Pleurotus nebrodensis strain as well as sporocarp and application thereof
By conducting single-single hybrid breeding of olive mushrooms and yellow white elixir mushrooms, a new variety with strong temperature adaptability, short production cycle, high mushroom yield rate and high bioconversion rate was obtained, which solved the problems of poor temperature adaptability and low production efficiency of existing white elixir mushrooms, and achieved efficient cultivation and large-scale promotion of white elixir mushrooms.
Patent Information
- Application Number
- CN202510188589.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-04-08
AI Technical Summary
The existing white ling mushrooms have poor temperature adaptability, long production cycle, low mushroom yield and low bioconversion rate, resulting in high cultivation costs, low economic efficiency, and limited resources, which cannot be promoted on a large scale.
Through hybrid breeding technology, the almond mushroom and yellow white lingmushroom were selected as parents, and single-single hybrid selection was carried out to obtain a new variety with strong temperature adaptability, short production cycle, high mushroom yield rate and high bioconversion rate - Xinghe white lingmushroom No. 1.
The temperature adaptability of white ling mushrooms has been improved, the production cycle has been shortened, and the mushroom yield and bioconversion rate has been significantly improved, which has reduced cultivation costs, improved economic efficiency, and increased resource utilization.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and more specifically to a Pleurotus nebrodensis strain, a fruiting body thereof and applications thereof. Background Art
[0002] The existing white mushroom (Pleurotus eryngii var.tuoliensis) has the following defects: (1) White mushroom has poor temperature adaptability and high requirements for cultivation conditions. It needs low temperature stimulation and temperature change to accelerate budding, and cannot be promoted on a large scale. White mushroom is cultured at a temperature of about 24°C. After 40 to 50 days, it will grow all over the cultivation material and enter the post-ripening stage. The temperature is lowered to 20 to 22°C to allow it to continue to grow and accumulate nutrients. After another 30 to 35 days of cultivation, low temperature stimulation is carried out. Stimulation is carried out at a low temperature of 0 to 3°C for 7 to 14 days, or the temperature is kept at 0 to 15°C, with a temperature difference of 10 to 15°C between day and night, and stimulation is carried out for 10 to 15 days. (2) The entire production cycle is long, requiring 90 to 100 days. (3) The fruiting rate of Pleurotus nebrodensis sticks is only 60-70%, and the bioconversion rate is only 40%. The fruiting rate is low. The existing patent has a fruiting rate of 100%, but the conversion rate is only 80%, which causes a waste of resources, makes the economic efficiency of cultivating Pleurotus nebrodensis low, and affects the production enthusiasm of growers. (4) The resources of wild Pleurotus nebrodensis are limited, and it cannot form a yield. The cultivation cost is high and cannot be promoted to ordinary people. (5) The color of the Pleurotus nebrodensis cap is commonly light yellow and white, and the color is single. Therefore, it is urgent to cultivate a new variety of Pleurotus nebrodensis with good temperature adaptability, low requirements for cultivation conditions, short production cycle, high fruiting rate and high bioconversion rate. Summary of the invention
[0003] In order to solve the above technical problems, the present invention utilizes hybrid breeding technology to carry out single-single hybrid breeding of Pleurotus eryngii and yellow Pleurotus nebrodensis, and obtains a new variety of brown Pleurotus nebrodensis No. 1, which has strong temperature adaptability, low requirements for cultivation conditions, short production cycle, high mushroom yield, high bioconversion rate, and striped cap.
[0004] The first aspect of the present invention provides a Pleurotus eryngii var.tuoliensis strain, whose deposit number is GDMCC No:64079.
[0005] The second aspect of the present invention further provides a Pleurotus eryngii fruiting body, which is obtained by cultivating the Pleurotus eryngii strain described in the first aspect of the present invention.
[0006] In a preferred embodiment, the cap length of the fruiting body is 4 to 8 cm, such as 4 cm, 5 cm, 7 cm or 8 cm.
[0007] In a preferred embodiment, the length of the fruiting body stipe is 4 to 8 cm, for example, 4 cm, 5 cm, 7 cm or 8 cm.
