An isolate of <i>Colletotrichum truncatum</i> (Schwein.) Andrus & W. D. Moore is a promising bioherbicide candidate against scentless chamomile (<i>Matricaria perforata</i> Mérat), a noxious weed in western Canada. A major constraint in the development of this bioherbicide is the inefficiency of inoculum production. The objective of this study was to explore submerged fermentation for mass production of <i>C. truncatum</i>.<p>A defined basal salts medium (DBSM) was used for liquid culture with glucose and casamino acids selected as the optimal carbon (C) and nitrogen (N) sources, respectively. Spore yield and biomass production were significantly higher when the DBSM glucose concentration was 35-40 g/L compared to lower concentrations, while inoculum efficacy was significantly greater when produced at 5-10 g/L than at 40 g/L of glucose. Spore yield in baffled flasks at 200 RPM shaker speed was significantly higher than in regular flasks at lower shaker speeds. Under conditions of high aeration, glucose concentration had a significant effect on spore yield, biomass production, and efficacy, whereas the effect was not significant at low aeration. Specific spore and biomass yields also increased significantly with increasing glucose concentrations at high aeration. The scale of submerged fermentation was increased to 20-L fermentors, with dO levels of 10%, 30%, and 60% maintained by agitation and airflow controls. Further study will be required to optimize spore yields at the large scale. This study led to development of a protocol for production of <i>C. truncatum</i> spores using submerged fermentation. Inoculum produced with this method can be used for laboratory, greenhouse, and field trials in development of the bioherbicide.

An isolate of <i>Colletotrichum truncatum</i> (Schwein.) Andrus & W. D. Moore is a promising bioherbicide candidate against scentless chamomile (<i>Matricaria perforata</i> Mérat), a noxious weed in western Canada. A major constraint in the development of this bioherbicide is the inefficiency of inoculum production. The objective of this study was to explore submerged fermentation for mass production of <i>C. truncatum</i>.<p>A defined basal salts medium (DBSM) was used for liquid culture with glucose and casamino acids selected as the optimal carbon (C) and nitrogen (N) sources, respectively. Spore yield and biomass production were significantly higher when the DBSM glucose concentration was 35-40 g/L compared to lower concentrations, while inoculum efficacy was significantly greater when produced at 5-10 g/L than at 40 g/L of glucose. Spore yield in baffled flasks at 200 RPM shaker speed was significantly higher than in regular flasks at lower shaker speeds. Under conditions of high aeration, glucose concentration had a significant effect on spore yield, biomass production, and efficacy, whereas the effect was not significant at low aeration. Specific spore and biomass yields also increased significantly with increasing glucose concentrations at high aeration. The scale of submerged fermentation was increased to 20-L fermentors, with dO levels of 10%, 30%, and 60% maintained by agitation and airflow controls. Further study will be required to optimize spore yields at the large scale. This study led to development of a protocol for production of <i>C. truncatum</i> spores using submerged fermentation. Inoculum produced with this method can be used for laboratory, greenhouse, and field trials in development of the bioherbicide.

No description available.

In soils of high matric potential, low oxygen conditions often develop that favour disease development by many soil-borne plant pathogens. The introduction of a third party, or biocontrol agent, to suppress disease development would require that the agent remains metabolically active under such conditions. In the rhizosphere, plant roots not only supply carbon as an electron donor but cause a localised lowering of oxygen concentrations, conditions favourable for nitrate respiration. The effect of the addition of nitrate on the activity of antagonistic strains of Bacillus subtilis, Pseudomonas fluorescens and P. corrugata was studied in vitro on agar plates, but no significant (P 0.05) quantitative effect was observed . A sealed plate method, using aerobic, anoxic, and anaerobic conditions with 0, 1, 2.5, 5, 10 and 100mM nitrate concentrations was investigated using the B. subtilis strains. This assay tested the activity of antifungal volatiles (AFV) produced by the bacteria. The results indicated that nitrate led to an increased AFV production and/or activity against fungal pathogens under anoxic conditions with nitrate at or above 10 mM. To investigate root colonisation and the establishment of biocontrol colonies in the rhizosphere, lux marking of the biocontrol bacterial strains was undertaken. The transformed bioluminescent B. subtilis strains lost the ability to antagonise the test fungi on agar plates. This loss of antagonism appeared to be due to luciferase utilising metabolites involved in antibiosis and producing a low, but significantly different (P ?0.05) from background and parental strains level of luminescence. The effects of nitrate on a soil based biocontrol system were studied in greenhouse trials. Unfortunately, disease failed to develop, and the effects of the addition of nitrate could not be assessed. The potential involvement of nitrate in maintaining certain biocontrol aspects under conditions that favour pathogen attack seems likely from in vitro based studies. The removal of the antagonistic phenotype, from lux marked B. subtilis strains, raised questions as to the suitability of luciferase for use in this system and highlighted the need for careful monitoring and screening of genetically modified organisms.

