Mushrooms. ) sing. Also known as Lentinus edodes, Lentinus edodes and Tricholoma matsutake (Japan). It belongs to Basidiomycotina, Laminaria, Ascomycetes, Agaricus, Agaricus and Lentinus. Annual or perennial edible and medicinal fungi. Mainly produced in Asia, it is one of the main edible fungi in Asia. The output accounts for about 15% of the total output of edible fungi in the world. At present, China's mushroom production has exceeded 1 10,000 tons, accounting for about 40% of the world's total mushroom production, ranking second in the world after Japan. Mushrooms play an important role in Japanese agriculture and are the main domestic products of Japanese farmers, such as Fujian, Zhejiang, Jiangsu, Jiangxi, Sichuan, Yunnan, Guizhou and Guangxi. Lentinus edodes is used as medicine with fruiting body and mycelium. According to analysis, the edible part of dried mushrooms accounts for 72%. Every100g of edible part contains water13g, fat1.8g, carbohydrate 54g, crude fiber 8g, ash 4.9g, calcium124mg, phosphorus 41.07mg and iron 25.3. The umami components of Lentinus edodes are some water-soluble substances, of which 5'- guanylic acid is the main component. Its aroma component is mainly Lentinus edodes essence produced by acid decomposition of Lentinus edodes. In addition, shiitake mushrooms are also rich in provitamin D and adenosine. It is mild in nature and sweet in taste, and has the effects of resolving phlegm and regulating qi, invigorating spleen and appetizing, treating wind and breaking blood, and protecting liver. Regular consumption can enhance human immune function and resist diseases, such as rickets, scurvy, various mucositis, skin inflammation, cirrhosis and atherosclerosis. Clinical tests in Japan in recent years show that lentinan can prolong the life of dying patients with gastric cancer. Pharmacological research results show that lentinan has the functions of anti-tumor, anti-virus, enhancing immunity and lowering cholesterol. Lentinus edodes polysaccharide extracted from fermented mycelium in China has achieved good results as an auxiliary treatment for chronic hepatitis, liver cancer, digestive tract malignant tumor and leukemia, as well as in the clinical application of preventing and treating colds.
I. Morphological characteristics
The fruiting bodies are clustered or clustered, with a cap of 3-20 cm, which gradually expands outward after protruding in the early stage, and sometimes the center is slightly concave, and turns purple-brown after light brown in the early stage, with yellowish brown or dark brown scales on the surface, and sometimes chrysanthemums or tortoise shells crack. The mushroom meat is thick, white and dense, and the mushroom folds are white, dense and curved, with a width of about 4mm, and red or black patches are produced when it is overcooked or injured. The stalk is medium or partial, white, solid, fibrous, often curved, and sometimes the lower part gradually tapers, cylindrical or slightly flat, 3-6 cm long and 0.6- 1 cm thick. The surface of the bacterial fold is covered with a layer of solid, and there are many basidiomycetes or sacs on the solid. There are four basidiomycetes densely planted on it, and each basidiomycete has a colorless and smooth oval spore with a size of about 6-7× 3.5-4 microns, and the spore print is white (Figure 2 1-8).
Fig. 2 1-8 morphological diagram of Lentinus edodes
1. fruiting body 2. Longitudinal section of fruiting body 3. Spore 4. responsibility
Spores germinate and grow at a certain temperature and humidity to form hyphae, which are pure white and fluffy, colorless and transparent under the microscope, with branches and transverse septa. Mycelium can be produced not only by spore germination, but also by any part of mushroom fruiting body. Therefore, Lentinus edodes strains can be isolated not only from spores, but also from any part of fruiting bodies.
Second, biological characteristics.
Lentinus edodes has different requirements on nutrition, temperature, humidity, air, sunlight, pH and so on in different development processes.
(1) nutrition
Mushrooms are wood rot fungi, so the main nutrients are cellulose, hemicellulose and lignin. Its mycelium can secrete enzymes to decompose cellulose and lignin for its own use.
