CN105684728A - Tricholoma matsutake liquid strain breeding method and field bionic cultivation method - Google Patents
Tricholoma matsutake liquid strain breeding method and field bionic cultivation method Download PDFInfo
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Abstract
The invention provides a tricholoma matsutake liquid strain breeding method, and further provides a tricholoma matsutake field bionic cultivation method. The tricholoma matsutake liquid strain breeding method comprises the steps that potatoes, saw dust water, glucose, magnesium sulfate, monopotassium phosphate, peptone, vitamin B1, yeast and agar are adopted for preparing a first culture solution, tissue at the joint of stipe and cap of tricholoma matsutake is placed into the first culture solution for culture to obtain a mother strain; then, potatoes, saw dust, corncob powder, water, glucose, magnesium sulfate, monopotassium phosphate, peptide and vitamin B1 are adopted for preparing a second culture solution, the second culture solution is subpackaged into shake flasks, the mother strain is inoculated into the shake flasks, and culture is performed to obtain a first liquid strain. The tricholoma matsutake field bionic cultivation method has the advantages of being short in production period, simple, low in infection rate and the like.
Description
Technical field
The present invention relates to the artificial breeding technique field of Tricholoma matsutake (lto et lmai) Singer, in particular to liquid spawn mating system and the land for growing field crops bionic cultivation method of a kind of Tricholoma matsutake (lto et lmai) Singer。
Background technology
Tricholoma matsutake (lto et lmai) Singer, formal name used at school Trichotoma matsutake, another name armillaria Matsutake, closes bacterium, platform bacterium, is subordinate to Basidiomycotina, Tricholomataceae, is the external mycorrhizal fungis of the trees such as Song Yue, have the strong fragrance of uniqueness, be rare famous and precious natural medicinal fungus in the world, be described as " king in bacterium "。But being currently limited to wild mushroom, yield is very limited, it is difficult to meet people's demand to Tricholoma matsutake (lto et lmai) Singer, so everybody a kind of always seeking artificial propagation Tricholoma matsutake (lto et lmai) Singer method。
The method not only complicated operation of existing artificial propagation Tricholoma matsutake (lto et lmai) Singer, production cycle are long, and infection rate is high。
Summary of the invention
It is an object of the invention to provide the liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, the liquid spawn mating system of described Tricholoma matsutake (lto et lmai) Singer has the advantages such as with short production cycle, method is simple, infection rate is low。
Further object is that the land for growing field crops bionic cultivation method providing a kind of Tricholoma matsutake (lto et lmai) Singer, the method has that cost is low, simple to operate, be easy to the advantages such as popularization。
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
The liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprises the following steps:
Female kind making step: under aseptic environment, take the tissue of the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively to be placed individually into the first culture medium and cultivate, then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 24-26 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging；
Wherein, by weight, the first culture medium is made up of following components: 180-220 part Rhizoma Solani tuber osi, 180-220 part wood flour, 1600-2400 part water, 18-22 part glucose, 1.8-2.2 part magnesium sulfate, 2.8-3.2 part potassium dihydrogen phosphate, 4.8-5.2 part peptone, 0.25-1 part vitamin B1 and 18-22 part agar；
Liquid spawn making step: accessing mother under gnotobasis in the second culture fluid and plant, carry out shaking table cultivation, the temperature that shaking table is cultivated is 24-26 DEG C, and the speed of shaking table is 350-500rpm, and shaking table obtains first liquid strain after cultivating 3-6 days；
Wherein, by weight, the second culture medium is made up of following components: 180-220 part Rhizoma Solani tuber osi, 180-220 part wood flour, 1600-2400 part water, 18-22 part glucose, 1.8-2.2 part magnesium sulfate, 2.8-3.2 part potassium dihydrogen phosphate, 4.8-5.2 part peptone and 0.25-1 part vitamin B1。
Present invention also offers the land for growing field crops bionic cultivation method of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
Bacterium bag making step: wood flour, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In mixture, the percetage by weight of each component is respectively as follows: wood flour 66-74%, Testa Tritici 18-22%, Gypsum Fibrosum 0.8-1.2%, Calx 0.8-1.2%, sugar 0.8-1.2%, Semen Maydis powder 4-6%, bean cake 1.8-2.2%；Then mixture packs and carry out heat sterilization process, then first liquid strain is inoculated into mixture in an aseptic environment, then cultivate under 25-30 DEG C of condition and within 28-35 days, obtain bacterium bag；
De-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, is then placed in heatable adobe sleeping platform after de-for bacterium bag bag, then bankets in heatable adobe sleeping platform and cover bacterium bag, then water to cultivation area。
Compared with prior art, the invention have the benefit that
(1) the liquid spawn mating system of this Tricholoma matsutake (lto et lmai) Singer, with short production cycle, the time that solid spawn plants cultigen three tier structure kind from mother is 3 months, and prepares liquid spawn less than 20 days, the time cultivating solid original seed or cultigen needs 90 days, and cultivates a set of liquid strain and only need 3-7 days。Make liquid spawn and substantially reduce the production of hybrid seeds time, it is possible to supply produces in time needs。
