CO_2倍增和施氮对水稻不同生长期土壤反硝化细菌丰度的影响
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摘要
随着全球气候变化的不断加剧,大气CO2浓度呈明显增加趋势,这将间接影响土壤-植物-微生物系统的氮循环过程.为研究典型水稻土壤反硝化细菌对CO2浓度升高的响应规律和机制,借助水稻密闭培养箱,运用实时荧光定量聚合酶链式反应(Real-Time q PCR)分子技术,设置不施氮(0 mg/kg)和常规施氮(100 mg/kg) 2个处理,研究CO2倍增对水稻不同生长期土壤关键反硝化功能细菌(nar G、nir K和nir S型)丰度的影响.结果表明:①在2种施氮水平,CO2倍增显著促进了水稻分蘖期、孕穗期、扬花期和成熟期水稻根系生长(增幅为2. 96%～28. 4%)、地上部生物量增加(增幅为7. 1%～107. 3%)以及成熟期籽粒干质量的增加(增幅为19. 5%和38. 0%),具有显著的增产效应.②反硝化细菌丰度对CO2倍增的响应与生育期及施氮水平有关,CO2倍增在2个施氮水平均抑制分蘖期反硝化细菌的繁殖,显著增加孕穗期反硝化细菌数量;在水稻扬花期,CO2倍增促进了施氮处理nar G和nir S型反硝化细菌数量的增加,在成熟期抑制未施氮处理下nar G、nir K和nir S型反硝化细菌的生长.另外,nar G、nir K、nir S型反硝化细菌丰度整体表现为nar G>nir S>nir K,且随水稻的生长,其在成熟期的丰度均呈降低趋势. nir K和nir S基因同属亚硝酸还原酶,但nir S基因丰度高于nir K,且对CO2倍增和施氮的响应有所差异.研究显示,CO2倍增可显著增加水稻生长和产量,不同施氮水平对稻田土壤反硝化细菌丰度的影响存在差异.
The atmospheric CO_2 concentration has an obvious rising trend with the increasing global climate change,which will indirectly affect the nitrogen cycling process of the soil-plant-microbe system. In order to investigate the mechanisms of the rising CO_2 effect on denitrifying bacteria in paddy soil,we used the airtight rice incubator and the real-time PCR technology to investigate the effect of the elevating CO_2 concentration on the abundance of nar G-,nir K-and nir S-containing bacterial communities at two nitrogen levels. The results showed that the elevating CO_2 concentration significantly increased the biomass of roots and aboveground parts( 2. 96%-28. 4%,7. 1%-107. 3%) at the tillering,booting,blooming and maturing stages either without nitrogen or with regular nitrogen application.Meantime,the dry weight of grains at the two levels of nitrogen application increased( 19. 5%,38. 0%) at the maturing stage,indicating that the rice yield was stimulated by the elevating CO_2 concentration; Response of denitrifying bacteria to elevating CO_2 concentration was related to growth stage and nitrogen application level: the elevating CO_2 decreased the number of denitrifying bacteria at tillering stage,but increased that at booting stage; The elevating CO_2 obviously increased the abundance of nar G-,nir S-containing communities in nitrogen application treatment at the booting stage,and decreased the abundance of nar G-,nir K-and nir S-containing communities at the maturing stage. In addition,the abundance of nar G-,nir K-and nir S-containing communities were nar G>nir S>nir K,and they decreased with the increase of rice growth. Both nir K and nir S genes belong to nitrite reductase,but the abundance of nir S gene was higher than that of nir K,and their responses to elevating CO_2 and nitrogen application were different. Overall,the results showed that the elevating CO_2 significantly increased rice growth and yield,and the effects of CO_2 on the abundance of denitrifying bacteria in paddy soil were different with different nitrogen application.
