Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges

  • 2952 Accesses

  • 123 Citations


The potential of nitrogen-fixing (NF) bacteria to form a symbiotic relationship with leguminous plants and fix atmospheric nitrogen has been exploited in the field to meet the nitrogen requirement of the latter. This phenomenon provides an alternative to the use of the nitrogenous fertiliser whose excessive and imbalanced use over the decades has contributed to green house emission (N2O) and underground water leaching. Recently, it was observed that non-leguminous plants like rice, sugarcane, wheat and maize form an extended niche for various species of NF bacteria. These bacteria thrive within the plant, successfully colonizing roots, stems and leaves. During the association, the invading bacteria benefit the acquired host with a marked increase in plant growth, vigor and yield. With increasing population, the demand of non-leguminous plant products is growing. In this regard, the richness of NF flora within non-leguminous plants and extent of their interaction with the host definitely shows a ray of hope in developing an ecofriendly alternative to the nitrogenous fertilisers. In this review, we have discussed the association of NF bacteria with various non-leguminous plants emphasizing on their potential to promote host plant growth and yield. In addition, plant growth-promoting traits observed in these NF bacteria and their mode of interaction with the host plant have been described briefly.

This is a preview of subscription content, log in to check access.


  1. Baldani VLD, Baldani JI, Döbereiner J (2000) Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.. Biol Fert Soils 30:485–491

  2. Barka EA, Nowak J, Clément C (2006) Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN. Appl Environ Microbiol 70:7246–7252

  3. Biswas JC, Ladha JK, Dazzo FB, Yanni YG, Rolfe BG (2000a) Rhizobial inoculation influences seedling vigor and yield of rice. Agronomy J 92:880–886

  4. Biswas JC, Ladha JK, Dazzo FB (2000b) Rhizobia inoculation improves nutrient uptake and growth of lowland rice. Soil Sci Soc Am J 64:1644–1650

  5. Boddey RM, Oliveira OCD, Urquiaga S, Reis VM, Olivares FLD, Baldani VLD, Döbereiner J (1995) Biological nitrogen fixation associated with sugarcane and rice: contributions and prospects for improvement. Plant Soil 174:195–209

  6. Bueno dos Reis Junior F, Massena Reis V, Urquiaga S, Döbereiner J (2000) Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp.). Plant Soil 219:153–159

  7. Cavalcante J, Vargas C, Nogueira E, Vinagre E, Schwarcz K, Baldani J, Ferreira P, Hemerly A (2007) Members of the ethylene signalling pathway are regulated in sugarcane during the association with nitrogen-fixing endophytic bacteria. J Exp Bot 58:673–686

  8. Chaintreuil C, Giraud E, Prin Y, Lorquin J, Ba A, Gillis M, de Lajudie P, Dreyfus B (2000) Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza breviligulata. Appl Environ Microbiol 66:5437–5447

  9. Chi F, Shen S-H, Cheng H-P, Jing J-X, Yanni YG, Dazzo FB (2005) Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology. Appl Environ Microbiol 71:7271–7278

  10. Compant S, Reiter B, Sessitsch A, Nowak J, Clément C, Barka EA (2005) Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl Environ Microbiol 71:1685–1693

  11. Conn VM, Franco CMM (2004) The effect of microbial inoculants on the indigenous actinobacterial endophyte population present in the roots of wheat as determined by terminal restriction fragment length polymorphism (T-RFLP). Appl Environ Microbiol 70:6407–6413

  12. Dalton DA, Kramer S (2006) Plant-associated bacteria. Springer, The Netherlands, pp 105–130

  13. de Campos SB, Roesch LFW, Zanettini MHB, Passaglia LMP (2006) Relationship between in vitro enhanced nitrogenase activity of an Azospirillum brasilense Sp7 mutant and its growth-promoting activities in situ. Curr Microbiol 53:43–47

  14. de Oliveira ALM, de Canuto EL, Urquiaga S, Reis VM, Baldani JI (2006) Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria. Plant Soil 284:23–32

  15. Dobbelaere S, Croonenborghs A, Thys A, Ptacek D, Vanderleyden J, Dutto P, Labandera-Gonzalez C, Caballero-Mellado J, Aguirre JF, Kapulnik Y, Brener S, Burdman S, Kadouri D, Sarig S, Okon Y (2001) Responses of agronomically important crops to inoculation with Azospirillum. Aust J Plant Physiol 28:871–879

  16. Egamberdiyeva D, Höflich G (2002) Root colonization and growth promotion of winter wheat and pea by Cellulomonas spp. at different temperatures. Plant Growth Regulation 38:219–224

