CN103725618B - Aperture single sheet black fungus bacterial classification - Google Patents
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Abstract
Aperture single sheet black fungus bacterial classification, relates to a strain black fungus bacterial classification.The present invention solves the black fungus bacterial classification used in existing production and there is aperture and go out that ear not easily forms monolithic, yields poorly, inferior problem.Aperture single sheet black fungus bacterial classification of the present invention is black fungus bacterial classification No. 15, black prestige (HW15), belong to black fungus and belong to (auricularia? auricula), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on November 27th, 2013, and deposit number is CGMCC8553.Sporophore is monolithic Dan Sheng, and aperture goes out ear monolithic rate and reaches more than 90%, and auricle is of moderate size, unrooted, in bowl-shape or conchoidal, and dry hard of hearing grey, outside of belly black, glossy, pros and cons color distinction is large, and ear edge rounding, back side muscle arteries and veins are not obvious; Dry otic vesicle is sent out rate and is reached 1:15 ~ 1:18.
Description
Technical field
The present invention relates to a strain black fungus bacterial classification.
Background technology
The Northeast is the main producing region of China's black fungus, and only black fungus ultimate production in Heilongjiang Province's accounts for more than 50% of the whole nation.Along with the raising of the production-scale expansion of black fungus and output, planting type and the market requirement are also changing, planting type gradually by great Kou, large go out that ear turns to aperture, monolithic goes out ear, aperture goes out ear mode and facilitates fruit-body formation monolithic, greatly can improve quality and the economic benefit of black fungus.But specific black fungus kind is the prerequisite that aperture goes out that ear forms monolithic, and the black fungus bacterial classification plurality shape used in producing at present is not suitable with the demand that aperture goes out ear.Be mainly manifested in: aperture goes out ear and do not form monolithic or monolithic rate low (lower than 50%), and sporophore muscle arteries and veins is many, poor morphology, and output (mean yield 35g ~ 40g/ bag) goes out ear lower than large mouth.Have impact on quality product and economic benefit.
Summary of the invention
The present invention will solve the black fungus bacterial classification used in existing production and there is aperture and go out that ear not easily forms monolithic, yields poorly, inferior technical problem, and provides a strain to be suitable for the black fungus bacterial classification No. 15, black prestige (HW15) of the Northeast's aperture monolithic cultivation.
Aperture single sheet black fungus bacterial classification of the present invention is black fungus bacterial classification No. 15, black prestige (HW15), belong to black fungus and belong to (auricularia auricula), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on November 27th, 2013, and deposit number is CGMCC8553.
Mycelium is in shape of crawling on PDA substratum for black prestige No. 15 bacterial classifications, and mycelia is denser, and neat in edge, branch are obvious.Sporophore is monolithic Dan Sheng, and aperture goes out ear monolithic rate and reaches more than 90%, and auricle is of moderate size, unrooted, in bowl-shape or conchoidal, and dry hard of hearing grey, outside of belly black, glossy, pros and cons color distinction is large, and ear edge rounding, back side muscle arteries and veins are not obvious; Dry otic vesicle is sent out rate and is reached 1:15 ~ 1:18.
No. 15, black prestige (HW15) is through artificial culture domestication, and the black fungus bacterial classification commonly used in producing with the Northeast carries out variety protection, determines the distinctiveness of No. 15, black prestige and existing kind and distinctive good character; And carry out Regional Cultural Experiment in different areas, determine that No. 15, black prestige is to the adaptability of the Northeast's weather and the stability of proterties.
The DNA(ISSR of aperture single sheet black fungus bacterial classification of the present invention) fingerprint polymorphism analysis result is that black prestige No. 15 coamplifications go out 7 bands, and molecular weight is respectively: 2111bp, 1346bp, 1091bp, 901bp, 795bp, 696bp, 489bp.The breed difference that banding pattern and quantity and Heilungkiang black fungus are conventional in producing is very large.Carry out UPGMA cluster analysis as shown in Figure 4 with NTsys_2.02 software, the genetic similarity of 27 strain fungus strains is between 0.63 ~ 0.97, and hereditary difference is larger.Be that 0.63 place will originally be divided into two large monoids for sample at similarity factor, 27 strain edible fungus kinshiies are comparatively far away, can be judged to be different fungus strains.Be 0.86 place at similarity factor, No. 15, black prestige and 981 divides a class into, but the two genetic distance is comparatively large, has bigger difference in sporophore shape, breeding time etc. in economical character.Thus, can judge that No. 15, black prestige is the novel species being different from existing kind.
