CN109355244A - Arbuscular mycorrhizal fungi spore enrichment screening method and device - Google Patents
Arbuscular mycorrhizal fungi spore enrichment screening method and device Download PDFInfo
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- CN109355244A CN109355244A CN201811537340.2A CN201811537340A CN109355244A CN 109355244 A CN109355244 A CN 109355244A CN 201811537340 A CN201811537340 A CN 201811537340A CN 109355244 A CN109355244 A CN 109355244A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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Abstract
Arbuscular mycorrhizal fungi spore enrichment isolation device of the present invention, is that filter cloth is respectively set in cabinet, and cabinet is made to form main culture vessel, main accumulation vessel, main accumulation vessel and secondary culture vessel, has drainage hole in main culture vessel and secondary bottom of culture vessel.Culture substrate is filled in main culture vessel, sterile host plant is planted in culture substrate；Aseptic substrate is filled in main accumulation vessel and secondary culture vessel and plants host plant；Sterile river sand is filled in secondary accumulation vessel and applies the nutrient solution containing the adverse circumstance factor, collects culture substrate and arbuscular mycorrhizal fungi spore largely with adverse-resistant characteristic is obtained by wet screening decantation.Material and training method required for the present invention can reach in general laboratory, compared with hairy root propagation arbuscular mycorrhizal fungi spore method, the device and method need not consider the problems of spore contamination, it operates with simply, and device is reusable, feasibility is high.
Description
Technical field
The present invention relates to a kind of screening technique and device, specifically a kind of arbuscular mycorrhizal fungi spore enrichment screening method and
Device belongs to microorganism field.
Background technique
Arbuscular mycorrhizal fungi is a kind of root system of plant symbiotic effects being widely present in soil, can be high with major part
Root system of plant forms symbiosis.Arbuscular mycorrhizal fungi spore is the organ of multiplication of arbuscular mycorrhizal fungi.In order to arbuscular mycorrhiza
Fungal spore is expanded, and expand numerous to arbuscular mycorrhizal fungi spore using artificial pure culture is most effective technology.At present
It was found that about 250 kinds of arbuscular mycorrhizal fungi spore type, be under the jurisdiction of the mould door of sacculus, can be seen in most of adverse circumstance environment
The trace of arbuscular mycorrhizal fungi spore.In order to probe into the characteristic of arbuscular mycorrhizal fungi spore and its degeneration-resistant machine in different adverse circumstance environment
It makes, a large amount of separation and screening have the arbuscular mycorrhizal fungi spore of adverse-resistant characteristic most important, this is also that subsequent monospore expansion is numerous
Technology and arbuscular mycorrhizal fungi strain are laid a good foundation.However arbuscular mycorrhizal fungi can only pass through living body host plant
Root system forms symbiosis, can complete reproductive process, is unable to obtain arbuscular mycorrhizal fungi spore by pure culture technigne
Son, this causes great difficulty for the separation screening of arbuscular mycorrhizal fungi microorganism resource.
Enriching apparatus is screened without specific arbuscular mycorrhizal fungi spore at present.Tradition screening enrichment arbuscular mycorrhizal fungi spore
Mainly by common pot experiment, field Rhizosphere Soil and host plant are subjected to long-term cultivation, to realize that arbuscular mycorrhiza is true
The expansion of bacterium spore is numerous.However this method tends not to access a large amount of arbuscular mycorrhizal fungi spore, and the spore of Partial Species
Son is unfavorable for carrying out production spore near root system due to host's root system.It also disclosed in recent years a kind of by hairy amplification
The method of arbuscular mycorrhizal fungi spore.It is to induce the root system of host at hairy, then by mycorrhizal fungi by agrobacterium rhizogenes
Spore be seeded in after surface sterilization on hairy near root system.However the arbuscular mycorrhizal fungi spore in field is often active
Lower, quantity is also fewer, is easy to introduce to pollute during the cultivation process to cause the failure of an experiment, is unfavorable for the big of arbuscular mycorrhizal fungi
Amount screening and enrichment.Not yet find that one kind can be enriched to a large amount of arbuscular mycorrhizal fungi spores at present, and Effective selection is anti-heavy
The method of the adverse circumstances adverse-resistant characteristic arbuscular mycorrhizal fungi spore such as metal, drought-resistant, salt resistance alkali.
Summary of the invention
The purpose of the invention is to overcome low efficiency present in current arbuscular mycorrhizal fungi spore screening technique and obtain
The few defect of arbuscular mycorrhizal fungi spore content, and a kind of arbuscular mycorrhizal fungi spore enrichment screening method and device are disclosed.
