CN106171515B - A kind of symbiosis method for building up of Applying Ectomycorrhizal Fungi and pine genus plant - Google Patents
A kind of symbiosis method for building up of Applying Ectomycorrhizal Fungi and pine genus plant Download PDFInfo
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- CN106171515B CN106171515B CN201610532499.XA CN201610532499A CN106171515B CN 106171515 B CN106171515 B CN 106171515B CN 201610532499 A CN201610532499 A CN 201610532499A CN 106171515 B CN106171515 B CN 106171515B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
- A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
- A01G9/029—Receptacles for seedlings
- A01G9/0299—Handling or transporting of soil blocks or seedlings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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Abstract
The present invention relates to two kinds of Applying Ectomycorrhizal Fungis such as wizened bacterium, matsutake several pine genus plant seedling are infected with a kind of method to form mycorhiza, belongs to artificial exogenous mycorrhizal fungi and infects nursery application technology and technical field of agricultural cultivation.Step of the invention includes the acquisition and culture of (1) pine genus plant aseptic seed；(2) selection and processing of cultivation matrix；(3) selection and processing of seedling culture hole plate；(4) preparation before infecting processing；(5) that dries up bacterium and matsutake infects processing；(6) seedling management of infecting that treated.It infects and is successfully formed the seedling of mycorhiza than the seedling robust growth that does not infect, leaf color is dark green, true leaf is grown in advance, disease resistance enhancing, it has innovatively been successfully established under controlled condition, wizened bacterium, matsutake and several pine genus plants symbiosis, case study can be not only provided for the symbiosis of plant and fungi in the research of Applying Ectomycorrhizal Fungi, and established solid scientific basic for its realization artificial cultivation.
Description
Technical field
The present invention relates to the Applying Ectomycorrhizal Fungis such as wizened bacterium, matsutake to infect to form the one of mycorhiza to several pine genus plant seedling
Kind method, belongs to artificial exogenous's VA Mycorrhizal Fungi and infects nursery application technology and technical field of agricultural cultivation.
Background technique
Wizened bacterium (ThelephoraganbajunZang) and matsutake (also known as Trichotoma matsutake) [Tricholomamatsutake
(lto et lmai) Singer] be two kinds of famous delicious wild mushrooms, nutritive value and medical value with higher, it is deep by
People's likes, as the improvement of people's living standards, supply falls short of demand currently on the market, expensive, bacterium fresh weight of drying up is most expensive
Up to every 1800 yuan of 1 Kg price.Wizened bacterium aromatic flavour, grow it is tender it is fragrant and sweet, meat is tough and tensile, dry, aftertaste mellowness is long, chews taste
It is infinite, and there is one to exactly like the dry strong fragrance of beefbacon, beefbacon is dry to be known as drying up by the local masses, thus this bacterium is gained the name
Wizened bacterium.Wizened bacterium is containing there are many amino acid, protein, vegetal pole horn of plenties；It is fragrant and mellow with fragrance, meat is tender and crisp, chew taste without
It thoroughly, is the top grade in wild edible fungus the characteristics of heterorexia Jiamei.
Matsutake has unique strong fragrance, is most rare rare natural medicinal fungi in the world, is known as " in bacterium
King ", be China's second level protection species in imminent danger；It is rich in protein, there is 18 kinds of amino acid, 14 kinds of micro elements needed by human,
49 kinds of active nutrients, 5 kinds of unsaturated fatty acids, nucleic acid derivative, the rare elements such as peptide matters.Separately contain 3 kinds of preciousnesses
Active material, be tricholoma matsutake polysaccharide, the unique cancer-resisting substance of tricholoma matsutake and the whole world --- matsutakealcohol respectively.Pine
Young pilose antler is regarded as " refreshing bacterium " in Japan.Japanese gets used to autumn edible matsutake cooking, believes in " mending shape with shape ", and having for food is strong smart
The effects of kidney tonifying, brain tonic and intelligence development and anticancer.
Wizened two kinds of wild mushrooms of bacterium and matsutake belong to typical Applying Ectomycorrhizal Fungi, not can be carried out artificial cultivation still at present,
It is difficult to develop and use, the growth and development of fructification is in close relations with aulophyte pine genus plant.Meanwhile dry up bacterium and matsutake
Facilitation is played to the growth of pine genus plant in several pine genus plant seedling raising process, pine genus plant well developed root system can be made, it is raw
Length is vigorous, and then accelerates the speed of growth of nursery stock.Realize that producing strains are infected in the success of wizened bacterium and matsutake and pine genus plant root
Root establishes symbiosis, not only can provide case study for the symbiosis of plant and fungi in the research of Applying Ectomycorrhizal Fungi,
And realize that artificial cultivation has established solid scientific basic for it.