(2) Temperature
The optimum temperature for spore germination is 22-26℃. The optimum temperature for mycelium growth is 25-26℃. The fruiting body can be formed at 3-22℃, but it grows fastest and best at 8- 16℃. When the temperature is lower than 0℃ or higher than 22℃, the fruiting body stops growing, but the high-temperature mushroom can still grow above 22℃. When the temperature is high, thin mushrooms will generally be produced; When the temperature is low, thicker mushrooms will be produced. Good quality mushrooms can be produced in dry weather around 4℃ and after snow. Low temperature stimulation and temperature change can promote the differentiation of mushroom fruiting body.
(3) Humidity
The optimum moisture content of mushroom wood is 45%, and it will stop growing when it is lower than 20%. 60% is the best fruiting body formation, and the difference between dry and wet mushrooms is beneficial to fruiting body formation. The optimum air relative humidity for mycelium growth is 7%; The optimum air relative humidity for the formation of fruiting bodies is 85-90%, and less than 5% or more than 90% is not conducive to the growth and development of fruiting bodies.
(4) Air
Mushrooms are aerobic fungi. Therefore, the mushroom farm needs frequent ventilation to promote the normal growth of hyphae and the formation of fruiting bodies.
(5) Lighting
Light will promote the formation of bacterial skin and primordium. Under the condition of astigmatism, the fruiting body has good quality and dark color. Mycelia growth does not need light.
(6) pH value
Lentinus edodes is an acid fungus. It can grow in the range of pH 3-7.5, and the optimum pH is 5-5.5.
Third, cultivation techniques.
China is the first country to cultivate mushrooms in the world. As early as more than 800 years ago, Wang Zhennong's book recorded the simple cultivation method of mushrooms. That is, cut the flower mouth on the trunk and inoculate wild mushroom spores to fall off naturally. This weather-dependent inoculation method has low yield. In recent years, pure mycelium inoculation cultivation has been adopted, with high yield. The cultivation methods include wood cutting cultivation, mushroom brick cultivation, plastic bag cultivation, raw material cultivation, and industrialized production of mushroom mycelium by fermentation engineering technology for clinical application.
Mushroom industry has developed rapidly in recent years. Due to the popularization of various substitute cultivation and plastic bag cultivation of Lentinus edodes, the production cycle is shortened, the method is simple, and various agricultural and sideline products can be used, which greatly improves the economic and social benefits.
At present, a large mushroom cultivation base and semi-automatic mushroom production workshop have been built in China, and the plastic bag cultivation of mushrooms has been successfully moved from indoor to outdoor and cultivated in gardens, which not only improves the yield, but also approaches the quality of mushrooms cultivated in wood.
At present, the more reliable cultivation methods that have been popularized are wood-saving cultivation method and outdoor plastic bag cultivation method.
(1) Cultivation method of Lentinus edodes segment wood
The biggest advantage of cultivating Lentinus edodes with segmented wood is that it can obtain high quality Lentinus edodes. In mountainous areas, the problem of raw materials can be solved by planting special fast-growing forests with thinned or cut branches. The dried Lentinus edodes can harvest about 10kg/m3 wood in three years, and the highest yield can reach 15kg, which is five times that of the traditional cut flower method. The export of shiitake mushrooms is mainly Duanmu shiitake mushrooms.
Isolation and culture of 1. strain
Refer to chapter 12, section 4, on tissue separation or spore separation.
2. Mushroom cultivation procedures
Comprises the following steps: cutting preparation, inoculation, rockery trapping, mountain trapping and mushroom production management.
