Development of Chromatography Techniques for Analysis and Preparative Isolation of Phytotoxic Metabolites Produced by Stagonospora cirsii

A simple HPLC technique for the quantitative determination of phytotoxins (stagonolide A and herbarumin I) in liquid culture of the fungus Stagonospora cirsii S-47 has been developed. Some examples of its application are given. The technique was tested in a study of effects of the concentration of refined sunflower oil and the cultivation duration on the production level of these toxins. At an oil concentration of 0.5% or higher in modified Czapek medium with vitamins, the accumulation of S. cirsii biomass and the concentration of stagonolide A increased, while the concentration of herbarumin I decreased. Maximal amounts of both phytotoxins were obtained on the tenth day of submerged S. cirsii cultivation in flasks. When S. cirsii was grown in the same medium supplied with 1% of oil in a laboratory bioreactor, the maximal concentration of stagonolide A (116 mg/L) in the liquid culture was achieved after 5 days of fermentation followed by its considerable reduction after 6 days, while after 7 days of the submerged growth the maximal content of herbarumin I (116 mg/L) increased sharply. The technique of the phytotoxins analysis was used to monitor them during their isolation, as well as for preparative HPLC.