Journal of Soils and Sediments

, Volume 19, Issue 2, pp 872–882 | Cite as

Nitrapyrin affects the abundance of ammonia oxidizers rather than community structure in a yellow clay paddy soil

  • Yan Gu
  • Wenhai Mi
  • Yinan Xie
  • Qingxu Ma
  • Lianghuan WuEmail author
  • Zhaoping Hu
  • Feng Dai
Soils, Sec 5 • Soil and Landscape Ecology • Research Article



Yellow clay paddy soil (Oxisols) is a low-yield soil with low nitrogen use efficiency (NUE) in southern China. The nitrification inhibitor nitrapyrin (2-chloro-6- (tricholoromethyl)-pyridine, CP) has been applied to improve NUE and reduce environmental pollution in paddy soil. However, the effects of nitrapyrin combined with nitrogen fertilizers on ammonia oxidizers in yellow clay paddy soil have not been examined.

Materials and methods

A randomized complete block design was set with three treatments: (1) without nitrogen fertilizer (CK), (2) common prilled urea (PU), and (3) prilled urea with nitrapyrin (NPU). Soil samples were collected from three treatments where CK, PU, and NPU had been repeatedly applied over 5 years. Soil samples were analyzed by quantitative PCR and 454 high-throughput pyrosequencing of the amoA gene to investigate the influence of nitrapyrin combined with nitrogen on the abundance and community structure of ammonia oxidizers in yellow clay paddy soil.

Results and discussion

The potential nitrification rate (PNR) of the soil was significantly correlated with the abundances of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Application of urea significantly stimulated AOA and AOB growth, whereas nitrapyrin exhibited inhibitory effects on AOA. Phylogenetic analysis showed that the most dominant operational taxonomic units (OTUs) of AOA and AOB were affiliated with the Nitrosotalea cluster and Nitrosospira cluster 12, respectively. AOA and AOB community structures were not altered by urea and nitrapyrin application.


Nitrogen fertilization stimulated nitrification and increased the population sizes of AOA and AOB. Nitrapyrin affected the abundance, but not community structure of ammonia oxidizers in yellow clay soil. Our results suggested that nitrapyrin improving NUE and inhibiting PNR was attributable to the inhibition of AOA growth.


454 pyrosequencing Ammonia-oxidizing archaea Ammonia-oxidizing bacteria Nitrapyrin Paddy soil 



We would like to thank Editage for English language editing.


