Composite Juncao of cold-region native plants in Xiaoxing'anling and its application in cultivating black fungus

Abstract

The present invention relates to the technical field of edible fungus cultivation, specifically to a composite mushroom grass of cold-region native plants of the Lesser Khingan Mountains and its application in cultivating black fungus. The composite mushroom grass of cold-region native plants of the Lesser Khingan Mountains is composed of burdock, Ligularia officinalis, sweet clover, Rumex crispus, and Sesamum indicum, with a mass ratio of 2:2:2:3:1. When using the composite mushroom grass to cultivate black fungus, the method includes the following steps: (1) pre-treating the composite mushroom grass: pre-wetting and fermenting; (2) preparing a planting culture medium, and cultivating the black fungus using a hanging bag cultivation method in a greenhouse; wherein the planting culture medium comprises 50% composite mushroom grass, 10% oak leaves, 24% sawdust, 12% bran or rice bran, 2.5% soy flour, 0.5% gypsum, and 1% lime. When using the method of the present invention to produce black fungus, the cost per bag is 1.65 yuan / bag, and the yield is 54g / bag, which is significantly better than the existing cultivation method.

Description

Technical Field

[0001] The present invention relates to the technical field of edible fungus cultivation, and in particular to a composite mushroom grass prepared from native plants of the cold region of Xiaoxing'anling Mountains and application thereof in cultivating black fungus. Background Art

[0002] Black fungus is a unique edible fungus and wild delicacy in my country. It has important medicinal and edible value and is also a world-recognized health food. It is deeply favored by consumers because of its rich nutrition and delicious taste.

[0003] Currently, the cultivation of black fungus still primarily relies on traditional broadleaf sawdust. However, the recent shortage of broadleaf sawdust and the resulting increase in raw material costs have impacted the entire black fungus industry. Straw and corncobs are widely researched as substitutes for black fungus, offering an effective solution to mitigate air pollution caused by straw burning. However, excessive removal of crop straw from the field can negatively impact the soil environment, reducing nutrient content. Therefore, crop straw should be returned to the field. Furthermore, crop straw is a residual residue left after grain harvesting, and its nutritional content is relatively low after harvest.

[0004] However, there are few reports and research on the use of Juncao as a substitute for wood chips in the cultivation of black fungus. Juncao is broadly defined as a grass that can be used to cultivate edible fungi. It is characterized by strong stress resistance, high yield, high crude protein and sugar content, and a well-developed root system. Juncao is a typical C4 plant with a high photosynthetic rate. Using Juncao as a sawdust substitute for black fungus cultivation and researching methods for this could address the raw material shortage issue in actual production.

[0005] Lin Zhanhui, Chief Scientist of the National Juncao Engineering Research Center and inventor of Juncao technology, introduced the plant to China in 1983. After over 20 years of cultivation, he developed a grass variety suited to my country's climate and soil conditions. Researcher Lin Zhanhui named it "Giant Juncao." Giant Juncao is a perennial upright, clumping grass of the Poaceae family with a strong tillering ability. It is a high-yielding, high-quality Juncao suitable for growth and cultivation in tropical, subtropical, and temperate zones. The plant is tall, resilient, and high in yield, boasting high crude protein and sugar content. It grows upright, clumping, and has a well-developed root system. The stem can reach 3.5 cm in diameter, with internodes 9 to 15 cm long and 15 active tillers. Each node bears an axillary bud enclosed by leaves. The leaves are alternate, 60 to 132 cm long, and 3.5 to 6 cm wide. A total of 35 leaves develop in 8 months.

[0006] However, due to the cold winter climate and short high temperature season in Northeast China, giant mushroom grass cannot grow normally in Northeast China and cannot grow large, so the effect of using mushroom grass is not good. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a composite mushroom grass of cold-region native plants of Xiaoxing'anling and its application in cultivating black fungus.

[0008] This method, developed to address the cold climate of Northeast China, selects wild plants native to the Lesser Khingan Mountains that are suitable for cold-weather growth, boast tall plants, high nutritional content, and high biomass yield. These plants are then propagated through tissue culture and then artificially cultivated under forests. The resulting composite Juncao serves as a substrate for black fungus cultivation, partially replacing sawdust, reducing costs and effectively resolving the conflict between the fungus and the forest. Furthermore, because Juncao has higher nutritional content (crude protein and sugar levels compared to straw and sawdust), the resulting black fungus possesses a higher nutritional content.

[0009] In order to make full use of the space under the forest, not occupy farmland, vigorously develop the forest economy, and practice the concept of forest food, the present invention adopts the method of under-forest cultivation and a mixed cultivation of multiple forests and grasses to promote synergy.

[0010] Specifically, this application provides the following technical solutions:

[0011] A composite mushroom grass of native plants from the cold regions of Xiaoxing'anling, composed of burdock Arctium lappa , Ligularia japonica Ligularia fischeri , sweet clover White sweet clover Rumex crispus Crispy Sorrel , Sesamum indica Deyeuxia purpurea The composition has a mass ratio of 2:2:2:3:1.

