Regulation of Biosurfactant Production by Quorum Sensing in Pseudomonas fluorescens 5064, the Cause of Broccoli Head Rot Disease

Abstract

Broccoli head rot is a destructive disease found in most broccoli production areas. The main pathogen is the bacterium Pseudomonas fluorescens. P. fluorescens 5064, which was first isolated from an infected broccoli head in SE Scotland, produces biosurfactants that are important for bacterial establishment on the plant surface prior to causing disease in broccoli. Preliminary experiments performed in this study showed that biosurfactant production in P. fluorescens 5064 was cell density dependent, which is a typical characteristic of the quorum sensing mechanism. Quorum sensing is a bacterial communication mechanism, which controls a number of key processes in growth, reproduction and virulence via signalling molecules (quorum sensing signal) in many gram-negative bacteria. One aim of this study was to determine if biosurfactant production in P. fluorescens 5064 is controlled via quorum sensing. To do this, 35 surfactant-minus Tn5 mutants of P. fluorescens 5064 were screened for their abilities to produce a quorum sensing signal. Six of these biosurfactant-deficient mutants showed a large reduction in quorum sensing signal production. In one mutant 6423, which contains a single Tn5 insertion, the production of the quorum sensing signal was almost eliminated. Addition of quorum sensing signal, either synthetic or extracted from wild type P. fluorescens 5064, was able to restore biosurfactant production in mutant 6423. This strongly suggests that quorum sensing regulates biosurfactant production in P. fluorescens 5064. Attempts were made to clone and sequence the Tn5 disrupted gene in mutant 6423, but the identity of the gene remains inconclusive. The quorum sensing signal in wild type P. fluorescens 5064 was identified in this study by High Pressure Liquid Chromatography and Mass Spectrometry as N-3-hydroxyoctanoyl-homoserine lactone, which has been shown by other researchers to be present in P. fluorescens strain 2-79, but not in the strains F113, 7-14 and NCIMB 10586. The discovery that biosurfactant production in P. fluorescens 5064 is regulated by quorum sensing opens up a possibility for novel control of broccoli head rot. Although only the control of biosurfactant production by quorum sensing was examined in this study, it is possible that other virulence factors, such as pectic enzyme production, are also controlled by quorum sensing as in other pathogenic bacteria. By blocking the quorum sensing system, the pathogenic P. fluorescens that use this mechanism to control virulence could potentially be rendered avirulent. In greenhouse pathogenicity tests, a quorum sensing signal-degrading bacterium, Bacillus sp. A24, was evaluated for biocontrol of head rot disease caused by P. fluorescens 5064 on broccoli. However, the Bacillus sp. A24 showed only limited control effects, despite its strong quorum sensing signal-degrading ability towards the pathogen in vitro. A subsequent test proved that Bacillus sp. A24 is a surfactant producer itself and this could explain its ineffectiveness in disease control. When screening the quorum sensing signals of the 35 biosurfactant mutants, mutant 6418 was found to produce a potent antibiotic-like compound. This was identified by thin-layer chromatography as pyrrolnitrin. Unlike wild-type P. fluorescens 5064, mutant 6418 has lost its ability to produce virulence factors and is thus non-pathogenic. It was therefore of interest to determine if mutant 6418 could be used as a biocontrol agent to control broccoli head rot disease. In greenhouse pathogenicity tests, mutant 6418 significantly reduced disease by 41 %. The practical application of this research to bacterial disease control – via the manipulation of quorum sensing to inhibit virulence gene expression – is discussed.