(2) liquid spawn makes simple, and the making of solid spawn to be passed through and to plant original seed again to cultigen three tier structure kind process from mother, and to change culture medium, and formality is numerous and diverse。The making of liquid spawn, as long as same culture medium, cultivates strain out, can not only be used for female kind and uses, it is possible to as the use of original seed, cultigen, decreases the replacing of culture medium and the change of culture environment。
(3) inoculation simplicity, liquid spawn can use the inoculation of the various ways such as injection, spice, easy, quickly, uniformly, be suitable to mechanization, automatic mass production use。
(4) grow fast, the mycelium pellet Dou Shiyige organic centre of each liquid spawn, owing to mycelium pellet quantity is many, be evenly distributed, after inoculation, mycelia can grow rapidly, completes vegetative growth phase in the short time, it is to avoid the generation of pollution and danger。The growing state mycelia winding power to cultigen of fungus ball can be found in advance。
(5) the liquid spawn mating system of this Tricholoma matsutake (lto et lmai) Singer is lower by about 50% than solid spawn infection rate。
(6) land for growing field crops bionic cultivation method reduces cost of investment, and processing ease is convenient, is more suitable for popularization, drives common people to build up the family fortunes。
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below。
Fig. 1 is the distribution schematic diagram of cultivation area in booth。
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, it will be appreciated by those skilled in the art that the following example is merely to illustrate the present invention, and are not construed as restriction the scope of the present invention。Unreceipted actual conditions person in embodiment, conventionally the condition of condition or manufacturer's suggestion carries out。Agents useful for same or the unreceipted production firm person of instrument, be and can pass through the commercially available conventional products bought and obtain。
The liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprises the following steps:
Female kind making step: 180-220 part Rhizoma Solani tuber osi and 180-220 part wood flour are used 800-1200 part decocting in water 20-40min respectively, then filters and two kinds of filtrates are mixed to get the first mixing water。Filter in order to convenient, first can obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 180-220 part Rhizoma Solani tuber osi 800-1200 part decocting in water 20-40min, then 180-220 part wood flour cloth bag is installed, again the wood flour installed with cloth bag is put into and take out after 800-1200 part water boils 20-40min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 18-22 part glucose, 1.8-2.2 part magnesium sulfate, 2.8-3.2 part potassium dihydrogen phosphate, 4.8-5.2 part peptone, 0.25-1 part vitamin B1 and 18-22 part agar are added in the first mixing water and boils 15-25min and obtain the first culture fluid。Wherein vitamin B1 and sheet yeast tablet are all in the form of sheets, and 0.5 refers to half, and 1.5 refer to 1 and add half, the like。Additionally, in order to make each nutritional labeling in the first culture fluid mix evenly, it is desirable to use after the first culture fluid is stirred。
Again the first culture fluid is divided in sterilization container and is heated sterilization treatment, then sterilization container stands and make the first culture fluid within cooled and solidified 3-5 days, obtain the first culture medium；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 24-26 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
As preferably, mother plants in making step, and Tricholoma matsutake (lto et lmai) Singer obtains after being processed by fresh Tricholoma matsutake (lto et lmai) Singer, and processing procedure is: pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, then fresh Tricholoma matsutake (lto et lmai) Singer is put into hermetic container or places 3-5 days under 18-22 DEG C of condition with after adhesive plaster parcel。
As preferably, the condition that female heat sterilization planted in making step processes is: the sterilizing 40-50min when pressure is 0.1-0.2Pa and temperature is 110-130 DEG C。
As preferably, sterilization container is test tube, is tilted by test tube and keep the angle of 15 ° to cool down with horizontal plane after heat sterilization process。Test tube capacity is little, and it is few that the first of every cuvette cartridge cultivates liquid measure, it is simple to is thoroughly killed by the pathogenic bacteria in the first culture fluid。Make test tube keep heeling condition to be the conventional means of this area, it is possible to increase the liquid level area of the first culture fluid, be conducive to obtaining more female plant in single test tube。Further, it is also possible to observe the growing state that mother plants, the female kind selecting wherein growing way good is purificated and rejuvenated。
Liquid spawn making step: 180-220 part Rhizoma Solani tuber osi and 180-220 part wood flour are used 800-1200 part decocting in water 20-40min respectively, then filters and two kinds of filtrates are mixed to get the second mixing water；Similar with the manufacturing process of the first mixing water, filter in order to convenient, first can obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 180-220 part Rhizoma Solani tuber osi 800-1200 part decocting in water 20-40min, then 180-220 part wood flour cloth bag is installed, again the wood flour installed with cloth bag is put into and take out after 800-1200 part water boils 20-40min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water。
Again by 18-22 part glucose, 1.8-2.2 part magnesium sulfate, 2.8-3.2 part potassium dihydrogen phosphate, 4.8-5.2 part peptone, 0.25-1 part vitamin B1 adds in the second mixing water and boils 15-25min and obtains the second culture fluid；Second culture fluid being dispensed into shaking flask and carries out heat sterilization process, treating that the second culture fluid is cooled to 24-26 DEG C, then accessing mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, at 24-26 DEG C, wave and culture obtains first liquid strain after 3-6 days。
