A pure strain of *Morchella asiatica* isolated from yellow lineage, its preparation method and application.

Abstract

This invention relates to the field of edible fungi spawn production technology, and more particularly to a yellow-lineage morel mushroom containing two mating type genes. Morchella sp. This invention discloses a pure strain of morel mushrooms, its preparation method, strain products containing the pure strain, and its use in the cultivation of *Morchella asiatica*. The genome of this pure strain contains both mating type genes MAT1-1-1 and MAT1-2-1. The advantage of this invention is that by placing multiple spores in a liquid culture medium for co-germination, the primary hyphae carrying different mating type genes will naturally fuse, forming visible antagonistic lines at the colony interface. Hyphae are picked from the antagonistic line region, and combined with PCR screening of the mating type genes, to efficiently obtain double-mating type strains. Furthermore, during the primary culture stage, covering the material surface with sterilized soil and controlling the temperature at 18-24℃ can induce the hyphae to expand in a directional, parallel manner from disordered twisting, and promote the differentiation of sclerotium-related structures at the casing soil-bottle wall interface, thereby significantly improving the quality of the primary culture.

Description

Technical Field

[0001] This invention relates to the field of edible fungi spawn production technology, and more particularly to a yellow-lineage morel mushroom containing two mating type genes. Morchella sp. Pure strains and their preparation methods, strain products containing the pure strains, and the use of the pure strains in the cultivation of yellow morel mushrooms. Background Technology

[0002] Morel mushrooms ( Morchella spp. Morels are a type of rare edible and medicinal fungus. Based on polygenic phylogenetic phylogenetic species identification (GCPSR), morels are classified into the yellow lineage (…). Esculenta clade ), Black Branch ( Elata clade ) and the reddening lineage ( Rufobrunnea clade my country has achieved the cultivation of black morel mushrooms (such as Morchella tessmannii). M. importuna Six Sister Morel Mushrooms M. sextelata While commercial cultivation of morels is possible, artificial cultivation of yellow morels remains a global challenge.

[0003] my country possesses extremely rich germplasm resources of yellow morel mushrooms; however, production practices have shown that their artificial cultivation suffers from serious instability. Regarding heterothallic mating (… heterothallic Using the same tissue isolation and propagation method for the yellow lineage of *Morchella esculenta* as for the black lineage significantly disrupts the integrity of the mating type genes in pure strains: the regenerated strains obtained from tissue isolation contain isotopic heterothallanes (…). idiomorph The proportion of genotypes was much lower than that of the black lineage control, causing most isolates to lose the binucleate / heterokine state necessary for heterothallic mating, thus failing to complete the formation of ascocarps in sexual life. In addition, the yeast and other associated microorganisms carried inside the fruiting bodies are difficult to remove through conventional surface disinfection, with a contamination rate of up to 100%, seriously hindering the efficient acquisition of pure mycelium.

[0004] Therefore, the tissue isolation method commonly used in the spawn production of black morel is not applicable to yellow morel, while the conventional multispore isolation method lacks effective technical means to screen double-crossing strains, and the original culture stage also lacks special culture conditions for the physiological characteristics of yellow morel. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pure strain of isolated yellow morel mushroom, its preparation method and application. The genome of this pure strain contains two mating type genes, MAT1-1-1 and MAT1-2-1, thereby solving the problems of high mating type gene deletion rate and serious contamination in tissue isolates, as well as disordered mycelial expansion and low sclerotium differentiation efficiency in the original culture.

[0006] This invention is achieved through the following technical solution: Firstly, it provides an isolated yellow morel mushroom (…). Morchella sp. A pure strain whose genome contains both mating type genes MAT1-1-1 and MAT1-2-1.

[0007] Furthermore, the yellow morel ( Morchella sp. It is a phylogenetic species of Mes-21.

[0008] Furthermore, when the pure strain was cultured on PDA medium in the dark at 24-26°C, the linear growth rate of its mycelium was 24-27 mm / d.

[0009] Furthermore, this pure strain possesses carbon and nitrogen source characteristics selected from at least one of the following:

[0010] (i) Using sucrose or soluble starch as the carbon source; (ii) Using beef extract or yeast extract as a nitrogen source; (iii) The carbon-to-nitrogen ratio is 30-40:1.

[0011] Furthermore, this pure strain exhibits growth capability on solid culture media with NaCl concentrations ≤150 mmol / L.

[0012] Secondly, a method for preparing the above-mentioned pure bacterial strain is provided, comprising the following steps: S1: Morel mushrooms of the yellow branch ( Morchella sp. Basidiospores were aseptically inoculated into liquid culture medium for germination culture to induce fusion between primary hyphae of different mating types. Hyphae were picked from the antagonistic line region formed after fusion and purified to obtain hybrid candidate strains. S2: Perform PCR typing of the hybrid candidate strains obtained in step S1 to screen out strains that simultaneously amplify double positive bands of MAT1-1-1 and MAT1-2-1, which are the pure strains according to any one of claims 1 to 5. S3: Aseptically inoculate the strains obtained from step S2 into a container containing the original culture medium, cover the surface of the material with a sterile soil layer, and culture in the dark at 18-24℃ until the mycelium fills the bottle to obtain the original culture.

[0013] Further, in step S1, the liquid culture medium is sterile water; and / or, the germination culture is carried out at 23-25°C in the dark for 6-7 days.

