A high-yield morchella strain mZ-12 and a cultivation method thereof
Patent Information
- Application Number
- CN202310699883.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-13
- Publication Date
- 2026-08-07
- Estimated Expiration
- 2043-06-13
AI Technical Summary
但是目前菌种生产大多数仍属于小工厂生产，生产技术不规范导致菌种总体质量低下，易出现杂菌、错种、批量污染、大幅度减产等不良事故，以及出现出菇不稳定、产量低、菌种容易退化、质量参差不齐、假菌种、劣质菌种等诸多问题
[0040]本发明利用化学试剂N-甲基-N’-硝基-N-亚硝基胍(NTG)诱变方式首次获得一株羊肚菌(Morchella esculenta)MZ-12，该菌株较原始羊肚菌菌株最短出菇时间缩短10天，产量更高，同时具有较强的抗链孢霉能力，且具有基因遗传稳定性，相较于现有已知的羊肚菌品种菇盖更大更厚，肉质更加厚实有弹性，香气更加浓郁，具备更好的商品性。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of edible fungi cultivation technology, specifically to a high-yielding morel mushroom strain MZ-12 and its cultivation method. Background Technology
[0002] Morel mushrooms have a distinct umami flavor and are often used as a food seasoning. Known as the "King of Mushrooms" for their delicious taste and unique aroma, they are one of China's rare edible fungi. According to statistics from the China Edible Fungi Association, China's morel mushroom production reached 239,583.45 tons in 2021, a year-on-year increase of 74.7%. In the first half of 2022, China exported 88,662 kilograms of dried morel mushrooms, with an export value of US$10,966,491. Artificial cultivation of morel mushrooms in my country, mimicking wild growth, originated in Sichuan. With continuous advancements in cultivation varieties and techniques, its development has been rapid in recent years. The main production areas have expanded from Sichuan and Hubei to provinces in the Central Plains such as Henan, Shandong, and Shaanxi. Due to significant climatic differences between the north and south, cultivation of morel mushrooms has only gradually become popular in recent years.
[0003] Morel spawn is the most fundamental production material in morel production, playing a crucial role in determining the yield and quality of morels. The main cost of morel cultivation lies in the spawn, and the core technology also lies in it. The spawn is the foundational factor and key to the success of morel cultivation, and superior spawn plays a vital role in the technological share of the industry's development. The selection and breeding of morel spawn in my country involved countless investigations of wild morels, specimen collection, and isolation and domestication. After seven years of exploration and experimentation, successful cultivation was finally achieved for the first time in 1992. However, currently, most spawn production still occurs in small factories. Unstandardized production techniques lead to low overall spawn quality, making it prone to problems such as contamination, incorrect spawn selection, batch contamination, and significant yield reductions. Other issues include unstable fruiting, low yield, easy spawn degeneration, inconsistent quality, fake spawn, and inferior spawn. In addition, morel cultivation requires land preparation and sowing every autumn and winter, and harvesting in spring. Extending the harvest period can significantly increase yield. However, the growth of morel mushrooms is greatly affected by temperature. Excessive temperature will affect the color and shape of the fruiting bodies. Once the temperature inside the greenhouse exceeds 25°C, the morel mushrooms will die directly and the fruiting bodies cannot be harvested, ending the production cycle. Therefore, shortening the fruiting time of morel mushrooms is a very favorable condition for increasing yield.
[0004] Hybrid breeding is a traditional breeding method for organisms, which combines the superior traits of two or more varieties through mating, followed by screening and cultivation to ultimately obtain the desired new variety. N-methyl-N'-nitro-N-nitrosoguanidine (NTG) is used for mutagenesis breeding and is a recognized highly effective chemical hypermutant. Its structural formula is: By combining gene recombination and induced mutation, various types of strains can be obtained, providing abundant materials for selection.
[0005] The selection method for new morel mushroom strains is natural breeding, which utilizes beneficial variations occurring under natural conditions to select new strains that meet production needs. The requirements for new strains are: robust mycelium with rapid growth; oval cap with a blunt, rounded apex and pitted surfaces resembling a sheep's stomach; large, round fruiting bodies; dense, non-breakable flesh; nearly cylindrical stipe, light in weight and brittle in texture; early and uniform fruiting with few deformed mushrooms, distinct flushes, high biological conversion rate, and stable and excellent traits.
