CN110106094A - High temperature resistant Stropharia rugoso-annulata bacterial strain and its application - Google Patents
High temperature resistant Stropharia rugoso-annulata bacterial strain and its application Download PDFInfo
- Publication number
- CN110106094A CN110106094A CN201910546457.5A CN201910546457A CN110106094A CN 110106094 A CN110106094 A CN 110106094A CN 201910546457 A CN201910546457 A CN 201910546457A CN 110106094 A CN110106094 A CN 110106094A
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- temperature resistant
- annulata
- high temperature
- temperature
- 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.)
- Granted
Links
Classifications
-
- 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
-
- 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
- A01G18/20—Culture media, e.g. compost
-
- 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
- A01G18/40—Cultivation of spawn
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H15/00—Fungi; Lichens
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Developmental Biology & Embryology (AREA)
- Mushroom Cultivation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses one plant of high temperature resistant Stropharia rugoso-annulata bacterial strain Stropharia rugosoannulata, entitled mountain agriculture spherical cap 3 of the bacterial strain, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC NO.17676.The bacterial strain has stable high-temperature stability and highly yielding ability, is new bacterial strain, and mycelia and fructification heat-resisting ability greatly improve, and solves the problems, such as production early sowing of upper early autumn mycelia non-refractory, the late fall sowing easy parachute-opening of the end of spring and the beginning of summer in coming year fructification.
Description
Technical field
The present invention relates to edible mushroom technical fields, and in particular to one plant of high temperature resistant Stropharia rugoso-annulata bacterial strain and its application.
Background technique
Stropharia rugoso-annulata (Strophariarugosoannulata) is a kind of important pharmaceutical fungi and edible fungi, and delicious color is excellent,
It is full of nutrition, there is enhancing human immunity, reduce the health care and medicinal values such as blood glucose.
Stropharia rugoso-annulata belongs to middle low temperature class edible mushroom, and mycelial growth temperature adaptation range is 5-32 DEG C, fruit-body formation
And the temperature range of growth is 10-20 DEG C, the easy parachute-opening of fructification when the temperature rises to 20 DEG C, quality variation, former base at 25 DEG C
It is undifferentiated.Therefore it can only be cultivated once in China East China open-air atmosphere next year, 10-11 month is typically scheduled in production
Sowing, early April in coming year ground temperature stabilization start fruiting at 12 DEG C or more, but since temperature, ground temperature go up quickly, in temperature height
Former base difficulty is broken up when 25 DEG C, and fructification easy parachute-opening when temperature is higher than 20 DEG C, yield and quality are decreased obviously, and seriously affects life
Produce benefit.If 8-9 month is sowed, good product quality when 11-12 month fruiting, shelf-stable, but after planting mycelia encounter 30 DEG C with
Upper high temperature easily causes " burning bacterium ", leads to cultivation failure or the underproduction.The shortage of high temperature resistant Stropharia rugoso-annulata bacterial strain has become industry development
Bottleneck.Therefore, it needs to filter out good high temperature resistant Stropharia rugoso-annulata bacterial strain.
The screening of edible mushroom high-temperature resistant strain at present mainly uses mycelium heat shock method, i.e. mycelium is being suitable for cultivating item
It is cultivated under part after a certain period of time, is put under hot conditions progress thermostimulation 1-8h, further taken out to be placed at 25 DEG C of normal temperature and carry out
Renewal cultivation measures the speed of growth of mycelia, eliminates the bacterial strain of heat-resisting ability difference.But the high temperature resistant bacterium that this method filters out
Strain lacks representative and stability, and with the increase of Subculture times, the heterogeneity of bacterial strain is also more and more obvious；And it is easy
Existing mycelium high temperature resistant, but under hot conditions cannot normal fruiting the problem of.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide one plant of high temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia
Rugosoannulata) mountain agriculture spherical cap 3.The bacterial strain has stable high-temperature stability and high yield characteristics, meanwhile, also have
The advantages that mushroom is early, fruiting phase is long, level-one mushroom ratio is high, anti-hybrid ability is strong is new bacterial strain by verifying, and mycelia and son are real
Body heat-resisting ability greatly improves, and efficiently solves production early sowing of upper early autumn mycelia non-refractory, sowing summer late spring in the coming year in late fall
The problem of first fructification easy parachute-opening.
Specifically, the present invention relates to following technical schemes:
High temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia rugosoannulata) provided by the invention, is named as mountain agriculture
Spherical cap 3, which is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on April 28th, 2019
The heart (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number are as follows: CGMCC
NO.17676。
High temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia rugosoannulata) mountain agriculture spherical cap 3 of the invention, mycelia
5~42 DEG C of growth temperature range, 24~32 DEG C of mycelia optimum growth temperature range, 45 DEG C of resistance to maximum temperature；Fruit-body formation temperature
10~35 DEG C of range, 18~30 DEG C of fruit-body formation preference temperature scope of degree.
The claimed high temperature resistant Stropharia rugoso-annulata bacterial strain of the present invention includes bacterium corresponding to CGMCC NO.17676
Strain also includes the offspring caused by bacterial strain breeding with the bacterial strain with identical science of heredity and/or Morphologic Characters.
Described (Stropharia rugosoannulata) the mountain agriculture of high temperature resistant Stropharia rugoso-annulata bacterial strain spherical cap 3 are used as parent
The application carried out in breeding also belongs to protection scope of the present invention.
Described (Stropharia rugosoannulata) the mountain agriculture of high temperature resistant Stropharia rugoso-annulata bacterial strain spherical cap 3 are cultivated to obtain
Fructification also belong to protection scope of the present invention.
Described (Stropharia rugosoannulata) the mountain agriculture of high temperature resistant Stropharia rugoso-annulata bacterial strain spherical cap 3 are cultivated to obtain
Mycelium and/or spore also belong to protection scope of the present invention.
According to an aspect of the present invention, the invention further relates to the high temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia
Rugosoan nulata) mountain agriculture spherical cap 3 cultural method, include the following steps:
(1) high temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia rugosoannulata) mountain agriculture No. 3 cultures of spherical cap are obtained
Original seed be inoculated into Cultivar culture medium, 29-31d is cultivated under the conditions of 25-27 DEG C, obtains cultivar；
(2) compost is built into heap fermentation, stone is sowed when material temperature is down to 30 DEG C or less, 1 layer of 1 layered material point of every paving cultivation
Kind, 2 layers of strain of totally 3 layered material, build up trapezoidal heap；
(3) start earthing when mycelia grows to 2/3 or more of compost thickness, the water content of cover soil material is maintained at 20-
25%, thickness of earth covering 3cm；
(4) moisturizing is taken the circumstances into consideration according to material heap situation after sowing 20d；Flood is filled after the completion of bacterium germination and urges mushroom, while increasing ventilation quantity,
Mycelia is promoted to twist together to form former base；
(5) air humidity is maintained 85-95% by fruiting phase, reinforces ventilation and disease and pest control.
