CN115895903A - Novel russule strain and application thereof - Google Patents

Abstract

The invention belongs to the field of edible fungi, and particularly relates to a new russula strain and application thereof. The classification name of the new strain of russula is pseudo-basidiolus (A) and (B)Pseudolagarobasidium acaciicola) The culture medium was deposited at 24.4.2022 with the culture Collection of microorganisms of Guangdong province, accession number GDMCC No.62414. The russula new strain GDMCC No.62414 provided by the invention can be cultivated artificially, has the characteristics of large fruiting quantity, high yield, good mushroom shape and the like, and is a russula new strain with great development potential.

Description

Novel russule strain and application thereof

Technical Field

The invention belongs to the field of edible fungi, and particularly relates to a new russula strain and application thereof.

Background

The russula pileus is like a dish, is thicker, has dark red or dark purple at the center of the top, is darker than the periphery of the pileus, and has the diameter of 3.5-6 cm; the mushroom stem is nearly cylindrical, thick, short and solid, and 3.5-6 cm long, and few mushroom feet have worm mouths; the fungus folds are radial and thick, and are gray and slightly dark blue; the color of the pileus fades after the fruit body is soaked and is light red brown, but red water leached by the pileus is not uniform when dispersed and is bright and transparent.

In terms of nutritional value, the Ming Dynasty ' Bamintongzhi ' lists the russula to the vegetable, has edible and medicinal values, rich nutrition, fat and tender meat quality, fresh and sweet taste, and is a good name of ' pure natural high-grade wild mountain delicacies ' in China '. In recent years, the russula sporocarp is found to contain rich amino acids, polysaccharides, various mineral elements essential to human body, and various components such as fatty acid, sterol and the like. The russula contains 16 amino acids, essential amino acids account for 54.4% of the total amino acids, the polysaccharide content is 2.74%, and the russula contains various fatty acids such as oleic acid, linoleic acid and the like. The russule also contains 17 amino acids, the total content of the amino acids is 26.53%, and the russule also contains 7 mineral substances necessary for human bodies, such as Zn, fe, si, cu, mn, ni and Cr. The russula is also a natural biological medicine good product, and the polysaccharide in the russula can reduce the blood cholesterol content, so the russula becomes an ideal health product for people with hypertension and hyperlipidemia. Most russules exhibit a characteristic mushroom flavor because they contain specific volatile components. The russula volatile substance contains acids, esters, hydrocarbons and heterocyclic derivatives, is the main component of the Chinese herbal medicine volatile oil of equisetum hiemale, fructus forsythiae and herba houttuyniae, and has pharmacological effects of clearing heat, removing toxicity, inhibiting bacteria and the like. Part of russule has the effects of oxidation resistance, beautifying and nourishing, aging resistance and the like, and related experiments prove that: the russula sporocarp contains antioxidant substances, and the damage of free radicals to an antioxidant system is relieved by participating in intracellular antioxidation, so that oxidative damage caused by formaldehyde inhalation is effectively repaired. The other part of russula species contains anticancer substances, and ergosterol compounds are separated from russula fruiting body, and have important effects of resisting cancer, suppressing immunity, promoting platelet aggregation, etc.

In the aspect of economic value, since russula can not be cultivated artificially at present, the yield is relatively limited, but the price is increased year by year. For example, the yield of russula vinosa of the professional cooperative society of the eight peak natural fungus industry in the county is about 2000 yuan per kilogram in 2011 and is increased to 3000 yuan in 2012; the sale price of russula vinosa from shoyuncu in Mingxi county is about 3000 yuan per kg in 2011 and is increased to 4000 yuan in 2012. Sanming city in Fujian province is one of the main producing areas of russula, and twelve counties (cities and districts) have rich russula resources. About 5333 hm of the current full-market russula vinosa forest land, the annual output is nearly 40t. If the method can be used for managing and cultivating 40 ten thousand mu of russula forest land, the yield of russula per mu (dry mushroom) is calculated according to 1 kilogram and 4000 yuan per kilogram, the yield value can be more than 16 hundred million annually, and the method is a huge under-forest economic growth project and has a very wide development prospect.

