Plant and Soil

, Volume 426, Issue 1–2, pp 413–428 | Cite as

Changes in 15N natural abundance of biologically fixed N2 in soybean due to shading, rhizobium strain and plant growth stage

  • Karla E. C. Araujo
  • Carlos Vergara
  • Ana Paula Guimarães
  • Janaina R. C. Rouws
  • Claudia P. Jantalia
  • Segundo Urquiaga
  • Bruno J. R. Alves
  • Robert M. Boddey
Regular Article



The evaluation of 15N abundance of N derived from biological N2 fixation (BNF) in legume shoots (‘B S value) is essential for quantifying BNF inputs to field-grown legumes. The aim of this study was to investigate the impact of shading, development stage of soybean (Glycine max) and rhizobium strain on the ‘B S value.


Soybean plants were grown in pots of autoclaved sand/perlite mixture in the open field. Plants were harvested at weekly intervals from 46 days after planting (DAP) to 75 DAP. All material was analysed for N and 15N abundance. ‘B S was calculated assuming 50% of seed N was translocated to the shoots.


‘B S was stable until 60 DAP but subsequently increased for the three strains tested. Nodule efficiency (N2 fixed g DM nodule−1) was greatly increased by shading and was significantly different between Bradyrhizobium species. ‘B S was greatly increased by shading.


We recommend that ‘B S should be evaluated on plants of the same development stage and light intensity as those where BNF is quantified in the field. Different Bradyrhizobium strains make a large impact on ‘B S and may lead to considerable errors in estimation of BNF inputs to plants with high %N derived from BNF.


15N natural abundance B value Biological nitrogen fixation Bradyrhizobium spp. Light intensity Soybean 



% N derived from air


Biological nitrogen fixation


B value of shoot tissue


B value of whole plant


Days after planting


dry matter



The authors thank Dr Nivaldo Schultz for arranging the irrigation/nutrient solution system, Alderi F. da Silva, Aurelio de S. Chagas, Cláudio P. Ferreira, Enivaldo Maia, Ernani C. de Meirelles and Roberto C. da S. Ramos for help in setting up and tending to the plants and to Dr Renato M. da Rocha for meticulous work on the isotope-ratio mass spectrometers. The authors KECA e CV gratefully acknowledge postgraduate fellowships from the Ministry of Education (CAPES) and APG, CPJ, SU, BJRA and RMB fellowships from the National Research Council (CNPq) and the Rio State Research Foundation (FAPERJ). The work was funded by CNPq, FAPERJ and Embrapa.

Supplementary material

11104_2018_3627_MOESM1_ESM.docx (21 kb)
ESM 1 (DOCX 21 kb)
11104_2018_3627_MOESM2_ESM.docx (23 kb)
ESM 2 (DOCX 22 kb)
11104_2018_3627_MOESM3_ESM.docx (24 kb)
ESM 3 (DOCX 24 kb)
11104_2018_3627_MOESM4_ESM.docx (24 kb)
ESM 4 (DOCX 23 kb)
11104_2018_3627_MOESM5_ESM.docx (23 kb)
ESM 5 (DOCX 22 kb)
11104_2018_3627_MOESM6_ESM.docx (21 kb)
ESM 6 (DOCX 20 kb)


