Microbial colonization and competition on the marine alga Ulva australis

Abstract

Pseudalteromonas tunicata and Roseobacter gallaeciensis are biofilm-forming marine bacteria that are often found in association with the surface of the green alga Ulva australis. They are thought to benefit the plant host by producing inhibitory compounds that are active against common fouling organisms. We investigated factors that influence the ability of P. tunicata and R. gallaeciensis to attach to and colonize the plant surface and also the competitive interactions that occur between these organisms and other isolates from U. australis during biofilm formation on the plant surface. A surprisingly high number of P. tunicata cells, at least 10(8) cells ml(-1), were required for colonization and establishment of a population of cells that persists on axenic surfaces of U. australis. Factors that enhanced colonization of P. tunicata included inoculation in the dark and pregrowth of inocula in medium containing cellobiose as the sole carbon source (cellulose is a major surface polymer of U. australis). It was also found that P. tunicata requires the presence of a mixed microbial community to colonize effectively. In contrast, R. gallaeciensis effectively colonized the plant surface under all conditions tested. Studies of competitive interactions on the plant surface revealed that P. tunicata was numerically dominant compared with all other bacterial isolates tested (except R. gallaeciensis), and this dominance was linked to production of the antibacterial protein AlpP. Generally, P. tunicata was able to coexist with competing strains, and each strain existed as microcolonies in spatially segregated regions of the plant. R. gallaeciensis was numerically dominant compared with all strains tested and was able to invade and disperse preestablished biofilms. This study highlighted the fact that microbial colonization of U. australis surfaces is a dynamic process and demonstrated the differences in colonization strategies exhibited by the epiphytic bacteria P. tunicata and R. gallaeciensis.

FIG. 1.

Effect of axenic treatment on the U. australis epiphytic community. (A) Nonaxenic U. australis with the natural community present. (B) Axenic U. australis with very few bacteria remaining. Only plant cells are visible. Specimens were stained with 0.01% acridine orange. Scale bars = 50 μm.

FIG. 2.

Comparison of attachment and colonization by GFP-labeled P. tunicata grown in cellobiose (A) and glucose (B). Magnification, ×600. Scale bars = 50 μm.

FIG. 3.

Comparison of biofilm formation by mixed-species biofilms containing P. tunicata cells suspended sterile seawater, in natural seawater, or a mixture of 17 epiphytic strains. P. tunicata inoculated into filtered seawater did not persist for more than 7 days (A). P. tunicata inoculated into natural seawater was able to attach to U. australis and form biofilms that persisted for up to 3 weeks (B). P. tunicata inoculated with a mixture of 17 strains isolated from U. australis formed a complex biofilm, which was also able to persist for up to 3 weeks (C). GFP-labeled P. tunicata cells were enumerated under an epifluorescence microscopy (Table 1) before being stained with 0.01% acridine orange as shown here. Scale bars = 50 μm.

FIG. 4.

Competitive biofilm development on the surface of U. australis in preestablished biofilms containing P. tunicata. In competitive biofilm development P. tunicata (green) was able to dominate in certain situations, but more commonly it was able to coexist with P. gracilis (red) (A). P. tunicata (green) was, however, outcompeted by R. gallaeciensis (red) (B). The P. tunicata AlpP mutant (green) was less competitive than the wild-type P. tunicata strain, as shown by competitive biofilm development with P. gracilis (red) (C). Scale bars = 50 μm.