CN104686196B - Method for preserving and separating toadstool strain through sporocarp dried in shade - Google Patents
Method for preserving and separating toadstool strain through sporocarp dried in shade Download PDFInfo
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- CN104686196B CN104686196B CN201510106417.0A CN201510106417A CN104686196B CN 104686196 B CN104686196 B CN 104686196B CN 201510106417 A CN201510106417 A CN 201510106417A CN 104686196 B CN104686196 B CN 104686196B
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- 238000004321 preservation Methods 0.000 claims abstract description 11
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention belongs to the technical field of microorganisms, relates to a method for preserving and separating a toadstool strain through sporocarp dried in the shade, and aims to solve the technical problems that a conventional toadstool strain preservation method is limited by season, complex to operate, easy in causing pollution and relatively high in cost. According to the technical scheme, the method for preserving and separating toadstool strain through sporocarp dried in the shade comprises the following steps: a, preserving the strain; b, separating the strain. The method provided by the invention is low in cost, simple to operate, unlimited by season, capable of effectively preserving activity of the strain, and convenient in transporting and preserving the strain.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of to be preserved by the sporophore that dries in the shade and separate Morchella esculenta (L.) Perss bacterium
The method planted.
Background technology
Morchella esculenta (L.) Perss are a kind of famous and precious Rare edible fungus, although artificial field production can be carried out at present, yield extremely unstable,
Its price is caused to remain high always.The acquisition of strain is the Morchella esculenta (L.) Perss Production of Large Fields first step, is also a critical step.At present
Morchella esculenta (L.) Perss strain separating is mainly with new fresh sporophore as separation material, and particular technique route has 2 kinds:In Morchella esculenta (L.) Pers sporophore
In output season (annual 3-4 months), new fresh sporophore is taken, is hung by the feet above culture medium, its ascospore falls to culture naturally
On base (spore separation method is commonly used)；Or new fresh sporophore tissue is taken, plus sterilized water grinding, prepare coating culture after bacteria suspension
Base (tissue isolation is of little use).After above method grows bacterium colony, by purification repeatedly for several times, obtain pure free of contamination
Strain, 4 DEG C of preservations are used when production.As new fresh sporophore easily rots, it is impossible to preserved for a long time, therefore Morchella esculenta (L.) Perss
Strain obtain with very strong seasonality, miss production season, cannot just obtain strain.And strain is preserved using the method
4 DEG C of refrigerator-freezers or Refrigerator store, high cost need to be placed in using test tube；Restored in test tube strain, needs subculture once every certain hour, in case
Only culture medium nutrient depletion, spawn activity are degenerated, complex operation；The insects such as new fresh sporophore upper band nematicide, and a large amount of miscellaneous bacterias,
Separate seriously polluted during strain.
The content of the invention
The technical problem to be solved in the present invention is that existing Morchella esculenta (L.) Perss preiservative methods are subject to seasonal restrictions, and is operated multiple
Easily pollute during miscellaneous, relatively costly, separation.
A kind of method for preserving and separating Morchella esculenta (L.) Perss strain by the sporophore that dries in the shade is the technical scheme is that, including such as
Lower step：
A, fungi preservation：In the 3-4 months, choose that maturation, stalwartness, no disease and pests harm, cap be full, rib and pit are complete on cap
Launch, stem is milky, the Morchella esculenta (L.) Perss that form is normal, volume is moderate, in well-ventilated, 10～15 DEG C of temperature, dark, drying
Place, spreads out and puts, stir frequently, be completely dried until drying in the air to sporophore；With paper bag packing, dark shady place is dried indoors and is protected
Deposit；
B, strain separating：When needing using strain, under aseptic condition, 0.5-1cm is taken2The sporophore that dries in the shade of size
Cap is organized, and is soaked with sterilized water, after softening completely, is put into sterilized mortar, is added 1 milliliter of sterilized water, grinds preparation group
Suspension is knitted, tissue suspension is taken and is inoculated in wheat grain culture medium central；In 15～25 DEG C of lucifuge cultures 10-20 days to the raw bacterium of Morchella esculenta (L.) Perss gas
Silk or sclerotium grow, and then with Inoculating needle picking aerial hyphae or sclerotium, are inoculated in PDA culture medium, cultivate again, hereafter exist
Purification culture repeatedly is carried out in PDA culture medium, till obtaining free of contamination strain.
