Plant and Soil

, Volume 419, Issue 1–2, pp 25–39 | Cite as

Legume inoculant application methods: effects on nodulation patterns, nitrogen fixation, crop growth and yield in narrow-leaf lupin and faba bean

  • Matthew D DentonEmail author
  • Lori A Phillips
  • Mark B Peoples
  • David J Pearce
  • Antony D Swan
  • Pauline M Mele
  • John Brockwell
Regular Article



Liquid and granular rhizobial inoculants have some practical advantages for delivering rhizobial inoculants to legume crops in terms of ease-of-use and in separating rhizobia from potentially harmful seed-applied pesticides. The aim of this research was to determine whether inoculant application methodologies altered the patterns of nodulation on roots, inputs of symbiotic nitrogen (N2) fixation, the accumulation of legume shoot dry matter (DM), grain yield, and grain nitrogen (N).


Eight field experiments were established at four different locations in south-eastern Australia to quantify the response of lupin (Lupinus angustifolius L.) and faba bean (Vicia faba L.) to three inoculant application methods (on-seed application as a peat slurry, in-furrow peat inoculant delivered as a liquid suspension at seeding, in-furrow peat granules delivered at seeding) compared with uninoculated treatments. N2 fixation was assessed using the 15N natural abundance method and canola was included as a non-legume reference.


Inoculation significantly improved crown nodulation, from 0.05 to 13 nodules plant−1 in lupin at two sites and from 0.17 to 21.3 nodules plant−1 in faba bean at three sites. Nodulation responses were decreased for faba bean treatments at sites with low pH, and for both lupin and faba bean at sites where soils contained large populations of naturally-occurring rhizobia. Inoculation increased grain yield from 0.48 to 1.94 t ha−1 in faba bean relative to uninoculated treatments at two sites; N2 fixation increased by 175 kg N ha−1 in lupin at one site and by 46 to 280 kg N ha−1 in faba bean at two sites. The different inoculant application methods led to minor differences in crown and lateral root nodulation patterns but only impacted N2 fixation and grain yield at one site with faba bean, where peat slurry treatments had 186 to 195 kg N ha−1 more N2 fixation than other treatments and peat slurry and granules provided 0.8 to 1.0 t ha−1 more grain yield than liquid inoculants.


On-seed application of peat slurry always provided the best nodulation, grain yield and N2 fixation. Small changes in nodulation patterns using in-furrow inoculants only resulted in reduced N2 fixation in faba bean at one site. At that site faba bean grain yield was reduced by 1.0 t ha−1 in liquid inoculant treatments, compared with on-seed peat slurry treatments.


15N natural abundance Faba bean Inoculation Lupin Nodulation Rhizobia 



This work was funded through the Grains Research and Development Corporation (GRDC) as part of the National Rhizobium Program and Nitrogen Fixation Program (UMU00032 and UA000138) and crop sequencing project (CSP000146). We thank John and Jan Harris, Boorhaman North, and Andrew Godde, Culcairn, who generously provided land for the study sites. Bernadette Carmody (Agriculture Victoria) and Laura Goward (CSIRO) provided assistance with field work and 15N analyses and are gratefully acknowledged. Thang Lai provided assistance with Fig. 1.

Supplementary material

11104_2017_3317_MOESM1_ESM.docx (19 kb)
ESM 1 (DOCX 19 kb)


