CN1979127A - Improved method for quick detection of Ramaria root spore density - Google Patents
Improved method for quick detection of Ramaria root spore density Download PDFInfo
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- CN1979127A CN1979127A CN 200610165325 CN200610165325A CN1979127A CN 1979127 A CN1979127 A CN 1979127A CN 200610165325 CN200610165325 CN 200610165325 CN 200610165325 A CN200610165325 A CN 200610165325A CN 1979127 A CN1979127 A CN 1979127A
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- 241000959624 Ramaria Species 0.000 title description 3
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- 239000002689 soil Substances 0.000 claims description 32
- 238000012216 screening Methods 0.000 claims description 18
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- 238000011081 inoculation Methods 0.000 description 7
- 241001123597 Funneliformis mosseae Species 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 241000317178 Claroideoglomus etunicatum Species 0.000 description 4
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- 238000007605 air drying Methods 0.000 description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to mycorhiza biotechnology that is a new method of ecological management for coal field. Mycorhiza spore density is a standard to measure mycorhiza annidation. It adopts optimized method--wet sieve decantation decoration method to test spore density. It could improve testing accuracy and speed, and lowers the human error in spore density testing process. It supplies a spore density rapid testing method for ecological application of mycorhiza biotechnology.
Description
Technical field
The present invention relates to a kind of microbial bacteria mycorrhiza fungi and learn the miospore density inspect method, be specifically related to a kind of AMF spore density rapid assay methods.
Background technology
AMF is the ubiquitous a kind of soil microorganism of occurring in nature (being called for short AM).The Anthophyta of land more than 90% can both form mycorrhizas homobium with it, and restoration of the ecosystem has tangible ecological effect to mycorhiza to coal field.AMF is made up of mycorhiza spore (really), Cong Zhiti, vesicle, mycelia, can be as brood body.Spore is not only the organ that stores as AMF nutrition, also is a kind of stable brood body, and its morphological feature is still the taxonomic important evidence of AMF at present.Select for use the AMF spore as inoculation source, not only can obtain single mycorhiza, and can understand the mycorrhizal fungi adaptedness of condition to external world by the sprouting of mycorrhizal fungal spore.Therefore, the screening of suitable mycorrhizal fungal spore of giving birth to separates for the distribution of research mycorrhizal fungal spore in soil, have value volume and range of product and study mycorhiza and have great importance in the adaptability of coal field ecological recovery.The method of spore collection at present mainly contains the wet screening decantation, post is analysed method, floating method and centrifuge method, but method commonly used is still wet screening decantation and centrifuge method.The common ground of these methods all is mycorhiza spore richness from growth matrix can be amassed, but particularly grains of sand color is close for the granule of spore and matrix, is not easy to distinguish.Therefore, mycorhiza is difficult to reach the mycorhiza spore density fast measuring of batch samples under the more and more wider applicable cases in aspects such as ECOLOGICAL REHABILITATION OF MINING AREAS, soil remediation and agro-ecology fertility.
Summary of the invention
At above-mentioned deficiency of the prior art, the present invention will provide a kind of method of energy fast measuring batch samples mycorhiza spore density, promptly spore density conventional determining method wet screening decantation has been done improvement, present technique provides scientific approach for fast measuring batch samples spore density.
The scheme of finishing the foregoing invention task is: a kind of improved method for quick detection of Ramaria root spore density may further comprise the steps:
(1) takes by weighing the soil sampling of constant weight, be placed on and be soaked in water 20-30 minute in the container, make looseness of soil,, can add conventional soil dispersing agent if soil stickiness is very big;
(2) select for use a cover clean, have the soil sieve that the aperture is the 0.5-0.034 micron, stack successively from top to bottom by aperture order from large to small, the bottom cushions with a solids, and compass screen surface is tilted a little;
(3) stir the aqueous solution of soaking soil with glass bar, after parked several seconds, big chad and foreign material are precipitated down, then the soil liquid that suspends is poured at leisure on the soil sieve of last layer, when toppling over, concentrate to be poured on the point of compass screen surface, avoid whole compass screen surface all to speckle with the soil liquid;
(4) with clear water successively gently flushing rest on outsifting on the compass screen surface, in order to avoid residual in the residue of upper strata scalping face the AMF spore arranged, will rest on the double dish the inside that outsifting on the compass screen surface is flushed to a cleaning gently with wash bottle;
(5) with wash bottle will rest on outsifting on the compass screen surface be flushed to gently one in vitro clean, drip the blue coloring agent of Qu Liben, be placed in 90 ℃ of water-baths or the baking oven and heat half an hour, dye;
(6) filtrate after will dyeing is by dusting cover, and the filter thing on the washing screen, the flush away coloring agent, outsifting is placed in the clean double dish, in the outsifting under flushing, except that many thin grit and impurity, all the other are the spore of the AMF of different-diameter;
(7) outsifting is placed on observation under the binocular entity dissecting microscope, can observe the purple spore.
