Spatial Organization of Dual-Species Bacterial Aggregates on Leaf Surfaces

Abstract

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% ؎ 8.2%) than that in monospecific aggregates of these two strains (1.6% ؎ 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.Leaf surfaces are colonized by a variety of microorganisms, and many factors have been suggested to be involved in dictating the composition and organization of such communities. While evidence of direct interactions among particular populations has been provided by studies of the biological control of plant-pathogenic fungi and bacteria (1,11,19) and by studies of coexistence of particular epiphytic bacterial strains (33), the factors determining interactions between epiphytic bacteria are poorly understood. The efficacy of biological control of foliar disease, especially under variable field conditions, is often inconsistent (1, 13, 15). While antibiosis, hyperparasitism, and resource competition are believed to play essential roles in such interactions, how spatially and temporally variable such interactions are between populations remains unclear. Incomplete biological control by applied antagonists has been proposed to be due...

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