Construct The T-DNA Insert Mutation Library Of Colletotrichum Higginsianum And Pathogenicity Study Of Pathopoiesis Mechanism

Colletotrichum higginsianum Sacc. is a kind of worldwide distribution of plant pathogenic fungi.and produce anthracnose pathogen of cruciferous vegetables. In this paper, the conditions of transform C. higginsianum by Agrobactirium tumfacience mediated-transformant(ATMT) have been studied.And the C. higginsianum T-DNA mutant library was constructed. In this study, it was high transform efficiency used the Ch-1 spores which had been growing for 17 days and the density was 106/ml were cultured 2 days at 23?. In this way, a mutant library contains more than 2000 T-DNA transformants was successfully constructedSelect 8 transformants, PCR amplification T-DNA on the hyg gene and dot blot analysis of transformants, the result showed that transformants did contain T-DNA insert fragment. Transformants were inoculation cultured to determine the inherited stability for 4 generations on PDA containing 20ug/mL hygromycin B and the result showed that all tested transformants could grow on hygromycin B containing PDA plate, but the primitive strain could not grow. Comparison of growth and sporulation, the result showed T-DNA insertion did't affect the normal growth of transformants but increased their sporulation capacity.According to growth characteristics of mutants, selected the three mutants (No.157, No.329 and No.506) to compare with Ch-1 strain on growth rate, sporulation ability and pathogenicity. Different species of Arabidopsis were inoculated, the result showed that No.329 didn't produce pathopoiesis, No.157 and No.506 could infest in the mutants jarl and pad4. Through Southern blot analysis, showed that No.329 and No.157 were T-DNA single-copied but No.506 was double-copied for the two copies.