CN109486682A - A kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method - Google Patents
A kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method Download PDFInfo
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Abstract
The invention discloses a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture methods, belong to VA Mycorrhizal Fungi and manually cultivate field.The following steps are included: 1) take Chinese chestnut VA Mycorrhizal Fungi fructification, longitudinal sectional is two halves, acquires the intersection of cap and stem, obtains meat bacteria organization's block；2) meat bacteria organization's block is placed in slant medium, after culture sprouts mycelia out to meat bacteria organization's block at 25~28 DEG C, after mycelia tube, culture covers with inclined-plane to mycelia at 25~28 DEG C, obtains pure mycelia；3) by pure mycelium inoculation into the inoculation hole for expanding compost, culture to mycelia covers with culture vessel at 25~28 DEG C, obtains Chinese chestnut VA Mycorrhizal Fungi.The artificial culture method of Chinese chestnut VA Mycorrhizal Fungi of the present invention, easy to operate, the strain tieback Chinese Chestnut Seedlings obtained using present invention culture, mycorhiza Infection Rate reaches as high as 50.3%, and average Infection Rate is 46.5%.
Description
Technical field
The invention belongs to VA Mycorrhizal Fungis manually to cultivate field, and in particular to a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method.
Background technique
Mycorhiza is edaphon and the most common symbiosis of plant compatibility, and the formation of exotrophic mycorrhiza relies on trees
The help of VA Mycorrhizal Fungi improves the absorbability to soil moisture and nutrient.The organic phosphorus catabolic enzyme energy of ectotrophic mycorrhiza secretion
Organic phosphorus in dry branches and fallen leaves and the soil organism is decomposed to absorb and is transferred directly to trees utilization, to play drought resisting, promote
Raw, diseases prevention effect.After Applying Ectomycorrhizal Fungi infects host plant root system, Hartig net and bacterium set, another party are on the one hand formed
Face mycelia extends outward to form fine and close mycelia net, or even forms shoestring.Since most Applying Ectomycorrhizal Fungis do not have host
Stringent specificity, touch in extension mycelia stretching process it is other can with after the root system of the host plant of its symbiosis also once again
It infects, forms the Hyphal links between root.The foundation of Hyphal links makes to form source and sink relation between different plants, makes to transmit between plant
C, the substances such as N, P and moisture, so that making the nutrition condition of recipient plant is improved.
There is also a kind of symbiosis for Chinese chestnut root system and ectotrophic mycorrhiza.In Chestnut Orchard hayashishita, it can be seen that different shape
With the fructification of the Chinese chestnut VA Mycorrhizal Fungi of color.Investigation discovery, the fungi that can form exotrophic mycorrhiza with Chinese chestnut have 29 kinds of 13 category,
The dominant fungi of distribution has Lycoperdon (Lycoperdon), must Hymenogaster (Rhizopogon), Calvatia (Calvatia), red
Mushroom category (Russula), Boletus (Boletus), Amanita (Amanita), Strobilomyces (Strobilomyces)
Deng.Wherein, Lasiosphaera fenzlii class and palpus false truffle and Chinese chestnut symbiosis are best, are secondly Russula and Boletus.
Applying Ectomycorrhizal Fungi has extremely important effect to the normal growth of Chinese chestnut, can improve Chinese chestnut root system in soil
The absorbability of moisture and nutrient.If lacking VA Mycorrhizal Fungi in soil, Chinese chestnut cannot normal growth and development.Chinese chestnut is grown in more
Mountain area, in the barren Chestnut Orchard of soil fertility, soil nutrient content is very low, and fertilising is difficult, therefore, the Chinese chestnut Chang Yin in mountain area it is raw
Position, strain age different manifestations go out nutrition status of the plant and growth potential very big difference.In Chinese chestnut routine graft seedling growth, due to seedbed
Or in culture medium lack can with the VA Mycorrhizal Fungi of Chinese chestnut Root Symbiont, so, be transplanted to the conventional grafting in crop field, survival rate is low,
Growth retardation, and to there is also the dead phenomenon of big tree after 2~3 years.
However, the symbiosis of Chinese chestnut root system and ectotrophic mycorrhiza is sufficiently complex, many mycorrhizal fungis are separately cultured ratio
It is more difficult.Chinese chestnut Mycorrhizal research has a reported success, but what the microbial inoculum being inoculated with was all made of is external strain, and effect of inoculation is very not
It is ideal.Although Qinling Mountains et al., which also succeeds, acquires a variety of fungus sporophores from the soil of Chinese chestnut producing region, not isolating can success
The strain of tieback.