[0012] burdock Arctium lappaBiennial herb with a thick, fleshy taproot up to 15 cm long and 2 cm in diameter, bearing branched lateral roots. The stem is erect, up to 2 meters tall, stout, and up to 2 cm in diameter at the base, often purplish-red or pale purplish-red, with numerous raised ridges. The branches are numerous and obliquely ascending, and the entire stem is covered with sparse, short, papillate hairs and long, spidery hairs interspersed with small, brownish-yellow glandular dots. The basal leaves are broadly ovate, up to 30 cm long and 21 cm wide, with sparse, shallowly wavy teeth or cusps along the margins. The base is cordate, bearing a petiole up to 32 cm long, with different colors on both sides: green above, sparsely short, rough hairs and yellow glandular dots; grayish-white or light green below, thinly or sparsely tomentose, with yellow glandular dots. The petiole is grayish-white, densely covered with spidery hairs and yellow glandular dots, but often hairless in the middle and lower parts. The stem leaves are identical or nearly identical to the basal leaves, with equal and numerous hairs. The leaves at the base of the inflorescence are smaller, truncate or shallowly cordate at the base. The inflorescence heads are numerous or few, arranged in loose corymbs or conical corymbs at the top of the stems and branches, with a stout peduncle. The involucre is ovate or ovoid, 1.5-2 cm in diameter. The bracts are multi-layered and numerous, the outer ones triangular or lanceolate-subulate, about 1 mm wide, while the middle and inner ones are lanceolate or linear-subulate, 1.5-3 mm wide. All bracts are nearly equal in length, about 1.5 cm long, with cartilaginous hooks at the apex. The florets are purplish-red, with a corolla 1.4 cm long, the tubules 8 mm long, the limbs 6 mm long, and no glandular dots on the outside. The corolla lobes are about 2 mm long. Achenes are oblong-ovate or obliquely oblong-ovate, 5-7 mm long and 2-3 mm wide, flattened on both sides, light brown with numerous fine veins and dark brown spots or no spots. The pappus is multi-layered and light brown; the bristles are coarse and hairy, unequal in length, up to 3.8 mm long, not united at the base into a ring, and fall off individually. Flowering and fruiting occur from June to September.

[0013] Ligularia truncatula Ligularia fischeriPerennial herb. Numerous, fleshy, dark brown roots. Tall, erect stems, 80–200 cm tall. Yellowish-brown, short, internoded pubescence covers the upper part and inflorescence, while the lower part is smooth, 0.5–1 cm in diameter at the base, and surrounded by brown, withered petiole fibers. The clustered leaves and lower stem leaves are petiolate, 18–59 cm long, smooth, and sheath-like at the base. The leaf blades are reniform, 10–30 cm long and 13–40 cm wide, with a rounded, sometimes pointed apex and regularly serrated margins. The base is broadly curved, 1 / 3 the length of the leaf blade. The lobes are nearly rounded and not spreading, green above and light green below, smooth on both surfaces, with palmate venation and 5–7 prominent primary veins. Leaves on the stem and upper and middle parts are short-petiolated, with swollen sheaths. The leaf blades are reniform, 4.5–5.5 cm long and 5–6 cm wide. The raceme is 25-75 cm long. The bracts are herbaceous, ovate or ovate-lanceolate, up to 6 cm long and 2 cm wide at the bottom, tapering upwards with a short apice at the apex and serrated margins. The peduncle is thin, up to 9 cm long at the bottom and gradually shortening upwards. The inflorescence is numerous and radiate. The bracteoles are narrowly lanceolate to linear. The involucre is bell-shaped, 7-20 mm long and 5-14 mm wide. The bracts are 8-9, 2-layered, oblong, 3-5 mm wide, with an acute apex, smooth dorsally, and a broad membranous margin on the inner layer. The ligulate flowers are 5-6 (9), yellow, with oblong ligules 15-25 mm long and 8 mm wide, obtuse at the apex, and 5-11 mm long. The tubular flowers are numerous, 10-17 mm long and 5-9 mm long, with reddish-brown pappus shorter than the tube. The achenes are cylindrical, 6-11 mm long, and smooth. Flowering and fruiting period is July-October.

[0014] Sweet clover White sweet clover : Annual or biennial herb, 70-200 cm tall. Stem erect, cylindrical, hollow, much branched, almost glabrous. Leaves pinnate-trifoliate; stipules spinulose-conical, 6-10 mm long, entire; petioles shorter than leaflets, slender; leaflets oblong or oblanceolate-oblong, 15-30 cm long, (4) 6-12 mm wide, obtuse at apex, cuneate at base, margin sparsely shallowly serrated, glabrous above, finely pubescent below, lateral veins 12-15 pairs, parallel to the apex of the leaf margin, not raised on either side, terminal leaflet slightly larger, with longer petiole, lateral leaflets with shorter petiole. The inflorescence is axillary, 9-20 cm long, with 40-100 loosely arranged flowers. Bracts are linear, 1.5-2 mm long; flowers are 4-5 mm long; pedicels are short, about 1-1.5 mm long; calyx is campanulate, about 2.5 mm long, slightly pubescent, with triangular-lanceolate calyx teeth shorter than the calyx tube; corolla is white, with elliptic stigma slightly longer than the lateral lobes, and keel as long as or slightly shorter than the lateral lobes; ovary is ovate-lanceolate, tapering to the style at the distal end, glabrous, and contains 3-4 ovules. Pods are elliptic to oblong, 3-3.5 mm long, with an acute apex and a pointed beak. Veins are finely veined, reticulate, and brown, turning dark brown with maturity. One or two seeds are present. Seeds are ovate, brown, and covered with small tubercles. Flowering occurs from May to July, and fruiting occurs from July to September.

