In-situ chemical imaging method and system applied to soil drilosphere

Abstract

An in-situ chemical imaging method and system applied to a soil drilosphere. The method comprises: S1, preparing an earthworm burrowing box and filling same with soil; S2, selecting appropriate earthworms and placing same in the soil for culturing, such that sufficient earthworm pores are generated in the soil; S3, sequentially providing a filter membrane (9), a DGT adsorption membrane (107) and a pH planar optode membrane (106) on a soil profile of a drilosphere which needs to be imaged, such that the three membranes can all be fully attached to the soil profile of the drilosphere, applying ultraviolet light under dark conditions, culturing for a period of time, and capturing a fluorescence signal emitted by the pH planar optode membrane (106), so as to obtain a pH fluorescence image; S4, performing color development on the DGT adsorption membrane (107); and S5, capturing a color-developed image of the DGT adsorption membrane (107) to obtain a DGT image, and performing picture processing on the DGT image and the pH fluorescence image to obtain spatial distribution images of available phosphorus and pH in the drilosphere. The problem of difficulty in in-situ detection of a biogeochemical cycling process driven by earthworm activities in soil is solved.

Description

An in-situ chemical imaging method and system for soil vermicontinence Technical Field

[0001] This invention belongs to the field of agricultural experimental technology, specifically relating to an in-situ chemical imaging method and system for soil vermiconium contact zones. Background Technology

[0002] Phosphorus (P) is an essential nutrient element in soil, playing a crucial role in increasing crop yields, maintaining terrestrial ecosystem functions, and improving global health. Soil phosphorus exists in two forms: inorganic and organic. Inappropriate agronomic practices and overplanting lead to a decline in available phosphorus in the soil, which is detrimental to crop growth and soil health. The concept of "sustainable agriculture" emphasizes the importance of increasing phosphorus availability while maintaining the function of agricultural ecosystems and has been increasingly applied to agricultural practices in recent years. Earthworms are widely distributed soil animals, currently believed to have existed on Earth for over 500 million years. They play a positive role in improving soil fertility and health, nutrient cycling, and pollutant remediation through processes such as carbon stabilization. Earthworms play a crucial role in shaping the physical, chemical, and biological properties of soil, earning them the title of "ecosystem engineers" (Vidal, A., Blouin, M., Lubbers, I., Capowiez, Y., Sanchez-Hernandez, JC, Calogiuri, T., van Groenigen, JW, 2023. The role of earthworms in agronomy: Consensus, novel insights and remaining challenges, Advances in Agronomy. Academic Press, pp. 1-78). Therefore, strengthening the ecological role of earthworms is beneficial for increasing crop yields while maintaining the basic functions of the ecosystem.

[0003] Earthworm-mediated phosphorus cycling has been a subject of extensive research. For example, Vos et al. studied the changes in available phosphorus levels in vermicompost and reference soils of eight earthworm species and found that earthworm activity increased soil phosphorus availability (Vos, HMJ, Koopmans, GF, Beezemer, L., de Goede, RGM, Hiemstra, T., van Groenigen, JW, 2019. Large variations in readily-available phosphorus in casts of eight earthworm species are linked to cast properties. Soil Biology and Biochemistry 138, 107583). Van Groenigen et al. conducted a meta-analysis to assess the role of earthworm castings in phosphorus activation, confirming that earthworms have a positive impact on increasing soil nutrients (Van Groenigen, J., Van Groenigen, K., Koopmans, G., Stokkermans, L., Vos, H., Lubbers, I., 2019. How fertile are earthworm casts? A meta-analysis. Geoderma 338, 525-535). The mechanism of earthworm-mediated phosphorus activation has been a focus of attention. This mainly includes: (1) earthworms stimulate soil microbial activity and increase phosphatase activity; (2) microbial activity leads to the release of soluble organic matter, which then competes with soil orthophosphate on clay and metal oxides for adsorption, replacing inorganic phosphorus; (3) earthworm activity changes soil pH, thereby affecting the form and solubility of phosphorus (Vidal, A., Blouin, M., Lubbers, I., Capowiez, Y., Sanchez-Hernandez, JC, Calogiuri, T., van Groenigen, JW, 2023. The role of earthworms in agronomy: Consensus, novel insights and remaining challenges, Advances in Agronomy. Academic Press, pp. 1-78).

[0004] Although extensive chemical studies have been conducted on earthworm-mediated soil phosphorus activation, the role of earthworms as a driver of soil microbial activity, similar to that of roots and litter in the rhizosphere, forms a microbial hotspot defined as the "vermicompost." Further exploration is needed due to the lack of reliable methods to study the biogeochemical processes of phosphorus in this unique soil environment (Kuzyakov Y, Blagodatskaya E, 2015. Microbial hotspots and hot moments in soil: Concept & review. Soil Biology and Biochemistry, 83:184-199). In-situ chemical imaging tools, including planar optical imaging for in-situ detection of spatial variations in soil pH and diffusion gradient film (DGT) for bioavailable chemicals, have been used to study biogeochemical processes in the rhizosphere and detritus, providing information on nutrient dynamics and the spatial distribution of pH. For example, Fang et al. used high-resolution zirconia DGT adsorption membranes to assess phosphorus availability in the rhizosphere and humic layer (dead roots) of rice, thereby gaining a deeper understanding of the spatial distribution and dynamics of phosphorus in these microenvironments (Fang, W., Williams, PN, Zhang, H., Yang, Y., Yin, D., Liu, Z., Sun, H., Luo, J., 2021. Combining multiple high-resolution in situ techniques to understand phosphorous availability around rice roots. Environmental Science & Technology. 55, 13082-13092). Blossfeld et al. used planar optodes to analyze the spatial dynamics of rhizosphere soil pH over time, thus providing a theoretical basis for crop root nutrient migration and utilization (Blossfeld, S., Schreiber, CM, Liebsch, G., Kuhn, AJ, Hinsinger, P., 2013. Quantitative imaging of rhizosphere pH and CO2 dynamics with planar optodes. Annals of Botany 112, 267–276). However, DGT and planar optodes techniques have not yet been applied to the study of biogeochemical cycling in the vermisphere. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems in the aforementioned related technologies.

