Adaptive laboratory evolution reveals regulators involved in repressing biofilm development as key players in root colonization.

Root-associated microorganisms play an important role in plant health, such as plant growth-promoting rhizobacteria (PGPR) from the and genera. Although bacterial consortia including these two genera would represent a promising avenue to efficient biofertilizer formulation, we observed that root colonization is decreased by the presence of and . To determine if can adapt to the inhibitory effect of on roots, we conducted adaptative laboratory evolution experiments with in mono-association or co-cultured with on tomato plant roots. Evolved isolates with various colony morphology and stronger colonization capacity of both tomato plant and roots emerged rapidly from the two evolution experiments. Certain evolved isolates also had better fitness on the root in the presence of other species. In all independent lineages, whole-genome resequencing revealed non-synonymous mutations in genes or encoding regulators involved in repressing biofilm development, suggesting their involvement in enhanced root colonization. These findings provide insights into the molecular mechanisms underlying 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 to colonize plant roots in the presence of competing bacteria. To achieve this, we conducted adaptive laboratory experiments, allowing to evolve in a defined environment. We successfully obtained strains of 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 isolates also displayed the remarkable ability to outcompete species. Our research sheds light on the mutational paths selected in 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.