The making of existing solid spawn to be passed through and to plant original seed again to cultigen three tier structure kind process from mother, and to change culture medium, and formality is numerous and diverse。And the making of the liquid spawn of the present invention, as long as same culture medium, cultivate strain out, namely female kind of gram conduct uses, it is possible to as original seed, the use of cultivation, decrease the replacing of culture medium and the change of culture environment。
Liquid spawn grows fast, the mycelium pellet Dou Shiyige organic centre of each liquid spawn, owing to mycelium pellet quantity is many, be evenly distributed, inoculate after mycelia can grow rapidly, complete vegetative growth phase in the short time, it is to avoid the generation of pollution and harm。The growing state mycelia winding power to cultigen of fungus ball can be found in advance。Additionally, the various culture fluid of the present invention always have employed the carbon-nitrogen ratio of the best, adopting the liquid spawn mating system of the Tricholoma matsutake (lto et lmai) Singer of the present invention, the mycelia length of playing a ball game is consistent, it is vigorous to grow, growing way good, vitality good。
The block vertical spread of planting that the growth of solids manufacture kind is from inoculation starts, and the mycelia in whole culture bottle (bag) differs 20～30 days from top to bottom cell age, and its vitality difference is bigger。Each mycelia bead of liquid spawn is substantially initially form the same time, and cell age is consistent, and the incubation time of whole strain is very short, if using liquid spawn in mycelial growth exponential phase (incubation time was at 5～7 days), i.e. best seed stage, mycelia can quickly field planting, vigorous growth。
As preferably, the condition that the heat sterilization in liquid spawn making step processes is: the sterilizing 40-50min when pressure is 0.1-0.2Pa and temperature is 110-130 DEG C。
If producing on a small quantity, it is possible to directly make bacterium bag with first liquid strain and carry out land for growing field crops bionic cultivation。
A kind of land for growing field crops bionic cultivation method of Tricholoma matsutake (lto et lmai) Singer, including the step of liquid spawn mating system and the following steps of Tricholoma matsutake (lto et lmai) Singer:
Bacterium bag making step: wood flour, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In mixture, the percetage by weight of each component is respectively as follows: wood flour 66-74%, cotton seed hulls 38-42%, Testa Tritici 18-22%, Gypsum Fibrosum 0.8-1.2%, Calx 0.8-1.2%, sugar 0.8-1.2%, Semen Maydis powder 4-6%, bean cake 1.8-2.2%；Then mixture packs and carry out heat sterilization process, then first liquid strain is inoculated into mixture in an aseptic environment, then cultivate under 25-30 DEG C of condition and within 28-35 days, obtain bacterium bag；
De-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, is then placed in heatable adobe sleeping platform after de-for bacterium bag bag, then bankets in heatable adobe sleeping platform and cover bacterium bag, then water to cultivation area。
As preferably, the condition that the heat sterilization in bacterium bag making step processes is: the sterilizing 4-6h when pressure is 0.1-0.2Pa and temperature is 110-130 DEG C。
As preferably, the degree of depth of heatable adobe sleeping platform is 12-18cm's, and the humidity of the soil of the cultivation area after watering to cultivation area is 85-96%, interval 3-5cm between adjacent bacterium bag, banketing in heatable adobe sleeping platform and cover the 1/4-3/4 of bacterium bag, the soil thickness at the top of bacterium bag is 3-5cm。
As preferably, cultivation area is multiple and is distributed in booth, and the temperature in booth is 25-30 DEG C, and the air humidity in booth is 70-90%。
As preferably, booth projection in the horizontal plane is rectangle, the main aisle of the length direction along booth it is provided with in booth, cultivation area is distributed in the both sides of main aisle, the width of main aisle is 1-1.5m, leaves the auxiliary passageway that width is 0.15-0.25m between the adjacent cultivation area of the same side of main aisle。
As preferably, booth is domed, the apogee distance ground 2-2.4m of booth, plastic house heat-insulating film, the plastic house sunshade net that light transmittance is 25%-35% or puggaree。
As it is preferred that, the light transmittance of sunshade net or puggaree is 30%。
Tricholoma matsutake (lto et lmai) Singer likes growth under dark environment, so requiring over sunshade net or the irradiation dose of puggaree minimizing sunlight。Saying popular in industry be 7 second 3 points of sun best。
If industrialization produces, in addition it is also necessary to be enlarged first liquid strain cultivating, to increase the amount of liquid spawn。
As preferably, the liquid spawn mating system of Tricholoma matsutake (lto et lmai) Singer also includes amplification culture step；
Amplification culture step is: first liquid strain is accessed fermentation tank in an aseptic environment, passes into filtrated air and the 3rd culture fluid in fermentation tank carries out stirring of blowing, obtain second liquid strain after cultivating 3-6 days in fermentation tank；
According to the ratio with the volume of fermentation tank, described 3rd culture fluid is made up of following components: wood flour 40-60g/100L, vitaminB10 .06-0.2g/100L, yeast tablet 0.25-0.35g/100L, starch 1.2-1.8kg/100L, magnesium sulfate 0.8-1.2kg/100L, potassium dihydrogen phosphate 0.1-0.3kg/100L, glucose 0.4-0.6kg/100L, white sugar 0.4-0.6kg/100L, peptone 0.2-0.4kg/100L and edible oil 0.01-0.03kg/100L, and surplus is water。