Preferably, in step a, the remaining ascospore quantity of sporophore of having dried in the shade before preserving, is checked, method is as follows：Take
0.5—1cm2The cap piece of tissue of size, clear water immersion, after softening completely, are put into mortar, plus a small amount of clear water, grind preparation group
Suspension is knitted, under the microscope microscopy, do not have ascospore or ascospore sporophore to be very little discarded, select ascospore many
Sporophore preserve.
Preferably, in step b, cultivation temperature is 20 DEG C.
Preferably, in step b, incubation time is 15d.
Preferably, in step b, the sclerotium that inoculum used by second purification is obtained when being and separating for the first time.
During step a fungi preservation, if the higher or moist environment of improper ventilation, temperature, sporophore is easy
Occur to rot, and light note causes sporophore to launch ascospore in a large number, it is therefore desirable to low temperature, ventilation, drying, dark surrounds.
As part ascospore may have been launched before sporophore harvesting and in dry run, therefore residue can be first carried out before preservation
Ascospore volume check.Should perform during preservation prevents insect pest plague of rats measure.
During step b strain separating, sporophore amount is dried in the shade very little, the spore quantity that can be sprouted is very little, it is not easy to long
Go out aerial hyphae, amount is too many, although the spore of sprouting is more, and miscellaneous bacteria is also more, 0.5--1cm2It is appropriate；The aseptic water yield very little, no
Bacteria suspension easily being ground to form, the aseptic water yield is too many, water layer is formed in wheat grain bottom, accelerating antibacterial diffusion, 1ml is proper.Cause
For dry in the shade sporophore cap tissue it is very thin, very gently, thus cartographic represenation of area commonly used in the art dry in the shade sporophore tissue it is big
It is little.
The making of wheat grain culture medium in step b：No disease and pests harm, full, comparatively fresh, the Semen Tritici aestivi wheat grain of the belt leather that shells are chosen,
Clear water soaks 24 hours, enters pot and adds clear water to boil to wheat grain without the white heart, without broken skin, pulls dewatering out, contain into plate, sterilizes in 121 DEG C
It is 30 minutes, standby after cooling.
The making of PDA culture medium in stepb：200 grams of Rhizoma Solani tuber osis cut fritter, and liquor is filtered, and glucose 20 is added in filtrate
Gram, 7 grams of agar powder, clear water are settled to 1000 milliliters, and 121 DEG C sterilize 30 minutes, sterilized plate, cooled and solidified standby
With.
In the present invention, according to conventional cognition, after sporophore is dried, ascospore therein can also be dried, and lose mycelia
Sprouting ability.And the present invention picks and much dries in the shade, carries out spore germination experiment containing a large amount of thecasporous sporophore, it is found that
Although many spores have lost sprouting ability, but still has the spore imbibition of about 40-70%, mycelia is sprouted, bacterium colony is formed,
This result is exactly the technical foundation of the present invention.And the sporophore of high temperature drying is lost by high temperature injury due to spore therein
Activity, therefore be not easy to sprout mycelia again.
In the present invention, the culture medium of the conventional agar+nutrient+water of conventional research carries out Morchella esculenta (L.) Perss strain separating, mainly
It is conventional PDA culture medium.As Morchella esculenta (L.) Perss take from land for growing field crops, therefore carry a large amount of miscellaneous bacterias, including antibacterial and funguses.It is raw on PDA
When long, miscellaneous bacteria, especially antibacterial usually grow up to a thick layer of lawn, cover Morchella esculenta (L.) Pers. Mycelium, cause Morchella esculenta (L.) Pers. Mycelium to be difficult to grow
Go out, even if Morchella esculenta (L.) Perss grow aerial hyphae, when cultivating on picking aerial hyphae to new PDA, the antibacterial length that aerial hyphae carries
Gesture usually can cover Morchella esculenta (L.) Pers. Mycelium again, so repeatedly, cause to separate failure.This problem is in fresh Morchella esculenta (L.) Perss using spore point
Cannot solve when separating strain from method, during using tissue isolation, although with carrying out flat board training after stepwise dilution bacteria suspension respectively
The method of foster base coating can be with pollution remission situation, but this method is applied to new fresh sporophore, and is not suitable for dry sporophore.
