河南黄河湿地放线菌多样性及植物病害生防放线菌的筛选
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摘要
【目的】探究河南黄河湿地放线菌多样性,筛选对植物病原菌有拮抗活性的放线菌菌株。【方法】基于Illumina HiSeq技术的高通量测序分析了河南黄河湿地放线菌物种多样性及其分布特点;利用8种分离培养基对采集自三门峡黄河湿地、郑州黄河湿地和开封黄河湿地21份土壤样品中的放线菌进行了分离纯化,通过16S rRNA基因序列分析对分离株进行了初步的分类鉴定和系统学研究;以7种植物病原菌为靶标筛选有拮抗活性的分离株,并对活性菌株进行了聚酮合酶、非核糖体多肽合成酶和安莎类化合物等基因筛查。【结果】高通量测序结果表明,河南黄河湿地放线菌物种多样性丰富,且不同湿地区域以及同一湿地不同生境间放线菌多样性存在显著差异,放线菌优势属物种依次为Nocardioides、Streptomyces、CL500-29 marine group、Fodinicola、Mycobacterium和Micromonospora,此外,还具有大量的未知类群;通过分离培养共获得了261株纯培养菌株,分布于放线菌门中的7个目9个科9个属,包含97个可能的已知物种和8个潜在新物种,对植物病原菌有拮抗活性的放线菌86株,其中74株中至少含有一种活性代谢物质合成相关基因。【结论】河南黄河湿地放线菌物种多样性丰富,具有发掘新物种和新型植物病害生防资源的潜力。
[Objective] This study is aimed to analyze the diversity of actinobacteria in soils of the Henan Yellow River wetland and screen actinobacteria strains which have antagonistic activity against plant pathogens.[Methods] High-throughput sequencing based on Illumina HiSeq technology was used to analyze the actinobacterial diversity and distribution in the Henan Yellow River wetland. We used eight selective media to isolate actinobacteria from 21 soil samples collected from Sanmenxia, Zhengzhou and Kaifeng Yellow River wetland. The cultivable actinobacteria were identified and phylogeny analyzed based on the 16S rRNA gene sequences. The active isolates were screened using seven plant pathogens as targets, and 5 functional genes were assayed, including polyketide synthases gene(PKS-I, PKS-II), nonribosomal peptide synthetases gene(NRPS),3-amino-5-hydroxybenzoic acid synthases gene(AHBA) and 3-hydroxy-3-methylglutaryl coenzyme A gene(HMG CoA). [Results] The high-throughput sequencing results indicated that the actinobacteria were abundant in Henan Yellow River wetland and there were significant differences among different wetland areas and different habitats in the same wetland. Nocardioides was the most frequently isolated genus, followed by the genera Streptomyces,CL500-29 marine group, Fodinicola, Mycobacterium and Micromonospora. In addition, there are still a lot of unknown groups. We isolated a total of 261 dereplicated strains, belonging to 7 orders, 9 families and 9 genera of the phylum Actinobacteria. They were including 97 putative species and eight unknown species. Among them, 86 actinobacteria strains have antagonistic activity against plant pathogens; 74 strains contain at least one gene which involved active metabolites synthesis. [Conclusion] Actinobacteria species diversity was abundant in Henan Yellow River wetland and new species have potential as biocontrol agents against plant diseases.
[Objective] This study is aimed to analyze the diversity of actinobacteria in soils of the Henan Yellow River wetland and screen actinobacteria strains which have antagonistic activity against plant pathogens.[Methods] High-throughput sequencing based on Illumina HiSeq technology was used to analyze the actinobacterial diversity and distribution in the Henan Yellow River wetland. We used eight selective media to isolate actinobacteria from 21 soil samples collected from Sanmenxia, Zhengzhou and Kaifeng Yellow River wetland. The cultivable actinobacteria were identified and phylogeny analyzed based on the 16S rRNA gene sequences. The active isolates were screened using seven plant pathogens as targets, and 5 functional genes were assayed, including polyketide synthases gene(PKS-I, PKS-II), nonribosomal peptide synthetases gene(NRPS),3-amino-5-hydroxybenzoic acid synthases gene(AHBA) and 3-hydroxy-3-methylglutaryl coenzyme A gene(HMG CoA). [Results] The high-throughput sequencing results indicated that the actinobacteria were abundant in Henan Yellow River wetland and there were significant differences among different wetland areas and different habitats in the same wetland. Nocardioides was the most frequently isolated genus, followed by the genera Streptomyces,CL500-29 marine group, Fodinicola, Mycobacterium and Micromonospora. In addition, there are still a lot of unknown groups. We isolated a total of 261 dereplicated strains, belonging to 7 orders, 9 families and 9 genera of the phylum Actinobacteria. They were including 97 putative species and eight unknown species. Among them, 86 actinobacteria strains have antagonistic activity against plant pathogens; 74 strains contain at least one gene which involved active metabolites synthesis. [Conclusion] Actinobacteria species diversity was abundant in Henan Yellow River wetland and new species have potential as biocontrol agents against plant diseases.