[0014] Further, in step S3, the covering thickness of the sterilized soil is 1-3 cm; and / or, the relative humidity of the culture is 50%-60%.

[0015] Thirdly, a method for cultivating yellow-lined morels is provided ( Morchella sp. The microbial product comprises the above-mentioned pure microbial strain, as well as the original culture medium and a sterilized soil layer covering the surface of the original culture medium.

[0016] Fourthly, the use of the aforementioned pure strains in the cultivation of yellow morel mushrooms is provided.

[0017] Beneficial effects This invention is the first to obtain a pure strain of *Morchella asiatica* containing two mating type genes in its genome, overcoming the high mating type deletion rate in tissue isolates. Casing culture shortens the mycelial full-body time by approximately 27%, increases the number of sclerotia by more than two times, and improves cultivation yield by approximately 198%. The process is simple, cost-effective, and suitable for large-scale production. Attached Figure Description

[0018] Figure 1 Effects of different temperature-controlled soil covering on mycelial growth and sclerotium formation in *Morchella esculenta* YF2 primary culture bottles (left side of the figure shows no soil covering, right side shows soil covering. A: 18℃, B: 20℃, C: 22℃, D: 24℃, E: 26℃, F: 28℃); Figure 2 YF2 mycelial growth after 10 days of culture (A in the figure: PDA medium, B: PDA medium diluted 5 times, C: PDA medium diluted 5 times + exogenous nutrient module). Figure 3 Photographs of YF2 mycelium at different temperatures (A: 0℃; B: 1℃; C: 2℃; D: 3℃; E: 4℃). Figure 4 Effects of different carbon sources on the mycelial growth of YF2-1 (A: control; B: fructose; C: galactose; D: glucose; E: arabinose; F: lactose; G: maltose; H: sucrose; I: soluble starch). Figure 5 Effects of different nitrogen sources on the mycelial growth of YF2-1 (A: Control; B: Urea; C: Sodium nitrate; D: Glycine; E: Phenylalanine; F: Peptone; G: Yeast extract; H: Beef extract). Figure 6 Effects of different NaCl concentrations on the mycelial growth of *Morchella esculenta* (A: 0 mmol / L; B: 20 ​​mmol / L; C: 50 mmol / L; D: 100 mmol / L; E: 150 mmol / L; F: 200 mmol / L; G: 300 mmol / L). Figure 7Effects of different NaCl concentrations on the mycelial growth of YF2 (A: 0 mmol / L; B: 20 ​​mmol / L; C: 50 mmol / L; D: 100 mmol / L; E: 150 mmol / L; F: 200 mmol / L; G: 300 mmol / L) Figure 8 Mycelial growth in cultivation bags with and without soil covering (A) after 30 days. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0021] Unless otherwise specified, the reagents and raw materials used in the embodiments and comparative examples of this invention are commercially available.

[0022] The pure strain YF2 of yellow morel mushroom involved in this invention has been deposited in the germplasm resource bank of Henan Edible Fungi Research Institute.

[0023] The MAT1-1-1 gene has the sequence shown in GenBank accession number MN513844; The MAT1-2-1 gene has the sequence shown in GenBank accession number MN513927.

[0024] Example 1: Isolation and Identification of Yellow Morel Strains 1.1 Material Collection In April 2025, ascocarps of *Morchella esculenta* var. *fuxing*, *fuxing*, and *fuxing* var. *fuxing* were collected from the artificial cultivation base of Luoyang Dahuangye Ecological Agriculture Co., Ltd. in Luoyang City, Henan Province. The selected ascocarps were morphologically intact and free from pests and diseases, and were used as test specimens.

[0025] 1.2. Morphological observation Macroscopic morphological records were made for fresh ascocarps: The ascocarps of all three strains were solitary or clustered, medium to small in size, with an overall height of 3.0 cm–12 cm; the caps were nearly round, oval, pagoda-shaped, or nearly columnar, 2.0 cm–9.0 cm high, and 2.0 cm–6.5 cm at their widest point. The cap surface had distinct longitudinal ridges, interlacing longitudinal and transverse ridges; the ridges were pale yellow to pale gray in color when mature; the pits were irregular in shape and size. The stipe was cylindrical, hollow, with a base that was not swollen, slightly swollen, or significantly swollen, white to pale yellow, with pale yellowish-brown or ochre-yellow ridges that did not darken in color upon maturity. These macroscopic morphological characteristics are consistent with those of the *Morchella spp.* lineage (…). Esculenta clade It is of the same species, but is clearly different from the black morel lineage.

[0026] Mature ascocarp cap tissue was manually sectioned, mounted with 15% glycerol, and its microstructure was observed under an inverted biological microscope: the asci were octosporangia, long club-shaped to nearly cylindrical, with a foot-like base, measuring (178.18–263.02) µm × (18.13–26.80) µm; the ascospores were elliptical, transparent and colorless, with a smooth surface, measuring (19.82–26.20) µm × (11.95–15.49) µm, aspect ratio 1.64±0.14; lateral filaments rod-shaped or nearly cylindrical, colorless and transparent, septate, size (61.96–103.26) µm × (8.75–12.46) µm; sterile ridges with rod-shaped setae, tapering to a thin rod shape at the top, size (65.52–115.42) µm × (13.65–31.64) µm; no color development in 3% KOH solution. Microscopic characteristics further confirmed that the three strains belonged to the *Morchella spp.* lineage.

