CN113444650B

CN113444650B - Pleurotus pulmonarius and application thereof

Info

Publication number: CN113444650B
Application number: CN202110719877.6A
Authority: CN
Inventors: 陈躬国
Original assignee: FUZHOU INSTITUTE OF AGRICULTURAL SCIENCES
Current assignee: FUZHOU INSTITUTE OF AGRICULTURAL SCIENCES
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2022-10-14
Anticipated expiration: 2041-06-28
Also published as: CN113444650A

Abstract

The present invention providesPleurotus pulmonarius and application thereof. The Pleurotus pulmonarius strain is P1.P-H215, which is classified and named as Pleurotus pulmonarius (A)Pleurotus pulmonarius) In 15 days 1 month 2021, the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.21450, and the preservation address is No. 3 Xilu No. 1 Beijing Hospital, beijing, chaoyang, north Chen. The strain pileus is brown, the mushroom shape is round, the pileus edge is not wavy after the fruiting body is mature, and the commercial property is good; not easy to grow natural mushroom, regular sprouting after cold stimulation, strong stress resistance and the like, and is more suitable for out-of-season temperature control facility cultivation in summer.

Description

Pleurotus pulmonarius and application thereof

Technical Field

The invention belongs to the technical field of edible fungi, and particularly relates to pleurotus pulmonarius and application thereof.

Background

Lung shaped side ear [ 2 ]Pleurotus pulmonarius (Fr.) Quél.]The product name is Pleurotus geesteranus belonging to Basidiomycetes (Basidiomycetes), agaricales (Agaricales), pleurotaceae (Pleurotaceae), pleurotus (A)Pleurotus). The pleurotus pulmonarius is rich in various proteins, essential amino acids, polysaccharides, important minerals and vitamins, has high nutritive value, also has pharmacological activities of resisting oxidation, reducing blood sugar level, assisting tumor chemotherapy and the like, and has wide market prospect.

At present, the development of the lung-shaped pleurotus good variety breeding research work is still weak, the existing main cultivated varieties are the introduced pleurotus geesteranus series varieties, the variety updating is lagged, and some varieties are degraded after years of introduction and are shown as delayed fruiting period, irregular fruiting, poor disease resistance and the like, so that the yield and quality of the pleurotus geesteranus fluctuate, the breeding research strength of the good varieties, particularly the out-of-season varieties suitable for summer cultivation is not developed enough, and the conditions become important factors for restricting the sustainable development of the lung-shaped pleurotus industry. Development of superior variety breeding of Pleurotus pulmonarius which can satisfy the requirements of facility cultivation and high temperature out-of-season has great significance for promoting sustainable development of industry.

Disclosure of Invention

The invention provides pleurotus pulmonarius and application thereof, aiming at the problems that most main cultivars used by pleurotus pulmonarius are introduced series and some cultivars are degenerated after being introduced for years. The Pleurotus pulmonarius strain provided by the invention can meet the seed demand of Pleurotus pulmonarius facility cultivation, especially high-temperature out-of-season facility cultivation.

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

a Pleurotus pulmonarius strain is P1.P-H215, classified and named as pleuromutilin: (Pleurotus pulmonarius) The culture medium has been preserved in China general microbiological culture Collection center (CGMCC) No.21450 at 15 days 1 month 2021, and the preservation address is No. 3 Siro-1 of Beijing province in the morning of the Yangxi district.

The Pleurotus pulmonarius strain P1.P-H215 is obtained by crossing strain P8 and strain P19 as parents and performing systematic breeding.

The Pleurotus pulmonarius strain P1.P-H215 is used for out-of-season edible fungus facility cultivation in summer.

The invention has the advantages that:

the Pleurotus pulmonarius strain P1.P-H215 has the following excellent properties: the pileus of the fruiting body of the strain P1.P-H215 is brown, the mushroom shape is round, the pileus edge is not wavy after the fruiting body is mature, and the commercial property is good. The temperature difference required by fruiting is large, primordium is not easy to generate when the temperature difference is small, and natural mushroom is not easy to generate; the growth is rapid, the fruiting is dense after cold stimulation, and the tide times are very obvious.

Compared with the existing Pleurotus pulmonarius strain mainly cultivated at present, the strain H215 has the excellent characteristics of high yield, good commodity, difficult natural mushroom growing, regular budding after cold stimulation, strong stress resistance and the like, and is more suitable for anti-season temperature control facility cultivation in summer.

