植物病原真菌拮抗细菌的筛选鉴定及活性成分分析

摘要

In order to screen the broad-spectrum antifungl bacteria in plant protection , 103 bacterial strains were isolated from soil samples from different agricultural sites in Beijing.Bacteria-pathogen co-cultured method and filtrated-metablites meth-od were used to screen antifungal bacteria.The bacteria with the highest antifungal activity were identified with their bio-chemical characteristics and were selected for further identification by partial sequence analysis of their 16 S rRNA genes. The bacteria of RN-61and RN-88 were the effective antifungal strains against ten kind pathogens between all the samples. 16 S rRNA squence of RN-61 shared 99%similarity with that of Bacillus subtilis subsp.subtilis str, named Bacillus subtilis RN-61(GenBank accession no.KC840668), and the16 S rRNA squence of RN-88 also shared 99%similarity with that of P seudomonas fluorescens, named Pseudomonas fluorescens RN-88.The optimum saturation of the solution (NH4)2SO4 for the antifungal protein extracting was also studied.The protein from the saturation of 80%(NH4)2SO4 showed the best an-tifungal effect (P<0.05).The diameterof the pathogen was 29.0 mmafter 96 h.%为了获得应用于植物病害防治的高效广谱拮抗细菌菌株，从采自北京不同农田区的土样中分离得到103株细菌，采用平板对峙培养法、发酵产物活性测定法，并根据16 S rRNA基因序列分析以及生理生化反应鉴定、筛选了拮抗菌株。选用不同饱和度的（ NH4）2 SO4溶液提取拮抗蛋白并分析其抗菌活性。结果表明：分离的菌株中，菌株RN-61和RN-88对供试的10种病原真菌均具有明显的抑制作用；菌株RN-61和RN-88的发酵液对桃褐腐病菌的抑制率较大，分别为87．31％和80．15％（ P＜0．05）；对棉花红腐病菌、辣椒炭疽病菌的抑制率低于40％（P＜0．05）。 RN-61与枯草芽孢杆菌相似度最高，为99％，确定其为芽孢杆菌属的枯草芽孢杆菌（Bacillus subtilis RN-61），NCBI登录号为GenBank accession no．KC840668。菌株RN-88与Pseudomonas fluorescens F113等相似度为99％，确定其为假单胞菌属的荧光假单胞杆菌（Pseudomonas fluorescens RN-88）。对不同盐浓度提取的菌株RN-61抗菌蛋白活性研究发现，效果最好的是饱和度为80％的（ NH4）2 SO4溶液提取的蛋白。蛋白原液对桃褐腐病菌的抑菌活性最强，96 h后的病原真菌直径为29．0 mm（P＜0．05）。