引文
[1]Li WJ,Zhi XY,Tang SK.Actinobacterial systematics in China:past,present and future.Microbiology China,2013,40(10):1860-1873.(in Chinese)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.微生物学通报,2013,40(10):1860-1873.
[2]Genilloud O.Actinomycetes:still a source of novel antibiotics.Natural Product Reports,2017,34(10):1203-1232.
[3]Law WF,Ser HL,Khan TM,Chuah LH,Pusparajah P,Chan KG,Goh BH,Lee LH.The potential of Streptomyces as biocontrol agents against the rice blast fungus,Magnaporthe oryzae(Pyricularia oryzae).Frontiers in Microbiology,2017,8:3.
[4]Tang J,Xu QR,Wang LM,Ding X,Tang B,Wu LS,Feng S,Sun Q,Yang ZR,Zhang J.Soil bacterial community diversity under different stages of degradation in Zoige wetland.Microbiology China,2011,38(5):677-686.(in Chinese)唐杰,徐青锐,王立明,丁祥,汤博,吴俐莎,冯甦,孙群,杨志荣,张杰.若尔盖高原湿地不同退化阶段的土壤细菌群落多样性.微生物学通报,2011,38(5):677-686.
[5]Feng G,Xu SS,Wang C,Yan LE,Zhang X,Li J.Actinobacterial diversity in a coastal wetland of the Yellow River delta revealed by culture-independent approach.Microbiology China,2013,40(9):1570-1579.(in Chinese)冯鸽,许姗姗,王超,严立恩,张璇,李静.黄河三角洲滨海湿地非培养放线菌多样性.微生物学通报,2013,40(9):1570-1579.
[6]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.Antonie van Leeuwenhoek,2010,98(2):213-223.
[7]Huang J,Yan BF,Huang Y,Diversity of culturable actinobacteria from soils collected in Ali,Naqu and Haixi districts on the Qinghai-Tibet Plateau.Acta Microbiologica Sinica,2017,57(9):1342-1351.(in Chinese)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.微生物学报,2017,57(9):1342-1351.
[8]Zhai YY,Wang WF,Tan HM,Cao LX.A new approach to analyzing endophytic actinobacterial population in the roots of banana plants(Musa sp.,AAA).Journal of Biochemistry and Molecular Biology Research,2016,2(3):180-184.
[9]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.Microbiology China,2016,43(7):1473-1484.(in Chinese)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.微生物学通报,2016,43(7):1473-1484.
[10]Xue D,Zhao ZG,Yao Q,Zhao HQ,Zhu HH.Diversity of cultivable actinobacteria in Xinghu wetland sediments.Acta Microbiologica Sinica,2015,55(11):1485-1494.(in Chinese)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.微生物学报,2015,55(11):1485-1494.
[11]Sigmund JM,Clark DC,Rainey FA,Anderson AS.Detection of eubacterial 3-hydroxy-3-methylglutaryl coenzyme a reductases from natural populations of actinomycetes.Microbial Ecology,2003,46(1):106-112.
[12]Edgar RC.Search and clustering orders of magnitude faster than BLAST.Bioinformatics,2010,26(19):2460-2461.
[13]Stach JEM,Maldonado LA,Masson DG,Ward AC,Goodfellow M,Bull AT.Statistical approaches for estimating actinobacterial diversity in marine sediments.Applied and Environmental Microbiology,2003,69(10):6189-6200.
[14]He JQ,Zhang GJ,Yue HM.Diversity and bioactivity analysis of actinomycetes isolated from Lhalu wetland nature reserve.Journal of Microbiology,2009,29(4):6-10.(in Chinese)何建清,张格杰,岳海梅.拉鲁湿地自然保护区放线菌组成分析及生物活性测定.微生物学杂志,2009,29(4):6-10.
[15]Guan XY,Han JB,Wang B,Dong Y,Jiang B,Chen Z,Yang AF,Wang ZH,Zhou ZC.Analysis of bacterial communities in Liaodong Bay Dalinghe estuarine wetland.Ecology and Environmental Sciences,2012,21(6):1063-1070.(in Chinese)关晓燕,韩家波,王摆,董颖,姜北,陈仲,杨爱馥,王召会,周遵春.辽东湾大凌河口湿地土壤微生物群落分析.生态环境学报,2012,21(6):1063-1070.