Starch, magnesium sulfate, potassium dihydrogen phosphate, glucose, white sugar, peptone are all relevant to the volume of the 3rd culture fluid that preparation obtains with the consumption of edible oil, the volume of usual fermentation tank is 100L, 200L, 400L and 500L, and correspondingly the volume of the 3rd culture fluid is generally 100L, 200L, 400L and 500L。Starch, magnesium sulfate, potassium dihydrogen phosphate, glucose, white sugar, peptone and edible oil all can directly use。Under wood flour is more special relative to other components, wood flour needs to add decocting in water 25-35min, is then passed through filtering removal wood flour and obtains wood flour water, then adds in fermentation tank and the mixing of other components by wood flour water。Starch preferably first adds suitable quantity of water moistening and becomes pasty state starch to use。Edible oil can select the edible oil that Oleum Brassicae campestris, Semen sojae atricolor wet goods are common。
During industrialization production, being make bacterium bag with second liquid strain, concrete manufacturing process is just the same with first liquid strain making bacterium bag。
The land for growing field crops bionic cultivation method of a kind of Tricholoma matsutake (lto et lmai) Singer, comprises the following steps:
Bacterium bag making step: wood flour, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In mixture, the percetage by weight of each component is respectively as follows: wood flour 66-74%, cotton seed hulls 38-42%, Testa Tritici 18-22%, Gypsum Fibrosum 0.8-1.2%, Calx 0.8-1.2%, sugar 0.8-1.2%, Semen Maydis powder 4-6%, bean cake 1.8-2.2%；Then mixture packs and carry out heat sterilization process, then second liquid strain is inoculated into mixture in an aseptic environment, then cultivate under 25-30 DEG C of condition and within 28-35 days, obtain bacterium bag；
De-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, is then placed in heatable adobe sleeping platform after de-for bacterium bag bag, then bankets in heatable adobe sleeping platform and cover bacterium bag, then water to cultivation area。
Embodiment 1
Present embodiments provide the liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water, wood flour water and corn cob water being mixed, obtain the first mixing water。
Then 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 and 20g agar are added in the first mixing water and boils 20min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 4 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 4 days under 20 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 25 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 adds in the second mixing water and boils 20min and obtains the second culture fluid；It is dispensed into shaking flask, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C after being stirred by second culture fluid；Treating that the second culture fluid is cooled to 25 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 350, and at 25 DEG C, wave and culture obtains first liquid strain after 5 days。
Embodiment 2
Present embodiments provide the liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 and 20g agar are added in the first mixing water and boils 20min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 4 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 4 days under 20 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 24-26 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 adds in the second mixing water and boils 20min and obtains the second culture fluid；It is dispensed into shaking flask, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C after being stirred by second culture fluid；Treating that the second culture fluid is cooled to 25 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 350rpm, and at 25 DEG C, wave and culture obtains first liquid strain after 5 days。
(3) amplification culture step is: is cleaned by the fermentation tank that capacity is 400L and sterilizes that (process of concrete cleaning and sterilizing is, after fermentation tank cleans up, each switch valve is carried out check errorless after, fermentation tank is carried out sky and disappears, add water at fermentation tank outer layer, then heat, when internal pressure reaches 0.1Pa, design temperature, the time sets 35 minutes, open outer layer simultaneously and connect three switches of inner layer, with steam, internal layer and pipeline are sterilized 35 minutes)；Then the wood flour water of Masson Pine will be filtrated to get after the wood flour 1000mL decocting in water 30min of 200g Masson Pine, wood flour water, 1 vitamin B1,4 tablets of yeast tablets, 6kg pasty state starch, 4kg magnesium sulfate, 0.8kg potassium dihydrogen phosphate, 2kg glucose, 2kg white sugar, 0.8kg peptone and 0.08kg edible oil are added fermentation tank, in fermentation tank, add sterilized water again to the heap(ed) capacity scale place of fermentation tank, after stirring, obtain 400L the 3rd culture fluid；3rd culture fluid is heated sterilizing, after the 3rd culture fluid is cooled to 25 DEG C, first liquid strain is accessed fermentation tank in an aseptic environment, in fermentation tank, passes into filtrated air and the 3rd culture fluid is carried out stirring of blowing, after cultivating 5 days, obtaining second liquid strain。Wherein the weight of every vitamin B1 is 0.5g, and the weight of every tablet of yeast tablet is 0.3g。
Embodiment 3
Present embodiments provide the land for growing field crops bionic cultivation method of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 and 20g agar are added in the first mixing water and boils 20min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 4 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 4 days under 20 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 25 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 adds in the second mixing water and boils 20min and obtains the second culture fluid；It is dispensed into shaking flask, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C after being stirred by second culture fluid；Treating that the second culture fluid is cooled to 25 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 350rpm, and at 25 DEG C, wave and culture obtains first liquid strain after 5 days。
(3) bacterium bag making step: weed tree sawdust, the wood flour of Masson Pine, cotton seed hulls, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In mixture, the percetage by weight of each component is respectively as follows: the wood flour 30% of Masson Pine, weed tree sawdust 40%, Testa Tritici 20%, Gypsum Fibrosum 1%, Calx 1%, sugar 1%, Semen Maydis powder 5%, bean cake 2%；Then by mixture pack, then when temperature 120 degree, pressure 0.15PA sterilizing 5h；With inoculating gun, first liquid strain is inoculated into mixture in an aseptic environment, then cultivates under 28 DEG C of conditions and obtain bacterium bag in 30 days。
