, Volume 76, Issue 1, pp 41–49 | Cite as

The benefits of foliar inoculation with Azospirillum brasilense in soybean are explained by an auxin signaling model

  • Mariana L. PuenteEmail author
  • José L. Gualpa
  • Gastón A. Lopez
  • Romina M. Molina
  • Susana M. Carletti
  • Fabricio D. Cassán


Azospirillum sp. is one of the most studied genera of plant growth-promoting rhizobacteria (PGPR). The ability of Azospirillum sp. to promote plant growth has been associated with its ability to produce several phytohormones, such as auxins, gibberellins and cytokinins, but mainly indole-3-acetic acid (IAA). It has been propoosed that the production of IAA explains the positive effects of co-inoculation with Azospirillum sp. on the rhizobia-legume symbiosis. In this study, we constructed an IAA-deficient mutant of A. brasilense Az39 (ipdC ) by using a restriction-free cloning method. We inoculated soybean seeds with 1·106 cfu·seed−1 of Bradyrhizobium japonicum E109 and co-inoculating leaves at the V3 stage with 1·108 cfu.plant−1 of A. brasilense Az39 wt or ipdC or inoculated leaves with 20 μg.plant−1 synthetic IAA. The results confirmed soybean growth promotion as there was increased total plant and root length, aerial and root dry weight, number of nodules on the primary root, and an increase in the symbiosis established with B. japonicum E109. Nodule weight also increased after foliar co-inoculation with the IAA- producer A. brasilense Az39. The exogenous application of IAA decreased aerial and root length, as well as the number of nodules on primary roots in comparison with the Az39 wt strain. These results allow us to propose a biological model of response to foliar co-inoculation of soybean with IAA-producing rhizobacteria. This model clearly shows that both the presence of microorganism as part of the colonization process and the production of IAA in situ are co-responsible, via plant signaling molecules, for the positive effects on plant growth and symbiosis establishment.


Azospirillum Indole-3-acetic acid Soybean Foliar inoculation Bradyrhizobium 



We thank Universidad Nacional de Río Cuarto, Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Fondo Nacional de Ciencia y Tecnología (FONCyT) and Instituto Nacional de Tecnología Agropecuaria (INTA, Argentina). Fabricio Cassán is a Researcher of CONICET at the Universidad Nacional de Río Cuarto. Gaston Lopez is a postdoctoral researcher; José Gualpa and Romina Molina are PhD students at the Universidad Nacional de Río Cuarto and granted by CONICET.

Compliance with ethical standards

Conflicts of interest

The authors report no conflicts of interest.

Supplementary material

13199_2017_536_MOESM1_ESM.docx (15 kb)
ESM 1 Table S1 Primers used in this study. (DOCX 14 kb)
13199_2017_536_MOESM2_ESM.docx (757 kb)
ESM 2 Fig. S1: Scanning electron microscopy (SEM) of soybean leaf showing A. brasilense Az39 colonizing the surface of leaves 48 h after inoculation. The white arrows indicate the presence of bacteria on the plant tissue. A and B represent different fractions of the leaf. Colonization ability was confirmed for both A. brasilense Az39 wt and the IAA–deficient mutant ipdC (not shown). (DOCX 756 kb)


