Morchella esculenta and breeding method and application thereof

Abstract

The invention relates to the technical field of edible fungus strain breeding, in particular to morchella esculenta and a breeding method and application thereof. The strain (Morchella importuna) Hunan microsheep 74 has the advantages of high primordium survival rate, heat resistance, long mushroom picking period, high average yield and the like, meanwhile, the high temperature resistance and the white mold resistance of ascos of the Hunan microsheep 74 strain are obviously higher than those of an original strain, and the strain can be used for cultivation and breeding of morchella esculenta.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of strain breeding, in particular to a mutagenized strain Xiangweixiang 74 of Morchella importuna and its breeding method and application. BACKGROUND

[0002] Morchella spp. is a delicious and nutritious edible fungus, which is deeply loved by consumers. The commercial cultivation of Morchella spp. in China includes Morchella importuna, M. sextelata and M. eximia. Morchella spp. is a low-temperature fungus as a whole, and the formation and development temperature of ascocarps is 6-16℃. The yield of artificial cultivation of Morchella spp. is affected by climate and region. High temperature can easily cause strain degeneration and outbreak of pests and diseases during the cultivation of Morchella spp., which has a great impact on stable and high yield.

[0003] The artificial cultivation of Morchella spp. has considerable benefits. In recent years, the Morchella industry in China has developed rapidly, and the cultivation area has spread to all parts of the country except Hainan. However, the yield and quality of Morchella cultivation are still unstable. In addition to the factors such as the cultivation management mode being unable to resist abnormal climate change, the serious mixing of Morchella cultivation varieties and the scarcity of excellent varieties are also important reasons. At present, there are few related researches on the breeding of Morchella spp. at home and abroad. Systematic breeding is a common way of breeding Morchella spp., which mainly collects wild Morchella spp. germplasm resources, and obtains new varieties of Morchella spp. through physiological index detection and domestication cultivation. A new breeding technology of Morchella spp. is established, which creates new germplasm with high primordium survival rate, high temperature resistance, high yield, and good disease resistance, etc., and helps to realize the high-efficiency cultivation of Morchella spp. in multiple seasons, which has important practical value and application prospect. SUMMARY

[0004] The primary object of the present application is to provide a mutagenized strain Xiangweixiang 74 of Morchella importuna, which has the advantages of high primordium survival rate, long harvesting time, high yield, high temperature resistance, and strong disease resistance. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 30, 2025, with the preservation number of CGMCC No. 42020, and named as Morchella importuna.

[0005] The average primordium survival rate of the strain is 83.45% at 10-22 DEG C, the average primordium survival rate is 73.6% after continuously experiencing 2 days of the highest outdoor temperature of 28 DEG C and the highest indoor temperature of 36 DEG C, the normal ascospore fruit development temperature is 4-28 DEG C, the average yield of mycelium of the strain is 411.69 kg / 667m 2 , the average harvesting time is 42 days, the white mold disease can be resisted, and the average incidence is 0.08%.

[0006] The second aspect of the present application is to provide a breeding method of Morellea esculenta, comprising the following steps:

[0007] (1) inoculating the mycelium block of the original strain of Morellea esculenta on PDA culture medium for culture, collecting the mycelium, and then obtaining protoplasts by enzymolysis through a cell wall enzyme mixed solution after washing;

[0008] (2) after the protoplast suspension is treated by ARTP mutagenesis breeding mutagenesis, culturing in a regeneration medium until single colonies are formed by germination;

[0009] (3) picking single colonies for preservation and propagation, carrying out high-temperature resistance screening experiments, and obtaining heat-resistant Morellea esculenta mutant strains;

[0010] (4) carrying out cultivation experiments and agronomic trait evaluation on the wild type original strain and the heat-resistant mutant strains, and screening the mutant strains with high yield, agronomic traits, heat resistance and disease resistance;

[0011] (5) identifying the screened mutant strains.

[0012] Further,

[0013] Step (1) inoculates the mycelium block of the original strain of Morellea esculenta on PDA culture medium containing glass paper for culture, collects the mycelium, and then obtains protoplasts by enzymolysis through a cell wall enzyme mixed solution after washing.