[0027] 1.3. Tissue isolation and mycelial culture Under aseptic conditions, a 2mm × 2mm piece of cap tissue from the dried ascocarp was dissected with a scalpel and inoculated onto a slant of CYM solid medium (20g / L glucose, 2g / L peptone, 2g / L yeast extract, 0.5g / L MgSO4·7H2O, 0.46g / L KH2PO4, 1g / L K2HPO4, 20g / L agar, pH natural) and incubated at 25℃ in the dark. When the hyphae reached the other end of the slant, the tip hyphae were transferred to a new CYM slant and purified to obtain a pure mycelial culture. The isolates from Fuxing No. 2, Fuxing No. 3, and Fuxing No. 5 were named FX-2, FX-3, and FX-5, respectively, and stored at 4℃ for later use.

[0028] 1.4. Genomic DNA Extraction and Multigene Amplification Mycelia of each strain were collected, ground into powder with liquid nitrogen, and genomic DNA was extracted using the CTAB method. PCR amplification was performed using the specific primers shown in Table 1 (ITS: ITS1 / ITS4; EF1-α: EF1-α-F / EF1-α-R; RPB1: RPB1-F / RPB1-R; RPB2: RPB2Y-F / RPB2Y-R). After passing 1% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing.

[0029] Table 1. Primer sequences for gene amplification

[0030] 1.5. Sequence alignment and phylogenetic analysis After assembling and proofreading the sequenced data, the sequences were submitted to the NCBI GenBank database to obtain accession numbers (FX-2: ITS PP949374, EF1-α PP965574, RPB1 PP999011, RPB2 PP999008; FX-3: ITS PP952081, EF1-α PP999006, RPB1 PP999012, RPB2 PP999009; FX-5: ITS PP949386, EF1-α PP999007, RPB1 PP999013, RPB2 PP999010). Morel reference sequences with greater than 99% homology were downloaded from the NCBI database to identify *Morchella esculenta* (red morel). M. rufobrunnea The YAASMVR strain was identified as an outgroup. Using MEGA11 software, based on the combined sequences of the four genes ITS, EF1-α, RPB1, and RPB2 (total length 2930 bp), the neighbor-joining method was employed. Neighbor-Joining Construct a phylogenetic tree with a bootstrap value of 1000 repetitions.

[0031] Phylogenetic tree analysis showed that the sequences of the three tested strains (FX-2, FX-3, and FX-5) clustered with the reference sequences of 10 morel strains in the yellow lineage (…). Esculenta clade Within the major branch, and forming a minor branch with the reference specimen Mes-21FCNU1082, the node support rate was 100%. Combining the macroscopic and microscopic morphological characteristics of the ascocarps, and based on the four-gene GCPSR analysis, it was finally confirmed that FX-2, FX-3, and FX-5 all belong to the phylogenetic species of morel mushroom Mes-21 in the yellow lineage.

[0032] Example 2 Using the yellow morel strain Mes-21 of the Fuxing 2 (FX-2) lineage, which was identified and confirmed in Example 1, as the test strain, pure strains were prepared by tissue isolation method and multispore isolation method, respectively, and the obtained strains were subjected to mating type gene detection and ITS identification.

[0033] 2.1 Tissue Separation Method Take 70% mature Fuxing No. 2 ascocarps, remove surface impurities, wipe with 75% ethanol for disinfection, and under aseptic conditions, use a scalpel to cut out internal tissue blocks (approximately 2mm × 2mm) from the cap. Inoculate one tissue block onto a CYM solid medium slant, for a total of 50 tissue blocks. Incubate at 25℃ in the dark.

[0034] After 3-5 days of cultivation, all 50 tissue blocks showed yeast-like colony contamination, with a contamination rate of 100%. After morel mycelia grew from the edges of the tissue blocks, the tips of the mycelia were picked up with an inoculation needle and transferred to a fresh CYM slant. The purification was repeated three times to finally obtain a pure morel mycelial culture, which was numbered YF2.

[0035] Genomic DNA was extracted from strain YF2, and PCR detection was performed using specific primers for the mating type genes MAT1-1-1 and MAT1-2-1. The results showed that YF2 only amplified the MAT1-1-1 band, indicating a single mating type strain. To further analyze the double mating type acquisition rate using tissue isolation, 50 regenerated strains were obtained from repeated tissue isolation of ascocarps of *Morchella fuxingii* var. *fuxingii*. Mating type detection revealed that only one strain contained both mating type genes (2%), while the remaining 49 strains were single mating type strains (26 strains of type MAT1-1-1 and 23 strains of type MAT1-2-1). As a control, tissue isolation was performed on ascocarps of *Morchella fuxingii* var. * ...

[0036] The above results indicate that tissue isolates of the yellow lineage morel mushroom Mes-21 exhibit severe segregation of mating-type genes, with the rate of obtaining double-mating-type strains being significantly lower than that of the black lineage.

[0037] 2.2 Multispore Isolation Method Spore collection: Take mature Fuxing No. 2 ascocarps, place them cap-down on clean, dry red paper, and let them stand at 25℃ for 24 hours to collect the naturally ejected basidiospores.

