蕲艾内生细菌的分离鉴定及抑菌活性分析

Isolation, identification, and analysis on antibacterial activities of endophytic bacteria from Artemisia argyi var. argyi ‘Qiai'

关键词:  蕲艾, 内生细菌, 产酶, 抑菌活性, 16S rDNA

Key words:  Artemisia argyi var. argyi ‘Qiai', endophytic bacteria, enzyme-production, antibacterial activity, 16s rDNA

摘要:

为探究蕲艾(Artemisia argyi var. argyi ‘Qiai')不同组织内生菌组成及其次级代谢产物的抑菌活性,该研究采用组织培养法对蕲艾根、茎和叶内生细菌进行分离,用滤纸片法检测内生菌发酵液挥发物对6种常见病原菌的抑菌活性,并分别对其最低抑菌浓度(MICs)和最低杀菌浓度(MBCs)进行测定,结合形态观察、生理生化性质及16S rDNA序列测定对分离菌株进行鉴定。结果表明:(1)菌株lzy-21、lzy-20和lzy-1分别具有较强的分泌纤维素酶、蛋白酶和脂肪酶的能力。(2)菌株lzy-20和wnn4-3发酵液挥发物对大肠杆菌、产气肠杆菌和枯草芽孢杆菌的MICs均为16 μg·mL^-1; 对三者的MBCs依次为32、32、16 μg·mL^-1和16、32、32 μg·mL^-1。(3)菌株lzy-12对金黄色葡萄球菌和小肠结肠炎耶尔森氏菌的MICs均为16 μg·mL^-1,对二者的MBCs分别为32 μg·mL^-1和16 μg·mL^-1。(4)菌株lzy-17和lzy-21对小肠结肠炎耶尔森氏菌的MICs均为16 μg·mL^-1,MBCs分别为16 μg·mL^-1和32 μg·mL^-1。其中,菌株lzy-1为首次从植物中分离到的皮提不动杆菌(Acinetobacter pittii),菌株lzy-20、lzy-21和wnn4-3依次为高地芽孢杆菌(Bacillus altitudinis)、韩国芽孢杆菌(B. koreensis)和暹罗芽胞杆菌(B. siamensis),菌株lzy-12和lzy-17分别为松果黄体杆菌(Luteibacter pinisoli)和嗜烟节杆菌(Paenarthrobacter nicotinovorans)。该研究结果为充分利用内生菌资源生产工业用酶以及深入探究蕲艾功能内生菌及其作用机制奠定了基础。

Abstract:

The composition of endophytic bacteria in different tissues of Artemisia argyi var. argyi ‘Qiai', and the antibacterial activities of their secondary metabolites were the main focus of this study. The tissue culture method was used to isolate the endophytic bacteria from roots, stems and leaves of Artemisia argyi var. argyi ‘Qiai' and then, the antibacterial activity of the volatiles from endophytic bacterial fermentation broth against six common pathogenic bacteria was assayed by the disk diffusion test. Meanwhile, the minimum inhibitory concentrations(MICs)and the minimum bactericidal concentrations(MBCs)were also determined. Finally, the isolated strains were identified based on phenotypical and biochemical properties as well as 16S rRNA gene sequencing.The results showed that,(1)among the thirteen strains isolated, strains lzy-21, lzy-20 and lzy-1 have strong capacity of producing cellulase, protease and lipase respectively.(2)The MICs of the volatiles of lzy-20 and wnn4-3 to Escherichia coli, Enterobacter aerogenes and Bacillus subtilis were all 16 μg·mL^-1. The MBCs of the them were 32, 32, 16 μg·mL^-1 and 16, 32, 32 μg·mL^-1, respectively.(3)The MICs of lzy-12 against Staphylococcus aureus and Yersinia enterocolitica were 16 μg·mL^-1, and the MBCs of them were 32 μg·mL^-1 and 16 μg·mL^-1, respectively.(4)The MICs of both lzy-17 and lzy-21 against Y. enterocolitica were 16 μg·mL^-1, and the MBCs were 16 μg·mL^-1 and 32 μg·mL^-1 respectively. Strain lzy-1 was identified as Acinetobacter pittii, which was isolated from plants for the first time. lzy-20, lzy-21, wnn4-3, lzy-12, and lzy-17 were identified as Bacillus altitudinis, B. koreensis, B. siamensis, Luteibacter pinisoli, and Paenarthrobacter nicotinovorans, respectively. The above results lay a foundation for making full use of endophytic bacteria resources to produce industrial enzymes, and further exploring the mechanisms of how endophytic bacteria interact with Artemisia argyi var. argyi ‘Qiai'.