植物病原真菌拮抗细菌的筛选鉴定及活性成分分析

Isolation and Identification of Antagonistic Bacteria(RN-61,RN-88) against Ten Different Plant Pathogens

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)2SO4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)2SO4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)。

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 fihrated-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 （ GerlBank accession no. KC840668）, and the 16 S rRNA squence of RN-88 also shared 99% similarity with that of Pseudomonas fluorescens, named Pseudomonas fluorescens RN-88. The optimum saturation of the solution （ NH4 ） 2 SO4 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 diameter of the pathogen was 29.0 mm after 96 h.