Morchella esculenta breeding method, obtained Morchella esculenta strain and application

Abstract

The invention aims to provide a morchella esculenta breeding method, an obtained morchella esculenta strain and application, the morchella esculenta is named as Morchella sextella NYD1, the taxonomic name of the morchella esculenta is Morchella sextella NYD1, the morchella esculenta is preserved in Guangdong Microbial Culture Collection Center, the preservation number of the morchella esculenta is GDMCC No: 66082, and the preservation date of the morchella esculenta is April 21, 2025. The morchella strain is further utilized as a mother strain to carry out domestication and cultivation, the morchella with the fruiting yield up to 400 kg / mu or above, excellent agronomic characters and rich nutritional ingredients such as crude protein, total sugar, iron, zinc and calcium is finally obtained, the content of the crude protein is not lower than 23 g / 100 g, the content of the total sugar is not lower than 55 g / 100 g, the content of the iron is not lower than 240 mg / kg, the content of the zinc is not lower than 70 mg / kg, and the content of the calcium is not lower than 76 mg / kg.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus cultivation, and in particular to a Morchella breeding method and an obtained Morchella strain and application thereof. Background Art

[0002] Morels are delicious, unique in flavor, crisp and tender in texture, and rich in nutrients. They are rich in protein, crude fiber, amino acids, unsaturated fatty acids, and mineral elements. They have high nutritional, health, and medicinal value. As an edible and medicinal fungus, they are deeply loved by consumers. In recent years, with the emergence and continuous improvement of foreign-aided nutrient bag technology, the selection and production of high-quality fungus strains, and the continuous integration and innovative development of cultivation and management technologies, the industrial cultivation area of morels in my country has increased rapidly. In 2022, the cultivation area of morels in my country reached 23,000 hectares. 2 , mainly distributed in Sichuan, Guizhou, Henan, Hebei, Yunnan, Gansu and other provinces.

[0003] The Liupanshan region (including Jingyuan and Longde counties in Ningxia) has an average annual temperature of 5–8°C, with a winter low of -15°C and an average summer temperature of 18°C. Annual precipitation is 600–800 mm, and humidity is 70–85%, meeting the cool-weather requirements of morels. The soil is primarily yellow-brown earth with a pH of 6.5–7.2 and an organic matter content of ≥3%. This soil is rich in selenium, which facilitates nutrient absorption by the mycelium and the development of flavor in the fruiting bodies. The high altitude offers low pollution and pest and disease incidence, making it suitable for cultivating organic fungus strains. This advantageous natural geographical environment makes the region an ideal location for the cultivation of morels. In 2020, the Liupanshan region began experimenting with the introduction and cultivation of morels, with some success. However, due to the varying sources of fungus strains used in cultivation in the Liupanshan region, the quality of the produced morels varies greatly, and yields are unstable, hindering the healthy and sustainable development of the local morel industry. Summary of the Invention

[0004] In order to obtain high-quality Morchella strains and explore domestication methods that can improve the cultivation characteristics of the strains, the present invention optimizes the breeding method of Morchella and screens out high-quality and high-yield Morchella strains. The Morchella strains are further used as mother strains for domestication and cultivation, ultimately obtaining Morchella with high fruiting rate, excellent agronomic traits, and rich nutritional components, providing a theoretical basis and technical support for the stable and high yield of local Morchella. The technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of Morchella edulis, which is named Morchella sextelata NYD1, whose taxonomic name is Morchella sextelata , deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit number is GDMCC NO: 66082, and the deposit date is April 21, 2025.

[0005] In a second aspect, the present invention provides a method for breeding Morchella edulis, comprising the following steps: Step 1: using the fruiting body of Morchella liumei as the mother strain, separating tissue from the fruiting body by a tissue separation method, and performing tissue culture to obtain a purified strain; Step 2: Perform mating type gene detection on the purified strain to screen out an effective cultivation strain containing both MAT1-1 and MAT1-2 mating type genes; the effective cultivation strain includes the Morchella according to claim 1.

[0006] Preferably, in step 1, the tissue is separated from the fruiting body by a tissue separation method, comprising: after surface disinfection of the fruiting body of Morchella liumei, cutting out a tissue block at the junction of the cap and the stipe; and / or, the tissue culture comprises: Step 1) Inoculate the tissue block onto PDA medium and culture at a constant temperature of 25±1°C until mycelium grows; Step 2), picking uncontaminated mycelium for purification and culture; Further preferably, the Liumei Morchella comes from Liupan Mountain Liumei Morchella.

