Niche separation in cross-feeding sustains bacterial strain diversity across nutrient environments and may increase chances for survival in nutrient-limited leaf apoplasts.

The apoplast is an important site of plant-microbe interactions where nutrients are tightly regulated. While leaf pathogens have evolved elaborate strategies to obtain nutrients there, it is not yet clear how commensals survive without most of these adaptations. Here, we investigated whether and how the nutrient environment might influence metabolic exchange and assembly of bacterial communities in Flaveria trinervia and F. robusta leaves. We enriched bacteria from both plant species in-vitro in minimal media with sucrose as a carbon source, and with or without amino acids. After enrichment, we studied the genetic and metabolic diversity within the communities. Enriched Pseudomonas koreensis strains could cross-feed from diverse leaf bacteria. Although P. koreensis could not utilize sucrose, cross-feeding diverse metabolites from Pantoea sp ensured their survival in the sucrose-only enrichments. The Pseudomonas strains had high genetic similarity but still displayed clear niche partitioning, enabling them to simultaneously cross-feed from Pantoea. Interestingly, cross-feeders were only enriched from F. robusta and not from F. trinervia. Untargeted metabolomics analysis of the leaf apoplasts revealed contrasting nutrient environments, with greater concentrations of high-cost amino acids in F. trinervia. Additionally, P. koreensis strains were better able to survive without a cross-feeding partner in these richer apoplasts. Thus, cross feeding might arise as an adaptation to cope with nutrient limitations in the apoplast. Understanding how apoplast resources influence metabolic interactions could therefore provide plant breeders targets to manipulate leaf microbiome shape and stability.