*2.4. Tricyclazole Inhibition Assay and Measurement of Flaviolin in Mycelia*

*A. resinae* was subcultured on potato dextrose agar for 10 days. Spores were then collected in 0.02% Tween-80, and their concentrations were adjusted to 1 × <sup>10</sup><sup>6</sup> spores/mL. Afterward, cellophane membrane disks were inoculated with 10 μL of spore suspension and placed on potato dextrose agar (PDA) supplemented with different concentrations of tricyclazole ranging from 0 to 100 ug/mL. Colony sizes and colors were evaluated after 10 days via microscope imaging.

The secondary metabolites in the grown mycelia were extracted as described by Lisec et al. [26]. The mycelia on the cellophane membrane were harvested and flash-frozen with liquid nitrogen. The frozen mycelia were then ground, after which 100 mg of sample was transferred to 1.5 mL microcentrifuge tubes containing 1.0 mL of methanol to extract the metabolites. After adding 60 μL of a ribitol solution (0.2 mg/mL), the sample was incubated for 10 min at 70 ◦C with mild mixing. The incubated sample was centrifuged at 3500 rpm for 20 min, and the supernatant was transferred to a new tube. The supernatant was then mixed with 750 μL of chloroform and 1400 μL of distilled water, and the mixture was thoroughly vortexed. The mixed solution was centrifuged at 3500 rpm for 20 min, after which 150 μL of the supernatant was transferred to a new 1.5 mL tube. The extract was then fully dried and stored at −80 ◦C. Synthetic flaviolin was also prepared according to a previous study, and stock solutions were fully dried prior to analysis [27].

To derivatize both the synthetic and naturally occurring flaviolin in the mycelia, the samples were mixed with 40 μL methoxyamination reagent (20 mg/mL of methoxyamine hydrochloride dissolved in pyridine) and incubated for 2 h at 37 ◦C with constant mixing (150 rpm). After incubation, 70 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and incubated at 60 ◦C for 1 h. The concentrations of the resulting products were then determined via gas chromatography-mass spectrometry (GC–MS) (Agilent 6890 N GC equipped with a quadrupole Agilent 5973N MS spectrometer; Santa Clara, CA, USA). The samples were injected in splitless mode at 230 ◦C. The GC oven was held at an initial temperature of 80 ◦C for 2 min, which was increased to 325 ◦C at a 15 ◦C/min rate, then held at this temperature for 6 min. The separation process was carried out using a 25 m × 0.25 mm (inner diameter) × 0.25 μm DB–5MS UI capillary column with helium as the carrier gas at a 2 mL/min flow rate. Full scan acquisitions were performed over an m/z 50–800 range. Mass spectrometry was conducted at a 70 eV ionization energy, 230 ◦C source temperature, and 290 ◦C transfer line temperature. Automatic tuning of the instrument was conducted according to the manufacturer's instructions.
