水仙内生真菌的分离鉴定及聚类分析  

Isolation and cluster analysis of endophytic fungi in Narcissus tazetta var. chinensis

作　　者：杨明俊[1] 李娟[1] 王永刚[1] 薛鸿燕[1] 马雪青[2] 杨林[1] 

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中草药》2014年第11期1625-1630,共6页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(31060041);甘肃省自然科学基金资助项目(0908ZTB090;1108ZSB095;2011GS04244);兰州理工大学优秀青年资助计划(1008ZCX007);兰州理工大学教学研究项目(JY2012045)

摘　　要：目的分离纯化水仙根茎中的内生真菌,并进行菌群组成及其多样性分析。方法采用表面消毒、划线法从水仙鳞茎和根中分离内生真菌,基于形态学特征和核糖体转录间隔区（ITS-rDNA）序列分析,对分离株进行分类鉴定;以内生真菌的多样性指数（H）和均匀度指数（E）为指标,分析水仙内生真菌的菌群组成及其多样性。结果共分离到18株内生真菌,其中16株来自根部,2株来自鳞茎,分别占总分离菌株的88.89%和11.11%。PCR扩增其中14株真菌ITS-rDNA,得到大小约500 bp片段,用BLAST软件对其测序结果进行相似性比对,发现4株为青霉属Penicillium,相似性95%-99%;3株为曲霉属Aspergillus,相似性99%-100%;3株为喙枝孢属Rhinocladiella,相似性为99%;其他4株分别为木霉属Trichoderma、链格孢属Alternaria、赤霉属Gibberella和镰孢霉属Fusarium,相似性都为99%。青霉菌为水仙内生真菌优势菌属。通过构建系统发育树发现这14株内生菌明显分为2大类群,Shannon多样性指数为1.778,均匀度为0.913 7。结论水仙内生真菌菌群组成具有一定程度的多样性和差异性。Objective To isolate and purify endophytic fungi from the roots and stems of Narcissus tazetta var.chinensis,and to analyze the composition and diversity of endophytic fungi.Medthods Endophytic fungi were isolated from the bulb and root of N.tazetta var.chinensis by surface sterilization and streaking on plate.Then,the isolates were identified based on morphological characteristics and internal transcribed spacer-rDNA（ITS-rDNA） sequences analysis.Besides,Shannon-Wiener biodiversity（H） and evenness（E） were selected as two key indexes,and the composition and diversity of endophytic fungi were analyzed.Results Eighteen strains of endophytic fungi were isolated,in which 16 strains were obtained from the root（88.89%）,the other two strains were obtained from the bulb（11.11%）.ITS-rDNA fragments from 14 samples were respectively amplified by PCR method with their genomic DNA as templates.The sequencing results showed that most of these DNA fragments were about 500 bp.In addition,according to sequence analysis and comparison with the data in GenBank by BLAST,four strains with the similarity of 95% to 99% were classified as Penicillium,three strains were classified as Aspergillus with the similarity of 99% to 100%,three strains were classified as Rhinocladiella with the similarity of 99%,and the other four strains were classified as Trichoderma,Alternaria,Gibberella,and Fusarium,respectively,with the similarities about 99%.Therefore,it was concluded that Penicillium was the dominant endophytic fungi genus in N.tazetta var.chinensis under our experimental conditions.The phylogenic tree showed that these 14 endophytic fungi were clustered into two main branches,by which factors H and E were determined as 1.778 and 0.913 7,respectively.Conclusion The composition of endophytic fungi community has a certain diversity and difference.

关 键 词：水仙 内生真菌 分离 鉴定 聚类分析 

分 类 号：R282.12[医药卫生—中药学]