Adaptative Laboratory Evolution reveals biofilm regulating genes as key players in B. subtilis root colonization

Abstract Root-associated microorganisms play an important role in plant health, such as plant growth-promoting rhizobacteria 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 B. subtilis root colonization is decreased by the presence of P. fluorescens and P. 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 A. thaliana roots emerged rapidly from the two evolution experiments. Certain evolved isolates had also a better fitness on root in presence of other Pseudomonas species. Whole genome sequencing revealed that single nucleotide polymorphism (SNPs) in negative biofilm regulator genes ywcC or sinR were found in all independent lineages, 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 beneficial traits of PGPRs.