In-situ culture device and method for analyzing ecological effect of root system and mycorrhiza

Abstract

The invention discloses an in-situ culture device and method for analyzing the ecological effect of a root system and mycorrhiza, the in-situ culture device comprises a first culture device, a second culture device and a third culture device, and the three culture devices are mesh bags made of nylon or polyester high polymer materials with acid and alkali resistance and ultraviolet ray resistance; the mesh bag is filled with carbon source soil subjected to standardized treatment; the three groups of mesh bags are simultaneously arranged in the field in situ; and the apertures of the three groups of mesh bags are respectively 1 mu m, 38 mu m and 1000 mu m. According to the device, separation and comparative analysis of different underground carbon input paths are realized through multi-aperture design, so that the relative contribution of a root system and mycorrhiza in the soil carbon immobilization and nutrient circulation process is quantitatively analyzed. The device is simple in structure, high in repeatability, small in disturbance to an ecological system and suitable for long-term in-situ microorganism-plant interaction research in forest, grassland and farmland ecological systems.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of ecological and soil microbiology experimental equipment, and particularly relates to an in-situ culture device and method for analyzing ecological effects of root systems and mycorrhizae, which is suitable for mycorrhizal symbiotic relationship research and soil carbon cycle mechanism analysis in scenes such as forest ecological systems and agricultural planting systems. BACKGROUND

[0002] In natural ecosystems, plant communities use 20-70% of photosynthetic products for the construction and maintenance of underground root systems and mycorrhizal symbionts. Plant roots and mycorrhizal fungi play an important role in soil carbon allocation, nutrient cycling and plant nutrient acquisition. Existing root research devices, such as root drills, root boxes and culture columns, usually have the following problems when quantitatively distinguishing the relative contributions of root systems and hyphae: ① It is difficult to achieve selective isolation of fine roots and hyphae; ② There is large disturbance in the field and poor sample repeatability; ③ There is a lack of dynamic observation capability at multiple time points in situ; ④ The culture medium is not uniform, resulting in large errors in isotope or biomarker analysis. Therefore, there is an urgent need for an experimental device that can operate in an in-situ environment for a long time, has repeatability and can effectively separate the contributions of root systems and hyphae. SUMMARY

[0003] In view of the deficiencies of the prior art, the present application provides an in-situ culture device and method for analyzing ecological effects of root systems and mycorrhizae, to realize accurate quantification of underground carbon input pathways, and quantitative evaluation of root-mycorrhizal-soil interactions and their relative contributions to soil carbon cycling and nutrient dynamics.

[0004] The present application is realized by the following technical scheme: an in-situ culture device for analyzing ecological effects of root systems and mycorrhizae, comprising a first culture device, a second culture device and a third culture device, the three groups of culture devices are net bags made of nylon or polyester high molecular materials with acid and alkali resistance and ultraviolet resistance; the net bags are filled with standardized carbon source soil inside; the three groups of net bags are simultaneously laid in-situ in the field; the pore sizes of the three groups of net bags are 1 μm, 38 μm and 1000 μm respectively.

[0005] In the device, the net bag with a pore size of 1 μm only allows dissolved leachate to enter; the net bag with a pore size of 38 μm allows the extension hyphae of mycorrhizae to pass through while blocking plant fine roots; the net bag with a pore size of 1000 μm allows fine roots and hyphae to enter simultaneously; the three groups of net bags realize different functions through the difference in mesh pore size, so as to realize the separation and quantitative analysis of root systems, hyphae and dissolved carbon input effects.

[0006] The height of the net bag is 15±1 cm, the inner diameter is 3.5±0.2 cm, and the mass of the carbon source soil filled in each bag is 50-60 g.

[0007] The net bag is sealed by hot melting process, the sealing temperature is 180-200℃, the sealing width is 5±1mm, and the leakage rate of the sealing part is less than or equal to 1%.

[0008] The carbon source soil is the surface soil of long-term planting of C4 crops to provide natural 13 C-rich tracer substrate.

[0009] The net bag is provided with an ultraviolet-resistant and acid-and-alkali-resistant label outside, which marks the number, aperture, filling date and experimental information of the net bag.

