Analysis of Sesquiterpenoids Using GC-MS

For the analysis of (+)-valencene and α-cuprenene, 10 mL of cells grown to an OD₆₀₀ of approximately 6 were harvested after 48 h. To trap the secreted sesquiterpenoids, 500 μL of n-dodecane (D0968, TCI Deutschland GmbH, Eschborn, Germany) were added to the shaking culture. To ensure phase separation, the n-dodecane containing layer was centrifuged twice at 16,000 × g for 10 min. The n-dodecane phase-containing sesquiterpenoids were analyzed using a GC/MS-QP2010 (Shimadzu, Tokyo, Japan) equipped with a FS-Supreme-5 column (30 m × 0.25 mm × 0.25 μm; Chromatographie Service GmbH, Langerwehe, Germany). The GC-MS conditions were adopted from a previous study (Schulz et al., 2015). Temperatures of the injector and interface were set at 250°C and 285°C, respectively. The carrier gas was helium and its velocity was set to 30 cm sec^–1. 1 μL of the sample was injected with a split ratio of 10. The column temperature was sequentially changed and maintained at 130°C for 3 min, ramped to 260°C at a rate of 10°C min^–1, held at 260°C for 1 min, ramped to 300°C at a rate of 40°C min^–1 and held at 300°C for 1 min. In the case of (+)-valencene, a purchased reference compound (75056, Merck) was diluted as 50 μM and 200 μM in n-dodecane samples of the negative control. The retention time and fragmentation pattern of the mass spectrum obtained with the U. maydis sample were compared with the reference. In addition, the fragmentation patterns of (+)-valencene and α-cuprenene were compared with the previously reported data (Agger et al., 2009; Troost et al., 2019).