  1. Arnold S, Schepers J (2004) A simple roller-mill grinding procedure for plant and soil samples. Commun Soil Sci Plant Anal 35:537–545. CrossRefGoogle Scholar
  2. Bergersen F, Turner G, Amarger N, Mariotti F, Mariotti A (1986) Strain of rhizobium lupini determines natural abundance of 15N in root nodules of Lupinus spp. Soil Biol Biochem 18:97–101. CrossRefGoogle Scholar
  3. Bergersen F, Peoples M, Turner G (1988) Isotopic discriminations during the accumulation of nitrogen by soybeans. Aust J Plant Physiol 15:407–420. CrossRefGoogle Scholar
  4. Boddey RM, Urquiaga S (1992) Calculations and assumptions involved in the use of the ‘A-value’and 15N isotope dilution techniques for the estimation of the contribution of plant-associated biological N2 fixation. Plant Soil 145:151–155. CrossRefGoogle Scholar
  5. Boddey RM, Müller SH, Alves BJ (1995) Estimation of the contribution of biological N2 fixation to twoPhaseolus vulgaris genotypes using simulation of plant nitrogen uptake from 15N-labelled soil. Fert Res 45:169–185. CrossRefGoogle Scholar
  6. Boddey RM, Peoples MB, Palmer B, Dart PJ (2000) Use of the 15N natural abundance technique to quantify biological nitrogen fixation by woody perennials. Nutr Cycl Agroecosyst 57:235–270. CrossRefGoogle Scholar
  7. Cadisch G, Hairiah K, Giller K (2000) Applicability of the natural 15N abundance technique to measure N2 fixation in Arachis hypogaea grown on an Ultisol NJAS-Wageningen. J Life Sci 48:31–45. Google Scholar
  8. Chalk PM (1985) Estimation of N2 fixation by isotope dilution: an appraisal of techniques involving 15N enrichment and their application. Soil Biol Biochem 17:389–410. CrossRefGoogle Scholar
  9. Delamuta JRM, Ribeiro RA, Ormeño-Orrillo E, Melo IS, Martínez-Romero E, Hungria M (2013) Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov. Int J Syst Evol Microbiol 63:3342–3351. CrossRefPubMedGoogle Scholar
  10. Döbereiner J, Franco AA, Guzmán I (1970) Estirpes de Rhizobium japonicum de excepcional eficiência. Pesq Agrop Brasileira 5:155–161Google Scholar
  11. Doughton J, Vallis I, Saffigna P (1992) An indirect method for estimating 15N isotope fractionation during nitrogen fixation by a legume under field conditions. Plant Soil 144:23–29. CrossRefGoogle Scholar
  12. Dudman WF, Brockwell J (1968) Ecological studies of root-nodule bacteria introduced into field environments. I. A survey of field performance of clover inoculants by gel immune diffusion serology. Aust J Agr Res 19:739–747. CrossRefGoogle Scholar
  13. Egli D (1997) Cultivar maturity and response of soybean to shade stress during seed filling. Field Crop Res 52:1–8. CrossRefGoogle Scholar
  14. Giongo A, Ambrosini A, Freire JRJ, Zanettini MHB, Passaglia LMP (2007) Amplification of 16S rRNA gene sequences to differentiate two highly related bradyrhizobia species. Pesq Agrop Brasileira 42:1361–1364. CrossRefGoogle Scholar
  15. Guimarães AP, RFde M, Urquiaga S, Boddey RM, BJR A (2008) Bradyrhizobium strain and the 15N natural abundance quantification of biological N2 fixation in soybean. Sci Agric 65:516–524. CrossRefGoogle Scholar
  16. Hansen AP, Gresshoff PM, Pate JS, Day DA (1990) Interactions between irradiance levels, nodulation and nitrogenase activity of soybean cv. Bragg and a supernodulating mutant. J Plant Physiol 136:172–179. CrossRefGoogle Scholar
  17. Hanway JJ, Thompson HE (1967) How a soybean plant develops. Special report 53 rev. Iowa State University of Science and Technology, cooperative extension service, Ames, IO, 18p. (available at:
  18. Hardarson G, Zapata F, Danso S (1988) Dinitrogen fixation measurements in alfalfa- ryegrass swards using nitrogen-15 and influence of the reference crop. Crop Sci 19:101–105. CrossRefGoogle Scholar
