CN103250564B - 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 VA Mycorrhizal Fungi and manually cultivate field, be specifically related to a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method.
Background technology
Mycorhiza is the most general symbiosis of edaphon and plant compatibility, and the formation of ectotrophic mycorrhiza makes trees rely on the help of VA Mycorrhizal Fungi to improve absorbing capacity to soil moisture and nutrient.Organic phosphorus in dry branches and fallen leaves and the soil organic matter can be decomposed absorption and directly be transferred to trees utilization by the organic phosphorus catabolic enzyme of ectotrophic mycorrhiza secretion, thus plays the effect of drought resisting, growth-promoting, diseases prevention.After Applying Ectomycorrhizal Fungi infects host plant root system, form Hartig net and bacterium cover on the one hand, another aspect mycelia stretches out and forms fine and close mycelia net, even forms shoestring.Because most Applying Ectomycorrhizal Fungi does not have strict selectivity to host, touch in 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 Hyphal links between root.The foundation of Hyphal links makes to define source and sink relation between different plants, makes can transmit the material such as C, N, P and moisture between plant, thus the nutrition condition of recipient plant is improved.
Also there is a kind of symbiotic relation in Chinese chestnut root system and ectotrophic mycorrhiza.In Chestnut Orchard sylvan life, the fruit body of the Chinese chestnut VA Mycorrhizal Fungi of difformity and color can be seen.Investigation finds, the fungi that can form ectotrophic mycorrhiza with Chinese chestnut has 13 genus 29 kinds, the dominant 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 must false truffle and Chinese chestnut symbiotic relation best, next is Russula and Boletus.
The normal growth of Applying Ectomycorrhizal Fungi to Chinese chestnut has extremely important effect, can improve the absorbing capacity of Chinese chestnut root system to soil moisture and nutrient.If lack VA Mycorrhizal Fungi in soil, Chinese chestnut just can not grow by normal growth.The many growths of Chinese chestnut are in mountain area, and at the Chestnut Orchard that soil fertility is barren, soil nutrient content is very low, and fertilising difficulty, therefore, the Chinese chestnut in mountain area is often because adnation position, strain different manifestations in age go out nutrition status of the plant and growth potential very big difference.In the conventional graft seedling growth of Chinese chestnut, due to lack in seedbed or medium can with the VA Mycorrhizal Fungi of Chinese chestnut Root Symbiont, so be transplanted to the conventional grafting in land for growing field crops, survival rate is low, growth retardation, and also there will be the dead phenomenon of large tree after 2 ~ 3 years.
But the symbiotic relation of Chinese chestnut root system and ectotrophic mycorrhiza is very complicated, the separation of many mycorrhizal fungis is cultivated more difficult.The research of Chinese chestnut Mycorrhizal has reported success, but inoculation microbial inoculum all adopts is external bacterial classification, effect of inoculation is very undesirable.Although the people such as the Qinling Mountains also success 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 a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method, cultivate and obtain the Chinese chestnut VA Mycorrhizal Fungi having good symbiotic relation with Chinese chestnut root system.
The present invention is achieved through the following technical solutions:
A kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method, comprises the following steps:
1) tissue is separated
Get Chinese chestnut VA Mycorrhizal Fungi fruit body, rip cutting is two halves, gathers meat bacteria organization's block of cap and stem intersection;
2) cultural hypha and purification
Meat bacteria organization's block of collection is placed in slant medium, is cultured to after meat bacteria organization's block sprouts mycelia at 25 ~ 28 DEG C, mycelia is forwarded in slant medium, at 25 ~ 28 DEG C, is cultured to mycelia covers with inclined-plane, obtain pure mycelia;
Being made as of described slant medium: first, filters with after boiling water boiling potato, reclaims filtrate and obtains potato fruit; Secondly, Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil Aqueous extracts and water are added after in potato fruit, adds agar, heat while stirring after melting completely to agar, then add glucose, fully cooling after mixing; Finally, adjust ph 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 Aqueous extracts and water is (2 ~ 5): (1 ~ 2): (2 ~ 5);
3) bacterial classification expands cultivation
By pure mycelium inoculation in expansion composts or fertilisers of cultivating, at 25 ~ 28 DEG C, be cultured to mycelia cover with culture vessel, obtain Chinese chestnut VA Mycorrhizal Fungi.
