A new species of russula and application thereof

Abstract

The present application belongs to the field of edible fungi, and particularly relates to a new species of Russula and application thereof. Pseudolagarobasidium acaciicola The taxonomic name of the new species of Russula is Pseudoguignardia sp., which has been registered and preserved in the Guangdong Microbial Culture Collection Center on April 24, 2022, and the preservation number is GDMCC No. 62414. The new species of Russula GDMCC No. 62414 provided in the present application can be artificially cultivated, and has the characteristics of large number of fruiting bodies, high yield, good mushroom shape and the like, and is a new species of Russula with great development potential.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi, specifically relating to a new strain of Russula and its applications. Background Technology

[0002] The cap of the red mushroom is shaped like a bowl or saucer, relatively thick, with a deep red or dark purple center at the top, slightly darker than the surrounding area, and a diameter of 3.5-6 cm. The stem is nearly cylindrical, short and thick, relatively solid, and 3.5-6 cm long, with a few mushrooms having insect bites at the base. The gills are radial, relatively thick, and grayish-white with a slight dark blue tinge. After soaking, the cap color fades to a light reddish-brown, but the red liquid extracted from the cap is not uniformly distributed and is bright and transparent.

[0003] In terms of nutritional value, the Ming Dynasty's *Eight Min General Records* classified red mushrooms as vegetables, highlighting their edible and medicinal value. Rich in nutrients, with tender, sweet, and delicious flesh, they are renowned as a "pure, natural, high-grade wild mountain delicacy of China." Recent studies have found that red mushroom fruiting bodies are rich in amino acids, polysaccharides, various essential minerals, and also contain various fatty acids and sterols. Red mushrooms contain 16 amino acids, with essential amino acids accounting for 54.4% of the total amino acids. The polysaccharide content is 2.74%, and they contain various fatty acids such as oleic acid and linoleic acid. Red mushrooms also contain 17 amino acids, with a total amino acid content of 26.53%, and seven essential minerals: Zn, Fe, Si, Cu, Mn, Ni, and Cr. Red mushrooms are also a valuable natural medicinal product; their polysaccharides can lower blood cholesterol levels, making them an ideal health supplement for people with hypertension and hyperlipidemia. Most red mushrooms exhibit a unique mushroom aroma due to their special volatile components. The volatile substances in *Russula rubra* contain acids, esters, hydrocarbons, and heterocyclic derivatives, and are the main components of the volatile oils of traditional Chinese medicinal herbs such as *Ephedra sinica*, *Forsythia suspensa*, and *Houttuynia cordata*, possessing pharmacological effects such as clearing heat, detoxifying, and inhibiting bacteria. Some *Russula rubra* species have antioxidant, beautifying, and anti-aging effects. Related experiments have shown that the fruiting bodies of *Russula rubra* contain antioxidants that, by participating in intracellular antioxidant processes, reduce the damage of free radicals to the antioxidant system and effectively repair oxidative damage caused by inhaled formaldehyde. Other *Russula rubra* species contain anticancer substances; ergosterol compounds have been isolated from the fruiting bodies of *Russula rubra*, which have important functions such as anticancer activity, immunosuppression, and promoting platelet aggregation.

[0004] In terms of economic value, the production of red mushrooms is currently limited due to the inability to cultivate them artificially, but the price has been rising year by year. For example, the red mushrooms from the Bafeng Natural Fungus Professional Cooperative in Datian County sold for about 2,000 yuan per kilogram in 2011, rising to 3,000 yuan per kilogram in 2012; the red mushrooms from Ziyun Village in Mingxi County sold for about 3,000 yuan per kilogram in 2011, rising to 4,000 yuan per kilogram in 2012. Sanming City in Fujian Province is one of the main production areas of red mushrooms, with abundant red mushroom resources in all twelve counties (cities and districts). Currently, the city has about 5,333 hectares of red mushroom forests, with an annual output of nearly 40 tons. If 400,000 mu of red mushroom forests can be managed and cultivated, with a yield of 1 kilogram of dried red mushrooms per mu and a price of 4,000 yuan per kilogram, the annual output value could exceed 1.6 billion yuan, representing a huge economic growth project under the forestry sector with very broad development prospects.