[0006] In the production of morel spawn, the mother spawn is the spawn obtained directly from the fruiting body (tissue or spore) or the wood (ear wood), also known as the primary spawn; the pure culture of mycelium obtained by transplanting and expanding the mother spawn is called the original spawn, also known as the secondary spawn; since the quantity of the original spawn is limited, in order to meet the needs of large-scale production, the original spawn must be expanded and cultured again, and the resulting spawn is called the cultivation spawn, also known as the tertiary spawn. The original spawn can also be used directly as the cultivation spawn for actual production. Summary of the Invention
[0007] To address the shortcomings of existing technologies, this invention provides a morel mushroom strain MZ-12 and its cultivation method. Morel mushroom MZ-12 was obtained through chemical mutagenesis screening, exhibiting excellent traits and achieving the goal of high yield and superior quality.
[0008] The technical solution of this invention is as follows:
[0009] A morel mushroom (Morchella esculenta) MZ-12 was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40394.
[0010] The preparation method of the above-mentioned morel mushroom MZ-12 mother culture specifically includes the following steps:
[0011] S1. Prepare solid culture medium, fill test tubes, sterilize, cool, and then arrange on slant plates;
[0012] S2. Under aseptic conditions, morel mushroom MZ-12 is inoculated onto the slant of a test tube and placed in a constant temperature incubator for cultivation until the mycelium has fully grown in the test tube, which is the mother culture of morel mushroom MZ-12.
[0013] S3. Place the test tubes covered with mycelium at a temperature of -12 to 0°C for cryopreservation.
[0014] Preferably, the solid culture medium in S1 is PDA solid culture medium, the components of which include: 200g peeled potato, 20g agar, 20g glucose, 1000mL water, and natural pH.
[0015] Preferably, the sterilization conditions in S1 are sterilization at 121°C for 30 minutes.
[0016] Preferably, the culture conditions in S2 are 10–18°C for 7 days.
[0017] The preparation method of the above-mentioned morel mushroom MZ-12 original strain specifically includes the following steps:
[0018] (1) Mix all components in the microbial culture medium evenly;
[0019] (2) Pack the culture medium into a culture bottle or culture bag, sterilize it, and inoculate the above morel MZ-12 mother culture in a sterile environment.
[0020] (3) Cultivate at 12-18℃ until fully grown, which is the original morel strain MZ-12.
[0021] Preferably, the microbial culture medium in step (1) is one of the following formulations, and the components of the microbial culture medium are as follows by weight percentage:
[0022] Formula 1: 70-72% wheat, 19-21% sawdust, 4-6% wheat bran, 2-4% cornmeal, 1-2% lime;
[0023] Formula 2: 60-62% wheat, 29-31% corn cob, 4-6% wheat bran, 2-4% cornmeal, 1-2% lime;
[0024] Formula 3: 60-62% wheat, 34-36% cottonseed hulls, 2-4% cornmeal, 1-2% lime;
[0025] Formula 4: 79-81% wheat, 14-16% rice bran, 3-5% cornmeal, 1-2% gypsum;
[0026] Formula 5: Rice bran 48-50%, wheat 39-41%, soybean flour 9-11%, gypsum 1-2%;
[0027] Formula 6: Rice bran 59-61%, wheat 34-36%, soybean flour 3-5%, gypsum 1-2%;
[0028] Formula 7: 77-79% sawdust, 19-21% wheat bran, 1-2% sucrose, and 1-2% gypsum.
[0029] Preferably, in step (1), the wheat in the microbial culture medium is soaked in advance.
[0030] Preferably, the sterilization conditions in step (2) are sterilization at 121°C for 30 minutes.
[0031] The cultivation method for morel mushroom MZ-12 described above specifically includes the following steps:
[0032] (i) Morel MZ-12 original strain is further expanded to obtain morel MZ-12 cultivar. The amount of cultivar used per mu is 150-200 kg. After crushing the morel MZ-12 cultivar, it is evenly spread on the soil and then immediately covered with 2-4 cm of soil.
[0033] (ii) Covering the soil with mulch;
[0034] (iii) Spawning and inducing mushroom growth, during which a large amount of white powdery asexual spore layer will appear on the soil surface during the spawning period;
[0035] (iv) Formation of fruiting body primordia, which grow into young mushrooms under suitable conditions.
[0036] Preferably, during step (i), the inoculum should be protected from direct sunlight.
[0037] Preferably, in step (iii), mushroom cultivation is achieved by rapidly increasing the soil temperature and watering the soil thoroughly.
