CN103250564A - Artificial cultivating method for chestnut mycorrhiza fungi - Google Patents
Artificial cultivating method for chestnut mycorrhiza fungi Download PDFInfo
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Abstract
The invention discloses an artificial cultivating method for chestnut mycorrhiza fungi and belongs to the field of mycorrhiza fungi artificial cultivating. The artificial cultivating method for the chestnut mycorrhiza fungi includes the following steps: (1) taking a chestnut mycorrhiza fungi fruiting body, cutting the chestnut mycorrhiza fungi fruiting body into two pieces, and collecting intersecting parts of fungus caps and fungus stipes to obtain fungus context issue blocks; (2) putting the fungus context issue blocks in an inclined plane culture medium, culturing the fungus context issue blocks under the temperature of 25-28 DEG C until mycelia are germinated by the fungus context issue blocks, then the mycelia are cultivated under the temperature of 25-28 DEG C until the mycelia grow to cover the inclined plane after the mycelia is through tube turning to obtain pure mycelia; (3) inoculating the pure mycelia to an inoculating hole of an enlarged cultivating material, and cultivating the pure mycelia under the temperature of 25-28 DEG C until the pure mycelia grow all over a cultivating container to obtain the chestnut mycorrhiza fungi. The artificial cultivating method for the chestnut mycorrhiza fungi is simple in operation, strains cultivated and obtained through the artificial cultivating method for the chestnut mycorrhiza fungi is inoculated to chestnut seedlings, a mycorrhiza impregnating rate can reach 50.3% to the maximum degree, and an average impregnating rate is 46.5%.
Description
Technical field
The invention belongs to the mycorhiza bacterium and manually cultivate the field, be specifically related to the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium.
Background technology
Mycorhiza is edaphon and the most general symbiosis of plant compatibility, and the formation of ectotrophic mycorrhiza makes trees rely on the help of mycorhiza bacterium to improve moisture in the soil and nutrient absorbing ability.The organic phosphorus catabolic enzyme of ectotrophic mycorrhiza secretion can decompose the organic phosphorus in dry branches and fallen leaves and the soil organic matter to absorb and directly be transferred to trees and utilize, thereby plays drought resisting, shortly gives birth to, the effect of diseases prevention.Behind the ectotrophic mycorrhiza fungal infection host plant root system, form on the one hand and breathe out Di Shi net and bacterium cover, mycelia stretches out and forms fine and close mycelia net on the other hand, even forms shoestring.Because most ectotrophic mycorrhiza fungies do not have strict selectivity to the host, touch in the extension mycelia stretching process other can with the root system of the host plant of its symbiosis after also can infect once again, form the mycelia bridge between root.The foundation of mycelia bridge makes and has formed the source base relation between the different plants, makes between plant and can transmit materials such as C, N, P and moisture, thereby the nutrition condition of recipient plant is improved.
Also there are a kind of symbiotic relation in Chinese chestnut root system and ectotrophic mycorrhiza.Under the Chinese chestnut gardens, can see the fruit body of the Chinese chestnut mycorhiza bacterium of difformity and color.Investigation is found, can have 13 to belong to 29 kinds with the fungi that Chinese chestnut forms ectotrophic mycorrhiza, the advantage fungi of distribution have Lycoperdon (Lycoperdon), must Hymenogaster (Rhizopogon), Calvatia (Calvatia), Russula (Russula), Boletus (Boletus), Amanita (Amanita), Strobilomyces (Strobilomyces) etc.Wherein, Lasiosphaera fenzlii class and palpus false truffle and Chinese chestnut symbiotic relation are best, are Russula and Boletus secondly.
The ectotrophic mycorrhiza fungi has important role to the normal growth of Chinese chestnut, can improve the Chinese chestnut root system to moisture in the soil and nutrient absorbing ability.If lack the mycorhiza bacterium in the soil, Chinese chestnut just can not grow by normal growth.Chinese chestnut is grown in the mountain area more, and in the barren Chinese chestnut garden of soil fertility, soil nutrient content is very low, fertilising difficulty, and therefore, the Chinese chestnut in mountain area is often revealed very big difference of nutrition status of the plant and growth potential because of adnation position, strain different table in age.In the conventional graft seedling growth of Chinese chestnut and since lack in seedbed or the medium can with the mycorhiza bacterium of Chinese chestnut root system symbiosis, so, be transplanted to the conventional grafting in land for growing field crops, survival rate is low, growth retardation, and the dead phenomenon of big tree also can occur after 2～3 years.
