A red-stemmed dictyophora wumeng 2 and application thereof

Abstract

The application discloses a Dictyophora rubrovolvata strain Wumeng No.2 belonging to the field of edible fungi, which is preserved in China Center for Type Culture Collection and has a preservation number of CCTCC NO:M2025781. The fruit body of the strain Wumeng No.2 has the advantages of white mycelium, large flower type, thick and compact fruit body, tight texture, crisp taste and high commodity value. In addition, the cultivation cycle of the strain is short, the cultivation cycle from sowing to the first harvest is only 60 days, the average yield of fresh mushroom reaches 2115kg / 667m 2 , and the selenium content of dried mushroom reaches 0.27mg / kg. The strain also has the advantages of single-mushroom harvest cycle, strong synchronism and the like, and is suitable for factory annual cultivation.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi, specifically relating to a strain of *Dictyophora indicum* and its uses. Background Technology

[0002] Red-topped bamboo fungus (Dictyophora rubrovolvata) belongs to the genus Dictyophora in the family Phallaceae, and is mainly distributed in Guizhou, Sichuan, and Yunnan provinces of my country. Red-topped bamboo fungus has a delicious flavor, a crisp and tender texture, and is rich in amino acids, vitamins, and various minerals. Furthermore, the polysaccharides extracted from red-topped bamboo fungus exhibit anti-cancer activity far superior to that of cordyceps and shiitake mushrooms. Therefore, red-topped bamboo fungus has high commercial and medicinal value. With the improvement of people's living standards, the demand for red-topped bamboo fungus has greatly increased, but the current production capacity is far from meeting the demand.

[0003] Currently, the main problems in the production of red-topped bamboo fungus are: (1) low yield. The current average yield of red-topped bamboo fungus is 500-600 kg / 667 m³. 2 (2) Long cultivation cycle. The cultivation cycle of the main red-topped bamboo fungus strain from sowing to the first harvest is generally 90 to 120 days. The long cultivation cycle means facing more climate risks and production costs. (3) Uneven fruiting and few varieties suitable for industrial production.

[0004] There are abundant wild edible fungi resources, including red-topped bamboo fungus, in southwestern my country, such as Guizhou. Through screening and domestication, it is possible to obtain red-topped bamboo fungus strains with short cultivation cycles, high yields, and excellent quality. This can not only enrich people's dining tables but also create higher economic benefits for mushroom farmers. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Dictyophora indica*.

[0006] Another object of the present invention is to provide the use of the above-mentioned *Dictyophora indicum* strain.

[0007] The third objective of this invention is to provide the molecular markers and methods for identifying the above-mentioned *Dictyophora indicum* strains.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a strain of Dictyophora rubrovolvata, Wumeng No. 2, which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M2025781.

[0009] This invention also provides the application of the above-mentioned strain Wumeng No. 2 in food.

[0010] The present invention also provides a food product containing the above-mentioned strain Wumeng No. 2 or an extract of Wumeng No. 2.

[0011] This invention also provides the application of the above-mentioned strain Wumeng No. 2 in beverages.

[0012] The present invention also provides a beverage containing the above-mentioned strain Wumeng No. 2 or an extract of Wumeng No. 2.

[0013] This invention also provides the application of the above-mentioned strain Wumeng No. 2 in the preparation of drugs that enhance human immunity.

[0014] The present invention also provides a molecular marker for identifying the above-mentioned strain Wumeng 2, wherein the molecular marker is a DNA molecule fragment; the nucleotide sequence of the DNA molecule fragment is shown in SEQ ID NO: 1.

[0015] This invention also provides a method for identifying the above-mentioned strain Wumeng 2, comprising using the genomic DNA of the strain to be tested as a template and universal primers LROR and LR5 as primers for PCR amplification; if the obtained amplification product is a DNA molecule fragment with a nucleotide sequence as shown in SEQ ID NO: 1, it is strain Wumeng 2; wherein the primers are as follows: LROR: 5'-ACCCGCTGAACTTAAGC-3', LR5: 5'-TCCTGAGGGAAACTTCG-3'.

