Molecular mechanisms involved in secondary metabolite production and biocontrol of Pseudomonas chlororaphis PA23
Poritsanos, Nicole Joanna
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ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.