  17. Egener T, Hurek T, Reinhold-Hurek B (1999) Endophytic expression of nif genes of Azoarcus sp. strain BH72 in rice roots. Mol Plant–Microbe Interact 12:813–819

  18. Elbeltagy AK, Sato NT, Suzuki H, Ye B, Hamada T, Isawa T, Mitsui H, Minamisawa K (2001) Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl Environ Microbiol 67:5285–5293

  19. Engelhard M, Hurek T, Reinhold-Hurek B (2000) Preferential occurrence of diazotrophic endophyte, Azoarcus sp. in wild rice species and land races of Oryza sativa in comparison with modern races. Environ Microbiol 2:131–141

  20. Estrada P, Mavingui P, Cournoyer B, Fontaine F, Balandreau J, Caballero-Mellado J (2005) A N2-fixing endophytic Burkholderia sp. associated with maize plants cultivated in Mexico. Int J Syst Evol Microbiol 55:1233–1237

  21. Feng Y, Shen D, Song W (2006) Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates. J Appl Microbiol 100:938–945

  22. Fuentes-Ramírez LE, Caballero-Mellado J, Sepúlveda J, Martinez-Romero E (1999) Colonization of sugarcane by Acetobacter diazotrophicus is inhibited by high N-fertilization. FEMS Microbiol Ecol 29:117–128

  23. Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41:109–117

  24. Govindarajan M, Balandreau J, Muthukumarasamy R, Revathi G, Lakshminarasimhan C (2006) Improved yield of micropropagated sugarcane following inoculation by endophytic Burkholderia vietnamiensis. Plant Soil 280:239–252

  25. Govindarajan M, Kwon S-W, Weon H-Y (2007) Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. World J Microbiol 23:997–1006

  26. Govindarajan M, Balandreau J, Muthukumarasamy R, Kwon S-W, Weon H-Y, Lakshminarasimhan C (2008) Effects of the inoculation of Burkholderia vietnamiensis and related endophytic diazotrophic bacteria on grain yield of rice. Microbial Ecol 55:21–37

  27. Gutierrez-Zamora ML, Martinez-Romero E (2001) Natural endophytic association between Rhizobium etli and maize (Zea mays L.). J Biotechnol 91:117–126

  28. Gyaneshwar P, James EK, Mathan N, Reddy PM, Reinhold-Hurek B, Ladha JK (2001) Endophytic colonization of rice by a diazotrophic strain of Serretia marcessens. J Bacteriol 183:2634–2635

  29. Gyaneshwar P, James EK, Reddy PM, Ladha JK (2002) Herbaspirillum colonization increases growth and nitrogen accumulation in aluminium-tolerant rice varieties. New Phytol 154:131–145

  30. Hilali A, Prévost D, Broughton WJ, Antoun H (2001) Effets de l’inoculation avec des souches de Rhizobium leguminosarum biovar trifolii sur la croissance du blé dans deux sols du Maroc. Can J Microbiol 47:590–59

  31. Hurek T, Handley LL, Reinhold-Hurek B, Piché Y (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant–Microbe Interact 15:233–242

  32. Iniguez AL, Dong Y, Triplett EW (2004) Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342. Mol Plant–Microbe Interact 17:1078–1085

  33. James EK, Olivares FL, de Oliveira ALM, dos Reis FB Jr, da Silva LG, Reis VM (2001) Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions. J Exp Bot 52:747–760

  34. James EK, Gyaneshwar P, Mathan N, Barraquio WL, Reddy PM, Iannetta PPM, Olivares FL, Ladha JK (2002) Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Mol Plant–Microbe Interact 15:894–906

  35. Khalid A, Arshad M, Zahir Z (2005) Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. J Appl Microbiol 96:473–480

  36. Knauth S, Hurek T, Brar D, Reinhold-Hurek B (2005) Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environmental Microbiol 7:1725–1733

  37. Legaz M-E, de Armas R, Barriguete E, Vicente C (2000) Binding of soluble glycoproteins from sugarcane juice to cells of Acetobacter diazotrophicus. Int Microbiol 3:177–182

  38. Matthews SS, Sparkes DL, Bullard MJ (2001) The response of wheat to inoculation with the diazotroph Azorhizobium caulinodans. Aspects Appl Biol 63:35–42

  39. Mishra RPN, Singh RK, Jaiswal HK, Kumar V Maurya S (2006) Rhizobium-mediated induction of phenolics and plant growth promotion in rice (Oryza sativa L.). Curr Microbiol 52:383–389

