目的 结合高通量测序技术和传统可培养技术对虎耳草Saxifraga stolonifera内生真菌类群及多样性进行系统分析，并对其抗氧化活性进行初筛，为虎耳草内生真菌资源的开发与利用奠定基础，也为天然抗氧剂的筛选提供了菌种资源。方法 虎耳草内生真菌多样性的分析采用Illumina Miseq高通量测序技术和组织分离法，利用化学法检测虎耳草内生真菌对1,1-二苯基-2-苦基肼自由基（2,2-diphenyl-1-picrylhydrazyl radical，DPPH）、羟基自由基（hydroxyl radical, OH）和2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)，ABT] 等自由基的清除率，进而筛选出具有抗氧化活性的菌株。结果 高通量测序分析发现虎耳草内生真菌隶属于347属，其中Dactylonectria（15.68%）为根部优势菌属，茎部的优势菌属为Humicola（9.57%），而叶部优势菌属为炭疽菌属Colletotrichum（5.33%）。利用组织分离法从虎耳草分离到381株内生真菌，隶属于31个属，主要类群为炭疽菌属（19.42%）、篮状菌属Talaromyces（10.76%）和曲霉菌Aspergillus（8.66%）。基于高通量测序的和组织分离法分析的多样性指数显示虎耳草根部内生真菌的多样性明显高于茎部和叶部，而且虎耳草内生真菌总体多样性指数均高于多数已报道的其他药用植物。24株代表性菌株发酵液的抗氧化活性初筛结果显示：对DPPH、OH和ABTS自由基清除率高于50%的菌株分别占总数的91.67%、79.17%和83.33%，其中菌株Talaromyces sp. HECS10对DPPH、OH和ABTS 3种自由基的清除率均高于75%。结论 虎耳草内生真菌的类群分布具有较高的多样性，且蕴含着较高比例的抗氧化活性菌种资源，为筛选微生物来源的天然抗氧化剂提供了重要资源库。

Objective To analyze the taxonomic structure and diversity of endophytic fungi of Huercao (Saxifraga stolonifera) by high-throughput sequencing technology and traditional culturing techniques, and screen the endophytic fungi strains with antioxidant activity, which laid the foundation for the utilization of the endophytic fungi of S. stolonifera and also provided microbial resources for screening of natural antioxidants. Methods The diversity of endophytic fungi in S. stolonifera was analyzed using Illumina Miseq high-throughput sequencing technology and tissue isolation method. The scavenging rate of endophytic fungi on DPPH, OH and ABTS was detected by chemical method for screening the endophytic strains with antioxidant activity. Results The diversity of endophytic fungi in S. stolonifera based on high-throughput sequencing showed that the endophytic fungi of S. stolonifera belonged to 347 genera. The Dactylonectria (15.68%) was the dominant genera in roots, and the dominant taxa in stems was Humicola (9.57%), while the dominant genera in leave was Colletotrichum (5.33%). A total of 381 endophytic fungi belonging to 31 genera were isolated from S. stolonifera using the tissue isolation method and the main taxa were Colletotrichum (19.42%), Talaromyces (10.76%) and Aspergillus (8.66%). Diversity indices analyzed based on high-throughput sequencing and the tissue isolation found that the diversity of endophytic fungi in S. stolonifera roots was significantly higher than those in stems and leaves, and that the total diversity index of fungal endophytes in S. stolonifera was higher than those in most other medicinal plants that have been reported. The results of the antioxidant activity of 24 representative endophytic strains showed that stains with free radical scavenging rates higher than 50% for DPPH, OH and ABTS accounted for 91.67%, 79.17% and 83.33%, respectively. Strain Taromyces sp. HECS10 had scavenging rates of over 75% for all of DPPH, OH and ABTS. Conclusion The endophytic fungi of S. stolonifera had a high diversity and contained a high proportion of antioxidant active bacterial resources, which provides an important resource library for screening natural antioxidants derived from microorganisms.

R286.2

国家自然科学基金项目（31971384）