CN110184201A - A kind of true pleurotus cornucopiae bacterial strain and its selection - Google Patents
A kind of true pleurotus cornucopiae bacterial strain and its selection Download PDFInfo
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- CN110184201A CN110184201A CN201910538171.2A CN201910538171A CN110184201A CN 110184201 A CN110184201 A CN 110184201A CN 201910538171 A CN201910538171 A CN 201910538171A CN 110184201 A CN110184201 A CN 110184201A
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- 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
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
- C12N15/04—Fungi
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
It is to hybridize to obtain hybrid strain by more spores of seafood mushroom and crab flavour mushroom the present invention provides a kind of true pleurotus cornucopiae bacterial strain and its selection, in the true Ji of true pleurotus cornucopiae kind No. 1 that more spores selfing using hybrid strain obtains；The present invention obtains true a Ji No. 1 by hybridizing and being selfed breeding, in the speed of growth compared with parent strain, the purseful time shifts to an earlier date 5d than starting strain seafood mushroom, growth cycle does sth. in advance 10d than parent seafood mushroom and is bred as harvesting, average stem length 14.4cm, average bacteria cover diameter 1.4cm, average single bottle yield 330.5g, average organism efficiency 110.1%, is better than parent's starting strain seafood mushroom and crab flavour mushroom, and economic benefit improves.
Description
Technical field
The present invention relates to a kind of true pleurotus cornucopiae bacterial strain and its selections, belong to the Breeding of Edible Mushroom technical field.
Background technique
True pleurotus cornucopiae (Hypsizigus marmoreus), also known as spot jade gill fungus, beautiful gill fungus, white jade, seafood mushroom, letter like mushroom etc.,
Taste is fresher than oyster mushroom, and matter is more tough than mushroom, and meat is thicker than sliding mushroom, has the fragrant unique taste of crab.Its Amino Acids in Proteins A wide selection of colours and designs,
Including 8 kinds of essential amino acids, wherein lysine and arginine content is higher than general mushroom class.Since the 1980s, very
Pleurotus cornucopiae, which is introduced a fine variety to China, starts commercialization plantation, has become important one of the batch production product of national edible mushroom.It is given birth in cultivation
Its production cycle is longer (120-140d) in production, and the speed of growth of thallus is poor compared with slow, inhibition miscellaneous bacteria ability, easy microbiological contamination, and
And the influence vulnerable to planting environment and cultivation condition, it is subject to certain restrictions in production.Having for cultivating at present is light grey and pure white
Two strains of color, White strain claim " white beech mushroom ", " seafood mushroom "；Grey strain claims " crab flavour mushroom ", due to true pleurotus cornucopiae delicious flavour,
Unique oral sensations and nutritive value and medical value are high, to be received by the market.
From in the 1980s, true pleurotus cornucopiae is introduced a fine variety to the manual cultivation in China, research is started late, wherein genetic breeding
Aspect is less, and true pleurotus cornucopiae cultivar is more single currently on the market, and homonymus or synonymum phenomenon is serious, therefore the cultivation of known kind
The breeding of method and new varieties is always the research important topic of true pleurotus cornucopiae research, production field.
Summary of the invention
The purpose of the present invention is provide one to overcome the problems, such as that existing factory culture commonly uses true pleurotus cornucopiae strain single variety
Kind of true pleurotus cornucopiae candida species of edible mushroom, specifically it is a kind of with merit true pleurotus cornucopiae (Hypsizygus marmoreus) Ji No. 1 true.This kind is on April 16th, 2019 in China Committee for Culture Collection of Microorganisms's commonly micro- life
The preservation of object center, deposit number are as follows: CGMCC NO. 17598, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
To achieve the above object, the present invention adopts the following technical solutions:
A kind of the true pleurotus cornucopiae candida species of edible mushroom true a Ji No. 1, obtained by following technical method crossbreeding and selfed breeding:
(1) basidiospore of seafood mushroom bacterial strain and crab flavour mushroom bacterial strain is collected respectively；
(2) single spore separation and confirmation: under aseptic condition, the spore that step (1) is collected into is diluted to and is seen under 40 power microscopes
After having observed 2-3 spore, be coated culture in PDA culture medium, picking single bacterium colony, switching, microscopy to get；
(3) under aseptic condition, by the pairing hybridization of two class monospores obtained by step (2), sealing is cultivated, and microscopy, purifying obtains different
Double-core hybridization；
(4) pairing is hybridized into successfully dicaryon hybridization and carries out cultural hypha, and according to factory culture process fruiting；
(5) yield, commodity property strain excellent are selected, the load of hybrid strain Jun lid parachute-opening ejection is collected according to step (1) method
Spore；
(6) single spore separation, coating culture, switching, microscopy are carried out according to basidiospore of the step (2) to hybridization daughter bacteria strain；
(7) monospore pairing selfing, sealing culture are carried out according to step (3), microscopy purifies up to the true Ji No. 1.
