CN107449647A - A kind of method that AMF spore is screened from soil - Google Patents
A kind of method that AMF spore is screened from soil Download PDFInfo
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- CN107449647A CN107449647A CN201710912405.6A CN201710912405A CN107449647A CN 107449647 A CN107449647 A CN 107449647A CN 201710912405 A CN201710912405 A CN 201710912405A CN 107449647 A CN107449647 A CN 107449647A
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- 239000002689 soil Substances 0.000 title claims abstract description 103
- 238000000034 method Methods 0.000 title claims abstract description 36
- 240000008042 Zea mays Species 0.000 claims abstract description 37
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 37
- 238000007873 sieving Methods 0.000 claims abstract description 32
- 230000001954 sterilising effect Effects 0.000 claims abstract description 18
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims abstract description 12
- 235000009973 maize Nutrition 0.000 claims abstract description 12
- 239000004576 sand Substances 0.000 claims abstract description 10
- 235000001405 Artemisia annua Nutrition 0.000 claims abstract description 6
- 240000000011 Artemisia annua Species 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims description 45
- 239000000203 mixture Substances 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 25
- 235000005822 corn Nutrition 0.000 claims description 25
- 239000012153 distilled water Substances 0.000 claims description 21
- 238000001914 filtration Methods 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 17
- 238000004140 cleaning Methods 0.000 claims description 15
- 241000196324 Embryophyta Species 0.000 claims description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 230000001186 cumulative effect Effects 0.000 claims description 10
- 239000011521 glass Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 230000034303 cell budding Effects 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 239000006059 cover glass Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- 244000068988 Glycine max Species 0.000 claims description 5
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 claims description 5
- 229960002327 chloral hydrate Drugs 0.000 claims description 5
- 238000000151 deposition Methods 0.000 claims description 5
- 239000011630 iodine Substances 0.000 claims description 5
- 229910052740 iodine Inorganic materials 0.000 claims description 5
- 239000008267 milk Substances 0.000 claims description 5
- 210000004080 milk Anatomy 0.000 claims description 5
- 235000013336 milk Nutrition 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Substances [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000005201 scrubbing Methods 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims 1
- 238000012216 screening Methods 0.000 abstract description 29
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 18
- 238000010908 decantation Methods 0.000 description 4
- 238000002224 dissection Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- 241000773655 Ambispora fennica Species 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 238000011017 operating method Methods 0.000 description 2
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Immunology (AREA)
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Abstract
The invention belongs to microbial technology field, specifically discloses a kind of method that AMF spore is screened from soil.The screening technique of the present invention comprises the following steps：(1) source soil collection：Gather artemisia annua root soil；(2) numerous culture is expanded：After root soil in (1) is mixed with sterilizing sand, cultivated maize germinated seeds；(3) collection of soil sample：Gather maize seedling root system surrounding soil；(4) collection of sieving sample：Three layers of screen are crossed after soil sample is dissolved；(5) centrifuge：Centrifuged twice；(6) spore collection and counting；(7) film-making.The screening technique of the present invention has the characteristics of human error is small, simple and easy to do, screening efficiency is high, spore Loss Rate is low, and the AMF spore population density obtained by its screening is uniform.
Description
【Technical field】
The present invention relates to microbial technology field, and in particular to a kind of side that AMF spore is screened from soil
Method.
【Background technology】
Mycorhiza is a kind of mutualistic symbiosis body established by fungi and root system of plant, wherein with arbuscular mycorrhiza
(Arbuscular Mycorrhizal Fungi, AMF) is distributed most wide in nature.Either monocotyledon or Shuangzi
Leaf plant, AMF symbiosis can be formed mostly.AMF can not only promote crop to absorb mineral nutrient and moisture,
The especially absorption of P elements, so as to improve the yield and quality of plant, and also have as multi-functional high-performance bio fertilizer
There is improved soil structure, reduce the administration of chemical fertilizer, agricultural chemicals, help recovers the impaired ecosystem, to environmental protection, afforestation
And the sustainable development of agricultural has far-reaching significance.And the spore of AMF is important numerous of AMF
Organ is grown, therefore, carries out that expansion of the research observation for AMF is numerous and it is being made to the spore of AMF
Application, preservation and production in species plant etc. have great importance.