This work was supported by the National Key Research and Development Program of China (2016YFD0200102), the National Key Basic Research Program of China (2015CB150502), and the Key Research And Development Program of Zhejiang Province (2015C03011).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Ai C, Liang G, Sun J, Wang X, He P, Zhou W (2013) Different roles of rhizosphere effect and long-term fertilization in the activity and community structure of ammonia oxidizers in a calcareous fluvo-aquic soil. Soil Biol Biochem 57:30–42CrossRefGoogle Scholar
  2. Arp DJ, Sayavedra-Soto LA, Hommes NG (2002) Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. Arch Microbiol 178:250–255CrossRefGoogle Scholar
  3. Berg C, Vandieken V, Thamdrup B, Jürgens K (2015) Significance of archaeal nitrification in hypoxic waters of the Baltic Sea. ISME J 9:1319–1332CrossRefGoogle Scholar
  4. Bierman PM, Rosen CJ, Venterea RT, Lamb JA (2012) Survey of nitrogen fertilizer use on corn in Minnesota. Agric Syst 109:43–52CrossRefGoogle Scholar
  5. Brookes P, Landman A, Pruden G, Jenkinson D (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem 17:837–842CrossRefGoogle Scholar
  6. Burton SAQ, Prosser JI (2001) Autotrophic Ammonia oxidation at low pH through urea hydrolysis. Appl Environ Microbiol 67:2952–2957CrossRefGoogle Scholar
  7. Burzaco JP, Ciampitti IA, Vyn TJ (2014) Nitrapyrin impacts on maize yield and nitrogen use efficiency with spring-applied nitrogen: field studies vs. meta-analysis comparison. Agron J 106:753CrossRefGoogle Scholar
  8. Cantú RR, Aita C, Doneda A, Giacomini DA, Dessbesell A, Arenhardt M, de Bastiani GG, Pujol SB, Rochette P, Chantigny MH, Giacomini SJ (2017) Alternatives to regular urea for abating N losses in lettuce production under sub-tropical climate. Biol Fertil Soils 53:589–599CrossRefGoogle Scholar
  9. Chen D, Suter H, Islam A, Edis R, Freney JR, Walker CN (2008) Prospects of improving efficiency of fertiliser nitrogen in Australian agriculture: a review of enhanced efficiency fertilisers. Soil Res 46:289–301CrossRefGoogle Scholar
  10. Chen X, Zhang L-M, Shen J-P, Wei W-X, He J-Z (2011) Abundance and community structure of ammonia-oxidizing archaea and bacteria in an acid paddy soil. Biol Fertil Soils 47:323–331CrossRefGoogle Scholar
  11. Chen Y, Xu Z, Hu H, Hu Y, Hao Z, Jiang Y, Chen B (2013) Responses of ammonia-oxidizing bacteria and archaea to nitrogen fertilization and precipitation increment in a typical temperate steppe in Inner Mongolia. Appl Soil Ecol 68:36–45CrossRefGoogle Scholar
  12. Chen QH, Qi LY, Bi QF, Dai PB, Sun DS, Sun CL, Liu WJ, Lu LL, Ni WZ, Lin XY (2015) Comparative effects of 3,4-dimethylpyrazole phosphate (DMPP) and dicyandiamide (DCD) on ammonia-oxidizing bacteria and archaea in a vegetable soil. Appl Microbiol Biotechnol 99:477–487CrossRefGoogle Scholar
  13. Chu HY, Morimoto S, Fujii T, Yagi K, Nishimura S (2009) Soil ammonia-oxidizing bacterial communities in paddy rice fields as affected by upland conversion history. Soil Sci Soc Am J 73:1–6CrossRefGoogle Scholar
  14. Cui PY, Fan FL, Yin C, Li ZJ, Song AL, Wan YF, Liang YC (2013) Urea- and nitrapyrin-affected N2O emission is coupled mainly with ammonia oxidizing bacteria growth in microcosms of three typical Chinese arable soils. Soil Biol Biochem 66:214–221CrossRefGoogle Scholar
  15. Di HJ, Cameron KC (2005) Reducing environmental impacts of agriculture by using a fine particle suspension nitrification inhibitor to decrease nitrate leaching from grazed pastures. Agric Ecosyst Environ 109:202–212CrossRefGoogle Scholar
  16. Faeflen SJ, Li S, Xin X, Wright AL, Jiang X (2016) Autotrophic and heterotrophic nitrification in a highly acidic subtropical pine Forest soil. Pedosphere 26:904–910CrossRefGoogle Scholar
  17. Francis CA, Roberts KJ, Beman JM, Santoro AE, Oakley BB (2005) Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proc Natl Acad Sci U S A 102:14683–14688CrossRefGoogle Scholar
  18. Galloway JN (2008) An earth-system perspective of the global nitrogen cycle. Nature 451:293CrossRefGoogle Scholar
  19. Gao S, Cao W, Zou C, Gao J, Huang J, Bai J, Zeng N, K-y S, Wright A, Dou F (2018) Ammonia-oxidizing archaea are more sensitive than ammonia-oxidizing bacteria to long-term application of green manure in red paddy soil. Appl Soil Ecol 124:185–193CrossRefGoogle Scholar
  20. Goring CA (1962) Control of nitrification by 2-chloro-6-(trichloro-methyl) pyridine. Soil Sci 93:211–218CrossRefGoogle Scholar
  21. Hart SC, Stark JM, Davidson EA, Firestone MK (1994) Nitrogen mineralization, immobilization, and nitrification. Method Soil Anal. Microbiol Biochem Prop. Part 2, SSSA, Madison 985–1018Google Scholar