Because new its spore germination rate of fresh sporophore is almost up to 100%, after a suitable dilution level is obtained Jing test, can be by
The dilution level is applied to the individual separation of different sporophore；And spore germination rate difference compares between dry sporophore Different Individual
Greatly, it is required for carrying out a series of dilution experiment for each sporophore, is only possible to the dilution level for obtaining being adapted to the individuality,
Workload is very big.And the research of the present invention finds, when the tissue suspension that stem organization makes is inoculated in wheat grain culture medium, Gaster caprae seu Ovis
Bacterium can grow a large amount of aerial hyphaes, and healthy and strong dense, or even develop sclerotium tissue, and growing way is significantly better than PDA culture medium.This is
As antibacterial volume is little more than Gaster caprae seu Ovis mycelia, they extend in culture medium and need continuous media, such as agar culture medium, and wheat grain
Between culture medium wheat grain, space is very big, hinders significantly the speed of antibacterial extension, and can not form lawn, and Gaster caprae seu Ovis mycelia is thin
Long, space is grown beneficial to which to its almost no inhibition, and good permeability, therefore growth diffusion velocity is faster than antibacterial,
Although there is mould contamination, do not have antibacterial strong the rejection ability of Morchella esculenta (L.) Perss yet, therefore on wheat broth, Morchella esculenta (L.) Pers. Mycelium
It is than growing more preferably on PDA, more healthy and stronger, and when taking these aerial hyphaes and carrying out purification culture to PDA, although also there is miscellaneous bacteria
Pollution, but Morchella esculenta (L.) Pers. Mycelium growing way is better than miscellaneous bacteria, accelerates purifying agaric process.Further, since wheat grain culture medium living contaminantses
Rate is lower than PDA, as long as therefore sporophore tissue amount and sterilized water amount control in a proportion substantially, all may be used
To grow the aerial hyphae of stalwartness, it is not necessary to do that too complicated dilution water is plain to be tested, be very suitable for spore germination rate inconsistent
Dry sporophore.It is exactly the technical foundation of the present invention above.
Beneficial effects of the present invention：
1st, the inventive method is adopted, at any time can be from the isolated strain of dry sporophore.In dry sporophore
Dry ascospore can keep sprouting vigor at least 48 months (because the preservation for not carrying out the longer time is tested, it is possible to son
The cystospore vigor retention time can also be longer), obtain strain.Thus breach the seasonal restriction of Morchella esculenta (L.) Perss strain production；This
Outward, excellent sporophore is obtained in field, can be by the strain that preserve for a long time that dries in the shade；
2nd, the isolated test tube strains of new fresh sporophore must be preserved in 4 DEG C of refrigerator ice cabinets.Dry sporophore can be in 4 DEG C of ice
Case refrigerator-freezer is preserved, it is also possible to be dried shady place preservation, low cost in room temperature dark；
3rd, the detached test tube strains of new fresh sporophore need new test tube of regularly transferring, and prevent nutrient depletion, spawn degeneration.
