CN104585058B - Pleurotus nebrodensis that a kind of stem is short and cultural method thereof - Google Patents
Pleurotus nebrodensis that a kind of stem is short and cultural method thereof Download PDFInfo
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Abstract
The invention discloses and belong to the Pleurotus nebrodensis bacterial strain that a kind of stem in edible fungi field is short, Pleurotus nebrodensis of the present invention (Pleurotus eryngii var.tuoliensis) bacterial strain middle peasant Bai Ling 3, in December in 2014 12 days in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, its deposit number is CGMCC No.10193.The invention also discloses the cultural method of middle peasant Bai Ling 3, plant breeding, breeding stock, cultigen breeding and bacterium, low temperature stimulation and management of producing mushroom and the step such as gather including female.The invention provides a kind of new Pleurotus nebrodensis bacterial strain, enrich the kind of Pleurotus nebrodensis, secondly, the sporophore stem of this bacterial strain is short, can not cut handle, directly pack；Middle peasant Bai Ling 3 work song physical commodity of the present invention is worth height, and its cap is scalloped shaped, and color canescence, quality is hard；The single mushroom weight of middle peasant Bai Ling of the present invention 3 is high.
Description
Technical field
The invention belongs to edible fungi field, be specifically related to the Pleurotus nebrodensis bacterial strain that a kind of stem is short, and the cultivation of this Pleurotus nebrodensis
Culture method.
Background technology
Pleurotus nebrodensis (Pleurotus eryngii var.tuoliensis, have another name called asafoetida mushroom, pleurotus eryngii Resina Ferulae mutation,
Pleurotus eryngii var. nebrodensis, pleurotus eryngii white mutation, wing abalone mushroom, beautiful snow Pleurotus ferulae Lanzi, the white Ganoderma in Western Paradise, Tianshan Mountains god mushroom etc.) belong to pleurotus
(Pleurotus), Chinese formal name used at school is that Pleurotus nebrodensis is (or by Classification system Pleurotus eryngii var.tuoliensis
Literal translation is: the mutation of pleurotus eryngii Tuoli).Pleurotus nebrodensis sporophore plumpness is pure white, exquisite quality is tasty and refreshing, nutritious, is a kind of pole
For precious large edible bacterium.Pleurotus nebrodensis facultative parasitism on the basal part of stem and root of half withered Ferula sinkiangensisK.M.Shen, wild resource pole
It is rare.
" Pleurotus nebrodensis " title buys asafoetida mushroom bacterium bag from Xinjiang, in north from Beijing Jinxin Edible Fungi Co., Ltd. in 1997
Capital large area fruiting success, classification of fungi scholar fourth of the twelve Earthly Branches Mr. morning mist wherein literature name will be set to Pleurotus nebrodensis, and Classification system is set to
Pleurotus nebrodensis, and be recorded in " Macrofungi From China " (fourth of the twelve Earthly Branches morning mist edit, Henan science tech publishing house,
2000).But the Pleurotus nebrodensis that the Pleurotus nebrodensis of the China of research discovery later is real from Europe is different,
The Pleurotus nebrodensis formal name used at school of China should be Pleurotus eryngii var.tuoliensis (.mycoscience such as Kawai,
2008,49:75-79；Huang Chenyang etc. plant genetic resources journal, 2011,12 (5): 825-827,823).And obtained authority
International mycology name database (http://www.indexfungorum.org/) recognize, i.e. the formal name used at school of Pleurotus nebrodensis is
Pleurotus eryngii var.tuoliensis。
Pleurotus nebrodensis and Pleurotus eryngii (Pleurotus eryngii var.eryngii) are mutually of the same race on taxonomy
Different mutation under (Pleurotus eryngii), sporophore mouthfeel is close.But, owing to being rushed by Pleurotus eryngii industrial cultivation
Hitting, Cultivation of Pleurotus nebrodensis amount is fewer and feweri.It is longer very than Pleurotus eryngii that the reason causing this phenomenon is mainly the cultivation period of Pleurotus nebrodensis
Many, but yield is more much lower than Pleurotus eryngii, and both are more or less the same at price.It addition, need overwhelming majority bacterium of pruning before Pleurotus nebrodensis packaging
Handle, and have only to, before Pleurotus eryngii packaging, root sub-fraction of pruning.Therefore, the Pleurotus nebrodensis kind that stem is short, sporophore may utilize
Part is more, more meets the demand of Producer.
