CN104805000B - 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
CN104805000B
CN104805000B CN201510215791.4A CN201510215791A CN104805000B CN 104805000 B CN104805000 B CN 104805000B CN 201510215791 A CN201510215791 A CN 201510215791A CN 104805000 B CN104805000 B CN 104805000B
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China
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growth
root
groove
sampling
wetland plant
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CN104805000A (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

A kind of sampler of the same thickness rhizosphere microorganism of wetland plant root different parts and Sampling method
Technical field
The present invention relates to microbe sampling technical field is and in particular to a kind of same thickness root of wetland plant root different parts The sampler of border microorganism and sampling method.
Background technology
It is different to there is mutually point oxidation-reduction that is different and maintaining close ties with wetland plant rhizosphere-deposit (rhizosphere circle) micro-interface , the processes such as violent exchange, degraded, conversion and deposition in matter environment, often organic matter degradation, material circulation and life there is Activity place the strongest, the metabolic activity of wherein rhizosphere microorganism is considered as the foundation machine of rhizospheric environment material circulation System, plays central role in the absorption and degraded of pollutant, the research of wetland plant rhizosphere microorganism also thus become focus.
There is part research that wetland plant is placed in culture in soil at present, and with reference to terrestrial plant rhizosphere microorganism sampling side Method, that is, uproot plants when sampling, shake off loose soil around root, be left as Rhizosphere Soil and carry out microbiological analysiss, Actually wetland plant is generally grown in deposit, thus these researchs lack practical significance, because deposition water content of matter is remote Higher than normal soil, terrestrial plant rhizosphere microorganism sampling method is simultaneously inapplicable, thus wetland plant is often given birth to by existing research The deposit in long region carries out microbiological analysiss as rhizodeposition thing sample, and actually rhizosphere micro-interface thickness only has number milli Rice, the sediment sample of said method collection is too big relative to yardstick for Root of Wetland Plants, all difficult title rhizodeposition thing, and Easily it is subject to the interference of root system external deposit it is impossible to the reality of true quantitative response wetland plant rhizosphere micro-interface microorganism in sampling process Border situation.
Content of the invention
The technical problem to be solved is for the above-mentioned state of the art, provide a kind of can in situ quantitation be noiseless adopts Collection wetland plant root different parts rhizosphere negligible deposition thing the growth in the different parts diameter design corresponding groove footpath according to root Groove, it is big that root slightly locates to grow the strain of groove groove footpath phase, and root is carefully located to grow groove groove footpath and accordingly diminished it is ensured that rhizodeposition thing thickness one The sampler of wetland plant rhizosphere microorganism causing and sampling method.
The technical scheme that present invention solution above-mentioned technical problem is adopted is:
A kind of sampler of the same thickness rhizosphere microorganism of wetland plant root different parts, grows groove including by several The growth groove body that module is spliced successively, growth groove body is provided with the growth groove axially extending, and growth groove is trapezoidal Groove, and grow groove groove footpath gradually successively decrease away from the distance of wetland plant foundation with it, each growth channel mould block on be respectively provided with There is a fixing hole axially penetrating through, the fixing hole that at least one fixing axle sequentially passes through each growth channel mould block will grow channel mould block Series connection forms growth groove body, and sampler also includes organic film, and it is big that organic film can cover obstruct diameter on growth groove In membrane aperture particulate matter enter growth groove, stretch into growth groove in plant roots diameter with its away from the distance of foundation by Gradually successively decrease, the successively decrease diameter amplitude of successively decreasing of amplitude and plant roots of the groove footpath of growth groove is adapted.
For optimizing technique scheme, the concrete measure taken also includes:
After fixing axle sequentially passes through the fixing hole of each growth channel mould block, it is spun in fixing axle by nut and will grow groove Module interfixes.
Above-mentioned organic film is cellulose acetate film.
The quantity of above-mentioned fixing axle is two, and correspondingly, the fixing hole number of each growth channel mould block is two, and two Individual fixing hole is symmetrically arranged on growth channel mould block.