[0016] The advantages and beneficial technical effects of this invention are as follows: (1) The stipe and veil of the red-topped bamboo fungus strain Wumeng No. 2 of this invention are white, cylindrical and hollow, and bell-shaped with an average length of 8.92 cm. The fruiting bodies are large and beautiful, and have high commercial value. (2) The fruiting bodies of strain Wumeng No. 2 of this invention are thick and firm, with an average stipe wall thickness of 7.14 mm, which is thicker than the 4-6 mm thickness of other red-topped bamboo fungus stipe walls. The texture is also dense, and the taste is crisper. In addition, the thick stipe wall is not easy to break, which makes it easier to transport fresh products. (3) strain Wumeng No. 2 of this invention can form a knot in about 10 days after sowing. The fruiting bodies grow rapidly, the fruiting cycle of single-flush mushrooms is concentrated, the strain has strong synchronicity, and it is suitable for factory year-round cultivation and production, which gets rid of the traditional seasonal field cultivation and has high economic value. (4) The cultivation cycle of strain Wumeng No. 2 of this invention is short, with a cultivation cycle of 60 days from sowing to the first harvest of mushrooms, which is shorter than the 90-120 day cultivation cycle of other existing red-topped bamboo fungus strains. (5) Strain Wumeng No. 2 of this invention has a high yield, with an average fresh mushroom yield of 2115 kg / 667 m³. 2 This is significantly higher than other existing red-topped bamboo fungus varieties, which produce 500-600 kg / 667m³. 2(6) The selenium content of the strain Wumeng No. 2 of this invention is high. The selenium content in the dried mushroom of this strain is 0.27 mg / kg, which is much higher than the selenium content of other bamboo fungus, which is 0.04 mg / kg to 0.07 mg / kg. (7) The molecular markers provided by this invention provide accurate and reliable identification results for strain Wumeng No. 2, providing a rapid and reliable identification method for handling infringement matters of Wumeng No. 2.

[0017] Biological Preservation: The *Dictyophora rubrovolvata* strain Wumeng No. 2 of this invention is a new strain obtained by the inventors in April 2019 from wild *Dictyophora rubrovolvata* strains collected in Nayong County, Bijie City, Guizhou Province, and then through screening and domestication. This strain was deposited on April 14, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025781. Attached Figure Description

[0018] Figure 1 Photograph of the fruiting body of the *Dictyophora indicum* strain Wumeng 2 of this invention.

[0019] Figure 2 Phylogenetic tree diagram of the *Dictyophora indicum* strain Wumeng 2 of this invention. Detailed Implementation

[0020] The present invention will be further illustrated by the following examples, but these examples do not constitute any limitation on the present invention.

[0021] Example 1: Breeding process of the *Dictyophora indicum* strain Wumeng 2 of this invention In April 2019, the inventors collected wild *Dictyophora indicum* fruiting bodies in Nayong County, Bijie City, Guizhou Province. Mycelia (strains) were obtained through tissue isolation from the fruiting bodies. The isolated strains were then used for cultivation and fruiting experiments. The strains with good fruiting body shape were further isolated and cultured on a mother culture medium. Strains with fast mycelial growth and uniform growth were selected. The strains were then cultivated and domesticated. After several generations of cultivation and repeated verification of their trait stability, a *Dictyophora indicum* strain with stable genetic traits, short fruiting cycle, high yield, and high nutritional value was finally selected and named: Wumeng No. 2.