[0010] The surface soil is selected from the soil with a thickness of 0-20cm below the surface.

[0011] The net bag is vertically buried by field drilling or hole digging method when laid out, the aperture of the drilling or hole digging is 4cm, the depth is 15cm, the top end of the net bag is flush with or 0-2cm higher than the ground surface, and the surrounding soil is backfilled to make the net bag in close contact with the parent soil; the recovery time of the net bag is 60-720 days after laying out to analyze the dynamic effect of root system and mycelium.

[0012] The filling operation of the carbon source soil in the net bag is carried out in an ultraviolet-sterilized clean bench, and the soil is filled in multiple times, 10-15g each time, and the soil is compacted by a sterile glass rod with a pressure of 1-2N to keep consistent with the density of the original soil.

[0013] A method based on an in-situ culture device for analyzing the ecological effect of root system and mycorrhizae, comprising the following steps:

[0014] Step one, batch preparation of cylindrical net bags with cutting templates, bottom hot sealing, and top opening to be sealed after soil filling;

[0015] Step two, collecting the surface 0-20cm soil in the soil of long-term planting of C4 crops;

[0016] Step three, soil impurity removal and screening: after removing visible roots, stones and dry branches, the soil sample is sieved through a 2mm metal sieve and naturally air-dried at room temperature;

[0017] Step four, homogenization treatment: the air-dried soil is placed in a pulverizer and stirred at 1500-2000rpm for 10-15 minutes;

[0018] Step five, sterilization: the soil is sterilized by Co-gamma rays, the radiation dose is 25kGy, the sterilization time is 48h, and the sterilization temperature is 27℃;

[0019] Step 6: Fill the mesh bags in the clean bench. First, wipe the bench surface with 75% ethanol and sterilize it with ultraviolet light for 20 minutes. Fill the sterilized soil into the mesh bags multiple times, each time with 10-15g. Use a sterile glass rod to gently press the soil into the target density, with a pressing force of 1-2N.

[0020] Step 7: Seal the mesh bag and perform an appearance seal test and a leak test. After passing the test, affix a label and prepare for deployment.

[0021] Step 8: Select woodland or farmland as the test site. Set up multiple homogeneous replicate quadrats for each test site type. Set up three sets of devices in each quadrat. Each set of devices contains three net bags.

[0022] Step 9: At the deployment location, use a soil drill or hole digging method to vertically insert the net bag to the top or 0-2cm above the ground surface, backfill and gently press the surrounding soil to make it in close contact, record the GPS location and number, and take photos / record the vegetation and soil surface characteristics of each sampling point after deployment.

[0023] Step 10: The mesh bags are recycled 60-720 days after being laid. Aseptic operation is used during recycling. After the mesh bags are retrieved, they are brought back to the laboratory for microscopic examination of root fragments and hyphae to verify the treatment effect. Alternatively, the samples can be refrigerated or freeze-dried. If refrigerated, they should be analyzed as soon as possible. The mesh bags are opened in a clean bench and the samples are taken for testing or analysis.

[0024] In situ culture devices are used for root biomass determination, mycelial marker analysis, DNA sequencing, stable isotope detection, enzyme activity determination, or physicochemical property analysis.

[0025] Compared with the prior art, the advantages of this invention are as follows:

[0026] (1) Multi-gradient pore size and in-situ culture design can distinguish between three sources of carbon input in natural environment: roots, mycelium and dissolved carbon, and analyze their relative contributions to carbon cycling.

[0027] (2) Utilization 13 Natural soils rich in C (such as sugarcane plantations) can be used as tracer substrates to avoid the disturbance and high cost of artificial labeling;

[0028] (3) It can realize dynamic recycling at multiple time points to study the temporal changes of underground ecological processes, effectively solving the dilemma of poor representativeness and comparability caused by single sampling in existing studies;

[0029] (4) The equipment materials are standardized, have good repeatability, low cost, and minimal disturbance to the ecosystem;

[0030] (5) It can be combined with a variety of biochemical and molecular ecological analysis techniques, thus expanding its applications. Attached Figure Description

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the accompanying drawings needed in the embodiments will be briefly introduced. Obviously, the accompanying drawings in the following description only represent some embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Fig. 1 The figure is a structural schematic diagram of the embodiment of the present application.