Preparation of (1) section wood
There are not less than 200 species of trees suitable for cultivating Lentinus edodes. Generally speaking, except pine, Chinese fir, camphor tree and Phoebe bournei, most broad-leaved trees and some coniferous trees can cultivate Lentinus edodes, mainly distributed in Fagaceae, betulaceae, Elaeagnus and Hamamelidaceae. Common and commonly used tree species in China are Castanea henryi, Castanea henryi, Castanea henryi, Liquidambar formosana, Castanea henryi and Quercus acutissima. Quercus variabilis Quercus adenophyllum. Wait. After the tree species are determined, the trees with less heartwood and more sapwood will grow on sunny slopes and fertile soil, and the diameter should be cut down to 8-20 cm, and the bark should not be too thick or too thin. In addition, we should take into account the protection of natural resources, not cut down all the mountains, but make full use of branches. Where conditions permit, special mushroom forests and fast-growing forests should be built in a planned way. Theoretically, the most suitable cutting period is from winter to the next year. At this time, the tree has high nutrients and the bark is not easy to fall off. After logging, the degree of pruning depends on the water content of the tree species, the slope direction and the shading degree of the site. Usually, the water content of newly cut trees is 40-45%, and it is most suitable for inoculation when the water content drops by 5% after drying for about half a month. If the moisture content of wood is lower than 30%, which is not conducive to planting mushrooms, measures such as soaking should be taken. Trees are generally cut into 1m long sections and transported to inoculation sites or fake mountain fields for stacking. In the case of loose texture, thin bark and small diameter, the drying time of trees should be shortened and inoculated quickly after cutting.
(2) Inoculation
At present, there are two kinds of strains used for mushroom cultivation: wood species and sawdust species. Seed inoculation is convenient and fast, but the cost is high; Sawdust has low cost and convenient production, but the inoculation efficiency is low. The local high-quality products with early, middle and late maturity should be selected according to the wood condition, local climate and market conditions. The strains should be stored in a cool and ventilated place to prevent the strains from aging. High-quality and high-yield strains are the premise to achieve high yield of mushrooms. Trunk inoculation is to inoculate mushroom mycelium into the cambium of trunk, which can be punched with a special punch or electric drill. If the parent wood species is used, the aperture and depth should be consistent with the size of the parent wood. If sawdust is used, the pore size is generally 1.2- 1.4 cm and the hole depth is 2-2.5cm.. The holes are arranged in a plum blossom shape, with the distance between two holes in the longitudinal direction of 25-30cm and the distance between two holes in the longitudinal direction of 3-5cm. The first holes at both ends of the section are 5cm away from the section, which is beneficial to prevent the invasion of miscellaneous bacteria from the section. Vaccination should choose sunny and cold weather, and the inoculation site should be open and clean. Don't rub sawdust into the hole too much. Select bark as the covering, the covering is slightly larger than the hole diameter, and the thickness of bark should be above 0.5cm. In addition, it can also be sealed with wax. The formula is paraffin 70%, rosin 20% and animal and vegetable oil 10%.
(3) False trapped mountain
Concentrate the grafted trees in a place suitable for mycelium recovery, which is called "fake trapped mountain". The suitable temperature is about 65438 08℃ and the suitable humidity is 70-80%. Special care should be taken not to expose the wood directly to the sun. The cross section of wood has high water content, so the "well" stacking method can be used. For wood segments that need moisture preservation, firewood can be stacked, wood segments can be placed horizontally and closely arranged, and wood segments can also be stacked vertically. No matter what method is adopted, branches and leaves or grass must be covered on the top of the pile in severe cold, and then plastic film should be used to keep warm and moist, and the method of uncovering the film should be adopted according to the temperature situation.
The length of holiday in the mountains varies with inoculation time and climatic conditions. Generally, a week after inoculation, white hyphae will grow on the edge of the strain block. One month after inoculation, a few pieces of wood should be randomly selected as samples. If there is white hypha spreading around the inoculation hole, it means that it is alive. If the fungus block is still yellow, it will break when touched, indicating that the part is too dry, and moisturizing measures such as spraying water should be taken. If the bacterial mass is black and sticky, it means that miscellaneous bacteria have invaded. Measures should be taken in time to fill holes and re-inoculate.