(4) de-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, the degree of depth of heatable adobe sleeping platform is 15cm's, then it is placed in heatable adobe sleeping platform after de-for bacterium bag bag, interval 4cm between adjacent bacterium bag, banket in heatable adobe sleeping platform again and cover the half of bacterium bag, second half of bacterium bag is in naked state, and the soil thickness at the top of bacterium bag is 4cm, and the humidity of soil then watered to cultivation area to cultivation area is 85-96%。
As shown in Figure 1, cultivation area 1 is multiple and is distributed in booth, booth projection in the horizontal plane is rectangle, the main aisle 2 of the length direction along booth it is provided with in booth, cultivation area 1 is distributed in the both sides of main aisle 2, the width of main aisle 2 is 1.2m, leaves the auxiliary passageway 3 that width is 0.2m between the adjacent cultivation area 1 of the same side of main aisle 2。Booth is domed, the apogee distance ground 2.2m of booth, plastic house heat-insulating film, the plastic house sunshade net that light transmittance is 30% or puggaree。Temperature in booth is 25-30 DEG C, and the air humidity in booth is 70-90%。
Embodiment 4
Present embodiments provide the land for growing field crops bionic cultivation method of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 and 20g agar are added in the first mixing water and boils 20min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 4 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 4 days under 20 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 25 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 adds in the second mixing water and boils 20min and obtains the second culture fluid；It is dispensed into shaking flask, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C after being stirred by second culture fluid；Treating that the second culture fluid is cooled to 25 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 350rpm, and at 25 DEG C, wave and culture obtains first liquid strain after 5 days。
(3) amplification culture step is: is cleaned by the fermentation tank that capacity is 400L and sterilizes that (process of concrete cleaning and sterilizing is, after fermentation tank cleans up, each switch valve is carried out check errorless after, fermentation tank is carried out sky and disappears, add water at fermentation tank outer layer, then heat, when internal pressure reaches 0.1Pa, design temperature, the time sets 35 minutes, open outer layer simultaneously and connect three switches of inner layer, with steam, internal layer and pipeline are sterilized 35 minutes)；Then after the wood flour 1000mL decocting in water 30min of 200g Masson Pine, it is filtrated to get wood flour water, wood flour water, 1 vitamin B1,4 tablets of yeast tablets, 6kg pasty state starch, 4kg magnesium sulfate, 0.8kg potassium dihydrogen phosphate, 2kg glucose, 2kg white sugar, 1.2kg peptone and 0.08kg edible oil are added fermentation tank, in fermentation tank, add sterilized water again to the heap(ed) capacity scale place of fermentation tank, after stirring, obtain 400L the 3rd culture fluid；3rd culture fluid is heated sterilizing, after the 3rd culture fluid is cooled to 25 DEG C, first liquid strain is accessed fermentation tank in an aseptic environment, in fermentation tank, passes into filtrated air and the 3rd culture fluid is carried out stirring of blowing, after cultivating 5 days, obtaining second liquid strain。Wherein the weight of every vitamin B1 is 0.5g, and the weight of every tablet of yeast tablet is 0.3g。
(4) bacterium bag making step: weed tree sawdust, the wood flour of Masson Pine, cotton seed hulls, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In mixture, the percetage by weight of each component is respectively as follows: the wood flour 30% of Masson Pine, weed tree sawdust 40%, Testa Tritici 20%, Gypsum Fibrosum 1%, Calx 1%, sugar 1%, Semen Maydis powder 5%, bean cake 2%；Then by mixture pack, then when temperature 120 degree, pressure 0.15PA sterilizing 5h；With inoculating gun, second liquid strain is inoculated into mixture in an aseptic environment, then cultivates under 28 DEG C of conditions and obtain bacterium bag in 30 days。
(5) de-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, the degree of depth of heatable adobe sleeping platform is 15cm's, then it is placed in heatable adobe sleeping platform after de-for bacterium bag bag, interval 4cm between adjacent bacterium bag, banket in heatable adobe sleeping platform again and cover the 1/4 of bacterium bag, other the 3/4 of bacterium bag is in naked state, and the soil thickness at the top of bacterium bag is 4cm, and the humidity of soil then watered to cultivation area to cultivation area is 85-96%。
As shown in Figure 1, cultivation area 1 is multiple and is distributed in booth, booth projection in the horizontal plane is rectangle, the main aisle 2 of the length direction along booth it is provided with in booth, cultivation area 1 is distributed in the both sides of main aisle 2, the width of main aisle 2 is 1.2m, leaves the auxiliary passageway 3 that width is 0.2m between the adjacent cultivation area 1 of the same side of main aisle 2。Booth is domed, the apogee distance ground 2.2m of booth, plastic house heat-insulating film, the plastic house sunshade net that light transmittance is 30% or puggaree。Temperature in booth is 25-30 DEG C, and the air humidity in booth is 70-90%。
Embodiment 5
