*2.2. Fermentation, Extraction, and Isolation*

The ex-type strain of *Jugulospora vestita* CBS 135.91, which was isolated from soil from Nepal (https://wi.knaw.nl/page/fungal\_table), was grown on potato dextrose agar (PDA; HiMedia, Mumbai, India) plates for 7 days at 23 ◦C; then, the fungal colonies on the culture medium were cut into pieces (1 × 1 cm) and transferred into two 250 mL Erlenmeyer flasks, each containing 100 mL of yeast-malt extract broth (YM broth; 4 g/L yeast extract, 10 g/L malt extract, 4 g/L d-glucose and pH 6.3 [3]). The seed fungus was incubated for 5 days at 23 ◦C under shake condition at 140 rpm. Fermentation

was carried out in 20 × 1 L Erlenmeyer flasks, each containing 400 mL of YM broth and inoculated with 5.0 mL of the mycelial suspension and cultivated for 13 days at 26 ◦C on a rotary shaker at 108 rpm.

The mycelium and the supernatant were separated by filtration via gauze. The mycelium was macerated three times by acetone and put in an ultrasonic water bath for 30 min at 40 ◦C. The supernatant was mixed with 275 g of adsorbent resin (Amberlite XAD-16 N, Sigma-Aldrich, Deisenhofen, Germany) and stirred for 2 h. The Amberlite® resin was then filtered and eluted three times with acetone. The resulting acetone extracts were dried in vacuo at 40 ◦C and the remaining aqueous residue was diluted with the same amounts of ethyl acetate (EtOAc) and extracted three times. The mycelium and the supernatant extracts were combined according to their chromatographic homogeneity to afford 738 mg of an oily crude extract.

The total extract was dissolved in MeOH and subjected to preparative reverse phase HPLC (PLC 2020, Gilson, Middleton, WI, USA). As stationary phase, VP Nucleodur 100-5 C18 ec column (250 × 40 mm, 7 μm, Macherey-Nagel, Düren, Germany) was used, while the mobile phase consisted of: solvent A, deionized water; solvent B, ACN. Purification of the crude extract was performed by using a linear gradient elution of 35–80% aqueous ACN with 0.05% formic acid at a flow rate of 45 mL/min for 55 min, 80–100% solvent B in 5 min, and finally, isocratic elution at 100% solvent B for 5 min to afford compounds **2** (*t*R: 13.4–13.5 min, 34 mg), **3** (*t*R: 13.2–13.3 min, 13 mg), and other observed peaks (F1–F8). Compounds **8** (*t*R: 10.6–10.8 min, 18.5 mg) and **7** (*t*R: 11.4–11.5 min, 5.5 mg) were obtained from purification of fraction F4 with the elution gradient 65–75% solvent B for 23 min, followed by isocratic elution with 100% B for 10 min. With the same method as for fraction F4, compounds **4** (*t*R: 11.6–11.7 min, 9.5 mg), **6** (*t*R: 12.5–12.6 min, 2 mg), and **5** (*t*R: 12.2–12.3 min, 3.3 mg) were obtained from fraction F5 and F6, as well as **1** (*t*R: 13.5–13.6 min, 7.5 mg) from fraction F8.

For comparison of secondary metabolite production of strains of *Jugulospora*, *Jugulospora vestita* strain CBS 135.91 and *Jugulospora rotula* strains CBS 110112, CBS 110113, FMR 12691, and FMR 12781, as well as *Triangularia backusii* FMR 12439 (a species that was previously also placed in the genus *Jugulospora*), were grown on PDA at 23 ◦C and the well-grown cultures were cut into small pieces using a cork borer (7 mm). Subsequently, a 200 mL Erlenmeyer flask containing 100 mL of YM were inoculated using five of those pieces and incubated at 23 ◦C on a rotary shaker (140 rpm). The growth of the fungus was monitored by constantly checking the amount of free glucose using Medi-test Glucose (Macherey-Nagel, Düren, Germany), and the fermentation was terminated 3 days after glucose depletion. Then, the mycelium and the supernatant were separated by filtration via gauze. The mycelia were extracted one time with acetone in an ultrasonic bath at 40 ◦C for 30 min. The resulting acetone extracts were dried in vacuo at 40 ◦C. The remaining aqueous residues were diluted with the same amounts of ethyl acetate and extracted one time. The supernatants were extracted with the same amount of EtOAc. The solvents were dried in vacuo at 40 ◦C.