The atmospheric CO_2 concentration has an obvious rising trend with the increasing global climate change,which will indirectly affect the nitrogen cycling process of the soil-plant-microbe system. In order to investigate the mechanisms of the rising CO_2 effect on denitrifying bacteria in paddy soil,we used the airtight rice incubator and the real-time PCR technology to investigate the effect of the elevating CO_2 concentration on the abundance of nar G-,nir K-and nir S-containing bacterial communities at two nitrogen levels. The results showed that the elevating CO_2 concentration significantly increased the biomass of roots and aboveground parts( 2. 96%-28. 4%,7. 1%-107. 3%) at the tillering,booting,blooming and maturing stages either without nitrogen or with regular nitrogen application.Meantime,the dry weight of grains at the two levels of nitrogen application increased( 19. 5%,38. 0%) at the maturing stage,indicating that the rice yield was stimulated by the elevating CO_2 concentration; Response of denitrifying bacteria to elevating CO_2 concentration was related to growth stage and nitrogen application level: the elevating CO_2 decreased the number of denitrifying bacteria at tillering stage,but increased that at booting stage; The elevating CO_2 obviously increased the abundance of nar G-,nir S-containing communities in nitrogen application treatment at the booting stage,and decreased the abundance of nar G-,nir K-and nir S-containing communities at the maturing stage. In addition,the abundance of nar G-,nir K-and nir S-containing communities were nar G>nir S>nir K,and they decreased with the increase of rice growth. Both nir K and nir S genes belong to nitrite reductase,but the abundance of nir S gene was higher than that of nir K,and their responses to elevating CO_2 and nitrogen application were different. Overall,the results showed that the elevating CO_2 significantly increased rice growth and yield,and the effects of CO_2 on the abundance of denitrifying bacteria in paddy soil were different with different nitrogen application.
引文
[1] PRENTICE I C,FARQUHAR G D,FASHAM M J R,et al. The carbon cycle and atmospheric carbon dioxide.the Third Assessment Report of Intergovernmental Panel on Climate Change(IPCC)[M].New York:Cambridge University Press,2001:159-165.
[2] MONTEALEGRE C M,KESSEL C V,RUSSELLE M P,et al.Changes in microbial activity and composition in a pasture ecosystem exposed to elevated atmospheric carbon dioxide[J].Plant and Soil,2002,243(2):197-207.
[3] ALBERTO A M P,ZISKA L H,CERVANCIA C R,et al. The influence of increasing carbon dioxide and temperature on competitive interactions between a C3crop,rice(Oryza sativa)and a C4weed(Echinochloa glabrescens)[J]. Functional Plant Biology,1996,23(6):795-802.
[4] KIM H Y,HORIE T,NAKAGAWA H. Effects of elevated CO2concentration and high temperature on growth and yield of rice:Ⅱ.the effect on yield and its components of Akihikari rice[J].Japanese Journal of Crop Science,2008,65(4):644-651.
[5]王大力,林伟宏.CO2浓度升高对水稻根系分泌物的影响:总有机碳,甲酸和乙酸含量变化[J].生态学报,1999,19(4):570-572.WANG Dali,LIN Weihong. Effects of CO2elevation on root exudates in rice[J].Acta Ecologica Sinica,1999,19(4):570-572.
[6]陈利军,武志杰,黄国宏,等.大气CO2增加对土壤脲酶、磷酸酶活性的影响[J].应用生态学报,2002,13(10):1356-1357.CHEN Lijun,WU Zhijie,HUANG Guohong,et al.Effect of elevated atmospheric CO2on soil urease and phosphatase activities[J].Chinese Journal of Applied Ecology,2002,13(10):1356-1357.
[7] CARNOL M,HOGENBOOM L,JACH M E,et al. Elevated atmospheric CO2in open top chambers increases net nitrification and potential denitrification[J]. Global Change Biology,2002,8(6):590-598.
[8] PHILIPPOT L,HOJBERG O. Dissimilatory nitrate reductases in bacteria[J]. Biochimica Et Biophysica Acta-Gene Structure and Expression,1999,1446(1/2):1-23.
[9] ZUMFT W G,KORNER H. Enzyme diversity and mosaic gene organization in denitrification[J]. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology,1997,71(1/2):43-58.
