A Grifola frondosa strain, spore, protoplast, fruiting body and their applications

Abstract

The present invention provides a Grifola frondosa strain, spores, protoplasts, fruiting bodies and applications thereof. The Grifola frondosa strain is named Finc‑G‑B1, belongs to Grifola frondosa, and is preserved in Guangdong Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a preservation number of GDMCC NO.62551 and a preservation date of June 20, 2022. The Grifola frondosa strain provided by the present invention solves the problem of poor stability and consistency of Grifola frondosa varieties in the factory bottle cultivation mode in the prior art.

Description

Technical Field

[0001] The present invention belongs to the field of edible fungi and relates to a Grifola frondosa strain, spores, protoplasts, fruiting bodies and their applications. Background Art

[0002] Grifola frondosa is a rare edible and medicinal fungus, belonging to the Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, Grifola. In China, Grifola frondosa mostly grows on chestnut trees, with layers overlapping like chrysanthemums and emitting a fragrant smell, so it is also known as chestnut mushroom, lotus mushroom, thousand-buddha mushroom, cloud mushroom, etc. Grifola frondosa has a unique taste, flavor, rich nutrition and medicinal value, and is one of the common mushroom varieties in the market.

[0003] Since the wild resources of Grifola frondosa are extremely scarce, artificial cultivation has become an important way to meet the demand since the efficacy of this fungus was recognized. The large-scale cultivation of Grifola frondosa in China started in Qingyuan County, Zhejiang Province and Qianxi County, Hebei Province. According to the production mode of Grifola frondosa, it can be divided into soil-covered cultivation and soil-free cultivation, and the latter includes bag cultivation and bottle cultivation. According to the color of the fruiting body, it can be divided into three types: brown, gray and white. The brown variety is the main type most used in current production, followed by the gray variety, and the white variety is the rarest.

[0004] Different domestic and foreign maitake mushroom production units use different main cultivated varieties, but the vast majority use brown or gray varieties suitable for bag cultivation. MH180211 is a commercial bag-cultivated brown maitake mushroom (MAITAKE) variety commonly used by Japanese companies. The main cultivated variety of Hokuto Co., Ltd., a leading Japanese edible fungus company, is HOKTUNT-100 (US PP17, 984P3). In recent years, it has also cultivated GRIFON120 (US 2009 / 0055986P1), GRIFON7 (US PP25, 856P3), GRIFON-8GO (US 2020 / 0100415P1) and other varieties through wild domestication and conventional hybrid breeding techniques. The domestic variety of Grifola frondosa "Qingyuan 151" bred by the Qingyuan County Edible Fungi Research Center in Zhejiang Province has an approval number of Zhejiang (non-) approval fungi 2013004. It has become the main variety of Grifola frondosa in Qingyuan County, with an annual cultivation volume of millions of bags, accounting for more than 90% of the total production of Grifola frondosa in the country. Qianxi County, Hebei Province is known as the "Hometown of Chinese Chestnut Mushrooms". The local variety used is "Qianxi No. 2" which is isolated and domesticated from wild strains. "Yongda No. 1" of Shanghai Yongda Mushroom Industry is approved as Shanghai Agricultural Products Edible Fungi 2021 No. 006 and is bred by the JH01 system. Most of the above varieties have the advantages of high yield in bag cultivation, delicious taste, unique fragrance, and rich nutrition. However, the above varieties are difficult to match with intensive and large-scale bottle cultivation models. Their mushroom production characteristics are usually characterized by insufficient stability and consistency, which directly affects the quality, efficiency and cost of factory production. Summary of the invention

[0005] The object of the present invention is to provide a Grifola frondosa strain, spores, protoplasts, fruiting bodies and applications thereof, aiming to solve the problems of poor stability and consistency of Grifola frondosa varieties in factory-scale bottle cultivation mode in the prior art.

[0006] To solve the above technical problems, the present invention provides a Grifola frondosa strain, named Finc-G-B1, which belongs to Grifola frondosa and is preserved in Guangdong Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the preservation number is GDMCC NO.62551, and the preservation date is June 20, 2022.

[0007] The invention also provides a kind of Grifola frondosa spores, which are spores obtained by culturing the Finc-G-B1 strain.

[0008] The invention also provides a Grifola frondosa (monokaryon) protoplast, which is the protoplast obtained by culturing the Finc-G-B1 strain.