Preferably, the basidiospore acquisition and collection method in step (1) are as follows: respectively by the seafood mushroom bacterial strain of parachute-opening and crab
Taste mushroom bacterial strain cap is removed, and sterile empty culture dish is placed into, and is enabled cap independently launch spore to empty culture dish surface, is then used
1 mL distilled water is collected into 2 mL PE pipe；
Preferably, the specific method is as follows for step (2):
1) first aseptically, the spore being collected into is diluted to after having observed 2-3 spore under 40 power microscopes
PDA culture medium is coated culture, when there is aristiform single bacterium colony, the Tip Splitting of picking single bacterium colony to plate
It is cultivated in PDA culture medium, 25 DEG C of 120~168 h of constant temperature incubation；
2) the lactic acid carbolic acid cotton indigo plant dyeing liquor of 5~10 μ L aseptically, is drawn on glass slide, is trained in picking step 1)
The mycelia supported is placed in the dyeing liquor, covered, dyes 1 minute, and rubber stopper flattens, film-making mirror under the microscope
Inspection, using no clamp connection as the screening criteria of mononuclear bacterial strain.
Preferably, the specific method is as follows for step (3):
1) under aseptic condition, the mononuclear bacterial strain of seafood mushroom is hybridized with the pairing of the mononuclear bacterial strain of crab flavour mushroom, its mononuclear bacterial strain of picking
Fungus block is inoculated into plate PDA culture medium, and two fungus block spacing distances are 1cm, 25 DEG C of 168 h of constant temperature incubation~240h；
2) when two mycelia are crosslinked together, the mycelia of picking crosslinking is in the lactic acid carbolic acid cotton indigo plant dyeing liquor of glass slide, lid
Upper coverslip dyes 1 minute, and rubber stopper flattens, the microscopy under field of microscope, to be judgement mark with the presence or absence of clamp connection
It is quasi-；If picking crosslinking mycelia is transferred in plate PDA culture medium, 25 DEG C of constant temperature incubations 168 it was found that there is the bacterial strain of clamp connection
H, then picking colony Tip Splitting is to test tube slant PDA culture medium culture to get the hybrid strain of purifying；
3) hybrid strain is verified: carrying out the shape difference of Antagonistic reaction experiment and fruiting experiment check cross kind and parent strain.
Preferably, the specific method is as follows for step (4):
1) it aseptically, is inoculated with after true pleurotus cornucopiae hybrid strain and test tube is put into 25 DEG C of insulating box cultures, until mycelia is covered with tiltedly
Face, it is spare as parent species；
2) clinker planting type is used, raw material will guarantee drying, without mould corruption, and sawdust needs to add water to prewet for 24 hours in advance, fill after spice
Bag, water content control, naturally, after 121 DEG C of high pressure sterilization 4h, cool in 60%~65%, pH and are followed by parent species, be put into bacterium germination
Room culture, controls 20~25 DEG C of room temperature, cultivates 40-55d；
3) after cultivar mycelia is covered with, bacterium bag is put into shady place, After-mature cultivation 60d or so, after opening sack, uses sterile razor blade
Mycelium stimulation is carried out, is poured out within 5 minutes after filling bag water, by 16 DEG C of mushroom house management of producing mushroom of cultivating bag dislocation；
4) mushroom house relative humidity 85%~95% is kept during fruiting, 12~16 DEG C of temperature, daily 200~800lx scatters illumination
Penetrate 2h.
Beneficial effects of the present invention:
The present invention is hybridized to true pleurotus cornucopiae, selfed breeding using mononuclear bacterial strain hybridization technique, breeding and obtain, be one kind
True pleurotus cornucopiae new varieties with excellent performance --- a true Ji No. 1.A true Ji No. 1 is used as a kind of true pleurotus cornucopiae new varieties of edible mushroom, property
It can optimize, the raising of biologicak efficiency and artificial cultivation yield is current research emphasis.
