CN111109009A - Armillaria mellea SWFU-09 and application thereof - Google Patents
Armillaria mellea SWFU-09 and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses armillaria mellea SWFU-09 and application thereof, and belongs to the technical field of microorganisms. The preservation number of the armillaria mellea SWFU-09 is as follows: CGMCC No. 18149; the Armillaria mellea provided by the invention has the advantages of good comprehensive properties, high growth speed, excellent properties, strong practicability and good growth promotion effect on the gastrodia elata, the SWFU-09 strain is used for cultivating the gastrodia elata, and the gastrodia elata is artificially cultivated indoors through links such as mother culture, liquid fermentation culture, bacterial material culture, gastrodia elata cultivation and the like, so that efficient production is realized; experiments carried out in greenhouse and small grass dam field district of southwest forestry university and Zhaotong Gastrodia elata research institute show that the method has remarkable and stable effect, can effectively cultivate Gastrodia elata in high-yield places, and has high content of medicinal components in commodity hemp.
Description
Technical Field
The invention relates to armillaria mellea SWFU-09 and application thereof, belonging to the technical field of microorganisms.
Background
Gastrodia elata Bl, a perennial heterotrophic herb of the genus Gastrodia of the family Orchidaceae. The rhizome is thick, the capsule is oval and egg-shaped, and has no green leaves, and the seed or tuber is usually used for propagation. Listed in the national emphasis protection wild plant directory (second batch), belonging to II-class protection plants, is one of the famous and precious Chinese medicinal materials, and is superior to Yunan Zhaotongxiaocao product. Digging in winter until Qingming festival of 2 years, cleaning, steaming, and drying at low temperature.
Modern pharmaceutical analysis shows that chemical components such as phenols, glycosides (gastrodin, gastrodine, etc.), benzyl alcohol ester glycosides (parishin A, B, C), nitrogenous alkaloids, organic acids (citric acid, succinic acid, etc.), sterols, trace elements (chromium, manganese, iron, copper, etc.), polysaccharide compounds, etc. mainly exist in rhizoma gastrodiae. The chemical components of gastrodia tuber are affected by bacteria, variety, producing area, harvesting period and other factors and closely related to the subsequent processing method.
The medicinal value and the edible nutritive value of the gastrodia elata are high. Has the effects of calming liver-yang, calming wind, relieving spasm, tranquilizing mind, improving intelligence, nourishing brain, enhancing immunity, relieving dizziness, and protecting against radiation, and can be used for improving memory, treating hypertension and senile dementia, relieving spasm, tranquilizing mind, and relieving pain, and treating convulsion, cancer, aging, rheumatism, epilepsy and depression. The gastrodia elata has unique flavor, fleshy texture, fresh and crisp taste and convenient eating, so that the food processed by taking the gastrodia elata as a main raw material has high nutritional value, is a special medicated diet which can treat diseases and nourish health, not only meets the pursuit of modern people on delicious foods, but also can delay aging, enhance memory, improve myocardial blood circulation and promote the enhancement and recovery of immune functions, and can treat cardiovascular and cerebrovascular diseases such as rheumatic arthralgia, hemiplegia, dizziness, hypertension and the like.
In the cultivation and production of the gastrodia elata, as the gastrodia elata has no roots and leaves with assimilation function, nutrients cannot be manufactured and nutrients in the surrounding environment cannot be directly absorbed, except for the flowering and fruiting period, the processes of formation of gastrodia elata seedlings, renewal of nutrimental organs, turning of nutritional growth to reproductive growth and the like all need to depend on germination bacteria and Armillaria mellea to provide nutrients, the whole process of completing the life history is that the gastrodia elata seeds germinate and depend on the germination bacteria of the seeds of the lentinus sp to provide nutrients, the germinated protocorm can normally grow only by assimilation Armillaria mellea (Valla) P.Kumm.), the Armillaria mellea can generate rhizomes in the growth process, the rhizomes can extend out of trees providing nutrients for the rhizomes and enter the protocorm of the gastrodia elata to grow, and the Armillaria mellea can normally grow only by decomposing and supplying nutrients to lignin through cellulose, therefore, the gastrodia elata has extremely close nutritional relationship with the germination bacteria and the armillaria mellea, the growth characteristics of the armillaria mellea have great influence on the yield and quality of the gastrodia elata, and therefore, the search for new armillaria mellea suitable for cultivating the gastrodia elata is necessary.