The described time with boiling water boiling potato is 30 ~ 50min; Lmol/L NaOH or lmol/L HCL adjust ph is adopted to be 5 ~ 6.
Described Chinese chestnut dry branches and fallen leaves water cooking liquid is: with boiling water boiling Chinese chestnut dry branches and fallen leaves 30 ~ 50min, leave standstill and filter the filtrate obtained afterwards.
Described rhizosphere soil Aqueous extracts is: with boiling water boiling Chinese chestnut rhizosphere soil 30 ~ 50min, leave standstill and filter the filtrate obtained afterwards.
Described expansion composts or fertilisers of cultivating is made into 10:2 ~ 3 in mass ratio by matrix and nutrient solution.
Described matrix prunes the crushed material of branch or chestnut bud and corn flour by volume (80 ~ 85) by Chinese chestnut: (20 ~ 15) mix and are made into.
Described nutrient solution is by water, white sugar, KH
2pO
4, NH
4cl, CaSO
42H
2o, FeCl
380:10:3:5:1:1 is formulated in mass ratio, and adjust ph is 5 ~ 6.
Described expansion composts or fertilisers of cultivating containing water quality be 60 ~ 65%.
Described Chinese chestnut VA Mycorrhizal Fungi fruit body also comprises pretreatment operation before tissue is separated, by the Wild Chinese Chestnut VA Mycorrhizal Fungi fruit body of fresh, sturdy, the medium maturation gathered, first through sterilized antistaling agent washing, then through 75% alcohol-pickled sterilization, finally by sterile water wash.
Described sterilized antistaling agent is: mass concentration be 0.02 ~ 0.06% Vitamin C acid solution and mass concentration be the citric acid solution of 0.01 ~ 0.03% (1 ~ 5) by volume: the mixed liquor that (1 ~ 3) is made into.
Compared with prior art, the present invention has following useful technique effect:
Chinese chestnut VA Mycorrhizal Fungi artificial culture method of the present invention, utilize the narrow spectrum attribute of VA Mycorrhizal Fungi nutrition, select Chinese chestnut dry branches and fallen leaves, rhizosphere soil etc. to obtain nutrient solution, through tissue separations, purifying, cultivation, breeding, acquisition can form the mycorhiza bacterial classification of best symbiotic relation with Chinese chestnut.The present invention is simple to operate, is tested by tieback, and the bacterial classification tieback Chinese Chestnut Seedlings utilizing the present invention to cultivate to obtain, proving effect is very good, and mycorhiza Infection Rate reaches as high as 50.3%, and average Infection Rate is 46.5%.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
Embodiment 1
A kind of Chinese chestnut mycorhiza artificial culture method, comprises the following steps:
1) collection of wild mushroom root fungus fruit body
Chestnut Orchard sylvan life more than 10 ages, gather the wild mushroom root fungus fruit body of fresh, sturdy, medium maturation, after removing surface irregularities, soak 1 ~ 2 minute in previously prepared good sterilized antistaling agent, pull out and drain, load in sterilized antistaling bag, sealing, in 75% alcohol, soak sterilization in 5 seconds after taking back in 48 hours, use sterile water wash twice more afterwards.
Wherein, described sterilized antistaling agent is by mass concentration 0.04% Vitamin C acid solution and the mass concentration 0.02% citric acid solution mixed liquor that is made into of 1:1 by volume.
