CN101328464B - Synthetic method of truffle and bacteriorhiza - Google Patents
Synthetic method of truffle and bacteriorhiza Download PDFInfo
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- CN101328464B CN101328464B CN2008100584479A CN200810058447A CN101328464B CN 101328464 B CN101328464 B CN 101328464B CN 2008100584479 A CN2008100584479 A CN 2008100584479A CN 200810058447 A CN200810058447 A CN 200810058447A CN 101328464 B CN101328464 B CN 101328464B
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Abstract
The invention provides a method for synthesizing Chinese truffles and mycorhiza of China pinus montana. The method comprises the following steps of: filtration of China pinus montana seeds and truffle seed entities; culture of aseptic seedlings; preparation of microbial inoculum; arrangement, optimal selection and inoculation of culture substrates; culture of mycorrhizal seedlings; anatomization of mycorhiza forms; detection and confirmation of molecules and transplanting culture, and finally, the culture of truffles is realized.
Description
Technical field:
The invention belongs to biological technical field, be specifically related to the synthetic method of India truffle (Chinese ferfas) and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza.
Background technology:
Ferfas (truffles) belongs to the ectomycorrhiza edible fungus (edible ectomycorrhizal fungi) of giving birth in the sylvan life soil such as pine, oak, owing to be rich in nutritive substance and special desire compounds and the human immunity system adjusting materials of urging such as protein, become the green ecological functional foodstuff that high protein, lower fat, dietotherapy are taken into account, in Europe so the whole world gain great popularity, being described as " black diamond " in the kitchen, is the most expensive so far in the world whole food.China annual outlet India truffle (Chinese ferfas) more than 360 ton becomes the novel wild mycorhiza edible mushrooms of shaking off poverty and setting out on the road to prosperity in the forest zone, mountain region.And more and more be subjected to people's popular welcome, demand is vigorous day by day.The sylvan life natural resources because the ferfas of being gathered places one's entire reliance upon, supply falls short of demand for natural production; The acquisition mode of commercialization blanket type particularly, natural survival state are subjected to disturbing greatly and destroying, and have caused natural production to reduce significantly in the last few years; If do not take corresponding measure, must become endangered species after several years, so that the extinction that disappears.How to realize that ferfas class mycorhiza wild edible fungus sustainable utilization has become the hot focal issue of global problem and innovative research.Therefore, at first the synthesis bacterium offspring under the condition of coercing with ferfas bacterial classification and aseptic sapling on artificial mycorhiza synthetic basis, carries out the inevitable requirement and the only way of ferfas artificial culture plantation having become ferfas sustainable development.Wherein ferfas seedling mycorhiza synthetic becomes the gordian technique of ferfas cultivation.So far, do not see the report and the record of India truffle is arranged (Chinese ferfas) and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza synthetic method in the prior art.
