Abstract:

Based on the establishment of the T-DNA insertion mutant library,in order to screen out the mutants with significantly decreased pathogenicity and clarify the insertion site information,the phenotypic and pathogenicity differences of the mutants were tested.The results laid a foundation for further elucidating the pathogenic molecular mechanism of Fusarium oxysporum.In our previous work,a T-DNA insertion library of F.oxysporum HS2 was constructed via optimized Agrobacterium tumefaciens-mediated transformation(ATMT).In the present study,totally 300 transformants generated by T-DNA insertions were randomly selected and the alien gene was detected via PCR.The colony morphology,growth rate,sporulation,mitosis stability and other biological traits were observed and measured. The pathogenicity differences of mutants were compared by begonia radicle inoculation method. The flanking sequence analysis of insertion site and Southern Blot analysis were performed on the mutants with significantly weakened pathogenicity.The results showed that 283 tested mutants had inserted the target gene with green fluorescence.After the T-DNA insertion mutant was continuously transferred for five generations,it could be stably inherited on the medium containing hygromycin B,and the colony morphology and color had no significant change.In the pathogenicity test of 283 mutants,the pathogenicity of 87.9% mutant strains was weakened.Among the attenuated mutants,HS2-520,HS2-1016 and HS2-2109 almost lost pathogenicity.Eight mutants with significantly weakened pathogenicity were selected for Southern Blot analysis.The results showed that six mutants were single-copy inserted strains and one was double-copy inserted strains.The TAIL-PCR flanking sequences of the eight mutants were amplified.After sequencing by the company,12 flanking sequences were obtained after comparison with the genomic sequence of wild strain HS2.And three of them were identified as gene sequences of F.oxysporum.Six single copy insertion mutants with significantly reduced pathogenicity were obtained,and flanking sequence information of insertion sites of two mutants were confirmed.

Key words: Fusarium oxysporum, T-DNA insertion mutant, Southern hybridization, TAIL-PCR, Pathogenicity