Disclosure of Invention
The invention aims to provide armillaria mellea SWFU-09, and the classification and the name of the strain areArmillaria melleaThe preservation number is: CGMCC No. 18149; the Armillaria mellea is separated from the small grass dam town of Yi-Liang county in Showtong City of Yunnan province, and the Armillaria mellea SWFU-09 is preserved in the general microbiological center of the China Committee for culture Collection of microorganisms in 8.7.2019, wherein the preservation unit addresses are as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The method comprises the steps of using SWFU-09 strains for cultivating the gastrodia elata, selecting halimasch strains with thick and long rhizomorph, multiple branches, large biomass and high robustness, performing amplification propagation on halimasch mother strains, and inoculating the halimasch strains to thick and strong logs.
The invention also aims to use the SWFU-09 pure strain for cultivating the gastrodia elata, and specifically comprises the following steps:
(1) and (3) strain culture: the halimasch rhizomorph is washed clean, and a purified mother strain is obtained after separation and purification.
(2) And (3) strain amplification culture: inoculating the cut mother strain into liquid culture medium, and allowing mycelia and mycelia of Armillaria mellea to overgrow in the culture medium.
(3) Culturing a bacterial material: selecting broad-leaved tree species (selected from Fagaceae, Betulaceae, Rosaceae, such as cyclobalanopsis glauca, peach, pear, apple, wild cherry, Chinese chestnut, water white gourd, locust, quercus acutissima, birch, French phoenix tree, oak, bamboo willow, etc.) as log of fungus material, punching or cutting wound on the log, soaking in water thoroughly, taking out, air drying until no water drops, placing into polypropylene bag, covering with sealing cover, autoclaving, cooling, opening the sealing cover on an ultraclean bench, inoculating liquid strain of Armillaria mellea in the hole or wound, and culturing for a period of time to obtain Armillaria mellea fungus material.
(4) And (3) gastrodia elata cultivation: laying a layer of basic culture material at the bottom of the cultivation container, laying a layer of leaves, laying a layer of Armillaria mellea material, placing hemp seeds at two ends of each Armillaria mellea material, laying a layer of basic culture material, laying leaves at the uppermost layer, watering thoroughly, and capping with soil; culturing at 20-25 deg.C and humidity of 80-90% for 3-5 months to obtain good rhizoma Gastrodiae.
Preferably, the specific process of strain isolation and purification in step (1) of the present invention is as follows: cleaning Armillaria mellea, washing with sterile water for 3 times, soaking in 75% ethanol for 1min, washing with sterile water for 3 times, cutting into 0.5-1cm segments, soaking in 20ug/ml penicillin solution for 3-5s, removing surface water with sterilized filter paper, and inoculating to culture medium; when the halimasch on the culture medium produces hypha branches, cutting the hypha branches with length of 2-3mm and vigorous and tender hypha branches with an inoculating shovel, transferring the hypha branches into another culture medium (the formula is the same as the above), and culturing at constant temperature of 25 ℃ until the hypha branches and the hypha branches overgrow the culture medium, thus obtaining the purified mother strain.
Preferably, the formulation of the culture medium in step (1) of the present invention is: 300g/L of potato 200-,Magnesium sulfate 0.1-5g/L and vitamin B15-10mg/L, agar 15-20g/L, pH value 5.5-7.
Preferably, the formula of the liquid medium in step (2) of the present invention is: wheat bran 10-60g/L, corn flour 10-30g/L, sucrose 5-20g/L, potassium dihydrogen phosphate 0.5-5g/L, magnesium sulfate 0.5-5g/L, vitamin B110-20mg/L, pH value 5.5-7; the culture conditions were: shaking culture is carried out for 10-20d under the constant temperature condition of 25 ℃, and the rotation speed is 120-.
Preferably, in the step (3) of the invention, the diameter of the log is 5-15cm, and the length is 10-25 cm; punching or cutting the cut on the log to expose 0.4-0.6cm of xylem.
Preferably, the culture conditions in step (3) of the present invention are: culturing at 20-25 deg.C and humidity of 80-90% for 3-5 months.
