Plant and Soil

, Volume 425, Issue 1–2, pp 507–525 | Cite as

Above- and belowground nitrogen distribution of a red clover-perennial ryegrass sward along a soil nutrient availability gradient established by organic and conventional cropping systems

  • Andreas Hammelehle
  • Astrid Oberson
  • Andreas Lüscher
  • Paul Mäder
  • Jochen MayerEmail author
Regular Article



Belowground legume nitrogen (N) composed of roots and rhizodeposition is an important N input to soils, but published data of belowground N vary broadly, probably due to extrapolation from short-term experiments and dissimilar growing conditions. We quantified belowground N inputs of red clover (Trifolium pratense L.) during two consecutive years in a clover-grass sward along a soil nutrient availability gradient.


We established a red clover-perennial ryegrass (Lolium perenne L.) model sward in microplots located in field plots of the DOK experiment, which has a 33-year history of organic and conventional cropping, resulting in a soil nutrient availability gradient. Four treatments were examined: the zero fertilisation control, bio-organic with half and full dose manure application, and the conventional system with mineral fertilisation at full dose. We studied the development of clover aboveground and belowground N using multiple pulse 15N urea leaf labelling.


Belowground clover N increased over time and with rising nutrient availability and was proportional to aboveground clover N at all times. Belowground clover N amounted to 40% of aboveground clover N during two consecutive years, irrespective of the nutrient availability status. Belowground clover N development was initially dominated by fast root growth, followed by enhanced root turnover during the second year. Potassium availability limited clover growth and total N accumulation in treatments with low nutrient availability.


Belowground red clover N inputs could be estimated from aboveground N by a constant factor of 0.4, regardless of the nutrient availability and cultivation time. Root turnover led to a distinct absolute increase of N rhizodeposition over time. Hence, N rhizodeposition, with an 80% share of belowground N, was the predominant N pool at the end of the second year.


Rhizodeposition 15N leaf labelling Cropping systems Belowground to aboveground N ratio Nutrient availability 



Aboveground N


Belowground N, comprising physically recoverable root N at the time of excavation plus NdfR


Bio-organic treatment of the DOK experiment with half dose fertilisation


Bio-organic treatment of the DOK experiment with full dose fertilisation


Conventional treatment of the DOK experiment with full dose sole mineral fertilisation


Chloroform fumigation extraction


Long-term experiment comparing Bio-Dynamic, Bio-Organic, and conventional (K) cropping systems


Excess atom fraction


Labelled microplot, delimiting the 15N labelled plant-soil system (excavated after t months of sward cultivation)


Nitrogen derived from rhizodeposition


Unfertilised control treatment of the DOK experiment


Reference microplot, delimiting the unlabelled plant-soil system (excavated after t months of sward cultivation)


Time from planting of red clover and perennial ryegrass until microplot excavation in months



We warmly thank the Agroscope field team, especially their head Ernst Brack, Lucie Gunst and Monika Schnider from Agroscope for their versatile help in the DOK experiment and in the lab, the FiBL field team for their help in the DOK experiment, Stephano Bernasconi from the Geological Institute at ETH Zurich for isotopic analysis, Claude Renaux from the statistical consulting service of the seminar of statistics at the ETH Zürich, and Juliane Hirte from Agroscope for the final internal review of the manuscript. The work was funded by the Swiss National Science Foundation Grant 205321_132770 / 1.

Supplementary material

11104_2018_3559_MOESM1_ESM.docx (99 kb)
ESM 1 (DOCX 98 kb)


