Construction And Characterization Of Two Respective T-DNA Insertional Mutation Population For Two Pathogens Fungus,Botryosphaeria Dothidea And Alternaria Alternata

The two worldwide fruit tree pandemics, caused respectively by Botryosphaeria dothidea and Alternaria alternata, are two major diseases that affect the production of fruits in China, resulting in huge economic loss. Currently only preliminary studies were conducted on these two pathogens. Although there are some progresses made in studying the synthetic genes of AM-toxins(toxins produced in Alternaria mali), function of most other genes remains unknown. The random T-DNA insertional mutation mediated by Agrobacterium tumefaciens is a widely used and yet efficient approach in genetic study on fungi and plants.This study aims to establish an A. tumefaciens-mediated transformation(ATMT) platform, to construct and characterize mutation population of B. dothidea and A. alternata by ATMT, to screen mutants with impaired pathogenicity. Our results, as descripted below, would be helpful for investigating pathogenicity-related gene functions and revealing the interaction between the pathogens and their respective hosts.1. An A. tumefaciens-mediated B. dothidea protoplast transformation platform was established, with a transformation efficiency of 220 transformants per 106 protoplasts; A mini-population with 901 B. dothidea mutants was constructed and insertion of eGFP gene and hygromycin B gene into the genome was verified by PCR.2. Phenotypic analysis of 120 abovementioned mutants found that, 43.3%of the mutants showed increased pigment sedimentation, while 14.2%decreased, and that hypha growth rate for 59.3%of the mutants was faster than that of the wild type, while 6.2%was slower.3. Pathogenicity screening on 500 mutants found 16(3.2%) mutants with obviously weakened pathogenicity.4. An ATMT procedure for A. alternata spore transformation was established and optimized. Spores produced by a 36-48 hours A. alternata culture were cold-treated at 4℃for6 hours before transformation, resulting in a high transformation efficiency at 200 transformants per 106 spores.5. A mini-population with 880 mutants of A. alternata strain XP-1was constructed. PCR and southern blot verified that insertion of hygromycin-resistant gene into the genome of the pathogen was successful, with 63.6%of the obtained mutants as single copy insertion.6. 7 out of 100 mutants with decreased pathogenicity were obtained by pathogenicity screening.