The present invention obtains true a Ji No. 1, in the speed of growth compared with parent strain, purseful time by hybridizing and being selfed breeding
Shift to an earlier date 5d than starting strain seafood mushroom, growth cycle does sth. in advance 10d than parent seafood mushroom and is bred as harvesting, average stem length
14.4cm, average bacteria cover diameter 1.4cm, average single bottle yield 330.5g, average organism efficiency 110.1% are set out better than parent
Bacterial strain seafood mushroom and crab flavour mushroom, economic benefit improve；Two parent of starting strain is shown from sporophore shape feature
Complementary traits；From color, cap and stem are whiter than starting strain seafood mushroom, than going out bacterium germination from the stem degree of packing
Strain seafood mushroom more consolidation, is more received by the market.
Detailed description of the invention
Fig. 1 is antagonistic experiment result.
Fig. 2 is the sub- sporophore shape of hybridization.
Fig. 3 is hybridization son selfing F2 for sporophore shape.
Specific embodiment
The present invention will be further elaborated with reference to the accompanying drawings and examples, it should which explanation, following the description is only
It is not to be defined to its content to explain the present invention.
True pleurotus cornucopiae bacterial strain of the present invention be commodity production bacterial strain, seafood mushroom bacterial strain (University Of Agriculture and Forestry In Fujian's genome with
Biotech research center preservation strain, strain number are Hy88(rocs, and Wang Yanfeng, Shi Lei, Liu's appearance is red, Pan Chunlei,
Sheng Chun dove, Wang Jinhe, Dong Xuemei, Yu Haiyang: 4 plants of true pleurotus cornucopiae Strain comparisons testChinese Forest by-product and speciality2016(1):
19-21.)) with crab flavour mushroom bacterial strain purchased from University Of Agriculture and Forestry In Fujian's genome and biotech research center preservation strain, strain number
For Hy61(rocs, Wang Yanfeng, Shi Lei, Liu's appearance is red, Pan Chunlei, Sheng Chun dove, Wang Jinhe, Dong Xuemei, Yu Haiyang: 4
The true pleurotus cornucopiae Strain comparison of strain testsChinese Forest by-product and speciality2016 (1): 19-21.)).
Reagent needed for the present invention is as follows:
PDA culture medium: potato 200g, glucose 20g, agar powder 20g, distilled water are settled to 1000mL, 121 DEG C of high steams
Sterilize 30min, and cooling is spare.
Cultivation matrix: thin sawdust 78%, wheat bran 22%, gypsum 1%, quick lime 1% add water to adjust water content to 60%~65%,
121 DEG C of high pressure steam sterilization 3h, cooling are spare.
Lactic acid carbolic acid cotton indigo plant dyeing liquor: carbolic acid 20g, cotton indigo plant 0.05g, glycerol 40mL, lactic acid 20mL.Distilled water
20mL, it is ready-to-use.
Embodiment 1:
A kind of true pleurotus cornucopiae new varieties of edible mushroom, are to hybridize by the following method and obtain:
(1) cap of the seafood mushroom bacterial strain of parachute-opening and crab flavour mushroom is removed respectively, is placed into sterile empty culture dish, enables cap
Then autonomous ejection spore is collected into 2 mL PE pipe with 1 mL distilled water to empty culture dish surface；
(2) first aseptically, the spore that step (1) is collected into is diluted to and has observed 2-3 under 40 power microscopes
It is coated culture in PDA culture medium after spore, when there is aristiform single bacterium colony, the Tip Splitting of picking single bacterium colony
It is cultivated into plate PDA culture medium, 25 DEG C of constant temperature incubation 150h；
(3) aseptically, the lactic acid carbolic acid cotton indigo plant dyeing liquor of 7uL is drawn on glass slide, is cultivated in picking step (2)
Good mycelia is placed in the dyeing liquor, covered, dyes 1min, and rubber stopper flattens, and film-making microscopy under the microscope is chosen
Select the mononuclear bacterial strain without clamp connection；
(4) under aseptic condition, the mononuclear bacterial strain of seafood mushroom is hybridized with the pairing of the mononuclear bacterial strain of crab flavour mushroom, its mononuclear bacterial strain of picking
Fungus block is inoculated into plate PDA culture medium, and two fungus block spacing distances are 1cm, 25 DEG C of constant temperature incubation 200h；
(5) when two mycelia are crosslinked together, the mycelia of picking crosslinking is in the lactic acid carbolic acid cotton indigo plant dyeing liquor of glass slide, lid
Upper coverslip dyes 1 minute, and rubber stopper flattens, the microscopy under field of microscope, to be judgement mark with the presence or absence of clamp connection
