*2.5. Isolation and Characterization of Antifungal Compounds*

The antifungal compounds were isolated using an in vitro antifungal activity-guided method [47]. The active isolates were inoculated into 250 mL flask containing 50 mL of tryptone soy broth (TSB) [48] and cultivated for two days at 28 ◦C with shaking at 200 rpm. Then, 12.5 mL of the seed culture was transferred into 1 L Erlenmeyer flask containing 250 mL of the fermentation medium (soluble starch 1%, dextrose 2%, tryptone 0.5%, yeast extract 0.5%, NaCl 0.4%, K2HPO4 3H2O 0.05%, MgSO4 7H2O 0.05%, CaCO3 0.5%, pH 7.2–7.4) and incubated at 28 ◦C for seven days with shaking at 200 rpm. The fermentation broth (20 L) was centrifuged (4000 rev/min, 20 min), and the supernatant and bacterial biomass were extracted with ethylacetate and methanol, respectively. Both extracts were concentrated by a rotary evaporator under reduced pressure until dry and mixed after dissolving their dried residues with methanol.

The crude extracts were divided into seven fractions using column fractionation packed with silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) eluting with petroleum ether/ethylacetate (20:1, 10:1, 5:1, 3:1, 2:1, 1:1 and 0:1). The bioactive fractions were then subjected to Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and eluted with methanol to obtain several subfractions. The active subfractions were further separated by semipreparative HPLC (Hitachi-DAD, Tokyo, Japan) using a YMC-Triart C18 column (250 × 10 mm i.d., 5 μm) at a flow rate of 3.0 mL/min, and the potent active principles were finally isolated.

Structural determination of the active compounds were made according to spectroscopic analysis. NMR spectra were measured with a Bruker Avance III-600 spectrometer in CDCl3 or CD3OD using TMS as internal standard. The ESI-MS spectrum was taken on a Waters Xevo TQ-S ultrahigh pressure liquid chromatography triple quadrupole mass spectrometer. The HR-ESI-MS spectrum was acquired with an Agilent G6230 Q-TOF mass instrument. The UV spectrum was recorded in chloroform using a Shimadzu UV-2401PC UV-VIS spectrophotometer. The IR spectrum was obtained using a Bruker Tensor 27 FTIR. Optical rotation was determined in chloroform using a JASCO P-1020 polarimeter. The ECD spectrum was measured on a Chirascan circular dichroism spectrometer (Applied Photophysics Corporation Limited, Leatherhead, UK).

#### *2.6. Antifungal Assay of Elucidated Bioactive Compounds*

The active compounds were dissolved in methanol and diluted to different concentrations, which were then added to PDA medium. A fresh fungal plug of the fungus (5 mm in diameter) was placed in the center of the agar plate and incubated at 20 ◦C. Experiments were performed in triplicate, and the plate with the same amount of methanol was used as control. When the control plate was covered completely with fungal mass, the percentage of inhibition was calculated with the formula as follows:

$$\text{Inhibition } (\%) = (1 - D/D\_c) \times 100\tag{2}$$

where *D* is average diameter of the treatment and *Dc* is average diameter of the control. Data were subjected to linear regression analysis, and the effective concentrations required for 50% inhibition (EC50) were calculated.