However, the russula fungus is ectomycorrhizal fungi, the forest land capable of growing the russula needs to have a certain proportion of Fagaceae trees, the population drop is difficult to cultivate and copy, and the special growth environment and the physiological characteristics of the russula fungus are difficult to form a fruit body under artificial conditions, so that large-scale artificial cultivation cannot be carried out. The artificial propagation promoting method can improve the yield of the russula, and the soil with the russula is transplanted to the soil with similar vegetation and without russula, so that the mycelium of the russula is transplanted, and suitable microorganisms are carried, because root microorganisms play an important role in the aspects of participating in substance metabolism or promoting growth of ectomycorrhizal fungi and mycorrhizal plants, but if only the mycelium of the russula is added, sporocarp is difficult to form. The invention separates and purifies a new russula strain which can be artificially cultured from the soil in the russula growing area so as to provide a research foundation for the subsequent research of the growth conditions and the growth mechanism of the russula and the large-scale artificial culture of the russula and lay a foundation stone for the popularization of the russula.

Disclosure of Invention

The invention aims to provide a new russula strain and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention firstly provides a new strain of russula, the classification name of the new strain of russula is pseudo-clavulans (Pseudolagarobasidium acaciicola) The culture is registered and preserved in Guangdong province microorganism culture collection center 24.4.2022, the preservation address is No. 59, no. 5, building of Michelia Tokyo No. 100, guangzhou city, and the preservation number is GDMCC No.62414.

The new russula strain is collected from Changkou village in le county in Sanming City of Fujian province by the inventor, and has the following characteristics:

morphological characteristics of mycelium: the colony on the PDA solid culture medium is white, mellow and smooth, has no obvious smell, and has luxuriant aerial hyphae which are milky white;

morphological characteristics of fruiting bodies: the pileus is red, and the average diameter is 10.23cm; the stipe is red, nearly cylindrical, and is grown in the center, and the average length is 5.13cm; the mushroom meat is white and the meat quality is thick; the hypha radiate radially and are gray white and slightly dark blue.

Molecular biological characteristics: the ITS sequence is shown as SEQ ID NO.1, and is identified as pseudo-clavulans through comparison analysis in Genbank database (Pseudolagarobasidium acaciicola）；

The yield characteristics are as follows: the fruiting quantity is 1185/mu, the fresh weight yield is 30.67 kg/mu, and the dry weight yield is 3.7 kg/mu.

The new russula strain can be applied to artificial cultivation of russula.

The new russula strain can be applied to russula breeding.

The new russula strain can be applied to food or medicine production.

The invention has the following remarkable advantages:

the russule is rich in various nutritional ingredients such as oleic acid, linoleic acid, amino acid, polysaccharide, vitamin and the like, has extremely high nutritional value and delicious taste, also has various health-care effects of enriching blood, strengthening body, delaying senility, nourishing blood, beautifying, preventing and resisting cancer, reducing cholesterol and the like, and is rare pure natural health-care edible fungus. Because of the special conditions and physiology required by the growth of the russula, the russula is mostly collected in wild and can not be planted manually, and only an artificial propagation promoting method is adopted to transplant the soil with the russula to similar vegetation and the soil without russula, so that the russula is transplantedThe mycelium of the mushroom is also accompanied by suitable microorganisms, because the root microorganisms play an important role in the participation of the ectomycorrhizal fungi and mycorrhizal plants in the aspects of substance metabolism or growth promotion, etc., but if only the mycelium of the russula vinosa is added, the fruiting body is often difficult to form. The invention separates a new russula strain from the soil in the growth area of russula from Changkoucun, san Ming city, fujian province, and the new russula strain is identified as pseudo-basidiomycetes (A. Pseudopodophyllum)Pseudolagarobasidium acaciicola) The accession number of the culture collection of microorganisms of Guangdong province is GDMCC No.62414. The invention inoculates the mycelium of russula GDMCC No.62414 to the soil without russula, the russula fruiting body can occur, and the mushroom shape is good, and the fruiting quantity, the fresh weight yield and the dry weight yield are respectively improved by 38.34%, 51.53% and 74.53% compared with the control group. As can be seen from the above, the russula vinosa GDMCC No.62414 provided by the invention is a new russula vinosa strain which can be artificially cultivated and has great development potential, and is beneficial to the artificial cultivation and popularization of the subsequent russula vinosa fruiting body.