It is quasi-；If picking crosslinking mycelia is transferred in plate PDA culture medium, 25 DEG C of constant temperature incubation 168h it was found that there is the bacterial strain of clamp connection,
Then picking colony Tip Splitting is to test tube slant PDA culture medium culture to get the hybrid strain of purifying；
(6) aseptically, it is inoculated with after true pleurotus cornucopiae hybrid strain and test tube is put into 25 DEG C of insulating box cultures, until mycelia is covered with
Inclined-plane, it is spare as parent species；
(7) clinker planting type is used, raw material will guarantee drying, without mould corruption, and sawdust needs to add water to prewet for 24 hours in advance, after spice
Pack, original seed, the pack of cultivar 15cm × 28cm Polypropylene Bag, water content control is in 65%, pH naturally, by 121 DEG C of high pressures
It sterilizes after 5h, cools and be followed by parent species, be put into bacteria-producing room culture, control 25 DEG C of room temperature, culture 50d or so；
(8) after cultivar mycelia is covered with, bacterium bag is put into shady place, After-mature cultivation 60d, after opening sack, with sterile razor blade into
Row mycelium stimulation, 5min is poured out after filling bag water, by 16 DEG C of mushroom house management of producing mushroom of cultivating bag dislocation；
(9) mushroom house relative humidity 90% is kept during fruiting, 14 DEG C of temperature, daily 400lx scattering light irradiates 2h；
(10) bag management of producing mushroom 15d or so is opened, quality, character strain excellent is selected, is named as Hyz5；
(11) it is collected according to step (1) method and hybridizes sub- Hyz5 basidiospore；
(12) basidiospore selfing, purifying, cultural hypha are carried out to get selfing bacterial strain true a Ji No. 1 according to (2) to (9) step.
Hybrid strain verifying
1, Antagonistic reaction is tested
Using two o'clock inocalation method respectively by seafood mushroom double-core bacterial strain (Hy88), crab flavour mushroom double-core bacterial strain (Hy61) and embodiment 1
The isolated true Ji of bacterial strain No. 1 of selfing connect in the same plate containing PDA culture medium, 25 DEG C of constant temperature incubations are observed and are clapped
According to the antagonism recorded between each bacterial strain, the result is shown in Figure 1 (is seafood mushroom bacterial strain Hy88 and the true Ji 1 of bacterial strain respectively from left to right
Number, crab flavour mushroom Hy61 and the true Ji of bacterial strain No. 1)；
2, fruiting experiment 1
In order to detect the Traits change of cenospecies and parent strain, preliminary screening identification is carried out to the hybrid strain of acquisition, is carried out
The experiment of cultivating bag fruiting, 10 bags of each bacterial strain, according to 1 step of embodiment (7)-(10) method cultivation management.It is measured when harvesting
The fructification fresh weight of each bacterial strain calculates its biological efficiency (biological efficiency=fructification fresh weight/compost dry weight × 100%),
The shapes such as bacteria cover diameter, cap color, stem length, stem diameter, stem color and the toughness of each bacterial strain fructification of Observe and measure
State feature (Fig. 2 is followed successively by seafood mushroom Hy88, hybrid strain Hyz5, crab flavour mushroom Hy61 from left to right), the results are shown in Table 1 and table 2.
1. fruiting experimental result of table
2. fruiting experimental result of table
2, fruiting body yield of table and biological efficiency compare
3, fruiting experiment 2
In order to detect the Traits change of the true Ji of selfed seed No. 1 with parent strain, preliminary screening mirror is carried out to the hybrid strain of acquisition
It is fixed, carry out the experiment of cultivating bag fruiting, 10 bags of each bacterial strain, according to 1 step of embodiment (7)-(10) method cultivation management.Harvesting
When measure the fructification fresh weight of each bacterial strain, calculate its biological efficiency (biological efficiency=fructification fresh weight/compost dry weight ×
100%), Observe and measure each bacterial strain purseful time, the bacteria cover diameter of whole breeding time and fructification, cap color, stem length,
(Fig. 3 is followed successively by a true Ji No. 1, seafood mushroom Hy88, crab flavour mushroom to the morphological features such as stem diameter, toughness and stem color from left to right
Hy61), it the results are shown in Table 3 and table 4.