Summary of the invention
The purpose of the present invention is to provide the Applying Ectomycorrhizal Fungis such as wizened bacterium, matsutake to one kind of several pine genus plant seedling
Exhaust processes establish the symbiosis of Applying Ectomycorrhizal Fungi Yu several pine genus plants, promote the growth of nursery stock, and artificial to realize
It cultivates wizened bacterium, matsutake etc. and establishes solid scientific basic.
The present invention adopts the following technical scheme that realization:
A kind of symbiosis method for building up of Applying Ectomycorrhizal Fungi and pine genus plant, method of the invention include following six
Step: (1) Pinus (Pinus) plant aseptic seed acquisition and culture；(2) selection and processing of cultivation matrix；(3) nursery cave
The selection and processing of disk；(4) preparation before infecting processing；(5) Applying Ectomycorrhizal Fungi infects processing；(6) infect that treated
Seedling management.
Applying Ectomycorrhizal Fungi of the present invention are as follows: when wizened bacterium；The pine genus plant are as follows: pinus yunnanensis
(Pinusyunnanensis) or pinus khasys (Pinuskesiya var. langbianensis) or Huashan pine
(Pinusarmandii).
Applying Ectomycorrhizal Fungi of the present invention are as follows: when matsutake；The pine genus plant are as follows: pinus yunnanensis
(Pinusyunnanensis) or Pinuns densata (Pinusdensata) or Japanese red pine (Pinusdensiflora).
The content of step (1) of the present invention is the acquisition and culture of (1) pine genus plant aseptic seed: selection it is full, without disease pest
Harmful pine genus plant seed is placed in 75% ethanol solution, is rocked and is impregnated 30 s, takes out seed, with aseptic water washing 3 times, often
Secondary 30 s of washing time；Seed is placed in 0.1%HgCl again2It in solution, rocks and impregnates 8 min, take out seed, use sterile water
It rinses 6 times, each 1 min of washing time；The seed disinfected is placed in the sterile culture for being lined with filter paper and sterile water
It is cultivated in ware.Culture environment: 2500 ~ 3000 Lux of illumination, temperature 25(± 1) DEG C.After 10 ~ 15 days, radicle growth to 5 ~ 15
It is spare when mm.Wherein, when spare radicle when using radicle growth to 11 ~ 15 mm, the later period infect success rate up to 85% with
On；When spare radicle when using radicle growth to 5 ~ 10 mm, the later period infects success rate up to 92% or more.Through test result
Show: when radicle growth is to 5 ~ 10 mm, wizened bacterium infects success rate up to 95.2%, and matsutake infects success rate up to 92.1%；Embryo
When root is less than 5 mm, the success rate that infects of wizened bacterium and matsutake is respectively less than 30%；When radicle growth is to 11 ~ 15 mm, bacterium of drying up
Success rate is infected 85.7% or more, and matsutake infects success rate 85.1% or more；When radicle is greater than 15 mm, dry up bacterium and pine
The fine and soft success rate that infects is respectively less than 50%.
The content of step (2) to step (4) of the present invention is the selection and processing of (2) cultivation matrix: matrix is selected less than 3
The fine grained vermiculite of mm；By fine grained vermiculite with originally water saturates, 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot,
After cooling 24 h, then it is placed in 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot, it is spare after cooling.
(3) selection and processing of seedling culture hole plate: seedling culture hole plate selection is deepened to thicken 50 hole Tree growing containers, bore open 5
× 5 cm, deep 8.5 cm；The seedling culture hole plate is placed in 75% ethanol solution, rocks and impregnates 10 min, take out the nursery cave
Disk, it is spare with aseptic water washing 1 time.
(4) the processed fine grained vermiculite of step (2) preparation before infecting processing: is placed in step (3) is processed to educate
It in seedling disk point, compresses in fact, depth standards are advisable with vermiculite from 0.5 cm of aperture, the centre diameter in the disk cave equipped with vermiculite
The hole of 11 ~ 1.5 cm of depth of metal bastinade of 0.5 cm；It is spare to prepare some sterile petri dish again simultaneously.
The content of step (5) of the present invention is that (5) infect processing: such as by Applying Ectomycorrhizal Fungi: wizened bacterium or tricholoma matsutake mycelium
It is placed in different sterile petri dish, then step (1) is handled into cultured radicle growth to 5 ~ 15 mm seeds and has been placed in bacterium
In the culture dish of filament, jiggle after 10 ~ 15 min that (purpose jiggled is the embryo for making mycelium Yu pine genus plant seed
Root comes into full contact with and is adhered on radicle, and success rate is infected in raising), the seed infected is planted ready in step (4)
In seedling-culturing dish cave equipped with fine grained vermiculite, 1, every cave, radicle covers seed downward, with a small amount of fine grained vermiculite, then with sterile
Water saturates vermiculite.
