Fungal Secondary Metabolism Methods and Protocols
Filamentous fungi have long been known for their ability to produce an enormous range of unusual chemical compounds known as secondary metabolites, many of which have potentially useful antibiotic or pharmacological properties. Recent focus on fungal geno
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1. Introduction Fungi produce an astonishing variety of secondary metabolites, some of which belong to the most toxic compounds in the living world. However, the benefits of secondary metabolites for fungi are often obscure. In contrast to the gap in knowledge of the biological relevance of secondary metabolites, enormous research has been devoted to identifying biochemical pathways and the underlying genetic mechanisms leading to the biosynthesis of fungal secondary metabolites (1). It is, however, likely that the fungal ability to regulate secondary metabolism reflects an evolutionary adaptation to ensure efficient exploitation of environmental resources and to synthesize secondary metabolite only when the ecological conditions demand it against natural enemies and competitors (2–4). It should be noted, however, that in addition to secondary metabolites, fungal trypsin-specific inhibitors and lectins have been reported as defense molecules towards competitors and predators (5–7).
Nancy P. Keller and Geoffrey Turner (eds.), Fungal Secondary Metabolism: Methods and Protocols, Methods in Molecular Biology, vol. 944, DOI 10.1007/978-1-62703-122-6_21, © Springer Science+Business Media, LLC 2012
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U. Fohgrub and F. Kempken
Previously performed food choice experiments with springtails support a role for secondary metabolism in predator defense. In Aspergillus nidulans the protein LaeA is a global regulator of secondary metabolite production (8–10). Using a mutant deleted for LaeA, it was demonstrated that springtails prefer the LaeA mutant for feeding in food choice experiments. Springtails feeding on the LaeA mutant produced more offspring than the wild type (3). These results demonstrate that fungal secondary metabolites shape food choice behavior and affect population dynamics of fungivores. That study also provides evidence for a selective force favoring secondary metabolite synthesis in fungi (3). Competition studies using insects and molds may provide important insight into the biological functions of fungal metabolites and their role in evolution. Here we describe a simple method for such competition experiments.
2. Materials 2.1. Drosophila and Aspergillus Medium
1. Drosophila standard medium (11, 12). 62.5 g untreated cornmeal, 62.5 g saccharose, 62.5 g yeast extract, 12.5 g agar, 1 L distilled H2O. 2. Drosophila feeding medium (11, 12). Drosophila standard medium, with 10 g cane syrup, 30 mL 10% (w/v) methyl parahydroxybenzoate or nipagin M (Merck catalog number: 106757) (dissolved in EtOH), 10 mL 10% (v/v) propionic acid (1 N diluted in distilled water). 3. Drosophila breeding medium (11, 12). Drosophila standard medium; add 1 mL 5% (v/v) acetic acid (1 N diluted in distilled water). 4. Drosophila standard medium plus foil (sterile). Drosophila standard medium with 1% (w/v) KOH treated foil (DESAGA GmbH, catalog number: SI-Nr.4-42284-01-0), distilled H2O wetted Whatman paper (Whatman, catalog number: 1001917), and a pair of tweezers (sterile, autoclaved) are necessary for sterile transfer. 5. As
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