Molecular Characterization of Indigenous Rhizobia from Kenyan Soils Nodulating with Common Beans

Abstract

Kenya is the seventh most prominent producer of common beans globally and the second leading producer in East Africa. However, the annual national productivity is low due to insufficient quantities of vital nutrients and nitrogen in the soils. Rhizobia are symbiotic bacteria that fix nitrogen through their interaction with leguminous plants. Nevertheless, inoculating beans with commercial rhizobia inoculants results in sparse nodulation and low nitrogen supply to the host plants because these strains are poorly adapted to the local soils. Several studies describe native rhizobia with much better symbiotic capabilities than commercial strains, but only a few have conducted field studies. This study aimed to test the competence of new rhizobia strains that we isolated from Western Kenya soils and for which the symbiotic efficiency was successfully determined in greenhouse experiments. Furthermore, we present and analyze the whole-genome sequence for a promising candidate for agricultural application, which has high nitrogen fixation features and promotes common bean yields in field studies. Plants inoculated with the rhizobial isolate S3 or with a consortium of local isolates (COMB), including S3, produced a significantly higher number of seeds and seed dry weight when compared to uninoculated control plants at two study sites. The performance of plants inoculated with commercial isolate CIAT899 was not significantly different from uninoculated plants (p > 0.05), indicating tight competition from native rhizobia for nodule occupancy. Pangenome analysis and the overall genome-related indices showed that S3 is a member of R. phaseoli. However, synteny analysis revealed significant differences in the gene order, orientation, and copy numbers between S3 and the reference R. phaseoli. Isolate S3 is phylogenomically similar to R. phaseoli. However, it has undergone significant genome rearrangements (global mutagenesis) to adapt to harsh conditions in Kenyan soils. Its high nitrogen fixation ability shows optimal adaptation to Kenyan soils, and the strain can potentially replace nitrogenous fertilizer application. We recommend that extensive fieldwork in other parts of the country over a period of five years be performed on S3 to check on how the yield changes with varying whether conditions.

Keywords: comparative genomics; pangenome; rhizobia; symbiotic potential; synteny.

Figure 1

Number of nodules per plant in Kakamega (A) and Busia (B) soils and dry weight of the shoots from Kakamega (C) and Busia (D) soils 28 days before planting. Diamonds represent outliers in the data while different lower-case letters above bars indicate significant differences (p ≤ 0.05).

Figure 2

Yield of common beans in terms of the number of seeds per plant in Kakamega (A) and Busia (B) and dry weight of seeds per plant in Kakamega (C) and Busia (D). Diamonds represent outliers in the data while different lower-case letters above bars indicate significant differences (p ≤ 0.05).

Figure 3

Subsystem classification of the annotated isolate S3 genes.

Figure 4

Venn diagram showing the intersection of orthologous groups resulting from COGtriangle and orthoMCL outputs.

Figure 5

The most parsimonious pangenome tree. The nodes are colored in relation to the legend and represent standard bootstrap support values computed by seqboot from the PHYLIP package. Highlighted taxa represent isolate S3’s closest neighbors.

Figure 6

Synteny blocks (regions of chromosomes or plasmids between genomes that share a common order of homologous genes believed to have been derived from a common ancestor) between R. phaseoli and isolate S3SC1 and RC1 represents, respectively, represents chromosomes from isolates S3 and R. phaseoli, SP1–4 and RP1–4 represents plasmids from S3 and R. phaseoli, respectively. Similar color bands point to similar blocks representing synteny blocks in identified between S3 and R. phaseoli.