The content of step (6) of the present invention is, (6) seedling management of infecting that treated: by step (5) infect it is processed simultaneously
The pine genus plant seed planted is placed in Small plastic shed together with seedling culture hole plate, before placement, is killed by spraying in Small plastic shed with 75% ethyl alcohol
Bacterium processing.Arch can use the bamboo chip of wide 2~3 cm, and length and width is general to encircle depending on sagitta and the size of seedling culture hole plate
High 60~80 cm, the white film of plastic film 0.08 mm thickness；The sunshade net that one layer of light transmittance is 50% is covered again, is reduced
Rising dehydration is moved back except sunshade net for 40 days；Every 3~5 days check an arched shed in pine genus plant seedling subenvironment, humidity with
Having droplet to be advisable on film without landing, (humidity is 60 ~ 80% at this time, which can seriously affect anaphase, and humidity is lower than
Infection rate is low when 60%, and rotten seedling is easy when higher than 80%, and survival rate is low, and it is low to also result in infection rate).If it is overly moist in arched shed or
Temperature is excessively high, to take off film ventilation at both ends, and overdrying waters in time, but should not be overly moist in order to avoid rotten seedling.Start to grow after 70 days true
Leaf removes Small plastic shed at this time, forms exotrophic mycorrhiza after 80~90 days.Formed exotrophic mycorrhiza infect success rate up to 85% with
On.
It is currently preferred, it is spare when radicle growth is to 5 ~ 10 mm in step (1).
The seedling of exotrophic mycorrhiza is successfully formed than the seedling robust growth that does not infect, leaf color the beneficial effects of the present invention are: infecting
It is dark green, grow true leaf in advance, disease resistance enhancing, be innovatively successfully established under controlled condition Applying Ectomycorrhizal Fungi with it is several
The symbiosis of Pinus is such as: wizened bacterium and pinus yunnanensis, pinus khasys, Huashan pine and matsutake and pinus yunnanensis, Pinuns densata, Japanese red pine are total to
Raw body system provides the research method of symbiosis under the conditions of Applying Ectomycorrhizal Fungi and plant experimental, is other external bacterium of large size
The research of mycorrhiza fungi and aulophyte provides method reference, and to realize that it is solid that the wizened bacterium of artificial cultivation, matsutake have been established
Scientific basic.
Specific embodiment
Embodiment below is only to further explanation of the invention, is not the limit to technical solution of the present invention protection scope
System.
A kind of symbiosis method for building up of Applying Ectomycorrhizal Fungi and pine genus plant, method of the invention include following six
Step: (1) acquisition and culture of pine genus plant aseptic seed；(2) selection and processing of cultivation matrix；(3) choosing of seedling culture hole plate
It selects and handles；(4) preparation before infecting processing；(5) Applying Ectomycorrhizal Fungi infects processing；(6) the seedling stage pipe that infects that treated
Reason.
Applying Ectomycorrhizal Fungi of the present invention are as follows: when wizened bacterium；The pine genus plant are as follows: pinus yunnanensis or pinus khasys or
Huashan pine.
Applying Ectomycorrhizal Fungi of the present invention are as follows: when matsutake；The pine genus plant are as follows: pinus yunnanensis or Pinuns densata are red
Pine.
The content of step (1) of the present invention is the acquisition and culture of (1) pine genus plant aseptic seed: selection it is full, without disease pest
Harmful pine genus plant seed is placed in 75% ethanol solution, is rocked and is impregnated 30 s, takes out seed, with aseptic water washing 3 times, often
Secondary 30 s of washing time；Seed is placed in 0.1%HgCl again2It in solution, rocks and impregnates 8 min, take out seed, use sterile water
It rinses 6 times, each 1 min of washing time；The seed disinfected is placed in the sterile culture for being lined with filter paper and sterile water
It is cultivated in ware.Culture environment: 2500 ~ 3000 Lux of illumination, temperature 25(± 1) DEG C.After 10 ~ 15 days, radicle growth to 5 ~ 15
It is spare when mm.Wherein, when spare radicle when using radicle growth to 11 ~ 15 mm, the later period infect success rate up to 85% with
On；When spare radicle when using radicle growth to 5 ~ 10 mm, the later period infects success rate up to 92% or more.Through test result
Show: when radicle growth is to 5 ~ 10 mm, wizened bacterium infects success rate 95.2%, and matsutake infects success rate 92.1%；Embryo
When root is less than 5 mm, the success rate that infects of wizened bacterium and matsutake is respectively less than 30%, only 28.1% or so；Radicle growth is to 11 ~ 15
When mm, wizened bacterium infects success rate 85.7%, and matsutake infects success rate 85.1%；When radicle is greater than 15 mm, bacterium of drying up
50% is respectively less than with the success rate that infects of matsutake.
