Detailed Explanation of Practical Technical Points for Mushroom Spawn Production
With the vigorous development of the edible mushroom industry, the demand for spawn has increased accordingly, while the number of existing spawn production units is clearly insufficient, and many mushroom growers have no choice but to introduce spawn from elsewhere. Long-distance purchase and transport of spawn is not only expensive and costly, but the spawn is also prone to contamination by unwanted microorganisms due to bottle breakage, thereby seriously affecting the economic benefits of cultivating edible mushrooms. If mushroom growers can master the spawn production procedures and techniques, carry out their own spawn production, and gradually develop into specialized spawn producers, this is also an excellent path to prosperity. Spawn production procedures are usually divided into three levels: mother spawn, stock spawn, and cultivation spawn, which are what are commonly called first-level, second-level, and third-level spawn. Among these, the isolation and selection of mother spawn is highly technical and requires refinement; whereas the making of stock spawn and cultivation spawn is relatively simple. The production techniques for each level of spawn are introduced below.
Isolation and Selection of Mother Spawn
Mother spawn is mainly obtained through artificial selection, mutation breeding, crossbreeding, and protoplast fusion. As an ordinary specialized spawn producer, one can use artificial selection to isolate and cultivate mother spawn. The specific steps and operating methods are as follows.
1. Collecting the source material. From wild or artificially cultivated populations, select representative superior fruiting bodies as the source material. The standard for the seed mushroom is: eight-tenths mature, round and regular in shape, thick and fleshy, free of pests and diseases. Collect 1-2 seed mushrooms meeting the above standards and number them as material for isolating mother spawn. Those collected from the cultivation room should be marked with the original strain code.
2. Medium preparation. Agar medium is also called PDA medium. The commonly used formula is: potato 200-250 g, agar 15-20 g, glucose 20-25 g, clean water 1000 mL. Magnesium sulfate 1-1.5 g, a trace amount of vitamin B1, and potassium dihydrogen phosphate 2-3 g may also be added. First wash and peel the potatoes, cut them into thin slices, place them in an aluminum pot with water and boil for 30 minutes, then remove them and filter through 4 layers of gauze to obtain the juice. Then add the agar to the juice, stirring while heating, so that the agar dissolves fully; then add the glucose and the rest, boil for a few more minutes, and likewise filter through 4 layers of gauze to obtain the liquid. Fill the liquid while hot into glass test tubes. Fill to 1/5 of the tube length, and plug the tube mouth tightly with cotton, or fill the liquid into a glass Erlenmeyer flask in an amount of 20 mL. Then place it in an autoclave and sterilize for about 30 minutes under a pressure of 98-108 kPa/cm. After sterilization, remove it promptly, and while hot arrange the test tubes in a slanted row on the table; after cooling, they become solid slanted medium.
3. Mother spawn isolation methods include three methods: spore discharge, tissue isolation, and isolation from within the substrate. (1) Spore discharge method. Disinfect the surface of the seed mushroom, blot dry the water, then suspend the fruiting body inside an Erlenmeyer flask containing agar medium, allowing the spores within the fruiting body to naturally fall onto the medium and germinate into mycelium. Alternatively, cut off a small piece of the fruiting body and attach it to the surface of the slanted medium in a test tube, allowing the spores to fall onto the medium and germinate into mycelium, thus obtaining mother spawn; (2) Tissue isolation method. In an inoculation box, use your hands to split the disinfected seed mushroom in half from the stem, or cut it open with a blade, so that the fruiting body is split in two. At the junction of the cap and stem or at the gills, use an inoculation knife to cut out a small piece of the fruiting body, then cut it longitudinally into small thin slices of 5 mm x 10 mm, use an inoculation needle to pick up one thin slice and inoculate it into the center of the slanted medium, inoculating one small thin slice per test tube; wait for the mycelium to germinate, and mother spawn is obtained; (3) Isolation from within the substrate method. Select a wood segment that has already produced mushrooms, shave off the bark and the surface xylem, disinfect with 70% alcohol, saw it into thin slices 1 cm thick, place them in 0.1% mercuric chloride water to disinfect for 1-2 minutes, then wash off the residual liquid with sterilized water. Then split the small slices into small strips 0.5-1 cm wide, and inoculate them into the center of the slanted medium; after the mycelium grows, mother spawn is obtained. Alternatively, from a spawn bag that has already produced mushrooms, after disinfection treatment, use an inoculation needle to hook out mycelium from inside the bag that is pure in color and vigorous in growth, and inoculate it into the center of the slanted medium in a test tube; after the mycelium germinates, spawn is likewise obtained.