Summary of the invention:
The object of the present invention is to provide a kind of India truffle (Chinese ferfas) and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza synthetic method.This method relates to a series of programsteps, be cultivation, the fungicide preparation of the Pinus armandi Franch-P. Komavovii Lavl. seed and the screening of ferfas bacterial classification sporophore, aseptic sapling, the configuration of culture medium, preferred and inoculation, the detection and the affirmation of the cultivation of mycorhiza seedling and dissection of mycorhiza form and molecule, implanting and cultivating is with final realization artificial culture ferfas.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
China ferfas and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza synthetic method comprise the steps:
(1) Pinus armandi Franch-P. Komavovii Lavl. is sowed and grows seedlings,
A, get in the 4-8 ℃ of refrigerator preservation 2-12 month Pinus armandi Franch-P. Komavovii Lavl. seed, 30%H
2O
2Soaked 2 hours, aseptic water washing discards the buoyant seed for several times, soaks 48 hours with sterilized water again, pulls out, makes the slight cut of seed with mechanical system, and multiplexing 75% alcohol cotton yarn wiping sterilization is put in the culture dish standby;
B, matrix are standby, get vermiculite and perlite 1/1 mixing by volume that do not sieve, and add entry and make its water content reach 30%, 121-126 ℃ of following high pressure steam sterilization 1 hour; Perhaps get the vermiculite and the perlite of new production;
C, sowing with the seed of getting ready in (1) A step, are broadcast into matrix with the downward direction of opening end, and depth of planting is 1.5-2cm;
D, grow seedlings;
(2) Chinese ferfas fungicide preparation and inoculation,
A, fungicide preparation, select the ripe Chinese ferfas that produces in 2-3 month, microscopically is observed spore shape, the ratio of chocolate spore and prematurity tawny spore reaches 95: 5 and is ripe ascoma, after ripe ascoma cleans up with tap water, aseptic water washing 3 times, 4-8 ℃ refrigeration 10-20 days after, again-24 ℃ of freezing preservations be no more than 18 months standby; Before the fungicide preparation freezing ascoma is taken out, thaw naturally, each ascoma is cut to several piece, be positioned in the stirrer, add sterilized water and pulverize the microscopically microscopy, determine that 95% ascus is free, till 5% spore was isolated from ascus, the concentration of microbial inoculum was every gram ascoma/water=1/3-5;
B, inoculation matrix preparation and the adjustment of pH value, muck soils sieves, and admixes water in right amount, makes moisture content 30%, and autoclaving 3 hours is standby; Soil ulmin, water, vermiculite add lime powder with the mixed of volume ratio 1/1/1, and mixing is adjusted pH and is 7, and is standby;
C, inoculation are by every strain inoculation 5 * 10
7The amount of individual spore is converted out the volume of the microbial inoculum that every strain need inoculate, and gets microbial inoculum and pours into matrix, mixes thoroughly; Step (1) gained seedling is taken out in the matrix from growing seedlings, remove the root end, take out the inoculation matrix of implantation step (2) B after the root system of bacterium all being immersed the microbial inoculum of step (2) A preparation;
(3) mycorrhiza fungi seeding cultivating, seedling place the natural ventilation booth, and the tap water pouring is done temperature and alternately cultivated, and culture temperature 10-30 ℃, solar irradiation 10-14 hour.
The proposition of technical solution of the present invention is based on following truffle mycorrhiza fungi composition principle: India truffle (Chinese ferfas) is the underground living fungi of symbiosis that a kind of and woody (being mainly Pinaceae Pinaceae, Fagaceae Fagaceae, corylaceae Corylaceae, Betulaceae Betulaceae etc.) plant forms ectomycorrhiza, and it grows and sporophore forms and all must could realize with the root system symbiosis of these plants.Therefore adopt the matrix of synthetic to grow saplings, tissue and spore with ferfas are made the inoculation of bacterial classification microbial inoculum, under the condition of artificial control, satisfy the spore germination of ferfas microbial inoculum and infect the demand of sapling young root, facilitate the formation of mycorhiza, synthetic mycorhiza sapling growing and cultivation in suitable mountain region (pH value 7-7.6, carbon-nitrogen rate C/N=10), to realize the artificial culture of ferfas, is guaranteed sustainable development.
India truffle of the present invention (Chinese ferfas) and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza synthetic method have been optimized cultivation, culture medium and the microbial inoculum making of mycorhiza sapling and mycorhiza is synthetic and the detection and the affirmation of mycorhiza.
Synthesis step and technological method that the present invention is concrete are as follows:
1. matrix prepares and grows seedlings
The matrix preparation: vermiculite and perlite be 1/1 mixing by volume, adds tap water and makes its water content reach 30%, 1 hour postcooling standby (if the vermiculite of new production and perlite also can be sterilized, can directly use) of 121-126 ℃ of following high pressure steam sterilization.
Sowing with grow seedlings: select the seed of mature and plump, with 75% alcohol surface sterilization, make the little mouth of its cracking with the method for machinery, opening end is broadcast downwards into above-mentioned matrix.Depth of planting 1.5-2cm is advisable; To water in good time; After planting the 10th day begins to sprout, and almost whole seeds sprout and finish after 30 days, and the statistics of germination rate was from the 6th day to the 30th day; Seedling age can be inoculated during the month at 1.5-3.