Black prestige No. 15 bacterial classifications are medium variety, adopt the general aperture in current the Northeast to go out ear management process and cultivate, 10 ~ 12 days existing ear buds after acanthopore.The more existing kind of growth cycle shifts to an earlier date 7 ~ 10 days; Sprout neat, go out ear rate and reach more than 95%, sporophore easily forms monolithic, monolithic rate more than 90%.Be suitable for the Northeast's spring and autumn cultivation.
Black prestige No. 15 cultivating methods carry out according to the following steps:
1, substratum make: take by mass percentage the broad-leaved weed tree sawdust of 85%, 8% rice bran, 5% wheat bran, the bean powder of 1%, the gypsum of 0.5% and 0.5% lime, then with water mixing, make water content 60% ~ 65%, obtain substratum wet feed; Cultivar adopts the slim bacterium bag of adherent good polyethylene of 16.5cm × 33cm specification, every packed substratum wet feed 1.0 ~ 1.1kg, and 19cm ~ 20cm is high, adopts plunger sealing.
2, sterilizing and inoculation: cultivar adopts normal pressure moist heat sterilization (at 100 DEG C sterilizing 8h); Every bottle of original seed connects 40 ~ 50 cultivating bacteria bags.
3, culture condition: indoor bacteria, room temperature 20 ~ 25 DEG C, relative air humidity 50% ~ 55%, indoor lucifuge, well-ventilated; After mycelia covers with bag, and give suitable light scattering; Cultivar incubation period controlled at 40 ~ 50 days.
4, scarfing: utilize opening machinery to carry out acanthopore in the bacterium bag covering with mycelia, pass is " O " shape or " Y " shape, aperture 0.4 ~ 0.5cm, hole depth 0.5 ~ 1.0cm, every bag of acanthopore 100 ~ 160.The size in different acanthopore modes and hole can be adopted to the quantity of the auricle size and auricle that control auricularia auriculajudae
5, ear is urged: the bacterium bag after opening adopts the indoor mode combined with outdoor to concentrate vernalization, and temperature controls below 20 DEG C, and humidity should control more than 80%.Point bed after ear bud all sprouts and grows to 1 ~ 2cm, bag spacing 12cm during point bed.
6, go out syrinx reason: will urge the bacterium bag of bud, dispersion is put on bacterium bed, adopt outdoor " full light " open go out ear, take micro-spray to feed water, alternation of wetting and drying, when watering, relative air humidity is 90% ~ 95%, and when not watering, relative air humidity is 70% ~ 85%.
7, gather: when auricle fully unfold, root shrinks, color and luster turns light and grows to medium well, about will pluck in time during 3 ~ 4cm, controls ear type and thickness well.Can gather 4 ~ 5 tide.
8, airing: the auricularia auriculajudae after harvesting is placed on gauze, by daylight natural drying, waits to do and does not stir before shaping; Within 1 ~ 2 day, parch and can pack storage.
Black prestige No. 15 cultivars adopt the bacterium bag of 16.5cm × 33cm specification, every packed substratum wet feed 1.0 ~ 1.1kg, and dry ear output is 50g ~ 65g/ bag; Proterties is stablized, strong resistance, and quality is better than other kinds, has good commodity, ear type, color and luster, quality compares and accords with the demands of the market; The more existing kind of auricularia auriculajudae price improves 15% ~ 30%.
Black fungus bacterial classification of the present invention No. 15, black prestige (HW15) belongs to black fungus and belongs to (auriculariaauricula), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on November 27th, 2013, and deposit number is CGMCC8553.
Accompanying drawing explanation
Fig. 1 is black prestige No. 15 hypha form; Fig. 2 is black prestige 15 work song entity morphology; Fig. 3 is agarose gel electrophoresis result; Fig. 4 is UPGMA cluster analysis result.
Embodiment
Aperture single sheet black fungus bacterial classification in embodiment one, present embodiment is black fungus bacterial classification No. 15, black prestige (HW15), belong to black fungus and belong to (auricularia auricula), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on November 27th, 2013, and deposit number is CGMCC8553.
Mycelium is in shape of crawling on PDA substratum for black prestige No. 15 bacterial classifications, and mycelia is denser, and neat in edge, branch are obvious.Sporophore is monolithic Dan Sheng, and aperture goes out ear monolithic rate and reaches more than 90%, and auricle is of moderate size, unrooted, in bowl-shape or conchoidal, and dry hard of hearing grey, outside of belly black, glossy, pros and cons color distinction is large, and ear edge rounding, back side muscle arteries and veins are not obvious; Dry otic vesicle is sent out rate and is reached 1:15 ~ 1:18.
