In the first part of this thesis the metabolism of the phytotoxins destruxin B and sirodesmin PL in crucifers and non-crucifers was studied using HPLC-ESI-MSn. Destruxin B and sirodesmin PL are phytotoxins produced by the phytopathogenic fungi Alternaria brassicae (Berk.) Sacc. (causative agent of blackspot disease) and Leptosphaeria maculans (Desm) Ces. et de Not.[asexual stage Phoma lingam (Tode ex Fr) Desm.] (causative agent of blackleg disease). Five cruciferous species were used in this study: Arabidopsis thaliana L., Brassica rapa L., B. napus L., Thellungiella salsuginea Pallas and Erucastrum gallicum O.E. Schulz. In addition, the cereals Avena sativa L. and Triticum aestivum L. were studied similarly. Destruxin B was metabolized by all crucifers to hydroxydestruxin B, a transformation similar to previously reported reactions in other crucifers. In addition, destruxin B elicited production of phytoalexins in A. thaliana, T. salsuginea and E. gallicum, while no phytoalexins were detected in case of B. rapa and B. napus. In cereals destruxin B was transformed differently. Several metabolites were detected and identified by HPLC-ESI-MSn analyses: hydroxydestruxin B, two isomers of dehydrodestruxin B and desmethyldestruxin B. On the other hand, no metabolites related to transformation of sirodesmin PL were detected in crucifers; however, in cereals sirodesmin PL was transformed to deacetylsirodesmin PL. In all crucifers sirodesmin PL was found to be a stronger elicitor of phytoalexin production than destruxin B. In the second part of this thesis, mycelia from different pathogenic fungi were screened for indolyl-3-acetaldoxime dehydratase. L. maculans isolate Laird 2 was chosen for isolation, characterization and substrate specificity of aldoxime dehydratase, as it showed the highest specific activity among the tested pathogens. The enzyme was partially purified using three chromatographic steps. It showed Michaelis–Menten kinetics and an apparent molecular mass of about 40 kDa. Based on its substrate specificity, the enzyme appears to be an indolyl-3-acetaldoxime dehydratase

The phytopathogenic fungus <i>Leptosphaeria maculans</i> can cause blackleg disease on crucifers, which results in significant yield losses. Fungal diseases involve interactions between pathogenic fungi and host plants. One aspect of these interactions is mediated by secondary metabolites produced by both fungi and host plants. Phytotoxins and elicitors as well as phytoanticipins and phytoalexins are metabolites produced by fungi and plants, respectively. This thesis describes and discusses the isolation, structure, biological activity and biosynthesis of the secondary metabolites produced by L. maculans.<p> The elicitor-toxin activity bioassay guided isolation of elicitors and phytotoxins produced by <i>L. maculans</i> in a chemically defined medium lead to the isolation of general elicitors, <i>sirodesmin PL</i> (165) and <i>deacetylsirodesmin PL</i> (166), and specific elicitors, <i>cerebrosides C</i> (14) and D (31) from minimum medium (MM) culture under standard conditions. The known phytotoxins sirodesmin PL (165) and deacetylsirodesmin PL (166) induced the production of <i>phytoalexin spirobrassinin</i> (122) in both resistant plant species (brown mustard, <i>Brassica juncea</i> cv. Cutlass) and susceptible plant species (canola, B. napus cv. Westar). A mixture of cerebrosides C (14) and D (31) induced the production of the phytoalexin rutalexin (127) in resistant plant species (brown mustard, B. juncea cv. Cutlass) but not in susceptible plant species (canola, B. napus cv. Westar). New metabolites leptomaculins A-E (267-269, 272 and 274) and deacetylleptomaculins C-E (270, 273 and 275) were isolated from elicitor-phytotoxin active fractions but did not display detectable elicitor activity or phytotoxicity after purification.<p> New metabolites maculansins A (299) and B (300), which were not detected in cultures of L. maculans incubated in MM, were isolated from cultures of <i>L. maculans</i> incubated in potato dextrose broth (PDB). Maculansins A (299) and B (300) displayed higher phytotoxicity on brown mustard than on canola and white mustard (<i>Sinapis alba cv. Ochre</i>) but did not elicit detectable production of phytoalexins in either brown mustard or canola. Metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde (212) was produced in higher amount in cultures of L. maculans incubated in PDB than in MM and displayed strong inhibition effect on the root growth of brown mustard and canola. <i>L. maculans</i> incubated in MM amended with high concentration of NaCl produced a new metabolite, 8-hydroxynaphthalene-1-sulfate (293), and a known metabolite, bulgarein (294), which are likely involved in the self-protection. The potential intermediates involved in the biosynthesis of sirodesmin PL (165) were investigated using deuterium labeled precursors: [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b), [5,5-2H2]phomamide (171a), [2,3,3-2H3]-L-serine (233d) and [5,5-2H2]cyclo-L-tyr-L-ser (252a). Intact incorporation of [5,5-2H2]phomamide (171a) into sirodesmin PL (165) suggested that leptomaculin D (272) and E (274), and deacetylleptomaculin D (273) and E (275) are not intermediates in the biosynthesis of sirodesmin PL (165). They are more likely the catabolic metabolites of sirodesmin PL (165). Phomamide (171), the intermediate in the biosynthetic pathway of sirodesmin PL (165), is likely biosynthesized by coupling of prenyl tyrosine (312) with serine (233) rather than prenylation of cyclo-L-tyr-L-ser (252). When [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), and E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b) were fed into cultures of L. maculans, a â proton exchange was detected by 1H NMR through intrinsic steric isotope effect, which occurs before the formation of phomamide (171). The biosynthesis and catabolism of sirodesmin PL (165) were proposed based on the results obtained in this work.

