Plant and Soil

, Volume 369, Issue 1–2, pp 187–197 | Cite as

Use of a 15N2 labelling technique to estimate exudation by white clover and transfer to companion ryegrass of symbiotically fixed N

Regular Article


Backgrounds and aims

N rhizodeposition by legumes leads to enrichment of N in soils and in companion plants. N rhizodeposition can be divided into two major components, root exudation and root senescence. Our aim was to quantify N root exudation in white clover (Trifolium repens L.) through an estimation of short-term N rhizodeposition and to assess its impact on N transfer to companion perennial ryegrass (Lolium perenne L.) grown in mixture with clover.


15N2 provided in the root atmosphere for 3 days was used to estimate transfer of symbiotically fixed nitrogen (SFN) to the growing medium by clover grown in pure stand and to ryegrass by clover grown in mixture for 2 months.


The proportion of N rhizodeposited over the 3 days increased from 3.5 % of SFN in pure stand to 5.3 % in mixture. The 15N-enrichment of ammonium from the adhering substrate shows that a part of the rhizodeposited N was released in the form of ammonium. 4 % of the rhizodeposited N was taken up by ryegrass during the labelling period.


This study showed a significant contribution of root N exudation to the total N rhizodeposition of legumes and in the transfer of N to grasses.


Rhizodeposition Nitrogen Clover Ryegrass Transfer Ammonium 



We thank Anne-Françoise Ameline for technical assistance during the labeling experiment and Marie-Paule Bataille for ammonium and isotopic analysis. We are grateful to Dr Laurence Cantrill for English improvement of the manuscript.