Black prestige No. 15 cultivating methods carry out according to the following steps:
1, substratum make: take by mass percentage the broad-leaved weed tree sawdust of 85%, 8% rice bran, 5% wheat bran, the bean powder of 1%, the gypsum of 0.5% and 0.5% lime, then with water mixing, make water content 60% ~ 65%, obtain substratum wet feed; Cultivar adopts the slim bacterium bag of adherent good polyethylene of 16.5cm × 33cm specification, every packed substratum wet feed 1.0 ~ 1.1kg, and 19cm ~ 20cm is high, adopts plunger sealing.
2, sterilizing and inoculation: cultivar adopts normal pressure moist heat sterilization (at 100 DEG C sterilizing 8h); Every bottle of original seed connects 40 ~ 50 cultivating bacteria bags.
3, culture condition: indoor bacteria, room temperature 20 ~ 25 DEG C, relative air humidity 50% ~ 55%, indoor lucifuge, well-ventilated; After mycelia covers with bag, and give suitable light scattering; Cultivar incubation period controlled at 40 ~ 50 days.
4, scarfing: utilize opening machinery to carry out acanthopore in the bacterium bag covering with mycelia, pass is " O " shape or " Y " shape, aperture 0.4 ~ 0.5cm, hole depth 0.5 ~ 1.0cm, every bag of acanthopore 100 ~ 160.The size in different acanthopore modes and hole can be adopted to the quantity of the auricle size and auricle that control auricularia auriculajudae
5, ear is urged: the bacterium bag after opening adopts the indoor mode combined with outdoor to concentrate vernalization, and temperature controls below 20 DEG C, and humidity should control more than 80%.Point bed after ear bud all sprouts and grows to 1 ~ 2cm, bag spacing 12cm during point bed.
6, go out syrinx reason: will urge the bacterium bag of bud, dispersion is put on bacterium bed, adopt outdoor " full light " open go out ear, take micro-spray to feed water, alternation of wetting and drying, when watering, relative air humidity is 90% ~ 95%, and when not watering, relative air humidity is 70% ~ 85%.
7, gather: when auricle fully unfold, root shrinks, color and luster turns light and grows to medium well, about will pluck in time during 3 ~ 4cm, controls ear type and thickness well.Can gather 4 ~ 5 tide.
8, airing: the auricularia auriculajudae after harvesting is placed on gauze, by daylight natural drying, waits to do and does not stir before shaping; Within 1 ~ 2 day, parch and can pack storage.
Table 1: black prestige No. 15 different areas cultivation yield statistics
DNA(ISSR) fingerprint polymorphism analysis
26 kinds that No. 15, black prestige (HW15) and Heilongjiang Province's black fungus are commonly used in producing carry out DNA(ISSR) fingerprint polymorphism analysis, compares their othernesses:
Conventional bacterial classification produced by table 2 Heilongjiang Province (identification) black fungus
| Strain number | Title | Strain number | Title |
| 1 | No. one, male ear | 15 | 9202 |
| 2 | No. 1, Suihua | 16 | No. 2, special product |
| 3 | No. 2, Suihua | 17 | Black greatly No. 1 |
| 4 | Black lotus | 18 | Prolong rich No. 1 |
| 5 | Long Bin | 19 | Green rich 007 |
| 6 | No. 1, Xingan | 20 | No. 16, black prestige |
| 7 | No. 2, Xingan | 21 | No. 15, black prestige |
| 8 | No. 3, Xingan | 22 | Black 29 |
| 9 | No. 3, woods section | 23 | 981 |
| 10 | No. 6, woods section | 24 | Male ear-08-19 |
| 11 | New millennium No. 3 | 25 | No. 4, Xingan |
| 12 | Snow plum No. 1 | 26 | DH1 |
| 13 | Agriculture 06 | 27 | DH2 |
| 14 | Hongda 2009 |
1, STb gene is extracted
Prepared by a, mycelium: get cPDA liquid nutrient medium 80mL and load 250mL triangular flask, sterilizing 30min at 121 DEG C, after cooling, obtain liquid nutrient medium, then by No. 15, black prestige (HW15) with compared with bacterial strain transfer respectively in liquid nutrient medium, static gas wave refrigerator 2 ~ 3 days at 25 DEG C, then with the rotating speed of 130r/min, constant-temperature table is cultivated 2 ~ 5 days at 25 DEG C, obtains mycelium (non-pigment generation).