The spore of AMF is distributed in the soil of host plant rhizosphere, is observed in the research of AMF
In, often need to separate spore from soil, to carry out research observation to spore.At present, generally use wet screening, which inclines, washes
Method is screened clump branch fungal spore from soil, and the clump branch that this method has that human error is big, screening efficiency is low, screening obtains
Fungal spore population density the shortcomings that totally Loss Rate bigger than normal or less than normal and spore is high, it is unfavorable for extensively and profoundly carrying out clump branch bacterium
The research of mycorrhiza fungi.
【The content of the invention】
The goal of the invention of the present invention is：For above-mentioned problem, there is provided one kind screens arbuscular mycorrhiza from soil
The method of fungi.The screening technique of the present invention has that human error is small, simple and easy to do, screening efficiency is high, spore Loss Rate is low
Feature, and the AMF spore population density obtained by its screening is uniform.
To achieve these goals, the technical solution adopted by the present invention is as follows：
A kind of method that AMF spore is screened from soil, comprises the following steps：
(1) source soil collection：The whole strain of artemisia annua and its root soil are gathered, is placed in depositing in 25 DEG C, it is standby；
(2) numerous culture is expanded：By the above-mentioned root soil collected and sterilizing husky by volume 1:1 is well mixed, must mix
Soil, the mixed soil is put into flowerpot by the amount of 3Kg/ basins；Planted into the flowerpot into the consistent corn budding of growing way
Seed, and 4/basin；After the emergence of corn germinated seeds, the consistent maize seedling of 2 plants of growing ways is retained per disc floret basin, and as greenhouse
Middle cultivation 95-100d；
(3) collection of soil sample：The soil around the above-mentioned maize seedling root system by cultivation is gathered, that is, obtains the soil
Earth sample, it is standby；
(4) collection of sieving sample：By above-mentioned soil sample and clear water in mass ratio 1:3-4 is mixed and is put into soy bean milk making machine
After being stirred until homogeneous, mixed liquor is obtained；The mixed liquor is poured into three layers of screen after carrying out filtering 5-8min, remove upper two layers of filter
Sieve, the sandy soil mixture obtained in orlop screen is rinsed with distilled water to rinse obtained filtrate it is limpid after, by the
Sandy soil mixture on three layers of screen is transferred in centrifuge tube, produces sieving sample, standby；Wherein, three layers of screen is by 20
Mesh sieve, 120 mesh sieves and 400 mesh sieves stack composition successively by order from top to bottom；
(5) centrifuge：
Centrifuge for the first time：Above-mentioned sieving sample is put into the supercentrifuge that rotating speed is 3400-3600r/min and centrifuges 3-
After 5min, the supernatant in centrifuge tube is poured into 400 mesh screens of cleaning and filtered, then scrubs and inclines by using distilled water
The spore being attached to after filtering on screen is transferred in culture dish by oblique screen, obtains centrifugation spore mixed liquor for the first time；
Second of centrifugation：The sucrose solution that mass concentration is 70% is added into the above-mentioned centrifuge tube poured out after supernatant,
The cumulative volume of material is the 2/3 of centrifugation pipe volume in centrifuge tube, and the material mixing rocked in centrifuge tube to centrifuge tube is uniform, will
Centrifuge tube is put into the supercentrifuge that rotating speed is 1400-1600r/min after centrifugation 3-5min, and the supernatant in centrifuge tube is fallen
Enter and filtered in 400 mesh screens of cleaning, screen will be attached to after filtering by then scrubbing inclined screen by using distilled water
On spore be transferred in culture dish, obtain second of centrifugation spore mixed liquor；
(6) spore collection and counting：It is micro- that the above-mentioned culture dish for being loaded with centrifugation spore mixed liquor for the first time is placed in dissection
Under mirror, under the observation of disecting microscope, after being acquired with spore collector to the spore in mixed liquor, line surface is put into
In ware, after the completion of collection, line surface plate is placed under disecting microscope and counted；
(7) film-making：The single taking-up of spore in line surface plate after counting is placed on slide, with the lid glass of cleaning
After piece covers slide, the liquid around cover glass is blotted with blotting paper, and finds the spore on slide under the microscope, is used
Tweezers, which press pressure, ruptures spore；After being dyed with Mei Zeer reagents to the spore after rupture, you can obtain sample slide；
(8) second centrifugation spore mixed liquor repeat the above steps (6) and (7) handled, you can.