The present invention has fully excavated brood body spore (really) in the mycorrhizal fungi, mycelia can empurpled characteristics with the blue dyeing of Qu Liben, morphological character and undersize heterochromia through the mycelia after the dyeing, mycorhiza spore are obvious, under microscopic, be easy to identification and identification, can improve precision and speed that spore density is measured significantly.
Conventional wet screening decantation; it is close with grains of sand matrix on every side to observe mycorhiza spore and mycelia color and luster under binocular entity dissecting microscope; be pale brown look; be not easy to distinguish and measure; and utilize observed spore of decoration method and mycelia to be purple, with grains of sand color and luster contrast is bigger on every side, spore is more obvious from morphological character observation; identification to the arbuscular mycorrhiza spore then is very clear, and the conventional wet screening decantation of decoration method is easier to identification and identification mycorhiza spore.Add up the spore density of testing sample with traditional wet screening decantation, 350 of 10 gram sandy soil sample spore count average out to, and utilize decoration method to measure 380 of the spore count average out to of same sample, each pedotheque precision has improved 8%, has improved the precision that spore density is measured.
Utilize conventional wet screening decantation to measure spore density, need 45-50 branch clock time to count under this test condition and finish a sandy soil sample (350 spores of 10 gram soil).Count and finish 1 sample (380 spores of 10 gram soil) and utilize the dyeing rule need spend 25-30 branch clock time, 1 sample has shortened half the time (25-30 minute) on the finding speed, and, on finding speed, saved 1000-1200 minute promptly 20 hours to open-air 40 soil samples of this test.40 samples can once dye in water-bath or baking oven 30 minutes in dyeing course, and are consuming time few, can greatly improve the speed that spore density is measured after the dyeing.
Utilize this decoration method, can observe tangible mycelia and spore shape, be easy to identification, identification and observation, can greatly improve precision and speed that spore density is measured, dynamic monitoring to the mycorhiza spore density of mycorhiza after open-air large-scale application such as ECOLOGICAL REHABILITATION OF MINING AREAS, soil remediation and agro-ecology fertility provides a kind of assay method of science fast, especially for a kind of especially feasible rapid assay methods of the people who begins to learn the mycorhiza technology.
Specific embodiments
Embodiment 1:(microbial inoculum culture matrix)
Culture matrix: pick up from ground, sandy beach, north, Beijing building sandy soil, (121 ℃, 2h), natural air drying is used for pot experiment through the high temperature and high pressure steam sterilization before the dress basin.
For the examination bacterial classification: fungi strain Moses blastocyst (G.mosseae), face of land blastocyst (G.versiforme) and (G.etunicatum) respectively by
Microbial room of Inst. of Plant Nutrition ﹠. Resource, Beijing City Academy of Agricultural ﹠. Fo provides.
For studying thing: corn, Chinese sorghum are provided by middle peasant seeds company of the Chinese Academy of Agricultural Sciences.Seed 10%H before the sowing
2O
2Soaked 10 minutes, deionized water cleans several standby all over draining repeatedly.
The test basin: the white plastic basin of 20cm * 20cm * 15cm (diameter at the bottom of height * basin mouth diameter * basin) specification is adopted in test with basin.After the basin alms bowl drains with the tap water cleaning, 75% alcohol disinfecting, every basin is adorned air-dry matrix 2.5kg.
Testing program: 4 of this research and design are handled mycorhiza and are handled, promptly inoculate the mix bacterium agent (handling 4) of G.mosseae (handling 1), inoculation G.versiforme (handling 2), inoculation G.mosseae and G.versiforme mix bacterium agent (handling 3), inoculation G.mosseae, G.versiforme and G.etunicatum, 2 kinds of test plants, 8 processing altogether.Wherein handle 1 and to handle 2 are 4 repetitions, handling 3, handling 4 is 10 repetitions, 56 basins altogether.
Test is in sowing on July 5, and every basin final singling to 2 strain applies the Hoagland nutrient solution at regular time and quantity after 1 month, keeps the matrix water holding capacity between 60%-80%, results after 3 months.The AMF spore count is measured with wet screening decantation and wet screening decant decoration method in the air-dry back of matrix.
Add up spore density with traditional wet screening decantation, 350 of 10 gram sandy soil sample spore count average out to need 45-50 minute time counting to finish a sample (350 spores of 10 gram soil); And utilize 380 of wet screening decant dyeing rule same sample spore count average out to, need 25-30 minute time counting of cost to finish 1 sample (380 spores of 10 gram soil), each pedotheque has on average improved 8% on the precision, 1 sample has shortened half the time (25-30 minute) from the finding speed, one has 76 samples, can economize altogether 1900～2280 minutes, promptly save 33～38 hours, measure spore density with wet screening decant decoration method and improved precision and speed that spore density is measured greatly, this method is suitable for the spore density of batch samples and measures.