The technical solution of arbuscular mycorrhizal fungi spore enrichment isolation device of the present invention is:
3 400 mesh filter clothes 6 of setting are respectively perpendicular in open cabinet above, make the main culture vessel 1 of cabinet self-assembling formation, master
Accumulation vessel 3, main accumulation vessel 3 and secondary culture vessel 2 have in main culture vessel 1 with 2 bottom of pair culture vessel several
A drainage hole.
The method screened using above-mentioned arbuscular mycorrhizal fungi spore enrichment isolation device is as follows:
A. the fresh host's Rhizosphere Soil acquired in field adverse circumstance environment is filled in main culture vessel 1 as culture substrate, by nothing
The host plant of bacterium is planted in culture substrate；
B. aseptic substrate that partial size is 1-2mm is filled in main accumulation vessel 3 to volume half；
C. the aseptic substrate that partial size is 1-2mm is filled in secondary culture vessel 2, and sterile host plant is planted in sterile base
In matter；
D. sterile river sand that partial size is 1-2mm is filled in secondary accumulation vessel 4 to accumulation vessel volume half；
It is dense that e poured 1/10th times of phosphorus into main culture vessel 1 and secondary culture vessel 2 every 15 days in plant culture 60 days
The Hoagland nutrient solution of degree keeps culture substrate wet；
F. applying Hoagland nutrient solution to culture substrate in 60 days backward main accumulation vessels 1 is moisture state, by accumulation vessel
It is sealed with PE preservative film, and is spaced 30 days supplement one time of nutrition liquid；
G. apply the Hoagland nutrient solution containing the adverse circumstance factor into secondary accumulation vessel 4 after plant is cultivated 120 days to culture
Matrix be it is wet, by accumulation vessel with PE preservative film seal, and be spaced Hoagland nutrient solution of 30 days supplements；
H. the semiarid state of secondary culture vessel 2 is kept after plant is cultivated 240 days, and secondary accumulation vessel is collected after the 270th day
A large amount of arbuscular mycorrhizal fungi spores with adverse-resistant characteristic can be obtained by wet screening decantation in culture substrate in 4.
The present invention is compared to common potting enrichment method, by setting up phosphorus gradient between culture vessel and accumulation vessel,
Arbuscular mycorrhizal fungi big volume production spore in accumulation vessel is induced, since the breeding of arbuscular mycorrhizal fungi needs sufficient air, because
This has reserved the space of half in main accumulation vessel and secondary accumulation vessel, in order to the aerobic respiration of arbuscular mycorrhizal fungi
Journey.For plant in secondary culture vessel due to the influence not being forced, photosynthesis of plant is high-efficient, can hold for secondary enrichment
Fungi in device provides sufficient carbon source.Also contain a degree of environment stress in secondary accumulation vessel at this time, pair can be enriched with
Arbuscular mycorrhizal fungi in container produces spore and plays screening effect, to obtain arbuscular mycorrhizal fungi spore largely with adverse-resistant characteristic
Son, in order to the separation and research of subsequent strain.Material and training method required for the present invention are in general laboratory item
It can reach under part, compared with hairy root propagation arbuscular mycorrhizal fungi spore method, which need not consider spore dirt
The problem of dye, operates with simply, and device is reusable, and feasibility is high.
Detailed description of the invention
Attached drawing 1 is front view structure diagrammatic cross-section of the present invention.
Attached drawing 2 is overlooking structure diagram of the present invention.
The reference numerals are as follows:
Main culture vessel 1, secondary culture vessel 2, main accumulation vessel 3, secondary accumulation vessel 4, drainage hole 5, filter cloth 6.
Specific embodiment
Combined with specific embodiments below and attached drawing is described in further details the present invention, and the present embodiment is intended merely to explain
The of the invention rather than limitation present invention:
The technical solution of arbuscular mycorrhizal fungi spore enrichment isolation device of the present invention is:
3 400 mesh filter clothes 6 of setting are respectively perpendicular in open cabinet above, make the main culture vessel 1 of cabinet self-assembling formation, master
Accumulation vessel 3, main accumulation vessel 3 and secondary culture vessel 2；It is dry that with secondary 2 bottom of culture vessel six are had in main culture vessel 1
A drainage hole.