[0008] In a preferred embodiment, the cap of the Pleurotus nebrodensis strain is brown with stripes.
[0009] A third aspect of the present invention provides a Pleurotus nebrodensis mycelium, which is obtained by culturing the Pleurotus nebrodensis strain described in the first aspect of the present invention.
[0010] A fourth aspect of the present invention provides a Pleurotus eryngii bacterial liquid, which is obtained by culturing the Pleurotus eryngii strain described in the first aspect of the present invention.
[0011] The fifth aspect of the present invention provides a method for culturing the Pleurotus nebrodensis fruiting body as described in the second aspect of the present invention, comprising culturing and cultivating the Pleurotus nebrodensis strain on a cultivation medium.
[0012] The sixth aspect of the present invention provides a method for culturing the Pleurotus nebrodensis mycelium as described in the third aspect of the present invention or the Pleurotus nebrodensis bacterial liquid as described in the fourth aspect of the present invention, which comprises culturing the Pleurotus nebrodensis strain in a liquid culture medium or a solid culture medium.
[0013] Another aspect of the present invention further provides the use of the Pleurotus nebrodensis strain as described in the first aspect of the present invention in preparing any one of Pleurotus nebrodensis spores, Pleurotus nebrodensis mycelium, Pleurotus nebrodensis liquid and Pleurotus nebrodensis fruiting bodies.
[0014] Another aspect of the present invention also provides the use of the Pleurotus eryngii fruiting body as described in the third aspect of the present invention in food processing.
[0015] On the other hand, the present invention also provides the use of the Pleurotus eryngii strain as described in the first aspect of the present invention, the Pleurotus eryngii fruiting body as described in the second aspect of the present invention, the Pleurotus eryngii mycelium as described in the third aspect of the present invention, or the Pleurotus eryngii liquid as described in the fourth aspect of the present invention in the preparation of any one of immunity enhancing drugs, cholesterol lowering drugs, anti-aging drugs, analgesic drugs, sedative drugs, cough and phlegm-relieving drugs, laxative and detoxification drugs and blood pressure lowering drugs.
[0016] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0017] Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.
[0018] The positive and progressive effects of the present invention are:
[0019] (1) The present invention provides a new Pleurotus eryngii strain, whose cap is brown with spots, which is obviously different from existing strains. The mycelium of the strain grows fast. After the mycelium is fully grown in 20 to 40 days, primordia can be formed without low temperature stimulation or temperature change to promote budding. The strain has strong adaptability to temperature, which reduces the difficulty of cultivation technology.
[0020] (2) The entire production cycle of the present invention is short, the cultivation of mycelium takes 20 to 40 days, the cultivation of mushrooms takes 13 to 20 days, and one cultivation cycle is completed in 33 to 60 days.
[0021] (3) The present invention achieves a mushroom yield rate of 100% and a bioconversion rate of 90-100%, thereby saving production costs, improving mass production, and greatly increasing the economic efficiency of Pleurotus nebrodensis cultivation and the production enthusiasm of growers.
[0022] Biomaterial Deposit Information
[0023] The Pleurotus eryngii var.tuoliensis strain of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on November 28, 2023. The deposit address is: 5th Floor, Building 59, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 64079. The culture name is Pleurotus eryngii var.tuoliensis. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is a photo of the fruiting body of Xinghe Bailing Mushroom No. 1.
[0025] Figure 2 This is a photo of the fruiting body of Pleurotus eryngii, the parent of Xinghe Bailing Mushroom No. 1.
[0026] Figure 3 This is a photo of the fruiting body of the yellow pleurotus eryngii, the parent of Xinghe pleurotus eryngii No. 1.
[0027] Figure 4 It is a schematic diagram of antagonistic reaction, where number 1 represents Xinghe Bailing Mushroom No. 1.