[0007] Further preferably, the preparation method of the PDA culture medium comprises: Step 1-1), peel the potatoes and cut them into pieces or slices, boil them in water, separate the solid and liquid, and retain the liquid; In step 1-2, the liquid obtained in step 1-1) is heated to boiling, agar is added, and the mixture is mixed until the agar is completely melted. After cooling to 50-60° C., glucose is added and dissolved to obtain a mixed solution; Step 1-3), sterilizing the mixed solution obtained in step 1-2) to obtain the PDA culture medium; More preferably, the raw materials of the PDA culture medium are composed of 20 parts of potatoes, 2 parts of glucose, 1.8-2 parts of agar, 100 parts of water, and natural pH.

[0008] In a third aspect, the present invention provides the use of the wild Morchella strain NYD1 described in the first aspect in improving the cultivation characteristics of Morchella.

[0009] Preferably, the cultivation traits include at least morel yield, agronomic traits and nutrient content, wherein the agronomic traits include but are not limited to cap length, cap diameter, cap color, stipe color, and fruiting body shape; and the nutrient content includes but is not limited to water, crude protein, total sugar, iron, zinc, and calcium.

[0010] In a fourth aspect, the present invention provides the domestication and cultivation method of Morchella according to the first aspect, characterized in that the domestication and cultivation method comprises the following steps: Step i, carrying out multiplication and cultivation of the Morchella; Step ii, preparing the stock of the expanded cultured strain and inoculating it; Step iii, further preparing the cultivar from the original seed and inoculating it; then covering with soil and film, placing a nutrient bag after the mycelium has fully grown on the mushroom bed, and controlling the temperature, humidity and light conditions during the spawning and fruiting periods for cultivation.

[0011] Preferably, in the step ii, the expanded cultured strain is prepared as a stock, comprising: first boiling the wheat until there is no white core after breaking it apart and then mixing it with other raw materials with water according to the stock culture material formula: 80-90% wheat, 5-18% corn cobs, 1-2% quicklime, and 1-2% gypsum, and controlling the moisture content at 75%-80%; after sterilizing the culture solution, the expanded cultured strain is aseptically inoculated and cultured at a constant temperature of 20±1°C in the dark.

[0012] Preferably, in step iii, the original seed is further prepared into a cultivar, comprising: first boiling the wheat until the wheat is broken open and no white core is left, and then mixing it with the other raw materials with water, with the moisture content controlled at 50% to 60%, according to the cultivar culture medium formula: 55-65% wheat, 25-30% rice husk, 7-12% corn cob, 1-2% quicklime, and 1-2% gypsum; sterilizing the culture medium, inoculating the original seed, and culturing at a constant temperature of 16-18°C in the dark, and waiting for the sclerotia to grow fully, thereby obtaining the cultivar.

[0013] Further preferably, the formula of the nutrient bag is: 68-72% wheat, 16-20% rice husk, 8-12% corn cob, 1-2% quicklime, and 1-2% gypsum.

[0014] Preferably, in step iii, the culture is carried out in a dark and ventilated environment during the spawning period, the temperature is controlled at 10-18°C, the relative humidity is 80%-90%, and the carbon dioxide concentration is controlled below 3000 ppm; and / or, During the fruiting period, light ventilation culture is adopted, the temperature is controlled at 12~20℃, the relative humidity of the air is 80%~90%, and the light intensity is 2000~3000lx.

[0015] Compared with the prior art, the technical effects of the present invention are: The present invention conducts research on a breeding method for wild morels, and can obtain strains with both MAT1-1 and MAT1-2 mating type genes. Finally, a morel strain NYD1 is screened out, and domestication cultivation is carried out using the morel strain as a mother strain. Finally, morels with a mushroom yield of more than 400 kg / mu, excellent agronomic traits, and rich in nutrients such as crude protein, total sugar, iron, zinc, and calcium are obtained. Among them, the crude protein content is not less than 23 g / 100 g, the total sugar content is not less than 55 g / 100 g, the iron content is not less than 240 mg / kg, the zinc content is not less than 70 mg / kg, and the calcium content is not less than 76 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a physical picture of the Morchella fruiting body collected from Liupan Mountain in Example 1 of the present invention.