[0006] Therefore, the purpose of this invention is to provide an in-situ chemical imaging method and system for soil vermiconceptions, which can solve the problem of difficulty in in-situ detection of biogeochemical cycles driven by earthworm activity in soil.

[0007] To solve the above-mentioned technical problems, the present invention is implemented as follows:

[0008] This invention provides an in-situ chemical imaging method for soil vermicontinence, the method comprising the following steps:

[0009] S1. Prepare the worm box and fill it with soil that meets the requirements;

[0010] S2. Select suitable earthworms and place them in the soil of the worm box for a period of time to cultivate the earthworms and create enough earthworm pores in the soil.

[0011] S3. A filter membrane, a DGT adsorption membrane, and a pH planar photoelectric membrane are sequentially placed at the soil profile of the worm contact circle to be imaged, so that the filter membrane, the DGT adsorption membrane, and the pH planar photoelectric membrane can all be fully attached to the soil profile of the worm contact circle. Ultraviolet light is applied to the worm digging box under light-proof conditions, and the worms are cultured for a period of time. The fluorescence signal emitted by the pH planar photoelectric membrane is captured by a camera to obtain a pH fluorescence image.

[0012] S4. Perform a colorimetric reaction on the DGT adsorption membrane;

[0013] S5. Use a flatbed scanner to capture the color image of the DGT adsorption membrane after color development to obtain the DGT image. Then, perform image processing on the DGT image and the pH fluorescence image obtained in S3 to obtain the spatial distribution image of available phosphorus in the worm contact zone.

[0014] In addition, the in-situ chemical imaging method for soil vermiconium according to the present invention may also have the following additional technical features:

[0015] In some of these embodiments, the soil in S1 is soil with low to medium available phosphorus content (5–40 mg kg). -1 Available phosphorus).

[0016] In some of these embodiments, the soil moisture in S1 is 60% to 70% of field capacity.

[0017] In some embodiments, a certain amount of deionized water is added between the pH plane photoelectric membrane and the worm contact zone soil profile to ensure that there are no air bubbles between the pH plane photoelectric membrane and the worm contact zone soil profile.

[0018] In some embodiments, after adding deionized water between the pH plane photoelectric film and the worm contact soil profile, the soil system is allowed to stand for more than 5 minutes to achieve complete stability.

[0019] In some embodiments, the camera device has a 370nm bandpass filter in front to avoid interference of the excitation light with the fluorescence signal.

[0020] In some embodiments, the method further includes the following steps:

[0021] S6. Establish effective phosphorus-gray value calibration curves and / or pH-gray value calibration curves, and use the established calibration curves to perform quantitative analysis on the imaging analysis results.

[0022] In some of the embodiments, the color development of the DGT adsorption membrane includes: after the DGT adsorption membrane is attached to the soil profile of the vermiconductor ring for 12 hours, it is peeled off, the soil particles adhering to the surface of the DGT adsorption membrane are rinsed with deionized water, and then immersed in molybdenum blue color development solution, reacted at 35°C for 30 minutes, the reacted DGT adsorption membrane is washed with deionized water and the moisture is absorbed with lint-free paper.

[0023] In some embodiments, the molybdenum blue colorimetric solution is prepared as follows:

[0024] A certain amount of concentrated sulfuric acid is poured into a certain amount of deionized water and stirred until homogeneous to obtain solution A.

[0025] A certain amount of ammonium molybdate was weighed and placed in a certain amount of deionized water, and heated and stirred to obtain solution B.

[0026] A certain amount of potassium antimony tartrate was dissolved in a certain amount of deionized water to obtain solution C.

[0027] Mix solutions A, B, and C thoroughly, then bring the volume up to the preset level to obtain the colorimetric reagent stock solution.

[0028] Weigh a certain amount of ascorbic acid and dissolve it in a certain amount of the color reagent stock solution, then add a certain amount of deionized water and mix well to obtain the molybdenum blue color reagent solution.

[0029] In some of these embodiments, the earthworms in S2 are 3 cm long, number 2 to 3, and cultured for 7 to 14 days.

[0030] The present invention also discloses an in-situ chemical imaging system for soil worm touch zones, the system being used to implement the in-situ chemical imaging method for soil worm touch zones as described above.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] In this embodiment of the invention, the in-situ chemical imaging method for soil vermicontinence is a vermicontinence soil in-situ chemical imaging technique that combines DGT technology and pH planar optical technology, in order to reveal the influence of earthworm activity on the spatial distribution characteristics of available phosphorus and pH in the soil.