The content of step (2) to step (4) of the present invention is the selection and processing of (2) cultivation matrix: matrix is selected less than 3
The fine grained vermiculite of mm；By fine grained vermiculite with originally water saturates, 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot,
After cooling 24 h, then it is placed in 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot, it is spare after cooling.
(3) selection and processing of seedling culture hole plate: seedling culture hole plate selection is deepened to thicken 50 hole Tree growing containers, bore open 5
× 5 cm, deep 8.5 cm；The seedling culture hole plate is placed in 75% ethanol solution, rocks and impregnates 10 min, take out the nursery cave
Disk, it is spare with aseptic water washing 1 time.
(4) the processed fine grained vermiculite of step (2) preparation before infecting processing: is placed in step (3) is processed to educate
It in seedling disk point, compresses in fact, depth standards are advisable with vermiculite from 0.5 cm of aperture, the centre diameter in the disk cave equipped with vermiculite
The hole of 11 ~ 1.5cm of depth of metal bastinade of 0.5 cm；It is spare to prepare some sterile petri dish again simultaneously.
The content of step (5) of the present invention is that (5) infect processing: such as by Applying Ectomycorrhizal Fungi: wizened bacterium or tricholoma matsutake mycelium
It is placed in different sterile petri dish, then step (1) is handled into cultured radicle growth to 5 ~ 15 mm seeds and has been placed in bacterium
In the culture dish of filament, jiggle after 10 ~ 15 min that (purpose jiggled is the embryo for making mycelium Yu pine genus plant seed
Root comes into full contact with and is adhered on radicle, and success rate is infected in raising), the seed infected is planted ready in step (4)
In seedling-culturing dish cave equipped with fine grained vermiculite, 1, every cave, radicle covers seed downward, with a small amount of fine grained vermiculite, then with sterile
Water saturates vermiculite.
The content of step (6) of the present invention is, (6) seedling management of infecting that treated: by step (5) infect it is processed simultaneously
The pine genus plant seed planted is placed in Small plastic shed together with seedling culture hole plate, before placement, is killed by spraying in Small plastic shed with 75% ethyl alcohol
Bacterium processing.Arch can use the bamboo chip of wide 2~3 cm, and length and width is general to encircle depending on sagitta and the size of seedling culture hole plate
High 60~80 cm, the white film of plastic film 0.08 mm thickness；The sunshade net that one layer of light transmittance is 50% is covered again, is reduced
Rising dehydration is moved back except sunshade net for 40 days；Every 3~5 days check an arched shed in pine genus plant seedling subenvironment, humidity with
Having droplet to be advisable on film without landing, (humidity is 60 ~ 80% at this time, which can seriously affect anaphase, and humidity is lower than
Infection rate is low when 60%, and rotten seedling is easy when higher than 80%, and survival rate is low, and it is low to also result in infection rate).If it is overly moist in arched shed or
Temperature is excessively high, to take off film ventilation at both ends, and overdrying waters in time, but should not be overly moist in order to avoid rotten seedling.Start to grow after 70 days true
Leaf removes Small plastic shed at this time, forms exotrophic mycorrhiza after 80~90 days.Formed exotrophic mycorrhiza infect success rate up to 85% with
On.
It is currently preferred, it is spare when radicle growth is to 5 ~ 10 mm in step (1).
Wizened bacterium infects embodiment one
(1) acquisition and culture of pinus yunnanensis aseptic seed: full, no disease and pests harm the pinus yunnanensis vegetable seeds of selection is placed in
It in 75% ethanol solution, rocks and impregnates 30 s, take out seed, with aseptic water washing 3 times, each 30 s of washing time；Again by seed
It is placed in 0.1%HgCl2It in solution, rocks and impregnates 8 min, take out seed, with aseptic water washing 6 times, each washing time 1
min；The seed disinfected is placed in the sterile petri dish for being lined with filter paper and sterile water and is cultivated.Culture environment: illumination
2500 ~ 3000 Lux, temperature 25(± 1) DEG C.It is spare when radicle growth is to 5 mm after 10 ~ 15 days.
