CN105917972A - Synthesis method of ectotrophic mycorrhiza - Google Patents
Synthesis method of ectotrophic mycorrhiza Download PDFInfo
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- CN105917972A CN105917972A CN201610516887.9A CN201610516887A CN105917972A CN 105917972 A CN105917972 A CN 105917972A CN 201610516887 A CN201610516887 A CN 201610516887A CN 105917972 A CN105917972 A CN 105917972A
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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
- A01G18/10—Mycorrhiza; Mycorrhizal associations
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Abstract
The invention discloses a rapid synthesis method of ectotrophic mycorrhiza. The method comprises the following steps: taking a fungus cap tissue of an ectotrophic mycorrhiza fungus under an aseptic condition by using a tissue separation method, then inoculating the fungus cap tissue into an MMN culture medium for dark culture, purely culturing mycelia obtained by separating in a mycorrhiza synthesis bag, then inoculating cultured mycelia to tree seedlings in a seedling age of 2-3 months, then putting the tree seedlings in an illumination incubator, and culturing for half of one month to three months to obtain mycorrhiza. The method is capable of conveniently screening the optimal tree seedlings in a paragenetic combination with the ectotrophic mycorrhiza fungus; the period is shortened; the efficiency is improved; the cost is reduced; the artificial cultivation of the mycorrhiza type edible fungi can be guided.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of exotrophic mycorrhiza synthetic technology, particularly relate to a kind of the highest
The exotrophic mycorrhiza synthetic technology of effect.
Background technology
After Applying Ectomycorrhizal Fungi forms exotrophic mycorrhiza with trees, make exotrophic mycorrhiza have various functions, such as promote that forest is raw
Long, strengthen alimentation, enhance disease resistance, drought resistance and improve Soil structure, for protection bio-diversity and
Maintaining ecological balance has positive meaning.Research in recent years there are some synthesize about Lactrarius hatsudake and Delicious lactarius mycorhiza
Method, but the cycle is generally the longest and process is complicated, the invention discloses a kind of exotrophic mycorrhiza and synthesizes fast and efficiently
Method, the artificial culture for Applying Ectomycorrhizal Fungi is laid a good foundation.
Summary of the invention
It is an object of the invention to provide a kind of exotrophic mycorrhiza fast synthesis method, in order to screen the suitableeest seeds with external
Mycorrhizal fungi paragenetic association, shortens the cycle, improves efficiency, reduces cost, instructs artificial culture.
In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme:
The synthetic method of a kind of exotrophic mycorrhiza, comprises the following steps:
(1) use tissue isolation, aseptic under conditions of, take the cap bacterial context of Applying Ectomycorrhizal Fungi and be inoculated in MMN training
Support lucifuge on base to cultivate；
(2) the symbiosis seeds seedling of the mycelium pure culture of step (1) isolated with three months seedling ages is inoculated in mycorhiza
Inoculate in Dai, add MMN nutritional solution, be placed in illumination box cultivation two weeks-three months, obtain mycorhiza.
According to the synthetic method of described exotrophic mycorrhiza, in wherein said step (1), MMN culture medium prescription is
MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, maltose 10g, VB10.01g,
NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA 24g, agar 7g (1L)；Described
MMN nutrient solution prescription is: MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, wheat
Bud sugar 10g, VB10.01g, NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA24g.
It it is to use according to the acquisition of tree seedling in the synthetic method of described exotrophic mycorrhiza, wherein said step (2): symbiosis
Seeds tap water cleans after three times, within alcohol-pickled 30 minutes, carries out surface sterilization with 75%, broadcasts after aseptic water washing three times
In equipped with in the Vermiculitum of 1:1 and perlitic plastic crate, at essence temperature-controlling chamber condition air themperature 20-25 DEG C, soil humidity
Cultivating under 60-70%, sterilized water waters；Seedling medium Vermiculitum and perlite need first at 121 DEG C, sterilizing 1h in 0.1MPa,
After sterilizing is placed 3 days after terminating, sterilizing 1 time again in similarity condition.
