*2.2. Isolation of Endophytic Actinobacteria*

Three groups of root samples were used for isolation of endophytic actinobacteria. The root samples were air dried for 24 h at room temperature and then washed in water with an ultrasonic step (160 W, 15 min) (KH-160TDV, Hechuang, China) to remove the surface soils and adherent epiphytes completely. After drying, the sample was cut into pieces of 5–10 mm in length and then subjected to a seven-step surface sterilization procedure: A 60-sec wash in sterile tap water containing cycloheximide (100 mg/L) and nalidixic acid (20 mg/L), followed by a wash in sterile water, a 5 min wash in 5% (v/v) NaOCl, a 10 min wash in 2.5% (w/v) Na2S2O3, a 5 min wash in 75% (v/v) ethanol, a wash in sterile water and a final rinse in 10% (w/v) NaHCO3 for 10 min. After being thoroughly dried under sterile

conditions, the surface-sterilized samples were subjected to continuous drying at 100 ◦C for 15 min. The sample was then cut up in a commercial blender and ground with a mortar and pestle, employing 1 mL of 0.5 M potassium phosphate buffer (pH 7.0) per 100 mg tissue. Tissue particles were allowed to settle down at 4 ◦C for 20–30 min, and an aliquot of 200 μL supernatants were spread on a series of isolation media and incubated at 28 ◦C for 2–3 weeks. Each isolation medium was supplemented with nalidixic acid (20 mg/L) and cycloheximide (50 mg/L) to inhibit the growth of Gram-negative bacteria and fungi. Five isolation media: Humic acid-vitamin (HV) agar [33], Gause's synthetic agar no. 1 [34], dulcitol-proline agar (DPA) [35], cellulose-proline agar [36], and amino acid agar (serine 0.05%, threonine 0.05%, alanine 0.05%, arginine 0.05%, agar powder 2%, pH 7.2–7.4) were selected for the isolation. After 14 days of aerobic incubation at 28 ◦C, the actinobacterial colonies were transferred onto oatmeal agar (International *Streptomyces* Project medium 3, ISP3) [37] and repeatedly re-cultured until pure cultures were obtained, and maintained as glycerol suspensions (20%, v/v) at −80 ◦C.