[0015] Rumex crispus Crispy Sorrel Perennial herb. Roots are stout, yellowish-brown. Stems are erect, 50–120 cm tall, unbranched or branched at the distal end with shallow grooves. Basal leaves are lanceolate or narrowly lanceolate, 10–25 cm long and 2–5 cm wide, acute at the apex, cuneate at the base, and wrinkled at the margins. Stem leaves are smaller and narrowly lanceolate. Petioles are 3–10 cm long. Stipules are membranous and easily broken. Inflorescence is narrowly conical, with branches that are suberect or ascending. Flowers are bisexual, light green. Pedicels are slender, articulated at the mid-lower part, and slightly swollen when fruiting. There are 6 tepals, the outer ones elliptic and about 1 mm long, while the inner ones enlarge in fruiting, becoming broadly ovate and 4–5 mm long, with distinct reticulate veins, slightly obtuse at the apex, nearly truncate at the base, and nearly entire at the margins. All tepals are tuberculate, with only one tepal tuberculate, ovate and 1.5–2 mm long. Achenes are ovate, acute at the apex, with three sharp edges, dark brown, and shiny. Flowering period is May-June, and fruiting period is June-July.

[0016] Big Leaf Seal Deyeuxia purpurea Perennial, with short rhizomes. Culms erect, smooth, glabrous, 30-100 cm tall, with 3-4 nodes. Leaf sheaths smooth, often shorter than internodes; ligule membranous, 3-5 mm long, obtuse or fragmented at apex; leaf blades longitudinally rolled, linear, 10-25 cm long, 1.5-2 (4) mm wide, slightly rough on both surfaces. The panicle is slightly loose, 5-12 cm long and about 5 cm wide, with rough, obliquely ascending branches. The spikelets are 2-3.5 mm long, yellow-green or lavender. The glumes are narrow-lanceolate, acuminate at the apex, smooth, and sub-membranous. The two glumes are nearly equal in length, the first with one vein, the second with three, and the midrib scabrous. The lemma is 1.5-2.5 mm long, membranous, and finely toothed at the apex. The pubescence on either side of the base is equal to or slightly longer than the lemma. The awn extends from near the middle of the dorsal part of the lemma, is thin and straight, and 1-2 mm long. The palea is about 1 / 2 shorter than the lemma. The extended spikelet rachis is 0.8-1 mm long, and together with the pubescence it is 2-3 mm long. The anthers are 1-1.5 mm long. Flowering occurs in July and August.

[0017] The method for preparing the composite mushroom grass of the Lesser Khingan Mountains cold-region native plants of the present invention comprises the following steps:

[0018] (1) Collect burdock, Ligularia officinalis, sweet clover, Rumex crispus and Sesamum indicum from the wild, propagate them through tissue culture, and then move them to the forest for artificial cultivation when they grow into seedlings;

[0019] (2) Harvest to 2 cm above the ground, dry and crush for later use;

[0020] (3) The mixture is prepared by mixing burdock, Ligularia japonica, sweet clover, Rumex truncatula and Calamagrostis chinensis in a mass ratio of 2:2:2:3:1.

[0021] Wherein, the specific method of artificial cultivation under the forest is:

[0022] Choose open woodlands or forest edges with flat terrain, fertile soil, no waterlogging, and a certain degree of canopy density. First, clear the forest of weeds, branches, and stones. When preparing the land, apply 1,000-2,000 kg of farmyard manure per mu. Deeply plow the land 20-25 cm deep, and make ridges or beds. The ridges should be 50-60 cm wide and 20-30 cm high. The beds should be 110 cm wide, flat or semi-high, and 12-15 cm high. The length of the beds depends on the conditions of the forest.

[0023] On the prepared bed, eucalyptus plants are planted at a row spacing of about 20×50cm. The planting depth should be such that 2cm of soil is covered on the top after planting. The soil should be compacted after covering to increase the contact between the rhizome and the soil, and then watered thoroughly. The seedlings will survive after 3-5 days of acclimatization. If the plants are divided in autumn, water them with frozen water once before wintering, apply soil fertilizer on the bed surface, and cover with sawdust and anti-freeze grass to prevent wintering.

[0024] The method of cultivating black fungus using the composite mushroom grass plant native to the cold regions of Xiaoxing'anling Mountains of the present invention comprises the following steps:

[0025] (1) Pretreatment of composite Juncao: pre-wetting and fermentation;

[0026] (2) preparing a planting culture medium and cultivating black fungus in a greenhouse by using a hanging bag cultivation method; wherein the formula of the planting culture medium is 50% of composite grass, 10% of oak leaves, 24% of sawdust, 12% of bran or rice bran, 2.5% of soybean powder, 0.5% of gypsum, and 1% of lime, and the moisture content is 60%.

[0027] Oak leaves: also known as Mongolian oak Mongolian oak This deciduous tree reaches up to 30 meters tall, with gray-brown, longitudinally fissured bark. Young branches are purple-brown, ribbed, and glabrous. The terminal bud is long ovate, slightly ribbed, and the bud scales are purple-brown and ciliate. The leaves are obovate to long-obovate, 7-19 cm long and 3-11 cm wide, with a short obtuse or shortly pointed apex and a narrowly rounded or auriculate base. The leaf margin has 7-10 pairs of blunt or coarse teeth, hairy along the veins when young, gradually glabrescent. The lateral veins have 7-11 on each side. The petiole is 2-8 mm long and glabrous. The male inflorescence grows at the base of new branches, 5-7 cm long, with a nearly glabrous rachis. The perianth is 6-8 lobed, with 8-10 stamens. The female inflorescence grows in the axils of new branches at the upper end, about 1 cm long, and bears 4-5 flowers, though usually only 1-2 develop. The perianth is 6-lobed, the style is short, and the stigma is 3-lobed. The cup is cup-shaped, enclosing 1 / 3-1 / 2 of the nut, 1.5-1.8 cm in diameter and 0.8-1.5 cm high. The bracteoles on the outer wall of the cup are triangular-ovate, with hemispherical, warty protuberances densely covered with short, off-white hairs, extending outwards to form a fringed margin at the mouth. The nut is ovate to oblong-ovate, 1.3-1.8 cm in diameter and 2-2.3 cm high, glabrous, with a slightly raised navel. Flowering occurs in April and May, and fruiting occurs in September.