Present embodiments provide the liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 180g Rhizoma Solani tuber osi 800mL decocting in water 20min, then the wood flour cloth bag of 180g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 800 water boil 20-40min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 18g glucose, 1.8g magnesium sulfate, 2.8g potassium dihydrogen phosphate, 4.8g peptone, 0.5 vitamin B1 and 18g agar are added in the first mixing water and boils 15min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 50min when pressure is 0.15Pa and temperature is 110 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 3 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 5 days under 18 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 24 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 180g Rhizoma Solani tuber osi 800mL decocting in water 20-40min, then the wood flour cloth bag of 180g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 800mL water boils 20min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 18g glucose, 1.8g magnesium sulfate, 2.8g potassium dihydrogen phosphate, 4.8g peptone, 0.5 vitamin B1 adds in the second mixing water and boils 15min and obtains the second culture fluid；Second culture fluid is dispensed into shaking flask, the sterilizing 50min when pressure is 0.15Pa and temperature is 110 DEG C；Treating that the second culture fluid is cooled to 24 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 400rpm, and at 24 DEG C, wave and culture obtains first liquid strain after 6 days。
Embodiment 6
Present embodiments provide the liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 220g Rhizoma Solani tuber osi 1200mL decocting in water 40min, then the wood flour cloth bag of 220g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1200mL water boils 40min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 22g glucose, 2.2g magnesium sulfate, 3.2g potassium dihydrogen phosphate, 5.2g peptone, 1.5 vitamin B1s and 22g agar are added in the first mixing water and boils 25min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 40min when pressure is 2Pa and temperature is 130 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 5 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 3 days under 22 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 26 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 220g Rhizoma Solani tuber osi 1200mL decocting in water 40min, then the wood flour cloth bag of 220g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1200mL water boils 40min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 22g glucose, 2.2g magnesium sulfate, 3.2g potassium dihydrogen phosphate, 5.2g peptone, 1.5 vitamin B1s add in the second mixing water and boil 25min and obtain the second culture fluid；Second culture fluid is dispensed into shaking flask, the sterilizing 40min when pressure is 2Pa and temperature is 130 DEG C；Treating that the second culture fluid is cooled to 26 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 450rpm, and at 26 DEG C, wave and culture obtains first liquid strain after 3 days。
Embodiment 7
Present embodiments provide the liquid spawn mating system of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 and 20g agar are added in the first mixing water and boils 20min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 4 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 4 days under 20 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 25 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 adds in the second mixing water and boils 20min and obtains the second culture fluid；It is dispensed into shaking flask, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C after being stirred by second culture fluid；Treating that the second culture fluid is cooled to 25 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 500rpm, and at 25 DEG C, wave and culture obtains first liquid strain after 5 days。
(3) amplification culture step is: is cleaned by the fermentation tank that capacity is 400L and sterilizes that (process of concrete cleaning and sterilizing is, after fermentation tank cleans up, each switch valve is carried out check errorless after, fermentation tank is carried out sky disappear, add water at fermentation tank outer layer, then heat, when internal pressure reaches 0.1Pa, design temperature, time sets 35 minutes, open outer layer simultaneously and connect three switches of inner layer, with steam, internal layer and pipeline are sterilized 35 minutes), the wood flour water of Masson Pine will be filtrated to get after the wood flour 1200mL decocting in water 32min of 220g Masson Pine, by wood flour water, 1.5 vitamin B1s, 4.5 tablets of yeast tablets, 7.2kg pasty state starch, 4.8kg magnesium sulfate, 1.2kg potassium dihydrogen phosphate, 2.4kg glucose, 2.4kg white sugar, 1.6kg peptone and 0.12kg edible oil add fermentation tank, the sterilized water heap(ed) capacity scale place to fermentation tank is added again in fermentation tank, 400L the 3rd culture fluid is obtained after stirring；3rd culture fluid is heated sterilizing, after the 3rd culture fluid is cooled to 26 DEG C, first liquid strain is accessed fermentation tank in an aseptic environment, in fermentation tank, passes into filtrated air and the 3rd culture fluid is carried out stirring of blowing, after cultivating 5 days, obtaining second liquid strain。Wherein the weight of every vitamin B1 is 0.5g, and the weight of every tablet of yeast tablet is 0.3g。
Embodiment 8
Present embodiments provide the land for growing field crops bionic cultivation method of a kind of Tricholoma matsutake (lto et lmai) Singer, comprise the following steps:
(1) female kind making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the first mixing water。
Then 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 and 20g agar are added in the first mixing water and boils 20min and obtain the first culture fluid, standby after the first culture fluid is stirred。Wherein the weight of every vitamin B1 is 0.5g。
Again by the first culture fluid subpackage in vitro, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C；Then test tube is tried slant setting and keeps the angle of 15 ° to cool down under shady and cool environment with horizontal plane；The first culture medium is obtained after first culture fluid cooled and solidified 4 days；Pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, place 4 days under 20 DEG C of conditions after then fresh Tricholoma matsutake (lto et lmai) Singer adhesive plaster being wrapped up；Under aseptic environment, the tissue taking the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively is placed individually into the first culture medium, and then by the stem after cultivation, cap and bacterium foot Mixed culture under the environment of 25 DEG C, the Hyphal form of stem, cap and bacterium foot obtains female kind after merging。
