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Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications


The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA3), and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA3 were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 μg ml−1), USDA110 (2.5 μg ml−1), and E109 (0.87 μg ml−1), respectively. ABA biosynthesis was detected only in USDA110 (0.019 μg ml−1). Ethylene was found in all three strains, with highest production rate (18.1 ng ml−1 h−1) in E109 cultured in yeast extract mannitol medium plus l-methionine. This is the first report of IAA, GA3, zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.

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  1. Antoun H, Beauchamp C, Goussard N, Chabot R, Lalande R (1998) Potential of Rhizobium and Bradyrhizobium species as plant growth promoting rhizobacteria on non-legumes: effect on radishes (Raphanus sativus L.). Plant Soil 204:57–67

  2. Atzorn R, Crozier A, Wheeler C, Sandberg G (1988) Production of gibberellins and indole 3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots. Planta 175:532–538

  3. Bashan Y, Holguín G (1998) Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrol-PGPB (plant growth promoting bacteria) and PGPB. Soil Biol Biochem 30:1225–1228

  4. Bastian F, Cohen A, Piccoli P, Luna V, Baraldi R, Bottini R (1998) Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media. Plant Growth Regul 24:7–11

  5. Bohlool B (1990) Introduction to nitrogen fixation in agriculture and industry: contribution of BNF to sustainability of agriculture. In: Gresshoff P, Roth L, Stacey G, Newton W (eds) Nitrogen fixation: achievements and objectives. Chapman & Hall, New York, pp 613–616

  6. Bottini R, Fulchieri M, Pearce D, Pharis R (1989) Identification of gibberellins A1, A3, and iso-A3 in cultures of A. lipoferum. Plant Physiol 90:45–47

  7. Bric J, Bostock R, Silverstone S (1991) Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535–538

  8. Cassán F, Bottini R, Schneider G, Piccoli P (2001) Azospirillum brasilense and Azospirillum lipoferum hydrolize conjugates of GA20 and metabolize the resultant aglycones to GA1 in seedlings of rice dwarf mutants. Plant Physiol 125:2053–2058

  9. Cassán F, Paz R, Maiale S, Masciarelli O, Vidal A, Luna V, Ruíz O (2005) Cadaverine production by Azospirillum brasilense Az39. A new plant growth promotion mechanism. In: XVth annual meeting of the Cordoba Biology Society, Sociedad de Biología de Córdoba, Argentina, p 103

  10. Cattelan A, Hartel P, Fuhrmann J (1999) Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J 63:1670–1680

  11. Chabot R, Antoun H, Cescas M (1996) Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar phaseoli. Plant Soil 184:311–321

  12. Foster R (1988) Micro-environments of soil microorganisms. Biol Fertil Soils 6:189–203

  13. Fukuhara H, Minakawa Y, Akao S, Minamisawa K (1994) The involvement of indole-3-acetic acid produced by Bradyrhizobium elkanii in nodule formation. Plant Cell Physiol 35:1261–1265

  14. Garate A, Bonilla I (2000) Nutrición mineral y producción vegetal. In: Azcón-Bieto J, Talón E (eds) Fundamentos de Fisiología Vegetal. McGraw-Hill Interam, Madrid, pp 113–130

  15. Gaur Y, Sen A, Subba Rao N (1980) Improved legume–Rhizobium symbiosis by inoculating preceding cereal crop with Rhizobium. Plant Soil 54:313–316

  16. Hablieb CM, Luden PW (2000) Regulation of biological nitrogen fixation. J Nutr 130:1081–1084

  17. Höflich G, Wiehe W, Kühn G (1994) Plant growth stimulation by inoculation with symbiotic and associative rhizosphere microorganisms. Experientia 50:897–905

  18. Hume D, Blair D (1992) Effect of numbers of Bradyrhizobium japonicum applied in commercial inoculants on soybean yield in Ontario. Can J Microbiol 38:588–593

  19. Hunter W (1987) Indole-3-acetic acid production by bacteroids from soybean root nodules. Physiol Plant 76:73–77

  20. Jaiswal V, Rizvi S, Mukerji D, Mathur S (1982) Nitrogenase activity in root nodules of Vigna mungo: the role of nodular cytokinins. Angew Bot 56:143–148

  21. Kaneshiro T, Kwoleck W (1985) Stimulated nodulation of soybean by Rhizobium japonicum mutant (B-14075) that catabolizes the conversion of tryptophan to indol-3yl-acetic acid. Plant Sci 42:141–146

  22. Katznelson H, Bose B (1959) Metabolic activity and phosphate-dissolving capability of bacterial isolates from wheat roots, rhizosphere, and non-rhizosphere soil. Can J Microbiol 5:79–85

  23. Katznelson H, Cole S (1965) Production of gibberellin-like substances by bacteria and actinomycetes. Can J Microbiol 11:733–741

  24. Kloepper J, Lifshitz R, Zablotowicz R (1989) Free-living bacteria inocula for enhancing crop productivity. Trends Biotechnol 7:39–44

  25. Kobats E (1958) Gas-Cromatographische Charakterisierung organischer Verbindungen. Teil 1: Retention Indices aliphatischer Halogenide, Alkohole, Aldehyde, und Ketone. Helvetica Chimica Acta 41:1915–1932

  26. Lucangelli C, Bottini R (1996) Reversion of dwarfism in dwarf-1 Maize (Zea mays L.) and dwarf-x Rice (Oryza sativa L.) mutants by endophytic Azospirillum spp. Biocell 20:221–226