Summary of the invention
The purpose of the present invention is to provide a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method, culture obtains having with Chinese chestnut root system good
The Chinese chestnut VA Mycorrhizal Fungi of good symbiosis.
The present invention is to be achieved through the following technical solutions:
A kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method, comprising the following steps:
1) tissue separation
Chinese chestnut VA Mycorrhizal Fungi fructification is taken, longitudinal sectional is two halves, acquires meat bacteria organization's block of cap and stem intersection；
2) cultural hypha and purification
Meat bacteria organization's block of acquisition is placed in slant medium, culture to meat bacteria organization's block is sprouted out at 25~28 DEG C
After mycelia, mycelia is forwarded in slant medium, culture covers with inclined-plane to mycelia at 25~28 DEG C, obtains pure mycelia；
The production of the slant medium are as follows: firstly, recycling filtrate obtains potato with filtering after boiling water boiled potatoes
Juice；Secondly, agar is added after Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil Aqueous extracts and water are added in potato fruit,
It heats while stirring to agar after melting completely, adds glucose, it is cooling after mixing well；Finally, adjusting pH value is 5~6；
Wherein, the mass ratio of potato, glucose and agar is (10~20): (1~2): (1~1.5), Chinese chestnut deadwood are fallen
The volume ratio of leaf water cooking liquid, rhizosphere soil Aqueous extracts and water is (2~5): (1~2): (2~5)；
3) strain expands culture
By pure mycelium inoculation to expanding in compost, culture to mycelia covers with culture vessel at 25~28 DEG C, obtains plate
Chestnut VA Mycorrhizal Fungi.
The time with boiling water boiled potatoes is 30~50min；It is adjusted using lmol/L NaOH or lmol/L HCL
PH value is 5~6.
The Chinese chestnut dry branches and fallen leaves water cooking liquid are as follows: use boiling water boiling Chinese chestnut 30~50min of dry branches and fallen leaves, be obtained by filtration after standing
Filtrate.
The rhizosphere soil Aqueous extracts are as follows: use boiling water boiling Chinese chestnut 30~50min of rhizosphere soil, the filter being obtained by filtration after standing
Liquid.
The expansion compost is made by matrix and nutrient solution 10:2~3 in mass ratio.
The matrix trims the crushed material of branch or chestnut bud with corn flour by volume (80~85) by Chinese chestnut: (20~
15) it mixes and is made into.
The nutrient solution is by water, white sugar, KH2PO4, NH4Cl, CaSO4 2H2O, FeCl3 80:10:3:5 in mass ratio:
1:1 is formulated, and adjusting pH value is 5~6.
The expansion compost is 60~65% containing water quality.
The Chinese chestnut VA Mycorrhizal Fungi fructification tissue separation before further include pretreatment operation, be will acquire it is fresh, strong
The Wild Chinese Chestnut VA Mycorrhizal Fungi fructification of real, medium maturation, is first washed through sterilized antistaling agent, then through 75% alcohol soaking disinfection,
Most afterwards through sterile water wash.
The sterilized antistaling agent are as follows: the Vitamin C acid solution and mass concentration that mass concentration is 0.02~0.06% are 0.01
~0.03% citric acid solution is by volume (1~5): the mixed liquor that (1~3) is made into.
Compared with prior art, the invention has the following beneficial technical effects:
Chinese chestnut VA Mycorrhizal Fungi artificial culture method of the invention selects Chinese chestnut withered using the attribute of VA Mycorrhizal Fungi nutrition specificity
Branch fallen leaves, rhizosphere soil etc. obtain nutrient solution, and by tissue separation, purifying, culture, breeding, acquisition can be formed most preferably with Chinese chestnut
The mycorhiza strain of symbiosis.Operation of the present invention is simple, is tested by tieback, the strain tieback plate obtained using present invention culture
Li Youmiao, it was demonstrated that effect is very good, and mycorhiza Infection Rate reaches as high as 50.3%, and average Infection Rate is 46.5%.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Embodiment 1
A kind of Chinese chestnut mycorhiza artificial culture method, comprising the following steps:
1) acquisition of wild VA Mycorrhizal Fungi fructification
In 10 Chestnut Orchard hayashishita more than age, the wild VA Mycorrhizal Fungi fructification of fresh, sturdy, medium maturation is acquired, is removed
It after surface irregularities, is impregnated 1~2 minute in sterilized antistaling agent well prepared in advance, pulls out and drain, be packed into sterilized antistaling bag,
Sealing is impregnated 5 seconds in 75% alcohol after taking back within 48 hours and is sterilized, uses twice of sterile water wash again later.