  1. Agroscope (1996) Schweizerische Referenzmethoden der Forschungsanstalt Agroscope. Band 1: Bodenuntersuchung und Substratuntersuchung zur Düngeberatung. Zürich-ReckenholzGoogle Scholar
  2. Aitchison J (1982) The statistical analysis of compositional data. J R Stat Soc Series B Stat Methodol 44:139–177. Google Scholar
  3. Almeida JPF, Lüscher A, Frehner M, Oberson A, Nösberger J (1999) Partitioning of P and the activity of root acid phosphatase in white clover (Trifolium Repens L.) are modified by increased atmospheric CO2 and P fertilisation. Plant Soil 210:159–166. CrossRefGoogle Scholar
  4. Bailey JS, Cushnahan A, Beattie JAM (1997) The diagnosis and recommendation integrated system (DRIS) for diagnosing the nutrient status of grassland swards: II. Model calibration and validation. Plant Soil 197:137–147. CrossRefGoogle Scholar
  5. Boller BC, Nösberger J (1987) Symbiotically fixed nitrogen from field- grown white and red clover mixed with ryegrasses at low levels of 15N-fertilization. Plant Soil 104:219–226. CrossRefGoogle Scholar
  6. Bolton J, Nowakowski TZ, Lazarus W (1976) Sulphur–nitrogen interaction effects on the yield and composition of the protein-N, non-protein-N and soluble carbohydrates in perennial ryegrass. J Sci Food Agric 27:553–560. CrossRefGoogle Scholar
  7. Bowley SR, Taylor NL, Dougherty CT (1984) Physiology and morphology of red clover. Adv Agron 37:317–347. CrossRefGoogle Scholar
  8. Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem 17:837–842. CrossRefGoogle Scholar
  9. Butterbach-Bahl K, Baggs EM, Dannenmann M, Kiese R, Zechmeister-Boltenstern S (2013) Nitrous oxide emissions from soils: how well do we understand the processes and their controls? Phil Trans R Soc B Biol Sci 368:20130122. CrossRefGoogle Scholar
  10. Cabrera ML, Beare MH (1993) Alkaline persulfate oxidation for determining total nitrogen in microbial biomass extracts. Soil Sci Soc Am J 57:1007–1012. CrossRefGoogle Scholar
  11. Cakmak I, Hengeler C, Marschner H (1994) Partitioning of shoot and root dry matter and carbohydrates in bean plants suffering from phosphorus, potassium and magnesium deficiency. J Exp Bot 45:1245–1250. CrossRefGoogle Scholar
  12. Chen SM, Lin S, Loges R, Reinsch T, Hasler M, Taube F (2016) Independence of seasonal patterns of root functional traits and rooting strategy of a grass-clover sward from sward age and slurry application. Grass Forage Sci 71:607–621. CrossRefGoogle Scholar
  13. Coplen TB (2011) Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Commun Mass Spectrom 25:2538–2560. CrossRefPubMedGoogle Scholar
  14. Dahlin AS, Mårtensson AM (2008) Cutting regime determines allocation of fixed nitrogen in white clover. Biol Fertil Soils 45:199–204. CrossRefGoogle Scholar
  15. Dahlin AS, Stenberg M (2010a) Transfer of N from red clover to perennial ryegrass in mixed stands under different cutting strategies. Eur J Agron 33:149–156. CrossRefGoogle Scholar
  16. Dahlin AS, Stenberg M (2010b) Cutting regime affects the amount and allocation of symbiotically fixed N in green manure leys. Plant Soil 331:401–412. CrossRefGoogle Scholar
  17. Dampney PMR (1992) The effect of timing and rate of potash application on the yield and herbage composition of grass grown for silage. Grass Forage Sci 47:280–289. CrossRefGoogle Scholar
  18. Davis MR (1991) The comparative phosphorus requirements of some temperate perennial legumes. Plant Soil 133:17–30. CrossRefGoogle Scholar
  19. Fließbach A, Oberholzer HR, Gunst L, Mäder P (2007) Soil organic matter and biological soil quality indicators after 21 years of organic and conventional farming. Agric Ecosyst Environ 118:273–284. CrossRefGoogle Scholar
