The cultivation of Agaricus blazei three-grade strains typically relies on wheat kernels as the primary raw material. However, in actual production, a high pollution rate or abnormal growth conditions are often observed. These issues usually stem from non-standardized strain production procedures. To address this, we have developed detailed specifications and operating techniques that can be used as a reference by farmers and growers to improve their practices.
1. **Raw Material Preparation**: Cow manure should be sifted and mixed with water in a 1:1.2 ratio. The mixture is then fermented and turned daily for about 20 days until it turns brown and emits a normal fermented forage scent. Once dried, it is ready for use. For wheat grains, they should be soaked thoroughly, drained, and dried in a shaded, well-ventilated area. When the outer layer dries slightly, mist them lightly with water to promote germination. After 24 hours, when the sprouts reach 1–2 cm, they are ready for use.
If the wheat has been worm-eaten, it can still be used, but the damaged grains must be washed multiple times. Soak them in lime water (pH 8–9) for 6 hours, then rinse with cold water until there's no white core left. This helps prevent starch loss and improves quality.
2. **Basic Formula**:
- Wheat grain (dry weight): 100 kg
- Cow dung powder: 30 kg
- Gypsum: 1 kg
- Calcium carbonate: 1.5 kg
3. **Substrate Mixing**: Mix wheat kernels with cow dung powder in a 1:0.2 ratio. Add other auxiliary materials while ensuring the cow dung fully adheres to the grains. The remaining cow dung is reserved for later use.
4. **Bottling Process**: In small-scale operations, standard bottles may not be used. Instead, 500 ml infusion bottles are commonly used. First, place a 1–1.5 cm layer of cow dung at the bottom. Then fill the bottle with wheat grains up to 1 cm below the neck. Add 1–2 pieces of cow dung, flatten, compact, and insert a tampon.
5. **Sterilization and Cooling**: Autoclave the filled bottles at 0.15 MPa for 2–2.5 hours. After sterilization, wait until the pressure drops to zero before removing the bottles. Transfer them to a clean cooling room or inoculation area. Disinfect the space with a disinfectant solution beforehand. Once cooled to 30°C, proceed with inoculation.
6. **Inoculation**: Spray the room with Saibai 09 solution, then wipe the original strain with 75% alcohol. Wait 10 minutes before starting the inoculation. Each 500 ml bottle can receive more than 40 inoculations.
7. **Cultivation**: Clean the culture room in advance, water the floor, and spray a disinfectant solution. After 2 days, sprinkle lime powder on the ground and transfer the inoculated bottles. Maintain a temperature of around 25°C during the growth phase, not exceeding 28°C. If possible, lower the temperature to 22°C. For farmers without climate control, place bottles vertically in groundwater covered with plastic, changing the water twice daily to keep temperatures under 25°C. Apply disinfectants every 3 days, alternating between Silly Solution and Saibai 09. If pests like fungus gnats appear, spray with high-efficiency cypermethrin.
8. **Contamination Control**: On the third day after inoculation, check for contamination. Remove any bottles showing signs of Streptomyces or other unwanted bacteria. Use a sealed bag to carefully remove contaminated bottles. For general bacterial contamination, spray the affected area, drain, dry, and reuse the bottle. For Clostridium spp., isolate and burn or bury the contaminated bottles. Before reuse, heat empty bottles above 70°C and wash them thoroughly.
9. **Temporary Storage**: After 35–45 days, the strain should be mature. Store unused strains at low temperatures, ideally between 2°C and 4°C.
10. **Quality Check**: Healthy Agaricus bisporus mycelium should be thick, white, and vigorous, with clear, fan-shaped growth and fine hyphae. The "2796" strain, which is semi-aerobic, should show consistent and robust growth. Avoid strains with yellow or white bacterial spots, irregular growth patterns, or unusual odors such as bleach or decay. If the mycelium is weak, yellowish, or shows signs of drying or uneven growth, it may indicate improper soaking, high temperature, or aging. Such strains should not be used for planting.
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