[0028] Figure 5 It is the technical route for bacterial strain identification and detection. DETAILED DESCRIPTION
[0029] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The data collection and analysis methods of the following examples are as follows:
[0030] Example 1 Origin process of Xinghe Bailing Mushroom No. 1
[0031] (1) Parent selection: Pleurotus eryngii and Pleurotus eryngii were selected as parents. Pleurotus eryngii is the fruiting body of Pleurotus eryngii brought back by the inventor from Guangdong Muhe Ecological Agriculture Development Co., Ltd. in December 2021. The parent strain Pleurotus eryngii strain with good mushroom production was obtained by fruiting body tissue separation. Pleurotus eryngii is the fruiting body of Pleurotus eryngii purchased by the inventor in Japan in December 2018. The parent strain Pleurotus eryngii strain with good mushroom production was obtained by fruiting body tissue separation.
[0032] (2) Single spore collection: Select fruiting bodies with good mushroom shape and caps about to open, cut the caps with gills from the edge of the cap to the place where the stipe is attached, hang them in a sterile and dry triangular flask, and culture them in the dark for 1 to 3 days at a temperature of 20 to 22° C. and a relative humidity of 60 to 70%. After white spore powder imprints are produced, spore dilution and plate coating operations are performed. The coated plates are cultured at a constant temperature and in the dark at a temperature of 20 to 22° C. and a relative humidity of 60 to 70%. The temperature is kept at 20 to 22° C. and the relative humidity is 60 to 70%. The single spores are collected. When the daughter hyphae grow to 0.5-1 cm, plate transfer culture is carried out, and the culture is carried out at a constant temperature and light-proof at a temperature of 20-22° C. and a relative air humidity of 60-70%. The culture is carried out for 10-20 days, and an advantageous plate with fast growth is selected for test tube transfer. At the same time, the hyphae of the plate that has completed the transfer is examined under a microscope. If there is no lock-like joint structure under the microscope, it is a single spore. The single spore test tube continues to be cultured at a constant temperature and light-proof at a temperature of 20-22° C. and a relative air humidity of 60-70%. After the hyphae cover the inclined surface, they are preserved at a storage temperature of 4-10° C.
[0033] (3) Single-spore hybridization: Pair the single spore of Pleurotus eryngii with the single spore of Pleurotus eryngii for single-single hybridization. Cut the edge of the activated single spore colony and inoculate it into a plate. The two inoculation blocks are separated by 3 cm and placed 1.5 cm from the center of the plate, respectively, with the mycelium facing upward. The culture is carried out at a constant temperature and in the dark at a temperature of 20-22° C. and a relative humidity of 60-70%. After the mycelium of the two single spore colonies come into contact, the culture is continued in the dark for 5-10 days.
[0034] (4) Hybrid strains: The hyphae at the junction of two monosporic colonies were selected for microscopic examination, and the plates with lock-like joint structures were transferred to the plates with the hyphae at the junction. The plates were cultured at a constant temperature and in the dark at a temperature of 20-22°C and a relative humidity of 60-70%. The culture was carried out for 10-15 days, and the dominant plates with fast growth were selected for test tube transfer. At the same time, the hyphae of the plates that have completed the transfer were examined under a microscopic examination. The hybrid strains with lock-like joint structures under the microscopic examination were continued to be cultured in the test tubes at a constant temperature and in the dark at a temperature of 20-22°C and a relative humidity of 60-70%. After the hyphae have covered the slope, they were preserved at a storage temperature of 4-10°C.
[0035] (5) Cultivation and screening: The hybrid strain obtained was subjected to cultivation experiments and screening according to conventional methods. After fruiting test, a new variety of white mushroom with strong temperature adaptability, low requirements for cultivation conditions, short production cycle, high fruiting rate, high bioconversion rate, and brown white mushroom with stripes on the cap was obtained - Xinghe White Mushroom No. 1.