The method screened using above-mentioned arbuscular mycorrhizal fungi spore enrichment isolation device is as follows:
A. fresh host's Rhizosphere Soil that heavy metal pollution area, arid and semi-arid area or salt-soda soil are filled in main culture vessel 1 is made
For culture substrate, host plant is planted in culture substrate；
B. aseptic substrate that partial size is 1-2mm is filled in main accumulation vessel 3 to volume half；
C. the aseptic substrate that partial size is 1-2mm is filled in secondary culture vessel 2, and sterile host plant is planted in sterile base
In matter；
D. sterile river sand that partial size is 1-2mm is filled in secondary accumulation vessel 4 to accumulation vessel volume half；
It is dense that e poured 1/10th times of phosphorus into main culture vessel 1 and secondary culture vessel 2 every 15 days in plant culture 60 days
The Hoagland nutrient solution 20ml of degree keeps culture substrate wet；
F. applying Hoagland nutrient solution 50ml in 60 days backward main accumulation vessels 1 to culture substrate is moisture state, will be enriched with
Container is sealed with PE preservative film, and is spaced 30 days supplement one time of nutrition liquid 20ml；
G. apply after plant is cultivated 120 days into secondary accumulation vessel 4 and contain high concentration heavy metal ion (such as lead concentration 10g/
L), PEG6000(15%) or high concentration sodium chloride (100mmol) Hoagland nutrient solution 50ml to culture substrate be wet shape
Accumulation vessel PE preservative film is sealed, and is spaced Hoagland nutrient solution 20ml of 30 days supplements by state；
H. the semiarid state of secondary culture vessel 2 is kept after plant is cultivated 240 days, and secondary accumulation vessel is collected after the 270th day
Culture substrate in 4, can be obtained largely by wet screening decantation has resistance to heavy metal, drought-resistant or saline-alkali tolerant characteristic clump branch
Mycorrhizal fungal spore.
Claims (2)
1. arbuscular mycorrhizal fungi spore enrichment isolation device, it is characterised in that its technical solution is: in open cabinet above
3 400 mesh filter clothes (6) of setting are respectively perpendicular, the main culture vessel of cabinet self-assembling formation (1), main accumulation vessel (3), main enrichment are made
Container (3) and secondary culture vessel (2) have several drainings in main culture vessel (1) and secondary culture vessel (2) bottom
Hole；
The method screened using above-mentioned arbuscular mycorrhizal fungi spore enrichment isolation device is as follows:
A. the fresh host's Rhizosphere Soil acquired in filling field adverse circumstance environment in main culture vessel (1), will as culture substrate
Sterile host plant is planted in culture substrate；
B. the aseptic substrate that filling partial size is 1-2mm in the main accumulation vessel (3) is to volume half；
C. the aseptic substrate that filling partial size is 1-2mm in secondary culture vessel (2), sterile host plant is planted in sterile
In matrix；
D. the sterile river sand that filling partial size is 1-2mm in the secondary accumulation vessel (4) is to accumulation vessel volume half；
E is cultivated in 60 days in plant and was poured 1/10th times into main culture vessel (1) and secondary culture vessel (2) every 15 days
The Hoagland nutrient solution of phosphorus concentration keeps culture substrate wet；
F. applying Hoagland nutrient solution to culture substrate in 60 days backward main accumulation vessels (1) is moisture state, and enrichment is held
Device is sealed with PE preservative film, and is spaced 30 days supplement one time of nutrition liquid；
G. apply the Hoagland nutrient solution containing the adverse circumstance factor into secondary accumulation vessel (4) after plant is cultivated 120 days to training
Support matrix be it is wet, by accumulation vessel with PE preservative film sealing, and interval Hoagland nutrient solution of 30 days supplements；
H. secondary culture vessel (2) semiarid state is kept after plant is cultivated 240 days, and is collected secondary enrichment after the 270th day and held
A large amount of arbuscular mycorrhizal fungi spores with adverse-resistant characteristic can be obtained by wet screening decantation in culture substrate in device (4).
2. arbuscular mycorrhizal fungi spore enrichment isolation device according to claim 1, it is characterised in that: open above
Be respectively perpendicular in cabinet setting 3 400 mesh filter clothes (6), make the main culture vessel of cabinet self-assembling formation (1), main accumulation vessel (3),
Main accumulation vessel (3) and secondary culture vessel (2)；Six rows are had in main culture vessel (1) and secondary culture vessel (2) bottom
Water hole；
The method screened using above-mentioned arbuscular mycorrhizal fungi spore enrichment isolation device is as follows:
A. fresh host's Rhizosphere Soil in heavy metal pollution area, arid and semi-arid area or salt-soda soil is filled in main culture vessel (1)
As culture substrate, host plant is planted in culture substrate；
B. the aseptic substrate that filling partial size is 1-2mm in the main accumulation vessel (3) is to volume half；
C. the aseptic substrate that filling partial size is 1-2mm in secondary culture vessel (2), sterile host plant is planted in sterile
In matrix；
D. the sterile river sand that filling partial size is 1-2mm in the secondary accumulation vessel (4) is to accumulation vessel volume half；
E is cultivated in 60 days in plant and was poured 1/10th times into main culture vessel (1) and secondary culture vessel (2) every 15 days
The Hoagland nutrient solution 20ml of phosphorus concentration keeps culture substrate wet；
F. apply in 60 days backward main accumulation vessels (1) Hoagland nutrient solution 50ml to culture substrate be moisture state, will be rich
Collect container to be sealed with PE preservative film, and is spaced 30 days supplement one time of nutrition liquid 20ml；
G. apply after plant is cultivated 120 days into secondary accumulation vessel (4) and contain high concentration heavy metal ion (such as lead concentration
10g/l), PEG6000(15%) or high concentration sodium chloride (100mmol) Hoagland nutrient solution 50ml to culture substrate be it is wet
Accumulation vessel PE preservative film is sealed, and is spaced Hoagland nutrient solution 20ml of 30 days supplements by profit state；
H. the semiarid state of secondary culture vessel 2 is kept after plant is cultivated 240 days, and secondary accumulation vessel is collected after the 270th day
(4) culture substrate in, can be obtained by wet screening decantation largely has resistance to heavy metal, drought-resistant or saline-alkali tolerant characteristic clump
Mycorrhizal fungi spore.