Summary of the invention
For the shortcoming that existing Pleurotus nebrodensis sporophore stem is longer, present invention aim at providing a kind of stem short white
Spirit mushroom.
Another object of the present invention is to provide the cultural method of above-mentioned Pleurotus nebrodensis.
The purpose of the present invention realizes by following technical solution:
One Pleurotus nebrodensis of the present invention (Pleurotus eryngii var.tuoliensis) bacterial strain middle peasant Bai Ling 3,
In December in 2014 12 days the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms (preservation address is:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode 100101), its deposit number is
CGMCC No.10193。
The cultural method of above-mentioned Pleurotus nebrodensis bacterial strain middle peasant Bai Ling 3, comprises the steps:
(1), female kind is bred: in the weight ratio ratio of 1:80～100 by the middle peasant Bai Ling 3 of diameter 0.3～0.5cm
Strain is inoculated on mother culture media, 22～28 DEG C, relative air humidity be 40～60%, cultivate 10～12 under the conditions of lucifuge
My god, obtain the female kind of middle peasant Bai Ling 3；
(2), Primary spawn: according to percentage by weight be 3～5% ratio by the middle peasant Bai Ling of gained 3 in step (1)
Female kind is inoculated on cultivation compost, 22～28 DEG C, relative air humidity be 40～60%, cultivate 28～35 under the conditions of lucifuge
My god, obtain No. 3 original seeds of middle peasant Bai Ling；
(3), cultigen cultivate: according to percentage by weight be 3～5% ratio by the middle peasant Bai Ling 3 of gained in step (2)
Number original seed is inoculated on cultivation compost, 22～28 DEG C, relative air humidity be 40～60%, cultivate under the conditions of lucifuge 28～
35 days, obtain No. 3 cultigens of middle peasant Bai Ling；
(4), cooling inoculation: according to percentage by weight be 5～8% ratio by the middle peasant Bai Ling of gained 3 in step (3)
Cultigen is inoculated on cultivation compost, 22～28 DEG C, relative air humidity be 40～60%, cultivate under the conditions of lucifuge 30～
40 days, mycelia covered with compost；
(5), mycelium stimulation: being opened by bag mouth, remove the mycoderma on charge level with sterilization spatula, pine is pricked suitable for reading, then by bacterium bag pine
On bundle, continue to cultivate 3～5 days, until mycelia recovers；
(6), low temperature stimulation: bacterium bag is placed under 1～3 DEG C of low temperature stimulation 7～14 days；Or greenhouse temperature is when 0～15 DEG C,
Keep day and night temperature 10～15 DEG C, stimulate 10～15 days；
(7), management of producing mushroom and gathering: after former base occurs, in 5～18 DEG C (agricultural formula cultivations) or 12～14 DEG C of (industry formulas
Cultivation), relative air humidity is 75～85%, illumination 500～800 lux, well-ventilated, environment CO2≤1000×10-6Bar
Cultivate 20～30 days under part；When sporophore cap is sufficiently spread out, but still there is slight crimping, sporophore of gathering when spore does not discharges.
Strain described in above-mentioned cultural method step (1) refers to mycelia.
Raw material constituent and the part by weight thereof of the mother culture media described in above-mentioned cultural method step (1) be: goes
Skin potato ball 180～300g (extracting juice), glucose 15～20g, potassium dihydrogen phosphate 0.5～3g, magnesium sulfate 0.5～3g, agar
15～25g, water 1000ml, pH value is natural.
The raw material constituent of the cultivation compost described in above-mentioned cultural method step (2), (3) or (4) and weight thereof
Percentage ratio is: cotton seed hulls 75～81%, Testa Tritici 8～15%, Semen Maydis powder 2～8%, Calx 1～3%；Again with material-water ratio as 1:1.8
～the ratio of 2.3 adds water.