  22. Hu B, Shuai L, Wei W, Shen L, Lou L, Liu W, Tian G, Xu X, Ping Z (2015) pH-dominated niche segregation of ammonia-oxidising microorganisms in Chinese agricultural soils. FEMS Microbiol Ecol 90:290–299Google Scholar
  23. Le C, Zha Y, Li Y, Sun D, Lu H, Yin B (2010) Eutrophication of lake waters in China: cost, causes, and control. Environ Manag 45:662–668CrossRefGoogle Scholar
  24. Lehtovirta-Morley LE, Verhamme DT, Nicol GW, Prosser JI (2013) Effect of nitrification inhibitors on the growth and activity of Nitrosotalea devanaterra in culture and soil. Soil Biol Biochem 62:129–133CrossRefGoogle Scholar
  25. Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol G, Prosser J, Schuster S, Schleper C (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442:806–809CrossRefGoogle Scholar
  26. Li B, Fan CH, Xiong ZQ, Li QL, Zhang M (2015) The combined effects of nitrification inhibitor and biochar incorporation on yield-scaled N2O emissions from an intensively managed vegetable field in southeastern China. Biogeosci 12:2003–2017CrossRefGoogle Scholar
  27. Liu Z, Zhou W, Shen J, Li S, Ai C (2014) Soil quality assessment of yellow clayey paddy soils with different productivity. Biol Fertil Soils 50:537–548CrossRefGoogle Scholar
  28. Liu T, Liang Y, Chu G (2017) Nitrapyrin addition mitigates nitrous oxide emissions and raises nitrogen use efficiency in plastic-film-mulched drip-fertigated cotton field. PLoS One 12Google Scholar
  29. Long X, Chen C, Xu Z, Linder S, He J (2012) Abundance and community structure of ammonia oxidizing bacteria and archaea in a Sweden boreal forest soil under 19-year fertilization and 12-year warming. J Soils Sediments 12:1124–1133CrossRefGoogle Scholar
  30. Lu R (2000) Methods of soil and agrochemistry analysis. China Agricultural Science and Technology Press, Beijing, pp 62–141Google Scholar
  31. Lu L, Jia Z (2013) Urease gene-containing archaea dominate autotrophic ammonia oxidation in two acid soils. Environ Microbiol 15:1795–1809CrossRefGoogle Scholar
  32. McCarty G (1999) Modes of action of nitrification inhibitors. Biol Fertil Soils 29:1–9CrossRefGoogle Scholar
  33. Morimoto S, Hayatsu M, Takada Hoshino Y, Nagaoka K, Yamazaki M, Karasawa T, Takenaka M, Akiyama H (2011) Quantitative analyses of Ammonia-oxidizing archaea (AOA) and Ammonia-oxidizing Bacteria (AOB) in fields with different soil types. Microbes Environ 26:248–253CrossRefGoogle Scholar
  34. Muema EK, Cadisch G, Musyoki MK, Rasche F (2016) Dynamics of bacterial and archaeal amoA gene abundance after additions of organic inputs combined with mineral nitrogen to an agricultural soil. Nutr Cycl Agroecosyst 104:143–158CrossRefGoogle Scholar
  35. Norman RJ, Edberg JC, Stucki JW (1985) Determination of nitrate in soil extracts by dual-wavelength ultraviolet spectrophotometry. Soil Sci Soc Am J 49:1182–1185CrossRefGoogle Scholar
  36. Ouyang Y, Norton JM, Stark JM, Reeve JR, Habteselassie MY (2016) Ammonia-oxidizing bacteria are more responsive than archaea to nitrogen source in an agricultural soil. Soil Biol Biochem 96:4–15CrossRefGoogle Scholar
  37. Parkin TB, Hatfield JL (2010) Influence of nitrapyrin on N2O losses from soil receiving fall-applied anhydrous ammonia. Agric Ecosyst Environ 136:81–86CrossRefGoogle Scholar
  38. Ravishankara A, Daniel JS, Portmann RW (2009) Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326:123–125CrossRefGoogle Scholar
  39. Ren B, Zhang J, Dong S, Liu P, Zhao B, Li H (2017) Nitrapyrin improves grain yield and nitrogen use efficiency of summer maize waterlogged in the field. Agron J 109:185–192CrossRefGoogle Scholar
  40. Rotthauwe J-H, Witzel K-P, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63:4704–4712Google Scholar
  41. Rovita D, Killorn R (2007) Kinetics of nitrification in selected Iowa soils treated with stay-N 2000. Commun Soil Sci Plant Anal 38:1949–1963CrossRefGoogle Scholar
  42. Schleper C, Nicol GW (2010) Ammonia-Oxidising archaea – physiology, ecology and evolution. Adv Microb Physiol 57:1–41CrossRefGoogle Scholar
  43. Shen JP, Zhang LM, Di HJ, He JZ (2012) A review of ammonia-oxidizing bacteria and archaea in Chinese soils. Front Microbiol 3:296Google Scholar
  44. Shen T, Stieglmeier M, Dai J, Urich T, Schleper C (2013) Responses of the terrestrial ammonia-oxidizing archaeon ca. Nitrososphaera viennensis and the ammonia-oxidizing bacterium Nitrosospira multiformis to nitrification inhibitors. FEMS Microbiol Lett 344:121–129CrossRefGoogle Scholar
  45. Shi X, Hu HW, Kelly K, Chen D, He JZ, Suter H (2017) Response of ammonia oxidizers and denitrifiers to repeated applications of a nitrification inhibitor and a urease inhibitor in two pasture soils. J Soils Sediments 17:974–984CrossRefGoogle Scholar