[0009] The invention also provides a Grifola frondosa mycelium, which is the mycelium obtained by culturing the Finc-G-B1 strain.

[0010] The present invention also provides a fruiting body of Grifola frondosa, which is a fruiting body obtained by cultivating the above-mentioned Finc-G-B1 strain.

[0011] The present invention also provides the application of the above-mentioned Grifola frondosa fruiting body in food processing.

[0012] The present invention also provides the application of a Grifola frondosa strain in breeding, using the Finc-G-B1 as a parent for breeding.

[0013] The present invention also provides the application of a Grifola frondosa strain in cross-breeding, using the Finc-G-B1 as a parent for cross-breeding.

[0014] The present invention also provides the application of a Grifola frondosa strain in breeding, performing molecular editing breeding on the Finc-G-B1.

[0015] Compared with the prior art, the Grifola frondosa strain Finc-G-B1 provided by the present invention has the following advantages:

[0016] 1. The fruiting bodies cultured from the strain have good stability and repeatability, and the differences in the traits of the fruiting bodies between batches are small, with strong consistency, ensuring the scale benefit of industrial production;

[0017] 2. The fruiting body emergence rate is high, with an emergence rate of over 90% per batch, high biological conversion efficiency, and good economic benefits;

[0018] 3. The growth cycle is short, with an average cycle of ≤ 40 days, improving the production efficiency of the factory and effectively saving energy and reducing consumption;

[0019] 4. The fruiting body has a long shelf life. Under the condition of a 4°C cold storage, the average shelf life cycle is ≥ 40 days, which is conducive to long-distance transportation and extends the shelf life.

[0020] In summary, the comprehensive traits of Finc-G-B1 are excellent, and it has a broad market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 and Figure 2 are respectively the morphological diagrams of the fruiting bodies of the Finc-G-B1 strain and one of its parental strains, the MH160820 strain, provided by an embodiment of the present invention;

[0022] Figure 3 and Figure 4 are respectively the front colony diagram and the back colony diagram of the Finc-G-B1 strain and its two parental strains cultured on the same culture medium provided by an embodiment of the present invention;

[0023] Figures 5 - 6The graphs of the single mushroom yields and the average graphs of the single yield distributions for the F-G-B1 strain and the MH160820 strain respectively.

[0024] Figure 7 The distribution graphs of the mushroom yields for the F-G-B1 strain and the MH160820 strain.

[0025] Figure 8 The distribution graphs of the mycelium ages and cycles for the F-G-B1 strain and the MH160820 strain. Detailed implementation manners

[0026] The following further elaborates in detail on a Grifola frondosa strain, spores, protoplasts, fruiting bodies and their applications proposed by the present invention in conjunction with the accompanying drawings and specific embodiments. According to the following description, the advantages and features of the present invention will be clearer. It should be noted that the accompanying drawings are all in a very simplified form and use non-precise scales, only for conveniently and clearly assisting in explaining the purpose of the embodiments of the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0027] The present invention provides a Grifola frondosa strain named Finc-G-B1, belonging to Grifola frondosa (Latin scientific name: Grifola Frondosa), deposited in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, deposit number: GDMCC NO.62551, deposit date: June 20, 2022.

[0028] The Finc-G-B1 Grifola frondosa strain of the present invention selects the brown Grifola frondosa strain from Niigata Prefecture, Japan (number M2019-5) and the gray Grifola frondosa strain HOKTU NT-100 (number MH160820) as parents and is obtained through cross-breeding and selection.

[0029] The present invention also provides a Grifola frondosa spore, which is the spore obtained after culturing the above-mentioned Finc-G-B1 strain.

[0030] The present invention also provides a Grifola frondosa (mononuclear) protoplast, which is the protoplast obtained after culturing the above-mentioned Finc-G-B1 strain.

[0031] The present invention also provides a Grifola frondosa mycelium, which is the mycelium obtained after culturing the above-mentioned Finc-G-B1 strain.

[0032] The present invention also provides the application of the above-mentioned Grifola frondosa mycelium in the extraction of active substances and secondary metabolites, and fermentation engineering.

[0033] The present invention also provides a Grifola frondosa fruit body, which is a fruit body obtained by cultivating the above-mentioned Finc-G-B1 strain.

[0034] The present invention also provides the application of the above-mentioned Grifola frondosa fruit body in food processing.

[0035] The present invention also provides the application of a Grifola frondosa strain in breeding, using the Finc-G-B1 as a parent for breeding.