Constituent and the percentage by weight thereof of the cultivation compost described in above-mentioned cultural method are preferably: cotton seed hulls
81%, Testa Tritici 12.5%, Semen Maydis powder 3.5%, Calx 3%, material-water ratio 1:1.86, i.e. water content are 65%.
Temperature described in above-mentioned cultural method step (1), (2), (3) or (4) is preferably 24～26 DEG C, further preferably
It it is 25 DEG C.
Relative air humidity described in above-mentioned cultural method step (1), (2), (3) or (4) is preferably 60%.
Inoculation method described in above-mentioned cultural method step (4) is first to extract the hollow plastics that bacterium bag central authorities are previously inserted
Rod, accesses cultigen in the hole stayed after extracting hollow plastic stick.
The preparation method of the mother culture media described in above-mentioned cultural method step (1), including proportionally removing the peel horse
Bell potato block (diameter about 1cm) is placed in 600ml water, boiling water boiling 30min, 4 layers of filtered through gauze, taking juice；Add in gained juice
Entering less than 400ml water, then add glucose, potassium dihydrogen phosphate, magnesium sulfate, agar, be settled to 1000ml, the least fire adds
Heat, is completely dissolved to above composition, subpackage test tube (specification is 20mm × 200mm), 121 DEG C of sterilizing 30min；Pendulum inclined-plane, standby.
Agar used can be agar strip or agar powder.
The preparation method of the cultivation compost described in above-mentioned cultural method step (2), (3) is: proportionally by each former
Material component mixing, stirs；Adding water according still further to material-water ratio, stir, fill bottle/bag, cultivation compost uses high pressure to go out
Bacterium, keeps 150min at 125 DEG C.
The preparation method of the cultivation compost described in above-mentioned cultural method step (4) is: proportionally by each raw material group
Divide mixing, stir；Adding water according still further to material-water ratio, stir, cultivating bag uses polypropylene or Polythene Bag, cultivating bag
General 5～6 of thickness, bacterium bag folding footpath 17cm, long 35cm；The cultivation compost prepared is loaded in bag, it is desirable to degree of tightness appropriateness,
Install in the hole in the middle of the press-in of rear bag mouth varus, and insert hollow bar, be placed in disinfection basket, i.e. carry out sterilizing.Typically adopt
With normal-pressure sterilization, in steamer after air, keep 12～14 hours at 100 DEG C, inoculate after cooling.
The separation domestication process of Pleurotus nebrodensis middle peasant Bai Ling of the present invention 3:
In May, 2009, the present inventor's Gobi desert near the brick field of Yumin County, Tacheng area collects and colonizes in Xinjiang
The wild Pleurotus nebrodensis sporophore of Resina Ferulae root, obtains mycelia (strain) by fruit body tissue separation, detects mycelia rDNA-ITS
Size and sequence, be identified as Pleurotus nebrodensis (Pleurotus eryngii var.tuoliensis, Pleurotus nebrodensis), will separate
The strain arrived, in cultivation in 2009～2011, carries out separate tissue every year, all trains at difcoPDA the sporophore that mushroom shape is good
Support the upper cultivation of base (its constituent is BDdifcoPDA powder 39g, distilled water 1000ml), therefrom filter out mycelial growth rate
Hurry up, robust growth, the bacterial strain of bacterium colony rounding.2012, experiment in cultivation in 2013 finds that No. 1590 bacterial strains filtered out for 2011 go out
Mushroom performance excellence, named middle peasant Bai Ling 3.
Advantages of the present invention and beneficial effect: (1), the invention provides a kind of new Pleurotus nebrodensis bacterial strain, enrich Bai Ling
The kind of mushroom；(2) the sporophore stem of middle peasant Bai Ling of the present invention 3 is short, average long 1.9cm, can not cut handle, directly pack；
(3), the sporophore commodity value of middle peasant Bai Ling of the present invention 3 high, its cap is scalloped shaped, and color canescence, quality is very
Firmly, stem length only has 1.9cm, the averagely thick 4.1cm of bacterial context；(4), single mushroom weight of Pleurotus nebrodensis of the present invention high, factory culture is put down
All single mushroom weighs 245.5 ± 44.6g, and the average single mushroom of agricultural formula cultivation weighs 260.5 ± 40.1g, and agricultural formula, factory culture are the suitableeest
Close.