[0038] Preferably, in step (iv), the primordia growth temperature is 5–12°C, the humidity is 85–95%, and the young mushroom growth temperature is 8–20°C.
[0039] The beneficial effects of this invention are:
[0040] This invention utilizes the chemical reagent N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis to obtain a new strain of morel mushroom (Morchella esculenta) MZ-12 for the first time. This strain has a shorter fruiting time of 10 days compared to the original morel strain, higher yield, stronger resistance to Neurospora, and genetic stability. Compared to existing known morel varieties, it has a larger and thicker cap, a more substantial and elastic flesh, a richer aroma, and better marketability. Attached Figure Description
[0041] Figure 1 Photographs of the antagonistic confrontation experiment between morel MZ-12 and primitive morel;
[0042] Figure 2 Photo of morel mushroom MZ-12 fruiting after 30 days of cultivation;
[0043] Figure 3 Comparative photographs of the morphology of harvested and dried morel mushroom MZ-12 and the original morel mushroom. Detailed Implementation
[0044] The following description is based on specific embodiments:
[0045] Strain source: Original morel (Morchella esculenta): obtained by collection and isolation from the wild.
[0046] Example 1:
[0047] (1) Select naturally mature, high-quality original morel fruiting bodies, collect spores using the spore printing method in a sterile environment, and transfer the collected spores to sterile water and dilute them 10 times after 10 hours. 5 Dip an inoculation loop into the spore solution and spread it on PDA solid medium. Seal and incubate in a dark environment at 23±1℃. When the hyphae grow to 3-6 mm, select small hyphae from independent colonies and place them in the center of the PDA solid medium. Incubate at 25℃ and select 5-8 colonies with the most vigorous hyphal growth. When the hyphae are about to cover the plate, examine each colony under a microscope and select monokaryotic hyphae.
[0048] The PDA solid culture medium consists of the following components: 200g potato, 20g glucose, 20g agar, and water to a final volume of 1L.
[0049] (2) Place N-methyl-N'-nitro-N-nitrosoguanidine (NTG) crystals with a diameter of 2 mm in the center of the culture medium for later use. Select 20 monokaryotic mycelia and pair them up. Inoculate them in the center of the culture dish at a distance of about 30 mm. Incubate at a constant temperature of about 18-20℃ in the dark. On the 5th, 10th and 15th days, check the growth distance between the two ends of the mycelia. Take the mycelia with a growth rate ≥4.5 mm / d for microscopic examination and discard the rest. After microscopic examination, select the mycelia with clamp connections that are close to the outer periphery of NTG for transplantation and propagation culture.
[0050] (3) Detoxification treatment: Cut off the front 3cm of the PDA solid medium slant in the test tube with an inoculation palladium and leave a 1cm blank area. Inoculate morel mycelium normally and culture at 18-20℃ in the dark until the mycelium crosses the blank area and fills the cut-off medium. Take the mycelium on the cut-off medium and inoculate it into the same treated blank test tube slant PDA solid medium. Repeat this treatment 4 times to remove the pathogens carried by morel.
[0051] (4) Inoculate the mycelium detoxified in step (3) into the prepared culture medium. The composition of the culture medium by weight percentage is: sawdust 78%, wheat bran 20%, sucrose 1%, and gypsum 1%.
[0052] (5) Once the mycelium in the spawn culture medium has fully grown, it is machine-crushed in a sterile environment, and then sown, mycelium inoculated, mushroomed, young mushrooms grow and develop, and fruiting culture is carried out.
[0053] The mushroom yield, characteristics, and cultivation cycle were detected and recorded. Finally, a new morel strain with a larger and thicker cap and a shorter mycelial growth time was selected compared to the original strain. The shortest fruiting period cultivation time was 39 days, which was 10 days shorter than the original morel strain. It was named MZ-12.
[0054] Validation and identification of the screened strains: The screened strains were identified by ITS sequence analysis. The comparative identification results showed that the strains screened in this invention belong to the kingdom Fungi, phylum Ascomycota, class Discomycetes, order Pezizales, family Morohellaceae, genus Morchella, and species Morchella esculenta. Their ITS sequences did not differ significantly from those of the original morel, indicating a close phylogenetic relationship. However, it cannot be denied that the fruiting bodies of the two strains differ in appearance, yield, and other agronomic traits, even showing significant differences, i.e., differences between varieties.
[0055] Morel (Morchella esculenta) MZ-12 was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40394.