  1. Bashan Y, de Bashan L (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth. A critical assessment. Adv Agron 108:77–136. CrossRefGoogle Scholar
  2. Bottini R, Fulchieri M, Pearce D, Pharis R (1989) Identification of gibberellins A1, A3, and Iso-A3 in cultures of a. Lipoferum. Plant Physiol 90(1):45–47. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Burdmann S, Kigel J, Okon Y (1997) Effects of Azospirillum brasilense on nodulation and growth of common bean (Phaseolus vulgaris L.) Soil Biol Biochem 29(5-6):923–929. CrossRefGoogle Scholar
  4. Cacciari I, Lippi D, Pietrosanti T, Pietrosanti W (1989) Phytohormone-like substances produced by single and mixed diazotrophic cultures of Azospirillum and Arthrobacter. Plant Soil 115(1):151–153. CrossRefGoogle Scholar
  5. Campo RJ, Araujo RS, Hungria M (2009) Nitrogen fixation with the soybean crop in Brazil: compatibility between seed treatment with fungicides and bradyrhizobial inoculants. Symbiosis 48(1):154–163. CrossRefGoogle Scholar
  6. Carreño-Lopez R, Campos-Reales N, Elmerich C, Baca B (2000) Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense. Mol Gen Genet 2 64(4):521–530Google Scholar
  7. Cassán F, Díaz-Zorita M (2016) Azospirillum sp. in current agriculture: From the laboratory to the field. Soil Biol Biochem 103:117–130. CrossRefGoogle Scholar
  8. Cassán F, Maiale S, Masciarelli O, Vidal A, Luna V, Ruiz O (2009) Cadaverine production by Azospirillum brasilense and its possible role in plant growth promotion and osmotic stress mitigation. Eur J Soil Biol 45(1):12–19. CrossRefGoogle Scholar
  9. Cassán F, Penna C, Creus C, Radovancich D, Monteleone E. et al. (2013) Inoculantes forulados con Azospirillum sp. In: Albanesi, A; Benintende, S; Cassán F. y Perticari A. (Eds). Manual de procedimientos microbiológicos para la evaluación de inoculantes. pp 25–34. Editorial: Asociación Argentina de Microbiología. ISBN: 978-987-26716-4-8Google Scholar
  10. Cassán F, Vanderleyden J, Spaepen S (2014) Physiological and agronomical aspects of phytohormone production by model plant-growth-promoting rhizobacteria (PGPR) belonging to the genus Azospirillum. Plant Growth Regul 33(2):440–459. CrossRefGoogle Scholar
  11. Costacurta A, Keijers V, Vanderleyden J (1994) Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3-pyruvate deccarboxylase. Mol Gen Genet 243(4):463–472PubMedGoogle Scholar
  12. Crozier A, Arruda P, Jasmim JM, Monteiro AM, Sandberg G (1988) Analysis of indole-3-acetic acid and related indoles in culture medium from Azospirillum lipoferum and Azospirillum brasilense. Appl Environ Microbiol 54(11):2833–2837PubMedPubMedCentralGoogle Scholar
  13. Dart P (1977) Infection and development of leguminous nodules. In: Hardy RWI, Silver WS (eds) A tratise of dinitrogen fixation section III-biology. Wiley, New York, pp 367–372Google Scholar
  14. Di Rienzo J.A.; Casanoves, F; Balzarini, M.G; Gonzalez, L; Tablada, M; Robledo, C.W. InfoStat versión 2014. Córdoba: Grupo InfoStact, FCA, Universidad Nacional de Córdoba, Argentina. URL
  15. Dobbelaere S, Croonenborghs A, Thys A, Broek AV et al (1999) Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat. Plant Soil 212(2):153–162. CrossRefGoogle Scholar
  16. Döbereiner J, Baldani VLD, Baldani JI (1995) Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Brasília: EMBRAPA-SPI: EMBRAPA-CNPAB, Itaguaí, RJ, 60pGoogle Scholar
  17. El-Saeid H, Abou-Hussein S, El-Tohamy W (2010) Growth characters, yield and endogenous hormones of cowpea plants in response to IAA application. Res J Agric Biol Sci 6:27–31Google Scholar
  18. Fehr, WR, Caviness CE (1977) Stages of soybean development. Cooperative Extension Service, Agriculture and Home Economics Experiment Station. Iowa State University, Ames, IowaGoogle Scholar
  19. Frugier F, Kosuta S, Murray JD, Crespi M, Szczyglowski K (2008) Cytokinin: secret agent of symbiosis. Trends Plant Sci 13(3):115–120. CrossRefPubMedGoogle Scholar