The whole process of the method is finished under the condition of artificially controlling the temperature and the humidity indoors, the used cultivation materials are disinfected and sterilized, and each operation procedure is guaranteed to be carried out in an ultra-clean working environment, so that the production benefit can be improved, the cultivation time of the gastrodia elata is greatly shortened, the pollution problem in the cultivation process is avoided, the efficient production is realized, the market demand is met, and the economic development is driven.
The colony characteristics of the armillaria mellea SWFU-09 are as follows:
the colony is white and round at the initial stage, is medium in thickness, is villous, and becomes dark in color at the later stage; the hyphae are thin and dense, and the average germination rate is 57%; the stropharia rugoso is milk white when tender, is brown to reddish brown after mature, is root-shaped, round or flat, has multiple branches, is 0.1-0.4cm thick, has an average germination rate of 100 percent, and grows over a dish after 15-20 days, as shown in figure 1.
Molecular identification test of Armillaria mellea SWFU-09 provided by the invention
(1) Materials:
a flameless high temperature sterilizer, a centrifuge tube, a pipette gun, a pipette tip, scissors, a conical flask, a 1200ml big bottle, a microwave oven, distilled water, an ITS4 primer, an ITS5 primer, an experimental clothes, a mask, rubber gloves, agarose, BM2000+ DNAaker reagent, a fluorescent dye, a bromophenol blue dye solution, a TAE electrophoresis buffer solution, mix, sterile water, absolute ethyl alcohol, liquid nitrogen, a mortar, a grinding rod, a scalpel, disinfectant alcohol, degreased cotton, an electronic scale, a PCR instrument and a D3195-01HP Fungal DNA MiniKit (50) kit.
(2) DNA extraction
And (3) extracting DNA by a CTAB method.
a. And putting the sample into a mortar, and adding a proper amount of liquid nitrogen to carry out grinding and wall breaking treatment. Put into a 2.0ml centrifuge tube of the corresponding number.
b. 600. mu.l of BufferCP reagent and 10. mu.l of β -mercaptoethanol (operating in a fume hood) were added, respectively.
c.65 ℃ for 30 minutes, shaking up every 10 minutes.
d. Adding 600 mul (chloroform: isoamyl alcohol 24: 1) mixed solution, and turning over for 5-10 seconds; 10000 rpm for 5 minutes.
e. The supernatant was pipetted into a centrifuge tube containing a mixed solution of 150. mu.l BufferCXD and 300. mu.l absolute ethanol, and mixed using a pipette gun.
f. Sucking 600 mul of the mixed solution to a HiBindDNA adsorption column; centrifuge at 10000 rpm for 1 min.
g. The liquid in the collection tube was discarded and 650. mu.l of SPW Wash Buffer reagent was added and centrifuged for 10000 cycles for 1 minute, and this procedure was repeated twice.
And h.12000-rotation centrifugal drying treatment is carried out for 5 minutes.
i. Adding 50 μ l of Elution Buffer reagent, standing for 5 minutes, and then centrifuging for 2 minutes at 12000 rpm; this step was repeated twice.
j. And opening the cover and standing for 5 minutes to volatilize the absolute ethyl alcohol in the mixture.
k samples were stored at-20 ℃.
(3) PCR amplification and electrophoresis detection
The PCR amplification reaction system was 25. mu.l (as shown in FIG. 2), which included 10 Xamplification buffer containing 12.5. mu.l mix reagent, 0.5. mu.l each of forward and reverse primers, 1.0. mu.l DNA sample, and 10.5. mu.l ddH2O 10.5 to 25. mu.l volume.
And (3) uniformly mixing 3.0 mu l of the extracted and amplified DNA with 0.3 mu l of bromophenol blue solution (40% of sucrose and 0.25% of bromophenol blue), carrying out electrophoresis in 1% agarose gel containing 5% of Super GelRed nucleic acid dye in 1.0 XTAE buffer solution at an electrophoresis voltage of 90V for 30 minutes, stopping electrophoresis, taking out the gel, observing the molecular weight of the obtained PCR on a gel imaging system, recording and storing the result, wherein the partial electrophoresis detection chart of the PCR sample is shown in figure 3.
