This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

No description available.

No description available.

Allium white rot (AWR) is a soilborne disease that seriously damages commercial cultivation of onion (Allium cepa) and garlic (Allium sativum) crops. The disease has been found everywhere onions are cultivated and at present no system of control has been found that fully prevents the occurrence of the disease. The fungus responsible for the disease, Sclerotium cepivorum, uses oxalic acid to kill Allium bulb and root tissue in growing onion and garlic plants. Research suggests recombinant oxalate oxidase and oxalate decarboxylase enzymes may be able to degrade this acid and confer resistance against pathogens that rely on it, such as Sm. cepivorum or Sclerotinia sclerotiorum. To test the efficacy of these enzymes against white rot pathogens, three transgenes for wheat oxalate oxidase, barley oxalate oxidase and Flammulina oxalate decarboxylase were transformed into onions and garlic by Agrobacterium-mediated transformation. Allium species are highly recalcitrant to transformation, so these three transgenes were also transformed into tobacco to provide fast-recovering, easy to test transformants to assess the efficacy of the transgenes. Transformed garlic and tobacco lines were analysed to assess the integration and expression of the transgenes, then challenged with Sm. cepivorum or Sa. sclerotiorum, respectively, to assess the bioactivity of recombinant wheat oxalate oxidase, barley oxalate oxidase, and Flammulina oxalate decarboxylase against oxalic acid-dependent pathogens. Results show that one line of tobacco expressing the Flammulina oxalate decarboxylase enzyme was found to be consistently resistant to Sclerotinia sclerotiorum. Garlic lines transformed with this transgene failed to display stable transgene expression or disease resistance, possibly due to silencing of the transgene in recovered transformant tissue.

Soybean (Glycine max (L.) Merr.) is one the major crops produced worldwide. However, soybean is susceptible to many diseases. Sclerotinia stem rot (SSR) disease caused by Sclerotinia sclerotiorum (Lib.) de Bary is considered one of the most important fungal diseases of soybean. It can be controlled by chemicals (e.g. fungicides), by breeding cultivars with disease resistance and by cultural control (e.g. increasing the width between rows, reducing plant populations). A promising and complementary method of controlling SSR disease in the field is the application of biological control agents. Biological control agents introduced in a soil environment will interact with other soil food web organisms, as do the pathogenic organism and infected plants, which may change the genetic and functional diversity in soil microbial communities. Profiling these changes may lead to an improved understanding of the interactions between these players (biological control agents, pathogens, soil biota and plants) in the biological control phenomenom, permiting us to exploit naturally-occurring ecological relationships and develop more sustainable approaches to control soybean diseases. Fatty acid biomarkers analysis was used to profile microbial communities in soils. Two laboratory studies were conducted to evaluate the methods used for extraction and profiling the fatty acid biomarkers from soil samples with a range of soil properties (clay content, organic matter content), The first study investigated the best solvent mixture for recovering fatty acid biomarkers from soil using an automated pressurized solvent extraction (PSE) system. Solvent mixtures containing chloroform and methanol were more efficient at extracting fatty acids from agricultural soils than hexane:2-propanol and acetone. The second study presented an exploratory pyrolysis-mass spectrometry technique to rapidly fingerprint soil lipids extracted from different agroecosystems. Pyrolysis-mass spectrometry discriminated among soils and crop production systems in the same way as the fatty acid profiling. I also report on the efficicacy of biological control agents to control Sclerotinia stem rot disease in soybean. A two-year study was conducted in soybean fields under conventional or no tillage to determine whether Trichoderma virens (SoilGard(TM)) and arbuscular mycorrhizal fungi (a mixture of Glomus intraradices and G. mosseae ), used alone or in combination, could reduce sclerotinia stem rot (SSR) disease incidence. Generally, SSR disease indicators, as well as the soybean yield, were not affected significantly by the biological control treatments. I then studied whether changes in microbial community composition were related to the inoculation of the biological control agents and the disease incidence in soybean fields. Inoculation of biological control agents changes the expression of many soil fatty acids during both years of the trial. Also, in the plots with severely diseased plants, fatty acids biomarkers of gram positive and actinomycetes bacteria were significantly greater than in plots with healthy plants. I conclude that further improvement in laboratory techniques and procedures will permit researchers to efficiently extract and characterize soil lipids, providing new insight into soil organic matter dynamics and soil microbial ecology. Further study will be needed to verify the efficacy and optimize the application method, dose and timing of biocontrol agents to provide protection against SSR disease in soybean fields.