2) tissue is separated
Aseptically, be two halves by the fruit body vertical profile after cleaning, sterilization, cut cap and stem intersection with scalpel, obtain meat bacteria organization's block (mung bean size);
3) slant medium is made
The formula of slant medium is: potato 200g, glucose 20g, agar 15g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 400ml, rhizosphere soil leach cooking liquid 200ml, running water 400ml, and pH value is 5;
The making step of slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boils 40min, by four layers of filtered through gauze, obtains Chinese chestnut dry branches and fallen leaves water cooking liquid, for subsequent use;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boils 30min, by four layers of filtered through gauze, obtains rhizosphere soil Aqueous extracts, for subsequent use;
3. by peeling potatoes and eye, stripping and slicing or section, take 200g, add water 1000ml, boils 30min, can be poked and be advisable, by three layers of filtered through gauze, obtain potato fruit to potato by glass bar, for subsequent use;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 400ml, rhizosphere soil leach cooking liquid 200ml, running water 400ml, join in potato fruit, add agar 15g again, continue heating, heat while stirring, dissolve completely to agar, add glucose 20g again, slightly cold, then complement to 1000ml with water; Be 5 by lmol/L NaOH or lmol/L HCL solution adjust ph;
5. the above-mentioned medium prepared is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Then tampon beyond the Great Wall, wrapping, puts into autoclave, upper cover 2 layers of newspaper, sterilizing 30 minutes under 121 DEG C or 0.103MPa, takes out test tube, is put into inclined-plane, cooling when pressure reduces to zero, for subsequent use;
6. insulating box slant medium test tube being placed in 28 DEG C is cultivated 3 days, as inclined-plane is still smooth, occurs without miscellaneous bacteria, just can be used as qualified slant medium and uses.
The above-mentioned slant medium test tube of separating making of organizing should be tried one's best many, is no less than 50, then cultivates in 28 DEG C.From the 4th day, checked 1 time every 3 days, brush in time and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, be cultured to meat bacteria organization block sprouting mycelia at 25 DEG C after, after aseptically carrying out tube (in new slant medium), move into incubator, temperature controls, at 28 DEG C, to cover with behind inclined-plane until mycelia, namely obtains 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 expands cultivation
By pure mycelium inoculation in the inoculation hole expanding composts or fertilisers of cultivating, slightly compacting, makes bacterial classification contact with composts or fertilisers of cultivating, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, at 25 DEG C, be cultured to mycelia cover with blake bottle, obtain and expand kind of (second class inoculum), i.e. a Chinese chestnut VA Mycorrhizal Fungi.
Wherein, described expansion composts or fertilisers of cultivating by matrix and nutrient solution in mass ratio 10:2 be made into, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume 85:15 mix and be made into, nutrient solution is by water, white sugar, KH
2pO
4, NH
4cl, CaSO
42H
2o, FeCl
380:10:3:5:1:1 is formulated in mass ratio, and adjust ph is 5 ~ 6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. Chinese chestnut is pruned branch or chestnut bud crushed material and corn flour by volume 85:15 mix, then nutrient solution is admixed matrix, the mass ratio of matrix and nutrient solution is 10:2;
2., after matrix and nutrient solution fully mix, regulating composts or fertilisers of cultivating moisture (with holding material, having water to ooze out between webs and being advisable) between 60 ~ 65% by suitable quantity of water, mixing evenly, can packing seed bottle immediately; (seed bottle can select the wide-mouth bottle of 75ml, limit rim compacting, and seed bottle installs rear top and flattens real, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. clean up inside and outside bottleneck with clear water, with one deck Polypropylence Sheet two layers of newspaper tying or directly use cotton plug mouth, after sealing, fill pot immediately, 121 DEG C of sterilizings, for subsequent use.
By above step 1) ~ 4) just can obtain secondary expansion bacterial classification, according to need of production, can also continue to expand acquisition three-class strain.Utilize second class inoculum or three-class strain can produce dissimilar mycorhiza preparation or Chinese chestnut root fungus special fertilizer.
