CN102757901B - Microorganism collection method in ecological environment survey - Google Patents
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- CN102757901B CN102757901B CN2012101351059A CN201210135105A CN102757901B CN 102757901 B CN102757901 B CN 102757901B CN 2012101351059 A CN2012101351059 A CN 2012101351059A CN 201210135105 A CN201210135105 A CN 201210135105A CN 102757901 B CN102757901 B CN 102757901B
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- 241000894006 Bacteria Species 0.000 claims description 122
- 239000002689 soil Substances 0.000 claims description 59
- 241000196324 Embryophyta Species 0.000 claims description 44
- 239000011159 matrix material Substances 0.000 claims description 41
- 239000000463 material Substances 0.000 claims description 11
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- 238000002360 preparation method Methods 0.000 claims description 4
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- 238000012216 screening Methods 0.000 abstract description 7
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a microorganism collection method in an ecological environment survey. The microorganism for the microorganism collection method is arbuscular mycorrhizal fungi. The collection method comprises the following steps: manufacturing a collection culture bag, preparing experimental matrixes, and packaging the experimental matrixes to the culture bag and sealing, and launching the culture bag. By the microorganism collection method in the ecological environment survey, the culture bag can be enriched in spore and mycelium, and the screening range is narrowed greatly and the workload is reduced.
Description
Technical field
The present invention relates to a kind of microorganism acquisition method, be specifically related to the microorganism acquisition method in a kind of ecotope prospecting.
Background technology
Bush mycorrhizal fungi (AMF) is a kind of ubiquitous endophytic fungi, and it can form symbiote with the terrestrial plant more than 80%.Bush mycorrhizal fungi is playing vital effect aspect the resistance of remaining the growth of stablizing, promote plant of the plant rhizosphere ecosystem, raising host plant and raising degenerated soil quality.Bush mycorrhizal fungi is comprised of mycorhiza spore (really), Cong Zhiti, vesicle, mycelia, and they can be as propagulum.At present, the separation and purification of mycorhiza mainly adopts spore as donor, and in soil, microsporocrap, spore and autochthonal helper utilize the wet screening collection usually.Utilize single spore, as the bacterium source, many unfavorable factors are arranged, at first, the spore infection rate is medium, and the accumulation of culture propagation body quantity is slower; Secondly, in the few soil of wet screening process miospore amount, due to the interference of grogs and organic granular, be not easy to separate spore; In the wet screening process, the larger or less particle of volume may be lost simultaneously.Although utilize spore can obtain purebred as propagulum, but be difficult to realize maximizing in the occurring in nature effect by the microbial inoculum that single bacterium source produces, the change of field environment, the Antagonism between bacterial classification and host plant all can impact single bacterium source the selectivity of bacterial classification etc., and the microbial inoculum field that produces thus is difficult to apply.Bush mycorrhizal fungi is obligate biotroph bacterial parasite, only after on the live plant root, forming mycorhiza, just can be multiplied into spore and sporocarp, in laboratory, be difficult to copy rhizosphere environment between mycorhiza and host plant, in addition the quantity of mycorhiza propagulum in soil, survival time, active size, to infect the opposite sex that is wanting in such as speed large, indoor be difficult to filter out be suitable for field growth mycorhiza flora.Therefore, along with mycorhiza under the more and more wider applicable cases in the aspects such as ECOLOGICAL REHABILITATION OF MINING AREAS, soil remediation and agro-ecology fertility, field in-situ acquisition arbuscular mycorrhiza flora and utilize composite flora to produce most important that microbial inoculum just becomes.
Summary of the invention
For the deficiency of bush mycorrhizal fungi acquisition method in prior art, the invention provides the microorganism acquisition method in a kind of ecotope prospecting.
The invention provides the microorganism acquisition method in a kind of ecotope prospecting, described microorganism is bush mycorrhizal fungi, and described acquisition method comprises the following steps:
(1) make to gather the bacterium bag: choose aperture and be the cloth of 30 μ m as the bacterium bag material; With this bacterium bag material, make the bacterium bag; One end of described bacterium bag is provided with opening;
(2) preparation is for trying matrix: for examination matrix, be the soil of target acquisition zone, described soil crossed to the sieve that aperture is 1mm, soil disinfection drying for standby after then sieving;
(3) by packing in the bacterium bag of making in step (1) for examination matrix of obtaining in step (2), load complete after, by the closure of openings of described bacterium bag;
(4) the bacterium bag that supplies to try matrix that is equipped with that obtains in step (3) is placed near the host plant root, after placement completes, mulching soil.
