Polyspore hybridization method of edible fungus monokaryotic strain breeding material and parent and application thereof

Abstract

The invention relates to a method for hybridizing an edible mushroom mononuclear strain breeding material and parent polyspores. The method specifically comprises the following steps: activating and culturing a mononuclear strain to be tested, collecting and activating parent spores, and mixing and hybridizing the polyspores. According to the method, a monokaryotic strain breeding material and parent polyspore hybridization technology system is constructed, polyspore directional hybridization with the monokaryotic strain breeding material as the core is achieved, the excellent hybrid strains which grow fast and are high in stress resistance can be efficiently screened out by applying the method, the bacterial pollution problem caused by conventional polyspore hybridization is avoided, and the method is suitable for large-scale popularization and application. The hybridization efficiency is obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus genetic breeding, and particularly relates to a method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polyspore, and an application thereof in screening excellent strains resistant to foreign substances. Background Art

[0002] As an important global food resource and agricultural industry, edible fungi require significant research on variety improvement and selection. Among edible fungi breeding techniques, hybridization can effectively integrate the superior traits of different parents and create new genetic combinations.

[0003] Single-single hybridization is a commonly used hybridization method. Its basic principle is to culture single spores of different strains in a confrontation culture to form new dikaryotic mycelium, thereby achieving the exchange and recombination of genetic material. However, due to the complexity and randomness of genetic trait separation and combination, it is difficult to accurately predict and control the expression of offspring traits. Traditional hybridization techniques require a large number of hybrid combinations to obtain offspring with desired traits. The experimental workload and resource investment are large, and a considerable amount of time and effort is required for repeated testing and screening.

[0004] On the other hand, the purpose of traditional hybridization is relatively weak, and it is difficult to accurately predict and control the separation and combination of offspring traits during the hybridization process, which makes it difficult to screen out excellent strains that meet the requirements from a large number of hybrid offspring, and the efficiency needs to be improved.

[0005] In edible fungus cultivation, the resistance of cultivated strains is particularly important. When screening hybrid strains, resistance to Trichoderma is a key indicator for evaluating superior strains. Therefore, improving hybridization efficiency, enhancing the effectiveness of the hybridization process, and efficiently selecting hybrid strains with superior resistance have become key issues in breeding. Summary of the Invention

[0006] The present invention aims to provide a method for the multispore hybridization of edible fungi with strong purpose and high efficiency, and specifically relates to a method for the multispore hybridization of a breeding material of a monokaryotic strain of an edible fungus and a parent.

[0007] The present invention adopts the following technical solutions: A method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polysporic strain comprises the following steps: (1) Collection of parent spores: Collect mature fruiting bodies of edible fungi, dry the surface moisture with toilet paper, hang them in a sterile "wide-mouth bottle", store them at room temperature in a sterile room for 3 hours, hang them in another sterile "wide-mouth bottle", and allow the spores to eject for 6 hours. The "wide-mouth bottle" for the second collection of spores was refrigerated at -20℃ for later use; (2) Activation of the test monokaryotic strain breeding materials: Take the test monokaryotic strain breeding materials stored in a 4°C refrigerator, transfer them into a test tube containing PDA culture medium, and culture at a constant temperature of 25°C until the tube is full; (3) Solid culture of the monokaryotic strain breeding material: inoculate the activated monokaryotic strain breeding material into a test tube containing nutrient sawdust culture medium and culture at a constant temperature of 25°C for 10 days; (4) Liquid culture of the test monokaryotic strain breeding material: prepare a conical flask containing PD liquid culture medium and a magnetic rotor, with a filling volume of 80 ml / 250 ml; inoculate the solid mycelium of the cultured monokaryotic strain breeding material into the PD liquid culture medium with an inoculum size of 20 g / L; after standing at 25°C for 2 days, break the mycelium with a magnetic stirrer and culture at a constant temperature of 80 rpm and 25°C for 5 days to obtain the liquid culture of the test monokaryotic strain breeding material; (5) Activation culture of parent spores: Sterile water was added to the "wide-mouth bottle" prepared in step (1), and gradient dilution was performed to prepare a spore culture containing 1×10 6 ~10 7 Take 200 μl of a spore suspension (100 μl / ml) and spread it on a culture dish containing spore activation medium. Incubate at 25°C for 5 days, observing bacterial growth every 12 hours. Once bacterial colonies are found, use a sterile loop or sterile punch to remove the area of ​​bacterial growth to prepare a spore activation culture free of other bacteria. (6) Multispore hybridization: Cut the spore-activated culture without foreign bacteria into 1 cm 2 The fragments were added to the shake flask containing the liquid culture material of the test monokaryotic strain breeding material prepared in step (4), and the mixture was broken up and evenly mixed by magnetic stirring. The culture was cultured at 80 rpm and 25°C for 3 days. 0.5 ml was spread on a 90 mm diameter culture dish containing PDA culture medium and cultured at 25°C for 12-17 days.