[0014] The configuration method of the cell wall enzyme mixed solution in step (1) is that 2% (W / V) cellulase, 2% (W / V) snail enzyme and 2% (W / V) cell wall enzyme are dissolved in a 0.6M mannitol solution.

[0015] The ARTP mutagenesis parameters in step (2) are as follows: distance 2mm, air pressure 0.1-0.2MPa, temperature 20 DEG C, power 120W, and air flow 10SLM.

[0016] The regeneration medium formula in step (2) is as follows: yeast extract 3 g, acid hydrolysis casein 3 g, sucrose 200 g, agar 10 g, and deionized water to 1 L.

[0017] Step (3) first inoculate the activated strain of step (2) into a 10cm square dish containing CYM medium in one corner, respectively, under different temperature conditions, and screen out the strains that can grow continuously to full dish to identify the heat-resistant Morchella importuna mutant strains.

[0018] The application further provides application of the Morchella importuna Xiangweiyang 74 in production of Morchella.

[0019] The identification process of the high-temperature-resistant Morchella importuna mutant strain Xiangweiyang 74 is as follows: first, amplify the ITS of the mutant strain Xiangweiyang 74 and the original strain Morchella importuna M04, and the sequencing comparison result shows that the sequences of the two are completely consistent, and are both compared to Morchella importuna in NCBI, and the sequence consistency is > 99%. Then, by observing the mycelium morphology of the mutant strain Xiangweiyang 74 and the original strain after 7d and 10d of culture at 20℃, it is found that compared with the original strain, the aerial mycelium of Xiangweiyang 74 is more dense, the range of sclerotium generation is wider, the mycelium density is higher, and the colony color is deeper. Finally, the genetic diversity of the mutant strain Xiangweiyang 74 and the original strain is detected by ISSR primer, and the result shows that there are polymorphic bands in the electrophoresis bands, and there is genetic diversity between Xiangweiyang 74 and the original strain.

[0020] Advantages of the application

[0021] The application obtains a high-temperature-resistant Morchella importuna mutant strain Xiangweiyang 74 based on the ARTP mutagenesis breeding technology, and the typical characteristics of the strain are that the mycelium can grow continuously at a high temperature environment of 31℃, which is 2℃ higher than that of the original strain Morchella importuna M04; the ascocarp development temperature is 4℃-28℃, and the highest temperature is 4℃ higher than that of the original strain; the average primordium survival rate of the strain is 83.45% at 10℃-22℃, and especially after encountering an abnormally high temperature in spring, the primordium survival rate is 1.53 times that of the original strain.

[0022] Compared with the original strain Morchella importuna M04, the Xiangweiyang 74 has a full chamber time of 5d, high mushrooming uniformity, and a mushrooming density of 49.94 / m 2 , which is 2.29 times that of the original strain, the mushrooming period is prolonged by 11 days, the average yield is 411.69 kg per mu, which is 1.7 times that of the original strain. Meanwhile, the ascocarp high-temperature resistance and resistance to white mold of the Xiangweiyang 74 strain are obviously stronger than those of the original strain.

[0023] In summary, the strain has the advantages of high primordium survival rate, long harvesting time, high yield, high-temperature resistance, strong disease resistance and the like, and has a good application prospect in Morchella cultivation and breeding. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1The mycelium of the high-temperature type Morchella prataevalis mutant strain Xiangweiyang 74 and the original strain M04 after being continuously cultured at 30℃ and 31℃ for 14d;

[0025] Figure 2 Comparison of the ITS sequences of the high-temperature type Morchella prataevalis mutant strain Xiangweiyang 74 and the original strain M04;

[0026] Figure 3 Mycelium traits (A) and growth speed (B) of the high-temperature type Morchella prataevalis mutant strain Xiangweiyang 74 and the original strain M04;

[0027] Figure 4 ISSR genetic diversity analysis of the high-temperature type Morchella prataevalis mutant strain Xiangweiyang 74 and the original strain M04;

[0028] M lane is 200 bp DNA Ladder, from top to bottom, the band size is 4000bp, 2200bp, 2000bp, 1800bp, 1600bp, 1400bp, 1200bp, 1000bp, 800bp, 600bp, 400bp, and 200bp; 1 lane is the mutant strain Xiangweiyang 74; 2 lane is the original strain M04; the primer group is from left to right: P17, P15, P12, P7, P1; wherein the characteristic band is indicated by an arrow;

[0029] Figure 5 Growth state of the high-temperature type Morchella prataevalis mutant strain Xiangweiyang 74 and the original strain M04 in the base. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with examples, without forming a limitation to the present application.

