旱地作物根际和非根际土壤硝酸盐异化还原成铵细菌群落组成的研究
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摘要
为研究典型旱地农田土壤硝酸盐异化还原成铵过程(Dissimilatory nitrate reduction to ammonium,DNRA)的群落组成,针对DNRA过程的功能基因nrfA进行高通量测序.根际和非根际、4种典型农作物共16个样品,质控后每个样品得到87000条序列,在相似度≥90%下划分到27952个OTUs,选取其中丰度较高的258个代表OTUs进行生态学分析.多样性分析(OTUs水平)结果表明:3/4的作物根际土壤样品中的DNRA群落丰富度、物种多样性和物种均匀度高于相应非根际样品,对比4种作物,粟作物根部土壤DNRA群落多样性最高,玉米作物非根际土壤最低.对代表OTUs进行分类,共定义到6个门(Phylum),19个属(Genus).其中相对丰度最高的3个属为Hyalangium(29.31%)、Chthoniobacter(20.33%)和Nitrospira(13.41%),表明三者在群落组成中占主导地位.结合土壤理化因子分析,DNRA群落相对丰度与NO~-_2-N、TN、含水率、TOM、pH及温度呈显著相关关系.本研究在一定程度上揭示了旱地农田土壤DNRA细菌的群落组成、多样性及与土壤环境因子的关系,为提高氮肥的利用效率和减小环境污染提供理论依据.
To explore the community composition of dissimilatory nitrate reduction to ammonium(DNRA) process in typical agricultural dryland soil, high-throughput sequencing targeting on the functional gene nrfA of DNRA bacteria was carried out. A total of 16 samples of rhizosphere and non-rhizosphere soils from 4 typical agricultural lands were collected, and 87000 sequences were obtained for each sample after quality control. Then these sequences were clustered to 27952 OTUs with similarity above 90%, of which 258 representative OTUs with higher abundance were selected for community analysis. Results of diversity analysis(OTUs level) demonstrated that 3/4 of the samples in rhizosphere soil had a higher diversity, richness, and species uniformity of DNRA community than those in the corresponding non-rhizosphere soils. Moreover, it was found that rhizosphere soil of millet crops had the highest diversity of DNRA community while non-rhizosphere soil of maize crops had the lowest one among the investigated crops. Furthermore, the representative OTUs was classified into 6 phyla and 19 genera, and, the three highest richness genera were Hyalangium(29.31%), Chthoniobacter(20.33%) and Nitrospira(13.41%), indicating their dominant role in DNRA community composition. Combined with soil physicochemical factor analysis, the relative abundance of DNRA community was significantly correlated with NO~-_2-N, TN, moisture content, TOM, pH and temperature in the four crops. To some extent, this study revealed the community composition, diversity of DNRA bacteria, and their relationship with soil environmental factors in agricultural dryland soil, providing theoretical basis for improving the utilization efficiency of nitrogen fertilizer and reducing environmental pollution.
To explore the community composition of dissimilatory nitrate reduction to ammonium(DNRA) process in typical agricultural dryland soil, high-throughput sequencing targeting on the functional gene nrfA of DNRA bacteria was carried out. A total of 16 samples of rhizosphere and non-rhizosphere soils from 4 typical agricultural lands were collected, and 87000 sequences were obtained for each sample after quality control. Then these sequences were clustered to 27952 OTUs with similarity above 90%, of which 258 representative OTUs with higher abundance were selected for community analysis. Results of diversity analysis(OTUs level) demonstrated that 3/4 of the samples in rhizosphere soil had a higher diversity, richness, and species uniformity of DNRA community than those in the corresponding non-rhizosphere soils. Moreover, it was found that rhizosphere soil of millet crops had the highest diversity of DNRA community while non-rhizosphere soil of maize crops had the lowest one among the investigated crops. Furthermore, the representative OTUs was classified into 6 phyla and 19 genera, and, the three highest richness genera were Hyalangium(29.31%), Chthoniobacter(20.33%) and Nitrospira(13.41%), indicating their dominant role in DNRA community composition. Combined with soil physicochemical factor analysis, the relative abundance of DNRA community was significantly correlated with NO~-_2-N, TN, moisture content, TOM, pH and temperature in the four crops. To some extent, this study revealed the community composition, diversity of DNRA bacteria, and their relationship with soil environmental factors in agricultural dryland soil, providing theoretical basis for improving the utilization efficiency of nitrogen fertilizer and reducing environmental pollution.
