河南省不同类型土壤可培养放线菌多样性及拮抗活性菌株筛选
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摘要
分别从河南省濮阳、南阳、信阳、三门峡和商丘等地区采集潮土、黄棕壤、水稻土、褐土、盐碱土等土壤样品45份,采用8种不同的分离培养基对土壤放线菌进行分离和培养;通过16S rRNA基因序列分析对可培养放线菌进行分类鉴定;以7种植物病原菌为靶标,通过对峙培养筛选拮抗活性菌株,并对具有拮抗活性菌株的次级代谢产物合成相关基因进行初步筛查.结果表明,从5种类型土壤中分离得到放线菌分离物955株,分布于放线菌门的10个目12个科17个属,包含626个物种,其中9个分离物为潜在的新种;分离物中链霉菌属菌株751株,占分离菌株的78.64%,为最主要的优势菌属,稀有放线菌204株,占21.36%;不同类型土壤放线菌的分离频率存在一定的差异,其中褐土分离的放线菌数量最多,水稻土的分离数量最少,但是各类型土壤分离的可培养放线菌在属水平上差异不大;筛选获得对植物病原菌有拮抗活性的菌株392株,占总分离物的41%,其中链霉菌332株,占总拮抗活性菌株的84.7%,稀有放线菌60株,占总拮抗活性菌株的15.3%,且多数活性菌株含有多种生物活性物质合成相关的功能基因.由此可见,河南省土壤放线菌多样性丰富,并蕴藏丰富的稀有放线菌和拮抗活性放线菌资源,具有发掘放线菌新物种和新活性化合物的潜力.(图4表6参33附表1)
To investigate the diversity of culturable actinomycetes in different soil types in the Henan Province of China,and screen active actinomycete strains for potential biocontrol on plant diseases, 45 soil samples were analyzed. The samples included fluvo-aquic soil, yellow-brown soil, paddy soil, cinnamon soil, and saline-alkali soil and were collected from the districts of Puyang, Nanyang, Xinyang, Sanmenxia, and Shangqiu, respectively. Culturable actinomycete strains were isolated by eight different separation mediums. The isolated strains were classified and identified based on the systematic analysis of the 16 S rRNA gene sequence. Antagonistic activity strains were screened by dual culture using seven types of plant pathogenic fungi as targets, and a preliminary screening of genes related to the secondary metabolite synthesis of antagonistic strains was conducted. Nine hundred and fifty-five strains of actinomycete isolates were isolated from 45 soil samples from five soil types and belonged to 10 orders, 12 families, and 17 genera. The strains included 626 operational taxonomic units(OTUs), which contained nine putative species. Among the isolates, 751 strains, representing 78.64% of the total strains isolated, belonged to the genus Streptomyces sp. and 204 rare actinomycetes represented the remaining 21.36%. There are some differences in screening frequency of actinomycetes among the different soil types; cinnamon soil contained the highest amount of actinomycetes isolates, and paddy soil contained the lowest amount. However, the number of culturable actinomycetes strains isolated from different soil types was not significantly different at the genus level. Three hundred and ninety-two strains with antagonistic activity against plant pathogens were obtained, accounting for 41% of the total isolates. Of these strains, 332 were Streptomyces sp., accounting for 84.7% of the total antagonistic strains, and the remaining 60 strains were rare actinomycetes,accounting for 15.3%. The majority of active strains contained multiple functional genes involved in the synthesis of biologically active substances. Different types of soil in the Henan Province contain a high diversity of actinomycetes, and the various soils are rich in rare actinomycetes and antagonistic actinomycetes resources, which are proved to have a great potential to discover novel actinomycetes species and new active compounds.
To investigate the diversity of culturable actinomycetes in different soil types in the Henan Province of China,and screen active actinomycete strains for potential biocontrol on plant diseases, 45 soil samples were analyzed. The samples included fluvo-aquic soil, yellow-brown soil, paddy soil, cinnamon soil, and saline-alkali soil and were collected from the districts of Puyang, Nanyang, Xinyang, Sanmenxia, and Shangqiu, respectively. Culturable actinomycete strains were isolated by eight different separation mediums. The isolated strains were classified and identified based on the systematic analysis of the 16 S rRNA gene sequence. Antagonistic activity strains were screened by dual culture using seven types of plant pathogenic fungi as targets, and a preliminary screening of genes related to the secondary metabolite synthesis of antagonistic strains was conducted. Nine hundred and fifty-five strains of actinomycete isolates were isolated from 45 soil samples from five soil types and belonged to 10 orders, 12 families, and 17 genera. The strains included 626 operational taxonomic units(OTUs), which contained nine putative species. Among the isolates, 751 strains, representing 78.64% of the total strains isolated, belonged to the genus Streptomyces sp. and 204 rare actinomycetes represented the remaining 21.36%. There are some differences in screening frequency of actinomycetes among the different soil types; cinnamon soil contained the highest amount of actinomycetes isolates, and paddy soil contained the lowest amount. However, the number of culturable actinomycetes strains isolated from different soil types was not significantly different at the genus level. Three hundred and ninety-two strains with antagonistic activity against plant pathogens were obtained, accounting for 41% of the total isolates. Of these strains, 332 were Streptomyces sp., accounting for 84.7% of the total antagonistic strains, and the remaining 60 strains were rare actinomycetes,accounting for 15.3%. The majority of active strains contained multiple functional genes involved in the synthesis of biologically active substances. Different types of soil in the Henan Province contain a high diversity of actinomycetes, and the various soils are rich in rare actinomycetes and antagonistic actinomycetes resources, which are proved to have a great potential to discover novel actinomycetes species and new active compounds.