Wherein, the sterilized antistaling agent is by 0.04% Vitamin C acid solution of mass concentration and 0.02% lemon of mass concentration
The acid solution mixed liquor that 1:1 is made by volume.
2) tissue separation
Aseptically, it is two halves by the fructification vertical profile after cleaning, disinfection, cuts cap with scalpel and stem is handed over
At remittance, meat bacteria organization's block (mung bean size) is obtained；
3) slant medium is made
The formula of slant medium are as follows: potato 200g, glucose 20g, agar 15g, Chinese chestnut dry branches and fallen leaves leach cooking liquid
400ml, rhizosphere soil leach cooking liquid 200ml, tap water 400ml, pH value 5；
The making step of slant medium are as follows:
1. taking Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 40min, with four layers of filtered through gauze, obtains Chinese chestnut deadwood and fall
Leaf water cooking liquid, it is spare；
2. taking Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 30min, with four layers of filtered through gauze, obtains rhizosphere soil water
Extract, it is spare；
3. peeling potatoes and bud eye, stripping and slicing or slice are weighed 200g, add water 1000ml, boil 30min, until Ma Ling
Potato can be poked by glass bar to be advisable, and with three layers of filtered through gauze, obtains potato fruit, spare；
4. measuring above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 400ml, rhizosphere soil leach cooking liquid 200ml, tap water 400ml add
Enter into potato fruit, add agar 15g, continue to heat, heat while stirring, until agar dissolves completely, then plus grape
Sugared 20g, it is slightly cold, then 1000ml is complemented to water；Adjusting 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 the 1/4 of test tube length；Then beyond the Great Wall
Tampon, wrapping, is put into autoclave, 2 layers of newspaper of upper cover sterilize 30 minutes under 121 DEG C or 0.103MPa, when pressure is reduced to zero
Test tube is taken out, inclined-plane is put into, it is cooling, it is spare；
It is cultivated 3 days 6. slant medium test tube is placed in 28 DEG C of insulating box, if inclined-plane is still smooth, no miscellaneous bacteria occurs,
It just can be used as qualified slant medium to use.
The slant medium test tube of above-mentioned tissue separating making should be more as far as possible, no less than 50, then cultivate in 28 DEG C.
It from the 4th day, was checked 1 time every 3 days, brushes pollution pipe in time, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, after culture sprouts mycelia to meat bacteria organization's block at 25 DEG C, in nothing
After carrying out tube (in new slant medium) under the conditions of bacterium, incubator is moved into, temperature is controlled at 28 DEG C, covers with inclined-plane to mycelia
Afterwards, that is, it obtains pure mycelia (first class inoculum), and sticks strain name, bacterial strain number, the label for being inoculated with the date outside pure mycelia test tube,
In order to save and expanding propagation.
5) strain expands culture
By pure mycelium inoculation into the inoculation hole for expanding compost, slightly it is compacted, strain is made to be in contact with compost, it will be wait train
Feeding seed bottle moves into the culturing room of incubator or dim light, and culture covers with culture bottle to mycelia at 25 DEG C, is expanded
Kind (second class inoculum), i.e. Chinese chestnut VA Mycorrhizal Fungi.
Wherein, the expansion compost is made by matrix and nutrient solution 10:2 in mass ratio, and matrix trims branch by Chinese chestnut
Or the 85:15 mixing by volume of the crushed material of chestnut bud and corn flour is made into, nutrient solution by water, white sugar, KH2PO4, NH4Cl,
CaSO4 2H2O, FeCl3 80:10:3:5:1:1 in mass ratio are formulated, and adjusting pH value is 5~6.
The preparation step for expanding compost are as follows:
1. Chinese chestnut is trimmed branch or chestnut bud crushed material, and 85:15 is uniformly mixed by volume with corn flour, then by nutrient solution
Admix matrix, the mass ratio of matrix and nutrient solution is 10:2；
After 2. matrix and nutrient solution mix well, with suitable quantity of water adjust compost moisture between 60~65% (with
Material is held, has water exudation to be advisable between webs), after mixing uniformly, seed bottle can be dispensed immediately；(the wide of 75ml can be selected in seed bottle
Mouth bottle, the compacting of side rim, seed bottle install rear top and flatten in fact, and an inoculation hole is made a call among compost.)
3. will be cleaned up inside and outside bottleneck with clear water, cotton mouth with one layer of plastic cloth, two layers of newspaper tying or is directly used,
Pot is filled after sealing immediately, is sterilized at 121 DEG C, it is spare.