引文
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Deng Y, Jiang Y H, Yang Y, et al. 2012. Molecular ecological network analyses[J]. BMC Bioinformatics, 13(1):113
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Uldahl A. 2011. Alternative nitrate reduction pathways in experimentally fertilized new england salt marshes: removal versus recycling of biologically available N[C]. 21st Biennial Conference of the Coastal and Estuarine Research Federation
Ushiki N, Fujitani H, Shimada Y, et al. 2017. Genomic Analysis of Two Phylogenetically Distinct Nitrospira Species Reveals Their Genomic Plasticity and Functional Diversity[J]. Frontiers in Microbiology, 8:1-7
Wang B Z, Zhao J, Guo Z Y, et al. 2015. Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils[J]. The ISME Journal, 9:1062-1075
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Bergmann G T, Bates S T, Eilers K G, et al. 2011. The under-recognized dominance of Verrucomicrobia in soil bacterial communities[J]. Soil Biology & Biochemistry, 43(7):1450-1455
Blackmer A M, Bremner J M. 1978. Inhibitory effect of nitrate on reduction of N2O to N2 by soil microorganisms[J]. Soil Biology and Biochemistry, 10(3):187-191
Bonin P. 1996. Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: a dissimilatory pathway[J]. FEMS Microbiology Ecology, 19(1):27-38
Deng Y, Jiang Y H, Yang Y, et al. 2012. Molecular ecological network analyses[J]. BMC Bioinformatics, 13(1):113
都韶婷, 章永松, 林咸永, 等. 2007. 蔬菜积累的硝酸盐及其对人体健康的影响[J]. 中国农业科学, 40(9):2007-2014
Edgar R C. 2010. Search and clustering orders of magnitude faster than blast[J]. Bioinformatics, 26(19):2460
范晓晖, 刘芷宇. 1992. 根际pH环境与磷素利用研究进展[J]. 土壤通报, 23(5):238-240
傅利剑, 郭丹钊, 史春龙, 等. 2005. 碳源及碳氮比对异养反硝化微生物异养反硝化作用的影响[J]. 生态与农村环境学报, 21(2):42-45
Guo G X, Deng H, Qiao M, et al. 2013. Effect of long-term wastewaterirrigation on potential denitrification and denitrifying communitiesin soils at the watershed scale[J]. Environmental Science&Technology, 47(7):3105-3113
Güven D, Dapena A, Kartal B, et al. 2005. Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria[J]. Applied & Environmental Microbiology, 71(2):1066-1071
韩江培. 2015. 设施栽培条件下±壤酸化与盐渍化耦合发生机理研[M]. 杭州:浙江大学
贺纪正, 张丽梅. 2013. 土壤氮素转化的关键微生物过程及机制[J]. 微生物学通报, 40(1):98-108
Houba V J G, Novozamsky I, Huybregts A W M, et al. 1986. Comparison of soil extractions by 0.01 M, CaCl2, by EUF and by some conventional extraction procedures[J]. Plant & Soil, 96(3):433-437
刘佳. 2007. 微生物好氧硝酸盐还原产铵研究[D]. 成都: 四川大学
蒋自立. 1991. 农作物积累硝酸盐的农业生态因素[J]. 农业环境与发展, 1:24-26
J?rgensen K S, Jensen H B, S?rensen J. 1984. Nitrous oxide production from nitrification and denitrification in marine sediment at low oxygen concentrations[J]. Canadian Journal of Microbiology, 30(8):1073-1078
Kant R, Passel M W J V, Palva A, et al. 2011. Genome sequence of Chthoniobacter flavus Ellin428, an aerobic heterotrophic soil bacterium[J]. Journal of Bacteriology, 193(11):2902-2903
Kartal B, Kuypers M M, Lavik G, et al. 2007. Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium[J]. Environmental Microbiology, 9(3):635-642
Kraft B, Strous M, Tegetmeyer H E. 2011. Microbial nitrate respiration--genes, enzymes and environmental distribution[J]. Journal of Biotechnology, 155(1):104-117
刘文国, 范学科, 马安良. 2002. 植物体对氮吸收和同化过程的研究进展[J]. 杨凌职业技术学院学报, (2):17-19
Lu W W, Zhang H L, Shi W M. 2013. Dissimilatory nitrate reduction to ammonium in an anaerobic agricultural soil as affected by glucose and free sulfide[J]. European Journal of Soil Biology, 58(58):98-104
Lundberg D S, Lebeis S L, Paredes S H, et al. 2012 Defining the core Arabidopsis thaliana root microbiome[J]. Nature, 488(7409):86
马冬云, 郭天财, 宋晓, 等. 2007. 尿素施用量对小麦根际土壤微生物数量及土壤酶活性的影响[J]. 生态学报, 27(12):5222-5228
Maag M, Vinther F P. 1996. Nitrous oxide emission by nitrification and denitrification in different soil types and at different soil moisture contents and temperatures[J]. Applied Soil Ecology, 4(1):5-14
Mroz G D, Reed D D. 1991. Forest soil sampling efficiency: Matching laboratory analyses and field sampling procedures[J]. Soil Science Society of America Journal, 55(5):1413-1416
Müller M M, Sundman V, Skujin? J. 1980. Denitrification in low pH spodosols and peats determined with the acetylene inhibition method[J]. Applied and Environmental Microbiology, 40(2):235-239