引文
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7 Bonaldi M,Kunova A,Saracchi M,Sardi P,Cortesi P.Streptomycetes as biological control agents against basal drop[J].Acta Horticult,2014,1044:313-318
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9阮继生.“伯杰氏系统细菌学手册(第二版)”第5卷与我国的放线菌系统学研究[J].微生物学报,2013,53(6):521-530[Ruan JS.Bergey’s Manual of Systematic Bacteriology(2th edition)volume 5 and the study of actinomycetes systematic in China[J].Acta Microbiol Sin,2013,53(6):521-530]
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11 Bates ST,Clemente JC,Flores GE,Walters WA,Parfrey LW,Knight R,Fierer N.Global biogeography of highly diverse protistan communities in soil[J].ISME J,2013,7(3):652-659
12李文均,徐丽华,姜成林.云南大学放线菌资源研究历史回顾、发展现状及未来展望[J].云南大学学报(自然科学版),2013,35(3):428-440[Li WJ,Xu LH,Jiang CL.Actinobacterial resource and utilization research in Yunnan University:past,present and future[J].J.Yuannan Univ(Nat Sci Ed),2013,35(3):428-440]
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18 Wen CY,Zheng DG,Shen SS,Chen JG,Liu WC,Liu T.Streptomyces scabiei subsp.x u cha nge n si s,a novel st re pt omycet e isolat e for staurosporine production and a wheat take-all control agent[J].Int JMicrobiol Res,2012,4(7):282-289
19李小俊,吴越,张伟铭,李静,刘少伟,蒋忠科,黄大林,孙承航.河北九莲城淖尔可培养放线菌多样性及抗菌活性筛选[J].微生物学通报,2016,43(7):1473-1484[Li XJ,Wu Y,Zhang WM,Li J,Liu SW,Jiang ZK,Huang DL,Sun CH.Biodiversity and antimicrobial activity of culturable actinobacteria isolated from Jiuliancheng Nur in Hebei province[J].Microbiol China,2016,43(7):1473-1484]
20 Ayuso SA,Genilloud O.New PCR primers for the screening of NRPSand PKS-I systems in actinomycetes:detection and distribution of these biosynthetic gene sequences in major taxonomic groups[J].Microb Ecol,2005,49(1):10-24
21薛冬,赵国振,姚青,赵海泉,朱红惠.肇庆星湖湿地可培养放线菌多样性[J].微生物学报,2015,55(11):1485-1494[Xue D,Zhao GZ,Yao Q,Zhao HQ,Zhu HH.Diversity of cultivable actinobacteria in Xinghu wetland sediments[J].Acta Microbiol Sin,2015,55(11):1485-1494]
22 Mets?-Ketel?M,Salo V,Halo L,Hautala A,Hakala J,M?nts?l?P,Ylihonko K.An efficient approach for screening minimal PKS genes from Streptomyces[J].FEMS Microbiol Lett,1999,180(1):1-6
23 He W,Wu L,Gao Q,Du Y,Wang Y.Identification of AHBAbiosynthetic genes related to geldanamycin biosynthesis in Streptomyces hygroscopicus 17997[J].Curr Microbiol,2006,52(3):197-203
24凡琴.放线菌产安莎类和环醇类抗生素合成基因资源的挖掘[D].厦门:厦门大学,2014[Fan Q.Gene mining of ansamycin and aminocyclitol antibiotics in actinomycetes[D].Xiamen:Xiamen University,2014]
25 Sigmund JM,Clark DC,Rainey FA,Anderson AS.Detection of eubacterial 3-hydroxy-3-methylglutaryl coenzyme a reductases from natural populations of actinomycetes[J].Microb Ecol,2003,46(1):106-112
26 Chun J,Oren A,Ventosa A,Christensen H,Arahal DR,da Costa MS,Rooney AP,Yi H,Xu XW,De Meyer S,Trujillo ME.Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes[J].Int J Syst Evol Microbiol,2018,68(1):461-466
27王保军,刘双江.环境微生物培养新技术的研究进展[J].微生物学通报,2013,40(1):6-17[Wang BJ,Liu SJ.Perspectives on the cultivability of environmental microorganisms[J].Microbiol China,2013,40(1):6-17]