According to the synthetic method of described exotrophic mycorrhiza, by seedling main root root before wherein said step (2) middle tree seedling inoculation
Point cuts off 1-2cm, in order to the growth of side root and infecting.
According to the synthetic method of described exotrophic mycorrhiza, wherein said step (2) middle inoculation bag is long 25cm, wide 15cm
Polypropylene culture bag, cut out sizeable filter paper in bag, the left and right sides, bottom of bag places long 3cm, width respectively
3cm, the sponge of thick 3cm, it is simple to later stage water suction moisturizing.
During inoculation, tree seedling is placed in culture bag according in the synthetic method of described exotrophic mycorrhiza, wherein said step (2)
Centre position, chooses the truffle split and is attached to symbiosis seeds seedling side root, and a Seedling needs 3 truffles blocks, adds
50ml nutritional solution, sealing；Described truffle refers to aseptically, will cover with mycelial culture medium vaccinating lancet and divide
Become the positive square truffle of 1cm × 1cm size.
According to the synthetic method of described exotrophic mycorrhiza, in wherein said step (2), illumination box design temperature is 23
DEG C, humidity is 55%, 12h illumination, and 12h is dark.
According to the synthetic method of described exotrophic mycorrhiza, the method comprises the steps:
(1) separation of external fungus strain and purification: take the cap of external fungus under sterile conditions close to group at lamella
Knitting, be inoculated in 28 DEG C of lucifuges on MMN culture medium flat plate and cultivate, MMN culture medium prescription is: MgSO4.7H2O:1.5
G, KH2PO4: 3g, yeast extract: 5g, glucose: 10g, maltose: 10g, VB1: 0.01g, NaCl:
0.058g, CaCl2: 1.11g, ferric citrate: 1.5 × 10-2G, PDA24g, agar: 7g (1L)；MMN seeks
Nutrient solution formula is: MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, maltose 10g,
VB10.01g, NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA24g.
(2) Aseptic seedling culture: after symbiosis seeds cleans three times with tap water, carry out for alcohol-pickled 30 minutes with 75%
Surface sterilization, aseptic water washing three times, seedling medium Vermiculitum and perlite press 1:1 proportioning, and sterilizing is placed in plastic crate,
Institute's nursery is all cultivated under the conditions of plastic greenhouse 10-30 DEG C, and tap water waters；Seedling medium Vermiculitum and perlite exist in advance
121 DEG C, sterilizing 2h under 0.1MPa, sterilizing places 3 days after terminating, similarity condition sterilizing again afterwards；
(3) inoculation: after symbiosis seeds 3 first quarter moons of cultivation, prepare inoculation, by the symbiosis seeds seedling main root tip of a root before inoculation
Cut off 1-2cm, it is simple to the growth of side root and infecting；After external fungal mycelium flat board covers with, aseptically,
The square that mycelium is divided into 1cm × 1cm size with vaccinating lancet is standby；Inoculation bag is long 25cm, wide 17cm
Valve bag, cuts out sizeable filter paper in bag, and the left and right sides, bottom of bag places long 3cm, wide 3cm, thickness respectively
The sponge of 3cm, it is simple to later stage water suction moisturizing；Seedling is placed in inoculation bag centre position, choose split external very
Bacterium truffle is attached to seedling side root, and a Seedling needs 3 truffles blocks, adds 50ml MMN nutritional solution, described MMN
Nutrient solution prescription is: MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, maltose
10g, VB10.01g, NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA24g；Sealing；
Being placed in incubator design temperature is 23 DEG C, and humidity is 55%, 12h illumination, and 12h is dark, cultivates 3 months, from aseptic
The mycorhiza that shoot root is fastened with external fungus；
(4) mycorhiza is identified: the mycorhiza obtained carries out DNA extraction, and checks order its PCR primer, it was demonstrated that institute
Obtaining mycorhiza is external fungus.