2. microbial inoculum and preparation thereof
Sporophore screening and preservation: at first microscope inspection is identified India truffle (Chinese ferfas), confirms that kames is accurate, avoids other ferfas to sneak into; Therefrom choose sophisticated India truffle, reaching with the spore amount ratio of pitch black brown more than 95% is ripe ascoma; Flush clean is used aseptic water washing after removing earth; 4-8 ℃ of refrigerator preservation moved into-24 ℃ of refrigeration chambers and preserves standby after 10-20 days.
Fungicide preparation: freezing ascoma thaws naturally, with pocket knife each ascoma is cut to several piece; Little stripping and slicing is positioned in the stirrer, adds sterilized water and pulverize, the microscopically microscopy to determine no macroscopic tissue block, dissociates 95% ascus, till about 5% spore is isolated from ascus.The concentration of microbial inoculum is advisable with every gram ascoma/water=1g/3-5ml.
3. inoculation
Matrix preparation and the adjustment of pH value: muck soils sieves, and admixes water in right amount, moisture content 30%, and autoclaving 3 hours is standby; Soil ulmin after the sterilization and vermiculite are mixed with the ratio of volume ratio 1/1, add lime powder, and abundant mixing can be standby when adjusting the pH value for 7-7.6.
Inoculation and cultivation: contain 5 * 10 according to every strain inoculation
720 times of above-mentioned microbial inoculum stoste dilutions of making are got in the dosage requirement of individual spore microbial inoculum, get 5ml dilution bacterium liquid and move into matrix, stir; The seedling of getting above-mentioned cultivation cuts off butt, and whole root system is immersed the grafting matrix cultivation afterwards of dilution microbial inoculum.Postvaccinal seedling places the natural ventilation booth to cultivate.
4. mycorhiza form and molecular level checking:
External appearance characteristic: it is bar-shaped or closely cylindric that mycorhiza forms back mycorhiza final stage branch, and smooth surface is had the extension mycelia sometimes, and least significant end is faint yellow to near-white, and is gradually dark to the base portion color, often forms the alternate snake sample spot of the depth; Bacterium cover obviously, surface coverage extension mycelia sometimes, the bacterium amphicyte is opaque.The every strain mycorhiza of sampling statistics quantity is 700-800, and individual plant root system infection rate reaches more than 95%.
Anatomical features: non-gelatinize is seen, paraplectenchyma (pseudoparenchymatous tissue) M type, surperficial tool epidermal-like cell (epidermal cells) in outer bacterium cover plane.Interior bacterium cover plane is seen between prosoplectenchyma type (pletenchymatous tissue) and paraplectenchyma's type, nearly H type, and Di Shi shape (Hartig net-like) is closely breathed out at most positions, and mycelia is arranged tightr, thick 2-4 μ m, thin-walled, colourless; The elongation of extension mycelia, the near grade slightly, cylindric, thin-walled, few branch, colourless.
Molecular level checking: through the extraction of bacteriostatic agent sporophore and the total DNA of intercepting Pinus armandi Franch-P. Komavovii Lavl. mycorhiza sapling mycorhiza, improved CTAB method and fungal DNA test kit, PCR amplification and ITS1/ITS4 and ITS4/ITS5 comparative analysis have been adopted, all obtain 100% consistence respectively, show that the Pinus armandi Franch-P. Komavovii Lavl. seedling mycorhiza that obtains of cultivating is formed by the intrusion of India truffle mycelia; Confirm that the Pinus armandi Franch-P. Komavovii Lavl. India truffle mycorrhiza fungi synthesizes successfully.
Embodiment:
In order to understand the present invention better, demonstrate,prove with embodiments of the invention below and further specify content of the present invention, but content of the present invention is not limited thereto.
