CN100434506C - Screening for strain of steepletop hickory chick and process for preparing strain thereof - Google Patents
Screening for strain of steepletop hickory chick and process for preparing strain thereof Download PDFInfo
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- CN100434506C CN100434506C CNB2005100486792A CN200510048679A CN100434506C CN 100434506 C CN100434506 C CN 100434506C CN B2005100486792 A CNB2005100486792 A CN B2005100486792A CN 200510048679 A CN200510048679 A CN 200510048679A CN 100434506 C CN100434506 C CN 100434506C
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Abstract
The present invention relates to a screening and preparation method for a morchella conica pers strain, which belongs to the technical field of microorganisms. The strain Morchella conica M0503 of the method is preserved in the common microorganism center of the China Committee for Culture Collection of Microorganisms on August 11th, 2005, and the preservation number of the strain Morchella conica M0503 is CGMCC NO. 1437. Liquid and solid excellent strains prepared by the strain Morchella conica M0503 are applied to the protection and the reproduction of the wild resources of conica pers in an origin place, and the natural output and the quality of the concia pers are enhanced. The present invention has marked social, economical and ecological benefit and wide application prospect.
Description
Technical field:
The invention belongs to microbial technology field, be specifically related to bacterial strain screening and the process for preparing strain thereof of morel.
Background technology:
Morel Morchella is world-renowned famous and precious wild edible fungus, and Morchellaconica Morchellaconica is one of main kind of Yunnan product morel.The artificial culture lab scale idol of morel has the report of success, but the poor repeatability of experiment in cultivation still can not be carried out the commercialization cultivation so far, and the morel on the market is all from wild.Wild toadstool causes natural production to decline to a great extent owing to excessively gather and acquisition mode science and do not have corresponding technical measures not in recent years.Studies show that, under the natural environmental condition of producing region, adopt artificial bacterial classification and supporting technical measures, is one of most economical valid approach that solves the morel imbalance between supply and demand.
In morel protection multiplication technique, the acquisition of excellent species is a key technique.The acquisition of morel bacterial strain utilizes normally that sporophore or spore separate, purifying, directly applies to cultivation or the mycelium production of morel.The deficiencies in the prior art are that the morel bacterial classification can not carry out strain identification with the method for fruiting, can only tentatively determine whether to be the morel pure growth according to mycelia form and isolate; The bacterial classification of producing is generally used solid spawn.It is not extensive that numerous application is expanded in the protection of at present adopting liquid spawn to carry out the morel resource at home.
Summary of the invention:
Main purpose of the present invention overcomes the prior art deficiency exactly; utilize country of origin, Yunnan Morchellaconica sporophore to filter out strain excellent; adopt biotechnology to carry out strain identification, preparation morel liquid and solid excellent species are for numerous excellent species that provides is provided the protection of morel wild resource.
The fungi that the present invention adopts is Morchellaconica Morchella conica M0503; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on 08 11st, 2005; The numbering CGMCC NO.1437 that preservation is registered on the books.
The Morchellaconica sporophore is less, high 5～8cm.Ascoma is closely cylindrical, and the pinnacle is light brown, and the microscler pit of surperficial recessed formation is vertically arranged.Stem white has irregular longitudinal furrow.Ascus is closely cylindrical, 8 of thecaspores, and single file is arranged.The mycelial growth initial stage is close to substratum, and is colourless, glossy, along with the increase of bacterium colony, has forked or dendroid branch, and the later stage produces sclerotium and light brown pigment.
Technical scheme of the present invention:
1. gather the wild toadstool sporophore;
2. separate tissue or spore separation;
3. purifying bacterial strain and identification of strains;
4. biological characteristics relatively reaches the production traits relatively;
5. determine strain excellent;
6. bacterial classification production and bacterial classification are used;
Through repeated screening, obtain mycelium production height, resistant to pollution morel liquid spawn special strain therefore M0503.
Fungi Morchella conica cultural method of the present invention (below be weight percentage):
Strain separating purifying substratum: 2% morel tankage, 0.001%V
B1, 2% glucose, 2% agar;
Morel strains separation and authentication method:
1, Morchella esculenta (L.) Pers sporophore tissue block or spore liquid are inoculated on the above-mentioned test tube nutrient agar inclined-plane, cultivated 5-10 days down in 18-24 ℃, test tube observed and recorded every day to separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain morel and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned nutrient agar, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 5-7 days down, obtain morel purifying test tube kind in 18-24 ℃.
3, adopt for examination sporophore and corresponding mycelia isolate, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, by the ITS sequence of Blast comparative sample and Morchellaconica (Morchella.conica) Identities=488/522 (93%) among the GENBANK, Gaps=9/522 (1%) analyzes and determines that separating the pure growth that obtains is the Morchellaconica mycelium.
The preparation method of morel bacterial classification of the present invention is divided into: two kinds of liquid culture and solid culture.
The liquid spawn preparation method:
The liquid culture based formulas is: 5-10% wheat bran, 0.5-1% analysis for soybean powder, 0.5-1% maltose, 1-3% Semen Maydis powder, remainder is a water, the pH nature.
