CN114350528B - Excellent strain of pholiota nameko, and specific molecular marker and application thereof - Google Patents
Excellent strain of pholiota nameko, and specific molecular marker and application thereof Download PDFInfo
- Publication number
- CN114350528B CN114350528B CN202210096154.XA CN202210096154A CN114350528B CN 114350528 B CN114350528 B CN 114350528B CN 202210096154 A CN202210096154 A CN 202210096154A CN 114350528 B CN114350528 B CN 114350528B
- Authority
- CN
- China
- Prior art keywords
- pholiota nameko
- strain
- pholiota
- nameko
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 244000168667 Pholiota nameko Species 0.000 title claims abstract description 123
- 235000014528 Pholiota nameko Nutrition 0.000 title claims abstract description 123
- 239000003147 molecular marker Substances 0.000 title claims abstract description 13
- 241000233866 Fungi Species 0.000 claims abstract description 39
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims abstract description 17
- 238000005520 cutting process Methods 0.000 claims abstract description 10
- 241000894007 species Species 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 241001123663 Penicillium expansum Species 0.000 claims abstract description 7
- 241000223260 Trichoderma harzianum Species 0.000 claims abstract description 7
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 241000223218 Fusarium Species 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 235000013339 cereals Nutrition 0.000 claims description 20
- 238000012258 culturing Methods 0.000 claims description 17
- 241000209140 Triticum Species 0.000 claims description 16
- 235000021307 Triticum Nutrition 0.000 claims description 16
- 241000222351 Pleurotus cornucopiae Species 0.000 claims description 11
- 239000002994 raw material Substances 0.000 claims description 9
- 239000002023 wood Substances 0.000 claims description 9
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 8
- 229910052602 gypsum Inorganic materials 0.000 claims description 8
- 239000010440 gypsum Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 6
- 210000003608 fece Anatomy 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 235000008733 Citrus aurantifolia Nutrition 0.000 claims description 4
- 235000011941 Tilia x europaea Nutrition 0.000 claims description 4
- 244000052616 bacterial pathogen Species 0.000 claims description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 4
- 239000000428 dust Substances 0.000 claims description 4
- 239000004571 lime Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 238000009402 cross-breeding Methods 0.000 claims description 3
- 238000012364 cultivation method Methods 0.000 claims description 3
- 230000001902 propagating effect Effects 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 2
- 241001149959 Fusarium sp. Species 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 238000012239 gene modification Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 241000243190 Microsporidia Species 0.000 claims 1
- 241000222350 Pleurotus Species 0.000 claims 1
- 230000005017 genetic modification Effects 0.000 claims 1
- 235000013617 genetically modified food Nutrition 0.000 claims 1
- 230000001717 pathogenic effect Effects 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 238000003306 harvesting Methods 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract description 4
- 208000035240 Disease Resistance Diseases 0.000 abstract description 4
- 230000003042 antagnostic effect Effects 0.000 abstract description 4
- 230000001775 anti-pathogenic effect Effects 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 22
- 239000012634 fragment Substances 0.000 description 18
- 244000061456 Solanum tuberosum Species 0.000 description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 description 9
- 239000002609 medium Substances 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- 244000053095 fungal pathogen Species 0.000 description 5
- 235000013372 meat Nutrition 0.000 description 5
- 241001238187 Pholiota microspora Species 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 240000001462 Pleurotus ostreatus Species 0.000 description 2
- 235000001603 Pleurotus ostreatus Nutrition 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000222485 Agaricales Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 241000801118 Lepidium Species 0.000 description 1
- 235000007685 Pleurotus columbinus Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000002956 ash Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 235000004213 low-fat Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000007837 multiplex assay Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Abstract
The invention discloses a pholiota nameko excellent strain, and a specific molecular marker and application thereof. The excellent strain is Pholiota nameko 682 (preservation number CGMCCNo: 23243) with high yield, quick fruiting and strong disease resistance. The mycelium of the pholiota nameko 682 has the advantages of fast growth, short post-ripening period, fast fruiting, dense mushroom buds, over 80 percent of survival rate of the mushroom buds, light yellow to yellow brown fungus caps, strong adaptability and short harvesting period; the biological conversion rate is high; the anti-pathogenic bacteria capability is strong, and the anti-pathogenic bacteria agent has strong antagonistic capability to Trichoderma harzianum, penicillium expansum and Fusarium. The invention obtains the pholiota nameko strain 682 through wild specimen separation, develops a new variety of edible fungi, obtains a specific DNA sequence and a specific enzyme cutting site of the pholiota nameko 682, and has important significance for protecting the germplasm resources of the species, enriching the edible fungi variety resources and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a pholiota nameko excellent strain, a specific molecular marker thereof and application thereof.
