CN104805000A - Sampling device and sampling method for rhizospheric microorganisms at different positions and same thickness of wetland plant root - Google Patents

Sampling device and sampling method for rhizospheric microorganisms at different positions and same thickness of wetland plant root Download PDF

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Publication number
CN104805000A
CN104805000A CN201510215791.4A CN201510215791A CN104805000A CN 104805000 A CN104805000 A CN 104805000A CN 201510215791 A CN201510215791 A CN 201510215791A CN 104805000 A CN104805000 A CN 104805000A
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growth
root
groove
sampling
wetland plant
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CN104805000B (en
Inventor
王文林
刘波
万寅婧
唐晓燕
李文静
李强
盛明
庄巍
何斐
梁斌
李维新
邹长新
潘国权
王昌群
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Nantong University
Nanjing Institute of Environmental Sciences MEP
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Nantong University
Nanjing Institute of Environmental Sciences MEP
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Abstract

The invention discloses a sampling device for rhizospheric microorganisms at different positions and the same thickness of a wetland plant root. Growth grooves extending in an axial direction are arranged on a growth groove main body; the growth grooves adopt dovetail grooves; axially through fixed holes are formed in growth groove modules respectively; at least a fixed shaft sequentially passes through the fixed holes of each growth groove module to connect the growth groove modules in series, so as to form the growth groove main body; the sampling device further comprises a piece of organic thin film; the piece of organic thin film covers the growth grooves, so as to prevent particulate matters with the diameters greater than the pore sizes of the film from entering the growth grooves; the diameters of the plant roots extending into the growth grooves are gradually reduced along with the distance of the plant roots away from root foundations; the declining rate of the groove diameters of the growth grooves is matched with the declining rate of the diameters of the plant roots. The sampling device disclosed by the invention has the advantages of quantitatively sampling trace rhizospheric sediments at the different positions of the wetland plant root in situ without interference and ensuring that the thicknesses of the rhizospheric sediments at the different positions of the root are consistent and comparable by designing the growth grooves with the corresponding groove diameters according to the diameters of the different positions of the root.

Description

The sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites and sampling method
Technical field
The present invention relates to microbe sampling technical field, be specifically related to sampling unit and the sampling method of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites.
Background technology
There is point different oxidation-reduction heterogeneous environment maintained close ties with again mutually in wetland plant rhizosphere-settling (rhizosphere circle) micro-interface, there is the processes such as violent exchange, degraded, conversion and deposition, the place that organic matter degradation, material cycle and vital movement are the strongest often, wherein the Metabolic activity of rhizospheric microorganism is considered to the base mechanisms of rhizospheric environment material cycle, in the absorption and degraded of pollutent, play central role, thus the research of wetland plant rhizospheric microorganism also become focus.
Have part research that wetland plant is placed in soil at present to cultivate, and with reference to terrestrial plant rhizospheric microorganism sampling method, namely uproot plants during sampling, shake off soil loose around root, be left for Rhizosphere Soil and carry out microbiological analysis, in fact wetland plant generally grows in settling, thus these researchs lack practical significance, because settling water ratio is far above normal soil, terrestrial plant rhizospheric microorganism sampling method is also inapplicable, thus the settling of wetland plant growth district is often carried out microbiological analysis as rhizodeposition matter sample by existing research, in fact rhizosphere micro-interface thickness only has several millimeters, the sediment sample of aforesaid method collection is too large relative to Root of Wetland Plants yardstick, equal difficulty claims rhizodeposition thing, and in sampling process, be subject to the interference of root system external deposit, can not the actual state of true quantitative response wetland plant rhizosphere micro-interface microorganism.
