and Verticillium dahliae was 4 d,and the culture temperature after transformation was 26℃GFP gene was successfully transferred into Fusarium oxysporum f.sp.vasinfectum,and the optimized transformation efficiency could reach287.33 transformants/10⁵spores.（3）After optimization,more than 1600 transformants were obtained,and the mutant library of cotton Fusarium wilt was preliminarily constructed.According to the phenotypic analysis of 232 mutant strains randomly selected,they were divided into mycelium type I,mycelium type II and bacterial membrane type.Among them,the proportion of mycelial type I was58.37%,the proportion of mycelial type II was 27.35%,and the proportion of membrane type was 14.29%.The pathogenicity of some mutants was tested.The results of southern hybridization showed that the single copy rate was 62.5%,and the mutants had genetic stability.According to the correlation of colony diameter,sporulation and pathogenicity,pathogenicity and sporulation were significantly correlated.（4）Finally,17 mutants with different colony phenotypes were selected,and the colony growth of the four mutants was slow.2 dark purple;4 strains pale purple;10 mutants light yellow;The growth rates of mutants A-7 and A-10 increased;The pathogenicity of A-1 increased,the pathogenicity of A-3,A-7 and C-4 decreased,the sporulation of A-1 increased,and the sporulation of C-6 and C-7 decreased.At the same time,a mutant A-6 similar to wild type was screened.（5）In this paper,the roots and stems were taken as the observation points,and the slice observation showed that the resistant materials could significantly inhibit the infection and expansion of pathogens.The response of resistant sea Gossypium barbadense to Fusarium oxysporum is reflected in the initial infection of root to pathogen,that is,the adsorption amount of conidia in root and germination time after inoculation;It is also reflected in the inhibition of xylem on systemic infection of hyphae or spores.