(4) Sequencing
And sending the ITS amplified fragment sample successfully obtained by PCR to Beijing Optimalaceae biotechnology Limited for sequencing to finally obtain a bidirectional ITS sequence fragment file of the sample.
(5) Data analysis
After a sequencing file is obtained, firstly opening observation peak values through BioEdit software, wherein each peak value corresponds to one base, and screening a sequence without impurity peak pollution for copying; each species sequence was compared to all other sequences of the species under study by estimating its genetic distance using NCBI website (https:// www.ncbi.nlm.nih.gov /) BLAST, downloading and saving the alignment gene bank values used in NCBI.
The results show that the similarity of the strain and other armillaria mellea is below 99 percent; using molecular data of this strain of Armillaria mellea SWFU-09 (M-9) to construct a phylogenetic tree in which the strain has the closest relationship to Armillaria mellea, as shown in FIG. 4; the results show that the armillaria mellea has a far distant relationship with other armillaria mellea, which indicates that various armillaria mellea can form a symbiotic relationship with gastrodia elata.
The invention has the beneficial effects that:
(1) the Armillaria mellea provided by the invention has the advantages of good comprehensive properties, high growth speed, excellent properties, strong practicability, good growth promotion effect on the Gastrodia elata, and suitability for production of Gastrodia elata.
(2) The method is efficient, nontoxic, safe, simple and convenient, and the gastrodia elata is artificially cultivated indoors through links such as mother culture, liquid fermentation culture, fungus culture and gastrodia elata cultivation, so that efficient production is realized; experiments carried out in greenhouse and small grass dam field district of southwest forestry university and Zhaotong Gastrodia elata research institute show that the method has remarkable and stable effect, can effectively cultivate Gastrodia elata in high-yield places, and has high content of medicinal components in commodity hemp.
Drawings
FIG. 1 is a characteristic diagram of colonies of Armillaria mellea SWFU-09;
FIG. 2 shows a PCR reaction system;
FIG. 3 is a partial electrophoretic detection map of a PCR sample;
FIG. 4 is a phylogenetic tree;
FIG. 5 is a schematic view of a Armillaria mellea log according to example 1;
FIG. 6 is a schematic view of a Armillaria mellea log according to example 2;
FIG. 7 is a schematic view of a Armillaria mellea fungus material in example 3.
Detailed Description
The present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited to the above description.
Example 1
The method for cultivating the gastrodia elata by using the SWFU-09 pure strain specifically comprises the following steps:
(1) strain culture
Cleaning Armillaria mellea rhizomorph, washing with sterile water for 3 times, soaking in 75% ethanol for 1min, washing with sterile water for 3 times, cutting into 0.5cm small segments, soaking in penicillin solution (20ug/ml) for 5s, removing surface water with sterile filter paper, and inoculating to culture medium.
The formula of the culture medium is as follows: 200g/L of potato, 15g/L of glucose, 4g/L of peptone, 4g/L of yeast extract, 0.1g/L of monopotassium phosphate, 0.1g/L of magnesium sulfate and vitamin B15mg/L, agar 15g/L, pH value 7.
When the halimasch on the culture medium produces rhizomorph branches, cutting the vigorous and tender rhizomorph with the length of 2mm by using an inoculating shovel, transferring the rhizomorph to another culture medium (the formula is the same as above), and culturing at 25 ℃ until the culture medium is full of hypha and rhizomorph, namely the purified mother strain.
(2) Culture of bacterial strain
Inoculating the minced mother seed into liquid culture medium, culturing at 25 deg.C and 160r/min with shaking for 10-20d, and allowing Armillaria mellea mycelium and mycelium to grow in the culture medium.
Culture solution formula: 10g/L of wheat bran, 10g/L of corn flour, 20g/L of cane sugar, 0.5g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and vitamin B110mg/L, pH value 6.5.
(3) Cultivation of bacterial material
In the embodiment, the log used as the fungus material is cyclobalanopsis glauca, the diameter of the log is 5-10cm, the length of the log is 10-25cm, and after being inoculated with armillaria mellea, rhizomorph is grown, so that the rhizomorph can be used for culturing the gastrodia elata.