[0038] Spore germination and fusion culture: Under aseptic conditions, scrape approximately 2 mm² of spores using an inoculation needle and inoculate them into a 250 mL Erlenmeyer flask containing 100 mL of sterile water. Shake to mix thoroughly to prepare a spore suspension. Transfer 1 mL of the spore suspension to a sterile petri dish (90 mm in diameter) and incubate at 23-25°C in the dark for 7 days. Observe daily during the culture period: On days 3-4, basidiospores begin to germinate and form primary hyphae; on days 6-7, primary hyphae of different mating types come into contact and fuse, forming a visible white raised antagonistic line area on the surface of the petri dish.

[0039] Antagonistic line region selection and purification: Hyphae were picked from each antagonistic line region using a sterile inoculation needle and transferred to fresh PDA plates for purification culture. A total of 45 hybridization fusion candidate strains were obtained.

[0040] Mating type gene identification: Genomic DNA was extracted from 45 candidate strains and PCR amplification was performed using MAT1-1-1 and MAT1-2-1 specific primers (annealing temperatures of 60℃ and 55℃, respectively, for 39 cycles). Electrophoresis results are shown in Table 2-1: Among the 45 candidate strains, 19 strains simultaneously amplified double-positive bands for MAT1-1-1 and MAT1-2-1 (accounting for 42.2%), of which 14 strains contained only MAT1-1-1 and 12 strains contained only MAT1-2-1. The 19 strains containing both mating type genes were designated as target pure strains and numbered YF2-1 to YF2-19.

[0041] ITS identification: Some double-mating strains (such as YF2-1, YF2-3, and YF2-5) were selected for ITS region amplification and sequencing. The sequences were confirmed to be Mes-21 phylogenetic species after BLAST alignment.

[0042] 2.3 Comparison of results between tissue isolation method and multispore isolation method The experimental results of the two methods are summarized in Table 2.

[0043] Table 2 Comparison of results between tissue isolation method and multispore isolation method

[0044] Control experiment: The same multispore isolation method was used to treat the black branch of morel (Chuan 6), and 33 candidate strains were obtained. The mating type test showed that all of them were double mating type and no contamination was found.

[0045] The above results confirm that for morel mushroom Mes-21, the tissue isolation method has the drawbacks of high contamination rate and extremely low yield of double-cross hybrid strains; while the multispore isolation method established in this invention can stably and efficiently obtain double-cross hybrid pure strains with a yield of over 42%, which is significantly better than the tissue isolation method.

[0046] 2.4 Validation of fruiting by a single mating strain Three strains of single-mating type (MAT1-1-1 and MAT1-2-1) and three strains of double-mating type (YF2-1, YF2-3 and YF2-5) were taken respectively and original seeds were prepared according to the method of Example 3 (covered with soil, 22℃), and fruiting test was carried out.

[0047] The results showed that none of the six single-mating strains formed ascocarps, and only a few strains produced sporadic primordia before ceasing development; while the three double-mating strains all produced fruiting normally, with an average yield of 1.85 kg / m². This proves that the double-mating strains obtained by the present invention through multispore isolation combined with mating type screening have complete fruiting ability.

[0048] Example 3 The pure strain of double mating type obtained by multispore isolation and mating type screening in Example 2.2 (represented by YF2-1) was used for original culture experiment.

[0049] 3.1 Preparation of original culture medium The original culture medium formula, by weight percentage, includes 55% wheat grains, 21.5% corn cobs, 21.5% sawdust, 1% gypsum powder, and 1% lime. Water is added to adjust the moisture content to about 60%, and the mixture is poured into 750mL incubator bottles and autoclaved at 121℃ for 2 hours.

[0050] 3.2 Soil Covering and Temperature Setting YF2-1 mycelium, inoculated aseptically into 5mm diameter mycelial blocks, was transferred to the surface of the substrate in the inoculum bottle. Two treatment groups were set up: Control group without soil covering: directly cultured after inoculation without adding any covering material.

[0051] Soil Covering Experiment Group: After inoculation, a layer of garden soil sterilized at 121℃ for 30 minutes was evenly covered on the surface of the material, with a covering thickness of 2cm.

[0052] Each treatment group was placed in one of six constant temperature incubators at 18℃, 20℃, 22℃, 24℃, 26℃, and 28℃, with relative humidity controlled at 55% and incubated in the dark. Each treatment was replicated at each temperature.

[0053] 3.3 Observation of hyphal expansion and sclerotium differentiation Observation record after 15 days of cultivation: (1) Hyphae expansion morphology (e.g.) Figure 1 (As shown) No soil covering treatment ( Figure 1 (Left A): At various temperatures, the mycelia in the bottle all exhibited obvious morphological heterogeneity—the mycelial bundles were twisted, the aerial mycelia were well-developed, the growth front was irregular, and the mycelia could not form an orderly expansion front.

[0054] Soil covering treatment ( Figure 1 (Right A): The hyphae form a relatively consistent advancing front inside the substrate, with significantly enhanced directionality, extending towards the bottom of the bottle in a "parallel" manner. The hyphae bundles are neatly arranged, and aerial hyphae are sparse.

