Sclerotinia sclerotiorum : phytotoxins and metabolism of phytoalexins
Ahiahonu, Pearson William Kwaku
Sclerotinia sclerotiorum (Lib.) de Bary is a plant pathogenic fungus causing serious yield losses in a broad range of cultivated plants, excluding cereals. Most of the economically important brassicas such as canola, rapeseed, mustards, cabbages and others such as sunflower, peanut, bean, soybean, lettuce, and carrot are susceptible to this pathogen. No host specificity has been demonstrated in S. sclerotiorum and there is no specific resistance known in the host species. The main trust of this research project was to establish biotransformation pathways used by S. sclerotiorum to detoxify phytoalexins produced by host plants. As well, the potential production of phytotoxins and cytotoxic compounds by S. sclerotiorum was analyzed. The metabolite sclerin was isolated from cultures of S. sclerotiorum and its phytotoxicity to crucifers established for the first time. Fatty acids isolated from sclerotia of S. sclerotiorum of which oleic acid was a major component were found to be cytotoxic to the brine shrimp (Artemia salina). Chemical defences, i.e. phytoalexins, were elicited and isolated from a resistant plant (Erucastrum gallicum, dog mustard): indole-3-acetonitrile, arvelexin, 1-methoxyspirobrassinin and erucalexin (new phytoalexin, a structural isomer of 1-methoxyspirobrassinin). As well, the biotransformations of the phytoalexins brassinin, produced by rapeseed, canola and brown mustard plants, camalexin, and 6-methoxycamalexin, produced by wild crucifers like Arabidopsis thaliana, Capsella bursapastoris and Camelina sativa, were investigated. It was established that S. sclerotiorum could efficiently metabolize these phytoalexins using a remarkable glucosylation reaction of their indole ring. Overall, results of these biotransformation studies followed by antifungal bioassays indicated that metabolism of brassinin, camalexin, and 6-methoxycamalexin were detoxification processes. Analogues of these phytoalexins were designed based on structures of the detoxification products to probe the specificity or otherwise of the enzyme(s) involved in the metabolism of the phytoalexins. All the analogues tested were metabolized by the fungus though at slower rates. 6-Fluorocamalexin, one of the analogues, significantly slowed down the metabolism of brassinin both in cell cultures and in enzymatic assays with cell homogenates. Partial purification (five fold) of the brassinin detoxifying enzyme (brassinin glucosyltransferase) of S.sclerotiorum was achieved.