Drawings

FIG. 1 is the DNA agarose gel electrophoresis of russula GDMCC No.62414.

FIG. 2 is a colony morphology diagram of russula GDMCC No.62414 on PDA medium.

FIG. 3 is a morphological diagram of fruiting body of russula GDMCC No.62414.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Example 1 isolation, purification and Classification of novel Rhododendron strain

The novel russula strain is deposited in the Guangdong province microbial strain preservation center with the preservation number of GDMCC No.62414, the preservation date of 2022, 4 months and 24 days, and the preservation address of No. 59 Lou 5 Lou of Michelia Zhonglu 100 Mr. The method for collecting the new russula strains comprises the following steps:

1) Soil sample collection: randomly selecting three parts in the growth area of russula vinosa in Chankou village of le county in Sanming City of Fujian province, and randomly digging three parts of soil with the depth of 0.2-0.3 m below the ground into a preservation bag for separating and screening strains;

2) Preparation of a culture medium: preparing a PDA solid culture medium according to a conventional method;

3) Separating soil fungi by a dilution plate method: preparing a soil dilution solution by using sterile water to obtain soil solutions with different dilutions; spreading soil solutions with different dilutions on a prepared PDA solid culture medium, and performing inversion culture at constant temperature of 22 ℃ for 2-3d in the dark; observing colony morphology, selecting single pure colonies with different morphologies, performing multiple streaking separation on a PDA solid culture medium, determining the pure colonies as pure strains, inoculating and storing in the PDA solid culture medium, and storing in a refrigerator at 4 deg.C after the culture medium is full of mycelia;

4) And (3) morphological characteristic observation: the colony of GDMCC No.62414 on PDA solid culture medium is white, mellow and smooth, has no obvious smell, and has luxuriant aerial hypha and milky white color (figure 1);

5) Molecular biological identification:

extracting mycelium DNA of the new russula strain GDMCC No.62414 as a template, and carrying out PCR amplification on ITS sequences of the strain by using ITS universal primers, wherein the primers are as follows:

forward primer ITS1:5 'TCCGTAGGTGAACCTGCGG-3',

reverse primer ITRS4:5 'TCCTCCGCTTATTGATATGC-3'.

The total volume of the PCR amplification reaction system is 10 mul: 1 mu l of DNA template, 1 mu l of BigDye Terminator v3.1 mu l of forward and reverse primers (3.2 mu M) and 1 mu l of ddH ₂ O 6µl。

The PCR amplification reaction conditions were as follows:

the amplified product was subjected to 1% agarose gel electrophoresis (FIG. 2), and sequenced, resulting in an ITS sequence fragment of 638bp (SEQ ID NO. 1). Comparing the sequencing result with other strains in the Genbank database, and identifying the strains as pseudo-pot cross armsBacteria (A), (B)Pseudolagarobasidium acaciicola）。

Example 2 Pseudoceromyces pseudo-Alternaria (C.), (Pseudolagarobasidium acaciicola) Artificial cultivation experiment of GDMCC No.62414

Selecting the following steps: a forest land which is suitable for the growth of the russula vinosa but has not generated the russula vinosa is selected from Chang Kou village of le county in san Ming city of Fujian province, and is divided into two parts with the same size (10 mx10 m) for subsequent artificial cultivation test.