[16]Hong K.Actinomycetes from mangrove and their secondary metabolites.Acta Microbiologica Sinica,2013,53(11):1131-1141.(in Chinese)洪葵.红树林放线菌及其天然产物研究进展.微生物学报,2013,53(11):1131-1141.
[17]Xu SQ,Wang YD,Guo CC,Zhang ZG,Shang YT,Chen Q,Wang ZL.Comparison of microbial community composition and diversity in native coastal wetlands and wetlands that have undergone long-term agricultural reclamation.Wetlands,2017,37(1):99-108.
[18]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets.Molecular Biology and Evolution,2016,33(7):1870-1874.
[19]Labeda DP,Dunlap CA,Rong XY,Huang Y,Doroghazi JR,Ju KS,Metcalf WW.Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis.Antonie van Leeuwenhoek,2017,110(4):563-583.
[20]Kumar Y,Aiemsum-ang P,Ward AC,Goodfellow M.Diversity and geographical distribution of members of the Streptomyces violaceusniger 16S rRNA gene clade detected by clade-specific PCR primers.FEMS Microbiology Ecology,2007,62(1):54-63.
[21]KyselkováM,KopeckyJ,Felf?ldi T,?ermák L,Omelka M,Grundmann GL,Mo?nne-Loccoz Y,Ságová-Mare?kováM.Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera.Antonie van Leeuwenhoek,2008,94(3):439-453.
[22]VětrovskyT,Baldrian P.An in-depth analysis of actinobacterial communities shows their high diversity in grassland soils along a gradient of mixed heavy metal contamination.Biology and Fertility of Soils,2015,51(7):827-837.
[23]Tang B,Yu YC,Zhi XY,Yang LL,Cen XF,Zhao GP,Ding XM.Streptomyces yangpuensis sp.nov.,isolated from soil.International Journal of Systematic and Evolutionary Microbiology,2016,66(3):1224-1229.
[24]Ravi L,Krishnan K.Benzoyloxy-ethyl-carbamic acid:a novel anticancerous secondary metabolite produced by Streptomyces globosus VITLGK011.Indian Journal of Experimental Biology,2017,55:411-420.
[25]Saeed EE,Sham A,Salmin Z,Abdelmowla Y,Iratni R,El-Tarabily K,AbuQamar S.Streptomyces globosus UAE1,a potential effective biocontrol agent for black scorch disease in date palm plantations.Frontiers in Microbiology,2017,8:1455.
[2]Genilloud O.Actinomycetes:still a source of novel antibiotics.Natural Product Reports,2017,34(10):1203-1232.
[3]Law WF,Ser HL,Khan TM,Chuah LH,Pusparajah P,Chan KG,Goh BH,Lee LH.The potential of Streptomyces as biocontrol agents against the rice blast fungus,Magnaporthe oryzae(Pyricularia oryzae).Frontiers in Microbiology,2017,8:3.
[4]Tang J,Xu QR,Wang LM,Ding X,Tang B,Wu LS,Feng S,Sun Q,Yang ZR,Zhang J.Soil bacterial community diversity under different stages of degradation in Zoige wetland.Microbiology China,2011,38(5):677-686.(in Chinese)唐杰,徐青锐,王立明,丁祥,汤博,吴俐莎,冯甦,孙群,杨志荣,张杰.若尔盖高原湿地不同退化阶段的土壤细菌群落多样性.微生物学通报,2011,38(5):677-686.
[5]Feng G,Xu SS,Wang C,Yan LE,Zhang X,Li J.Actinobacterial diversity in a coastal wetland of the Yellow River delta revealed by culture-independent approach.Microbiology China,2013,40(9):1570-1579.(in Chinese)冯鸽,许姗姗,王超,严立恩,张璇,李静.黄河三角洲滨海湿地非培养放线菌多样性.微生物学通报,2013,40(9):1570-1579.
[6]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.Antonie van Leeuwenhoek,2010,98(2):213-223.
[7]Huang J,Yan BF,Huang Y,Diversity of culturable actinobacteria from soils collected in Ali,Naqu and Haixi districts on the Qinghai-Tibet Plateau.Acta Microbiologica Sinica,2017,57(9):1342-1351.(in Chinese)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.微生物学报,2017,57(9):1342-1351.