(4) Trapped mountain
The original intention of the trapped mountain is to arrange the wood blocks with hyphae in the mountain. The key to the mountain-trapped stage is to choose a good mushroom farm. Usually, we will choose a well-ventilated, open and cool hillside in summer, and it is best to sit on a gentle slope facing south or southeast. Mushroom farm should be shaded by trees, so it is advisable to "divide the sun into three parts, divide the sun into seven parts and let the flowers shine in". It is best to be near the water source. Mushroom piles vary according to mushroom farm conditions. Generally, the degree of shading is high, and wood can be thinned in wet sites in the forest. On the contrary, the wood blocks should be densely arranged and covered with branches and leaves to prevent direct sunlight. Trapped in the mountains for a month, you need to turn over the pile and change the cross section of the wood from head to head. In the trapped mountain stage, attention should be paid to ventilation and grass planting in the rainy season in the south, and attention should be paid to sun protection and crack prevention of bark in the high temperature season.
(5) Mushroom management
After the management in spring and summer, the hyphae in the artificially inoculated wood have fully diffused and become mushroom wood that can produce mushrooms. At this time, mushrooms can be classified according to their maturity, diameter and variety, so as to control the production of mushrooms manually. The management of mushroom wood in the fruiting period is mainly water management. At present, the more advanced management method of mushroom farm is mushroom soaking method. The soaking time varies with temperature, variety, wood segment thickness and tree species. Generally, mushrooms are soaked for 36-46 hours in the first year, 24-36 hours in the second year and 0/2-24 hours in the third year. After soaking, pile up and drain. Before soaking in water, you can let the mushrooms dry for a week without spraying water. The temperature difference between air temperature and water temperature should be as large as possible to stimulate the formation of mushroom buds. Some people fish out the mushroom wood and knock it on the ground a few times, which is called "knocking the wood out of the mushroom", which is beneficial to the formation of mushroom buds. After soaking in water to produce mushrooms, it should be managed normally for half a month to January, then dried and soaked in water to produce mushrooms.
(2) outdoor bag cultivation of mushrooms
Substitute cultivation of Lentinus edodes is a new technology developed in recent years. Because of its simple cultivation technique, short growth cycle and high economic benefit, it has developed rapidly in the vast rural areas. Besides using sawdust to cultivate mushrooms, we also successfully cultivated mushrooms with bagasse, cottonseed hull, corncob, sorghum stalk, soybean stalk, peanut seedling and reed.
There are many alternative cultivation methods, such as brick cultivation, box cultivation, plastic bag cultivation, etc. In recent years, raw material cultivation has also been developed. Judging from the current cultivation practice in various places, the success rate of plastic bag cultivation is large, and the output is high and stable. The following mainly introduces the cultivation techniques of mushroom outdoor plastic bags.
1. formula
(1) Broad-leaved tree species despise 78%, wheat bran 16%, sucrose 1.2%, gypsum 2-2.5%, urea 0.3%, calcium superphosphate 0.5%, and corn flour (or rice bran) with proper amount of water, with a pH of 5.5-6.5.
(2) hardwood sawdust 66%, cottonseed hull 20%, bran 10%, sucrose 1.2%, gypsum powder 2-2.5%, urea 0.3%, calcium superphosphate 0.5%, and proper amount of water, with a pH value of 5.5-6.5.
2. Inoculation and spawning
According to the above formula, high-pressure polyethylene plastic film tube (suitable for rural atmospheric sterilization) or low-pressure polyethylene polypropylene plastic film tube (width 15cm, length 50-55cm, thickness 0.05—0.07mm) is used for material preparation and filling, and bagging machine can be used to improve bagging efficiency. After sterilization, it can be inoculated when the temperature in the cylinder drops below 30℃. Inoculation is carried out in a sterile room, and 3-4 people cooperate with each other. One person wipes the barrel with 70% alcohol gauze, and then punches 4-6 holes in the bag with a hollow iron drill with a diameter of 1.5-2 cm at equal intervals. One person took out the fungus block with a racket and quickly inserted it into the hole. One person prepares a 3.5×3.5cm square tape in advance and quickly sticks it on the hole where bacteria have been attached. Each bottle of original seed can be connected with 80- 120 holes, and the holes should be filled with strains as much as possible, and the strains should protrude slightly from the holes, so as not to break the strains, so as to improve the survival rate of mycelium.