Pass through above step 1)~4) be obtained with second level expand strain can also continue to expand according to production needs
Obtain three-class strain.Different types of mycorhiza preparation can be produced using second class inoculum or three-class strain or Chinese chestnut root fungus is dedicated
Fertilizer.
The Chinese chestnut VA Mycorrhizal Fungi obtained to present invention culture carries out tieback effect test, and specific content of the test and test result are such as
Under:
1) test method
It is designed using randomized block experiment, when chestnut container seeding just grows a pair of of true leaf, is directly connect using solid fungicide
Kind, not connect bacterium as control.Solid fungicide inoculum concentration is 10g/ plants, and point 3 groups are repeated 3 times, each group 30 plants of repeated inoculation.
After inoculation 1 month, Mycorrhizal Infection Incidence is investigated using sarranine-pale green decoration method.After dyeing, root cells core is dyed scarlet or purplish red
Color, mycelia green or blue-green calculate infection rate whether can differentiating root dip dyeing using this.Calculation formula: Mycorrhizal Infection Incidence
(%)=infect radical/whole radical 100%.
3 months after seedling root Mycorrhizal, the growth indexes such as height of seedling, ground diameter, the ratio of height to diameter of nursery stock are measured.
2) test result
1. Mycorrhizal Infection Incidence, as shown in table 1:
Chestnut Seedlings mycorhiza Infection Rate after 1 tieback bush mycorrhiza agent of table
| First group | Second group | Third group | It compares (CK) | |
| Mycorhiza Infection Rate (%) | 41.4 | 50.3 | 47.7 | 0.0 |
In terms of measurement result: utilizing the strain tieback Chinese Chestnut Seedlings, mycorhiza Infection Rate reaches as high as 50.3%, average to disseminate
Rate is 46.5%.
2. Va Mycorrhiza Seedling and common seedling growing state comparing result, as shown in table 2:
2 Va Mycorrhiza Seedling of table and common seedling growing state compare
| Height of seedling (cm) | Ground diameter (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 |
In terms of measurement result: the height of seedling of Va Mycorrhiza Seedling, ground diameter are all bigger than common seedling, but the ratio of height to diameter of nursery stock is less than commonly
Seedling illustrates that seedling growth amount can not only be improved by being inoculated with bacterium, additionally aid nurturing staff.
Embodiment 2
A kind of Chinese chestnut mycorhiza artificial culture method, comprising the following steps:
1) acquisition of wild VA Mycorrhizal Fungi fructification
In 10 Chestnut Orchard hayashishita more than age, the wild VA Mycorrhizal Fungi fructification of fresh, sturdy, medium maturation is acquired, is removed
It after surface irregularities, is impregnated 2 minutes in sterilized antistaling liquid well prepared in advance, pulls out and drain, be packed into sterilized antistaling bag, it is close
Envelope is impregnated 5 seconds in 75% alcohol after taking back within 48 hours and is sterilized, uses twice of sterile water wash again later.
The sterilized antistaling agent is by 0.02% Vitamin C acid solution of mass concentration and 0.01% citric acid solution of mass concentration
1: 3 mixed liquor being made by volume.
2) tissue separation
Aseptically, it is two halves by the fructification vertical profile after cleaning, disinfection, cuts cap with scalpel and stem is handed over
At remittance, meat bacteria organization's block (mung bean size) is obtained；
3) slant medium is made
The formula of slant medium are as follows: potato 100g, glucose 10g, agar 10g, Chinese chestnut dry branches and fallen leaves leach cooking liquid
200ml, rhizosphere soil leach cooking liquid 100ml, tap water 200ml, adjusting pH value is 6；
The making step of slant medium are as follows:
1. taking Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 30min, with four layers of filtered through gauze, obtains Chinese chestnut deadwood and fall
Leaf water cooking liquid, it is spare；
2. taking Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 40min, with four layers of filtered through gauze, obtains rhizosphere soil water
Extract, it is spare；
3. peeling potatoes and bud eye, stripping and slicing or slice are weighed 100g, add water 500ml, boils 30 minutes or so, until
Potato can be poked by glass bar to be advisable, and with four layers of filtered through gauze, obtains potato fruit, spare；
4. measuring above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 200ml, rhizosphere soil leach cooking liquid 100ml, tap water 200ml add
Enter into potato fruit, add agar 10g, continue to heat, heat while stirring, until agar dissolves completely, then plus grape
Sugared 10g, it is slightly cold, then 500ml is complemented to water；PH value is adjusted 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 the 1/4 of test tube length；Then beyond the Great Wall
Tampon, wrapping, is put into autoclave, 2 layers of newspaper of upper cover sterilize 30 minutes under 121 DEG C or 0.103MPa, when pressure is reduced to zero
Test tube is taken out, inclined-plane is put into, it is cooling, it is spare；
It is cultivated 3 days 6. slant medium test tube is placed in 25 DEG C of insulating box, if inclined-plane is still smooth, no miscellaneous bacteria occurs,
It just can be used as qualified slant medium to use.