Background
Pholiota nameko (Pholiota microspora) belongs to the kingdom fungi, basidiomycetes, agaricales, the family of the Strophaceae, and the genus Lepidium. The aliases include pholiota nameko, oyster mushroom, pholiota nameko and pholiota nameko. The pholiota nameko has extremely high edible and nutritional values, contains rich nutrients such as crude proteins, crude fibers, polysaccharides, fats, minerals, ash, vitamins and the like, and has the effects of resisting tumors, resisting oxidation, enhancing immunity and the like. The pholiota nameko is one of ten mushrooms in the world mushroom trade market, and is also one of edible fungus types recommended to be cultivated by the united nations grain and agricultural organization (FAO) to developing countries. The pholiota nameko has the characteristics of gorgeous color, crisp and tender mushroom meat, delicious taste, smooth, fresh, tender and crisp taste, rich nutrition, good additive of soup stock, welcome by people, sticky matter secreted by the surface of the mushroom cover, efficacy of inhibiting tumor, capability of improving organism immunity, great benefit for improving brain and physique of human body, and low-calorie and low-fat health food.
At present, the varieties of the main production area of the domestic pholiota nameko mainly comprise Zaofeng 112 and C3, the strains are not updated for many years, the original strains are aged gradually, the fruiting capacity is weakened, and the number of mushroom buds is reduced gradually. Although easy to cultivate, the strain aging problem of the pholiota nameko is not well solved, and strain improvement is needed to promote the development of the pholiota nameko industry.
Disclosure of Invention
The invention aims to provide a pholiota nameko excellent strain and a specific molecular marker and application thereof.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a pholiota nameko (Pholiota microspora) 682, wherein the strain 682 is a pholiota nameko strain with high yield and strong disease resistance, which is obtained from wild pholiota nameko fruiting bodies collected from Tibetan ink drop through separation, purification and domestication culture. The strain 682 is preserved in China general microbiological culture Collection center, north West Lu No. 1, 3 of the area of Chaoyang in Beijing, and the post code 100101 of the national academy of sciences of China, with a preservation number CGMCC No. 23243, and a preservation date 2021, 8, 27.
In a second aspect, the invention provides a specific DNA molecular marker of pholiota nameko 682, and the nucleotide sequence is shown as SEQ ID NO. 4.
In a third aspect, the invention provides PCR primers for detecting pholiota nameko 682, comprising an upstream primer as shown in SEQ ID NO. 1 and a downstream primer as shown in SEQ ID NO. 2.
In a fourth aspect, the invention provides a method for detecting pholiota nameko 682, wherein the primer pair SEQ ID NO. 1 and SEQ ID NO. 2 are used for detection; the specific DNA molecular sequence of Pholiota nameko 682 was digested with XbaI.
The method comprises the following steps: extracting genome DNA of the to-be-detected pholiota nameko as a template, performing PCR amplification by using an upstream primer and a downstream primer, and judging the to-be-detected pholiota nameko as pholiota nameko 682 if the amplified product contains a specific DNA molecular marker shown by SEQ ID NO. 4 and a specific enzyme cutting site on the specific DNA molecular marker.
In a fifth aspect, the present invention provides the use of pholiota nameko 682 for combating pathogenic bacteria and plant diseases caused by pathogenic bacteria.
Such pathogens include, but are not limited to, trichoderma harzianum (Trichoderma harzianum), penicillium expansum (Penicillium expansum), and Fusarium sp.
In a sixth aspect, the present invention provides the use of pholiota nameko 682 as a parent for cross breeding (development of new pholiota nameko varieties), genetic engineering.
