*2.7. In Vitro Antibacterial Activity Test*

The antibacterial activity of strain NEAU-SSA 1T against two pathogenic bacteria (*Micrococcus luteus* and *Ralstonia solanacearum*) was evaluated using the agar well diffusion method [44] with the cultures growth on ISP 3 medium at 28 ◦C for four weeks as follows: All the spores and mycelia were collected from one ISP 3 plate (diameter, 9mm) and then extracted using 1 mL methanol with an ultrasonic step (300 W, 30–60 min). Afterwards, 200 μL methanol extract or methanol was added to the agar well, and methanol was used as the control. To further investigate the antibacterial components produced by NEAU-SSA 1T, the strain was cultured in tryptone-glucose-soluble starch-yeast extract medium (tryptone 0.2%, glucose 1%, soluble starch 0.5%, yeast extract 0.2%, NaCl 0.4%, K2HPO4 0.05%, MgSO4.7H2O 0.05%, CaCO3 0.2%, *w*/*v*, pH 7.0–7.4), and the inhibitory activity was tested. Briefly, strain NEAU-SSA 1<sup>T</sup> was inoculated into MB medium and incubated at 28 ◦C for seven days in a rotary shaker. The supernatant (100 mL for this study) was obtained via centrifugation at 8000 rpm and 4 ◦C for 10 min and subsequently extracted by using an equal volume of ethyl acetate. Then, the extract was dried in a rotary evaporator at 40 ◦C and eluted with proper volume methanol (1 mL used in this study). The cell precipitate was extracted with an equal volume of methanol and also

condensed as above. After that, the antibacterial activity was evaluated using the agar well diffusion method, and each well contained 200 μL of the methanol extract. To examine the effect of temperature on antibacterial activity, the ten-fold dilution methanol extract was placed in a water bath at 40, 60, 80, and 100 ◦C for 30 min, and then cooled to room temperature. The antibacterial activity was evaluated using the agar well diffusion method.