[0055] (2) Location and number of sclerotia (e.g.) Figure 1 (as shown in B) No soil covering treatment ( Figure 1 (Left B): Sclerotium-related structures (sclerotium initiators formed by hyphal entanglement) mainly appear at the bottle mouth and in the bottle wall area without substrate attachment; sclerotia are extremely rare inside the substrate. The average number of sclerotia per bottle at various temperatures was 2.1 ± 1.0.

[0056] Soil covering treatment ( Figure 1 (B, right): After about 10 days of cultivation, the sclerotium differentiation hotspot shifted from the bottle mouth and concentrated at the interface between the casing soil and the bottle wall. At this interface, a large number of dense sclerotium initiators were visible, some of which had developed into mature sclerotia. The average number of sclerotia per bottle at various temperatures was 6.8 ± 1.5, which was more than twice that of the uncovered treatment.

[0057] (3) The effect of temperature on sclerotium differentiation (e.g.) Figure 1 (as shown in C) The sclerotium differentiation under different temperature covering treatments is shown in Table 3 below. Figure 1 C. Within the range of 18℃ to 22℃ ( Figure 1 (C, third from left) The sclerotium-related structures are relatively obvious, the number of sclerotia is large (6.8-7.5 per bottle), and the degree of differentiation is high (+++); when the temperature rises to 24℃ ( Figure 1 (C, right) The number of sclerotia decreased to 5.1 per bottle (++); at 26℃~28℃ ( Figure 1 (C second from right and far right) Although the mycelial growth rate is fast, sclerotium differentiation is significantly reduced, with only 1.2 sclerotia / bottle at 26℃ (+) and almost no sclerotium formation at 28℃ (0.3 sclerotia / bottle, –).

[0058] Table 3. Effects of soil covering culture at different temperatures on sclerotium differentiation (15 days of culture)

[0059] Note: +++ for large amounts, ++ for medium amounts, + for small amounts, – for almost none.

[0060] The results showed that sclerotium-related structures were more pronounced in the 18-24℃ range, with the highest number and most complete differentiation of sclerotia occurring at 18-22℃. While mycelial growth was rapid at 26-28℃, sclerotium differentiation significantly decreased, and almost no sclerotia formed at 28℃. Therefore, for the purpose of obtaining spawn with energy storage structures and high subsequent fruiting potential, the optimal spawn culture temperature should be 18-24℃, more preferably 20-22℃.

[0061] 3.4 Verification of the effect of soil-covered original seed cultivation The original spawn obtained from soil-covered cultivation at 22℃ (experimental group) and the original spawn obtained from uncovered cultivation (control group) were transferred to cultivation bags (with the same cultivation substrate formula as the original spawn) using conventional methods. After cultivation at 22℃ and 60% relative humidity for 30 days, a fruiting experiment was conducted. The fruiting experiment was carried out at the cultivation base of Luoyang Dahuangye Company, using conventional field cultivation management methods for black morel mushrooms. Each treatment consisted of 3 plots, each with an area of ​​5m², arranged in a randomized block design.

[0062] The results are shown in Table 4.

[0063] Table 4 Comparison of cultivation yields between soil-covered and uncovered spawn.

[0064] Compared with the control group without soil covering, the original seed treated with soil covering increased the cultivation yield by about 198% (P<0.01), advanced the fruiting time by about 6 days, and significantly improved the uniformity of fruiting.

[0065] Example 4 Using YF2-1 obtained in Example 2 as a representative (it was determined that its biological characteristics were not significantly different from those of the double-crossing strain YF2-35 obtained from tissue isolation under the same culture conditions, and the following data use the measurement results of YF2-35 as the representative of YF2-1), the temperature adaptability, optimal carbon source, optimal nitrogen source, optimal carbon-nitrogen ratio and salt tolerance of its mycelial growth were systematically measured to clarify the physiological and metabolic characteristics of this pure strain.

[0066] 4.1 Effect of temperature on mycelial growth YF2-1 mycelial blocks (5 mm in diameter) were inoculated into the center of plates using PDA medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L, natural pH). The plates were then incubated in constant temperature incubators at 0℃, 1℃, 2℃, 3℃, 4℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, and 35℃ in the dark, with three replicates for each temperature. A control group was also included, treated at 48℃ for 2 hours and then transferred to 24℃. After 7 days of incubation, colony diameter was measured, the linear mycelial growth rate (mm / d) was calculated, and mycelial color and growth were observed.

[0067] The results are shown in Table 4-1.

[0068] Table 4-1 Effects of different temperatures on the mycelial growth of YF2-1 (see table 4-1) Figure 3 )

[0069] Note: "–" indicates that the hyphae have not grown or formed colonies; "+" indicates the growth of the hyphae, and the more "+", the denser the hyphae.

[0070] The results showed that YF2-1 could still grow slowly at low temperatures of 1-4℃, but the hyphae were sparse; the rapid growth range was 20-28℃, with the highest hyphal growth rate at 24-26℃ (26.2 mm / d at 24℃ and 25.1 mm / d at 26℃), and the hyphae were white and dense (+++); at 30-35℃, the spores could only germinate but could not form stable and expanding colonies; after treatment at 48℃ for 2 hours, the germination ability was completely lost. Therefore, the optimal growth temperature for this pure strain is 24-26℃.

