| Banana fusarium wilt, a soil-borne vascular disease caused by Fusarium oxysporum f. sp. cubense race 4, is considered as one of the most destructive diseases in banana production due to its fast spread and the difficulty of control with chemical pesticides. In this paper, we first collected the pathogen from different cities in Hainan province, and after confirming their pathogenicity, we chose an isolate as the wild strain that was used in the Agrobacterium tumefaciens-mediated transformation. In the second part, we optimized the factors which affected genetic transformation of Fusarium oxysporum f·sp·cubense (Foc) race 4 resulting in transformation efficiency in the range of 700-800 transformants per 106 conidia of Foc race 4. PCR analysis of the mutants showed that the T-DNA had been successfully integrated into the genome of the fungus. A total of 2520 mutants were obtained in the study, and the single-spore isolation and pahtogenicity tests of all the mutants were completed. In the third part, we developed an effective procedure to clone the sequences flanking the T-DNA inserted sites on the genome DNA, and then finished the cloning of sequences flanking the T-DNA inserted sites of our best mutants. The results are shown as follows:1. We first collected and isolated the pathogen from the banana plants showing fusarium wilt disease symptoms from different cities in Hainan province. After microscopic observation and pathogenicity tests, we identified the pathogen as Fusarium oxysporum f.sp. cubense, and found 10 isolates belonged to Fusarium oxysporum f.sp. cubense (Foc) race4. Later we chose isolate 193-6 as our wild-type strain for ATMT.2. Before the establishment of T-DNA insert mutants library, we optimized the transformation system of Focr4 as follows:co-cultivation with 150μmol/L acetosyringone for 48 h, induction cultivation at pH 5.5 and 25℃,7 hours for Agrobacterium induction by AS and 0.15 for the Agrobacterium OD600 before IM culture3. Hygromycin gene using a strong promoter of the plasmid pBHt2 successfully expressed in our mutants, and a total of 2520 transformants were obtained in the study, which provided a foundation for better understanding of the molecular mechanism of Foc pathogenicity. Also we obtained single spore cultures of the mutants, and completed the pathogenicity tests. 4. An efficient and large-scale pathogenicity testing method was developed for our mutants. We first tested the pathogenicity of our mutants by using banana leaves in vitro, then tested the good mutants by using banana plants growing in tissue culture bottles, and finally we tested the best mutants in the field.5. After the pathogenicity tests, we obtained 3 completely non-pathogenic mutants, 7 almost non-pathogenic mutants,11 mutants with decreased pathogenicity,4 mutants with increased pathogenicity, and 1 mutants with strongly increased pathogenicity and some mutants with different colony morphology and colony colour.6. The sequences flanking the T-DNA inserted sites on the genome DNA of our 18 pathogenicity-related mutants were amplified by DW-ACP-PCR technology. The specific fragments were amplified from all these mutants. These fragments were recovered, cloned, and successfully sequenced. After comparison with the Focr4 genome, we found the sequence to be the same as that flanking the T-DNA inserted sites, and the DW-ACP-PCR technology could be used effectively to clone the flanking sequence of our mutants. |
