设施菜田不同碳氮管理对反硝化菌结构和功能的影响
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摘要
【目的】通过6年长期定位试验,比较设施菜田不同碳氮管理下反硝化菌结构和功能的差异。【方法】采用末端限制性片段多态性(T-RFLP)和变性梯度凝胶电泳(DGGE)方法分别分析nir K/nir S和nos Z型反硝化菌群结构特征,利用自动连续在线培养监测体系(Robot系统)测定分析NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比,并通过乙炔抑制法测定反硝化酶活性。【结果】传统施肥处理(CN)显著改变了nir K和nos Z型反硝化菌的结构,增加了NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比。nir S型菌受碳氮管理影响较小。减氮(RN)和添加秸秆处理(RN+S)的nir K和nos Z型反硝化菌结构与CN处理的差异性显著,且会显著降低NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比;与CN和RN相比,RN+S显著增加反硝化酶活性。【结论】设施菜田长期传统施肥措施改变了反硝化菌的结构和功能,增加土壤自身的NO产生能力并减弱了N2O还原N2的能力。减氮和添加秸秆管理能形成自身的反硝化菌群结构,并降低NO和N2O排放风险;秸秆的添加会促进反硝化潜在速率,降低菜田NO3-淋洗风险。
[Objective] Based on a 6-years greenhouse cropping field,the objective of the present study was to find out the differences of the structure and function of denitrifier community under different carbon and nitrogen managements.[Methods] We used terminal restriction fragment length polymorphism(T-RFLP) and denaturing gradient gel electrophoresis(DGGE) methods to analyze the structure of microbial communities containing nir K/nir S and nos Z,respectively,a robotized incubation system to measure NO/(NO3-+NO2-) and N2O/(N2O+N2) product ratio and an acetylene inhibition method to determine denitrification enzyme activity.[Results] Conventional N management(CN) significantly changed the structure of denitrifier communities containing nir K and nos Z and increased the NO/(NO3-+NO2-) and N2O/(N2O+N2) product ratio.nir S-denitrifiers were less affected by carbon and nitrogen managements.Reduced N(RN) and reduced N plus straw(RN+S) management had significant changed the denitrifier communities containing nir K and nos Z and reduced NO/(NO3-+NO2-) and N2O/(N2O+N2) product ratio compared with CN treatment.In contrast to CN and RN treatments,RN+S significantly increased denitrification enzyme activity.[Conclusion] Conventional N management significantly changed the structure and function of denitrifier community,and promoted the formation of NO and N2 O.RN and RN+S managements could form new denitrifier community structure and reduced the risk of NO and N2 O emissions.In addition,straw application promoted potential denitrification rates and reduced the risk of NO3-leaching in greenhouses.
[Objective] Based on a 6-years greenhouse cropping field,the objective of the present study was to find out the differences of the structure and function of denitrifier community under different carbon and nitrogen managements.[Methods] We used terminal restriction fragment length polymorphism(T-RFLP) and denaturing gradient gel electrophoresis(DGGE) methods to analyze the structure of microbial communities containing nir K/nir S and nos Z,respectively,a robotized incubation system to measure NO/(NO3-+NO2-) and N2O/(N2O+N2) product ratio and an acetylene inhibition method to determine denitrification enzyme activity.[Results] Conventional N management(CN) significantly changed the structure of denitrifier communities containing nir K and nos Z and increased the NO/(NO3-+NO2-) and N2O/(N2O+N2) product ratio.nir S-denitrifiers were less affected by carbon and nitrogen managements.Reduced N(RN) and reduced N plus straw(RN+S) management had significant changed the denitrifier communities containing nir K and nos Z and reduced NO/(NO3-+NO2-) and N2O/(N2O+N2) product ratio compared with CN treatment.In contrast to CN and RN treatments,RN+S significantly increased denitrification enzyme activity.[Conclusion] Conventional N management significantly changed the structure and function of denitrifier community,and promoted the formation of NO and N2 O.RN and RN+S managements could form new denitrifier community structure and reduced the risk of NO and N2 O emissions.In addition,straw application promoted potential denitrification rates and reduced the risk of NO3-leaching in greenhouses.