[8]Zhai YY,Wang WF,Tan HM,Cao LX.A new approach to analyzing endophytic actinobacterial population in the roots of banana plants(Musa sp.,AAA).Journal of Biochemistry and Molecular Biology Research,2016,2(3):180-184.
[9]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.Microbiology China,2016,43(7):1473-1484.(in Chinese)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.微生物学通报,2016,43(7):1473-1484.
[10]Xue D,Zhao ZG,Yao Q,Zhao HQ,Zhu HH.Diversity of cultivable actinobacteria in Xinghu wetland sediments.Acta Microbiologica Sinica,2015,55(11):1485-1494.(in Chinese)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.微生物学报,2015,55(11):1485-1494.
[11]Sigmund JM,Clark DC,Rainey FA,Anderson AS.Detection of eubacterial 3-hydroxy-3-methylglutaryl coenzyme a reductases from natural populations of actinomycetes.Microbial Ecology,2003,46(1):106-112.
[12]Edgar RC.Search and clustering orders of magnitude faster than BLAST.Bioinformatics,2010,26(19):2460-2461.
[13]Stach JEM,Maldonado LA,Masson DG,Ward AC,Goodfellow M,Bull AT.Statistical approaches for estimating actinobacterial diversity in marine sediments.Applied and Environmental Microbiology,2003,69(10):6189-6200.
[14]He JQ,Zhang GJ,Yue HM.Diversity and bioactivity analysis of actinomycetes isolated from Lhalu wetland nature reserve.Journal of Microbiology,2009,29(4):6-10.(in Chinese)何建清,张格杰,岳海梅.拉鲁湿地自然保护区放线菌组成分析及生物活性测定.微生物学杂志,2009,29(4):6-10.
[15]Guan XY,Han JB,Wang B,Dong Y,Jiang B,Chen Z,Yang AF,Wang ZH,Zhou ZC.Analysis of bacterial communities in Liaodong Bay Dalinghe estuarine wetland.Ecology and Environmental Sciences,2012,21(6):1063-1070.(in Chinese)关晓燕,韩家波,王摆,董颖,姜北,陈仲,杨爱馥,王召会,周遵春.辽东湾大凌河口湿地土壤微生物群落分析.生态环境学报,2012,21(6):1063-1070.
[16]Hong K.Actinomycetes from mangrove and their secondary metabolites.Acta Microbiologica Sinica,2013,53(11):1131-1141.(in Chinese)洪葵.红树林放线菌及其天然产物研究进展.微生物学报,2013,53(11):1131-1141.
[17]Xu SQ,Wang YD,Guo CC,Zhang ZG,Shang YT,Chen Q,Wang ZL.Comparison of microbial community composition and diversity in native coastal wetlands and wetlands that have undergone long-term agricultural reclamation.Wetlands,2017,37(1):99-108.
[18]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets.Molecular Biology and Evolution,2016,33(7):1870-1874.
[19]Labeda DP,Dunlap CA,Rong XY,Huang Y,Doroghazi JR,Ju KS,Metcalf WW.Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis.Antonie van Leeuwenhoek,2017,110(4):563-583.
[20]Kumar Y,Aiemsum-ang P,Ward AC,Goodfellow M.Diversity and geographical distribution of members of the Streptomyces violaceusniger 16S rRNA gene clade detected by clade-specific PCR primers.FEMS Microbiology Ecology,2007,62(1):54-63.
[21]KyselkováM,KopeckyJ,Felf?ldi T,?ermák L,Omelka M,Grundmann GL,Mo?nne-Loccoz Y,Ságová-Mare?kováM.Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera.Antonie van Leeuwenhoek,2008,94(3):439-453.
[22]VětrovskyT,Baldrian P.An in-depth analysis of actinobacterial communities shows their high diversity in grassland soils along a gradient of mixed heavy metal contamination.Biology and Fertility of Soils,2015,51(7):827-837.
[23]Tang B,Yu YC,Zhi XY,Yang LL,Cen XF,Zhao GP,Ding XM.Streptomyces yangpuensis sp.nov.,isolated from soil.International Journal of Systematic and Evolutionary Microbiology,2016,66(3):1224-1229.
[24]Ravi L,Krishnan K.Benzoyloxy-ethyl-carbamic acid:a novel anticancerous secondary metabolite produced by Streptomyces globosus VITLGK011.Indian Journal of Experimental Biology,2017,55:411-420.
[25]Saeed EE,Sham A,Salmin Z,Abdelmowla Y,Iratni R,El-Tarabily K,AbuQamar S.Streptomyces globosus UAE1,a potential effective biocontrol agent for black scorch disease in date palm plantations.Frontiers in Microbiology,2017,8:1455.