Secondary metabolites in fungal biotic interactions
by Yi Kuang
Date of Examination:2014-05-09
Date of issue:2014-05-22
Advisor:Prof. Dr. Petr Karlovsky
Referee:Prof. Dr. Petr Karlovsky
Referee:Prof. Dr. Matthias Hahn
Referee:PD Dr. Michael Hoppert
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Abstract
English
Fungi are known for their production of chemical and biological diversity of secondary metabolites. To understand the structure, biological activity and function of fungal secondary metabolites contribute to the development of natural product chemistry and mycology. The aim of this study was to investigate the fungal secondary metabolites involved in different fungal interactions. In this work, HPLC-MS based metabolic profiling was used to study the induction, inhibition and degradation of secreted fungal metabolites in the co-culture of phytopathogen Fusarium verticillioides with mycoparasite Gliocladium roseum. The results indicated that several metabolites were induced or suppressed in the co-cultures; moreover, specific metabolites which only occurred in the dual cultures were found. Large-scale co-cultures of F. verticillioides and G. roseum (80 Liter) were prepared for the isolation of interaction-specific metabolites. After extraction, selected metabolites were purified by using combinations of different chromatography methods. Four selected metabolites were obtained from the co-cultures. All of them turned out to be derivatives of fusaric acid (FA). Two of the metabolites (compound 2 and 3) have not been described before. In addition, the production of known mycotoxins in dual cultures was studied. The amount of fumonisins and FA were decreased in dual cultures with G. roseum as compared to F. verticillioides single cultures. The comparison of fungal biomass of F. verticillioides in single and co-cultures showed that after 20 days the growth of F. verticillioides was suppressed by G. roseum. Two experiments were carried out to identify the origin of the FA derivatives. Firstly, both F. verticillioides and G. roseum were co-cultivated with other ten different fungal species. Secondly, biotransformation of FA by G. roseum was studied. The results showed that all four FA derivatives only produced in dual cultures of F. verticillioides with G. roseum. The two new FA derivatives were biotransformation products of FA by G. roseum. Furthermore, we synthesized all the isolated metabolites to test their biological activities against model fungi Botrytis cinerea and Aspergillus niger. The bioassay indicated that the FA derivatives were less toxic than FA; compound 2 was the least toxic FA derivative. We thus showed that the detoxification of FA is involved in fungal interactions. Another part of this study was on the metabolic profiling of Botrytis species. Metabolic profiling helps to understand how differences in the secondary metabolites among different fungal strains and explain differences in their biological properties. Ten different Botrytis strains, including B. cinerea, B. pseudocinerea, B. fabae and B. group Sa, were incubated for 3 days in Gamborg’s B5 medium. After 3 days, the culture supernatants were analyzed by HPLC-MS with full-scan detection and data dependent fragmentation (TurboDDS). Totally 50 metabolic signals were detected and made the comparison of the metabolites such as species-specific or species-nonspecific metabolites, which were used to (i) explore variation in secondary metabolites produced by isolates of Botrytis spp. specialized to different host plants, and (ii) identify changes in metabolic profiles caused by disruption of selected genes of secondary metabolism synthesis. Similarities among aligned metabolic profiles for all tested strains were investigated by hierarchical cluster analysis (HCA) and non-metric multidimensional scaling (MDS). The results support the classification of tested Botrytis strains based on their morphological characteristic and indicate that Botrytis group Sa (D08_H_8 and G09_S33) and B. cinerea were closely related based on their metabolite profiles. The slightly slower growth rate of B. cinerea B05.10pks6bot2 and B. cinerea B05.10bot2 than the wild-type B. cinerea B05.10, suggest the phytotoxin botrydial might cause effect in saprophytic growth. Turbo data dependent scanning (TurboDDS) was used for identification of secondary metabolites. Some botrydial-related metabolites were identified by the comparison with the published tandem mass spectrometry data.
Keywords: Secondary metabolites, Fusarium verticillioides, Gliocladium roseum, Botrytis cinerea, Fungal interactions