Genomic and Metabolic Characterization of Plant Growth-Promoting Rhizobacteria Isolated from Nodules of Clovers Grown in Non-Farmed Soil

Abstract

The rhizosphere microbiota, which includes plant growth-promoting rhizobacteria (PGPR), is essential for nutrient acquisition, protection against pathogens, and abiotic stress tolerance in plants. However, agricultural practices affect the composition and functions of microbiota, reducing their beneficial effects on plant growth and health. Among PGPR, rhizobia form mutually beneficial symbiosis with legumes. In this study, we characterized 16 clover nodule isolates from non-farmed soil to explore their plant growth-promoting (PGP) potential, hypothesizing that these bacteria may possess unique, unaltered PGP traits, compared to those affected by common agricultural practices. Biolog profiling revealed their versatile metabolic capabilities, enabling them to utilize a wide range of carbon and energy sources. All isolates were effective phosphate solubilizers, and individual strains exhibited 1-aminocyclopropane-1-carboxylate deaminase and metal ion chelation activities. Metabolically active strains showed improved performance in symbiotic interactions with plants. Comparative genomics revealed that the genomes of five nodule isolates contained a significantly enriched fraction of unique genes associated with quorum sensing and aromatic compound degradation. As the potential of PGPR in agriculture grows, we emphasize the importance of the molecular and metabolic characterization of PGP traits as a fundamental step towards their subsequent application in the field as an alternative to chemical fertilizers and supplements.

Keywords: Rhizobium; genomic characteristics; metabolic profiling; plant growth-promoting rhizobacteria (PGPR); plant microbiota.

Figure 1

Taxonomic classification of bacterial communities at the phylum and the genus level in soil rhizosphere of clovers grown in non-farmed (A) and agricultural soil (B).

Figure 2

Heat maps of carbon utilization patterns of 71 substrates ((A)—carbohydrates and their derivatives, (B)—organic acids and their derivatives, (C)—amino acids, (D)—other compounds) and sensitivity to 23 stressors (E) from Biolog GEN III arrays used for the metabolic profiling of the obtained isolates. The results are presented as standardized data of absorbance measurement at 590 nm, where higher values indicate greater functional activity (A–D) or an increased tolerance level (E). The control group comprised previously characterized symbionts of red clover that grew in arable lands (K2.9, K3.6, K4.15, and K5.4).

Figure 3

ACC deaminase synthesis, metallophore production, and phosphate solubilization activity of R. leguminosarum KB and KC isolates. ACC deaminase synthesis is indicated by growth in the medium containing ACC as the sole nitrogen source. Chelation of metal ions is visible as color changes to yellow-orange and purple in and around the bacterial colonies. Tricalcium phosphate dissolution zones around bacterial colonies point out phosphate solubilization properties.

Figure 4

Symbiotic performance of R. leguminosarum KB and KC isolates. Symbiotic performance was assessed by measuring the fresh masses of shoots (depicted as green bars) and roots (depicted as yellow bars), and the number of induced nodules (depicted as blue triangles). The bars represent the mean values for 10 groups, each consisting of 30 plants: 7 groups were inoculated with the different strains under study (KB3, KB7, KB8, KB10, KB12, KC4, and KC5), 2 groups were inoculated with K2.9 and K4.15 as positive controls, and 1 group served as the negative control (NC) with non-inoculated plants. Extended black segments represent standard deviation. Statistical analyses were conducted using ANOVA and post hoc Tukey tests. Bars labeled with the same lowercase letters represent values with no significant differences, while those marked with different lowercase letters indicate mean values that exhibit statistically significant differences at p < 0.05.

Figure 5

Maximum-likelihood phylogenomic tree based on 70 concatenated core genes, constructed using the autoMLST tool, which automates the extraction and analysis of multilocus sequence typing (MLST) data from genomic sequences. The tree includes both query strains (QSs) and type strains (TSs), which serve as references. Dietzia cinnamea NBRC 102147 was used as an outgroup (OG) to provide context for the diversity of Rhizobium species. The GenBank genome assembly accession numbers are shown in brackets. Bootstrap values greater than 70 are indicated in the nodes. The bar indicates sequence divergence.

Figure 6

Pangenome analysis of KB, KC, and reference strains of Rlt and R. gallicum. Predicted protein sequences from the CDSs of all genomes analyzed were grouped into orthologous gene clusters. The presence of genes belonging to a particular cluster in a given genome is indicated with a colored bar. Each ring represents an individual genome. Genomes of strains derived from white clover (group KB) are marked in red and from red clover (group KC) in green. In addition, clusters of genes that are unique to the KB, KC, and both groups, were highlighted. The similarity matrix on the right shows the average nucleotide identity (ANI) comparisons between the analyzed genomes, providing an insight into the genetic relatedness between them, where the color scale reflects a similarity level ranging from 70 (grey) to 100% (red).

Figure 7

KEGG enrichment analysis results of KB (A) and KC (B) unique gene clusters. The bar graph illustrates the KEGG pathway enrichment analysis results for a set of genes. Each bar represents a KEGG pathway category, and the color intensity corresponds to the enrichment significance level (p-value). Reddish colors indicate more significant enrichment, highlighting pathways of potential biological importance.