Further, in step (2), the sterilizing sand is prepared by following methods：River sand is gathered, the river sand is put
At 120-125 DEG C after sterilization 4h, it is placed at 25 DEG C and deposits, you can it is husky obtains the sterilizing.
Further, in step (2), the corn germinated seeds are cultivated to obtain by following methods：The corn that will have been chosen
Seed be first put into mass concentration be 2% hydrogen peroxide in soaks 2min, then be placed in water seed soaking 12h after, the vernalization one at 30 DEG C
In week, produce the corn germinated seeds.
Further, in step (4), the cumulative volume of the sandy soil mixture loaded in the centrifuge tube is no more than centrifugation body
Long-pending 2/3.
Further, in step (6), glass tip of the spore collector by rubber head dropper and with the rubber head dropper
The pipettor gun head composition of socket.
Further, in step (7), the Mei Zeer reagents are by chloraldurate, chloral hydrate, distilled water, iodine
With KI in mass ratio 200:200:200:3:10 well mixed are prepared.
Further, the sample slide is in faint yellow.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
(1) sieving sample of the invention collects low-density spore and high density spore respectively using centrifugation twice, the
When once centrifuging, the liquid in centrifuge tube is water, therefore, is centrifuged by first time, population density is less than 1g/cm3Spore float
In the supernatant of centrifuge tube, low-density spore is can obtain by filtering, and population density is more than 1g/cm3Spore then with
Sandy soil particle falls to centrifugation bottom of the tube；In second centrifuges, the liquid in centrifuge tube is the sucrose that mass concentration is 70%
Solution, its density are 1.35g/cm3, the spore density of centrifugation bottom of the tube is deposited in being centrifuged higher than first time, now, by the
Secondary centrifuging, being deposited on the high density spore of centrifugation bottom of the tube can float in the supernatant of centrifuge tube, i.e. available by filtering
High density spore；And in conventional wet screening inclines the method for washing, directly the sieving sample for handling to obtain by sieving is carried out further
Cleaning filtering, the outsifting obtained by collecting are AMF spore that population density size mixes, it is necessary to test behaviour
Make personnel further to judge the density size of spore according to experience, so as to which the spore being collected into is close by individual
Degree carries out classification collection, and therefore, wet screening method of washing of inclining requires that the operating personnel of sieve spore have a high proficiency, and would generally be because of
The criterion of screening person is different and spore population density totally situation bigger than normal or less than normal occurs, while spore Loss Rate is high.
To sum up, method of the invention is for wet screening inclines the method for washing, have it is easy to operate, be affected by human factors the characteristics of small, and
Screen obtained spore density uniformly and spore collection rate is high.
(2) present invention since sieving sample collection untill film-making, it is only necessary to 20-25min, greatly improve spore sieve
The operating efficiency of choosing.Wherein, conventional wet screening inclines the method for washing when carrying out the collection of sieving sample, it usually needs soil sample is entered
20~30min of row immersion, and pedotheque directly can be stirred dissolving, taken short by the method for the present invention without immersion；
Meanwhile spore collector of the present invention effectively to spore can catch and grab collection, picking rate is fast, takes short.
(3) the sample slide that is prepared of the present invention can be observed preferably the form of spore and the diameter to spore
It is measured.