Embodiment 1:
India truffle (Chinese ferfas) and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza seedling synthetic method:
1, Pinus armandi Franch-P. Komavovii Lavl. is grown seedlings:
(1) makes preparations for sowing
Gather ripe Pinus armandi Franch-P. Komavovii Lavl. seed autumn, preservation in the 4-8 ℃ of refrigerator, preservation term is no more than 1 year.Use preceding taking-up, 30%H
2O
2Soaked 2 hours, stirring several → aseptic water washing several → discarding buoyant seed → sterilized water soaked 48 hours, pulled → use mechanical system out therebetween, made the slight cut of seed → multiplexing 75% alcohol cotton yarn wiping sterilization, put in the culture dish standby.
(2) matrix
Vermiculite (not sieving) and perlite be 1/1 mixing by volume, adds tap water and makes its water content reach 30%, 121-126 ℃ of following high pressure steam sterilization 1 hour; If the vermiculite of new production and perlite also can unsterilised direct uses.
(3) sowing
With the seed of getting ready, broadcast into matrix with the direction that opening end is downward.Depth of planting is 1.5-2cm; Container is the permeable plastic crate of 425 * 340 * 200mm.
(4) grow seedlings
The tap water pouring; After planting the 10th day begins to sprout, and almost whole seeds sprout and finish after 30 days, and the statistics of germination rate was from the 6th day to the 30th day, and percentage of germination is about 80%; Seedling age 1.5-3 can inoculate during the month.
2, microbial inoculum
Select homemade ripe India truffle of 2-3 month (Chinese ferfas), be regardless of size; The exactness of taxonomy features such as microscopically microscopy spore shape to determine to plant; The spore ripening degree, promptly the ratio with chocolate spore (maturation) and tawny spore (prematurity) reaches 95: 5.Surperficial earth scrubbed in tap water by scrub-brush and cleaning is residual to there not being earth, aseptic water washing 3 times; 4-8 ℃ refrigeration 10-20 days after ,-24 ℃ of freezing preservations; Preservation period should be above 18 months.
Before the fungicide preparation freezing ascoma is taken out in refrigerator, thaw naturally.With pocket knife each ascoma is cut to several piece after thawing; Stripping and slicing is positioned in the two-in-one stirrer of PHILIPS HR2839 type, put the about 200g of ascoma at every turn, adding sterilized water pulverizes, add at first water a little, after increase the water yield gradually, pulverized about 3-4 minute, the microscopically microscopy, to determine no macroscopic tissue block, 95% ascus is separated from one another, till about spore more than 5% is isolated in the ascus.The concentration of microbial inoculum is advisable with every gram ascoma/water=1g/3-5ml.
The spore counting: microbial inoculum stoste fully stirs evenly, and respectively gets 1ml at container upper, middle and lower position, places in 3 graduated cylinders, and 1ml is diluted to 10ml in the graduated cylinder, stirs evenly; Each graduated cylinder upper, middle and lower portion respectively gets one, puts blood counting chamber; Calculate unit volume stoste reduced spore number; Simultaneously, get stoste 10ml microbial inoculum to dip in root in 20 times of rearmounted beakers of sterilized water dilution during for inoculation.
3. inoculation
(1) matrix preparation and pH value are determined
Soil ulmin 2 orders sieve, and admix tap water in right amount, make moisture content about 30%, 121-126 ℃ of high pressure steam sterilization 3 hours; Standby after soil ulmin behind the above autoclaving and vermiculite (sterilization or not) the mixed with volume ratio 1/1;
Above matrix is got certain mass (as 100g), is divided into 4 equal portions, adds the dry hydrate of different mass in each equal portions respectively, fully mixing is got 30ml for every part, adds 150ml distilled water, stir for several times therebetween, after 1 hour, record the pH value of every part of matrix with accurate pH test paper (5.5-9.0).The mass ratio of matrix and lime when calculating or reckoning pH are 7-7.6.In matrix, add lime in above ratio, fully standby behind the mixing.