Preferably, described microorganism acquisition method specifically comprises the following steps:
(1) make to gather the bacterium bag: choose aperture and be the nylon cloth of 30 μ m as the bacterium bag material; With this bacterium bag material, make the bacterium bag; The length of described bacterium bag is that 5～15cm, width are that 4～12cm, thickness are 2～5cm; One end of bacterium bag is provided with opening;
(2) preparation is for trying matrix: for examination matrix, be the soil of target acquisition zone, described soil crossed to the sieve that aperture is 1mm, soil disinfection drying for standby after then sieving;
(3) by packing in the bacterium bag of making in step (1) for examination matrix of obtaining in step (2), the amount of fill of each bacterium bag is 50～150g, load complete after, by the closure of openings of described bacterium bag;
(4) the bacterium bag that supplies to try matrix that is equipped with that obtains in step (3) is placed near the host plant root, the modes of emplacement of described bacterium bag is vertical the placement, after placement completes, and mulching soil.
In step (4), be equipped with for the bacterium bag of examination matrix and place and comprise near the host plant root: the soil that will press close to described host plant root cuts out, and then described being equipped with for the bacterium bag of examination matrix is placed near the host plant root.
In step (1), described bacterium bag has anti-corrosion function, namely in one of host plant growth season, and this bacterium bag imputrescibility in soil.
Preferably, in step (1), the length of described bacterium bag is that 10cm, width are that 8cm, thickness are 3cm; And in step (3), the amount of fill of each bacterium bag is 100g.
Preferably, in step (1), an end of described bacterium bag has traction rope.During collection, described traction rope ties up to the stem section of host plant, is convenient to reclaim described bacterium bag.
It will be understood by those skilled in the art that described traction rope preferably is arranged on the end that described bacterium bag has opening for convenient.
Further, in order to make described bacterium bag, can vertically place, described traction rope more preferably is arranged on the middle part of an end of opening.
In step (2), describedly sterilizingly can adopt any mode well known by persons skilled in the art to carry out; Preferably, described sterilizing employing high temperature and high pressure steam is sterilizing.
In step (3), the sealing of described opening can adopt any mode well known by persons skilled in the art to carry out, and for example adopts the mode of sewing up by closure of openings.
Microorganism acquisition method in ecotope prospecting provided by the invention, further comprising the steps:
After one of described host plant is grown season, remove the soil on described bacterium bag top, take out described bacterium bag, then the index that comprises spore density, mycelial density, Hyphal length in described bacterium bag is measured.These measuring methods can adopt any method well known by persons skilled in the art to carry out, and for example spore density adopts wet screening tilt-pour process, mycelial density to adopt the Box junction method.
Microorganism acquisition method in ecotope prospecting of the present invention, utilizing aperture is nylon cloth making bacterium bag collection mycelium and the spore of 30 μ m.The mycelium diameter is 3～7 μ m, and mycelium can freely enter the bacterium bag like this, and very difficult the entering such as host plant root system, in such cases, the host plant Local Root is to obtain nutrient, just forces its symbiote mycorhiza and appurtenant thereof to enter the bacterium bag.Sieving with sterilizing for examination matrix in the bacterium bag, to a certain degree improved the quality for examination matrix, got rid of simultaneously the interference of other microorganism, more is conducive to mycorhiza and appendicular growth thereof.In prior art, arbuscular mycorrhiza separates every sample need gather soil sample 1.5～2kg, and then from these soil samples, separating spore, workload is often larger like this.Utilize the microorganism acquisition method in ecotope prospecting of the present invention to gather the difficult problem that arbuscular mycorrhiza has largely solved field arbuscular mycorrhiza collection, in the bacterium bag for the examination matrix 100g that only has an appointment, changed artificially simultaneously in the bacterium bag for physicochemical character and the quality thereof of trying matrix, more be conducive to spore and mycelial enrichment, dwindled so to a great extent the screening scope and reduced workload.Utilize the microorganism acquisition method in ecotope prospecting of the present invention, wherein said bacterium bag can enrichment spore and mycelium, and the bacterium source that it is produced, this just provides technical support for field produces the arbuscular mycorrhiza microbial inoculum, also for microbial inoculum, in different zones, applies and provides fundamental basis.