This method is easy to screen the suitableeest seeds and Applying Ectomycorrhizal Fungi paragenetic association, shortens the cycle, improves efficiency, reduction
Cost, instructs artificial culture.
Accompanying drawing illustrates:
Fig. 1 is that mycorhiza of the present invention synthesizes bag schematic diagram, and in figure: 1 is valve bag, 2 is Masson Pine Seedling, and 3 is the mycelia of inoculation
Body block, 4 is sponge block, and 5 is chromatographic paper.
Detailed description of the invention
Below in conjunction with the accompanying drawings, further illustrate the essentiality content of the present invention with embodiments of the invention, but not with this
Limit the present invention.
Embodiment 1
Milk cattle liver (Suillus bovinus) and the quick mycorhiza synthetic method of larch (Larix gmelinii):
The sporophore of milk cattle liver differentiates: Suillus granulatus (L. Ex Franch.) Ktze. sporophore is medium greatly.Cap flat hemispherical or the most flat, diameter
5.2-10cm, faint yellow or yellowish-brown, stick-slip time wet, dry rear glossy.Bacterial context is faint yellow, injured rear invariant color.Bacterium
Manage faint yellow, directly give birth to or slightly prolong life, mouth of pipe triangle.The nearly cylindricality of stem, solid, khaki, long 3-10cm,
Thick 0.8-1.6cm, the even reticulate pattern having about 1cm length in top, stem half or all have dark-coloured body of gland and gland point, have when hindering
Milk flows out.Spore is colourless to faint yellow, oblong, (6.5-9.1) μ m (2.6-3.9) μm.
Suillus bovines kind separates, purification: take the cap of milk cattle liver (Suillus bovinus) under sterile conditions close to bacterium
Organizing at pleat, be inoculated in 28 DEG C of lucifuges on MMN culture medium flat plate and cultivate, MMN culture medium prescription is: MgSO4.7H2O:
1.5g, KH2PO4: 3g, yeast extract: 5g, glucose: 10g, maltose: 10g, VB1: 0.01g, NaCl:
0.058g, CaCl2: 1.11g, ferric citrate: 1.5 × 10-2G, PDA 24g, agar: 7g (1L).MMN seeks
Nutrient solution formula is: MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, maltose 10g,
VB10.01g, NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA 24g.
Aseptic seedling culture: after Larch Seed cleans three times with tap water, carry out surface in alcohol-pickled 30 minutes with 75% and disappear
Poison, aseptic water washing three times, seedling medium Vermiculitum and perlite press 1:1 proportioning, and sterilizing is placed in plastic crate, is educated
Miao Jun cultivates (10-30 DEG C) under the conditions of plastic greenhouse, and tap water waters.
Further scheme may select: seedling medium Vermiculitum and perlite in advance at 121 DEG C, sterilizing 2h under 0.1MPa,
Sterilizing is placed 3 days after terminating, similarity condition sterilizing again afterwards.
Inoculation: after larch 3 first quarter moons of cultivation, prepare inoculation.Need before inoculation to cut off the Larch Seedling main root tip of a root
1-2cm, in order to the growth of side root and infecting；After Suillus bovines filament flat board covers with, aseptically, with inoculation
The square that mycelium is divided into 1cm × 1cm size by cutter is standby；Inoculation bag is long 25cm, the valve bag of wide 17cm,
Cutting out sizeable filter paper in bag, the left and right sides, bottom of bag places long 3cm, wide 3cm, the sea of thick 3cm respectively
Continuous, it is simple to later stage water suction moisturizing；Larch Seedling is placed in inoculation bag centre position, chooses the Suillus bovines split
Block is attached to Masson Pine seedling side root, and a Seedling needs 3 truffles blocks, and (every liter contains to add 50ml MMN nutritional solution
MgSO4.7H2O:1.5g, KH2PO4: 3g, yeast extract: 5g, glucose: 10g, maltose: 10g, VB1:
0.01g, NaCl:0.058g, CaCl2: 1.11g, ferric citrate: 1.5 × 10-2G, PDA24g), sealing；It is placed in
Incubator design temperature is 23 DEG C, and humidity is 55%, 12h illumination, and 12h is dark, cultivates 3 months, from larch without
Band Suillus bovines root is obtained on vaccine root system.