4. Cultivation at suitable temperature. After isolating and inoculating the spawn into test tubes by the various methods above, promptly transfer the test tubes into a disinfected incubator or culture room for cultivation, with the temperature controlled at about 25°C, so that the isolated spores or mycelium develop at a suitable temperature. Generally, after spore discharge, the spores germinate into colonies after 3-4 days, and the mycelium fills the tube after 10 days; after tissue isolation and inoculation, the mycelium germinates within 2-3 days and spreads and grows over the medium; after mushroom wood isolation and inoculation, the mycelium resumes growth within 7 days.
5. Selection, breeding, and purification. The mycelium obtained by the above methods is not necessarily all high quality and still needs selection, breeding, and purification. Therefore, after the mycelium germinates, observe carefully, select mycelium that is pure in color, robust, normal in growth, and uninterrupted, and in the inoculation box hook out the mycelium together with the medium and inoculate it onto separately prepared test tube medium. Under constant temperature conditions of 23-25°C, cultivate for 7-10 days; after the mycelium fills the tube, observe again, and select the best from among them for use; this is the mother-generation mother spawn.
6. Tube transfer and expansion. The mother-generation mother spawn can be transferred and expanded into daughter-generation mother spawn. Using the same slanted medium, each tube can be expanded into 30-50 tubes of daughter-generation mother spawn. What is supplied in production is mostly daughter-generation mother spawn. It can be transferred and expanded again. Generally, each tube can be expanded into 20-25 tubes of daughter-generation mother spawn, but the number of tube transfers must not exceed 5 times.
7. Fruiting test. The mother spawn obtained by isolation and selection must also undergo a fruiting test. The method is: inoculate the mother spawn onto sawdust medium in bottles or bags, and according to the temperature requirements of the various mushroom and fungus species, carry out cultivation at a suitable temperature; only after fruiting does it prove usable for production.
Propagation and Cultivation of Stock Spawn
Inoculate the mother spawn onto sawdust medium in bottles or bags; through cultivation, it becomes stock spawn. The technical requirements are as follows.
1. Formula of sawdust medium for stock spawn: mixed hardwood sawdust 77.5%, wheat bran 20%, sucrose 1%, gypsum powder 1.2%, magnesium sulfate 0.3%. pH (before sterilization) 6.5-7, water content adjusted to 60%. Mix the above raw and auxiliary materials evenly, and fill them into 750 mL glass spawn bottles; the filling requirement is loose at the bottom and firm at the top. Plug the bottle mouth tightly with cotton, then wrap the bottle neck and cotton plug with kraft paper. Then place it in an autoclave and sterilize under a pressure of 147 kPa/sq cm for 2-2.5 hours. Normal-pressure sterilization may also be used, maintaining 100°C for 10-11 hours; after reaching the standard, remove while hot and let it cool.
2. Inoculating the spawn. When the material temperature drops below 25°C, under sterilized conditions, divide the test-tube spawn into several pieces and quickly inoculate them onto the stock spawn medium through the flame of an alcohol lamp; each tube of mother spawn can inoculate 4-6 bottles of stock spawn.
3. Mycelial growth cultivation. After inoculation, promptly transfer the glass spawn bottles into a 25°C spawn culture room for cultivation. The relative humidity of the space should be controlled above 70°C, and strong light should be avoided. The stock spawn cultivation time generally requires 40-50 days for mushroom species, while straw mushroom and silver ear only need 15-20 days.
4. Removing contaminants and selecting the superior. During the stock spawn cultivation process, observe every day; if contamination by unwanted microorganisms is found, it should be eliminated immediately.
Expanded Cultivation of Cultivation Spawn
Transfer and expand the stock spawn again onto the same sawdust medium, and cultivate under suitable temperature conditions for 30-35 days to obtain cultivation spawn (also called production spawn), which can be used for production cultivation. For cultivation spawn, 750 mL special spawn bottles may be used, but because the amount used is large, polypropylene spawn bags are usually mostly used. Bag specifications include 12 cm x 24 cm, 15 cm x 28 cm, 17 cm x 33 cm, etc., which can be selected for use according to the characteristics of the various mushrooms and fungi and local habits. For medium sterilization, a normal-pressure sterilization stove is used, requiring the sterilization temperature to reach 100°C and be maintained for 8-10 hours; after reaching the standard, unload the bags while hot and cool them. When the material temperature drops below 25°C, inoculate the selected variety of stock spawn under aseptic conditions. Each bottle of stock spawn can be expanded into 50-60 bottles of cultivation spawn. The spawn culture room should be clean, protected from strong light, with the room temperature kept at 23-25°C, and frequent inspections should be carried out; contamination by unwanted microorganisms should be eliminated promptly, and continual selection of the superior and removal of the inferior should be practiced. When the mycelium fills the bag and the mycelium at the tip reverses and climbs upward 1-3 cm, it is cultivation spawn of the right age.