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温郁金内生拮抗细菌B-11的分离及其抑菌活性
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浙江省“三农六方”科技协作项目(CTZB-F160728AWZ-SNY1-37)


Antimicrobial activity of endophytic bacterium strain B-11 isolated from Curcuma wenyujin
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    摘要:

    【背景】植物内生菌广泛分布在自然界中,具有巨大的潜在开发与应用价值。【目的】对温郁金内生细菌进行分离鉴定,并从中筛选出具有生防潜能的菌株。【方法】采用常规组织分离法对温郁金根茎内生细菌进行分离并利用16S rRNA基因序列分析进行初步鉴定;采用平板对峙法以铁皮石斛炭疽病菌(Colletotrichum gloeosporioides)为供试菌株对温郁金内生细菌进行拮抗菌的筛选;通过形态学鉴定、生理生化鉴定以及16S rRNA基因序列分析,确定拮抗菌株B-11的分类地位;以6个不同属的植物病原真菌为供试菌株对拮抗菌株B-11的抑菌谱进行测定,并研究其对病原菌菌丝的影响;利用特异性平板和MALDI-TOF-MS检测技术对拮抗菌株B-11产生的抑菌物质进行检测;采用离体叶片接种法研究拮抗菌株B-11对铁皮石斛炭疽病菌的防治效果。【结果】从温郁金根茎中分离得到25株内生细菌菌株,这些细菌菌株分属于12个属,其中芽孢杆菌属为优势菌属,占分离菌株的28%;通过初筛获得8株对C. gloeosporioides有抑菌活性的菌株,其中菌株B-11的抑菌活性最强,经鉴定该菌为贝莱斯芽孢杆菌(Bacllius velezensis);抑菌谱测定发现菌株B-11能够对供试的属于不同属的6种植物病原真菌的菌丝生长具有抑制作用,显微观察发现经对峙培养6 d后这6种病原菌的菌丝体出现畸形膨大、分枝增多等现象;特异性平板检测结果表明拮抗菌株B-11能够产生蛋白酶、β-葡聚糖酶和嗜铁素,但不产生几丁质酶;MALDI-TOF-MS检测结果表明拮抗菌株B-11能够产生伊枯草素、丰源素和表面活性素3种脂肽类抗生素,其中伊枯草素的产量最高;离体叶片接种实验表明,拮抗菌B-11的离心去菌发酵液对铁皮石斛炭疽病的防治效率可达64%。【结论】温郁金根茎含有丰富的内生细菌资源,其内生细菌菌株B-11有潜力作为开发抗真菌代谢物和新药物的重要微生物资源。

    Abstract:

    [Background] Endophytes are widely distributed in nature, and have great potential for further exploration and application. [Objective] To isolate and identify endophytic bacteria of Curcuma wenyujin and evaluate their antimicrobial activity. [Methods] Endophytic bacteria from rhizomeof C. wenyujin were separated using routine tissue-separation method and identified by 16S rRNA gene sequences analysis. Their antimicrobial activity against Colletotrichum gloeosporioides was tested by confront-culture method. Strain B-11. C. gloeosporioides was identified through morphological, physiological, biochemical characteristics and 16S rRNA gene sequences analysis. Its influence on the hyphae of 6 pathogenic fungi, belonging to different families, was detected. Its antimicrobial components were determined by 4 specific culture mediums and MALDI-TOF-MS. The control efficacy against C. gloeosporioides on Dendrobium officinale was analyzed by inoculation of leaves in vitro. [Results] Twenty-five strains of endophytic bacteria, belonging to 12 families, were isolated and Bacillus spp. were the dominant species account for 28%. Eight strains showed antagonistic effect against C. gloeosporioides. Bacillus velezensis strain B-11 inhibited the growth of 6 different pathogenic fungi, with the hyphae inflated, deformed and branches increased. It can produce diverse antifungal substances including protease, β-gluconase and siderophore and lipopeptide. Inoculation experiment in vitro found that the cell-free culture of it had a control efficacy of 64% against C. gloeosporioides on D. officinale. [Conclusion] Endophytic bacterium strain B-11 in the rhizome of Curcuma wenyujin is an efficient biocontrol agent with potential for antibiotics development.

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曾欣,张亚惠,迟惠荣,孙钟毓,陈卫良,毛碧增. 温郁金内生拮抗细菌B-11的分离及其抑菌活性[J]. 微生物学通报, 2019, 46(5): 1018-1029

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  • 在线发布日期: 2019-04-30
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