Dry sporophore is now with strain is now separated, without test tube of transferring, easy to operate；
4th, dry sporophore volume ratio test tube is much smaller, and can crumb and deposit for fine grained chippings, even if preserving many kinds,
Too large space is not needed；
5th, the test tube strains that new fresh sporophore is obtained easily are mutated during preservation, and good strains of seeds is degenerated.In dry sporophore
Dry ascospore Mutation probability it is very low, good strains of seeds can be kept for a long time；
If the 6, introduced a fine variety from other places, new fresh sporophore long-distance transport is perishable, and test tube kind easily pollutes, and packs complicated, such as
, in high temperature season, strain can also be dead for fruit.Dry sporophore is simple during long-distance transport to be packed, and activity is not limited by temperature
System, and will not rot；
7th, the sporophore that dries in the shade is compared new fresh sporophore and separates strain, can pollution abatement to a certain extent.Nematicide etc. is miniature
Insect is mainly derived from the soil of sporophore growth, and they need to gnaw fresh soft tender sporophore and survive, and is separating strain
When, the freely activity in the medium of these insects is propagated miscellaneous bacteria, greatly increases the probability of pollution.And the sporophore being dried is hardened,
It is unfavorable for that insect is gnawed with parasitism wherein, their meeting death, escape or dormancy, although when separating afterwards, the polypide of dormancy can
Can survive again, but quantity greatly reduces than new fresh sporophore, so as to reduce pollution probability；The mycelial growth of miscellaneous bacteria is needed
Environment to be moistened, when environment desiccation, the also dehydration atrophy of miscellaneous bacteria mycelia loses growth activity, even if portion when separating later
Divide miscellaneous bacteria mycelia to run into wet environment to survive again, but quantity is reduced, pollution probability is also few than new fresh sporophore；
8th, first wheat broth when separating, rather than conventional PDA culture medium, because Semen Tritici aestivi intergranular pore it is very big, significantly
The extension of antibacterial is hindered, is thus advantageous to reduce pollution, and it is ventilative, grow beneficial to Morchella esculenta (L.) Pers. Mycelium, accelerate purifying agaric and enter
Journey.
Specific embodiment
The dry sporophore separation strain of 1 different holding times of embodiment separates strain with new fresh sporophore and compares
3-4 month fields choose that maturation, stalwartness, no disease and pests harm, cap be full, on cap rib and pit be fully deployed, stem
For milky, the Morchella esculenta (L.) Perss that form is normal, volume is moderate, 2 parts are averagely allocated as, 1 part carries out strain separating immediately, and another 1 part logical
Wind is good, temperature relatively low (10-15 DEG C), dark, be dried at, spread out and put, stir frequently, it is completely dry to sporophore until drying in the air
It is dry, a small amount of cap tissue is taken, after clear water immersion softens, mortar is milled making tissue suspension, after microscopy, selects more than ascospore
Dry sporophore, fills envelope, and dry dark shady place disposed within is preserved, and performs and prevent insect pest plague of rats measure.
Due to wanting microscopy spore germination situation, therefore PDA culture medium when separating here.PDA culture medium makes：200 grams
Rhizoma Solani tuber osi cuts fritter, and liquor is filtered, and adds 20 grams of glucose, 7 grams of agar powder, clear water to be settled to 1000 milliliters in filtrate, and 121 DEG C go out
Bacterium 30 minutes, sterilized plate are standby after cooling.
New fresh sporophore spore separation strain method (spore separation method)：On superclean bench, by new fresh sporophore bacterium
Lid is downward, and stem upwards, is hung by the feet above culture medium, notices that sporophore should not contact culture medium, while irradiation of turning on light, promotes son
Entity launches ascospore.After about 12 hours, ware of making even, in 20 DEG C of lucifuge cultures, timing microscopy, records spore germination situation.
After bacterium colony grows, the mycelia of a little colony edge of Inoculating needle picking is inoculated in another PDA culture medium central authorities, cultivates again.So
Purification culture is repeated, till obtaining free of contamination strain.
It is dried sporophore and separates strain method：The dry sporophore of 12,24,36,48 months is saved in aforementioned method of drying in the shade,
0.5-1cm is taken respectively2The cap piece of tissue of size, is soaked with sterilized water on superclean bench, after softening completely, is put into sterilized
The mortar of bacterium, plus 1ml sterilized water, grinding prepare tissue suspension, are added in PDA culture medium with sterilized dropper, use L-type glass
Rod coating makes tissue suspension be evenly distributed in media surface.In 20 DEG C of lucifuge cultures, timing microscopy, spore germination feelings are recorded
Condition.After bacterium colony grows, the mycelia of a little colony edge of Inoculating needle picking is inoculated in another PDA culture medium central authorities, cultivates again.