Punching or cutting wound (fish scale/ring/strip) according to length and diameter of the log (depth to expose xylem 0.5cm), soaking in water thoroughly (36h), taking out, air drying until no water drops, placing into polypropylene bag, covering with sealing cover, and sterilizing in autoclave (0.15MPa, 121 deg.C) for 2 h. After cooling, the sealing cover is opened on the superclean bench, and the armillaria liquid strain is inoculated in the hole or the wound.
Culturing at constant temperature (25 deg.C) and humidity (humidity 90%) for 2 months to obtain mycelia and cut wood with mycelia and mycelia of Armillaria mellea, and collecting Armillaria mellea mycelia as shown in FIG. 5.
(4) Gastrodia elata cultivation method
The base compost with good air permeability is selected, a matrix with strong water retention, rich organic matters, looseness, easy water seepage and proper pH value is required, and sandy loam, humus, sand-added sawdust, sand-added rotten fallen leaves and the like can be selected and prepared according to a certain proportion.
The culture method comprises the following steps: selecting a cultivation container with the depth of 40cm and the length and width of 50cm, paving a layer of basic culture material at the bottom of the container, paving a layer of leaves, laying a layer of fungus material, placing hemp seeds at two ends of each fungus material, paving a layer of basic culture material, paving leaves with the thickness of 6cm at the uppermost layer, thoroughly watering, and then capping with soil; culturing at 20 deg.C and 90% humidity for 3-5 months to obtain good rhizoma Gastrodiae.
Example 2
The method for cultivating the gastrodia elata by using the SWFU-09 pure strain specifically comprises the following steps:
(1) strain culture
Cleaning Armillaria mellea rhizomorph, washing with sterile water for 3 times, soaking in 75% ethanol for 1min, washing with sterile water for 3 times, cutting into 1cm long segments, soaking in penicillin solution (20ug/ml) for 3s, removing surface water with sterile filter paper, and inoculating to culture medium for culturing as shown in FIG. 1.
The formula of the culture medium is as follows: 200g/L of potato, 20g/L of glucose, 2g/L of peptone, 1g/L of yeast extract, 5g/L of monopotassium phosphate, 5g/L of magnesium sulfate and vitamin B110mg/L, agar 15g/L, pH value 5.5.
When the halimasch on the culture medium produces hypha branches, cutting the hypha branches with the length of 3mm and the tender hypha branches with the inoculation shovel, transferring the hypha branches into another culture medium (the formula is the same as the above), and culturing at the constant temperature of 25 ℃ until the hypha branches and the hypha branches overgrow the culture medium, so that the purified mother strain is obtained.
(2) Culture of bacterial strain
Inoculating the minced mother strain into liquid culture medium, culturing at 25 deg.C and constant temperature at rotation speed of 120r/min for 10-20 days under shaking, and allowing Armillaria mellea mycelium and funicularis to overgrow in the culture solution.
The formula of the culture solution is as follows: 60g/L of wheat bran, 30g/L of corn flour, 15g/L of cane sugar, 0.5g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and vitamin B110mg/L, pH value 7.
(3) Cultivation of bacterial material
In the embodiment, the log used as the fungus material is an apple, the diameter of the log is 10-15cm, the length of the log is 10-20cm, and after the armillaria mellea is inoculated, a rhizomorph is grown, and the rhizomorph can be used for culturing the gastrodia elata.
Punching or cutting a wound (fish scale opening/annular opening/strip-shaped opening) on the log according to the length and diameter of the log (the depth is 0.5cm higher than the xylem), fully soaking in water (48h), taking out, airing until no water drops, putting into a polypropylene bag, covering with a sealing cover, and putting into an autoclave (0.15MPa, 121 ℃) for sterilization for 2 h; after cooling, the sealing cover is opened on the superclean bench, and the armillaria liquid strain is inoculated into the hole or the wound.
Culturing at constant temperature (25 deg.C) and humidity (humidity 90%) for 3 months to obtain mycelia and cut wood with mycelia and mycelia of Armillaria mellea, and collecting Armillaria mellea mycelia as shown in FIG. 6.
(4) Gastrodia elata cultivation method
The base compost with good air permeability is selected, a matrix with strong water retention, rich organic matters, looseness, easy water seepage and proper pH value is required, and sandy loam, humus, sand-added sawdust, sand-added rotten fallen leaves and the like can be selected and prepared according to a certain proportion.