In a seventh aspect, the present invention provides a cultivation method of pholiota nameko 682, comprising the steps of:
(1) Expanding propagation of mother strain of pholiota nameko 682 strain: inoculating the Pleurotus cornucopiae 682 strain on the mother culture medium, and culturing at 18-25deg.C in dark for 10-15 days to obtain Pleurotus cornucopiae 682 strain mother strain; wherein the mother culture medium is PDA culture medium;
the PDA culture medium comprises the following raw materials: 200g of potato, 20g of glucose, 18g of agar and 1000mL of water; wherein the potato is prepared by decocting 200g of fresh potato in 1000mL boiling water for 20-30 min, filtering with four layers of gauze, and collecting juice;
(2) Propagating stock strain of pholiota nameko 682: inoculating the mother strain of the pholiota nameko 682 obtained in the step (1) to a stock culture medium, and culturing for 20-30 days under dark conditions at 18-25 ℃ to obtain the stock strain of the pholiota nameko 682; wherein the stock culture medium comprises the following raw materials: 93% of wheat grains, 5% of cow dung, 1% of gypsum, 0.9% of calcium carbonate and 0.1% of lime, and the water content is 50% -60%;
(3) Preparation of a cultivation seed of a pholiota nameko 682 strain: inoculating the stock strain of the pholiota nameko 682 obtained in the step (2) onto a culture medium of the cultivated species, and culturing for 20-30 days under dark conditions at a temperature of 20-23 ℃ to obtain the cultivated species of the pholiota nameko 682; wherein the culture medium for cultivars comprises the following raw materials: 80% of miscellaneous wood dust, 18% of bran and 2% of gypsum, and 50% -60% of water content;
(4) Cultivation: after-ripening for 10-30 days at 15-25deg.C under dark condition; uniformly placing the after-ripe fungus bags on a layer rack or the ground, cutting two ends of the fungus bags, culturing for 8-10 days, and atomizing and spraying water for 3-4 times each day; the temperature difference between day and night is 5-15 ℃, the material surface forms a pale yellow rice grain primordium after 3-5 days, the indoor temperature is 5-30 ℃, the air relative humidity is 85-90%, and the culture is carried out for 2-3 days under the condition of a small amount of scattered light to form pale yellow young buds; after 5-7 days, the mushroom buds grow to the diameter of the mushroom caps of 2cm, spraying water is reduced to 1-2 times per day, the space is sprayed with atomized water, and the relative humidity of the space is 85-90%; picking when the diameter of the fungus cover reaches 1.5-3.5cm and the fungus curtain is not opened.
Mycelium obtained by culturing the pholiota nameko 682 is all within the protection scope of the invention.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the mycelium of the pholiota nameko 682 grows fast, the after-ripening time is short, the fruiting body obtained by cultivation has early primordium emergence time (fast fruiting), the buds are dense, the survival rate of the buds is more than 80%, the adaptability is strong, and the harvesting period is short; the biological conversion rate is high (the biological conversion rate reaches about 112 percent); the anti-pathogenic bacteria capability is strong, and the anti-pathogenic bacteria agent has strong antagonistic capability to Trichoderma harzianum, penicillium expansum and Fusarium. The invention obtains the pholiota nameko strain 682 through the separation of wild strains, develops a new variety of edible fungi, obtains a specific DNA sequence of the pholiota nameko 682, and has important significance for protecting the germplasm resources of the species, enriching the edible fungi variety resources and the like.
Drawings
FIG. 1 shows fruiting bodies produced by laboratory cultivation of Pleurotus cornucopiae 682 strain in a preferred embodiment of the present invention.
FIG. 2 shows fruiting bodies produced by laboratory cultivation of the Pholiota nameko strain purchased in the preferred embodiment of the present invention.
FIG. 3 shows the growth of mycelium of Pholiota nameko 682 after 10 days of PDA plating in a preferred embodiment of the present invention.
FIG. 4 shows the status of the flower bud of the Pleurotus ostreatus 682 strain in the preferred embodiment of the present invention.
Fig. 5 shows fruiting status of fruiting body of Pleurotus cornucopiae 682 in the preferred embodiment of the present invention.
FIG. 6 shows a cultivation of a strain 682 of Pholiota nameko against pathogenic fungi according to a preferred embodiment of the present invention.
FIG. 7 is an electropherogram of a specific DNA fragment of Pholiota nameko 682 strain according to a preferred embodiment of the present invention.
FIG. 8 is a sequence alignment of DNA fragments specific to Pholiota nameko 682 strain in a preferred embodiment of the present invention.
FIG. 9 is a diagram showing the enzyme digestion electrophoresis of DNA fragments specific to Pholiota nameko 682 strain in the preferred embodiment of the present invention.
FIG. 10 shows the results of a comparison of the Pholiota nameko 682 and the commercial Pholiota nameko strains C3, 112 by Tian Pin in a preferred embodiment of the present invention.
Detailed Description
The invention provides a pholiota nameko strain 682 with high yield, fast fruiting and strong disease resistance, and the preservation number is CGMCC No. 23243.
The pholiota nameko strain 682 is obtained by separating, purifying and domesticating fruiting bodies of wild pholiota nameko.
The invention also provides application of the pholiota nameko strain 682 serving as a parent in cross breeding and gene modification.
