Alginate genes are required for optimal soil colonization and persistence by Pseudomonas fluorescens Pf0-1

Abstract

Pseudomonas fluorescens strains are important candidates for use as biological control agents to reduce fungal diseases on crop plants. To understand the ecological success of these bacteria and for successful and stable biological control, determination of how these bacteria colonize and persist in soil environments is critical. Here we show that P. fluorescens Pf0-1 is negatively impacted by reduced water availability in soil, but adapts and persists. A pilot transcriptomic study of Pf0-1 colonizing moist and dehydrated soil was used to identify candidate genetic loci, which could play a role in the adaptation to dehydration. Genes predicted to specify alginate production were identified and chosen for functional evaluation. Using deletion mutants, predicted alginate biosynthesis genes were shown to be important for optimal colonization of moist soil, and necessary for adaptation to reduced water availability in dried soil. Our findings extend in vitro studies of water stress into a more natural system and suggest alginate may be an essential extracellular product for the lifestyle of P. fluorescens when growing in soil.

Keywords: Pseudomonas fluorescens; biological control; soil colonization; water stress.

Fig. 1.

Effect of dehydration on P. fluorescens Pf0-1 fitness in soil. Five gram soil samples were hydrated to 50 % WHC and inoculated with Pf0-1. After 2 days, samples were dried to adjust the degree of hydration to WHC of 20, 30 and 40 %. Control samples were left at 50 % WHC. The population of Pf0-1 in experimental and control samples was determined immediately after treatment (day 0), and 1, 3 and 5 days post dehydration. Experiments were carried out at least three times. Data were analysed using one-way ANOVA with Tukey’s multiple comparison test. Error bars show standard error.

Fig. 2.

Response of the P. fluorescens Pf0-1 alginate biosynthetic locus to dehydration stress. Dark grey arrows show alginate genes annotated with locus tags and alg gene names. Genomic coordinates in the Pf0-1 genome are shown above the genes. Below each gene is the transcriptional response to dehydration expressed as fold increase in RPKM (reads per kilobase of transcript, per million mapped reads) relative to controls. Bold numbers indicate significant changes in expression. Bars under alg44 and algD indicate regions that were deleted when constructing mutants.

Fig. 3.

Populations of Pf0-1 and alg mutants in moist soil (a) and soil dried to 30 % WHC 2 days after inoculation (b). Each soil sample was inoculated with the same number of bacteria. Both panels show that each alg mutant was defective in colonization of moist soil (day 1 samples). Dehydration caused a decline in wild-type and mutant populations, with mutants being more dramatically affected. Experiments were carried out at least three times. Data were analysed by two-way ANOVA with post-hoc Dunnett’s multiple comparison test. Error bars show the standard error.