Exploring Pseudomonas syringae Ecology via Direct Microscopic Observations of the Leaf Surface

Abstract

While the factors affecting the relative abundance of bacterial populations have been addressed for entire leaves or plants, little is know about how they affect the density and spatial distribution of micro-organisms at small scales corresponding to their size. The development of new approaches that combine molecular biological tools with light microscopy techniques has enabled us to extend our scale of investigation of epiphytic communities to small scales and has revealed unanticipated features of leaf surface microbial communities. We examined Pseudomonas syringae pv. syringae strain B728a inoculated onto bean leaves. Cells genetically marked with genes conferring fluorescent proteins of different colours were visualised using epifluorescence microscopy directly on leaf surfaces. Cell viability was also assessed following propidium iodide staining. Bacterial cells are not randomly distributed on the leaf surface but occur in a wide range of cluster sizes. Large cell aggregates are not frequent on a given leaf but often account for the majority of the cells present. Aggregated cells are much more capable of tolerating desiccation stress on leaves than solitary cells and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaves. The fate of immigrant. cells-depends on both anatomical features of the leaf and the number of resident cells at their landing site. The relatively low occurrence of mixed species. aggregates on leaves reveals that bacterial colonisation of leaves is characterised by a high level of spatial segregation and direct bacterial interactions between two populations is limited to only those few cells in direct contact. Description of the spatial distribution of epiphytic bacteria at small scales corresponding to their size has provided us with important biological and ecological information on processes enabling growth and survival of epiphytes and their interactions on leaf surfaces.