Yet the symbiotic relation of Chinese chestnut root system and ectotrophic mycorrhiza is very complicated, and relatively difficulty is cultivated in the separation of many mycorrhizal fungis.The research of Chinese chestnut Mycorrhizal has successfully report, is external bacterial classification but the microbial inoculum of inoculation all adopts, and effect of inoculation is very undesirable.Though people such as the Qinling Mountains also successfully gather multiple fungus sporophore from the soil of Chinese chestnut producing region, do not isolate the bacterial classification of the successful tieback of energy.
Summary of the invention
The object of the present invention is to provide the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium, cultivate the Chinese chestnut mycorhiza bacterium that obtains having with the Chinese chestnut root system good symbiotic relation.
The present invention is achieved through the following technical solutions:
The artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium may further comprise the steps:
1) tissue separates
Get Chinese chestnut mycorhiza mushroom entity, rip cutting is two halves, gathers meat bacteria organization's piece of cap and stem intersection;
2) cultural hypha and purification
Meat bacteria organization's piece of gathering is placed slant medium, after being cultured to meat bacteria organization's piece under 25～28 ℃ and sprouting mycelia, mycelia is forwarded in the slant medium, under 25～28 ℃, be cultured to mycelia and cover with the inclined-plane, obtain pure mycelia;
Being made as of described slant medium: at first, with filtering after the boiling water boiled potatoes, reclaim filtrate and obtain potato fruit; Secondly, add Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil water extract and water in the potato fruit after, add agar, heat while stirring melt fully to agar after, add glucose again, fully cool off behind the mixing; At last, regulating the pH value is 5～6;
Wherein, the mass ratio of potato, glucose and agar is (10～20): (1～2): (1～1.5), and the volume ratio of Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil water extract and water is (2～5): (1～2): (2～5);
3) bacterial classification enlarges cultivation
Pure mycelium inoculation to enlarging in the composts or fertilisers of cultivating, is cultured to mycelia and covers with culture vessel under 25～28 ℃, obtain Chinese chestnut mycorhiza bacterium.
The described time with the boiling water boiled potatoes is 30～50min; Adopting lmol/L NaOH or lmol/L HCL to regulate the pH value is 5～6.
Described Chinese chestnut dry branches and fallen leaves water cooking liquid is: boil Chinese chestnut dry branches and fallen leaves 30～50min with boiling water, leave standstill the back and filter the filtrate that obtains.
Described rhizosphere soil water extract is: boil Chinese chestnut rhizosphere soil 30～50min with boiling water, leave standstill the back and filter the filtrate that obtains.
Described expansion composts or fertilisers of cultivating is made into by mass ratio 10:2～3 by matrix and nutrient solution.
Described matrix is pruned the crushed material of branch or chestnut bud and corn flour (80～85) by volume by Chinese chestnut: (20～15) mixing is made into.
Described nutrient solution is by water, white sugar, KH
2PO
4, NH
4Cl, CaSO
42H
2O, FeCl
3It is formulated to press mass ratio 80:10:3:5:1:1, and regulating the pH value is 5～6.
The water quality that contains of described expansion composts or fertilisers of cultivating is 60～65%.
Described Chinese chestnut mycorhiza mushroom entity also comprises pretreatment operation before tissue separates, it is the wild Chinese chestnut mycorhiza mushroom entity of fresh, sturdy, the medium maturation of will gather, earlier through sterilized antistaling agent washing, again through 75% alcohol-pickled sterilization, finally by sterile water wash.
Described sterilized antistaling agent is: mass concentration is that 0.02～0.06% ascorbic acid liquid and mass concentration are 0.01～0.03% citric acid solution (1～5) by volume: the mixed liquor that (1～3) is made into.
Compared with prior art, the present invention has following beneficial technical effects:
The artificial cultural method of Chinese chestnut mycorhiza bacterium of the present invention, utilize the narrow spectrum attribute of mycorhiza bacterium nutrition, select for use Chinese chestnut dry branches and fallen leaves, rhizosphere soil etc. to obtain nutrient solution, through organizing separation, purifying, cultivation, breeding, acquisition can form the mycorhiza bacterial classification of best symbiotic relation with Chinese chestnut.The present invention is simple to operate, by tieback test, and the bacterial classification tieback Chinese chestnut seedling that utilizes the present invention to cultivate to obtain, proving effect is very good, and mycorhiza dip-dye rate reaches as high as 50.3%, and on average dip-dye rate is 46.5%.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment, and the explanation of the invention is not limited.