[0071] 4.2 Effects of carbon source on mycelial growth Using a carbon-source basal medium (2 g / L peptone, 3 g / L KH₂PO₄, 1.5 g / L MgSO₄, 18 g / L agar, natural pH) as a base, 20 g / L of fructose, galactose, glucose, arabinose, lactose, maltose, sucrose, or soluble starch were added as carbon sources, with a medium without added carbon sources serving as a control. YF2-1 mycelial blocks were inoculated and cultured at 24°C in the dark for 7 days, and the colony diameter was measured.

[0072] The results are shown in Table 4-2.

[0073] Table 4-2 Effects of different carbon sources on the mycelial growth of YF2-1 (see Table 4-2) Figure 4 )

[0074] The results showed that sucrose had the highest mycelial growth rate (21.25 mm / d), with dense (+++) hyphae and neat edges; soluble starch was the second highest (20.5 mm / d); glucose and fructose both grew at 18.75 mm / d. The control (no carbon source) had a mycelial growth rate of only 16.25 mm / d and sparse hyphae. Therefore, sucrose or soluble starch is the optimal carbon source for YF2-1.

[0075] 4.3 Effects of nitrogen source on mycelial growth Using a nitrogen-based basal medium (20 g / L glucose, 3 g / L KH₂PO₄, 1.5 g / L MgSO₄, 18 g / L agar, natural pH) as a base, urea, sodium nitrate, glycine, phenylalanine, peptone, yeast extract, or beef extract were added at 2 g / L as nitrogen sources, with a medium without added nitrogen sources serving as a control. YF2-1 mycelial blocks were inoculated and cultured at 24°C in the dark for 7 days, and the colony diameter was measured.

[0076] The results are shown in Table 4-3.

[0077] Table 4-3 Effects of different nitrogen sources on the mycelial growth of YF2-1 (see Table 4-3) Figure 5 )

[0078] The results showed that organic nitrogen sources (beef extract, yeast extract, and peptone) had significantly better growth-promoting effects than inorganic nitrogen sources (urea and sodium nitrate) and amino acid nitrogen sources (glycine and phenylalanine). Among these, the beef extract treatment resulted in the highest mycelial growth rate (20.25 mm / d), with dense (+++) hyphae and neat edges; yeast extract was the second best (18.5 mm / d). Therefore, the optimal nitrogen source for YF2-1 is either beef extract or yeast extract.

[0079] 4.4 Effect of carbon-nitrogen ratio on mycelial growth Using 20 g / L sucrose as the carbon source and beef extract as the nitrogen source, carbon-to-nitrogen (C / N) ratio gradients were set at 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, and 60:1. YF2-1 mycelial blocks were inoculated and cultured at 24℃ in the dark for 7 days, and colony diameters were measured.

[0080] The results are shown in Table 4-4.

[0081] Table 4-4 Effects of different carbon-nitrogen ratios on the mycelial growth of YF2-1

[0082] The results showed that the mycelial growth rate was highest (24 mm / d) at a carbon-to-nitrogen ratio of 35:1, with dense (+++) hyphae and neat edges. At a carbon-to-nitrogen ratio of 30:1, the growth rate was 23.6 mm / d, and the hyphal density was ++. When the carbon-to-nitrogen ratio was below 20:1 or above 40:1, the mycelial growth rate decreased, and the hyphal color became lighter or the density decreased. Therefore, the optimal carbon-to-nitrogen ratio for YF2-1 is 30:1 to 40:1, with 35:1 being the best.

[0083] 4.5 Effects of salt stress on mycelial growth NaCl was added to PDA medium at final concentrations of 0, 20, 50, 100, 150, 200, and 300 mmol / L. Mycelial blocks of YF2-1 and the black clado control strain *Morchella esculenta* (Chuan 6) were inoculated separately and cultured at 24°C in the dark for 10 days. The colony diameter was measured and the mycelial morphology was observed.

[0084] The results are shown in Tables 4-5 and 4-6.

[0085] Table 4-5 Effects of different NaCl concentrations on the mycelial growth of *Morchella esculenta* (Chuan 6) (see Table 4-5) Figure 6 )

[0086] Table 4-6 Effects of different NaCl concentrations on the mycelial growth of YF2-1 (see Table 4-6) Figure 7 )

[0087] The results showed that YF2-1 retained its growth capacity at NaCl concentrations not exceeding 150 mmol / L (growth rate of 4.75 mm / d at 150 mmol / L), while the black morel strain was severely inhibited at 100 mmol / L (4.1 mm / d) and completely stopped growing at 150 mmol / L and above. Therefore, the pure strain YF2-1 of yellow morel obtained in this invention exhibits significantly better salt tolerance than the black morel strain and can grow on media with NaCl concentrations ≤150 mmol / L.

[0088] Example 5 Using the double-cross pure strain YF2-1 obtained in Example 2 and the original strain obtained from the 22℃ soil covering culture in Example 3 as materials, further research was conducted on the propagation in cultivation bags and the characteristics of sclerotium formation to verify the applicability of the seed production method of the present invention in large-scale cultivation.