  1. Abdelhamid MT, Palta JA, Veneklaas EJ, Atkins C, Turner NC, Siddique KHM (2011) Drying the surface soil reduces the nitrogen content of faba bean (Vicia faba L.) through a reduction in nitrogen fixation. Plant Soil 339:351–362CrossRefGoogle Scholar
  2. Bergersen FJ, Turner GL, Peoples MB, Gault RR, Morthorpe LJ, Brockwell J (1992) Nitrogen fixation during vegetative and reproductive growth of irrigated soybeans in the field: application of δ15N methods. Aust J Agric Res 43:145–153CrossRefGoogle Scholar
  3. Bhuvaneswari TV, Bhagwat AA, Bauer WD (1981) Transient susceptibility of root cells in four common legumes to nodulation by rhizobia. Plant Physiol 68:1144–1149CrossRefPubMedPubMedCentralGoogle Scholar
  4. Brockwell J (1963) Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Appl Microb 11:377–383Google Scholar
  5. Brockwell J, Gault RR, Chase DL, Hely FW, Zorin M, Corbin JE (1980) An appraisal of practical alternatives to legume seed inoculation:field experiments on seed bed inoculation with solid and liquid inoculants. Aust J Agric Res 31:47–60CrossRefGoogle Scholar
  6. Brockwell J, Gault RR, Herridge DF, Morthorpe LJ, Roughley RJ (1988a) Studies on alternative means of legume inoculation: microbiological and agronomic appraisals of commercial procedures for inoculating soybeans with Bradyrhizobium japonicum. Aust J Agric Res 39:965–972CrossRefGoogle Scholar
  7. Brockwell J, Herridge DF, Morthorpe LJ, Roughley RJ (1988b) Numerical effects of rhizobium population on legume symbiosis. In: Beck DP, Materon LA (eds) Nitrogen fixation by legumes in Mediterranean Agriculture. International Centre for Agricultural Research in Dry Areas, Netherlands, pp 179–193CrossRefGoogle Scholar
  8. Brockwell J, Bottomley PJ, Thies JE (1995) Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant Soil 174:143–180CrossRefGoogle Scholar
  9. Deaker R, Roughley RJ, Kennedy IR (2004) Legume seed inoculation technology: a review. Soil Biol Biochem 36:1275–1288CrossRefGoogle Scholar
  10. Denton MD, Pearce DJ, Ballard RA, Hannah MC, Mutch LA, Norng S, Slattery JF (2009) A multi-site field evaluation of granular inoculants for legume nodulation. Soil Biol Biochem 41:2508–2516CrossRefGoogle Scholar
  11. Denton MD, Coventry DR, Bellotti WD, Howieson JG (2011) Nitrogen fixation in annual Trifolium species in alkaline soils as assessed by the 15N natural abundance method. Crop Pasture Sci 62:712–720CrossRefGoogle Scholar
  12. Denton MD, Pearce DJ, Peoples MB (2013) Nitrogen contributions from faba bean (Vicia faba L.) reliant on soil rhizobia or inoculation. Plant Soil 365:363–374CrossRefGoogle Scholar
  13. Drew E, Herridge D, Ballard R, O’Hara G, Deaker R, Denton M, Yates R, Gemell G, Hartley E, Phillips L, Seymour N, Howieson J, Balllard N (2014) Inoculating legumes: a practical guide. Grains Research and Development Corporation. Kingston, AustraliaGoogle Scholar
  14. Gan Y, Selles F, Hanson KG, Zentner RP, McConkey BG, McDonald CL (2005) Effect of formulation and placement of Mesorhizobium inoculants for chickpea in the semiarid Canadian prairies. Can J Plant Sci 85:555–560CrossRefGoogle Scholar
  15. Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron 4:1–9Google Scholar
  16. Hardarson G, Golbs M, Danso SKA (1989) Nitrogen fixation in soybean (Glycine max L. Merrill) as affected by nodulation patterns. Soil Biol. Biochemist 21:783–787Google Scholar
  17. Herridge DF (2008) Inoculation technology for legumes. In ‘Leguminous nitrogen-fixing symbioses’. Nitrogen fixation: origins, applications and research progress, vol. 7. (Eds MJ Dilworth, EK James, JI Sprent, WE Newton.) pp. 77-115. (springer Netherlands — springer Verlag: Heidelberg)Google Scholar
  18. Hungria M, Vargas MAT (2000) Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil. Field Crops Res 65:151–164CrossRefGoogle Scholar
  19. Isbell R (1996) The Australian soil classification. Australian soil and land survey handbook. CSIRO, MelbourneGoogle Scholar
  20. Khan DF, Peoples MB, Schwenke GD, Felton WL, Chen DL, Herridge DF (2003) Effects of below-ground nitrogen on N balances of field-grown fababean, chickpea, and barley. Aust J Agric Res 54:333–340CrossRefGoogle Scholar
  21. Kyei-Boahen S, Slinkard AE, Walley FL (2002) Evaluation of rhizobial inoculation methods for chickpea. Agron J 94:851–859CrossRefGoogle Scholar