Embodiment 1
The artificial cultural method of a kind of Chinese chestnut mycorhiza may further comprise the steps:
1) collection of wild mycorhiza mushroom entity
Under the Chinese chestnut gardens more than 10 ages, gather wild mycorhiza mushroom entity fresh, sturdy, medium maturation, after removing surface irregularities, in the sterilized antistaling agent for preparing in advance, soaked 1～2 minute, pull out and drain, in the sterilized antistaling bag of packing into, sealing, in 48 hours, take back the back and in 75% alcohol, soak sterilization in 5 seconds, use twice of sterile water wash afterwards again.
Wherein, described sterilized antistaling agent is by mass concentration 0.04% ascorbic acid liquid and the mass concentration 0.02% citric acid solution mixed liquor that is made into of 1:1 by volume.
2) tissue separates
Under aseptic condition, be two halves with the fruit body vertical profile after cleaning, sterilizing, cut cap and stem intersection with scalpel, obtain meat bacteria organization's piece (mung bean size);
3) make slant medium
The prescription of slant medium is: potato 200g, and glucose 20g, agar 15g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 400ml, rhizosphere soil leach cooking liquid 200ml, running water 400ml, the pH value is 5;
The making step of slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 40min, with four layers of filtered through gauze, get Chinese chestnut dry branches and fallen leaves water cooking liquid, standby;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 30min, with four layers of filtered through gauze, get rhizosphere soil water extract, standby;
3. with peeling potatoes and eye, stripping and slicing or section, take by weighing 200g, add water 1000ml, boil 30min, can be poked by glass bar to potato and be advisable, with three layers of filtered through gauze, get potato fruit, standby;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 400ml, rhizosphere soil leach cooking liquid 200ml, running water 400ml joins in the potato fruit, add agar 15g again, continue heating, heat while stirring, dissolve fully to agar, add glucose 20g again, cold slightly, water complements to 1000ml again; Regulating the pH value with lmol/L NaOH or lmol/L HCL solution is 5;
5. above-mentioned prepared culture medium is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Tampon beyond the Great Wall then, autoclave is put in wrapping, 2 layers of newspaper of loam cake, sterilization is 30 minutes under 121 ℃ or 0.103MPa, treats to take out test tube when pressure reduces to zero, puts into the inclined-plane, and cooling is standby;
6. the slant medium test tube is placed 28 ℃ insulating box to cultivate 3 days, still smooth as the inclined-plane, do not have assorted bacterium and occur, just can be used as qualified slant medium and use.
The above-mentioned slant medium test tube of separating making of organizing should be no less than 50 as much as possible, cultivates in 28 ℃ then.From the 4th day, checked 1 time every 3 days, in time brush and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's piece is placed slant medium, after being cultured to meat bacteria organization piece sprouting mycelia under 25 ℃, after under aseptic condition, carrying out tube (in the new slant medium), move into incubator, temperature control is at 28 ℃, treat that mycelia is covered with the inclined-plane after, namely obtain pure mycelia (first class inoculum), and outside pure mycelia test tube, stick the label on strain name, bacterial strain number, inoculation date, so that preserve and expanding propagation.
5) bacterial classification enlarges cultivation
In the inoculation hole that enlarges composts or fertilisers of cultivating, compacting slightly makes bacterial classification contact with composts or fertilisers of cultivating with pure mycelium inoculation, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, under 25 ℃, be cultured to mycelia and cover with blake bottle, obtain to enlarge kind of (second class inoculum), i.e. a Chinese chestnut mycorhiza bacterium.
Wherein, described expansion composts or fertilisers of cultivating is made into by mass ratio 10:2 by matrix and nutrient solution, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume the 85:15 mixing be made into, nutrient solution is by water, white sugar, KH
2PO
4, NH
4Cl, CaSO
42H
2O, FeCl
3It is formulated to press mass ratio 80:10:3:5:1:1, and regulating the pH value is 5～6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. Chinese chestnut is pruned branch or tremble luxuriant crushed material and corn flour by volume 85:15 mix, again nutrient solution is admixed matrix, the mass ratio of matrix and nutrient solution is 10:2;
2. behind the abundant mixing of matrix and nutrient solution, between 60～65%, (with holding material, having water to ooze out between webs and be advisable) regulating composts or fertilisers of cultivating moisture with suitable quantity of water, mix even after, packing seed bottle immediately; (seed bottle can be selected the wide-mouth bottle of 75ml for use, limit rim compacting, and seed bottle installs the top, back and flattens in fact, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. will clean up inside and outside the bottleneck with clear water, with two layers of newspaper tying of one deck Polypropylence Sheet or directly use the cotton plug mouth, dress pot immediately after sealing, 121 ℃ of sterilizations, standby.