[0028] The pre-wetting method is specifically as follows: pre-wetting the mixed raw materials with water, spraying the water evenly with a tap, and after the upper layer of the pile is moistened, turning the pile after leaving it for 30-60 minutes, and repeating the pre-wetting steps after turning the pile; until the pile is fully pre-wetted. If the moisture content is too high after pre-wetting, drain it naturally for 12-24 hours to make the moisture content reach 70-75%, ensuring that the pile is evenly pre-wetted.

[0029] The fermentation method specifically comprises the following steps: building a pile of pre-wetted raw materials for fermentation, wherein the size of the pile is determined according to the amount of raw materials; if the pile is large, it may not be covered with a film; if the pile is small, a layer of plastic sheet may be covered on the surface of the pile;

[0030] After the pile is built, use a stick to poke holes in the upper part of the pile, rolling it diagonally until it reaches the ground. Poke holes every 1.5-2m, with a diameter of 5-10cm. Place a thermometer at the hole to monitor the temperature.

[0031] When the daytime temperature is above 23℃, the pile can be built for fermentation. The optimal fermentation temperature is 55-60℃. Turn the pile for the first time after 3 days and the second time after 2-3 days. Whether the third turning is needed depends on the fermentation situation. The fermentation time should not be too long and the maximum fermentation time should not exceed 7 days.

[0032] After fermentation, the fermentation material is grayish white in color and has a light fruity sour taste; the hardness of the composite mushroom grass becomes soft, and there is no prickly feeling when grabbing the culture material with one hand, and the fermentation is completed.

[0033] The process of the black fungus greenhouse hanging bag cultivation method is as follows: preparation of planting culture medium → bagging → sterilization → cooling → inoculation → cultivation → entering the greenhouse → germination → fungus management → harvesting → storage.

[0034] Compared with the prior art, the composite mushroom grass of the Lesser Khingan Mountains cold-region native plants and its application in cultivating black fungus of the present invention have at least the following beneficial effects:

[0035] (1) In view of the cold climate characteristics of Northeast China, the present invention screened the native plants in the cold regions of Lesser Khingan Mountains and obtained plants that are suitable for growing in cold regions, have tall plants and high biomass yield as the substrate for black fungus cultivation, which partially replaces sawdust, reduces costs, and effectively solves the contradiction between fungus and forest.

[0036] (2) Because the nutrient content of mushroom grass is higher than that of straw and sawdust, the black fungus produced has a higher nutrient content.

[0037] (3) Using the existing conventional production method (composition of sawdust 84%, bran (rice bran) 12%, soy flour 2.5%, gypsum 0.5%, lime 1%), the dry material weight per bag is 500g, the cost is 1.8 yuan / bag, and the yield is 50g / bag. However, using the method of the present invention to produce black fungus, the cost per bag is 1.65 yuan / bag, and the yield is 54g / bag, which is significantly better than the existing cultivation method.

[0038] (4) The mushroom grass used in the present invention is cultivated under the forest, which does not occupy farmland and makes full use of the space under the forest. It can vigorously develop the forest economy and practice the concept of forest food. DETAILED DESCRIPTION

[0039] Example 1 Preparation of composite mushroom grass of native plants in the cold region of Xiaoxing'anling

[0040] (1) From the list of wild plants in Xiaoxing'anling, select five plant species suitable for wild growth in cold regions - Burdock Arctium lappa , Ligularia japonica Ligularia fischeri , sweet clover White sweet clover Rumex crispus Crispy Sorrel , Sesamum indica Deyeuxia purpurea The five varieties have tall plants, high nutrient content and high biomass yield. They are collected from the wild, propagated through tissue culture, and then moved to the forest for artificial cultivation when they grow into seedlings.

[0041] 1) Tissue culture propagation

[0042] Plant tissue culture (in a broad sense), also known as in vitro culture, refers to the technique of isolating desired tissues, organs, cells, protoplasts, etc. from a plant and cultivating them under aseptic conditions and controlled conditions to regenerate intact plants or produce other economically valuable products. Plant tissue culture (in a narrow sense) refers to the use of various plant tissues, such as the cambium, parenchyma, mesophyll, and endosperm, to regenerate plants. It also refers to the cultivation of callus tissue generated from various organs during the culture process, which then undergoes redifferentiation to form regenerated plants.

[0043] The specific steps are:

[0044] ① Culture medium preparation

[0045] There are two ways to prepare culture media: one is to purchase all the chemicals needed for the culture medium and prepare it yourself as needed; the other is to purchase pre-mixed powders of the basic culture medium components, such as MS and B5. Self-preparation can save money, but it wastes time and manpower, and sometimes causes problems with the quality of the chemicals, which can lead to problems with the experiment.

[0046] This example uses MS culture medium as an example to introduce the main process of preparing the culture medium.

[0047] (A) Preparation of mother liquor

[0048] a. Prepare MS macroelement stock solutions: Generally, macroelements are prepared into 100-fold stock solutions, which are then diluted 100-fold before use.

[0049] b. Prepare MS trace element mother solution: generally prepare the trace elements into 100 times mother solution.

[0050] c. Prepare MS organic mother liquor: generally prepare it into 100 times of MS organic mother liquor.