GDMCC No.62414 hypha inoculation test: inoculating GDMCC No.62414 into PDA solid culture medium, culturing at 22 deg.C in dark, and storing in 4 deg.C refrigerator after the culture medium is full of mycelia; selecting a proper amount of hyphae from a pseudoalterobasidium plate colony stored at 4 ℃ to scribe on a PDA solid culture medium, culturing for 2-3d in a constant temperature incubator at 22 ℃, then selecting a proper amount of hyphae on a PDA culture substrate on which the hyphae are newly grown by using a sterilization gun head, adding the hyphae into 500 microliter PDA liquid culture medium, carrying out shaking culture for 12h at 22 ℃ and 220r/min, then sucking 50-100 microliter culture solution, inoculating into 500ml PDA liquid culture medium, homogenizing for 5min by using a floor centrifuge (4000 r/min), preparing hyphae suspension, and adjusting the concentration of the hyphae suspension to OD ₆₀₀ Is 1.0; digging soil of the sample plot by using an iron shovel, uniformly pouring the hypha suspension on the soil according to the depth of 0.2-0.3 m according to 1.5L/sample plot, then backfilling all the dug soil in the original place, and observing whether the russula sporocarp can occur.

Control test: diluting the soil sample used for separating and purifying GDMCC No.62414 in example 1, inoculating the diluted soil sample to a PDA liquid culture medium, and performing shaking culture at 22 ℃ and 220r/min for 12h to obtain a mixed bacterial liquid; then 50-100 microliters of the mixed bacterial liquid is sucked and inoculated into a new PDA liquid culture medium, homogenate is carried out for 5min by a floor centrifuge (4000 r/min), and the concentration of the mixed bacterial liquid is adjusted to OD ₆₀₀ Is 1.0; and (3) digging soil of the sample plot by using an iron shovel, uniformly irrigating the mixed bacterial liquid on the soil according to the depth of 0.2 to 0.3 m in 1.5L/sample plot, then backfilling the dug soil in situ, and observing whether russula sporocarp can occur.

The inoculation time is 6 late months of the lunar calendar, and the hyphae inoculated with different treatments are subjected to fruiting quantity and quality statistics in 8 middle months of the lunar calendar, and the yield and quality of the fruiting bodies of russula vinosa are analyzed and compared in a GDMCC No.62414 hypha inoculation test and a comparison test.

Inoculation of GDMCC No.62414 mycelium can result in russula fruiting bodies (fig. 3), pileus being red; stipe is red, nearly cylindrical, and is grown in the center; the mushroom meat is white and the meat quality is thick; the funguses are radial and gray with slight dark blue.

Compared with the control test (table 1), the average diameter of the canopy of the russula vinosa fruiting body generated by the inoculation of the GDMCC No.62414 mycelium is increased by 16.38 percent, the average length of the stem of the russula vinosa fruiting body is increased by 5.56 percent, the fruiting number is increased by 38.43 percent, the single fresh weight is increased by 9.43 percent, the single dry weight is increased by 25.81 percent, the fresh weight per mu is increased by 51.53 percent, and the dry weight per mu is increased by 74.53 percent.

To sum up, pseudomonadaceae (Amycolatopsis pseudomonadacea) (II)Pseudolagarobasidium acaciicola) GDMCC No.62414 is a new strain of russula vinosa with good artificial cultivation character and great development potential.

TABLE 1 statistical results of the Artificial cultivation experiment

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (5)

1. A new russule strain is characterized in that: the classification name of the new strain of russula is pseudo-basidiolus (A) and (B)Pseudolagarobasidium acaciicola) The culture medium has been registered and preserved in Guangdong provincial microorganism culture collection center at 24.4.2022, with the preservation address of No. 59, no. 5, building of Michelia Tokyo No. 100, guangzhou city, and the preservation number of GDMCC No.62414.

2. The novel russule species of claim 1, wherein: the ITS sequence of the new russula species is shown as SEQ ID NO. 1.

3. The use of the novel russula species of claim 1 in artificial cultivation of russula.

4. The use of a new species of russula as claimed in claim 1 in russula breeding.

5. Use of a new species of russula as claimed in claim 1 in the production of a food or pharmaceutical product.

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