By above step 1)～4) just can obtain secondary expansion bacterial classification, according to producing needs, can also continue to enlarge the acquisition three-class strain.Utilize second class inoculum or three-class strain can produce dissimilar mycorhiza preparations or the special-purpose fertilizer of Chinese chestnut root fungus.
The present invention is cultivated the Chinese chestnut mycorhiza bacterium that obtains carry out the tieback effect test, concrete content of the test and test result are as follows:
1) test method
The design of employing randomized block experiment when the Chinese chestnut container seedling has just grown a pair of true leaf, utilizes solid fungicide directly to inoculate, and is contrast not connect bacterium.The solid fungicide inoculum concentration is the 10g/ strain, divides 3 groups, repeats each group repeated inoculation 30 strain 3 times.Inoculate after 1 month, adopt sarranine-pale green decoration method investigation mycorhiza infection rate.After dyed, root cells nuclear is dyed scarlet or aubergine, and mycelia green or blue-green are utilized this can differentiate root and whether contaminated, and calculate infection rate.Computing formula: radicals * 100% of mycorhiza infection rate (%)=infect radical/all.
Treated after the seedling root Mycorrhizal 3 months, and measured the growth indexes such as height of seedling, leading thread, ratio of height to diameter of nursery stock.
2) result of the test
1. mycorhiza infection rate, as shown in table 1:
Chinese chestnut seedling mycorhiza dip-dye rate behind the table 1 tieback bush mycorrhiza agent
| ? | First group | Second group | The 3rd group | Contrast (CK) |
| Mycorhiza dip-dye rate (%) | 41.4 | 50.3 | 47.7 | 0.0 |
From measurement result: utilize this bacterial classification tieback Chinese chestnut seedling, mycorhiza dip-dye rate reaches as high as 50.3%, and on average dip-dye rate is 46.5%.
2. mycorhiza seedling and common seedling growing state comparing result, as shown in table 2:
Table 2 mycorhiza seedling and the contrast of common seedling growing state
| ? | Height of seedling (cm) | Leading thread (cm) | Ratio of height to diameter |
| The mycorhiza seedling | 43.6 | 0.81 | 53.83 |
| Common seedling | 32.3 | 0.53 | 60.94 |
| Increment | +11.3 | +0.28 | -7.11 |
From measurement result: the height of seedling of mycorhiza seedling, leading thread are all big than common seedling, but the ratio of height to diameter of nursery stock is less than common seedling, illustrate that the inoculation bacterium can not only improve the seedling growth amount, also help to cultivate strong sprout.
Embodiment 2
The artificial cultural method of a kind of Chinese chestnut mycorhiza may further comprise the steps:
1) collection of wild mycorhiza mushroom entity
Under the Chinese chestnut gardens more than 10 ages, gather wild mycorhiza mushroom entity fresh, sturdy, medium maturation, after removing surface irregularities, in the sterilized antistaling liquid for preparing in advance, soaked 2 minutes, pull out and drain, in the sterilized antistaling bag of packing into, sealing, in 48 hours, take back the back and in 75% alcohol, soak sterilization in 5 seconds, use twice of sterile water wash afterwards again.
Described sterilized antistaling agent is by mass concentration 0.02% ascorbic acid liquid and the mass concentration 0.01% citric acid solution mixed liquor that is made into of 1:3 by volume.
