Biologicals-based plant protection relies on the use of safe microbial strains. During application of biologicals to the rhizosphere, microbes adapt to the niche, including genetic mutations shaping the physiology of the cells.

Although certain isolates from the Bacillus cereus group (Bacillus cereus sensu lato) are used as probiotics, safety concerns remain due to pathogenic traits. For example, toxin production might shift as an adaptive survival strategy in natural niches (the soil and plant rhizosphere). Therefore, it is crucial to explore bacterial evolutionary adaptation to the environment. Herein, we investigated Bacillus thuringiensis (Cry-) adaptation to the colonisation of Arabidopsis thaliana roots, and monitored changes in cellular differentiation in experimentally evolved isolates. Isolates from two populations displayed improved iterative ecesis on roots and increased toxicity against insect larvae. Molecular dissection and recreation of a causative mutation revealed the importance of a non-sense mutation in the rho transcription terminator gene. Transcriptome analysis revealed how Rho impacts various B. thuringiensis genes involved in carbohydrate metabolism and virulence. Our work suggests that evolved multicellular aggregates have a fitness advantage over single cells when colonising plants, creating a trade-off between swimming and multicellularity in evolved lineages, in addition to unrelated alterations in pathogenicity.