1, the mycelium with morel is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 5-7 days down in 18-24 ℃, obtains test tube strains.
2, the test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, culture medium prescription is the aforementioned liquids culture medium prescription, cultivates in 20-26 ℃ of following shaking table, and rotating speed is 120-160r/min, incubation time 5-7 days.
3, will shake bottle bacterial classification inoculation in the fermentor tank of the automatic fermentative production line of 25L, air flow is 1: 0.8v/ (vmin); Mixing speed is 120-160r/min; Inoculum size is 5-10%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 20-26 ℃; Aerated culture 72-96 hour, obtain the morel liquid spawn.
The solid spawn preparation method:
The solid culture based formulas is: wood chip 75-90%, wheat bran 5-20%, phosphate fertilizer 1%, gypsum 1%, morel be vegetable mould 3% vegetatively, water content 60%, pH nature.
1, the mycelium with morel is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 5-7 days down in 18-24 ℃, obtains test tube strains.
2, the test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, culture medium prescription is the aforementioned liquids culture medium prescription, cultivates in 20-26 ℃ of following shaking table, and rotating speed is 120-160r/min, incubation time 5-7 days.
3, adopt aforementioned solid culture based formulas.After culture material mixed, after adding water and mixing evenly, in the jar of the 750ml that packs into, compress and seal the back, insert liquid spawn, cultivated 15-20 days in 20-26 ℃, cover with up to mycelia 120 ℃ of sterilizations 60 minutes.
Embodiment:
Embodiment one:
1, Morchellaconica sporophore tissue block is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 10 days down in 18 ℃, test tube observed and recorded every day to separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain morel and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned nutrient agar, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 7 days down, obtain morel purifying test tube kind in 18 ℃.
3, adopt for examination sporophore and corresponding mycelia isolate, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, by the ITS sequence of Blast comparative sample and Morchellaconica (Morchella.conica) Identities=499/510 (97%) among the GENBANK, Gaps=2/510 (0%) analyzes and determines that separating the pure growth that obtains is the Morchellaconica mycelium.
4, the mycelium with Morchella conica M0503 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 7 days down, obtains the test tube kind for 18 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 5% wheat bran, 0.5% analysis for soybean powder, 0.5% maltose, 1% Semen Maydis powder, and remainder is a water, the pH nature.Cultivate in 20 ℃ of following shaking tables, rotating speed is 120rpm, and incubation time 7 days obtains the level liquid bacterial classification.
In the fermentor tank of the automatic fermentative production line of 25L, air flow is 1 with the level liquid bacterial classification inoculation: 0.8v/ (vmin); Mixing speed is 120r/min; Inoculum size is 5%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 22 ℃; Aerated culture 96 hours obtains the morel liquid spawn.
Embodiment two:
The step of strain preparation is identical with embodiment one.
1, Morchellaconica sporophore tissue block is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 6 days down in 22 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain morel and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned nutrient agar, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 6 days down, obtain morel purifying test tube kind in 22 ℃.
3, adopt for examination sporophore and corresponding mycelia isolate, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, by the ITS sequence of Blast comparative sample and Morchellaconica (Morchella.conica) Identities=499/510 (97%) among the GENBANK, Gaps=2/510 (0%) analyzes and determines that separating the pure growth that obtains is the Morchellaconica mycelium.
4, the mycelium with Morchella conica 0503 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 6 days down, obtains the test tube kind for 22 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 6% wheat bran, 0.6% analysis for soybean powder, 0.6% maltose, 1.2% Semen Maydis powder, and remainder is a water, the pH nature.Cultivate in 22 ℃ of following shaking tables, rotating speed is 140rpm, and incubation time 6 days obtains the level liquid bacterial classification.
In the fermentor tank of the automatic fermentative production line of 25L, air flow is 1 with the level liquid bacterial classification inoculation: 0.8v/ (vmin); Mixing speed is 140r/min; Inoculum size is 8%; Tank pressure: 0.04Mpa; PH is 6.0; Temperature is 24 ℃; Aerated culture 84 hours obtains the morel liquid spawn.
Embodiment three:
1, Morchellaconica sporophore tissue block is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 5 days down in 24 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain morel and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned nutrient agar, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 5 days down, obtain morel purifying test tube kind in 24 ℃.
3, adopt for examination sporophore and corresponding mycelia isolate, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, by the ITS sequence of Blast comparative sample and Morchellaconica (Morchella.conica) Identities=499/510 (97%) among the GENBANK, Gaps=2/510 (0%) analyzes and determines that separating the pure growth that obtains is the Morchellaconica mycelium.
4, the mycelium with Morchella conica 0503 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 5 days down, obtains the test tube kind for 24 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 10% wheat bran, 0.5% analysis for soybean powder, 0.5% maltose, 1% Semen Maydis powder, and remainder is a water, the pH nature.Cultivate in 26 ℃ of following shaking tables, rotating speed is 160rpm, and incubation time 5 days obtains the level liquid bacterial classification.