The Chinese chestnut VA Mycorrhizal Fungi obtained is cultivated to the present invention and carries out tieback effect test, concrete content of the test and test result as follows:
1) test method
Employing randomized block experiment designs, and when chestnut container seeding just grows a pair true leaf, utilizes solid fungicide directly to inoculate, not connect bacterium for contrast.Solid fungicide inoculum concentration is 10g/ strain, and point 3 groups, repeat 3 times, the strain of each group of repeated inoculation 30.Inoculate after 1 month, adopt sarranine-pale green decoration method investigation Mycorrhizal Infection Incidence.After dyed, root cells core dyes scarlet or aubergine, and mycelia green or blue-green, utilize this can differentiate root and whether contaminate, and calculates infection rate.Computing formula: Mycorrhizal Infection Incidence (%)=infect radical/all radical × 100%.
After seedling root Mycorrhizal 3 months, measure the growth indexes such as height of seedling, leading thread, ratio of height to diameter of nursery stock.
2) result of the test
1. Mycorrhizal Infection Incidence, as shown in table 1:
Chestnut Seedlings mycorhiza Infection Rate after table 1 tieback bush mycorrhiza agent
| ? | First group | Second group | 3rd group | Contrast (CK) |
| Mycorhiza Infection Rate (%) | 41.4 | 50.3 | 47.7 | 0.0 |
From measurement result: utilize this bacterial classification tieback Chinese Chestnut Seedlings, mycorhiza Infection Rate reaches as high as 50.3%, and average Infection Rate is 46.5%.
2. Va Mycorrhiza Seedling and common seedling growing state comparing result, as shown in table 2:
Table 2 Va Mycorrhiza Seedling and common seedling growing state contrast
| ? | Height of seedling (cm) | Leading thread (cm) | Ratio of height to diameter |
| Va Mycorrhiza 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: height of seedling, the leading thread of Va Mycorrhiza Seedling are all large than common seedling, but the ratio of height to diameter of nursery stock is less than common seedling, illustrate that inoculation bacterium can not only improve seedling growth amount, also contribute to nurturing staff.
Embodiment 2
A kind of Chinese chestnut mycorhiza artificial culture method, comprises the following steps:
1) collection of wild mushroom root fungus fruit body
Chestnut Orchard sylvan life more than 10 ages, gather the wild mushroom root fungus fruit body of fresh, sturdy, medium maturation, after removing surface irregularities, soak 2 minutes in previously prepared good sterilized antistaling liquid, pull out and drain, load in sterilized antistaling bag, sealing, in 75% alcohol, soak sterilization in 5 seconds after taking back in 48 hours, use sterile water wash twice more afterwards.
Described sterilized antistaling agent is by mass concentration 0.02% Vitamin C acid solution and the mass concentration 0.01% citric acid solution mixed liquor that is made into of 1:3 by volume.
2) tissue is separated
Aseptically, be two halves by the fruit body vertical profile after cleaning, sterilization, cut cap and stem intersection with scalpel, obtain meat bacteria organization's block (mung bean size);
3) slant medium is made
The formula of slant medium is: potato 100g, glucose 10g, agar 10g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 200ml, rhizosphere soil leach cooking liquid 100ml, running water 200ml, and adjust ph is 6;
The making step of slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boils 30min, by four layers of filtered through gauze, obtains Chinese chestnut dry branches and fallen leaves water cooking liquid, for subsequent use;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boils 40min, by four layers of filtered through gauze, obtains rhizosphere soil Aqueous extracts, for subsequent use;
3. by peeling potatoes and eye, stripping and slicing or section, take 100g, add water 500ml, boils about 30 minutes, can be poked and be advisable, by four layers of filtered through gauze, obtain potato fruit to potato by glass bar, for subsequent use;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 200ml, rhizosphere soil leach cooking liquid 100ml, running water 200ml, join in potato fruit, add agar 10g again, continue heating, heat while stirring, dissolve completely to agar, add glucose 10g again, slightly cold, then complement to 500ml with water; By lmol/L NaOH or lmol/L HCL solution adjust ph to 6;
5. the above-mentioned medium prepared is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Then tampon beyond the Great Wall, wrapping, puts into autoclave, upper cover 2 layers of newspaper, sterilizing 30 minutes under 121 DEG C or 0.103MPa, takes out test tube, is put into inclined-plane, cooling when pressure reduces to zero, for subsequent use;
6. insulating box slant medium test tube being placed in 25 DEG C is cultivated 3 days, as inclined-plane is still smooth, occurs without miscellaneous bacteria, just can be used as qualified slant medium and uses.