The spawning ground should be in a cool, dry and ventilated place, and the temperature should be controlled at about 25℃. The bacteria tanks are arranged in a "well" shape, with 4 bags in each layer, and 5- 10 layers are stacked according to different temperatures. Don't move for 5 days at the beginning. On the sixth day, turn over the pile to check whether the mycelium has been eaten and whether there is mixed bacteria infection. After that, check the pile every 5-6 days to remove the seriously polluted bacteria tank. Trace amounts of bacteria can be injected with formalin around bacterial colonies. After 20-25 days, the distance between the hyphae of the two holes is about 2cm. It is necessary to tear a small hole and increase oxygen to promote the growth of hyphae. After about 50 days of culture from inoculation, when the hyphae are covered with bacteria tubes and scattered primordia appear under the membrane, they can be moved out of the room and bagged for finishing.
3. Outdoor cultivation management
It is very important to choose an outdoor mushroom farm. It should be sunny, ventilated and close to the water source, and the water in the water source should be clean. You can use the open space in front of the house or paddy field or dry land. Weeds and insects must be removed from the site. The frame width is 1.2m, and the frame spacing is 0.3m. At the same time, a soaking ditch with a depth of 0.8m and a width of 0.6m should be dug, and the length depends on the site and the number of fungus barrels. Build a shade shed on site, 2m high, covered with grass or branches for shade, which can penetrate 30% sunlight. Mushroom farms are also surrounded by grass to keep out the wind. At the same time, stick or bamboo is fixed along the frame, 25cm away from the ground, and then small wooden strips with the same width as the frame are fixed perpendicular to the stick, with a spacing of 20cm, for placing mushroom tubes.
Before putting the fungus tube into the bed, the bag must be removed, that is, the film is cut off with a blade, and then the mycelium tube is placed on the branch at an angle of 70-80 degrees with the bed surface. The distance between hyphal tubes is 2-3 cm, which is in a cross arrangement. After arrangement, cover the whole mycelium tube with a film. Do not flip the film for 3-5 days unless it is in a high temperature climate. When the surface of the mycelium tube is covered with white Lentinus edodes mycelium, the difference between dry and wet on the surface of the mycelium tube should be widened, and the number and time of membrane extraction should be appropriately increased, so as to force the villous mycelium to gradually lodging and secrete pigment, and finally form a reddish-brown membrane.
15- 20 days after opening the bag, the color of the hyphae tube is complete. At this time, it is necessary to stimulate the mycelium tube with a certain humidity and temperature difference to promote the mycelium tube to enter the reproductive growth period from the vegetative growth period. The specific method is to cover the mushroom bed tightly during the day, reduce ventilation to improve the bed temperature, and uncover the film in the middle of the night to make the temperature of the mushroom bed drop suddenly. This can produce fruiting body primordium for 3-4 days continuously. When the base grows to the size of the thumb, you can spray water. The frequency of spraying depends on the weather. Do not spray on rainy days, spray less on cloudy days and spray more on sunny days. When the temperature is high, water should be sprayed in the morning and evening to strengthen shade and ventilation. After the first batch of mushrooms are harvested, stop spraying water for a few days, widen the temperature difference, and let the hyphae recover and produce mushroom buds. Generally, 2-3 tidal mushrooms can be harvested in autumn and winter. Mushrooms are generally not produced in cold season (except for a few low-temperature varieties).