[0033] In this embodiment of the invention, the in-situ chemical imaging method applied to the soil vermicontinuum enables in-situ monitoring of the nutrient cycling process caused by earthworm activity in the soil. Unlike conventional chemical testing methods (changes in soil mineral composition or content before and after the addition of earthworms), this invention conducts in-situ monitoring of the earthworm activity micro-zone (a 2mm range near earthworm pores, also known as the vermicontinuum), that is, directly imaging and detecting nutrients and pH in key soil areas during earthworm activity.

[0034] In this embodiment of the invention, the in-situ chemical imaging method for soil vermicontinence provides visualization of the biochemical reaction process in vermicontinence. Previous visualization methods for vermicontinence mainly reflect the degree of possible biochemical reactions and lack quantitative analysis. This invention applies two chemical imaging technologies to the vermicontinence, a key area of ​​soil, to visualize and monitor the available phosphorus content and pH value during earthworm activity, intuitively show the degree of biochemical reactions in the area, and perform quantitative analysis.

[0035] In this embodiment of the invention, the in-situ chemical imaging method for soil worm contact zone provides the simultaneous monitoring of multiple substances during the mineral-solution interface reaction process; by combining DGT and pH planar optical polarization technology, the simultaneous monitoring of available phosphorus and pH in this key area of ​​soil worm contact zone is achieved.

[0036] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0037] Figure 1 is a front view of the worm-digging box construction components disclosed in an embodiment of the present invention;

[0038] Figure 2 is a side view of the worm-digging box construction component disclosed in an embodiment of the present invention;

[0039] Figure 3 is a schematic diagram of the detachable acrylic front panel and imaging system structure of the vermicomposting box disclosed in an embodiment of the present invention; from top to bottom, there are filter membrane, DGT adsorption membrane, pH photoelectric membrane and transparent acrylic plate, forming a layered structure that is imaging the soil profile.

[0040] Figure 4 is a schematic diagram of a worm box filled with soil and incorporating earthworms, as disclosed in an embodiment of the present invention;

[0041] Figure 5 is a standard curve diagram disclosed in an embodiment of the present invention; wherein, (a) is the effective phosphorus-ash value standard curve, and (b) is the pH-ash value standard curve;

[0042] Figure 6 shows the soil imaging results of the low-phosphorus vermiconductor pores (earthworm pores) disclosed in an embodiment of the present invention; wherein, (a) is a soil profile and (b) is a spatial distribution map of available phosphorus diffusion flux.

[0043] Figure 7 shows the soil imaging results of the medium phosphorus vermiconductor contact zone (earthworm pores) disclosed in an embodiment of the present invention; wherein, (a) is a soil profile and (b) is a spatial distribution map of effective phosphorus diffusion flux.

[0044] Figure 8 shows the soil imaging results of the medium-phosphorus vermicontinuum (earthworm pores) disclosed in another embodiment of the present invention; wherein, (a) is a soil profile, (b) is a spatial distribution map of available phosphorus diffusion flux, and (c) is a spatial distribution map of pH.

[0045] Explanation of reference numerals in the attached figures:

[0046] 1. Removable acrylic front panel; 101. Front panel screws; 102. Front panel side holes; 103. Imaging film support plate; 104. Front panel rear groove; 105. Front panel screw holes; 106. pH planar photoelectric film; 107. DGT adsorption film; 108. Front panel screw holes; 2. Removable acrylic upper panel; 201. Upper panel screws; 202. Ventilation holes; 203. Upper panel screw holes; 3. Soil; 301. Earthworm pores; 302. Earthworm; 4. Rear of the worm digging box; 401. Front screw hole of the worm digging box; 402. Top screw hole of the worm digging box; 5. Wire; 6. Thermometer and hygrometer; 601. Thermometer and hygrometer display; 602. Thermometer and hygrometer sensor; 7. Humidifier; 701. Water pipe; 702. Shower head; 8. Exhaust fan; 801. Exhaust fan shaft; 802. Exhaust fan blades; 9. Filter membrane. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and specific examples and application scenarios.

[0049] To address the challenge of in-situ detection of earthworm-driven biogeochemical cycles in soil, this invention provides an imaging system based on diffuse gradients thin-film technique (DGT) and pH planar optical imaging. This system enables in-situ detection of available phosphorus (labile-P) and pH in the earthworm contact zone (a micro-region within the earthworm's activity area), thus achieving in-situ analysis of earthworm-mediated soil biogeochemical processes. Unlike traditional potted soil sampling methods, this invention overcomes the potential for changes in soil physicochemical properties caused by destructive sampling. Furthermore, it significantly reduces detection time and costs in terms of operation and economic efficiency. Moreover, compared to the poor precision of traditional potted experiments, this invention achieves sub-millimeter-scale detection, greatly improving detection precision.

