一株香樟炭疽病拮抗菌的鉴定及其发酵条件优化

Identification of an Antagonistic Bacterium Against Camphor Anthracnose and Optimization of Fermentation Conditions for It

为寻获香樟炭疽病生物防治的优良菌种资源,通过形态学观察、生理生化检测和基于16S rDNA系统发育分析,对一株香樟炭疽病拮抗菌SWUJ1进行了菌种鉴定;通过单因素试验优化该菌株产生抑菌活性物质的发酵条件,并釆用抑菌圈法检测其发酵液抑菌活性;进而利用菌丝生长速率法测定该菌株的抑菌谱.菌种鉴定结果表明SWUJ1为革兰氏阳性杆状菌株、产芽孢,在LB固体培养基上菌落呈圆形、边缘整齐光滑、湿润、呈乳白色黏稠状,过氧化氢酶呈阳性且具运动性;基于16S rDNA序列的系统发育分析结果显示该菌株与登录号为NR116240的甲基营养型芽孢杆菌( Bacillus methylotrophicus )的亲缘关系最近,且处于系统发育树的同一分枝,故将SWUJ1菌株鉴定为甲基营养型芽孢杆菌,命名为 B. methylotrophicus SWUJ1;发酵条件优化结果表明该菌株产抑菌活性物质的最佳氮源为酵母粉,碳源为乳糖,无机盐离子为MgSO 4,初始pH值为5.0,培养温度为25 ℃,接种量为1.0%,发酵时间为96 h,优化后拮抗细菌 B. methylotrophicus SWUJ1等量发酵上清液对香樟炭疽病菌的拮抗作用显著提高,且其发酵上清液对核盘菌( Sclerotinia sclerotiorum )及旋孢腔菌( Cochiobolus sativus )等10余种常见植物病原菌具不同程度的抑制作用.研究结果表明, B. methylotrophicus SWUJ1菌株可作为开发香樟炭疽病生防制剂的候选菌株.

In order to prepare strain resources for the biological control of camphor anthracnose, an antagonistic bacterium SWUJ1 against camphor anthracnose was identified by morphological features, physiological and biochemical characteristics and 16 S rDNA phylogenetic analysis. Further, the fermentation conditions and medium composition were optimized through a single-factor experiment, and the antimicrobial activity of the cell-free fermentation supernatant was determined with the inhibition zone method. Moreover, the antimicrobial spectrum of the cell-free fermentation supernatant was assayed with the method of mycelia growth rate. The results of identification showed that SWUJ1 was Gram-positive, rod-shaped and able to form spores. The colony of SWUJ1 was round, neat, smooth, moist, milky white and viscous on the LB solid medium. Its catalase and motility were positive. Based on 16 S rDNA phylogenetic analysis, SWUJ1 was close to Bacillus methylotrophicus and in the same minimal clade with B.methylotrophicus(accession number: NR116240). Therefore, strain SWUJ1 was identified as a strain of B. methylotrophicus, and named B. methylotrophicus SWUJ1. The results of fermentation optimization showed that optimal nitrogen source for the antibacterial substances of strain SWUJ1 was yeast powder, the carbon source was lactose, and the inorganic ion was MgSO4 and the optimal culture conditions were inoculation size of 1.0% for 94 h at 25 ℃ with an initial pH of 5.0. Equal fermentation supernatant of the antagonistic bacterium B. methylotrophicus SWUJ1 significantly improved its antagonistic activity against camphor anthracnose via fermentation optimization and showed antagonistic activity in different degrees against more than 10 plant pathogens such as Sclerotinia sclerotiorum and Cochiobolus sativus. The above results indicated that B. methylotrophicus SWUJ1 could be a candidate strain for future biological control of camphor anthracnose.