[0036] Example 2 Morphological characteristics and biological properties
[0037] (1) Morphological characteristics: The fruiting body photo of Xinghe Bailing Mushroom No. 1 is as follows Figure 1 When Xinghe Bailing Mushroom No. 1 matures, the fruiting body has a white stipe, which is subcentral or lateral, rod-shaped, and 4 to 8 cm long. The gills are brown, the cap is brown with spots, the cap has a hard texture, and the cross-section is fan-shaped or lateral, 4 to 8 cm long, 4 to 8 cm wide, and 2 to 3 cm thick. The fruiting body photos of Xinghe Bailing Mushroom No. 1's parents, Pleurotus eryngii and Yellow Pleurotus eryngii, can be found at Figure 2 , Figure 3 ,Depend on Figure 1 , Figure 2 , Figure 3 It can be seen that the color and pattern of the cap of Xinghe Bailing Mushroom No. 1 are obviously different from those of Pleurotus eryngii and yellow Pleurotus eryngii, which shows that the Xinghe Bailing Mushroom No. 1 strain of the present invention is a new strain that is different from its parent in terms of morphological characteristics.
[0038] (2) Biological characteristics
[0039] Nutrition: The main nutrients needed are carbohydrates, proteins, cellulose, hemicellulose, vitamins, minerals, etc.
[0040] Temperature: The mycelium culture temperature is 20-25℃, and the mushroom cultivation temperature is 15-25℃.
[0041] Light: Mycelium does not require light, and the light intensity required during the fruiting period is 0 to 800 lux.
[0042] Moisture: The relative humidity of the air in mycelium culture is 60-70%, the moisture content of the mycelium culture medium is 60-65%, and the relative humidity of the air in the cultivation room is 70-90%.
[0043] Air: Good ventilation and fresh air are required, with a carbon dioxide concentration of 1000-3000ppm.
[0044] Time requirement: It usually takes 33 to 60 days from cultivation inoculation to completion of mushroom production.
[0045] Example 3 Antagonistic reaction
[0046] Testing basis: Inoculation, cultivation and observation of samples to be tested are carried out in accordance with the agricultural industry standard of the People's Republic of China "NY / T1845-2010 Differential Identification and Antagonistic Reaction of Edible Fungi Species".
[0047] The Xinghe Pleurotus eryngii No. 1 strain obtained in Example 1 was subjected to an antagonistic reaction with the parent Pleurotus eryngii strain and the yellow Pleurotus eryngii strain.
[0048] (1) Culture medium: PDA medium (potato dextrose agar medium), 200 g potato (with extract), 20 g glucose, 20 g agar, 1000 ml water, natural pH, introduced into a culture dish and cooled into a flat plate.
[0049] (2) Inoculation and culture: The inoculation combinations were 5 pairs, Pleurotus eryngii and Pleurotus eryngii, Yellow Pleurotus eryngii and Yellow Pleurotus eryngii, Xinghe Pleurotus eryngii No. 1 and Xinghe Pleurotus eryngii No. 1, Xinghe Pleurotus eryngii No. 1 and Pleurotus eryngii, Xinghe Pleurotus eryngii No. 1 and Yellow Pleurotus eryngii. Strictly follow the aseptic operation, cut the 0.2 cm × 0.2 cm inoculation block from the edge of the activated bacterial colony with an inoculation shovel, inoculate one inoculation block each time, with a 3 cm interval between the two inoculation blocks, and place them 1.5 cm from the center of the plate, with the mycelium facing upwards, with 3 replicates in each group, and culture them in confrontation, at a temperature of 24-26°C, with ventilation and light protection for 14 days.
[0050] (3) Observation and judgment of antagonistic reactions
[0051] Under the light, observe the hyphae reaction at the junction of the two strains on the culture surface. The antagonistic reaction results are shown in Figure 4 . The mycelium on the culture surfaces of Pleurotus eryngii and Pleurotus eryngii, Yellow Pleurotus eryngii and Yellow Pleurotus eryngii, Xinghe Pleurotus eryngii No. 1 and Xinghe Pleurotus eryngii No. 1 did not show any of the three reactions of ridge type, groove type and isolation type, indicating that Pleurotus eryngii, Yellow Pleurotus eryngii and Xinghe Pleurotus eryngii No. 1 themselves will not show antagonistic reactions. Xinghe Pleurotus eryngii No. 1 and Pleurotus eryngii, Xinghe Pleurotus eryngii No. 1 and Yellow Pleurotus eryngii showed isolation type reactions on the culture surfaces of Pleurotus eryngii, Xinghe Pleurotus eryngii No. 1 and Yellow Pleurotus eryngii, and both antagonistic reactions indicate that Xinghe Pleurotus eryngii No. 1, Pleurotus eryngii and Yellow Pleurotus eryngii are different varieties.