[0033] Fig. 2 The figure is a field layout schematic diagram of the embodiment of the present application.

[0034] In the figure, 1 is a first culture device, 2 is a second culture device, and 3 is a third culture device. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort fall within the scope of the present application.

[0036] Embodiment:

[0037] It should be noted that the terms "first", "second", and the like in the specification and claims of the present application and the above-mentioned accompanying drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or a chronological sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof in the embodiments of the present application are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not necessarily limit to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0038] It should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0039] In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise explicitly specified and limited. In addition, unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0040] In the present application, unless otherwise explicitly specified and limited, the first feature is "on" or "under" the second feature, which can be direct contact between the first and second features, or indirect contact between the first and second features through an intermediate medium. Moreover, the first feature "above", "above" and "above" the second feature can be directly above or obliquely above the first feature, or only indicate that the horizontal height of the first feature is higher than that of the second feature. The first feature "below", "below" and "below" the second feature can be directly below or obliquely below the first feature, or only indicate that the horizontal height of the first feature is less than that of the second feature.

[0041] Referring to Figs. 1-2 It is a kind of for resolving rhizosphere and mycorrhizal ecological effect in situ culture device, including first culture device 1, second culture device 2 And third culture device 3, three groups of culture devices are the net bag made of nylon or polyester high molecular material with acid and alkali resistance and ultraviolet resistance;The inside of net bag is filled with standardization treatment carbon source soil;Three groups of net bags are simultaneously laid in situ in the field;The aperture of three groups of net bags is 1 μm, 38 μm, 1000 μm respectively.

[0042] In the device, the first culture device 1 adopts a mesh bag with a pore size of 1 μm, only allowing the dissolved extract to enter; the second culture device 2 adopts a mesh bag with a pore size of 38 μm, allowing the extension mycelium of mycorrhiza to pass through while blocking the plant fine roots; the third culture device 3 adopts a mesh bag with a pore size of 1000 μm, allowing the fine roots and mycelium to enter at the same time; the three groups of mesh bags realize different functions through the difference in mesh hole pore size, so as to realize the separation and quantitative analysis of the effects of root system, mycelium and soluble carbon input. In the embodiment, the mesh bag adopts a polyester high molecular material, preferably polyethylene terephthalate (PET).

[0043] The height of the mesh bag is 15±1 cm, the inner diameter is 3.5±0.2 cm, and the mass of the carbon source soil filled in each bag is 50-60 g.

[0044] The mesh bag is sealed by a hot melting process, the sealing temperature is 180-200 ℃, the sealing width is 5±1 mm, and the leakage rate at the sealing position is ≤1%.

[0045] The carbon source soil adopts the surface soil of long-term planting of C4 crops to provide natural 13 C-enriched tracer substrate. In the embodiment, the C4 crop is preferably sugarcane.

[0046] The mesh bag is provided with a label resistant to ultraviolet rays and acid and alkali outside, and the label marks the number, pore size, filling date and experimental information of the mesh bag. In the embodiment, the labels of mesh bags with different pore sizes adopt different colors, and the label adopts a polyvinyl chloride (PVC) label.

[0047] The surface soil selects the soil with a thickness of 0-20 cm below the surface, i.e. 20 cm below the ground.

[0048] The mesh bag is vertically buried by field drilling or hole digging method when laid out, the drilling or hole digging has a diameter of 4 cm and a depth of 15 cm, the top end of the mesh bag is flush with or 0-2 cm higher than the ground surface, and the surrounding soil is backfilled to make the mesh bag closely contact with the parent soil; the recovery time of the mesh bag is 60-720 days after laying out to analyze the dynamic effect of the root system and mycelium. In the embodiment, multiple regular recovery time points such as 90, 180, 360 and 720 days are preferred.

[0049] The filling operation of the carbon source soil in the mesh bag is carried out in an ultraviolet disinfected super-clean workbench, and the soil is filled in multiple times, 10-15 g each time, and the soil is compacted by a sterile glass rod with a pressure of 1-2 N to keep the same density as the original soil.

