Genomic and Phenotypic Insights Into the Potential of Rock Phosphate Solubilizing Bacteria to Promote Millet Growth in vivo

Abstract

Rock phosphate (RP) is a natural source of phosphorus for agriculture, with the advantage of lower cost and less impact on the environment when compared to synthetic fertilizers. However, the release of phosphorus (P) from RP occurs slowly, which may limit its short-term availability to crops. Hence, the use of P-solubilizing microorganisms to improve the availability of P from this P source is an interesting approach, as microorganisms often perform other functions that assist plant growth, besides solubilizing P. Here, we describe the characterization of 101 bacterial isolates obtained from the rhizosphere and endosphere of maize plants for their P solubilizing activity in vitro, their growth-promoting activity on millet plants cultivated in soil amended with RP, and their gene content especially associated with phosphate solubilization. For the in vitro solubilization assays, two mineral P sources were used: rock phosphate from Araxá (Brazil) mine (AP) and iron phosphate (Fe-P). The amounts of P released from Fe-P in the solubilization assays were lower than those released from AP, and the endophytic bacteria outperformed the rhizospheric ones in the solubilization of both P sources. Six selected strains were evaluated for their ability to promote the growth of millet in soil fertilized with a commercial rock phosphate (cRP). Two of them, namely Bacillus megaterium UFMG50 and Ochrobactrum pseudogrignonense CNPMS2088, performed better than the others in the cRP assays, improving at least six physiological traits of millet or P content in the soil. Genomic analysis of these bacteria revealed the presence of genes related to P uptake and metabolism, and to organic acid synthesis. Using this approach, we identified six potential candidates as bioinoculants, which are promising for use under field conditions, as they have both the genetic potential and the experimentally demonstrated in vivo ability to improve rock phosphate solubilization and promote plant growth.

Keywords: bacteria; maize; phosphate; plant growth promotion; solubilization.

FIGURE 1

P solubilization from Araxá Phosphate (AP) (A), and Iron Phosphate (Fe–P) (C) by planktonic cells and from AP by sessile cells (E) of the endophyte and rhizosphere bacteria. The bacteria were clustered into three groups according to their P solubilization efficiency, as follows: Group 1- high P solubilization; 2- medium P solubilization; and 3- low P solubilization. The average soluble P-values recorded as well as the best solubilizer in each group are indicated. The graphs to the right show the endophyte and rhizosphere bacterial genera distribution in each group in assays with AP (B) and Fe–P (D) under agitation and AP with sessile cells (F). The identification of other isolates of each group obtained by K-means analysis is shown in the Supplementary Tables S2–S4.

FIGURE 2

Relationship between soluble P-values and final pH in the assays of Araxá Phosphate solubilization under agitation for the endophyte and rhizosphere bacteria studied. The data show negative and significant correlation based on Pearson’s correlation coefficient for the endophytic and rhizospheric bacteria belonging to Group 1 (high solubilization) (r: –0.47; p = 6.2 × 10^–7).

FIGURE 3

Presence or absence of genes related to the synthesis of organic acids: (A) Gluconic, (B) ketogluconic, (C) Formic, (D) Acetic, (E) Glyoxylic, (F) Lactic, and (G) Glycolic in the six bacterial genomes (B. megaterium UFMG50, K. variicola UFMG51, P. ananatis UFMG54, Microbacterium sp. UFMG61, Pseudomonas sp. UFMG81 and O. pseudogrignonense CNPMS2088). Pyrroloquinoline quinone biosynthesis protein B (pqqB), pyrroloquinoline-quinone synthase (pqqC), pyrroloquinoline quinone biosynthesis protein D (pqqD), PqqA peptide cyclase (pqqE), quinoprotein glucose dehydrogenase (gcd), glucose 1-dehydrogenase (gdh), gluconolactonase (gnl), 2-dehydro-3-deoxygluconokinase (kdgK), 6-phosphogluconate dehydrogenase (gnd), 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (kduD), gluconate:H+ symporter (gntP), gluconate operon transcriptional repressor (gntR), aldose sugar dehydrogenase (yliI), gluconate 2-dehydrogenase alpha chain (gadα), gluconate 2-dehydrogenase gamma chain (gadγ), 2-keto-D-gluconate reductase-gluconate 2-dehydrogenase (ghrB), oxalate decarboxylase (oxdD), pyruvate dehydrogenase (poxB), aldehyde dehydrogenase (aldB), acetyl-CoA synthetase (acs), propionyl-CoA carboxylase alpha chain (pccA), Isocitrate lyase (aceA), aconitate hydratase A (acnA), aconitate hydratase B (acnB), citrate synthase (gltA), malate synthase (glcB), malate dehydrogenase (mdh1), L-lactate dehydrogenase complex protein (lldE), L-lactate dehydrogenase complex protein (lldF), L-lactate dehydrogenase complex protein (lldG), L-lactate dehydrogenase operon regulator (lldR), lactate permease (lctP), L-lactate dehydrogenase (ldh), Na+:H+ antiporter NhaA family (nhaA), Na+:H+ antiporter NhaB family (nhaB), malate-2H(+)/Na(+)-lactate antiporter (nhaC), glyoxylate/hydroxypyruvate/2-ketogluconate reductase (ghrB), glyoxylate/hydroxypyruvate reductase (ghrA), glycerate dehydrogenase (hpra), glyoxylate reductase (gyaR). A list with KO and GOG numbers can be accessed in Supplementary Table S6.