[0052] Example 4 Bacterial species identification and detection
[0053] The whole genomes of Xinghe Bailing Mushroom No. 1 and Xinghe Bailing Mushroom No. 2 (with the accession number of GDMCC No: 64080), the parent yellow Bailing Mushroom, and Bailing Mushroom were sequenced, and the sequencing results were compared with the Bailing Mushroom genome on NCBI (GENE ID NO: ASM3687298v1). Bailing Mushroom is the fruiting body of Kitajima Bailing Mushroom purchased by the inventor in Japan in December 2018. The Bailing Mushroom strain with good mushroom production was obtained through fruiting body tissue separation.
[0054] (1) The experimental instruments, reagents and consumables involved are shown in Table 1.
[0055] Table 1 Main instruments, reagents and consumables
[0056]
[0057] (2) The technical route used is shown in Figure 5 .
[0058] (3) Genome comparison results
[0059] The sequencing results of Xinghe Bailing Mushroom No. 1, Xinghe Bailing Mushroom No. 2, the parent yellow Bailing Mushroom, and Bailing Mushroom were compared with the Bailing Mushroom genome on NCBI. The results are shown in Table 2. The comparison found that the comparison rates of all samples were between 81.39% and 93.87%, all higher than 75% and the data had large differences, indicating that the four samples all belonged to the Bailing Mushroom species, but did not belong to the same strain. The genome comparison results directly indicate that the Xinghe Bailing Mushroom No. 1 strain of the present invention belongs to the Bailing Mushroom species, and is a new strain different from Xinghe Bailing Mushroom No. 2, the parent yellow Bailing Mushroom, and Bailing Mushroom.
[0060] Table 2 Genome alignment
[0061]
[0062] Note: (1) Sample: sample name.
[0063] (2) Mapped reads: The number of reads mapped to the reference (including single-end and double-end alignments).
[0064] (3)Total reads: The total number of reads of valid sequencing data.
[0065] (4) Mapping rate: the number of reads mapped to the reference genome divided by the number of reads in the effective sequencing data.
[0066] (5) Average depth: Average sequencing depth, the total number of bases aligned to the reference genome divided by the size of the genome covered.
[0067] (6) Coverage at least 1X: The percentage of sites in the reference genome that are covered by at least 1 base.
[0068] (7) Coverage at least 4X: The percentage of sites in the genome that are covered by at least 4 bases in the reference genome.
[0069] Example 5 Cultivation Experiment
[0070] Xinghe Bailing Mushroom No. 1 and its parents, Pleurotus eryngii and Yellow Bailing Mushroom, were cultivated in bottles to produce mushrooms.
[0071] (1) Plate culture: The bacteria were inoculated on a PDA plate and cultured at a constant temperature of 20-25° C. and a relative humidity of 60-70% for 10-15 days to obtain plate bacteria.
[0072] (2) Liquid primary seed culture: The plate strain obtained in step (1) is inoculated into a triangular flask liquid culture medium and cultured in the dark at a temperature of 20 to 25° C. for 5 to 10 days to obtain a liquid primary seed. The triangular flask liquid culture medium is formulated as follows: 5% glucose, 5% white sugar, 0.5% soybean meal, 0.5% peptone, 0.2% magnesium sulfate, 0.2% potassium dihydrogen phosphate, and natural pH.
[0073] (3) Liquid secondary seed culture: The liquid primary seed obtained in step (2) is inoculated into a fermentation tank liquid culture medium and cultured in the dark at a temperature of 20 to 25° C. for 5 to 10 days to obtain a liquid secondary seed. The formula of the fermentation tank liquid culture medium is the same as that of the flask liquid culture medium.
[0074] (4) Inoculation and culture: The liquid secondary seed is inoculated into the culture medium of the culture bottle and cultured in the dark, the culture temperature is 20-25°C, the relative humidity of the air is 60-70%, the carbon dioxide concentration is 2000-3000ppm, and the mycelium is fully grown in the culture bottle after 40 days of culture. The culture medium of the culture bottle is formulated as follows: 21% corn cobs, 20% sawdust, 19% cottonseed hulls, 19% bran, 16% rice bran, 5% corn flour, 64.5% water content, and 530g material weight.
