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

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.