The material of above-mentioned growth groove body is lucite.
Sampling method to the same thickness rhizosphere microorganism of wetland plant root different parts, comprises the following steps:
Step one, stripping Root of Wetland Plants, and will intend measuring its root diameter after mensure root stretches from foundation, obtain This plan measures root diameter change rate, sets the groove footpath rate of change of growth groove by this diameter change rate, then will intend surveying root water Keep flat and be placed in growth groove, make the both sides cell wall of growth groove and intend remaining predetermined same distance between mensure root;
Step 2, growth groove in add predetermined thickness culture this Root of Wetland Plants deposit, cover intend measure Root;
Step 3, on growth groove, it is coated with machine thin film, and add on organic film and cultivate this Root of Wetland Plants Deposit, to cover organic film, cultivates the scheduled time;
Step 4, the deposit removing on organic film, cut off intending mensure root from foundation, and organic film are removed, Take out growth groove body;
Step 5, by described intend measuring root cut off paragraph by paragraph at growth channel mould block joint gap, will grow channel mould block separately, Each is grown the deposit in channel mould block separate and extract microorganism, obtain described plan and measure root different parts same thickness root Border microorganism.
Intend measuring the diameter of root by vernier caliper measurement in step one.
Deposit is the culture through homogenize process of the plan mensure root of culture wetland plant.
Predetermined incubation time is more than seven days.
Total dna of microorganism in described rhizodeposition thing in step 5, is extracted using test kit.
Wetland plant is intended measuring root and being placed horizontally in the lucite growth groove of detachable segmentation covering mud training by the present invention Support, groove groove footpath is grown according to root different parts diameter design, the diameter according to plant roots is with it away from the distance of foundation gradually Growth groove is designed as groove footpath and gradually successively decreases away from the distance of wetland plant foundation with it by the characteristic successively decreased, and makes the two of growth groove Side channel wall and plan measure and remain preset distance between root;Because rhizosphere micro-interface thickness only has several millimeters, such as it is not provided with groove Footpath changes, and the meeting of the distance between mensure root is distant in intending for root end cell wall, and root different parts rhizodeposition thing is due to thickness Differ and lack comparability, after being provided with the change of groove footpath, root different parts rhizodeposition thing consistency of thickness can be made.The present invention also may be used Design growth groove groove footpath determines the rhizodeposition thing thickness intending collection, then covers the acetic acid with certain pore size on growth groove Fibrous membrane, this film has the large particulate matters such as obstruct silt but does not affect what the finely ground particle substances such as water, inorganic salt and antibacterial passed through Effect, makes the inside and outside Sediment environment of film approximately consistent, and after culture a period of time, carefully removing cellulose acetate film is that sectional is adopted Collection root different parts rhizodeposition thing.The present invention can be micro with situ quantitation noiseless collection wetland plant root different parts rhizosphere Deposit, the technology such as available t-rflp characterizes rhizosphere microorganism population structure multiformity and spatial diversity from molecular level, And then provide fundamental basis for inquiring into the influencing mechanism of rhizospheric environment material circulation.
Brief description
Fig. 1 is the structural representation of the present invention;
Fig. 2 is the structure broken away view of Fig. 1;
Fig. 3 is the front view of the present invention;
Fig. 4 is that wetland plant is intended measuring the structural representation that root is positioned in growth groove;
Fig. 5 is the front view of Fig. 4;
Fig. 6 is that the deposit adding predetermined thickness in growth groove covers the structural representation intending measuring root;
Fig. 7 is the structural representation being coated with machine thin film on growth groove;
Fig. 8 is to cut off intending measuring root paragraph by paragraph at growth channel mould block joint gap, grow the detached structure of channel mould block and show It is intended to;
Fig. 9 is rhizosphere bacteria group t-rflp display figure in Rhizoma Acori Graminei a part in embodiment;
Figure 10 be film outer-unrooted deposit bacterial community t-rflp display figure;
Figure 11 is-unrooted deposit bacterial community t-rflp display figure in film;
Figure 12 is every gram of deposit microbial total calculated by peak area value comparison schematic diagram.
