Root-associated microorganisms play an important role in plant health, such as plant growth-promoting rhizobacteria (PGPR) from the Bacillus and Pseudomonas genera. Although bacterial consortia including these two genera would represent a promising avenue to efficient biofertilizer formulation, we observed that Bacillus subtilis root colonization is decreased by the presence of Pseudomonas fluorescens and Pseudomonas protegens. To determine if B. subtilis can adapt to the inhibitory effect of Pseudomonas on roots, we conducted adaptative laboratory evolution experiments with B. subtilis in mono-association or co-cultured with P. fluorescens on tomato plant roots. Evolved isolates with various colony morphology and stronger colonization capacity of both tomato plant and Arabidopsis thaliana roots emerged rapidly from the two evolution experiments. Certain evolved isolates also had better fitness on the root in the presence of other Pseudomonas species. In all independent lineages, whole-genome resequencing revealed non-synonymous mutations in genes ywcC or sinR encoding regulators involved in repressing biofilm development, suggesting their involvement in enhanced root colonization. These findings provide insights into the molecular mechanisms underlying B. subtilis adaptation to root colonization and highlight the potential of directed evolution to enhance the beneficial traits of PGPR.IMPORTANCEIn this study, we aimed to enhance the abilities of the plant-beneficial bacterium Bacillus subtilis to colonize plant roots in the presence of competing Pseudomonas bacteria. To achieve this, we conducted adaptive laboratory experiments, allowing Bacillus to evolve in a defined environment. We successfully obtained strains of Bacillus that were more effective at colonizing plant roots than the ancestor strain. To identify the genetic changes driving this improvement, we sequenced the genomes of these evolved strains. Interestingly, mutations that facilitated the formation of robust biofilms on roots were predominant. Many of these evolved Bacillus isolates also displayed the remarkable ability to outcompete Pseudomonas species. Our research sheds light on the mutational paths selected in Bacillus subtilis to thrive in root environments and offers exciting prospects for improving beneficial traits in plant growth-promoting microorganisms. Ultimately, this could pave the way for the development of more effective biofertilizers and sustainable agricultural practices.
Keywords: Bacillus subtilis; Pseudomonas fluorescens; SinR; YwcC; adaptative laboratory evolution; biofilm regulation; root colonization.