In spring, the nutrients in the mycelium tube are consumed more, and the water content accounts for 30-35% of the mycelium tube. At this time, the mycelium tube should be soaked. Before soaking in water, use No.8 iron wire to make holes at both ends of the mycelium tube with a depth of 6- 15 cm, and then arrange them in the soaking ditch in turn, then put heavy objects on them and drain water to make the mycelium tube submerged by water. The soaking time depends on the water content of the mycelium tube, which is generally 5- 12 hours, so that the water content of the mycelium tube reaches 55-60%. After soaking, the mycelium tubes are also arranged on the edge, and then covered with plastic film, and ventilated 1-2 times a day, each time 1-2 hours, without spraying water. A few days later, small mushroom buds were produced, but the yield was already very low. If a large number of mushrooms are still produced after the first soaking, the second and third soaking can be carried out. Generally, there will be no more mushrooms in June-July.
The quality of spring mushrooms is generally poor, with thin cover and long handle. In the later stage of spring mushroom, it is mainly to do a good job of preventing high temperature.
(3) diseases and their prevention and control
Trichoderma viride ), Penicillium. Wait. Environmental discomfort or negligence in any link such as raw materials, inoculation and sterilization may cause mixed bacteria infection. Generally, it needs to be checked a few days after the outbreak. If the pollution is serious, it should be treated in time, picked out and away from the breeding ground, burned or buried deeply; Choosing low concentration of carbendazim has certain effect on the control of miscellaneous bacteria. Strict inspection is carried out in all production links to prevent mixed bacteria infection.
Fourth, harvesting and processing.
(1) Harvest
When the mushroom cover grows to 80%, it should be harvested in time. At this time, the mushroom cap is thick and the quality is good. When fully unfolded, the hat is of poor quality. Fungal folds are harvested when mottling occurs, turn black after baking and lose commercial value. When picking mushrooms in bags, the mushroom feet should be picked together, and the base of the mushroom stalk will stay on the mushroom block, which will cause mildew, but do not pick up the culture materials together, so as not to affect the next mushroom tide.
(2) Processing
When picking mushrooms on sunny days, you can put them in the sun first and then bake them when the skin is dry. The quality is good. When picking in rainy days, it should be baked in time, and the baking temperature should be gradually increased from low to promote the enzymatic reaction to synthesize aroma substances, but it should not exceed 65℃. Otherwise, the activity of the enzyme will be quickly destroyed and the aroma substance cannot be formed. The appearance and color of dried mushrooms are poor, so there can be no fire smoke. It should be turned frequently until the joint between the mushroom handle and the mushroom cover becomes hard. The mushrooms processed in this way are golden yellow, rich in flavor and good in quality.
(3) Two common methods for preserving strains
1.PDA slant culture medium preservation method
That is, PDA slant culture medium is stored at 4-5℃. Transplant every 3-6 months. It is best to replant when using it.
2. Preservation method of sawdust culture medium
That is, the sawdust and rice bran are inoculated with strains, cultured and stored at 4-5℃. Transplant every 3-6 months.
Stone thunder pill
(Wang Shufang)
Polyporus umbellatus Also known as Lei Wan, Zhu Ling, Schleswig, Zhu Lingzi, Lai Wan, etc. It belongs to Basidiomycotina, Laminaria, Agaricus, Agaricus blazei and Pleurotus. New Year's Eve or all the year round. Mainly distributed in the south-central provinces of China, such as Anhui, Zhejiang, Fujian, Sichuan, Guizhou, Yunnan, Guangxi, Hubei and Hunan. Also found in Shaanxi and Gansu. Taking sclerotia as medicine, it tastes bitter, cold and has little toxicity. It can help digestion, eliminate food accumulation, kill insects and remove heat. It can be used in combination with other Chinese medicines to treat schistosomiasis, cysticercosis and cerebral cysticercosis. Contains about 3% protease. It is a brownish-gray odorless and tasteless substance. Soluble in water and glycerol, insoluble in organic solvents such as methanol, ethanol, chloroform and ether. It has the strongest effect in intestinal weak alkaline (pH8) solution, and only 0.06μg can hydrolyze protein in 10ml weak alkaline solution, but it fails in acidic solution. In addition, it also contains 22% alcohol extract, 4% ash, 6.5% water, 0.06% ether extract and inorganic salts such as calcium, aluminum and magnesium. Because magnesium has a cathartic effect, it is not necessary to take laxatives when expelling tapeworms. In recent years, it has been reported that the inhibitory rate of its protease on mouse ascites carcinoma S- 180 is 33.3-69.7%, and intraperitoneal injection is better than intramuscular injection.