[10] BUSCH A,POHLMANN A,FRIEDRICH B,et al. A DNA region recognized by the nitric oxide responsive transcriptional activator norR is conserved in beta-and gamma-proteobacteria[J].Journal of Bacteriology,2004,186(23):7980-7987.
[11] HOLLOWAY P,MCCORMICK W,WATSON R J,et al.Identification and analysis of the dissimilatory nitrous oxide reduction genes,nosRZDFY,of Rhizobium meliloti[J]. Journal of Bacteriology,1996,178(6):1505-1514.
[12] LIU C Y,ZHENG X H,ZHOU Z X,et al.Nitrous oxide and nitric oxide emissions from an irrigated cotton field in northern China[J].Plant and Soil,332(1/2):123-134.
[13]郑燕,侯海军,秦红灵,等.施氮对水稻土N2O释放及反硝化功能基因(narG/nosZ)丰度的影响[J].生态学报,2012,32(11):3386-3393.ZHENG Yan,HOU Haijun,QIN Hongling,et al. Effect of N application on the abundance of denitrifying genes(nar G/nos Z)and N2O emission in paddy soil[J]. Acta Ecologica Sinica,2012,32(11):3386-3393.
[14]宋亚娜,林智敏,林艳.氮肥对稻田土壤反硝化细菌群落结构和丰度的影响[J].中国生态农业学报,2012,20(1):7-12.SONG Yana,LIN Zhimin,LIN Yan. Response of denitrifying bacteria community structure and abundance to nitrogen in paddy fields[J].Chinese Journal of Eco-Agriculture,2012,20(1):7-12.
[15]刘远,王光利,李恋卿,等.土壤硝化和反硝化微生物群落及活性对大气CO2浓度和温度升高的响应[J].环境科学,2017,38(3):1245-1252.LIU Yuan,WANG Guangli,LI Lianqing,et al. Community and activity to elevated atmospheric CO2concentration and temperature[J].Environmental Science,2017,38(3):1245-1252.
[16]王殳屹,韩琳,史奕,等.开放式大气CO2浓度增高对水稻土反硝化活性的影响[J].浙江大学学报(农业与生命科学版),2007,33(3):284-289.WANG Shuyi,HAN Lin,SHI Yi,et al. Effects of FACE denitrification activity in paddy-field soil[J]. Journal of Zhejiang University(Agricultural and Life Science),2007,33(3):284-289.
[17] OLSEN S R,COLE C V,WATANABE F S,et al. Estimation of available phosphorus in soils by extraction with sodium bicarbonate[M].Washington DC:USDA Circular,1954.
[18] CHEN Z,LUO X,HU R,et al.Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil[J].Microbial Ecology,2010,60(4):850-861.
[19] HALLIN S,LINGDGREN P E. PCR detection of genes encoding nitrite reductase in denitrifying bacteria PCR detection of genes encoding nitrite reductase in denitrifying bacteria[J]. Applied and Environmental Microbiology,1999,65(4):1652-1657.
[20] MICHOTEY V,MEJEAN V,BONIN P.Comparison of methods for quantification of cytochrome, cd1-denitrifying bacteria in environmental marine samples[J]. Applied and Environmental Microbiology,2000,66(4):1564-1571.
[21]杨连新,王云霞,朱建国,等.开放式空气中CO2浓度增高(FACE)对水稻生长和发育的影响[J].生态学报,2010,30(6):1573-1585.YANG Lianxin,WANG Yunxia,ZHU Jianguo,et al. What we have learned from 10 years of free-air CO2enrichment(FACE)experiments on rice? growth and development[J]. Acta Ecologica Sinica,2010,30(6):1573-1585.
[22]谢祖彬,朱建国,张雅丽,等.水稻生长及其体内C、N、P组成对开放式空气CO2浓度增高和N、P施肥的响应[J].应用生态学报,2002,13(10):1223-1230.XIE Zubin,ZHU Jianguo,ZHANG Yali,et al. Responses of rice(Oryza sativa)growth and its C,N and P composition to FACE(Free-air Carbon Dioxide Enrichment)and N,P fertilization[J].Chinese Journal of Applied Ecology,2002,13(10):1223-1230.