(2) selection and processing of cultivation matrix: matrix selects the fine grained vermiculite less than 3 mm；Fine grained vermiculite is used certainly
Carry out water saturates, after 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot, cooling 24 h, then is placed in 121 DEG C of high pressure and goes out
30 min of sterilization in bacterium pot, it is spare after cooling.
(3) selection and processing of seedling culture hole plate: seedling culture hole plate selection is deepened to thicken 50 hole Tree growing containers, bore open 5
× 5 cm, deep 8.5 cm；Seedling culture hole plate is placed in 75% ethanol solution, rocks and impregnates 10 min, takes out seedling culture hole plate, is used
Aseptic water washing 1 time spare.
(4) the processed fine grained vermiculite of step (2) preparation before infecting processing: is placed in step (3) is processed to educate
It in seedling disk point, compresses in fact, depth standards are advisable with vermiculite from 0.5 cm of aperture, the centre diameter in the disk cave equipped with vermiculite
The hole of 1 depth 1cm of metal bastinade of 0.5 cm；It is spare to prepare some sterile petri dish again simultaneously.
(5) it infects processing: Thelephora ganbajun mycelium is placed in sterile petri dish, then step (1) is handled into cultured embryo
Root growth to 5mm seed has been placed in mycelial culture dish, and after jiggling 10 ~ 15min, the seed infected is planted
In the ready seedling-culturing dish cave equipped with fine grained vermiculite of step (4), 1, every cave, radicle downward, with a small amount of fine grained vermiculite
Seed is covered, then with sterile water saturates vermiculite.
(6) seedling management of infecting that treated: by step (5) infect pinus yunnanensis seed that is processed and having planted together with
Seedling culture hole plate is placed in Small plastic shed, before placement, is handled in Small plastic shed with 75% ethyl alcohol spray disinfection.Arch can use wide 2~3 cm
Bamboo chip, length and width is depending on sagitta and the size of seedling culture hole plate, general 60~80 cm of sagitta, plastic film use
The white film of 0.08 mm thickness；The sunshade net that one layer of light transmittance is 50% is covered again, is reduced rising dehydration, is moved back within 40 days except screening
Screened postive；Check within every 3~5 days that the subenvironment of pinus yunnanensis seedling in an arched shed, humidity are without sliding to there is droplet on film
Preferably.If overly moist in arched shed or temperature is excessively high, film ventilation is taken off at both ends, overdrying waters in time, but should not be overly moist in order to avoid rotten seedling.70
Start to grow true leaf after it, removes Small plastic shed at this time, form exotrophic mycorrhiza after 80 days.Form exotrophic mycorrhiza infects success rate
Up to 86.3%.
Wizened bacterium infects embodiment two
(1) acquisition and culture of pinus yunnanensis aseptic seed: full, no disease and pests harm the pinus yunnanensis vegetable seeds of selection is placed in
It in 75% ethanol solution, rocks and impregnates 30 s, take out seed, with aseptic water washing 3 times, each 30 s of washing time；Again by seed
It is placed in 0.1%HgCl2It in solution, rocks and impregnates 8 min, take out seed, with aseptic water washing 6 times, each washing time 1
min；The seed disinfected is placed in the sterile petri dish for being lined with filter paper and sterile water and is cultivated.Culture environment: illumination
2500 ~ 3000 Lux, temperature 25(± 1) DEG C.It is spare when radicle growth is to 10 mm after 10 ~ 15 days.
(2) selection and processing of cultivation matrix: matrix selects the fine grained vermiculite less than 3 mm；Fine grained vermiculite is used certainly
Carry out water saturates, after 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot, cooling 24 h, then is placed in 121 DEG C of high pressure and goes out
30 min of sterilization in bacterium pot, it is spare after cooling.
(3) selection and processing of seedling culture hole plate: seedling culture hole plate selection is deepened to thicken 50 hole Tree growing containers, bore open 5
× 5 cm, deep 8.5 cm；Seedling culture hole plate is placed in 75% ethanol solution, rocks and impregnates 10 min, takes out seedling culture hole plate, is used
Aseptic water washing 1 time spare.
(4) the processed fine grained vermiculite of step (2) preparation before infecting processing: is placed in step (3) is processed to educate
It in seedling disk point, compresses in fact, depth standards are advisable with vermiculite from 0.5 cm of aperture, the centre diameter in the disk cave equipped with vermiculite
The hole of 1 depth 1cm of metal bastinade of 0.5 cm；It is spare to prepare some sterile petri dish again simultaneously.
(5) it infects processing: Thelephora ganbajun mycelium is placed in sterile petri dish, then step (1) is handled into cultured embryo
Root growth to 10 mm seeds have been placed in mycelial culture dish, and after jiggling 10 min, the seed infected is planted
In the ready seedling-culturing dish cave equipped with fine grained vermiculite of step (4), 1, every cave, radicle downward, with a small amount of fine grained vermiculite
Seed is covered, then with sterile water saturates vermiculite.