[0022] Example 2: Classification and identification of strain Wumeng 2 of the present invention: (1) Morphological identification of strain Wumeng No. 2 of the present invention Red-topped bamboo fungus, Wumeng No. 2: Buds are solitary or clustered, oval, with an average diameter of 48 mm. The mature fruiting body consists of a stipe, cap, veil, and volva. The stipe is white, columnar spongy, tender and brittle, hollow, 12.5–17.8 cm high, 2.31–3.98 cm thick, with an average stipe wall thickness of 7.14 mm, conical to cylindrical. The veil is white, brittle, with an average length of 8.92 cm, and has polygonal meshes, averaging per 25 cm. 2 The veil has 50 pores, and the lower edge of the veil does not easily dry out during the flowering period. The cap is bell-shaped with an internal perforation at the apex, and the spores are attached to the cap surface and appear dark green. The basidiospores are elliptical. Based on the morphological characteristics and photographs of *Dictyophora rubrovolvata* on page 1166 of *Illustrated Handbook of Macrofungi Resources of China* (Li Yu, Li Taihui, et al., Zhongyuan Farmers Publishing House, 2015), Wumeng No. 2 has the same morphological characteristics as *Dictyophora rubrovolvata*. This indicates that the strain Wumeng No. 2 of this invention belongs to the genus *Dictyophora* and species *Dictyophora rubrovolvata*.

[0023] (2) Molecular biological classification and identification of strain Wumeng No. 2 of the present invention Genomic DNA was extracted from strain 2 Wumeng using a plant genomic DNA extraction kit CW0531 (Beijing Kangwei Century Co., Ltd.). Then, using the genomic DNA as a template, PCR amplification was performed using universal primers LROR and LR5. The primers used are as follows: LROR: 5'-ACCCGCTGAACTTAAGC-3', LR5: 5'-TCCTGAGGGAAACTTCG-3'.

[0024] The PCR amplification system was as follows (20 μL): 1 μL of DNA template (20-50 ng / μL), 10× Buffer (with Mg) 2 + 2 μL of 2.5 mM dNTP, 0.5 μL of 5 U / μL DNA polymerase, and 0.5 μL of 0.2 μM primers were added to a final volume of 20 μL with double-distilled water. The reaction conditions were: pre-denaturation at 94℃ for 4 min; 35 cycles of 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 1 min; extension at 72℃ for 10 min. The amplified DNA fragment (also known as the LSU sequence) was 997 bp in length. The fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the nucleotide sequence of the LSU sequence is shown in SEQ ID NO: 1.

[0025] BLAST comparison revealed that the strain Wumeng 2 of this invention had the highest LSU sequence similarity to Dictyophora rubrovolvata, reaching 99.57%. A phylogenetic tree was constructed (see...). Figure 2 As can be seen from the above comparison results, the strain Wumeng No. 2 of the present invention belongs to the species Dictyophora rubrovolvata in the genus Dictyophora, and is a new strain, unlike any known Dictyophora rubrovolvata strain.

[0026] In addition, the above-mentioned DNA molecular fragments can also be used as molecular markers for identifying the strain Wumeng 2 of the present invention, and the identification results are accurate and reliable.

[0027] Example 3: Cultivation experiment of strain Wumeng 2 of the present invention Follow these steps: (1) Mother culture: Using a sterilized and cooled inoculation hook, a 5mm×5mm Wumeng No. 2 strain (the strain of this invention has been deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M2025781) was inoculated in the center of the mother culture medium plate and cultured at 22℃, relative humidity 65-75%, and in the dark for 20 days to obtain the Wumeng No. 2 mother culture. The mother culture medium was prepared as follows: 100g of potatoes were peeled and cut into cubes of about 1cm, boiled in 700 mL of RO water for 30min, filtered through 4 layers of gauze to obtain the extract, and then 5g of glucose, 25g of fructose, 15g of agar powder, 5g of peptone, 2.5g of ammonium sulfate, 20g of corn flour, and 20g of hardwood sawdust (particle size 100 mesh) were added. The volume was adjusted to 1000mL with RO water, sterilized at 121℃ for 30min, and poured into a 9cm diameter plate for later use.