[0005] However, Russula fungi are ectomycorrhizal fungi, and the woodlands where Russula can grow require a certain proportion of Fagaceae tree species. Such communities are difficult to cultivate and replicate, and their unique growth environment and physiological characteristics make it difficult for them to form fruiting bodies under artificial conditions, thus hindering large-scale artificial cultivation. Artificial propagation methods can increase Russula yield by transplanting soil already containing Russula to soil with similar vegetation that has never produced Russula. This not only transplants the Russula mycelium but also introduces suitable microorganisms. Root microorganisms play a crucial role in ectomycorrhizal fungi and mycorrhizal plants in terms of metabolism and growth promotion, but simply adding Russula mycelium often fails to form fruiting bodies. This invention isolates and purifies a new Russula strain suitable for artificial cultivation from the soil of Russula growing areas. This provides a research foundation for further investigation into the growth conditions and mechanisms of Russula, as well as for large-scale artificial cultivation, laying the groundwork for the promotion of Russula. Summary of the Invention

[0006] The purpose of this invention is to provide a new strain of Russula and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention first provides a new strain of Russula, the taxonomic name of which is *Pseudocytrophus*. Pseudolagarobasidium acaciicola It was registered and deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 24, 2022. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 62414.

[0009] The aforementioned new strain of *Russula rubra* was collected by the inventor in Changkou Village, Jianle County, Sanming City, Fujian Province. This new strain of *Russula rubra* has the following characteristics:

[0010] Mycelial morphological characteristics: Colonies on PDA solid medium are white, round and smooth, with no obvious odor, and the aerial mycelia are abundant and milky white;

[0011] Fruiting body morphological characteristics: The cap is red, with an average diameter of 10.23 cm; the stipe is red, nearly cylindrical, centrally located, with an average length of 5.13 cm; the flesh is white and relatively thick; the gills are radial, grayish-white with a slight dark blue tinge.

[0012] Molecular biological characteristics: The ITS sequence is shown in SEQ ID NO.1. After comparison and analysis in the Genbank database, it was identified as *Pseudocybata*. Pseudolagarobasidium acaciicola );

[0013] Yield characteristics: The number of mushrooms produced is 1185 per mu, the fresh weight yield is 30.67 kg per mu, and the dry weight yield is 3.7 kg per mu.

[0014] The aforementioned new strain of Russula can be applied to the artificial cultivation of Russula.

[0015] The aforementioned new strain of Russula can be applied to Russula breeding.

[0016] The aforementioned new strain of Russula can be used in food or pharmaceutical production.

[0017] The significant advantages of this invention are:

[0018] Red mushrooms are rich in oleic acid, linoleic acid, amino acids, polysaccharides, vitamins, and other nutrients, making them highly nutritious and delicious. They also possess various health benefits, including blood-tonifying, body-strengthening, anti-aging, blood-nourishing, skin-beautifying, anti-cancer, and cholesterol-lowering effects, making them a rare and valuable all-natural health-promoting edible fungus. Due to the specific conditions and physiological characteristics required for red mushroom growth, they are mostly collected wild and cannot be artificially cultivated. Artificial propagation methods involve transplanting soil already containing red mushrooms into similar vegetation but soil where red mushrooms have never grown before. This not only transplants the red mushroom mycelium but also introduces suitable microorganisms. Root microorganisms play a crucial role in the metabolism and growth promotion of ectomycorrhizal fungi and mycorrhizal plants, but simply adding red mushroom mycelium often fails to form fruiting bodies. This invention, however, isolated a new strain of red mushroom from the soil of the red mushroom growing area in Changkou Village, Jianle County, Sanming City, Fujian Province, which was identified as *Pseudohypophyllariae*. Pseudolagarobasidium acaciicolaThe strain, with accession number GDMCC No. 62414 at the Guangdong Provincial Center for Microbial Culture Collection, was used in this invention to inoculate the mycelium of *Russula rubra* GDMCC No. 62414 into soil where *Russula rubra* had never grown before. This resulted in the formation of *Russula rubra* fruiting bodies with good shape, and the number of fruiting bodies, fresh weight yield, and dry weight yield increased by 38.34%, 51.53%, and 74.53%, respectively, compared to the control group. Therefore, the *Russula rubra* GDMCC No. 62414 provided by this invention is a new *Russula rubra* strain that can be artificially cultivated and has great development potential, which is beneficial for the subsequent artificial cultivation and promotion of *Russula rubra* fruiting bodies. Attached Figure Description