Allium white rot (AWR) is a soilborne disease that seriously damages commercial cultivation of onion (Allium cepa) and garlic (Allium sativum) crops. The disease has been found everywhere onions are cultivated and at present no system of control has been found that fully prevents the occurrence of the disease. The fungus responsible for the disease, Sclerotium cepivorum, uses oxalic acid to kill Allium bulb and root tissue in growing onion and garlic plants. Research suggests recombinant oxalate oxidase and oxalate decarboxylase enzymes may be able to degrade this acid and confer resistance against pathogens that rely on it, such as Sm. cepivorum or Sclerotinia sclerotiorum. To test the efficacy of these enzymes against white rot pathogens, three transgenes for wheat oxalate oxidase, barley oxalate oxidase and Flammulina oxalate decarboxylase were transformed into onions and garlic by Agrobacterium-mediated transformation. Allium species are highly recalcitrant to transformation, so these three transgenes were also transformed into tobacco to provide fast-recovering, easy to test transformants to assess the efficacy of the transgenes. Transformed garlic and tobacco lines were analysed to assess the integration and expression of the transgenes, then challenged with Sm. cepivorum or Sa. sclerotiorum, respectively, to assess the bioactivity of recombinant wheat oxalate oxidase, barley oxalate oxidase, and Flammulina oxalate decarboxylase against oxalic acid-dependent pathogens. Results show that one line of tobacco expressing the Flammulina oxalate decarboxylase enzyme was found to be consistently resistant to Sclerotinia sclerotiorum. Garlic lines transformed with this transgene failed to display stable transgene expression or disease resistance, possibly due to silencing of the transgene in recovered transformant tissue.

The general aim of this research was to analyse the genetic diversity in Sclerotinia species. More specifically the aims of this research were: to separate the three species of Sclerotinia, S. sclerotiorum (Lib.) de Bary, S. minor Jagger and S. trifoliorum Erikss.; to determine the breeding mechanism in S. minor and S. sclerotiorum; to test S. minor for the possibility of causing head rot of sunflower; to examine isolate of S. sclerotiorum from sunflower for aggressiveness and to see if this correlates with underlying genetic diversity. Sclerotinia species were separated using a variety of morphological and molecular criteria. S. minor, S. sclerotiorum and S. trifoliorum were analyzed on characters including host, sclerotial diameters, ascospore morphism, breeding type and RFLP analysis. Cloned DNA fragments from S. sclerotiorum were used as probes, these were compared with a cloned rDNA probe from Neurospora. These probes enabled clear separation of the Sclerotinia species. Sclerotial diameters appear to be good criteria for separating S. minor from S. sclerotiorum and S. trifoliorum. Host species may be sufficient criteria for separating S. sclerotiorum and S. trifoliorum for the plant pathologist in the field, however it was inadequate to accurately separate all isolates. S. minor and S. sclerotiorum were found to be homothallic ascomycetes. Apothecia were raised from all eight ascospores of a single tetrad from four isolates of S. minor and from an isolate of S. sclerotiorum indicating that inbreeding may be the predominant breeding type mechanism of S. minor. Ascospores from asci of S. minor and S. sclerotiorum were predominantly monomorphic, but rare examples of ascospore dimorphism similar to S. trifoliorum were found. Ascospores of S. minor were shown to be capable of causing head rot of sunflower (Helianthus annuus L.) when inoculated onto the floral face during anthesis. This is the first record of the carpogenic germination of S. minor in Australia and demonstration of infectivity of the ascospores on sunflower. Isolates of S. sclerotiorum differ significantly in aggressiveness on sunflower. One hundred and twenty isolates were collected from head and basal stem rots of sunflower in two locations in south east Queensland. The inoculation of sunflower stems with mycelial plugs and the measurement of lesion development were found to be reliable and revealed differences in lesion lengths produced by the different isolates. The time of assessment after inoculation was found to be of significance. Assessment two days after inoculation was more reliable than after three days or the linear rate of lesion development. The significant differences between isolates indicated that the pathogenicity testing method would also be good for virulence testing. The significant differences between the isolates however, was not consistent with repetition and division of the isolates into groups with different aggressive levels was therefore not possible. Differences in aggressiveness were more indicative of a continuous variation in pathogenicity rather than discrete aggressive groups. The number of significantly different isolates was most associated with the statistical test employed. The different multiple comparison procedures used made more difference in interpretation of aggressiveness than the data itself. Isolate aggressiveness did not correspond to the location of collection. Isolates collected from both head and basal stem rots were capable of causing stem infections therefore no specificity for mode of reproduction or infection was found. S. sclerotiorum attacking sunflower in Queensland and New South Wales was found to belong to one large population. Hierarchical sampling only detected one example of a plant lesion infected by more than one genotype. Thus most diseased plants are the result of a single infection only. Population substructuring could not be detected using 11 single copy Restriction Fragment Length Polymorphism (RFLP) loci, suggesting gene flow occurs within the Australian population. Mycelial Compatibility Groups (MCGs), Random Amplified Polymorphic DNAs (RAPDs) single and multicopy RFLPs analysis indicated differences amongst the genotypes identified by each criteria. From 120 isolates the number of genotypes ranged from 13 to 24 depending on the marker used. However there were many similarities in the assemblages of isolates within each genotype. Genotypic diversity could not be correlated with aggressiveness. Initial mode of infection could not be correlated with genotypic differences. Genotypes were also not specific to geographic locations around Australia. However, genotypes identified in Australia were unique from genotypes identified in Canada and United States. Temporal studies also indicated a single population as genetic uniformity was maintained between years. Frequent recovery of the same genotypes around Australia suggests a clonal population structure. The Australian S. sclerotiorum population attacking sunflower appears to have a large asexual component most likely due to the sclerotial production and homothallic sexual reproduction. Gametic disequilibrium was found for all the populations. However, clonal correction of the samples meant that the majority of populations were not at gametic disequilibrium, indicating random associations among loci. Therefore genetic exchange and recombination would appear to be a component of the reproductive cycle of S. sclerotiorum in Australia.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.