  1. Abdallah M, Dubousset L, Meuriot F, Etienne P, Avice JC, Ourry A (2010) Effect of mineral sulphur availability on nitrogen and sulphur uptake and remobilization during the vegetative growth of Brassica napus L. J Exp Bot 61:2635–2646PubMedCrossRefGoogle Scholar
  2. Barea JM, Azcoz R, Azcon-Aguilar C (1989) Time-course of N2-fixation (15N) in the field by clover growing alone or in mixture with ryegrass to improve pasture productivity, and inoculated with vesicular-arbuscular mycorrhizal fungi. New Phytol 112:399–404CrossRefGoogle Scholar
  3. Bazot S (2005) Contribution à l’étude de l’allocation des photoassimilats récents dans la plante et la rhizosphère chez une graminée pérenne (Lolium perenne L.). Thèse de l’Institut National Polytechnique de Lorraine, 174 p.Google Scholar
  4. Bingham IJ, Rees RM (2008) Senescence and N release from clover roots following permanent excision of the shoot. Plant Soil 303:229–240CrossRefGoogle Scholar
  5. Brophy LS, Heichel GH (1989) Nitrogen release from roots of alfalfa and soybean grown in sand culture. Plant Soil 116:77–84CrossRefGoogle Scholar
  6. Cliquet JB, Murray PJ, Boucaud J (1997) Effect of the arbuscular mycorrhizal fungus Glomus fasciculatum on the uptake of amino nitrogen by Lolium perenne L. New Phytol 137:345–349CrossRefGoogle Scholar
  7. Dahlin AS, Mårtenson AM (2008) Cutting regimes determines allocation of fixed nitrogen in white clover. Biol Fertil Soil 45:199–204CrossRefGoogle Scholar
  8. Dahlin AS, Stenberg M (2010) Transfer of N from red clover to perennial ryegrass in mixed stands under different cutting strategies. Eur J Agron 33:149–156CrossRefGoogle Scholar
  9. Dubach M, Russelle MP (1994) Forage legume roots and nodules and their role nitrogen transfer. Agron J 86:259–266CrossRefGoogle Scholar
  10. Elgersma A, Schlepers H, Nassiri M (2000) Interactions between perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) under contrasting nitrogen availability: productivity, seasonal patterns of species composition, N2 fixation, N transfer and N recovery. Plant Soil 221:281–299CrossRefGoogle Scholar
  11. Fujita K, Ofosu-Budu KG, Ogata S (1992) Biological nitrogen fixation in mixed legume-cereal cropping systems. Plant Soil 141:155–175CrossRefGoogle Scholar
  12. Fustec J, Lesuffleur F, Mahieu S, Cliquet JB (2009) Nitrogen rhizodeposition of legumes. Agron Sustain Dev 30:57–66CrossRefGoogle Scholar
  13. Gylfadóttir T, Helgadóttir A, Høgh-Jensen H (2007) Consequences of including adapted white clover in northern european grassland: transfer and deposition of nitrogen. Plant Soil 297:93–104CrossRefGoogle Scholar
  14. Hamel C, Barrantes-Cartin U, Furlan V, Smith DL (1991) Endomycorrhizal fungi in nitrogen transfer from soybean to maize. Plant Soil 138:33–40CrossRefGoogle Scholar
  15. Hartwig UA (1998) The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the novel ecosystem to the gene expression level. Perspect Plant Ecol Evol System 1(1):92–120CrossRefGoogle Scholar
  16. Haynes RJ (1980) Competitive aspects of the grass/legume association. Adv Agron 33:227–261CrossRefGoogle Scholar
  17. Haystead A, Malaczuck N, Grove T (1988) Underground transfer of nitrogen between pasture plants infected with vesicular-arbuscular mycorrhizal fungi. New Phytol 108:417–423CrossRefGoogle Scholar
  18. He X, Xu M, Qiu GY, Zhou J (2009) Use of 15N stable isotope to quantify nitrogen transfer between mycorrhizal plants. J Plant Ecol 2:107–118CrossRefGoogle Scholar
  19. Hertenberger G, Wanek W (2004) Evaluation of methods to measure differential 15N labeling of soil and root N pools for studies of root exudation. Rapid Comm Mass Spectrom 18:2415–2425CrossRefGoogle Scholar
  20. Høgh-Jensen H, Schjoerring JK (1994) Measurement of biological dinitrogen fixation in grassland: Comparison of the enriched 15N dilution and the natural 15N abundance methods at different nitrogen application rates and defoliation frequencies. Plant Soil 166:153–163CrossRefGoogle Scholar
