Agrobacterium Tumefaciens-mediated Mutants Library Construction Of Penicillium Digitatum And Their Pathogenicity Analysis

Green mold caused by Penicillium digitatum is one of the most serious fungal diseases of postharvest citrus.At present,the main citrus prevention and control methods are still relying on chemical control.However,the environmental pollution of chemical agents,the amount of residual pesticides and the emergence of drug-resistant pathogens have forced us to find safer methods of prevention and control.Therefore,the pathogenic mechanism of P.digitatum is studied and it has important theoretical and practical value for its better prevention and control.The project was conducted by the Agrobacterium tumefaciens-mediated transformation of P.digitatum to establish an expanded transformant mutant library.From the physiological level,the phenotypic differences between non-pathogenic and wild strains were analyzed.At the molecular level,the mutated gene responsible for the phenotypic changes was determined,knocked out of the mutated genes,and analyzed for gene function.The main findings are as follows:1.The construction of the mutant library.More than 1,000 transformants were obtained through Agrobacterium tumefaciens-mediated transformation of P.digitatum,and they could stably grow on hygromycin.The phosphotransferase gene was amplified by PCR.2.Compare the difference in physiological levels between mutant and wild strains.(1)Scanning electron microscopy and projection electron microscopy were performed on mutant and wild strains.Scanning electron microscopy showed that the wild plants had obvious wormlike structures.The mutants had only mycelium and no wormlike structures.Under the projection electron microscope,the conidia cell structure of the wild strain was intact,the cell wall,septum,cell membrane and internal organelles were obvious.The mutant cell wall was markedly blackened and thickened.(2)The pH of the surface of the mutant and wild citrus was inoculated.Distilled water,wild spore suspension and mutant spore suspension were inoculated into Wenzhou mandarin and Newhall navel oranges respectively.The surface pH of the wound was measured with a flat pH agent.On the third day,the pH at the wounds of inoculated wild strains of Satsuma mandarin decreased from 5.5 to 3.9,and the pH of Newhall navel oranges dropped from 5.3 to 3.3.However,there was no significant change in the results of the inoculation of mutant strains and inoculated water.(3)The growth rate and spore yield of wild and mutant strains were analyzed.On the 8th day,the growth diameter of the wild strains was 5 times that of the mutant strains.On the 13 th day,the diameter of the wild strains was nearly 4 times that of the mutant strains.The number of spores of wild bacteria was nearly 7 times that of the mutant strains.The growth rate and spore yield of the mutant strains were found to be significantly lower than those of wild strains.3.The molecular level analysis of mutants.(1)The T-DNA copy numbers and insertion sites of the mutants were determined by the re-sequencing technique.The re-sequencing results were consistent with the previous Southern blot and Tail-PCR results.The mutants had three T-DNA copies inserted,two T-DNA insertions resulted in two gene mutations,named genes 269 and 275,and another T-DNA insertion site was in the two gene gaps.(2)For the two mutant genes 269?275,the relationship between the mutated gene and the pathogenicity of P.digitatum was studied by the gene knockout method.