The culture method comprises the following steps: selecting a cultivation container with the depth of 50cm and the length and width of 60cm, paving a layer of basic culture material at the bottom of the container, paving a layer of leaves, laying a layer of fungus material, placing hemp seeds at two ends of each fungus material, paving a layer of basic culture material, paving 10 cm-thick leaves at the uppermost layer, thoroughly watering, and then sealing the top with soil; culturing at 25 deg.C and humidity of 80% for 5 months to obtain good rhizoma Gastrodiae.
Example 3
The method for cultivating the gastrodia elata by using the SWFU-09 pure strain specifically comprises the following steps:
(1) strain culture
Cleaning Armillaria mellea rhizomorph, washing with sterile water for 3 times, soaking in 75% ethanol for 1min, washing with sterile water for 3 times, cutting into 0.7cm small segments, soaking in penicillin solution (20ug/ml) for 4s, removing surface water with sterile filter paper, and inoculating to culture medium.
The formula of the culture medium is as follows: 250g/L of potato, 18g/L of glucose, 2g/L of peptone, 3g/L of yeast extract, 4g/L of monopotassium phosphate, 4g/L of magnesium sulfate and vitamin B18mg/L agar 15g/L, pH value 6.
When the halimasch on the culture medium produces hypha branches, cutting the hypha branches with length of 2-3mm and vigorous and tender hypha branches with an inoculating shovel, transferring the hypha branches into another culture medium (the formula is the same as above), and culturing at constant temperature of 25 ℃ until the hypha branches and the hypha branches overgrow the culture medium, thus obtaining the purified mother strain.
(2) Culture of bacterial strain
Inoculating the minced mother strain into liquid culture medium, culturing at 25 deg.C and constant temperature at rotation speed of 150r/min under shaking for 10-20d, and allowing Armillaria mellea mycelium and funicularis to overgrow in the culture solution.
The formula of the culture solution is as follows: 20g/L of wheat bran, 20g/L of corn flour, 15g/L of cane sugar, 2g/L of monopotassium phosphate, 3g/L of magnesium sulfate and vitamin B115mg/L, pH value 6.
(3) Cultivation of bacterial material
The log used as the fungus material in the embodiment is bamboo willow, the diameter of the log is 5cm, the length of the log is 10cm, and after armillaria mellea is inoculated, a fungus rope grows out, and the fungus rope can be used for cultivating the gastrodia elata.
Punching or cutting a wound (fish scale opening/annular opening/strip-shaped opening) on the log according to the length and diameter of the log (the depth is 0.5cm higher than the xylem), fully soaking in water (40h), taking out, airing until no water drops, putting into a polypropylene bag, covering with a sealing cover, and putting into an autoclave (0.15MPa, 121 ℃) for sterilization for 2 h; after cooling, the sealing cover is opened on the superclean bench, and the armillaria liquid strain is inoculated into the hole or the wound.
Culturing at constant temperature (25 deg.C) and humidity (humidity 90%) for 2 months to obtain mycelia and cut wood with mycelia and mycelia of Armillaria mellea, and collecting Armillaria mellea (Armillaria mellea) material as shown in FIG. 7.
(4) Gastrodia elata cultivation method
The base compost with good air permeability is selected, a matrix with strong water retention, rich organic matters, looseness, easy water seepage and proper pH value is required, and sandy loam, humus, sand-added sawdust, sand-added rotten fallen leaves and the like can be selected and prepared according to a certain proportion.
The culture method comprises the following steps: selecting a cultivation container with the depth of about 45cm and the length and width of about 55cm, paving a layer of basic culture material at the bottom of the container, paving a layer of leaves, laying a layer of fungus material, placing hemp seeds at two ends of each fungus material, paving a layer of basic culture material, paving leaves with the thickness of 6-10cm at the uppermost layer, thoroughly watering, and capping with soil. Culturing at 28 deg.C and humidity of 90% for 3 months to obtain good rhizoma Gastrodiae.
