Milli q

Fungal cultures were maintained on PDA at 22 °C for 14 days as pre-cultures. Pre-cultures were separately inoculated onto PDA in triplicates and cultivated for 21 days at 22 °C in the dark. The cultures were extracted with EtOAc in a 1:4 (agar:solvent) ratio. To achieve the highest extraction efficiency, agar cultures were first sliced and homogenized with an ultra-turrax in the extracting solvent. The EtOAc extracts were sequentially washed with an equal volume of Milli-Q^® (Arium^®, Sartorius) water in a liquid–liquid partitioning experiment to deplete salts from the extracts. The clearly separated EtOAc layer was then evaporated to dryness. Dried extracts were solubilized in MeOH and filtered (0.2 μm filter) into storage vials and dried in vacuo. Aliquots of 1 mg were transferred into separate vials for bioactivity testing and UPLC-MS/MS profiling.

In this study, we used three Z. tritici isolates that were previously described (26 (link)). Previous studies using axenic propagation of Z. tritici have used fungal cells propagated on yeast-malt-sucrose (YMS) medium (21 (link)), and similar growth conditions were used in this study. YMS-agar medium was prepared with 0.4% (wt/vol) yeast extract (Bacto yeast extract; Thermofisher), 0.4% (wt/vol) malt extract, and 0.4% (wt/vol) sucrose supplemented with 2% (wt/vol) Bacto agar. The fungal isolates were incubated at 18°C for 15 consecutive days prior to extractions.
We pooled four solid cultures of Z. tritici and extracted metabolites with 400 mL ethyl acetate (EtOAc) (Pestinorm; VWR Chemicals, Leuven, Belgium) after homogenizing by an Ultra Turrax at 19,000 rpm. Each EtOAc extract was washed twice with 200 mL of Milli-Q (Arium, Sartorius) water in a separatory funnel to remove salts. The EtOAc layer was then evaporated to dryness by a rotary evaporator (150 rpm at 40°C). Dried extracts were solubilized in methanol and filtered (0.2-μm filter) into storage vials and dried in vacuo. Each fungal strain was extracted in triplicate (biological replicates).