Shen Hong Miao, Li Zheng Nan, Jia Zhao Shan, Yang Jia Yao, Ran Long Xian
College of Forestry, Hebei Agricultural University, Baoding 071000, China.
Mountain Area Research Institute of Hebei Province, Baoding 071000, China.
Ying Yong Sheng Tai Xue Bao. 2016 Dec;27(12):4022-4028. doi: 10.13287/j.1001-9332.201612.038.
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 3.6×10, 2.7×10 and 3.1×10 CFU·g at 0, 3 and 7 days after inoculation, and the population density inside the leaf tissue reached a maximum of 9.6×10 CFU·g 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 10 CFU·g was a prerequisite for this Bacillus strain to present efficient control effects.
为了解枯草芽孢杆菌JL4在葡萄叶片表面和内部的定殖模式及其对葡萄霜霉病的防治效果，将携带绿色荧光蛋白（GFP）基因的穿梭载体pGFP78转化到枯草芽孢杆菌JL4中，成功获得了绿色荧光蛋白标记的转化子，命名为JL4-gfp。检测了JL4-gfp标记的稳定性及其对葡萄霜霉病菌的拮抗活性。将JL4-gfp喷雾接种到葡萄园的葡萄叶片上，通过在选择性培养基上稀释平板计数来研究其在叶片上的定殖情况。采集经JL4-gfp处理的叶片带回实验室，接种葡萄霜霉病菌的孢子囊悬浮液，以测定其对葡萄霜霉病的防治效果。标记菌株的绿色荧光至少在连续传代10次后仍保持稳定，JL4-gfp对葡萄霜霉病菌保持野生型拮抗活性。通过在添加抗生素的培养基上稀释平板计数，从葡萄叶片中回收JL4-gfp。接种后0、3和7天从葡萄叶片表面回收的菌数分别为3.6×10、2.7×10和3.1×10 CFU·g，叶片组织内部的菌量在接种后3天达到最高，为9.6×10 CFU·g，但在14天后无法回收。接种后3天，标记菌株对霜霉病的防治效果超过88.0%，但7天后未观察到显著的防治效果。我们的结果表明，JL4-gfp的菌量密度与葡萄霜霉病的防治效果呈正相关，10 CFU·g的定殖水平阈值是该芽孢杆菌菌株发挥高效防治效果的前提条件。