Preferably, in step (1), the Cultivar culture medium is made of the raw material of following mass percent:
Sawdust 30%, cotton seed hulls 30%, rice husk 20%, wheat bran 18%, gypsum 1%, quick lime 1%；
The water content for adding water to Cultivar culture medium is 75%.
Preferably, in step (2), the compost is made of the raw material of following mass percent:
Rice husk 45%, weed tree sawdust 28%, ramulus mori 10%, corncob 10%, rural area soil 5%, quick lime 2%；
The water content for adding water to compost is 75%.
Preferably, in step (3), the cover soil material is mixed simultaneously by the topsoil and turfy soil shone by weight 1:1
Water wetting is added to be prepared.
According to an aspect of the present invention, the invention further relates to above-mentioned high temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia
Rugosoan nulata) application of No. 3 obtained fructifications of mountain agriculture spherical cap in food processing.
According to an aspect of the present invention, the invention further relates to a kind of food, contain bacterial strain as described above；
And/or:
The mycelium and/or fructification that the bacterial strain is cultivated.
Beneficial effects of the present invention:
High temperature resistant Stropharia rugoso-annulata bacterial strain (Stropharia rugosoannulata) mountain agriculture spherical cap 3 provided by the present invention
It number is to be obtained using monospore crossbreeding technology, compared with conventional Stropharia rugoso-annulata kind, mycelia and fructification heat-resisting ability are obtained
To greatly improving, it is easy to efficiently solve production early sowing of upper early autumn mycelia non-refractory, sowing late fall, fructification the end of spring and the beginning of summer in the coming year
The problem of parachute-opening, biological efficiency are producing upper application value with higher 85% or more.It can be used as and be suitble to early
Mycelia high temperature resistant, the stropharia rugoso-annulata plantation kind of good quality and high output of autumn cultivation, reach time of the year when autumn changes into winter current year fruiting, crop
Fructification high, quality is good；It can also be cultivated in the time of the year when autumn changes into winter, the fructification that early summer in coming year season crop is high, quality is good,
It is second-rate to solve the problems, such as that conventional Stropharia rugoso-annulata kind season early summer goes out massee fruiting bodies.
Detailed description of the invention
Fig. 1: restoration ecosystem lab diagram after partial hybridization bacterial strain heat shock.
Fig. 2: the affiliation figure of hybrid strain and parent strain.
Fig. 3: primer pair Me5-Em2 corresponding 10 plants of bacterial strains SRAP map.
Fig. 4: restoration ecosystem lab diagram after bacterial strain heat shock of the invention.
Fig. 5: ground temperature and temperature change curve in spring brooder.
Fig. 6: ground temperature and temperature change curve in winter brooder.
Fig. 7: Stropharia rugoso-annulata fruiting photo.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
Term explanation:
Fructification: it is the production spore construction of higher fungus, i.e. sporocarp, is made of the mycelium texturized.
Former base: before edible mushroom, the little particle one by one formed by mycelia winding kink, is one after growing up
One mushroom.
Mycelia: single tubular filament is the structural units of most of fungies.
Mycelium: many mycelia, which flock together, forms the trophosome of fungi, i.e. mycelium.
Biological efficiency: referring to edible mushroom fresh weight and the ratio between compost dry weight used, and commonly using percentage indicates.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.If the experiment actual conditions being not specified in embodiment, usually according to
Normal condition, or the condition recommended according to Reagent Company；Reagent as used in the following examples, consumptive material etc., such as without special
Illustrate, can be obtained through commercial channels.
Culture medium used in the embodiment of the present invention is as follows:
Regeneration culture medium: potato 200.0g, sucrose 20.0g, peptone 2.0g, yeast powder 2.0g, potassium dihydrogen phosphate
1.5g, dipotassium hydrogen phosphate 1.5g, magnesium sulfate 1.5g, homeo-osmosis agent 1000.0mL, agar 20.0g.
Using the mannitol of 0.6mol/L as homeo-osmosis agent.
Mother culture media: potato 200.0g, glucose 20.0g, magnesium sulfate 1.5g, peptone 3.0g, potassium dihydrogen phosphate
3.0g, agar 20.0g, distilled water 1000mL.
Fluid nutrient medium: potato 200.0g, glucose 20.0g, distilled water 1000mL.
Pedigree seed culture medium: wheat 96%, wheat bran 2%, gypsum 2%；It is mass percent.Add water to pedigree seed culture medium
Water content is 75%.
Cultivar culture medium: sawdust 30%, cotton seed hulls 30%, rice husk 20%, wheat bran 18%, gypsum 1%, quick lime 1%；
It is mass percent.The water content for adding water to Cultivar culture medium is 75%.
Compost: rice husk 45%, weed tree sawdust 28%, ramulus mori 10%, corncob 10%, vegetable garden soil 5%, quick lime 2%；?
For mass percent.The water content for adding water to compost is 75%.
Embodiment 1: the breeding and identification of high temperature resistant Stropharia rugoso-annulata
1. the selection of parent:
The high yield Stropharia rugoso-annulata bacterial strain S26 of microorganism key lab, Shandong Agricultural University breeding (fruiting is early, and yield is high)
With R10 (level-one mushroom ratio is high, is not easy parachute-opening).
2. obtaining spore:
In experimental field, select the first damp fruiting, kind mushroom medium well, growing way is neat, mushroom shape is dignified, former base time of occurrence
Fructification short, mushroom cap is thick, stem is slightly grown.
In superclean bench, the fructification for being suitable for use as parent of picking is cut into stem, is thrown aside.Lamella is placed in downwards
In sterile petri dish, places for 24 hours, obtain spore print.Cap is taken out later, covers culture dish, has marked the title of bacterial strain, with guarantor
Fresh film sealing, spore print are spare.
3. single spore separation:
Using plating dilutions partition method, in superclean bench, sterile water is picked with aseptic filter paper item, paper slip wetting is dipped in
Spore is taken, is moved into the centrifuge tube with sterile water, the concentration after mixing well at this time is denoted as a, then draws 100 μ L spore liquids,
Injection is repeatedly inhaled and is beaten equipped in the centrifuge tube of 900 μ L sterile waters, and spore liquid concentration is 10 at this time-1A, repeatedly aforesaid operations later,
It is 10 that concentration is made in 5 centrifuge tubes-2、10-3、10-4、10-5、10-6The dilution of a, it is noted that the replacement of pipette tips when dilution,
Prevent the concentration of spore liquid from generating variation.The spore liquid of various concentration is drawn into the plating medium that 100 μ L injection diameter is 9cm
Middle coating, each concentration are coated with 3 plates, record data after sealing.It is suitable according to the selection of the result of spore germination after coating
Concentration is 10-4a.Single bacterium colony is made in spread plate at this concentration later.Culture dish is placed under 26 DEG C of dark conditions and is trained
Support, spore germination after 4d, the well-balanced quick single bacterium colony of development is selected in observation, in superclean bench picking spore, switching in
Slant medium, purifying culture, tentatively obtains mononuclear bacterial strain.
4. the identification of uninucleate hyphae
When the bacterium colony that single spore is sprouted grows to diameter greater than 1cm, picking colony edge mycelia is moved on glass slide,
Carry out microscopy, if without clamp connection, can initial guess its be uninucleate hyphae, be determined as being linked into inclined-plane training after uninucleate hyphae
Feeding base carries out purifying culture.