2) tissue separates
Under aseptic condition, be two halves with the fruit body vertical profile after cleaning, sterilizing, cut cap and stem intersection with scalpel, obtain meat bacteria organization's piece (mung bean size);
3) make slant medium
The prescription of slant medium is: potato 100g, and glucose 10g, agar 10g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 200ml, rhizosphere soil leach cooking liquid 100ml, running water 200ml, regulating the pH value is 6;
The making step of slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 30min, with four layers of filtered through gauze, get Chinese chestnut dry branches and fallen leaves water cooking liquid, standby;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 40min, with four layers of filtered through gauze, get rhizosphere soil water extract, standby;
3. with peeling potatoes and eye, stripping and slicing or section, take by weighing 100g, add water 500ml, boil about 30 minutes, can be poked by glass bar to potato and be advisable, with four layers of filtered through gauze, get potato fruit, standby;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 200ml, rhizosphere soil leach cooking liquid 100ml, running water 200ml joins in the potato fruit, add agar 10g again, continue heating, heat while stirring, dissolve fully to agar, add glucose 10g again, cold slightly, water complements to 500ml again; Regulate pH value to 6 with lmol/L NaOH or lmol/L HCL solution;
5. above-mentioned prepared culture medium is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Tampon beyond the Great Wall then, autoclave is put in wrapping, 2 layers of newspaper of loam cake, sterilization is 30 minutes under 121 ℃ or 0.103MPa, treats to take out test tube when pressure reduces to zero, puts into the inclined-plane, and cooling is standby;
6. the slant medium test tube is placed 25 ℃ insulating box to cultivate 3 days, still smooth as the inclined-plane, do not have assorted bacterium and occur, just can be used as qualified slant medium and use.
The above-mentioned slant medium test tube of separating making of organizing should be no less than 50 as much as possible, cultivates in 25 ℃ then.From the 4th day, checked 1 time every 3 days, in time brush and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's piece is placed slant medium, after being cultured to meat bacteria organization piece sprouting mycelia under 27 ℃, after under aseptic condition, carrying out tube (in the new slant medium), move into incubator, temperature control is at 25 ℃, treat that mycelia covers with the inclined-plane and namely obtain pure mycelia (first class inoculum), and outside pure mycelia test tube, stick strain name, bacterial strain number, the label on inoculation date so that preserve and expanding propagation.
5) bacterial classification enlarges cultivation
In the inoculation hole that enlarges composts or fertilisers of cultivating, compacting slightly makes bacterial classification contact with composts or fertilisers of cultivating with pure mycelium inoculation, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, under 25 ℃, be cultured to mycelia and cover with blake bottle, obtain to enlarge kind of (second class inoculum), i.e. a Chinese chestnut mycorhiza bacterium.
If do not see mycelium germination yet after inoculating 5 days then should in time mend inoculation, as find that assorted bacterium should in time remove.To often change the position of bottle simultaneously, be beneficial to the mycelial growth unanimity, after mycelia is covered with bottle, in time use.If short-term is preserved under the environmental condition of temperature, drying, the lucifuge temporarily need not lowerd.
Above-mentioned expansion composts or fertilisers of cultivating is made into by mass ratio 10:2.5 by matrix and nutrient solution, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume the 80:20 mixing be made into, nutrient solution is by water, white sugar, KH
2PO
4, NH
4Cl, CaSO
42H
2O, FeCl
3It is formulated to press mass ratio 80:10:3:5:1:1, and regulating the pH value is 5～6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. earlier Chinese chestnut is pruned branch or tremble luxuriant crushed material, corn flour by volume 80:20 mix, again nutrient solution is admixed matrix, the mass ratio of matrix and nutrient solution is 10:2.5.
2. matrix and nutrient solution full and uniform after, between 60～65%, (with holding material, having water to ooze out between webs and be advisable) regulating composts or fertilisers of cultivating moisture with suitable quantity of water, mix even after, packing seed bottle immediately.(seed bottle can be selected the wide-mouth bottle of 75ml for use, limit rim compacting, and seed bottle installs the top, back and flattens in fact, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. will clean up inside and outside the bottleneck with clear water, with two layers of newspaper tying of one deck Polypropylence Sheet or directly use the cotton plug mouth, dress pot immediately after sealing, 121 ℃ of sterilizations, standby.
By above step 1)～4) just can obtain secondary expansion bacterial classification, according to producing needs, can also continue to enlarge the acquisition three-class strain.
Embodiment 3
The artificial cultural method of a kind of Chinese chestnut mycorhiza may further comprise the steps:
1) collection of wild mycorhiza mushroom entity
Under the Chinese chestnut gardens more than 10 ages, gather wild mycorhiza mushroom entity fresh, sturdy, medium maturation, after removing surface irregularities, in the sterilized antistaling liquid for preparing in advance, soaked 1 minute, pull out and drain, in the sterilized antistaling bag of packing into, sealing, in 48 hours, take back the back and in 75% alcohol, soak sterilization in 5 seconds, use twice of sterile water wash afterwards again.Described sterilized antistaling agent is by mass concentration 0.06% ascorbic acid liquid and the mass concentration 0.03% citric acid solution mixed liquor that is made into of 3:2 by volume.