[0050] In some embodiments of the present invention, an in-situ chemical imaging method for soil vermicomposting is provided, the process of which mainly includes five steps: (1) filling the vermicomposting box with soil; (2) earthworm culture; (3) attaching an imaging membrane to the soil profile; (4) chemical imaging experiment; and (5) image processing. The basic principle can be summarized as follows: (1) DGT: The high-resolution adsorption membrane adsorbs soluble and exchangeable phosphorus in the soil within a certain time. The phosphorus content is characterized by molybdenum blue colorimetric solution, and the corresponding gray value and phosphorus standard curve are obtained based on the scanner to quantitatively analyze the phosphorus content in each area of ​​the image. In addition, in order to accurately detect the content of available phosphorus in the soil, the adsorption membrane still needs to meet the requirement that the adsorbent particles are small enough (≤10μm) and uniformly distributed in the membrane; (2) The surface of the pH photoelectric membrane is coated with a pH-sensitive fluorescent dye, which is excited by ultraviolet light. + Changes in induced membrane fluorescence intensity. The combined use of these two imaging techniques enables visualization and quantitative analysis of the spatial distribution of available phosphorus and pH in soil. The specific scheme of this technique is described in detail below.

[0051] The first step is the construction of the worm-digging box: To balance the free movement of earthworms with the adhesion of the detection membrane to the soil profile, as shown in Figures 1-4, a transparent acrylic square box (hereinafter referred to as the worm-digging box) is designed as a container for earthworm activity. The worm-digging box has a square box structure, which mainly includes a detachable acrylic front panel 1, a detachable acrylic top panel 2, a rear part of the worm-digging box 4, wires 5, a thermometer and hygrometer 6, a humidifier 7, and an exhaust fan 8. On the detachable acrylic front panel 1, front panel screws 101 are used to fix the detachable acrylic front panel 1 to the rear part of the worm-digging box 4. The side of the detachable acrylic front panel 1 has a front panel side hole 102, which is used to insert the imaging membrane carrier plate 103, that is, the imaging membrane carrier plate 103 is inserted into the front panel side hole 102. On the detachable acrylic upper plate 2, upper plate screws 201 are used to fix the detachable acrylic upper plate 2 to the detachable acrylic front plate 1 and the rear part 4 of the worm box. Several ventilation holes 202 are provided on the detachable acrylic upper plate 2 to allow air inside the worm box to communicate with the outside air. One end of the wire 5 is connected to the power supply and the other end is connected to the ventilation fan 8 to supply power to the ventilation fan 8. The thermometer and hygrometer 6, humidifier 7 and ventilation fan 8 are all provided on the detachable acrylic upper plate 2. The thermometer and hygrometer 6 includes a thermometer and hygrometer reading 601 and a thermometer and hygrometer sensor 602. The thermometer and hygrometer reading 601 is located above the detachable acrylic upper plate 2, allowing external viewing of the current temperature and humidity readings. The thermometer and hygrometer sensor 602 extends into the internal space of the worm box to measure the temperature and humidity inside the worm box. The humidifier 7 is used to periodically water the soil to maintain soil moisture. The ventilation fan 8 is used to accelerate air circulation inside the worm box. The upper plate screw hole 203 and the front plate screw hole 108 are connected and fixed by the upper plate screw 201. The upper wall of the rear part 4 of the worm box is provided with a worm box upper screw hole 402, and the upper plate screw hole 203 and the worm box upper screw hole 402 are connected and fixed by the upper plate screw 201. The filter membrane 9 is vertically arranged between the detachable acrylic front plate 1 and the rear part 4 of the worm box. The rear side of the detachable acrylic front plate 1 has a front plate rear groove 104, which facilitates the formation of images after the pH planar photoelectric membrane 106 and DGT adsorption membrane 107 on the imaging membrane carrier plate 103 come into contact with the soil 3. The front plate rear groove 104 is a groove opened inside the front plate and is connected to the soil inside the worm box. By filling the groove with soil, each membrane has enough space to contact the soil. The membranes used for soil imaging are arranged in the following order from the outside to the inside: DGT adsorption membrane 107 and pH planar photoelectric membrane 106. The front plate screw hole 105 and the front screw hole 401 of the worm box are fixed with front plate screws 101.

[0052] In some embodiments of the present invention, the humidifier 7 includes a water pipe 701 and a shower head 702. The water pipe 701 is used to draw in deionized water and transport it to the shower head 702, which is used to spray the deionized water evenly onto the surface of the soil 3. The ventilation fan 8 includes a shaft 801 and fan blades 802, which is driven by electricity transmitted by wire 5 to ensure the oxygen content inside the worm box.

[0053] The second step is earthworm cultivation: Select a slightly neutral soil (3) rich in organic matter as the substrate for earthworm cultivation. Before filling the worm box with soil, the soil should be air-dried and sieved through a 2mm sieve. To further provide a suitable soil environment for earthworm growth, the soil moisture should be adjusted to 60%–70% of field capacity using a humidifier (7) during or after filling, and measured using a thermometer and hygrometer (6). Select mature and vigorous earthworms (302) and cultivate them in the soil (3). After 7–14 days of cultivation, stable and sufficiently numerous earthworm pores (301) will be formed. During the cultivation process, wrap the worm box around its sides and top and bottom with aluminum foil to simulate a dark environment in the soil. Place the worm box in a greenhouse at 25℃ and 60% humidity for cultivation.