5. the hybridization of uninucleate hyphae is matched
The uninucleate hyphae of two different parents is inoculated on plating medium simultaneously, at a distance of 1.0cm-1.5cm, cultivates 7d
Left and right, after the contact of two mycelia, the part of picking intersection carries out microscopy, such as finds clamp connection, is transferred immediately in new
Slant medium on, 7d-10d or so is cultivated at 25 DEG C, chooses mycelia and grows vigorous, the uniform bacterial strain of colonial morphology, choose
A little mycelia is taken to be placed on microscopically observation, identification really after nucleated mycelium, carries out PDA slant tube and expands culture, by miscellaneous
It hands over and obtains 228 plants of hybrid strains altogether, be put into preservation in refrigerator, give over to screening test use.
6. the screening of hybrid strain:
6.1 primary dcreening operations:
(1) Heat thermostability
The plating medium of 228 plants of nucleated myceliums is set in full-automatic biochemical incubator, is arranged parameter (38 DEG C, for 24 hours), knot
Restore 26 DEG C after beam to continue to cultivate.Colony radius is measured with crossing method, it is extensive to calculate mycelia for the growth position after recording heat shock
Multiple growth rate and growth rate carry out Integrated Selection by index of growth rate, colony colour and the sturdy degree of mycelia.
Test result: colonial morphology is inhomogenous, mycelia grows weaker, growth recovery rate and is slower than eliminating for parent, most
49 plants of excellent heterocaryotic myceliums are filtered out afterwards, the results are shown in Table 1.
1:38 DEG C of heat shock screening gained high temperature resistant of table hybridizes hypha growth condition
Note :+growing way is general ++ grow fine +++ growing way is vigorous
As can be seen from Table 1,49 plants of screenings obtain the colony radius of bacterial strain, and RCM15 (No. 40) colony radius is 13mm,
It is minimum in 49 plants.J7L1 (No. 17) is held pride of place with the colony radius of 35mm, wherein 2 plants be less than 15mm accounting 4.08%, 13 plants
In 15~20mm accounting 26.53%, 10 plants in 20~25mm accounting 20.41%, 13 plants in 25~30mm accounting 26.53%, 10
Strain is greater than 30mm accounting 20.41%.Colony radius distribution is relatively uniform.Come top ten be J6L8, J8L8,20, J2L8,
J6L7、J7L5、L20、J5L4、J8L4、J8L3。
(2) the high temperature resistant domestication of hybrid strain
By observing mycelia upgrowth situation, the hybrid strain filtered out after Heat thermostability is transferred, each bacterial strain is made
It is handled for one, 3 repetitions of each processing, and is control with the hybrid strain of 26 DEG C of constant temperature incubations.Bacterial strain is placed in automatically
In biochemical cultivation case, setting parameter be (33 DEG C, 8h；26 DEG C, 16h), cultivate 14d.Selection grow fine bacterial strain switching, be placed in 25
5d is cultivated at DEG C, again be placed in biochemical cultivation case in, setting parameter be (38 DEG C, 8h；26 DEG C, 16h), cultivate 14d.Reject disadvantage
After bacterial strain, transfer, culture.5d is placed in full-automatic biochemical incubator, setting parameter be (42 DEG C, 8h；26 DEG C, 16h), culture
14d.Mycelia bacterial strain that is sturdy, pure white, growing fine, be resistant to 42 DEG C is filtered out to transfer.
Test result:
Table 2: high temperature resistant acclimation and screening obtains hybridization mycelia upgrowth situation
Note :+growing way is general ++ grow fine +++ growing way is vigorous
As shown in table 2, it recycles and tames by high temperature resistant, it is long with bacterium colony mean radius, the per day speed of growth of mycelia, mycelia
Gesture, mycelia color are that index comprehensive screens to obtain 27 plants of high-temperature resistant strains.
Colony radius relatively in, J8L4 has maximum colony radius 21mm, J8L7 to have minimum colony radius 7mm.Have 9 in 27 plants
Strain in 15~20mm accounting 33.33%, 15 plants in 10~15mm accounting 55.56%, 3 plants in 5~10mm accounting 11.11%, greatly
Part is in 10~15mm.Top ten J8L4, J8L3, M1, J6L8, Y1, J8L7, L20, J6L4, J8L8, J2L8.
(3) antagonistic effect
It the high-temperature resistant strain that selects and starting strain (or two parents) while being inoculated in culture dish, at a distance of 20mm, often
A processing is repeated 3 times, and is cultivated under the conditions of 26 DEG C, and whether observation bacterium colony intersection generates antagonism line.
Test result: the high-temperature resistant strain and starting strain (or two parents) selected has apparent antagonism.
(4) squamous subculture is tested
The high-temperature resistant strain selected carries out squamous subculture under the conditions of 26 DEG C, chooses the 5th generation, the 10th generation, the 15th generation work
For identifying object.The bacterial strain newly transferred is placed in (26 DEG C, 16h；38 DEG C, 8h) the lower culture 5d of circulation, mycelial growth rate is calculated,
It makes comparisons with mycelial growth rate primary, the results are shown in Table 3.
Table 3: high-temperature resistant strain genetic stability test result
7. secondary screening:
(1) after heat shock hybrid strain second level kind restoration ecosystem rate
Secondary screening is carried out to the high temperature resistant mycelia that primary dcreening operation obtains.By hybrid strain and parent strain switching equipped with 90% wheat,
8% sawdust, 2% gypsum 25 × 200mm test tube in, be placed in 38 DEG C of incubators heat shock for 24 hours until mycelia is colonized completely,
It marks in the growth position of mycelia.Restore 25 DEG C of cultures after heat shock, measures the growth recovery rate of mycelia.
Experimental result such as table 4:
Table 4: the strain growth rate after Heat thermostability
From table 4, it can be seen that J8L4 is most fast in 12 plants of hybrid strain growth rates, it is higher than starting strain and other hybridization bacterium
Strain, illustrates in second level cultivar, J8L4 also shows optimal.
The lab diagram of restoration ecosystem is as shown in Figure 1, as seen from Figure 1 after partial hybridization bacterial strain heat shock, bacterial strain J8L4 with
Other hybrid strains are compared, and after heat shock, faster, mycelia is relatively white sturdy for the growth rate of mycelia, and growing way is better than bacterium germination out
Strain and other hybrid strains.
8. bacterial strain genetic diversity and Genetic relationship
By primary dcreening operation, secondary screening selection growth rate is most fast, growing way is best, mycelia is most sturdy, the strongest 8 plants of bacterial strains of high temperature resistant
SRAP analysis is done with parent, analyzes its affiliation distance.