Purification culture is repeated so, till obtaining free of contamination strain.
Purification strain inoculation PDA derived above, 3 wares/process are recorded into mycelial growth situation.
The dry sporophore of the different holding times of table 1 and growth situation compares obtained by new fresh sporophore
Note：In upper table, "+" is the quantitative expression to index, and "+" is more, represents that growing way is better, otherwise then poorer.Similarly hereinafter.
From 1 result of table, although xerospore is longer than Fresh spores due to Imbibing, sprout relatively slow, but its mycelia and
Sclerotium growing way does not have difference with Fresh spores.And xerospore its mycelia of different holding times, sclerotium growing way difference are also less, can
See that xerospore at least can maintain vigour in 48 months.
2 isolation medium of embodiment separates the impact of strain to xerospore
In view of in testing, find routine PDA because nutrient is sufficient in the past, while Morchella esculenta (L.) Pers. Mycelium grows, other are miscellaneous
Bacterium, especially antibacterial also raised growth, it is seriously polluted, therefore do following experiment, with the pure agar culture medium without any nutrient,
Wheat grain culture medium and conventional PDA culture medium are contrasted.Make following 3 kinds of culture medium：
Culture medium 1：Conventional PDA culture medium, composition and manufacture method are with embodiment 1.
Culture medium 2：Pure agar culture medium, compared with conventional PDA culture medium, without murphy juice and glucose, remaining composition
It is identical with manufacture method.
Culture medium 3：Wheat grain culture medium, chooses no disease and pests harm, full, comparatively fresh, the Semen Tritici aestivi wheat grain of the belt leather that shells, clear water leaching
Bubble 24 hours, enters pot and adds clear water to boil to wheat grain without the white heart, without broken skin, pull dewatering out, contain into plate, sterilizes 30 minutes in 121 DEG C,
It is standby after cooling.
Strain is separated with the dry sporophore for preserving 12 months, method is inoculated with 3 kinds of culture medium, 3 wares/place respectively with embodiment 1
When reason, wherein inoculation medium 1 and 2, it is that bacterium is hanged when being and bacteria suspension being uniformly coated on media surface, inoculation medium 3
Liquid is inoculated in culture medium central.Spore germination situation after inoculation in 20 DEG C of lucifuge cultures, timing microscopy culture medium 1 and 2.Bacterium
Fall after growing, record aerial hyphae growing way.
On 2 different isolation mediums of table, Morchella esculenta (L.) Pers. Mycelium growing state compares
If no nutrient from result above, culture medium, although miscellaneous bacteria is little, beneficial to purification, and Morchella esculenta (L.) Perss spore
Son can also be sprouted, but after sprouting to a certain extent, the nutrient depletion that spore is carried, mycelia just stop growing, it is impossible to further give birth to
A length of bacterium colony.And nutritious PDA is used, germ contamination is very serious, although can grow aerial hyphae, but growing way is weak, is unfavorable for
Purification.And although wheat grain culture medium aerial hyphae substantially grows that the time is longer than PDA, mycelia is sturdy, well-grown, or even can send out
Sclerotium is bred as, and pollutes little, therefore, strain is separated for the first time and needs for wheat grain culture medium.
3 cultivation temperature of embodiment separates the impact of growth to xerospore.
Strain is separated with the dry sporophore for preserving 12 months, method is inoculated with wheat grain culture medium central, respectively with embodiment 1
In 15 DEG C, 20 DEG C, 25 DEG C of lucifuge cultures, 3 wares/process, mycelia sclerotium growing state is recorded.
3 different cultivation temperature of table separate the impact of growth to xerospore
Although mycelial growth rate is very fast when 25 DEG C, its growing way is not so good as 20 DEG C, and sclerotium is less.Because Morchella esculenta (L.) Perss are low
Warm mushroom, temperature is too high to be easily caused mycelia premature aging.And although 15 DEG C of spores are also sprouted, and bacterium colony, sclerotium growing way,
Due to mycelial growth slowly, easily by living contaminantses, therefore, 20 DEG C is optimum growth temperature.