Wherein, reference is: growth groove body 1, growth channel mould block 2, growth groove 3, fixing hole 4, fixing axle 5, organic Thin film 6.
Specific embodiment
Below in conjunction with accompanying drawing, embodiments of the invention are described in further detail.
As shown in Figures 1 to 8, the sampler of the same thickness rhizosphere microorganism of a kind of wetland plant root different parts, bag Including is spliced successively by several growth channel mould blocks 2 grows groove body 1, and growth groove body 1 is provided with the life axially extending Elongated slot 3, growth groove 3 is dovetail groove, and gradually successively decreases away from the distance of wetland plant foundation with it, often in the groove footpath of growth groove 3 The fixing hole 4 axially penetrating through is provided with individual growth channel mould block 2, at least one fixing axle 5 sequentially passes through each and grows groove The fixing hole 4 of module 2 will grow channel mould block 2 and connect to be formed and grow groove body 1, and sampler also includes organic film 6, organic thin Film 6 can cover to intercept on growth groove 3 and enter growth groove 3 with diameter greater than the particulate matter of membrane aperture, stretch into the plant in growth groove 3 The diameter of thing root gradually successively decreases with its distance away from foundation, and the successively decrease diameter of amplitude and plant roots of the groove footpath of growth groove 3 is passed Amount of decrease degree is adapted.After fixing axle 5 sequentially passes through the fixing hole 4 of each growth channel mould block 2, fixing axle 5 is spun on by nut On by growth channel mould block 2 interfix.
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 fixing axle 5 is two, and correspondingly, fixing hole 4 quantity of each growth channel mould block 2 is two Individual, and two fixing holes 4 are symmetrically arranged on growth channel mould block 2.
In embodiment, the material of growth groove body 1 is lucite.
Method to the sampling of wetland plant root different parts rhizosphere microorganism, comprises the following steps:
Step one, stripping Root of Wetland Plants, and will intend measuring its root diameter after mensure root stretches from foundation, obtain This plan measures root diameter change rate, sets the groove footpath rate of change of growth groove 3 by this diameter change rate, then will intend surveying root water Keep flat and be placed in growth groove 3, make the both sides cell wall of growth groove 3 and intend remaining predetermined same distance between mensure root;As figure 4th, shown in Fig. 5;
Step 2, growth groove 3 in add predetermined thickness culture this Root of Wetland Plants deposit, cover intend measure Root;As shown in Figure 6;
Step 3, growth groove 3 on be coated with machine thin film 6, and on organic film 6 add cultivate this Root of Wetland Plants Deposit to cover organic film 6, cultivate the scheduled time;As shown in Figure 7;
Step 4, the deposit removing on organic film 6, cut off intending mensure root from foundation, and organic film 6 are moved Remove, take out growth groove body 1;
Step 5, by described intend measure root growth channel mould block 2 joint gap at cut off paragraph by paragraph, 2 points of channel mould block will be grown From, by each grow channel mould block 2 in deposit separates and extracts microorganism, obtain described intend mensure root different parts rhizosphere Microorganism.As shown in Figure 8.
Intend measuring the diameter of root by vernier caliper measurement in step one.
Deposit is the culture through homogenize process of the plan mensure root of described culture wetland plant.
Predetermined incubation time is more than seven days.
In step 5, the microbial total dna in described rhizodeposition thing is extracted using test kit.
Below taking Rhizoma Acori Graminei as a example, specifically tell about the sampling method of the present invention:
First, experiment material: Rhizoma Acori Graminei, eutrophic lake bed mud.