[0008] Furthermore, the edible fungi include but are not limited to shiitake mushrooms and oyster mushrooms.

[0009] Furthermore, in step (3), the nutrient sawdust culture medium comprises the following components: 819.85 g / kg of fine sawdust, 150 g / kg of bran, 10 g / kg of sucrose, 20 g / kg of glucose, 0.03 g / kg of KH2PO4, 0.1 g / kg of MgSO4·7H2O, 10.02 g / kg of VB1, 58% water content, and natural pH.

[0010] Furthermore, in step (4), the PD liquid culture medium includes the following components: 200 g / L potato, 20 g / L glucose, and natural pH.

[0011] Furthermore, in step (5), the spore activation medium comprises the following components: 200 g / L potato, 20 g / L glucose, 3 g / L beef extract, 2 g / L peptone, 12 g / L agar, and natural pH.

[0012] Furthermore, it also includes step (7): resistance screening.

[0013] Furthermore, the resistance screening method in step (7) is a punching method.

[0014] The invention relates to an application of the above-mentioned edible fungus monokaryotic strain breeding material and the parent polyspore hybridization method in resistance screening.

[0015] The invention relates to an application of the above-mentioned edible fungus monokaryotic strain breeding material and parent polyspore hybridization method in Trichoderma resistance screening.

[0016] In the application, the specific steps of screening the resistant Trichoderma strain are as follows: a multispore hybrid colony of edible fungi is punched with a hole puncher to prepare a 3 mm diameter bacterial cake, which is inoculated into a PDA culture medium containing a sterile fermentation filtrate of Trichoderma, cultured at a constant temperature of 25°C for 7 days, and the hyphae at the edge of the colony are picked and transferred into a PDA test tube for culture, and cultured at a constant temperature of 25°C until the tube is full.

[0017] The method for preparing the culture medium containing the sterile fermentation filtrate of Trichoderma is as follows: green Trichoderma mycelium is inoculated into a shake flask containing Trichoderma fermentation liquid culture medium, cultured at a constant temperature of 25°C, allowed to stand for 24 hours, and then cultured on a shaker at 80 rpm for 5 days. The Trichoderma fermentation broth is centrifuged, the supernatant is taken, and filtered through 0.45 μm and 0.22 μm sterile filter membranes in sequence to obtain the sterile fermentation filtrate of Trichoderma. PDA culture medium containing 20 g / L agar is prepared, cooled to 50°C, and the mixture is mixed in a ratio of 1:2 between the sterile fermentation filtrate of Trichoderma and the PDA culture medium, poured into a culture dish, and cooled.

[0018] The formula of the Trichoderma fermentation liquid culture medium is: peptone 5g / L, sucrose 30g / L, MgSO40.3g / L, KH2PO40.5g / L, CaCO31g / L, and VB1 0.02g / L.

[0019] The invention relates to an application of the above-mentioned edible fungus monokaryotic strain breeding material and the parent polyspore hybridization method in edible fungus breeding.