[0036] The present invention also provides the application of a Grifola frondosa strain in cross-breeding, using the Finc-G-B1 as a parent for cross-breeding.

[0037] The present invention also provides the application of a Grifola frondosa strain in breeding, performing molecular editing breeding on the Finc-G-B1.

[0038] The Grifola frondosa mycelia cultured from the Finc-G-B1 strain of the present invention are thick, white, with underdeveloped aerial mycelia, the colony surface is smooth, the thickness is moderate, there is no pigment, and the edge is irregular.

[0039] As Figure 1 and Figure 2 shown, they are respectively the morphological diagrams of the fruit bodies cultivated from the Finc-G-B1 strain of the present invention and the fruit bodies cultivated from its parent strain MH160820. It can be seen that the fruit bodies cultivated from the Finc-G-B1 strain of the present invention have full and upright shapes, dense slices, arranged in a shingled manner, the overall color of the fruit body is yellowish-brown to brown, and the smell is fragrant. The cap is fleshy, fan-shaped to spatulate, without obvious notches on the periphery, and the vertical section is nearly horizontal; the front of the cap is yellowish-brown to brown, with a brown ring pattern at the edge, the thickness is medium to thick, and the hardness is medium; the pores are attached to the back of the cap, and the attachment range extends to the base, the color is white to milky white, and the density is medium; the stipe is fleshy, yellowish-brown to brown, the length is short to medium, the diameter is medium, and the base is thin to medium.

[0040] In order to further highlight the advantages of the Grifola frondosa strain provided by the present invention, the following several experiments are used to further and specifically compare and explain the strain and its applications.

[0041] Example 1 Hybrid breeding of the Finc-G-B1 strain

[0042] In this example, the specific breeding method of the Grifola frondosa strain Finc-G-B1 is as follows:

[0043] 1) Select parent strains: Select MH160820 and M2019-5 as cross-breeding parents, and both of these parent materials are preserved in the Shanghai Academy of Agricultural Sciences Culture Collection Center.

[0044] 2) Prepare the mother spawn medium, original spawn medium and cultivated spawn medium: The formula of each medium is as follows:

[0045] Mother spawn medium (PDA medium): For every 1 L of medium, use 200 g of potatoes, 20 g of glucose, and 20 g of agar.

[0046] Original spawn medium: By mass percentage, it includes 35 - 40% of the original spawn culture material and 60 - 65% of water. The original spawn culture material, by mass percentage, includes 80 - 90% of wood chips and 10 - 20% of wheat bran.

[0047] Cultivated spawn medium: By mass percentage, it includes 35 - 40% of the cultivated spawn culture material and 60 - 65% of water. The cultivated spawn culture material, by mass percentage, includes 20 - 25% of chestnut wood chips, 20 - 25% of oak wood chips, 15 - 20% of cottonseed hulls, 10% of wheat bran, 5 - 10% of brewer's grains, and 5 - 10% of bean dregs.

[0048] 3) Obtain spores: Activate the two parental strains through test tube spawn, transfer to Erlenmeyer flask spawn, transfer to original spawn, transfer to cultivated spawn, then conduct mycelium culture, raking the mycelium surface, and fruiting after cultivation. After the mushroom pores open, collect the spores by the spread plate method and place them at 21°C for 2 days to obtain the spore print.

[0049] 4) Spore spreading: Dilute the spore print with sterile water to obtain a spore suspension. Use 1 ml of the spore suspension per petri dish and evenly spread it on the PDA medium with a spreader.

[0050] 5) Obtain monokaryotic mycelium: Culture the PDA medium in step 4 at 21°C for about 15 days. Pick single spore colonies at sparse areas and transfer them to the PDA medium for cultivation to obtain the monokaryotic mycelium corresponding to the two parental strains.

[0051] 6) Microscopic examination of monokaryotic mycelium: In the microscope field of view at 10×40 magnification, search for 3 - 5 fields of view in different areas. The absence of clamp connections indicates monokaryotic mycelium (referred to as single spores).

[0052] 7) Pairing and hybridization: Select single spores with fast mycelial growth rate, white and dense mycelium from the monokaryotic mycelium of the two parental strains respectively for pairing and culture on the PDA medium;

[0053] 8) Microscopic examination of heterozygotes: Select the mycelium on both sides of the paired monokaryons. In the microscope field of view at 10×40 magnification, search for 3 - 5 fields of view in different areas. If there are clamp connections, it is a heterozygote.

