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Biosurfactant production by bacteria on leaf surfaces is poorly documented, and its role in this habitat has not been explored. Therefore, we investigated the production and fitness benefits of syringafactin by Pseudomonas syringae pv. syringae B728a on leaves. Syringafactin largely adsorbed to the waxy leaf cuticle both when topically applied and when produced by cells on plants. Syringafactin increased the rate of diffusion of water across isolated cuticles and attracted water to hydrophobic surfaces exposed to high relative humidity due to its hygroscopic properties. While a wild-type and syringafactin mutant exhibited similar fitness on bean leaves incubated in static conditions, the fitness of the wild-type strain was higher under fluctuating humidity conditions typical of field conditions. When co-inoculated onto either the host plant bean or the non-host plant romaine lettuce, the proportion of viable wild-type cells recovered from plants relative to that of a mutant unable to produce syringafactin increased 10% over 10 days. The number of disease lesions incited by the wild-type strain on bean was also significantly higher than that of the syringafactin mutant. The production of hygroscopic biosurfactants on waxy leaf surfaces apparently benefits bacteria by both attracting moisture and facilitating access to nutrients., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Biosurfactants are diverse molecules with numerous biological functions and industrial applications. A variety of environments were examined for biosurfactant-producing bacteria including soil, water and leaf surfaces. Biosurfactant production was assessed with an atomized oil assay for a large number of bacterial isolates and compared with a commonly used drop collapse assay from broth and plate cultures. The atomized oil assay detected every strain that produced a biosurfactant detectable by the drop collapse test, and also identified additional strains that were not detected with the drop collapse assay because they produced low levels of surfactant or hydrophobic (low water solubility) surfactants such as pumilacidins. Not all strains that produced a biosurfactant detectable by the drop collapse when cultured on agar surfaces produced surfactants detectable by drop collapse when cultured in broth, and vice versa. Many bacterial strains exhibited preferential production of surfactants when grown on an agar surface compared with broth cultures, and such surface enhancement of production could also be stimulated by increasing the viscosity of liquid culture media. Surface induction of surfactant production in the epiphyte Pseudomonas syringae was regulated at the transcriptional level., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Leaf surfaces harbour bacterial epiphytes that are capable of influencing the quorum sensing (QS) system, density determination through detection of diffusible signal molecules, of the plant-pathogen Pseudomonas syringae pv. syringae (Pss) which controls expression of extracellular polysaccharide production, motility and other factors contributing to virulence to plants. Approximately 11% of the bacterial epiphytes recovered from a variety of plants produced a diffusible factor capable of inhibiting the QS system of Pss as indicated by suppression of ahlI. Blockage of QS by these interfering strains correlated strongly with their ability to limit iron availability to Pss. A direct relationship between the ability of isogenic Escherichia coli strains to sequester iron via their production of different siderophores and their ability to suppress QS in Pss was also observed. Quorum sensing induction was inversely related to iron availability in culture media supplemented with iron chelators or with FeCl(3). Co-inoculation of interfering strains with Pss onto leaves increased the number of resultant disease lesions over twofold compared with that on plants inoculated with Pss alone. Transposon-generated mutants of interfering strains in which QS inhibition was blocked did not increase disease when co-inoculated with Pss. Increased disease incidence was also not observed when a non-motile mutant of Pss was co-inoculated onto plants with QS interfering bacteria suggesting that these strains enhanced the motility of Pss in an iron-dependent manner, leading to an apparent increase in virulence of this pathogen. Considerable cross-talk mediated by iron scavenging apparently occurs on plants, thereby altering the behaviour of bacteria such as Pss that exhibit important QS-dependent traits in this habitat.

The foliar pathogen and ice nucleator, Pseudomonas syringae pv. syringae B728a, demonstrates a high level of epiphytic fitness on plants. Using a promoter-trapping strategy termed habitat-inducible rescue of survival (HIRS), we identified genes of this organism that are induced during colonization of healthy bean leaf surfaces. These plant-inducible genes (pigs) encode diverse cellular functions including virulence, transcription regulation, transport, nutrient acquisition and other known and unknown loci, some of which may result in antisense transcripts to annotated P. syringae genes. Prominent among the pigs was ssuE, a gene in the sulfate-starvation regulon, indicating that sulfate is not abundant on leaf surfaces. inaZ reporter gene fusion assays of the plant-inducible loci revealed up to 300-fold higher levels of pig transcriptional activity on plant leaves compared with minimal medium. However, the maximum levels of pig transcriptional activity were typically too weak to be measured using a gfp reporter gene. One exception was orf6 in the hrp/hrc pathogenicity island which was highly induced in epiphytic P. syringae cells. Four pigs were disrupted by insertional mutagenesis. While growth of the ssuE mutant was impaired under certain conditions in laboratory medium, the epiphytic and virulence properties of the mutants on bean plants were identical to wild-type P. syringae. Our results demonstrate the utility of HIRS to identify genes expressed on leaves and provide new insight into the leaf surface environment.