[0051] d. Prepare MS iron salt mother liquor: generally prepare it into 100 times MS iron salt mother liquor.

[0052] e. Preparation of hormone mother solution

[0053] (B) Preparation of culture medium

[0054] Taking the preparation of 1L MS culture medium as an example, perform the following steps in order:

[0055] a. First put some distilled water in a beaker.

[0056] b. Take 10ml of each of the above eight mother solutions and pour them in.

[0057] c. Generally weigh 30g of sucrose and pour it in, stirring to dissolve.

[0058] d. Add distilled water and use a graduated cylinder to dilute to 1 L.

[0059] e. Add various hormones according to the designed plan. Since the amount of hormones used is very small and hormones are crucial to the growth of tissue culture plants, it is best to use a micro-adjustable pipette if possible to reduce errors.

[0060] f. Adjust the pH to 5.7-5.8 using precision test paper or a pH meter. (If possible, use a pH meter for greater accuracy.) You can also prepare 1 equivalent of HCl and 1 equivalent of NaOH to adjust the pH of the solution.

[0061] ②Sterilization

[0062] Sterilization refers to the use of physical or chemical methods to kill all microorganisms or organisms on the surface and in the pores of objects, that is, to kill all living substances.

[0063] Culture media are generally sterilized by moist heat. The sterilization process should be completed within 24 hours of preparation.

[0064] The principle of autoclaving is this: within a sealed steamer, steam cannot escape, and the pressure continues to rise, causing the boiling point of water to rise continuously, and thus the temperature inside the steamer. At a pressure of 0.1 MPa, the temperature inside the steamer reaches 121°C. At this steam temperature, various bacteria and their highly heat-resistant spores can be quickly killed.

[0065] Completely expel air from the pot, ensuring it's completely filled with steam, for complete sterilization. There are several different ways to vent air during autoclave sterilization, but the goal is to expel all air, evenly heat the pot, and ensure thorough sterilization. A common method is to close the vent valve, turn on the power, and when the pressure reaches 0.05 MPa, open the vent valve to release the air. Once the pressure gauge returns to zero, close the vent valve.

[0066] After closing the valve and then turning on the power, when the pressure gauge rises to 0.1MPa, start timing and maintain the pressure at 0.1~0.15MPa for 20 minutes.

[0067] ③ Vaccination

[0068] Sterile inoculation steps:

[0069] (A) Place the preliminarily washed and cut materials in a beaker, bring it to the clean bench, sterilize it with disinfectant, rinse it with sterile water, drain the water, take it out and place it on sterilized gauze or filter paper.

[0070] (B) After the material has been blotted dry, use forceps in one hand and scissors or a scalpel in the other to cut the material appropriately. For example, cut leaves into 0.5 cm square pieces; cut stems into segments containing a single node. Peel the micro-stem tips to the size of a stem tip containing only 1-2 young leaves. During the inoculation process, frequently scorch the inoculation equipment to prevent cross-contamination.

[0071] (C) Use sterilized instruments to insert or place the explants onto the culture medium. The specific procedure (using a test tube as an example) is as follows: First, unwrap the paper covering the tube. Hold the tube nearly horizontally, bring the tube mouth close to the flame of an alcohol burner, and rotate the tube mouth over the flame, burning the inside and outside of the tube mouth for several seconds. If the tube mouth is covered with cotton plugs, burn the outside of the tube mouth first, remove the cotton plug, and then burn the inside of the tube mouth. Then, use tweezers to pick up a piece of the explant, place it into the test tube, and gently insert it into the culture medium. If the leaves are directly attached to the culture medium, place 1 to 3 pieces. There are no uniform requirements for material placement, except that the stem tip and stem segment should be placed upright (pointy end up). After inoculation, hold the tube mouth over the flame again for a few seconds. Then, use cotton plugs, wrap the cover with the paper covering, and also burn the inside of the paper covering.

[0072] ④ Cultivation

[0073] Cultivation refers to the process of placing the culture material in a culture room (no light, suitable temperature, sterile) to allow it to grow, divide and differentiate to form callus tissue, and further differentiate into regenerated plants under light conditions.

[0074] Cultivation method:

[0075] 1. Solid culture method: This is the method of using agar solidified culture medium to culture plant materials. It is the most commonly used method. Although this method is simple and easy to use, it has uneven nutrient distribution, uneven growth rate, and often causes browning poisoning.

[0076] 2. Liquid culture: This method involves culturing plant materials in a liquid culture medium without a solidifying agent. Because liquids contain relatively little oxygen, agitation or vibration of the culture medium is usually necessary to ensure an oxygen supply. This is accomplished using a reciprocating or rotary shaker, typically at a speed of 50 to 100 rpm. This periodic immersion ensures both uniformity and a stable oxygen supply.

[0077] Culture steps: 1. Primary culture; 2. Subculture; 3. Rooting culture.

[0078] ⑤Aclimation and transplanting

[0079] Before transplanting, the culture can be moved to natural light for 2-3 days without opening, so that the test tube seedlings can be exposed to strong light and grow strong. Then, the seedlings can be opened for 1-2 days and subjected to lower humidity treatment to adapt to the natural humidity conditions in the future.

[0080] 2) Artificial cultivation under forests

[0081] Choose open woodlands or forest edges with flat terrain, fertile soil, no waterlogging, and a certain degree of canopy density. First, clear the forest of weeds, branches, and stones. When preparing the land, apply 1,000-2,000 kg of farmyard manure per mu. Deeply plow the land 20-25 cm deep, and make ridges or beds. The ridges should be 50-60 cm wide and 20-30 cm high. The beds should be 110 cm wide, flat or semi-high, and 12-15 cm high. The length of the beds depends on the conditions of the forest.