[0031] Example 1 Breeding of Xiangweiyang 74 strain

[0032] The present application also provides a breeding method of the Morchella prataevalis mutant strain Xiangweiyang 74, comprising the following steps:

[0033] 1) Mycelium collection: inoculate the original strain Morchella prataevalis M04 (preserved in China Center for Type Culture Collection, strain preservation number: CCTCC AF 2021045) in PDA medium, and culture at 20℃ for 4-5d. After the mycelium grows on the plate, punch and pick 4 pieces of mycelium block to inoculate on the PDA culture dish paved with glass paper, and culture at 20℃ for 48h. Collect the young mycelium of Morchella esculenta in the clean bench for standby.

[0034] 2) Mycelium washing: The collected young mycelium of Morchella was immersed in sterile ddH2O, and washed for 1 min with stirring, then filtered. After washing 2-3 times with ddH2O, the mycelium was washed again with 0.6M mannitol solution in the same way to remove the impurities on the surface of the mycelium.

[0035] 3) Protoplast preparation: A certain amount of snail enzyme, lywallzyme and cellulose were weighed and added to 0.6M mannitol solution to prepare a composite lysis solution. The final concentration of the three enzymes was 2% (W / V). The lysis solution was divided into 5mL centrifuge tubes, and the washed Morchella mycelium was added. After sealing, it was placed in a shaker for enzymolysis for 3h at 32℃ and 100 rpm. After enzymolysis, the mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was removed to obtain the protoplast precipitate. After washing twice with 0.6M mannitol, the protoplast was resuspended with 0.6M mannitol for standby use.

[0036] 4) Protoplast mutagenesis: The above protoplast suspension was diluted to about 1×10 6 uL, and 10uL of the diluted protoplast suspension was evenly spread on the ARTP mutagenesis special metal sheet. Then the metal sheet was placed in the ARTP mutagenesis instrument, and the program was set for mutagenesis treatment. The ARTP mutagenesis parameters were: distance 2mm, air pressure 0.1-0.2MPa, temperature 20℃, power 120W, air flow 10SLM. The mutagenesis treatment time was 0s, 10s, 20s, 30s, 40s, 50s and 60s, respectively. After mutagenesis, the protoplasts on the metal sheet were eluted with 0.6M mannitol to obtain the mutagenized protoplast suspension. The suspension was added to the melted 45℃ regeneration medium, mixed thoroughly, and then poured into a petri dish. It was placed at 20℃ for 3-4d of culture. The preparation method of the regeneration medium was as follows: yeast extract 3g, acid hydrolyzed casein 3g, sucrose 200g, agar 10g, and deionized water 1L.

[0037] 5) High-temperature-resistant mutagenized strain screening: The Morchella single colony after protoplast germination was inoculated on a PDA plate, and cultured at 20℃. After the mycelium covered the plate, a puncher was used to punch a hole, and the mycelial block was inoculated in the corner of a 12cm square plate containing CYM medium (formula: glucose 20g, peptone 2g, yeast extract 1g, magnesium sulfate 0.5g, potassium dihydrogen phosphate 0.46g, dipotassium hydrogen phosphate 1g, agar 20g, distilled water 1000mL). It was placed in a 30℃ and 31℃ incubator for static culture, and the mycelium coverage of the plate was observed after 14d. Full plate was identified as heat-resistant strain, and non-full plate was identified as non-heat-resistant strain.

[0038] The original strain of Morchella stephaniana M04 grew almost nothing at 31℃, and grew for a distance and then stopped growing at 30℃. The mutant strain Xiangweishang 74 could grow continuously at 30℃ and 31℃, and grew full of the culture dish at 30℃ on the 12th day and at 31℃ on the 14th day, which was identified as the high-temperature mutant strain of Morchella stephaniana Figure 1 ).

