中国主要疯草内生真菌分离鉴定及其抑菌活性研究

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (9): 1471-1478.

中国主要疯草内生真菌分离鉴定及其抑菌活性研究

陈基萍，赵宝玉^*，路浩^*，丁来弟，张广芳，李小蒙，高源，周启武，杨晓雯

（西北农林科技大学动物医学院，杨凌 712100）

收稿日期:2012-02-14 出版日期:2012-09-25 发布日期:2012-10-26
通讯作者: 赵宝玉，E-mail: zhaobaoyu12005@163.com；路浩,E-mail: luhao@nwsuaf.edu.cn
作者简介:陈基萍(1986-)，女，广西浦北人，硕士生，主要从事动物中毒病与毒理学研究，E-mail: cjp04205153@163.com
基金资助:
国家自然科学基金项目（31072175；31201958）；国家“十二五”公益性行业（农业）科研专项（201203062）；教育部高校博士点基金新教师项目（20100204120018）；中央高校基本科研业务费（QN2011108）

Study on Isolation, Identification and the Antibacterial Activity of Endophytic
Fungi from the Major Locoweed in China

CHEN Ji-ping, ZHAO Bao-yu^*, LU Hao^*, DING Lai-di, ZHANG Guang-fang,
LI Xiao-meng, GAO Yuan, ZHOU Qi-wu, YANG Xiao-wen

（College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China）

Received:2012-02-14 Online:2012-09-25 Published:2012-10-26

摘要/Abstract

摘要： 探明中国西部草地主要疯草内生真菌的种类及其分布，并通过内生真菌次生代谢产物抑菌活性筛选，为疯草内生真菌次生代谢产物开发利用提供参考。将采自青海和内蒙古草地的小花棘豆、黄花棘豆、甘肃棘豆、急弯棘豆、兰花棘豆和变异黄芪，运用表面消毒法进行内生真菌分离纯化；采用形态学和结合rDNA-ITS序列分析技术种属鉴定，NJ法构建系统发育树并分析其亲缘关系；以金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌和沙门菌为测试菌株，用纸片法进行抑菌活性筛选。结果从中国6种主要疯草中分离获得265株内生真菌，合并得到38株，30株分属12科19属，8株未确定科。其中镰刀菌属、链格孢属和拟青霉属是优势种群，分别占总菌株的16.98%、11.32%和9.81%；系统发育分析显示，38株内生真菌绝大部分具有较高亲缘关系，仅少数亲缘关系偏低，其亲缘关系的远近受疯草种类、部位及生态环境等多种因素的影响；抑菌试验结果表明，38株内生真菌有29株次生代谢产物至少对1种测试病原菌有抑菌活性，占总分离菌株的76.32 %，其中有2株次生代谢产物对4种测试病原菌均有较强抑菌作用。中国西部草地疯草含有丰富的内生真菌，不同生态地域的疯草内生真菌分布存在空间差异性，其次生代谢产物中存在丰富的天然抑菌活性物质，可作为筛选抑菌活性物质的新资源。

关键词: 疯草, 内生真菌, 分离, 鉴定, 抑菌活性

Abstract: The objective of this study was to ascertain the species, distribution and genetic development of the endophytic fungus from the major locoweed in the west meadow of China and detect the antibacterial activity of their secondary metabolites. The study collected Oxytropis flabra, Oxytropis Ochrocephala Bunge, Oxytropis Kansuensis, Oxytropis Deflexa, Oxytropis Coerulea and Astragalus variabilis samples to isolate and purify the endophytic fungi in the different tissues, then used the morphology techniques and ITS sequence analysis to identify their species, and built the phylogenetic tree by Neighbor-Joining method. Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella were selected as indicators to do the antibacterial tests of the fungi secondary metabolites by paper disc diffusion method. 265 fungal colonies were isolated from the collected locoweed, which were combined to 38 kinds of different endophytic fungus. The distribution of the endophytic fungus in the tissues of locoweed was different; Fusarium sp. Alternaria sp. and Paecilomyces sp. were the dominant strains, which occupied 16.98%, 11.33% and 9.81% of the total isolated colonies respectively. Phylogenetic analysis showed that 38 kinds of endophytic fungus could be divided into two populations based on the evolutionary kinship. The endophyte species and the distance of genetic relationship belonged to locoweed species, tissues, ecological environment and other factors. The antibacterial tests results showed that the secondary metabolites of 29 strains at least (76.32% of the total strains isolated) presented the antibacterial activity against one of the tested bacteria, of which the secondary metabolites of two strains were found to have stronger antibacterial activity against the four tested bacteria. There are various species of endophytic fungus in the major locoweed in the west meadow of China, the endophytes in different ecological environment have a certain kinship but also with some differences. Diverse antibacterial natural products are existed in the secondary metabolites of endophytic fungus from locoweed, which could be a new resource to screen active substance with antibacterial activity.

Key words: locoweed, endophytic fungi, isolation, identification, antibacterial activity

中图分类号:

陈基萍, 赵宝玉, 路浩, 丁来弟, 张广芳, 李小蒙, 高源, 周启武, 杨晓雯. 中国主要疯草内生真菌分离鉴定及其抑菌活性研究[J]. 畜牧兽医学报, 2012, 43(9): 1471-1478.

CHEN Ji-Ping, ZHAO Bao-Yu, LU Hao, DING Lai-Di, ZHANG Guang-Fang, LI Xiao-Meng, GAO Yuan, ZHOU Qi-Wu, YANG Xiao-Wen. Study on Isolation, Identification and the Antibacterial Activity of Endophytic
Fungi from the Major Locoweed in China[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1471-1478.

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