  21. Høgh-Jensen H, Schjoerring JK (1997) Interaction between white clover and ryegrass under contrasting nitrogen availability: N2 fixation, N fertilizer recovery, N transfer and water-use efficiency. Plant Soil 197:187–199CrossRefGoogle Scholar
  22. Høgh-Jensen H, Schjoerring JK (2000) Below-ground nitrogen transfer between different grassland species: direct quantification by 15N leaf feeding compared with indirect dilution of soil 15N. Plant Soil 227:171–183CrossRefGoogle Scholar
  23. Høgh-Jensen H, Schjoerring JK (2001) Rhizodeposition of nitrogen by red clover, white clover and ryegrass leys. Soil Biol Biochem 33:439–448CrossRefGoogle Scholar
  24. Jalonen R, Nygren P, Sierra J (2009) Transfer of nitrogen from a tropical legume tree to an associated fodder grass via root exudation and common mycelial network. Plant Cell Environ 32:1366–1376PubMedCrossRefGoogle Scholar
  25. Janzen HH, Gilbertson C (1994) Exchange of N-15 among plants in controlled environments studies. Can J Soil Sci 74:109–110CrossRefGoogle Scholar
  26. Jensen ES (1996) Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops. Plant Soil 182:25–38CrossRefGoogle Scholar
  27. Jeudy C, Ruffel S, Freixes S, Tillard P, Santoni AL, Morel S, Journet EP, Duc G, Gojon A, Lepetit M, Salon C (2010) Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses. New Phytol 185:817–828PubMedCrossRefGoogle Scholar
  28. Johansen A, Jensen ES (1996) Transfer of N and P from intact or decomposing roots of pea to barley inter connected by an arbuscular mycorrhizal fungus. Soil Biol Biochem 28:73–81CrossRefGoogle Scholar
  29. Jones DL, Darrah PR (1993) Influx and efflux of amino acids from Zea mays L. Roots and their implication for N nutrition and the rhizosphere. Plant Soil 155(156):87–90CrossRefGoogle Scholar
  30. Jørgensen FV, Jensen ES, Schjoerring JK (1999) Dinitrogen fixation in white clover grown in pure stand and mixture with ryegrass estimated by the immobilized 15N isotope dilution method. Plant Soil 208:293–305CrossRefGoogle Scholar
  31. Kurppa M, Leblanc HA, Nygren P (2010) Detection of nitrogen transfer from N2-fixing shade trees to cacao saplings in 15 labelled soil: ecological and experimental considerations. Agrofores Syst 80:223–229CrossRefGoogle Scholar
  32. Lainé P, Bigot J, Ourry A, Boucaud J (1994) Effects of low temperature on nitrate uptake, and xylem and phloem flows of nitrogen, in Secale cereale L. and Brassica napus L. New Phytol 127:675–683CrossRefGoogle Scholar
  33. Ledgard SF, Steele KW (1992) Biological nitrogen fixation in mixed legume/grass pastures. Plant Soil 141:137–153CrossRefGoogle Scholar
  34. Lesuffleur FS, Cliquet JB (2010) Characterisation of root amino acid exudation in white clover. Plant Soil 333:191–201CrossRefGoogle Scholar
  35. Lipson DA, Raab TK, Schmidt SK, Monson RK (1999) Variation in competitive abilities of plants and microbes for specific amino acids. Biol Fertil Soils 29:257–261CrossRefGoogle Scholar
  36. Macduff JH, Jackson SB (1992) Influx and efflux of nitrate and ammonium in italian ryegrass and white clover roots: comparisons between effects of darkness and defoliation. J Exp Bot 43:525–535CrossRefGoogle Scholar
  37. Marty C, Pornon A, Escaravage N, Winterton P, Lamaze T (2009) Complex interactions between a legume and two grasses in a subalpine meadow. Am J Bot 96:1814–1820PubMedCrossRefGoogle Scholar
  38. McNeill AM, Hood RC, Wood M (1994) Direct measurement of nitrogen fixation by Trifolium repens L. and Alnus glutinosa L. using 15N2. J Exp Bot 45:749–755Google Scholar
  39. Moyer-Henry KA, Burton JW, Israel DW, Rufty TW (2006) Nitrogen transfer between plants: a 15N natural abundance study with crop and weed species. Plant Soil 282:7–20CrossRefGoogle Scholar
  40. N’Guyen C (2003) Rhizodeposition of organic C by plants: mechanisms and controls. Agron Sustain Dev 23:375–396Google Scholar
  41. 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 140:155–163CrossRefGoogle Scholar
  42. Ofosu-Budu KG, Fujita K, Ogata S (1990) Excretion of ureide and other nitrogenous compounds by the root system of soybean at different growth stages. Plant Soil 128:135–142CrossRefGoogle Scholar
