Experimental evolution ofBacillus subtilisonArabidopsis thalianaroots reveals fast adaptation and improved root colonization in the presence of soil microbes

Abstract

The soil ubiquitous Bacillus subtilis is known to promote plant growth and protect plants against disease. These characteristics make B. subtilis highly relevant in an agricultural perspective, fueling the interest in studying B. subtilis-plant interactions. Here, we employ an experimental evolution approach to explore adaptation of B. subtilis to Arabidopsis thaliana roots. B. subtilis rapidly adapts to the plant root environment, as evidenced by improved root colonizers observed already after 12 consecutive transfers between seedlings in a hydroponic setup. Further phenotypic characterization of evolved isolates from transfer 30 revealed that increased root colonization was associated with robust biofilm formation in response to the plant polysaccharide xylan. Additionally, several evolved isolates across independent populations were impaired in motility, a redundant trait in the selective environment. Interestingly, two evolved isolates outcompeted the ancestor during competition on the root but suffered a fitness disadvantage in non-selective environment, demonstrating an evolutionary cost of adaptation to the plant root. Finally, increased root colonization by a selected evolved isolate was also demonstrated in the presence of resident soil microbes. Our findings provide novel insights into how a well-known PGPR rapidly adapts to an ecologically relevant environment and reveal evolutionary consequences that are fundamental to consider when evolving strains for biocontrol purposes.

Adaptation strategies of mutualistic rhizobacteria on Arabidopsis roots

“…Similar to the present research, altered bacterial motility of evolved isolates during adaptation was reported in an evolution experiment involving the gram-positive rhizobacterium, B. subtilis DK1042 (DK1042) (Nordgaard et al, 2021). DK1042 rapidly adapted to Arabidopsis following 12 consecutive transfers from seedling to seedling in a hydroponic system.…”

“…Evolved DK1042 isolates displayed increased rootcolonization ability, which was associated with robust biofilm formation in response to the plant polysaccharide xylan. Surprisingly, in contrast with the Pseudomonas mutants from the present study, DK1042 mutants were significantly impaired in both swimming and swarming motility (Nordgaard et al, 2021). In another experimental evolutionary study involving Bacillus thuringiensis 407 Cry -(Bt407) and Arabidopsis, and using the same hydroponic setup as described by Nordgaard et al (2021) but performed over 40 cycles, led to the emergence of isolates with reduced swimming and enhanced swarming motility in two out of five lineages (Lin et al, 2021).…”

Bacillus cereus sensu lato biofilm formation and its ecological importance

“…4 ) [ 30 ]. Directed laboratory evolution of Bacillus root colonizers in planta has additionally provided a robust methodology for studying root-associated biofilms and the evolutionary path connected to this colonization setup [ 30 , 107 , 108 ]. This approach allowed the identification of several genes related to efficient root colonization by B. cereus .…”

Establishment of a transparent soil system to study Bacillus subtilis chemical ecology

“…Subsequently, seedlings were soaked on a P5_B1gfp bacterial solution (OD600 = 0.1) for 10 minutes and placed in LEGO brick boxes containing 50 g of beads (35). The root colonization was tracked by confocal laser scanning microscopy imaging (CLSM) as described previously (15,(36)(37)(38)(39). Colonized roots were washed twice with sterile ddH2O and placed onto microscope slides.…”