[0050] A method based on an in-situ culture device for analyzing the ecological effect of root system and mycorrhiza, comprising the following steps:

[0051] Step one, cut the template to prepare cylindrical mesh bags in batches, seal the bottom by hot melting, and seal the top after filling the soil. Step two, fill the carbon source soil in the mesh bag.

[0052] Step two, collect the surface 0-20 cm soil in the long-term planting C4 crops soil;

[0053] Step three, soil impurity removal and screening: after removing visible roots, stones and branches, the soil sample is screened through a 2 mm metal sieve and naturally air-dried at room temperature;

[0054] Step four, homogenization treatment: the air-dried soil is placed in a pulverizer and stirred at 1500-2000 rpm for 10-15 minutes;

[0055] Step five, sterilization: the soil is sterilized by Co-gamma rays, with a radiation dose of 25 kGy, a sterilization time of 48 h, and a sterilization temperature of 27°C;

[0056] Step six, net bag filling in a clean bench: first wipe the table surface with 75% ethanol and sterilize it with ultraviolet light for 20 minutes; sterilize the soil and fill it into the net bag multiple times, with each time filling 10-15 g, and gradually using a sterile glass rod to press to the target density, with a compaction force of 1-2 N;

[0057] Step seven, seal the net bag and perform appearance sealing detection and leakage detection, and then label it after passing the test;

[0058] Step eight, select forest land or farmland as the test site, set multiple homogeneous repeated sample plots for each test site type, and arrange three groups of devices in each sample plot, with each group of devices containing three net bags;

[0059] Step nine, vertically insert the net bag into the top or 0-2 cm above the ground surface by using a soil drill or hole digging method at the arrangement position, backfill and lightly press the surrounding soil to make it contact closely, record the GPS position and number, and take a photo / record the vegetation and soil surface characteristics of each sample point after arrangement;

[0060] Step ten, after 60-720 days of net bag arrangement, perform recovery using sterile operation, take back the net bag to the laboratory after recovery, and perform root fragment and mycelium microscope detection on the recovered net bag to verify the treatment effect; or, store the sample in a refrigerator or freeze-dry, and analyze as soon as possible in the case of refrigeration, open the net bag in a clean bench, and take the sample for determination or analysis.

[0061] The in-situ culture device is applied to cooperate with root biomass determination, mycelium marker analysis, DNA sequencing, stable isotope detection, enzyme activity determination, or physicochemical property analysis.

[0062] The preparation of the device of this embodiment is as follows:

[0063] 1. Net bag and material preparation

[0064] 1.1 Bag material: Nylon unidirectional woven mesh, thickness 0.15-0.4mm, pore size 1 pm, 38 pm and 1000 pm respectively. To ensure uniformity, bag size is prepared in batches using cutting templates.

[0065] 1.2 Bag specifications: cylindrical, 15 cm high, 3.5 cm inner diameter. The bottom of both ends is heat-sealed, and the top is left open for sealing after filling with soil.

[0066] 1.3 Sealing process: using heat-sealing integrated die-casting technology, the temperature is controlled at 190°C during heat-sealing, the sealing width is about 5mm, and the air tightness and leakage of the seam are checked after each batch of sealing.

[0067] 1.4 Label: use soil-resistant and UV-resistant PVC labels on the top of the bag, and tie the label to the stitched part with nylon rope.

[0068] 2. Bag and material preparation

[0069] 2.1 Collection: collect surface 0-20 cm soil in long-term C4 crop (such as sugarcane) agricultural systems, avoid obvious pollution sources and stones at the sampling points, and mix several points to ensure representativeness.

[0070] 2.2 Preliminary treatment: manually remove visible roots, stones and dry branches, and after sieving the soil sample through a 2mm metal sieve, air dry at room temperature 25-28°C.

[0071] 2.3 Homogenization treatment: place the air-dried soil in a grinder and mix thoroughly at 2000 rpm for 10 minutes.

[0072] 2.4 Sterilization: use 60Co-γ ray sterilized soil, radiation dose 25kGy, sterilization time 48h, sterilization temperature 27°C.

[0073] 3. Bag filling and clean operation

[0074] 3.1 Bag filling in super workbench, first wipe the table with 75% ethanol and sterilize with ultraviolet light for 20 minutes.

