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

Abstract

Aims

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.

Methods

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.

Results

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.

Conclusions

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.

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Abbreviations

PGPR:

Plant-growth-promoting rhizobacterium (PGPR)

BNF:

Biological nitrogen fixation

GS:

Glutamine synthetase

GOGAT:

Glutamate synthase

GUS:

β-glucuronidase

CFU:

Colony forming units

D.a.i:

Days after inoculation

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Acknowledgements

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.

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Correspondence to E. M. Souza.

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Responsible Editor: Euan K. James.

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Santos, K.F.D.N., Moure, V.R., Hauer, V. et al. Wheat colonization by an Azospirillum brasilense ammonium-excreting strain reveals upregulation of nitrogenase and superior plant growth promotion. Plant Soil 415, 245–255 (2017). https://doi.org/10.1007/s11104-016-3140-6

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Keywords

  • Azospirillum brasilense
  • Ammonium-excreting mutant
  • Biofertilizer
  • Glutamine synthetase
  • glnA gene