The invention also provides a fruiting body obtained by cultivating the pholiota nameko strain 682.
The formula of a culture medium for culturing the pholiota nameko and the preparation method thereof are as follows:
PDA medium (stock medium): 200g of potato, 20g of glucose, 18g of agar powder and 1000mL of water. Wherein the potato is prepared by decocting 200g of fresh potato in 1000mL boiling water for 20-30 min, filtering with four layers of gauze, and collecting juice.
Wheat grain medium (stock medium): 93% of wheat grains, 5% of cow dung, 1% of gypsum, 0.9% of calcium carbonate and 0.1% of lime.
The wheat grains in the wheat grain culture medium are soaked overnight before being precooked, and cow dung is prewetted in advance. The wheat grains are washed clean, fished out and drained, soaked in water and boiled for about 20min, so that the wheat grains are changed from beige to light brown, and the anti-cracking effect is achieved. And (3) fishing out the cooked wheat grains, spreading and airing the wheat grains in a clean large basin, evaporating the moisture on the surface of the wheat grains until the wheat grains are not scalded, mixing other auxiliary materials while the wheat grains are hot, wherein the moisture content is 50-60%, and the pH is natural.
Wood chip compost (cultivar medium): 80% of miscellaneous wood dust, 18% of bran and 2% of gypsum. The water content is 50% -60%. Before the wood chip culture material is mixed with the material, the wood chips are required to be soaked overnight for prewetting.
The pholiota nameko strain provided by the invention is a Tibetan ink-drop wild specimen isolated strain, and fruiting bodies can be obtained after the strain is cultivated. Fruiting body obtained by cultivating the strain has a height of 5.5-20cm and a fruiting body weight of 8-20 g; the fungus cover is round and bell-shaped; the outer surface of the fungus cover is yellow and has mucus and meat; the stem is made of medium, fibrous, with the diameter of 0.5-1.3 cm, and solid; the fungus pleat and the fungus handle are separated from each other, and the fungus meat is light yellow, has a fungus ring structure and membranous, is connected with the fungus cover when the umbrella is not opened, and is easy to loose and slide after the fungus cover is ripe and the umbrella is opened.
The invention adopts PCR technology to carry out a large number of screening tests. Wherein, the specific DNA fragment of the pholiota nameko 682 is obtained by adopting primers (6063-1027F: 5'-AAATGGTAACGTCGAGTGCG-3' and 6063-1027R:5 '-CGTTTGACGTGATCGCAATGTT-3'), the size is about 1027bp, and the same gene fragment is different from other strains and can be used as the specific molecular marker of the pholiota nameko strain 682.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The percentage "%" referred to in the present invention refers to mass percent unless otherwise specified; however, the percentage of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of solution.
Example 1 identification of parent Strain
The fruiting body of wild Pholiota nameko is obtained from Tibetan ink drop, and morphological observation and cell nucleus rDNA-ITS sequencing (SEQ ID NO: 3) prove that the strain is a Pholiota nameko strain. Separating and purifying the wild pholiota nameko fruiting body, culturing strain 682, and culturing, comparing and screening.
The specific breeding method is as follows:
cultivation of wild strains: the wild isolated strain is cultivated, a PDA culture medium is used for mother strain cultivation, a wheat grain culture medium is used for stock strain cultivation, and a wood chip culture medium is used for cultivated species. After the hypha grows fully on the culture material, the bag opening, soil covering and fruiting are directly carried out. The 682 strain has regular fruiting and basically consistent maturity.
