施肥制度对水稻土壤nosZ型反硝化细菌群落的影响
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摘要
由含氧化亚氮还原酶(NOS)的反硝化细菌驱动的氧化亚氮(N2O)还原成氮气(N2)的过程是N2O排放的重要调控途径。为探明施肥对稻田土壤nosZ型反硝化细菌群落的影响,采用荧光定量PCR和高通量测序等方法,研究了湖南省宁乡县长达30年的定位试验条件下4种施肥制度[不施肥(CK)、化肥(CF)、70%化肥+30%有机肥(CFM1)和40%化肥+60%有机肥(CFM2)]对水稻土壤nosZ型反硝化细菌数量和群落结构的影响。结果表明:不同施肥处理nosZ基因丰度为2.14×10~8～6.09×10~8copies·g~(-1)干土,施肥处理nosZ基因拷贝数比对照低47.3%～64.8%(P<0.05),但不同有机肥配施比例处理间nosZ基因拷贝数差异不显著;变形菌门是优势门水平类群,占总序列的60.2%～77.5%; Bacteria_unclassified、Proteobacteria_unclassified、Betaproteobacteria_unclassified和根瘤菌目为优势目水平类群,占总序列的93.6%～95.9%。施肥显著降低了Proteobacteria_unclassified的相对丰度(P<0.01),但显著提高了根瘤菌目、environmental_samples和红环菌目的相对丰度(P<0.05);施肥显著改变nosZ型反硝化细菌的群落结构,但有机肥配施比例对其影响较弱;除碳氮比外,其他土壤理化性质均显著影响nosZ型反硝化细菌的数量和群落结构,其中,硝态氮和土壤p H是驱动nosZ型反硝化细菌群落变化的主要因子;施肥显著影响nosZ型反硝化细菌数量和群落结构,有机肥配施比例对nosZ型反硝化细菌群落的影响较弱,研究结果为进一步阐述施肥制度对土壤反硝化微生物的影响提供依据。
The reduction of nitric oxide( N_2 O) to dinitrogen( N_2) in denitrification is performed by the nosZ-type denitrifying bacteria,which plays a key role in regulating N_2 O emission. To verify the effects of fertilization on the nosZ-type denitrifying bacterial communities in paddy soils,real-time PCR and high-throughput sequencing approaches were applied to investigate the abundance and structure of the nosZ-type denitrifying bacterial communities in a 30-year fertilized paddy soil in Hunan Province,China. The abundances of nosZ gene ranged from 2. 14 × 10~8 to6.09×10~8 copies·g~(-1) dry soil. The nosZ gene abundances in the fertilized treatments( chemical fertilizer,70% chemical fertilizer + 30% organic manure,and 40% chemical fertilizer + 60%organic manure) were 47.3%-64.8% lower than that of CK( P<0.01). Organic manure application rates rarely affected the nosZ gene abundance. Proteobacteria was the predominant phylum,accounting for 60.2%-77.5% of the total reads. Bacteria_unclassified,Proteobacteria_unclassified,Betaproteobacteria_unclassified,and Rhizobiales were the abundant order groups that represented 93.6%-95.9% of the total reads. Fertilization significantly decreased the Proteobacteria_unclassified abundances( P<0.01) and significantly increased the relative abundance of Rhizobiales,environmental _ samples, and Rhodocyclales in the fertilized soils compared with CK( P<0.05). Fertilization altered the nosZ-type denitrifying bacterial community structure. Organic manure application rates had minor effects on the community structure. All the examined soil properties,expect for C/N ratio,were significantly correlated with the abundance,relative abundance of some order groups and structure of the nosZ-type denitrifying bacterial communities,with nitrate and soil pH were the most important factors affecting the nosZ-type denitrifying bacterial community structure. These results indicated that fertilization significantly changed the abundance and structure of the nosZ-type denitrifying bacterial communities,while organic manure application rates showed limited effect. Our results provided basis for further research on the effects of fertilization on the denitrifying bacterial community.
