*2.6. In Vitro Antifungal Activity Test*

Antifungal screening was performed against 10 different phytopathogenic fungi: *Sclerotinia sclerotiorum*, *Exserohilum turcicum*, *Colletotrichum orbiculare*, *Corynespora cassiicola*, *Rhizoctonia solani*, *Fusarium graminearum*, *Fusarium oxysporum*, *Sphacelotheca reiliana*, *Curvularia lunata*, and *Helminthosporium maydis*. Ten phytopathogenic fungi were preserved in the Key Laboratory of Agricultural Microbiology within the Heilongjiang province, China. Antifungal activity of strain NEAU-H2T was assessed using the dual culture plate assay [44]. The strain was point-inoculated at the margin of potato dextrose agar (PDA) [45] plates and cultivated for three days at 28 ◦C, after which a fresh mycelial PDA agar plug of the fungus was transferred into the opposite margin of the corresponding plate. Inhibition of hyphal growth of the fungus was recorded after incubated for seven days at 28 ◦C. The percentage inhibition rates were calculated using the formula: Inhibition rate (%) = Wi/W × 100%, where Wi is the width of inhibition and W is the width between the pathogen and actinobacteria. The assay was repeated three times and the average was calculated.

#### *2.7. Isolation and Characterization of Secondary Metabolites*

Strain NEAU-H2T was grown on ISP 3 agar plates for five days at 28 ◦C. Then, it was inoculated into 250-mL baffle Erlenmeyer flasks containing 50 mL of tryptone soy broth (TSB) and cultivated for one day at 28 ◦C with shaking at 250 rpm. After that, aliquots (15 mL) of the culture were transferred into 1-L baffled Erlenmeyer flasks filled with 250 mL of the production medium (tryptone 0.1%, glucose 3%, beef extract 0.5%, 0.25% CaCO3, 0.5% NaCl, 0.1% minor elements concentrate (FeSO4·7H2O 1.0 g, CuSO4·5H2O 0.45 g, ZnSO4·7H2O 1.0 g, MnSO4·4H2O 0.1 g, K2MoO4 0.1 g, distilled water 1 L), pH 7.2–7.4), and cultured at 30 ◦C for six days with shaking at 250 rpm.

The fermentation broth (25 L) was centrifuged (4000 rev/min, 20 min), and the supernatant was extracted with ethylacetate three times. The ethylacetate extract was evaporated under reduced pressure at temperatures within 40 ◦C to yield an oily crude extract (5.0 g). The mycelia were extracted with methanol (1 L) and then concentrated in vacuo to remove the methanol to yield the aqueous concentrate. The mycelia concentrate was extracted with ethylacetate (1 L × 3) to afford 1.0 g of crude extract after removing the ethylacetate. Both extracts displayed most of the similar secondary metabolites based on HPLC analyses. Thus, they were combined for further purification. The samples were applied to reverse-phase HPLC analysis eluted with a flow rate of 1 mL·min−<sup>1</sup> over a 28 min gradient with water and methanol (T = 0 min, 10% methanol; T = 20.0 min, 100% methanol; T = 24.0 min, 100% methanol; T = 24.1 min, 10% methanol; T = 28.0 min, 10% methanol) and at 25 ◦C.

The crude extract (6.1 g) was subjected to silica gel CC using a successive elution of petroleum ether/ethylacetate (1:0, 10:1, 5:1, 1:1 and 0:1, *v*/*v*) to yield A−F fractions. Fraction D (petroleum ether/ ethylacetate = 1:1, *v*/*v*) was subjected to semipreparative HPLC (YMC- Hydrosphere C18 column, 250 mm × 10 mm i.d., 5 μm, 0–20.0 min CH3OH:H2O = 35:55, *v*/*v*, 3 mL/min) to afford **1** (*t*<sup>R</sup> = 25.4 min, 2.2 mg) and **2** (*t*<sup>R</sup> = 30.4 min, 2.6 mg). Compound **4** (*t*<sup>R</sup> = 17.2 min, 5.5 mg) was obtained from the fraction C (petroleum ether/ethylacetate = 5:1, *v*/*v*) by semipreparative HPLC (YMC-Triart C18 column, 250 mm × 10 mm i.d., 5 μm, CH3OH:H2O = 25:75, *v*/*v*, 3 mL/min). Fraction E (petroleum ether/ ethylacetate = 0:1, *v*/*v*) was further purified by semipreparative HPLC (YMC-Hydrosphere C18 column, 250 mm × 10 mm i.d., 5 μm, 0–15.0 min, CH3OH:H2O = 25:75; 15.1–30 min, CH3OH:H2O = 35:65, *v*/*v*, 3 mL/min) to give compound **3** (*t*<sup>R</sup> = 21.4 min, 2.0 mg).

NMR spectra were recorded in methanol-d4 or DMSO-d6 using a Bruker AVANCE III-600 or a Bruker AVANCE III-400 spectrometer (Bruker Corp., Karlsruhe, Germany) and TMS was used as the internal standard. HR-ESI-MS data were obtained using an Agilent G6230 Q-TOF mass instrument (Agilent Technologies Inc. Santa Clara, CA, USA). Thin-layer chromatography (TLC) was performed using precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China), and spots were visualized by UV light (254 nm) and colored by iodine, or by spraying heated silica gel plates with 10% H2SO4 in ethanol. Semipreparative HPLC was conducted on a HITACHI Chromaster system (Hitachi-DAD, Tokyo, Japan).