Embodiment
The following examples are only be used to explaining the present invention, and unrestricted the present invention.
Nylon cloth is purchased from Chongwen, Beijing development wire cloth shop, and aperture is 30 μ m.
Spore density adopts the wet screening tilt-pour process to measure; Mycelial density adopts the Box junction method to measure.
The in-situ acquisition of subsidence area, live chickens rabbit ore deposit, embodiment 1:(Yulin bush mycorrhizal fungi)
Choose aperture and be the nylon cloth of 30 μ m as the bacterium bag material.This bacterium bag has anti-corrosion function, in a growth season of host plant, and this bacterium bag imputrescibility in soil.The length of bacterium bag is that 10cm, width are 8cm, and the thickness of bacterium bag is 3cm; One end of bacterium bag is provided with opening; The middle part of opening end is provided with traction rope.
For examination matrix collecting location, be positioned at the Daliuta Town Gao Jiapan of Shenmu County, Yulin, belong to Cai Kong subsidence area, live chickens rabbit ore deposit, Shen Dong mining area, for examination matrix, be the degenerated soil of this subsidence area, and aperture is the sieve of 1mm excessively, soil after sieving before the above-mentioned bacterium bag of packing into through high temperature and high pressure steam sterilizing (121 ℃, 2h), natural air drying.
By above-mentioned, for examination matrix, pack in above-mentioned bacterium bag, each bacterium is packed to be entered for examination matrix 100g, load complete after, the opening of described bacterium bag is sewed up.
Host plant is false indigo, and false indigo is selected from subsidence area, live chickens rabbit ore deposit microbe restoration Demonstration Base.Average plant height for the examination false indigo is 43.3cm, and the hat width is 26cm.
Tested on April 17th, 2011 and carry out, the soil thawing in Shen Dong mining area at this moment, host plant is in the season of turning green.20 repetitions of test design, namely select 20 strain false indigos as host plant, according to S type curve, chooses false indigo, and selected 20 strain false indigo sizes are close.The soil that to press close to the false indigo root cuts out, then above-mentioned being equipped with for the bacterium bag of examination matrix vertically placed near the false indigo root, the placement degree of depth is 15cm, mulching soil after placement completes, and the traction rope of bacterium bag is tied up to the stem section of false indigo, in order to reclaim the bacterium bag.
At the beginning of 10 months, fetch described bacterium bag and gather the host plant rhizosphere soil.October is study area freezing season just, and the plant in this district stops growing, and this test is a growth season of host plant.In bacterium bag to the rhizosphere soil that gathers and recovery, for examination matrix, measure.Host plant rhizosphere soil miospore density, mycelial density and 100g soil Hyphal length are respectively 15/g, 3.19mg
-1And 319m.Spore density, mycelial density and Hyphal length in the bacterium bag that reclaims are measured, and the spore density in the bacterium bag is 6.04mg up to 33/g, mycelial density
-1, total Hyphal length is 604m in the bacterium bag.Spore density and mycelial density in described bacterium bag exceed respectively 18/g and 2.85mg than false indigo rhizosphere soil
-1.Illustrate that microorganism acquisition method of the present invention can improve collection capacity and the picking rate of field bush mycorrhizal fungi greatly.
The in-situ acquisition of subsidence area, Bu Lianta ore deposit, embodiment 2:(Shen Dong mining area bush mycorrhizal fungi)
Choose aperture and be the nylon cloth of 30 μ m as the bacterium bag material.This bacterium bag has anti-corrosion function, in a growth season of host plant, and this bacterium bag imputrescibility in soil.The length of bacterium bag is that 10cm, width are 8cm, and the thickness of bacterium bag is 3cm; One end of bacterium bag is provided with opening; The middle part of opening end is provided with traction rope.
Experimental field be selected in Bu Lianta ore deposit, Shen Dong mining area, for the examination matrix be this subsidence area, ore deposit degenerated soil, and the mistake aperture be the sieve of 1mm, the soil after sieving through high temperature and high pressure steam sterilizing (121 ℃, 2h), natural air drying.
