水生植物小叶满江红内生真菌与古菌的发现及基于高通量测序的群落组成分析  

Discovery of Endo-Fungi and Archaea Within Water Fern Azolla microphylla and Their Community Analyses Based on High Throughput Sequencing

作　　者：陈坚[1] 郑伟文[1] 郑益平[1] 陈彬[1] 郑斯平[1] 朱炳耀[1] CHEN Jian;ZHENG Wei-Wen;ZHENG Yi-Ping;CHEN Bin;ZHENG Si-Ping;ZHU Bin-Yao(Institute of Biotechnology,Fujian Academy of Agricultural Sciences,Fuzhou 350003,China)

机构地区：[1]福建省农业科学院生物技术研究所

出　　处：《农业生物技术学报》2019年第11期2063-2072,共10页Journal of Agricultural Biotechnology

基　　金：福建省自然科学基金(No.2017J01053);福建省科技计划项目(No.2017N1001);福建省农科院科技计划项目(No.A2012-2)

摘　　要：水生植物满江红(Azolla)是一种具代表性的植物和蓝细菌的共生体。利用常规技术,可以发现固氮蓝细菌和众多细菌栖居于满江红的叶腔中。本研究旨在应用扫描电镜(scanning electron microscopy,SEM),荧光原位杂交(fluorescence in situ hybridization,FISH)和高通量测序技术探讨满江红微生物群落的遗传多样性,尤其揭示叶腔中真菌和古菌的存在。以新鲜健康的小叶满江红(A.microphylla,IRRI accesion No.4018)萍体为材料,对从120个叶腔中分离到的250个微生物样本进行荧光显微镜和电镜的镜检,在230个样本中发现了真菌状的结构特征,包括了菌丝,子囊,子囊孢子,分生孢子和担子等结构。80%的真菌状结构可以定位于第七片至第十五片叶的叶腔中,且叶龄越大,被检出的真菌数量越多。86%的样本检测为古菌阳性,但其丰度与叶龄无关。随后通过高通量测序,对其群落组成进行分析。结果表明,满江红体内有子囊菌门(Ascomycota)(67.39%),拟杆菌门(Phragmoplastophyta)(31.72%),担子菌门(Cordycipitaceae)(0.56%),虫菌门(Entomophthoromycota)(0.33%)4个真菌门和广古菌门(Euryarchaeota)(99.68%)为主的两2个古菌门共存。本研究结果表明满江红体内的微生态系统比我们原先想象的复杂得多,也再次证明满江红是植物-微生物相互作用研究颇具价值的模式植物。Water fern Azolla is a striking plant-cyanobacteria mutualistic association.N2-fixing cyanobacteria and numerous bacteria have been found within the leaf cavities of Azolla using traditional techniques.The aim of the present study is to explore the genetic diversity of microbial community,particularly fungi and archaea,employing scanning electron microscopy(SEM),fluorescence in situ hybridization(FISH)and high throughout sequencing technique.250 microbial samples isolated from 120 leaf cavities of fresh and healthy Azolla microphylla fronds were examined with both fluorescence microscopy and electron microscopy.Fungi-like structures,including hyphae,ascus,ascospore,conidium and basidium,were found in 230 samples.Eighty percent of fungi-like structures were positioned in leaf 7th to leaf 15th,indicating the older of leaf age,the more number of fungi detected.Archaea was investigated positively in 86%of the samples,and there was no correlation between the abundance and the leaf age.Through high throughout sequencing both fungi and archaea were identified qualitatively and quantitatively.The results showed that there were Ascomycota(67.39%),Phragmoplastophyta(31.72%),Cordycipitaceae(0.56%),Entomophthoromycota(0.33%),totally 4 fungal phyla,and 2 archaeal phyla with dominance of Euryarchaeota(99.68%)co-existing in Azolla.The results of this study implied that the microecosystem within Azolla was much more complex than we originally thought.It also indicates that Azolla is a valuable model plant for the study of plant-microbe interaction.

关 键 词：满江红 真菌 古菌 高通量测序 群落 

分 类 号：S184[农业科学—农业基础科学] S188