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.

The stringent response (SR) is a global regulatory mechanism that allows bacteria to survive starvation. The plant surface is one environment where a fluctuation in nutrient availability is experienced. Because both Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23 are able to protect canola from the fungal pathogen Sclerotinia sclerotiorum when applied as a foliar spray, we sought to investigate the impact of this response on the antifungal activities of these two biocontrol strains. The SR exerts its effects on gene transcription through production of the alarmone(p)ppGpp. Metabolism of (p)ppGpp is governed by two enzymes; RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. To investigate how the SR affects the ability of strains DF41 and PA23 to inhibit the fungal pathogen,relA and relAspoT mutants were generated through allelic exchange. Strain DF41 relA and relAspoT mutants were found to exhibit increased antifungal activity due to enhanced lipopeptide (LP) antibiotic production. Addition of relA, but not spoT in trans restored the mutant phenotype to that of the parent. The influence of the SR on the regulatory mechanisms governing strain DF41 biocontrol was also investigated. It was determined that relA forms part of the Gac regulon while RpoS is under SR control. In fact, addition of rpoS in trans restored protease activity to wild-type levels, but did not attenuate antifungal activity. The SR mutants PA23relA and PA23relAspoT, also exhibited increased growth inhibition of S. sclerotiorum in vitro compared to the wild type. Both mutants showed enhanced production of the antifungal factors pyrrolnitrin, lipase and protease and were complemented by the addition of relA but not spoT. Herein, the SR was found to regulate that Gac system, QS, and RpoS. The presence of gacS or rpoS in multicopy restored the mutant phenotype to that of the wild type.In summary, these findings suggest that the SR negatively influences the biocontrol activities of strains DF41 and PA23. It is evident that the SR is merely one mechanism by which DF41 and PA23-mediated antagonism is regulated.

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.

The stringent response (SR) is a global regulatory mechanism that allows bacteria to survive starvation. The plant surface is one environment where a fluctuation in nutrient availability is experienced. Because both Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23 are able to protect canola from the fungal pathogen Sclerotinia sclerotiorum when applied as a foliar spray, we sought to investigate the impact of this response on the antifungal activities of these two biocontrol strains. The SR exerts its effects on gene transcription through production of the alarmone(p)ppGpp. Metabolism of (p)ppGpp is governed by two enzymes; RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. To investigate how the SR affects the ability of strains DF41 and PA23 to inhibit the fungal pathogen,relA and relAspoT mutants were generated through allelic exchange. Strain DF41 relA and relAspoT mutants were found to exhibit increased antifungal activity due to enhanced lipopeptide (LP) antibiotic production. Addition of relA, but not spoT in trans restored the mutant phenotype to that of the parent. The influence of the SR on the regulatory mechanisms governing strain DF41 biocontrol was also investigated. It was determined that relA forms part of the Gac regulon while RpoS is under SR control. In fact, addition of rpoS in trans restored protease activity to wild-type levels, but did not attenuate antifungal activity. The SR mutants PA23relA and PA23relAspoT, also exhibited increased growth inhibition of S. sclerotiorum in vitro compared to the wild type. Both mutants showed enhanced production of the antifungal factors pyrrolnitrin, lipase and protease and were complemented by the addition of relA but not spoT. Herein, the SR was found to regulate that Gac system, QS, and RpoS. The presence of gacS or rpoS in multicopy restored the mutant phenotype to that of the wild type.In summary, these findings suggest that the SR negatively influences the biocontrol activities of strains DF41 and PA23. It is evident that the SR is merely one mechanism by which DF41 and PA23-mediated antagonism is regulated.

No description available.

No description available.