[23] ALLEN L H,BOOTE K J,JONES J W,et al.Response of vegetation to rising carbon dioxide:photosynthesis,biomass,and seed yield of soybean[J].Global Biogeochemical Cycles,2012,1(1):1-14.
[24] LAREEN A,BURTON F,SCHAFER P. Plant root-microbe communication in shaping root microbiomes[J]. Plant Molecular Biology,2016,90(6):575-587.
[25] CHAPARRO J M,BADRI D V,VIVANCO J M. Rhizosphere microbiome assemblage is affected by plant development[J]. Isme Journal,2014,8(4):790-803.
[26] KANDELER E,DEIGLMAYR K,TSCHERKO D,et al.Abundance of narG,nir S,nirK,and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland[J]. Applied and Environmental Microbiology,2006,72(9):5957-5962.
[27] STITT M,KRAPP A. The interaction between elevated carbon dioxide and nitrogen nutrition:the physiological and molecular background[J].Plant Cell&Environment,1999,22(6):583-621.
[28] CARNOL M,HOGENBOOM L,JACH M E,et al. Elevated atmospheric CO2in open top chambers increases net nitrification and potential denitrification[J]. Global Change Biology,2010,8(6):590-598.
[29] KAISER E A,KOHRS K,KUCKE M,et al. Nitrous oxide release from arable soil:importance of N-fertilization,crops and temporal variation[J].Soil Biology and Biochemistry,1998,30(12):1553-1563.
[30] KIM S,DALE B E. Effects of nitrogen fertilizer application on greenhouse gas emissions and economics of corn production[J].Environmental Science&Technology,2008,42(16):6028-6033.
[31]高嵩涓,曹卫东,白金顺,等.湘南红壤稻田AOA-amo A、narG、nosZ基因丰度及其环境影响因子[J].中国土壤与肥料,2017(1):21-27.GAO Songjuan,CAO Weidong,BAI Jinshun,et al. Abundances of AOA-amoA,narG,nosZ genes and their relationships with soil environment factors in red paddy soils in south Hunan Province[J].Soil and Fertilizer Science in China,2017(1):21-27.
[32] JONES C M,STRES B,ROSENQUIST M,et al. Phylogenetic analysis of nitrite,nitric oxide,and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification[J].Molecular Biology and Evolution,2008,25(9):1955-1966.
[33]陈哲.长期施肥对水稻土反硝化作用和反硝化功能微生物的影响机理[D].北京:中国科学院研究生院,2010.
[34] YOSHIDA M,ISHII S,OTSUKA S,et al. Temporal shifts in diversity and quantity of nirS and nir K in a rice paddy field soil[J].Soil Biology and Biochemistry,2009,41(10):2044-2051.
[35] YOSHIDA M,ISHII S,OTSUKA S,et al.nirK-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.
[2] MONTEALEGRE C M,KESSEL C V,RUSSELLE M P,et al.Changes in microbial activity and composition in a pasture ecosystem exposed to elevated atmospheric carbon dioxide[J].Plant and Soil,2002,243(2):197-207.
[3] ALBERTO A M P,ZISKA L H,CERVANCIA C R,et al. The influence of increasing carbon dioxide and temperature on competitive interactions between a C3crop,rice(Oryza sativa)and a C4weed(Echinochloa glabrescens)[J]. Functional Plant Biology,1996,23(6):795-802.
[4] KIM H Y,HORIE T,NAKAGAWA H. Effects of elevated CO2concentration and high temperature on growth and yield of rice:Ⅱ.the effect on yield and its components of Akihikari rice[J].Japanese Journal of Crop Science,2008,65(4):644-651.
[5]王大力,林伟宏.CO2浓度升高对水稻根系分泌物的影响:总有机碳,甲酸和乙酸含量变化[J].生态学报,1999,19(4):570-572.WANG Dali,LIN Weihong. Effects of CO2elevation on root exudates in rice[J].Acta Ecologica Sinica,1999,19(4):570-572.