[0075] (5) Management of mushroom production: Open the bottle mouth, remove the mushroom skin on the surface of the material, and transfer it to the mushroom room. The culture temperature is 15-25°C, the relative humidity of the air is 70-90%, and the carbon dioxide concentration is 1000-3000ppm. The light intensity is set to 0-100 lux in the first 1-7 days, and then set to 200-800 lux. Turn on the shelf lights intermittently every day. After 16-23 days of cultivation, the fruiting body caps are fully expanded, but the edges of the caps are still slightly curled, and then harvested.
[0076] The results of the bottle cultivation experiment of Xinghe Bailing Mushroom No. 1 and its parents, Pleurotus eryngii and Pleurotus eryngii, are shown in Table 3. The photos of their respective fruiting bodies are shown in Figure 1 , Figure 2 , Figure 3 .
[0077] Table 3 Cultivation experimental results of Xinghe Bailing Mushroom No. 1 and its parents
[0078]
[0079] As shown in Table 3, under the same culture cycle and control parameters, Xinghe Bailing Mushroom No. 1 has the shortest total production cycle, the highest bioconversion rate, and the fruiting rate is the same as that of Pleurotus eryngii, which can reach 100%. Yellow Bailing Mushroom has the longest total production cycle, the lowest fruiting rate and bioconversion rate. Figure 1 , Figure 2 , Figure 3 It can be seen that the cap colors and patterns of the three mushrooms are different. This shows that the Xinghe Bailing Mushroom No. 1 strain of the present invention is a new strain different from the parent, has obvious advantages in mushroom yield and bioconversion rate, and has a short cultivation period.
Claims
1. A Pleurotus eryngii var. tuoliensis strain, whose deposit number is GDMCC No: 64079.
2. A Pleurotus eryngii fruiting body, characterized in that: The Pleurotus nebrodensis fruiting body is obtained by cultivating the Pleurotus nebrodensis strain according to claim 1.
3. The Pleurotus eryngii fruiting body according to claim 2, characterized in that The cap of the fruiting body is 4 to 8 cm in length, and / or the stipe is 4 to 8 cm in length, and / or the cap is brown with stripes.
4. A Pleurotus nebrodensis mycelium, characterized in that: The Pleurotus nebrodensis mycelium is obtained by culturing the Pleurotus nebrodensis strain according to claim 1.
5. A white mushroom liquid, characterized in that: The Pleurotus nebrodensis liquid is obtained by culturing the Pleurotus nebrodensis strain as claimed in claim 1.
6. The method for cultivating the fruiting body of Pleurotus eryngii according to claim 2 or 3, characterized in that: The method comprises culturing and cultivating the Pleurotus nebrodensis strain on a cultivation medium.
7. The method for culturing the Pleurotus nebrodensis mycelium according to claim 4 or the Pleurotus nebrodensis liquid according to claim 5, characterized in that: The method comprises culturing the Pleurotus nebrodensis strain on a liquid culture medium or a solid culture medium to obtain the strain.
8. Use of the Pleurotus nebrodensis strain according to claim 1 in preparing any one of Pleurotus nebrodensis spores, Pleurotus nebrodensis mycelium, Pleurotus nebrodensis liquid and Pleurotus nebrodensis fruiting bodies.
9. Use of the Pleurotus nebrodensis fruiting body as claimed in claim 2 or 3 in food processing.
10. Use of the Pleurotus eryngii strain according to claim 1, the Pleurotus eryngii fruiting body according to claim 2 or 3, the Pleurotus eryngii mycelium according to claim 4, or the Pleurotus eryngii liquid according to claim 5 in the preparation of any one of immunity enhancing drugs, cholesterol lowering drugs, anti-aging drugs, analgesic drugs, sedative drugs, cough and phlegm-relieving drugs, laxative and detoxifying drugs, and antihypertensive drugs.