Summary of the invention
Technical problem to be solved by this invention is for the above-mentioned state of the art, there is provided a kind of can in situ quantitation noiseless collection wetland plant root different sites rhizosphere negligible deposition thing the growth groove in different sites diameter design respective grooves footpath according to root, root is slightly located growth groove groove footpath and is strained greatly mutually, root carefully locates growth, and groove groove footpath is corresponding diminishes, and ensures sampling unit and the sampling method of the wetland plant rhizospheric microorganism of rhizodeposition thing consistency of thickness.
The present invention solves the problems of the technologies described above adopted technical scheme:
The sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites, comprise the growth groove body be spliced successively by several growth channel mould blocks, growth groove body is provided with the growth groove extended vertically, growth groove is dovetail groove, and successively decrease away from the distance of wetland plant foundation gradually with it in the groove footpath of growth groove, each growth channel mould block is provided with the fixed orifices axially run through, at least one stationary shaft will grow groove block coupled in series formation growth groove body through the fixed orifices of each growth channel mould block successively, sampling unit also comprises organic film, organic film can cover and growth groove intercept the particulate matter that diameter is greater than membrane pore size and enter growth groove, the diameter stretching into the roots of plants in growth groove successively decreases away from the distance of foundation gradually with it, the successively decrease diameter amplitude of successively decreasing of amplitude and roots of plants of the groove footpath of growth groove adapts.
For optimizing technique scheme, the concrete measure taked also comprises:
After the fixed orifices of stationary shaft successively through each growth channel mould block, be spun on stationary shaft by nut and growth channel mould block is interfixed.
Above-mentioned organic film is cellulose acetate film.
The quantity of above-mentioned stationary shaft is two, and correspondingly, the fixed orifices quantity of each growth channel mould block is two, and two fixed orificess are symmetrically arranged on growth channel mould block.
The material of above-mentioned growth groove body is synthetic glass.
To the sampling method of the same thickness rhizospheric microorganism of wetland plant root different sites, comprise the following steps:
Step one, stripping Root of Wetland Plants, and by plan measure root from foundation place its root diameter of stretching rear measurement, obtain this plan and measure root diameter velocity of variation, by the groove footpath velocity of variation of this diameter velocity of variation setting growth groove, then plan is measured root to be placed horizontally in growth groove, make the both sides cell wall of growth groove and intend measuring between root remaining predetermined same distance;
Step 2, in growth groove, add the settling of this Root of Wetland Plants of cultivation of pre-determined thickness, cover and intend measuring root;
Step 3, on growth groove, cover organic film, and add on organic film and cultivate the settling of this Root of Wetland Plants to cover organic film, the cultivation scheduled time;
Step 4, the settling removed on organic film, measure root by plan and cut off from foundation, and removed by organic film, takes out growth groove body;
Step 5, described plan is measured root cut off piecemeal at growth channel mould block joint gap place, will grow the separation of channel mould block, the settling in each growth channel mould block is separated and extracts microorganism, and obtain the same thickness rhizospheric microorganism of described plan mensuration root different sites.
Intend by vernier caliper measurement the diameter measuring root in step one.
Settling is that the plan of cultivating wetland plant measures the process of root and to homogenize the culture of process.
Predetermined incubation time is greater than seven days.
In step 5, test kit is adopted to extract the STb gene of microorganism in described rhizodeposition thing.
