基于高通量测序的连续传代富集土壤可培养菌菌群变化规律研究  

High-throughput Sequencing Technology Based Research on the Changes of Culturable Bacteria Communities in Soil during Serial Enrichment

作　　者：文昌丽 曹伟伟 唐雪莲 赵雯淑 贾仲君[2,3] 孟磊 WEN Chang-li;CAO Wei-wei;TANG Xue-lian;ZHAO Wen-shu;JIA Zhong-jun;MENG Lei(College of Tropical Crops,Hainan University,Haikou 570228,China;State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences,Nanjing 210008,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]海南大学热带作物学院,海南海口570228 [2]中国科学院南京土壤研究所土壤与农业可持续发展国家重点实验室,江苏南京210008 [3]中国科学院大学,北京100049

出　　处：《生态与农村环境学报》2023年第1期123-135,共13页Journal of Ecology and Rural Environment

基　　金：中国科学院战略性先导科技专项(A类)(XDA28020203);国家自然科学基金(42067008);海南省自然科学基金高层次人才项目(320RC493)。

摘　　要：利用高通量测序技术研究连续传代富集过程中,固体和液体牛肉膏蛋白胨可培养土壤细菌多样性的变化规律,量化可培养菌占水稻土本底微生物群落的比例。通过设置常规营养和1/10低营养处理,包括常规牛肉膏蛋白胨固体培养(NA)、液体培养(NB),低营养固体培养(1/10 NA)和低营养液体培养(1/10 NB),开展连续传代富集10次,获得第1、3、5、7、10代细菌培养物并提取DNA,同时直接提取水稻土中所有微生物基因组DNA并对16S rRNA基因进行高通量测序,分析水稻土可培养菌群落变化规律及其占本底土著微生物的比例。结果表明,水稻土本底微生物多样性Chao指数为4806,在连续传代培养10次过程中,降幅最高为98.9%,其中,固体和液体可培养微生物Chao指数以第1代为最低,分别为49.7和142.0,低营养1/10固体和液体培养下,Chao指数分别为75.1和531.0。高通量测序16S rRNA基因后发现水稻土本底土著微生物共713属,连续10次传代富集培养过程中,固体和液体常规培养基中分别检测到52属和600属,低营养1/10固体和液体培养下,分别为62属和597属,可培养菌占比最高分别为8.7%和83.7%。连续第1、3、5、7和10次传代培养过程中,固体培养基每代独有微生物属分别为7、2、3、4和3属,液体培养基独有属则分别为5、1、102、44和24属,低营养1/10培养也得到了类似结果。假单胞菌属(Pseudomonas)是水稻土连续10次传代过程中绝对的优势类群,特别在固体培养基中相对丰度变幅范围为97.70%~99.47%,与水稻土本底土壤中相对丰度相比,增幅最高为74倍。低营养1/10固体条件下,随着传代次数的增加,杆菌属(Bacillus)和代尔夫特菌属(Delftia)成为优势类群,在第10代的相对丰度最高分别为18.07%和13.82%。液体连续传代10次过程中,假单胞菌属也是绝对优势类群,但与固体可培养菌相比略下降为45.48%~55.99%,梭菌属(Clostridium)则是第2大优势类群,其�High-throughput sequencing technology was used to study the changes in the diversity of culturable bacteria in solid and liquid beef extract peptone soil during serial enrichment,and to quantify the proportion of culturable bacteria in the background microbial community of a paddy soil.Serial passages were performed by setting up treatments of conventional nutrient and 1/10 low nutrient treatments,including conventional beef extract peptone solid(NA) and liquid(NB) cultures,as well as low nutrient solid(1/10 NA) and low nutrient liquid(1/10 NB) cultures.The cultures were enriched 10 times to obtain the 1^(st),3^(rd),5^(th),7^(th),and 10^(th) generation of bacterial cultures and then to extract their DNAs.At the same time,all microbial genomic DNAs in the paddy soil were directly extracted and high-throughput sequencing 16S rRNA gene was used to analyze the change of culturable microorganisms communities in the paddy soil and their proportions to the background indigenous microorganisms.The results show that the background Chao index of the microbial diversity of the paddy soil was 4 806,with a maximum reduction of 98.9% in 10 consecutive generations.Among them,the Chao index of solid and liquid culturable microorganisms was the lowest in the first generation,which were 49.7 and 142,respectively.Under 1/10 solid and 1/10 liquid cultures,the Chao index was 75.1 and 531,respectively.High-throughput sequencing of 16S rRNA genes revealed a total of 713 genera of background indigenous microorganisms in paddy soil.During the enrichment culture process of 10 consecutive passages,52 and 600 genera were detected in solid and liquid conventional culture,respectively.However,62 and 597 genera were detected in 1/10 solid and 1/10 liquid cultures,respectively.The highest proportion of culturable bacteria was 8.7% and 83.7%,respectively.During the 1^(st),3^(rd),5^(th),7^(th) and 10^(th) consecutive subcultures,7,2,3,4 and 3 genera were unique to each passage in solid medium,while the unique genera in liquid culture medium were

关 键 词：微生物可培养法 连续传代 固液体培养基 氮素营养 高通量测序 

分 类 号：S154.36[农业科学—土壤学]