I. Morphological characteristics
In nature, Wan Lei mainly appears in the form of sclerotia, and its fruiting body is not easy to find. Its two scientific names belong to two different families and genera. At the end of 1980, Plant magazine published fresh thunder pills collected in Beishan, Jinhua, Zhejiang. The fruiting body germinated after culture is Agaricus blazei, with mushroom folds, and the diameter of mushroom umbrella is 2-3 cm. When they are all spread out, the middle part is slightly sunken and emits white powdery basidiospores. Sclerotinia is irregular spherical or massive with different sizes, and its diameter is generally 1-3.5 cm. When excavated for the first time, the surface was reddish brown or dark brown, and the surface had tiny protruding longitudinal wrinkles or was slightly smooth. The profile is white to grayish white, sometimes pale yellow or orange-brown. Slices are translucent. After drying, the appearance is purplish brown or dark brown, and the texture is hard. The newly unearthed sclerotia often has one or several white or yellowish rope-like cords in the surface depression, and some hyphae are entangled, so it is the vegetative organ of Wan Lei.
Fig. 2 1-9 schematic diagram of thunderbolt morphology
Second, biological characteristics.
(A) growth and development and its requirements for environmental conditions
Wan Lei is a kind of facultative weak parasitic fungus, mainly saprophytic, which often grows beside rotten roots such as mixed bamboo forest, tung tree, Liquidambar formosana and Elaeagnus Elaeagnus, and likes to grow in sandy soil with good permeability. On the hillside near the foot of the mountain or in the valley with short sunshine; There are many in the topsoil at a depth of 10- 15 cm above the ground. In autumn, Sclerotinia sclerotiorum of different sizes can often be seen in the above woodland.
1. temperature
Mycelia can tolerate low temperature below 0℃ in host rotten wood, and some strains of inclined mother species lose their vitality after being stored at 2-4℃ for two weeks, but sclerotia has strong cold resistance. The optimum temperature for mycelium growth is 25-30℃. When cultured at 25℃ and 30℃, the colony expanded greatly at 30℃, indicating that 30% was more conducive to mycelium growth.
2. Nutrition
In agar medium containing 1.5% bean cake powder and 0.5% wheat bran cooking juice, adding 1% glucose, sucrose and alcohol respectively, compared with sodium acetate, it was found that adding glucose promoted the initial mycelium growth slightly. After two weeks of culture, there was no difference between sugar-free control and sucrose treatment. Adding alcohol is unfavorable to its growth, and adding 1% sodium acetate inhibits its growth. At 25℃, using 2% glucose as the basic medium and adding 0.5% peptone, fish meal, ammonium sulfate and ammonium nitrate, the mycelium growth is obviously better than inorganic nitrogen.
3. Moisture and air
In the matrix of sawdust and wheat bran (3∶ 1), the material-water ratio is 1∶ 1.5— 1.8. When wheat bran is cultured at 25℃, the water content of wheat bran is less than 60%, so it is difficult to grow. Huang Youzhi of Hubei reported that excessive drying of the upper part of box culture would affect the formation of sclerotia. However, when the water content exceeds 300%, the mycelium is not easy to swell due to lack of air.
4.ph value
Generally, the soil in southern China is acidic, which is suitable for mycelium germination and growth. The natural pH value of artificial culture medium is mostly below 6.5, and it can grow. Previously, the natural pH of submerged culture was 5.8, and it was adjusted to 6.4, 6.8, 7.0 and 7.6 * * 5 groups with 10% anhydrous sodium carbonate. The general growth trend is that the natural pH is pH5.8, the growth becomes worse above 6.4, and it is not easy to grow at 7.6.
lamplight
Both hyphae and sclerotia in Wan Lei are below the surface of soil, that is, they grow and develop under the condition of no light. Cultured on artificial plate, the growth is normal without light, and there is no abnormality with light. Only some strains are pale yellow, while some strains are still white. Fruit bodies rarely appear, and whether it is related to insufficient light is still inconclusive.