【Embodiment】
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1
A kind of method that AMF spore is screened from soil, comprises the following steps：
(1) source soil collection：From the whole strain of Chongzuo somewhere collection artemisia annua and its root soil, it is placed in depositing in 25 DEG C,
It is standby；
(2) numerous culture is expanded：By the above-mentioned root soil collected and sterilizing husky by volume 1:1 is well mixed, must mix
Soil, the mixed soil is put into flowerpot by the amount of 3Kg/ basins；Planted into the flowerpot into the consistent corn budding of growing way
Seed, and 4/basin；After the emergence of corn germinated seeds, the consistent maize seedling of 2 plants of growing ways is retained per disc floret basin, and as greenhouse
Middle cultivation 95d；
Wherein, the husky river sand for after 120 DEG C of sterilization 4h of sterilizing；
The corn germinated seeds are cultivated to obtain by following methods：The corn seed chosen first is put into mass concentration is
2min is soaked in 2% hydrogen peroxide, then after being placed in water seed soaking 12h, vernalization one week at 30 DEG C, produces corn budding and plants
Son；
(3) collection of soil sample：The soil around the above-mentioned maize seedling root system by cultivation is gathered, that is, obtains the soil
Earth sample, it is standby；
(4) collection of sieving sample：By above-mentioned soil sample and clear water in mass ratio 1:3 mix and are put into soy bean milk making machine and stir
Mix to uniformly after, obtain mixed liquor；The mixed liquor is poured into three layers of screen after carrying out filtering 5min, removes upper two layers of screen,
The sandy soil mixture obtained in orlop screen is rinsed with distilled water to rinse obtained filtrate it is limpid after, by third layer
Sandy soil mixture on screen is transferred to centrifuge tube, and the cumulative volume of sandy soil mixture is no more than the 2/3 of centrifugation pipe volume, produces
Sieve sample, standby；Wherein, three layers of screen by 20 mesh sieves, 120 mesh sieves and 400 mesh sieves by from top to bottom order successively
Stack composition；
(5) centrifuge：
Centrifuge for the first time：Above-mentioned sieving sample is put into after centrifuging 3min in the supercentrifuge that rotating speed is 3400r/min,
Supernatant in centrifuge tube is poured into 400 mesh screens of cleaning and filtered, then scrubs inclined filter by using distilled water
The spore being attached to after filtering on screen is transferred in culture dish by sieve, obtains centrifugation spore mixed liquor for the first time；
Second of centrifugation：The sucrose solution that mass concentration is 70% is added into the above-mentioned centrifuge tube poured out after supernatant,
The cumulative volume of material is the 2/3 of centrifugation pipe volume in centrifuge tube, and the material mixing rocked in centrifuge tube to centrifuge tube is uniform, will
Centrifuge tube is put into the supercentrifuge that rotating speed is 1400r/min after centrifugation 3min, and the supernatant in centrifuge tube is poured into cleaning
400 mesh screens in filtered, then scrub the spore that inclined screen will be attached on screen after filtering by using distilled water
Son is transferred in culture dish, obtains second of centrifugation spore mixed liquor；
(6) spore collection and counting：It is micro- that the above-mentioned culture dish for being loaded with centrifugation spore mixed liquor for the first time is placed in dissection
Under mirror, under the observation of disecting microscope, with the pipettor being socketed by rubber head dropper and with the glass tip of the rubber head dropper
After the spore collector of pipette tips composition is acquired to the spore in mixed liquor, it is put into line surface plate, will after the completion of collection
Line surface plate, which is placed under disecting microscope, to be counted；
(7) film-making：The single taking-up of spore in line surface plate after counting is placed on slide, with the lid glass of cleaning
After piece covers slide, the liquid around cover glass is blotted with blotting paper, and finds the spore on slide under the microscope, is used
Tweezers, which press pressure, ruptures spore；With by chloraldurate, chloral hydrate, distilled water, iodine and KI in mass ratio 200:
200:200:3:After the 10 well mixed Mei Zeer reagents being prepared dye to the spore after rupture, you can obtain in light
The sample slide of yellow；
(8) second centrifugation spore mixed liquor repeat the above steps (6) and (7) handled, you can.