[0089] 5.1 Preparation of cultivation bags and verification of soil covering effect (see...) Figure 1 A and Figure 1 B) The cultivation substrate formula was the same as the original culture medium in Example 3 (55% wheat grains, 21.5% corn cobs, 21.5% sawdust, 1% gypsum powder, 1% lime, 60% moisture content), and it was filled into polypropylene cultivation bags (15cm × 30cm). The filling height was set at 15cm and 20cm respectively, with each bag containing approximately 1.0kg (wet weight). The original culture (YF2-1 original culture) obtained from the 22℃ soil covering culture in Example 3 was inoculated onto the surface of the cultivation bag material using conventional methods. Two treatment groups were set up: Control group without soil covering: directly cultured after inoculation without adding any covering material.

[0090] Soil Covering Experiment Group: After inoculation, a layer of garden soil sterilized at 121℃ for 30 minutes was covered on the surface of the material, with a covering thickness of 2cm.

[0091] A separate black lineage control was established: Morel 6-62 (tissue-isolated double-cross hybrid type) was inoculated using the same method, with treatments including both covered and uncovered soil. Each treatment contained 10 bags per filling height and was cultured in the dark at 25℃ and 60% relative humidity.

[0092] Mycelial expansion and sclerotium formation were observed after 20 and 30 days of cultivation. The results are as follows: (1) Mycelial expansion morphology (after 20 days of culture, see...) Figure 1 A) YF2-1 without soil covering (see...) Figure 1 (Left A): The mycelial bundles are twisted and grow in an irregular direction. The aerial mycelium is well-developed and the mycelial tip extends irregularly in a wavy pattern. A yellowish-brown mycelial layer appears on the surface of some bags.

[0093] YF2-1 backfill (see) Figure 1 (Right A): The hyphae spread uniformly and with significantly enhanced directionality within the substrate, advancing downwards in an orderly manner, with few aerial hyphae; a large number of dense white hyphae can be seen below the casing layer.

[0094] Sichuan 6-62 (Six Sister Morel): Regardless of whether it was covered with soil or not, the mycelium showed a normal downward gradual spread, and there was no obvious difference between treatments, indicating that the black branch is not sensitive to soil covering.

[0095] (2) Sclerotium differentiation location (after 20 days of culture, see Figure 1 B): A concentrated distribution of sclerotia initiators was also observed at the interface between the soil covering and the bottle wall of the cultivation bag (see [link]). Figure 1 (B right) In the uncovered soil treatment, sclerotia only appeared at the bag opening and in the bag wall area where no substrate was attached (see B right). Figure 1 (B left), consistent with the phenomenon in the original seed bottle.

[0096] (3) Mycelial aging and hardening phenomena (after 30 days of culture, see...) Figure 8 ) YF2-1 without soil covering (see...) Figure 8 B): The outer layer of mycelium in the cultivation bag turns a darker yellowish-brown and hardens. After peeling off the bag, the surface mycelium layer is dense, hard, and difficult to break apart (forming a "hard shell"); the internal mycelium is relatively sparse.

[0097] YF2-1 backfill (see) Figure 8 A): The mycelium is yellowish in color and relatively loose in texture, and it is not easy to form an obvious hard shell on the surface with the naked eye; the mycelium is evenly distributed throughout the substrate.

[0098] Chuan 6-62: No obvious hardening was observed under either treatment, and the mycelium color was normal.

[0099] (4) Effect of substrate loading height on mycelial colonization Cultivation bags with a substrate height of 15cm: Whether or not they are covered with soil, the mycelium can basically fill the entire bag in about 30 days, with even distribution.

[0100] In uncovered cultivation bags with a substrate height of 20cm: mycelium colonized only to a depth of approximately 15cm, with the bottom 5cm of substrate unoccupied by mycelium, resulting in a sour odor; the upper colonized area showed no obvious odor. In the soil-covered treatment group, the mycelial colonization depth increased slightly (to approximately 17cm) in the 20cm substrate height, but a slight sour odor still appeared in the lower part. Analysis of the causes of the sour odor: excessive substrate height led to decreased substrate porosity, restricting oxygen diffusion and creating a localized hypoxic microenvironment; bacterial / yeast fermentation and metabolism occurred in the lower substrate unoccupied by mycelium, producing organic acids. Furthermore, YF2 may release metabolic products in the upper colonized area, which migrate downwards with moisture, accumulating under hypoxic conditions and amplifying the acidic odor.

[0101] The above results indicate that YF2 also requires soil covering during the cultivation bag stage to maintain orderly mycelial expansion and prevent surface aging and hardening; the filling height should not exceed 15cm, i.e., adopting a "low temperature and low material" seed production strategy.

[0102] 5.2 Observation of sclerotia initiators on culture dishes To investigate the sclerotium differentiation characteristics of YF2 under plate culture conditions, the following three culture media were set up: Group A: Conventional PDA culture medium (potato 200g / L, glucose 20g / L, agar 20g / L); Group B: PDA medium diluted 5 times (reducing the concentration of all components of conventional PDA to 1 / 5); Group C: PDA medium diluted 5 times + exogenous nutrient module (pour 10 mL of PDA diluted 5 times into a 60 mm petri dish, and after solidification, cut out a 1 cm × 1 cm module and place it in the center of a 90 mm petri dish as an exogenous nutrient source).

[0103] YF2-1 mycelial blocks, 5 mm in diameter, were inoculated in the center of each plate and cultured at 24°C in the dark for 10 days. The mycelial morphology and sclerotium formation were then observed.

