In order to understand the colonization pattern of Bacillus subtilis JL4 both on and inside grape leaves, and its control of grape downy mildew, a shuttle vector pGFP78, carrying the GFP gene, was transformed into B. subtilis JL4, and a GFP-labelled transformant designated as JL4-gfp was obtained successfully. The stability of the marker and antagonistic activity to Plasmopara viticola of JL4-gfp were tested. JL4-gfp was spray inoculated on grape leaves in a vine yard and colonization of the leaves was investigated by dilution plating on selective medium. Leaves treated with JL4-gfp were collected and taken to the laboratory for inoculation of a sporangial suspension of P. viticola, to determine its control effect on grape downy mildew. The green fluorescence of the marked strain was stable for at least 10 subcultures, and JL4-gfp maintained wild type antagonistic activity against P. viticola. JL4-gfp was recovered from the grape leaves by dilution plating on medium supplemented with antibiotics. Numbers recovered from the leaf surface of grape leaves were 3.6×105, 2.7×105 and 3.1×103 CFU·g-1 at 0, 3 and 7 days after inoculation, and the population density inside the leaf tissue reached a maximum of 9.6×104 CFU·g-1 at 3 days after inoculation, but could not be recovered after 14 days. The efficiency of downy mildew control by the marked strain was more than 88.0% at 3 days after inoculation, but no significant control effect was observed after 7 days. Our results suggested that there was a positive correlation between the JL4-gfp population density and control efficiency of grape downy mildew, and a threshold colonization level at 105 CFU·g-1 was a prerequisite for this Bacillus strain to present efficient control effects.
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3.6×105、2.7×105和3.1×103 CFU·g-1;叶片内部的定殖在接种3 d后达到最大(9.6×104 CFU·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105 CFU·g-1.
Keywords: Bacillus subtilis; biocontrol; colonization threshold; endophytic bacteria; green fluorescent protein.