[0054] Step 3, solution preparation:

[0055] Preparation of molybdenum blue colorimetric solution: Slowly pour 194.6 mL of concentrated sulfuric acid into 500 mL of deionized water and stir with a glass rod to prepare solution A; weigh 20.00 g of ammonium molybdate, add 200 mL of deionized water, heat and stir to dissolve, and prepare solution B; weigh 0.50 g of potassium antimony tartrate, dissolve in 50 mL of deionized water, and prepare solution C; after solutions A and B have cooled to room temperature, mix solutions A, B, and C, dilute to 1000 mL with deionized water, and transfer to a brown reagent bottle to protect from light and store at 4°C. This solution serves as the stock solution for the colorimetric reagent; weigh 0.60 g of ascorbic acid, dissolve in 40 mL of the stock solution, and then add 400 mL of deionized water and mix thoroughly to obtain the phosphorus colorimetric reagent for the molybdenum blue colorimetric method.

[0056] The fourth step is the construction of the imaging system: A filter membrane 9, a DGT adsorption membrane 107, and a pH planar optical electrode membrane 106 are sequentially attached to a soil profile with earthworm pores. A plastic partition is placed between the pH planar optical electrode membrane and the imaging membrane support plate 103 to ensure close adhesion between the combined membrane and the soil profile, thereby improving the reliability of solute imaging by the DGT high-resolution adsorption membrane and the pH planar optical electrode membrane. The optimal placement time for the DGT and planar optical electrodes needs to be determined during the experimental period. Images are processed using ImageJ software, and the spatial resolution of the DGT and pH planar optical electrode images is confirmed. The filter membrane, as part of the diffusion layer in the DGT device, reduces radial diffusion to maintain stable diffusion and adsorption of the dissolved substances; it was purchased from Whatman (Nuclepore Track-Etch Membrane, 0.2 μm pore size). The DGT high-resolution adsorption membrane, used for the enrichment of available phosphorus, was purchased from Nanjing Weishen Environmental Protection Technology Co., Ltd., model GDZR precipitated zirconia adsorption membrane, with a thickness of 0.1 mm. The pH planar photoelectric membrane was purchased from Zhongke Zhigan (Nanjing) Environmental Technology Co., Ltd. After reaction time, the DGT membrane and pH planar photoelectric membrane were imaged separately. (a) DGT membrane imaging: The DGT high-resolution adsorption membrane was attached to the soil profile for 12 hours, then peeled off. The surface soil particles were rinsed with deionized water, and the membrane was immersed in molybdenum blue colorimetric solution. After reacting at 35℃ for 30 minutes, it was washed with deionized water and dried with lint-free paper. The resulting image was then scanned using a scanner. (b) pH photoelectric membrane imaging: The pH photoelectric membrane was attached to the soil profile. To avoid air bubbles between the membrane and the soil profile, a small amount of deionized water was added between the membrane and soil. The membrane was allowed to stand for 5 minutes to allow the soil system to stabilize completely. The worm box was placed in front of a planar ultraviolet light source with a wavelength of 355 nm. A camera was used to capture the fluorescence signal emitted by the pH photoelectric membrane, and a 370 nm bandpass filter was placed in front of the camera lens to filter ultraviolet light and prevent it from interfering with the fluorescence signal emitted by the pH photoelectric membrane.

[0057] The fifth step is to establish a standard curve for quantitative analysis of the results of the previous image processing.

[0058] The imaging shows the two-dimensional distribution of available phosphorus and pH in all pixels within the membrane area, not just the average concentration in that area or the concentration change in a certain dimension; the data shown without a standard curve is just the image grayscale value identified by the software, and the experimental results obtained after calibration and analysis using a standard curve are meaningful.

[0059] Please refer to Figure 5, where (a) is the standard curve of effective phosphorus-ash value. Phosphorus concentrations of 0, 20, 50, 100, 200, 500, 750, 1000, and 2000 μg / L were prepared using phosphorus standard materials. -1A phosphorus standard solution was used to immerse a circular DGT membrane in the P solution. After adsorption for 12 hours, the membrane was developed in molybdenum blue solution. The stained DGT membrane was immediately rinsed several times with deionized water at 4°C to remove any residual developer, and then soaked in deionized water for 5 minutes (to stop the colorimetric reaction). The DGT membrane was then removed, wiped dry with filter paper, and placed face down on a scanner. The resolution was set to 1200 dpi. After scanning and obtaining the image, ImageJ software was used to convert the image to grayscale. The exponential function with the highest correlation was selected to fit the cumulative amount of P per unit area on the membrane and its corresponding grayscale intensity, which is the standard curve (G(f)) of the cumulative amount of analyte per unit area (f) - grayscale value (G). (b) pH-grayscale standard curve: Prepare pH standard solutions using sodium acetate-acetic acid solution (pH 4–6) and potassium dihydrogen phosphate-borax solution (pH 6–9). Place a long strip of pH photoelectric standard film in a cuvette and immerse the film in deionized water to ensure complete adhesion. Then, pour in standard solutions representing different pH values ​​sequentially, develop the color under 355nm ultraviolet light, and record the light intensity using a camera equipped with a pre-filter. Measure the concentration values ​​using ImageJ software and plot the standard curve. (c) Select the Calibration bar function option in ImageJ software to insert the effective phosphorus concentration and pH value bars into the image.

