Abstract
Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.
Highlights
Successful establishment of a biocontrol agent in a particular environment depends upon a number of factors including competition with indigenous microflora for available resources and resisting the deleterious effects of grazing predators
Pseudomonas strains were cultured on Lysogeny Broth (LB) or King’s B (KB) [21] medium at 28°C or in M9 minimal salts medium amended with 0.4% glucose and 1mM magnesium sulfate (MgSO4)
We were interested to learn whether PA23 exhibited lethality to C. elegans through one or both of these means
Summary
Successful establishment of a biocontrol agent in a particular environment depends upon a number of factors including competition with indigenous microflora for available resources and resisting the deleterious effects of grazing predators. Bacteria fall prey to a number of organisms; among these, bacterivorous nematodes are thought to play a major role in shaping the microbial community structure. The model organism Caenorhabditis elegans is frequently employed for studies of bacterial-nematode interactions. For bacteria that exhibit pathogenicity towards C. elegans, nutrient availability plays a significant role in determining how these harmful effects are mediated. Under nutrient-limiting conditions, sublethal levels of toxic bacterial metabolites are produced and nematode death proceeds over the course of days. In this case, the so-called “slow-killing” effect is reliant upon bacterial colonization of the nematode intestine [8]. For the well-studied pathogen Pseudomonas aeruginosa, different mechanisms of fast-killing have been reported depending on the bacterial strain in question and the growth medium employed. Glucose and sorbitol (PGS) medium, phenazines (PHZ) are responsible for PA14-mediated intoxication [7,8]
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