  20. Gruodien J, Zvironaite V (1971) Effect of IAA on growth and synthesis of N compounds in Lucerne. Luk TSR Aukstuju Mosklo Darbai Biologia 17:77–87Google Scholar
  21. Hirsch A, Fang Y, Asad S, Kapulnik Y (1997) The role of phytohormones in plant-microbe symbioses. Plant Soil 194(1/2):171–184. CrossRefGoogle Scholar
  22. Hoagland D, Arnon D (1950) The Water-Culture Method for Growing Plants without Soil. California Agricultural Experiment Station. Circular 347:1–32Google Scholar
  23. Horemans S, Koninck K, Neuray J, Hermans R et al (1986) Production of plant growth substances by Azospirillum sp. and other rhizophere bacteria. Symbiosis 2:341–346Google Scholar
  24. Hubbell D, Tien T, Gaskins M, Lee J (1979) Physiological interaction in the Azospirillum-grass root association. In: Vose P, Ruschel A (eds) Associative N2-fixation. CRC Press, Boca Raton, pp 1–6Google Scholar
  25. Hungria M, Nogueira M, Araujo R (2015) Soybean seed co-inoculation with Bradyrhizobium spp. and Azospirillum brasilense: a new biotechnological tool to improve yield and sustainability. Am J Plant Sci 6(06):811–817. CrossRefGoogle Scholar
  26. Hussain K, Hussain M, Nawaz K, Majeed A et al (2011) Morphochemical response of Chaksu (Cassia absus L.) to different concentrations of indole acetic acid (IAA). Pak J Bot 43:1491–1493Google Scholar
  27. Janzen R, Rood S, Dormar J, McGill W (1992) Azospirillum brasilense produces gibberellins in pure culture and chemically-medium and in co-culture on straw. Soil Biol Biochem 24(10):1061–1064. CrossRefGoogle Scholar
  28. Kloepper J, Schroth M (1978) Plant growth-promoting rhizobacteria on radishes. In: Proceedings of the 4th international conference on plant pathogenic bacteria Vol, 2, pp 879–882Google Scholar
  29. Kolb W, Martin P (1985) Response of plant roots to inoculation with Azospirillum brasilense and to application of indole acetic acid. In: Azospirillum III. Springer Berlin Heidelberg, pp 215–221.
  30. Lodeiro AR (2015) Interrogantes en la tecnología de la inoculación de semillas de soja con Bradyrhizobium spp. Rev Argent Microbiol 47(3):261–273. PubMedCrossRefGoogle Scholar
  31. López-García S, Perticari A, Piccinetti C, Ventimiglia L et al (2009) In-furrow inoculation and selection for higher motility enhances the efficacy of Bradyrhizobium japonicum nodulation. Agron J 101(2):357–363. CrossRefGoogle Scholar
  32. Mathesius U, Schlaman H, Spaink H, Of Sautter C et al (1998) Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides. Plant J 14(1):23–34. CrossRefPubMedGoogle Scholar
  33. Miles A, Misra S (1938) The estimation of the bactericidal power of blood. J Hyg 38(06):732–737. CrossRefPubMedGoogle Scholar
  34. Muniralzaman M (2000) Effect of CyCoCel (CCC) on the growth and yield manipulations of vegetable soybean. ARG Traininy. Kasetsart university kamphaen sean. Nakhon phathom, Thailand, pp 1–16Google Scholar
  35. Patten C, Glick B (1996) Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol 42(3):207–220. CrossRefPubMedGoogle Scholar
  36. Patten C, Glick B (2002) The role of Pseudomonas putida indoleacetic acid in the development of the host plant root system. Appl Environ Microbiol 68(8):3795–3801. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Perrig D, Boiero M, Masciarelli O, Penna C et al (2007) Plant-growth-promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and implications for inoculant formulation. Appl Microbiol Biotechnol 75(5):1143–1150. CrossRefPubMedGoogle Scholar
  38. Piccoli P, Bottini R (1996) Gibberellins production in A. lipoferum cultures y enhanced by light. Biocell 20:185–190Google Scholar
  39. Prinsen E, Costacurta A, Michiels K, Vanderleyden J et al (1993) Azospirillum brasilense indole-3-acetic acid biosynthesis: evidence for a non-tryptophan dependent pathway. Mol Plant-Microb Interact 6(5):609–615. CrossRefGoogle Scholar
  40. Remans R, Beebe S, Blair M, Manrique G, Tovar E, Rao I, Croonenborghs A, Torres-Gutierrez R, el Howeity M, Michiels J, Vanderleyden J (2008) Physiological and genetic analysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.) Plant Soil 302(1-2):149–161. CrossRefGoogle Scholar
