生防枯草芽孢杆菌Kct99的GFP标记及其在甘蓝根部的定殖示踪

Colonization Observation and Control Efficiency Test against Cabbage Wilt Disease of GFP Tagged BacillussubtilisKct99

将含有绿色荧光蛋白基因的质粒pGFP4412,电击转入生防菌株枯草芽孢杆菌Kct99,获得具有良好发光表型的荧光标记菌株gfp-Kct99。经稳定性测定表明,质粒pGFP4412在gfp-Kct99中的遗传稳定性为89%。平板对峙培养和温室盆栽接种试验显示,标记菌株gfp-Kct99对甘蓝枯萎病菌保持了原有的拮抗活性;以标记菌株和野生型菌株培养液对甘蓝盆栽苗灌根处理5 d后再接种病原菌,对甘蓝枯萎病的防治效果分别为87.7%,90.2%,二者无显著性差异,但均显著高于同时接种生防菌和病原菌、接种病原菌5 d后再接种生防菌2种处理,说明,绿色荧光标记菌株gfp-Kct99抗甘蓝枯萎病的活性未受标记基因的影响。借助荧光显微镜观察表明,标记菌株可以在甘蓝根部表皮内大量定殖,从而阻止病原菌的侵入。

Marked strain gfp-Kct99 with luminous phenotype fluorescent was obtained by transmitting plasmid pGFP4412 containing green fluorescent protein gene.The heredity test showed that the stability of plasmid pGFP4412 in engineering strains was 89%;The confrontation culture and pot experiment proved that marked strain gfp-Kct99 maintained the original antagonistic activity to cabbage fusarium oxysporium;The control efficiency of the marked and wild strains were 87.7% and 90.2% against cabbage wilt disease respectively when they were vaccinated 5 d ahead of pathogenic bacteria,no significant differences between the two strains,but significantly superior to that of the two treatments when the antagonistic and Pathogenic bacteria were vaccinated simultaneously or the antagonistic bacteria was vaccinated 5 d later.The results showed that the activity of the marked strain gfp-Kct99 against cabbage blight was not influenced by GFP marking.Observing by fluorescence microscope showed that the marked strain was able to colonize in the root skin of the cabbage to stop the invasion of the pathogen.