Accompanying drawing illustrates:
Fig. 1. middle peasant Bai Ling 3 work song entity photo.
Fig. 2. use 5 Pleurotus nebrodensis bacterial strain ISSR electrophoresis patterns of primer P4 amplification.Wherein M is Marker, and 1 is that China is miscellaneous
13,2 is middle peasant wing Bao, and 3 is KH2, and 4 is middle peasant No. 1, and 5 is middle peasant Bai Ling 3.
Fig. 3. use 5 Pleurotus nebrodensis bacterial strain ISSR electrophoresis patterns of primer P24 amplification；Wherein M is Marker, and 1 is that China is miscellaneous
13,2 is middle peasant wing Bao, and 3 is KH2, and 4 is middle peasant No. 1, and 5 is middle peasant Bai Ling 3.
Fig. 4. the IGS2 that middle peasant Bai Ling 3 is through Hap II restriction enzyme digestion and electrophoresis collection of illustrative plates；Wherein 1 is middle peasant No. 1, and 2 is middle peasant Bai Ling 3
Number.
Fig. 5. the IGS2 that middle peasant Bai Ling 3 is through Rsa I restriction enzyme digestion and electrophoresis collection of illustrative plates；Wherein 1 is middle peasant No. 1, and 2 is middle peasant Bai Ling 3
Number.
Detailed description of the invention:
Below by way of example, the invention will be further described, but does not constitutes any limitation the present invention.
The taxonomic identification of embodiment 1 middle peasant of the present invention Bai Ling 3
(1) morphological characteristic of middle peasant Bai Ling 3 and taxonomic identification:
No. 3 former bases of middle peasant Bai Ling are scattered, white；Time ripe, cap is light gray, mussel shape, and quality is hard, and major diameter is average
12.7 centimetres, average 10.1 centimetres of minor axis, average 4.1 centimetres of thickness；In stem raw, white, basic wait thick, quality is hard, surface
Smooth, stem averagely grows 1.9 centimetres, the thickest 2.7 centimetres；Lamella is creamy white, and has reticulate pattern.Cap length-width ratio about 1.16:1,
The ratio about 7.2:1 of the length-width ratio of stem about 0.7:1, cap length and stem length.According to " Macrofungi From China " (fourth of the twelve Earthly Branches morning mist edit,
Henan science tech publishing house, 2000), middle peasant Bai Ling 3 and page 66 photo about Pleurotus nebrodensis and corresponding morphological characteristic
Description is consistent, it is known that it belongs to Pleurotus nebrodensis in classification, and as the Classification system change of the Pleurotus nebrodensis that technical background is introduced, it is just
True Classification system is Pleurotus eryngii var.tuoliensis.
(2) the ISSR collection of illustrative plates with 4 the Pleurotus nebrodensis bacterial strains assert by country contrasts identification and analysis
(1), test strain: control strain: the Pleurotus nebrodensis bacterial strain assert by country only has 4, the most magnificent miscellaneous No. 13, middle peasant
Wing Bao, KH2 and middle peasant No. 1；Bacterial strain of the present invention: middle peasant Bai Ling 3
(2), test method: with the DNA of bacterial strain as template, carry out ISSR amplification with P4 or P24 for primer respectively, obtain ISSR
Amplified production；Described primer sequence is as follows:
P4:5 '-GGATGCAACACACACACAC-3 ' (SEQ ID No.1)
P24:5 '-CACGAGAGAGAGAGAGA-3 ' (SEQ ID No.2)
ISSR amplification system is: dNTP (2.5mM) 1.5 μ L, primer (10 μm ol) 1 μ L, Ex TaqTMArchaeal dna polymerase
0.5U, 10 × Ex Taq PCR buffer 2 μ L, DNA profiling 4 μ L, use ddH2O polishing is to 20 μ L；ISSR reaction condition: 94 DEG C
4min；94 DEG C of 50s, 55 DEG C of 50s, 72 DEG C of 2min, totally 35 circulations；72 DEG C of polishing 7min.By amplified production at 1.0% agarose
Carry out electrophoresis detection on gel, detect ISSR amplification.