Ogilvie B G, Rutter M, Nedwell D B. 1997. Selection by temperature of nitrate-reducing bacteria from estuarine sediments: species composition and competition for nitrate[J]. FEMS Microbiology Ecology, 23(1):11-22
Patra A K, Abbadie L, Clays-Josserand A, et al. 2006. Effects ofmanagement regime and plant species on the enzyme activity andgenetic structure of N-fixing, denitrifying and nitrifying bacterialcommunities in grassland soils[J]. Environmental Microbiology, 8(6):1005-1016
Rehr B, Klemme J H. 1989. Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii, and competition with denitrifying bacteria[J]. Antonie Van Leeuwenhoek, 56(4):311-321
Sangwan P, Chen X, Hugenholtz P, et al. 2004. Chthoniobacter flavus gen. nov. sp nov. the first pure-culture representative of subdivision two, Spartobacteria classis nov. of the phylum Verrucomicrobia[J]. Applied & Environmental Microbiology, 70(10):5875-5881
Senga Y, Mochida K, Fukumori R, et al. 2006. N2O accumulation in estuarine and coastal sediments: The influence of H2S on dissimilatory nitrate reduction[J]. Estuarine, Coastal and Shelf Science, 67(1/2):231-238
Schloss P D. 2009. A high-throughput DNA sequence aligner for microbial ecology studies[J]. Plos One, 4(12):e8230
Sher Y, Schneider K, Schwermer C U, et al. 2008. Sulfide-induced nitrate reduction in the sludge of an anaerobic digester of a zero-discharge recirculating mariculture system[J]. Water Research, 42(16):4386-4392
Shu D, He Y, Yue H, et al. 2015. Metagenomic insights into the effects of volatile fatty acids on microbial community structures and functional genes in organotrophic anammox process[J]. Bioresource Technology, 196:621-633
Smalla K, Wieland G, Buchner A, et al. 2001. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed[J]. Applied and Environmental Microbiology, 67(10): 4742-4751
Song B, Lisa J A, Tobias C R. 2014. Linking DNRA community structure and activity in a shallow lagoonal estuarine system[J]. Frontiers in Microbiology, 5:460
Steenkamp D J, Peck H D Jr. 1981. Proton translocation associated with nitrite respiration in Desulfovibrio desulfuricans[J]. The Journal of Biological Chemistry, 256(11):5450-5458
Su Y, Wang W, Wu D, et al. 2018. Stimulating ammonia oxidizing bacteria (AOB) activity drives the ammonium oxidation rate in a constructed wetland (CW)[J]. Science of the Total Environment, 624: 87-95
孙羲. 1997. 植物营养原理, 农学, 园艺, 桑茶等专业用[M]. 北京:中国农业出版社
Tiedje J M, Sexstone A J, Myrold D D, et al. 1983. Denitrification: ecological niches, competition and survival[J]. Antonie van Leeuwenhoek, 48(6):569-583
Thomas E F, Lisa C H, Christpher D C, et al. 2005. Influence of inorganic nitrogen management regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils[J]. Applied & Environmental Microbiology, 71:8323-8334
Uldahl A. 2011. Alternative nitrate reduction pathways in experimentally fertilized new england salt marshes: removal versus recycling of biologically available N[C]. 21st Biennial Conference of the Coastal and Estuarine Research Federation
Ushiki N, Fujitani H, Shimada Y, et al. 2017. Genomic Analysis of Two Phylogenetically Distinct Nitrospira Species Reveals Their Genomic Plasticity and Functional Diversity[J]. Frontiers in Microbiology, 8:1-7
Wang B Z, Zhao J, Guo Z Y, et al. 2015. Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils[J]. The ISME Journal, 9:1062-1075
Wang S, Wang W, Liu L, et al. 2018. Microbial nitrogen cycle hotspots in the plant-bed/ditch system of a constructed wetland with N2O mitigation[J]. Environmental Science & Technology, 52(11):6226-6236
韦宗敏. 2012. 微好氧环境中硝酸盐异化还原成铵的影响研究[D]. 广州:华南理工大学
Welsh A, Chee-Sanford J C, Connor L M, et al. 2014. Refined nrfA phylogeny improves PCR-based nrfA gene detection[J]. Applied & Environmental Microbiology, 80(7):2110-2119
Wood G A, Taylor J C, Godwin R J. 2003. Calibration Methodology for Mapping Within-field Crop Variability using Remote Sensing[J]. Biosystems Engineering, 84(4):409-423
Yamulki S, Harrison R M, Goulding K W T, et al. 1997. N2O, NO and NO2 fluxes from a grassland: effect of soil pH[J]. Soil Biology and Biochemistry, 29(8):1199-1208
雍太文, 杨文钰, 向达兵, 等. 2012. 不同种植模式对作物根系生长、产量及根际土壤微生物数量的影响[J]. 应用生态学报, 23(1):125-132
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