By above-mentioned, for examination matrix, pack in above-mentioned bacterium bag, each bacterium is packed to be entered for examination matrix 100g, load complete after, the opening of described bacterium bag is sewed up.
Host plant is salix monogolica.The average plant height of salix monogolica is 136.5cm, and the hat width is 96.2cm.
Tested on April 18th, 2011 and carry out, the soil thawing in Shen Dong mining area at this moment, host plant is in the season of turning green.20 repetitions of test design, namely select 20 strain salix monogolicas as host plant, according to S type curve, chooses false indigo, and selected 20 strain false indigo sizes are close.The soil that to press close to the salix monogolica root cuts out, and then above-mentioned being equipped with for the bacterium bag of examination matrix is vertically placed near the salix monogolica root, and the placements degree of depth is 15cm, place complete after mulching soil, and the traction rope of bacterium bag is tied up to the stem section of salix monogolica, so that recovery bacterium bag.
At the beginning of 10 months, fetch described bacterium bag and gather the host plant rhizosphere soil, to measuring for Hyphal length in examination matrix and salix monogolica rhizosphere soil miospore density, mycelial density and 100g soil in the bacterium bag that reclaims.As a result, spore density, mycelial density and the Hyphal length in the bacterium bag reaches respectively 21/g, 4.74mg
-1And 4.74m, and rhizosphere soil miospore density, mycelial density and Hyphal length are respectively 11/g, 1.91mg
-1And 1.91m.Spore density and mycelial density in described bacterium bag exceed 10/g and 2.83mg than salix monogolica rhizosphere soil
-1.Illustrate that microorganism acquisition method of the present invention can significantly improve the acquisition rate of field bush mycorrhizal fungi, shorten the acquisition time of bush mycorrhizal fungi.
The in-situ acquisition of embodiment 3:(Ningxia coal gangue hill Subsided Reclamation Region bush mycorrhizal fungi)
Choose aperture and be the nylon cloth of 30 μ m as the bacterium bag material.This bacterium bag has anti-corrosion function, in a growth season of host plant, and this bacterium bag imputrescibility in soil.The length of bacterium bag is that 10cm, width are 8cm, and the thickness of bacterium bag is 3cm; One end of bacterium bag is provided with opening; The middle part of opening end is provided with traction rope.
Test site is at Da Wukou coal washery, Ningxia coal gangue hill, and this ground is positioned at Ningxia Hui Autonomous Region's Shizuishan City, belongs to dry climate district, middle temperate zone, and precipitation is rare and concentrate, the illumination abundance, evaporation strongly, dry air, the utmost point is unfavorable for the growth of plant.This test site is selected on coal gangue hill, the coal gangue hill upper berth the thick river sand of 0.8～1m as growth matrix, test site is China Mining Univ.'s mining area microbe restoration Demonstration Base.
For examination matrix, be discarded chiltern on waste dump, and to cross aperture be the sieve of 1mm, sieve by high temperature and high pressure steam sterilizing (121 ℃, 2h), natural air drying.
By above-mentioned, for examination matrix, pack in above-mentioned bacterium bag, each bacterium is packed to be entered for examination matrix 100g, load complete after, the opening of described bacterium bag is sewed up.
Host plant is local pioneer plant Chinese wax, and the average plant height of Chinese wax is 217cm, and the hat width is 151cm.
Tested on April 25th, 2011 and carry out, the soil thawing of Ningxia, China at this moment, host plant is in the season of turning green.20 repetitions of test design, namely select 20 strain Chinese wax as host plant, according to S type curve, chooses Chinese wax, and selected 20 strain Chinese wax sizes are close.The soil that to press close to the Chinese wax root cuts out, and then above-mentioned being equipped with for the bacterium bag of examination matrix is vertically placed near the Chinese wax root, and the placements degree of depth is 15cm, place complete after mulching soil, and the traction rope of bacterium bag is tied up to the stem section of Chinese wax, so that recovery bacterium bag.