引文
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[14]宋贺,徐新超,王敬国,等.设施菜田土壤剖面中的反硝化特征[J].植物营养与肥料学报,2012,18(4):860-867.
[15]Braker G,Fesefeldt A,Witzel KP.Development of PCR primer systems for amplification of nitrite reductase genes(nir K and nir S)to detect denitrifying bacteria in environmental samples[J].Applied and Environmental Microbiology,1998,64(10):3769-3775.
[16]Kloos K,Mergel A,Rosch C,et al.Denitrification within the genus Azospirillum and other associative bacteria[J].Australian Journal of Plant Physiology,2001,28(9):991-998.
[17]Throbck IN,Enwall K,Jarvis A,et al.Reassessing PCR primers targeting nir S,nir K and nos Z genes for community surveys of denitrifying bacteria with DGGE[J].FEMS Microbiology Ecology,2004,49(3):401-417.
[18]Page AL,Miller RH,Keeney DR.Methods of soil analyses.part 2.Chemical and Microbiological Properties[M].Madison:American Society of Agronomy,1982:1011-1026.
[19]imek M,Cooper JE,Picek T,et al.Denitrification in arable soils in relation to their physico-chemical properties and fertilization practice[J].Soil Biology and Biochemistry,2000,32(1):101-110.
[20]张真和.我国蔬菜产业发展中的问题与对策[J].中国农业科技导报,2001,3(4):57-60.
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[22]董章杭.山东省寿光市集约化蔬菜种植区农用化学品使用及其对环境影响的研究[D].北京:中国农业大学博士学位论文,2006.
[23]Wertz S,Dandie CE,Goyer C,et al.Diversity of nir K denitrifying genes and transcripts in an agricultural soil[J].Applied and Environmental Microbiology,2009,75(23):7365-7377.
[24]Yoshida M,Ishii S,Otsuka S,et al.nir K-harboring denitrifiers are more responsive to denitrification-inducing conditions in rice paddy soil than nir S-harboring bacteria[J].Microbes and Environments,2010,25(1):45-48.
[25]Cui F,Yan GX,Zhou ZX,et al.Annual emissions of nitrous oxide and nitric oxide from a wheat-maize cropping system on a silt loam calcareous soil in the North China Plain[J].Soil Biology and Biochemistry,2012,48(3):10-19.
[26]Ding WX,Cai Y,Cai ZC,et al.Nitrous oxide emissions from an intensively cultivated maize-wheat rotation soil in the North China Plain[J].Science of the Total Environment,2007,373(2/3):501-511.
[27]Dambreville C,Hallet S,Nguyen C,et al.Structure and activity of denitrify community in a maize-cropped field fertilized with composted pig manure or ammonium nitrate[J].FEMS Microbiology Ecology,2006,56(1):119-131.
[28]Paul EA.Soil microbiology,ecology,and biochemistry[M].Oxford:Academic Press,2007:341-361.
[29]Mathieu O,Léveque J,Hénault C,et al.Emissions and spatial variability of N2O,N2 and nitrous oxide mole fraction at the field scale,revealed with 15N isotopic techniques[J].Soil Biology and Biochemistry,2006,38(5):941-951.
[2]王敬国.设施菜田退化土壤修复与资源高效利用[M].北京:中国农业大学出版社,2011:1-18.
[3]Guo JH,Liu XJ,Zhang Y,et al.Significant acidification in major Chinese croplands[J].Science,2010,327(5968):1008-1010.
[4]刘兆辉,江丽华,张文君,等.山东省设施蔬菜施肥量演变及土壤养分变化规律[J].土壤学报,2008,45(2):296-302.
[5]Song H,Guo JH,Ren T,et al.Increase of soil p H in a solar greenhouse vegetable production system[J].Soil Science Society of America Journal,2012,76(6):2074-2082.
[6]He FF,Jiang RF,Chen Q,et al.Nitrous oxide emissions from an intensively managed greenhouse vegetable cropping system in Northern China[J].Environmental Pollution,2009,157(5):1666-1672.
[7]Schepers JS,Raun W.Nitrogen in agricultural systems[M].Madison:ASA-CSSSA-SSSA,2008:197-249.
[8]Philippot L,Hallin S,Schlote M.Ecology of denitrifying prokaryotesin agricultural soil[J].Advances in Agronomy,2007,96:250-287.