[0028] (2) Seed culture: The mother seed of Wumeng No. 2 obtained in step (1) was inoculated onto the seed culture medium at a mass ratio of 1:30. The culture was carried out at 18℃, relative humidity of 65-75%, and in the dark for 80 days until the bag was full, thus obtaining the original seed of Wumeng No. 2. The composition and weight percentage of the seed culture medium were as follows: 71.5% broad-leaved hardwood sawdust (3-5mm in diameter), 10% corn flour, 10% wheat bran, 0.2% potassium dihydrogen phosphate, 0.3% magnesium sulfate, 5% soybean meal, 1% brown sugar (added after dissolving in water), 1% gypsum, and 1% lime. Water was then added according to the proportion to make the water content of the culture medium 55-60%, and the mixture was stirred evenly. The mixture was then placed into a polypropylene plastic bag (16cm×35cm×0.005cm), tied with a polypropylene rope, sterilized at 121℃ for 2 hours, and cooled for later use.

[0029] (3) Cultivation of spawn: The original spawn of Wumeng No. 2 obtained in step (2) is inoculated onto the spawn culture medium at a mass ratio of 1:30. It is then cultured for 80 days at 18°C, relative humidity of 60-75%, and in the dark. Once the mycelium has fully grown on the spawn bag, the spawn of Wumeng No. 2 is obtained. The composition and preparation method of the spawn culture medium are the same as those of the original spawn culture medium in step (2).

[0030] (4) Cultivation site and sowing: The cultivation site is located in Nayong County, Bijie City, Guizhou Province, and facility-based tiered cultivation is carried out in temperature-controlled greenhouses. The amount of peat moss was calculated, and the moisture content of the peat moss was adjusted to about 70% as required. The peat moss and soil were mixed at a volume ratio of 1:1, and after being stirred evenly with a rotary tiller, it was spread evenly on the tiered shelves. The sowing time was March 15, 2025. The surface of the bags of Wumeng No. 2 spawn obtained in step (3) was cut open with a knife, the bags were removed and the cylindrical spawn sticks were taken out. The spawn sticks were placed along the width of the tiered shelves, parallel to the shelves, with a spacing of 10-12cm between the sticks. An average of 3,000 bags of spawn were placed per mu. After sowing, a layer of soil was evenly covered immediately, with the soil covering thickness 3-4cm higher than the spawn sticks. After the spawn sticks were removed from the bags and covered with soil, the soil was watered in time, and the soil moisture was controlled at 65-75%.

[0031] (5) Bud nurturing and bamboo egg management: After sowing, the mycelium is cultured at a low temperature, initially maintained at 15℃, and increased by 1℃ each day (too rapid an increase will cause severe soil water loss, and the mycelium will react with stress, turning purple and making it susceptible to disease). This process is repeated for 5 days, until the temperature reaches 20℃, at which point the temperature increase is stopped. The culture continues for another 5 days, during which primordia form. In total, it takes 10 days from sowing to the formation of primordia.

[0032] During the growth period of bamboo eggs, the temperature should be 21-23℃, the humidity 80%-85%, and the CO2 concentration should be controlled to ≤2000ppm through internal and external circulation. During the harvest period, the key is space moisture management. Bamboo eggs require a large amount of space moisture to flower; otherwise, the bamboo eggs will dry out and crack, and the bamboo flowers will break. At this time, the frequency of humidification is artificially controlled to ensure the space humidity. The cultivation continues until the bamboo eggs mature, and this stage of cultivation lasts for 50 days.

[0033] (6) Harvesting mushrooms By May 15, 2025, mature bamboo fungus eggs will sprout into fruiting bodies from the top. The process from the fruiting body breaking through the bamboo egg to maturity takes only a few hours. The first harvest is carried out when the veil is exposed at one-third of the cap length. A total of 4 flushes of mushrooms can be harvested. The flushes are quick, with distinct flushes, and each flush is about 20 days apart.

