Sample preparation and gas chromatography-mass spectrometry analysis

To an aliquot of 50 μL of the cell content or the fungal supernatant was added 200 μL methanol containing 12.5 μg/mL myristic-1,2-¹³C₂ acid as an internal standard (IS). The mixture was vortexed for 5 min, placed at 4°C for 1 h, and centrifuged at 20,000 g for 10 min at 4°C. An aliquot of 100 μL supernatant was transferred into a GC vial and evaporated to dryness under vacuum (Thermo Fisher Scientific, Asheville, NC, USA). An aliquot of 30 μL methoxyamine in pyridine (10 mg/mL) was added and vortexed for 3 min. After 16 h of methoximation reaction at room temperature, 30 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane was added, vortexed for 1 min, and placed at room temperature for 1 h for trimethylsilylation. Finally, 30 μL methyl myristate in heptane (30 μg/mL) was added and vortexed, and an aliquot of 0.5 μL was used for the GC/MS analysis.

The analysis of metabolites was conducted by a GC/MS system (SHIMADZU GC/MS QP2010Ultra/SE, Kyoto, Japan) fitted with a RTx-5MS capillary column (30 m×0.25 mm, Agilent J&W Scientific, Folsom, CA, USA). The conditions used for GC/MS analysis were as follows: Helium as carrier gas at flow rate of 1.5 mL/min; initial column temperature of 70°C for 3 min followed by increasing from 70°C to 300°C at a rate of 20°C/min and held for 3 min; transfer line temperature of 205°C and ion source temperature of 200°C; ion source voltage of −70 eV and acceleration voltage of −950 V; and solvent delay of 300 s. The MS data were acquired over the range between m/z 50 – 800 [12].