[6]陈利军,武志杰,黄国宏,等.大气CO2增加对土壤脲酶、磷酸酶活性的影响[J].应用生态学报,2002,13(10):1356-1357.CHEN Lijun,WU Zhijie,HUANG Guohong,et al.Effect of elevated atmospheric CO2on soil urease and phosphatase activities[J].Chinese Journal of Applied Ecology,2002,13(10):1356-1357.
[7] CARNOL M,HOGENBOOM L,JACH M E,et al. Elevated atmospheric CO2in open top chambers increases net nitrification and potential denitrification[J]. Global Change Biology,2002,8(6):590-598.
[8] PHILIPPOT L,HOJBERG O. Dissimilatory nitrate reductases in bacteria[J]. Biochimica Et Biophysica Acta-Gene Structure and Expression,1999,1446(1/2):1-23.
[9] ZUMFT W G,KORNER H. Enzyme diversity and mosaic gene organization in denitrification[J]. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology,1997,71(1/2):43-58.
[10] BUSCH A,POHLMANN A,FRIEDRICH B,et al. A DNA region recognized by the nitric oxide responsive transcriptional activator norR is conserved in beta-and gamma-proteobacteria[J].Journal of Bacteriology,2004,186(23):7980-7987.
[11] HOLLOWAY P,MCCORMICK W,WATSON R J,et al.Identification and analysis of the dissimilatory nitrous oxide reduction genes,nosRZDFY,of Rhizobium meliloti[J]. Journal of Bacteriology,1996,178(6):1505-1514.
[12] LIU C Y,ZHENG X H,ZHOU Z X,et al.Nitrous oxide and nitric oxide emissions from an irrigated cotton field in northern China[J].Plant and Soil,332(1/2):123-134.
[13]郑燕,侯海军,秦红灵,等.施氮对水稻土N2O释放及反硝化功能基因(narG/nosZ)丰度的影响[J].生态学报,2012,32(11):3386-3393.ZHENG Yan,HOU Haijun,QIN Hongling,et al. Effect of N application on the abundance of denitrifying genes(nar G/nos Z)and N2O emission in paddy soil[J]. Acta Ecologica Sinica,2012,32(11):3386-3393.
[14]宋亚娜,林智敏,林艳.氮肥对稻田土壤反硝化细菌群落结构和丰度的影响[J].中国生态农业学报,2012,20(1):7-12.SONG Yana,LIN Zhimin,LIN Yan. Response of denitrifying bacteria community structure and abundance to nitrogen in paddy fields[J].Chinese Journal of Eco-Agriculture,2012,20(1):7-12.
[15]刘远,王光利,李恋卿,等.土壤硝化和反硝化微生物群落及活性对大气CO2浓度和温度升高的响应[J].环境科学,2017,38(3):1245-1252.LIU Yuan,WANG Guangli,LI Lianqing,et al. Community and activity to elevated atmospheric CO2concentration and temperature[J].Environmental Science,2017,38(3):1245-1252.
[16]王殳屹,韩琳,史奕,等.开放式大气CO2浓度增高对水稻土反硝化活性的影响[J].浙江大学学报(农业与生命科学版),2007,33(3):284-289.WANG Shuyi,HAN Lin,SHI Yi,et al. Effects of FACE denitrification activity in paddy-field soil[J]. Journal of Zhejiang University(Agricultural and Life Science),2007,33(3):284-289.
[17] OLSEN S R,COLE C V,WATANABE F S,et al. Estimation of available phosphorus in soils by extraction with sodium bicarbonate[M].Washington DC:USDA Circular,1954.
[18] CHEN Z,LUO X,HU R,et al.Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil[J].Microbial Ecology,2010,60(4):850-861.
[19] HALLIN S,LINGDGREN P E. PCR detection of genes encoding nitrite reductase in denitrifying bacteria PCR detection of genes encoding nitrite reductase in denitrifying bacteria[J]. Applied and Environmental Microbiology,1999,65(4):1652-1657.
[20] MICHOTEY V,MEJEAN V,BONIN P.Comparison of methods for quantification of cytochrome, cd1-denitrifying bacteria in environmental marine samples[J]. Applied and Environmental Microbiology,2000,66(4):1564-1571.
[21]杨连新,王云霞,朱建国,等.开放式空气中CO2浓度增高(FACE)对水稻生长和发育的影响[J].生态学报,2010,30(6):1573-1585.YANG Lianxin,WANG Yunxia,ZHU Jianguo,et al. What we have learned from 10 years of free-air CO2enrichment(FACE)experiments on rice? growth and development[J]. Acta Ecologica Sinica,2010,30(6):1573-1585.
[22]谢祖彬,朱建国,张雅丽,等.水稻生长及其体内C、N、P组成对开放式空气CO2浓度增高和N、P施肥的响应[J].应用生态学报,2002,13(10):1223-1230.XIE Zubin,ZHU Jianguo,ZHANG Yali,et al. Responses of rice(Oryza sativa)growth and its C,N and P composition to FACE(Free-air Carbon Dioxide Enrichment)and N,P fertilization[J].Chinese Journal of Applied Ecology,2002,13(10):1223-1230.
[23] ALLEN L H,BOOTE K J,JONES J W,et al.Response of vegetation to rising carbon dioxide:photosynthesis,biomass,and seed yield of soybean[J].Global Biogeochemical Cycles,2012,1(1):1-14.
[24] LAREEN A,BURTON F,SCHAFER P. Plant root-microbe communication in shaping root microbiomes[J]. Plant Molecular Biology,2016,90(6):575-587.
[25] CHAPARRO J M,BADRI D V,VIVANCO J M. Rhizosphere microbiome assemblage is affected by plant development[J]. Isme Journal,2014,8(4):790-803.
[26] KANDELER E,DEIGLMAYR K,TSCHERKO D,et al.Abundance of narG,nir S,nirK,and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland[J]. Applied and Environmental Microbiology,2006,72(9):5957-5962.
[27] STITT M,KRAPP A. The interaction between elevated carbon dioxide and nitrogen nutrition:the physiological and molecular background[J].Plant Cell&Environment,1999,22(6):583-621.
[28] CARNOL M,HOGENBOOM L,JACH M E,et al. Elevated atmospheric CO2in open top chambers increases net nitrification and potential denitrification[J]. Global Change Biology,2010,8(6):590-598.
[29] KAISER E A,KOHRS K,KUCKE M,et al. Nitrous oxide release from arable soil:importance of N-fertilization,crops and temporal variation[J].Soil Biology and Biochemistry,1998,30(12):1553-1563.
[30] KIM S,DALE B E. Effects of nitrogen fertilizer application on greenhouse gas emissions and economics of corn production[J].Environmental Science&Technology,2008,42(16):6028-6033.
[31]高嵩涓,曹卫东,白金顺,等.湘南红壤稻田AOA-amo A、narG、nosZ基因丰度及其环境影响因子[J].中国土壤与肥料,2017(1):21-27.GAO Songjuan,CAO Weidong,BAI Jinshun,et al. Abundances of AOA-amoA,narG,nosZ genes and their relationships with soil environment factors in red paddy soils in south Hunan Province[J].Soil and Fertilizer Science in China,2017(1):21-27.
[32] JONES C M,STRES B,ROSENQUIST M,et al. Phylogenetic analysis of nitrite,nitric oxide,and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification[J].Molecular Biology and Evolution,2008,25(9):1955-1966.
[33]陈哲.长期施肥对水稻土反硝化作用和反硝化功能微生物的影响机理[D].北京:中国科学院研究生院,2010.
[34] YOSHIDA M,ISHII S,OTSUKA S,et al. Temporal shifts in diversity and quantity of nirS and nir K in a rice paddy field soil[J].Soil Biology and Biochemistry,2009,41(10):2044-2051.
[35] YOSHIDA M,ISHII S,OTSUKA S,et al.nirK-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.