[0082] On the prepared bed, eucalyptus plants are planted at a row spacing of about 20×50cm. The planting depth should be such that 2cm of soil is covered on the top after planting. The soil should be compacted after covering to increase the contact between the rhizome and the soil, and then watered thoroughly. The seedlings will survive after 3-5 days of acclimatization. If the plants are divided in autumn, water them with frozen water once before wintering, apply soil fertilizer on the bed, and cover it with some sawdust and anti-cold grass to prevent wintering.

[0083] (2) Harvest burdock, Ligularia officinalis, sweet clover, Rumex crispus and Sesamum indica to 2 cm above the ground, dry them in the sun and crush them for later use;

[0084] (3) The mixture is prepared by mixing burdock, Ligularia japonica, sweet clover, Rumex truncatula and Calamagrostis chinensis in a mass ratio of 2:2:2:3:1.

[0085] Table 1 Screening experiment of mixed Juncao formula (amount added is mass percentage)

[0086]

[0087] From the results in Table 1, it can be seen that the best addition amount of Juncao is 10% of burdock, 10% of Ligularia officinalis, 10% of Sweet Sweet Meadow, 15% of Rumex crispus and 5% of Salix babylonica, that is, the addition ratio of the five kinds of Juncao is 2:2:2:3:1. The best formula is: Juncao is 10% of burdock, 10% of Ligularia officinalis, 10% of Sweet Sweet Meadow, 15% of Rumex crispus, 5% of Salix babylonica, 34% of sawdust, 12% of bran (rice bran), 2.5% of soy flour, 0.5% of gypsum and 1% of lime; the yield of black fungus is the highest and the nutrient content is the highest, with a yield of 51g / bag.

[0088] Example 2 Cultivation of black fungus using composite mushroom grass, a native plant of the cold region of Xiaoxing'anling

[0089] (1) Pretreatment of composite Juncao

[0090] Pre-wetting: Pre-wet the mixed raw materials with water. Spray the water evenly using a tap. Once the upper layer of the pile is moistened, let it sit for 30-60 minutes before turning it over. Repeat the pre-wetting process until the pile is fully moistened. If the moisture content is too high after pre-wetting, drain it naturally for 12-24 hours until the moisture content reaches 70-75%. Ensure that the pile is evenly pre-wetted.

[0091] Fermentation: The pre-wetted raw materials are piled for fermentation. The size of the pile is determined by the amount of raw materials. Large piles can be left uncovered. For smaller piles, it is recommended to cover the surface with a layer of plastic sheeting. After the pile is built, use a stick to poke holes in the top of the pile, rolling diagonally down to the ground. Poke holes approximately every 1.5-2 meters, with a diameter of 5-10 cm. Place a thermometer at the hole to monitor the temperature.

[0092] When the daytime temperature is above 23°C, the fermentation can be started. The optimal fermentation temperature is 55-60°C. The first turning of the pile should be done after 3 days, and the second turning after 2-3 days. The need for a third turning will be determined based on the fermentation progress. The fermentation time should not be too long, and the maximum fermentation time should not exceed 7 days.

[0093] After fermentation, the fermented material is off-white in color and has a light, fruity aroma. The mixed grass becomes soft and can be grasped with one hand without any prickling. Fermentation is complete.

[0094] (2) Cultivation of black fungus in hanging bags in greenhouses

[0095] Process flow: culture medium preparation → bagging → sterilization → cooling → inoculation → cultivation → entering the greenhouse → germination → ear management → harvesting → storage.

[0096] 1) Prepare planting culture medium

[0097] Cultivation experiments were conducted using the composite Juncao prepared in Example 1 to replace 10%, 20%, 30%, 40%, 50%, 60%, and 70% of the sawdust, and then using oak leaves to replace 10% of the sawdust. The experimental results are shown in Table 2.

[0098] Table 2 Planting culture material formula screening experiment (amount added is mass percentage)

[0099]

[0100] From the results in Table 2, it can be seen that the best addition amount of Juncao is 50%, and the best formula is: mixed Juncao 50%, oak leaves 10%, sawdust 24%, bran (rice bran) 12%, bean powder 2.5%, gypsum 0.5%, lime 1%; the yield of black fungus is the highest and the nutrient content is the highest. The cost per bag is 1.65 yuan / bag and the yield is 54g / bag.

[0101] The existing conventional method for producing black fungus has a formula of 84% sawdust, 12% bran (rice bran), 2.5% soy flour, 0.5% gypsum, and 1% lime. The final dry material weight per bag is 500g, the cost is 1.8 yuan / bag, and the output is 50g / bag.

[0102] 2) Bagging: Use a bagging machine to put the material into polyethylene plastic bags and tie the mouth with a hole-tying stick.

[0103] 3) Sterilization

[0104] High-pressure steam sterilization: First, release the cold air in the pot, close the air release valve, 121℃-126℃, and the pressure reaches 1.2 kg / cm 2 Keep it for 3 hours and stop heating, let the pressure drop naturally, when the pressure drops by 0.5, open the air release valve to release the air, and remove the product from the pot when the pressure drops to 0. The sterilization pressure and holding time vary depending on the volume of the sterilized object and the medium.

[0105] Note: The cold air in the autoclave must be completely removed, otherwise a "false pressurization phenomenon" will occur; follow the principle of slow air intake and slow exhaust. Use polypropylene bags.