Mycorhiza is identified: the mycorhiza obtained carries out DNA extraction, and checks order its PCR primer, it was demonstrated that gained bacterium
Root is milk cattle liver.
Embodiment 2
Lactrarius hatsudake (Lactarius hatsudake) and the quick mycorhiza synthetic technology of Pinus densiflora (Pinus densiflora):
Lactrarius hatsudake sporophore differentiates: basidiocarps median size is to less.Bacteria cover diameter 3-7cm, edge is involute, center
Depression；Surface water soaking mode, shoal after bois de rose, pale red, wound dark blue-green, has faint ring grain or does not have ring grain.Bacterium
Meat pale red, has aubergine point.Lamella width 1.5-5mm, close, the closeest directly give birth to the dilutest, closely prolong life, claret,
After wound or old after deepen aeruginous.Stem 1.5-4 × 0.5-1 (3) cm, solid during children, hollow after maturation, the most cylindric,
Etc. thick or the most tapered, middle life；Smooth surface, without nest speckle, pale red, deepens aeruginous after wound；Base portion tool is pale yellow brown
Color strigose.Hypogalactia, claret, the milk of stem base portion is more orange, and invariant color is soft.Without special odor.Spore
Son print whipping cream brown.
Lactarius hatsutake separates, purification: take the bacterium of Lactrarius hatsudake (Lactarius hatsudake) under sterile conditions
Covering tissue at lamella and be inoculated in 28 DEG C of lucifuges cultivations on MMN culture medium flat plate, MMN culture medium prescription is:
MgSO4.7H2O:1.5g, KH2PO4: 3g, yeast extract: 5g, glucose: 10g, maltose: 10g, VB1:
0.01g, NaCl:0.058g, CaCl2: 1.11g, ferric citrate: 1.5 × 10-2G, PDA 24g, agar: 7g (1L).
Aseptic seedling culture: after Chinese pine seeds cleans three times with tap water, carry out surface sterilization in alcohol-pickled 30 minutes with 75%,
Aseptic water washing three times, seedling medium Vermiculitum and perlite press 1:1 proportioning, and sterilizing is placed in plastic crate, and institute's nursery is equal
Cultivating (10-30 DEG C) under the conditions of plastic greenhouse, tap water waters.
Seedling medium Vermiculitum and perlite are in advance at 121 DEG C, sterilizing 2h under 0.1MPa, and sterilizing is placed 3 days after terminating, it
The sterilizing again of rear similarity condition.
Inoculation: after Pinus tabuliformis 2 first quarter moons of cultivation, prepare inoculation.Need before inoculation the Pinus tabuliformis seedling main root tip of a root is cut off 1-2cm,
So that the growth of side root and infecting；After Lactrarius hatsudake mycelium flat board covers with, under sterile conditions, will be with inoculation
The square that mycelium is divided into 1cm × 1cm size by cutter is standby；Inoculation bag is long 25cm, the valve bag of wide 17cm,
Cutting out sizeable chromatographic paper in bag, the left and right sides, bottom of bag places long 3cm, wide 3cm, thick 3cm respectively
Sponge, it is simple to later stage water suction moisturizing；Pinus tabuliformis seedling is placed in inoculation bag centre position, chooses the Lactrarius hatsudake split
Truffle is attached to Masson Pine seedling side root, and a Seedling needs 3 truffles blocks, and (every liter contains to add 20ml MMN nutritional solution
MgSO4.7H2O:1.5g, KH2PO4: 3g, yeast extract: 5g, glucose: 10g, maltose: 10g, VB1:
0.01g, NaCl:0.058g, CaCl2: 1.11g, ferric citrate: 1.5 × 10-2G, PDA 24g), sealing；It is placed in
Incubator design temperature is 23 DEG C, and humidity is 55%, 12h illumination, and 12h is dark, cultivates 3 months, aseptic from Pinus tabuliformis
Shoot root fastens to obtain the mycorhiza of band Lactrarius hatsudake.
