Directed Evolution of Plant-Associated Bacteria Enhances Plant Holobiont Stress Tolerance

Plant stress responses are shaped by both the plant genome and its associated microbial communities, which together form the plant holobiont. Given the rapid adaptive potential of plant-associated microbes, we hypothesised that directed evolution under selective pressure can accelerate the development of holobiont-level stress tolerance. Directed evolution of the plant-associated bacterium Bacillus subtilis MR21 produced three evolved strains (EV1-EV3) with distinct plant-beneficial traits. These strains enhanced coriander growth under conditions of lead (Pb) toxicity and micronutrient deficiency. The evolved bacteria improved rhizosphere conditions by reducing Pb availability and increasing the availability of essential nutrients, including phosphorus, iron and zinc, thereby enhancing plant holobiont stress tolerance. In addition, rhizosphere detoxification and elevated production of organic acids, such as succinic and glutamic acid, contributed to improved soil chemical conditions. Together, these processes promoted a by-product mutualism within the rhizosphere, in which detoxifying bacteria indirectly supported less beneficial members of the microbial community. We conclude that directed evolution of plant-associated bacteria provides a natural and efficient strategy to enhance plant stress tolerance at the holobiont level, offering a complementary approach to conventional breeding and genetic engineering.