The above-mentioned slant medium test tube of separating making of organizing should be tried one's best many, is no less than 50, then cultivates in 25 DEG C.From the 4th day, checked 1 time every 3 days, brush in time and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, be cultured to meat bacteria organization block sprouting mycelia at 27 DEG C after, after aseptically carrying out tube (in new slant medium), move into incubator, temperature controls at 25 DEG C, treat that mycelia is covered with inclined-plane and namely obtains 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 expands cultivation
By pure mycelium inoculation in the inoculation hole expanding composts or fertilisers of cultivating, slightly compacting, makes bacterial classification contact with composts or fertilisers of cultivating, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, at 25 DEG C, be cultured to mycelia cover with blake bottle, obtain and expand kind of (second class inoculum), i.e. a Chinese chestnut VA Mycorrhizal Fungi.
If inoculate and have not yet to see mycelium germination after 5 days, should mend inoculation in time, as found, miscellaneous bacteria should be removed in time.Often to exchange the position of bottle simultaneously, be beneficial to mycelial growth consistent, use in time after mycelia covers with bottle.If short-term preservation under the environmental condition of temperature, drying, the lucifuge temporarily need not lowerd.
Above-mentioned expansion composts or fertilisers of cultivating by matrix and nutrient solution in mass ratio 10:2.5 be made into, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume 80:20 mix and be made into, nutrient solution is by water, white sugar, KH
2pO
4, NH
4cl, CaSO
42H
2o, FeCl
380:10:3:5:1:1 is formulated in mass ratio, and adjust ph is 5 ~ 6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. first Chinese chestnut is pruned branch or chestnut bud crushed material, corn flour by volume 80:20 mix, then 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, regulating composts or fertilisers of cultivating moisture (with holding material, having water to ooze out between webs and being advisable) between 60 ~ 65% by suitable quantity of water, mixing evenly, can packing seed bottle immediately.(seed bottle can select the wide-mouth bottle of 75ml, limit rim compacting, and seed bottle installs rear top and flattens real, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. clean up inside and outside bottleneck with clear water, with one deck Polypropylence Sheet two layers of newspaper tying or directly use cotton plug mouth, after sealing, fill pot immediately, 121 DEG C of sterilizings, for subsequent use.
By above step 1) ~ 4) just can obtain secondary expansion bacterial classification, according to need of production, can also continue to expand acquisition three-class strain.
Embodiment 3
A kind of Chinese chestnut mycorhiza artificial culture method, comprises the following steps:
1) collection of wild mushroom root fungus fruit body
Chestnut Orchard sylvan life more than 10 ages, gather the wild mushroom root fungus fruit body of fresh, sturdy, medium maturation, after removing surface irregularities, soak 1 minute in previously prepared good sterilized antistaling liquid, pull out and drain, load in sterilized antistaling bag, sealing, in 75% alcohol, soak sterilization in 5 seconds after taking back in 48 hours, use sterile water wash twice more afterwards.Described sterilized antistaling agent is by mass concentration 0.06% Vitamin C acid solution and the mass concentration 0.03% citric acid solution mixed liquor that is made into of 3:2 by volume.
2) tissue is separated
Aseptically, be two halves by the fruit body vertical profile after cleaning, sterilization, cut cap and stem intersection with scalpel, obtain meat bacteria organization's block (mung bean size);
3) slant medium is made
The formula of slant medium is: potato 150g, glucose 7.5g, agar 10g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, and rhizosphere soil leach cooking liquid 60ml, running water 180ml, adjust ph is 5 ~ 6.