[0020] The beneficial effects of the present invention are: (1) A hybridization technology system between the breeding materials of specific test monokaryotic strains and the parent polyspores was constructed. The breeding materials of the test monokaryotic strains were fully mixed with the parent polyspores, which significantly improved the hybridization efficiency and filled the gap in mono-polyspore hybridization.

[0021] (2) By collecting parental spores and activating spores in solid culture medium with beef extract and peptone, early identification and removal of bacterial colonies can be achieved, thus avoiding the bacterial contamination problem that occurs in conventional multi-spore hybridization.

[0022] (3) Multispore hybrid colonies were cultured in a culture medium containing sterile fermentation filtrate of Trichoderma, which promoted the proliferation of hybrid strains that were resistant to Trichoderma and grew fast, and could effectively screen out hybrid strains with excellent stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Flow chart of the method of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] Example 1 Liquid culture of the tested monokaryotic strain breeding material (1) Activation of the breeding materials of the monokaryotic strains The monokaryotic strain breeding materials of Lentinus edodes B5.14, B5.40, RX.10 and Y22.63 stored in a 4°C refrigerator were transferred into test tubes containing PDA culture medium. Each strain was transferred to 3 test tubes and cultured at a constant temperature of 25°C until the tubes were full.

[0026] (2) Solid culture of the breeding materials of the monokaryotic strains to be tested Prepare test tubes containing different solid culture media, as shown in Table 1. Inoculate the mononuclear Lentinus edodes strain breeding material activated in step (1) into each solid culture medium test tube and culture at 25°C for 10 days. Observe the mycelial growth.

[0027] Table 1 Three solid culture media used for cultivating monokaryotic strains of Lentinula edodes for breeding .

[0028] (3) Liquid culture of the breeding materials of the monokaryotic strains for testing Prepare a conical flask containing PD liquid culture medium and a magnetic rotor, with a capacity of 80 ml / 250 ml. Inoculate the solid mycelium of the monokaryotic strain breeding material cultured in step (2) into the PD liquid culture medium at an inoculum size of 20 g / L. After standing at a constant temperature of 25°C for 2 days, break up the inoculum with a magnetic stirrer and incubate at a constant temperature of 25°C at 80 rpm for 6 days. Observe the mycelial growth and determine the mycelial mass.

[0029] The formula of PD liquid culture medium is: potato 200g / L, glucose 20g / L, natural pH.

[0030] The method for determining the amount of mycelium is as follows: filter the edible mycelium culture solution with filter paper, transfer the filtered mycelium into a culture dish and air-dry for 6 hours, and then weigh the fresh weight of the mycelium.

[0031] (4) Results and analysis The solid culture results of the tested monokaryotic strain breeding materials showed (Table 2) that all four tested monokaryotic strain breeding materials could grow in PDA and nutrient-added sawdust medium, while only the monokaryotic strain Y22.63 could grow in ordinary sawdust medium.

[0032] Liquid culture results of the tested monokaryotic strain breeding materials (Table 3) showed that mycelium cultured in PDA medium had fewer germination points in the liquid medium, tended to sink to the bottom due to hypoxia and discoloration, and aggregated into clumps rather than extending outward. Mycelium cultured in nutrient sawdust medium remained well suspended in the liquid medium, exhibited more germination points, grew faster, and produced a large mycelial mass.

[0033] Table 2 Growth of the tested monokaryotic strains in different activation media .

[0034] Table 3 The amount of mycelia transferred from different activation culture media to liquid culture media .

[0035] Example 2 Activation of Lentinus edodes parent spores (1) Collection of spores from Lentinus edodes parents Mature fruiting bodies of strains 0912, 610 and Qihe No. 9 were collected, the surface moisture was absorbed with toilet paper, and the bottles were hung in a sterile "wide-mouth bottle". After being stored at room temperature in a sterile room for 3 hours, the bottles were hung in another sterile "wide-mouth bottle" to allow spores to eject for 6 hours. The "wide-mouth bottle" for the second spore collection was refrigerated at -20℃ for later use.

[0036] (2) Spore activation medium Prepare a 90 mm diameter culture dish containing 20 ml of spore activation medium. The formula of the spore activation medium is shown in Table 4.