(1) bacterial strain activation, culture and DNA are extracted
It is inoculated on mother culture media after activating 10 plants of Stropharia rugoso-annulata bacterial strains before DNA is extracted, 26 DEG C of culture 7d take side
The eugonic mycelia of edge is transferred to rejuvenation on new mother culture media.Bacterial strain after rejuvenation is transferred to the mother for being covered with glassine paper again
On kind culture medium, 26 DEG C of constant temperature dark culturing 10d.Every bacterial strain scrapes the fresh mycelia of 200mg, is put into mortar, it is fast to pour into liquid nitrogen
Fast grind into powder, using plant genome DNA extracts kit (Beijing CoWin Bioscience Co., Ltd.) according to behaviour
Make step, extracts total DNA and take pictures in Labworks image acquisition and analysis software after 1.0% agarose gel electrophoresis, be stored in -20
It is spare in DEG C refrigerator.
(2) SRAP amplification reaction system and response procedures
SRAP amplification reaction system: 25 μ L of reaction volume, wherein 2 × Tap PCR Master Mix 12.5 μ L, 100 μ
Each 1 μ L of the upstream and downstream mo1/L primer, template are 50ng/ μ L DNA1 μ L, supply volume with distilled water.
SRAP expands benchmark program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min, 35 DEG C of renaturation 1min, 72 DEG C extend
1min, 5 circulations；94 DEG C of denaturation 1min, 50 DEG C of renaturation 1min, 72 DEG C of extension 1min, 35 recycle；72 DEG C of extension 10min, 4
DEG C save.
7 μ L of amplified production is taken to carry out 1.5% agarose gel electrophoresis, while with DNA Ladder Marker2000bp work
For molecular weight marker, 100V constant pressure 40~-60min of electrophoresis is observed and is taken pictures by ultraviolet gel imaging system.
(3) SRAP primer screening
The primer delivered using Li et al.2001, by Shanghai, Bo Shang Bioisystech Co., Ltd synthesizes (table 5).Just
To primer 16 (SEQ ID NO.1-SEQ ID NO.16), reverse primer 18 (SEQ ID NO.17-SEQ ID NO.34),
288 pairs of primers are obtained using the combination representation of Me-Em.3 parts of Stropharia rugoso-annulata DNA are selected to screen 288 pairs of primers,
By reproducible, intensity is higher, it is clear and legible be confirmed as slug band without dispute, the band that can embody bacterium strain difference, carry out
Corresponding statistics and analysis is to filter out optimal combination primer.
The selection result: the primer that 38 pairs reproducible, polymorphism ratio is high is filtered out altogether from 288 pairs of primers.Utilize 38
SRAP amplification is carried out to 10 parts of Stropharia rugoso-annulata genomic DNAs of primer pair and electrophoresis detection, coamplification go out 218 sites, band
Molecular size range is between 100-2000bp.Wherein polymorphic site 144, polymorphism ratio 66.1% are averagely each pair of to draw
Object amplifies 5.73 sites, and average polymorphic position points are 3.78.
Table 5:SRAP primer sequence
(4) SRAP electrophoresis result statistics and processing analysis
The consistent slug band of electrophoretic mobility in same primers pcr amplification product is confirmed as same band, amplification sun
Property be recorded as " 1 ", amplification feminine gender is recorded as " 0 ", and graphic documentation is converted into data information using Excel, and statistics forms " 0/1 "
Phenotypic data matrix calculates polymorphic site number, total number of sites and polymorphic site ratio P according to the data matrix
(Percentage of polymorp hic sites), estimates the level of genetic variation and genetic differentiation.
Calculation formula: P=k/n (100%)
In formula: P is polymorphic site ratio, and k is polymorphic position points, and n is amplification number of sites.
Using NTSY S-PC software carry out bacterial strain between non-weighting group average method (UPGMA) clustering, generate heredity away from
From dendrogram 2.
As seen from Figure 2, when likeness coefficient be 0.86 when, 10 plants of high-temperature resistant strains are distinguished, illustrate this 10
Strain high-temperature resistant strain is suitable as the strains tested of experiment in cultivation.Bacterial strain M1, L20 and its starting strain S26 gather in same branch,
Bacterial strain Y1, J8L3 and starting strain R10 gather at one, and J8L7 and J8L4 gather at one, and J6L4, J6L8 gather at one, but relationship
Relationship farther out, illustrates obvious with parent's difference, produces different with starting strain, meets screening and requires.
The present invention selects high-temperature resistant result and the optimal bacterial strain J8L4 of high yield characteristics carries out biological deposits, bacterial strain J8L4 identification
For Stropharia rugoso-annulata bacterial strain (Stropharia rugosoannulata), it is named as mountain agriculture spherical cap 3.After measured, breeding of the present invention
5~42 DEG C of mycelial growth temperature range of high-temperature resistant strain (strain number J8L4), mycelia optimum growth temperature range 24
~32 DEG C, 45 DEG C of resistance to maximum temperature；10~35 DEG C of fruit-body formation temperature range, fruit-body formation preference temperature scope 18~30
℃。
Restoration ecosystem lab diagram is as shown in Figure 4 after No. 3 heat shocks of mountain agriculture spherical cap.
The preservation information of bacterial strain J8L4 is as follows:
Join the biomaterial (strain) of evidence: mountain agriculture spherical cap 3
Classification naming: Stropharia rugoso-annulata Stropharia rugosoannulata
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: 2019.4.28.
Collection is registered on the books number: CGMCC NO.17676.
Embodiment 2: the cultivation of high temperature resistant Stropharia rugoso-annulata
1. the preparation of parent species
By No. 3 strain inoculateds of activated high temperature resistant Stropharia rugoso-annulata mountain agriculture into mother culture media, it is placed under the conditions of 26 DEG C
Constant temperature incubation 15d.
2. the preparation of liquid spawn
Parent species are inoculated into the conical flask equipped with 100mL aseptic liquid nutrient medium, the bacterium of 7 diameter 5mm of every bottle of inoculation
Cake is placed in 160r/min in 26 DEG C of constant-temperature tables and cultivates 7d.
3. the preparation of original seed
Appropriate wheat is weighed, 2% quick lime is added and impregnates 12h, is boiled by fire, is boiled to major part after pulling rinsing 1~2 time out
Wheat is transparent without the white heart, pours into cold water cooled down immediately.Excessive moisture is fallen in drip, and 2% wheat bran is added and the stirring of 2% gypsum is equal
It is even.With original seeds bottle dispense wheat, per it is bottled enter 2/3 wheat, sealed with breathable sealing film.Sterilize 120min under the conditions of 126 DEG C,
Every bottle of access liquid spawn 20mL, is cultivated under the conditions of being placed in 26 DEG C after cooling, spare as original seed.
4. the preparation of cultivar
Cultivar culture medium is fitted into polypropylene bacterium bag the 120min that sterilizes under the conditions of 126 DEG C, accesses original seed after cooling
It is spare that 30d is cultivated under the conditions of being placed in 26 DEG C.
5. building heap fermentation
The compost prewetted is uniformly mixed, bottom width 1.5m or so, top width 0.8m or so, high 1.5m or so are built up, it is long
The suitable fermentation heap of degree.It punches every the 20cm bamboo punt-pole of diameter 8cm or so, to improve the gas permeability of material heap, prevents in material heap
Portion and bottom anoxic cause anaerobic fermentation.It builds heap and pays attention to moisturizing in the process, make water content 80% or more, build heap after the completion with grass
Curtain or sunshade net cover moisturizing, when necessary covered plastic film.The temperature at the place measurement material heap 20cm or so daily, if plastic covering film
Ventilation 2~3 times daily increase oxygen content under film.