The cultivation method of the pholiota nameko 682 comprises the following steps:
(1) Expanding propagation of mother strain of pholiota nameko 682 strain: inoculating the Pleurotus cornucopiae 682 strain on the mother culture medium, and culturing at 18-25deg.C in dark for 10-15 days to obtain Pleurotus cornucopiae 682 strain mother strain; wherein the mother culture medium is PDA culture medium, and the raw materials comprise: 200g of potato, 20g of glucose, 18g of agar and 1000mL of water; wherein the potato is prepared by decocting 200g of fresh potato in 1000mL boiling water for 20-30 min, filtering with four layers of gauze, and collecting juice;
(2) Propagating stock strain of pholiota nameko 682: inoculating the mother strain of the pholiota nameko 682 obtained in the step (1) to a stock culture medium, and culturing for 20-30 days under dark conditions at 18-25 ℃ to obtain the stock strain of the pholiota nameko 682; wherein the stock culture medium comprises the following raw materials: 93% of wheat grains, 5% of cow dung, 1% of gypsum, 0.9% of calcium carbonate and 0.1% of lime, and the water content is 50% -60%;
(3) Preparation of a cultivation seed of a pholiota nameko 682 strain: inoculating the stock strain of the pholiota nameko 682 obtained in the step (2) onto a culture medium of the cultivated species, and culturing for 20-30 days under dark conditions at a temperature of 20-23 ℃ to obtain the cultivated species of the pholiota nameko 682; wherein the culture medium for cultivars comprises the following raw materials: 80% of miscellaneous wood dust, 18% of bran and 2% of gypsum, and the water content is 50% -60%;
(4) Cultivation: after-ripening for 10-30 days at 15-25deg.C under dark condition; uniformly placing the after-ripe fungus bags on a layer rack or the ground, cutting two ends of the fungus bags, culturing for 8-10 days, and atomizing and spraying water for 3-4 times each day; the temperature difference between day and night is 5-15 ℃, the material surface forms a pale yellow rice grain primordium after 3-5 days, the indoor temperature is 5-30 ℃, the air relative humidity is 85-90%, and the culture is carried out for 2-3 days under the condition of a small amount of scattered light to form pale yellow young buds; after 5-7 days, the mushroom buds grow to the diameter of the mushroom caps of 2cm, spraying water is reduced to 1-2 times per day, the space is sprayed with atomized water, and the relative humidity of the space is 85-90%; picking when the diameter of the fungus cover reaches 1.5-3.5cm and the fungus curtain is not opened.
Fruiting bodies produced by cultivation of the Pleurotus cornucopiae 682 strain are shown in figure 1.
The fruiting body of the purchased Pholiota nameko strain (Zaofeng 112) is shown in fig. 2.
Compared with Zaofeng 112, the pholiota nameko 682 has dense buds, quick fruiting, short harvesting period and high yield.
The growth of mycelium of Pholiota nameko 682 strain after 10d of PDA plate culture is shown in FIG. 3.
The status of the flower bud of the Pleurotus cornucopiae 682 strain is shown in FIG. 4.
Fruiting status of fruiting body of Pleurotus cornucopiae 682 strain is shown in figure 5.
Example 2 Pholiota nameko strain quality comparison experiment
1. Quality control test of Pholiota nameko strain
Bacterial strain 682 and commercial pholiota nameko C3, 112 bacterial strains are cultivated, PDA culture medium is used for mother strain cultivation, wheat grain culture medium is used for stock strain cultivation, and wood chip culture medium is used for cultivated species. After the mycelium grows Man Muxie on the culture medium, the color is changed for one month, and then the bag is opened, soil is covered and mushroom is produced. And comparing fruiting conditions and yields of the mushrooms.
The results are shown in Table 1, the strain 682 is more than the commercial strain control, the mushroom buds are dense, the fruiting is quick, the harvesting period is short, and only 26+/-1 d from earthing to harvesting; the biological conversion rate is high and reaches 112+/-0.9 percent.
Table 1 results of the ratio of strain 682 to other Pholiota nameko strains
Note that: the same letter in the same row indicates that there is no significant difference in multiplex assay (P.ltoreq.0.05).
2. Pholiota nameko strain and pathogenic bacteria antagonism test
Bacterial strain 682, commercial pholiota nameko bacterial strain and pathogenic fungi are subjected to counter culture, and antagonistic capability of the bacterial strain 682 and commercial pholiota nameko bacterial strain on the pathogenic fungi is compared.
The method for culturing the pholiota nameko strains and pathogenic fungi in the opposite direction comprises the following steps: selecting a pholiota nameko strain fungus block to one side of a PDA flat plate, placing the pholiota nameko strain fungus block in a 20 ℃ incubator for 3d, taking out the pholiota nameko strain fungus block, respectively transferring trichoderma harzianum, penicillium expansum and fusarium to the other side of the PDA flat plate, placing the pholiota nameko strain block in the 20 ℃ incubator for continuous culture, and observing antagonistic capability of the pholiota nameko 682 strain to pathogenic fungi. Has strong disease resistance and strong antagonism to Trichoderma harzianum, penicillium expansum and Fusarium (figure 6).