The result ISSR collection of illustrative plates (see Fig. 2) with P4 as primer amplification and the ISSR collection of illustrative plates (see Fig. 3) with P24 as primer amplification
In all can be seen that middle peasant Bai Ling 3 and China is miscellaneous 13, all there is notable difference band, explanation in middle peasant wing Bao, KH2, middle peasant No. 1
Middle peasant Bai Ling 3 is a kind of new Pleurotus nebrodensis (Pleurotus eryngii var.tuoliensis) bacterial strain.
(3) utilize IGS2-RFLP collection of illustrative plates that middle peasant Bai Ling 3 is carried out taxonomic identification
Test strain: middle peasant Bai Ling 3 and middle peasant No. 1
Test method: (1), DNA extraction: extract No. 3 DNA of middle peasant Bai Ling according to a conventional method.
(2), primer: InvSR1R and 5SRNAR.
InvSR1R:5 '-ACTGGCAGAATCAACCAGGTA-3 ' (SEQ ID No.3)
5SRNAR:5 '-ACCGCATCCCGTCTGAT-3 ' (SEQ ID No.4)
(3), restricted enzyme: Hap II, Rsa I.
IGS2-PCR amplification system: dNTP (2.5mM each) 4 μ L, each 2.5 μ L of InvSR1R, 5SRNAR, Ex TaqTM
Archaeal dna polymerase 1.25U, 1 × PCR buffer, template 25ng, use ddH2O polishing is to 50 μ L.IGS2-RFLP reaction condition: 94 DEG C
1min, 60 DEG C of 1min, 72 DEG C of 3min, totally 35 circulations；72 DEG C, 7min.Enzyme action system: restricted enzyme (10U/ μ L) 1 μ L,
PCR primer 6 μ L, 10 × buffer 1 μ L, with water polishing to 10 μ L.It is placed in enzyme action 4 hours at 37 DEG C.1.2% agarose gel
Electrophoresis, detects endonuclease reaction result under ultraviolet gel imaging system.Data process and use Quantity One to analyze software.
It is 609bp, 1821bp, 2561bp that the IGS2 of result middle peasant Bai Ling 3 generates size through Hap II enzyme action (see Fig. 4)
3 fragments；5 sheets that size is 510bp, 1051bp, 1118bp, 2262bp, 2679bp are generated through Rsa I enzyme action (see Fig. 5)
Section.Find that through contrast the banding pattern of middle peasant Bai Ling 3 is the most different from other Pleurotus nebrodensis bacterial strains, illustrate that middle peasant Bai Ling 3 is a kind of
New Pleurotus nebrodensis bacterial strain.
The experiment in cultivation of embodiment 2 Pleurotus nebrodensis of the present invention middle peasant Bai Ling 3
(1) female breeding planted: in the weight ratio ratio of 1:100 by No. 3 strains of middle peasant Bai Ling of 0.3～0.5cm size (
In December in 2014 within 12nd, at the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, its deposit number it is
CGMCC No10193) be inoculated on mother culture media, 25 DEG C, relative air humidity is 60%, cultivates 12 under the conditions of lucifuge
My god, cover with test tube (test tube specification 20mm × 200mm) inclined-plane, obtain the female kind of middle peasant Bai Ling 3；The wherein preparation of mother culture media
Method is: by peeled potatoes stripping and slicing (diameter about 1cm) 200g, be placed in about 600ml water, boiling water boiling 30min, 4 layers of gauze mistake
Filter, taking juice；Add less than 400ml cold water in gained juice, then add glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate
0.5g, agar powder 15g, be settled to 1000ml, and stirring is to agar powder without conglomeration, and little fire heating is also stirred, to above composition simultaneously
It is completely dissolved, subpackage test tube (specification 20mm × 200mm), 121 DEG C of sterilizing 30min；Pendulum inclined-plane, obtains mother culture media.