[0060] Example 1: Visualization of the spatial distribution of available phosphorus in the vermicompost of low-phosphorus soil

[0061] (1) Earthworm culture: Select low-phosphorus soil (available phosphorus content of 8.75 mg / kg) -1 The earthworms were placed in a designed worm-burrowing box, and three Eisenia fetidae earthworms, each 3 cm long and weighing 0.5 g, were cultured in the box for 7 days to create sufficient worm pores. During the cultivation process, the worm-burrowing box was wrapped with aluminum foil to simulate the dark environment of soil. The worm-burrowing box was then placed in a greenhouse at 25°C and 60% humidity. During the cultivation process, a suitable amount of water was sprinkled on the top of the box periodically to maintain soil moisture.

[0062] (2) Material preparation: Cut DGT high-resolution adsorption membranes with a diameter of 2.5 cm for soaking in adsorbed phosphorus standard solutions and plotting a standard curve of available phosphorus concentration-grayscale value. Simultaneously, cut several 5 cm × 5 cm DGT membranes for attaching to worm palpation profiles to image the spatial distribution of available phosphorus. Prepare filter membranes slightly larger than the imaging membrane to maintain the stability of the soil-solution interface. Prepare phosphorus standard solutions with specific concentration gradients and molybdenum blue colorimetric solution for subsequent reaction and color development of the phosphorus standard adsorption membrane and the imaging membrane.

[0063] (3) Imaging Operation: After a period of cultivation, the detachable acrylic front plate of the worm box was removed. A filter membrane was attached to the desired soil profile for imaging, and the DGT high-resolution adsorption membrane was placed on the imaging support plate. The acrylic front plate was then replaced, and the imaging support plate was inserted. To ensure a tight fit between the acrylic plate and the DGT membrane, a thin plastic sheet was inserted between the DGT membrane and the imaging support plate, allowing the DGT membrane and filter membrane to fully adhere to the soil profile. After 12 hours of cultivation in the dark, the imaging support plate was removed, the DGT membrane was peeled off, and the surface of the DGT membrane was washed with deionized water. The membrane was then placed in a molybdenum blue developing solution for 30 minutes. After rinsing the surface of the DGT membrane with deionized water to remove any remaining developing solution, a color image was captured using a planar scanner. Imaging of the standard phosphorus adsorption membrane was performed using the same method.

[0064] (4) Image processing: Import the captured image into ImageJ and convert it into an 8-bit grayscale image. Select an appropriate pseudocolor to assign to the grayscale image. The pseudocolor displayed on the image corresponds one-to-one with the grayscale value. By using the calibration curve to match the effective phosphorus concentration value with the pseudocolor displayed on the image, the spatial distribution image of effective phosphorus in the earthworm contact ring can be obtained, as shown in Figure 6.

[0065] Example 2: Visualization of the spatial distribution of available phosphorus in the vermicompost of medium-phosphorus soil

[0066] (1) Earthworm culture: Select medium-phosphorus soil (available phosphorus content of 40 mg / kg) -1 The earthworms were placed in a designed worm-burrowing box, and three Eisenia fetidae earthworms, each 3 cm long and weighing 0.5 g, were cultured in the box for 7 days to create sufficient worm pores. During the cultivation process, the worm-burrowing box was wrapped with aluminum foil to simulate the dark environment of soil. The worm-burrowing box was then placed in a greenhouse at 25°C and 60% humidity. During the cultivation process, a suitable amount of water was sprinkled on the top of the box periodically to maintain soil moisture.

[0067] (2) Material preparation: Cut DGT high-resolution adsorption membranes with a diameter of 2.5 cm for soaking in the adsorption phosphorus standard solution and plotting the effective phosphorus concentration-grayscale value standard curve. Simultaneously, cut several 5 cm × 5 cm DGT high-resolution adsorption membranes for attaching to the vermicompost profile for imaging the spatial distribution of effective phosphorus. Prepare a filter membrane slightly larger than the imaging membrane to maintain the stability of the soil-solution interface. Prepare phosphorus standard solutions with a certain concentration gradient and molybdenum blue colorimetric solution for subsequent reaction and color development of the phosphorus standard adsorption membrane and the imaging membrane.

[0068] (3) Imaging Operation: After a period of cultivation, the detachable acrylic front plate of the worm box was removed. A filter membrane was attached to the desired soil profile for imaging, and the DGT high-resolution adsorption membrane was placed on the imaging support plate. The acrylic front plate was then replaced, and the imaging support plate was inserted. To ensure a tight fit between the acrylic plate and the DGT membrane, a thin plastic sheet was inserted between the DGT membrane and the imaging support plate, allowing the DGT membrane and filter membrane to fully adhere to the soil profile. After 12 hours of cultivation in the dark, the imaging support plate was removed, the DGT membrane was peeled off, and the surface of the DGT membrane was washed with deionized water. The membrane was then placed in a molybdenum blue developing solution for 30 minutes. After rinsing the surface of the DGT membrane with deionized water to remove any remaining developing solution, a color image was captured using a planar scanner. Imaging of the standard phosphorus adsorption membrane was performed using the same method.

[0069] (4) Image processing: Import the two captured images into ImageJ and convert them into 8-bit grayscale images. Select appropriate false colors to assign to the grayscale images, and the false colors displayed on the images correspond one-to-one with the grayscale values. By using the calibration curve to match the concentration values ​​of fluorescent substances and available phosphorus with the false colors displayed on the images, the distribution images of phosphatase and available phosphorus at the interface can be obtained, as shown in Figure 7.