[9]Bothe H,Ferguson SJ,Binaldo S,et al.Biology of the nitrogen cycle[M].Newton:Elsevier,2007:37-55.
[10]Philippot L,Andert J,Jones CM,et al.Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil[J].Global Change Biology,2011,17(3):1497-1504.
[11]Enwall K,Philippot L,Hallin S.Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization[J].Applied and Environmental Microbiology,2005,71(6):8335-8343.
[12]Ren T,Christie P,Wang JG,et al.Root zone soil nitrogen management to maintain high tomato yields and minimum nitrogen losses to the environment[J].Scientia Hotriculturea,2010,125(1):22-33.
[13]Molstad L,Drsch P,Bakken LR.Robotized incubation system for monitoring gases(O2,NO,N2O,N2)in denitrifying cultures[J].Journal of Microbiological Methods,2007,71(3):202-211.
[14]宋贺,徐新超,王敬国,等.设施菜田土壤剖面中的反硝化特征[J].植物营养与肥料学报,2012,18(4):860-867.
[15]Braker G,Fesefeldt A,Witzel KP.Development of PCR primer systems for amplification of nitrite reductase genes(nir K and nir S)to detect denitrifying bacteria in environmental samples[J].Applied and Environmental Microbiology,1998,64(10):3769-3775.
[16]Kloos K,Mergel A,Rosch C,et al.Denitrification within the genus Azospirillum and other associative bacteria[J].Australian Journal of Plant Physiology,2001,28(9):991-998.
[17]Throbck IN,Enwall K,Jarvis A,et al.Reassessing PCR primers targeting nir S,nir K and nos Z genes for community surveys of denitrifying bacteria with DGGE[J].FEMS Microbiology Ecology,2004,49(3):401-417.
[18]Page AL,Miller RH,Keeney DR.Methods of soil analyses.part 2.Chemical and Microbiological Properties[M].Madison:American Society of Agronomy,1982:1011-1026.
[19]imek M,Cooper JE,Picek T,et al.Denitrification in arable soils in relation to their physico-chemical properties and fertilization practice[J].Soil Biology and Biochemistry,2000,32(1):101-110.
[20]张真和.我国蔬菜产业发展中的问题与对策[J].中国农业科技导报,2001,3(4):57-60.
[21]张真和,陈青云,高丽红,等.我国设施蔬菜产业发展对策研究(上)[J].蔬菜,2010,5:1-4.
[22]董章杭.山东省寿光市集约化蔬菜种植区农用化学品使用及其对环境影响的研究[D].北京:中国农业大学博士学位论文,2006.
[23]Wertz S,Dandie CE,Goyer C,et al.Diversity of nir K denitrifying genes and transcripts in an agricultural soil[J].Applied and Environmental Microbiology,2009,75(23):7365-7377.
[24]Yoshida M,Ishii S,Otsuka S,et al.nir K-harboring denitrifiers are more responsive to denitrification-inducing conditions in rice paddy soil than nir S-harboring bacteria[J].Microbes and Environments,2010,25(1):45-48.
[25]Cui F,Yan GX,Zhou ZX,et al.Annual emissions of nitrous oxide and nitric oxide from a wheat-maize cropping system on a silt loam calcareous soil in the North China Plain[J].Soil Biology and Biochemistry,2012,48(3):10-19.
[26]Ding WX,Cai Y,Cai ZC,et al.Nitrous oxide emissions from an intensively cultivated maize-wheat rotation soil in the North China Plain[J].Science of the Total Environment,2007,373(2/3):501-511.
[27]Dambreville C,Hallet S,Nguyen C,et al.Structure and activity of denitrify community in a maize-cropped field fertilized with composted pig manure or ammonium nitrate[J].FEMS Microbiology Ecology,2006,56(1):119-131.
[28]Paul EA.Soil microbiology,ecology,and biochemistry[M].Oxford:Academic Press,2007:341-361.
[29]Mathieu O,Léveque J,Hénault C,et al.Emissions and spatial variability of N2O,N2 and nitrous oxide mole fraction at the field scale,revealed with 15N isotopic techniques[J].Soil Biology and Biochemistry,2006,38(5):941-951.