[0037] Table 4 Three spore activation media .

[0038] (3) Parent spore activation culture Sterile water was added to the "wide-mouth bottle" containing the spores of Lentinus edodes parent, and gradient dilution was performed to prepare a spore containing 1×10 6 ~1×10 7Spread 200 μl of a spore suspension (100 μl / ml) onto a culture dish containing spore activation medium. Incubate at 25°C for 6 days, observing bacterial growth every 12 hours. Once bacterial colonies are detected, remove the area of ​​bacterial growth with a sterile loop or sterile borer to prepare a spore activation culture free of contaminants.

[0039] (4) Results and analysis The results in Table 5 show that the bacterial development time in PDA medium is 72 hours, and the bacterial colonies are lightly transparent and closely match the color of the culture medium, making them difficult to detect. The addition of antibiotics has a certain inhibitory effect on bacteria, but bacterial growth begins around day 6 of culture. Furthermore, the culture medium supplemented with antibiotics has a certain inhibitory effect on the germination and growth of Lentinus edodes colonies. However, PDA medium supplemented with beef extract and peptone provides favorable growth conditions for bacteria, shortening the development time. Bacterial colonies can be detected as early as 36 hours. Furthermore, bacterial colonies in PDA medium supplemented with beef extract and peptone appear yellow and thicker, allowing for more distinct observation of their size and morphology, making them easier to pick.

[0040] Table 5 Bacterial growth in different culture media .

[0041] Example 3 Multispore hybridization of Lentinus edodes and screening of Trichoderma-resistant strains (1) Collection of spores from Lentinus edodes parents Mature fruiting bodies of strains 0912, 610 and Qihe No. 9 were collected, the surface moisture was absorbed with toilet paper, and the bottles were hung in a sterile "wide-mouth bottle". After being stored at room temperature in a sterile room for 3 hours, the bottles were hung in another sterile "wide-mouth bottle" to allow spores to eject for 6 hours. The "wide-mouth bottle" for the second spore collection was refrigerated at -20℃ for later use.

[0042] (2) Activation of the breeding materials of the monokaryotic strains The monokaryotic strain breeding materials of Lentinus edodes B5.14, B5.40, RX.10 and Y22.63 stored in a 4°C refrigerator were transferred into test tubes containing PDA culture medium. Each strain was transferred to 3 test tubes and cultured at a constant temperature of 25°C until the tubes were full.

[0043] (3) Solid culture of the breeding materials of the monokaryotic strains to be tested The activated mononuclear Lentinus edodes strain breeding materials were inoculated into a test tube containing nutrient sawdust culture medium and cultured at a constant temperature of 25°C for 10 days.

[0044] (4) Liquid culture of the monokaryotic strain breeding material Prepare a conical flask containing PD liquid medium and a magnetic rotor, with a capacity of 80 ml / 250 ml. Inoculate the cultured solid mycelium of the breeding material of the test monokaryotic strain into the PD liquid medium at an inoculum rate of 20 g / L. After incubating at 25°C for 2 days, break up the inoculum with a magnetic stirrer and incubate at 80 rpm at 25°C for 6 days to prepare a liquid culture containing the breeding material of the test monokaryotic strain.

[0045] The formula of PD liquid culture medium is: potato 200g / L, glucose 20g / L, natural pH.

[0046] (5) Activation of parent spores Sterile water was added to the "wide-mouth bottle" containing the spores of Lentinus edodes parent, and gradient dilution was performed to prepare a spore containing 1×10 6 ~1×10 7 Spread 200 μl of a spore suspension containing 100 μl / ml onto a culture dish containing spore activation medium. Incubate at 25°C for 6 days, observing bacterial growth every 12 hours. Once bacterial colonies are detected, use a sterile loop or sterile borer to remove any areas of bacterial growth to prepare a spore activation culture free of contaminants.

