Chemostat glucose-limited synthetic minimal medium containing (per liter) 0.1 g calcium chloride, 0.1 g sodium chloride, 0.5 g magnesium sulfate, 1 g potassium dihydrogen phosphate, 5 g ammonium sulfate, 500 μg boric acid, 40 μg sulfuric acid Copper, 100 μg potassium iodide, 200 μg ferric chloride, 400 μg manganese sulfate, 200 μg sodium molybdate, 400 μg zinc sulfate, 1 μg biotin, 200 μg calcium pantothenate, 1 μg folic acid, 1 mg inositol, 200 μg smoke Acid, 100 μg p-aminobenzoic acid, 200 μg pyridoxine, 100 μg riboflavin, 200 μg thiamine, and 0.08% glucose. calcium pantothenate
Prepare 10 L of medium, mix well, filter sterilize and place in autoclaved glass bottles. The bottom of the Carboy has an outlet port that leads to a short length of tubing with a Luer lock connector on the end. All inlets and outlets are covered with aluminum foil prior to autoclaving. The outflow tube is sealed with a metal clip before filling. Carboy is placed on a shelf above the chemostat area.
Purification maintenance
1.
Prepare media for purification and maintenance.
2.
TWYE and 1/2 ISP-2 agars were used for the purification of actinomycetes, LB agar was used for bacteria, and PDA was used for fungi. TSA (tryptone 15 g, soy peptone (enzymatic digest of soybean meal) 5 g, sodium chloride 5 g, agar 15 g) and modified ISP-2 (YIM 38 medium, 10 g malt extract, 4 g yeast extract Glucose, 4 g, vitamin blend (0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine-HCl, inositol, calcium pantothenate, and p-aminobenzoic acid, and 0.25 mg biotin), 20 g agar; pH 7.2) (Jiang et al., 2007) for the maintenance of actinomycetes.
3.
After 3 days to 12 weeks of incubation, pick colonies from the isolation plates.
4.
Streak colonies formed on plates containing purification medium.
5.
The purity of the isolate was checked visually and microscopically. When necessary, mixed endophytic cultures can be purified by diluting cultures grown on selective media to isolate different microorganisms.
6.
Grow purified single colonies on selected media, PDA for fungi, LB agar for bacteria, TSA, and modified ISP-2 for actinomycetes.
7.
Depending on the endophyte species and growth conditions, maintain growing strains on slants of PDA, LB agar, TSA, or modified ISP-2.
8.
Store mature strains (spores or mycelia) in 20% glycerol at −70 °C and store in sealed freeze-dried milk tubes at 4 °C.