  40. Mirza MS, Rasul G, Mehnaz S, Ladha JK, So RB, Ali S, Malik KA (2000) Beneficial effects of inoculated nitrogen-fixing bacteria on rice. In: Ladha JK, Reddy PM (eds) The quest for nitrogen fixation in rice. International Rice Research Institute, Los Baños, pp 191–204

  41. Mirza MS, Ahmad W, Latif F, Haurat J, Bally R, Normand P, Malik KA (2001) Isolation, partial characterization, and effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro. Plant Soil 237:47–54

  42. Muñoz-Rojas J, Caballero-Mellado J (2003) Population dynamics of Gluconacetobacter diazotrophicus in sugarcane cultivars and its effect on plant growth. Microb Ecol 46:454–464

  43. Muthukumarasamy R, Revathi G, Lakshminarasimhan C (1999) Diazotrophic associations in sugar cane cultivation in South India. Trop Agric 76:171–178

  44. Muthukumarasamy R, Revathi G, Loganathan P (2002) Effect of inorganic N on the population, in vitro colonization and morphology of Acetobacter diazotrophicus (syn Gluconacetobacter diazotrophicus). Plant Soil 243:91–102

  45. Muthukumarasamy R, Cleenwerck I, Revathi G, Vadivelu M, Janssens D, Hoste B, Ui Gum K, Park K, Son CY, Sa T, Caballero-Mellado J (2005) Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice. Syst Appl Microbiol 28:277–286

  46. Muthukumarasamy R, Kang UG, Park KD, Jeon W-T, Park CY, Cho YS, Kwon S-W, Song J, Roh D-H, Revathi G (2007) Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants. J Appl Microbiol 102:981–991

  47. Oliveira ALM, Urquiaga S, Döbereiner J, Baldani JI (2002) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 242:205–215

  48. Ormeño-Orrillo E, Rosenblueth M, Luyten E, Vanderleyden J, Martínez-Romero E (2008) Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899. Environ Microbiol 10:1271–1284

  49. Peng SB, Biswas JC, Ladha JK, Yaneshwar PG, Chen Y (2002) Influence of rhizobial inoculation on photosynthesis and grain yield of rice. Agron J 94:925–929

  50. Perrine-Walker FM, Hocart CH, Hynes MF, Rolfe BG (2005) Plasmid-associated genes in the model micro-symbiont Sinorhizobium meliloti 1021 affect the growth and development of young rice seedlings. Environ Microbiol 7:1826–1838

  51. Perrine-Walker FM, Gartner E, Hocart CH, Becker A, Rolfe BG (2007a) Rhizobium-initiated rice growth inhibition caused by nitric oxide accumulation. Mol Microbe Plant Interact 20:283–292

  52. Perrine-Walker FM, Prayitno J, Rolfe BG, Weinman JJ, Hocart CH (2007b) Infection process and the interaction of rice roots with rhizobia. J Exp Bot 58:3343–50

  53. Pieterse CMJ, Van Wees SCM, Ton J, Van Pelt JA, van Loon LC (2002) Signalling in rhizobacteria-induced systemic resistance in Arabidopsis thaliana. Plant Biol (Stuttgart) 4:535–544

  54. Pinheiro RD, Boddey LH, James EK, Sprent JI, Boddey RM (2002) Adsorption and anchoring of Azospirillum strains to roots of wheat seedlings. Plant Soil 246:151–166

  55. Prayitno J, Stefaniak J, McIver J, Weinmen JJ, Dazzo FB, Ladha JK, Barraquio W, Yanni YG, Rolfe BG (1999) Interactions of rice seedlings with bacteria isolated from rice roots. Aust J Plant Physiol 26:521–535

  56. Reinhold-Hurek B, Hurek T (1997) Azoarcus spp. and their interactions with grass roots. Plant Soil 194:57–64

  57. Reinhold-Hurek B, Hurek T (1998) Life in grasses: diazotrophic endophytes. Trends Microbiol 6:139–144

  58. Rejesus RM, Hornbaker RH (1999) Economic and environmental evaluation of alternative pollution-reducing nitrogen management practices in central Illinois. Agric Ecosyst Environ 75:41–53

  59. Riggs PJ, Chelius MK, Iniguez AL, Kaeppler SM, Triplett EW (2001) Enhanced maize productivity by inoculation with diazotrophic bacteria. Aust J Plant Physiol 28:829–836