The reduction of nitric oxide( N_2 O) to dinitrogen( N_2) in denitrification is performed by the nosZ-type denitrifying bacteria,which plays a key role in regulating N_2 O emission. To verify the effects of fertilization on the nosZ-type denitrifying bacterial communities in paddy soils,real-time PCR and high-throughput sequencing approaches were applied to investigate the abundance and structure of the nosZ-type denitrifying bacterial communities in a 30-year fertilized paddy soil in Hunan Province,China. The abundances of nosZ gene ranged from 2. 14 × 10~8 to6.09×10~8 copies·g~(-1) dry soil. The nosZ gene abundances in the fertilized treatments( chemical fertilizer,70% chemical fertilizer + 30% organic manure,and 40% chemical fertilizer + 60%organic manure) were 47.3%-64.8% lower than that of CK( P<0.01). Organic manure application rates rarely affected the nosZ gene abundance. Proteobacteria was the predominant phylum,accounting for 60.2%-77.5% of the total reads. Bacteria_unclassified,Proteobacteria_unclassified,Betaproteobacteria_unclassified,and Rhizobiales were the abundant order groups that represented 93.6%-95.9% of the total reads. Fertilization significantly decreased the Proteobacteria_unclassified abundances( P<0.01) and significantly increased the relative abundance of Rhizobiales,environmental _ samples, and Rhodocyclales in the fertilized soils compared with CK( P<0.05). Fertilization altered the nosZ-type denitrifying bacterial community structure. Organic manure application rates had minor effects on the community structure. All the examined soil properties,expect for C/N ratio,were significantly correlated with the abundance,relative abundance of some order groups and structure of the nosZ-type denitrifying bacterial communities,with nitrate and soil pH were the most important factors affecting the nosZ-type denitrifying bacterial community structure. These results indicated that fertilization significantly changed the abundance and structure of the nosZ-type denitrifying bacterial communities,while organic manure application rates showed limited effect. Our results provided basis for further research on the effects of fertilization on the denitrifying bacterial community.
引文
黄莹,李雅颖,姚槐应.2013.强酸性茶园土壤中添加不同肥料氮后N2O释放量变化.植物营养与肥料学报,19(6):1533-1538.
李香兰,徐华,蔡祖聪.2009.水分管理影响稻田氧化亚氮排放研究进展.土壤,41(1):1-7.
宋亚娜,林智敏,林艳.2012.氮肥对稻田土壤反硝化细菌群落结构和丰度的影响.中国生态农业学报,20(1):7-12.
孙翼飞,沈菊培,张翠景,等.2018.不同放牧强度下土壤氨氧化和反硝化微生物的变化特征.生态学报,38(8):2874-2883.
王海斌,张清旭,陈晓婷,等.2017.动物源有机肥对茶树根际土壤酸度及微生物的影响.中国农业科技导报,19(5):115-122.
王亚男,曾希柏,王玉忠,等.2015.施肥模式对设施菜地根际土壤微生物群落结构和丰度的影响.生态学杂志,34(3):826-834.
杨亚东,宋润科,赵杰,等.2018.长期不同施肥制度对水稻土氨氧化微生物数量和群落结构的影响.应用生态学报,29(11):3829-3837.
张晶,林先贵,尹睿,等.2009.参与土壤氮素循环的微生物功能基因多样性研究进展.中国生态农业学报,17(5):1029-1034.
郑燕,候海军,秦红灵,等.2012.施氮对水稻土N2O释放及反硝化功能基因(nar G/nos Z)丰度的影响.生态学报,32(11):3386-3393.
Bateman EJ,Baggs EM.2005.Contributions of nitrification and denitrification to N2O emissions from soils at different water-filled pore space.Biology and Fertility of Soils,41:379-388.
Caporasp JG,Kuczynski J,Stombaugh J.2010.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,7:335-336.
Chen Z,Liu JB,Wu MN,et al.2012.Differentiated response of denitrifying communities to fertilization regime in paddy soil.Microbial Ecology,63:446-459.
Cui PY,Fan FL,Yin C,et al.2016.Long-term organic and inorganic fertilization alters temperature sensitivity of potential N2O emissions and associated microbes.Soil Biology and Biochemistry,93:131-141.
Duan R,Long XE,Tang YF,et al.2018.Effects of different fertilizer application methods on the community of nitrifiers and denitrifiers in a paddy soil.Journal of Soils and Sediments,18:24-38.
Edgar RC.2013.UPARSE:Highly accurate OTU sequences from microbial amplicon reads.Nature Methods,10:996-998.
Enwall K,Philippot L,Hallin S.2005.Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization.Applied and Environmental Microbiology,71:8335-8343.
FAO.1974.FAO-UNESCO Soil Map of the World.Paris:UNESCO.
FAO.2013.Food and Agriculture Organization statistical database.http://faostat3.fao.org.
