Metabolism of cruciferous phytoalexins by Alternaria brassicicola: metabolites and pathways

Phytoalexins are plant metabolites produced in response to different kinds of stress like pathogen attack, while not present in healthy plants. Cruciferous phytoalexins are produced under pathogen attack and different types of stress by crucifers such as oilseed crops (canola, rapeseed), vegetables (rutabaga, cauliflower, turnip) and condiments (wasabi, mustard). The fungal species Alternaria brassicicola (Schwein.) Wiltshire is an economically important pathogen that causes Alternaria black spot in many important crucifer species, namely in Brassica species. Although resistance to A. brassicicola within the commercially available Brassica species is not known, some ecotypes of the wild species Arabidopsis thaliana are resistant to this pathogen. In the first part of this thesis, synthesis of some important cruciferous phytoalexins (camalexin, 1-methylcamalexin, 6-methoxycamalexin, cyclobrassinin, brassilexin, rutalexin and rapalexin A), their antifungal activity and metabolism by the crucifer pathogen A. brassicicola was investigated. Almost all tested phytoalexins showed substantial activity against A. brassicicola except cyclobrassinin with moderate activity. Metabolic investigation has shown that A. brassicicola was able to detoxify camalexin to indole-3-thiocarboxamide (> 5 days), which was transformed further to indole-3-carbonitrile and carboxylic acid, respectively. With the aim of obtaining mechanistic clue on the biotransformation of camalexins, some camalexin derivatives and analogues were synthesized and their antifungal activity as well as metabolism by A. brassicicola was investigated. All camalexin derivatives and analogues showed strong antifungal activity against A. brassicicola. Metabolic investigation has shown that substituent at C-4′ or C-5′ positions of thiazolyl ring of camalexin stopped oxidative degradation of thiazolyl ring by A. brassicicola. In addition, it was obtained that oxidative degradation of the thiazolyl ring requires it to be attached to indole by C-2′. In comparison to camalexins, cyclobrassinin was quickly (ca. 8 hours) detoxified by A. brassicicola to S-methyl [(2-sulfanyl-1H-indolyl-3)methyl]carbamothioate, which was further oxidized to sulfinic and sulfonic acid derivatives. Similarly, A. brassicicola was able to detoxify brassilexin to 3-aminomethylinindole-2-thione (24 hours). Rutalexin was detoxified by A. brassicicola to a highly reactive metabolite that reacted with phomapyrone G, a secondary metabolite produced by A. brassicicola, to yield a stable adduct. All metabolites from biotransformation of mentioned phytoalexins were synthesized and their metabolism as well as antifungal activity against A. brassicicola was investigated. It was indicated that A. brassicicola can detoxify the cruciferous phytoalexins camalexin, 1-methylcamalexin, 6-methoxycamalexin, cyclobrassinin, brassilexin, and rutalexin. Rapalexin A with strong activity against A. brassicicola was resistant to metabolism. In the second part of this thesis, inhibition of brassinin detoxification by Leptosphaeria maculans was investigated. Potential inhibitors were designed and synthesized based on the camalexin scaffold and their inhibitory activity against BOLm was determined using cell-free extracts. Almost all tested compounds showed inhibitory activity against BOLm, however their activity was weaker than camalexin