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为了明确枯草芽孢杆菌JL4在葡萄叶表面和内部的定殖情况,研究定殖与防治效果的关系,采用电击转化的方法将含有GFP基因的质粒pGFP78导入枯草芽孢杆菌JL4中,并得到成功表达GFP的生防菌JL4-gfp,测试了标记菌株的稳定性及其对葡萄霜霉病菌的抑制作用.采用叶片喷雾法接种,用抗生素平板稀释分离回收,检测生防菌JL4-gfp在葡萄叶片的定殖情况,并将采回的叶片在室内接种葡萄霜霉菌孢子囊悬浮液进行生防测定.结果表明:标记菌株在经过10次传代培养后,仍具有良好的发光表型,能稳定表达GFP蛋白,并且标记菌株JL4-gfp对葡萄霜霉菌保持了原有的抑菌作用;用抗生素平板稀释分离回收,检测到JL4-gfp菌株在葡萄叶片表面的定殖量在接种后的0、3和7 d分别为3.6×10~5、2.7×10~5和3.1×10~3CFU·g~(-1);叶片内部的定殖在接种3 d后达到最大(9.6×10~4CFU·g~(-1)),然后下降,14 d后已经检测不到接种菌株;室内生防测定结果显示,喷雾后3 d对葡萄霜霉病的防治效果达88.0%以上,但7 d后则无明显防效JL4-gfp的定殖量与其防治葡萄霜霉病的效果呈正相关,其有效定殖量临界值为10~5CFU·g~(-1).
In order to clarify the colonization of Bacillus subtilis JL4 on the surface and inside of grape leaves, the relationship between colonization and control effect was studied. Plasmid pGFP78 containing GFP gene was introduced into Bacillus subtilis JL4 by electroporation and successfully expressed GFP The biocontrol strain JL4-gfp was tested for the stability of the marker strain and its inhibitory effect on Plasmopara viticola.The leaf spay method was used to inoculate and the antibiotic was used for dilution and recovery. The biocontrol strain JL4-gfp The results showed that the labeled strain still had a good luminescent phenotype after 10 subcultures and could stably express GFP Protein, and the marker strain JL4-gfp retained the original bacteriostatic effect on grape downy mildew; the antibiotic plate dilution was used for isolation and recovery, and the colonization amount of JL4-gfp strain on grape leaf surface was detected at 0, 3 and The colonization rate in leaves was 3.6 × 10 ~ 5, 2.7 × 10 ~ 5 and 3.1 × 10 ~ 3CFU · g ~ (-1) (-1)), and then decreased, after 14 d has not detected the connection The results of indoor biocontrol test showed that the control effect on grape downy mildew was above 88.0% at 3 days after spray, but no obvious control effect after 7 days was observed. The colonization amount of JL4-gfp and its control effect on grape downy mildew There was a positive correlation between them. The critical value of effective colonization was 10 ~ 5CFU · g ~ (-1).