[0056] Example 2:
[0057] The preparation method of morel mushroom MZ-12 mother culture is as follows:
[0058] (1) Prepare PDA solid culture medium, fill the test tube to 1 / 2 full, sterilize at 121℃ for 30 minutes, cool and place on slant; under aseptic conditions, inoculate morel MZ-12 onto the slant of the test tube, stopper the test tube, and culture at 18℃ until the mycelium covers the slant.
[0059] (2) Detoxification: Cut off the front 3cm of the PDA solid medium slant in the test tube with an inoculation palladium and leave a 1cm blank area. Inoculate morel MZ-12 mycelium and culture the mycelium at 18℃ in the dark until it grows over the blank area and fills the cut-off medium block. Take the cut-off medium block and inoculate it into other blank test tubes that have been treated in the same way. Repeat this treatment 4 times to remove the pathogens carried by morel MZ-12.
[0060] The preparation method of morel mushroom MZ-12 original strain is as follows:
[0061] The detoxified mycelium was inoculated into the prepared inoculum culture medium and cultured at 18°C. The inoculum culture medium consisted of the following components by weight percentage: wheat 71%, sawdust 20%, wheat bran 5%, cornmeal 3%, and lime 1%.
[0062] The cultivation method for morel mushroom MZ-12 is as follows:
[0063] (i) Morel MZ-12 original strain is further expanded to obtain morel MZ-12 cultivar. During the sowing period, the cultivar should be protected from direct sunlight. The amount of cultivar used per mu is 150-200 kg. After crushing the morel MZ-12 cultivar, it is evenly spread on the soil and then immediately covered with 2-4 cm of soil.
[0064] (ii) Covering the soil with mulch;
[0065] (iii) Promote mycelium growth and mushroom development by watering the mushrooms thoroughly.
[0066] (iiii) Fruiting body primordia are formed at temperatures of 5–12℃ and humidity of 85–95%, and the primordia grow into young mushrooms at temperatures of 8–20℃.
[0067] Comparative example:
[0068] The preparation method is the same as described in Example 1, except that the strain used is the original morel mushroom, and the shortest fruiting period cultivation time is 49 days.
[0069] Experimental example:
[0070] (1) Strain antagonism experiment:
[0071] Morel MZ-12 and original morel were used as experimental materials, and PDA solid medium was used as the culture medium.
[0072] Morel MZ-12 and original morel were inoculated and cultured separately on PDA solid medium, with the inoculation points 3 cm apart, and cultured at a constant temperature of 20℃.
[0073] Results analysis:
[0074] Culture results as follows Figure 1 As shown, the left side represents the original morel mushroom, and the right side represents morel mushroom MZ-12. At the boundary between the opposing colonies, the antagonistic line between morel mushroom MZ-12 and the original morel mushroom is obvious, indicating that the screened morel mushroom MZ-12 has undergone fundamental differences from the original morel mushroom and is a new morel mushroom variety.
[0075] (2) Disease resistance test:
[0076] Neurospora is a major disease in morel cultivation. Using the pathogenic fungus of morels and Neurospora as indicators, a confrontation culture was conducted. The disease resistance of morel MZ-12 and the original morel was determined by observing colony growth and the width of the antagonistic band.
[0077] Strain activation: Morel MZ-12 strain and Alternaria strain were placed in PDA solid medium and activated at 20°C. After the culture dish was filled, it was used for later use. Before use, mycelial cakes were prepared using a 0.5cm diameter punch.
[0078] Methods for determining resistance to Neurospora:
[0079] Using 90 mm diameter culture dishes, 40 mL of PDA solid medium was added to each dish using a peristaltic pump and allowed to solidify. A 0.5 cm diameter *Neurospora* spore cake was inoculated 1.7 cm from the center. On the opposite side, 1.7 cm from the center, activated *Morchella mongolica* strain MZ-12 and the original *Morchella mongolica* strain were inoculated using an inoculation loop. After incubation at 20°C for 4 days, the width of the antagonistic line and the colony growth width were observed and measured.
[0080] The results of the strains' disease resistance are shown in Table 1:
[0081] Table 1. Anti-Nematode Properties of Morel MZ-12 and Original Morel
[0082]
[0083] The data above show that, against *Nepeta tenuifolia*, the antagonistic line width of *Morchella mongolica* MZ-12 is 4.6 mm, which is narrower than the original *Morchella mongolica* antagonistic line width of 4.9 mm; the colony width of *Morchella mongolica* MZ-12 is 3.7 mm, which is also narrower than the original *Morchella mongolica* colony width of 3.9 mm; indicating that the disease resistance of *Morchella mongolica* MZ-12 is superior to that of the original *Morchella mongolica*.