Material heap temperature is raised to 65 DEG C or so, keeps carrying out the 1st turning for 24 hours.Pay attention to mixing inside and outside layered material when turning, if
It was found that material heap overdrying can supplement appropriate clarification limewash, it is consistent that remaining operation with the 1st time builds heap.Rise to 65 DEG C of left sides again to temperature
Keep 24d behind the right side, carry out the 2nd turning, build running check Compost fermentation situation after heap heating, it is ensured that material heap without acid smell,
Compost can be used for sowing when soft, brown.
6. stropharia rugoso-annulata plantation and management
1. being sprinkled with quick lime in entire cultivation area before sowing sowing, material temperature drops to 30 DEG C or so stones and sows, every square metre
Sowing quantity is 500g.11 layer of strain of layered material point of every paving, totally 3 layers of stock strain, builds up trapezoidal heap, facilitates side earthing.Sowing
Afterwards with the wooden stick punching of diameter 4cm or so, increase oxygen content.Aerial temperature and humidity and soil temperature-moisture sensor are installed, to
Monitoring record entirely cultivate during temperature condition.
2. earthing and covering build the heap straw that after planting timely bedding uses limewash to impregnate as covering, thick 3cm
Left and right.2/3 or more fermentation material is grown to mycelia and starts earthing, and cover soil material selects the topsoil shone to mix with turfy soil 1:1
And add water wetting, so that water content is kept 20%~25%.Thickness of earth covering 3cm or so, is punched again after earthing.
3. being not necessarily to moisturizing bacterium germination period management bacterium germination early period, moisturizing is taken the circumstances into consideration according to material heap situation after sowing 20d.It is seasonable when moisturizing
The case where often paying attention to material heap side, controls water speed and water, prevents side earthing washed away.After the completion of bacterium germination, fills flood and urge
Mushroom, while increasing ventilation quantity, promote mycelia to twist together to form former base.
4. fruiting period management fruiting phase air humidity maintains 90% or so, sprayed in time at noon when fine
Water, it is a small amount of multiple, increase air humidity and covering humidity.(more than 30 DEG C), ventilation is sprayed water in time when air temperature is too high, really
Protect mushroom body normal growth.
It should be noted that intensity of illumination, will cover sunshade net in time when necessary, prevent illumination too strong, cause to burn during fruiting,
Reduce mushroom matter.Influence former base if illumination is too weak and form differentiation, fruit-body color shoals, influence yield and quality, according to weather and
When control greenhouse rolling blind situation.It should reinforce divulging information during fruiting, reduce gas concentration lwevel, good ventilation and penetrating light situation energy
Enough make fructification healthy and strong, uniformity is higher.
5. bacteria developing period mainly prevents and treats miscellaneous bacteria dirt without using pesticide in the entire cultivation of prevention and control of plant diseases, pest control Stropharia rugoso-annulata
Dye is pulled out in time if there are the miscellaneous bacterias such as terrible umbrella, cup fungi, Acarasiales, if timely when discovery local infection mould, green mold or Mucor
Infection site is dug up, and spills a small amount of lime, supplements new soil, holds together flat furrow face.Mycelia hangs yellow plate after covering in time, lures winged
Worm prevents the harm of mushroom mosquito, mushroom fly etc., while luring drosophila, black cutworm etc. using sugar-vinegar liquid.
6. harvesting and change of tide management when fructification is mature on the whole, and velum not yet ruptures, harvest in time.One hand when harvesting
It pinches mushroom foot and gently rotates and lift then up, another hand is pressed around mushroom foot, and charge level and soil layer are consolidated, and is prevented near drive
Mushroom flower bud.Each collecting time is fixed on 16:00 or so, passes through the megathermal period at noon at this time, and temperature is gradually fallen after rise, and high temperature can promote son
The rapid mature, parachute-opening of entity.Harvesting statistics level-one mushroom yield can relatively accurately reflect Stropharia rugoso-annulata fructification high temperature resistant situation at this time.
After one damp mushroom harvests, bacterium bed is cleared up, filling-in earthing, cut off the water 5~7d of bacteria, fills big again after mycelia restores
Water increases ventilation and carries out urging mushroom.
Embodiment 3: fruiting experiment
The high-temperature resistant strain filtered out by monospore crossbreeding, high temperature resistant domestication, primary dcreening operation, secondary screening, is tried through indoor antagonism
Test, 8 plants of high-temperature resistant strains (J8L4, J8L3, J8L7, J6L8, J6L4, M1, Y1, L20) are obtained in the verifying of the methods of squamous subculture,
Experiment in cultivation (cultural method is with embodiment 2) is carried out to it, (out with SM (national authentication kind), S26 (starting strain) and R10
Bacterium germination strain) as control.
Test one: spring brooder fruiting
September in 2018 15 days warms up progress stone sowing in greenhouse in the spring, and ground temperature is 26 DEG C when cultivation.After cultivation in time
Air, soil temperature sensor are installed, the measurement spring warms up temperature of shed, records a temperature data per hour, and make big spherical cap
Ground temperature and temperature change curve (Fig. 5) in spring brooder during mushroom is cultivated.Strain sprouting, field planting, bacterium germination number of days and each producing strain
6 and table 7 are shown in Table with level-one mushroom ratio.
Table 6: the Hypha Growth of Stropharia Rugosa-annulata time counts in spring brooder
Table 7: spring brooder Stropharia rugoso-annulata fruiting situation and output statistics
As seen from Figure 5, spring warm temperature of shed is obvious with Changes in weather, and temperature fluctuation is larger, and material temperature is comparatively steady
It is fixed.It is after planting strain sprouting and setting date in 4d, during which the highest temperature is 44 DEG C, and temperature on average is 33.5 DEG C, highest material temperature
It is 37 DEG C, average material temperature is 32 DEG C.It is sprouted after it can be seen that bacterial strain J8L3, J8L4 sowing 1d in table 6 and table 7,3d material feeding is fixed
It plants, is secondly J8L7, J6L4, L20, after planting 2d sprouts, and 4d field planting, material feeding field planting is slower for SM (CK), S26, R10, Y1,
It is sprouted after sowing 3d, 5d field planting.Although all bacterial strains sprout field planting in 5d, bacterial strain SM, Y1 mycelium germination time is slow, and
Mycelia is more thin and delicate, illustrates that the growth of the bacterial strain under the above cultivation condition is suppressed.Bacterial strain J8L3, J8L4, at sowing initial stage
Just it sprouts rapidly, illustrates mycelia not to be inhibited to grow in the above cultivation condition, mycelia has certain heat-resisting ability.