[0106] Normal pressure sterilization: Start with high heat and keep the water boiling. Remember to replenish the water in the pot. The temperature in the sterilizer must reach 100°C within 4 hours. The sterilization temperature usually fluctuates between 100-103°C. Start counting the time when the temperature reaches 100°C, keep it for 10 hours, then stop the heat and simmer for another 3 hours before removing from the pot to achieve the sterilization effect.

[0107] 4) Cooling: After sterilization, place the fungus bags in a cooling room and wait until the temperature drops below 25°C before inoculation.

[0108] 5) Inoculation: Specific steps: Wear latex gloves, light an alcohol burner, and wipe the gloves and tools with 75% alcohol. Then, heat the inoculation hoe under the outer flame of the alcohol burner. After cooling, remove the cotton from the mother seed tube and the stick from the fungus bag. Place the fungus into the inoculation hole and plug it with cotton. After inoculating one bag, reheat the inoculation hoe before continuing.

[0109] 6) Cultivation:

[0110] ① In dark conditions, the temperature of the material in the bag should be 28℃ for 1-10 days after inoculation, which is conducive to the resurrection and stabilization of mycelium and reduces the contamination rate. The incubation room does not need to be ventilated for the first three days.

[0111] ②11-25 days after inoculation, the temperature of the material in the bag should drop to 22°C. 26 days after inoculation, when the mycelium has fully grown in the bag, the temperature of the material in the bag should drop to 20°C. Increase ventilation daily to keep the indoor air fresh and dry.

[0112] ③After-ripening: After the mycelium has grown all over the bag, maintain the material temperature in the bag at 20℃ and continue to cultivate for 15-20 days until the mycelium reaches physiological maturity.

[0113] ④ It is best to keep the temperature of the fungus in the bag with mycelium growing all over it at around 10℃.

[0114] 7) Moving into the greenhouse: Disinfect and open the bags filled with fungi. Place 45-50 bags per square meter on a bed covered with specialized mulch film for growing fungi, with 2-3 cm spacing between bags. Cover with plastic film, shade netting (75%), or straw mats (pre-soaked in thiophanate-methyl to control moisture). Ventilation holes should be installed at both ends and along the sides of the bed every 4-5 meters. Drill 180-220 nail holes per bag, 0.5-0.8 cm deep and evenly spaced.

[0115] 8) Germination: During this period, maintain a temperature between 15-24°C. If the temperature exceeds 24°C, remove the plastic film and allow ventilation. Maintain a relative humidity of 80%-85%. Primordia will form within 7-10 days.

[0116] 9) Ear management:

[0117] ①Fruiting body differentiation period

[0118] The coral-like auricle, formed from the primordium, grows to the size of an apricot kernel, and small auricles begin to sprout from it. This is called the differentiation stage. Management during this period is essentially the same as during the initial stages of auricle formation, much like the "settling" phase of a crop. After the auricle is formed, maintain a certain temperature, humidity, temperature range, and ventilation to allow for gradual differentiation.

[0119] During this stage, avoid heavy watering and water frequently. The relative humidity of the air should be 85-90% during this stage, which lasts for about 5-7 days.

[0120] ②Fruiting body growth period

[0121] When the fruiting body grows to the size of an apricot kernel, many auricles will differentiate on the edge and gradually extend outward, completely sealing the incision. At this stage, you should gradually increase watering, improve ventilation, and spray water as much as possible. The relative humidity of the air should be 90-95%. The fruiting body growth period lasts about 7-10 days.

[0122] In order to ensure that the ear pieces are black and thick, the growth rate of the ear pieces should be properly controlled. In principle, no watering is done when the temperature in the greenhouse exceeds 25℃. In early spring, intermittent watering is generally done from 3 pm to 9 am the next day. After May, watering is generally done from 5 pm to 7 am the next day to keep the relative humidity of the air at 90-95%.

[0123] ③Watering management methods

[0124] Watering must be done flexibly according to the growth conditions.

[0125] ⑴ Spray more if there are many ear buds, spray less if there are few; spray more if the ears are withered, spray less if the ears are wet; spray more on sunny days, spray less on rainy days; spray more when the temperature is high, spray less when the temperature is low.

[0126] ⑵ When the fruiting body enters the maturity stage, the ear pieces are fully expanded, the ear roots are contracted, and the ground-grown wood ear mushrooms reach "80% ripe" and the shed-type hanging bag wood ear mushrooms reach "50% ripe", you can stop spraying water and start harvesting.

[0127] ⑶Control the temperature: high temperature will make the fruiting body grow faster, mature earlier, and have lower yield. Low temperature will make the fruiting body grow slower, the growth period will be longer, but the total yield will be higher. If the temperature is too high or too low, watering should be reduced or stopped, otherwise it will easily cause ear runny nose.

[0128] ⑷ Alternating between dry and wet: dry conditions grow mycelium, wet conditions grow fruiting bodies, alternating between dry and wet is beneficial to growing ears.

[0129] ④Watering control principles

[0130] In principle, do not water when the temperature is above 25℃ and below 15℃. The cultivation of black fungus should be managed in a "dry-dry-wet-wet" manner, and watering should depend on the watering conditions:

[0131] During the ear-stimulating period, the humidity should be controlled at 70-75%;

[0132] Humidity should be controlled at 75-80% during the bud stage;

[0133] Humidity should be controlled at 80-85% during the young ear stage;

[0134] Humidity should be controlled at 85-90% during the ear growth period;

[0135] When the ears are almost ripe, the humidity should be controlled at 90-95%.

[0136] The temperature is low in early spring, so water during the day and less at night. In spring, water should be applied between 3 pm and 7 am the next day; in summer, water should be applied between 5 pm and 3 am the next day.