[0054] 9) Pick heterozygotes: Transfer the mycelium on the side with clamp connections to the PDA medium for cultivation.

[0055] 10) Cultivation experiment: Conduct cultivation experiments and screening on the obtained heterozygotes according to conventional methods.

[0056] 11) 500 heterozygotes were obtained through the hybridization of M2019-5 and MH160820. From the 500 heterozygotes, the target strains suitable for industrialized bottle cultivation, with high fruiting rate and good stability, were screened and named "F-G-B1".

[0057] 12) Strain preservation: The new Grifola frondosa strain F-G-B1 of the present invention was preserved in the Guangdong Microbial Culture Collection Center on June 20, 2022, with the preservation number GDMCC NO.62551. The strain was detected to be viable, and its taxonomic status is Grifola.

[0058] Example 2 Antagonism experiment

[0059] Using the new Grifola frondosa strain F-G-B1 of the present invention and its parents M2019-5 and MH160820 as experimental materials, the medium used was PDA medium.

[0060] Control group: The three strains were cultured separately. Only one strain was cultured on one plate, with 3 inoculation points on each plate, arranged in a triangle. The distance between the inoculation points of the same material was 1.5 cm. They were cultured at a constant temperature of 21°C for 18 days, and the culture results were used as a reference for non-antagonistic phenomena.

[0061] Experimental group: The different strains were co-cultured. There were 3 inoculation points on each plate, arranged in a triangle. The distance between the inoculation points of different varieties was 1.5 cm. They were cultured at a constant temperature of 21°C for 18 days.

[0062] The culture results of the experimental group were compared with those of the control group respectively to determine whether there was an antagonistic phenomenon between F-G-B1 and the parental strains.

[0063] For the test results, please refer to Figures 3 to 4 and Table 1 below.

[0064] Table 1 Antagonistic test results among three Grifola frondosa strains

[0065]

[0066]

[0067] Note: "+" indicates the presence of an antagonistic phenomenon.

[0068] Figure 3 and Figure 4 are the results of culturing the three strains on the same medium. Figure 3 is the front view of the colony on the medium. Figure 4This is the back view of the colonies. It can be seen that on the front of the culture medium, the colonies of the three strains do not intersect. On the back, dark grooves are formed between the colonies of the two strains, indicating that there is an antagonistic phenomenon between F-G-B1 and both M2019-5 and MH160820. Combining the experimental results in Table 1, it can be concluded that the strains Finc-G-B1, M2019-5, and MH160820 are different Grifola frondosa strains.

[0069] Example 3 Cultivation Test Experiment

[0070] Continuous multi-batch fruiting tests were conducted on the F-G-B1 strain of the present invention at different times and seasons. At the same time, the MH160820 strain, which showed better cultivation performance among the parents, was used as a control group for cultivation comparison tests.

[0071] The cultivation conditions adopted in this example are as follows:

[0072] For the culture medium, refer to the culture medium for cultivated strains, which will not be elaborated here.

[0073] Volume of cultivation bottle: 850 mL.

[0074] Mycelium culture conditions: temperature 20 - 25 °C, humidity 70 - 75%, intermittent scattered light, light intensity 50 - 100 lx, CO2 concentration controlled below 1500 ppm. The mycelium culture period is the number of days from inoculation to raking the mycelium.

[0075] Fruiting body cultivation conditions: the temperature in the cultivation room is set at 15 - 18 °C, humidity 85 - 95%, CO2 concentration controlled below 1200 ppm, light intensity 50 - 100 lx, and the fruiting bodies are harvested in time when they are nearly mature. The fruiting body cultivation period is the number of days from raking the mycelium to harvesting.

[0076] Figures 5 - 6 These are the graphs of the single-yield data and the average single-yield distribution of the F-G-B1 strain and the MH160820 strain respectively.

[0077] From Figure 5 the comparison of 8 batches of tests, it can be seen that the single yield of the F-G-B1 strain is significantly higher than that of the MH160820 strain. Figure 6 From the single-yield distribution graph, it can be seen that the single yield of the F-G-B1 strain is mainly distributed in the range of 41 - 100 g, while the single yield of the MH160820 strain is distributed in the range of 11 - 90 g. Moreover, the proportion of the single yield of MH160820 in the two ranges of 41 - 50 g and 51 - 60 g is relatively large, while the F-G-B1 strain has a larger proportion of the single yield in the two ranges of 61 - 70 g and 71 - 80 g, indicating that the single yield of the F-G-B1 strain is more stable and has higher consistency than that of the MH160820 strain, and is more suitable for industrial production.