Embodiment 2
A kind of method that AMF spore is screened from soil, comprises the following steps：
(1) source soil collection：From the whole strain of Chongzuo somewhere collection artemisia annua and its root soil, it is placed in depositing in 25 DEG C,
It is standby；
(2) numerous culture is expanded：By the above-mentioned root soil collected and sterilizing husky by volume 1:1 is well mixed, must mix
Soil, the mixed soil is put into flowerpot by the amount of 3Kg/ basins；Planted into the flowerpot into the consistent corn budding of growing way
Seed, and 4/basin；After the emergence of corn germinated seeds, the consistent maize seedling of 2 plants of growing ways is retained per disc floret basin, and as greenhouse
Middle cultivation 95-100d；
Wherein, the husky river sand for after 122 DEG C of sterilization 4h of sterilizing；
The corn germinated seeds are cultivated to obtain by following methods：The corn seed chosen first is put into mass concentration is
2min is soaked in 2% hydrogen peroxide, then after being placed in water seed soaking 12h, vernalization one week at 30 DEG C, produces corn budding and plants
Son；
(3) collection of soil sample：The soil around the above-mentioned maize seedling root system by cultivation is gathered, that is, obtains the soil
Earth sample, it is standby；
(4) collection of sieving sample：By above-mentioned soil sample and clear water in mass ratio 1:4 mix and are put into soy bean milk making machine and stir
Mix to uniformly after, obtain mixed liquor；The mixed liquor is poured into three layers of screen after carrying out filtering 5-8min, remove upper two layers of filter
Sieve, the sandy soil mixture obtained in orlop screen is rinsed with distilled water to rinse obtained filtrate it is limpid after, by the
Sandy soil mixture on three layers of screen is transferred to centrifuge tube, and the cumulative volume of sandy soil mixture is no more than the 2/3 of centrifugation pipe volume,
Sieving sample is produced, it is standby；Wherein, three layers of screen by 20 mesh sieves, 120 mesh sieves and 400 mesh sieves by order from top to bottom
Composition is stacked successively；
(5) centrifuge：
Centrifuge for the first time：Above-mentioned sieving sample is put into after centrifuging 4min in the supercentrifuge that rotating speed is 3500r/min,
Supernatant in centrifuge tube is poured into 400 mesh screens of cleaning and filtered, then scrubs inclined filter by using distilled water
The spore being attached to after filtering on screen is transferred in culture dish by sieve, obtains centrifugation spore mixed liquor for the first time；
Second of centrifugation：The sucrose solution that mass concentration is 70% is added into the above-mentioned centrifuge tube poured out after supernatant,
The cumulative volume of material is the 2/3 of centrifugation pipe volume in centrifuge tube, and the material mixing rocked in centrifuge tube to centrifuge tube is uniform, will
Centrifuge tube is put into the supercentrifuge that rotating speed is 1500r/min after centrifugation 4min, and the supernatant in centrifuge tube is poured into cleaning
400 mesh screens in filtered, then scrub the spore that inclined screen will be attached on screen after filtering by using distilled water
Son is transferred in culture dish, obtains second of centrifugation spore mixed liquor；
(6) spore collection and counting：It is micro- that the above-mentioned culture dish for being loaded with centrifugation spore mixed liquor for the first time is placed in dissection
Under mirror, under the observation of disecting microscope, with the pipettor being socketed by rubber head dropper and with the glass tip of the rubber head dropper
After the spore collector of pipette tips composition is acquired to the spore in mixed liquor, it is put into line surface plate, will after the completion of collection
Line surface plate, which is placed under disecting microscope, to be counted；
(7) film-making：The single taking-up of spore in line surface plate after counting is placed on slide, with the lid glass of cleaning
After piece covers slide, the liquid around cover glass is blotted with blotting paper, and finds the spore on slide under the microscope, is used
Tweezers, which press pressure, ruptures spore；With by chloraldurate, chloral hydrate, distilled water, iodine and KI in mass ratio 200:
200:200:3:After the 10 well mixed Mei Zeer reagents being prepared dye to the spore after rupture, you can obtain in light
The sample slide of yellow；
(8) second centrifugation spore mixed liquor repeat the above steps (6) and (7) handled, you can.