2nd, experimental technique:
First, peel off the root system of Rhizoma Acori Graminei, and be placed horizontally at lucite growth after root stretches from foundation by intending measuring In groove.Lucite growth groove groove footpath at foundation is big, little away from groove footpath at foundation, then adds rich battalion in growth groove 3 Fosterization bottom mud in lake, as deposit, keeps the groove footpath on same tangent plane to be all higher than root diameter 2mm, intends mensure rhizodeposition thing and is 1mm is thick.Then, the cellulose acetate film being 0.8 μm in growth groove 3 overlying cap bore footpath, and add deposit on cellulose acetate film, After thing to be deposited covers growth groove and cellulose acetate film, cultivate 7 days.
After culture 7 days, remove the deposit on cellulose acetate film, the plan of Rhizoma Acori Graminei is measured root and cuts off from foundation, and by vinegar Sour fibrous membrane removes, and takes out growth groove 3.Finally, cut off intending mensure root paragraph by paragraph, and groove 3 will be grown and separate paragraph by paragraph, by every section of life Deposit in elongated slot separates and extracts microorganism, can use at once after scraping the deposit in different slots section Powersoil dna isolation kit test kit (mobio, U.S.) extracts the microbial total dna in rhizodeposition thing.
With t-rflp technology (integrated use pcr technology, dna Restriction Enzyme incision technology, fluorescent labelling techniques and dna Sequence automatic analysis technology), by analyzing and comparing Rhizoma Acori Graminei to the mensure of specific nucleic acid fragment length polymorphism in dna level The structure of community of root different parts rhizosphere microorganism and population quantity.
Microorganism in the deposit that comparative examples are detected be cellulose acetate film outer-unrooted deposit (compareing outside film) With in film-unrooted deposit (film internal reference).
3rd, 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) comparison outside film, film internal reference microbe species are (it is generally acknowledged that each peak on peak value figure represents one kind Bacterium) and quantity (peak area at each peak) no significant difference, show that cellulose acetate film has to large particulate matter in deposit Iris action but finely ground particle substance such as microorganism etc. simultaneously do not make significant difference, and the iris action hence with cellulose acetate film eliminates and adopts Root external sediment thing interference during collection rhizodeposition thing is feasible;
(2) Rhizoma Acori Graminei does not make significant difference to deposit microbial diversity, in film-rhizosphere, comparison outside film, right in film Basically identical according to microbe species;
(3) Rhizoma Acori Graminei is notable on the impact of different types of microorganisms population quantity, compared with comparison outside film, film internal reference, right As the Partial Species growth of microorganism such as t-rf121bp, 466 bp, 488 bp have facilitation, to such as t-rf 61 bp, 507 The Partial Species growth of microorganism such as bp has inhibitory action, to Partial Species growth of microorganism such as such as t-rf 286bp, 462 bp then Do not make significant difference.
As shown in figure 12, from microbial population, there is significant space in Rhizoma Acori Graminei rhizosphere microbial population Difference, assumes the phenomenon being gradually reduced from the middle part of root to foundation, the tip of a root, but each each position rhizosphere microorganism population quantity is all aobvious Write and compare higher than outside film internal reference and film, and film internal reference with compare deposit microbial population no significant difference outside film.
According to above-described embodiment as can be seen that adopting a kind of same thickness rhizosphere of present invention wetland plant root different parts micro- Biological sampler and sampling method gather and detect that the wetland plant rhizosphere microorganism type and quantity obtaining are significantly more than Non-rhizospheric microbe, energy direct reaction wetland plant rhizosphere microorganism population structure multiformity and spatial diversity, truly Realize rhizosphere microorganism research, the technology such as available t-rflp characterizes rhizosphere microorganism population structure multiformity from molecular level And spatial diversity, and then provide fundamental basis for inquiring into the influencing mechanism of rhizospheric environment material circulation.
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment, All technical schemes belonging under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art For those of ordinary skill, some improvements and modifications without departing from the principles of the present invention, should be regarded as the protection of the present invention Scope.