[0070] Example 3: Visualization of the spatial distribution of pH and available phosphorus in the vermicompost of medium-phosphorus soil

[0071] (1) Earthworm culture: Select medium-phosphorus soil (available phosphorus content of 40 mg / kg) -1 The earthworms were filled into a designed worm-burrowing box, and three Eisenia fetidae earthworms, each 3 cm long and weighing 0.5 g, were selected and cultured in the box for 7 days to generate sufficient worm pores. During the cultivation process, the worm-burrowing box was wrapped with aluminum foil to simulate the dark environment in the soil. The worm-burrowing box was then placed in a greenhouse at 25°C and 60% humidity, and a suitable amount of water was sprinkled on the top of the box periodically to maintain soil moisture.

[0072] (2) Material preparation: (a) Cut a standard pH photosensitive membrane with a size of 1cm × 3cm for immersion imaging in standard pH solution, and prepare pH standard solutions with a certain concentration gradient. After immersing the above pH photosensitive membrane in the standard solution for 5 minutes, excite it under 355nm ultraviolet light for imaging, and capture the fluorescence image with a camera with a 370nm bandpass filter in front of the lens. In addition, cut a 5cm × 5cm pH photosensitive membrane and attach it to the imaging membrane support plate, ensuring full contact with the soil profile, for in-situ determination of the spatial distribution of pH in the soil profile; (b) Cut a DGT circular membrane with a diameter of 2.5cm for immersion in adsorbed phosphorus standard solution and plot the effective phosphorus concentration-gray value standard curve. At the same time, cut several 5cm × 5cm DGT square membranes for attachment to the vermicompost profile for imaging of the spatial distribution of effective phosphorus. Prepare a filter membrane slightly larger than the size of the imaging membrane to maintain the stability of the soil-solution interface. Phosphorus standard solutions and molybdenum blue colorimetric solutions with specific concentration gradients were prepared and subsequently used for the reaction and color development of phosphorus standard adsorption membranes and imaging membranes.

[0073] (3) Imaging Operation: After a period of cultivation, the detachable acrylic front plate of the worm box was removed. A filter membrane was then attached to the desired soil profile for imaging. The pH photoelectric membrane and DGT square membrane were placed sequentially on the imaging membrane support plate. A plastic sheet was placed between the pH photoelectric membrane and the imaging membrane support plate. The acrylic front plate was then covered, and the imaging membrane support plate was inserted into the acrylic front plate to ensure the pH photoelectric membrane, DGT membrane, and filter membrane were fully adhered to the soil profile. After 12 hours of cultivation in the dark, the worm box was first placed directly under a UV lamp for in-situ pH imaging. Then, the imaging membrane support plate was removed, and the pH photoelectric membrane and DGT membrane were peeled off. The surface of the DGT membrane was washed with deionized water and reacted in molybdenum blue chromogenic solution for 30 minutes. After rinsing the surface of the DGT membrane with deionized water to remove any residual chromogenic solution, a color image was captured using a planar scanner. Imaging of the standard phosphorus adsorption membrane was performed using the same method. The planar photoelectric membrane was used to capture fluorescence images under 355 nm UV light using a camera with a front-lens filter.

[0074] (4) Image processing: Import the two captured images into ImageJ and convert them into 8-bit grayscale images. Select appropriate false colors to assign to the grayscale images, and the false colors displayed on the images correspond one-to-one with the grayscale values. By using the calibration curve to match the concentration of fluorescent substances and the concentration of available phosphorus with the false colors displayed on the images, the pH value and available phosphorus distribution images at the interface can be obtained, as shown in Figure 8.

[0075] Although planar optical transillumination combined with DGT technology is widely used for in-situ detection of the spatial distribution of solutes (heavy metals) in rhizosphere soil, there are fundamental differences between the rhizosphere environment and the vermicompostor environment. These differences include variations in the growth environments of plants and earthworms, differences in the turnover of soil nutrients by plant roots and earthworms, and differences in the micro-regions formed by root growth and earthworm movement. Specifically:

[0076] 1) Plant growth depends on a large amount of soil moisture to supply the plant's metabolism. Rice plants, in particular, rely on flooded soil environments, where soil moisture reaches or exceeds 100%. However, earthworms generally require soil moisture of 60-70% or even lower to survive.

[0077] 2) The rhizosphere plays a vital role in plant growth, such as absorbing mineral nutrients from the soil. Consequently, the nutrient content in the rhizosphere is often lower than in undisturbed soil. Earthworm activity (such as secreting coelomic fluid and excreting vermicompost) directly increases the nutrient content in the soil within the vermicomposted area and promotes soil nutrient activation by supplying nutrients to soil microorganisms.

[0078] 3) The relatively fixed growth of the root system and the randomness of earthworm activity lead to the disorder of earthworm pore formation.

[0079] Therefore, the advantages and features of this invention are reflected in the following aspects:

[0080] 1) The devices used in traditional soil in-situ imaging technology are applied to soil-plant systems and are often set up in a semi-open manner to facilitate plant growth. However, this method is not suitable for earthworm contact soil systems, which can lead to earthworms escaping and thus reducing the number of stable earthworm gaps in the device. Therefore, the soil in-situ imaging technology device in this invention differs from the root box in that it is equipped with a detachable acrylic upper plate to prevent earthworms from escaping during soil cultivation.

