Skip to main content

Advertisement

SpringerLink
Log in

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

  • Published:
Symbiosis Aims and scope Submit manuscript

Abstract

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.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bashan Y, de Bashan L (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth. A critical assessment. Adv Agron 108:77–136. https://doi.org/10.1016/S0065-2113(10)08002-8

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1104/pp.90.1.45

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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. https://doi.org/10.1016/S0038-0717(96)00222-2

    Article  Google Scholar 

  • 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. https://doi.org/10.1007/BF02220706

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1007/BF03179994

    Article  CAS  Google Scholar 

  • 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):521–530

    Google Scholar 

  • Cassán F, Díaz-Zorita M (2016) Azospirillum sp. in current agriculture: From the laboratory to the field. Soil Biol Biochem 103:117–130. https://doi.org/10.1016/j.soilbio.2016.08.020

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1016/j.ejsobi.2008.08.003

    Article  CAS  Google Scholar 

  • 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: 978-987-26716-4-8

  • 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. https://doi.org/10.1007/s00344-013-9362-4

    Article  CAS  Google Scholar 

  • 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):463–472

    PubMed  CAS  Google Scholar 

  • 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):2833–2837

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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 367–372

    Google Scholar 

  • 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 http://www.infostat.com.ar

  • 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. https://doi.org/10.1023/A:1004658000815

    Article  Google Scholar 

  • Döbereiner J, Baldani VLD, Baldani JI (1995) Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Brasília: EMBRAPA-SPI: EMBRAPA-CNPAB, Itaguaí, RJ, 60p

  • 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 6:27–31

    CAS  Google Scholar 

  • Fehr, WR, Caviness CE (1977) Stages of soybean development. Cooperative Extension Service, Agriculture and Home Economics Experiment Station. Iowa State University, Ames, Iowa

  • Frugier F, Kosuta S, Murray JD, Crespi M, Szczyglowski K (2008) Cytokinin: secret agent of symbiosis. Trends Plant Sci 13(3):115–120. https://doi.org/10.1016/j.tplants.2008.01.003

    Article  PubMed  CAS  Google Scholar 

  • Gruodien J, Zvironaite V (1971) Effect of IAA on growth and synthesis of N compounds in Lucerne. Luk TSR Aukstuju Mosklo Darbai Biologia 17:77–87

    Google Scholar 

  • Hirsch A, Fang Y, Asad S, Kapulnik Y (1997) The role of phytohormones in plant-microbe symbioses. Plant Soil 194(1/2):171–184. https://doi.org/10.1023/A:1004292020902

    Article  CAS  Google Scholar 

  • Hoagland D, Arnon D (1950) The Water-Culture Method for Growing Plants without Soil. California Agricultural Experiment Station. Circular 347:1–32

  • Horemans S, Koninck K, Neuray J, Hermans R et al (1986) Production of plant growth substances by Azospirillum sp. and other rhizophere bacteria. Symbiosis 2:341–346

    CAS  Google Scholar 

  • 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 1–6

    Google Scholar 

  • 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. https://doi.org/10.4236/ajps.2015.66087

    Article  CAS  Google Scholar 

  • 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 43:1491–1493

    Google Scholar 

  • 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. https://doi.org/10.1016/0038-0717(92)90036-W

    Article  CAS  Google Scholar 

  • 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 879–882

  • 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. https://doi.org/10.1007/978-3-642-70791-9_20

  • Lodeiro AR (2015) Interrogantes en la tecnología de la inoculación de semillas de soja con Bradyrhizobium spp. Rev Argent Microbiol 47(3):261–273. https://doi.org/10.1016/j.ram.2015.06.006

    Article  PubMed  Google Scholar 

  • 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. https://doi.org/10.2134/agronj2008.0155x

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1046/j.1365-313X.1998.00090.x

    Article  PubMed  CAS  Google Scholar 

  • Miles A, Misra S (1938) The estimation of the bactericidal power of blood. J Hyg 38(06):732–737. https://doi.org/10.1017/S002217240001158X