[0137] 10) Harvesting: When the black fungus ear pieces grow to 3-5cm and the edges droop, they can be harvested (50-60 minutes ripe). After harvesting the fungus, roll up the plastic film and shade net of the greenhouse to the roof, dry the bags for 5-7 days, and then water them, using a "dry-dry, wet-wet" moisture management method.

[0138] Harvesting Method: Use your fingers to pinch the entire ear, including the base, and twist slightly to remove the entire ear. Place it in a container. This method will ensure the ear is well-shaped and high-quality. When harvesting, be sure to remove the ear roots thoroughly to prevent them from rotting and attracting insects and bacteria. However, be sure to leave the ear buds.

[0139] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A composite mushroom grass of cold-region native plants in Lesser Khingan Mountains, characterized by: It is composed of burdock, Ligularia japonica, sweet clover, Rumex crispus and Sesamum indica, and the mass ratio is 2:2:2:3:

1.

2. The method for preparing the composite Juncao of the cold-region native plants of Xiaoxing'anling according to claim 1, characterized in that: The following steps are involved: (1) Collect burdock, Ligularia officinalis, sweet clover, Rumex crispus and Sesamum indicum from the wild, propagate them through tissue culture, and then transplant them into artificial cultivation under the forest when they grow into seedlings; (2) Harvest to 2 cm above the ground, dry and crush for later use; (3) The mixture is prepared by mixing burdock, Ligularia japonica, sweet clover, Rumex truncatula and Calamagrostis chinensis in a mass ratio of 2:2:2:3:

1.

3. The method for preparing the composite Juncao of the cold-region native plants of Xiaoxing'anling according to claim 2, characterized in that: The specific method of artificial cultivation under the forest is: Choose open woodlands or forest edges with flat terrain, fertile soil, no waterlogging, and a certain degree of canopy density. First, clear the forest of weeds, branches, and stones. When preparing the land, apply 1,000-2,000 kg of farmyard manure per mu. Deeply plow the land 20-25 cm deep, and make ridges or beds. The ridges should be 50-60 cm wide and 20-30 cm high. The beds should be 110 cm wide, flat or semi-high, and 12-15 cm high. The length of the beds depends on the conditions of the forest. On the prepared bed, eucalyptus plants are planted at a row spacing of about 20×50cm. The planting depth should be such that 2cm of soil is covered on the top after planting. The soil should be compacted after covering to increase the contact between the rhizome and the soil, and then watered thoroughly. The seedlings will survive after 3-5 days of acclimatization. If the plants are divided in autumn, water them with frozen water once before wintering, apply soil fertilizer on the bed surface, and cover with sawdust and anti-freeze grass to prevent wintering.

4. The application of the composite grass of the cold-region native plant of Xiaoxing'anling in claim 1 in the cultivation of black fungus is characterized in that: The following steps are involved: (1) Pretreatment of composite Juncao: pre-wetting and fermentation; (2) preparing a planting culture medium and cultivating black fungus in a greenhouse using a hanging bag cultivation method; wherein the formula of the planting culture medium is 50% of composite mushroom grass, 10% of oak leaves, 24% of sawdust, 12% of bran or rice bran, 2.5% of soybean powder, 0.5% of gypsum, and 1% of lime.

5. The use of the composite mushroom grass of the cold-region native plant of Xiaoxing'anling in the cultivation of black fungus according to claim 4, characterized in that: The pre-wetting method specifically comprises the following steps: adding water to the mixed raw materials for pre-wetting, spraying the water evenly with a tap, and after the upper layer of the pile is moistened, turning the pile after standing for 30-60 minutes, and repeating the pre-wetting steps after turning the pile until the pile is fully pre-wetted. If the moisture content is too high after pre-wetting, drain the pile naturally for 12-24 hours to reduce the moisture content to 70-75%, thereby ensuring that the pile is evenly pre-wetted.

6. The use of the composite grass of the Lesser Khingan Mountains cold region native plant in the cultivation of black fungus according to claim 4, characterized in that: The fermentation method specifically comprises the following steps: building a pile of pre-wetted raw materials for fermentation, wherein the size of the pile is determined according to the amount of the raw materials; if the pile is large, the film may not be covered; if the pile is small, a layer of plastic sheet may be covered on the surface of the pile; After the pile is built, use a stick to poke holes in the upper part of the pile, rolling it diagonally until it reaches the ground. Poke holes every 1.5-2m, with a diameter of 5-10cm. Place a thermometer at the hole to monitor the temperature. When the daytime temperature is above 23℃, the pile can be built for fermentation. The optimal fermentation temperature is 55-60℃. Turn the pile for the first time after 3 days and the second time after 2-3 days. Whether the third turning is needed depends on the fermentation situation. The fermentation time should not be too long and the maximum fermentation time should not exceed 7 days. After fermentation, the fermentation material is grayish white in color and has a light fruity sour taste; the hardness of the composite mushroom grass becomes soft, and there is no prickly feeling when grabbing the culture material with one hand, and the fermentation is completed.

7. The use of the composite mushroom grass of the Lesser Khingan Mountains cold region native plant in the cultivation of black fungus according to claim 6, characterized in that: The process of the black fungus greenhouse hanging bag cultivation method is as follows: preparing planting culture medium → bagging → sterilizing → cooling → inoculating → cultivating → entering the greenhouse → germinating → fungus management → harvesting → storing.

8. The use of the composite mushroom grass of the cold-region native plants of Lesser Khingan Mountains in the cultivation of black fungus according to claim 7, characterized in that: The moisture content of the planting culture medium is 60%.

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