Thin‐layer chromatography (TLC) analysis was performed as described earlier (Bok and Keller, 2004 ; Reyes‐Dominguez et al., 2010 ) with several modifications: Briefly, samples were collected from agar plates in the same size, growth stage and location as used for qRT‐PCR (Fig. 3 ). Agar pieces from at least three plates were pooled and two independent biological replicates were made. Samples were homogenized in liquid nitrogen and resuspended in acetone : water (1:1; v/v; chemicals from Roth). For extraction of metabolites from mycelia and agar, samples were stored at 4°C for several hours and thereafter chloroform was added. The organic phase (chloroform) was transferred to a conical tube. After complete evaporation of the solvent, the samples were resuspended in 160 μl chloroform and 8 μl was spotted on a TLC plate (HPTLC silica gel 60 F254s, Merck 1.15696.0001) using the CAMAG Automatic TLC sampler 4 (CAMAG, Muttenz, Switzerland). This amount was chosen in order to detect smaller changes in metabolite patterns without overloading the plate. Separation was performed in a saturated twin trough chamber with chloroform : formic acid 7:1 (v/v). The plates were analyzed under ultraviolet light (254 nm and 366 nm) using a CAMAG visualizer (CAMAG). Additionally, the plates were derivatized with p‐anisaldehyde : sulfuric acid and evaluated again with white and ultraviolet light. Results were visualized using the software visionCATS 1.4.14017.1 (CAMAG).
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