    Article  PubMed  CAS  Google Scholar 

  • 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 1–16

    Google Scholar 

  • Patten C, Glick B (1996) Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol 42(3):207–220. https://doi.org/10.1139/m96-032

    Article  PubMed  CAS  Google Scholar 

  • 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. https://doi.org/10.1128/AEM.68.8.3795-3801.2002

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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. https://doi.org/10.1007/s00253-007-0909-9

    Article  PubMed  CAS  Google Scholar 

  • Piccoli P, Bottini R (1996) Gibberellins production in A. lipoferum cultures y enhanced by light. Biocell 20:185–190

    CAS  Google Scholar 

  • 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. https://doi.org/10.1094/MPMI-6-609

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1007/s11104-007-9462-7

    Article  CAS  Google Scholar 

  • 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–e00614

    Article  PubMed  PubMed Central  Google Scholar 

  • Rodríguez Cáceres E (1982) Improved medium for isolation of Azospirillum spp. Appl Environ Microbiol 44:990–991

    Google Scholar 

  • Sadasivan L, Neyra C (1985) Floculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation. J Bacteriol 163(2):716–723

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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 92–100

    Google Scholar 

  • 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. https://doi.org/10.1038/nbt1183-784

    Article  CAS  Google Scholar 

  • Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol 31(4):425–448. https://doi.org/10.1111/j.1574-6976.2007.00072.x

    Article  CAS  Google Scholar 

  • 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. https://doi.org/10.1007/s11104-008-9560-1

    Article  CAS  Google Scholar 

  • Srinivasan P, Gopal K (1977) Effect of plantofix and NAA formulation on groundnut var TMU-7. Curr Sci 46:119–120

    CAS  Google Scholar 

  • 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. https://doi.org/10.1139/m96-129

    Article  CAS  Google Scholar 

  • Thimann K (1936) Auxins and the growth of roots. Am J Bot 23(8):561–569. https://doi.org/10.2307/2436087

    Article  CAS  Google Scholar 

  • 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):1016–1024

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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–e01514

    Article  PubMed  PubMed Central  Google Scholar 

  • 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. https://doi.org/10.1016/j.jsb.2010.06.016

    Article  PubMed  CAS  Google Scholar 

  • 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, Cham

  • Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell Scientific Publications, Oxford

    Google Scholar 

  • 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. https://doi.org/10.1139/m90-003

    Article  Google Scholar 

  • 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. https://doi.org/10.1590/S0100-06832009000400016

    Article  CAS  Google Scholar 

  • Zimmer W, Bothe H (1988) The phytohormonal interactions between Azospirillum and wheat. Plant Soil 110(2):239–247. https://doi.org/10.1007/BF02226804

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Laboratorio de Bacterias Promotoras del Crecimiento Vegetal, Instituto de Microbiología y Zoología Agrícola IMYZA, INTA Castelar, Buenos Aires, Argentina

    Mariana L. Puente

  2. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo, Departamento de Ciencias Naturales, FCEFQyN, Universidad Nacional de Río Cuarto, Córdoba, Argentina

    José L. Gualpa, Gastón A. Lopez, Romina M. Molina & Fabricio D. Cassán

  3. Departamento de Ciencias Básicas, Universidad Nacional de Luján, Buenos Aires, Argentina

    Susana M. Carletti

Authors
  1. Mariana L. Puente
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. José L. Gualpa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gastón A. Lopez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Romina M. Molina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susana M. Carletti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fabricio D. Cassán
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mariana L. Puente.

Ethics declarations

Conflicts of interest

The authors report no conflicts of interest.

Electronic supplementary material

ESM 1

Table S1 Primers used in this study. (DOCX 14 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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Puente, M.L., Gualpa, J.L., Lopez, G.A. et al. The benefits of foliar inoculation with Azospirillum brasilense in soybean are explained by an auxin signaling model. Symbiosis 76, 41–49 (2018). https://doi.org/10.1007/s13199-017-0536-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13199-017-0536-x

Keywords

Search

Navigation