[0084] Morel mushrooms are prone to bacterial diseases during cultivation, but the MZ-12 strain of morel mushrooms has a strong resistance to Trichoderma, which can reduce the incidence of diseases and has broad application prospects.
[0085] (3) Cultivation experiment:
[0086] A cultivation cycle optimization experiment was conducted at a morel cultivation enterprise using morel mushroom MZ-12 and the original morel mushroom. 2000 bags of fruiting mushrooms were produced from each of the two types. The entire cultivation cycle, biological conversion rate, and yield were compared.
[0087] The cultivation steps are as follows:
[0088] The substrate for microbial culture before cultivation is composed of the following components by weight percentage: wheat 71%, sawdust 20%, wheat bran 5%, cornmeal 3%, and lime 1%.
[0089] (i) Site selection and land management
[0090] Choose a location with convenient transportation and flat ground for morel mushroom cultivation. Loamy soil is preferred, with high humus content and good water retention and aeration. It should be near a water source with convenient drainage to prevent waterlogging and drought. Weeds should be removed one month before sowing using a weeding machine or harvester. After weeding, apply lime powder at a rate of 75-100 kg per acre to effectively kill or repel most insects and appropriately adjust the soil pH.
[0091] (ii) Sowing and covering
[0092] Till the land to a depth of 25-30cm. After tilling, level the ground appropriately and begin sowing. The amount of spawn sown is about 150kg / mu. Crush the spawn into spawn blocks with a diameter of 1.0-1.5cm and scatter them on the soil surface. Use a small trenching machine to dig trenches 20-25cm wide and about 20cm deep. The soil ridges should be 0.8-1.5m wide. Turn the soil to both sides to cover the spawn. For areas that are not adequately covered, manually cover them with soil.
[0093] There are two main methods for sowing morel mushrooms: broadcasting and row sowing. Broadcasting is the primary method used in large-scale cultivation. If the crushed spawn is too dry, it can be mixed with a 0.1-0.5% potassium dihydrogen phosphate solution and pre-moistened to a moisture content of 65%-70%. If the soil is short of water, irrigate the land before tilling and digging ditches, and let it dry for 7 days. If the soil is not severely short of water, replenish the soil moisture by irrigation or spraying after sowing.
[0094] (iii) Covering
[0095] After morel mushrooms are sown, a layer of black mulch is placed on the seedbed. This helps to retain moisture, prevent waterlogging, block light, suppress weeds, accelerate heat accumulation, promote mushroom growth, control excessive growth of mycelial frost, increase the flexibility of cultivation, direct fruiting, and save production costs.
[0096] (iv) Apply exogenous nutrient bags
[0097] The spawn blocks sown in the soil germinate within 7-20 days, and the mycelium begins to spread, initially forming a morel mycelial network in the soil. A layer of white mycelial bloom forms on the soil surface. During this stage, morel mushrooms mainly absorb the remaining nutrients from the soil and the spawn. At this time, an external nutrient bag is applied to the soil, coming into contact with the already formed morel mycelial network. The morel mycelium gradually spreads into the nutrient bag, making full use of the nutrients inside for growth, and transferring the nutrients accumulated in the mycelium back to the mycelial network in the soil. This greatly increases the nutrients stored in the sclerotia and mycelial network, thus continuously supplying morel primordia differentiation and young mushroom development in the later stages, thereby ensuring increased and stable morel mushroom yield.
[0098] When using exogenous nutrient bags, remove the soil clods from one side of the mulch film, and lay the mulch film flat on the other side of the bed. Apply the sterilized and cooled exogenous nutrient bags at a rate of 1600-1800 bags per acre. Make slits on the side of the bags with a knife or punch holes with nails, with the slits and holes facing down, and cover the mycelium-covered bed. Then replace the mulch film. The nutrient bags should be spaced 20-30 cm apart, the row spacing 30-40 cm, and the density 4-5 bags / m². 2 They are arranged in a plum blossom pattern.
[0099] (v) Erecting shade netting canopies
[0100] After applying the exogenous nutrient bags, cover the mulch film with the shade netting to be used for building the flat shed. Covering the mulch film with the shade netting helps to keep the soil warm and moist. Before the mushrooms are promoted, the mulch film with the shade netting is built.