The making step of described slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boils 50min, by four layers of filtered through gauze, obtains Chinese chestnut dry branches and fallen leaves water cooking liquid, for subsequent use;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boils 50min, by four layers of filtered through gauze, obtains rhizosphere soil Aqueous extracts, for subsequent use;
3. by peeling potatoes and eye, stripping and slicing or section, take 150g, add water 750ml, boils about 30 minutes, can be poked and be advisable, by four layers of filtered through gauze, obtain potato fruit to potato by glass bar, for subsequent use;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 60ml, running water 180ml, add in potato fruit, add agar 10g, continue heating, heat while stirring, dissolve completely to agar, add glucose 7.5g again, slightly cold, then complement to 540ml with water; By lmol/L NaOH or lmol/L HCL solution adjust ph to 6;
5. the above-mentioned medium prepared is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Then tampon beyond the Great Wall, wrapping, puts into autoclave, upper cover 2 layers of newspaper, sterilizing 30 minutes under 121 DEG C or 0.103MPa, takes out test tube, is put into inclined-plane, cooling when pressure reduces to zero, for subsequent use; Insulating box slant medium test tube being placed in 27 DEG C is cultivated 3 days, as inclined-plane is still smooth, occurs without miscellaneous bacteria, just can be used as qualified slant medium and uses.
The above-mentioned slant medium test tube of separating making of organizing should be tried one's best many, is no less than 50, then cultivates in 27 DEG C.From the 4th day, checked 1 time every 3 days, brush in time and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, be cultured to meat bacteria organization block sprouting mycelia at 28 DEG C after, after aseptically carrying out tube (in new slant medium), move into incubator, temperature controls at 27 DEG C, treat that mycelia is covered with inclined-plane and namely obtains 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 expands cultivation
By pure mycelium inoculation in the inoculation hole expanding composts or fertilisers of cultivating, slightly compacting, makes bacterial classification contact with composts or fertilisers of cultivating, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, at 27 DEG C, be cultured to mycelia cover with blake bottle, obtain and expand kind of (second class inoculum), i.e. a Chinese chestnut mycorhiza.
If inoculate and have not yet to see mycelium germination after 5 days, should mend inoculation in time, as found, miscellaneous bacteria should be removed in time.Often to exchange the position of bottle simultaneously, be beneficial to mycelial growth consistent, use in time after mycelia covers with bottle.If short-term preservation under the environmental condition of temperature, drying, the lucifuge temporarily need not lowerd.
Above-mentioned expansion composts or fertilisers of cultivating by matrix and nutrient solution in mass ratio 10:3 be made into, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume 85:15 mix and be made into, nutrient solution is by water, white sugar, KH
2pO
4, NH
4cl, CaSO
42H
2o, FeCl
380:10:3:5:1:1 is formulated in mass ratio, and adjust ph is 5 ~ 6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. first Chinese chestnut is pruned branch or chestnut bud crushed material, corn flour by volume 85:15 mix, then 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, regulating composts or fertilisers of cultivating moisture by suitable quantity of water at 60 ~ 65%(with holding material, having water to ooze out between webs and being advisable), mix evenly, can packing seed bottle immediately.(seed bottle can select the wide-mouth bottle of 75ml, limit rim compacting, and seed bottle installs rear top and flattens real, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. clean up inside and outside bottleneck with clear water, with one deck Polypropylence Sheet two layers of newspaper tying or directly use cotton plug mouth, after sealing, fill pot immediately, 121 DEG C of sterilizings, for subsequent use.
By above step 1) ~ 4) just can obtain secondary expansion bacterial classification, according to need of production, can also continue to expand acquisition three-class strain.
Embodiment 4
A kind of Chinese chestnut mycorhiza artificial culture method, comprises the following steps:
1) collection of wild mushroom root fungus fruit body
Chestnut Orchard sylvan life more than 10 ages, gather the wild mushroom root fungus fruit body of fresh, sturdy, medium maturation, after removing surface irregularities, soak 1 minute in previously prepared good sterilized antistaling liquid, pull out and drain, load in sterilized antistaling bag, sealing, in 75% alcohol, soak sterilization in 5 seconds after taking back in 48 hours, use sterile water wash twice more afterwards.Described sterilized antistaling agent is by mass concentration 0.04% Vitamin C acid solution and the mass concentration 0.02% citric acid solution mixed liquor that is made into of 5:1 ~ 3 by volume.