  20. Flisch R, Sinaj S, Charles R, Richner W (2009) Grundlagen für die Düngung im Acker- und Futterbau (GRUDAF). Agrarforsch Schweiz 16:1–97Google Scholar
  21. Fustec J, Lesuffleur F, Mahieu S, Cliquet JB (2010) Nitrogen rhizodeposition of legumes. A review. Agron Sustain Dev 30:57–66. CrossRefGoogle Scholar
  22. Fystro G, Nesheim L, Bakken AK (2008) The N:P ratio in plant tissues as a diagnostic tool for P supply. NJF report. Accessed 24 Apr 2017
  23. Galloway JN, Townsend AR, Erisman JW, Bekunda M, Cai Z, Freney JR, Martinelli LA, Seitzinger SP, Sutton MA (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320:889–892. CrossRefPubMedGoogle Scholar
  24. Gardner M, Peoples M, Condon J, Li G, Conyers M, Dear B (2014) Evaluating the importance of a potential source of error when applying shoot 15N labelling techniques to legumes to quantify the belowground transfer of nitrogen to other species. Proceedings of the 16th Australian agronomy conference, Armidale, NSW, AustraliaGoogle Scholar
  25. Gasser M, Hammelehle A, Oberson A, Frossard E, Mayer J (2015) Quantitative evidence of overestimated rhizodeposition using 15N leaf-labelling. Soil Biol Biochem 85:10–20. CrossRefGoogle Scholar
  26. Gooding MJ, Davies WP (1992) Foliar urea fertilization of cereals: a review. Fertil Res 32:209–222. CrossRefGoogle Scholar
  27. Gylfadóttir T, Helgadóttir Á, Høgh-Jensen H (2007) Consequences of including adapted white clover in northern European grassland: transfer and deposition of nitrogen. Plant Soil 297:93–104. CrossRefGoogle Scholar
  28. Haase S, Ruess L, Neumann G, Marhan S, Kandeler E (2007) Low-level herbivory by root-knot nematodes (Meloidogyne Incognita) modifies root hair morphology and rhizodeposition in host plants (Hordeum Vulgare). Plant Soil 301:151–164. CrossRefGoogle Scholar
  29. Hamilton EW, Frank DA, Hinchey PM, Murray TR (2008) Defoliation induces root exudation and triggers positive rhizospheric feedbacks in a temperate grassland. Soil Biol Biochem 40:2865–2873. CrossRefGoogle Scholar
  30. Hart PBS, Rayner JH, Jenkinson DS (1986) Influence of pool substitution on the interpretation of fertilizer experiments with 15N. Eur J Soil Sci 37:389–403. CrossRefGoogle Scholar
  31. Haystead A, Marriott C (1979) Transfer of legume nitrogen to associated grass. Soil Biol Biochem 11:99–104. CrossRefGoogle Scholar
  32. Herridge D, Peoples M, Boddey R (2008) Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 311:1–18. CrossRefGoogle Scholar
  33. Hill JO, Simpson RJ, Moore AD, Chapman DF (2006) Morphology and response of roots of pasture species to phosphorus and nitrogen nutrition. Plant Soil 286:7–19. CrossRefGoogle Scholar
  34. Høgh-Jensen H, Schjoerring JK (2001) Rhizodeposition of nitrogen by red clover, white clover and ryegrass leys. Soil Biol Biochem 33:439–448. CrossRefGoogle Scholar
  35. Huss-Danell K, Chaia E, Carlsson G (2007) N2 fixation and nitrogen allocation to above and belowground plant parts in red clover-grasslands. Plant Soil 299:215–226. CrossRefGoogle Scholar
  36. Janzen HH, Bruinsma Y (1989) Methodology for the quantification of root and rhizosphere nitrogen dynamics by exposure of shoots to 15N-labelled ammonia. Soil Biol Biochem 21:189–196. CrossRefGoogle Scholar
  37. Jensen ES (1996) Rhizodeposition of N by pea and barley and its effect on soil N dynamics. Soil Biol Biochem 28:65–71. CrossRefGoogle Scholar
  38. Khan WDF, Peoples MB, Herridge DF (2002) Quantifying below-ground nitrogen of legumes. Plant Soil 245:327–334. CrossRefGoogle Scholar
  39. Ledgard SF, Freney JR, Simpson JR (1985) Assessing nitrogen transfer from legumes to associated grasses. Soil Biol Biochem 17:575–577. CrossRefGoogle Scholar