[0047] (6) Polyspore hybridization Cut the spore activation medium without foreign bacteria into pieces of about 1 cm 2 The fragments were added into a shake flask containing the liquid culture of the tested monokaryotic strain breeding material, broken up and mixed evenly by magnetic stirring, and cultured at 80 rpm and 25°C for 3 d and 15 d, respectively, to prepare edible fungus multispore mixed hybrid cultures.

[0048] Hybrid combinations: Hybrid combination A: B5.14×0912 multisporic; Hybrid combination B: B5.14×Qihe No. 9 multisporic; Hybrid combination C: B5.40×0912 multisporic; Hybrid combination D: Y22.63×0912 multisporic; Hybrid combination E: RX.10×610 multisporic.

[0049] (7) Resistance screening Prepare PDA culture medium containing sterile fermentation filtrate of Trichoderma. Trichoderma viride ) The mycelium was inoculated into a shake flask containing Trichoderma fermentation liquid culture medium and cultured at a constant temperature of 25°C. After standing for 24 hours, the culture was shaken at 80 rpm for 5 days. The Trichoderma fermentation liquid was centrifuged and the supernatant was taken and passed through 0.45 μm and 0.22 μm sterile filter membranes in sequence to obtain the Trichoderma sterile fermentation filtrate.

[0050] Trichoderma viride ( Trichoderma viride ) was isolated and preserved at the Institute of Biology, Hebei Academy of Sciences.

[0051] The formula of Trichoderma fermentation liquid culture medium is: peptone 5g / L, sucrose 30g / L, MgSO40.3g / L, KH2PO40.5g / L, CaCO31g / L, VB1 0.02g / L.

[0052] Preparation of PDA culture medium containing sterile fermentation filtrate of Trichoderma: First, prepare PDA culture medium containing 20 g / L agar, cool to 50°C, mix the sterile fermentation filtrate of Trichoderma:PDA culture medium in a volume ratio of 1:2, pour into a culture dish, and cool.

[0053] Take 0.5 ml of the multispore mixed hybrid culture cultured for 3 days and spread it on PDA solid medium. After constant temperature cultivation at 25℃ for 12-17 days, use a 3mm puncher to prepare bacterial cakes, and transfer them to PDA medium and PDA medium containing sterile fermentation filtrate of Trichoderma, respectively. Transfer 30 culture dishes to each medium, and culture at a constant temperature of 25℃ for 7 days to observe the growth of the colonies.

[0054] Lentinus edodes cakes were prepared using a 3mm borer at the edge of colonies on both cultures. These cakes were then inoculated into 90mm Petri dishes containing PDA medium, with the inoculation point 15mm from the center of the dish. The dishes were incubated at 25°C for 5 days. Activated Trichoderma cakes with a diameter of 3mm were then inoculated symmetrically with the inoculation point. Incubation was continued at 25°C for another 7 days, and the antagonistic radius of Lentinus edodes was measured. The antagonistic radius is the radius of mycelial growth from the Lentinus edodes inoculation point to the Trichoderma inoculation point. A larger antagonistic radius indicates a stronger resistance to Trichoderma.

[0055] (8) Results and analysis The results in Table 6 indicate that the average antagonistic radius of the multisporic hybrid strains grown in PDA medium containing Trichoderma sterile fermentation filtrate was significantly greater than that of the multisporic hybrid strains grown in PDA medium alone. The Trichoderma sterile fermentation filtrate may inhibit the growth of Lentinus edodes mycelia with poor Trichoderma resistance by releasing secondary metabolites, thereby selecting strains with strong Trichoderma resistance and rapid growth. After 15 days of shake flask culture of the multisporic hybrid culture, the liquid was turbid, but the mycelium within the culture clumped into agglomerates, some of which condensed into granules and sank to the bottom, while others died due to lack of oxygen.

[0056] Table 6 Resistance determination of hybrid strains of Lentinus edodes .

[0057] Example 4 Multispore hybridization of Pleurotus ostreatus and screening of Trichoderma-resistant strains (1) Collection of spores from Pleurotus ostreatus parent plants Mature fruiting bodies of strain 2028 were collected, and the surface moisture was absorbed with toilet paper. The fruiting bodies were hung in a sterile "wide-mouth bottle". After being stored at room temperature in a sterile room for 3 hours, the fruiting bodies were hung in another sterile "wide-mouth bottle" to allow spores to eject for 6 hours. The "wide-mouth bottle" for the second spore collection was refrigerated at -20℃ for later use.

