A red-stemmed dictyophora wumeng no. 1 and application thereof

Abstract

The application discloses a Dictyophora rubrovolvata strain Wumeng No.1 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:M2025780. The fruit body of the strain Wumeng No.1 has the advantages of white mycelium, large flower type, thick and compact fruit body, short time of appearing primordium, rapid growth of fruit body, concentrated single-mushroom fruiting period, strong synchronism, short cultivation period, high yield and high selenium content, and the like. The fresh mushroom yield of the strain can be up to 2025 kg / 667 m 2 The selenium content of the dried mushroom can be up to 0.224 mg / kg, and the strain has high nutritional value.

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 longer cultivation cycle means facing more climate risks and higher production costs. (3) Few varieties are suitable for factory cultivation.

[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 a molecular marker and method for identifying the above-mentioned *Dictyophora indicum* strain.

[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. 1, which is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO:M2025780 and deposit date of April 14, 2025.

[0009] This invention also provides the application of the above-mentioned strain Wumeng No. 1 in food.

[0010] The present invention also provides a food product containing the above-mentioned strain Wumeng No. 1 or an extract of Wumeng No. 1.

[0011] This invention also provides the application of the above-mentioned strain Wumeng No. 1 in beverages.

[0012] The present invention also provides a beverage containing the above-mentioned strain Wumeng No. 1 or an extract of Wumeng No. 1.

[0013] The present invention also provides a molecular marker for identifying the above-mentioned strain Wumeng 1, wherein the molecular marker is a DNA molecule fragment; the nucleotide sequence of the DNA molecule fragment is shown in SEQ ID NO: 1.

[0014] This invention also provides a method for identifying the above-mentioned strain Wumeng No. 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 Wumeng No. 1 strain; wherein the primers are as follows: LROR: 5'-ACCCGCTGAACTTAAGC-3', LR5: 5'-TCCTGAGGGAAACTTCG-3'.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial technical effects: (1) The red-topped bamboo fungus strain Wumeng No. 1 of the present invention has a white stipe and veil, a cylindrical and hollow stipe, a bell-shaped veil, and a large fruiting body; its fruiting body is thick and firm, with an average stipe wall thickness of 7.11 mm, which is thicker than the 5-6 mm thickness of other red-topped bamboo fungus, with a dense texture, crisp taste, and thick stipe walls that are not easily broken, making it easier to transport fresh products; its veil has an average length of 91.29 mm, a beautiful fruiting body, and high commercial value. (2) The strain Wumeng No. 1 of the present invention can produce primordia about 10 days after being planted with soil, and the fruiting body grows rapidly. The fruiting cycle of single-flush mushrooms is concentrated, and the strain has strong synchronicity, making it suitable for year-round industrial cultivation and production, getting rid of the traditional seasonal field cultivation, and having high economic value. (3) Short cultivation cycle. The cultivation cycle of strain Wumeng No. 1 of this invention from sowing to the first harvest of mushrooms is 60 days, which is significantly shorter than the 90-120 day cultivation cycle of other existing red-topped bamboo fungus varieties. (4) Strain Wumeng No. 1 of this invention has a high yield. Its average fresh mushroom yield can reach 2025 kg / 667 m³. 2 It is significantly higher than other red-topped bamboo fungus varieties, which have a yield of 500-600 kg / 667m³. 2(5) The selenium content of the dried mushroom of the strain of the present invention is 0.224 mg / kg, which is much higher than the selenium content of other red-topped bamboo fungus strains of 0.04 mg / kg to 0.07 mg / kg. (6) The molecular markers provided by the present invention for identifying strain Wumeng No. 1 are accurate and reliable, providing a rapid and reliable identification method for handling the infringement identification of strain Wumeng No. 1.

[0016] Biological Preservation: The *Dictyophora rubrovolvata* strain Wumeng No. 1 of this invention is a new strain obtained by the inventors in April 2020 from wild *Dictyophora rubrovolvata* fruiting bodies collected in Nayong County, Bijie City, Guizhou Province, and subsequently screened and domesticated. This strain was deposited on April 14, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M2025780. Attached Figure Description

[0017] Figure 1 Photograph of the fruiting body of the *Dictyophora indicum* strain Wumeng 1, as described in this invention. Figure 2 Phylogenetic tree diagram of the *Dictyophora indicum* strain Wumeng 1 of this invention. Detailed Implementation

[0018] The present invention will be further illustrated by the following examples, but these examples do not constitute any limitation on the present invention.