  19. Herridge DF, Peoples MB, Boddey RM (2008) Marschner review: global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 311:1–18. CrossRefGoogle Scholar
  20. Hobbie EA, Ouimette AP (2009) Controls of nitrogen isotope patterns in soil profiles. Biogeochemistry 95:355–371. CrossRefGoogle Scholar
  21. Högberg P (1997) Tansley review no. 95. 15N natural abundance in soil–plant systems. New Phytol 137:179–203. CrossRefGoogle Scholar
  22. Hungria M, Boddey LH, Santos MA, Vargas MAT (1998) Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains. Biol Fertil Soils 127:393–399. CrossRefGoogle Scholar
  23. Hungria M, Franchini JC, Campo RJ, Crispino CC, Moraes JZ, Sibaldelli RNR, Mendes IC, Arihara J (2006) Nitrogen nutrition of soybean in Brazil: contributions of biological N2 fixation and N fertilizer to grain yield. Can J Plant Sci 86:927–939. CrossRefGoogle Scholar
  24. IBGE-LSPA (2017) Levantamento Sistemático da Produção Agrícola:setembro 2017 Accessed 30 octuber 2017
  25. Kakiuchi J, Kobata T (2004) Shading and thinning effects on seed and shoot dry matter increase in determinate soybean during the seed-filling period. Agron J 96:398–405. CrossRefGoogle Scholar
  26. Kakiuchi J, Kobata T (2006) The relationship between dry matter increase of seed and shoot during the seed-filling period in three kinds of soybeans [Glycine max] with different growth habits subjected to shading and thinning. Plant Prod Sci 9:20–26. CrossRefGoogle Scholar
  27. Kaschuk G, Kuyper TW, Leffelaar PA, Hungria M, Giller KE (2009) Are the rates of photosynthesis stimulated by the carbon sink strength of rhizobial and arbuscular mycorrhizal symbioses? Soil Biol Biochem 41:1233–1244. CrossRefGoogle Scholar
  28. Kaschuk G, Hungria M, Leffelaar PA, Giller KE, Kuyper TW (2010) Differences in photosynthetic behaviour and leaf senescence of soybean (Glycine max [L.] Merril) dependent on N2 fixation or nitrate supply. Plant Biol 12:60–69. CrossRefPubMedGoogle Scholar
  29. Kaschuk G, Xinyou Y, Hungria M, Leffelaar PA, Giller KE, Kuyper TW (2012) Photosynthetic adaptation of soybean due to varying effectiveness of N2 fixation by two distinct Bradyrhizobium japonicum strains. Environ Exp Bot 76:1–6. CrossRefGoogle Scholar
  30. Kishinevsky B, Gurfel D (1980) Evaluation of enzyme-linked immunosorbent assay (ELISA) for serological identification of different rhizobium strains. J Appl Bacteriol 149:517–526. CrossRefGoogle Scholar
  31. Kurosaki H, Yumoto S (2003) Effects of low temperature and shading during flowering on the yield components in soybeans. Plant Prod Sci 6:17–23. CrossRefGoogle Scholar
  32. Means UM, Johnson HW, Date RA (1964) Quick serological method of classifying strains of' Rhizobium japonicum in nodules. J Bacteriol 87:547–553PubMedPubMedCentralGoogle Scholar
  33. Nascimento EC do (2011) Potencial desnitrificador de estirpes de Bradyrhizobium recomendadas para a cultura da soja. MSc Dissertation, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, BrazilGoogle Scholar
  34. Neves MC, Didonet AD, Duque FF, Döbereiner J (1985) Rhizobium strain effects on nitrogen transport and distribution in soybeans. J Exp Bot 36:1179–1192. CrossRefGoogle Scholar
  35. Norris D, Date R (1976) Legume bacteriology Tropical Pastures Research Principles and Methods p:134–174, DOI:
  36. Odeleye et al (2001) The effect of light intensity on the growth, development and yield of soybean in southwest Nigeria. Afr Crop Sci J 9:577–590. CrossRefGoogle Scholar
  37. Okito A, Alves B, Urquiaga S, Boddey R (2004) Isotopic fractionation during N2 fixation by four tropical legumes. Soil Biol Biochem 36:1179–1190. CrossRefGoogle Scholar
  38. Osei O, Simões-Araújo JL, Zilli JE, Boddey RM, Ahiabor BDK, Abaidoo RC, Rouws LFM (2017) PCR assay for direct specific detection of Bradyrhizobium elite strain BR 3262 in root nodule extracts of soil-grown cowpea. Plant Soil 417:535–548. CrossRefGoogle Scholar
