Knock Out And Preliminary Study Of Gene Acetolactate Synthase ALS In Fusarium Oxysporum F.Sp.Cubense

Banana vascular wilt is a kind of solid transfer vascular bundle systemic disease caused by Fusarium oxysporum f.sp. cubense. Pathogens infect the plant roots by blocking the vascular bundle and secrete toxic substance, which result in the leaves wilt and the whole plant dying of wilt. However, there is no ideal chemical to control fusarium wilt happening or effective resistant variety at present, and the research of biological control is just stated. Therefore, it is especially important to understand the related pathogenic gene of Fusarium oxysporum f.sp. cubense about controlling banana vascular wilt. Now, it is a main way to know if gene function is relative to the pathogenicity through DNA inserted mutation producing pathogenic defect mutants.In this research, a Fusarium oxysporum f.sp. cubense transfer system has been used and the transformant phenotype was analyzed. The preparation of fusarium protoplasts following program:Fusarium oxysporum f.sp. cubense is inoculated in PDA medium with cultivating for5days, and then use sterile toothpicks to pick a few mycelium to PD liquid medium with cultivating for2days under28℃、200r/min. Spore suspension was obtained by filtrating mycelium. Inoculating1～2ml (the concertration of spore suspension is1×107cfu/mL) to PD liquid medium and sharking cultivated for12-14h under28℃、120r/min, following by filtrating, washing and collecting the mycelium.10mg/ml of lytic enzymes and10mg/ml of driselase are mixed with the mycelium to form enzyme solution, the enzymolysis condition is70r/min shaking,with room temperature for2-3h. Then the protoplasts has been collect after filtration and washing by the0.8mol/mL NaCl. This program could be obtained800μl protoplasts with100ml (2-5)×107cfu/mL mycelium culture solution.In this study, the transfer method mediated by CaCl2-PEG (polythylene glycol) has been used, and the representative strain of Fusarium oxysporum f.sp. cubense B2was labeled with ALS.5obtain transformants and the wild-type strain are no significant difference in colony morphology, mycelial morphology, colony growth rate, terms of biocontrol Bacillus antagonistic effect and pathogenic to healthy Brazilian banana seedlingsare.