Embodiment 3
A kind of method that AMF spore is screened from soil, comprises the following steps：
(1) source soil collection：From the whole strain of Chongzuo somewhere collection artemisia annua and its root soil, it is placed in depositing in 25 DEG C,
It is standby；
(2) numerous culture is expanded：By the above-mentioned root soil collected and sterilizing husky by volume 1:1 is well mixed, must mix
Soil, the mixed soil is put into flowerpot by the amount of 3Kg/ basins；Planted into the flowerpot into the consistent corn budding of growing way
Seed, and 4/basin；After the emergence of corn germinated seeds, the consistent maize seedling of 2 plants of growing ways is retained per disc floret basin, and as greenhouse
Middle cultivation 100d；
Wherein, the husky river sand for after 125 DEG C of sterilization 4h of sterilizing；
The corn germinated seeds are cultivated to obtain by following methods：The corn seed chosen first is put into mass concentration is
2min is soaked in 2% hydrogen peroxide, then after being placed in water seed soaking 12h, vernalization one week at 30 DEG C, produces corn budding and plants
Son；
(3) collection of soil sample：The soil around the above-mentioned maize seedling root system by cultivation is gathered, that is, obtains the soil
Earth sample, it is standby；
(4) collection of sieving sample：By above-mentioned soil sample and clear water in mass ratio 1:4 mix and are put into soy bean milk making machine and stir
Mix to uniformly after, obtain mixed liquor；The mixed liquor is poured into three layers of screen after carrying out filtering 8min, removes upper two layers of screen,
The sandy soil mixture obtained in orlop screen is rinsed with distilled water to rinse obtained filtrate it is limpid after, by third layer
Sandy soil mixture on screen is transferred to centrifuge tube, and the cumulative volume of sandy soil mixture is no more than the 2/3 of centrifugation pipe volume, produces
Sieve sample, standby；Wherein, three layers of screen by 20 mesh sieves, 120 mesh sieves and 400 mesh sieves by from top to bottom order successively
Stack composition；
(5) centrifuge：
Centrifuge for the first time：Above-mentioned sieving sample is put into after centrifuging 5min in the supercentrifuge that rotating speed is 3600r/min,
Supernatant in centrifuge tube is poured into 400 mesh screens of cleaning and filtered, then scrubs inclined filter by using distilled water
The spore being attached to after filtering on screen is transferred in culture dish by sieve, obtains centrifugation spore mixed liquor for the first time；
Second of centrifugation：The sucrose solution that mass concentration is 70% is added into the above-mentioned centrifuge tube poured out after supernatant,
The cumulative volume of material is the 2/3 of centrifugation pipe volume in centrifuge tube, and the material mixing rocked in centrifuge tube to centrifuge tube is uniform, will
Centrifuge tube is put into the supercentrifuge that rotating speed is 1600r/min after centrifugation 3-5min, the supernatant in centrifuge tube is poured into clean
Filtered in 400 net mesh screens, then scrubbing inclined screen by using distilled water will be attached on screen after filtering
Spore is transferred in culture dish, obtains second of centrifugation spore mixed liquor；
(6) spore collection and counting：It is micro- that the above-mentioned culture dish for being loaded with centrifugation spore mixed liquor for the first time is placed in dissection
Under mirror, under the observation of disecting microscope, with the pipettor being socketed by rubber head dropper and with the glass tip of the rubber head dropper
After the spore collector of pipette tips composition is acquired to the spore in mixed liquor, it is put into line surface plate, will after the completion of collection
Line surface plate, which is placed under disecting microscope, to be counted；
(7) film-making：The single taking-up of spore in line surface plate after counting is placed on slide, with the lid glass of cleaning
After piece covers slide, the liquid around cover glass is blotted with blotting paper, and finds the spore on slide under the microscope, is used
Tweezers, which press pressure, ruptures spore；With by chloraldurate, chloral hydrate, distilled water, iodine and KI in mass ratio 200:
200:200:3:After the 10 well mixed Mei Zeer reagents being prepared dye to the spore after rupture, you can obtain in light
The sample slide of yellow；
(8) second centrifugation spore mixed liquor repeat the above steps (6) and (7) handled, you can.
Comparative example 1：Using traditional wet screening decantation come pair with the soil sample identical soil sample in embodiment 1
The screening (identical in quality) for carrying out AMF spore, is comprised the following steps that：
(1) after the soil sample weighed being placed in clean container into the 20~30min that is soaked in water, 2- is stirred with glass bar
3min, stop stirring, stand into soil sample big chad and debris is deposited in container bottom, obtain sediment and soil is molten
Liquid；The soil liquid is poured into inclined three layers of screen into (identical with three layers of screen of the present invention) to be filtered, obtains filtrate
And outsifting；
(2) outsifting on compass screen surface is will stay on distilled water flushing compass screen surface to be transferred in the culture dish of cleaning；
(3) filtrate obtained in (1) is filtered again, and the outsifting being filtrated to get is transferred to the training in (2)
Support in ware, produce spore mixed liquor；
(4) the spore mixed liquor that (3) obtain is placed under disecting microscope, is acquired counting.
(5) above-mentioned experimental procedure (1)-(4) are repeated respectively totally 3 times, and calculate the average spore number of 3 sieving gained.