  40. Liebisch F, Bünemann EK, Huguenin-Elie O, Jeangros B, Frossard E, Oberson A (2013) Plant phosphorus nutrition indicators evaluated in agricultural grasslands managed at different intensities. Eur J Agron 44:67–77. CrossRefGoogle Scholar
  41. Maeder P, Fliessbach A, Dubois D, Gunst L, Fried P, Niggli U (2002) Soil fertility and biodiversity in organic farming. Science 296:1694–1697. CrossRefGoogle Scholar
  42. Mayer J, Buegger F, Jensen ES, Schloter M, Heß J (2003) Estimating N rhizodeposition of grain legumes using a 15N in situ stem labelling method. Soil Biol Biochem 35:21–28. CrossRefGoogle Scholar
  43. Mayer J, Gunst L, Mäder P, Samson MF, Carcea M, Narducci V, Thomsen IK, Dubois D (2015) Productivity, quality and sustainability of winter wheat under long-term conventional and organic management in Switzerland. Eur J Agron 65:27–39. CrossRefGoogle Scholar
  44. McNaught KJ, During C (1970) Relations between nutrient concentrations in plant tissues and responses of white clover to fertilisers on a gley podzol near westport. New Zeal J Agr Res 13:567–590. CrossRefGoogle Scholar
  45. McNeill AM, Zhu C, Fillery IRP (1997) Use of in situ 15N-labelling to estimate the total below-ground nitrogen of pasture legumes in intact soil–plant systems. Aust J Agric Res 48:295–304. CrossRefGoogle Scholar
  46. Mengel K, Steffens D (1985) Potassium uptake of rye-grass (Lolium Perenne) and red clover (Trifolium Pratense) as related to root parameters. Biol Fertil Soils 1:53–58. CrossRefGoogle Scholar
  47. Nesheim L, Øyen J (1994) Nitrogen fixation by red clover (Trifolium Pratense L.) grown in mixtures with timothy (Phleum Pratense L.) at different levels of nitrogen fertilization. Acta Agr Scand B- S P 44:28–34. Google Scholar
  48. Neumann G, Römheld V (2012) Rhizosphere chemistry in relation to plant nutrition. In: Marschner P (ed) Marschner's mineral nutrition of higher plants, 3rd edn. Academic, San Diego, pp 347–368CrossRefGoogle Scholar
  49. Nyfeler D, Huguenin-Elie O, Suter M, Frossard E, Lüscher A (2011) Grass-legume mixtures can yield more nitrogen than legume pure stands due to mutual stimulation of nitrogen uptake from symbiotic and non-symbiotic sources. Agric Ecosyst Environ 140:155–163. CrossRefGoogle Scholar
  50. Oberson A, Nanzer S, Bosshard C, Dubois D, Mäder P, Frossard E (2007) Symbiotic N2 fixation by soybean in organic and conventional cropping systems estimated by 15N dilution and 15N natural abundance. Plant Soil 290:69–83. CrossRefGoogle Scholar
  51. Oberson A, Frossard E, Bühlmann C, Mayer J, Mäder P, Lüscher A (2013) Nitrogen fixation and transfer in grass-clover leys under organic and conventional cropping systems. Plant Soil 371:237–255. CrossRefGoogle Scholar
  52. Rasmussen J, Eriksen J, Jensen ES, Esbensen KH, Høgh-Jensen H (2007) In situ carbon and nitrogen dynamics in ryegrass–clover mixtures: transfers, deposition and leaching. Soil Biol Biochem 39:804–815. CrossRefGoogle Scholar
  53. Robertson GP, Vitousek PM (2009) Nitrogen in agriculture: balancing the cost of an essential resource. Annu Rev Environ Resour 34:97–125. CrossRefGoogle Scholar
  54. Römheld V (2012) Diagnosis of deficiency and toxicity of nutrients. In: Marschner P (ed) Marschner's mineral nutrition of higher plants, 3rd ed. academic, San Diego, pp 299–312CrossRefGoogle Scholar
  55. Russell CA, Fillery IRP (1996) In situ 15N labelling of lupin below-ground biomass. Aust J Agric Res 47:1035–1046. CrossRefGoogle Scholar
  56. Sawatsky N, Soper RJ (1991) A quantitative measurement of the nitrogen loss from the root system of field peas (Pisum avense L.) grown in the soil. Soil Biol Biochem 23:255–259. CrossRefGoogle Scholar