Wetland plant plan mensuration root is placed horizontally in the synthetic glass growth groove of detachable segmentation by the present invention covers mud cultivation, according to root different sites diameter design growth groove groove footpath, with its characteristic of successively decreasing gradually away from the distance of foundation, growth groove is designed to groove footpath according to the diameter of roots of plants successively decrease gradually away from the distance of wetland plant foundation with it, makes the both sides cell wall of growth groove and intend measuring between root remaining predetermined distance; Because rhizosphere micro-interface thickness only has several millimeters, if do not arranged the change of groove footpath, root end cell wall can be distant in intending the distance measured between root, root different sites rhizodeposition thing lacks comparability due to variable thickness, after being provided with the change of groove footpath, root different sites rhizodeposition thing consistency of thickness can be made.The present invention also can design the rhizodeposition thing thickness that growth groove groove footpath is determined to intend gathering, then on growth groove, cover the cellulose acetate film with certain pore size, this film has and intercepts the large particulate matter such as silt but do not affect the effect that the finely ground particle substances such as water, inorganic salt and bacterium pass through, make the inside and outside Sediment environment of film approximate consistent, after cultivating for some time, carefully removing cellulose acetate film can piecewise acquisition root different sites rhizodeposition thing.The present invention can in situ quantitation noiseless collection wetland plant root different sites rhizosphere negligible deposition thing, the technology such as T-RFLP can be utilized to characterize rhizospheric microorganism population structure diversity and spatial diversity from molecular level, and then be that the influencing mechanism inquiring into rhizospheric environment material cycle is provided fundamental basis.
Accompanying drawing explanation
Fig. 1 is structural representation of the present invention;
Fig. 2 is the structure broken away view of Fig. 1;
Fig. 3 is front view of the present invention;
Fig. 4 is the structural representation that wetland plant plan mensuration root is positioned in growth groove;
Fig. 5 is the front view of Fig. 4;
Fig. 6 is that the settling adding pre-determined thickness in growth groove covers the structural representation intending measuring root;
Fig. 7 is the structural representation covering organic film on growth groove;
Plan is measured the structural representation that root cut off at growth channel mould block joint gap place, grow the separation of channel mould block piecemeal by Fig. 8;
Fig. 9 is Rhizoma Acori Graminei a part rhizosphere bacteria group T-RFLP display figure in embodiment;
Figure 10 be film outer-unrooted settling bacterial flora T-RFLP display figure;
Figure 11 is-unrooted settling bacterial flora T-RFLP display figure in film;
Figure 12 is that every gram of settling microbial total calculated by peak area value compares schematic diagram.
Wherein, Reference numeral is: growth groove body 1, growth channel mould block 2, growth groove 3, fixed orifices 4, stationary shaft 5, organic film 6.
Embodiment
Below in conjunction with accompanying drawing, embodiments of the invention are described in further detail.
As shown in Figures 1 to 8, the sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites, comprise and to be spliced successively growth groove body 1 by several growth channel mould blocks 2, growth groove body 1 is provided with the growth groove 3 extended vertically, growth groove 3 is dovetail groove, and successively decrease away from the distance of wetland plant foundation gradually with it in the groove footpath of growth groove 3, each growth channel mould block 2 is provided with the fixed orifices 4 axially run through, growth channel mould block 2 is connected to be formed through fixed orifices 4 of each growth channel mould block 2 and is grown groove body 1 by least one stationary shaft 5 successively, sampling unit also comprises organic film 6, organic film 6 can cover and growth groove 3 intercept the particulate matter that diameter is greater than membrane pore size and enter growth groove 3, the diameter stretching into the roots of plants in growth groove 3 successively decreases away from the distance of foundation gradually with it, the successively decrease diameter amplitude of successively decreasing of amplitude and roots of plants of the groove footpath of growth groove 3 adapts.After the fixed orifices 4 of stationary shaft 5 successively through each growth channel mould block 2, be spun on stationary shaft 5 by nut and growth channel mould block 2 is interfixed.
In embodiment, organic film 6 is cellulose acetate film.The aperture of organic film 6 is 0.8 μm, and thickness is 0.03mm.
In embodiment, the quantity of stationary shaft 5 is two, and correspondingly, fixed orifices 4 quantity of each growth channel mould block 2 is two, and two fixed orificess 4 are symmetrically arranged on growth channel mould block 2.
In embodiment, the material of growth groove body 1 is synthetic glass.
