内生枯草芽孢杆菌JL4在葡萄叶上的定殖及其对葡萄霜霉病的防治

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.

Colonization of grape leaves by endophytic Bacillus subtilis JL4 and its control of grape downy mildew

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.