(2) Primary spawn: female for step (1) gained middle peasant Bai Ling 3 kind is inoculated according to the ratio of percentage by weight 5%
On cultivation compost, 25 DEG C, relative air humidity 60%, cultivate 30 days under the conditions of lucifuge, obtain No. 3 original seeds of middle peasant Bai Ling；Institute
The cultivation compost stated is prepared as follows: according to percentage by weight ratio by cotton seed hulls 81%, Testa Tritici 12.5%, Semen Maydis
Powder 3.5%, Calx 3% mixes, and stirs；Part by weight according still further to material-water ratio 1:1.86 adds water, stirs, and fills bacterium
Bag, bacterium bag specification is 17cm × 33cm, the Polypropylene Bag of thick 4, pack height 10cm, and rubber band tying uses autoclaving,
150min is kept at 125 DEG C.After bacterium bag sterilizing, take out spreading for cooling.
(3) cultigen is cultivated: connect by step (2) gained No. 3 original seeds of middle peasant Bai Ling according to the ratio that percentage by weight is 5%
Kind in cultivation compost (its constituent and the same step of ratio (2) thereof), 25 DEG C, relative air humidity 60%, lucifuge bar
Cultivate 28 days under part, obtain No. 3 cultigens of middle peasant Bai Ling.
(4), cultivation compost preparation: with reference to step (2) prepare cultivation compost, pack, bacterium bag specification be 17cm ×
35cm, the Polypropylene Bag of thick 4, expect high about 16cm, the middle edible fungi plastic hollow rod inserting long 13cm, use diameter
The 3.5cm collar and the sealing of supporting tampon lid；Use normal pressure bacterium sterilizing, keep 14 hours at 100 DEG C.
(5), cooling inoculation: sterilize after cooling chamber is cleaned up, step (4) gained bacterium bag is put into cooling chamber to temperature
It is reduced to room temperature.Aseptically, take out the edible fungi hollow plastic stick in bacterium bag, according to the ratio of percentage by weight 5%,
No. 3 cultigens of middle peasant Bai Ling are inoculated in the hole of bacterium bag central authorities, seal with the collar and supporting tampon lid；At 25 DEG C, air
Relative humidity 60%, half-light, well-ventilated, cultivates and covers with bacterium bag in 30 days.
(6), mycelium stimulation: being opened by bag mouth, remove the mycoderma of 3 square centimeters of sizes of charge level with sterilization spatula, pine is pricked suitable for reading, so
After by bacterium bag pine prick on, continue cultivate 5 days, until mycelia recover.
(7), low temperature stimulation: bacterium bag is put into low temperature stimulation 7 days under the conditions of 1 DEG C of freezer.
(8), management of producing mushroom and gathering: the bacterium bag that will process through step (7), proceed to mushroom room, control temperature at 12 DEG C,
Relative air humidity 85%, illumination 500～800 lux, well-ventilated, environment CO2≤1000×10-6Under the conditions of cultivate 28
My god；When sporophore cap is sufficiently spread out, but still there is slight crimping, sporophore of gathering when spore does not discharges；From sporophore when gathering
Root is cut off, and cases after wrapping up with tin foil.
No. 3 stems of result middle peasant Bai Ling are short, only 1.9cm, the averagely thick 4.1cm of bacterial context；The single mushroom of agricultural formula cultivation weighs 260.5
± 40.1g, cultivation period 100-110 days.Sporophore (see Fig. 1) commodity value of middle peasant Bai Ling of the present invention 3 is higher, its bacterium
Lid is scalloped shaped, color canescence, and quality is stone, and agricultural formula, factory culture are suitable for.
Claims (1)
1. Pleurotus nebrodensis (Pleurotus eryngii var.tuoliensis) bacterial strain middle peasant Bai Ling 3, in 2014
In the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, its deposit number was CGMCC to December on 12nd
No.10193。
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