An investigation of the mechanisms underlying biological control activity of a novel canola-associated bacterial isolate, Pseudomonas species DF41
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Abstract The ability of several plant-associated bacteria to inhibit the proliferation of root-pathogens has been well established whereas considerably less has been reported about bacterial species inhibiting pathogens on the phylloplane. Sclerotinia sclerotiorum is the fungal causative agent of stem rot and is capable of infecting over 400 plant species, including flowering canola plants. For this reason, there is a need for disease management strategies targeted at preventing sclerotinia infection. Pseudomonas species DF41 was isolated from the canola rhizosphere and found to be an excellent antagonist of sclerotinia stem rot. Therefore, research efforts turned towards elucidating the mechanisms underlying DF41 antifungal (AF) activity. A random transposon mutagenesis approach facilitated the identification of genes essential for DF41 fungal antagonism. One gene that was identified, gacS, encodes the sensor kinase of the Gac two-component signal transduction system. Characterization of the DF41 gacS mutant revealed that this regulator is essential for secondary metabolite production. In other bacteria, the Gac system activates target gene expression by upregulating the transcription of small, untranslated RNA molecules (sRNA). A sRNA molecule called RsmZ was found to act as a downstream regulatory element in the DF41 Gac regulatory cascade. Furthermore, we discovered that DF41 is producing acyl homoserine lactone (AHL) signalling molecules. This prompted us to investigate the effect of quorum sensing (QS) on phenotypes contributing to AF activity. In DF41, AHL- signalling is not important for secondary metabolite production but does influence motility and may indirectly govern gene expression by controlling other regulatory elements Screening of our transposon library led to the identification of a non-ribosomal peptide synthetase gene involved in synthesis of a cyclic lipopeptide (CLP) molecule. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) enabled the identification of an unusual CLP and we propose a preliminary structure containing some unique features. The role of this molecule in Pseudomonas sp. DF41 AF activity was also elucidated. Altogether, this investigation has revealed a number of important findings regarding how DF41 functions as a biocontrol agent. This information will allow us to use DF41 more effectively in the future in managing sclerotinia stem rot on canola plants.