(2) inoculation
Method: by every strain inoculation 5 * 10
7The amount of individual spore is converted out the volume of the microbial inoculum stoste that every strain need inoculate, and after 20 times of microbial inoculum stoste dilutions, pours into matrix after measuring the 5ml microbial inoculum with graduated cylinder, mixes thoroughly microbial inoculum and matrix standby; Seedling is taken out in the matrix of growing seedlings, and the end that cuts off root with scissors bears more lateral root to impel; Take out after root system all being submerged into the dilution microbial inoculum of step 2 preparation, container bottom 1/5 after putting into inoculation matrix is implanted seedling; Matrix accounts for 4/5 of vessel volume.
4, mycorrhiza fungi seeding cultivating
The inoculation seedling places the natural ventilation booth, and the tap water pouring is done temperature alternately, and culture temperature 10-30 ℃, illumination every day 10-14 hour.
5, mycorhiza form
Need 3-5 month after the inoculation approximately, the ferfas mycelia is invaded seedlings root and forms mycorhiza.Confirm whether mycorhiza forms, need add their confirmation from mycorhiza form and anatomical features thereof and molecular level (being paternity test).
External appearance characteristic: mycorhiza system (mycorrhizal systems) two forked (dichotomous), sometimes near coralliform (coralloid) or between above two types, idol is branch (unramified) not simply, length overall 2-4.6mm, the secondary branch of tool 1-4 level, the branched mycorhiza of several tools 3-4 level is closely arranged along root and is formed near bunch of shape sometimes; The final stage branch is bar-shaped or closely cylindric, the top is straight, long 0.8-3.9mm, top end diameter (220) 350-500 μ m, the thick 250-350 μ of base portion m, smooth surface is had the extension mycelia sometimes, and least significant end is faint yellow to near-white, less tawny or sorrel, gradually dark to the base portion color, even top and base portion are homochromy, often form the alternate snake sample of the depth; The bacterium cover is obvious, and the extension mycelia is covered on the surface sometimes, and the bacterium amphicyte is opaque; Tegumental cell is invisible; The extension mycelia lacks or only limits to the last grade branch, and is long, white, obviously.The shoestring end is seen.The sclerotium end is seen.
Anatomical features: non-gelatinize is seen on outer bacterium cover plane, paraplectenchyma (pseudoparenchymatous tissue) M type (according to Agerer, 1987-2002), surperficial tool epidermal-like cell (epidermal cells), do not have a mycelia net (hyphalnets), the cell thin-walled, tawny, normal local mycelia color is darker, smooth surface does not have appurtenant, long 10-25 μ m, 7-10 cell in thick (5) the 8-20 μ m, per 20 μ m * 20 μ m squares (comprise complete with the cell that only partly is positioned at sample prescription).Interior bacterium cover plane is seen between prosoplectenchyma type (pletenchymatous tissue) and paraplectenchyma's type, near the H type (according to Agerer, 1987-2002), Di Shi shape (Hartig net-like) is closely breathed out at most positions, and mycelia is arranged tightr, and thick 2-4 μ m; thin-walled is colourless; The elongation of extension mycelia, the near grade slightly, cylindric, thick 2-3 μ m, thin-walled, few branch, colourless.Clamp connexion lacks.Cystidium and chlamydospore lack.
Molecular level checking: through the extraction of bacteriostatic agent sporophore and the total DNA of intercepting Pinus armandi Franch-P. Komavovii Lavl. mycorhiza sapling mycorhiza, improved CTAB method and fungal DNA test kit, PCR amplification and ITS1/ITS4 and ITS4/ITS5 comparative analysis have been adopted, all obtain 100% consistence respectively, show that the Pinus armandi Franch-P. Komavovii Lavl. seedling mycorhiza that obtains of cultivating is formed by the intrusion of India truffle mycelia; Confirm that the Pinus armandi Franch-P. Komavovii Lavl. India truffle mycorrhiza fungi synthesizes successfully.
Mycorhiza quantity: the every strain mycorhiza of sampling statistics quantity is 700-800, and (mycorhiza number/(not infecting tip of a root number+mycorhiza number) is 95% to individual plant root system infection rate.