  43. Parsons R, Stanforth A, Raven JA, Sprent JI (1993) Nodule growth and activity may be regulated by a feedback mechanism involving phloem nitrogen. Plant Cell Environ 16:125–136CrossRefGoogle Scholar
  44. Paynel F, Cliquet JB (2003) N transfer from white clover to perennial ryegrass, via exudation of nitrogenous compounds. Agron Sustain Dev 23:503–510Google Scholar
  45. Paynel F, Lesuffleur F, Bigot J, Diquélou S, Cliquet JB (2008) A study of 15N transfer between legumes and grasses. Agron Sustain Dev 28:281–290CrossRefGoogle Scholar
  46. Paynel F, Murray PJ, Cliquet JB (2001) Root exudates: a pathway for short-term N transfer from clover and ryegrass. Plant Soil 229:235–243CrossRefGoogle Scholar
  47. Pirhofer-Walzl K, Rasmussen J, Høgh-Jensen H, Eriksen J, Søegaard K, Rasmussen J (2012) Nitrogen transfer from forage legumes to nine neighbouring plants in a multi-species grassland. Plant Soil 350:71–84CrossRefGoogle Scholar
  48. 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 39:804–815CrossRefGoogle Scholar
  49. Rogers JB, Scott Laidlaw A, Christie P (2001) The role of arbuscular mycorrhizal fungi in the transfer of nutrients between white clover and perennial ryegrass. Chemosphere 42:153–159PubMedCrossRefGoogle Scholar
  50. Russelle MP, Allan DL, Gourley CJP (1994) Direct assessment of symbiotically fixed nitrogen in the rhizosphere of alfalfa. Plant Soil 159:233–243CrossRefGoogle Scholar
  51. Shepherd T, Davies HV (1994) Patterns of short-term amino acid accumulation and loss in the root-zone of liquid-cultured forage rape (Brassica napus L.). Plant Soil 158:99–109CrossRefGoogle Scholar
  52. Sierra J, Nygren P (2006) Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system. Soil Biol Biochem 38:1893–1903CrossRefGoogle Scholar
  53. Sokal RR, Rohlf FJ (1995) In: Freeman WH, Company (eds) Biometry; the principles and practice of statistics in biology research, 3rd edn. Freeman, New York, p 897Google Scholar
  54. Soussana JF, Hartwig UA (1996) The effect of elevated CO2 on symbiotic N2 fixation: a link between carbon and nitrogen cycles in grassland ecosystems. Plant Soil 187:321–332CrossRefGoogle Scholar
  55. Sturite I, Henriksen TM, Breland TA (2007) Longevity of White Clover (Trifolium repens) leaves, stolons and roots, and consequences for nitrogen dynamics under northern temperate climatic conditions. Ann Bot 100:33–40PubMedCrossRefGoogle Scholar
  56. Ta TC, Faris M (1987) Species variation in the fixation and transfer of nitrogen from legumes to associated grasses. Plant Soil 98:265–274CrossRefGoogle Scholar
  57. Ta TC, Macdowall FDH, Faris MA (1986) Excretion of nitrogen assimilated from N2 fixed by nodulated roots of alfalfa (Medicago sativa). Can J Bot 64:2063–2067CrossRefGoogle Scholar
  58. Thomas BD, Bowman WD (1998) Influence of a N2-fixing Trifolium on plant species composition and biomass production in alpine tundra. Oecologia 115:26–31CrossRefGoogle Scholar
  59. Voisin AS, Salon C, Jeudy C, Warembourg F (2003) Symbiotic Nitrogen Fixation in relation to C economy of Pisum sativum L. as a function of plant phenology. J Exp Bot 54:2733–2744PubMedCrossRefGoogle Scholar
  60. White J, Prell J, James EK, Poole P (2007) Nutrient sharing between symbionts. Plant Physiol 144:604–614PubMedCrossRefGoogle Scholar
  61. Wacquant JP, Ouknider M, Jacquart M (1989) Evidence for a periodic excretion of nitrogen by roots of grass-legume associations. Plant Soil 116:57–58CrossRefGoogle Scholar
  62. Wichern F, Eberhardt E, Mayer J, Joergensen RG, Müller T (2008) Nitrogen rhizodeposition in agricultural crops. Soil Biol Biochem 40:30–48CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • F. Lesuffleur
    • 1
    • 2
  • C. Salon
    • 3
  • C. Jeudy
    • 3
  • J. B. Cliquet
    • 1
    • 2
  1. 1.INRA UMR 950, EVA Ecophysiologie Végétale, Agronomie et nutritions N,C,S, Université de CaenCaen CedexFrance
  2. 2.Université de Caen UMR 950, EVA Ecophysiologie Végétale, Agronomie et nutritions N,C,S, Université de CaenCaen CedexFrance
  3. 3.UMR1347 Agroécologie AgroSup/INRA/uBDijon CedexFrance

Personalised recommendations