In-situ chemical imaging method and system for earthworm contact circles in soil

Abstract

The present invention discloses an in-situ chemical imaging method and system for soil earthworm haptospheres, belonging to the field of agricultural experimental technology. The method comprises: S1, preparing an earthworm digging box and filling it with soil; S2, selecting suitable earthworms and placing them in the soil for cultivation, so that sufficient earthworm pores are generated in the soil; S3, sequentially placing a filter membrane, a DGT adsorption membrane, and a pH plane photoelectrode membrane on the soil profile of the earthworm haptosphere to be imaged, so that the three membranes can fully adhere to the soil profile of the earthworm haptosphere, applying ultraviolet light under light-proof conditions, and cultivating for a period of time; capturing the fluorescence signal emitted by the pH plane photoelectrode membrane to obtain a pH fluorescence image; S4, coloring the DGT adsorption membrane; S5, capturing the color image of the DGT adsorption membrane to obtain a DGT image, performing image processing on the DGT image and the pH fluorescence image, and obtaining a spatial distribution image of available phosphorus and pH in the earthworm haptosphere. The present invention can solve the problem of difficulty in in-situ detection of biogeochemical cycle processes driven by earthworm activity in soil.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural experiments, and in particular relates to an in-situ chemical imaging method and system applied to soil earthworm contact circles. Background Art

[0002] Phosphorus (P) is an important nutrient element in the soil and plays a key role in increasing crop yields, maintaining terrestrial ecosystem functions, and improving the health of the Earth. Soil phosphorus includes two forms: inorganic phosphorus and organic phosphorus. Due to improper agronomic practices and over-cultivation, the available phosphorus in the soil decreases, which is not conducive to crop growth and soil health. The concept of "sustainable agriculture" emphasizes the importance of increasing phosphorus availability while maintaining the functions of agricultural ecosystems, and has gradually been applied to agricultural practices in recent years. Earthworms are a widely distributed soil animal that is currently believed to have existed on Earth for more than 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. Because of their crucial role in shaping the physical, chemical, and biological properties of soil, earthworms have been dubbed "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 could help increase crop yields while maintaining essential ecosystem functions.

[0003] Earthworm-mediated phosphorus cycling has been a subject of extensive research. For example, Vos et al. studied the variations in available phosphorus levels in the casts of eight earthworm species and in reference soils 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 evaluate the role of earthworm castings in phosphorus mobilization and confirmed 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 earthwormcasts? A meta-analysis. Geoderma 338, 525-535). The mechanism of earthworm-mediated phosphorus activation has always been a focus of attention, which 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 for adsorption on clay and metal oxides, 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 the process of soil phosphorus activation mediated by earthworms has been widely studied by a large number of chemical methods, the role of earthworms as a driving factor in stimulating soil microbial activity is similar to that of roots in the rhizosphere and litter in the detritus layer, forming a microbial hot zone defined as the “earthworm touch circle”. Due to the lack of reliable methods to study the biogeochemical processes of phosphorus in this unique soil environment, further exploration is still needed (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 photoelectrode technology for in situ detection of spatial changes in soil pH and diffusion gradient film technology (DGT) for available chemical substances, have been used to study the biogeochemical processes of the rhizosphere and detritus layer, providing information on the spatial distribution of nutrient dynamics and pH. For example, Fang et al. used high-resolution zirconia DGT adsorption film to evaluate the phosphorus availability in the rice rhizosphere and humus (dead roots), thereby gaining in-depth 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 optode technology to analyze the spatial dynamics of soil pH changes over time near wheat roots, providing a theoretical basis for nutrient migration and utilization by crop roots (Blossfeld, S., Schreiber, CM, Liebsch, G., Kuhn, AJ, Hinsinger, P., 2013. Quantitative imaging of rhizosphere pH and CO2 dynamics with planaroptodes. Annals of Botany 112, 267–276). However, DGT technology and planar optode technology have not yet been applied to the study of biogeochemical cycles in the earthworm haptosphere. Summary of the Invention

[0005] The present invention aims to solve one of the technical problems in the above-mentioned related art at least to a certain extent.

[0006] To this end, the purpose of the present invention is to provide an in situ chemical imaging method and system for soil earthworm contact circles, which can solve the problem of difficulty in in situ detection of biogeochemical cycle processes driven by earthworm activity in soil.