[0017] Figure 2 This is a phylogenetic tree constructed using the maximum likelihood (ML) method based on the ribosomal gene internal transcribed spacer sequence (nDNA-ITS) of wild Morchella in Example 1 of the present invention.

[0018] Figure 3 This is the colony and mycelial growth process of specimen 2956 in Example 1 of the present invention, where B1, B2, B3, B4, B5, B6, B7, B8, B9, and B10 are the colony morphologies of Morchella nyd1 on the 1st, 3rd, 6th, 9th, 12th, 15th, 18th, 21st, 25th, and 28th day of culture, respectively; B11, B12, B13, and B14 are the mycelial morphologies on the 3rd, 6th, 9th, and 12th day of culture, respectively. DETAILED DESCRIPTION

[0019] A strain of Morchella siliqua obtained in the embodiment of the present invention ( Morchella sextelata NYD1), deposited in Guangdong Provincial Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; deposit date: April 21, 2025; deposit number: GDMCC NO: 66082; taxonomic name: Morchella sextelata .

[0020] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1:

[0023] This embodiment carries out the research of wild Morchella breeding method, specifically as follows: 1. Identification and screening of wild morels A total of 7 wild Morchella specimens were collected in the Liupan Mountain area (Jingyuan County and Longde County). The specific habitat information of the specimens is shown in Table 1. Each collected specimen was numbered, placed in a specimen bag, and brought back to the laboratory for identification and isolation on the same day.

[0024] Table 1 Detailed information of Morchella specimens

[0025] (1) Morphological description The surface characteristics of fresh Morchella were observed using the classic morphological identification method, including the shape, size, color and stipe characteristics of the cap. The specific morphological characteristics of the specimens are as follows (according to Figure 1 Summarize): Specimens 1493 and 1494: The fruiting body is 10-12.5 cm long, with a conical cap with a sharp or slightly blunt tip, a cap diameter of 6.5 cm, brown in color, and a reticular surface with irregularly distributed grooves; the flesh is light yellow with a fragrant smell; the stipe is irregular, about 6.5 cm long, swollen at the base, 5 cm wide, light yellow, with a rough texture, no hairs on the surface, naturally connected to the cap, and a hollow structure inside.

[0026] Specimen 2956: The fruiting body is 9 cm long, with a conical cap with a sharp tip and a diameter of 5-6.3 cm. The color ranges from brown to gray to gray-brown, with grooves and longitudinal ridges in a regular distribution. The flesh is white to light yellow with a fragrant smell. The stipe is cylindrical with a slightly swollen base, 2-2.3 cm wide and 2-2.7 cm long, light yellow, with a rough texture, no hairs on the surface, and a natural connection with the cap. The interior is a hollow structure.

[0027] Specimen 2959: The fruiting body is 8-10.5 cm long, with a conical cap with a sharp tip and a diameter of 6-7.5 cm. The color ranges from brown to gray to gray-brown, with grooves and longitudinal ridges in a regular distribution. The flesh is white to light yellow with a fragrant smell. The stipe is cylindrical, 3-3.5 cm long, slightly swollen at the base, 2-2.5 cm wide, light yellow, with a rough texture, no hairs on the surface, and a natural connection with the cap. The interior is a hollow structure.

[0028] Specimens 2964, 2965, and 2966: The average total length is approximately 9-11 cm. The fruiting body is approximately oval in shape, with a cap diameter of 6.5 cm and a yellow-brown color. The surface has typical reticular grooves, forming a honeycomb structure. These grooves are arranged in an irregular manner, with uniform density and no sterile folds. The flesh is light yellow with a fragrant smell. The stipe is cylindrical, 4-4.5 cm long, slightly swollen at the base, about 2.5-3 cm wide, white to light yellow, with a rough texture, no hairs on the surface, and a natural connection with the cap. The interior is a hollow structure, and the stipe and cap have no special attachments, which conforms to the common morphological characteristics of morels.

