Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces

Abstract

The survival of individual Pseudomonas syringae cells was determined on bean leaf surfaces maintained under humid conditions or periodically exposed to desiccation stress. Cells of P. syringae strain B728a harboring a GFP marker gene were visualized by epifluorescence microscopy, either directly in situ or after recovery from leaves, and dead cells were identified as those that were stained with propidium iodide in such populations. Under moist, conducive conditions on plants, the proportion of total live cells was always high, irrespective of their aggregated state. In contrast, the proportion of the total cells that remained alive on leaves that were periodically exposed to desiccation stress decreased through time and was only approximately 15% after 5 days. However, the fraction of cells in large aggregates that were alive on such plants in both condition was much higher than more solitary cells. Immediately after inoculation, cells were randomly distributed over the leaf surface and no aggregates were observed. However, a very aggregated pattern of colonization was apparent within 7 days, and >90% of the living cells were located in aggregates of 100 cells or more. Our results strongly suggest that, although conducive conditions favor aggregate formation, such cells are much more capable of tolerating environmental stresses, and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaf surfaces.

Fig. 1.

Visualization of viable and nonviable cells of P. syringae strain B728a directly on bean leaf surfaces. P. syringae cells harbored a gfp marker gene and propidium iodide was used as a cell viability indicator. P. syringae cells appear green, and nonviable cells appear red. A bright-field image of the leaf surface provides spatial context for cell location. The arrow points at one of the two stomates present in the field of view. (Inset) Aggregated cells (detail of a larger field of view). (Magnification, ×500.)

Fig. 2.

Average fraction of living cells in relation to the total number of cells per aggregate on plants maintained under humid conditions (A) or periodically exposed to low RH (B), 0 (black diamonds), 3 (open diamonds), 4 (triangles), 5 (circles), and 7 (squares) days after inoculation. The standard errors of the average fractions of living cells per aggregate were removed for clarity but averaged ≈0.06.

Fig. 3.

Cumulative fraction of living P. syringae cells as a function of aggregate size. Leaves of plants periodically exposed to desiccation stress were visualized at 0 (triangles), 3 (squares), 4 (circles), 5 (crosses), and 7 (diamonds) days after inoculation for plants periodically exposed to stress. Plants were exposed three times to low RH for 8 h prior sampling at 3, 4, and 5 days and otherwise maintained under humid conditions.

Fig. 4.

Fraction of living cells in relation to the total number of cells in the aggregate in which they occurred for cell aggregates artificially generated from cultured cells on slides (circles) or for aggregates recovered from plants and deposited onto membranes filters (squares). Slides and filters were exposed to low RH for 8 h after cell deposition. Vertical bars represent the standard error of the mean fraction of living cells in aggregates of different sizes.