  46. Singh S, Shivay YS (2003) Coating of prilled urea with ecofriendly neem (Azadirachta indica a. Juss. ) formulations for efficient nitrogen use in hybrid rice. Acta Agron Hung 51:53–59CrossRefGoogle Scholar
  47. Singh S, Verma A (2007) The potential of nitrification inhibitors to manage the pollution effect of nitrogen fertilizers in agricultural and other soils: a review. Environ Pract 9:266–279CrossRefGoogle Scholar
  48. Song H, Che Z, Cao W, Huang T, Wang J, Dong Z (2016) Changing roles of ammonia-oxidizing bacteria and archaea in a continuously acidifying soil caused by over-fertilization with nitrogen. Environ Sci Pollut Res Int 23:11964–11974CrossRefGoogle Scholar
  49. Subbarao GV, Ito O, Sahrawat KL, Berry WL, Nakahara K, Ishikawa T, Watanabe T, Suenaga K, Rondon M, Rao IM (2006) Scope and strategies for regulation of nitrification in agricultural systems—challenges and opportunities. Crit Rev Plant Sci 25:303–335CrossRefGoogle Scholar
  50. Subbarao GV, Nakahara K, Ishikawa T, Yoshihashi T, Ito O, Ono H, Ohnishi-Kameyama M, Yoshida M, Kawano N, Berry WL (2008) Free fatty acids from the pasture grass Brachiaria humidicola and one of their methyl esters as inhibitors of nitrification. Plant Soil 313:89–99CrossRefGoogle Scholar
  51. Subbarao GV, Nakahara K, Hurtado MP, Ono H, Moreta DE, Salcedo AF, Yoshihashi AT, Ishikawa T, Ishitani M, Ohnishi-Kameyama M, Yoshida M, Rondon M, Rao IM, Lascano CE, Berry WL, Ito O (2009) Evidence for biological nitrification inhibition in Brachiaria pastures. Proc Natl Acad Sci U S A 106:17302–17307CrossRefGoogle Scholar
  52. Sun H, Zhang H, Powlson D, Min J, Shi W (2015) Rice production, nitrous oxide emission and ammonia volatilization as impacted by the nitrification inhibitor 2-chloro-6-(trichloromethyl)-pyridine. Field Crop Res 173:1–7CrossRefGoogle Scholar
  53. Vetsch JA, Scherder EF, Ruen DC (2017) Does liquid swine manure application timing and nitrapyrin affect corn yield and inorganic soil nitrogen? Agron J 109:2358–2370CrossRefGoogle Scholar
  54. Walker C, De La Torre J, Klotz M, Urakawa H, Pinel N, Arp D, Brochier-Armanet C, Chain P, Chan P, Gollabgir A (2010) Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. PNAS 107:8818–8823CrossRefGoogle Scholar
  55. Wang Y, Ke X, Wu L, Lu Y (2009) Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization. Syst Appl Microbiol 32:27–36CrossRefGoogle Scholar
  56. Wan-Tai Y, Yong-Gang X, Ming-Li B, Qiang M, Hua Z (2010) Activity and composition of ammonia-oxidizing bacteria in an aquic brown soil as influenced by land use and fertilization. Pedosphere 20:789–798CrossRefGoogle Scholar
  57. Wu J, Joergensen R, Pommerening B, Chaussod R, Brookes P (1990) Measurement of soil microbial biomass C by fumigation-extraction—an automated procedure. Soil Biol Biochem 22:1167–1169CrossRefGoogle Scholar
  58. Wu Y, Lu L, Wang B, Lin X, Zhu J, Cai Z, Yan X, Jia Z (2011) Long-term field fertilization significantly alters community structure of Ammonia-oxidizing Bacteria rather than archaea in a Paddy soil. Soil Sci Soc Am J 75:1405–1413CrossRefGoogle Scholar
  59. Xi R, Long XE, Huang S, Yao H (2017) pH rather than nitrification and urease inhibitors determines the community of ammonia oxidizers in a vegetable soil. AMB Express 7:1–14CrossRefGoogle Scholar
  60. Zhang LM, Hu HW, Shen JP, He JZ (2012) Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils. ISME J 6:1032–1045CrossRefGoogle Scholar
  61. Zhang Y, Chen L, Sun R, Dai T, Tian J, Wen D (2015) Ammonia-oxidizing bacteria and archaea in wastewater treatment plant sludge and nearby coastal sediment in an industrial area in China. Appl Microbiol Biotechnol 99:4495–4507CrossRefGoogle Scholar
  62. Zhou ZF, Wang MX, Liu WL, Li ZL, Luo F, Xie DT (2016) A comparative study of ammonia-oxidizing archaea and bacteria in acidic and alkaline purple soils. Ann Microbiol 66:615–623CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yan Gu
    • 1
    • 2
  • Wenhai Mi
    • 3
  • Yinan Xie
    • 1
    • 2
  • Qingxu Ma
    • 1
    • 2
  • Lianghuan Wu
    • 1
    • 2
    • 4
    Email author
  • Zhaoping Hu
    • 4
  • Feng Dai
    • 5
  1. 1.Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, College of Environmental and Resource SciencesZhejiang UniversityHangzhouChina
  2. 2.Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource SciencesZhejiang UniversityHangzhouChina
  3. 3.College of Environmental Science and EngineeringYangzhou UniversityYangzhouChina
  4. 4.State Key Laboratory of Nutrition Resources Integrated UtilizationKingenta Ecological Engineering Group Co. Ltd.LinyiChina
  5. 5.Zhejiang Aofutuo Chemical Co. Ltd.ShangyuChina

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