The phyllosphere -the aerial parts of plants -is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random, and correlates with leaf microscopic features. Yet, the role of microscale interactions between bacterial cells therein is not well understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined community. By means of live imaging in a simplified in vitro system, we studied the spatial organization, at the micrometer scale, of the bio-control agent Pseudomonas fluorescens A506 and the plant pathogen P. syringae B728a when introduced to pear and bean leaf microbiota (the corresponding native plants of these strains). We found significant co-localization of immigrant and resident microbial cells at distances of a few micrometers, for both strains. Interestingly, this co-localization was in part due to preferential attachment of microbiota cells near newly formed P. fluorescens aggregates. Our results indicate that two-way immigrant bacteria -resident microbiota interactions affect the leaf's microscale spatial organization, and possibly that of other surface-related microbial communities..

The phyllosphere -the aerial parts of plants -is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random and correlates with leaf microscopic features. Yet the role of microscale interactions between cells therein is not well-understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined population. By means of live imaging on a simplified in vitro system, we studied the microscale spatial organization of the plant pathogen Pseudomonas syringae B728a and the bio-control agent P. fluorescens A506 when introduced to both native and non-native leaf microbiota (bean and pear). We revealed that both strains preferentially attach to the surface in locations adjacent to microbiota aggregates. Interestingly, preferential attachment of microbiota cells near newly formed P. fluorescens aggregates was also observed. Our results indicate that twoway immigrant bacteria -resident microbiota interactions affect the microscale spatial organization of leaf microbial communities; and that preferential attachmentpreviously suggested as a general strategy that increases fitness under periodic stressis a common surface colonization strategy. The implications of this study are likely relevant to other surface-associated microbial habitats. DiscussionIn summary, through live imaging and spatial analyses at microscale, we show that immigrant bacteria display a non-random spatial colonization pattern with respect to the spatial organization of natural resident leaf microbiota cells. This non-random organization is not necessarily observable by eye, but was revealed by a rigorous image and spatial analysis. As our simplified system excludes the heterogeneity of