(2) liquid spawn making step: first obtain Rhizoma Solani tuber osi water by filtering removal Rhizoma Solani tuber osi after 200g Rhizoma Solani tuber osi 1000mL decocting in water 30min, then the wood flour cloth bag of 200g Masson Pine is installed, again the wood flour of the Masson Pine installed with cloth bag is put into and take out after 1000mL water boils 30min and obtain wood flour water, after then Rhizoma Solani tuber osi water and wood flour water being mixed, obtain the second mixing water；
Again by 20g glucose, 2g magnesium sulfate, 3g potassium dihydrogen phosphate, 5g peptone, 1 vitamin B1 adds in the second mixing water and boils 20min and obtains the second culture fluid；It is dispensed into shaking flask, the sterilizing 45min when pressure is 0.15Pa and temperature is 120 DEG C after being stirred by second culture fluid；Treating that the second culture fluid is cooled to 25 DEG C, then access mother in an aseptic environment and plant, then shaking flask is positioned over shaking table, the speed of shaking table is 500rpm, and at 25 DEG C, wave and culture obtains first liquid strain after 5 days。
(3) amplification culture step is: is cleaned by the fermentation tank that capacity is 400L and sterilizes that (process of concrete cleaning and sterilizing is, after fermentation tank cleans up, each switch valve is carried out check errorless after, fermentation tank is carried out sky disappear, add water at fermentation tank outer layer, then heat, when internal pressure reaches 0.1Pa, design temperature, time sets 35 minutes, open outer layer simultaneously and connect three switches of inner layer, with steam, internal layer and pipeline are sterilized 35 minutes), it is filtrated to get wood flour water after the wood flour 1200mL decocting in water 32min of 220g Masson Pine, by wood flour water, 1.5 vitamin B1s, 4.5 tablets of yeast tablets, 7.2kg pasty state starch, 4.8kg magnesium sulfate, 1.2kg potassium dihydrogen phosphate, 2.4kg glucose, 2.4kg white sugar, 1.6kg peptone and 0.12kg edible oil add fermentation tank, the sterilized water heap(ed) capacity scale place to fermentation tank is added again in fermentation tank, 400L the 3rd culture fluid is obtained after stirring；3rd culture fluid is heated sterilizing, after the 3rd culture fluid is cooled to 26 DEG C, first liquid strain is accessed fermentation tank in an aseptic environment, in fermentation tank, passes into filtrated air and the 3rd culture fluid is carried out stirring of blowing, after cultivating 5 days, obtaining second liquid strain。Wherein the weight of every vitamin B1 is 0.5g, and the weight of every tablet of yeast tablet is 0.3g。
(4) bacterium bag making step: weed tree sawdust, the wood flour of Masson Pine, cotton seed hulls, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In mixture, the percetage by weight of each component is respectively as follows: the wood flour 28% of Masson Pine, weed tree sawdust 38%, Testa Tritici 18%, Gypsum Fibrosum 0.8%, Calx 0.8%, sugar 0.8%, Semen Maydis powder 4%, bean cake 1.8%；Then mixture is packed, then temperature be 120 DEG C, pressure 0.15PA when sterilizing 5h；With inoculating gun, second liquid strain is inoculated into mixture in an aseptic environment, then cultivates under 28 DEG C of conditions and obtain bacterium bag in 30 days。
(5) de-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, the degree of depth of heatable adobe sleeping platform is 18cm's, then it is placed in heatable adobe sleeping platform after de-for bacterium bag bag, interval 5cm between adjacent bacterium bag, banket in heatable adobe sleeping platform again and cover the half of bacterium bag, second half of bacterium bag is in naked state, and the soil thickness at the top of bacterium bag is 5cm, and the humidity of soil then watered to cultivation area to cultivation area is 85-96%。
As shown in Figure 1, cultivation area 1 is multiple and is distributed in booth, booth projection in the horizontal plane is rectangle, the main aisle 2 of the length direction along booth it is provided with in booth, cultivation area 1 is distributed in the both sides of main aisle 2, the width of main aisle 2 is 1.5m, leaves the auxiliary passageway 3 that width is 0.25m between the adjacent cultivation area 1 of the same side of main aisle 2。Booth is domed, the apogee distance ground 2.4m of booth, plastic house heat-insulating film, the plastic house sunshade net that light transmittance is 25% or puggaree。Temperature in booth is 25-30 DEG C, and the air humidity in booth is 70-90%。
Although illustrate and describing the present invention with specific embodiment, however it will be appreciated that may be made that when without departing substantially from the spirit and scope of the present invention many other change and amendment。It is, therefore, intended that include all such changes and modifications belonging in the scope of the invention in the following claims。
Claims (10)
1. the liquid spawn mating system of a Tricholoma matsutake (lto et lmai) Singer, it is characterised in that comprise the following steps:
Female kind making step: under aseptic environment, take the tissue of the stem of Tricholoma matsutake (lto et lmai) Singer, cap and bacterium foot respectively to be placed individually into the first culture medium and cultivate, then by the stem after cultivation, described cap and described bacterium foot Mixed culture under the environment of 24-26 DEG C, the Hyphal form of described stem, described cap and described bacterium foot obtains female kind after merging；
Wherein, by weight, described first culture medium is made up of following components: 180-220 part Rhizoma Solani tuber osi, 180-220 part wood flour, 1600-2400 part water, 18-22 part glucose, 1.8-2.2 part magnesium sulfate, 2.8-3.2 part potassium dihydrogen phosphate, 4.8-5.2 part peptone, 0.25-1 part vitamin B1 and 18-22 part agar；