The phytopathogenic fungus <i>Leptosphaeria maculans</i> can cause blackleg disease on crucifers, which results in significant yield losses. Fungal diseases involve interactions between pathogenic fungi and host plants. One aspect of these interactions is mediated by secondary metabolites produced by both fungi and host plants. Phytotoxins and elicitors as well as phytoanticipins and phytoalexins are metabolites produced by fungi and plants, respectively. This thesis describes and discusses the isolation, structure, biological activity and biosynthesis of the secondary metabolites produced by L. maculans.<p> The elicitor-toxin activity bioassay guided isolation of elicitors and phytotoxins produced by <i>L. maculans</i> in a chemically defined medium lead to the isolation of general elicitors, <i>sirodesmin PL</i> (165) and <i>deacetylsirodesmin PL</i> (166), and specific elicitors, <i>cerebrosides C</i> (14) and D (31) from minimum medium (MM) culture under standard conditions. The known phytotoxins sirodesmin PL (165) and deacetylsirodesmin PL (166) induced the production of <i>phytoalexin spirobrassinin</i> (122) in both resistant plant species (brown mustard, <i>Brassica juncea</i> cv. Cutlass) and susceptible plant species (canola, B. napus cv. Westar). A mixture of cerebrosides C (14) and D (31) induced the production of the phytoalexin rutalexin (127) in resistant plant species (brown mustard, B. juncea cv. Cutlass) but not in susceptible plant species (canola, B. napus cv. Westar). New metabolites leptomaculins A-E (267-269, 272 and 274) and deacetylleptomaculins C-E (270, 273 and 275) were isolated from elicitor-phytotoxin active fractions but did not display detectable elicitor activity or phytotoxicity after purification.<p> New metabolites maculansins A (299) and B (300), which were not detected in cultures of L. maculans incubated in MM, were isolated from cultures of <i>L. maculans</i> incubated in potato dextrose broth (PDB). Maculansins A (299) and B (300) displayed higher phytotoxicity on brown mustard than on canola and white mustard (<i>Sinapis alba cv. Ochre</i>) but did not elicit detectable production of phytoalexins in either brown mustard or canola. Metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde (212) was produced in higher amount in cultures of L. maculans incubated in PDB than in MM and displayed strong inhibition effect on the root growth of brown mustard and canola. <i>L. maculans</i> incubated in MM amended with high concentration of NaCl produced a new metabolite, 8-hydroxynaphthalene-1-sulfate (293), and a known metabolite, bulgarein (294), which are likely involved in the self-protection. The potential intermediates involved in the biosynthesis of sirodesmin PL (165) were investigated using deuterium labeled precursors: [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b), [5,5-2H2]phomamide (171a), [2,3,3-2H3]-L-serine (233d) and [5,5-2H2]cyclo-L-tyr-L-ser (252a). Intact incorporation of [5,5-2H2]phomamide (171a) into sirodesmin PL (165) suggested that leptomaculin D (272) and E (274), and deacetylleptomaculin D (273) and E (275) are not intermediates in the biosynthesis of sirodesmin PL (165). They are more likely the catabolic metabolites of sirodesmin PL (165). Phomamide (171), the intermediate in the biosynthetic pathway of sirodesmin PL (165), is likely biosynthesized by coupling of prenyl tyrosine (312) with serine (233) rather than prenylation of cyclo-L-tyr-L-ser (252). When [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), and E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b) were fed into cultures of L. maculans, a â proton exchange was detected by 1H NMR through intrinsic steric isotope effect, which occurs before the formation of phomamide (171). The biosynthesis and catabolism of sirodesmin PL (165) were proposed based on the results obtained in this work.