To the method for wetland plant root different sites rhizospheric microorganism sampling, comprise the following steps:
Step one, stripping Root of Wetland Plants, and by plan measure root from foundation place its root diameter of stretching rear measurement, obtain this plan and measure root diameter velocity of variation, by the groove footpath velocity of variation of this diameter velocity of variation setting growth groove 3, then plan is measured root to be placed horizontally in growth groove 3, make the both sides cell wall of growth groove 3 and intend measuring between root remaining predetermined same distance; As shown in Figure 4, Figure 5;
Step 2, in growth groove 3, add the settling of this Root of Wetland Plants of cultivation of pre-determined thickness, cover and intend measuring root; As shown in Figure 6;
Step 3, on growth groove 3, cover organic film 6, and add on organic film 6 and cultivate the settling of this Root of Wetland Plants to cover organic film 6, the cultivation scheduled time; As shown in Figure 7;
Step 4, the settling removed on organic film 6, measure root by plan and cut off from foundation, and removed by organic film 6, takes out growth groove body 1;
Step 5, described plan is measured root cut off piecemeal at growth channel mould block 2 joint gap place, growth channel mould block 2 is separated, the settling in each growth channel mould block 2 is separated and extracts microorganism, obtain the rhizospheric microorganism of described plan mensuration root different sites.As shown in Figure 8.
Intend by vernier caliper measurement the diameter measuring root in step one.
Settling is that the plan of described cultivation wetland plant measures the process of root and to homogenize the culture of process.
Predetermined incubation time is greater than seven days.
In step 5, employing test kit extracts the microorganism total DNA in described rhizodeposition thing.
Below for calamus, specifically tell about sampling method of the present invention:
One, experiment material: calamus, eutrophic lake bed mud.
Two, experimental technique:
First, peel off the root system of calamus, and plan mensuration root is placed horizontally in synthetic glass growth groove after foundation place is stretching.Synthetic glass growth groove is large near groove footpath, foundation place, little away from groove footpath, foundation place, then in growth groove 3, adds eutrophic lake bed mud as settling, keeps the groove footpath on same tangent plane to be all greater than root diameter 2mm, and intending measuring rhizodeposition thing is that 1mm is thick.Then, growth groove 3 covers the cellulose acetate film that aperture is 0.8 μm, and add settling on cellulose acetate film, thing to be deposited is cultivated 7 days after covering growth groove and cellulose acetate film.
Cultivate after 7 days, remove the settling on cellulose acetate film, the plan of calamus is measured root and cuts off from foundation, and cellulose acetate film is removed, take out growth groove 3.Finally, plan is measured root cut off piecemeal, and growth groove 3 is separated piecemeal, settling in every section of growth groove is separated and extracts microorganism, can after the settling in different slots section be scraped, the microorganism total DNA in rhizodeposition thing is extracted in use PowerSoil DNA Isolation Kit test kit (MOBIO, the U.S.) at once.
Use T-RFLP technology (integrated use round pcr, DNA Restriction Enzyme incision technology, fluorescent labelling techniques and DNA sequence dna automatic analysis technology), by comparing structure of community and the population quantity of Rhizoma Acori Graminei different sites rhizospheric microorganism to the determination and analysis of specific nucleic acid fragment length polymorphism on DNA level.
Microorganism in the settling that comparative examples detects be cellulose acetate film outer-unrooted settling (film contrasts outward) and film interior-unrooted settling (film internal reference).
Three, experimental result:
Rhizoma Acori Graminei a part rhizosphere bacteria group T-RFLP collection of illustrative plates shows as shown in Figures 9 to 11:
(1) film contrasts outward, film internal reference microbe species (it is generally acknowledged that each peak on peak value figure represents a kind of bacterium) and quantity (peak area at each peak) is without significant difference, show that cellulose acetate film has iris action to large particulate matter in settling but finely ground particle substance does not make significant difference as microorganism etc., the root external sediment thing interference therefore utilizing the iris action of cellulose acetate film to eliminate when gathering rhizodeposition thing is feasible;
(2) Rhizoma Acori Graminei does not make significant difference to settling microbial diversity, in film-and rhizosphere, film contrast outward, film internal reference microbe species is basically identical;
(3) Rhizoma Acori Graminei on the impact of different types of microorganisms population quantity significantly, contrast outward with film, compared with film internal reference, promoter action is had to the such as Partial Species microorganism growth such as T-RF121bp, 466 bp, 488 bp, there is restraining effect to the such as Partial Species microorganism growth such as T-RF 61 bp, 507 bp, the such as Partial Species microorganism growth such as T-RF 286bp, 462 bp is not then made significant difference.