[0039] Example 2 Identification and genetic diversity analysis of Xiangweishang 74 strain

[0040] ①Molecular identification of mutant strain Xiangweishang 74

[0041] The mycelium was scraped from the PDA plate containing glass paper, frozen in liquid nitrogen, and ground into powder. The DNA was extracted using the Fungal DNA Kit (omega). The ITS sequences of the mutant strain Xiangweishang 74 and the original strain Morchella stephaniana M04 were amplified using the ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS-4 (5'-TCCTCCGCTTATTGATATGC-3') primers, respectively, with the extracted DNA as the template. The PCR products were sent to the company for bidirectional sequencing. The sequencing results were found to be completely consistent by comparison Figure 2 ). At the same time, the sequencing results were preliminarily compared by BLAST on the NCBI website, and the results showed that the sequences of both were compared to Morchella stephaniana, with a sequence consistency of >99%.

[0042] ②Mycelial characteristics and growth rate of mutant strain Xiangweishang 74

[0043] The activated mutant strain Xiangweishang 74 and the original strain Morchella stephaniana M04 were punched and inoculated in 9cm PDA plates, and cultured at 20℃ in the dark for 10d. The mycelial characteristics were observed and photographed on the 7th and 10th days, respectively Figure 3 A). The results showed that compared with the original, the aerial mycelium of the mutant strain Xiangweishang 74 was denser, the sclerotia were generated in a wider range, the mycelial density was higher, and the colony color was darker. In addition, the growth rate of the mutant strain Xiangweishang 74 and the original strain was measured by the "cross" method, and the results showed that the mycelial growth rate of the mutant strain Xiangweishang 74 was significantly higher than that of the original strain Figure 3 B).

[0044] ③Genetic diversity of mutant strain Xiangweishang 74

[0045] DNA was extracted from the mutant strain Xiangweiyang 74 and the original strain Morchella tiliacea M04 as described above, and the ISSR primer set was referenced (Lv Ruiling, Analysis of Genetic Diversity of Morchella Cultivar in my country Based on ISSR Markers. Northern Horticulture, 2023). PCR reaction system: 10× PCR Buffer (Mg 2- ) 2.5 μL, Mg 2- 20mmol·L -1 The total volume was 1 μL DNA template, 1 μL dNTP Mix, 0.4 μL Taq enzyme, 1 μL primers, and ddH₂O added to 25 μL. Amplification products were detected by 1.5% agarose gel electrophoresis and photographed. Amplification results showed that all five ISSR primer pairs amplified characteristic bands when DNA from strain Xiangweiyang 74 and the original strain was used as template.

[0046] Finally, the genetic diversity of the mutated strain Xiangweiyang 74 and the original strain was detected by ISSR primers, and the results showed that there were polymorphic bands in the electrophoresis bands. Figure 4 The results showed that all five pairs of ISSR primers could amplify the original M04 and the induced strain Xiangweiyang 74, of which M04 produced a total of 24 characteristic bands and the induced strain produced 26 characteristic bands. The amplified band sizes ranged from 400 bp to 1600 bp, and each set of primers amplified 4.8 characteristic bands on average. In primer P1, the induced strain Xiangweiyang 74 had an obvious polymorphic band at approximately 1300 bp and 1000 bp compared with the original strain M04, indicating that the strain bred by ARTP mutagenesis in the present invention was significantly different from the parent strain, and the primer combination and fingerprint map tested could be used for identification of the induced strain ( Figure 4 ).

[0047] Example 3 Cultivation experiment of Xiangweiyang 74 strain

[0048] Production of Xiangweiyang 74 strain

[0049] To prepare the mother culture: Boil 200g of potato juice, 20g of glucose, 20g of agar powder, and 1L of distilled water. Heat and mix the mixture, then divide it into 20mm x 200mm glass test tubes. Autoclave at 115°C for 30 minutes. The mother culture temperature is 20-23°C, and the incubation period is 5-7 days.