Annotate: " mycorhiza " is defined as herein: overlapped the unit that forms, zone that is covered by the discernible bacterium of successive.The mycorhiza of multi-branched if covered by successive bacterium cover, is considered as a mycorhiza.
Compared with prior art, the excellent benefit that possesses of the present invention is:
The present invention has established indispensable key technology for the artificial cultivation of realizing domestic India truffle and sibling species thereof. Realized the synthetic of domestic ferfas and Huashan pine mycorhiza by the present invention, utilized the in enormous quantities synthetic cultivation of scale mycorhiza sapling of this method, for the artificial growth industrialization of next step ferfas provides technical guarantee. The present invention can be applicable to reparation and the reconstruction of deserted mountain soil remediation and improvement, afforestation and vegetation, has great potential and wide application prospect for development and use limerock area and forest zone, remote mountain region. Method operation of the present invention is simple and easy, easily extensive use and popularization. The present invention is that biology (microbiology, bacteriology) intersects with agronomy and the technology that forestry (cultivation and forestry) and cross discipline mycorhiza thereof are learned the Mycorrhizology field, draw the multiple advantage of a plurality of subjects, had the advantage of practical simplicity.
Claims (1)
1. Chinese ferfas and Pinus armandi Franch-P. Komavovii Lavl. mycorhiza synthetic method comprise the steps:
(1) Pinus armandi Franch-P. Komavovii Lavl. is sowed and grows seedlings,
A, get in the 4-8 ℃ of refrigerator preservation 2-12 month Pinus armandi Franch-P. Komavovii Lavl. seed, 30%H
2O
2Soaked 2 hours, aseptic water washing discards the buoyant seed for several times, soaks 48 hours with sterilized water, pulls out, makes the slight cut of seed with mechanical system, with 75% alcohol cotton yarn wiping sterilization, puts in the culture dish standby again;
B, matrix are standby, get vermiculite and perlite 1/1 mixing by volume that do not sieve, and add entry and make its water content reach 30%, 121-126 ℃ of following high pressure steam sterilization 1 hour;
C, sowing with the seed of getting ready in (1) A step, are broadcast into matrix with the downward direction of opening end, and depth of planting is 1.5-2cm;
D, grow seedlings;
(2) Chinese ferfas fungicide preparation and inoculation,
A, fungicide preparation, select the ripe Chinese ferfas that produces in 2-3 month, microscopically is observed spore shape, the ratio of chocolate spore and prematurity tawny spore reaches 95: 5 and is ripe ascoma, after ripe ascoma cleans up with tap water, aseptic water washing 3 times, 4-8 ℃ refrigeration 10-20 days after, again-24 ℃ of freezing preservations be no more than 18 months standby; Before the fungicide preparation freezing ascoma is taken out, thaw naturally, each ascoma is cut to several piece, be positioned in the stirrer, add sterilized water and pulverize the microscopically microscopy, determine that 95% ascus is free, till 5% spore was isolated from ascus, the concentration of microbial inoculum was every gram ascoma/3-5ml water;
B, inoculation matrix preparation and the adjustment of pH value, muck soils sieves, and admixes water in right amount, makes moisture content 30%, and autoclaving 3 hours is standby; Soil ulmin, water, vermiculite add lime powder with the mixed of volume ratio 1/1/1, and mixing is adjusted p H and is 7, and is standby;
C, inoculation are by every strain inoculation 5 * 10
7The amount of individual spore is converted out the volume of the microbial inoculum that every strain need inoculate, and gets microbial inoculum and pours into matrix, mixes thoroughly; Step (1) gained seedling is taken out in the matrix from growing seedlings, remove the root end, take out the inoculation matrix of implantation step (2) B after the root system of bacterium all being immersed the microbial inoculum of step (2) A preparation;
(3) mycorrhiza fungi seeding cultivating, seedling place the natural ventilation booth, the tap water pouring, and alternation of wetting and drying is cultivated, and culture temperature 10-30 ℃, solar irradiation 10-14 hour.
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