Experimental evolution of plant rhizobacteria reveals emerging adaptive mutations.

The colonization capability of plant growth-promoting rhizobacteria (PGPRs) in the rhizosphere is essential for their beneficial effects on plant growth, yet the evolutionary processes and adaptive potential of PGPRs in this environment remain underexplored. Here, we established an experimental evolution system for the wheat rhizosphere using Pseudomonas bijieensis 2P24 (former name: Pseudomonas fluorescens 2P24) as a model, simulating its natural evolution and tracking its genomic alterations to uncover adaptive mutations. Whole-genome sequencing and single-nucleotide polymorphism analysis revealed the accumulation of mutations in the flagella-associated gene fleN during evolution, reaching 49% frequency in the final communities. These mutations increased the number of bacterial flagella, which in turn enhanced motility and colonization capacity in the wheat rhizosphere compared to the ancestral strain. This study represents the first investigation of PGPR adaptive evolution in the wheat rhizosphere, providing a system for studying plant-associated bacteria and insights into selecting and engineering PGPR strains for agricultural applications.IMPORTANCERoot colonization ability results from the long-term evolutionary adaptation of certain bacteria to the plant rhizosphere, involving extensive bacterial genetic resources. Understanding colonization mechanisms is crucial for fully exploiting the potential of PGPRs. Pseudomonas spp. are important biocontrol agents for plant diseases, with strong affinity for plant roots and large populations in the rhizosphere, making them key models for studying PGPR colonization mechanisms. Most studies on Pseudomonas colonization rely on molecular genetics and omics approaches, which reveal many bacterial traits and mechanisms involved in rhizosphere colonization but are limited in detecting continuous genomic changes and subtle nucleotide variations. In this study, we established an experimental evolution system using Pseudomonas bijieensis 2P24 in the wheat rhizosphere to simulate bacterial evolution in the plant rhizosphere. We observed that bacteria enhance colonization ability by fine-tuning the flagellar number, revealing a novel adaptive mutation in plant rhizobacteria.