[0007] In order to solve the above-mentioned technical problems, the present invention is achieved as follows:

[0008] An embodiment of the present invention provides an in-situ chemical imaging method for earthworm contact circles in soil, the method comprising the following steps:

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

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

[0011] S3. Place a filter membrane, a DGT adsorption membrane, and a pH plane optode membrane in sequence at the soil profile of the earthworm contact circle to be imaged, so that the filter membrane, the DGT adsorption membrane, and the pH plane optode membrane can all be fully attached to the soil profile of the earthworm contact circle, apply ultraviolet light to the earthworm digging box under light-proof conditions, and incubate for a period of time; and use a camera to capture the fluorescence signal emitted by the pH plane optode membrane to obtain a pH fluorescence image;

[0012] S4, performing color reaction on the DGT adsorption film;

[0013] S5. Use a flatbed scanner to capture the color image of the DGT adsorption film after color development to obtain a DGT image, and perform image processing on the DGT image and the pH fluorescence image obtained in S3 to obtain a spatial distribution image of available phosphorus in the earthworm tactile circle.

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

[0015] In some embodiments, the soil in S1 is a soil with low or medium available phosphorus content (5-40 mg / kg -1 available phosphorus).

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

[0017] In some embodiments, a certain amount of deionized water is added between the pH plane photoelectrode membrane and the earthworm contact circle soil profile to ensure that no bubbles exist between the pH plane photoelectrode membrane and the earthworm contact circle soil profile.

[0018] In some embodiments, deionized water is added between the pH plane photoelectrode membrane and the earthworm contact circle soil profile and then allowed to stand for more than 5 minutes to allow the soil system to reach complete stability.

[0019] In some embodiments, the imaging device is provided with a 370 nm bandpass filter in front to prevent the interference of the excitation light on the fluorescence signal.

[0020] In some embodiments, the method further comprises:

[0021] S6. Establishing an effective phosphorus-grayscale value calibration curve and / or a pH-grayscale value calibration curve, and using the established calibration curve to perform quantitative analysis on the imaging analysis results.

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

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

[0024] Measure a certain amount of concentrated sulfuric acid and pour it into a certain amount of deionized water, stir evenly to obtain solution A;

[0025] Weigh a certain amount of ammonium molybdate and add it to a certain amount of deionized water, heat and stir to obtain solution B;

[0026] Weigh a certain amount of potassium antimony tartrate and dissolve it in a certain amount of deionized water to obtain solution C;

[0027] Mix solution A, solution B and solution C evenly, and then dilute to the preset volume to obtain a color developer stock solution;

[0028] A certain amount of ascorbic acid is weighed and dissolved in a certain amount of the developer stock solution, and then a certain amount of deionized water is added and mixed uniformly to obtain the molybdenum blue developer solution.

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

[0030] The present invention also discloses an in-situ chemical imaging system applied to soil earthworm haptic circles, and the system is used to implement any of the above-described in-situ chemical imaging methods applied to soil earthworm haptic circles.

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

[0032] In an embodiment of the present invention, the provided in situ chemical imaging method for earthworm haptosphere in soil is a technology combining DGT technology and pH plane optode technology to reveal the influence of earthworm activity on the spatial distribution characteristics of available phosphorus and pH in soil.

[0033] In an embodiment of the present invention, an in situ chemical imaging method for earthworm contact circles in soil is provided, which enables in situ monitoring of the nutrient cycling process caused by earthworm activity in the soil. Unlike conventional chemical testing methods (which measure changes in soil mineral composition or content before and after the addition of earthworms), the present invention conducts in situ monitoring of the earthworm activity microzone (the 2 mm range around the earthworm pores, also known as the earthworm contact circle), that is, directly imaging and detecting nutrients and pH in key areas of the soil during earthworm activity.

[0034] In an embodiment of the present invention, an in-situ chemical imaging method for earthworm haptic circles in soil is provided, which realizes the visualization of the biochemical reaction process in earthworm haptic circles. Previous visualization methods for earthworm haptic circles mainly reflect the degree of possible biochemical reactions and lack quantitative analysis. The present invention applies two chemical imaging technologies to the earthworm haptic circle, a key area of ​​the soil, to visually monitor the available phosphorus content and pH value during earthworm activity, intuitively display the degree of biochemical reactions in this area, and perform quantitative analysis.

[0035] In an embodiment of the present invention, the provided in situ chemical imaging method applied to the soil earthworm haptic circle realizes the simultaneous monitoring of multiple substances during the mineral-solution interface reaction process; through the combination of DGT and pH planar photoelectrode technology, the simultaneous monitoring of available phosphorus and pH in the key area of ​​the soil earthworm haptic circle is realized.