Liquid spawn making step: accessing described female kind under gnotobasis in the second culture fluid, carry out shaking table cultivation, the temperature that shaking table is cultivated is 24-26 DEG C, and shaking table obtains first liquid strain after cultivating 3-6 days；
Wherein, by weight, described second culture medium is made up of following components: 180-220 part Rhizoma Solani tuber osi, 180-220 part wood flour, 1600-2400 part water, 18-22 part glucose, 1.8-2.2 part magnesium sulfate, 2.8-3.2 part potassium dihydrogen phosphate, 4.8-5.2 part peptone and 0.25-1 part vitamin B1。
2. the liquid spawn mating system of Tricholoma matsutake (lto et lmai) Singer according to claim 1, it is characterised in that the liquid spawn mating system of Tricholoma matsutake (lto et lmai) Singer also includes amplification culture step；
Described amplification culture step is: described first liquid strain is accessed fermentation tank in an aseptic environment, passes into filtrated air and the 3rd culture fluid in described fermentation tank carries out stirring of blowing, obtain second liquid strain after cultivating 3-6 days in described fermentation tank；
According to the ratio with the volume of described fermentation tank, described 3rd culture fluid is made up of following components: wood flour 40-60g/100L, vitaminB10 .06-0.2g/100L, yeast tablet 0.25-0.35g/100L, starch 1.2-1.8kg/100L, magnesium sulfate 0.8-1.2kg/100L, potassium dihydrogen phosphate 0.1-0.3kg/100L, glucose 0.4-0.6kg/100L, white sugar 0.4-0.6kg/100L, peptone 0.2-0.4kg/100L and edible oil 0.01-0.03kg/100L, and surplus is water。
3. the liquid spawn mating system of Tricholoma matsutake (lto et lmai) Singer according to claim 1, it is characterized in that, in described female kind making step, described Tricholoma matsutake (lto et lmai) Singer obtains after being processed by fresh Tricholoma matsutake (lto et lmai) Singer, processing procedure is: pluck the fresh Tricholoma matsutake (lto et lmai) Singer that growth is intact, strong, places 3-5 days after then fresh Tricholoma matsutake (lto et lmai) Singer being put into hermetic container or wrapping up with adhesive plaster under 18-22 DEG C of condition。
4. the liquid spawn mating system of Tricholoma matsutake (lto et lmai) Singer according to claim 1, it is characterized in that, the condition that the heat sterilization in described liquid spawn making step and in female kind making step processes is: the sterilizing 40-50min when pressure is 0.1-0.2Pa and temperature is 110-130 DEG C。
5. the liquid spawn mating system of Tricholoma matsutake (lto et lmai) Singer according to claim 1, it is characterised in that described first culture medium is slant medium。
6. the land for growing field crops bionic cultivation method of a Tricholoma matsutake (lto et lmai) Singer, it is characterised in that include the step as described in any one of claim 1-5 and following steps:
Bacterium bag making step: wood flour, Testa Tritici, Gypsum Fibrosum, Calx, sugar, Semen Maydis powder and bean cake are mixed to get mixture；In described mixture, the percetage by weight of each component is respectively as follows: described wood flour 66-74%, described Testa Tritici 18-22%, described Gypsum Fibrosum 0.8-1.2%, described Calx 0.8-1.2%, described sugar 0.8-1.2%, described Semen Maydis powder 4-6%, described bean cake 1.8-2.2%；Then described mixture packed and carry out heat sterilization process, again first liquid strain as claimed in claim 1 or second liquid strain as claimed in claim 2 are inoculated into described mixture in an aseptic environment, then cultivate under 25-30 DEG C of condition and within 28-35 days, obtain bacterium bag；
De-bag cultivating step: first excavate heatable adobe sleeping platform in cultivation area, is then placed in heatable adobe sleeping platform after de-for described bacterium bag bag, then bankets in heatable adobe sleeping platform and cover described bacterium bag, then water to cultivation area。
7. the land for growing field crops bionic cultivation method of Tricholoma matsutake (lto et lmai) Singer according to claim 6, it is characterized in that, the degree of depth of described heatable adobe sleeping platform is 12-18cm's, the humidity of the soil of the cultivation area after watering to described cultivation area is 85-96%, interval 3-5cm between adjacent bacterium bag, banketing in heatable adobe sleeping platform and cover the 1/4-3/4 of described bacterium bag, the soil thickness at the top of described bacterium bag is 3-5cm。
8. the land for growing field crops bionic cultivation method of Tricholoma matsutake (lto et lmai) Singer according to claim 6, it is characterised in that described cultivation area is multiple and is distributed in booth, and the temperature in booth is 25-30 DEG C, the air humidity in booth is 70-90%。
9. the land for growing field crops bionic cultivation method of Tricholoma matsutake (lto et lmai) Singer according to claim 8, it is characterized in that, the projection in the horizontal plane of described booth is rectangle, the main aisle of the length direction along booth it is provided with in described booth, described cultivation area is distributed in the both sides of described main aisle, the width of described main aisle is 1-1.5m, leaves the auxiliary passageway that width is 0.15-0.25m between the adjacent described cultivation area of the same side of described main aisle。
10. the land for growing field crops bionic cultivation method of Tricholoma matsutake (lto et lmai) Singer according to claim 9, it is characterized in that, described booth is domed, the apogee distance ground 2-2.4m of described booth, described plastic house heat-insulating film, described the plastic house sunshade net that light transmittance is 25%-35% or puggaree。
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| CN112961778A (en) * | 2021-03-25 | 2021-06-15 | 云南乌蒙山国家级自然保护区管护局 | Universal wild edible fungus separation culture medium and preparation method thereof |
| CN115104481A (en) * | 2021-12-31 | 2022-09-27 | 林礼 | Method for liquid culture of Huang cluster bacterium strain |
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