  22. McNeill AM, Fillery IRP (2008) Field measurement of lupin belowground nitrogen accumulation and recovery in the subsequent cereal-soil system in a semi-arid Mediterranean-type climate. Plant Soil 302:297–316CrossRefGoogle Scholar
  23. Nleya T, Walley F, Vandenberg A (2001) Response of four common bean cultivars to granular inoculant in a short-season dryland production system. Can J Plant Sci 81:385–390CrossRefGoogle Scholar
  24. Peoples MB, Brockwell J, Herridge DF, Rochester IJ, Alves BJR, Urquiaga S, Boddey RM, Dakora FD, Bhattarai S, Maskey SL, Sampet C, Rerkasem B, Khan DF, Hauggaard-Nielsen H, Jensen ES (2009) The contributions of nitrogen-fixing crop legumes to the productivity of agricultural systems. Symbiosis 48:1–17CrossRefGoogle Scholar
  25. Remmler L, Clairmont L, Rolland-Lagan A-G, Guinel FC (2014) Standardized mapping of nodulation patterns in legume roots. New Phytol 202:1083–1094CrossRefPubMedGoogle Scholar
  26. Rochester IJ, Peoples MB, Constable GA, Gault RR (1998) Faba beans and other legumes add nitrogen to irrigated cotton cropping systems. Aust J Exp Ag 38:253–260CrossRefGoogle Scholar
  27. Ronner E, Franke AC, Vanlauwe B, Dianda M, Edeh E, Ukem B, Bala A, van Heerwaarden J, Giller KE (2016) Understanding variability in soybean yield and response to P-fertilizer and rhizobium inoculants on farmers’ fields in northern Nigeria. Field Crops Res 186:133–145CrossRefGoogle Scholar
  28. Roughley RJ, Gemell LG, Thompson JA, Brockwell J (1993) The number of Bradyrhizobium sp. (Lupinus) applied to seed and its effect on rhizosphere colonization, nodulation and yield of lupin. Soil Biol Biochem 25:1453–1458CrossRefGoogle Scholar
  29. Thilakaranthna MS, Raizada MN (2017) A meta-analysis of the effectiveness of diverse rhizobia inoculants on soybean traits under field conditions. Soil Biol Biochem 105:177–196CrossRefGoogle Scholar
  30. Thomson BD, Siddique KHM, Barr MD, Wilson JM (1997) Grain legume species in low rainfall Mediterranean-type environments I. Phenology and seed yield. Field Crops Res 54:173–187CrossRefGoogle Scholar
  31. Turner NC, Asseng S (2005) Productivity, sustainability, and rainfall-use efficiency in Australia rainfed Mediterranean agricultural systems. Aust J Agric Res 56:1123–1136CrossRefGoogle Scholar
  32. Unkovich MJ, Pate JS, Sanford P, Armstrong EL (1994) Potential precision of the delta N15 natural abundance method in field estimates of nitrogen fixation by crop and pasture legumes in south-west Australia. Aust J Agric Res 45:119–132CrossRefGoogle Scholar
  33. Unkovich MJ, Herridge DF, Peoples MB, Cadisch G, Boddey RM, Giller KE, Alves B, Chalk PM (2008) Measuring plant-associated nitrogen fixation in agricultural systems. Australian Centre for International Agricultural Research (ACIAR), Canberra. ACIAR Monograph No. 136 pp. 258.Google Scholar
  34. Unkovich MJ, Baldock J, Peoples MB (2010) Prospects and problems of simple linear models for estimating symbiotic N2 fixation by crop and pasture legumes. Plant Soil 329:75–89CrossRefGoogle Scholar
  35. Valverde C, Wall LG (2002) Nodule distribution on the roots of actinorhizal Discaria trinervis (Rhamnaceae) in pots. Environ Exp Bot 47:95–100CrossRefGoogle Scholar
  36. Wadisirisuk P, Danso SKA, Hardarson G, Bowen GD (1989) Influence of Bradyrhizobium japonicum location and movement on nodulation and nitrogen fixation in soybeans. Appl Environ Microb 55:1711–1716Google Scholar
  37. Wichern F, Eberhardt E, Mayer J, Joergensen RG, Mueller T (2008) Nitrogen rhizodeposition in agricultural crops: Methods, estimates and future prospects. Soil Biol Biochem 40:30–48CrossRefGoogle Scholar
  38. Youseif SH, Fayrouz H, Abd El-Megeed FH, Saleh SA (2017) Improvement of faba bean yield using Rhizobium/Agrobacterium inoculant in low-fertility sandy soil. Agron 7:2. doi: 10.3390/agronomy7010002 CrossRefGoogle Scholar
  39. Zapata F, Danso SKA, Hardarson G, Fried M (1987) Time course of nitrogen fixation in field-grown soybean using nitrogen-15 methodology. Agron J 79:172–176CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Matthew D Denton
    • 1
    Email author
  • Lori A Phillips
    • 2
  • Mark B Peoples
    • 3
  • David J Pearce
    • 2
  • Antony D Swan
    • 3
  • Pauline M Mele
    • 2
  • John Brockwell
    • 3
  1. 1.School of Agriculture, Food and WineThe University of AdelaideGlen OsmondAustralia
  2. 2.AgriBio Agriculture Victoria & Latrobe UniversityBundooraAustralia
  3. 3.CSIRO Agriculture and Food, Black Mountain LaboratoriesCanberraAustralia

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