2) tissue separates
Under aseptic condition, be two halves with the fruit body vertical profile after cleaning, sterilizing, cut cap and stem intersection with scalpel, obtain meat bacteria organization's piece (mung bean size);
3) make slant medium
The prescription of slant medium is: potato 150g, and glucose 7.5g, agar 10g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 60ml, running water 180ml, regulating the pH value is 5～6.
The making step of described slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 50min, with four layers of filtered through gauze, get Chinese chestnut dry branches and fallen leaves water cooking liquid, standby;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 50min, with four layers of filtered through gauze, get rhizosphere soil water extract, standby;
3. with peeling potatoes and eye, stripping and slicing or section, take by weighing 150g, add water 750ml, boil about 30 minutes, can be poked by glass bar to potato and be advisable, with four layers of filtered through gauze, get potato fruit, standby;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 60ml, running water 180ml adds in the potato fruit, add agar 10g, continue heating, heat while stirring, dissolve fully to agar, add glucose 7.5g again, cold slightly, water complements to 540ml again; Regulate pH value to 6 with lmol/L NaOH or lmol/L HCL solution;
5. above-mentioned prepared culture medium is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Tampon beyond the Great Wall then, autoclave is put in wrapping, 2 layers of newspaper of loam cake, sterilization is 30 minutes under 121 ℃ or 0.103MPa, treats to take out test tube when pressure reduces to zero, puts into the inclined-plane, and cooling is standby; Place 27 ℃ insulating box to cultivate 3 days in the slant medium test tube, still smooth as the inclined-plane, do not have assorted bacterium and occur, just can be used as qualified slant medium and use.
The above-mentioned slant medium test tube of separating making of organizing should be no less than 50 as much as possible, cultivates in 27 ℃ then.From the 4th day, checked 1 time every 3 days, in time brush and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's piece is placed slant medium, after being cultured to meat bacteria organization piece sprouting mycelia under 28 ℃, after under aseptic condition, carrying out tube (in the new slant medium), move into incubator, temperature control is at 27 ℃, treat that mycelia covers with the inclined-plane and namely obtain pure mycelia (first class inoculum), and outside pure mycelia test tube, stick strain name, bacterial strain number, the label on inoculation date so that preserve and expanding propagation.
5) bacterial classification enlarges cultivation
In the inoculation hole that enlarges composts or fertilisers of cultivating, compacting slightly makes bacterial classification contact with composts or fertilisers of cultivating with pure mycelium inoculation, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, under 27 ℃, be cultured to mycelia and cover with blake bottle, obtain to enlarge kind of (second class inoculum), i.e. a Chinese chestnut mycorhiza.
If do not see mycelium germination yet after inoculating 5 days then should in time mend inoculation, as find that assorted bacterium should in time remove.To often change the position of bottle simultaneously, be beneficial to the mycelial growth unanimity, after mycelia is covered with bottle, in time use.If short-term is preserved under the environmental condition of temperature, drying, the lucifuge temporarily need not lowerd.
Above-mentioned expansion composts or fertilisers of cultivating is made into by mass ratio 10:3 by matrix and nutrient solution, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume the 85:15 mixing be made into, nutrient solution is by water, white sugar, KH
2PO
4, NH
4Cl, CaSO
42H
2O, FeCl
3It is formulated to press mass ratio 80:10:3:5:1:1, and regulating the pH value is 5～6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. earlier Chinese chestnut is pruned branch or tremble luxuriant crushed material, corn flour by volume 85:15 mix, again nutrient solution is admixed matrix, the part by weight of matrix and nutrient solution is 10:3.
2. matrix and nutrient solution fully rub with the hands and evenly after, regulate with suitable quantity of water composts or fertilisers of cultivating moisture at 60～65%(with holding material, have water to ooze out between webs and be advisable), mix even after, packing seed bottle immediately.(seed bottle can be selected the wide-mouth bottle of 75ml for use, limit rim compacting, and seed bottle installs the top, back and flattens in fact, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. will clean up inside and outside the bottleneck with clear water, with two layers of newspaper tying of one deck Polypropylence Sheet or directly use the cotton plug mouth, dress pot immediately after sealing, 121 ℃ of sterilizations, standby.