[0019] Figure 1 DNA agarose gel electrophoresis image of Russula ovata GDMCC No. 62414.

[0020] Figure 2 This is a colony morphology diagram of Russula griseus GDMCC No. 62414 on PDA medium.

[0021] Figure 3 This is a morphological diagram of the sub-entity of the red mushroom GDMCC No. 62414. Detailed Implementation

[0022] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] Example 1: Isolation, purification, classification, and identification of a new strain of Russula.

[0024] The new Russula rubra strain described in this invention has the accession number GDMCCNo.62414 at the Guangdong Provincial Center for Microbial Culture Collection, with an accession date of April 24, 2022. The accession address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The collection method for the new Russula rubra strain is as follows:

[0025] 1) Soil sampling: Three sites were randomly selected in the red mushroom growing area of ​​Changkou Village, Jianle County, Sanming City, Fujian Province. Three soil samples were randomly dug from each site at a depth of 0.2 to 0.3 meters below the ground and placed in a plastic bag for the isolation and screening of fungal strains.

[0026] 2) Preparation of culture medium: Prepare PDA solid culture medium according to conventional methods;

[0027] 3) Isolation of soil fungi by dilution plate method: Prepare soil dilution solutions with sterile water to obtain soil solutions of different dilutions; spread soil solutions of different dilutions onto prepared PDA solid medium and incubate at 22℃ in the dark for 2-3 days; observe colony morphology, pick single pure colonies of different morphologies, and perform multiple streaking isolation on PDA solid medium. After confirming that they are pure strains, inoculate and preserve them in PDA solid medium. After the medium is covered with mycelia, store them in a refrigerator at 4℃.

[0028] 4) Morphological observation: GDMCC No. 62414 colonies on PDA solid medium are white, round, smooth, and odorless, with abundant aerial hyphae that appear milky white. Figure 1 );

[0029] 5) Molecular biological identification:

[0030] Genomic DNA was extracted from the mycelium of the novel Russula vinifera strain GDMCC No. 62414 as a template. The ITS sequence of the strain was amplified by PCR using universal ITS primers. The primers used are as follows:

[0031] Forward primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3',

[0032] Reverse primer ITRS4: 5'-TCCTCCGCTTATTGATATGC-3'.

[0033] The total volume of the PCR amplification reaction system was 10µl: 1µl DNA template, 2µl BigDye® Terminator v3.1, 1µl each of forward and reverse primers (3.2µM), and 6µl ddH2O.

[0034] The PCR amplification reaction conditions are as follows:

[0035]

[0036] The amplification products were subjected to 1% agarose gel electrophoresis ( Figure 2 The sequence was then sequenced, and the resulting ITS sequence fragment was 638 bp (SEQ ID NO.1). The sequencing results were compared with other bacterial species in the Genbank database, and the species was identified as *Pseudocytrophomyces*. Pseudolagarobasidium acaciicola ).

[0037] Example 2: Pseudo-pochaete bacteria ( Pseudolagarobasidium acaciicola Artificial cultivation experiment of GDMCC No. 62414

[0038] Plot selection: A suitable forest land for the growth of Russula ovata, but which has not yet been planted in Changkou Village, Jianle County, Sanming City, Fujian Province, was selected and divided into two parts of the same size (10m x 10m) for subsequent artificial cultivation experiments.