[0078] In addition, in the single yield range of 61 - 100 g, the single yield of strain F-G-B1 is basically higher than that of strain MH160820, indicating that the single yield of strain F-G-B1 is also higher than that of MH160820.

[0079] Figure 7 The figure shows the fruiting rate distribution diagrams of strain F-G-B1 and strain MH160820. It can be seen that from the perspective of the fruiting rate, the fruiting rate of strain F-G-B1 is more stable than that of strain MH160820.

[0080] Figure 8 The figure shows the mycelium age (the cultivation period of the mycelium, from inoculation to raking the bacteria) and cycle (the cultivation period of the fruiting body, from raking the bacteria to harvesting) distribution diagrams of strain F-G-B1 and strain MH160820. The lower part of the bar chart is the mycelium cultivation period, that is, the mycelium age, and the upper part of the bar chart is the cultivation period. Figure 6 It can be seen that the mycelium age and cultivation period of strain F-G-B1 are shorter than those of MH160820.

[0081] Example 4 Preservation Experiment

[0082] In this example, a preservation experiment was carried out on 3 batches of F-G-B1 and MH160820. After harvesting, the whole mushroom was quickly packaged, with 8 packages for each material, and stored in a cold storage (4°C). Once a week was taken out after the 3rd week to record the quality changes of the fruiting body. The experimental results are shown in Table 2.

[0083] Table 2

[0084]

[0085]

[0086] From the results in Table 2, it can be seen that the preservation period of the fruiting body obtained from strain F-G-B1 is longer than that of strain MH160820, and the average preservation period can reach more than 45 days.

[0087] To sum up, the Grifola frondosa strain Finc-G-B1 provided by the present invention has excellent comprehensive properties and broad market prospects, and has the following advantages:

[0088] 1. The fruiting bodies cultured from the strain have good stability and repeatability, and the differences in the fruiting body traits between batches are small, with strong consistency, ensuring the scale benefits of industrial production;

[0089] 2. The fruiting rate of the fruiting body is high, with a fruiting rate of more than 90% for each batch, high biological conversion efficiency, and good economic benefits;

[0090] 3. The cultivation and growth cycle of the whole fruiting body is relatively short, with an average cycle of ≤ 40 days, which improves the production efficiency of the factory and effectively saves energy and reduces consumption.

[0091] 4. The fruiting body has a long fresh-keeping period. Under the condition of a 4°C cold storage, the average fresh-keeping cycle is ≥ 45 days, which is conducive to long-distance transportation and extends the shelf life.

[0092] The above description is only a description of the preferred embodiments of the present invention, and does not limit the scope of the present invention in any way. Any changes and modifications made by those of ordinary skill in the art of the present invention according to the above disclosure shall fall within the protection scope of the claims.

Claims

1. A Grifola Frondosa strain Finc-G-B1, characterized in that, Named Finc-G-B1, belonging to Grifola frondosa, preserved in Guangdong Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, preservation number GDMCC NO. 62551, preservation date June 20, 2022.

2. An Grifola frondosa spore, characterized in that, Spores obtained after culturing the Grifola frondosa strain Finc-G-B1 described in claim 1.

3. A Grifola frondosa protoplast, characterized in that, Protoplasts obtained after culturing the Grifola frondosa strain Finc-G-B1 described in claim 1.

4. A Grifola frondosa mycelium, characterized in that, Mycelia obtained after culturing the Grifola frondosa strain Finc-G-B1 described in claim 1.

5. An agrocybe cylindracea fruit body, characterized in that, Fruiting bodies obtained after cultivating the Grifola frondosa strain Finc-G-B1 described in claim 1.

6. Application of the Grifola frondosa fruiting body described in claim 5 in food processing.

7. Use of the Grifola frondosa strain according to claim 1 in breeding, characterized in that, Using the Grifola frondosa strain Finc-G-B1 as a parent for breeding.

8. Use of the Grifola frondosa strain according to claim 1 in cross breeding, characterized in that, Using the Grifola frondosa strain Finc-G-B1 as a parent for cross-breeding.

9. Use of the Grifola frondosa strain according to claim 1 in breeding, characterized in that, Performing molecular editing breeding on the Grifola frondosa strain Finc-G-B1.

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