By above step 1)～4) just can obtain secondary expansion bacterial classification, according to producing needs, can also continue to enlarge the acquisition three-class strain.
Embodiment 4
The artificial cultural method of a kind of Chinese chestnut mycorhiza may further comprise the steps:
1) collection of wild mycorhiza mushroom entity
Under the Chinese chestnut gardens more than 10 ages, gather wild mycorhiza mushroom entity fresh, sturdy, medium maturation, after removing surface irregularities, in the sterilized antistaling liquid for preparing in advance, soaked 1 minute, pull out and drain, in the sterilized antistaling bag of packing into, sealing, in 48 hours, take back the back and in 75% alcohol, soak sterilization in 5 seconds, use twice of sterile water wash afterwards again.Described sterilized antistaling agent is by mass concentration 0.04% ascorbic acid liquid and the mass concentration 0.02% citric acid solution mixed liquor that is made into of 5:1～3 by volume.
2) tissue separates
Under aseptic condition, be two halves with the fruit body vertical profile after cleaning, sterilizing, cut cap and stem intersection with scalpel, obtain meat bacteria organization's piece (mung bean size);
3) make slant medium
The prescription of slant medium is: potato 150g, and glucose 10g, agar 15g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 200ml, running water 500ml, regulating the pH value is 6.
The making step of described slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 45min, with four layers of filtered through gauze, get Chinese chestnut dry branches and fallen leaves water cooking liquid, standby;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 35min, with four layers of filtered through gauze, get rhizosphere soil water extract, standby;
3. with peeling potatoes and eye, stripping and slicing or section, take by weighing 150g, add water 750ml, boil about 30 minutes, can be poked by glass bar to potato and be advisable, with four layers of filtered through gauze, get potato fruit, standby;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 200ml, running water 500ml adds in the potato fruit, add agar 15g, continue heating, heat while stirring, dissolve fully to agar, add glucose 10g again, cold slightly, water complements to 1000ml again; Regulate pH value to 6 with lmol/L NaOH or lmol/L HCL solution;
5. above-mentioned prepared culture medium is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Tampon beyond the Great Wall then, autoclave is put in wrapping, 2 layers of newspaper of loam cake, sterilization is 30 minutes under 121 ℃ or 0.103MPa, treats to take out test tube when pressure reduces to zero, puts into the inclined-plane, and cooling is standby; Place 27 ℃ insulating box to cultivate 3 days in the slant medium test tube, still smooth as the inclined-plane, do not have assorted bacterium and occur, just can be used as qualified slant medium and use.
The above-mentioned slant medium test tube of separating making of organizing should be no less than 50 as much as possible, cultivates in 27 ℃ then.From the 4th day, checked 1 time every 3 days, in time brush and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's piece is placed slant medium, after being cultured to meat bacteria organization piece sprouting mycelia under 28 ℃, after under aseptic condition, carrying out tube (in the new slant medium), move into incubator, temperature control is at 27 ℃, treat that mycelia covers with the inclined-plane and namely obtain pure mycelia (first class inoculum), and outside pure mycelia test tube, stick strain name, bacterial strain number, the label on inoculation date so that preserve and expanding propagation.
5) bacterial classification enlarges cultivation
In the inoculation hole that enlarges composts or fertilisers of cultivating, compacting slightly makes bacterial classification contact with composts or fertilisers of cultivating with pure mycelium inoculation, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, under 27 ℃, be cultured to mycelia and cover with blake bottle, obtain to enlarge kind of (second class inoculum), i.e. a Chinese chestnut mycorhiza.
If do not see mycelium germination yet after inoculating 5 days then should in time mend inoculation, as find that assorted bacterium should in time remove.To often change the position of bottle simultaneously, be beneficial to the mycelial growth unanimity, after mycelia is covered with bottle, in time use.If short-term is preserved under the environmental condition of temperature, drying, the lucifuge temporarily need not lowerd.
Above-mentioned expansion composts or fertilisers of cultivating is made into by mass ratio 10:3 by matrix and nutrient solution, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume the 83:17 mixing be made into, nutrient solution is by water, white sugar, KH
2PO
4, NH
4Cl, CaSO
42H
2O, FeCl
3It is formulated to press mass ratio 80:10:3:5:1:1, and regulating the pH value is 5～6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. earlier Chinese chestnut is pruned branch or tremble luxuriant crushed material, corn flour by volume 83:17 mix, again nutrient solution is admixed matrix, the mass ratio of matrix and nutrient solution is 10:3.