28谭红妍,陈宝瑞,闫瑞瑞,辛小平,陶金.草地土壤微生物特性及其对人为干扰响应的研究进展[J].草地学报,2014,22(6):1163-1170[Tan HY,Chen BR,Yan RR,Xin XP,Tao J.Advances on soil microbiological characteristics of grassland ecosystems and its response to human disturbances[J].Acta Agrectir Sin,2014,22(6):1163-1170]
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33李威,肖熙鸥,李可,高晓敏,吕玲玲.娄彻氏链霉菌XL-6的抑菌活性及对茄子幼苗的防病促生效应[J].中国生物防治学报,2017,33(4):531-536[Li W,Xiao XO,Li K,Gao XM,LüLL.The antibiotic activity of fermentation broth produced from Streptomyces rochei XL-6 and its influence on bacterial wilt control and plant growth in eggplant seedling[J].Chin J Biol Control,2017,33(4):531-536]
2 Genilloud O.Actinomycetes:still a source of novel antibiotics[J].Nat Prod Rep,2017,349(10):1203-1232
3 Kunova A,Bonaldi M,Saracchi M,Pizzatti C,Chen X,Cortesi P.Selection of Streptomyces against soil borne fungal pathogens by a standardized dual culture assay and evaluation of their effects on seed germination and plant growth[J].BMC Microbiol,2016,16(1):272
4 Sharma M.Actinomycetes:source,identification,and their applications[J].Int J Curr Microbiol App Sci,2014,3(2):801-832
5 Barka EA,Vatsa P,Sanchez L,Gaveau-Vaillant N,Jacquard C,Klenk HP,Clément C,Ouhdouch Y,van Wezel GP.Taxonomy,physiology,and natural products of Actinobacteria[J].Microbiol Mol Biol R,2015,80(1):1-43
6 Koyama R,Matsumoto A,Inahashi Y,Omura S,Takahashi Y.Isolation of actinomycetes from the root of the plant,Ophiopogon japonicus,and proposal of two new species,Actinoallomurus liliacearum sp.nov.and Actinoallomurus vinaceus sp.nov.[J].J Antibiot,2012,65(7):335-340
7 Bonaldi M,Kunova A,Saracchi M,Sardi P,Cortesi P.Streptomycetes as biological control agents against basal drop[J].Acta Horticult,2014,1044:313-318
8李文均,职晓阳,唐蜀昆.我国放线菌系统学研究历史、现状及未来发展趋势[J].微生物学通报,2013,40(10):1860-1873[Li WJ,Zhi XY,Tang SK.Actinobacterial systematics in China:past,present and future[J].Microbiol China,2013,40(10):1860-1873]
9阮继生.“伯杰氏系统细菌学手册(第二版)”第5卷与我国的放线菌系统学研究[J].微生物学报,2013,53(6):521-530[Ruan JS.Bergey’s Manual of Systematic Bacteriology(2th edition)volume 5 and the study of actinomycetes systematic in China[J].Acta Microbiol Sin,2013,53(6):521-530]
10 Oren A.第七届全国微生物资源学术暨国际微生物系统与分类学研讨会:评论和结论[J].微生物学通报,2016,43(5):895-898[Oren A.The 7th National Conference of Microbial Resources&the International Symposium on Microbial Systematics and Taxonomy:comments and conclusions[J].Microbiol China,2016,43(5):895-898]
11 Bates ST,Clemente JC,Flores GE,Walters WA,Parfrey LW,Knight R,Fierer N.Global biogeography of highly diverse protistan communities in soil[J].ISME J,2013,7(3):652-659
12李文均,徐丽华,姜成林.云南大学放线菌资源研究历史回顾、发展现状及未来展望[J].云南大学学报(自然科学版),2013,35(3):428-440[Li WJ,Xu LH,Jiang CL.Actinobacterial resource and utilization research in Yunnan University:past,present and future[J].J.Yuannan Univ(Nat Sci Ed),2013,35(3):428-440]
13田新朋,张偲,李文均.海洋放线菌研究进展[J].微生物学报,2011,51(2):161-169[Tian XP,Zhang S,Li WJ.Advance in marine actinobacterial research-a review[J].Acta Microbiol Sin,2011,51(2):161-169]