[0058] (2) Activation of the breeding materials of the monokaryotic strains The monokaryotic strain breeding material of Pleurotus ostreatus, Kejia No. 1, which was stored in a 4°C refrigerator, was transferred into a test tube containing PDA culture medium. Three test tubes were transferred and cultured at a constant temperature of 25°C until the tube was full.

[0059] (3) Solid culture of the breeding materials of the monokaryotic strains to be tested The activated mononuclear Pleurotus ostreatus strain breeding materials were inoculated into a test tube containing nutrient sawdust culture medium and cultured at a constant temperature of 25°C for 10 days.

[0060] (4) Liquid culture of the monokaryotic strain breeding material Prepare a conical flask containing PD liquid medium and a magnetic rotor, with a capacity of 80 ml / 250 ml. Inoculate the cultured solid mycelium of the test monokaryotic strain breeding material, Kejia No. 1-1, into the PD liquid medium at an inoculum rate of 20 g / L. After incubating at 25°C for 2 days, break up the mycelium with a magnetic stirrer and incubate at 80 rpm at 25°C for 5 days to prepare a liquid culture containing the test monokaryotic strain breeding material.

[0061] (5) Activation of parent spores Sterile water was added to the "wide-mouth bottle" containing the parent Pleurotus ostreatus 2028 spores, and gradient dilution was performed to prepare a 1×10 6 ~1×10 7 Spread 200 μl of a spore suspension containing 100 μl / ml onto a culture dish containing spore activation medium. Incubate at 25°C for 5 days, observing bacterial growth every 12 hours. Once bacterial colonies are detected, use a sterile loop or sterile borer to remove any areas of bacterial growth to prepare a spore activation culture free of contaminants.

[0062] (6) Multi-spore mixture Cut the spore-activated culture without foreign bacteria into pieces of about 1 cm 2 The fragments were added into the shake flask containing the liquid culture of the tested monokaryotic strain breeding material, broken up and mixed evenly by magnetic stirring, and cultured at 80 rpm and 25°C for 3 days.

[0063] Hybrid combination F: Kejia No. 1-1×2028 polyspores.

[0064] (7) Resistance screening Prepare PDA medium containing sterile Trichoderma fermentation filtrate (same as in Example 3). Spread 0.5 ml of the multispore hybrid mixture, cultured for 3 days, onto PDA solid medium. After constant incubation at 25°C for 10-15 days, use a 3 mm borer to prepare bacterial cakes. Transfer the cakes to PDA medium and PDA medium containing sterile Trichoderma fermentation filtrate, respectively. Transfer 30 culture dishes to each medium. Incubate at 25°C for 5 days and observe colony growth.

[0065] Pleurotus ostreatus cakes were prepared using a 3mm borer at the edge of colonies on both cultures. These cakes were then inoculated into 90mm Petri dishes containing PDA medium, with the inoculation point 15mm from the center of the dish. The dishes were incubated at 25°C for 5 days. Activated Trichoderma spore cakes with a diameter of 3mm were then inoculated symmetrically with the inoculation point. Cultures were continued at 25°C for another 7 days, and the antagonistic radius of Pleurotus ostreatus was measured. The antagonistic radius of Pleurotus ostreatus is the radius of mycelial growth from the inoculation point of Pleurotus ostreatus to the inoculation point of Trichoderma spores. A larger antagonistic radius indicates a stronger resistance to Trichoderma spores.

[0066] (8) Results and analysis The results in Table 7 show that the multisporic hybrid strains grown in PDA medium containing Trichoderma sterile fermentation filtrate have an average antagonistic radius greater than that of the multisporic hybrid strains grown in PDA medium. Trichoderma sterile fermentation filtrate medium can more efficiently screen out multisporic hybrid strains of Pleurotus ostreatus that are highly resistant to Trichoderma and grow faster.