  57. Schipanski ME, Drinkwater LE (2012) Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient. Plant Soil 357:147–159. CrossRefGoogle Scholar
  58. Sierra J, Daudin D, Domenach A-M, Nygren P, Desfontaines L (2007) Nitrogen transfer from a legume tree to the associated grass estimated by the isotopic signature of tree root exudates: a comparison of the 15N leaf feeding and natural 15N abundance methods. Eur J Agron 27:178–186. CrossRefGoogle Scholar
  59. Smith GS, Cornforth IS, Henderson HV (1985) Critical leaf concentrations for deficiencies of nitrogen, potassium, phosphorous, sulphur, and magnesium in perennial ryegrass. New Phytol 101:393–409. CrossRefGoogle Scholar
  60. Snaydon R, Howe C (1986) Root and shoot competition between established ryegrass and invading grass seedlings. J Appl Ecol 23:667–674. CrossRefGoogle Scholar
  61. Suter D, Rosenberg E, Frick R, Mosimann E (2012) Swiss standard mixtures for ley farming, revision 2013-2016. Agrarforsch Schweiz 3:1–12Google Scholar
  62. Suter D, Frick R, Hirschi H, Aebi P (2014) Substantial progress in variety testing with red clover. Agrarforsch Schweiz 5:272–279Google Scholar
  63. Ta TC, Faris MA (1987) Effects of alfalfa proportions and clipping frequencies on timothy-alfalfa mixtures. II: nitrogen fixation and transfer. Agron J 79:820–824. CrossRefGoogle Scholar
  64. Thilakarathna MS, Papadopoulos YA, Rodd AV, Grimmett M, Fillmore SAE, Crouse M, Prithiviraj B (2016) Nitrogen fixation and transfer of red clover genotypes under legume–grass forage based production systems. Nutr Cycl Agroecosyst 106:233–247. CrossRefGoogle Scholar
  65. Thió-Henestrosa S, Barceló-Vidal C, Martín-Fernandez A, Pawlowsky-Glahn V (2009) CoDaPack. An excel and visual basic based software of compositional data analysis: current version and discussion for upcoming versions. In: proceedings of the 6th international workshop on compositional data analysis: Girona, June 1-5, 2015Google Scholar
  66. Trannin WS, Urquiaga S, Guerra G, Ibijbijen J, Cadisch G (2000) Interspecies competition and N transfer in a tropical grass-legume mixture. Biol Fertil Soils 32:441–448. CrossRefGoogle Scholar
  67. Tucker TC, Smith FW (1952) The influence of applied boron, magnesium, and potassium on the growth and chemical composition of red clover grown under greenhouse conditions1. Soil Sci Soc Am J 16:252–255. CrossRefGoogle Scholar
  68. Uren NC (2007) Types, amounts, and possible functions of compounds released into the rhizosphere by soil-grown plants. In: Pinton R, Varanini Z, Nannipieri P (eds) The Rhizosphere, 2nd edn. CRC Press, Boca Raton, pp 1–21Google Scholar
  69. Van Den Boogaart KG, Tolosana-Delgado R (2013) Analyzing compositional data with R. Springer, DordrechtCrossRefGoogle Scholar
  70. Wichern F, Mayer J, Joergensen RG, Muller T (2007) Release of C and N from roots of peas and oats and their availability to soil microorganisms. Soil Biol Biochem 39:2829–2839. CrossRefGoogle Scholar
  71. Wichern F, Eberhardt E, Mayer J, Joergensen RG, Müller T (2008) Nitrogen rhizodeposition in agricultural crops: methods, estimates and future prospects. Soil Biol Biochem 40:30–48. CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Agroscope, Water Protection and Substance FlowsZurichSwitzerland
  2. 2.Swiss Federal Institute of Technology (ETH), Institute of Agricultural Sciences, Group of Plant NutritionLindauSwitzerland
  3. 3.Agroscope, Forage Production and Grassland SystemsZurichSwitzerland
  4. 4.Research Institute of Organic Agriculture (FiBL)FrickSwitzerland

Personalised recommendations