华石斛内生真菌组织分布特点及其抑菌活性
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摘要
植物内生菌种类繁多,且具有较丰富的生物学功能。从海南特有药用植物华石斛(Dendrobium sinense)野生植株中分离内生真菌,初步探索其组织分布特点及抑菌活性。结果表明:从华石斛中分离内生真菌179株,通过ITS序列将之划为51个OTU,分属于25个属以上的类群,其中叶点霉属(Phyllosticta)为优势属。仅轮层炭壳属广泛存在于华石斛根、茎、叶中,其余类群皆存在一定的组织偏好性,其中18个类群只在一种组织中分布。叶中分离的菌株数最多,占比60.33%。不同培养基条件下分离得到的内生真菌种类也存在明显差异,以PDA和华石斛粉培养基分离到的内生菌类群最多,分别为12种,各占总分离类群的48%。12属36个菌株对至少1种供试菌具有抑制效果,其中抑菌生物活性最强的菌株为拟盘多毛孢属(Pestalotiopsis)1株和蓝状菌属(Talaromyces)1株。上述研究结果表明,华石斛内生真菌多样性丰富,部分菌株具有较强抑菌生物活性。这为华石斛内生菌资源的深入开发奠定理论基础,为进一步创制农用杀菌制剂提供依据。
Due to their species and functional diversity, plant endophytic fungi have gradually become a hot topic in current plant biotechnology. In this study, endophytic fungi were separated from an Hainan endemic medicinal plant Dendrobium sinense to explore their diversity, tissue distributing characteristics and antimicrobial funtions, in order to set a foundation for further invention of new agricultural fungicides. The results are presented as following: 179 strains of endophytic fungi were isolated and identified as 51 OTUs in 25 taxons based on ITS sequences analysis. Among them, Phyllosticta was the dominant genera. Except Daldinia which widely distributed in roots, stems and leaves, all of the other taxons showed tissue bias. 18 taxons only distributed in one kind of tissue. The number of strains isolated from leaves was accounted for the biggest isolation rate of 60.33%. The taxons of endophytic fungi isolated with different kinds of culture medium were varied. Among all of the five culture media, PDA and D. sinense natural medium had the best endophyte capture ability, with 12 taxons(48%of the total taxon number) being isolated from both of them separately. Antimicrobial activity assay showed that 36 strains in 12 taxons were antagonistic to at least one kind of the testing fungi. One strain of Pestalotiopsis and one strain of Talaromyces showed the strongest antifungal ability. Above all, the diversity of D. Sinense endophytic fungi was very rich. The antifungal bioactivity of some endophyte strains were strong, and worth further study in the future. The results in this study set a foundation for in-depth explosion of endophytic resources of D. sinense.
Due to their species and functional diversity, plant endophytic fungi have gradually become a hot topic in current plant biotechnology. In this study, endophytic fungi were separated from an Hainan endemic medicinal plant Dendrobium sinense to explore their diversity, tissue distributing characteristics and antimicrobial funtions, in order to set a foundation for further invention of new agricultural fungicides. The results are presented as following: 179 strains of endophytic fungi were isolated and identified as 51 OTUs in 25 taxons based on ITS sequences analysis. Among them, Phyllosticta was the dominant genera. Except Daldinia which widely distributed in roots, stems and leaves, all of the other taxons showed tissue bias. 18 taxons only distributed in one kind of tissue. The number of strains isolated from leaves was accounted for the biggest isolation rate of 60.33%. The taxons of endophytic fungi isolated with different kinds of culture medium were varied. Among all of the five culture media, PDA and D. sinense natural medium had the best endophyte capture ability, with 12 taxons(48%of the total taxon number) being isolated from both of them separately. Antimicrobial activity assay showed that 36 strains in 12 taxons were antagonistic to at least one kind of the testing fungi. One strain of Pestalotiopsis and one strain of Talaromyces showed the strongest antifungal ability. Above all, the diversity of D. Sinense endophytic fungi was very rich. The antifungal bioactivity of some endophyte strains were strong, and worth further study in the future. The results in this study set a foundation for in-depth explosion of endophytic resources of D. sinense.
引文
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[22]李向楠,周稚凡,李明勇,等.云南热区拟盘多毛孢抑菌活性筛选[J].生物学杂志,2015,32(6):65.
[23]刘韩,刘应高,李姝江,等.四川松树内生拟盘多毛孢的多样性及其防病效果[J].西北农林科技大学学报(自然科学版),2015,43(12):41.