The comparison that different vaccination thing is grown to Morchella esculenta (L.) Pers. Mycelium during 4 second purification of embodiment
In example 2, because the suitable Morchella esculenta (L.) Pers. Mycelium growth of wheat grain, Morchella esculenta (L.) Perss can develop sclerotium, this research point
The sclerotium of morchella esculenta that grows in wheat grain culture medium in other Example 2, mycelia, the wheat grain with Morchella esculenta (L.) Pers. Mycelium (1-2 grains/ware) connect
Plant in PDA culture medium, compare purification effect.3 wares/process.20 DEG C, after lucifuge culture 15 days, observe mycelial growth situation.
The impact that inoculum during 4 second purification of table is grown to Morchella esculenta (L.) Pers. Mycelium
As seen from Table 4, sclerotium is best inoculum, therefore when separating for second, if can send out in wheat grain culture medium
Sclerotium is brought out, most handy sclerotium inoculation, purification are fast, and gained strain is excellent, and living contaminantses are few.If no sclerotium, wheat grain can be used
Inoculation, will first observe the pollution situation of wheat grain, take Morchella esculenta (L.) Pers. Mycelium robust growth as far as possible, and the few wheat grain of miscellaneous bacteria is inoculated with.
Claims (4)
1. a kind of by sporophore preservation and the method for separating Morchella esculenta (L.) Perss strain of drying in the shade, it is characterised in that：Comprise the steps：
A, fungi preservation：In the 3-4 months, choose that maturation, stalwartness, no disease and pests harm, cap be full, rib and pit are opened up completely on cap
Open, stem is milky, the Morchella esculenta (L.) Perss that form is normal, volume is moderate, in well-ventilated, 10～15 DEG C of temperature, dark, drying
Place, spreads out and puts, stir frequently, be completely dried until drying in the air to sporophore, take 0.5-1cm2The cap piece of tissue of size, clear water leaching
Bubble, after softening completely, is put into mortar, plus a small amount of clear water, and grinding prepares tissue suspension, under the microscope microscopy, no ascus spore
Son or ascospore sporophore very little is discarded, and selects the sporophore more than ascospore to preserve, with paper bag packing, does indoors
Dry dark shady place is preserved；
B, strain separating：When needing using strain, under aseptic condition, 0.5-1cm is taken2The sporophore cap group of drying in the shade of size
Knit, soaked with sterilized water, after softening completely, be put into sterilized mortar, add 1 milliliter of sterilized water, grinding prepares tissue suspension,
Take tissue suspension and be inoculated in wheat grain culture medium central；In 15～25 DEG C of lucifuge cultures 10-20 days to Morchella esculenta (L.) Perss aerial hyphae or bacterium
Core grows, and then with Inoculating needle picking aerial hyphae or sclerotium, is inoculated in PDA culture medium, cultivates again, hereafter trains in PDA
Purification culture repeatedly is carried out on foster base, till obtaining free of contamination strain.
2. the method for claim 1, it is characterised in that：In step b, cultivation temperature is 20 DEG C.
3. the method for claim 1, it is characterised in that：In step b, incubation time is 15 days.
4. the method for claim 1, it is characterised in that in step b, inoculum used by second purification are first separation
When the sclerotium that obtains.
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| CN106282034A (en) * | 2016-08-30 | 2017-01-04 | 杨燕 | A kind of Morchella esculenta (L.) Pers strain fast breeding method |
| CN109837216B (en) * | 2017-11-27 | 2021-04-27 | 中国农业科学院烟草研究所 | Method for quickly separating tobacco endophytic fungi |
| CN110604004B (en) * | 2019-09-20 | 2021-08-03 | 新疆农垦科学院 | Method for separating strains by dried morchella sporocarp |
| CN112449988A (en) * | 2020-10-20 | 2021-03-09 | 安徽乐永园农业科技有限公司 | Breeding and transferring method suitable for morchella |
| CN112655468A (en) * | 2020-10-20 | 2021-04-16 | 安徽乐永园农业科技有限公司 | Process suitable for fungus picking spores to prevent mixed bacteria |
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