[0075] 3.2 Fill the sterilized soil into the bag in several times, about 10-15g each time, and use sterile glass rod to press gently to the target density. The total filling amount is recommended to be about 50g. The compaction force is recommended to be 1-2N.

[0076] 3.3 Seal and perform appearance sealing detection (manual visual inspection) and leakage detection (immersion), and then label after passing.

[0077] 4. Field layout and site plan

[0078] 4.1 Test site selection: forest or farmland, select representative plots according to experimental purposes.

[0079] 4.2 Plot layout: for example, set 5 5m x 5m repeated plots for each forest type, with a plot spacing of ≥50m; within each plot, lay out 3 groups of devices according to a 1.5m x 1.5m grid, with each group containing three mesh bags of different diameters.

[0080] 4.3 Installation: at the layout position, use a soil drill or dig a hole with a diameter slightly larger than the outer diameter of the mesh bag (hole diameter 4cm, depth 15cm), vertically insert the mesh bag to the top or slightly higher than the ground surface 0-2cm, backfill and lightly press the surrounding soil to make close contact, record the GPS position and number. After layout, it is recommended to take pictures / record the vegetation and soil surface characteristics of each sample point.

[0081] 5. Recovery time and sample processing

[0082] 5.1 Example of recovery time points: such as 90, 180, 360, 720 days (which can be adjusted according to experimental design). When recovering, use sterile operation (or clean tools on site) to carefully retrieve the mesh bag, and after bringing it back to the laboratory, open it on the clean bench and take samples for:

[0083] Root dry / fresh weight determination (weighing accuracy 0.001g);

[0084] Mycelial biomass (weighing method or biochemical markers);

[0085] DNA sequencing;

[0086] Isotope analysis;

[0087] High-throughput sequencing;

[0088] Soil enzyme activity determination;

[0089] Analysis of other target parameters or physicochemical properties.

[0090] 5.2 Sample preservation: microbial samples are quick-frozen at -80°C, biological markers are frozen or stored at -20°C, enzyme activity samples are refrigerated and analyzed as soon as possible or freeze-dried for isotope and other physicochemical property analysis.

[0091] 6. Isolation effect and quality control

[0092] 6.1 Mesh integrity testing: before layout, randomly select 5% of each batch of mesh bags for microscopic aperture measurement and comparison with factory parameters, with an aperture deviation of ≤±5%.

[0093] 6.2 Sealing tightness testing: after sealing each batch of mesh bags, perform a vacuum water immersion test to detect the leakage rate (target ≤1.0%.

[0094] 6.3 Sterilization confirmation: Take the soil sample after sterilization to count the colony forming units (CFU) to confirm that the biological load is significantly reduced (e.g. CFU reduction ≥ 99%).

[0095] Verification after recovery: Microscopic detection of root fragments and mycelium of the recovered mesh bag to verify the treatment effect (e.g. 1 µm mesh bag without roots / mycelium, 38 µm mesh bag with mycelium without roots, 1000 µm mesh bag with both roots and mycelium), and to check the isolation efficiency (target isolation efficiency ≥ 95%).

[0096] The device separates and compares different underground carbon input paths through multi-aperture design, thereby quantitatively analyzing the relative contribution of roots and mycorrhizae in the process of soil carbon fixation and nutrient cycling. The device has simple structure, high repeatability, and small disturbance to the ecosystem, and is suitable for long-term in-situ microbial-plant interaction research in forest, grassland and farmland ecosystems, and has strong popularization value.

[0097] The above examples are only for the purpose of illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the essence of the present application should be covered within the protection scope of the present application.

Claims

1. An in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects, characterized by: The application relates to a carbon source soil culture device, which comprises a first culture device, a second culture device and a third culture device, wherein the three culture devices are all net bags made of nylon or polyester high-molecular materials which are acid and alkali resistant and ultraviolet resistant; the net bags are filled with carbon source soil which is subjected to standardization treatment; the three net bags are simultaneously arranged in situ in the wild; the pore diameters of the three net bags are 1 mu m, 38 mu m and 1000 mu m respectively.

2. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects according to claim 1, characterized in that: The height of the net bag is 15+ / -1 cm, the inner diameter is 3.5+ / -0.2 cm, and the mass of the carbon source soil filled in each bag is 50-60 g.

3. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects of claim 1, wherein: The net bag is sealed by a hot melting process, the sealing temperature is 180-200 DEG C, the sealing width is 5+ / -1 mm, and the leakage rate of the sealing position is less than or equal to 1%.

4. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects of claim 1, wherein: The carbon source soil is surface soil which is planted with C4 crops for a long time.

5. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects of claim 1, wherein: The net bag is externally provided with a label which is resistant to ultraviolet and acid and alkali, and the label is marked with the number, pore diameter, filling date and experimental information of the net bag.

6. The in-situ culture device for resolving root system and mycorrhizal ecological effects according to claim 4, characterized in that: The surface soil selects soil with a thickness of 0-20 cm below the surface.

7. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects of claim 1, wherein: When the net bag is arranged, the net bag is vertically buried by a field drilling or hole digging method, the pore diameter of the drilling or hole digging is 4 cm, the depth is 15 cm, the top end of the net bag is flush with or higher than the ground surface by 0-2 cm, and the surrounding soil is backfilled to make the net bag closely contact with the parent soil; the recycling time of the net bag is 60-720 days after the net bag is arranged.

8. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects of claim 1, wherein: The filling operation of the carbon source soil in the net bag is carried out in an ultraviolet disinfected super-clean workbench, the soil is filled in multiple times, 10-15 g is filled in each time, and the soil is compacted by a sterile glass rod with a pressure of 1-2 N.

9. A method for resolving rhizosphere and mycorrhiza ecological effects based on the in-situ culture device according to claim 1, characterized in that, The application further discloses a preparation method of the carbon source soil culture device. Step one: cylindrical net bags are prepared in batches according to cutting templates, the bottom is sealed by hot melting, and the top is opened and then sealed after the soil is filled; Step two: surface soil with a thickness of 0-20 cm is collected from soil which is planted with C4 crops for a long time; Step three: soil impurity removal and sieving: after visible root systems, stones and dry branches are removed, the soil sample is sieved through a 2 mm metal sieve and naturally air-dried at room temperature; Step four: homogenization treatment: the air-dried soil is placed in a pulverizer and fully stirred at 1500-2000 rpm for 10-15 minutes; Step five: sterilization: the soil is sterilized by Co-gamma rays, the radiation dose is 25 kGy, the sterilization time is 48 h, and the sterilization temperature is 27 DEG C; Step six: the net bag is filled in a super-clean workbench, the workbench is wiped with 75% ethanol and sterilized by ultraviolet rays for 20 minutes; the sterilized soil is filled in the net bag multiple times, 10-15 g is filled in each time, and the soil is compacted by a sterile glass rod with a pressure of 1-2 N; Step seven: the net bag is sealed, appearance sealing detection and leakage detection are conducted, and the net bag is labeled after being qualified and then arranged; Step eight: a forest land or a farmland is selected as a test land, multiple homogeneous repeated sample plots are arranged in each test land type, three groups of devices are arranged in each sample plot, and each group of devices comprises three net bags; Step nine: the net bag is vertically inserted into the test land by a soil drill or a hole digging method to the top end or higher than the ground surface by 0-2 cm, the surrounding soil is backfilled and slightly pressed to make close contact, the GPS position and number are recorded, and the vegetation and soil surface features of each sample point are photographed / recorded after the net bag is arranged. Step ten, after 60-720 days of net bag layout, the net bag is recovered, sterile operation is used during recovery, the net bag is taken back to the laboratory after recovery, and root fragments and mycelium microscope detection are performed on the recovered net bag to verify the treatment effect; or the sample is refrigerated or freeze-dried for preservation, and in the case of refrigeration, analysis should be performed as soon as possible, the net bag is opened on a clean bench, and the sample is taken for determination or analysis.

10. The in-situ culture device for resolving rhizosphere and mycorrhizal ecological effects of claim 1, wherein: The in-situ culture device is applied to cooperate with biomass determination of root system, analysis of mycelium marker, DNA sequencing, stable isotope detection, determination of enzyme activity or analysis of physicochemical properties.

Images