The field studies of embodiment 2:(microbial inoculum in the coal gangue hill ecological reconstruction of Ningxia)
Practice ground
Da Wukou coal washery, Ningxia coal gangue hill, this ground is positioned at Ningxia Hui Autonomous Region's Shizuishan City, arid climate district, temperate zone in belonging to, precipitation is rare and concentrate, the illumination abundance, evaporation strongly, air drying, the utmost point is unfavorable for the coal gangue hill revegetation.This test site is selected on the coal gangue hill, the coal gangue hill upper berth the thick river sand of 0.8-1m as growth matrix.These sandy soil pH is 8.12, and conductivity EC is 6.67 μ S/cm, and maximum water-holding capacity is 14.48%, available phosphorus 2.9ppm, and available nitrogen 5.27ppm, effectively potassium 97ppm belongs to the lean sandy soil of extreme poverty.
Test material
For the examination bacterial classification is the Glomus mosseae and the Glomus etunicatum mix bacterium agent (being called for short G.spp) of this laboratory enrichment culture.Test plant is local pioneer plant Chinese wax seedling, bare-rooted planting, about 1 meter of plant height.
Test design and management
Test was planted on April 18th, 2005, established inoculation and did not inoculate two processing, inoculated 192 strains, did not inoculate 204 strains, altogether 396 strains.Every strain bunch planting inoculation 50g mixing bush mycorrhiza agent.The Chinese wax spacing in the rows is 1 meter, in inoculation with between not inoculating the isolation strip is arranged, and spacing is 2 meters, and ridge, about altogether 500m are arranged around the rand
2Mainly be water management after planting, water first and adopt broad irrigation to irrigate, water weekly 2 times, water weekly 1 time after 1 month, watered in 2 weeks 1 time after 2 months that water at every turn and all irritate, plant is exempted from the water management self-sow after 3 months.
3,6 and measure the spore density of collected specimens after 13 months, the each collection connects bacterium, do not connect each 10 sample of bacterium, three samplings are 60 samples altogether, adopt wet screening decant dyeing rule same sample spore count, 1 sample has shortened half the time (25-30 minute) from the finding speed, one has 60 samples, can economize altogether 1500～1800 minutes, promptly save 25～30 hours, 60 samples can once dye in water-bath or baking oven 30 minutes in dyeing course, measured spore density with wet screening decant decoration method and had greatly improved the spore density finding speed.
Test routine 3:(microbial inoculum in the eastern ecological recovery of god ground, Inner Mongol field studies)
Substantially the same manner as Example 2, but being ecological evergreen forest zone, group of the Shenhua refreshing eastern coal Da Liu of branch office tower Western Hills North Area, test and Selection ground supplies examination soil pH 8, test plant is the Xinjiang willow.For the examination bacterial classification is the Glomus mosseae and the Glomus etunicatum mix bacterium agent (being called for short G.spp) of this laboratory enrichment culture, the conventional maintenance of plant.
3 and measure the spore density of collected specimens after 6 months, each collection connects bacterium, does not connect each 10 sample of bacterium, three samplings are 60 samples altogether, adopt wet screening decant dyeing rule same sample spore count, 1 sample has shortened half the time (25-30 minute) from the finding speed, one has 60 samples, can economize altogether 1500～1800 minutes, promptly save 25～30 hours, 60 samples can once dye in water-bath or baking oven 30 minutes in dyeing course, measured spore density with wet screening decant decoration method and had greatly improved the spore density finding speed.
Claims (3)
1. AMF spore density rapid assay methods is characterized in that may further comprise the steps:
(1) takes by weighing the soil sampling of constant weight, be placed on and be soaked in water 20-30 minute in the container, make looseness of soil,, can add conventional soil dispersing agent if soil stickiness is very big;
(2) select for use a cover clean, have the soil sieve that the aperture is the 0.5-0.034 micron, stack successively from top to bottom by aperture order from large to small, the bottom cushions with a solids, and compass screen surface is tilted a little;
(3) stir the aqueous solution of soaking soil with glass bar, after parked several seconds, big chad and foreign material are precipitated down, then the soil liquid that suspends is poured at leisure on the soil sieve of last layer, when toppling over, concentrate to be poured on the point of compass screen surface, avoid whole compass screen surface all to speckle with the soil liquid;
(4) with clear water successively gently flushing rest on outsifting on the compass screen surface, in order to avoid residual in the residue of upper strata scalping face the AMF spore arranged, will rest on the double dish the inside that outsifting on the compass screen surface is flushed to a cleaning gently with wash bottle;
(5) with wash bottle will rest on outsifting on the compass screen surface be flushed to gently one in vitro clean, drip the blue coloring agent of Qu Liben, be placed in 90 ℃ of water-baths or the baking oven and heat half an hour, dye;
(6) filtrate after will dyeing is by dusting cover, and the filter thing on the washing screen, the flush away coloring agent, outsifting is placed in the clean double dish, in the outsifting under flushing, except that many thin grit and impurity, all the other are the spore of the AMF of different-diameter;
(7) outsifting is placed on observation under the binocular entity dissecting microscope, can observe the purple spore.