[0081] 2) The devices used in traditional soil in-situ imaging technology are applied to soil-plant systems under flooded conditions, where the soil moisture content often reaches or exceeds 100%. However, this growth environment is not suitable for earthworm cultivation. In order to further simulate a suitable soil environment for earthworm growth, the device of this invention is equipped with a thermometer / hygrometer and a humidifier in a detachable acrylic upper plate to regulate the soil moisture content in real time to ensure the normal survival of earthworms.

[0082] 3) Unlike traditional soil in-situ imaging devices, this experimental device has a rear groove cut into the detachable acrylic front plate for easier operation, and a movable imaging film support plate is installed to facilitate timely replacement of the pH plane photoelectric film and DGT film.

[0083] For any part of this invention that is not described in detail, please refer to the prior art or the art known to those skilled in the art, and will not be described in detail here.

[0084] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. An in-situ chemical imaging method applied to soil vermicontinence, characterized in that, The steps of the method include: S1. Prepare the worm box and fill it with soil that meets the requirements; the soil moisture content in S1 should be 60% to 70% of field capacity. S2. Select suitable earthworms and place them in the soil of the worm box for a period of time to cultivate the earthworms and create enough earthworm pores in the soil. The worm digging box is a square box structure, which includes a detachable acrylic front panel (1), a detachable acrylic upper panel (2), and a worm digging box rear part (4); on the detachable acrylic front panel (1), front panel screws (101) are used to fix the detachable acrylic front panel (1) and the worm digging box rear part (4); on the detachable acrylic upper panel (2), upper panel screws (201) are used to fix the detachable acrylic upper panel (2) and the detachable acrylic front panel (1) and the worm digging box rear part (4); the upper wall of the worm digging box rear part (4) is provided with a worm digging box upper screw hole (402), and the upper panel screw hole (203) and the worm digging box upper screw hole (402) are connected and fixed by the upper panel screw (201); Wrap the worm box around its sides and top and bottom with aluminum foil; S3. A filter membrane, a DGT adsorption membrane, and a pH planar optical membrane are sequentially placed at the soil profile of the worm-infested enclosure to be imaged, ensuring that all three membranes adhere fully to the soil profile. Ultraviolet light is applied to the worm-infested enclosure under dark conditions, and the enclosure is incubated for a period of time. The fluorescence signal emitted by the pH planar optical membrane is captured using a camera to obtain a pH fluorescence image. A certain amount of deionized water is added between the pH planar optical membrane and the soil profile to ensure that there are no air bubbles between them. After adding deionized water, the enclosure is allowed to stand for at least 5 minutes to allow the soil system to reach complete stability. S4. Perform a colorimetric reaction on the DGT adsorption membrane; S5. Use a flatbed scanner to capture the color image of the DGT adsorption membrane after color development to obtain the DGT image. Then, perform image processing on the DGT image and the pH fluorescence image obtained in S3 to obtain the spatial distribution image of available phosphorus and pH in the worm contact zone.

2. The in-situ chemical imaging method for soil vermicontinence as described in claim 1, characterized in that, The soil in S1 has a low to medium content of available phosphorus.

3. The in-situ chemical imaging method for soil vermicontinence as described in claim 1, characterized in that, The camera device is equipped with a 370nm bandpass filter to avoid interference from ultraviolet light on the fluorescence signal.

4. The in-situ chemical imaging method for soil vermicontinence as described in claim 1, characterized in that, The method further includes the following steps: S6. Establish effective phosphorus-gray value calibration curves and / or pH-gray value calibration curves, and use the established calibration curves to perform quantitative analysis on the imaging analysis results.

5. The in-situ chemical imaging method for soil vermicontinence as described in claim 1, characterized in that, The process for developing the color of the DGT adsorption membrane includes: attaching the DGT adsorption membrane to the soil profile of the vermiconductor for 12 hours, peeling it off, rinsing the soil particles adhering to the surface of the DGT adsorption membrane with deionized water, immersing it in molybdenum blue coloring solution, reacting it at 35°C for 30 minutes, washing the reacted DGT adsorption membrane with deionized water, and blotting off the moisture with lint-free paper.

6. The in-situ chemical imaging method for soil vermicontinence as described in claim 5, characterized in that, The molybdenum blue colorimetric solution is prepared as follows: A certain amount of concentrated sulfuric acid is poured into a certain amount of deionized water and stirred until homogeneous to obtain solution A. A certain amount of ammonium molybdate was weighed and placed in a certain amount of deionized water, and heated and stirred to obtain solution B. A certain amount of potassium antimony tartrate was dissolved in a certain amount of deionized water to obtain solution C. Mix solutions A, B, and C thoroughly, then bring the volume up to the preset level to obtain the colorimetric reagent stock solution. Weigh a certain amount of ascorbic acid and dissolve it in a certain amount of the color reagent stock solution, then add a certain amount of deionized water and mix well to obtain the molybdenum blue color reagent solution.

7. An in-situ chemical imaging system for use in soil vermicontinence, characterized in that, The system is used to implement the in-situ chemical imaging method for soil vermicomposts as described in any one of claims 1 to 6.

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