Approaches Optimization Of Agrobacterium Tumefaciens-mediated Transformation Of The Filamentous Fungus Penicillium Digitatum In Citrus And The Preliminary Research On The Pathogenic Mechanism

Penicillium digitatum is the most significant pathogen to post-harvest citrus losses. Therefore, studying its pathogenic mechanism and preventing the disease effectively caused by the pathogen are extremely imperative. In this work, establishing the Agrobacterium tumefaciens-mediated transformation system of Penicillium digitatum, stably and efficiently obtaining plenty of mutant transformants and eventually establishing the pathogen mutant library. At the same time, by comparing the pathogenic differences of transformants, an non-pathogenic mutant strain was obtained.In addition, molecular analysis(PCR analysis, Tail-PCR analysis, Southern blot, etc.) were conducted to identify T-DNA insertion site. RNAi technology was applied to explore the relationship between mutation gene and the pathogenicity of Penicillium digitatum. The main results are as follows:1.By optimizing the method of Agrobacterium tumefaciens-mediated transformation of Penicillium digitatum, the optimal transformation conditions were eventually found: The results showed that 20 to 50 transformants per dish could be obtained when nitrocellulose membrane as qualitative filters paper, Agrobacterium concentration up to an OD600 of 0.6 to 0.8, the pathogenic concentration up to 2×106 CFU/ml, and co-incubation at 25℃ for 48 h. The mutant library of Penicillium digitatum including more than one thousand of transformants has been established.2.An non-pathogenic mutant strain was obtained through comparative experiments that transformations were vaccinated in the citrus. two T-DNA insertion sites were found by Southern blot analysis and the method of Tail-PCR.3.The relationship between the two mutant genes and the pathogenicity of Penicillium digitatum by RNAi technology has been preliminary study. But further research is necessary.