Co-assembly of <i>Azospirillum–Pseudomonas</i> biofilms in the rhizosphere enhances lettuce root colonization, growth, and heat-stress resilience

Understanding plant growth-promoting bacteria interaction is essential for developing of effective multi-strain inoculants. Here, we investigated how Azospirillum baldaniorum Sp245 and Pseudomonas fluorescens A506 interact when establishing biofilms under rhizospheric conditions and its impact on root colonization and plant growth. Mixed biofilms assembled in vitro on root exudates revealed competition between both strains, with Sp245 outcompeting A506. On lettuce roots, they formed spatially segregated biofilms according to their individual niche preferences: Sp245 exhibited dense biofilms on and along the main root, while A506 grew preferentially associated to root hairs. Both strains co-localized only in certain hotspots on the root surface and hairs bases. Yet when colonizing roots in substrate, their colonization was mutually enhanced, suggesting that cooperation prevails under these conditions. Co-inoculation of Sp245 and A506 promoted lettuce growth synergistically, increasing leaf area, fresh and dry biomass, and root dry weight. Moreover, co-inoculated plants showed enhanced survival and growth after heat stress. Our findings unveil a complex yet complementary interaction between Sp245 and A506 in the rhizosphere, where their spatial segregation does not preclude cooperation and synergistic plant-beneficial effects. Likewise, the results highlight the potential of simplified two-strain synthetic communities for enhancing crop productivity and resilience under climatic stress.