猕猴桃溃疡病拮抗菌的抑菌物质分离及鉴定

Isolation and identification of antibacterial substances in antagonistic ofPseudomonas syringae pv. actinidiae

对前期筛选得到的猕猴桃溃疡病病原菌丁香假单胞菌亚种(Pseudomonas syringae pv.actinidiae,PSA)拮抗菌进行了鉴定和抗菌物质分离及分析。用革兰氏染色鉴别菌株;将硫酸铵沉淀溶液进行牛津杯抑菌试验,观察抑菌圈的大小;利用Tricine-SDS-PAGE电泳分析硫酸铵沉淀的抑菌物质;然后溶解沉淀进行去离子水透析脱盐,超滤浓缩后用葡聚糖凝胶柱进行分子筛凝胶过滤层析,最后采用高效液相色谱分析其分子量大小。结果发现:可判断有效抑制PSA生长的假单胞菌菌落形态特征为圆形菌落、乳白色、光滑、凸起、边缘整齐、不透明、不运动,为革兰氏阴性菌,菌体呈短杆状;其抑菌物质为细菌素,在40%和60%硫酸铵饱和度下提取效果最明显,抑菌作用最好,细菌素分子量大小为7 k Da左右。为研发细菌素产品提供了新思路,为防治猕猴桃溃疡病提供了理论基础和应用指导。

In this study,an antagonistic bacteria was identified as a potential biological agent to resist Pseudomonas syringaepv.actinidiae(PSA).The antimicrobial substances were isolated and analyzed.The morphology was observed by agar plate,then the strains were confirmed by Gram staining.The antibacterial activity tests of ammonium sulfate precipitation were carried out using Oxford cup assays to observe the size of the inhibition zone.The bacteriostatic substances in ammonium sulfate precipitates were analyzed by Tricine-SDS-PAGE electrophoresis.Then the precipitates were dissolved and desalted with deionized water and concentrated by ultrafiltration and further purified by chromatography.The gel filtration chromatography of molecular sieve was carried out with dextran gel column.According to morphological characteristics,the antagonistic bacteria are round colony,milky white,smooth,protruding,neat edge,opaque,motionless,gram-negative bacteria,short rod-shaped.Subsequent experiments showed that bacteriocin was the effective substance to resist PSA.As observed in the bacterial experiments,the elutes using 40%and 60%ammonium sulfate saturation had the highest activity against PSA,and the molecular weight of bacteriocin is about 7 kDa.The results suggested the potential to develop treatment using bacteriocin products in prevention and treatment of kiwifruit canker.