Description of the drawings:

FIG. 1: the strain P1.P-H215 of the invention is used for fruiting bodies and cultivation in summer facilities.

FIG. 2: the strain P1.P-H215 of the invention has different performance with the parent strain P19 fruiting body.

FIG. 3: the strain P1.P-H215 of the invention and two parents thereof have antagonistic reaction test results. Left panel: a front side; right panel: and (4) the reverse side.

FIG. 4: amplification map of primer ME9/Em3 to P1.P-H215 and its parent. M: DL2000 DNA Marker;1: p8;2: p1.P-H215;2: p19, the arrow indicates the specific band of the parent strain.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1 parental mating

Collecting Pleurotus pulmonarius strains in domestic main scientific research units and Pleurotus pulmonarius production areas, identifying and analyzing genetic diversity through molecular markers such as antagonistic reaction test, ITS-RFLP and RAPD, and performing comprehensive evaluation on agronomic characters by using grey correlation analysis evaluation. According to genetic diversity analysis and agronomic character evaluation, parents are selected, strains P8 and P19 with large genetic diversity and excellent agronomic characters are screened out to serve as the parents (Chen Du, chenjian, wuhan' qiong. Pleurotus ostreatus germplasm resource antagonism reaction and RAPD molecular marker genetic diversity analysis [ J ]. Southern agricultural science report, 2020, 51 (12): 2892-2900), genetic single spores are obtained, and single-spore hybridization breeding is developed.

Example 2 Single spore crossbreeding

(1) Selection of seed mushrooms: the parent strains P8 and P19 are cultivated to form mushrooms, when the fruiting bodies grow to seven or eight mature, the fruiting bodies with complete mushroom shapes and strong growth are selected as seed mushrooms, after the seed mushrooms are selected, the small mushrooms in the range of 6 cm-10 cm around are picked, the growth conditions of the seed mushrooms are checked at any time, and when the seed mushrooms grow to eight mature, the small mushrooms are picked in time for single spore separation.

(2) And (3) collecting spores: taking a culture dish with the diameter of 9cm, binding, sterilizing by high-pressure steam, drying, paving parchment on the bottom of the culture dish, cutting off stipe of the seed mushrooms, putting mushroom folds downwards on the parchment, slightly buckling the parchment on the parchment by a dish cover, and allowing a large amount of spores to fall on the parchment after 18-24 hours.

(3) And (3) spore separation: the separation of the spores adopts a continuous dilution method, a certain amount of spores are picked by an inoculating needle under the aseptic condition, injected into 10mL of aseptic water and shaken up, then lmL spore liquid is taken out, added into 9mL of aseptic water to obtain spore suspension, and then diluted by multiple times to diluted spore suspension containing less than 100 spores per milliliter. Sucking 100 mu L of diluted spore suspension by a liquid transfer machine, uniformly coating the diluted spore suspension on a blank PDA plate culture medium, sealing the culture medium by a sealing film, culturing in a constant-temperature incubator at 25 ℃, observing spore germination conditions every day, picking single spore colonies when the colonies are visible by naked eyes, and culturing in the constant-temperature incubator at 25 ℃.

(4) And (3) identifying the mononuclear hyphae: when the colony grows to 2cm in diameter, picking edge hyphae and placing the edge hyphae on a gossypol cotton blue staining solution, observing the edge hyphae by using a microscope, preliminarily regarding the edge hyphae as mononuclear hyphae generated by single spore germination if no latticed combination exists, and then transferring the mononuclear hyphae to a slant for culture.

(5) Single-spore hybridization: inoculating two different parent mononuclear hyphae onto a plate culture medium at the same time, culturing for 10 days at a distance of 1.5cm, after the two hyphaes are contacted, selecting partial hyphae at the junction, dyeing the part of the hyphae with gossypol blue lactate, performing microscopic examination, immediately transferring the hyphae onto a new slant culture medium if finding a locked union, culturing for 7-10 days at 25 ℃, selecting strains with vigorous hyphae growth and uniform colony morphology, selecting a small amount of hyphae, observing the hyphae under a microscope, identifying the hyphae as a binuclear hyphae, performing PDA slant test tube amplification culture, and eliminating the hyphae with nonuniform colony morphology and weaker hyphae growth. After the hyphae are filled in the tube, excellent heterocaryotic hyphae are selected and stored in a refrigerator for screening test.