  60. Roncato-Maccari LDB, Ramos HJO, Pedrosa FO, Alquini Y, Chubatsu LS, Yates MG, Rigo LU, Steffens MBR, Souza EM (2003) Endophytic Herbaspirillum seropedicae expresses nif genes in gramineous plants. FEMS Microbiol Ecol 45:39–47

  61. Saleh SA, Mekhemar GAA, El-Soud AAA, Ragab AA, Mikhaeel FT (2001) Survival of Azorhizobium and Azospirillum in different carrier materials: inoculation of wheat and Sesbania rostrata. Bulletin of Faculty of Agriculture, Cairo University 52:319–338

  62. Sevilla M, Kennedy C (2000) Genetic analysis of nitrogen fixation and plant-growth stimulating properties of Acetobacter diazotrophicus, an endophyte of sugarcane. In: Triplett EW (ed) Prokaryotic nitrogen fixation: a model system for analysis of a biological process. Horizon Scientific, Norwich, UK, pp 737–760

  63. Sevilla M, Gunapala N, Burris RH, Kennedy C (2001) Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and nif mutant strains. Mol Plant–Microbe Interact 14:358–366

  64. Shaharoona B, Arshad M, Zahir ZA (2006) Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.). Lett Appl Microbiol 42:155–59

  65. Suman A, Gaur A, Shrivastava AK, Yadav RL (2005) Improving sugarcane growth and nutrient uptake by inoculating Gluconacetobacter diazotrophicus. Plant Growth Regul 47:155–162

  66. Suman A, Gaur A, Shrivastava AK, Gaur A, Singh P, Singh J, Yadav RL (2007) Nitrogen use efficiency of sugarcane in relation to its BNF potential and population of endophytic diazotrophs at different N levels. Plant Growth Regul 54:1–11

  67. Trân Van V, Berge O, Kê SN, Balandreau J, Heulin T (2000) Repeated beneficial effects of rice inoculation with a strain of Burkholderia vietnamiensis on early and late yield components in low fertility sulphate acid soils of Vietnam. Plant Soil 218:273–284

  68. Verma SC, Ladha JK, Tripathi AK (2001) Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J Biotechnol 91:127–41

  69. Verma SC, Singh A, Paul Chowdhury S, Tripathi AK (2004) Endophytic colonization ability of two deep-water rice endophytes, Pantoea sp. and Ochrobactrum sp. using green fluorescent protein reporter. Biotechnol Lett 26:425–429

  70. Wang K, Conn K, Lazarovits G (2006) Involvement of quinolinate phosphoribosyl transferase in promotion of potato growth by a Burkholderia strain. Appl Environ Microbiol 72:760–768

  71. Yanni YG, Rizk RY, Corich V, Squartini A, Ninke K, Philip-Hollingsworth S, Orgambide G, de Bruinj F, Stoltzfus J, Buckley D, Schmidt TM, Mateos PF, Ladha JK, Dazzo FB (1997) Natural endophytic association between Rhizobium leguminosarum bv. trifolii and rice roots and assessment of its potential to promote rice growth. Plant Soil 194:99–114

  72. Yanni YG, Rizk RY, El-Fattah FKA, Squartini A, Corich V, Giacomini A, de Bruijn F, Rademaker J, Maya-Flores J, Ostrom P, Vega-Hernandez M, Hollingsworth RI, Martinez-Molina E, Mateos P, Velazquez E, Wopereis J, Triplett E, Umali-Garcia M, Anarna JA, Rolfe BG, Ladha JK, Hill J, Mujoo R, Ng PK, Dazzo FB (2001) The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots. Aust J Plant Physiol 28:845–870

  73. You M, Nishiguchi T, Saito A, Isawa T, Mitsui H, Minamisawa K (2005) Expression of the nifH gene of a Herbaspirillum endophyte in wild rice species: daily rhythm during the light–dark cycle. Appl Environ Microbiol 71:8183–8190

  74. Zhang L, Hurek T, Reinhold-Hurek B (2007) A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms. Microbiol Ecol 53:456–470

Download references


One of the authors, RBB, is thankful to Council of Scientific and Industrial Research, India, for the Ph.D. fellowship. We are thankful to Sujay Bhattacharjee for his editorial help.

Author information

Correspondence to Aqbal Singh.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bhattacharjee, R.B., Singh, A. & Mukhopadhyay, S.N. Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 80, 199–209 (2008). https://doi.org/10.1007/s00253-008-1567-2

Download citation


  • Biological nitrogen fixation (BNF)
  • Nitrogen-fixing (NF) bacteria
  • Endophyte
  • Rhizobium
  • Non-legume
  • Growth promotion