(6) seedling management of infecting that treated: by step (5) infect pinus yunnanensis seed that is processed and having planted together with
Seedling culture hole plate is placed in Small plastic shed, before placement, is handled in Small plastic shed with 75% ethyl alcohol spray disinfection.Arch can use wide 2~3 cm
Bamboo chip, length and width is depending on sagitta and the size of seedling culture hole plate, general 60~80 cm of sagitta, plastic film use
The white film of 0.08 mm thickness；The sunshade net that one layer of light transmittance is 50% is covered again, is reduced rising dehydration, is moved back within 40 days except screening
Screened postive；Check within every 3~5 days that the subenvironment of pinus yunnanensis seedling in an arched shed, humidity are without sliding to there is droplet on film
Preferably.If overly moist in arched shed or temperature is excessively high, film ventilation is taken off at both ends, overdrying waters in time, but should not be overly moist in order to avoid rotten seedling.70
Start to grow true leaf after it, removes Small plastic shed at this time, form exotrophic mycorrhiza after 80 days.Form exotrophic mycorrhiza infects success rate
Up to 95.2%.
Wizened bacterium infects embodiment three
(1) acquisition and culture of pinus yunnanensis aseptic seed: full, no disease and pests harm the pinus yunnanensis vegetable seeds of selection is placed in
It in 75% ethanol solution, rocks and impregnates 30 s, take out seed, with aseptic water washing 3 times, each 30 s of washing time；Again by seed
It is placed in 0.1%HgCl2It in solution, rocks and impregnates 8 min, take out seed, with aseptic water washing 6 times, each washing time 1
min；The seed disinfected is placed in the sterile petri dish for being lined with filter paper and sterile water and is cultivated.Culture environment: illumination
2500 ~ 3000 Lux, temperature 25(± 1) DEG C.It is spare when radicle growth is to 15 mm after 10 ~ 15 days.
(2) selection and processing of cultivation matrix: matrix selects the fine grained vermiculite less than 3 mm；Fine grained vermiculite is used certainly
Carry out water saturates, after 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot, cooling 24 h, then is placed in 121 DEG C of high pressure and goes out
30 min of sterilization in bacterium pot, it is spare after cooling.
(3) selection and processing of seedling culture hole plate: seedling culture hole plate selection is deepened to thicken 50 hole Tree growing containers, bore open 5
× 5 cm, deep 8.5 cm；Seedling culture hole plate is placed in 75% ethanol solution, rocks and impregnates 10 min, takes out seedling culture hole plate, is used
Aseptic water washing 1 time spare.
(4) the processed fine grained vermiculite of step (2) preparation before infecting processing: is placed in step (3) is processed to educate
It in seedling disk point, compresses in fact, depth standards are advisable with vermiculite from 0.5 cm of aperture, the centre diameter in the disk cave equipped with vermiculite
The hole of 1 depth 1cm of metal bastinade of 0.5 cm；It is spare to prepare some sterile petri dish again simultaneously.
(5) it infects processing: Thelephora ganbajun mycelium is placed in sterile petri dish, then step (1) is handled into cultured embryo
Root growth to 15 mm seeds have been placed in mycelial culture dish, and after jiggling 15 min, the seed infected is planted
In the ready seedling-culturing dish cave equipped with fine grained vermiculite of step (4), 1, every cave, radicle downward, with a small amount of fine grained vermiculite
Seed is covered, then with sterile water saturates vermiculite.
(6) seedling management of infecting that treated: by step (5) infect pinus yunnanensis seed that is processed and having planted together with
Seedling culture hole plate is placed in Small plastic shed, before placement, is handled in Small plastic shed with 75% ethyl alcohol spray disinfection.Arch can use wide 2~3 cm
Bamboo chip, length and width is depending on sagitta and the size of seedling culture hole plate, general 60~80 cm of sagitta, plastic film use
The white film of 0.08 mm thickness；The sunshade net that one layer of light transmittance is 50% is covered again, is reduced rising dehydration, is moved back within 40 days except screening
Screened postive；Check within every 3~5 days that the subenvironment of pinus yunnanensis seedling in an arched shed, humidity are without sliding to there is droplet on film
Preferably.If overly moist in arched shed or temperature is excessively high, film ventilation is taken off at both ends, overdrying waters in time, but should not be overly moist in order to avoid rotten seedling.70
Start to grow true leaf after it, removes Small plastic shed at this time, form exotrophic mycorrhiza after 80 days.Form exotrophic mycorrhiza infects success rate
Up to 85.7%.
