Plant and Soil

, Volume 253, Issue 2, pp 353–372 | Cite as

Nitrogen fixation in perennial forage legumes in the field

  • G. CarlssonEmail author
  • K. Huss-Danell


Nitrogen acquisition is one of the most important factors for plant production, and N contribution from biological N2 fixation can reduce the need for industrial N fertilizers. Perennial forages are widespread in temperate and boreal areas, where much of the agriculture is based on livestock production. Due to the symbiosis with N2-fixing rhizobia, perennial forage legumes have great potential to increase sustainability in such grassland farming systems. The present work is a summary of a large number of studies investigating N2 fixation in three perennial forage legumes primarily relating to ungrazed northern temperate/boreal areas. Reported rates of N2 fixation in above-ground plant tissues were in the range of up to 373 kg N ha−1 year−1 in red clover (Trifolium pratense L.), 545 kg N ha−1 year−1 in white clover (T. repens L.) and 350 kg N ha−1 year−1 in alfalfa (Medicago sativa L.). When grown in mixtures with grasses, these species took a large fraction of their nitrogen from N2 fixation (average around 80%), regardless of management, dry matter yield and location. There was a large variation in N2 fixation data and part of this variation was ascribed to differences in plant production between years. Studies with experiments at more than one site showed that also geographic location was an important source of variation. On the other hand, when all data were plotted against latitude, there was no simple correlation. Climatic conditions seem therefore to give as high N2 fixation per ha and year in northern areas (around 60°N) as in areas with a milder climate (around 40°N). Analyzing whole plants or just above-ground plant parts influenced the estimate of N2 fixation, and most reported values were underestimated since roots were not included. Despite large differences in environmental conditions, such as N fertilization and geographic location, N2 fixation (Nfix; kg N per ha and year) was significantly (P<0.001) correlated to legume dry matter yield (DM; kg per ha and year). Very rough, but nevertheless valuable estimations of Nfix in legume/grass mixtures (roots not considered) are given by Nfix = 0.026ċDM + 7 for T. pratense, Nfix = 0.031ċDM + 24 for T. repens, and Nfix = 0.021ċDM + 17 for M. sativa.

Medicago sativa methods N2 fixation review Trifolium pratense Trifolium repens 


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  1. Boller B C and 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.Google Scholar
  2. Boller B C and Nösberger J 1994 Differences in nitrogen fixation among field-grown red-clover strains at different levels of 15N fertilization. Euphytica 78, 167–174.Google Scholar
  3. Bremer E and van Kessel C 1990 Appraisal of the nitrogen-15 natural abundance method for quantifying dinitrogen fixation. Soil Sci. Soc. Am. J. 54, 404–411.Google Scholar
  4. Burity H A, Ta T C, Faris MA and Coulman B E 1989 Estimation of nitrogen fixation and transfer from alfalfa to associated grasses in mixed swards under field conditions. Plant Soil 114, 249–255.Google Scholar
  5. Carranca C, de Varennes A and Rolston D E 1999 Biological nitrogen fixation estimated by 15N dilution, natural 15N abundance, and N difference techniques in a subterranean clover—grass sward under Mediterranean conditions. Eur. J. Agron. 10, 81–89.Google Scholar
  6. Danso S K A 1995 Assessment of biological nitrogen fixation. Fertil. Res. 42, 33–41.Google Scholar
  7. Danso S K A, Hardarson G and Zapata F 1993 Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation. Plant Soil 152, 25–52.Google Scholar
  8. Danso S K A, Hardarson G and Zapata F 1988 Dinitrogen fixation estimates in alfalfa—ryegrass swards using different nitrogen-15 labeling methods. Crop Sci. 28, 106–110.Google Scholar
  9. Dear B S, Cocks P S, Peoples M B, Swan A D and Smith A B 1999 Nitrogen fixation by subterranean clover (Trifolium subterraneum L.) growing in pure culture and in mixtures with varying densities of lucerne (Medicago sativa L.) or phalaris (Phalaris aquatica L.). Aust. J. Agric. Res. 50, 1047–1058.Google Scholar
  10. Edmeades D C and Goh K M 1978 Symbiotic nitrogen fixation in a sequence of pastures of increasing age measured by a 15N dilution technique. N. Z. J. Agric. Res. 21, 623–628.Google Scholar
  11. Elgersma A and Hassink J 1997 Effects of white clover (Trifolium repens L.) on plant and soil nitrogen and soil organic matter in mixtures with perennial ryegrass (Lolium perenne L.). Plant Soil 197, 177–186.Google Scholar
  12. Elgersma A, NassiriM and Schlepers H 1998 Competition in perennial ryegrass-white clover mixtures under cutting. 1. Dry-matter yield, species composition and nitrogen fixation. Grass Forage Sci. 53, 353–366.Google Scholar
  13. Evans R D 2001 Physiological mechanisms influencing plant nitrogen isotope composition. Trends Plant. Sci. 6, 121–126.Google Scholar
  14. Fagerberg B and Sundqvist U 1994 Öjebynprojektet Vallarnas botaniska sammansättning 1992—93 samt symbiotiska kvävefixering 1990—93. Röbäcksdalen meddelar 9. Sveriges Lantbruksuniversitet, Umeå. 41 pp.Google Scholar
  15. Farnham D E and George J R 1993 Dinitrogen fixation and nitrogen transfer among red clover cultivars. Can. J. Plant Sci. 73, 1047–1054.Google Scholar
  16. Farnham D E and George J R 1994 Harvest management effects on dinitrogen fixation and nitrogen transfer in red cloverorchardgrass mixtures. J. Prod. Agric. 7, 360–364.Google Scholar
  17. Goodman P J 1988 Nitrogen fixation, transfer and turnover in upland and lowland grass—clover swards, using 15N isotope dilution. Plant Soil 112, 247–254.Google Scholar
  18. Granstedt A 1990 Fallstudier av kväveförsörjning i alternativ odling (Summary: Case studies on nitrogen supply in alternative farming). Alternativ odling 4. Sveriges Lantbruksuniversitet, Uppsala. 267 pp.Google Scholar
  19. Gustavsson A-M 1989 Kvävegödslingens och klöverns betydelse i vallen (Influence of N-fertilization and clover content in grassland for cutting). Grovfoder Forskning — Tillämpning (Grass and Forage Reports) 8, 25–44.Google Scholar
  20. Halliday J and Pate J S 1976 The acetylene reduction assay as a means of studying nitrogen fixation in white clover under sward and laboratory conditions. J. Br. Grassld. Soc. 31, 29–35.Google Scholar
  21. Hansen J P and Vinther F P 2001 Spatial variability of symbiotic N2 fixation in grass-white clover pastures estimated by the 15N isotope dilution method and the natural 15N abundance method. Plant Soil 230, 257–266.Google Scholar
  22. Hardarson G and Danso S K A 1993 Methods for measuring biological nitrogen fixation in grain legumes. Plant Soil 152, 19–23.Google Scholar
  23. Hardarson G, Danso S K A and Zapata F 1988 Dinitrogen fixation measurements in alfalfa—ryegrass swards using nitrogen-15 and influence of the reference crop. Crop Sci. 28, 101–105.Google Scholar
  24. Heichel G H 1987 Legume nitrogen: symbiotic fixation and recovery by subsequent crops In Energy in Plant Nutrition and Pest Control. Ed. Helsel Z R. pp. 63–80. Elsevier, Amsterdam & Oxford.Google Scholar
  25. Heichel G H and Henjum K I 1991 Dinitrogen fixation, nitrogen transfer, and productivity of forage legume-grass communities. Crop Sci. 31, 202–208.Google Scholar
  26. Heichel G H, Vance C P, Barnes D K and Henjum K I 1985 Dinitrogen fixation, and N and dry matter distribution during 4 year stands of birdsfoot trefoil and red clover. Crop Sci. 25, 101–105.Google Scholar
  27. Högberg P 1997 15N natural abundance in soil-plant systems. New Phytol. 137, 179–203.Google Scholar
  28. Høgh-Jensen H and Kristensen E S 1995 Estimation of biological N2 fixation in a clover—grass system by the 15N dilution method and the total-N difference method. Biol. Agric. Hort. 11, 203–219.Google Scholar
  29. Høgh-Jensen H and Schjørring J K 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, 153–163.Google Scholar
  30. Høgh-Jensen H and Schjørring J K 1997 Interactions between white clover and ryegrass under contrasting nitrogen availability: N2 fixation, N fertilizer recovery, N transfer and water use efficiency. Plant Soil 197, 187–199.Google Scholar
  31. Jacot K A, Lüscher A, Nösberger J and Hartwig UA 2000 Symbiotic N2 fixation of various legume species along an altitudinal gradient in the Swiss Alps. Soil Biol. Biochem. 32, 1043–1052.Google Scholar
  32. Jørgensen F V and Ledgard S F 1997 Contribution from stolons and roots to estimates of the total amount of N2 fixed by white clover (Trifolium repens L.). Ann. Bot. (Lond.) 80, 641–648.Google Scholar
  33. Jørgensen F V, Jensen E S and Schjørring J K 1999 Dinitrogen fixation in white clover grown in pure stand and in mixture with ryegrass estimated by the immobilized 15N isotope dilution method. Plant Soil 208, 293–305.Google Scholar
  34. Kristensen E S, Høgh-Jensen H and Kristensen I S 1995 A simple model for estimation of atmospherically-derived nitrogen in grass-clover systems. Biol. Agric. Hortic. 12, 263–276.Google Scholar
  35. Kumar K and Goh K M 2000 Biological nitrogen fixation, accumulation of soil nitrogen and nitrogen balance for white clover (Trifolium repens L.) and field pea (Pisum sativum L.) grown for seed. Field Crop Res. 68, 49–59.Google Scholar
  36. Lamb J F S, Barnes D K, Russelle M P, Vance C P, Heichel G H and Henjum K I 1995 Ineffectively and effectively nodulated alfalfas demonstrate biological nitrogen fixation continues with high nitrogen fertilization. Crop Sci. 35, 153–157.Google Scholar
  37. Ledgard S F and Steele K W 1992 Biological nitrogen fixation in mixed legume/grass pastures. Plant Soil 141, 137–153.Google Scholar
  38. Ledgard S F, Brier G J and Littler R A 1987 Legume production and nitrogen fixation in hill pasture communities. N. Z. J. Agric. Res. 30, 413–421.Google Scholar
  39. Ledgard S F, Brier G J and Upsdell M P 1990 Effect of white clover cultivar on production and nitrogen fixation in clover-ryegrass swards under dairy cow grazing. N. Z. J. Agric. Res. 33, 243–249.Google Scholar
  40. Ledgard S F, Sprosen M S, Penno J W and Rajendram G S 2001 Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization. Plant Soil 229, 177–187.Google Scholar
  41. Loiseau P, Soussanna J F, Lounault F and Delpy R 2001 Soil N contributes to the oscillations of the white clover content in mixed swards of perenial ryegrass under conditions that simulate grazing over five years. Grass Forage Sci. 56, 205–217.Google Scholar
  42. Lüscher A, Hartwig U A, Suter D and Nösberger J 2000 Direct evidence that symbiotic N2 fixation in fertile grassland is an important trait for a strong response of plants to elevated atmospheric CO2. Glob. Change Biol. 6, 655–662.Google Scholar
  43. Marschner H 1995 Mineral Nutrition of Higher Plants. 2nd Edition. Academic Press, London.Google Scholar
  44. Mårtensson A M and Ljunggren H D 1984 Nitrogen fixation in an establishing alfalfa (Medicago sativa L.) ley in Sweden, estimated by three different methods. Appl. Environ. Microbiol. 48, 702–707.Google Scholar
  45. Masterson C L and Murphy P M 1976 Application of the acetylene reduction technique to the study of nitrogen fixation by white clover in the field. In Symbiotic Nitrogen Fixation in Plants. Ed. Nutman P. S. pp. 299–316. Cambridge University Press, Cambridge.Google Scholar
  46. McNeill A M and Wood M 1990 15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.). Plant Soil 128, 265–273.Google Scholar
  47. Minchin F R, Witty J F, Sheehy J E and Müller M 1983 A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. J. Exp. Bot. 34, 641–649.Google Scholar
  48. Munro J M M and Davies D A 1974 Potential pasture production in the uplands of Wales. J. Br. Grassld. Soc. 29, 213–223.Google Scholar
  49. Myrold D D, Ruess R W and Klug M J 1999 Dinitrogen fixation In Standard Soil Methods for Long-Term Ecological Research. Eds. Robertson G P, Coleman D C, Bledsoe C S and Sollins P pp 241–257. Oxford University Press, New York, NY.Google Scholar
  50. Nesheim L and 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 Agric. Scand., Sect. B, Soil Plant Sci. 44, 28–34.Google Scholar
  51. Parsons A J, Orb R J, Penning P D and Lockyer D R 1991 Uptake, cycling and fate of nitrogen in grass-clover swards continuously grazed by sheep. J. Agric. Sci. 116, 47–61.Google Scholar
  52. Peoples M B, Herridge D F and Ladha J K 1995 Biological nitrogen fixation: an efficient source of nitrogen for sustainable agricultural production? Plant Soil 174, 3–28.Google Scholar
  53. Rice WA 1980 Seasonal patterns of nitrogen fixation and dry matter production by clovers grown in the Peace river region. Can. J. Plant Sci. 60, 847–858.Google Scholar
  54. Riffkin P A, Quigley P E, Kearney G A, Cameron F J, Gault R R, Peoples M B and Thies J E 1999 Factors associated with biological nitrogen fixation in dairy pastures in south-western Victoria. Aust. J. Agric. Res. 50, 261–272.Google Scholar
  55. Seresinhe T, Hartwig U A, Kessler W and Nosberger J 1994 Symbiotic nitrogen-fixation of white clover in a mixed sward is not limited by height of repeated cutting. J. Agron. Crop Sci. 172, 279–288.Google Scholar
  56. Shearer G and Kohl D H 1986 N2-fixation in field settings: estimations based on natural 15N abundance. Aust. J. Plant Physiol. 13, 699–756.Google Scholar
  57. Sparrow S D, Cochran V L and Sparrow E B 1995 Dinitrogen fixation by seven legume crops in Alaska. Agron. J. 87, 34–41.Google Scholar
  58. Spehn E M, Joshi J, Schmid B, Alphei J and Korner C 2000 Plant diversity effects on soil heterotrophic activity in experimental grassland ecosystems. Plant Soil 224, 217–230.Google Scholar
  59. Unkovich M J and Pate J S 2000 An appraisal of recent field measurements of symbiotic N2 fixation by annual legumes. Field Crop Res. 65, 211–228.Google Scholar
  60. Vance C P 1997 Enhanced agricultural sustainability through biological nitrogen fixation In Biological Fixation of Nitrogen for Ecology and Sustainable Agriculture. Eds. Legocki A, Bothe H and Pühler A. pp. 179–186. Springer-Verlag, Berlin.Google Scholar
  61. Vessey J K 1994 Measurement of nitrogenase activity in legume root nodules: in defense of the acetylene reduction assay. Plant Soil 158, 151–162.Google Scholar
  62. Vinther F P 1998 Biological nitrogen fixation in grass-clover affected by animal excreta. Plant Soil 203, 207–215.Google Scholar
  63. Vinther F P and Jensen E S 2000 Estimating legume N2 fixation in grass-clover mixtures of a grazed organic cropping system using two 15N methods. Agric. Ecosyst. Environ. 78, 139–147.Google Scholar
  64. Walley F L, Tomm G O, Matus A, Slinkard A E and vanKessel C 1996 Allocation and cycling of nitrogen in an alfalfa-bromegrass sward. Agron. J. 88, 834–843.Google Scholar
  65. Warembourg F R, Lafont F and Fernandez M P 1997 Economy of symbiotically fixed nitrogen in red clover (Trifolium pratense L.). Ann. Bot. (Lond.) 80, 515–523.Google Scholar
  66. Weaver R W and Danso S K A 1994 Dinitrogen fixation In Methods of Soil Analysis Part 2 — Microbiological and Biochemical Properties. Eds. Weaver R W, Angle J S and Bottomley P S. pp. 557–573. Soil Science Society of America, Madison, WI.Google Scholar
  67. Whitehead D C 1995 Grassland Nitrogen. CAB International, Wallingford.Google Scholar
  68. Witty J F 1983a Estimating N2-fixation in the field using 15N fertilizer: some problems and solutions. Soil Biol. Biochem. 15, 631–639.Google Scholar
  69. Witty J F 1983b Measurement of N2 fixation by 15N fertiliser dilution; problems of declining soil enrichment In Temperate Legumes: Physiology, Genetics and Nodulation. Eds. Jones D G and Davies D R. pp. 253–267. Pitman Advanced Publishing Program, London.Google Scholar
  70. Witty J F and Minchin F R 1988 Measurement of nitrogen fixation by the acetylene reduction assay; myths and mysteries In Nitrogen Fixation by Legumes in Mediterranean Agriculture. Eds. Beck D P and Materon L A. pp. 331–344. Martinus Nijhoff Publishers, Dordrecht.Google Scholar
  71. Wivstad M, Mårtensson A M and Ljunggren H D 1987 Field measurement of symbiotic nitrogen fixation in an established lucerne ley using 15N and an acetylene reduction method. Plant Soil 97, 93–104.Google Scholar
  72. Yoneyama T, Fujita K, Yoshida T, Matsumoto T, Kambayashi I and Yazaki J 1986 Variation in natural abundance of 15N among plant parts and in 15N/14N fractionation during N2 fixation in the legume-rhizobia symbiotic system. Plant Cell Physiol. 27, 791–799.Google Scholar
  73. Zanetti S, Hartwig U A, Lüscher A, Hebeisen T, Frehner M, Fischer B U, Hendrey G R, Blum H and Nösberger J 1996 Stimulation of symbiotic N2 fixation in Trifolium repens L. under elevated atmospheric pCO2 in a grassland ecosystem. Plant Physiol. 112, 575–583.Google Scholar

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© Kluwer Academic Publishers 2003

Authors and Affiliations

  1. 1.Department of Agricultural Research for Northern Sweden, Crop Science SectionSwedish University of Agricultural SciencesUmeåSweden

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