*4.4. UPLC-HRMS*/*MS and Microfractionation*

UHPLC-HR-MS analysis was performed on a 1290 UHPLC system (Agilent, Santa Clara, CA, USA) equipped with DAD, ELSD, and maXis II™ (Bruker, Billerica, MA, USA) ESI-qTOF-UHRMS with the following gradient: 0 min: 95% A; 0.30 min: 95% A; 18.00 min: 4.75% A; 18.10 min: 0% A; 22.50 min: 0% A; 22.60 min: 95% A; 25.00 min: 95% A (A: H2O, 0.1% formic acid (FA); B: Acetonitrile, 0.1% FA; Flow: 600 μL/min). Column oven temperature: 45◦C. Column: Acquity UPLC BEH C18 1.7 μm (2.1x100 mm) with Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column (2.1 × 5 mm).

For microfractionation, the flow path was changed, so that 90% of the flow was collected with a custom made fraction collector (Zinsser–Analytik, Eschborn, Germany) while the rest was analyzed in MS/MS mode in maXis II™. Collision induced fragmentation was performed at 28.0–35.05 eV using argon at 10−<sup>2</sup> mbar.

Depending on the potency observed in the primary screening, microfractionation assay plates were prepared by injecting 1 and 2 μL or 2 and 5 μL of extract. A total of 159 fractions were generated per extract and collected on one 384 well plate (fraction length is 7 s, starting immediately after injection) (Figure S11). Plates were dried in vacuo using a HT12-II centrifugal concentrator (Genevac, Ipswitch, Suffolk, GB) at 35 ◦C before screening. Microfractionation assay volume of *S. aureus* and *C. albicans* was 20 μL, while volume of *S. tritici* assays was 50 μL.