Claims (8)
1. a synthetic method for exotrophic mycorrhiza, comprises the following steps:
(1) method using separate tissue, aseptically, the meat bacteria organization taking Applying Ectomycorrhizal Fungi is inoculated in MMN
In culture medium, lucifuge is cultivated；
(2) by step (1) in mycorhiza inoculation bag, with the symbiosis of the mycelium pure culture of isolated Yu three months seedling ages
Seedling inoculates seeds, add MMN nutritional solution, be placed in illumination box cultivation two weeks to three months,
Obtain mycorhiza.
Exotrophic mycorrhiza synthetic method the most according to claim 1, it is characterised in that MMN in described step (1)
Culture medium prescription is MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, maltose 10g,
VB10.01g, NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA 24g, agar 20g (1L)；
Described MMN nutrient solution prescription is: MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g,
Maltose 10g, VB10.01g, NaCl 0.058g, CaCl21.11g, ferric citrate 1.5 × 10-2G, PDA 24g.
Exotrophic mycorrhiza synthetic method the most according to claim 1, it is characterised in that symbiosis tree in described step (2)
The acquisition planting seedling is to use: after the seed tap water of tree cleans three times, within alcohol-pickled 30 minutes, carry out surface with 75%
Sterilization, is sowed in the Vermiculitum equipped with 1:1 and perlitic plastic crate after aseptic water washing three times, and in essence temperature-controlling chamber, condition is
Cultivating under air themperature 20-25 DEG C, soil humidity 60-70%, sterilized water waters；Seedling medium Vermiculitum and perlite need elder generation
At 121 DEG C, sterilizing 1h in 0.1MPa, after sterilizing is placed 3 days after terminating, sterilizing 1 time again in similarity condition.
The synthetic method of exotrophic mycorrhiza the most according to claim 1, it is characterised in that symbiosis in described step (2)
Before seeds seedling inoculation, the seedling main root tip of a root is cut off 1-2cm, in order to the growth of side root and infecting.
The synthetic method of exotrophic mycorrhiza the most according to claim 1, it is characterised in that inoculation in described step (2)
Bag be long 25cm, the polypropylene culture bag of width 15cm, cuts out sizeable filter paper in bag, about the bottom of bag two
Side places long 3cm, wide 3cm, the sponge of thick 3cm respectively, it is simple to later stage water suction moisturizing.
Exotrophic mycorrhiza synthetic method the most according to claim 1, it is characterised in that in described step (2) during inoculation
Symbiosis seeds seedling is placed in culture bag centre position, chooses the truffle split and be attached to set seedling side root, a Seedling
Need 3 truffles blocks, add 50ml nutritional solution, sealing；Described truffle refers to aseptically, will cover with mycelial
Culture medium vaccinating lancet is divided into the positive square truffle of 1cm × 1cm size.
The synthetic method of exotrophic mycorrhiza the most according to claim 1, it is characterised in that illumination in described step (2)
Incubator design temperature is 23 DEG C, and humidity is 55%, 12h illumination, and 12h is dark.
