Plant and Soil

, Volume 415, Issue 1–2, pp 245–255 | Cite as

Wheat colonization by an Azospirillum brasilense ammonium-excreting strain reveals upregulation of nitrogenase and superior plant growth promotion

  • K. F. D. N. Santos
  • V. R. Moure
  • V. Hauer
  • A. R. S Santos
  • L. Donatti
  • C. W. Galvão
  • F. O. Pedrosa
  • E. M. SouzaEmail author
  • R. Wassem
  • M. B. R. Steffens
Regular Article



In this work, an ammonium-excreting strain (HM053) of A. brasilense was further characterized genetically and biochemically, and its abilities to colonize and promote wheat growth were determined.


Immunoblot, reverse transcription-qPCR, and DNA sequencing were used for HM053 characterization. To analyze wheat-A. brasilense interaction nifH::gusA fusions in the wild-type FP2 (FP2-7) and HM053 (HM053-36) backgrounds were employed.


HM053 glutamine synthetase (GS) was not adenylylated in response to an ammonium shock or under any condition tested. Sequencing of the glnA gene revealed a substitution of a proline residue by a leucine at position 347 of the GS. Under axenic growth condition, HM053 was capable of colonizing the surface of wheat roots and increased by 30 and 49% the shoot and root dry weight, respectively, when compared with uninoculated plants, and by 30 and 31% when compared with the parental strain FP2. Although HM053-36 and FP2-7 showed GUS activity located mainly at lateral root emergence points, HM053-36 consistently showed stronger signals and expressed the nifH gene at a level 278 fold higher than strain FP2 in planta, according to qPCR data.


HM053, a spontaneous mutant in GS, increased wheat root and shoot dry weight when compared to the wild-type FP2. HM053 ability to excrete ammonium and fix nitrogen constitutively, even in the presence of high NH4 + concentration, could explain why this mutant has a higher potential to promote plant growth than FP2 and suggests HM053 as a potential nitrogen biofertilizer. However, HM053 should be tested under field conditions to evaluate its abilities to compete with indigenous microflora.


Azospirillum brasilense Ammonium-excreting mutant Biofertilizer Glutamine synthetase glnA gene 



Plant-growth-promoting rhizobacterium (PGPR)


Biological nitrogen fixation


Glutamine synthetase


Glutamate synthase




Colony forming units


Days after inoculation



This work was supported by the National Institute of Science and Technology on Biological Nitrogen Fixation (INCT/CNPq). We thank Roseli Prado, Valter A. Baura, and Marilza Doroty Lamour for the technical assistance.

Supplementary material

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Figure S2 (TIFF 143630 kb)
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Figure S3 (TIFF 147708 kb)


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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • K. F. D. N. Santos
    • 1
  • V. R. Moure
    • 1
  • V. Hauer
    • 2
  • A. R. S Santos
    • 1
  • L. Donatti
    • 3
  • C. W. Galvão
    • 4
  • F. O. Pedrosa
    • 1
  • E. M. Souza
    • 1
    Email author
  • R. Wassem
    • 2
  • M. B. R. Steffens
    • 1
  1. 1.Departamento de Bioquímica e Biologia MolecularUniversidade Federal do ParanáCuritibaBrazil
  2. 2.Departamento de GenéticaUniversidade Federal do ParanáCuritibaBrazil
  3. 3.Departamento de Biologia CelularUniversidade Federal do ParanáCuritibaBrazil
  4. 4.Departamento de Biologia Estrutural, Molecular e GenéticaUniversidade Estadual de Ponta GrossaPonta GrossaBrazil

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