[0029] (2) Molecular biological identification results 100 mg of fresh Morchella fruiting bodies were ground into powder using liquid nitrogen. DNA was extracted using a fungal DNA extraction kit (UN1Q-10 Column-based Fungal Genomic DNA Extraction Kit, Shanghai Biotechnology Co., Ltd.). PCR amplification of the ITS fragment with the highest resolution among fungal species was performed using universal primers ITS1F (5′-TCCGTAGGTGAACCTGCGG-3′, SEQ ID NO. 1) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′, SEQ ID NO. 2). The PCR reaction system (50 μL) consisted of 2 μL of genomic DNA, 1.5 μL of each primer (ITS1F and ITS4) at 10 mmol / L, 25 μL of 2× Hief PCR Master Mix, and ddH2O to 50 μL. The PCR amplification protocol was as follows: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 5 min, annealing at 52°C for 1 min, and extension at 72°C for 1 min; and extension at 72°C for 10 min. PCR products were detected by 1% agarose gel electrophoresis, and qualified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Amplified sequences were assembled and quality-controlled using Contig Express (V3.0.0) software and submitted to the NCBI (http: / / www.ncbi.nlm.nih.gov / ) and UNITE (https: / / unite.ut.ee) databases for BLAST sequence alignment analysis. Homologous sequences and pattern sequences with OTU similarity exceeding 97% were downloaded and aligned using MAFFT (https: / / mafft.cbrc.jp / alignment / software) software for manual correction. A phylogenetic tree was constructed using RAxML (Windows executables V8.2.10) software using the maximum likelihood (ML) method based on multi-gene sequence analysis to determine the taxonomic status of wild Morchella.

[0030] The phylogenetic tree constructed based on nDNA-ITS sequences using the maximum likelihood (ML) method ( Figure 2 ), Morchella specimens 1493 and 1494 clustered in the same branch and were the same as Morchella serrata in the database. Morchella importuna Gather together; specimen 2956 and the six-sister morel in the database Morchella sextelata Clustered in the same branch; specimen 2959 and the seventh sister morel in the database Morchella septimelata Clustered in the same branch; specimens 2964, 2965, and 2966 clustered together and were identical to the Morchella stoloniferum in the database. Morchella crassipes Gathered in the same branch.

[0031] The above analysis of the morphological characteristics and taxonomic status of wild morels found in the Liupanshan area reveals that, based on macroscopic and microscopic morphological characteristics, combined with molecular phylogenetic analysis data, the morel specimens collected from the Liupanshan area were identified as Morchella esculenta, Morchella esculenta (sixth sister), Morchella esculenta (seventh sister), and Morchella esculenta (thick leg). This indicates that the local Morchella resources are rich in variety and that the Liupanshan area provides relatively ideal environmental conditions for the growth and development of these morels. Of the four Morchella species found in the Liupanshan area, Morchella esculenta (thick leg) is a yellow morel strain, while Morchella esculenta, Morchella esculenta (sixth sister), and Morchella esculenta (seventh sister) are black morel strains. Since there have been no substantial breakthroughs in the domestication and cultivation of yellow morels in China, this paper will not further investigate the domestication and cultivation of Morchella esculenta. Although Morchella esculenta is a black strain and has been cultivated artificially, it has been gradually phased out of the Chinese morel cultivation market in recent years due to its low yield and lack of commercial advantages. Because the sixth-sister morel is more resistant to low temperatures than the seventh-sister morel, in view of the cold climate characteristics of Liupan Mountain, the present invention mainly conducts strain isolation and cultivation and domestication research on the sixth-sister morel collected in Liupan Mountain.

[0032] 2. Wild Morel Breeding This example adopts a systematic breeding method to breed the above-screened Morchella 6956, specifically as follows: (1) Tissue separation and purification culture of Morchella mother culture (1.1) Preparation of potato dextrose agar (PDA) medium Recipe: 200g potatoes, 20g glucose, 18g agar, 1000ml tap water, natural pH.

[0033] Preparation: Scrape the rough skin of the potatoes, remove the eyes, and cut into chunks or thin slices. Weigh the potatoes and place them in a pot. Add 1200-1300ml of water and boil until tender. Filter through double-layered gauze and collect 1000ml of the filtrate. Heat the filtrate until it is almost boiling, add agar, and stir continuously until the agar is completely dissolved. Be careful not to spill or burn the culture medium. After the culture medium cools to 50-60°C, add glucose and dissolve it (use hot water to fill the volume). While still hot, dispense the culture medium into test tubes using a funnel, filling approximately 1 / 4-1 / 3 of the tube's height. (This experiment used direct dispensing from a beaker). Be careful not to contaminate the tube or bottle openings with culture medium (wipe the inside of the tube opening with gauze). After plugging the tube openings with rubber stoppers, bundle seven tubes together and wrap the rubber stoppers with kraft paper. Autoclave at 121°C for 30 minutes.