The synthetic method of exotrophic mycorrhiza the most according to claim 1, it is characterised in that the method comprises the steps:
(1) separation of external fungus strain and purification: under aseptic condition, take the bacterium of external fungus cap and stem junction
Meat tissue, is inoculated in 28 DEG C of lucifuges in MMN culture medium and cultivates, and MMN culture medium prescription is: MgSO4.7H2O:1.5
G, KH2PO4: 3g, yeast extract: 5g, glucose: 10g, maltose: 10g, VB1: 0.01g, NaCl:0.058g,
CaCl2: 1.11g, ferric citrate: 1.5 × 10-2G, PDA 24g, agar: 20g (1L)；
(2) Aseptic seedling culture: after symbiosis seeds cleans three times with tap water, carry out for alcohol-pickled 30 minutes with 75%
Surface sterilization, aseptic water washing three times, seedling medium Vermiculitum and perlite press 1:1 proportioning, and sterilizing is placed in plastic crate,
Institute's nursery is all cultivated under the conditions of plastic greenhouse 10-30 DEG C, and tap water waters；Seedling medium Vermiculitum and perlite exist in advance
121 DEG C, sterilizing 2h under 0.1MPa, sterilizing places 3 days after terminating, similarity condition sterilizing again afterwards；
(3) inoculation: after seedling 3 first quarter moons of cultivation, prepare inoculation, before inoculation, the symbiosis seeds seedling main root tip of a root is cut off
1-2cm, it is simple to the growth of side root and infecting；After external fungal mycelium flat board covers with, aseptically, with inoculation
The square that mycelium is divided into 1cm × 1cm size by cutter is standby；Inoculation bag is long 25cm, the valve bag of wide 17cm,
Cutting out sizeable filter paper in bag, the left and right sides, bottom of bag places long 3cm, wide 3cm, the sea of thick 3cm respectively
Continuous, it is simple to later stage water suction moisturizing；Seeds seedling is placed in inoculation bag centre position, chooses the external fungus bacterium split
Block is attached to seedling side root, and a Seedling needs 3 truffles blocks, adds 50ml MMN nutritional solution, described MMN nutrition
Formula of liquid is: MgSO4.7H2O 1.5g, KH2PO43g, yeast extract 5g, glucose 10g, maltose 10g,
VB10.01g, NaCl 0.058g, CaCl2.11g, ferric citrate 1.5 × 10-2G, PDA 24g；Sealing；It is placed in cultivation
Case design temperature is 23 DEG C, and humidity is 55%, 12h illumination, and 12h is dark, cultivates 3 months, aseptic from symbiosis seeds
Shoot root fastens the mycorhiza obtaining external fungus；
(4) mycorhiza is identified: the mycorhiza obtained carries out DNA extraction, and checks order its PCR primer, it was demonstrated that institute
Obtaining mycorhiza is external fungus.
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Cited By (6)
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| CN108432547A (en) * | 2018-03-14 | 2018-08-24 | 云南上智科技有限公司 | A kind of mycorhiza synthesis cultural method |
| CN108541514A (en) * | 2018-03-21 | 2018-09-18 | 丽水市林业科学研究院 | A method of cultivating orange newborn mushroom offspring |
| CN110301290A (en) * | 2019-07-26 | 2019-10-08 | 金埔园林股份有限公司 | A kind of device and method of rapid synthesis exotrophic mycorrhiza |
| CN110604048A (en) * | 2018-06-14 | 2019-12-24 | 南京农业大学 | Woody plant mycorrhiza multi-inoculation method and application |
| CN111357566A (en) * | 2020-04-10 | 2020-07-03 | 福建农林大学 | Method for quickly cultivating mycorrhizal seedlings of ectomycorrhizal fungi |
| CN111742781A (en) * | 2020-07-31 | 2020-10-09 | 北京农学院 | Efficient and practical Chinese chestnut ectomycorrhiza soil basin inoculation system and application thereof |
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| CN110604048B (en) * | 2018-06-14 | 2021-05-04 | 南京农业大学 | Woody plant mycorrhiza multi-inoculation method and application |
| CN110301290A (en) * | 2019-07-26 | 2019-10-08 | 金埔园林股份有限公司 | A kind of device and method of rapid synthesis exotrophic mycorrhiza |
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