Sowing 7d-40d is bacteria developing period, and during which the highest temperature is 42 DEG C, and highest material temperature is 36 DEG C, and average material temperature is tieed up substantially
It holds at 23 DEG C or more.After planting 25d major part mycelia covers with the 2/3 of material heap, has carried out earthing at this time.This stage material temperature compared with
For stabilization, between 20-28 DEG C, bacterium germination is most fast for bacterial strain J8L4 in strains tested, and only 25d just covers with material heap, is secondly
J8L3, is J6L8, J8L7, J6L4, R10 and S26 that 35d covers with material heap later, and growing slower is Y1, M1, and control SM is
38d, and bacterial strain SM (CK), M1, L20 have more terrible umbrella, the pollution of alveolar substance cup fungi, illustrate that its anti-hybrid ability is weaker.
After planting 41d-60d is the first damp fruiting phase, and during which the temperature difference is larger, and after planting the lowest temperature is down to 0 after 60d
DEG C or so, material temperature is also gradually decreased to 15 DEG C or less.This stage highest temperature is 30 DEG C, and temperature on average is 11 DEG C, highest material temperature
25 DEG C, average material temperature is 19 DEG C.Bacterium germination speed speed and fruiting time have certain relationship sooner or later, and fruiting is earliest in strains tested
Be J8L4, be secondly J8L3 and J6L8, control strain 60d starts fruiting.Highest first damp mushroom yield is S20
(5.02kg/m2), it is secondly J8L3 (3.93kg/m2) and M1 (3.53kg/m2), control SM yield is minimum, is 0.95kg/m2, former
Because may be that fruiting time is later, material temperature is down to 10 DEG C and is formed hereinafter, being not suitable for former base later, and yield is relatively low.After planting 60d-
150d material temperature is substantially below 12 DEG C, the non-fruiting of each bacterial strain.
Sowing 150d-190d (the next spring) is the second damp fruiting phase, and during which the highest temperature is 40 DEG C, wherein having
40d temperature is higher than 25 DEG C (maintaining 3h or more daily), and material temperature is up to 26 DEG C；After planting 191d-210d is third tide fruiting
Phase, during which the highest temperature is 44 DEG C, and basic at least 4h or more daily is higher than 25 DEG C of temperature, and material temperature is up to 28.5 at this time
DEG C, average material temperature is 24.5 DEG C.Spring brooder Stropharia rugoso-annulata output statistics ended to April 16 (211d) in 2019, and the spring is warm at this time
The highest temperature is substantially at 40 DEG C or more in canopy, and for material temperature substantially at 25 DEG C or more, bacterial strain J8L4, J8L3 and J6L8 still have more original
Base differentiation.It is J8L4 (6.82kg/m that second and third damp mushroom yield is highest under this condition2), it is secondly J8L3 (5.52kg/m2) and
J8L7(5.42kg/m2), yield is minimum for L20 (3.85kg/m2) and CK (4.38kg/m2), starting strain S26 and R10 yield
Respectively 4.27kg/m2And 3.48kg/m2.It is J8L4 (11.84kg/m that yield is highest in strains tested2), biological efficiency is
70.27%.Secondly be J8L3 and M1, conversion ratio is respectively 66.23% and 65.20%, biological efficiency it is minimum be L20, only
21.71%.In addition to big L20, all high-temperature resistant strain biological efficiencies are above control strain (26.87%).Level-one mushroom ratio
Highest example is J8L4, is 70.9%, is much higher than control strain (33.45%), followed by bacterial strain J8L3 (63.59%) and J6L8
(62.69%).
As can be seen from the above results, bacterial strain J8L4, J8L3 mycelia and fructification high temperature resistant behave oneself best, at 30 DEG C of material temperature
It can sprout rapidly, be colonized under conditions of above, fruiting phase shifts to an earlier date 10d or more than control, and yield is much higher than control, in later period gas
Fructification quality is obviously better than compareing under warm 30 DEG C of hot conditions, still has former base to be formed under 25 DEG C of hot conditions of material temperature.
Test two: winter brooder fruiting
Stone sowing is carried out in greenhouse on October 15th, 2018,24 DEG C of ground temperature when cultivation.After cultivation in time
Air, soil temperature sensor are installed, the measurement spring warms up temperature of shed, records a temperature data per hour, and make big spherical cap
Ground temperature and temperature change curve (Fig. 6) in spring brooder during mushroom is cultivated.Strain sprouting, field planting, bacterium germination number of days and each producing strain
8 and table 9 are shown in Table with level-one mushroom ratio.
Table 8: the Hypha Growth of Stropharia Rugosa-annulata time counts in winter brooder
Table 9: winter brooder Stropharia rugoso-annulata fruiting situation and output statistics
From fig. 6, it can be seen that the highest temperature and highest material temperature are changed greatly with weather condition in winter brooder, but temperature on average
Peaceful material temperature is basicly stable.42 DEG C of the highest temperature of sprouting, setting date (1d-5d), 26 DEG C of temperature on average, 39 DEG C of highest material temperature,
Average 29 DEG C of material temperature.Similar with Stropharia rugoso-annulata sprouting, resident time in spring brooder, bacterial strain J8L3 and J8L4 sprout field planting earliest.
42 DEG C of the highest temperature of bacteria developing period (7d-40d), highest material temperature are 30 DEG C, and temperature on average peace material temperature is at 20 DEG C or so, temperature ring
Border is similar with spring brooder, and bacterial strain bacterium germination speed is also similar with spring brooder.Bacterium germination it is most fast be J8L4, after planting 26d covers with material heap,
Followed by J8L3 28d covers with material heap, bacterium germination it is most slow be CK, after planting 39d covers with material heap.Bacterial strain CK and L20 have a large amount of cup fungis
Pollution, remaining bacterial strain do not find miscellaneous bacteria, illustrate that bacterial strain CK and L20 anti-hybrid ability is poor.After planting 25d major part mycelia covers with material
The 2/3 of heap has carried out earthing.
It is the first damp fruiting phase from 45d-65d, 34 DEG C of the highest temperature, has 15d temperature that can maintain 5h at 20 DEG C or more.Winter
Fruiting order is similar to spring brooder fruiting order in brooder, fruiting it is earliest be J8L4, followed by J8L3 and J6L8, fruiting is the latest
Be bacterial strain CK, after planting 75d fruiting.81d-211d is second and third damp fruiting phase of Stropharia rugoso-annulata in winter brooder, can from figure
To find out, temperature is ascendant trend during this, and the highest temperature can rise to 44 DEG C or more, and the especially fruiting later period highest temperature is basic
30 DEG C or more are maintained, and the duration is longer.Winter brooder Stropharia rugoso-annulata output statistics was on May 1st, 2018, highest gas at this time
Temperature is at 35 DEG C or more, and 28 DEG C of material temperature or more, bacterial strain J8L4, J8L3, J6L8 still have former base.The highest bacterial strain of total output is J8L4,
Reach 14.05kg/m2, to compare SM (6.46kg/m2) 2.2 times, secondly be J8L3 (12.94kg/m2).Level-one mushroom ratio is most
High is J8L4 (68.68%), is 2.05 times of SM (33.45%).
