Genetic diversity and growth promoting characteristics of diazotrophic bacteria isolated from 20 genotypes of Brachiaria spp.


Background and aims

Diazotrophic bacteria, including those of the genus Azospirillum and Nitrospirillum, colonize Brachiaria genotypes and contribute to plant development through nitrogen fixation, production of phytohormones and bioavailability of nutrients. This study aimed to determine the phylogenetic positioning and evaluate the functional abilities of diazotrophic bacteria isolated from Brachiaria genotypes.


Diazotrophic bacterial counting and isolation were carried out with rhizosphere soil and root samples from 20 Brachiaria genotypes after inoculation in nitrogen-free semi-solid NFb and LGI media. The isolates were analyzed using 16S rRNA and nifH sequences, and tested for their functional abilities to produce auxin and siderophores, to solubilize phosphate and zinc, and to degrade cellulose.


The diazotrophic population ranged from 102 to 108 g−1 rhizosphere soil or roots. Sequencing of 16S rRNA from 213 isolates confirmed the presence of genera Azospirillum and Nitrospirillum, and revealed the presence of 14 other diazotrophic genera. The genus Nitrospirillum was detected colonizing all niches of most Brachiaria species. A PCA analysis showed a positive correlation between the ability to produce siderophores with the ability to produce IAA; and between phosphate and zinc solubilisation.


The results showed a high diversity of diazotrophic bacterial species colonizing 20 Brachiaria genotypes and revealed the presence of bacteria with variable growth-promoting characteristics, highlighting their potential as good candidates for the development of biofertilizers.

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This study was funded by the project Embrapa MP2 (n° The first author was supported by a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. José Ivo Baldani was supported by fellowship from CNPq (process n° 306011/2017-4) and a bench fee from FAPERJ–CNE (E26/203.011/2016). The authors thank Dr. Ederson da Conceição Jesus for the multivariate statistical analysis and PCA analysis.

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Figure S1

Venn diagram representation of the diazotrophic strains isolated from the different plant compartments (non and desinfected roots and rhizosphere soil) with respect to bacterial genera from the Brachiaria species: (A) B. brizantha, (B) B. humidicola, (C) B. decumbens and (D) Brachiaria spp. plus B. arrecta. Signals: no *: belongs to Brachiaria spp., *: belongs to B. arrecta and **: belongs to both Brachiaria spp. and B. arrecta. (PNG 2066 kb)

Figure S6

Detailed cluster of strains closely related to Azospirillum lipoferum from the phylogenetic tree presented in Fig. 2. (PNG 208 kb)

Figure S7

Detailed cluster of strains closely related to Azospirillum brasilense from the phylogenetic tree presented in Fig. 2. (PNG 154 kb)

Figure S8

Detailed cluster of strains closely related to Azospirillum formosense from the phylogenetic tree presented in Fig. 2. (PNG 150 kb)

Figure S9

Detailed cluster of strains closely related to Azospirillum melinis from the phylogenetic tree presented in Fig. 2. (PNG 178 kb)

Figure S10

Detailed cluster of strains closely related to Nitrospirillum amazonense from the phylogenetic tree presented in Fig. 2. (PNG 367 kb)

High Resolution (TIF 17044 kb)

Figure S2

Examples of in vitro halo formation characteristic of solubilization of inorganic phosphate (A) and zinc oxide (B) by strains NRB081 and NRB082 grown in NBRIP medium containing insoluble β-tricalcium phosphate (Ca3(PO4)2) or zinc oxide (ZnO). (JPG 79 kb)

Figure S3

. Production of siderophore in vitro of catecholate type by strains isolated from Brachiaria genotypes and evaluated in solid NFBb medium with 0.1% (NH4)2SO4 of CAS. The blue/yellow color change was observed 72 h after incubation at 30 °C. (A) strains with no production capacity; (B) strain with low production capacity; (C) strain with medium capacity; and (D) strain with high capacity of siderophore production. (JPG 47 kb)

Figure S4

Production of siderophore in vitro of hydroxamate type by strains isolated from Brachiaria genotypes and analysed in solid LGI medium, with 0.1% (NH4)2SO4 of CAS. The change of color blue/pink was observed 72 h after incubation at 30 °C. (A) strain with no production capacity; (B) strain with low production capacity; (C) strain with medium capacity; and (D) strain with high capacity of siderophore production. (JPG 40 kb)

Figure S5

Examples of in vitro cellulolytic activity of strains isolated from Brachiaria genotypes. Bacteria were inoculated onto CMC plate medium containing 0.2% of carboxymethylcellulose. (A) strain showing no activity; (B) strain with low activity; (C) strain with medium capacity; and (D) strain with high cellulose degradation capacity. (JPG 68 kb)

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Ribeiro, N.V.d.S., Vidal, M.S., Barrios, S.C.L. et al. Genetic diversity and growth promoting characteristics of diazotrophic bacteria isolated from 20 genotypes of Brachiaria spp.. Plant Soil 451, 187–205 (2020).

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  • Isolation
  • Functional capacity
  • Sequencing
  • Phylogenetic positioning