[0101] In northern regions, to ensure the relative humidity required for morel mushroom cultivation during primordia differentiation and young mushroom development, it is necessary to erect small arched sheds less than 50cm high on the raised beds within the flat shed. The frames of these small arched sheds are covered with black or white plastic film.
[0102] (vi) Management during the mycelium growth period
[0103] The period from sowing to fruiting is called the mycelial growth stage. The management of this stage is called the mycelial growth period management. The main goal of this stage is to make morel mycelium grow in large quantities and fill the top 20-30cm of soil. Attention should be paid to the management of temperature, moisture, air, pests, wind, rain, snow and miscellaneous fungi.
[0104] If the soil is short of water, you can replenish the water by irrigating the furrows, removing the mulch and spraying water onto the soil surface, or spraying water directly onto the mulch film without removing it, so that the water flows into the furrows and slowly seeps into the soil.
[0105] If sowing is early or the temperature is high, causing the temperature under the mulch or on the surface of the seedbed near the ground to remain above 25°C for an extended period, the mulch should be removed promptly for ventilation and cooling. If clay or plastic mulch is tightly covered, a hole with a diameter of 2-3 cm can be punched every 30 cm or so in the mulch to prevent damage to the fungus growth caused by excessive temperature and humidity.
[0106] If the soil surface is too moist, during the inoculum growth period, there may be excessive aerial mycelium on the soil surface, dense mycelium growing in large quantities on the soil surface, or too many conidia on the soil surface, resulting in a snow-white surface. It is necessary to improve ventilation and dehumidification and reduce the moisture content of the topsoil to improve the situation.
[0107] (vii) Mushroom cultivation management
[0108] ①Nutritional stimulation: When the external nutrient bag becomes noticeably lighter, it indicates that the nutrients have been transferred to the mycelial network in the soil. The bag can be removed. Removing the bag will provide mechanical and nutritional stimulation to the mycelium in the soil, which is conducive to the formation of primordia. Not removing the bag will not only hinder mushroom growth, but will also lead to the breeding of pests in the later stages.
[0109] ②Removing the film: Remove the covering film 15 days before the edible fungi begin to grow. The removal of the film should be done slowly to avoid causing the death of primordia or young mushrooms. The specific operation is as follows: First, make holes in the film so that the microenvironment under the film gradually approaches the macroenvironment. Then, remove the film after 3 days. Throughout the operation, ensure that the air humidity is maintained at 85-95%.
[0110] ③ Moisture stimulation: Use micro-spraying or sprinkler irrigation to water until the soil surface of 15cm is completely saturated. Repeat the watering process 3 times. Alternatively, water can be poured into the furrows and kept in the furrows for 24 hours to allow the water to seep into the soil. Needle-shaped primordia can be seen 1-3 days after watering (if the temperature is low, the primordia may appear about 2 weeks after water stimulation). Spherical primordia can be seen 3-5 days later.
[0111] ④ Humidity control: Maintain relative humidity of air at 85-95% and soil moisture content at 25-30%; erect small arched sheds on the raised beds and use such facilities for cultivation, which can basically meet the air humidity requirements for primordia differentiation. After erecting the small arched sheds, pay attention to the high temperature inside the sheds. If the temperature exceeds 20℃ during the fruiting period, lift the film at both ends of the small arched sheds to cool it down.
[0112] ⑤ Other management practices: Temperature difference stimulation of 10℃ or more, and lifting the film to increase light and oxygen stimulation are all beneficial to the differentiation of primordia. If conventional methods still cannot induce the formation of primordia, it may be due to the aging and degeneration of the strain. Extreme methods such as trampling the soil on the bed to create mechanical stimulation can be used to induce the growth of mushrooms.
[0113] (viii) Mushroom Management
[0114] During the differentiation of primordia into young mushrooms, maintain the stability of the microenvironment close to the ground as much as possible. Keep the temperature at 6-15℃, relative humidity at 85-95%, soil moisture at 25-28%, and ensure even and bright diffused light. Avoid large ventilation that could cause significant fluctuations in temperature and humidity, and do not spray water directly onto the primordia. If the temperature drops below 0℃, cover with straw or plastic film. After the young mushrooms have formed to a height of 1.5-3.0cm, maintain air humidity and reduce soil moisture to 20-25%. Appropriately increase the temperature inside the shed, but not exceeding 20℃, to accelerate the growth and development of the young mushrooms. In the later stage of rapid growth of young mushrooms, maintain the ground temperature at 12-16℃, relative humidity at 80-90%, and increase soil moisture to 25-28%, accelerating the air circulation inside the shed to promote the rapid growth and development of morel mushrooms. During the ascocarp maturation stage, reduce air humidity to 70-85%, decrease soil moisture, and accelerate air circulation.