As shown in figure 12, from microbial population, there is significant spatial diversity in calamus rhizosphere microbial population, present in the middle part of root to the phenomenon that foundation, the tip of a root decline gradually, but each each position rhizospheric microorganism population quantity is all significantly higher than film internal reference and film contrasts outward, and film internal reference and film contrast settling microbial population outward without significant difference.
Can find out according to above-described embodiment, adopt the sampling unit of the present invention's a kind of wetland plant root different sites same thickness rhizospheric microorganism and sampling method collection and detect the wetland plant rhizospheric microorganism kind and quantity that obtain obviously more than non-rhizospheric microbe, energy direct reaction wetland plant rhizospheric microorganism population structure diversity and spatial diversity, truly realize rhizospheric microorganism research, the technology such as T-RFLP can be utilized to characterize rhizospheric microorganism population structure diversity and spatial diversity from molecular level, and then be that the influencing mechanism inquiring into rhizospheric environment material cycle is provided fundamental basis.
Below be only the preferred embodiment of the present invention, protection scope of the present invention be not only confined to above-described embodiment, all technical schemes belonged under thinking of the present invention all belong to protection scope of the present invention.It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principles of the present invention, should be considered as protection scope of the present invention.

Claims (10)

1. the sampling unit of the same thickness rhizospheric microorganism of wetland plant root different sites, it is characterized in that: comprise by several growth channel mould block (2) be spliced successively growth groove body (1), described growth groove body (1) is provided with the growth groove (3) extended vertically, described growth groove (3) is dovetail groove, and successively decrease away from the distance of wetland plant foundation gradually with it in the groove footpath of described growth groove (3), each described growth channel mould block (2) is provided with the fixed orifices (4) axially run through, at least one stationary shaft (5) will grow channel mould block (2) series connection formation growth groove body (1) through the fixed orifices (4) of each growth channel mould block (2) successively, sampling unit also comprises organic film (6), described organic film (6) can cover that growth groove (3) is upper to be intercepted the particulate matter that diameter is greater than membrane pore size and enter growth groove (3), the diameter stretching into the roots of plants in growth groove (3) successively decreases away from the distance of foundation gradually with it, the successively decrease diameter amplitude of successively decreasing of amplitude and roots of plants of the groove footpath of described growth groove (3) adapts.
2. the sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites according to claim 1, it is characterized in that: after the fixed orifices (4) of described stationary shaft (5) successively through each growth channel mould block (2), be spun on by nut that stationary shaft (5) is upper will be grown channel mould block (2) and interfix.
3. the sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites according to claim 2, is characterized in that: described organic film (6) is cellulose acetate film.
4. the sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites according to claim 2, it is characterized in that: the quantity of described stationary shaft (5) is two, correspondingly, fixed orifices (4) quantity of each growth channel mould block (2) is two, and two fixed orificess (4) are symmetrically arranged in growth channel mould block (2).
5. the sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites according to claim 2, is characterized in that: the material of described growth groove body (1) is synthetic glass.