Thus, the spore number statistical form that embodiment 1-3 and the sieving of comparative example 1 obtain is as shown in table 1：
The embodiment 1-3 of table 1 and comparative example 1 are sieved resulting spore number statistical form
Conclusion 1：As shown in Table 1, the spore number of embodiment 1-3 sievings gained is above comparative example 1, and embodiment 1-3
The average spore number of sieving gained is 36, and 13 than comparative example more than 1, the spore number for sieving to obtain adds
56.5%.
Embodiment 4：Source soil collection in embodiment 1 is changed into Baise by Chongzuo, remaining screening step with implementation
Example 1 is identical.
Embodiment 5：Source soil collection in embodiment 2 is changed into Baise by Chongzuo, remaining screening step with implementation
Example 2 is identical.
Embodiment 6：Source soil collection in embodiment 3 is changed into Baise by Chongzuo, remaining screening step with implementation
Example 3 is identical.
Comparative example 2：Using traditional wet screening decantation come with the soil sample identical soil sample (matter in embodiment 4
Measure identical) screening of AMF spore is carried out, operating procedure is identical with comparative example 1.
Thus, the spore number statistical form that embodiment 4-6 and the sieving of comparative example 2 obtain is as shown in table 2：
The embodiment 4-6 of table 2 and comparative example 2 are sieved resulting spore number statistical form
Conclusion 2：As shown in Table 2, the spore number of embodiment 4-6 sievings gained is above comparative example 2, and embodiment 4-6
The average spore number of sieving gained is 35, and 12 than comparative example more than 1, the spore number for sieving to obtain adds
52.2%.
Embodiment 7：Source soil collection in embodiment 1 is changed into Rong'an by Chongzuo, remaining screening step with implementation
Example 1 is identical.
Embodiment 8：Source soil collection in embodiment 2 is changed into Rong'an by Chongzuo, remaining screening step with implementation
Example 2 is identical.
Embodiment 9：Source soil collection in embodiment 3 is changed into Rong'an by Chongzuo, remaining screening step with implementation
Example 3 is identical.
Comparative example 3：Using traditional wet screening decantation come pair with the soil sample identical soil sample in embodiment 7
The screening (identical in quality) for carrying out AMF spore, operating procedure are identical with comparative example 1.
Thus, the spore number statistical form that embodiment 7-9 and the sieving of comparative example 3 obtain is as shown in table 3：
The embodiment 7-9 of table 3 and comparative example 3 are sieved resulting spore number statistical form
Conclusion 3：As shown in Table 3, the spore number of embodiment 7-9 sieves gained is above comparative example 3, and embodiment 7-9 mistakes
The average spore number of sieve gained is 26, and 6 than comparative example more than 1, the spore number for sieving to obtain adds 30%..
To sum up, understood with reference to conclusion 1-3, the spore number of embodiment 1-9 sieving gained compares ratio 1-3 and adds 30%
More than, illustrate that the collection rate of the screen method spore of the present invention is high；Also, relative to comparative example tradition wet screening decantation once mistake
Filter collects that obtained spore population density is not of uniform size, the shortcomings that needing experiment operator empirically to carry out magnitude classification,
The spore that population density is low and population density is high is effectively carried out separate collection by the present invention using centrifugation sieving twice, each time
It is uniform to collect obtained spore population density, and method is easy, is affected by human factors that small, screening efficiency is high.
Described above is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, the equal change completed or modification change under the technical spirit suggested by all present invention, all should belong to
Cover the scope of the claims in the present invention.