[0019] Example 1: Breeding process of the *Dictyophora indicum* strain Wumeng No. 1 of this invention. In April 2020, the inventors collected wild *Dictyophora indicum* fruiting bodies in Nayong County, Bijie City, Guizhou Province. Mycelia (strains) were obtained through tissue isolation of the fruiting bodies. The strains obtained after tissue isolation 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 rich nutritional value was finally selected and named: Wumeng No. 1.

[0020] Example 2: Classification and identification of strain Wumeng 1 of the present invention (1) Morphological identification of Wumeng No. 1: The morphological characteristics of *Wumeng No. 1* are as follows: The buds are solitary or clustered, ovoid, with an average diameter of 52.51 mm. The mature fruiting body consists of a stipe, cap, veil, and volva. The stipe is white, columnar-spongy, tender and brittle, hollow, 12–22 cm high, and 2.9–3.9 cm thick, with an average stipe wall thickness of 7.11 mm, conical to cylindrical. The veil is white, brittle, with an average length of 91.29 mm, and has polygonal meshes with a mesh diameter of 2–7 mm, averaging per 25 cm. 2 The veil has an area of ​​44.5 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), the morphological characteristics of strain Wumeng No. 1 of this invention are consistent with those of *Dictyophora rubrovolvata*. This indicates that strain Wumeng No. 1 of this invention belongs to the genus *Dictyophora* and species *Dictyophora rubrovolvata*.

[0021] (2) Molecular biological classification and identification of Wumeng No. 1 Genomic DNA was extracted from strain 1 of the Wumeng plant strain using a plant genomic DNA extraction kit CW0531 (Beijing Kangwei Century Co., Ltd.). Then, PCR amplification was performed using the genomic DNA as a template and universal primers LROR and LR5. The universal primers are as follows: LROR: 5'-ACCCGCTGAACTTAAGC-3' LR5: 5'-TCCTGAGGGAAACTTCG-3' The PCR amplification system (20 μL) consisted of: 1 μL of DNA template at a concentration of 20-50 ng / μL, and 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. A DNA fragment of 923 bp (also known as the LSU sequence) was amplified and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The nucleotide sequence of this DNA fragment is shown in SEQ ID NO: 1.

[0022] BLAST alignment in GeneBank revealed that the LSU sequence of strain Wumeng 1 of this invention showed the highest similarity to the LSU sequence of Dictyophora rubrovolvata, with a similarity of up to 99.78%. A phylogenetic tree was constructed (see...). Figure 2 As can be seen from the above comparison results, the strain Wumeng No. 1 of the present invention belongs to the species Dictyophora rubrovolvata in the genus Dictyophora, and is a new strain, unlike any other existing Dictyophora rubrovolvata strains.

[0023] In addition, the specific DNA fragments of this strain can also be used as molecular markers for rapid identification of Wumeng No. 1, and the identification results are accurate and reliable.

[0024] Example 3: Cultivation experiment of the red-topped bamboo fungus strain Wumeng No. 1 of the present invention Follow these steps: (1) Mother culture: Using a sterilized and cooled inoculation hook, inoculate a 5mm×5mm strain of Wumeng No. 1 (the strain of this invention, Wumeng No. 1, has been deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025780) into the center of the mother culture medium plate. Culture for 20 days at 22℃, relative humidity of 65-75%, and in the dark to obtain the Wumeng No. 1 mother culture. The mother culture medium is prepared as follows: Peel 100g of potatoes and cut them into cubes of about 1cm. Boil them in 700 mL of RO water for 30min. Filter the extract through 4 layers of gauze. Add 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 (100 mesh). Make up the volume to 1000mL with RO water. Sterilize at 121℃ for 30min. Pour the solution into a 9cm diameter plate for later use.

[0025] (2) Seed culture: The mother seed of Wumeng No. 1 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. 1. 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 mixed 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.