2) tissue is separated
Aseptically, be two halves by the fruit body vertical profile after cleaning, sterilization, cut cap and stem intersection with scalpel, obtain meat bacteria organization's block (mung bean size);
3) slant medium is made
The formula of slant medium is: potato 150g, glucose 10g, agar 15g, Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, and rhizosphere soil leach cooking liquid 200ml, running water 500ml, adjust ph is 6.
The making step of described slant medium is:
1. get Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boils 45min, by four layers of filtered through gauze, obtains Chinese chestnut dry branches and fallen leaves water cooking liquid, for subsequent use;
2. get Chinese chestnut rhizosphere soil 100g, add water 500ml, boils 35min, by four layers of filtered through gauze, obtains rhizosphere soil Aqueous extracts, for subsequent use;
3. by peeling potatoes and eye, stripping and slicing or section, take 150g, add water 750ml, boils about 30 minutes, can be poked and be advisable, by four layers of filtered through gauze, obtain potato fruit to potato by glass bar, for subsequent use;
4. measure above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 200ml, running water 500ml, add in potato fruit, add agar 15g, continue heating, heat while stirring, dissolve completely to agar, add glucose 10g again, slightly cold, then complement to 1000ml with water; By lmol/L NaOH or lmol/L HCL solution adjust ph to 6;
5. the above-mentioned medium prepared is sub-packed in test tube while hot, charge weight is about 1/4 of test tube length; Then tampon beyond the Great Wall, wrapping, puts into autoclave, upper cover 2 layers of newspaper, sterilizing 30 minutes under 121 DEG C or 0.103MPa, takes out test tube, is put into inclined-plane, cooling when pressure reduces to zero, for subsequent use; Insulating box slant medium test tube being placed in 27 DEG C is cultivated 3 days, as inclined-plane is still smooth, occurs without miscellaneous bacteria, just can be used as qualified slant medium and uses.
The above-mentioned slant medium test tube of separating making of organizing should be tried one's best many, is no less than 50, then cultivates in 27 DEG C.From the 4th day, checked 1 time every 3 days, brush in time and pollute pipe, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, be cultured to meat bacteria organization block sprouting mycelia at 28 DEG C after, after aseptically carrying out tube (in new slant medium), move into incubator, temperature controls at 27 DEG C, treat that mycelia is covered with inclined-plane and namely obtains 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 expands cultivation
By pure mycelium inoculation in the inoculation hole expanding composts or fertilisers of cultivating, slightly compacting, makes bacterial classification contact with composts or fertilisers of cultivating, seed bottle to be cultivated is moved into the culturing room of incubator or dim light, at 27 DEG C, be cultured to mycelia cover with blake bottle, obtain and expand kind of (second class inoculum), i.e. a Chinese chestnut mycorhiza.
If inoculate and have not yet to see mycelium germination after 5 days, should mend inoculation in time, as found, miscellaneous bacteria should be removed in time.Often to exchange the position of bottle simultaneously, be beneficial to mycelial growth consistent, use in time after mycelia covers with bottle.If short-term preservation under the environmental condition of temperature, drying, the lucifuge temporarily need not lowerd.
Above-mentioned expansion composts or fertilisers of cultivating by matrix and nutrient solution in mass ratio 10:3 be made into, matrix by Chinese chestnut prune the crushed material of branch or chestnut bud and corn flour by volume 83:17 mix and be made into, nutrient solution is by water, white sugar, KH
2pO
4, NH
4cl, CaSO
42H
2o, FeCl
380:10:3:5:1:1 is formulated in mass ratio, and adjust ph is 5 ~ 6.