The slant medium test tube of above-mentioned tissue separating making should be more as far as possible, no less than 50, then cultivate in 25 DEG C.
It from the 4th day, was checked 1 time every 3 days, brushes pollution pipe in time, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, after culture sprouts mycelia to meat bacteria organization's block at 27 DEG C, in nothing
After carrying out tube (in new slant medium) under the conditions of bacterium, incubator is moved into, temperature is controlled at 25 DEG C, covers with inclined-plane to mycelia
It obtains pure mycelia (first class inoculum), and sticks strain name, bacterial strain number, the label for being inoculated with the date outside pure mycelia test tube, with
Convenient for preservation and expanding propagation.
5) strain expands culture
By pure mycelium inoculation into the inoculation hole for expanding compost, slightly it is compacted, strain is made to be in contact with compost, it will be wait train
Feeding seed bottle moves into the culturing room of incubator or dim light, and culture covers with culture bottle to mycelia at 25 DEG C, is expanded
Kind (second class inoculum), i.e. Chinese chestnut VA Mycorrhizal Fungi.
Inoculation should be mended in time if having not yet to see mycelium germination after inoculation 5 days, such as find that miscellaneous bacteria should remove in time.It wants simultaneously
The position of bottle is often exchanged, so that mycelia growth is consistent, is timely used after mycelia covers with bottle.If not having to temporarily to lower
Short-term preservation under warm, drying, the environmental condition being protected from light.
Above-mentioned expansion compost is made by matrix and nutrient solution 10:2.5 in mass ratio, and matrix trims branch or chestnut by Chinese chestnut
The crushed material of bud is made into corn flour 80:20 mixing by volume, and nutrient solution is by water, white sugar, KH2PO4, NH4Cl, CaSO4
2H2O, FeCl3 80:10:3:5:1:1 in mass ratio are formulated, and adjusting pH value is 5~6.
The preparation step for expanding compost are as follows:
1. Chinese chestnut is first trimmed branch or chestnut bud crushed material, corn flour, and 80:20 is uniformly mixed by volume, then by nutrient solution
Admix matrix, the mass ratio of matrix and nutrient solution is 10:2.5.
2. matrix and nutrient solution it is full and uniform after, with suitable quantity of water adjust compost moisture between 60~65% (with
Material is held, has water exudation to be advisable between webs), after mixing uniformly, seed bottle can be dispensed immediately.(the wide of 75ml can be selected in seed bottle
Mouth bottle, the compacting of side rim, seed bottle install rear top and flatten in fact, and an inoculation hole is made a call among compost.)
3. will be cleaned up inside and outside bottleneck with clear water, cotton mouth with one layer of plastic cloth, two layers of newspaper tying or is directly used,
Pot is filled after sealing immediately, is sterilized at 121 DEG C, it is spare.
Pass through above step 1)~4) be obtained with second level expand strain can also continue to expand according to production needs
Obtain three-class strain.
Embodiment 3
A kind of Chinese chestnut mycorhiza artificial culture method, comprising the following steps:
1) acquisition of wild VA Mycorrhizal Fungi fructification
In 10 Chestnut Orchard hayashishita more than age, the wild VA Mycorrhizal Fungi fructification of fresh, sturdy, medium maturation is acquired, is removed
It after surface irregularities, is impregnated 1 minute in sterilized antistaling liquid well prepared in advance, pulls out and drain, be packed into sterilized antistaling bag, it is close
Envelope is impregnated 5 seconds in 75% alcohol after taking back within 48 hours and is sterilized, uses twice of sterile water wash again later.Described is sterile
Antistaling agent be by 0.06% Vitamin C acid solution of mass concentration and 0.03% citric acid solution of mass concentration by volume 3:2 be made into it is mixed
Close liquid.
2) tissue separation
Aseptically, it is two halves by the fructification vertical profile after cleaning, disinfection, cuts cap with scalpel and stem is handed over
At remittance, meat bacteria organization's block (mung bean size) is obtained；
3) slant medium is made
The formula of slant medium are as follows: potato 150g, glucose 7.5g, agar 10g, Chinese chestnut dry branches and fallen leaves leach cooking liquid
300ml, rhizosphere soil leach cooking liquid 60ml, tap water 180ml, adjusting pH value is 5~6.