6. adopt sampling unit as claimed in claim 1 to the method for the same thickness rhizospheric microorganism sampling of wetland plant root different sites, it is characterized in that: comprise the following steps:
Step one, stripping Root of Wetland Plants, and by plan measure root from foundation place its root diameter of stretching rear measurement, obtain this plan and measure root diameter velocity of variation, by the groove footpath velocity of variation of this diameter velocity of variation setting growth groove (3), then plan is measured root to be placed horizontally in growth groove (3), make the both sides cell wall of growth groove (3) and intend measuring between root remaining predetermined same distance;
Step 2, add the settling of this Root of Wetland Plants of pre-determined thickness in the described growth groove (3), cover and intend measuring root;
Step 3, on described growth groove (3), cover organic film (6), and above add the settling of this Root of Wetland Plants to cover organic film (6) at described organic film (6), cultivate the scheduled time;
Step 4, the settling removed on described organic film (6), measure root by plan and cut off from foundation, and removed by described organic film (6), take out growth groove body (1);
Step 5, described plan is measured root cut off piecemeal at growth channel mould block (2) joint gap place, channel mould block (2) will be grown be separated, settling in each growth channel mould block (2) is separated and extracts microorganism, obtains the described rhizospheric microorganism intending measuring root different sites.
7. the sampling method of the same thickness rhizospheric microorganism of wetland plant root different sites according to claim 6, is characterized in that: intend by vernier caliper measurement the diameter measuring root in step one.
8. the sampling method of the same thickness rhizospheric microorganism of wetland plant root different sites according to claim 7, is characterized in that: described settling is that the plan of cultivating described wetland plant measures the process of root and to homogenize the culture of process.
9. the sampling method of the same thickness rhizospheric microorganism of wetland plant root different sites according to claim 7, is characterized in that: described predetermined incubation time is greater than seven days.
10. the sampling method of the same thickness rhizospheric microorganism of wetland plant root different sites according to claim 7, is characterized in that: in step 5, and employing test kit extracts the microorganism total DNA in described rhizodeposition thing.
CN201510215791.4A 2015-04-30 2015-04-30 Sampling device and sampling method for rhizospheric microorganisms at different positions and same thickness of wetland plant root Expired - Fee Related CN104805000B (en)

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CN1869631A (en) * 2006-05-31 2006-11-29 浙江大学 Sampling device of rhizosphere micro-region soil
CN202485937U (en) * 2012-03-01 2012-10-10 中国科学院南海海洋研究所 Handheld layering sampling device for mangrove forest sediment
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CN103624070A (en) * 2013-10-18 2014-03-12 上海市环境科学研究院 Polyaromatic hydrocarbon polluted soil phytoremediation simulation test system and simulation test method
CN204171073U (en) * 2014-11-10 2015-02-25 长江水利委员会长江科学院 The multiple water plant fast screening devices of a kind of organic contamination deposit efficient repairing
CN204241404U (en) * 2014-12-18 2015-04-01 环境保护部南京环境科学研究所 For the device of in-site detecting submerged plant rhizosphere micro-interface dissolved oxygen
CN204589181U (en) * 2015-04-30 2015-08-26 环境保护部南京环境科学研究所 The sampling unit of the same thickness rhizospheric microorganism of a kind of wetland plant root different sites

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1869631A (en) * 2006-05-31 2006-11-29 浙江大学 Sampling device of rhizosphere micro-region soil
CN202485937U (en) * 2012-03-01 2012-10-10 中国科学院南海海洋研究所 Handheld layering sampling device for mangrove forest sediment
CN103624070A (en) * 2013-10-18 2014-03-12 上海市环境科学研究院 Polyaromatic hydrocarbon polluted soil phytoremediation simulation test system and simulation test method
CN103604660A (en) * 2013-11-04 2014-02-26 浙江大学 Soil vertical section millimeter micro domain soil layer sampling method and sampling device used in method
CN204171073U (en) * 2014-11-10 2015-02-25 长江水利委员会长江科学院 The multiple water plant fast screening devices of a kind of organic contamination deposit efficient repairing
CN204241404U (en) * 2014-12-18 2015-04-01 环境保护部南京环境科学研究所 For the device of in-site detecting submerged plant rhizosphere micro-interface dissolved oxygen
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