CN113444650A - Pleurotus pulmonarius and application thereof - Google Patents
Pleurotus pulmonarius and application thereof Download PDFInfo
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Abstract
The invention provides pleurotus pulmonarius and application thereof. The Pleurotus pulmonarius strain is P1.p-H215, which is classified and named as Pleurotus pulmonarius (A)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 strain pileus is brown, the mushroom shape is round, the pileus edge is not wavy after the fruit body is mature, and the commercial property is good; not easy to grow natural mushroom, neat budding after cold stimulation, strong stress resistance and the like, and is more suitable for out-of-season temperature control facility cultivation in summer.
Description
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 good variety breeding research work of the pleurotus pulmonarius is still weak, the existing main cultivated varieties are still introduced pleurotus geesteranus series varieties, the variety updating is lagged, some varieties are degenerated after being introduced for years, the fruiting period is delayed, the fruiting is irregular, the disease resistance is poor, and the like, so that the fluctuation of the yield and the quality of the pleurotus geesteranus is caused, the breeding research strength of the good varieties, particularly the out-of-season varieties suitable for summer cultivation is insufficient, and the conditions become important factors for restricting the sustainable development of the pleurotus pulmonarius 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 achieve the purpose, the invention adopts the following technical scheme:
a Pleurotus pulmonarius strain P1.p-H215 which is classified and named as Pleurotus pulmonarius (A)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 is 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 temperature control facility cultivation in out-of-season 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 from the parent strain P19 in fruiting body.
FIG. 3: the antagonistic reaction test result of the strain P1.p-H215 and the two parents is shown. Left panel: a front side; right panel: and (4) the reverse side.
FIG. 4: the amplification map of the primer ME9/Em3 on P1.p-H215 and the parent thereof. M: DL2000 DNA Marker; 1: p8; 2: p1. p-H215; 2: p19, arrow indicates the parental strain-specific band.
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 trait evaluation, parents are selected, strains P8 and P19 with large genetic diversity and excellent agronomic traits are screened out to serve as the parents (Chen Pan, Chenjian, Wuhan Jong. Pleurotus pulmonarius germplasm resource antagonism reaction and RAPD molecular marker genetic diversity analysis [ J ]. southern agricultural science, 2020, 51(12): 2892-2900), genetic single spores are obtained, and single spore cross breeding is carried out.
Example 2 monospore crossbreeding
(1) Selection of seed mushrooms: parent strains P8 and P19 are cultivated for fruiting, when the fruiting bodies grow to seven-eight mature, 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 6-10 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 paper at the bottom of the culture dish, cutting off mushroom stipe of the seed mushroom, putting mushroom pleat downwards on the parchment paper, slightly covering the parchment paper by a dish cover, and observing that a large amount of spores fall on the parchment paper 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 evenly shaken, lmL spore liquid is taken out of the spores, and added into 9mL of aseptic water to obtain spore suspension, and then diluted by multiple times to dilute the 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 on a gossypol lactate blue staining solution, observing with a microscope, if no latticed combination exists, preliminarily considering that the hyphae are mononuclear hyphae generated by single spore germination, and then transferring to a slant for culturing.
(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 (shown 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 summer out-of-season temperature control facility cultivation tests, 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 value is natural.
Stock and cultivar media: 58wt% of cottonseed hulls, 20wt% of sawdust, 20wt% of bran, 1 wt% of brown sugar, 1 wt% 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, 1 wt% of lime, 1 wt% of gypsum and 60-62wt% of water content of compost.
According to the formula of a fruiting culture medium, cultivating and fruiting by using a polyethylene plastic bag with the thickness of 17cm, 33cm and 0.05cm, wherein the weight of dry materials in each bag is 400g, sterilizing at normal pressure, inoculating, culturing at the constant temperature of 24 ℃ for 30-40 d, transferring the fungus bags to a fruiting room for cooling after hyphae grow over the fungus bags and are physiologically mature, and performing fruiting management after cooling at the temperature of 8-10 ℃. 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 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 significantly higher than the first tide yields of its 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 from the first two tides of strain P1.P-H215 of the present invention 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 primordium is 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 and the tide frequency is very 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 commercial property 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 used culture medium is PDA culture medium.
Activating the strains, respectively pairing and combining P1.P-H215, P8 and P19 under an aseptic condition, inoculating the strains on a plate culture medium, placing the strains in an incubator at 25 ℃ with a distance of about 2cm, carrying out inverted dark light culture for 8 days, 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 and P19 on the PDA plate medium, hypha swelling occurred at the growth junctions of the strains, and pigment precipitation occurred on the back of the plate medium. The hybrid strain P1.p-H215 and hyphae of a parent strain have obvious antagonistic relationship, 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 strain, inoculating the activated test strain into a PD liquid culture medium, and culturing at the constant temperature of 150 r/min at 25 ℃ for 8 d. Collecting fresh mycelia, grinding the mycelia by liquid nitrogen, extracting genomic DNA of the mycelia by using E.Z.N.A. Plant DNA Kit, detecting the extraction effect of the mycelia by using 1% agarose gel electrophoresis, measuring the purity by using an ultraviolet spectrophotometer, and storing the mycelia at-20 ℃ for later use.
The SRAP primers are as follows: ME 9: 5'-TGAGTCCAAACCGGTCA-3' and Em 3: 5' -GACTGCGTACGAATTGThe AC-3' 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 H2O9.5. mu.L, template DNA 1. mu.L. The PCR amplification program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 30s, annealing at 34 deg.C for 30s, extension at 72 deg.C for 1min, and circulation for 5 times; denaturation at 94 deg.C for 30s, annealing at 48 deg.C for 30s, extension at 72 deg.C for 1min, and circulation for 35 times; extension at 72 ℃ for 5 min.
The SRAP detection result (figure 4) of the primer ME9/Em3 on the P1.P-H215 and the parent strains P8 and P19 shows that the strain P1.P-H215 contains a specific strip of the parent strain P8 and 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 P1.P-H215 from the P8 and P19 strains.
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.
SEQUENCE LISTING
<110> research institute of agricultural science in Fuzhou City
<120> pleurotus pulmonarius strain and application thereof
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213> ME9
<400> 1
tgagtccaaa ccggtca 17
<210> 2
<211> 18
<212> DNA
<213> Em3
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gactgcgtac gaattgac 18
Claims (2)
1. LungPleurotus ostreatus, characterized in that: the Pleurotus pulmonarius strain is P1.p-H215, which is classified and named as Pleurotus pulmonarius (A)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.
2. The pleurotus pulmonarius strain as claimed in claim 1 is used for edible fungus facility cultivation in out-of-season summer.
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| CN115710554A (en) * | 2022-12-28 | 2023-02-24 | 吉林农业大学 | Pleurotus pulmonarius strain for decoloring and removing COD (chemical oxygen demand) in sewage and application thereof |
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