  39. Pauferro N, Guimarães AP, Jantalia CP, Urquiaga S, Alves BJ, Boddey RM (2010) 15 N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant. Soil Biol Biochem 42:1694–1700. CrossRefGoogle Scholar
  40. Polthanee A, Promsaena K, Laoken A (2011) Influence of low light intensity on growth and yield of four soybean cultivars during wet and dry seasons of Northeast Thailand. Agric Sci 2:61–67. Google Scholar
  41. R Development Core Team (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing Vienna, Austria. Google Scholar
  42. Saito M, Kato T, Saito M (1994) Effects of low temperature and shade on relationships between nodulation, vesicular-arbuscular mycorrhizal infection, and shoot growth of soybeans. Biol Fertil Soils 17:206–211. CrossRefGoogle Scholar
  43. Santos VA, Neves MCP, Rumjanek NG (1997) Differential symbiotic efficiency by shading of soybean nodulated by B. japonicum and B. elkanii strains. Soil Biol Biochem 29:1015–1018. CrossRefGoogle Scholar
  44. Santos MA, Vargas MAT, Hungria M (1999) Characterization of soybean Bradyrhizobium strains adapted to the Brazilian savannas. FEMS Microbiol Ecol 30:261–272. CrossRefPubMedGoogle Scholar
  45. Scott AJ, Knott M (1974) A cluster analysis method for grouping means in the analysis of variance. Biometrics 30:507–512. CrossRefGoogle Scholar
  46. Shearer G, Kohl DH (1986) N2-fixation in field settings: estimations based on natural 15N abundance. Func Plant Biol 13:699–756. Google Scholar
  47. Steele K, Bonish P, Daniel RM, O'hara G (1983) Effect of rhizobial strain and host plant on nitrogen isotopic fractionation in legumes. Plant Physiol 72:1001–1004. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Unkovich M (2013) Isotope discrimination provides new insight into biological nitrogen fixation. New Phytol 198:643–646. CrossRefPubMedGoogle Scholar
  49. Unkovich MJ, Pate JS, Sanford P, Armstrong EL (1994) Potential precision of the δ15N natural abundance method in field estimates of nitrogen fixation by crop and pasture legumes in south-west. Aust J Agric Res 45:119–132. CrossRefGoogle Scholar
  50. Unkovich MJ, Herridge DF, Peoples MB, Cadisch G, Boddey RM, Giller KE, Alves BJR, Chalk PM (2008) Measuring plant-associated nitrogen fixation in agricultural systems. ACIAR monograph no. 136, Canberra, p 258Google Scholar
  51. Van Berkum P, Elia P, Song Q, Eardly BD (2012) Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). MPMI 25:321–330. CrossRefPubMedGoogle Scholar
  52. Yates RJ, Howieson JG, Hungria M, Bala A, O’Hara GW, Terpolilli J (2016) Authentication of rhizobia and assessment of the legume symbiosis in controlled plant growth systems. In: Howieson JG, Dilworth MJ (eds) Working with rhizobia. Australian Centre for International Agricultural Research, Canberra, pp 73–108 (Available at Google Scholar
  53. Yoneyama T, Fujita K, Yoshida T, Matsumoto T, Kambayashi I, 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. CrossRefGoogle Scholar
  54. Zotarelli L, Zatorre NP, Boddey RM, Urquiaga S, Jantalia CP, Franchini JC, Alves BJR (2012) Influence of no-tillage and frequency of a green manure legume in crop rotations for balancing N outputs and preserving soil organic C stocks. Field Crop Res 132:185–195. CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Karla E. C. Araujo
    • 1
  • Carlos Vergara
    • 2
  • Ana Paula Guimarães
    • 3
  • Janaina R. C. Rouws
    • 3
  • Claudia P. Jantalia
    • 3
  • Segundo Urquiaga
    • 3
  • Bruno J. R. Alves
    • 3
  • Robert M. Boddey
    • 3
  1. 1.Departamento de FitotecniaUniversidade Federal Rural do Rio de JaneiroSeropédicaBrazil
  2. 2.Departamento de SolosUniversidade Federal Rural do Rio de JaneiroSeropédicaBrazil
  3. 3.Embrapa AgrobiologiaSeropédicaBrazil

Personalised recommendations