[0034] (1.2) Tissue isolation and purification Take the fruiting bodies of the aforementioned Liumei Morchella 2956 wild Morchella mushrooms and wash them with sterile water to remove any surface residue. Excise the base of the stem and wipe it three times with 75% alcohol. Place it in an inoculation box and wipe it twice more with sterile water. Use a flame-sterilized scalpel or blade to make a longitudinal cut in the middle of the stem. Manually break the mushroom apart and use a razor blade to remove a mushroom fragment at the junction of the cap and stem. Place the fragment in a sterile Petri dish and use a razor blade to cut it into soybean-sized pieces. Under sterile conditions, select a small piece of tissue and quickly transfer it to a PDA slant culture medium, plugging it with cotton. Before removing the test tube from the inoculation box, wrap it tightly and affix a label on the top of the front with the strain number and inoculation date. Then, place the test tube in an incubator (room) and incubate at an appropriate temperature (25°C). Once the fragment grows mycelium and is free of contamination, it is considered a pure strain. A total of 32 pure strains were obtained in this experiment, including 25 samples of Morchella liumei.

[0035] (2) Determination of the production rate of Morchella strains Purified mycelia were inoculated onto PDA medium and cultured at 25°C. Colonies grew rapidly within the first 48 hours, reaching diameters of 3.5–6 cm. Most strains filled the plate within 96 hours. Mycelial morphology was observed and photographed on days 1, 3, 6, 9, 12, 15, 18, 21, 25, and 28 of culture. Figure 3 Figures B1, B2, B3, B4, B5, B6, B7, B8, B9, and B10 represent the colony morphology of Morchella siliquae NYD1 on days 1, 3, 6, 9, 12, 15, 18, 21, 25, and 28, respectively. With increasing culture time, the mycelial color of 2956 gradually darkened from white to brown. In the early stages of culture, there were no aerial hyphae, and the hyphae were sparse and had few branches. In the later stages, the aerial hyphae became dense and vigorous, with numerous branches and accompanied by the production of sclerotia. Microscopic examination of mycelium (10×4) at 3 days (B11), 6 days (B12), 9 days (B13), and 12 days (B14) of culture revealed that the mycelium had septa and a bamboo-like structure, which was narrowed at the nodes. On the 6th day, the mycelium began to fuse and connect with each other, forming bridge-like and network-like structures. On the 9th day, the mycelium condensed into a mass. On the 12th day, sclerotia were formed on the basis of the mycelial mass.

[0036] (3) Mating type gene testing Morchella is a heterothallic fungus, requiring strains containing both the MAT1-1 and MAT1-2 mating type genes to achieve sexual reproduction and form fertile fruiting bodies. Strains containing only a single mating type gene (e.g., only MAT1-1 or MAT1-2) are unable to self-fertilize and must be combined with a strain of the complementary mating type to reproduce. In production practice, production failures are common due to the presence of only one mating type gene in the parent strain.

[0037] To ensure that the selected strains contained both mating type genes, the mating type gene detection primers MAT1-1 and MAT1-2 were used in this example. 100 mg of mycelium was scraped from the plates, ground into a powder using liquid nitrogen, and DNA was extracted using a fungal DNA extraction kit. Conventional PCR amplification was performed using the MAT1-1T and MAT1-2 primers. The MAT1-1 forward primer (P8-4F: 5'-TTACCTTACTGGACTGGTTC-3', SEQ ID NO. 3) and reverse primer (P8-4R: 5'-AATGCAAGTAGGTGTCATTC-3', SEQ ID NO. 4) were used, while the MAT1-2 forward primer (P6-1F: 5'-TCCTATGAATGCGTAAGTTC-3', SEQ ID NO. 5) and reverse primer (P6-1R: 5'-GTATTATCACCAACCGTAGC-3', SEQ ID NO. 6) were used. PCR reaction system (50 μL): 2 μL of genomic DNA, 1.5 μL of each of 10 mmol / L primers (P8-4F / P8-4R and P6-1F / P6-1R), 25 μL of 2×Hief PCR Master Mix, and ddH2O to 50 μL. PCR amplification procedure: 94°C pre-denaturation for 3 min; 94°C denaturation for 1 min, 50°C annealing for 30 s, 72°C extension for 1 min, for a total of 35 cycles; 72°C extension for 10 min. PCR products were detected by 1% agarose gel electrophoresis. A single band indicated that only one mating type was present, while a double band indicated that two mating types were present, indicating that the strain was a valid cultivated strain. One of the strains was submitted for deposit. Morchella sextelata NYD1. The NYD1 strain is currently deposited in the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is April 21, 2025; the deposit number is GDMCC No: 66082; the taxonomic name is: Morchella sextelata .