14 Zhang YQ,Liu HY,Chen J,Yuan LJ,Sun W,Zhang LX,Zhang YQ,Yu LY,Li WJ.Diversity of culturable actinobacteria from Qinghai-Tibet plateau,China[J].Antonie van Leeuwenhoek,2010,98(2):213-223
15黄娇,闫兵法,黄英.青藏高原阿里、那曲和海西地区土壤可培养放线菌的多样性[J].微生物学报,2017,57(9):1342-1351[Huang J,Yan BF,Huang Y.Diversity of culturable actinobacteria from soils collected in Ali,Naqu and Haixi districts on the Qinghai-Tibet plateau[J].Acta Microbiol Sin,2017,57(9):1342-1351]
16徐丽华,刘春雷,常玉梅,梁利群,刘金亮,高国强,韩启霞.双标准曲线相对定量PCR试验原理与方法[J].生物技术通报,2011(1):70-75[Xu LH,Liu CL,Chang YM,Liang LQ,Liu JL,Gao QG,Han QM.Theory and method of double-standard curves method of relative quantification PCR[J].Biotechnol Bull,2011(1):70-75]
17卜春亚,孙晔,张天蔚,王帅,张英英,牛薇,官学琼,王有年,师光禄.一株草莓根腐尖孢镰刀菌拮抗内生细菌的分离鉴定及抑菌特性[J].应用与环境生物学报,2014,20(2):300-304[Bu CY,Sun Y,Zhang TW,Wang S,Zhang YY,Niu W,Guan XQ,Wang YN,Shi GL.Isolation and antagonistic character analysis of endophytic bacteria from the strawberry[J].Chin J Appl Environ Biol,2014,20(2):300-304]
18 Wen CY,Zheng DG,Shen SS,Chen JG,Liu WC,Liu T.Streptomyces scabiei subsp.x u cha nge n si s,a novel st re pt omycet e isolat e for staurosporine production and a wheat take-all control agent[J].Int JMicrobiol Res,2012,4(7):282-289
19李小俊,吴越,张伟铭,李静,刘少伟,蒋忠科,黄大林,孙承航.河北九莲城淖尔可培养放线菌多样性及抗菌活性筛选[J].微生物学通报,2016,43(7):1473-1484[Li XJ,Wu Y,Zhang WM,Li J,Liu SW,Jiang ZK,Huang DL,Sun CH.Biodiversity and antimicrobial activity of culturable actinobacteria isolated from Jiuliancheng Nur in Hebei province[J].Microbiol China,2016,43(7):1473-1484]
20 Ayuso SA,Genilloud O.New PCR primers for the screening of NRPSand PKS-I systems in actinomycetes:detection and distribution of these biosynthetic gene sequences in major taxonomic groups[J].Microb Ecol,2005,49(1):10-24
21薛冬,赵国振,姚青,赵海泉,朱红惠.肇庆星湖湿地可培养放线菌多样性[J].微生物学报,2015,55(11):1485-1494[Xue D,Zhao GZ,Yao Q,Zhao HQ,Zhu HH.Diversity of cultivable actinobacteria in Xinghu wetland sediments[J].Acta Microbiol Sin,2015,55(11):1485-1494]
22 Mets?-Ketel?M,Salo V,Halo L,Hautala A,Hakala J,M?nts?l?P,Ylihonko K.An efficient approach for screening minimal PKS genes from Streptomyces[J].FEMS Microbiol Lett,1999,180(1):1-6
23 He W,Wu L,Gao Q,Du Y,Wang Y.Identification of AHBAbiosynthetic genes related to geldanamycin biosynthesis in Streptomyces hygroscopicus 17997[J].Curr Microbiol,2006,52(3):197-203
24凡琴.放线菌产安莎类和环醇类抗生素合成基因资源的挖掘[D].厦门:厦门大学,2014[Fan Q.Gene mining of ansamycin and aminocyclitol antibiotics in actinomycetes[D].Xiamen:Xiamen University,2014]
25 Sigmund JM,Clark DC,Rainey FA,Anderson AS.Detection of eubacterial 3-hydroxy-3-methylglutaryl coenzyme a reductases from natural populations of actinomycetes[J].Microb Ecol,2003,46(1):106-112
26 Chun J,Oren A,Ventosa A,Christensen H,Arahal DR,da Costa MS,Rooney AP,Yi H,Xu XW,De Meyer S,Trujillo ME.Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes[J].Int J Syst Evol Microbiol,2018,68(1):461-466
27王保军,刘双江.环境微生物培养新技术的研究进展[J].微生物学通报,2013,40(1):6-17[Wang BJ,Liu SJ.Perspectives on the cultivability of environmental microorganisms[J].Microbiol China,2013,40(1):6-17]
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