  27. Monteleone M, Thuar A, Olmedo C (2003) Efecto de la promoción del crecimiento con PGPRs en un cultivo de trigo en presencia de B. japonicum. Actas de la IV Reunión Nacional Científico Técnica de Biología de Suelos, Santiago del Estero, Argentina. CD-ROM. ISBN 097-99083-6-8

  28. Noel T, Sheng C, Yost C, Pharis P, Hynes M (1996) Rhizobium leguminosarum as a plant growth-promoting rhizobacterium: direct growth promotion of canola and lettuce. Can J Microbiol 42:279–283

  29. Nukui N, Ezura H, Yuhashi K, Yasuta T, Minamisawa K (2000) Effects of ethylene precursor and inhibitors for ethylene biosynthesis and perception on nodulation in Lotus japonicus and Macroptilium atropurpureum. Plant Cell Physiol 41:893–897

  30. Perrig D, Masciarelli O, Perticari A, Cassán F, Luna V (2005) Caracterización de la capacidad promotora y biocontroladora de Azospirillum brasilense Az39, la cepa más utilizada en la formulación de inoculantes para gramíneas en Argentina. Actas de la V Reunión Nacional Científico Técnica de Biología del Suelo y FBN, Sociedad de Biología de Suelos, Jujuy, Argentina, p 70

  31. Perticari A, Parra R, Balatti P, Fiqueni M, Rodriguez Caceres E (1996) Selección de cepas de Bradyrhizobium japonicum, B. elkanii y Sinorhizobium fredii para la inoculación de soja. Memorias de la XVIII Reunión Latinoamericana de Rizobiología, Santa Cruz de La Sierra, Bolivia, pp 103–104

  32. Pierce M, Bauer W (1997) A rapid regulatory response governing nodulation in soybean. Plant Physiol 73:286–290

  33. Radley M (1961) Gibberellins-like substances in plants. Nature 191:684–685

  34. Ressia J, Lázaro L, Lett L, Mendivil G, Portela G, Balbuena R (2003) Tillage systems and inoculation in soybean. Effects on growth and yield. Agrociencia 37:167–176

  35. Ribaudo C, Krumpholz E, Cassán F, Bottini R, Cantore M, Curá A (2006) Azospirillum sp. promotes root hair development in tomato plants through a mechanism that involves ethylene. J Plant Growth Regul 24:175–185

  36. Schwyn B, Neilands J (1987) Universal assay for detection and determination of siderophores. Anal Biochem 160:47–56

  37. Seshadri S, Muthukumarasamy R, Lakshminarasimhan C, Ignacimuthu S (2000) Solubilization of inorganic phosphates by Azospirillum halopraeferans. Curr Sci 79:565–567

  38. Srinivasan M, Petersen D, Holl F (1996) Influence of indoleacetic acid-producing Bacillus isolates on the nodulation of Phaseolus vulgaris by Rhizobium etli under gnobiotic conditions. Can J Microbiol 42:1006–1014

  39. Strzelczyk E, Kamper M, Li C (1994) Cytokinin-like-substances and ethylene production by Azospirillum in media with different carbon sources. Microbiol Res 149:55–60

  40. Sturtevant D, Taller B (1989) Cytokinin production by a parasponia nodule bacterium. Abstr Annu Meet Am Soc Microbiol 89:300

  41. Suzuki A, Akune M, Kosigo M, Imagama K, Osuki K, Uchiumi T, Higashi S, Han S, Yoshida S, Asami T, Abe M (2005) Control of nodule number by phytohormone abscisic acid in the roots of two leguminous species. Plant Cell Physiol 45:914–922

  42. Taller B, Sturtevant D (1989) Modification of cytokinin production in Bradyrhizobium cultures. J Cell Biochem Suppl 12(Part C):274

  43. Tien T, Gaskin M, Hubbell D (1979) Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetumamericanum L.). Appl Environ Microbiol 37:1016–1024

  44. Vincent J (1970) A manual for the practical study of the root-nodule bacteria. International Biological Program Handbook no.15. Blackwell Scientific Publications, Oxford

  45. Yahalom E, Okon Y, Dovrat A (1990) Posible mode of action of Azospirillum brasilense strain Cd on the root morphology and nodule formation in burr medic (Medicago polymorpha). Can J Microbiol 36:10–14

  46. Yanni Y, Rizk R, Corich V, Squartini A, Ninke K, Philip-Hollingsworth S, Orgambide G, de Bruinj F, Stoltzfus J, Buckley D, Schmidt T, Mateos P, Ladha J, Dazzo F (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

  47. Zelena E, Kutacek M, Cermak V (1988) Fate of root applied indolyl-acetic acid and its influence on the growth of intact plants. In: Kutacek M, Bandurski R, Krekuble J (eds) Physiology and biochemistry of auxins in plants. SPB Academic Publishing. The Hauge, pp 371–376

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The authors are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas, Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto, and Nitragin Argentina SA for supporting this research. We also thank Dr. W. Giordano for critical review and comments on the manuscript and Dr. S. Anderson for English editing.

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Correspondence to V. Luna.

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Boiero, L., Perrig, D., Masciarelli, O. et al. Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications. Appl Microbiol Biotechnol 74, 874–880 (2007).

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  • Rhizobium
  • Ethylene Production
  • Zeatin
  • Siderophore Production
  • Japonicum Strain