At the beginning of 10 months, fetch the bacterium bag and gather the host plant rhizosphere soil, to reclaiming measuring for Hyphal length in examination matrix and Chinese wax rhizosphere soil spore density, mycelial density and 100g soil in the bacterium bag.As a result, bacterium bag Endospore density, mycelial density and Hyphal length are respectively 28/g, 5.66mg
-1And 566m, and Chinese wax rhizosphere soil miospore density, mycelial density and Hyphal length are respectively 13/g, 2.81mg
-1And 281m.Spore density and mycelial density in described bacterium bag exceed 15/g and 2.85mg than Chinese wax rhizosphere soil
-1.Illustrate that microorganism acquisition method of the present invention can significantly improve the acquisition rate of field bush mycorrhizal fungi, shorten the acquisition time of bush mycorrhizal fungi.
It will be understood by those skilled in the art that under the instruction of this specification sheets, can make some modifications or variation to the present invention.These modifications and variations also should be within the scope of the claims in the present invention.
Claims (8)
1. the microorganism acquisition method in ecotope prospecting, described microorganism is bush mycorrhizal fungi, described acquisition method comprises the following steps:
(1) make to gather the bacterium bag: choose aperture and be the cloth of 30 μ m as the bacterium bag material; With this bacterium bag material, make the bacterium bag; One end of described bacterium bag is provided with opening;
(2) preparation is for trying matrix: for examination matrix, be the soil of target acquisition zone, described soil crossed to the sieve that aperture is 1mm, soil disinfection drying for standby after then sieving;
(3) by packing in the bacterium bag of making in step (1) for examination matrix of obtaining in step (2), load complete after, by the closure of openings of described bacterium bag;
(4) the bacterium bag that supplies to try matrix that is equipped with that obtains in step (3) is placed near the host plant root, after placement completes, mulching soil;
Described microorganism acquisition method is further comprising the steps: after one of described host plant is grown season, remove the soil on described bacterium bag top, take out described bacterium bag.
2. microorganism acquisition method according to claim 1, is characterized in that, described acquisition method specifically comprises the following steps:
(1) make to gather the bacterium bag: choose aperture and be the nylon cloth of 30 μ m as the bacterium bag material; With this bacterium bag material, make the bacterium bag; The length of described bacterium bag is that 5～15cm, width are that 4～12cm, thickness are 2～5cm; One end of bacterium bag is provided with opening;
(2) preparation is for trying matrix: for examination matrix, be the soil of target acquisition zone, described soil crossed to the sieve that aperture is 1mm, soil disinfection drying for standby after then sieving;
(3) by packing in the bacterium bag of making in step (1) for examination matrix of obtaining in step (2), the amount of fill of each bacterium bag is 50～150g, load complete after, by the closure of openings of described bacterium bag;
(4) the bacterium bag that supplies to try matrix that is equipped with that obtains in step (3) is placed near the host plant root, the modes of emplacement of described bacterium bag is vertical the placement, after placement completes, and mulching soil.
3. microorganism acquisition method according to claim 2, it is characterized in that, in step (4), be equipped with for the bacterium bag of examination matrix and place and comprise near the host plant root: the soil that will press close to described host plant root cuts out, and then described being equipped with for the bacterium bag of examination matrix is placed near the host plant root.
4. microorganism acquisition method according to claim 3, is characterized in that, in step (1), the length of described bacterium bag is that 10cm, width are that 8cm, thickness are 3cm; And in step (3), the amount of fill of each bacterium bag is 100g.
5. microorganism acquisition method according to claim 4, is characterized in that, in step (1), an end of described bacterium bag has traction rope.
6. microorganism acquisition method according to claim 5, is characterized in that, described traction rope is arranged on the end that described bacterium bag has opening, and be arranged on described middle part with end of opening.
7. microorganism acquisition method according to claim 6, is characterized in that, in step (2), described sterilizing be to adopt high temperature and high pressure steam sterilizing.
8. the described microorganism acquisition method of according to claim 1～7 any one, is characterized in that, described microorganism acquisition method is further comprising the steps:
The index that comprises spore density, mycelial density, Hyphal length in described bacterium bag is measured.
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| Title |
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| 丛枝菌根对神东煤矿区塌陷地的修复作用与生态效应;杜善周等;《科技导报》;20101231;第28卷(第07期);41-44 * |
| 李振高等.第五章样品采集和保存处理.《土壤与环境微生物研究法》.科学出版社,2008,71-78. * |
| 杜善周等.丛枝菌根对神东煤矿区塌陷地的修复作用与生态效应.《科技导报》.2010,第28卷(第07期),41-44. |
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