The making step of the slant medium are as follows:
1. taking Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 50min, with four layers of filtered through gauze, obtains Chinese chestnut deadwood and fall
Leaf water cooking liquid, it is spare；
2. taking Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 50min, with four layers of filtered through gauze, obtains rhizosphere soil water
Extract, it is spare；
3. peeling potatoes and bud eye, stripping and slicing or slice are weighed 150g, add water 750ml, boils 30 minutes or so, until
Potato can be poked by glass bar to be advisable, and with four layers of filtered through gauze, obtains potato fruit, spare；
4. measuring above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 60ml, tap water 180ml are added
In potato fruit, agar 10g is added, continues to heat, heat while stirring, until agar dissolves completely, then plus glucose 7.5g,
It is slightly cold, then 540ml is complemented to water；PH value is adjusted 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 the 1/4 of test tube length；Then beyond the Great Wall
Tampon, wrapping, is put into autoclave, 2 layers of newspaper of upper cover sterilize 30 minutes under 121 DEG C or 0.103MPa, when pressure is reduced to zero
Test tube is taken out, inclined-plane is put into, it is cooling, it is spare；Slant medium test tube is placed in 27 DEG C of insulating box and is cultivated 3 days, such as inclined-plane
Still smooth, no miscellaneous bacteria occurs, so that it may use as qualified slant medium.
The slant medium test tube of above-mentioned tissue separating making should be more as far as possible, no less than 50, then cultivate in 27 DEG C.
It from the 4th day, was checked 1 time every 3 days, brushes pollution pipe in time, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, after culture sprouts mycelia to meat bacteria organization's block at 28 DEG C, in nothing
After carrying out tube (in new slant medium) under the conditions of bacterium, incubator is moved into, temperature is controlled at 27 DEG C, covers with inclined-plane to mycelia
It obtains pure mycelia (first class inoculum), and sticks strain name, bacterial strain number, the label for being inoculated with the date outside pure mycelia test tube, with
Convenient for preservation and expanding propagation.
5) strain expands culture
By pure mycelium inoculation into the inoculation hole for expanding compost, slightly it is compacted, strain is made to be in contact with compost, it will be wait train
Feeding seed bottle moves into the culturing room of incubator or dim light, and culture covers with culture bottle to mycelia at 27 DEG C, is expanded
Kind (second class inoculum), i.e. Chinese chestnut mycorhiza.
Inoculation should be mended in time if having not yet to see mycelium germination after inoculation 5 days, such as find that miscellaneous bacteria should remove in time.It wants simultaneously
The position of bottle is often exchanged, so that mycelia growth is consistent, is timely used after mycelia covers with bottle.If not having to temporarily to lower
Short-term preservation under warm, drying, the environmental condition being protected from light.
Above-mentioned expansion compost is made by matrix and nutrient solution 10:3 in mass ratio, and matrix trims branch or chestnut bud by Chinese chestnut
The 85:15 mixing by volume of crushed material and corn flour be made into, nutrient solution is by water, white sugar, KH2PO4, NH4Cl, CaSO4
2H2O, FeCl3 80:10:3:5:1:1 in mass ratio are formulated, and adjusting pH value is 5~6.
The preparation step for expanding compost are as follows:
1. Chinese chestnut is first trimmed branch or chestnut bud crushed material, corn flour, and 85:15 is uniformly mixed by volume, then by nutrient solution
Admix matrix, the weight ratio of matrix and nutrient solution is 10:3.
After 2. matrix and nutrient solution are sufficiently rubbed and are uniform, with suitable quantity of water adjusting compost moisture 60~65% (with
Material is held, has water exudation to be advisable between webs), after mixing uniformly, seed bottle can be dispensed immediately.(the wide of 75ml can be selected in seed bottle
Mouth bottle, the compacting of side rim, seed bottle install rear top and flatten in fact, and an inoculation hole is made a call among compost.)
3. will be cleaned up inside and outside bottleneck with clear water, cotton mouth with one layer of plastic cloth, two layers of newspaper tying or is directly used,
Pot is filled after sealing immediately, is sterilized at 121 DEG C, it is spare.
Pass through above step 1)~4) be obtained with second level expand strain can also continue to expand according to production needs
Obtain three-class strain.
