黄河三角洲不同盐碱农田生态系统中氮循环功能菌群研究
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摘要
采用Illumina Miseq对黄河三角洲盐碱农田5种典型农作物种植体系土壤中的氨氧化(amoA)和反硝化基因(nirS/nirK)进行测序,研究参与其氨氧化和反硝化过程的功能菌群落结构和多样性。结果表明,水稻土壤中的氨氧化和反硝化菌群落结构和多样性与其他4种农田土壤差异显著;土壤电导率、含水率及有效磷是造成群落结构差异的主要因子。另外,对不同作物种植体系中氨氧化和反硝化过程的优势菌研究表明,在大豆和小麦-玉米轮作中,AOB起主要的氨氧化作用,而在水稻土中是AOA; nirS和nirK型反硝化菌在水稻和大豆农田生态系统中起主要的反硝化作用,但是在不同农田生态系统中其优势菌明显不同。
The present paper is inclined to make an analysis of the α-diversity of the sequencial results as well as that of a nonmetric multi-dimensional scale( NMDS) β-diversity by using the MRPP function of the "Vegan" package in R( Ⅴ3. 5. 0). In addition,we have also toorder and sequence the ammonia oxidation( amoA) and denitrification genes( nirS/nirK) in the soil of the 5 typical agro-ecosystems in the aforementioned farmlands of the Yellow River estruarydelta,by using the Mothur( Ⅴ1. 39. 5) Illumina Miseq. What is more,we have also traced the influence of the soil factors on the structure differences of N-cycling functional microbial communities based on the sequential data and the soil 's physic-chemical properties through the redundancy analysis( RDA). At the same time,we have also analyzed the dominant microorganisms involved in the ammonia-oxidation and denitrification in the different agro-ecosystems through their main specific components. The results of our diversity identification prove that the greatest diversity of the ammonia-oxidizing microorganisms and denitrifying bacteria has been found in the rice planting fields. And,then,the sequential analysis results of α-diversity and β-diversity as well as that of the β-diversity in the non-metric multidimensional scale( NMDS) have been found significantly higher than the other 3 crops( p < 0. 05),that is,just a bit less seriously in the wheat-maize recycling growing soils. However,the community composition of the ammonia-oxidizing microorganisms and the denitrifying bacteria in the rice planting field tend to be quite different from that in other cropsgrowing soils. The RDA testing results have verified that RDA1 and RDA2 may account for 54. 12% and 43. 88% of differences in the community structure of the ammonia-oxidizing and denitrifying microorganisms in the soils of the different agro-ecosystems,respectively. Or,specifically speaking,the soil's electric conductivity rate,the moisture content rate and the available phosphorus content may all have their own significant effects on the composition of the ammonia-oxidizing and denitrifying microorganisms of the soils of different agro-ecosystems( p < 0. 05).Among them,most important influential factor that should be stressed is its electrical conductivity,which may account for 22. 1% and 17. 5% of the differences( p < 0. 01). The dominant microorganisms in the ammonia oxidation and the denitrification process in the different agro-ecosystems can verify that AOB has been playing a major role in ammonia oxidation in the soybean and wheat-maize rotated-growing system,whereas AOA—a major role in the paddy soil. As to the nirS-and nirK-type denitrifying bacteria,they prefer to play main role in the rice and soybean planting fields,though the dominant bacteria tend to be significantly different in the said 2 systems.