3. Fruiting body characteristics of inventive strains
The wild pholiota nameko strain 682 is successfully cultivated to obtain fruiting bodies. The fruiting body height is 5.5-20cm, and the fruiting body weight is 8-20 g; the fungus cover is round and clock; the fungus cover is light yellow to yellow brown, and the outer surface of the fungus cover is yellow and has mucus and meat quality; the stem is made of medium, fibrous, with the diameter of 0.5-1.3 cm, and solid; the fungus pleat and the fungus handle are separated from each other, and the fungus meat is light yellow, has a fungus ring structure and membranous, is connected with the fungus cover when the umbrella is not opened, and is easy to loose and slide after the fungus cover is ripe and the umbrella is opened.
Example 3 specific DNA molecular markers of Pholiota nameko 682
A number of screening assays were performed using PCR techniques. Wherein, the specific DNA fragment of the pholiota nameko strain 682 is obtained by adopting primers shown in SEQ ID NO. 1 and SEQ ID NO. 2 (6063-1027F: 5'-AAATGGTAACGTCGAGTGCG-3' and 6063-1027R:5 '-CGTTTGACGTGATCGCAATGTT-3'), the size of the specific DNA fragment is about 1027bp (SEQ ID NO. 4), and the same gene fragment is different from other pholiota nameko strains (SEQ ID NO. 4), namely the specific molecular marker of the pholiota nameko strain 682.
PCR reaction system (20. Mu.l): taq-Mix 10. Mu.l, each of the upstream and downstream primers 0.5. Mu.l, template DNA 2. Mu.l, ddH 2 O 7μl。
PCR reaction procedure: 95 ℃ for 5min;95℃30s,60℃30s,72℃15s,35 cycles; and at 72℃for 10min.
The electrophoresis pattern of specific DNA fragments of the Pholiota nameko 682 strain is shown in FIG. 7, wherein C3 is the Pholiota nameko C3 strain (commercial strain), 112 is the Pholiota nameko 112 strain (commercial strain), and the other is the Pholiota nameko wild strain (saved in the Dong rainbow subject group of the national academy of sciences of microbiology).
The sequence comparison result of the specific DNA fragment of the Pholiota nameko 682 strain is shown in FIG. 8.
The specific enzyme cutting site XbaI in the specific fragment of the pholiota nameko 682 strain is subjected to enzyme cutting, and the enzyme cutting system is as follows: DNA 1. Mu.g, 10 XrCutSmart Buffer 5. Mu.l (1X), xbaI 1.0. Mu.l (20 units), add ddH 2 O to 50. Mu.l.
After the specific DNA fragment XbaI of the pholiota nameko 682 strain is digested, the pholiota nameko 682 strain is divided into two fragments, wherein the large fragment is 777bp, and the small fragment is 250bp. The other strain fragments are 1027bp, have no XbaI enzyme cutting site and can not be cut into two fragments.
The enzyme digestion electrophoresis pattern of the specific DNA fragment of the Pholiota nameko 682 strain is shown in FIG. 9.
Example 4 greater Tian Pin than validated
The results of the large Tian Pin ratio test of the pholiota nameko 682 and the commercial pholiota nameko strains C3 and 112 in Wuchuan county of inner Mongolia show that the strain 682 is more than the commercial strain control, the mushroom buds are dense, the fruiting is quick, the harvesting period is short, and the yield is high (figure 10) which is 1.6 times that of the commercial strain.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of microorganisms at national academy of sciences
<120> Pholiota nameko excellent strain, and specific molecular marker and application thereof
<130> PI202110043