Embodiment 4
A kind of Chinese chestnut mycorhiza artificial culture method, comprising the following steps:
1) acquisition of wild VA Mycorrhizal Fungi fructification
In 10 Chestnut Orchard hayashishita more than age, the wild VA Mycorrhizal Fungi fructification of fresh, sturdy, medium maturation is acquired, is removed
It after surface irregularities, is impregnated 1 minute in sterilized antistaling liquid well prepared in advance, pulls out and drain, be packed into sterilized antistaling bag, it is close
Envelope is impregnated 5 seconds in 75% alcohol after taking back within 48 hours and is sterilized, uses twice of sterile water wash again later.Described is sterile
Antistaling agent is made into 5:1~3 by volume by 0.04% Vitamin C acid solution of mass concentration and 0.02% citric acid solution of mass concentration
Mixed liquor.
2) tissue separation
Aseptically, it is two halves by the fructification vertical profile after cleaning, disinfection, cuts cap with scalpel and stem is handed over
At remittance, meat bacteria organization's block (mung bean size) is obtained；
3) slant medium is made
The formula of slant medium are as follows: potato 150g, glucose 10g, agar 15g, Chinese chestnut dry branches and fallen leaves leach cooking liquid
300ml, rhizosphere soil leach cooking liquid 200ml, tap water 500ml, adjusting pH value is 6.
The making step of the slant medium are as follows:
1. taking Chinese chestnut dry branches and fallen leaves 200g, add water 1000ml, boil 45min, with four layers of filtered through gauze, obtains Chinese chestnut deadwood and fall
Leaf water cooking liquid, it is spare；
2. taking Chinese chestnut rhizosphere soil 100g, add water 500ml, boil 35min, with four layers of filtered through gauze, obtains rhizosphere soil water
Extract, it is spare；
3. peeling potatoes and bud eye, stripping and slicing or slice are weighed 150g, add water 750ml, boils 30 minutes or so, until
Potato can be poked by glass bar to be advisable, and with four layers of filtered through gauze, obtains potato fruit, spare；
4. measuring above-mentioned Chinese chestnut dry branches and fallen leaves leach cooking liquid 300ml, rhizosphere soil leach cooking liquid 200ml, tap water 500ml add
Enter in potato fruit, agar 15g is added, continues to heat, heat while stirring, until agar dissolves completely, then plus glucose
10g, it is slightly cold, then 1000ml is complemented to water；PH value is adjusted 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 the 1/4 of test tube length；Then beyond the Great Wall
Tampon, wrapping, is put into autoclave, 2 layers of newspaper of upper cover sterilize 30 minutes under 121 DEG C or 0.103MPa, when pressure is reduced to zero
Test tube is taken out, inclined-plane is put into, it is cooling, it is spare；Slant medium test tube is placed in 27 DEG C of insulating box and is cultivated 3 days, such as inclined-plane
Still smooth, no miscellaneous bacteria occurs, so that it may use as qualified slant medium.
The slant medium test tube of above-mentioned tissue separating making should be more as far as possible, no less than 50, then cultivate in 27 DEG C.
It from the 4th day, was checked 1 time every 3 days, brushes pollution pipe in time, uncontaminated pipe is continued to cultivate.
4) cultural hypha and purification
Meat bacteria organization's block is placed in slant medium, after culture sprouts mycelia to meat bacteria organization's block at 28 DEG C, in nothing
After carrying out tube (in new slant medium) under the conditions of bacterium, incubator is moved into, temperature is controlled at 27 DEG C, covers with inclined-plane to mycelia
It obtains pure mycelia (first class inoculum), and sticks strain name, bacterial strain number, the label for being inoculated with the date outside pure mycelia test tube, with
Convenient for preservation and expanding propagation.
5) strain expands culture
By pure mycelium inoculation into the inoculation hole for expanding compost, slightly it is compacted, strain is made to be in contact with compost, it will be wait train
Feeding seed bottle moves into the culturing room of incubator or dim light, and culture covers with culture bottle to mycelia at 27 DEG C, is expanded
Kind (second class inoculum), i.e. Chinese chestnut mycorhiza.
Inoculation should be mended in time if having not yet to see mycelium germination after inoculation 5 days, such as find that miscellaneous bacteria should remove in time.It wants simultaneously
The position of bottle is often exchanged, so that mycelia growth is consistent, is timely used after mycelia covers with bottle.If not having to temporarily to lower
Short-term preservation under warm, drying, the environmental condition being protected from light.