[0067] Table 7 Resistance determination of hybrid strains of Pleurotus ostreatus .

Claims

1. A method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polysporic strain, characterized in that: It includes the following steps: (1) Collection of parent spores: Collect mature fruiting bodies of edible fungi, dry the surface moisture with toilet paper, hang them in a sterile wide-mouth bottle, store them at room temperature in a sterile room for 3 hours, hang them in another sterile wide-mouth bottle, and allow the spores to eject for 6 hours. The wide-mouth bottle used for the second collection of spores was refrigerated at -20℃ for later use; (2) Activation of the test monokaryotic strain breeding materials: Take the test monokaryotic strain breeding materials stored in a 4°C refrigerator, transfer them into a test tube containing PDA culture medium, and culture at a constant temperature of 25°C until the tube is full; (3) Solid culture of the monokaryotic strain breeding material: inoculate the activated monokaryotic strain breeding material into a test tube containing nutrient sawdust culture medium and culture at a constant temperature of 25°C for 10 days; (4) Liquid culture of the test monokaryotic strain breeding material: prepare a conical flask containing PD liquid culture medium and a magnetic rotor, with a filling volume of 80 ml / 250 ml; inoculate the solid mycelium of the cultured monokaryotic strain breeding material into the PD liquid culture medium with an inoculum size of 20 g / L; after standing at 25°C for 2 days, break the mycelium with a magnetic stirrer and culture at a constant temperature of 80 rpm and 25°C for 5 days to obtain the liquid culture of the test monokaryotic strain breeding material; (5) Activation culture of parent spores: Sterile water was added to the wide-mouth bottle prepared in step (1) and gradient dilution was performed to prepare a spore culture containing 1×10 6 ~10 7 Take 200 μl of a spore suspension (100 μl / ml) and spread it on a culture dish containing spore activation medium. Incubate at 25°C for 5 days, observing bacterial growth every 12 hours. Once bacterial colonies are found, use a sterile loop or sterile punch to remove the area of ​​bacterial growth to prepare a spore activation culture free of other bacteria. (6) Multispore hybridization: Cut the spore-activated culture without foreign bacteria into 1 cm 2 The fragments were added to the shake flask containing the liquid culture material of the test monokaryotic strain breeding material prepared in step (4), and the mixture was broken up and evenly mixed by magnetic stirring. The culture was cultured at 80 rpm and 25°C for 3 days. 0.5 ml was spread on a 90 mm diameter culture dish containing PDA culture medium and cultured at 25°C for 12-17 days.

2. The method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polyspore according to claim 1, wherein: The nutrient sawdust culture medium in step (3) comprises the following components: 819.85 g / kg of fine sawdust, 150 g / kg of bran, 10 g / kg of sucrose, 20 g / kg of glucose, 0.03 g / kg of KH2PO4, 0.1 g / kg of MgSO4·7H2O, 0.02 g / kg of VB1, 58% water content, and natural pH.

3. The method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polyspore according to claim 1, wherein: The PD liquid culture medium in step (4) includes the following components: 200 g / L potato, 20 g / L glucose, and natural pH.

4. The method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polyspore according to claim 1, wherein: The spore activation medium in step (5) comprises the following components: 200 g / L potato, 20 g / L glucose, 3 g / L beef extract, 2 g / L peptone, 12 g / L agar, and natural pH.

5. The method for hybridizing a monokaryotic strain breeding material of an edible fungus with a parent polyspore according to claim 1, wherein: It also includes step (7): resistance screening.

6. Use of the edible fungus monokaryotic strain breeding material according to any one of claims 1 to 5 and the parent polyspore hybridization method in edible fungus resistance screening.

7. An application of the edible fungus monokaryotic strain breeding material according to any one of claims 1 to 6 and the parent polyspore hybridization method in the edible fungus Trichoderma resistance screening.

8. Use of the edible fungus monokaryotic strain breeding material according to any one of claims 1 to 6 and the parent polyspore hybridization method in edible fungus breeding.

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