B, get fresh mycelia and to weigh 0.5g, add liquid nitrogen, after liquid nitrogen evaporate to dryness, be ground to pasty state, extract mycelium STb gene with UNIQ-10 pillar plant genome DNA extraction agent box.
2, ISSR-PCR reaction system (20 μ L)
Be 0.1mmol/L MgCl containing 2 × PCRbuffer, concentration
2, 0.1mmol/L dNTP
s, 15ng ISSR primer (AGAGAGAGAGAGAGAGYT), 15 ~ 25ng template DNA and 1U Taq archaeal dna polymerase; The primer is synthesized by Shanghai Sheng Gong biotechnology Services Co., Ltd (Sagon).
3, response procedures is: 94 DEG C of denaturation 5min, then 94 DEG C of sex change 30s, 50 ~ 55 DEG C of annealing 30s, and 72 DEG C extend 120s, carry out 35 and follow bad, and last 72 DEG C extend 7min.4 DEG C of preservations.
4, ISSR-PCR amplified production agarose gel electrophoresis detects
ISSR-PCR reaction product is mixed with 3 μ L bromophenol blue indicators, with 1.0% (wt.) sepharose, 0.5 × tbe buffer liquid, 5V/cm electrophoresis, 0.5 μ g/mL EB dyeing 15min, with DL2000 (Plus) for Marker, gel imaging scanning record result also estimates the general band size of DNA.
5, data processing and analysis
Band in statistics electrophoretogram, utilizes NTsys_2.02 software to analyze, the genetic similarity between calculation sample, carries out cluster analysis obtain Dendrogram by UPGMA method.
DNA(ISSR) fingerprint polymorphism analysis result
ISSR-PCR amplified production agarose gel electrophoresis detects (strain number in table 2 and Fig. 3 in swimming lane numbering one_to_one corresponding) as shown in Figure 3, and the 21st swimming lane is No. 15, black prestige, and coamplification goes out 7 bands, and molecular weight is respectively: 2111bp, 1346bp, 1091bp, 901bp, 795bp, 696bp, 489bp.The breed difference that banding pattern and quantity and Heilungkiang black fungus are conventional in producing is very large.
Carry out UPGMA cluster analysis as shown in Figure 4 with NTsys_2.02 software, the genetic similarity of 27 strain fungus strains is between 0.63 ~ 0.97, and hereditary difference is larger.Be that 0.63 place will originally be divided into two large monoids for sample at similarity factor, 27 strain edible fungus kinshiies are comparatively far away, can be judged to be different fungus strains.Be 0.86 place at similarity factor, No. 15, black prestige and 981 divides a class into, but the two genetic distance is comparatively large, has bigger difference in sporophore shape, breeding time etc. in economical character: 981 sporophores are a shape consor, auricle back side muscle arteries and veins is many and obvious, No. 15, more black prestige breeding time about 7d in evening.Thus, can judge that No. 15, black prestige is the novel species being different from existing kind.
Claims (1)
1. aperture single sheet black fungus bacterial classification, it is characterized in that this bacterium be No. 15, the black prestige of black fungus bacterial classification belong to black fungus belong to (
auricularia auricula), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date is on November 27th, 2013, and deposit number is CGMCC No.8553.
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| CN104094770B (en) * | 2014-05-05 | 2016-05-18 | 广东省微生物研究所 | A kind of black fungus novel bacterial and artificial cultivation method thereof |
| CN104106375B (en) * | 2014-07-01 | 2015-12-02 | 云南大学 | One plant height warm type black fungus strain |
| CN106434365B (en) * | 2016-09-20 | 2019-04-05 | 黑龙江省科学院微生物研究所 | The black fungus strain of one plant of suitable black fungus bacterial slag for cultivating and its application |
| CN112568064B (en) * | 2020-11-23 | 2022-04-05 | 河北省科学院生物研究所 | Black fungus strain JH40 and application thereof |
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| CN1047200C (en) * | 1994-03-09 | 1999-12-08 | 刘永昶 | Earth-cultivated black fungus and its cultivating method by use of substitute material for plastic bag |
| CN101897299A (en) * | 2010-01-27 | 2010-12-01 | 黑龙江省科学院微生物研究所 | Black fungus strain suitable to be cultivated in northeast regions of China |
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