[0038] Example 2:

[0039] For the six-sister morel (number NYD1) obtained in Example 1, this example carried out domestication cultivation research and compared the cultivation characteristics of wild morel strains, as follows: 1. Control strain The mature Liumei varieties G8, Kunyang No. 1 and Qimei G10 were selected as comparison strains, among which Kunyang No. 1 was provided by the Culture Collection Center of Kunming Institute of Botany, and G8 and G10 were provided by Sichuan Jiarui Tiancheng Modern Agriculture Technology Co., Ltd.

[0040] 2. Culture medium and culture material formulation Mother culture medium: PDA medium (200 g peeled potatoes, 20 g glucose, 20 g agar, 1000 mL water).

[0041] Formula of original culture medium: 90% wheat, 8% corn cobs, 1% quicklime, and 1% gypsum.

[0042] Cultivated seed culture medium formula: wheat 60%, rice husk 28%, corn cob 10%, quicklime 1%, gypsum 1%.

[0043] Nutrition bag formula: wheat 70%, rice husk 18%, corn cob 10%, quicklime 1%, gypsum 1%.

[0044] 3. Experimental Design The experimental base is located at Huangda Biotechnology Co., Ltd. in Jingyuan County, Ningxia. The experimental cultivation and management period is from October 2023 to April 2024. The morel cultivation greenhouse is a steel frame arch shed with a height of 3.5 m. The top of the steel frame is covered with a sunshade net with a shading rate of 90%. The greenhouse is equipped with automatic spraying equipment. The experimental design is to treat each variety as one, for a total of four treatments, and conduct a large-scale comparative test without replication. The area of the large area is 100 m 2 .

[0045] 3.1 Cultivation methods Stock culture preparation: Weigh the dry weight of all ingredients strictly according to the recipe. Boil the wheat kernels until they are open and free of white core, then mix with the other ingredients. Add water and stir thoroughly, adjusting the moisture content to 75%-80%. Place the mixed culture medium into a 750mL culture bottle and sterilize at 120°C for 120 minutes. After cooling, inoculate a mother culture tissue approximately 1 cm in diameter and incubate at 20°C in the dark.

[0046] Preparation of cultivars: Select vigorous, infection-free stock and propagate it into cultivars. Weigh the dry weight of all ingredients according to the recipe. Boil the wheat kernels until they are free of white cores, then mix with the other ingredients. Adjust the moisture content to 50%-60%, stir evenly, and place into polypropylene bags (15 x 30 cm), approximately 750g per bag. Sterilize at 121°C for 120 minutes. Cool to room temperature, inoculate, and incubate at a constant temperature of 16-18°C in the dark until sclerotia are fully grown.

[0047] On October 7, 2023, the mother stock of morels was propagated, the stock seed inoculated on October 17, and the cultivar inoculated on November 10. Before sowing, the land was deep plowed and beds were prepared, with a width of 1.5 meters. Sowing took place on November 29, 2023, with a sowing rate of approximately 200 kg per mu of cultivar. After sowing, the soil was covered with film. Seven to ten days after sowing, once the mycelium had fully grown on the fungus bed, nutrient bags were placed. Nine bags were placed per square meter, with two or three rows per compartment, depending on the width of the fungus bed. The bags were spaced 0.45 meters apart, resulting in approximately 2,600 bags per mu. When placing the nutrient bags, make a 5 cm cut on the side of the bag and place them with the cut facing downward, allowing the base material inside the bag to contact the fungus bed.

[0048] During the incubation period, keep the soil in the bed moist, with a relative humidity of 80% to 90%. On February 25, 2024, spray the bed thoroughly with water, maintaining a relative humidity of 80% to 90%, to promote the formation of morel primordia. After water stimulation, the hyphae entangle and form a large number of primordia. At this time, when the relative humidity falls below 70%, mist spraying is implemented. The mist spraying time is generally between 8:00 AM and 11:00 AM, with the temperature controlled between 10°C and 18°C and the carbon dioxide concentration below 3000 ppm.

[0049] After the morels have fruited, the temperature should be controlled between 12°C and 20°C. Lighting should be primarily diffuse light, preferably between 2000 and 3000 lux. During harvest, the characteristics of the morels should be examined.

