微生物学通报 APR 20, 2008, 35(4): 512~518
Microbiology 2008 by Institute of Microbiology, CAS
曾庆桂 朱 笃* 颜日明 朱 飞 王志兴
(江西师范大学生命科学学院 江西省亚热带植物资源保护与利用重点实验室 南昌 330022)
摘 要: 从蛇足石杉(Huperzia serrata)叶片中分离到一株对小麦赤霉(Fusarium graminearum)等多种植物病原真菌有强拮抗作用的内生细菌H-6。通过形态和培养特性观察、生理生化实验及16S rDNA序列分析, 初步鉴定该菌属于伯克霍尔德属, 命名为Burkholderia sp. H-6。同时还对该菌培养条件进行了优化, 得出马铃薯浸出液中添加2.5%的甘露醇和0.1% NaNO3有利于细胞生长和抑菌活性的产生, 最适培养温度为28℃, 最佳初始pH4.0。
关键词: 蛇足石杉, 内生细菌, 拮抗真菌活性, 鉴定
Isolation and Identification of Endophytic Bacteria H-6 with High Antifungal Activity from Huperzia serrata
ZENG Qing-Gui ZHU Du* YAN Ri-Ming ZHU Fei WANG Zhi-Xing
(College of Life Sciences, Jiangxi Normal University, Jiangxi Provincial Key Lab of Protection and Utilization
of Subtropic Plant Resources, Nanchang 330022)
Abstract: In this study, nearly 200 endophytic bacteria were isolated from different part of Huperzia serrata, over 60 bacterium with clear antifungal activity were selected from those cultures. Among them, strain H-6 exhibited the highest antifungal activity which was strongly inhibits the growth of many plant pathogenic fungi such as Sclerotinia scleroliorum, Fusarium graminearumt, Sclerotinia libertiana, Phytophthora capsici Leonia and Sesame fusarium wilt. According to the characteristics of morphology, physiology and biochem-istry tests and the comparison of 16S rDNA sequence, the strain H-6 was similar to the Burkholderia. So strain H-6 was identified as Burkholderia sp. H-6. The results also showed that Burkholderia sp. H-6 was markedly different from Burkholderia cepacia that was applied widely in agriculture as antagonistic bacte-ria.
The medium and culture conditions of the strain all were optimized by single factor and orthogonal ex-periments. In the investigation of the culture condition, growth was carried out in a basal medium (potato juice) and gradually supplemented with the various ingredients to be investigated. The major ingredients be-ing investigated included carbon sources and nitrogen sources. The optimal antifungal activity production condition is growth in a medium (potato juice with 2.5% mannitol and 0.1% NaNO3), initial pH 4.0 at 28℃.
Keywords: Huperzia serrata, Endophytic bacterium, Antifungal activity, Identification
植物病害80%以上是由真菌引起的, 控制真菌病害对于提高农作物产量极其重要。目前真菌防治主要以化学农药为主, 而化学农药引起的环境污染已经对人类健康造成潜在的威胁, 因而从中寻找可用作生物农药或者生物肥料的菌种成为当前的研究热点[1]。
植物内生菌是一个多样性十分丰富的生物类群。在植物内生菌中, 有对植物病原菌产生拮抗而保护植物免受病原菌侵害的卫士, 有对宿主体内某些活性成分的形成有重要影响者, 也有能产生与宿主相同或相似的生理活性成分者, 还有与宿主只是共生或伴生关系而作用不明者等等[2]。迄今, 从植物中分离可用作生物农药菌种已有较多的研究, 已从裸子植物和被子植物中分离到较多能产拮抗病害真菌或细菌次级代谢产物的内生菌株[3-6]。但相对而言, 蕨类植物中的内生菌资源研究报道较少。
蛇足石杉( Huperzia serrata (Thunb.) Trev.) 是一种珍贵的药用蕨类植物, 属石杉科石杉属[7]。迄今有关蛇足石杉资源、药用成分等方面已有较多报道, 但有关蛇足石杉内生菌资源的研究方面, 仅见2005年石玮等从蛇足石杉分离出4株内生真菌的报道[8], 且尚未有对其价值进一步研究的报道。本实验室从蛇足石杉中分离到了近200株内生细菌、150株内生真菌。在内生菌分离的过程中发现, 在近三分之一的经过表面消毒的蛇足石杉碎片中都长出了周围有一黄绿色色素圈的细菌, 而且在分离平板上对长出的内生真菌有较强拮抗作用, 命名为H-6。对其进行了病原真菌的拮抗对峙实验, 发现它对小麦赤霉(Fusarium graminearum)等多种植物病原菌都有强拮抗作用。为此笔者对其进行了形态及生理生化特征分析以及16S rDNA分子鉴定, 并对其培养条件进行了初步优化, 为进一步研究H-6和将其应用于生物防治提供依据。
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