  41. Rivera D, Revale S, Molina R, Gualpa J et al (2014) Complete genome sequence of the model rhizosphere strain Azospirillum brasilense Az39, successfully applied in agriculture. Genome announcements 2(4):e00683–e00614CrossRefPubMedPubMedCentralGoogle Scholar
  42. Rodríguez Cáceres E (1982) Improved medium for isolation of Azospirillum spp. Appl Environ Microbiol 44:990–991Google Scholar
  43. Sadasivan L, Neyra C (1985) Floculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation. J Bacteriol 163(2):716–723PubMedPubMedCentralGoogle Scholar
  44. Schmidt W, Martin P, Omay H, Bangerth F (1988) Influence of Azospirillum brasilense on nodulation of legumes. In: Kling-Müller W (ed) Azospirillum IV. Genetics, physiology, ecology. Springer, Heidelberg, pp 92–100Google Scholar
  45. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1(9):784–790. CrossRefGoogle Scholar
  46. Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol 31(4):425–448. CrossRefGoogle Scholar
  47. Spaepen S, Dobbelaere S, Croonenborghs A, Vanderleyden J (2008) Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants. Plant Soil 312(1–2):15–23. CrossRefGoogle Scholar
  48. Srinivasan P, Gopal K (1977) Effect of plantofix and NAA formulation on groundnut var TMU-7. Curr Sci 46:119–120Google Scholar
  49. Srinivasan M, Holl F, Petersen D (1996) Influence of indoleacetic-acid-producing Bacillus isolates on the nodulation of Phaseolus vulgaris by Rhizobium etli under gnotobiotic conditions. Can J Microbiol 42(10):1006–1014. CrossRefGoogle Scholar
  50. Thimann K (1936) Auxins and the growth of roots. Am J Bot 23(8):561–569. CrossRefGoogle Scholar
  51. Tien T, Gaskina M, Hubbell D (1979) Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum Americanum L). Appl Environ Microbiol 37(5):1016–1024PubMedPubMedCentralGoogle Scholar
  52. Torres D, Revale S, Obando M, Maroniche G et al (2015) Genome sequence of Bradyrhizobium japonicum E109, one of the most agronomically used nitrogen-fixing rhizobacteria in Argentina. Genome announcements 3(1):e01566–e01514CrossRefPubMedPubMedCentralGoogle Scholar
  53. Unger T, Jacobovitch Y, Dantes A, Bernheim R (2010) Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172(1):34–44. CrossRefPubMedGoogle Scholar
  54. Vicario J, Gallarato L, Paulucci N, Perrig D et al (2015) Co-inoculation of legumes with Azospirillum and symbiotic rhizobia. In: Cassán F, Okon Y, Creus C (eds) Handbook for Azospirillum. Springer, ChamGoogle Scholar
  55. Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell Scientific Publications, OxfordGoogle Scholar
  56. Yahalom E, Okon Y, Dovrat A (1990) Possible mode of action of Azospirillum brasilense strain cd on the root morphology and nodule formation in burr medic (Medicago polymorpha). Can J Microbiol 36(1):10–14. CrossRefGoogle Scholar
  57. Zilli JÉ, Ribeiro KG, Campo RJ, Hungria M (2009) Influence of fungicide seed treatment on soybean nodulation and grain yield. Revista Brasileira de Ciência do Solo 33(4):917–923. CrossRefGoogle Scholar
  58. Zimmer W, Bothe H (1988) The phytohormonal interactions between Azospirillum and wheat. Plant Soil 110(2):239–247. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Mariana L. Puente
    • 1
    Email author
  • José L. Gualpa
    • 2
  • Gastón A. Lopez
    • 2
  • Romina M. Molina
    • 2
  • Susana M. Carletti
    • 3
  • Fabricio D. Cassán
    • 2
  1. 1.Laboratorio de Bacterias Promotoras del Crecimiento Vegetal, Instituto de Microbiología y Zoología Agrícola IMYZAINTA CastelarBuenos AiresArgentina
  2. 2.Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo, Departamento de Ciencias Naturales, FCEFQyNUniversidad Nacional de Río CuartoCórdobaArgentina
  3. 3.Departamento de Ciencias BásicasUniversidad Nacional de LujánBuenos AiresArgentina

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