[0104] The results show: Group A (Regular PDA, see below) Figure 2 A): The hyphae are white, with well-developed aerial hyphae, and no typical discrete sclerotia have formed. sclerotia However, a small number of dense, woolly-ball-like hyphal clusters appear at the colony edges and the ends of aerial hyphae, characterized by significant hyphal twisting and local aggregation, forming white to light yellow aerial hyphal clusters / hyphae aggregates. mycelialtufts / aggregates ).

[0105] Group B (PDA diluted 5 times, see...) Figure 2 B): The mycelial growth is relatively sparse, but the mycelial knotting phenomenon is more obvious, forming more mycelial aggregates, and the mycelial density increases in some areas.

[0106] Group C (PDA diluted 5 times + exogenous nutrient module, see...) Figure 2 C): A large number of hyphae aggregate around the exogenous nutrient module, with the most clusters formed by the twisting of hyphae. Some clusters have a pale yellow center and a relatively dense structure.

[0107] Based on macroscopic morphological observation, YF2 did not form the discrete, blocky mature sclerotia commonly seen in morel mushrooms under the above culture conditions, but instead tended to undergo hyphal nodulation. hyphalknotting ), forming sclerotium initiators ( sclerotialinitials This structure appears extensively at the "soil-bottle wall" interface in primary culture bottles with soil covering, and can further develop into mature sclerotia under suitable conditions. However, in primary culture bottles or cultivation bags without soil covering, the mycelium exhibits an overdeveloped aerial hyphae and disordered hyphal bundles, making it difficult to form effective sclerotia initiators. This phenomenon further explains the important role of soil covering in inducing orderly hyphal expansion and sclerotia differentiation in YF2.

[0108] 5.3 Verification of mushroom production in cultivation bags YF2-1 cultivation bags (15cm high, soil-covered) from section 5.1, which were cultured with soil for 30 days, and control bags (without soil covering) were transferred to a mushroom cultivation greenhouse for routine mushroom cultivation management (fruiting conditions: temperature 10-18℃, relative humidity 85-95%, diffused light stimulation). Each treatment was replicated in triplicate, with 10 bags per replicate, and the fruiting yield was recorded.

[0109] The results showed that: For cultivation bags with soil covering, primordia began to appear 50-55 days after inoculation, fruiting was uniform, and the ascocarps were of normal morphology (light yellow to light gray, consistent with the characteristics of the yellow lineage). The average yield of fresh mushrooms per bag (containing approximately 330g of dry substrate) was 185g, with a bioconversion rate of 55.5%, consistent with the yield in Example 3.4. For cultivation bags without soil covering, fruiting occurred sporadically 60-70 days after inoculation, with fewer primordia and some bags failing to produce fruiting. The average yield of fresh mushrooms per bag was only 62g, with a bioconversion rate of 18.6%. Furthermore, the ascocarps were smaller and the caps were darker in color.

[0110] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An isolated yellow morel ( Morchella sp. Pure strain, characterized in that, The genome of this pure strain contains both mating type genes MAT1-1-1 and MAT1-2-1.

2. The pure strain according to claim 1, characterized in that, The yellow morel ( Morchella sp. It is a phylogenetic species of Mes-21.

3. The pure strain according to claim 1 or 2, characterized in that, When this pure strain was cultured on PDA medium in the dark at 24-26℃, the linear growth rate of its mycelium was 24-27 mm / d.

4. The pure strain according to claim 1 or 2, characterized in that, This pure strain possesses carbon and nitrogen source characteristics selected from at least one of the following: (i) Using sucrose or soluble starch as the carbon source; (ii) Using beef extract or yeast extract as a nitrogen source; (iii) The carbon-to-nitrogen ratio is 30-40:

1.

5. The pure strain according to claim 1 or 2, characterized in that, This pure strain is capable of growth on solid culture media with NaCl concentration ≤150 mmol / L.

6. A method for preparing a pure bacterial strain according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Morel mushrooms of the yellow branch ( Morchella sp. Basidiospores were aseptically inoculated into liquid culture medium for germination culture to induce fusion between primary hyphae of different mating types. Hyphae were picked from the antagonistic line region formed after fusion and purified to obtain hybrid candidate strains. S2: Perform PCR typing of the hybrid candidate strains obtained in step S1 to screen out strains that simultaneously amplify double positive bands of MAT1-1-1 and MAT1-2-1, which are the pure strains according to any one of claims 1 to 5. S3: Aseptically inoculate the strains obtained from step S2 into a container containing the original culture medium, cover the surface of the material with a sterile soil layer, and culture in the dark at 18-24℃ until the mycelium fills the bottle to obtain the original culture.

7. The preparation method according to claim 6, characterized in that, In step S1, the liquid culture medium is sterile water; and / or, the germination culture is carried out at 23-25°C in the dark for 6-7 days.

8. The preparation method according to claim 6, characterized in that, In step S3, the covering thickness of the sterilized soil is 1-3 cm; and / or, the relative humidity of the culture is 50%-60%.

9. A method for cultivating yellow-branched morel mushrooms ( Morchella sp. The microbial product of ) is characterized by, The microbial product comprises the pure microbial strain as described in claim 1 or 2, as well as the original culture medium and a sterilized soil layer covering the surface of the original culture medium.

10. Use of the pure strain according to claim 1 or 2 in the cultivation of yellow morel mushrooms.

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