[0039] GDMCC No. 62414 mycelial inoculation test: The mother culture GDMCC No. 62414 was inoculated into PDA solid medium and incubated at 22℃ in the dark under inverted conditions. After the medium was fully colonized with mycelia, it was stored at 4℃ for later use. A suitable amount of mycelia was picked from the *Pseudomonas sylvestris* agar colonies stored at 4℃ and streaked onto PDA solid medium. After incubation at 22℃ for 2-3 days, a suitable amount of mycelia from the newly grown mycelia on the PDA medium plate was picked using a sterilized pipette tip and added to 500 μL of PDA liquid medium. The medium was incubated at 22℃ and 220 rpm with shaking for 12 h. Then, 50-100 μL of the culture solution was inoculated into 500 ml of PDA liquid medium and homogenized using a centrifuge (4000 rpm) for 5 min to prepare a mycelial suspension. The concentration of the mycelial suspension was adjusted to OD0.05. 600 The value is 1.0; the soil of the sample plot is dug up with an iron shovel to a depth of 0.2~0.3 meters, and the mycelial suspension is evenly poured onto the soil at a rate of 1.5L / plot. Then, the dug-up soil is completely backfilled into the original place, and it is observed whether red mushroom fruiting bodies can occur.

[0040] Control experiment: The soil sample used for the isolation and purification of GDMCC No. 62414 in Example 1 was diluted and inoculated into PDA liquid medium. After shaking culture at 22℃ and 220r / min for 12h, a mixed bacterial solution was obtained. Then, 50-100 μL of the mixed bacterial solution was inoculated into a new PDA liquid medium, homogenized in a floor centrifuge (4000r / min) for 5min, and the concentration of the mixed bacterial solution was adjusted to OD. 600 The value is 1.0; the soil of the sample plot is dug up with an iron shovel to a depth of 0.2~0.3 meters, and the mixed bacterial solution is evenly poured onto the soil at a rate of 1.5L / plot. Then, all the dug-up soil is backfilled into the original place, and it is observed whether red mushroom fruiting bodies can occur.

[0041] Inoculation was carried out in late June of the lunar calendar. In mid-August, the number and quality of fruiting bodies of the mycelium inoculated with different treatments were statistically analyzed and compared between the GDMCC No.62414 mycelium inoculation experiment and the control experiment.

[0042] GDMCC No. 62414 mycelial inoculation can produce Russula fruiting bodies ( Figure 3 The cap is red; the stem is red, nearly cylindrical, and centrally located; the flesh is white and relatively thick; the gills are radial, grayish-white with a slight dark blue tinge.

[0043] Compared with the control experiment (Table 1), the average diameter of the cap of the fruiting bodies of *Russula ovata* inoculated with GDMCC No. 62414 mycelium increased by 16.38 percentage points, the average length of the stem increased by 5.56 percentage points, the number of fruits increased by 38.43 percentage points, the fresh weight of a single fruiting body increased by 9.43 percentage points, the dry weight of a single fruiting body increased by 25.81 percentage points, the fresh weight per mu increased by 51.53 percentage points, and the dry weight per mu increased by 74.53 percentage points.

[0044] In summary, *Pseudocytrophus* ( Pseudolagarobasidium acaciicola GDMCC No. 62414 is a new Russula strain with excellent artificial cultivation characteristics and great development potential.

[0045] Table 1 Statistical results of artificial cultivation experiments

[0046]

[0047] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A new species of Russula, characterized by: The taxonomic name of the said Russula new species is Pseudoguignardia sp. Pseudolagarobasidium acaciicola , which has been deposited in the Guangdong Microbial Culture Collection Center on April 24, 2022, at 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100, with the deposit number of GDMCC No. 62414.

2. The new species of Russula as claimed in claim 1 is applied to artificial cultivation of Russula.

3. The new species of Russula as claimed in claim 1 is applied to breeding of Russula.

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