Claims (10)

1. a kind of sampler of the same thickness rhizosphere microorganism of wetland plant root different parts, is characterized in that: include by some Individual growth channel mould block is spliced growth groove body successively, and described growth groove body is provided with the growth groove axially extending, Described growth groove is dovetail groove, and the groove footpath of described growth groove is gradually passed away from the distance of wetland plant foundation with it Subtract, each described growth channel mould block is provided with the fixing hole axially penetrating through, at least one fixing axle sequentially passes through each The fixing hole of individual growth channel mould block will grow groove block coupled in series and form growth groove body, and sampler also includes organic film, institute The organic film stated can cover to intercept on growth groove and enter growth groove with diameter greater than the particulate matter of membrane aperture, stretch into growth groove In the diameter of plant roots gradually successively decrease with its distance away from foundation, successively decrease amplitude and plant for the groove footpath of described growth groove The diameter of root successively decrease amplitude be adapted.
2. the sampler of the same thickness rhizosphere microorganism of a kind of wetland plant root different parts according to claim 1, It is characterized in that: after described fixing axle sequentially passes through the fixing hole of each growth channel mould block, be spun in fixing axle by nut Growth channel mould block is interfixed.
3. the sampler of the same thickness rhizosphere microorganism of a kind of wetland plant root different parts according to claim 2, It is characterized in that: described organic film is cellulose acetate film.
4. the sampler of the same thickness rhizosphere microorganism of a kind of wetland plant root different parts according to claim 2, It is characterized in that: the quantity of described fixing axle is two, correspondingly, the fixing hole number of each growth channel mould block is two, and Two fixing holes are symmetrically arranged on growth channel mould block.
5. the sampler of the same thickness rhizosphere microorganism of a kind of wetland plant root different parts according to claim 2, It is characterized in that: the material of described growth groove body is lucite.
6. adopt sampler as claimed in claim 1 same thickness rhizosphere microorganism sampling to wetland plant root different parts Method, it is characterized in that: comprise the following steps:
Step one, stripping Root of Wetland Plants, and will intend measuring its root diameter after mensure root stretches from foundation, obtain this plan Measure root diameter change rate, set the groove footpath rate of change of growth groove by this diameter change rate, then put intending mensure root level It is placed in growth groove, make the both sides cell wall of growth groove and intend remaining predetermined same distance between mensure root;
Step 2, add in described growth groove predetermined thickness this Root of Wetland Plants deposit, cover and intend measuring root;
Step 3, on described growth groove, it is coated with machine thin film, and this wetland plant root is added on described organic film The deposit of system, to cover organic film, cultivates the scheduled time;
Step 4, the deposit removing on described organic film, cut off intending measuring root from foundation, and will be described organic thin Film removes, and takes out growth groove body;
Step 5, by described intend measuring root cut off paragraph by paragraph at growth channel mould block joint gap, will grow channel mould block and separate, will often Deposit in individual growth channel mould block separates and extracts microorganism, obtains the described rhizosphere microorganism intending mensure root different parts.
7. the sampling method of the same thickness rhizosphere microorganism of wetland plant root different parts according to claim 6, it is special Levying is: intends measuring the diameter of root by vernier caliper measurement in step one.
8. the sampling method of the same thickness rhizosphere microorganism of wetland plant root different parts according to claim 7, it is special Levying is: described deposit is the culture through homogenize process of the plan mensure root cultivating described wetland plant.
9. the sampling method of the same thickness rhizosphere microorganism of wetland plant root different parts according to claim 7, it is special Levying is: described predetermined incubation time is more than seven days.
10. the sampling method of the same thickness rhizosphere microorganism of wetland plant root different parts according to claim 7, it is special Levying is: in step 5, extracts the microbial total dna in described rhizodeposition thing using test kit.
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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CN100592060C (en) * 2006-05-31 2010-02-24 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
CN103624070B (en) * 2013-10-18 2015-04-01 上海市环境科学研究院 Polyaromatic hydrocarbon polluted soil phytoremediation simulation test system and simulation test method
CN103604660B (en) * 2013-11-04 2016-01-20 浙江大学 Soil vertical section millimeter micro domain soil layer sampling method and sampler used
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

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