[0050] Seed preparation: Mix 15% wheat, 83% sawdust, 1% lime, and 1% gypsum. Autoclave at 121°C for 150 minutes. Inoculate one mother seed tube with four bags of seed. Incubate the seed at 20-23°C for 14 days. The recommended seed dosage per mu is 2 kg.

[0051] Preparation of the cultivation medium: 35% of wheat, 33% of sawdust, 20% of chaff, 10% of humus, 1% of gypsum, 1% of lime, sterilized by high-pressure steam at 121 ℃ for 210 minutes. The cultivation medium is cultured at a temperature of 18-21 ℃ for 15-20 days, and the use amount of the cultivation medium per mu is 160 kg. One bag of the original medium is used for inoculation of 30-40 bags of the cultivation medium.

[0052] Preparation of the exogenous nutrition bag: 50% of wheat, 48% of chaff, 1% of gypsum, and 1% of lime; sterilized by high-pressure steam at 121 ℃ for 210 minutes; the use amount per mu is 1200 kg.

[0053] Cultivation of Xiangwei Yang 74 strain

[0054] Seeding: The seeding can be performed when the maximum temperature of the soil in autumn is lower than 16 ℃. The cultivation medium is taken out of the bag and rubbed to be scattered, and then is seeded by way of strip seeding or hole seeding. After the seeding, the seeding area is covered with soil with a thickness of 3-5 cm. After the seeding, the seeding area is covered with black plastic film with a thickness of 0.6-0.8 silk.

[0055] Placement of the exogenous nutrition bag: After the seeding, the mycelium growth is observed, and the exogenous nutrition bag is placed after the mycelium fills the chamber. The use amount of the exogenous nutrition bag per mu is 1200 kg.

[0056] Mycelial cultivation: The soil water content is maintained at 23%-26% and the soil temperature is 10 ℃-14 ℃ during the mycelial cultivation. The mycelial cultivation time is ensured to be greater than or equal to 45 days from the seeding to the mushroom cultivation.

[0057] Removal of the plastic film: The black plastic film is removed 10-15 days before the mushroom cultivation, and the ventilation and moisture removal are performed.

[0058] Mushroom cultivation technology: Whether the mushroom cultivation is performed is determined according to the 15-day weather forecast and the local climate conditions, and the water is supplemented to ensure that the soil layer below 30 cm is permeable. Generally, a small amount of water in the furrow is enough.

[0059] Primordium maintenance: The primordium formation can be induced 5-7 days after the mushroom cultivation. During the primordium formation stage, the temperature is maintained at 10 ℃-22 ℃, and the optimal temperature is 18 ℃. The air relative humidity is 85%-95%, the soil water content is 50%-60%, and the soil temperature is 4 ℃-10 ℃. The watering and strong wind are avoided during the primordium stage.

[0060] Mushroom maintenance: During the primordium differentiation period, the temperature in the shed is maintained at 10 ℃-22 ℃, the air relative humidity is 75%-95%, there is scattered light, and the soil water content is 25%-32%.

[0061] Ascocarp maturation: The temperature at a depth of 5 cm in the soil is 8 ℃-12 ℃, the soil water content is 25%-32%, and the air relative humidity in the shed is about 85% during the ascocarp maturation. During the mushroom period, the ventilation is performed in the middle of the day to prevent high temperature and high humidity and reduce the occurrence of diseases.

[0062] Cultivation evaluation test of original strain and mutant strain Xiangweishang 74

[0063] The original strain Morchella stepanowi M04 was used as a control to evaluate the agronomic traits of the strains. The test site was located at the experimental base in Heshan District, Yiyang City, Hunan Province, and four experimental plots were set up for each variety. As shown in Table 1, the original strain Morchella stepanowi M04 and the heat-resistant mutant strain Xiangweishang 74 could grow normally in the cultivation base. Figure 5

[0064] The cultivation test used the conventional field cultivation scheme of Morchella, including strain preparation, land arrangement, sunshade shed construction, seeding, exogenous nutrient bag feeding, mycelium cultivation, conservation and mushroom induction, mushroom management, and harvesting. The mycelial growth, primordium occurrence, yield, mushroom density, and disease resistance were recorded in detail during the cultivation process. The cultivation evaluation results are shown in Table 1.