3. fruiting experimental result of table
4. fruiting body yield of table and biological efficiency compare
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations, are all covered by the present invention.
Claims (2)
1. a kind of true pleurotus cornucopiae bacterial strain, it is characterised in that: the bacterial strain be true pleurotus cornucopiae (Hypsizygus marmoreus) a true Ji 1
Number, on April 16th, 2019 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number
Are as follows: CGMCC NO.17598.
2. a kind of selection of true pleurotus cornucopiae bacterial strain as described in claim 1, characterized by the following steps: (1) respectively
Collect the seafood mushroom bacterial strain of acquisition merit and the basidiospore of crab flavour mushroom bacterial strain；(2) under aseptic condition, the spore that will be collected into
It is diluted to after suitable concentration and is coated culture, picking single bacterium colony, switching, microscopy in PDA culture medium；(3) seafood is picked out
Mushroom, crab flavour mushroom single-ascospore strain carry out pairing hybridization, sealing culture, microscopy, purifying；(4) it is carried out according to the factorial production process
Fruiting；(5) spore of the excellent hybrid strain of character is collected；(6) be coated according to step (2), step (3), choose monospore,
Hybridization, inoculated and cultured, purifying to get.
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Cited By (7)
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| CN111084098A (en) * | 2020-01-02 | 2020-05-01 | 上海雪榕生物科技股份有限公司 | Hypsizygus marmoreus H18 and cultivation method thereof |
| CN111727809A (en) * | 2020-07-27 | 2020-10-02 | 平泉市希才应用菌科技发展有限公司 | Lentinus edodes strain and cultivation method and application thereof |
| CN112063534A (en) * | 2020-08-25 | 2020-12-11 | 福建农林大学 | Hypsizigus marmoreus strain |
| CN112063533A (en) * | 2020-08-25 | 2020-12-11 | 福建农林大学 | Hypsizigus marmoreus strain |
| CN112063535A (en) * | 2020-08-25 | 2020-12-11 | 福建农林大学 | Hypsizigus marmoreus strain |
| CN113637591A (en) * | 2020-04-27 | 2021-11-12 | 西充星河生物科技有限公司 | Hypsizigus marmoreus strain |
| WO2022170960A1 (en) * | 2021-02-10 | 2022-08-18 | 上海丰科生物科技股份有限公司 | Hypsizigus marmoreus strain, spore, fruiting body and use thereof |
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| CN105802862A (en) * | 2016-05-31 | 2016-07-27 | 上海丰科生物科技股份有限公司 | Hypsizigus marmoreus strain, and culture method, planting method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111084098A (en) * | 2020-01-02 | 2020-05-01 | 上海雪榕生物科技股份有限公司 | Hypsizygus marmoreus H18 and cultivation method thereof |
| CN111084098B (en) * | 2020-01-02 | 2021-11-05 | 上海雪榕生物科技股份有限公司 | Hypsizygus marmoreus H18 and cultivation method thereof |
| CN113637591A (en) * | 2020-04-27 | 2021-11-12 | 西充星河生物科技有限公司 | Hypsizigus marmoreus strain |
| CN113637591B (en) * | 2020-04-27 | 2023-10-03 | 西充星河生物科技有限公司 | Hypsizygus marmoreus strain |
| CN111727809A (en) * | 2020-07-27 | 2020-10-02 | 平泉市希才应用菌科技发展有限公司 | Lentinus edodes strain and cultivation method and application thereof |
| CN112063534A (en) * | 2020-08-25 | 2020-12-11 | 福建农林大学 | Hypsizigus marmoreus strain |
| CN112063533A (en) * | 2020-08-25 | 2020-12-11 | 福建农林大学 | Hypsizigus marmoreus strain |
| CN112063535A (en) * | 2020-08-25 | 2020-12-11 | 福建农林大学 | Hypsizigus marmoreus strain |
| WO2022170960A1 (en) * | 2021-02-10 | 2022-08-18 | 上海丰科生物科技股份有限公司 | Hypsizigus marmoreus strain, spore, fruiting body and use thereof |
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| CN110184201B (en) | 2021-01-29 |
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