Firestone MK,Firestone RB,Tiedje JM.1980.Nitrous oxide from soil denitrification:Factors controlling its biological production.Science,208:749-751.
Gao B,Ju XT,Zhang Q,et al.2011.New estimates of direct N2O emissions from Chinese croplands from 1980 to 2007using localized emission factors.Biogeosciences,8:3011-3024.
Gao N,Shen WS,Camargo E,et al.2017.Nitrous oxide(N2O)-reducing denitrifier-inoculated organic fertilizer mitigates N2O emissions from agricultural soils.Biology and Fertility of Soils,53:885-898.
Hallin S,Jones CM,Schloter M,et al.2009.Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment.The ISME Journal,3:597-605.
Hallin S,Lindgren PE.1999.PCR detection of genes encoding nitrile reductase in denitrifying bacteria.Applied and Environmental Microbiology,65:1652-1657.
IPCC.2013.Climate change 2013:The Physical Science Basis.Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change.Cambridge,UK:Cambridge University Press.
IPCC.2014.Climate Change 2014:Synthesis Report.Contribution of Working Groups I,II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change.Geneva,Switzerland.
Kloos K,Mergel A,Rosch C,et al.2001.Denitrification within the genus Azospirillum and other associative bacteria.Australian Journal of Plant Physiology,28:991-998.
Philippot L,Andert J,Jones CM,et al.2011.Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil.Global Change Biology,17:1497-1504.
Schloss PD,Gevers D,Westcott SL.2011.Reducing the effects of PCR amplification and sequencing artifacts on 16SrRNA-based studies.PLo S ONE,6:e27310.
Sun RB,Guo XS,Wang DZ,et al.2015.Effects of long-term application of chemical and organic fertilizers on the abundance of microbial communities involved in the nitrogen cycle.Applied Soil Ecology,95:171-178.
Tao R,Wakelin SA,Liang YC,et al.2018.Nitrous oxide emission and denitrifier communities in drip-irrigated calcareous soil as affected by chemical and organic fertilizers.Science of the Total Environment,612:739-749.
Throback IN,Enwall K,Jarvis A,et al.2004.Reassessing PCR primers targeting nir S,nir K and nos Z genes for community surveys of denitrifying bacteria with DGGE.FEMSMicrobiology Ecology,49:401-417.
Waibel AE,Peter T,Carslaw KS,et al.1999.Arctic ozone loss due to denitrification.Science,283:2064-2069.
Wang YY,Lu SE,Xiang QJ,et al.2017.Responses of N2Oreductase gene(nos Z)-denitrifer communities to long-term fertilization follow a depth pattern in calcareous purplish paddy soil.Journal of Integrative Agriculture,16:2597-2611.
Yang YD,Hu YG,Wang ZM,et al.2018.Variations of the nir S-,nir K-,and nos Z-denitrifying bacterial communities in a northern Chinese soil as affected by long-term different irrigation regimes.Environmental Science and Pollution Research,25:14057-14067.
Zumft WG.1997.Cell biology and molecular basis of denitrification.Microbiology and Molecular Biology Reviews,61:533-616.
李香兰,徐华,蔡祖聪.2009.水分管理影响稻田氧化亚氮排放研究进展.土壤,41(1):1-7.
宋亚娜,林智敏,林艳.2012.氮肥对稻田土壤反硝化细菌群落结构和丰度的影响.中国生态农业学报,20(1):7-12.
孙翼飞,沈菊培,张翠景,等.2018.不同放牧强度下土壤氨氧化和反硝化微生物的变化特征.生态学报,38(8):2874-2883.
王海斌,张清旭,陈晓婷,等.2017.动物源有机肥对茶树根际土壤酸度及微生物的影响.中国农业科技导报,19(5):115-122.
王亚男,曾希柏,王玉忠,等.2015.施肥模式对设施菜地根际土壤微生物群落结构和丰度的影响.生态学杂志,34(3):826-834.
杨亚东,宋润科,赵杰,等.2018.长期不同施肥制度对水稻土氨氧化微生物数量和群落结构的影响.应用生态学报,29(11):3829-3837.
张晶,林先贵,尹睿,等.2009.参与土壤氮素循环的微生物功能基因多样性研究进展.中国生态农业学报,17(5):1029-1034.
郑燕,候海军,秦红灵,等.2012.施氮对水稻土N2O释放及反硝化功能基因(nar G/nos Z)丰度的影响.生态学报,32(11):3386-3393.