(2) Comparison of different strains
The strains isolated from different sclerotia are different in growth rate and ability to form sclerotia. The ability of wild sclerotia to form sclerotia on artificial culture medium is strong. Four strains isolated from different areas in Wan Lei were compared on PSA plate at 25-30℃. The results are shown in table 2 1-2.
Table 2 1-2 Comparison of Different Strains on PSA Plate (unit: cm)
Sclerotinia sclerotiorum can be formed not only on PSA medium, but also on potato residue medium.
(3) profound cultural background
Most strains are easy to form mycelium balls, with less burrs, long culture time and fermentation time of 10 days. It is better to use sucrose and glucose as mixed carbon sources. There is no obvious lock-like association in submerged culture hyphae, and it is easier to find the lock-like association of aerial hyphae germinated on the surface after standing. The final pH value of acidic fermentation broth is low, which is not conducive to protease with high activity under alkaline conditions. For comprehensive utilization, it should be considered to improve the final pH value of submerged culture fermentation broth.
Third, cultivation techniques.
Wan Lei's life history, although it can send out fruiting bodies from sclerotia and produce mature spores. Spores germinate into mycelium and then form sclerotia. But it is mainly from mycelium to the formation of new sclerotia, and artificial culture is carried out according to the latter.
(1) Artificial isolation and culture of pure strain.
Wash the skin of fresh sclerotia with tap water, then soak it in 75% alcohol for 3 minutes, and then soak it in 0.2% chlortetracycline aqueous solution. Sterile operation to cut open the sclerotia, take about 3-5 mm2 tissue block, put it on PDA or PSA plate, and culture at 25℃. A week later, white mycelium grows, the top is removed, and it is transferred to a new inclined plane for culture to obtain the mother seed. Each sclerotia can be divided into one strain. Because of different producing areas, the age and tenderness of sclerotia are different, and the speed of mycelium germination is also different. The tender sclerotia is not as hard as the old sclerotia, but it is easy to split and separate. Old sclerotia can be soaked in clean water for 1-2 days, or buried in clean wet sand for two days before separation, which is convenient for operation.
(2) in-bottle culture
Hong Zhen used Fujian Yelei Pill to isolate the strain, PSA medium was used for the first mother seed, and sawdust medium was used for the second and third grades. The matrix consists of sawdust 70%, rice sugar (fresh) 18%, brown sugar 1.5-2.0% and calcium carbonate 1%. The mother seed was inoculated into sawdust culture medium and cultured in about 24% environment. After the mycelium is filled in the bottle, it is cultured at room temperature. Digging out sawdust after 4-5 months, you can see that there are 2-3 lightning balls buried in the sawdust, which are light yellow. Each one is the size of a thumb. After being cut open, it is white to light yellow, with rough texture and fine particles. It seems feasible to cultivate Sclerotinia sclerotiorum with sawdust indoors, but it takes a long time.
(3) Farmland and forest cultivation
Wan Lei was originally wild. In recent years, using local resources and using wild Wan Lei as primer, artificial cultivation experiments were carried out in some areas of Guizhou, and the cultivation was successful. The specific method is as follows:
1. Land selection
Loose and dry soil with good drainage should be selected, which can be planted in fields, corners, in front of and behind houses, but sticky yellow soil should not be selected.
Prepare materials
Besides pine, fir, camphor and cypress, other trees such as Liquidambar formosana, Cyclobalanopsis glauca, poplar, chestnut and coriaria are also acceptable, as are other vines, bamboo shoots and roots. Elaeagnus and cinnamon are better. Cut the tree into 70-90 cm pieces, half dry and half fresh. And collect a large number of litter and part of semi-rotten wood for later use. Where the thunder pill grows, it grows white.