Wizened bacterium infects example IV
The same wizened bacterium embodiment one of the content and parameter of step (1) to step (6), pine genus plant is pinus khasys, last shape
Success rate is infected up to 88.7% at exotrophic mycorrhiza.
Wizened bacterium infects embodiment five
The same wizened bacterium embodiment two of the content and parameter of step (1) to step (6), pine genus plant is pinus khasys, last shape
Success rate is infected up to 97.1% at exotrophic mycorrhiza.
Wizened bacterium infects embodiment six
The same wizened bacterium embodiment three of the content and parameter of step (1) to step (6), pine genus plant is pinus khasys, last shape
Success rate is infected up to 86.4% at exotrophic mycorrhiza.
Wizened bacterium infects embodiment seven
The same wizened bacterium embodiment one of the content and parameter of step (1) to step (6), pine genus plant is Huashan pine, last shape
Success rate is infected up to 85.4% at exotrophic mycorrhiza.
Wizened bacterium infects embodiment eight
The same wizened bacterium embodiment two of the content and parameter of step (1) to step (6), pine genus plant is Huashan pine, last shape
Success rate is infected up to 91.3% at exotrophic mycorrhiza.
Wizened bacterium infects embodiment nine
The same wizened bacterium embodiment three of the content and parameter of step (1) to step (6), pine genus plant is Huashan pine, last shape
Success rate is infected up to 85.1% at exotrophic mycorrhiza.
Another: for radicle less than 5 mm, the success rate that infects of pinus khasys and Huashan pine is below 30%；Radicle is greater than 15mm, Simao
Pine infects success rate and is below 50%, and Huashan pine is lower than 40%.
Matsutake infects embodiment one
The same wizened bacterium embodiment one of the content and parameter of step (1) to step (6), pine genus plant is pinus yunnanensis, last shape
Success rate is infected up to 85.8% at exotrophic mycorrhiza.
Matsutake infects embodiment two
The same wizened bacterium embodiment two of the content and parameter of step (1) to step (6), pine genus plant is pinus yunnanensis, last shape
Success rate is infected up to 92.1% at exotrophic mycorrhiza.
Matsutake infects embodiment three
The same wizened bacterium embodiment three of the content and parameter of step (1) to step (6), pine genus plant is pinus yunnanensis, last shape
Success rate is infected up to 85.1% at exotrophic mycorrhiza.
Matsutake infects example IV
The same wizened bacterium embodiment one of the content and parameter of step (1) to step (6), pine genus plant is Pinuns densata, last shape
Success rate is infected up to 87.5% at exotrophic mycorrhiza.
Matsutake infects embodiment five
The same wizened bacterium embodiment two of the content and parameter of step (1) to step (6), pine genus plant is Pinuns densata, last shape
Success rate is infected up to 96.2% at exotrophic mycorrhiza.
Matsutake infects embodiment six
The same wizened bacterium embodiment three of the content and parameter of step (1) to step (6), pine genus plant is Pinuns densata, last shape
Success rate is infected up to 85.7% at exotrophic mycorrhiza.
Matsutake infects embodiment seven
The same wizened bacterium embodiment one of the content and parameter of step (1) to step (6), pine genus plant is Japanese red pine, is eventually formed
Exotrophic mycorrhiza infects success rate up to 86.1%.
Matsutake infects embodiment eight
The same wizened bacterium embodiment two of the content and parameter of step (1) to step (6), pine genus plant is Japanese red pine, is eventually formed
Exotrophic mycorrhiza infects success rate up to 93.3%.
Matsutake infects embodiment nine
The same wizened bacterium embodiment three of the content and parameter of step (1) to step (6), pine genus plant is Japanese red pine, is eventually formed
Exotrophic mycorrhiza infects success rate up to 85.2%.
Another: for radicle less than 5 mm, the success rate that infects of Pinuns densata and Japanese red pine is below 30%；Radicle is greater than 15mm, Pinuns densata
50% is below with the success rate that infects of Japanese red pine.
