盐生海芦笋抗菌内生细菌的筛选与鉴定

Isolation and identification of endophytic bacteria from Salicornia bigelovii Torr. and their antimicrobial activities

以抗菌活性为指标,从野生海芦笋中筛选与鉴定具有抗菌活性的内生细菌。采用平板分离与斜面纯化的方法,从海芦笋中分离到13株内生细菌,经过对8株病原菌抗菌活性测试,菌株HLS-7和HLS-8对大肠杆菌(Escherichia coli)、金色葡萄球菌(Staphylococcus aureus)、耻垢分枝杆菌(Mycobacterium smegmatis)和白假丝酵母(Candida albicans)均显示了较强的抑制活性,其抑菌圈直径均>10 mm;对枯草芽孢杆菌(Bacillus subtilis)、产气肠杆菌(Enterobacter aerogenes)、单增李斯特菌(Listeria monocytogenes)和肺炎克雷伯菌(Klebsiella penumoniae)均显示了中等抗菌活性,表现出广谱的抑菌活性。PCR扩增这2株菌的16S r DNA区域,单克隆后测序进行同源性分析,构建系统发育树,结合形态学观察和生理生化实验,将菌株HLS-7和HLS-8分别鉴定庆生芽孢杆菌(Bacillus qingshengii)和甲基营养型芽孢杆菌(Bacillus methylotrophicus)。通过电喷雾质谱分析这2株菌的发酵产物,结果显示,菌株HLS-7可产生脂肽类化合物Fenycin和Iturin,菌株HLS-8可产生脂肽类化合物Iturin,提示这2株菌的抗菌活性可能源于脂肽化合物。

Based on the antimicrobial bioassay-guided screening,13 endophytic bacteria were isolated from Salicornia bigelovii Torr. by spread plate and slant culture methods. The antimicrobial activities of these isolates were tested against 8 strains of pathogens. Among the 13 tested isolates,the isolate HLS-7 and HLS-8 showed strong inhibitory activities against E. coli,S. aureus,M. smegmatis and C. albicans with the inhibition zone diameters more than10 mm,meanwhile showed a broad-spectrum antimicrobial activities against B. subtilis,E. aerogenes,L. monocytogenes and K. penumoniae. The 16 S r DNA region of these two isolates were amplified by polymerase chain reaction（ PCR）and were subjected to monoclone,sequencing and homology analysis,their phylogenetic trees were established as well. Combined with physiological and biochemical experiments,these two isolates were identified as B. qingshengii and B. methylotrophicus,respectively. The electrospray ionization mass spectrometry（ ESI-MS） analysis of the fermentant products indicated that the isolate HLS-7 could highly produced cyclic lipopeptides Fengycin and Iturin,whereas the strain HLS-8 produced Iturin,suggesting that the antimicrobial activities of these two isolates probably derived from cyclic lipopeptides. The current study lays the foundation for further revealing of the relationship between the endophytes and its host as well as mining of active components with antimicrobial activities.