[0065]

[0066] As shown in Table 1, compared with the original strain Morchella stepanowi M04, the high-temperature mutant strain Xiangweishang 74 had a significantly higher primordium survival rate after encountering high-temperature weather in spring (2025.3.1-2025.3.2, outdoor maximum temperature 28℃, shed maximum temperature 36℃); the ascocarp development temperature was 4℃-28℃, and the maximum temperature was 4℃ higher than that of the original strain, indicating that the high-temperature tolerance of Xiangweishang 74 primordium was improved. The average yield of the high-temperature mutant strain Xiangweishang 74 was 1.7 times higher than that of the original strain, with an average yield of 411.69 kg / 667m 2 ; the average mushroom density per unit area was 2.29 times that of the original strain; the total harvesting time was extended by 11 days; and the resistance of the mutant strain Xiangweishang 74 to white mold was also better than that of the original strain Morchella stepanowi M04.​

Claims

1. A strain of Morchella importuna, Xiangweiyang 74, with the accession number: CGMCC No. 42020.

2. The Morchella importuna Xiangweiyang 74 according to claim 1, characterized in that: The strain has high primordium survival rate, long harvesting time, high yield, high temperature resistance and strong disease resistance.

3. The Morchella importuna Xiangweiyang 74 according to claim 2, characterized in that: The strain had an average primordium survival rate of 83.45% at 10°C to 22°C. After two days of exposure to a maximum outdoor temperature of 28°C and a maximum indoor temperature of 36°C, the average primordium survival rate reached 73.6%. The normal development temperature of ascocarps is 4°C to 28°C. The mycelium of the strain can continue to grow at a high temperature of 31°C, with an average yield of 411.69 kg / 667m 2 The average harvest time is 42 days, and it is resistant to white mold with an average incidence rate of 0.08%.

4. The breeding method of Morchella tiliacea is characterized in that: The following steps are involved: (1) The mycelial block of the original strain of Morchella tiliacea was inoculated on PDA culture medium for cultivation, the mycelium was collected, and the mycelium was washed and then enzymatically hydrolyzed with a mixture of lytic enzymes to obtain protoplasts; (2) After the protoplast suspension is treated with atmospheric pressure and room temperature plasma mutagenesis, it is cultured in a regeneration medium until it germinates and forms single colonies; (3) Pick a single colony to preserve and expand the strain, carry out high temperature resistance screening experiments, and obtain the heat-resistant Morchella edulis mutant strain; (4) Conduct cultivation tests and agronomic trait evaluation on the original strain and the heat-resistant mutant strain; select the mutant strain with the best yield, agronomic traits, heat resistance and disease resistance; (5) Identify the mutagenic strains screened.

5. The breeding method according to claim 4, characterized in that: Step (1) inoculating a mycelial block of the original strain of Morchella tiliacea on a PDA culture medium containing cellophane for cultivation, collecting the mycelium, washing it, and then enzymatically hydrolyzing it with a wall-lytic enzyme mixture to obtain protoplasts.

6. The breeding method according to claim 4 or 5, characterized in that: Step (1) The preparation method of the lytic enzyme mixture is as follows: 2% (W / V) cellulase, 2% (W / V) snail enzyme and 2% (W / V) lytic enzyme are dissolved in 0.6M mannitol solution.

7. The breeding method according to claim 4, characterized in that: Step (2) ARTP mutagenesis parameters are: distance 2 mm, air pressure 0.1-0.2 MPa, temperature 20°C, power 120 W, and air flow 10 SLM.

8. The breeding method according to claim 4 or 7, characterized in that: Step (2) The regeneration medium formula is: 3 g yeast extract, 3 g acid hydrolyzed casein, 200 g sucrose, 10 g agar, and deionized water to 1 L.

9. The breeding method according to claim 4, characterized in that: Step (3) First, the activated strain of step (2) is inoculated into a corner of a 10 cm square dish containing CYM culture medium, and cultured under different temperature conditions. The strain that continues to grow to fill the dish is screened and identified as the heat-resistant Morchella edulis mutagenized strain.

10. Use of the Morchella importuna Xiangweiyang 74 according to any one of claims 1 to 3 in Morchella production.

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