(6) And (3) screening hybrids: the obtained hybrid is subjected to cultivation test and screening by a conventional method, and finally a new hybrid strain P1.P-H215 (marked as H215 in figure 1) with stable properties, excellent properties and high yield is screened out. The strain P1.P-H215 is high temperature resistant, and the phenomenon of burning the bacteria is not easy to occur in the process of culturing the bacteria; the temperature difference required by fruiting is large, and natural mushrooms are not easy to grow. The pileus is brown, semicircular, round, and good in commodity property. The mushroom is dense and has obvious tide times after cold stimulation, and the yield is high.

Example 3 cultivation test

The Pleurotus pulmonarius strain P1.P-H215 and parent strains P8 and P19 are subjected to out-of-season temperature control facility cultivation tests in summer, each strain is cultivated for 100 bags, and then the results of small-batch cultivation are evaluated and analyzed, so that the advantages and the characteristics of the Pleurotus pulmonarius strain P1.P-H215 are summarized.

The formulations of the various media used in the following examples are as follows:

PDA plate culture medium: 200g of potato (peeled), 20g of glucose, 20g of agar and 1000mL of water, and the pH is natural.

Stock and cultivar media: 58wt% of cottonseed hulls, 20wt% of sawdust, 20wt% of bran, 1wt% of brown sugar, 1wt% of gypsum and 60-62wt% of water content of culture materials.

And (3) mushroom culture medium growth: 58wt% of sawdust, 20wt% of cottonseed hulls, 20wt% of bran, 1wt% of lime, 1wt% of gypsum and 60-62wt% of water content of compost.

According to the formula of a fruiting culture medium, a polyethylene plastic bag with the thickness of 17cm, 33cm and 0.05cm is used for cultivating and fruiting, the weight of dry materials in each bag is 400g, the bag is sterilized and inoculated under normal pressure, the bag is cultured at the constant temperature of 24 ℃ for 30d to 40d, the bag is filled with mycelia, the bag is moved to a fruiting room for cooling after the mycelia are matured, the cooling temperature is 8 to 10 ℃, and fruiting management is carried out after cooling. The yield of single-weight, first two-tide mushrooms of P1.P-H215 and the parent strain were recorded and compared, and the biological conversion rate was calculated. The cultivation experiment was performed in a randomized block design with 3 replicates per strain.

As can be seen from Table 1, the total bag yield of the first two tides of the Pleurotus pulmonarius strain P1.P-H215 is 352.35 g/bag, the biological efficiency reaches 88.09%, and the LSD multiple comparison analysis shows that the yield of the strain P1.P-H215 is significantly higher than that of the parent strains P19 and P8. Wherein the first tide yield of the strain P1.P-H215 is 231.29 g/bag, which is obviously higher than the first tide yields of parent strains P8 and P19; the second tide yields of strain P1.P-H215 and other strains were not significantly different. The average yield of bags of the first two tides of the strain P1.P-H215 is significantly higher than that of the parent strains P8 and P19.

The strain P1.P-H215 does not grow mushrooms in the mycelium culture stage, so that primordia are prevented from being formed without opening bags, the yield is reduced, and grown mushrooms are deformed and poor in quality; the temperature difference required by the fruiting of the strain P1.P-H215 is large, when the temperature difference is insufficient, the fruiting is not realized or is less, the fruiting can be realized through proper chilling temperature, the seasonal overproduction caused by centralized marketing of regional fresh mushrooms is avoided, and the 'off-peak fruiting' is realized. After the strain P1.P-H215 is subjected to cold stimulation, the fruiting is dense, the germination is neat, the tide times are obvious, and the harvest is completed within 2 days in the first tide.

TABLE 1 expression of the yield of hybrid strains of Pleurotus pulmonarius P1.P-H215 and the like from the parent strains

Note: the bacteria-moving speed value is the average value plus or minus standard error; different lower case letters indicate significant difference (P < 0.05) and different upper case letters indicate significant difference (P < 0.01).

The morphological characteristics of the fruiting bodies of strain P1.P-H215 are shown in Table 2 and FIG. 2. As can be seen from Table 2 and FIG. 2, the sporomorph of the strain P1.P-H215 is semicircular or nearly circular, the diameter of the pileus is 51.62mm, the numerical value is the largest, the thickness of the pileus is 14.72mm, the diameter of the stipe and the length of the stipe are 9.09mm and 43.87mm respectively, the weight of a single flower is 6.26g, and each is small and medium. The cap edge is not wavy after the fruiting body of the strain P1.P-H215 is mature, the mushroom shape is round, the commodity character is good, and the cap edge is easy to appear wavy after the fruiting body of the parent strain P19 grows up.