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aaatggtaac gtcgagtgcg 20
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
cgtttgacgt gatcgcaatg tt 22
<210> 3
<211> 601
<212> DNA
<213> Pholiota nameko (Pholiota microspora)
<400> 3
aaaaacttgg ttggattgtt gctggcctga atgagggcat gtgcacatct gccatctttt 60
atctttccac ctgtgcacac tttgtaggtc tgggattaaa ctttctgagg tcaactcagt 120
tttgaggact gctgttagca ataatggctt tccttgtctt tccagatcta tgttttcata 180
tacaccataa aaatgtaata gaatgtgtta ataagccttg tgcttataaa ctatatacaa 240
ctttcagcaa cggatctctt ggctctcgca tcgatgaaga acgcagcgaa atgcgataag 300
taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacctt gcgctccttg 360
gtattccgag gagcatgcct gtttgagtgt cattaaatta tcaatctttg cagcttttgt 420
tgttaaagac ttggatgtgg gggttttatt tggaggcttt tcggagcgtc ctcccctaaa 480
atgtattagc tagtcgctcg tgcggacttg tctattggtg tgataattat ctacgccatg 540
gtcagactgc cattaagtag cactgcttct aatcgtcctt tactggacaa cttatgacaa 600
t 601
<210> 4
<211> 1027
<212> DNA
<213> Pholiota nameko (Pholiota microspora)
<400> 4
atttgattct tgagtttgca tactattact tatcccggat gatttacaag ggttttggcg 60
ggggaaaggc acgtgcacga cttatctacc ttgttacgca tctctcctcc tccttcctcc 120
ctccctcctc cctccccttc cctctcttcc cgccgactgg gatcaaggtt acgacttacg 180
acttcacgct agatagtttg agttcaaggg aggaagaaaa gagttgacgt caccccaatt 240
ggtttcagct ctagaccgcc tttgcctctt tttttacgcc gttcaaaccg ttcctaaccg 300
acacccgcag tattccattc gaggtgtttc tgttatcttc gaggtgtctg ttatcgccgc 360
cttgttcaca ttctttgcgt ctttgcgtcg ctgttctttg ttgagcattc actgtcgacg 420
ccatgcccta ttcgacgccc acatcaccta cctcgtctgg ggatattgcg ttcccctcca 480
cgggctcctc aggatactca ggatactcac aaggccgtcc agggcaccgt cgctcgtatt 540
cacacatcac cgcttccatg tatcctactt tgaatacgtc aacgtcaaca acgacgacgc 600
cgggtctcgc cacacttccc cgccgacggc cgactacgag cgcgccactt ccgccttcgt 660
tcgctttcgg cgggcctgca agggaaagta ggaaaccgac gttccagttg gggaaggatg 720
acgatgatga tgatgatgat tcggacgaaa atggggcagg gaagaaattt ggtgtagctg 780
cgaggagagt aggaggaatg gatggggagg gaagaggaag gccgcattct tcctccgctt 840
cggttgaaga cgaagcaggc gtcgttcgcg ctgcccgcag cggtgtcgcc gccgccgcct 900
gtgccggtga ttgcgctgca tccacccttc tggtggaagg tggagaggga ggagcaatac 960
catttccgcg ctcgtcgcgc tgtcgtcgcc gtcgtgaccg cgaccccccc cctatctcac 1020
tcatatt 1027
Claims (8)
1. Pleurotus cornucopiaePholiota microspora) 682, wherein the preservation number is CGMCC No. 23243.
2. The specific DNA molecular marker of pholiota nameko 682 of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 4.
3. The PCR primer for detecting the pholiota nameko 682 of claim 1, which is characterized by comprising an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2.
4. The method for detecting pholiota nameko 682 of claim 1, wherein the detection is performed using the primer of claim 3.
5. The method according to claim 4, characterized in that the method comprises: extracting genome DNA of the to-be-detected pholiota nameko as a template, and performing PCR amplification by using an upstream primer and a downstream primer, wherein if the amplified product contains a specific DNA molecular marker shown as SEQ ID NO. 4, or the amplified product has a specific characteristicXbaAnd (3) determining the to-be-detected pholiota nameko as pholiota nameko 682 at the enzyme cutting site.
6. Use of pholiota nameko 682 according to claim 1 for combating pathogenic bacteria, including trichoderma harzianum, and plant diseases caused by pathogenic bacteriaTrichoderma harzianum) Penicillium expansum (L.) KuntzePenicillium expansum) And fusarium are%Fusarium sp.）。