Bateman EJ,Baggs EM.2005.Contributions of nitrification and denitrification to N2O emissions from soils at different water-filled pore space.Biology and Fertility of Soils,41:379-388.
Caporasp JG,Kuczynski J,Stombaugh J.2010.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,7:335-336.
Chen Z,Liu JB,Wu MN,et al.2012.Differentiated response of denitrifying communities to fertilization regime in paddy soil.Microbial Ecology,63:446-459.
Cui PY,Fan FL,Yin C,et al.2016.Long-term organic and inorganic fertilization alters temperature sensitivity of potential N2O emissions and associated microbes.Soil Biology and Biochemistry,93:131-141.
Duan R,Long XE,Tang YF,et al.2018.Effects of different fertilizer application methods on the community of nitrifiers and denitrifiers in a paddy soil.Journal of Soils and Sediments,18:24-38.
Edgar RC.2013.UPARSE:Highly accurate OTU sequences from microbial amplicon reads.Nature Methods,10:996-998.
Enwall K,Philippot L,Hallin S.2005.Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization.Applied and Environmental Microbiology,71:8335-8343.
FAO.1974.FAO-UNESCO Soil Map of the World.Paris:UNESCO.
FAO.2013.Food and Agriculture Organization statistical database.http://faostat3.fao.org.
Firestone MK,Firestone RB,Tiedje JM.1980.Nitrous oxide from soil denitrification:Factors controlling its biological production.Science,208:749-751.
Gao B,Ju XT,Zhang Q,et al.2011.New estimates of direct N2O emissions from Chinese croplands from 1980 to 2007using localized emission factors.Biogeosciences,8:3011-3024.
Gao N,Shen WS,Camargo E,et al.2017.Nitrous oxide(N2O)-reducing denitrifier-inoculated organic fertilizer mitigates N2O emissions from agricultural soils.Biology and Fertility of Soils,53:885-898.
Hallin S,Jones CM,Schloter M,et al.2009.Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment.The ISME Journal,3:597-605.
Hallin S,Lindgren PE.1999.PCR detection of genes encoding nitrile reductase in denitrifying bacteria.Applied and Environmental Microbiology,65:1652-1657.
IPCC.2013.Climate change 2013:The Physical Science Basis.Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change.Cambridge,UK:Cambridge University Press.
IPCC.2014.Climate Change 2014:Synthesis Report.Contribution of Working Groups I,II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change.Geneva,Switzerland.
Kloos K,Mergel A,Rosch C,et al.2001.Denitrification within the genus Azospirillum and other associative bacteria.Australian Journal of Plant Physiology,28:991-998.
Philippot L,Andert J,Jones CM,et al.2011.Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil.Global Change Biology,17:1497-1504.
Schloss PD,Gevers D,Westcott SL.2011.Reducing the effects of PCR amplification and sequencing artifacts on 16SrRNA-based studies.PLo S ONE,6:e27310.
Sun RB,Guo XS,Wang DZ,et al.2015.Effects of long-term application of chemical and organic fertilizers on the abundance of microbial communities involved in the nitrogen cycle.Applied Soil Ecology,95:171-178.
Tao R,Wakelin SA,Liang YC,et al.2018.Nitrous oxide emission and denitrifier communities in drip-irrigated calcareous soil as affected by chemical and organic fertilizers.Science of the Total Environment,612:739-749.
Throback IN,Enwall K,Jarvis A,et al.2004.Reassessing PCR primers targeting nir S,nir K and nos Z genes for community surveys of denitrifying bacteria with DGGE.FEMSMicrobiology Ecology,49:401-417.
Waibel AE,Peter T,Carslaw KS,et al.1999.Arctic ozone loss due to denitrification.Science,283:2064-2069.
Wang YY,Lu SE,Xiang QJ,et al.2017.Responses of N2Oreductase gene(nos Z)-denitrifer communities to long-term fertilization follow a depth pattern in calcareous purplish paddy soil.Journal of Integrative Agriculture,16:2597-2611.
Yang YD,Hu YG,Wang ZM,et al.2018.Variations of the nir S-,nir K-,and nos Z-denitrifying bacterial communities in a northern Chinese soil as affected by long-term different irrigation regimes.Environmental Science and Pollution Research,25:14057-14067.
Zumft WG.1997.Cell biology and molecular basis of denitrification.Microbiology and Molecular Biology Reviews,61:533-616.