Analysis of toxigenic fungi and their mycotoxins in biotic interactions

Filamentous fungi are producers of a broad range of secondary metabolites with a high diversity of biological activities. It is generally assumed that the production of these metabolites leads to benefits which allow the fungi to survive in their ecological niche. The role of most secondary metabolites is still unknown, but many of them may be involved in biotic interactions. Understanding the function of secondary metabolites in biotic interactions requires experimental studies for which sensitive and accurate analytical techniques for the quantification of fungal biomass and secondary metabolites are necessary. The aim of this study was the development and validation of analytical detection methods based on real-time PCR and HPLC-MS/MS as well as the identification and investigation of toxic secondary metabolites involved in diverse fungal interactions. First of all a sensitive method for the simultaneous quantification of the six hexadepsipeptides beauvericin, enniatin A, A1, B and B1 and destruxin A in various matrices like asparagus, potato, maize, tomato, rice and wheat were developed and validated. Fragmentation of sodium adducts of the analytes and determination of their specific mass spectra with LC-ESI-MS/MS using an ion trap allowed the specific and sensitive identification of the mycotoxins. Furthermore, sample preparation steps, including choice of suitable organic solvents for extraction and defatting, were thoroughly investigated. A new solvent combination acetonitrile/isopropyl alcohol/water (70:15:15) lead to high efficiency rates and low matrix effects. The limits of quantification and limits of detection ranged from 1-12 ng g-1 and 0.3-4 ng g-1 respectively across all mycotoxins and matrices. Furthermore real-time PCR is a common method for species-specific quantification of fungal biomass in epidemiological studies. However, no methods for the determination of the two performance characteristic parameters in real time PCR assays, that is, the limit of quantification and the limit of detection, exist as they do in common chemical analytical techniques. In this study a method based on receiver operating characteristic (ROC) curve analysis in combination with Youden index was established to determine both performance characteristic parameters. The concept was applied to two species-specific real-time PCR assays which had been developed in earlier studies and which served the quantification of F. verticillioides and F. proliferatum. Apart from the establishment of analytical methods, studies on biotic interactions of fungi with a focus on the production of secondary metabolites were carried out. Interactions of two major causal agents of maize ear rot F. verticillioides and F. graminearum were investigated by inoculating maize ears with a spore suspension of F. verticillioides, F. graminearum and a mixture of both in field trials. Maize kernels were analyzed for their content of fungal biomass and mycotoxins. Amounts of F. verticillioides and fumonisin B1 either increased or were not affected by mixed inoculations. By contrast, the incidence of F. graminearum were either not affected or decreased in mixed inoculations. However, the amounts of mycotoxins relative to biomass revealed no differences between single and mixed inoculated treatments. Additionally, the effect of different temperature scenarios on fungal disease severity and interactions were investigated under controlled conditions. Overall, incidence and amounts of F. verticillioides and fumonisin B1 were either not affected or correlated positively with increased temperature in single and mixed inoculations. The results indicate that an increase of temperature due to climate warming may favor maize ear rot caused by F. verticillioides. The interaction with F. graminearum may additionally facilitate infestation by F. verticillioides. This may result in increased risk of fumonisin B1 contamination of maize in moderate climate areas. Another part of this dissertation deals with the production of secondary metabolites produced by Aspergillus nidulans as a putative chemical defense reaction against the fungivore Folsomia candida. Grazing of F. candida on A. nidulans induced significant up-regulation of the highly toxic mycotoxin sterigmatocystin. Furthermore, the enhanced production of two meroterpenoids austinol and dehydroaustinol as well as the cyclic nonribosomal depsipeptides emericellamides C, D, E and F was identified. These findings may indicate the involvement of these secondary metabolites in the defense reaction of A. nidulans to fungivores. Finally, the fungal strain Fusarium oxysporum f. sp. strigae Elzein et Thines, was analyzed for its production of the mycotoxins fumonisins, beauvericin, enniatins and moniliformin. It was found that the strain in question produced the mycotoxin beauvericin. Furthermore, the ability of the fungus to produce cancerogenic fumonisins was additionally excluded by genetic analysis. The fungal strain is highly host specific and aggressive towards the parasitic weed Striga hermonthica but is nonpathogenic towards sorghum. Beauvericin was obtained in Striga shoots but not in sorghum grains. The host-specific pathogenicity of the fungus towards S. hermonthica in combination with unaffected sorghum grains indicates that the fungus acting as biocontrol agent poses no risks for animal or human consumers of sorghum grains. The reported studies revealed the involvement of several secondary metabolites in diverse types of complex biotic interactions demonstrating the high diversity and biological activities of fungal secondary metabolites.