Test results and analysis
In examples 1 to 3, 3 kinds of trees including cyclobalanopsis glauca, apple and bamboo willow were used to culture Armillaria mellea, and the results of mass cultivation are shown in Table 1.
| Different fungus materials | Percentage of germination of fungal cord (%) | Gastrodia elata yield (Individual basin) | Gastrodia elata weight (g/basin) | Content of Gastrodin (%) | Total gastrodin content (%) |
| Apple (Malus pumila) | 59.1948 | 8 | 216.56 | 0.7815 | 1.2167 |
| Bamboo willow | 50.6795 | 6 | 131.41 | 0.5201 | 0.8955 |
| Cyclobalanopsis glauca | 42.0909 | 10 | 564.46 | 0.7848 | 1.1015 |
As can be seen from Table 1, after 3-5 months of culture, Armillaria mellea SWFU09 has good growth on 3 strains, the germination rate of rhizomorph is higher than 40.00%, the number of produced gastrodia elata is stable, 6-10 gastrodia elata can be collected in each pot, the weight of the gastrodia elata is 32 g/each on average, the total content of gastrodin is higher than 0.8%, and the method can be used for cultivation production of gastrodia elata and the content of medicinal components is not lower than the national standard (the total content of gastrodin is not less than 0.25%); compared with the local traditional mode, the method of the invention shortens the time by 2.4-4.8 times and improves the breeding efficiency by about 3 times.
Sequence listing
<110> southwest university of forestry
<120> armillaria mellea SWFU-09 and application thereof
<130>2019-12-09
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>819
<212>DNA
<213>Armillaria mellea?(Vahl) P. Kumm.
<400>1
aagatcatta tgaaacttga atcgtagcat cgagaactgt tgctgacctg ttaaagggta 60
tgtgcacgtt cgacgtgttg cgttctattc atccacctgt gcacctttgt agacttgatt 120
aactttcgct ttcgagcggt tagaagggtt gctttcgagc tccctttgtc tatcaagtct 180
atgtctatat aatctcttgt atgtctagaa tgtcttgttt atgggatgca agtcctttaa 240
atcttataca actttcaaca acggatctct tggctctcgc atcgatgaag aacgcagcga 300
aatgcgataa ctaatgtgaa ttgcagaatt cagtgaatca tcgagtcttt gaacgcacct 360
tgcgcccttt ggtattccga agggcatgcc tgtttgagtg tcattaaatt ctcaacctcc 420
ccttctttca ttaggagtgc ggcggattgg atatgggggt ttgctggttt ctaacgagat 480
cagctcctct gaaatgcatt agcagaaacc gtttgacttt ggctgctagg ctgtgataat 540
atctacgcct tgtagttggg tcggaatacg agtcatacag tggtaactaa tcaggctttc 600
gggtctggct tagaatcggt ttggaaggtg cttaacggct ccttctgctt tctccctttg 660
cggagatact tgtccaattc taagagagga gttgcttagc gcgggcttag ctttccttga 720
tatttccctt tgactttgta gaaggattca gcttctaacc gtccattgac ttggacaatt 780
tattgactat ttgacctcaa atcaggtagg actacccgc 819
Claims (9)
1. Armillaria mellea SWFU-09 is characterized in that: the preservation number of the strain is as follows: CGMCC No. 18149.
2. The use of Armillaria mellea SWFU-09 as claimed in claim 1, wherein: the SWFU-09 strain is used for cultivating rhizoma Gastrodiae.
3. The use of Armillaria mellea SWFU-09 as claimed in claim 2, wherein: the specific process of using the SWFU-09 pure strain for cultivating the gastrodia elata comprises the following steps:
(1) and (3) strain culture: cleaning halimasch strongylocentrotus, separating and purifying to obtain a purified mother strain;
(2) and (3) strain amplification culture: inoculating the cut mother seeds into a liquid culture medium, and allowing mycelia and rhizomorph of Armillaria mellea to overgrow in the culture solution;
(3) culturing a bacterial material: selecting broad-leaved tree species as a log of the fungus material, punching or cutting a wound on the log, fully soaking in water, taking out and airing until no water drops, then putting into a polypropylene bag, covering a sealing cover, performing high-pressure sterilization, after cooling, opening the sealing cover on an ultraclean workbench, inoculating an armillaria liquid strain into the hole or the wound, and culturing for a period of time to obtain the armillaria fungus material;
(4) and (3) gastrodia elata cultivation: laying a layer of basic culture material at the bottom of the cultivation container, laying a layer of leaves, laying a layer of armillaria mellea material, placing hemp seeds at two ends of each armillaria mellea material, laying a layer of basic culture material, laying leaves at the uppermost layer, watering thoroughly, and then sealing with soil; culturing at 20-25 deg.C and humidity of 80-90% for 3-5 months to obtain good rhizoma Gastrodiae, wherein all steps are performed under indoor artificial temperature and humidity control, and each operation is performed in ultra-clean working environment.