As can be seen from the above results, winter temperature is suitable in winter brooder, can significantly extend Stropharia rugoso-annulata fruiting phase, always
Yield is higher than spring brooder.Bacterial strain J8L4 mycelia and fructification high temperature resistant behave oneself best, can be under conditions of 35 DEG C of material temperature or more
It sprouts rapidly, field planting, fructification quality is obviously better than compareing under 30 DEG C of hot conditions of later period temperature, in 25 DEG C of high temperature items of material temperature
Still there is former base to be formed under part.
Comprehensive bacterial strain sprouting each under two kinds of spring brooder, winter brooder environment, field planting, bacterium germination and fruiting phase and fructification produce
It is stronger that amount and quality condition can be seen that bacterial strain J8L4 mycelia heat-resisting ability, can comparatively fast determine under conditions of 30 DEG C or more
Hair transplant bacterium, fruiting phase shift to an earlier date, and reduce nutrition loss to a certain extent, improve yield, and fructification is not easy at high temperature
Parachute-opening, level-one mushroom ratio is high, and quality is substantially better than control.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>high temperature resistant Stropharia rugoso-annulata bacterial strain and its application
<130> 2019
<160> 34
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>artificial sequence
<400> 1
tgagtccaaa ccggata 17
<210> 2
<211> 17
<212> DNA
<213>artificial sequence
<400> 2
tgagtccaaa ccggagc 17
<210> 3
<211> 17
<212> DNA
<213>artificial sequence
<400> 3
tgagtccaaa ccggaat 17
<210> 4
<211> 17
<212> DNA
<213>artificial sequence
<400> 4
tgagtccaaa ccggacc 17
<210> 5
<211> 17
<212> DNA
<213>artificial sequence
<400> 5
tgagtccaaa ccggaag 17
<210> 6
<211> 17
<212> DNA
<213>artificial sequence
<400> 6
tgagtccaaa ccggaca 17
<210> 7
<211> 17
<212> DNA
<213>artificial sequence
<400> 7
tgagtccaaa ccggacg 17
<210> 8
<211> 17
<212> DNA
<213>artificial sequence
<400> 8
tgagtccaaa ccggact 17
<210> 9
<211> 17
<212> DNA
<213>artificial sequence
<400> 9
tgagtccaaa ccggagg 17
<210> 10
<211> 17
<212> DNA
<213>artificial sequence
<400> 10
tgagtccaaa ccggaaa 17
<210> 11
<211> 17
<212> DNA
<213>artificial sequence
<400> 11
tgagtccaaa ccggaac 17
<210> 12
<211> 17
<212> DNA
<213>artificial sequence
<400> 12
tgagtccaaa ccggaga 17
<210> 13
<211> 17
<212> DNA
<213>artificial sequence
<400> 13
tgagtccaaa ccggaag 17
<210> 14
<211> 17
<212> DNA
<213>artificial sequence
<400> 14
tgagtccaaa ccggtaa 17
<210> 15
<211> 17
<212> DNA
<213>artificial sequence
<400> 15
tgagtccaaa ccggtcc 17
<210> 16
<211> 17
<212> DNA
<213>artificial sequence
<400> 16
tgagtccaaa ccggtgc 17
<210> 17
<211> 18
<212> DNA
<213>artificial sequence
<400> 17
gactgcgtac gaattaat 18
<210> 18
<211> 18
<212> DNA
<213>artificial sequence
<400> 18
gactgcgtac gaatttgc 18
<210> 19
<211> 18
<212> DNA
<213>artificial sequence
<400> 19
gactgcgtac gaattgac 18
<210> 20
<211> 18
<212> DNA
<213>artificial sequence
<400> 20
gactgcgtac gaatttga 18
<210> 21
<211> 18
<212> DNA
<213>artificial sequence
<400> 21
gactgcgtac gaattaac 18
<210> 22
<211> 18
<212> DNA
<213>artificial sequence
<400> 22
gactgcgtac gaattgca 18
<210> 23
<211> 18
<212> DNA
<213>artificial sequence
<400> 23
gactgcgtac gaattcaa 18
<210> 24
<211> 18
<212> DNA
<213>artificial sequence
<400> 24
gactgcgtac gaattcac 18
<210> 25
<211> 18
<212> DNA
<213>artificial sequence
<400> 25
gactgcgtac gaattcag 18
<210> 26
<211> 18
<212> DNA
<213>artificial sequence
<400> 26
gactgcgtac gaattcat 18
<210> 27
<211> 18
<212> DNA
<213>artificial sequence
<400> 27
gactgcgtac gaattcta 18
<210> 28
<211> 18
<212> DNA
<213>artificial sequence
<400> 28
gactgcgtac gaattctc 18
<210> 29
<211> 18
<212> DNA
<213>artificial sequence
<400> 29
gactgcgtac gaattctg 18
<210> 30
<211> 18
<212> DNA
<213>artificial sequence
<400> 30
gactgcgtac gaattctt 18
<210> 31
<211> 18
<212> DNA
<213>artificial sequence
<400> 31
gactgcgtac gaattgat 18
<210> 32
<211> 18
<212> DNA
<213>artificial sequence
<400> 32
gactgcgtac gaattgtc 18
<210> 33
<211> 18
<212> DNA
<213>artificial sequence
<400> 33
tgtggtccgc aaatttag 18
<210> 34
<211> 18
<212> DNA
<213>artificial sequence
<400> 34
aggcggttgt caattgac 18
Claims (10)
1. one plant of high temperature resistant Stropharia rugoso-annulata bacterial strain Stropharia rugosoannulata, the entitled mountain agriculture ball of the bacterial strain
Lid 3, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC
NO.17676。
2. application of the high temperature resistant Stropharia rugoso-annulata bacterial strain described in claim 1 in being cross-breeding as parent.
3. cultivating the fructification that high temperature resistant Stropharia rugoso-annulata bacterial strain described in claim 1 obtains.
4. cultivating mycelium and/or spore that high temperature resistant Stropharia rugoso-annulata bacterial strain described in claim 1 obtains.
5. the cultural method of high temperature resistant Stropharia rugoso-annulata bacterial strain described in claim 1, which comprises the following steps:
(1) obtained original is cultivated by (Stropharia rugosoannulata) the mountain agriculture of high temperature resistant Stropharia rugoso-annulata bacterial strain spherical cap 3
Kind is inoculated into Cultivar culture medium, and 29-31d is cultivated under the conditions of 25-27 DEG C, obtains cultivar；
(2) compost is built into heap fermentation, stone sowing when material temperature is down to 30 DEG C or less, 11 layer of cultivar of layered material point of every paving, totally 3
2 layers of strain of layered material, build up trapezoidal heap；
(3) starting earthing when mycelia grows to 2/3 or more of compost thickness, the water content of cover soil material is maintained at 20-25%,
Thickness of earth covering is 3cm；
(4) moisturizing is taken the circumstances into consideration according to material heap situation after sowing 20d；Flood is filled after the completion of bacterium germination and urges mushroom, while increasing ventilation quantity, is promoted
Mycelia twists together to form former base；
(5) air humidity is maintained 85-95% by fruiting phase, reinforces ventilation and disease and pest control.