Claims (4)
1. the symbiosis method for building up of a kind of Applying Ectomycorrhizal Fungi and pine genus plant, which is characterized in that this method includes following
Six steps: (1) acquisition and culture of pine genus plant aseptic seed；(2) selection and processing of cultivation matrix；(3) seedling culture hole plate
Selection and processing；(4) preparation before infecting processing；(5) Applying Ectomycorrhizal Fungi infects processing；(6) seedling that infects that treated
Period management；
The content of step (1) is the acquisition and culture of pine genus plant aseptic seed: full, no disease and pests harm the pine genus plant of selection
Seed is placed in 75% ethanol solution, is rocked and is impregnated 30 s, and seed, with aseptic water washing 3 times, each washing time 30 are taken out
s；Seed is placed in 0.1%HgCl again2It in solution, rocks and impregnates 8 min, take out seed, with aseptic water washing 6 times, rush every time
Wash 1 min of time；The seed disinfected is placed in the sterile petri dish for being lined with filter paper and sterile water and is cultivated；Cultivate ring
Border: 2500 ~ 3000 Lux of illumination, 25 ± 1 DEG C of temperature；It is spare when radicle growth is to 5 ~ 15 mm after 10 ~ 15 days；
The content of step (2) is the selection and processing of cultivation matrix: matrix selects the fine grained vermiculite less than 3 mm；By thin
Grain vermiculite is with originally water saturates, 30 min of sterilization in 121 DEG C of high-pressure sterilizing pot, after cooling 24 h, then is placed in 121
DEG C high-pressure sterilizing pot in 30 min of sterilization, it is spare after cooling；
The content of step (3) is the selection and processing of seedling culture hole plate: seedling culture hole plate selection is deepened to thicken 50 hole forest seedling growth caves
Disk, 5 × 5 cm of bore open, deep 8.5 cm；The seedling culture hole plate is placed in 75% ethanol solution, rocks and impregnates 10 min, take
The seedling culture hole plate out, it is spare with aseptic water washing 1 time；
The content of step (4) is the preparation infected before processing: the processed fine grained vermiculite of step (2) is placed at step (3)
In the seedling-culturing dish cave managed, compress in fact, depth standards: vermiculite is from 0.5 cm of aperture；It is standby to prepare some sterile petri dish again simultaneously
With；
The content of step (5) is that Applying Ectomycorrhizal Fungi infects processing: Applying Ectomycorrhizal Fungi mycelium being placed in different sterile
In culture dish, then step (1) is handled into cultured radicle growth to 5 ~ 15 mm seeds and has been placed in mycelial culture dish
In, after jiggling 10 ~ 15 min, by the seed infected plantation in ready the educating equipped with fine grained vermiculite of step (4)
In seedling disk point, 1, every cave, radicle covers seed downward, with a small amount of fine grained vermiculite, then with sterile water saturates vermiculite；
The content of step (6) is seedling management of infecting that treated: step (5) is infected Pinus that is processed and having planted
Vegetable seeds is placed in Small plastic shed together with seedling culture hole plate, before placement, is handled in Small plastic shed with 75% ethyl alcohol spray disinfection；Arch
With the bamboo chip of wide 2~3 cm, length and width is depending on sagitta and the size of seedling culture hole plate, 60~80 cm of sagitta, and plastics are thin
The white film of film 0.08 mm thickness；The sunshade net that one layer of light transmittance is 50% is covered again, is reduced rising dehydration, is moved back within 40 days
Except sunshade net；Check within every 3~5 days the subenvironment of pine genus plant seedling in an arched shed, humidity on film to have droplet without cunning
Falling is advisable；Start to grow true leaf after 70 days, removes Small plastic shed at this time, form exotrophic mycorrhiza after 80~90 days.
2. the symbiosis method for building up of a kind of Applying Ectomycorrhizal Fungi according to claim 1 and pine genus plant, feature
It is, the Applying Ectomycorrhizal Fungi are as follows: when wizened bacterium；The pine genus plant are as follows: pinus yunnanensis or pinus khasys or Huashan pine.
3. the symbiosis method for building up of a kind of Applying Ectomycorrhizal Fungi according to claim 1 and pine genus plant, feature
It is, the Applying Ectomycorrhizal Fungi are as follows: when matsutake；The pine genus plant are as follows: pinus yunnanensis or Pinuns densata or Japanese red pine.
4. the symbiosis method for building up of a kind of Applying Ectomycorrhizal Fungi according to claim 1 and pine genus plant, feature
It is, it is spare when radicle growth is to 5 ~ 10 mm in step (1).
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| JP2022503893A (en) | 2018-10-18 | 2022-01-12 | マイコワークス， インコーポレイテッド | Mycelium growth bed |
| CN110915546B (en) * | 2019-12-17 | 2021-11-02 | 云南大学 | Method for promoting plant to form ectomycorrhiza |
| CN117652343B (en) * | 2024-01-31 | 2024-04-16 | 云南上智科技有限公司 | Mycorrhiza synthesis method of target plant |
| CN117887595B (en) * | 2024-03-14 | 2024-05-17 | 云南省林业和草原科学院 | Phlebopus portentosus YAFMF008, separation method thereof and mycorrhizal seedling infection method |
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