4. The use of Armillaria mellea SWFU-09 as claimed in claim 3, wherein: the specific process of strain separation and purification in the step (1) is as follows: cleaning Armillaria mellea rhizomorph, washing with sterile water for 3 times, soaking in 75% ethanol for 1min, washing with sterile water for 3 times, cutting into 0.5-1cm segments, soaking in 20ug/ml penicillin solution for 3-5s, drying with sterile filter paper, and inoculating to culture medium; when the halimasch on the culture medium produces rhizomorph branches, cutting out rhizomorph with the length of 2-3mm by using an inoculating shovel, transferring into another culture medium, culturing at 25 ℃ until hypha and rhizomorph grow over the culture medium, and obtaining the purified mother strain.
5. The use of Armillaria mellea SWFU-09 as claimed in claim 4, wherein: the formula of the culture medium is as follows: potato 200-300g/L, glucose 15-20g/L, peptone 2-4g/L, yeast extract 1-4g/L, potassium dihydrogen phosphate 0.1-5g/L, magnesium sulfate 0.1-5g/L, vitamin B15-10mg/L, agar 15-20g/L, pH value 5.5-7.
6. The use of Armillaria mellea SWFU-09 as claimed in claim 3, wherein: the formula of the liquid culture medium in the step (2) is as follows: wheat bran 10-60g/L, corn flour 10-30g/L, sucrose 15-20g/L, potassium dihydrogen phosphate 0.5-5g/L, magnesium sulfate 0.5-5g/L, vitamin B110-20mg/L, pH value 5.5-7; the culture conditions were: shaking culture at 25 deg.C for 10-20d, rotation speed of 120-.
7. The use of Armillaria mellea SWFU-09 as claimed in claim 3, wherein: in the step (3), the diameter of the log is 5-15cm, and the length of the log is 10-25 cm; the cut is punched or cut out to a depth of 0.4-0.6cm to expose xylem.
8. The use of Armillaria mellea SWFU-09 as claimed in claim 3, wherein: the culture conditions in the step (3) are as follows: culturing at 20-25 deg.C and humidity of 80-90% for 3-5 months.
9. The use of Armillaria mellea SWFU-09 as claimed in claim 3, wherein: the broad-leaved tree in step (3) is Fagaceae, Betulaceae or Rosaceae.
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| CN114938760A (en) * | 2021-10-15 | 2022-08-26 | 黑龙江省林业科学院伊春分院 | Cultivation method of red mushroom |
| CN115336503A (en) * | 2022-08-30 | 2022-11-15 | 西南林业大学 | Preparation method of substitute bacterial material for gastrodia elata cultivation |
| CN116171800A (en) * | 2023-04-25 | 2023-05-30 | 云南省农业科学院质量标准与检测技术研究所 | Armillariella mellea growth matrix for gastrodia elata growth |
| CN117770140B (en) * | 2024-02-26 | 2024-04-23 | 云南省农业科学院质量标准与检测技术研究所 | Tissue culture and breeding method for gastrodia elata |
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| CN114938760A (en) * | 2021-10-15 | 2022-08-26 | 黑龙江省林业科学院伊春分院 | Cultivation method of red mushroom |
| CN115336503A (en) * | 2022-08-30 | 2022-11-15 | 西南林业大学 | Preparation method of substitute bacterial material for gastrodia elata cultivation |
| CN115336503B (en) * | 2022-08-30 | 2024-01-16 | 西南林业大学 | Preparation method of substitute bacteria for gastrodia elata cultivation |
| CN116171800A (en) * | 2023-04-25 | 2023-05-30 | 云南省农业科学院质量标准与检测技术研究所 | Armillariella mellea growth matrix for gastrodia elata growth |
| CN117770140B (en) * | 2024-02-26 | 2024-04-23 | 云南省农业科学院质量标准与检测技术研究所 | Tissue culture and breeding method for gastrodia elata |
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