The present paper is inclined to make an analysis of the α-diversity of the sequencial results as well as that of a nonmetric multi-dimensional scale( NMDS) β-diversity by using the MRPP function of the "Vegan" package in R( Ⅴ3. 5. 0). In addition,we have also toorder and sequence the ammonia oxidation( amoA) and denitrification genes( nirS/nirK) in the soil of the 5 typical agro-ecosystems in the aforementioned farmlands of the Yellow River estruarydelta,by using the Mothur( Ⅴ1. 39. 5) Illumina Miseq. What is more,we have also traced the influence of the soil factors on the structure differences of N-cycling functional microbial communities based on the sequential data and the soil 's physic-chemical properties through the redundancy analysis( RDA). At the same time,we have also analyzed the dominant microorganisms involved in the ammonia-oxidation and denitrification in the different agro-ecosystems through their main specific components. The results of our diversity identification prove that the greatest diversity of the ammonia-oxidizing microorganisms and denitrifying bacteria has been found in the rice planting fields. And,then,the sequential analysis results of α-diversity and β-diversity as well as that of the β-diversity in the non-metric multidimensional scale( NMDS) have been found significantly higher than the other 3 crops( p < 0. 05),that is,just a bit less seriously in the wheat-maize recycling growing soils. However,the community composition of the ammonia-oxidizing microorganisms and the denitrifying bacteria in the rice planting field tend to be quite different from that in other cropsgrowing soils. The RDA testing results have verified that RDA1 and RDA2 may account for 54. 12% and 43. 88% of differences in the community structure of the ammonia-oxidizing and denitrifying microorganisms in the soils of the different agro-ecosystems,respectively. Or,specifically speaking,the soil's electric conductivity rate,the moisture content rate and the available phosphorus content may all have their own significant effects on the composition of the ammonia-oxidizing and denitrifying microorganisms of the soils of different agro-ecosystems( p < 0. 05).Among them,most important influential factor that should be stressed is its electrical conductivity,which may account for 22. 1% and 17. 5% of the differences( p < 0. 01). The dominant microorganisms in the ammonia oxidation and the denitrification process in the different agro-ecosystems can verify that AOB has been playing a major role in ammonia oxidation in the soybean and wheat-maize rotated-growing system,whereas AOA—a major role in the paddy soil. As to the nirS-and nirK-type denitrifying bacteria,they prefer to play main role in the rice and soybean planting fields,though the dominant bacteria tend to be significantly different in the said 2 systems.
引文
[1] WANG Jingguo,LIN Shan,LI Baoguo. Nitrogen cycling and management strategies in Chinese agriculture[J].Scientia Agricultura Sinica,2016(3):503-517.
[2] HOU H J,QIN H L,CHEN C L,et al. Research progress of the molecular ecology on microbiological processes in soil nitrogen cycling[J]. Research of Agricultural Modernization,2014,35(5):588-594.
[3] WARD B B,JENSEN M M. The microbial nitrogen cycle[J]. Environmental Microbiology, 2008, 10(11):2903-2909.
[4] VENTER J C,REMINQTON K,HEIDELBERG J F,et al. Environmental genome shotgun sequencing of the Sargasso Sea[J]. Science,2013,304:66-74.
[5] YERGEAU E,HOGUES H,WHYTE L G,et al. The functional potential of high Arctic permafrost revealed by metagenomic sequencing,q PCR and microarray analyses[J]. Isme Journal,2010,4(9):1206-1214.
[6] DAIMS H,LEBEDEVA E V,PJEVAC P,et al. Complete nitrification by Nitrospira bacteria[J]. Nature,2015,528:504–509.
[7] LU Rukun(鲁如坤). Analysis method of soil agricultural chemistry(土壤农化分析)[M]. Beijing:Chinese Agricultural Science and Technology Press,2000.
[8] DING K,WEN X,LI Y,et al. Ammonia-oxidizing archaea versus bacteria in two soil aquifer treatment systems[J]. Applied Microbiology and Biotechnology,2015,99(3):1337-1347.
[9] SCHLOSS P D,WESTCOTT S L,RYABIN T,et al. Introducing mothur:open-source, platform-independent,community-supported software for describing and comparing microbial communities[J]. Applied and Environmental Microbiology,2009,75(23):7537-7541.
[10] BROCKETT B F T,PRESOTT C E,GRARYSTON S J.Soil moisture is the major factor influencing microbial community structure and enzyme activities across seven biogeoclimatic zones in western Canada[J]. Soil Biology&Biochemistry,2012,44(1):9-20.
[11] HEIJDEN M G A V D,BARDGETT R D,STRAALEN N M V. The unseen majority:soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems[J]. Ecology Letters,2008,11(3):296-310.
[12] CHU H,FIERER N,LAUBER C L,et al. Soil bacterial diversity in the Arctic is not fundamentally different from that found in other biomes[J]. Environmental microbiology,2010,12(11):2998-3006.
[13] MASSENSSINI,ANDRè M,BONDUKI V H A,et al.Relative importance of soil physico-chemical characteristics and plant species identity to the determination of soil microbial community structure[J]. Applied Soil Ecology,2015,91:8-15.
[14] HUANG K,HUI X,CUI G,et al. Effects of earthworms on nitrification and ammonia oxidizers in vermicomposting systems for recycling of fruit and vegetable wastes[J]. Science of the Total Environment,2016,578:337-345.
[15] PURCHASE B S. The influence of phosphate deficiency on nitrification[J]. Plant Soil,1974,41(3):541-547.
[16] HE L,BI Y,ZHAO J,et al. Population and community structure shifts of ammonia oxidizers after four-year successive biochar application to agricultural acidic and alkaline soils[J]. Science of the Total Environment,2018,619/620:1105-1115.
[17] KOWALCHUK G A,STIENSTRA A W,HEILIG G H J,et al. Molecular analysis of ammonia-oxidizing bacteria in soil of successional grasslands of the Drentsche A(The Netherlands)[J]. FEMS Microbiology Ecology,2000,31(3):207-215.
[18] HAMMER M. Water and wastewater technology:United States edition[M]. New York:Pearson Education Inc,2003.
[19] DHALIWAL S S,TOOR G S,RODRIGUEZ-JORQUERA I A,et al. Trace metals in the soils of water conservation area of Florida everglades:considerations for ecosystem restoration[J]. Journal of Soils and Sediments,2018,18(2):342-351.
[20] HIORNS W D,HASTINGS R C,HEAD I M,et al.Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment[J]. Microbiology,1995,141(11):2793-2800.
[21] ATHERTON P,MAHNE S,TIEDJE T,et al. Effects of p H and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater[J]. Applied&Environmental Microbiology,1998,64(10):3584-3590.
[22] PAUL E A. Soil microbiology,ecology and biochemistry[M]. New York:Academic Press,2014.
[23] FAULWETTER J L,GAGNON V,SUNDBERG C,et al. Microbial processes influencing performance of treatment wetlands:a review[J]. Ecological Engineering,2009,35(6):987-1004.
[24] LIU Ruoxuan(刘若萱), HE Jizheng(贺纪正),ZHANG Limei(张丽梅). Response of nitrification/denitrification and their associated microbes to soil moisture change in paddy soil[J]. Environmental Science(环境科学),2014,1(11):4275-4283.
[25] BEHRENDT U,K?MPFER P,GLAESER S P,et al.Characterisation of the N2O producing soil bacterium Rhizobium azooxidifex sp. nov[J]. International Journal of Systematic and Evolutionary Microbiology,2016,66(6):2354-2361.
[26] YOSHIDA M,ISHII S,OTSUKA S,et al. Temporal shifts in diversity and quantity of nir S and nir K in a rice paddy field soil[J]. Soil Biology&Biochemistry,2009,41(10):2044-2051.
[27] YU Z,LIU J,LI Y,et al. Impact of land use,fertilization and seasonal variation on the abundance and diversity of nir S-type denitrifying bacterial communities in a mollisol in northeast China[J]. European Journal of Soil Biology,2018,85:4-11.
[28] DANDIE C E,MILLER M N,BURTON D L,et al. Nitric oxide reductase-targeted real-time PCR quantification of denitrifier populations in soil[J]. Applied and Environmental Microbiology,2007,73(13):4250-4258.
[2] HOU H J,QIN H L,CHEN C L,et al. Research progress of the molecular ecology on microbiological processes in soil nitrogen cycling[J]. Research of Agricultural Modernization,2014,35(5):588-594.
[3] WARD B B,JENSEN M M. The microbial nitrogen cycle[J]. Environmental Microbiology, 2008, 10(11):2903-2909.
[4] VENTER J C,REMINQTON K,HEIDELBERG J F,et al. Environmental genome shotgun sequencing of the Sargasso Sea[J]. Science,2013,304:66-74.
[5] YERGEAU E,HOGUES H,WHYTE L G,et al. The functional potential of high Arctic permafrost revealed by metagenomic sequencing,q PCR and microarray analyses[J]. Isme Journal,2010,4(9):1206-1214.
[6] DAIMS H,LEBEDEVA E V,PJEVAC P,et al. Complete nitrification by Nitrospira bacteria[J]. Nature,2015,528:504–509.
[7] LU Rukun(鲁如坤). Analysis method of soil agricultural chemistry(土壤农化分析)[M]. Beijing:Chinese Agricultural Science and Technology Press,2000.
[8] DING K,WEN X,LI Y,et al. Ammonia-oxidizing archaea versus bacteria in two soil aquifer treatment systems[J]. Applied Microbiology and Biotechnology,2015,99(3):1337-1347.
[9] SCHLOSS P D,WESTCOTT S L,RYABIN T,et al. Introducing mothur:open-source, platform-independent,community-supported software for describing and comparing microbial communities[J]. Applied and Environmental Microbiology,2009,75(23):7537-7541.
[10] BROCKETT B F T,PRESOTT C E,GRARYSTON S J.Soil moisture is the major factor influencing microbial community structure and enzyme activities across seven biogeoclimatic zones in western Canada[J]. Soil Biology&Biochemistry,2012,44(1):9-20.
[11] HEIJDEN M G A V D,BARDGETT R D,STRAALEN N M V. The unseen majority:soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems[J]. Ecology Letters,2008,11(3):296-310.
[12] CHU H,FIERER N,LAUBER C L,et al. Soil bacterial diversity in the Arctic is not fundamentally different from that found in other biomes[J]. Environmental microbiology,2010,12(11):2998-3006.
[13] MASSENSSINI,ANDRè M,BONDUKI V H A,et al.Relative importance of soil physico-chemical characteristics and plant species identity to the determination of soil microbial community structure[J]. Applied Soil Ecology,2015,91:8-15.
[14] HUANG K,HUI X,CUI G,et al. Effects of earthworms on nitrification and ammonia oxidizers in vermicomposting systems for recycling of fruit and vegetable wastes[J]. Science of the Total Environment,2016,578:337-345.
[15] PURCHASE B S. The influence of phosphate deficiency on nitrification[J]. Plant Soil,1974,41(3):541-547.
[16] HE L,BI Y,ZHAO J,et al. Population and community structure shifts of ammonia oxidizers after four-year successive biochar application to agricultural acidic and alkaline soils[J]. Science of the Total Environment,2018,619/620:1105-1115.
[17] KOWALCHUK G A,STIENSTRA A W,HEILIG G H J,et al. Molecular analysis of ammonia-oxidizing bacteria in soil of successional grasslands of the Drentsche A(The Netherlands)[J]. FEMS Microbiology Ecology,2000,31(3):207-215.
[18] HAMMER M. Water and wastewater technology:United States edition[M]. New York:Pearson Education Inc,2003.
[19] DHALIWAL S S,TOOR G S,RODRIGUEZ-JORQUERA I A,et al. Trace metals in the soils of water conservation area of Florida everglades:considerations for ecosystem restoration[J]. Journal of Soils and Sediments,2018,18(2):342-351.
[20] HIORNS W D,HASTINGS R C,HEAD I M,et al.Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment[J]. Microbiology,1995,141(11):2793-2800.
[21] ATHERTON P,MAHNE S,TIEDJE T,et al. Effects of p H and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater[J]. Applied&Environmental Microbiology,1998,64(10):3584-3590.
[22] PAUL E A. Soil microbiology,ecology and biochemistry[M]. New York:Academic Press,2014.
[23] FAULWETTER J L,GAGNON V,SUNDBERG C,et al. Microbial processes influencing performance of treatment wetlands:a review[J]. Ecological Engineering,2009,35(6):987-1004.
[24] LIU Ruoxuan(刘若萱), HE Jizheng(贺纪正),ZHANG Limei(张丽梅). Response of nitrification/denitrification and their associated microbes to soil moisture change in paddy soil[J]. Environmental Science(环境科学),2014,1(11):4275-4283.
[25] BEHRENDT U,K?MPFER P,GLAESER S P,et al.Characterisation of the N2O producing soil bacterium Rhizobium azooxidifex sp. nov[J]. International Journal of Systematic and Evolutionary Microbiology,2016,66(6):2354-2361.
[26] YOSHIDA M,ISHII S,OTSUKA S,et al. Temporal shifts in diversity and quantity of nir S and nir K in a rice paddy field soil[J]. Soil Biology&Biochemistry,2009,41(10):2044-2051.
[27] YU Z,LIU J,LI Y,et al. Impact of land use,fertilization and seasonal variation on the abundance and diversity of nir S-type denitrifying bacterial communities in a mollisol in northeast China[J]. European Journal of Soil Biology,2018,85:4-11.
[28] DANDIE C E,MILLER M N,BURTON D L,et al. Nitric oxide reductase-targeted real-time PCR quantification of denitrifier populations in soil[J]. Applied and Environmental Microbiology,2007,73(13):4250-4258.