2. matrix and nutrient solution fully rub with the hands and evenly after, regulate with suitable quantity of water composts or fertilisers of cultivating moisture at 60～65%(with holding material, have water to ooze out between webs and be advisable), mix even after, packing seed bottle immediately.(seed bottle can be selected the wide-mouth bottle of 75ml for use, limit rim compacting, and seed bottle installs the top, back and flattens in fact, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. will clean up inside and outside the bottleneck with clear water, with two layers of newspaper tying of one deck Polypropylence Sheet or directly use the cotton plug mouth, dress pot immediately after sealing, 121 ℃ of sterilizations, standby.
By above step 1)～4) just can obtain secondary expansion bacterial classification, according to producing needs, can also continue to enlarge the acquisition three-class strain.
Claims (10)
1. the artificial cultural method of Chinese chestnut mycorhiza bacterium is characterized in that, may further comprise the steps:
1) tissue separates
Get Chinese chestnut mycorhiza mushroom entity, rip cutting is two halves, gathers meat bacteria organization's piece of cap and stem intersection;
2) cultural hypha and purification
Meat bacteria organization's piece of gathering is placed slant medium, after being cultured to meat bacteria organization's piece under 25～28 ℃ and sprouting mycelia, mycelia is forwarded in the slant medium, under 25～28 ℃, be cultured to mycelia and cover with the inclined-plane, obtain pure mycelia;
Being made as of described slant medium: at first, with filtering after the boiling water boiled potatoes, reclaim filtrate and obtain potato fruit; Secondly, add Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil water extract and water in the potato fruit after, add agar, heat while stirring melt fully to agar after, add glucose again, fully cool off behind the mixing; At last, regulating the pH value is 5～6;
Wherein, the mass ratio of potato, glucose and agar is (10～20): (1～2): (1～1.5), and the volume ratio of Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil water extract and water is (2～5): (1～2): (2～5);
3) bacterial classification enlarges cultivation
Pure mycelium inoculation to enlarging in the composts or fertilisers of cultivating, is cultured to mycelia and covers with culture vessel under 25～28 ℃, obtain Chinese chestnut mycorhiza bacterium.
2. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 1 is characterized in that the described time with the boiling water boiled potatoes is 30～50min; Adopting lmol/L NaOH or lmol/L HCL to regulate the pH value is 5～6.
3. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 1 is characterized in that described Chinese chestnut dry branches and fallen leaves water cooking liquid is: boil Chinese chestnut dry branches and fallen leaves 30～50min with boiling water, leave standstill the back and filter the filtrate that obtains.
4. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 1 is characterized in that described rhizosphere soil water extract is: boil Chinese chestnut rhizosphere soil 30～50min with boiling water, leave standstill the back and filter the filtrate that obtains.
5. the artificial cultural method of a kind of Chinese chestnut mycorhiza according to claim 1 is characterized in that described expansion composts or fertilisers of cultivating is made into by mass ratio 10:2～3 by matrix and nutrient solution.
6. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 5 is characterized in that, described matrix is pruned the crushed material of branch or chestnut bud and corn flour (80～85) by volume by Chinese chestnut: (20～15) mixing is made into.
7. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 5 is characterized in that described nutrient solution is by water, white sugar, KH
2PO
4, NH
4Cl, CaSO
42H
2O, FeCl
3It is formulated to press mass ratio 80:10:3:5:1:1, and regulating the pH value is 5～6.
8. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 5 is characterized in that, the water quality that contains of described expansion composts or fertilisers of cultivating is 60～65%.
9. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 1, it is characterized in that, described Chinese chestnut mycorhiza mushroom entity also comprises pretreatment operation before tissue separates, it is the wild Chinese chestnut mycorhiza mushroom entity of fresh, sturdy, the medium maturation of will gather, wash through the sterilized antistaling agent earlier, again through 75% alcohol-pickled sterilization, finally by sterile water wash.
10. the artificial cultural method of a kind of Chinese chestnut mycorhiza bacterium according to claim 9, it is characterized in that described sterilized antistaling agent is: mass concentration is that 0.02～0.06% ascorbic acid liquid and mass concentration are 0.01～0.03% citric acid solution (1～5) by volume: the mixed liquor that (1～3) is made into.
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