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[26]胡文丽.红豆杉根系多环芳烃降解菌的筛选及应用[D].大连:大连交通大学,2015.
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[2]Stierle A,Strobel G,Stierle D.Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Pacific yew[J].Science,1993,260(5 105):214.
[3]李艳玲,王德才,史仁玖,等.泰山黄精内生真菌的分离鉴定及抑菌活性研究[J].中草药,2013,44(11):1 490.
[4]周琢艳,刘玉寒,刘文洪,等.铁皮石斛内生真菌的分离及其体外抑菌和抗肿瘤活性初步研究[J].中草药,2017,48(3):533.
[5]孙瑞方,冉雪琴,王嘉福.一株产紫杉醇罗汉松内生真菌的分离和鉴定[J].微生物学报,2008,48(5):589.
[6]孙立军.植物内生菌Bacillus amyloliquefaciens ES-2的分离筛选及其抗菌物质的研究[D].南京:南京农业大学,2006.
[7]张玲琪,邵华,郭波,等.长春花内生真菌的分离及其发酵产生药用成分的初步研究[J].中草药,2000,31(11):805.
[8]陈贤兴,陈析丰,南旭阳,等.喜树果内生真菌的分离与鉴定[J].河南科学,2003,21(4):431.
[9]王晓鹏,钟佳,王兴红,等.桃儿七和南山荷叶内生真菌的生物活性研究[J].天然产物研究与开发,2006,18(1):15.
[10]郁美娟,孟庆华,黄德音,等.石斛属植物有效成分及药理作用研究[J].中成药,2003,25(11):918-921.
[11]宋希强.海南石斛属野生植物种质资源及华石斛保育生物学研究[D].北京:北京林业大学,2005.
[12]寸德志,朱国鹏,宋希强,等.海南黎族药用兰科植物的民族植物学研究[J].热带作物学报,2014,35(10):2 025-2 029.
[13]姜昊颖.海南特有兰科植物华石斛共生萌发技术研究[D].海口:海南大学,2011.
[14]李尚真.华石斛菌根真菌鉴定及其对华石斛与文心兰成苗影响研究[D].海口:海南大学,2015.
[15]Wang X,Li Y,Song X,et al.Influence of host tree species on isolation and communities of mycorrhizal and endophytic fungi from roots of a tropical epiphytic orchid,Dendrobium sinense(Orchidaceae)[J].Mycorrhiza,2017(10):1-10.
[16]陈秀娟.华石斛化学成分及其生物活性研究[D].海口:海南大学,2013.
[17]谭彩银,梅文莉,赵友兴,等.华石斛化学成分研究[J].热带亚热带植物学报,2017,25(2):189-194.
[18]陈娟,谭小明,邢咏梅,等.石斛属植物内生真菌及菌根真菌物种多样性研究进展[J].中国药学杂志,2013,48(19):1 649.
[19]胡克兴,侯晓强,郭顺星.铁皮石斛内生真菌分布[J].微生物学通报,2010,37(1):37.
[20]张瑞杰,张琼琼,黄兴如,等.基于分离培养方法的香蒲根内生细菌多样性[J].湿地科学,2016,14(2):201.
[21]褚晓玲,杨波,高丽,等.蕙兰根内可培养细菌的物种多样性[J].武汉植物学研究,2010,28(2):199.
[22]李向楠,周稚凡,李明勇,等.云南热区拟盘多毛孢抑菌活性筛选[J].生物学杂志,2015,32(6):65.
[23]刘韩,刘应高,李姝江,等.四川松树内生拟盘多毛孢的多样性及其防病效果[J].西北农林科技大学学报(自然科学版),2015,43(12):41.
[24]赵彩桂.山楝棒孢拟盘多毛孢和杂色曲霉化学成分研究[D].兰州:兰州大学,2014.
[25]白志强,林秀萍,刘永宏.内生拟盘多毛孢菌化学成分研究进展[J].天然产物研究与开发,2013,25(5):706.
[26]胡文丽.红豆杉根系多环芳烃降解菌的筛选及应用[D].大连:大连交通大学,2015.
[27]Li H,Huang H,Shao C,et al.Cytotoxic norsesquiterpene peroxides from the endophytic fungus Talaromyces flavus isolated from the mangrove plant Sonneratia apetala[J].Journal of Natural Products,2011,74(5):1 230.
[28]何军伟,高昊,刘杏忠,等.湿地真菌Talaromyces flavus中2个新的聚酯类化合物[J].中国中药杂志,2015,40(17):3 347.