The preparation process of described expansion composts or fertilisers of cultivating is:
1. first Chinese chestnut is pruned branch or chestnut bud crushed material, corn flour by volume 83:17 mix, then 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, regulating composts or fertilisers of cultivating moisture by suitable quantity of water at 60 ~ 65%(with holding material, having water to ooze out between webs and being advisable), mix evenly, can packing seed bottle immediately.(seed bottle can select the wide-mouth bottle of 75ml, limit rim compacting, and seed bottle installs rear top and flattens real, makes a call to an inoculation hole in the middle of composts or fertilisers of cultivating.）
3. clean up inside and outside bottleneck with clear water, with one deck Polypropylence Sheet two layers of newspaper tying or directly use cotton plug mouth, after sealing, fill pot immediately, 121 DEG C of sterilizings, for subsequent use.
By above step 1) ~ 4) just can obtain secondary expansion bacterial classification, according to need of production, can also continue to expand acquisition three-class strain.
Claims (6)
1. a Chinese chestnut VA Mycorrhizal Fungi artificial culture method, is characterized in that, comprises the following steps:
1) tissue is separated
Get Chinese chestnut VA Mycorrhizal Fungi fruit body, rip cutting is two halves, gathers meat bacteria organization's block of cap and stem intersection;
2) cultural hypha and purification
Meat bacteria organization's block of collection is placed in slant medium, is cultured to after meat bacteria organization's block sprouts mycelia at 25 ~ 28 DEG C, mycelia is forwarded in slant medium, at 25 ~ 28 DEG C, is cultured to mycelia covers with inclined-plane, obtain pure mycelia;
Being made as of described slant medium: first, filters with after boiling water boiling potato, reclaims filtrate and obtains potato fruit; Secondly, Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil Aqueous extracts and water are added after in potato fruit, adds agar, heat while stirring after melting completely to agar, then add glucose, fully cooling after mixing; Finally, adjust ph 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 Aqueous extracts and water is (2 ~ 5): (1 ~ 2): (2 ~ 5);
3) bacterial classification expands cultivation
By pure mycelium inoculation in expansion composts or fertilisers of cultivating, at 25 ~ 28 DEG C, be cultured to mycelia cover with culture vessel, obtain Chinese chestnut VA Mycorrhizal Fungi;
Described expansion composts or fertilisers of cultivating is made into 10:2 ~ 3 in mass ratio by matrix and nutrient solution, and expansion composts or fertilisers of cultivating is 60 ~ 65% containing water quality;
Wherein, described matrix prunes the crushed material of branch or chestnut bud and corn flour by volume (80 ~ 85) by Chinese chestnut: (20 ~ 15) mix and are made into;
Described nutrient solution is by water, white sugar, KH
2pO
4, NH
4cl, CaSO
42H
2o, FeCl
380:10:3:5:1:1 is formulated in mass ratio, and adjust ph is 5 ~ 6.
2. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, is characterized in that, the described time with boiling water boiling potato is 30 ~ 50min; 1mol/L NaOH or 1mol/LHCL adjust ph is adopted to be 5 ~ 6.
3. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, is characterized in that, described Chinese chestnut dry branches and fallen leaves water cooking liquid is: with boiling water boiling Chinese chestnut dry branches and fallen leaves 30 ~ 50min, leave standstill and filter the filtrate obtained afterwards.
4. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, it is characterized in that, described rhizosphere soil Aqueous extracts is: with boiling water boiling Chinese chestnut rhizosphere soil 30 ~ 50min, leave standstill and filter the filtrate obtained afterwards.
5. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, it is characterized in that, described Chinese chestnut VA Mycorrhizal Fungi fruit body also comprises pretreatment operation before tissue is separated, by the Wild Chinese Chestnut VA Mycorrhizal Fungi fruit body of fresh, sturdy, the medium maturation gathered, first wash through sterilized antistaling agent, again through 75% alcohol-pickled sterilization, finally by sterile water wash.
6. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 5, it is characterized in that, described sterilized antistaling agent is: mass concentration be 0.02 ~ 0.06% Vitamin C acid solution and mass concentration be the citric acid solution of 0.01 ~ 0.03% (1 ~ 5) by volume: the mixed liquor that (1 ~ 3) is made into.
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| CN106212042A (en) * | 2016-07-04 | 2016-12-14 | 李业武 | A kind of pseudo-wild cultivating method of Tricholoma matsutake (lto et lmai) Singer |
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