Pseudomonas chlororaphis PA23 is a promising biological control candidate against Sclerotinia sclerotiorum, a fungal pathogen that causes stem rot in canola. A library of transposon mutants was previously created to understand the molecular mechanisms underlying the antifungal capabilities of PA23. A novel LysR-type transcriptional regulator, called PtrA, was identified as a key global regulator involved in secondary metabolite production. The function of PtrA at the molecular level was investigated in this thesis. Solubility problems encountered during the purification of PtrA redirected efforts to studying a truncated version of the protein instead. A two-step purification of the truncated protein, involving streptomycin sulfate precipitation and immobilized metal-ion affinity chromatography, yielded a highly pure protein. Preliminary crystal growth was achieved for the effector binding domain portion of PtrA. Transcriptional fusions suggested that essential regulatory binding sites of ptrA may lie somewhere between 52 and 198 bp upstream of the translational start site. The research presented in this thesis will help guide future functional studies on PtrA.

Pseudomonas chlororaphis PA23 is a promising biological control candidate against Sclerotinia sclerotiorum, a fungal pathogen that causes stem rot in canola. A library of transposon mutants was previously created to understand the molecular mechanisms underlying the antifungal capabilities of PA23. A novel LysR-type transcriptional regulator, called PtrA, was identified as a key global regulator involved in secondary metabolite production. The function of PtrA at the molecular level was investigated in this thesis. Solubility problems encountered during the purification of PtrA redirected efforts to studying a truncated version of the protein instead. A two-step purification of the truncated protein, involving streptomycin sulfate precipitation and immobilized metal-ion affinity chromatography, yielded a highly pure protein. Preliminary crystal growth was achieved for the effector binding domain portion of PtrA. Transcriptional fusions suggested that essential regulatory binding sites of ptrA may lie somewhere between 52 and 198 bp upstream of the translational start site. The research presented in this thesis will help guide future functional studies on PtrA.

Universiẗat, Diss., 2002--München.

Pseudomonas chlororaphis strain PA23 has demonstrated biocontrol of Sclerotinia sclerotiorum (Lib.) de Bary, a fungal pathogen of canola (Brassica napus L.). The objectives of this research were two-fold: to optimize PA23 phyllosphere biocontrol and to investigate PA23’s influence in the rhizosphere. PA23 demonstrated longevity when inoculated on B. napus under greenhouse conditions. Carbon source differentially effected growth rate and antifungal metabolite production of PA23 in culture. Carbon source did not have a significant effect on in vivo biocontrol. PA23 demonstrated biocontrol ability of the fungal root pathogens Rhizoctonia solani J.G. Kühn and Pythium ultimum Trow in radial diffusion assays. PA23’s ability to promote seedling root growth was demonstrated in sterile growth pouches, but in a soil system these results were reversed. This research is essential for developing PA23 into an effective biocontrol agent in the phyllosphere and it opens the door for use of PA23 as a rhizosphere seed treatment. / October 2016

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. / February 2006

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

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.

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.

Burkholderia contaminans MS14 shows excellent antimicrobial activities against a wide range of pathogens. Complete sequence analysis reveals that the MS14 genome harbors multiple gene loci that contribute to its antimicrobial activities and lacks key virulence features commonly found in pathogenic Burkholderia species. A mutagenesis study identified the genes required for MS14 antibacterial activities and gene expression profiling targeted a polyketide synthase (PKS) gene cluster. Site-specific mutagenesis confirmed the PKS gene cluster is directly related to MS14 antibacterial activities and the PKS gene product is predicted to be the MS14 antibacterial compound. Strain UFB2 isolated from Mississippi shows significant antifungal and antibacterial activities. UFB2 was classified to be Pseudomonas chlororaphis and its complete genome sequence was reported in this study. Green house trails showed P. chlororaphis strain UFB2 could efficiently reduce the disease severity of bacterial canker of tomato, by significantly inhibiting the growth of the pathogen Clavibacter michiganensis subsp. michiganensis. The research findings of B. contaminans MS14 and P. chlororaphis UFB2 have provided insights into the development of MS14 antibacterial compound for agricultural application and potential use of strain UFB2 as a biocontrol agent.