2. require described AMF spore density rapid assay methods according to right 1, it is characterized in that, increased this step of dyeing than the wet screening decantation of routine.
3. require described AMF spore density rapid assay methods according to right 2, it is characterized in that, dyeing back spore is purple, is easy to distinguish morphological character.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006101653250A CN100545630C (en) | 2006-12-18 | 2006-12-18 | A kind of improved method for quick detection of Ramaria root spore density |
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| CNB2006101653250A CN100545630C (en) | 2006-12-18 | 2006-12-18 | A kind of improved method for quick detection of Ramaria root spore density |
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| CN1979127A true CN1979127A (en) | 2007-06-13 |
| CN100545630C CN100545630C (en) | 2009-09-30 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101148677B (en) * | 2007-09-19 | 2010-05-19 | 云南大学 | Method for fast extracting AM epiphyte environment DNA in plant rhizosphere soil |
| CN102759476A (en) * | 2012-07-20 | 2012-10-31 | 公安部禁毒情报技术中心 | Preparation method of cocaine hydrochloride standard substance |
| CN103396980A (en) * | 2013-06-18 | 2013-11-20 | 中国矿业大学(北京) | Field collection method of arbuscular mycorrhizal spores by rapid gathering |
| CN103411813A (en) * | 2013-05-28 | 2013-11-27 | 江苏大学 | Method for rapidly and efficiently dyeing arbuscular mycorrhizal fungi |
| CN108504726A (en) * | 2018-03-28 | 2018-09-07 | 华南农业大学 | The pretreated methods of PCR are carried out using AMF single spore minim DNAs |
| CN108844855A (en) * | 2018-04-12 | 2018-11-20 | 闫超良 | A kind of spore granule density and partial size monitoring analysis system |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6387973A (en) * | 1986-10-02 | 1988-04-19 | Kyowa Hakko Kogyo Co Ltd | Multiplication of mycorrhiza cell in sac-shaped state or in arborescent state |
| EP0558639A1 (en) * | 1990-11-23 | 1993-09-08 | Coulter Corporation | Method and apparatus for optically screening microscopic cells |
| CN100336913C (en) * | 2005-06-03 | 2007-09-12 | 厦门大学 | Proto green algae micro quantitation method |
-
2006
- 2006-12-18 CN CNB2006101653250A patent/CN100545630C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101148677B (en) * | 2007-09-19 | 2010-05-19 | 云南大学 | Method for fast extracting AM epiphyte environment DNA in plant rhizosphere soil |
| CN102759476A (en) * | 2012-07-20 | 2012-10-31 | 公安部禁毒情报技术中心 | Preparation method of cocaine hydrochloride standard substance |
| CN103411813A (en) * | 2013-05-28 | 2013-11-27 | 江苏大学 | Method for rapidly and efficiently dyeing arbuscular mycorrhizal fungi |
| CN103411813B (en) * | 2013-05-28 | 2017-02-08 | 江苏大学 | Method for rapidly and efficiently dyeing arbuscular mycorrhizal fungi |
| CN103396980A (en) * | 2013-06-18 | 2013-11-20 | 中国矿业大学(北京) | Field collection method of arbuscular mycorrhizal spores by rapid gathering |
| CN103396980B (en) * | 2013-06-18 | 2015-08-12 | 中国矿业大学(北京) | A kind of field collection method of fast enriching arbuscular mycorrhiza spore |
| CN108504726A (en) * | 2018-03-28 | 2018-09-07 | 华南农业大学 | The pretreated methods of PCR are carried out using AMF single spore minim DNAs |
| CN108504726B (en) * | 2018-03-28 | 2021-10-26 | 华南农业大学 | Method for carrying out PCR pretreatment by adopting AMF single spore trace DNA |
| CN108844855A (en) * | 2018-04-12 | 2018-11-20 | 闫超良 | A kind of spore granule density and partial size monitoring analysis system |
| CN108844855B (en) * | 2018-04-12 | 2021-07-13 | 闫超良 | Spore particle density and particle size monitoring and analyzing system |
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| Publication number | Publication date |
|---|---|
| CN100545630C (en) | 2009-09-30 |
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