Parallel genetic adaptation of Bacillus subtilis to different plant species.

Plant growth-promoting rhizobacteria benefit plants by stimulating their growth or protecting them against phytopathogens. Rhizobacteria must colonize and persist on plant roots to exert their benefits. However, little is known regarding the processes by which rhizobacteria adapt to different plant species, or behave under alternating host plant regimes. Here, we used experimental evolution and whole-population whole-genome sequencing to analyse how Bacillus subtilis evolves on Arabidopsis thaliana and tomato seedlings, and under an alternating host plant regime, in a static hydroponic setup. We observed parallel evolution across multiple levels of biological organization in all conditions, which was greatest for the two heterogeneous, multi-resource, spatially structured environments at the genetic level. Species-specific adaptation at the genetic level was also observed, possibly caused by the selection stress imposed by different host plants. Furthermore, a trade-off between motility and biofilm development was supported by mutational changes in motility- and biofilm-related genes. Finally, we identified several condition-specific and common targeted genes in different environments by comparing three different B. subtilis biofilm adaptation settings. The results demonstrate a common evolutionary pattern when B. subtilis is adapting to the plant rhizosphere in similar conditions, and reveal differences in genetic mechanisms between different host plants. These findings will likely support strain improvements for sustainable agriculture.

Experimental evolution of Bacillus subtilis on Arabidopsis thaliana roots reveals fast adaptation and improved root colonization.

Bacillus subtilis is known to promote plant growth and protect plants against disease. B. subtilis rapidly adapts to Arabidopsis thaliana root colonization, as evidenced by improved root colonizers already after 12 consecutive transfers between seedlings in a hydroponic setup. Re-sequencing of single evolved isolates and endpoint populations revealed mutations in genes related to different bacterial traits, in accordance with evolved isolates displaying increased root colonization associated with robust biofilm formation in response to the plant polysaccharide xylan and impaired motility. Interestingly, evolved isolates suffered a fitness disadvantage in a non-selective environment, demonstrating an evolutionary cost of adaptation to the plant root. Finally, increased root colonization by an evolved isolate was also demonstrated in the presence of resident soil microbes. Our findings highlight how a plant growth-promoting rhizobacterium rapidly adapts to an ecologically relevant environment and reveal evolutionary consequences that are fundamental to consider when evolving strains for biocontrol purposes.

Diversification of Bacillus subtilis during experimental evolution on Arabidopsis thaliana and the complementarity in root colonization of evolved subpopulations.

The soil bacterium Bacillus subtilis is known to suppress pathogens as well as promote plant growth. However, in order to fully exploit the potential as natural fertilizer, we need a better understanding of the interactions between B. subtilis and plants. Here, B. subtilis was examined for root colonization through experimental evolution on Arabidopsis thaliana. The populations evolved rapidly, improved in root colonization and diversified into three distinct morphotypes. In order to better understand the adaptation that had taken place, single evolved isolates from the final transfer were randomly selected for further characterization, revealing changes in growth and pellicle formation in medium supplemented with plant polysaccharides. Intriguingly, certain evolved isolates showed improved root colonization only on the plant species they evolved on, but not on another plant species, namely tomato, suggesting A. thaliana specific adaption paths. Finally, the mix performed better than the sum of its constituents in monoculture, which was demonstrated to be caused by complementarity effects. Our results suggest that genetic diversification occurs in an ecological relevant setting on plant roots and proves to be a stable strategy for root colonization.

Deletion of Rap-Phr systems in Bacillus subtilis influences in vitro biofilm formation and plant root colonization.

Natural isolates of the soil-dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap-Phr regulatory systems. Most Rap-Phr systems in B. subtilis have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap-Phr systems' impact on bacterial cell differentiation. Here, we investigated each of the 12 Rap-Phr systems of B.subtilis NCIB 3610 for their effect on biofilm formation. By studying single ∆rap-phr mutants, we show that despite redundancy between the cell-cell communication systems, deletion of each of the 12 Rap-Phr systems influences matrix gene expression. These Rap-Phr systems therefore enable fine-tuning of the timing and level of matrix production in response to specific conditions. Furthermore, some of the ∆rap-phr mutants demonstrated altered biofilm formation in vitro and colonization of Arabidopsis thaliana roots, but not necessarily similarly in both processes, indicating that the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under these distinct conditions.