[0115] Morel mushrooms are mature when the asci stop swelling, the cap ridges and pits are clearly defined, and the flesh is thick and elastic. Mature morel asci should be picked promptly; otherwise, overripe morels will have thinner flesh, spores will be released rapidly, the mushroom will collapse, the aroma will be reduced, and the market quality will be severely compromised. When picking, clean your hands and use a sharp knife to cut the stem horizontally near the ground to avoid contaminating the mushroom. After collecting morels, promptly remove any remaining stem bases from the soil. This not only promotes the growth of new morel primordia in the surrounding soil but also prevents pests from breeding at the stem base.
[0116] Figure 2 Photos showing the fruiting stage of morel mushroom MZ-12 after 30 days of cultivation; comparison photos of morel mushrooms MZ-12 and the original morel mushrooms, both cultivated for 40 days, after harvesting and drying. Figure 3 As shown in Table 2, the first and second from the left are the original morel mushrooms, and the one on the right is morel mushroom MZ-12. Morel mushrooms MZ-12 and the original morel mushrooms were harvested after maturity. Statistical analyses were performed on the fruiting rates and average yield per acre for both species. The results are shown in Table 2.
[0117] Table 2. Fruiting results of morel mushroom MZ-12 and original morel mushroom cultivation
[0118]
[0119] Depend on Figure 2 , Figure 3As shown in Table 2, the fruiting performance, yield, and overall evaluation of Morel MZ-12 all meet the screening requirements. Compared with the original Morel, the average fruiting time of Morel MZ-12 is significantly shortened, allowing the fruiting bodies more time to grow and be harvested. Therefore, within the same cultivation cycle, Morel MZ-12 achieves a higher yield, with larger and thicker caps and a more robust and elastic texture, showing a clear advantage over the original Morel. Morel MZ-12 is genetically stable, and its fruiting yield and quality do not degrade under long-term cultivation.
[0120] (4) Aroma component analysis experiment:
[0121] The quality of morel mushrooms is determined by their taste, appearance, aroma, texture, and color. The taste of food comes from soluble, non-volatile compounds, while the aroma comes from volatile compounds such as aldehydes, alcohols, esters, ketones, acids, sulfides, pyrazines, and terpenes. Many of these are C8 compounds, which are ubiquitous in mushrooms and give them their characteristic aroma. By using an electronic nose to mimic human olfaction, the aroma of morel mushrooms can be identified, allowing for the analysis of the overall flavor of the sample. GC-MS (gas chromatography-mass spectrometry) can detect the types and amounts of volatile components in the sample, providing a more complete description of the aroma of morel mushrooms.
[0122] (i) Select 500g each of morel mushroom MZ-12 and original morel mushroom, crush them separately and mix them thoroughly, and make three samples of each group of morel mushrooms;
[0123] (ii) Extraction of volatile substances from morel mushrooms based on HS-SPME method: 500 mg of crushed morel mushroom samples were placed in 30 mL headspace vials, heated in a constant temperature water bath at 60 °C for 10 min, adsorbed with an aged extraction head for 20 min, and then injected for detection. Desorption was performed for 3 min.
[0124] (iii) Electronic nose detection: Sample preparation time 5s, data acquisition time 120s, sensor rinsing time 120s, zeroing time 10s, internal flow rate 400mL / min, sample injection flow rate 400mL / min;
[0125] (iv) GC-MS detection: Chromatographic conditions: Non-polar capillary column SH-Rxi-5Sil MS (30m×0.25mm×0.25μm); carrier gas He, flow rate 1.78mL / min, injection port temperature 270℃, splitless injection; column oven temperature 40℃, hold for 3 min, increase to 160℃ at a rate of 6℃ / min, hold for 2 min, then increase to 280℃ at a rate of 10℃ / min, hold for 3 min; Mass spectrometry conditions: Ionization mode EI; ion source temperature 230℃; interface temperature 240℃; electron energy 70eV; scan mass range 35~500u;
[0126] (v) Data analysis: The electronic nose detection data and relative content data of volatile components between the two samples, morel MZ-12 and original morel, are shown in Table 3.