To solid medium, culture medium prescription is wood chip 75%, wheat bran 20%, phosphate fertilizer 1%, gypsum 1%, a morel vegetable mould 3% vegetatively with the level liquid bacterial classification inoculation, water content 60%, pH nature.Wood chip, wheat bran, phosphate fertilizer, gypsum, vegetable mould are mixed after the weighing in proportion, in the seed bottle of the 750ml that packs into behind the poach, every bottled 500ml, 120 ℃ of sterilizations are after 60 minutes, and inoculation is placed on 26 ℃ and cultivated 15 days, obtains the morel solid spawn.
Embodiment four:
1, Morchellaconica sporophore tissue block is inoculated on the above-mentioned strain separating purifying medium slant, cultivated 5 days down in 24 ℃, to test tube observed and recorded every day of separation and Culture, the test tube that rejecting has in time been polluted, pick out bacterial strain pollution-free, that grow fine, obtain morel and separate the test tube kind.
2, will separate test tube kind inoculated by hypha block and to the dull and stereotyped culture dish of above-mentioned nutrient agar, carry out the bacterial classification purifying, get most advanced and sophisticated inoculated by hypha block after 2 days on medium slant, cultivate 5 days down, obtain morel purifying test tube kind in 24 ℃.
3, adopt for examination sporophore and corresponding mycelia isolate, extract total DNA by the SDS method respectively, carry out pcr amplification and ITS sequencing, by the ITS sequence of Blast comparative sample and Morchellaconica (Morchella.conica) Identities=499/510 (97%) among the GENBANK, Gaps=2/510 (0%) analyzes and determines that separating the pure growth that obtains is the Morchellaconica mycelium.
4, the mycelium with Morchella conica 0503 is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is the PDA substratum, cultivates 5 days down, obtains the test tube kind for 24 ℃.
5, the test tube kind is inoculated in the 500ml triangular flask in (every bottled 200ml) liquid nutrient medium, culture medium prescription is 8% wheat bran, 0.5% analysis for soybean powder, 1% maltose, 1% Semen Maydis powder, and remainder is a water, the pH nature.Cultivate in 26 ℃ of following shaking tables, rotating speed is 160rpm, and incubation time 5 days obtains the level liquid bacterial classification.
To solid medium, culture medium prescription is wood chip 90%, wheat bran 5%, phosphate fertilizer 1%, gypsum 1%, a morel vegetable mould 3% vegetatively with the level liquid bacterial classification inoculation, water content 60%, pH nature.Wood chip, wheat bran, phosphate fertilizer, gypsum, vegetable mould are mixed after the weighing in proportion, in the seed bottle of the 750ml that packs into behind the poach, every bottled 500ml, 120 ℃ of sterilizations are after 60 minutes, and inoculation is placed on 20 ℃ and cultivated 20 days, obtains the morel solid spawn.
Claims (1)
1, a kind of strain of steepletop hickory chick is characterized in that described bacterial strain is (Morchella conica) M0503, and the preserving number of this bacterial strain is CGMCC NO.1437.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101926834B (en) * | 2010-06-13 | 2012-05-09 | 华南师范大学 | Morchella conica granules and preparation method and application thereof |
| CN102986536B (en) * | 2012-12-02 | 2016-12-21 | 中华全国供销合作总社昆明食用菌研究所 | A kind of flammulina velutipes strain and preparation method |
| CN103004465A (en) * | 2012-12-02 | 2013-04-03 | 中华全国供销合作总社昆明食用菌研究所 | Coprinus comatus strain and preparation method |
| CN102986452A (en) * | 2012-12-02 | 2013-03-27 | 中华全国供销合作总社昆明食用菌研究所 | Agrocybe aegerita KMFJ-FC and preparation method thereof |
| CN103923842B (en) * | 2014-04-20 | 2016-06-15 | 云南省农业科学院高山经济植物研究所 | The screening method of a kind of Morchellaconica quality strains |
| CN104137736A (en) * | 2014-08-08 | 2014-11-12 | 梁平县顶力羊肚菌种植基地 | Method for cultivating morchella esculenta strain |
| CN104686196B (en) * | 2015-01-29 | 2017-04-12 | 重庆市中药研究院 | Method for preserving and separating toadstool strain through sporocarp dried in shade |
| CN106665120A (en) * | 2016-12-15 | 2017-05-17 | 防城港市蓝瀚达科技有限公司 | Common morel sporophore artificial cultivation method |
| CN107711290B (en) * | 2017-09-28 | 2020-07-24 | 山西省农业科学院食用菌研究所 | Culture medium for mycorrhizal edible fungus symbiotic seedling and synchronous culture method thereof |
| CN110140591B (en) * | 2019-06-12 | 2021-06-25 | 山东农业大学 | Morchella high-yield strain and application thereof |
| CN114231419B (en) * | 2021-11-23 | 2022-07-12 | 贵州省土壤肥料研究所(贵州省生态农业工程技术研究中心)(贵州省农业资源与环境研究所) | High morchella esculenta with short production period and application thereof |
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