7. Use of pholiota nameko 682 according to claim 1 as parent for cross breeding and genetic modification.
8. The cultivation method of pholiota nameko 682 of claim 1, comprising the steps of:
(1) Expanding propagation of mother strain of pholiota nameko 682 strain: inoculating the Pleurotus cornucopiae 682 strain on the mother culture medium, and culturing at 18-25deg.C in dark for 10-15 days to obtain Pleurotus cornucopiae 682 strain mother strain; wherein the mother culture medium is PDA culture medium;
(2) Propagating stock strain of pholiota nameko 682: inoculating the mother strain of the pholiota nameko 682 obtained in the step (1) onto a stock culture medium, and culturing for 20-30 days under dark conditions at 18-25 ℃ to obtain stock strain of the pholiota nameko 682; wherein the stock culture medium comprises the following raw materials: 93% of wheat grains, 5% of cow dung, 1% of gypsum, 0.9% of calcium carbonate and 0.1% of lime, and the water content is 50% -60%;
(3) Preparation of a cultivation seed of a pholiota nameko 682 strain: inoculating the stock strain of the pholiota nameko 682 obtained in the step (2) onto a culture medium of the cultivated species, and culturing for 20-30 days under dark conditions at a temperature of 20-23 ℃ to obtain the cultivated species of the pholiota nameko 682; wherein the culture medium for cultivars comprises the following raw materials: 80% of miscellaneous wood dust, 18% of bran and 2% of gypsum, and 50% -60% of water content;
(4) Cultivation: after-ripening for 10-30 days at 15-25deg.C under dark condition; uniformly placing the after-ripe fungus bags on a layer rack or the ground, cutting two ends of the fungus bags, culturing for 8-10 days, and atomizing and spraying water for 3-4 times each day; the temperature difference between day and night is 5-15 ℃, the material surface forms a pale yellow rice grain primordium after 3-5 days, the indoor temperature is 5-30 ℃, the air relative humidity is 85-90%, and the culture is carried out for 2-3 days under the condition of a small amount of scattered light to form pale yellow young buds; after 5-7 days, the mushroom buds grow to have the diameter of the mushroom caps of 2cm, spraying water is reduced to be sprayed 1-2 times per day, atomized water is sprayed in space, and the relative humidity of the space is 85-90%; picking when the diameter of the fungus cover reaches 1.5-3.5 and cm and the fungus curtain is not opened.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210096154.XA CN114350528B (en) | 2022-01-26 | 2022-01-26 | Excellent strain of pholiota nameko, and specific molecular marker and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210096154.XA CN114350528B (en) | 2022-01-26 | 2022-01-26 | Excellent strain of pholiota nameko, and specific molecular marker and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114350528A CN114350528A (en) | 2022-04-15 |
| CN114350528B true CN114350528B (en) | 2024-02-20 |
Family
ID=81093400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210096154.XA Active CN114350528B (en) | 2022-01-26 | 2022-01-26 | Excellent strain of pholiota nameko, and specific molecular marker and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114350528B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881931A (en) * | 2014-04-04 | 2014-06-25 | 王会森 | Pholiota nameko and culture method thereof |
-
2022
- 2022-01-26 CN CN202210096154.XA patent/CN114350528B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881931A (en) * | 2014-04-04 | 2014-06-25 | 王会森 | Pholiota nameko and culture method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114350528A (en) | 2022-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103349166A (en) | Feed formula of taking cordyceps militaris culture medium residue as material, and preparation method thereof | |
| CN106613350B (en) | Schizophyllum commune and domestication and cultivation method and application thereof | |
| CN114250152B (en) | Six-sister morchella Guizhou MS630 and application thereof | |
| Erum et al. | Genetic divergence in amaranthus collected from Pakistan. | |
| CN107189947B (en) | Method for producing feed additive rich in heat-resistant protease and cordycepin by solid-state fermentation of cordyceps militaris | |
| CN114262668A (en) | Six-sister morchella Guizhou MS311 and application thereof | |
| US20230100319A1 (en) | A New Strain of Flammulina fennae and its molecular marker primers and molecular marker method | |
| CN111742778B (en) | Lepista sordida strain and method for cultivating fruiting bodies by using liquid strain of Lepista sordida strain | |
| CN114350528B (en) | Excellent strain of pholiota nameko, and specific molecular marker and application thereof | |
| Abdel-Azeem et al. | The Egyptian Ascomycota 1: Genus Aspergillus | |
| CN108770593B (en) | Lepista nuda strain and fruiting body cultivation method thereof | |
| Sharma et al. | Flammulina velutipes, the culinary medicinal winter mushroom | |
| CN113637594B (en) | Pholiota adiposa strain YX1, culture method and application thereof | |
| CN114085781B (en) | Ganoderma GZ and application thereof | |
| CN110583362A (en) | Black termitomyces albuminosus cultivation method | |
| CN109220514B (en) | Separation and artificial domestication cultivation method of new wild edible fungi | |
| CN112931059B (en) | Phellinus igniarius strain and cultivation method thereof | |
| CN112662570B (en) | Brown agaricus bisporus XZ2 strain and application thereof | |
| CN112673900B (en) | A strain of Rumex crispus and its cultivation, picking and preservation method | |
| CN108611279B (en) | Domestication method of sorangium cellulosum, domesticated sorangium cellulosum and application | |
| CN114145179B (en) | White lung-shaped pleurotus and cultivation method thereof | |
| CN116114534B (en) | Dictyophora rubrovalvata strain Qian PR20 and application thereof | |
| CN115948253B (en) | Dictyophora rubrovalvata strain Qian PR12 and application thereof | |
| CN106912293B (en) | Artificial culture method of cordyceps sobolifera | |
| CN116004400B (en) | Dictyophora rubrovalvata strain Qian PR23 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |