Dinitrogen fixation in white clover (Trifolium repens L.) grown in pure stand and mixture with perennial ryegrass (Lolium perenne L.) was determined in the field using 15N isotope dilution and harvest of the shoots. The apparent transfer of clover N to perennial ryegrass was simultaneously assessed. The soil was labelled either by immobilizing 15N in organic matter prior to establishment of the sward or by using the conventional labelling procedure in which 15N fertilizer is added after sward establishment. Immobilization of 15N in the soil organic matter has not previously been used in studies of N2 fixation in grass/clover pastures. However, this approach was a successful means of labelling, since the 15N enrichment only declined at a very slow rate during the experiment. After the second production year only 10–16% of the applied 15N was recovered in the harvested herbage. The two labelling methods gave, nonetheless, a similar estimate of the percentage of clover N derived from N2 fixation. In pure stand clover, 75–94% of the N was derived from N2 fixation and in the mixture 85–97%. The dry matter yield of the clover in mixture as percentage of total dry matter yield was relatively high and increased from 59% in the first to 65% in the second production year. The average daily N2 fixation rate in the mixture-grown clover varied from less than 0.5 kg N ha−1 day−1 in autumn to more than 2.6 kg N ha−1 day−1 in June. For clover in pure stand the average N2 fixation rate was greater and varied between 0.5 and 3.3 kg N ha−1 day−1, but with the same seasonal pattern as for clover in mixture. The amount of N fixed in the mixture was 23, 187 and 177 kg N ha−1 in the seeding, first and second production year, respectively, whereas pure stand clover fixed 28, 262 and 211 kg N ha−1 in the three years. The apparent transfer of clover N to grass was negligible in the seeding year, but clover N deposited in the rhizosphere or released by turnover of stolons, roots and nodules, contributed 19 and 28 kg N ha−1 to the grass in the first and second production year, respectively.
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Berg B, Müller M and Wessén B 1987 Decomposition of red clover (Trifolium pratense) roots. Soil Biol. Biochem. 19, 589–593.
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.
Boller B C and Nösberger J 1988 Influence of dissimilarities in temporal and spatial N-uptake patterns on 15N-based estimates of fixation and transfer of N in ryegrass-clover mixtures. Plant Soil 112, 167–175.
Breland T A 1994 Measured and predicted mineralization of clover green manure at low temperatures at different depths in two soils. Plant Soil 166, 13–20.
Chalk P M 1985 Estimation of N2 fixation by isotope dilution: An appraisal of techniques involving 15N enrichment and their application. Soil Biol. Biochem. 17, 389–410.
Chalk P M 1996 Nitrogen transfer from legumes to cereals in intercropping. In Dynamics of roots and Nitrogen in cropping Systems of the Semi-Arid Tropics. Eds. O Ito, J J Johansen, K Adu-Gyamfi, J V D K Katayama, K Rao and T J Rego. pp. 351–374. JIRCAS, Japan.
Crouchley G 1979 Nitrogen fixation in pasture. VIII. Wairarapa plains dryland. Masterton. N.Z.J. Exp. Agric. 7, 31–33.
Fried M and Middelboe V 1977 Measurement of the amount of nitrogen fixed by a legume crop. Plant Soil 47, 713–715.
Frame J and Newbould P 1986 Agronomy of white clover. Adv. Agron. 40, 1–89.
Giller K E and Witty J F 1987 Immobilized 15N-fertilizer sources improve the accuracy of field estimates of N2-fixation by isotope dilution. Soil Biol. Biochem. 19, 459–463.
Halberg N, Kristensen E S and Kristensen I S 1995 Nitrogen turnover on organic and conventional mixed farms. J. Agric. Environ. Ethics 8, 30–51.
Harris S L and Clark D A 1996 Effect of high rates of nitrogen fertiliser on white clover growth, morphology, and nitrogen fixation activity in grazed dairy pasture in northern New Zealand. N.Z.J. Agric. Res. 39, 149–158.
Hatch D J and Murray P J 1994 Transfer of nitrogen from damaged roots of white clover (Trifolium repens L.) to closely associated roots of intact perennial ryegrass (Lolium perenne L.). Plant Soil 166, 181–185.
Haystead A, Malajczuk N and Grove T S 1988 Underground transfer of nitrogen between pasture plants infected with vesiculararbuscular mycorrhizal fungi. New Phytol. 108, 417–423.
Høgh-Jensen H and Schjoerring J K 1994 Measurements 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.
Jensen E S 1991 Evaluation of automated analysis of 15N and total N in plant and soil. Plant Soil 133, 83–92.
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. 80, 641–648.
Ledgard S F 1991 Transfer of fixed nitrogen from white clover to associated grasses in swards grazed by dairy cows, estimated using 15N Methods. Plant and Soil 131, 215–223.
Ledgard S F, Freney J R and Simpson J R 1985 Assessing nitrogen transfer from legumes to associated grasses. Soil Biol. Biochem. 17, 575–577.
Ledgard S F, Sprosen M S and Steele K W 1996 Nitrogen fixation by nine white clover cultivars in grazed pasture, as affected by nitrogen fertilization. Plant Soil 178, 193–203.
Ledgard S F and Steele K W 1992 Biological nitrogen fixation in mixed legume/grass pastures. Plant Soil 141, 137–153.
Legg J O and Sloger C 1975 A tracer method for determining symbiotic nitrogen fixation in field studies. In Proc. Second Int. Conf. Stable Isotopes. Eds. E R Klein and P D Klein. pp 661–666. Oak Brook, Illinois.
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.
Murray P J and Hatch D J 1994 Sitona weevils (Coleoptera: Curculionidae) as agents for rapid transfer of nitrogen from white clover (Trifolium repens L.) to perennial ryegrass (Lolium perenne L.). Ann. Appl. Biol. 125, 29–33.
Nesheim L and Boller B C 1991 Nitrogen fixation by white clover when competing with grasses at moderately low temperatures. Plant Soil 133, 47–56.
Powlson D S and Barraclough D 1993 Mineralization and assimilation in soil-plant systems InNitrogen Isotope Techniques. Eds. R Knowles and T H Blackburn. pp 209–242. Academic Press, London, UK.
Russelle M P, Allan D L and Gourley C J P 1994 Direct assessment of symbiotically fixed nitrogen in the rhizosphere of alfalfa. Plant Soil 159, 233–243.
SAS 1989 SAS/STAT Users Guide, Version 6, Fourth edition, Volume 2. Cary, North Carolina, USA.
Svensson K S, Clarholm M 1994 Dynamics of biomass, nitrogen and carbon in shoots and roots of a ryegrass catch crop during winter. Swe. J. Agric. Res. 24, 77–84.
Ta T C and Faris M A 1987 Species variation in the fixation and transfer of nitrogen from legumes to associated grasses. Plant and Soil 98, 265–274.
Talbott H, Kenworthy W J and Legg J O 1982 Field comparison of the nitrogen-15 and difference methods of measuring nitrogen fixation. Agron. J. 74, 799–804.
Vallis I, Haydock K P, Ross P J and Henzell E F 1967 Isotopic studies on the uptake of nitrogen by pasture plants. III. The uptake of small additions of 15N-labelled fertilizer by Rhodes grass and Townsville lucerne. Aust. J. Agric. Res. 18, 865–877.
Vallis I, Henzell E F and Evans T R 1977 Uptake of soil nitrogen by legumes in mixed swards. Aust. J. Agric. Res. 28, 413–425.
van Veen J A, Ladd J N and Amato M. 1985 Turnover of carbon and nitrogen through the microbial biomass in a sandy loam and a clay soil incubated with [14C(U)]Glucose and [15N](NH4)2SO4 under different moisture regimes. Soil Biol. Biochem. 17, 747–756.
Wacquant J P, Ouknider M and Jacquard P 1989 Evidence for periodic excretion of nitrogen by roots of grass-legume associations. Plant Soil 116, 57–68.
Wardle D A and Greenfield L G 1991 Release of mineral nitrogen from plant root nodules. Soil Biol. Biochem. 23, 827–832.
Weaver R W 1986 Measurement of biological dinitrogen fixation in the field In Field Measurements of Dinitrogen Fixation and Denitrification. Eds. R D Hauck and R W Weaver. pp 1-10. SSSA Spec. Pub. no. 18. Madison, USA.
Werner W and Lopotz H 1995 Field and pot experiments on the influence of different N delivery potential of soil on symbiotic N2 fixation of red clover In Nitrogen Fixation: Fundamentals and Applications. Proceedings of the 10th International Congress on Nitrogen Fixation, St. Peterbourg, May 28-June 3, 1995. Eds. I Tikhonovich, N A Provorov, V I Romanov and W E Newton. pp 746. Klüwer Academic Publishers, Dordrecht, The Netherlands.
Witty J F 1983 Estimating N2-fixation in the field using 15Nlabelled fertilizer: Some problems and solutions. Soil Biol. and Biochem. 15, 631–639.
Witty J F, Rennie R J and Atkins C A 198815N addition methods for assessing N2 fixation under field conditions. In World Crops: Cool Season Food Legumes. Ed. R J Summerfield. Kluwer Academic Publishers, London UK.
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Jørgensen, F.V., Jensen, E.S. & Schjoerring, J.K. Dinitrogen fixation in white clover grown in pure stand and mixture with ryegrass estimated by the immobilized 15N isotope dilution method. Plant and Soil 208, 293–305 (1999). https://doi.org/10.1023/A:1004533430467
- grass/clover mixtures
- Lolium perenne L.
- 15N immobilization
- 15N isotope dilution
- N2 fixation
- Trifolium repens L