Above-mentioned expansion compost is made by matrix and nutrient solution 10:3 in mass ratio, and matrix trims branch or chestnut bud by Chinese chestnut
The 83:17 mixing by volume of crushed material and corn flour be made into, nutrient solution is by water, white sugar, KH2PO4, NH4Cl, CaSO4
2H2O, FeCl3 80:10:3:5:1:1 in mass ratio are formulated, and adjusting pH value is 5~6.
The preparation step for expanding compost are as follows:
1. Chinese chestnut is first trimmed branch or chestnut bud crushed material, corn flour, and 83:17 is uniformly mixed by volume, then by nutrient solution
Admix matrix, the mass ratio of matrix and nutrient solution is 10:3.
After 2. matrix and nutrient solution are sufficiently rubbed and are uniform, with suitable quantity of water adjusting compost moisture 60~65% (with
Material is held, has water exudation to be advisable between webs), after mixing uniformly, seed bottle can be dispensed immediately.(the wide of 75ml can be selected in seed bottle
Mouth bottle, the compacting of side rim, seed bottle install rear top and flatten in fact, and an inoculation hole is made a call among compost.)
3. will be cleaned up inside and outside bottleneck with clear water, cotton mouth with one layer of plastic cloth, two layers of newspaper tying or is directly used,
Pot is filled after sealing immediately, is sterilized at 121 DEG C, it is spare.
Pass through above step 1)~4) be obtained with second level expand strain can also continue to expand according to production needs
Obtain three-class strain.
Claims (6)
1. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method, which comprises the following steps:
1) tissue separation
Chinese chestnut VA Mycorrhizal Fungi fructification is taken, longitudinal sectional is two halves, acquires meat bacteria organization's block of cap and stem intersection；
2) cultural hypha and purification
Meat bacteria organization's block of acquisition is placed in slant medium, culture to meat bacteria organization's block sprouts mycelia out at 25~28 DEG C
Afterwards, mycelia is forwarded in slant medium, culture covers with inclined-plane to mycelia at 25~28 DEG C, obtains pure mycelia；Described
The production of slant medium are as follows: firstly, recycling filtrate obtains potato fruit with filtering after boiling water boiled potatoes；Secondly, by plate
After chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil Aqueous extracts and water are added in potato fruit, agar is added, heat while stirring to
After agar melts completely, glucose is added, it is cooling after mixing well；Finally, adjusting pH value is 5~6；Wherein, potato, Portugal
The mass ratio of grape sugar and agar is (10~20): (1~2): (1~1.5), Chinese chestnut dry branches and fallen leaves water cooking liquid, rhizosphere soil water mention
The volume ratio of liquid and water is (2~5): (1~2): (2~5)；
3) strain expands culture
By pure mycelium inoculation to expanding in compost, culture to mycelia covers with culture vessel at 25~28 DEG C, obtains Chinese chestnut bacterium
Root fungus；The expansion compost is made by matrix and nutrient solution 10:2~3 in mass ratio, and expand compost contain water quality
It is 60~65%；Wherein, the matrix trims the crushed material of branch or chestnut bud with corn flour by volume (80~85) by Chinese chestnut:
(20~15) it mixes and is made into；The nutrient solution by water, white sugar, KH2PO4, NH4Cl, CaSO42H2O, FeCl3 in mass ratio
80:10:3:5:1:1 is formulated, and adjusting pH value is 5~6.
2. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, which is characterized in that described uses boiling water boiling
The time of potato is 30~50min；1mol/L NaOH or 1mol/LHCL is used to adjust pH value as 5~6.
3. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, which is characterized in that the Chinese chestnut deadwood is fallen
Leaf water cooking liquid are as follows: use boiling water boiling Chinese chestnut 30~50min of dry branches and fallen leaves, the filtrate being obtained by filtration after standing.
4. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, which is characterized in that the rhizosphere soil water
Extract are as follows: use boiling water boiling Chinese chestnut 30~50min of rhizosphere soil, the filtrate being obtained by filtration after standing.
5. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 1, which is characterized in that the Chinese chestnut mycorhiza
Mushroom entity further includes pretreatment operation before tissue separation, is the Wild Chinese Chestnut bacterium for fresh, sturdy, the medium maturation that will be acquired
Root fungus fructification is first washed through sterilized antistaling agent, then through 75% alcohol soaking disinfection, most afterwards through sterile water wash.
6. a kind of Chinese chestnut VA Mycorrhizal Fungi artificial culture method according to claim 5, which is characterized in that the sterilized antistaling
Agent are as follows: the citric acid solution that the Vitamin C acid solution and mass concentration that mass concentration is 0.02~0.06% are 0.01~0.03% is by body
Product is than (1~5): the mixed liquor that (1~3) is made into.
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