Establishment Of Transformation System Of Colletotrichum Gloeosporioides

Li Wei Plant Protection College, Environment and Plant Protection Research Institute;CATAS & SCUTA Baodao Xjncun, Danzhou, Hainan, P.R. China, 571737Based on Agrobacterium-mediated T-DNA insertion, we established an efficient transformation system of collecotrichum gloeosporioides(CG) from mango and obtained some T-DNA insertion mutants of CG by this system in this study, aiming to clone the pathogenic-related genes from CG and for the further research on the CG functional genomics. The main results are as followings:1. 14 CG strains were isolated from the mango fruits with anthracnose symptoms in different locations on Guangdong and Hainan Province, China. The pathogenicity and sporulation capability of these isolates were compared, and the isolate LCG5 from CATAS, Danzhou City, Hainan Province, China were Chosen for further use in this study for its high virulence on mango fruits and also good sporulation capability.2. The key conditions and parameters for co-cultivation of LCG5 and Agrobacterium were tested and optimized, including: Agrobacterium in OD660=0.15 was the optimum density for LCG5 transformation; pH5.5 was most suitable acidity of the medium for the co-cultivation of LCG5 and Agrobaterium; Among 3 Agrobaterium isolates, AGL-1, EHA105 and LBA4404, AGL-1 resulted in highest transformation efficiency on IM medium with pH5.5 and 200ppm of Hygromycin B; Medium with 200umol/L of AS had a much higher transformation efficiency than the medium without AS.33. The above optimized conditions and parameters were integrated into a LCG5 tansformation system. This system was practically tested and the transformation quality was testified. The results were as followings: 30-40 of Hygromycin B resistant clones per Ixl05 conidia of LCG5 were obtained. The clones still maintained their Hygromycin B resistance after 6 continuous transfers and incubated on PDA medium that absent Hygromycin B, showing the stabilization of Hygromycin B resistance of the mutants obtained was stable. With a pair of primers designed by Hygromycin Phosphotransferase gene in plasmid used in the LCG5 transformation, a fragment completely homologous to this gene was separately amplified by PCR from all clones randomly selected from the LCG5 mutants, confirming the Hygromycin gene has really inserted into the LCG5 genome, and also indicated the low fake positive clones from the LCG5 transformation system.4. Many T-DNA insertion mutants LCG5 were obtained by using the above transformation system. The colony morphology and sporulation capability of the mutants and their wild type (LCG5) were compared and 11 morphology and sporulation capability mutants discovered, of which 3 mutants were significantly different from wild type in colony appearance and color mutants, 6 mutants produced distinctly less conidia.