Claims (7)
- A kind of 1. method that AMF spore is screened from soil, it is characterised in that comprise the following steps：(1) source soil collection：The whole strain of artemisia annua and its root soil are gathered, is placed in depositing in 25 DEG C, it is standby；(2) numerous culture is expanded：By the above-mentioned root soil collected and sterilizing husky by volume 1:1 is well mixed, obtains mixed soil, The mixed soil is put into flowerpot by the amount of 3Kg/ basins；Planted into the flowerpot into the consistent corn germinated seeds of growing way, And 4/basin；After the emergence of corn germinated seeds, retain the consistent maize seedling of 2 plants of growing ways per disc floret basin, and as being planted in greenhouse Train 95-100d；(3) collection of soil sample：The soil around the above-mentioned maize seedling root system by cultivation is gathered, that is, obtains the soil-like This, it is standby；(4) collection of sieving sample：By above-mentioned soil sample and clear water in mass ratio 1:3-4 is mixed and is put into soy bean milk making machine and stirs To after uniformly, mixed liquor is obtained；The mixed liquor is poured into three layers of screen after carrying out filtering 5-8min, removes upper two layers of screen, The sandy soil mixture obtained in orlop screen is rinsed with distilled water to rinse obtained filtrate it is limpid after, by third layer Sandy soil mixture on screen is transferred in centrifuge tube, produces sieving sample, standby；Wherein, three layers of screen by 20 mesh sieves, 120 mesh sieves and 400 mesh sieves stack composition successively by order from top to bottom；(5) centrifuge：Centrifuge for the first time：Above-mentioned sieving sample is put into the supercentrifuge that rotating speed is 3400-3600r/min and centrifuges 3-5min Afterwards, the supernatant in centrifuge tube is poured into 400 mesh screens of cleaning and filtered, then scrubbed by using distilled water inclined The spore being attached to after filtering on screen is transferred in culture dish by screen, obtains centrifugation spore mixed liquor for the first time；Second of centrifugation：Add the sucrose solution that mass concentration is 70% into the above-mentioned centrifuge tube poured out after supernatant, to from The cumulative volume of material is the 2/3 of centrifugation pipe volume in heart pipe, and the material mixing rocked in centrifuge tube to centrifuge tube is uniform, will centrifuge Pipe is put into the supercentrifuge that rotating speed is 1400-1600r/min after centrifugation 3-5min, the supernatant in centrifuge tube is poured into clean Filtered in 400 net mesh screens, then scrubbing inclined screen by using distilled water will be attached on screen after filtering Spore is transferred in culture dish, obtains second of centrifugation spore mixed liquor；(6) spore collection and counting：The above-mentioned centrifugation spore mixed liquor culture dish for the first time that is loaded with is placed under disecting microscope, Under the observation of disecting microscope, after being acquired with spore collector to the spore in mixed liquor, it is put into line surface plate, adopts After the completion of collection, line surface plate is placed under disecting microscope and counted；(7) film-making：The single taking-up of spore in line surface plate after counting is placed on slide, with the cover glass lid of cleaning After upper slide, the liquid around cover glass is blotted with blotting paper, and finds the spore on slide under the microscope, uses tweezers Pressing pressure ruptures spore；After being dyed with Mei Zeer reagents to the spore after rupture, you can obtain sample slide；(8) second centrifugation spore mixed liquor repeat the above steps (6) and (7) handled, you can.
- A kind of 2. method that AMF spore is screened from soil according to claim 1, it is characterised in that step (2) in, the sterilizing sand is prepared by following methods：River sand is gathered, the river sand is placed in sterilization at 120-125 DEG C After 4h, it is placed at 25 DEG C and deposits, you can it is husky obtains the sterilizing.
- A kind of 3. method that AMF spore is screened from soil according to claim 1, it is characterised in that step (2) in, the corn germinated seeds are cultivated to obtain by following methods：The corn seed chosen first is put into mass concentration is 2min is soaked in 2% hydrogen peroxide, then after being placed in water seed soaking 12h, vernalization one week at 30 DEG C, produces corn budding and plants Son.
- A kind of 4. method that AMF spore is screened from soil according to claim 1, it is characterised in that step (4) in, the cumulative volume of the sandy soil mixture loaded in the centrifuge tube is no more than the 2/3 of centrifugation pipe volume.
- A kind of 5. method that AMF spore is screened from soil according to claim 1, it is characterised in that step (6) in, the pipettor gun head that the spore collector is socketed by rubber head dropper and with the glass tip of the rubber head dropper forms.
- A kind of 6. method that AMF spore is screened from soil according to claim 1, it is characterised in that step (7) in, the Mei Zeer reagents are by chloraldurate, chloral hydrate, distilled water, iodine and KI in mass ratio 200: 200:200:3:10 well mixed are prepared.
- 7. a kind of method that AMF spore is screened from soil according to claim 1, it is characterised in that described Sample slide is in faint yellow.
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| CN111587727A (en) * | 2020-03-10 | 2020-08-28 | 绵阳师范学院 | Method for improving drought resistance of mulberry seedlings in seedling stage by using arbuscular mycorrhizal fungi in soil |
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