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| CN201811537340.2A CN109355244A (en) | 2018-12-15 | 2018-12-15 | Arbuscular mycorrhizal fungi spore enrichment screening method and device |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110476713A (en) * | 2019-09-26 | 2019-11-22 | 华南农业大学 | The continuous device and method for collecting arbuscular mycorrhizal fungi mycelia |
| CN113278530A (en) * | 2021-04-13 | 2021-08-20 | 湖北民族大学 | Method for promoting spore germination and hypha growth of arbuscular mycorrhizal fungi |
| CN114657074A (en) * | 2022-04-15 | 2022-06-24 | 北京市农林科学院 | Culture method for efficiently propagating arbuscular mycorrhizal fungal spores in layered manner by using matrix nutrients |
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| CN1354252A (en) * | 2000-11-21 | 2002-06-19 | 李晓林 | Culture of ramaria mycorrhizal fungi by using glass bead as culture medium |
| CN1548524A (en) * | 2003-05-16 | 2004-11-24 | 北京市农林科学院植物营养与资源研究 | Efficient drought-resisting and high phoshpate-tolerant nutritious bush mycorrhizal fungus and its production process |
| WO2009090220A1 (en) * | 2008-01-15 | 2009-07-23 | Universite Catholique De Louvain | Method and system for in vitro mass production of arbuscular mycorrhizal fungi |
| CN103125251A (en) * | 2013-03-18 | 2013-06-05 | 西南大学 | Application method of arbuscular mycorrhizal fungus in large-scale tobacco cultivation |
| CN106190944A (en) * | 2016-07-07 | 2016-12-07 | 西北农林科技大学 | A kind of layering cultivates the method being enriched with AMF spore |
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2018
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1354252A (en) * | 2000-11-21 | 2002-06-19 | 李晓林 | Culture of ramaria mycorrhizal fungi by using glass bead as culture medium |
| CN1548524A (en) * | 2003-05-16 | 2004-11-24 | 北京市农林科学院植物营养与资源研究 | Efficient drought-resisting and high phoshpate-tolerant nutritious bush mycorrhizal fungus and its production process |
| WO2009090220A1 (en) * | 2008-01-15 | 2009-07-23 | Universite Catholique De Louvain | Method and system for in vitro mass production of arbuscular mycorrhizal fungi |
| CN103125251A (en) * | 2013-03-18 | 2013-06-05 | 西南大学 | Application method of arbuscular mycorrhizal fungus in large-scale tobacco cultivation |
| CN106190944A (en) * | 2016-07-07 | 2016-12-07 | 西北农林科技大学 | A kind of layering cultivates the method being enriched with AMF spore |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110476713A (en) * | 2019-09-26 | 2019-11-22 | 华南农业大学 | The continuous device and method for collecting arbuscular mycorrhizal fungi mycelia |
| CN110476713B (en) * | 2019-09-26 | 2021-03-16 | 华南农业大学 | Device and method for continuously collecting arbuscular mycorrhizal fungi hyphae |
| CN113278530A (en) * | 2021-04-13 | 2021-08-20 | 湖北民族大学 | Method for promoting spore germination and hypha growth of arbuscular mycorrhizal fungi |
| CN114657074A (en) * | 2022-04-15 | 2022-06-24 | 北京市农林科学院 | Culture method for efficiently propagating arbuscular mycorrhizal fungal spores in layered manner by using matrix nutrients |
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Effective date of registration: 20200812 Address after: 510642 College of Forestry and Landscape Architecture, 483 Wushan Road, Tianhe District, Guangzhou City, Guangdong Province Applicant after: SOUTH CHINA AGRICULTURAL University Address before: 712100 Xianyang city of Shaanxi province Yangling Demonstration Zone Road No. 3 Tai Forestry College Applicant before: NORTHWEST A & F University |
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Application publication date: 20190219 |