[0026] (3) Cultivation of the spawn: The original spawn of Wumeng No. 1 obtained in step (2) is inoculated onto the spawn culture medium at a mass ratio of 1:30. The spawn is cultured for 80 days at 18°C, relative humidity of 60-75%, and in the dark. When the mycelium has fully grown into the spawn bag, the spawn of Wumeng No. 1 is obtained. The composition and preparation method of the spawn culture medium are the same as those of the original spawn culture medium described in step (2).

[0027] (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 for use. The specific sowing time was March 20, 2024. The surface of the spawn bags obtained in step (3) was cut open with a knife, the cylindrical spawn sticks were removed from the bags, and the spawn sticks were placed along the width of the tiered shelves, with the spawn sticks parallel to the shelves and the spacing between the sticks 10-12cm. An average of 3,000 spawn bags 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%.

[0028] (5) Bud nurturing and bamboo egg management: After sowing, cultivate the mycelium at a low temperature, initially maintaining 15℃, increasing it 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). Stop increasing the temperature when it reaches 20℃; continue cultivation for 5 days, allowing the mycelium to twist and form primordia. This stage, from sowing to the formation of primordia, takes 10 days.

[0029] During the growth period of bamboo eggs, the temperature should be 21-23℃, the humidity 80%-85%, and the carbon dioxide 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 process continues until the bamboo eggs mature, and this stage of cultivation takes 50 days.

[0030] (6) Harvesting mushrooms By May 20, 2024, mature bamboo fungus eggs will grow 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 will be carried out when the veil is exposed to one-third of the length of the cap.

[0031] (7) Observe and weigh the sub-entities.

[0032] Results (see) Figure 1This invention relates to the *Dictyophora indica* strain Wumeng No. 1, whose fruiting bodies have white stipes and veils. The stipes are cylindrical and hollow, while the veils are bell-shaped and the fruiting bodies are relatively large. The fruiting bodies are thick and firm, with an average stipe wall thickness of 7.11 mm, which is thicker than the 5-6 mm thickness of other *Dictyophora indica* strains. This results in a denser texture, a crisper taste, higher commercial value, and greater consumer appeal. Furthermore, the thick stipe wall makes it less prone to breakage, facilitating the transportation of fresh produce. The veils have an average length of 91.29 mm, a beautiful shape, and high commercial value.

[0033] During cultivation, the strain Wumeng No. 1 of this invention exhibits robust mycelium, strong adaptability, high tolerance to environmental changes, strong resistance to contamination by miscellaneous bacteria, and uniform fruiting.

[0034] As can be seen from steps (4) to (6) above, the primordia of strain Wumeng No. 1 of this invention appear early, about 10 days after soil covering and planting, while other red-topped bamboo fungus strains usually require 25 to 45 days to appear. The fruiting bodies of this strain grow rapidly, and the strain has strong synchronicity, making it suitable for industrialized cultivation and production.

[0035] The strain Wumeng No. 1 of this invention requires only 60 days from sowing to the first harvest, a significantly shorter cultivation cycle than other *Dictyophora indica* strains which have a cultivation cycle of 90–120 days. Furthermore, this strain exhibits rapid flushing, with distinct flushes approximately 20 days apart, resulting in a cumulative harvest of four flushes. The strain also boasts a high fresh mushroom yield, reaching up to 2025 kg / 667 m². 2 The yield of fresh mushrooms was significantly higher than that of other existing red-topped bamboo fungus strains.

[0036] Example 4: Selenium content determination test of strain Wumeng 1 of the present invention Follow these steps: The fruiting bodies of Wumeng No. 1 obtained in Example 3 were dried with hot air to obtain dried Wumeng No. 1 mushrooms; then the selenium content of dried Wumeng No. 1 mushrooms was determined according to the method of GB5009.93-2017 "National Food Safety Standard - Determination of Selenium in Food".

[0037] The results showed that the selenium content of the dried mushroom strain Wumeng No. 1 was 0.224 mg / kg, which is much higher than the selenium content of other *Dictyophora indica* strains (0.04 mg / kg to 0.07 mg / kg). This indicates that the dried mushroom strain Wumeng No. 1 has a high selenium content, resulting in high nutritional and commercial value.

Claims

1. A type of red-topped bamboo fungus ( Dictyophora rubrovolvata The strain Wumeng No. 1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M2025780.

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

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

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