[0127] Table 3. Analysis of Odor and Volatile Component Content
[0128]
[0129] As shown in Table 3, the content of volatile components in morel mushroom MZ-12 is generally higher than that in the original morel mushroom. Furthermore, the volatile substances α-terpineol and 2-undecone contained in morel mushroom MZ-12 can bring it a unique aroma; thus, the aroma of morel mushroom MZ-12 is more intense.
[0130] In summary, the MZ-12 strain of morel mushrooms has a significantly shorter fruiting time and higher yield compared to the original morel mushrooms. It also has a stronger resistance to Trichoderma. Compared to existing known morel mushroom varieties, it has a larger and thicker cap, a more substantial and elastic flesh, and a richer aroma, making it more marketable.
Claims
1. A morel mushroom ( Morchella esculenta MZ-12 was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40394.
2. The method for preparing the mother culture of Morel MZ-12 according to claim 1, characterized in that, Specifically, the steps include the following: S1. Prepare solid culture medium, fill test tubes, sterilize, cool, and then arrange on slant plates; S2. Under aseptic conditions, morel mushroom MZ-12 is inoculated onto the slant of a test tube and placed in a constant temperature incubator for cultivation until the mycelium has fully grown in the test tube, which is the mother culture of morel mushroom MZ-12. S3. Place the test tubes covered with mycelium at a temperature of -12 to 0°C for cryopreservation.
3. The preparation method according to claim 2, characterized in that, The sterilization conditions in S1 are sterilization at 121°C for 30 minutes.
4. The preparation method according to claim 2, characterized in that, The culture temperature in S2 is 10~18℃.
5. A method for preparing morel mushroom MZ-12 primary strain, characterized in that, Specifically, the steps include the following: (1) Mix all components in the microbial culture medium evenly; (2) The culture medium is put into a culture bottle or culture bag, sterilized, and inoculated in a sterile environment with the morel MZ-12 mother culture prepared by the preparation method described in claim 2; (3) Cultivate at 12~18℃ until fully grown, which is the original morel strain MZ-12.
6. The preparation method according to claim 5, characterized in that, The microbial culture medium in step (1) is one of the following formulations, and the components of the microbial culture medium are as follows by weight percentage: Formula 1: 70-72% wheat, 19-21% sawdust, 4-6% wheat bran, 2-4% cornmeal, 1-2% lime; Formula 2: Wheat 60-62%, corn cob 29-31%, wheat bran 4-6%, cornmeal 2-4%, lime 1-2%; Formula 3: Wheat 60-62%, cottonseed hulls 34-36%, cornmeal 2-4%, lime 1-2%; Formula 4: Wheat 79-81%, rice bran 14-16%, cornmeal 3-5%, gypsum 1-2%; Formula 5: Rice bran 48-50%, wheat 39-41%, soybean flour 9-11%, gypsum 1-2%; Formula 6: Rice bran 59-61%, wheat 34-36%, soybean flour 3-5%, gypsum 1-2%; Formula 7: 77-79% sawdust, 19-21% wheat bran, 1-2% sucrose, 1-2% gypsum.
7. The preparation method according to claim 6, characterized in that, In step (1), the wheat in the microbial culture medium is soaked in advance.
8. The preparation method according to claim 5, characterized in that, The sterilization conditions in step (2) are sterilization at 121°C for 30 minutes.
9. A method for cultivating morel mushroom MZ-12, characterized in that, Specifically, the steps include the following: (i) The morel mushroom MZ-12 original strain prepared by the preparation method described in claim 5 is further expanded to obtain morel mushroom MZ-12 spawn. The amount of spawn used per mu is 150-200 kg. After crushing the morel mushroom MZ-12 spawn, it is evenly spread on the soil and then immediately covered with 2-4 cm of soil. (ii) Covering the soil with mulch; (iii) Inoculation and induction of fruiting; (iv) Formation of fruiting body primordia, which grow into young mushrooms under suitable conditions.
10. The cultivation method according to claim 9, characterized in that, During the inoculation process, step (i) requires that the inoculum be protected from direct sunlight.
11. The cultivation method according to claim 9, characterized in that, In step (iii), mushroom cultivation is achieved by increasing the soil temperature or watering.
12. The cultivation method according to claim 9, characterized in that, In step (iv), the primordia growth temperature is 5~12℃, the growth humidity is 85~95%, and the young mushroom growth temperature is 8~20℃.