6. cultural method according to claim 5, which is characterized in that in step (1), the Cultivar culture medium is by following
The raw material of mass percent forms:
Sawdust 30%, cotton seed hulls 30%, rice husk 20%, wheat bran 18%, gypsum 1%, quick lime 1%；
The water content for adding water to Cultivar culture medium is 75%.
7. cultural method according to claim 5, which is characterized in that in step (2), the compost is by following quality hundred
Divide the raw material composition of ratio:
Rice husk 45%, weed tree sawdust 28%, ramulus mori 10%, corncob 10%, rural area soil 5%, quick lime 2%；
The water content for adding water to compost is 75%.
8. cultural method according to claim 5, which is characterized in that in step (3), the cover soil material is by the table that has shone
Layer soil and turfy soil mix by weight 1:1 and water wetting are added to be prepared.
9. application of the fructification as claimed in claim 3 in food processing.
10. a kind of food contains high temperature resistant Stropharia rugoso-annulata bacterial strain described in claim 1；
And/or:
The mycelium and/or fructification that the bacterial strain is cultivated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910546457.5A CN110106094B (en) | 2019-06-24 | 2019-06-24 | High-temperature-resistant stropharia rugosoannulata strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910546457.5A CN110106094B (en) | 2019-06-24 | 2019-06-24 | High-temperature-resistant stropharia rugosoannulata strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110106094A true CN110106094A (en) | 2019-08-09 |
| CN110106094B CN110106094B (en) | 2020-07-24 |
Family
ID=67495705
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910546457.5A Active CN110106094B (en) | 2019-06-24 | 2019-06-24 | High-temperature-resistant stropharia rugosoannulata strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110106094B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452823A (en) * | 2019-10-11 | 2019-11-15 | 鲁东大学 | A kind of fluid nutrient medium of stropharia rugosoannulata strain and preparation method thereof |
| CN111887096A (en) * | 2020-07-29 | 2020-11-06 | 云南省供销合作社科学研究所 | Golden stropharia rugoso-annulata strain and cultivation method thereof |
| CN112410226A (en) * | 2020-09-10 | 2021-02-26 | 云南菌视界生物科技有限公司 | Golden stropharia rugoso-annulata strain |
| CN114250151A (en) * | 2021-11-22 | 2022-03-29 | 海南省林业科学研究院(海南省红树林研究院) | Cross breeding method of tricholoma lobayense heim |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753676A (en) * | 2018-07-20 | 2018-11-06 | 山东农业大学 | A kind of selection of Stropharia rugoso-annulata high-temperature resistant strain |
| CN108967042A (en) * | 2018-10-18 | 2018-12-11 | 山东农业大学 | A method of utilizing 1-DNJ in Stropharia rugoso-annulata enrichment ramulus mori |
-
2019
- 2019-06-24 CN CN201910546457.5A patent/CN110106094B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753676A (en) * | 2018-07-20 | 2018-11-06 | 山东农业大学 | A kind of selection of Stropharia rugoso-annulata high-temperature resistant strain |
| CN108967042A (en) * | 2018-10-18 | 2018-12-11 | 山东农业大学 | A method of utilizing 1-DNJ in Stropharia rugoso-annulata enrichment ramulus mori |
Non-Patent Citations (1)
| Title |
|---|
| GRIT ET AL: "Fate of bisphenol A during treatment with the litter-decomposing fungi Stropharia rugosoannulata and Stropharia coronilla", 《CHEMOSPHERE》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452823A (en) * | 2019-10-11 | 2019-11-15 | 鲁东大学 | A kind of fluid nutrient medium of stropharia rugosoannulata strain and preparation method thereof |
| CN111887096A (en) * | 2020-07-29 | 2020-11-06 | 云南省供销合作社科学研究所 | Golden stropharia rugoso-annulata strain and cultivation method thereof |
| CN111887096B (en) * | 2020-07-29 | 2022-03-04 | 云南省供销合作社科学研究所 | Golden stropharia rugoso-annulata strain and cultivation method thereof |
| CN112410226A (en) * | 2020-09-10 | 2021-02-26 | 云南菌视界生物科技有限公司 | Golden stropharia rugoso-annulata strain |
| CN112410226B (en) * | 2020-09-10 | 2023-02-17 | 云南菌视界生物科技有限公司 | Golden stropharia rugoso-annulata strain |
| CN114250151A (en) * | 2021-11-22 | 2022-03-29 | 海南省林业科学研究院(海南省红树林研究院) | Cross breeding method of tricholoma lobayense heim |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110106094B (en) | 2020-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110106094A (en) | High temperature resistant Stropharia rugoso-annulata bacterial strain and its application | |
| CN107460133B (en) | Dark color has every endogenetic fungus HS40 and its application in dendrobium candidum production | |
| CN107529398B (en) | A kind of detoxicated ginger mating system | |
| CN107988087B (en) | Blueberry endophytic fungus with growth promoting effect and application thereof | |
| CN108753676A (en) | A kind of selection of Stropharia rugoso-annulata high-temperature resistant strain | |
| CN109430063B (en) | A kind of directed screening method of floorboard with high oil content peanut | |
| CN105918200A (en) | Crab-rice symbiotic planting-breeding pool and comprehensive temperature-adjusting method thereof | |
| CN107306786A (en) | A kind of breeding method of the anti-fall double-linear hybrid rice kind of high-grain weight excellent-quality blast resisting | |
| CN105340755A (en) | Method for continuously culturing high frequency regeneration plants through cereal crop single plant source microspores | |
| CN105453923A (en) | Method for promoting target rhizobium to migrate and colonize in alfalfa plant | |
| CN110140591A (en) | Hickory chick superior strain and its application | |
| CN110447532B (en) | Breeding method of high-temperature-resistant cotton | |
| CN108260470A (en) | A kind of method for improving matsutake mycorrhizal seedling raising and application | |
| CN110073898A (en) | A kind of ladder rib hickory chick strain the factorial production and northern facility cultivation technique | |
| CN107926549B (en) | Method for improving resistance of crops to herbicide bensulfuron methyl by utilizing Piriformospora indica | |
| CN106212277A (en) | A kind of method producing Rhizoma Zingiberis Recens mycorhiza tissue culture of sprout | |
| CN108913625A (en) | Salt tolerant streptomycete, its microbial inoculum and its microbial inoculum are promoting the application in plant growth | |
| CN107125084A (en) | A kind of implantation methods grown suitable for blueberry | |
| CN107099465B (en) | Growth-promoting bacterial strain and its microorganism seedling medium for promoting cuttage tea shoot to take root | |
| CN108203695B (en) | Rhododendron mycorrhizal fungi functional strain and application thereof | |
| CN1820581A (en) | Breeding method for chilli pepper nuclear male sterile dual purpose line and nuclear substance male sterile recovery line | |
| CN110218657A (en) | One plant of long shoot trichoderma MD30 and its biological organic fertilizer of development | |
| CN103444520A (en) | Breeding technology of novel strawberry virus-free seedling | |
| CN109943496A (en) | One plant of rhizobium seeks the method with the efficient symbiosis alfalfa variety of specificity | |
| CN116171808A (en) | Method for repairing degraded grassland by combining arbuscular mycorrhizal fungi and bacteria |
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 |