[0050] 3.2 Measurement items and methods For each treatment, 30 fruiting bodies were randomly selected from the first harvest. Cap color, stipe color, fruiting body shape, flesh quality, and pest and disease status were observed, and cap length and diameter were measured. A 500g sample was randomly selected from the first harvest of each variety, packaged, and sent to the Ningxia Tianhao Biological Testing Center on the same day for nutrient content determination. The moisture content is determined by referring to the direct drying method in (GB5009.3-2016 "National Food Safety Standard - Determination of Moisture in Foods"), the crude protein content is determined by referring to the Kjeldahl nitrogen method in (GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Foods"), the total sugar content is determined by referring to the phenol-sulfuric acid method in (GB / T 15672-2009), the iron content is determined by referring to the flame atomic absorption spectrometry method in (GB 5009.90-2016 "National Food Safety Standard - Determination of Iron in Foods"), and the zinc and calcium content are determined by referring to the flame atomic absorption spectrometry method in (GB5009.14-2017 "National Food Safety Standard - Determination of Zinc in Foods").

[0051] 4. Results and Analysis 4.1 Comparison of mushroom production Table 2 shows the fruiting status of four morel varieties. After sowing on November 29, 2023, watering was initiated on February 25, 2024, to promote fruiting. Primordia began forming in early March. Large-scale harvesting of Kunyang No. 1 began on March 28, and the remaining varieties were harvested on March 20. Fruiting varied significantly among varieties. G8 and NYD1 had the highest fruiting density and yield, followed by Kunyang No. 1, which had a denser fruiting density. G10 had less than ideal fruiting. No pests or diseases were observed for any of the varieties.

[0052] Table 2 Fruiting of different varieties

[0053] Note: “+” indicates sparse fruiting density, “++” indicates average fruiting density, “+++” indicates dense fruiting density, and “++++” indicates very dense fruiting density. Yields refer to fresh mushroom yields.

[0054] Subsequently, the same planting experiment was carried out on NYD1 alone, with 200 kg of cultivated seeds invested, and the yield of morels was above 400 kg per mu.

[0055] 4.2 Comparison of agronomic traits of different varieties Table 3 shows that the agronomic traits of the four fruiting morel varieties vary considerably. G8 had the shortest cap length, at 6.9 cm, significantly smaller than the other varieties; NYD1 had the smallest cap diameter, while 2959 had a significantly larger cap diameter than the other varieties. Regarding cap color, the three Liumei varieties were gray-brown, while the two Qimei varieties were brown or yellow-brown. The stipes were all white, with Jiangyou and Tiling having a creamy white to yellowish color. The remaining varieties were creamy white, with Kunyang No. 1 showing a yellowish tint. The fruiting bodies were conical in shape, with thick or medium-thick flesh.

[0056] Table 3 Comparison of agronomic traits of experimental varieties

[0057] Note: Data without the same lowercase letters in the same column indicate significant differences among different varieties (P < 0.05).

[0058] 4.3 Comparison of nutritional components of test varieties Table 4 shows a comparison of the nutritional composition of the fruiting bodies of different Morchella cultivars. The moisture content of fresh Morchella fruiting bodies ranged from 85.2% to 86%, with no significant differences among varieties. Crude protein content ranged from 23.5 to 23.9 g / 100 g, with no significant differences among varieties. Total sugar content ranged from 49.4 to 57.1 g / 100 g, with Kunyang No. 1 and NYD1 having significantly higher total sugar content than the other two varieties. Iron content ranged from 132 to 245 mg / kg, with NYD1 having the highest iron content and G8 having the lowest. Kunyang No. 1 and NYD1 had significantly higher iron content than the other two varieties. Zinc content ranged from 70.4 to 71.2 mg / kg, with NYD1 having significantly higher zinc content than the other four varieties. Calcium content ranged from 67 to 96 mg / kg, with G8 having the highest calcium content and G10 having the lowest. In a comprehensive comparison of different nutritional components, NYD1 performed best.

[0059] Table 4 Comparison of general nutritional components of different varieties

[0060] A comprehensive comparison of yield, agronomic traits, and nutritional content across different varieties revealed that NYD1 performed on par with the existing G8 variety, and both outperformed the other varieties. This suggests that NYD1 can be cultivated through domestication to produce superior Morchella varieties, demonstrating its potential for widespread adoption and demonstration.