[0034] (7) Observe and weigh the sub-entities.

[0035] Results (see) Figure 1The fruiting bodies of strain Wumeng No. 2 of this invention have white stipes and veils. The stipes are cylindrical and hollow, and the veils are bell-shaped with large fruiting bodies, resulting in high commercial value. The fruiting bodies are thick and firm, with an average stipe wall thickness of 7.14 mm, which is thicker than the 4-5 mm thickness of other existing red-topped bamboo fungus strains. This results in a denser texture, a crisper taste, and greater consumer appeal. The thicker stipe wall also makes it less prone to breakage, facilitating the transportation of fresh produce. The average veil length is 8.92 cm, which is relatively long and contributes to its high commercial value. Furthermore, the fruiting is uniform, with a short cycle, and performs exceptionally well at temperatures of 15-28℃, demonstrating promising prospects for industrial application. It is suitable for year-round cultivation and has high economic value.

[0036] As can be seen from steps (4) to (6) above, the primordia of Wumeng No. 2 appear early, and the fruiting bodies can form knots in as little as 10 days after soil covering and planting. The fruiting bodies grow rapidly, making it suitable for factory-scale cultivation and production. The strains exhibit strong synchronicity. The first harvest can be obtained 60 days after sowing. The turnover of the flush is rapid, and the number of flushes is distinct, with each flush occurring approximately 20 days apart, resulting in a total of 4 flushes of mushrooms. The final fresh mushroom yield can reach 2115 kg / 667 m³. 2 .

[0037] The above results indicate that, compared with other existing *Dictyophora indica* strains, the *Wumeng No. 2* strain of this invention has a shorter time for primordia to appear after sowing, a shorter cultivation cycle from sowing to the first harvest, and a higher yield.

[0038] Example 4: Selenium content detection test of strain Wumeng 2 of the present invention The following method was used: the fruiting bodies of Wumeng No. 2 harvested in Example 3 were dried with hot air to obtain dried mushrooms; then the selenium content was determined according to the standard GB 5009.93-2017 "National Food Safety Standard - Determination of Selenium in Food".

[0039] The results showed that the selenium content of the dried mushroom strain Wumeng No. 2 of this invention was 0.27 mg / kg, which is much higher than the selenium content of other existing red-topped bamboo fungus strains (0.04 mg / kg to 0.07 mg / kg). This indicates that the dried mushroom strain Wumeng No. 2 of this invention has a high selenium content, high nutritional value, and high commercial value.

Claims

1. A strain of Dictyophora rubrovolvata, Wumeng No. 2, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2025781.

2. The application of the strain Wumeng No. 2 as described in claim 1 in food.

3. A food product, characterized in that, The food contains the strain Wumeng No. 2 as described in claim 1 or an extract of Wumeng No.

2.

4. The application of the strain Wumeng No. 2 as described in claim 1 in beverages.

5. A beverage, characterized in that, The beverage contains the strain Wumeng No. 2 as described in claim 1 or an extract of Wumeng No.

2.

6. The application of the strain Wumeng No. 2 as described in claim 1 in the preparation of drugs that enhance human immunity.

7. A molecular marker for identifying strain Wumeng 2 of claim 1, characterized in that, The molecular marker is a DNA molecule fragment; the nucleotide sequence of the DNA molecule fragment is shown in SEQ ID NO:

1.

8. A method for identifying the strain Wumeng 2 as described in claim 1, comprising using the genomic DNA of the strain to be tested as a template and universal primers LROR and LR5 as primers for PCR amplification; if the obtained amplification product is a DNA molecule fragment with a nucleotide sequence as shown in SEQ ID NO: 1, it is identified as strain Wumeng 2; wherein the primers are as follows: LROR: 5'-ACCCGCTGAACTTAAGC-3', LR5: 5'-TCCTGAGGGAAACTTCG-3'.

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