TABLE 2 major agronomic traits of Pleurotus pulmonarius P1.P-H215 hybrid strains and parents

Note: the main agronomic trait values are mean ± sem.

In conclusion, the strain P1.P-H215 has the excellent characteristics of high yield of the first two tides, good commodity, difficult growth of natural mushrooms, regular budding after cold stimulation and the like in a cultivation test, and is more suitable for out-of-season temperature control facility cultivation in summer.

Example 4 antagonistic response assay

The Pleurotus pulmonarius strain P1.P-H215 and 2 parent strains P8 and P19 are used as test materials, and the culture medium is PDA culture medium.

Activating the strains, respectively pairing and combining P1.p-H215, P8 and P19 under the aseptic condition, inoculating the strains on a plate culture medium, placing the strains in an incubator at 25 ℃ for inverted dark light culture for 8d with the distance of about 2cm, and observing whether antagonistic lines (whether hypha bulges or pigment precipitates exist at hypha growth intersections of different strains) exist among the strains.

As can be seen from the results of the antagonistic reactions in FIG. 3, there were significant lines of antagonism between the Pleurotus pulmonarius hybrid strain P1.P-H215 and the parent strains P8, P19 on the PDA plate medium, hypha swelling occurred at the intersection of the growth of the strains, and pigment precipitation occurred on the back of the plate medium. The method shows that the hypha of the hybrid strain P1.P-H215 has obvious antagonistic relation with the hypha of the parent strain, and the hybrid strain is different from the parent strain and is a new strain generated by recombination of parent genetic materials in pairing hybridization of monospore strains.

Example 5 molecular biological identification

In this example, the genetic characteristics of the strain P1.P-H215 of the present invention were identified by SRAP molecular markers using the strain P1.P-H215 of the present invention and the parent strains P8 and P19 as the subjects of analysis.

Extraction of genomic DNA: activating the test strains, inoculating the activated test strains into a PD liquid culture medium, and culturing at a constant temperature of 150 r/min at 25 ℃ for 8 d. After collecting fresh mycelia and grinding through liquid nitrogen, extracting genomic DNA of the mycelia by using E.Z.N.A. Plant DNA Kit, detecting the extraction effect of the genomic DNA by using 1% agarose gel electrophoresis, and determining the purity of the mycelia by using an ultraviolet spectrophotometer and storing the mycelia at-20 ℃ for later use.

The SRAP primers are as follows: ME9:5' TGAGTCCAAAACCGGTCA-: 5 'GACTGCGTACGAATTGAC-3', the SRAP primer was synthesized by Shanghai Bioengineering Co., ltd. The SRAP-PCR reaction system is 25 mu L, wherein 2 xTaq PCR Master Mix is 12.5 mu L, the upstream primer is 1 mu L, the downstream primer is 1 mu L, and dd H ₂ O9.5. Mu.L, template DNA 1. Mu.L. The PCR amplification program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 34 ℃ for 30s, extension at 72 ℃ for 1min, and circulation for 5 times; denaturation at 94 ℃ for 30s, annealing at 48 ℃ for 30s, extension at 72 ℃ for 1min, and circulation for 35 times; extension at 72 ℃ for 5min.

The SRAP detection result (figure 4) of the primer ME9/Em3 on the P1.P-H215 and the parent strains P8 and P19 thereof shows that the strain P1.P-H215 of the invention not only contains a specific strip of the parent strain P8, but also contains a specific strip of the parent strain P19, the molecular marker level proves that the strain P1.P-H215 is a hybrid of the parent strains P8 and P19 and is a new strain of Pleurotus pulmonarius, and the primer ME9/Em3 can be used for distinguishing the strain P1.P-H215 from the strains P8 and P19.

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

SEQUENCE LISTING

<110> agricultural science institute of Fuzhou City

<120> pleurotus pulmonarius strain and application thereof

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<160> 2

<170> PatentIn version 3.3

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<212> DNA

<213> ME9

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tgagtccaaa ccggtca 17

<210> 2

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<212> DNA

<213> Em3

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gactgcgtac gaattgac 18

Claims

1. A Pleurotus pulmonarius strain is characterized in that: the Pleurotus pulmonarius strain is P1.P-H215, which is classified and named as Pleurotus pulmonarius (A)Pleurotus pulmonarius) In 15 days 1 month 2021, the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.21450, and the preservation address is No. 3 Xilu No. 1 Beijing Hospital, beijing, chaoyang, north Chen.