[0061] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent of the present invention. It should be pointed out that, without departing from the concept of the present invention, a number of variations and improvements may be made by those skilled in the art, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims.

Claims

1. A Morchella edodes, characterized in that: The morel is named Morchella sextelata NYD1, whose taxonomic name is Morchella sextelata , deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit number is GDMCC NO: 66082, and the deposit date is April 21, 2025.

2. A method for breeding morels, characterized in that: The following steps are involved: Step 1: using the fruiting body of Morchella liumei as the mother strain, separating tissue from the fruiting body by a tissue separation method, and performing tissue culture to obtain a purified strain; Step 2: Perform mating type gene detection on the purified strain to screen out an effective cultivation strain containing both MAT1-1 and MAT1-2 mating type genes; the effective cultivation strain includes the Morchella according to claim 1.

3. The method for breeding morels according to claim 2, wherein In step 1, separating the tissue from the fruiting body by tissue separation includes: disinfecting the surface of the fruiting body of Morchella liumei and then cutting out a tissue block at the junction of the cap and the stipe; and / or, the tissue culture includes: Step 1) Inoculate the tissue block onto PDA medium and culture at a constant temperature of 25±1°C until mycelium grows; Step 2), picking uncontaminated mycelium for purification and culture; Preferably, the Liumei Morchella is derived from Liupan Mountain Liumei Morchella.

4. The method for breeding Morchella according to claim 3, wherein The preparation method of the PDA culture medium comprises: Step 1-1), peel the potatoes and cut them into pieces or slices, boil them in water, separate the solid and liquid, and retain the liquid; In step 1-2, the liquid obtained in step 1-1) is heated to boiling, agar is added, and the mixture is mixed until the agar is completely melted. After cooling to 50-60° C., glucose is added and dissolved to obtain a mixed solution; Step 1-3), sterilizing the mixed solution obtained in step 1-2) to obtain the PDA culture medium; Preferably, the raw materials of the PDA culture medium are composed of 20 parts of potatoes, 2 parts of glucose, 1.8-2 parts of agar, 100 parts of water, and natural pH.

5. Use of the wild Morchella strain NYD1 according to claim 1 in improving the cultivation characteristics of Morchella.

6. The domestication and cultivation method of Morchella according to claim 1, characterized in that: The domestication cultivation method comprises the following steps: Step i, carrying out multiplication and cultivation of the Morchella; Step ii, preparing the stock of the expanded cultured strain and inoculating it; Step iii, further preparing the cultivar from the original seed and inoculating it; then covering with soil and film, placing a nutrient bag after the mycelium has fully grown on the mushroom bed, and controlling the temperature, humidity and light conditions during the spawning and fruiting periods for cultivation.

7. The acclimation cultivation method according to claim 6, characterized in that: In the step ii, the expanded cultured strain is prepared into a stock, comprising: first boiling the wheat until the wheat is broken open and no white core is left, and then mixing it with the other raw materials with water, with the moisture content controlled at 75% to 80%; after sterilizing the culture solution, the expanded cultured strain is aseptically inoculated and cultured at a constant temperature of 20±1°C in the dark.

8. The acclimation cultivation method according to claim 6, characterized in that: In step iii, the original seed is further prepared into a cultivar, comprising: first boiling the wheat until the wheat is broken open and no white core is left, and then mixing it with the other raw materials with water, with the moisture content controlled at 50% to 60%, according to the cultivar culture medium formula: 55-65% wheat, 25-30% rice husk, 7-12% corn cob, 1-2% quicklime, and 1-2% gypsum; sterilizing the culture medium, inoculating the original seed, and culturing at a constant temperature of 16-18° C. in the dark, and waiting for the sclerotia to grow fully, thereby obtaining the cultivar.

9. The acclimation cultivation method according to claim 6, characterized in that: The formula of the nutrient bag is: 68-72% wheat, 16-20% rice husk, 8-12% corn cob, 1-2% quicklime, and 1-2% gypsum.

10. The acclimation cultivation method according to claim 6, characterized in that: In step iii, the culture is carried out in a dark and ventilated environment during the incubation period, the temperature is controlled at 10-18° C., the relative humidity is 80%-90%, and the carbon dioxide concentration is controlled below 3000 ppm; and / or, During the fruiting period, light ventilation culture is adopted, the temperature is controlled at 12~20℃, the relative humidity of the air is 80%~90%, and the light intensity is 2000~3000lx.

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