[0036] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A front view of components of an earthworm digging box disclosed in one embodiment of the present invention;

[0038] Figure 2 A side view of components of an earthworm digging box disclosed in one embodiment of the present invention;

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

[0040] Figure 4A schematic diagram of a soil-filled earthworm digging box and earthworms introduced therein according to an embodiment of the present invention;

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

[0042] Figure 6 This is a diagram of low-phosphorus earthworm pore soil imaging results disclosed in one embodiment of the present invention; wherein (a) is a soil profile, and (b) is a spatial distribution diagram of effective phosphorus diffusion flux;

[0043] Figure 7 This is a soil imaging result diagram of the earthworm pores (earthworm pores) in the medium phosphorus environment disclosed in one embodiment of the present invention; wherein (a) is a soil profile diagram, and (b) is a spatial distribution diagram of the effective phosphorus diffusion flux;

[0044] Figure 8 This is a soil imaging result diagram of the earthworm apex (earthworm pores) disclosed in another embodiment of the present invention; wherein, (a) is a soil profile diagram, (b) is a spatial distribution diagram of the effective phosphorus diffusion flux, and (c) is a pH spatial distribution diagram.

[0045] Description of reference numerals:

[0046] 1. Removable acrylic front panel; 101. Front panel screws; 102. Front panel side holes; 103. Imaging film carrier plate; 104. Front panel rear slot; 105. Front panel screw holes; 106. pH planar photoelectrode membrane; 107. DGT adsorption membrane; 108. Front panel screw holes; 2. Removable acrylic top panel; 201. Top panel screws; 202. Ventilation holes; 203. Top panel screw holes; 3. Soil; 301. Earthworm pores; 302. Earthworms; 4. Rear of the earthworm burrowing box; 401. Front screw holes of the earthworm burrowing box; 402. Upper screw holes of the earthworm burrowing box; 5. Electrical wires; 6. Thermohygrometer; 601. Thermohygrometer indicator; 602. Thermohygrometer sensor; 7. Humidifier; 701. Water pipe; 702. Shower head; 8. Ventilation fan; 801. Ventilation fan shaft; 802. Ventilation fan blades; 9. Filter membrane. DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

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

[0049] In order to solve the problem of difficulty in in-situ detection of biogeochemical cycle processes driven by earthworm activity in soil, the present invention provides an imaging system based on diffuse gradient thin-film technology (DGT) and pH planar photoelectrode technology to achieve in-situ detection of soil available phosphorus (labile-P) and pH in the earthworm tactile circle (soil micro-area within the earthworm activity range), so as to achieve in-situ analysis of soil biogeochemical processes mediated by earthworm activity. Unlike traditional potted soil sampling methods, the design of the present invention overcomes the changes in soil physical and chemical properties that may be caused by destructive soil sampling, and in terms of operation and economic benefits, greatly shortens the detection time and reduces the detection cost. In addition, compared with the problem of poor precision of traditional potted experiments, the present invention achieves sub-millimeter scale detection, greatly improving the precision of detection.

[0050] In some embodiments of the present invention, an in-situ chemical imaging method for soil earthworm contact circles is provided, and the process mainly includes five processes: (1) filling the earthworm digging box with soil; (2) cultivating earthworms; (3) attaching imaging film to the soil profile; (4) chemical imaging experiment; (5) image processing. The basic principles can be summarized as follows: (1) DGT: The high-resolution adsorption film adsorbs soluble and exchangeable phosphorus in the soil within a certain period of time, uses molybdenum blue colorimetric solution to characterize the phosphorus content, and obtains the corresponding grayscale value and phosphorus standard curve 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 film still needs to meet the requirements that the adsorbent particles are small enough (≤10μm) and evenly distributed in the film; (2) The surface of the pH photoelectrode membrane is coated with a fluorescent dye sensitive to pH changes, which is excited by ultraviolet light. + Inducing changes in 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 protocol for this technology is detailed below.

[0051] Step 1: Construction of earthworm digging box: To take into account the free movement of earthworms and the attachment of detection membrane to the soil profile, please refer to Figures 1-4As shown, a transparent acrylic square box (hereinafter referred to as an earthworm digging box) is designed as a container for earthworm activities. The earthworm digging box is a square box structure, which mainly includes a detachable acrylic front panel 1, a detachable acrylic upper panel 2, an earthworm digging box rear portion 4, an electric wire 5, a thermometer and hygrometer 6, a humidifier 7 and a ventilation fan 8. On the detachable acrylic front panel 1, a front panel screw 101 is used to fix the detachable acrylic front panel 1 and the earthworm digging box rear portion 4. A front panel side hole 102 is provided on the side of the detachable acrylic front panel 1, and the front panel side hole 102 is used to insert an imaging film carrier plate 103, that is, the imaging film carrier plate 103 is inserted into the front panel side hole 102. On the detachable acrylic upper panel 2, an upper panel screw 201 is used to fix the detachable acrylic upper panel 2 to the detachable acrylic front panel 1 and the earthworm digging box rear portion 4. Several ventilation holes 202 are provided on the removable acrylic upper panel 2, allowing the air inside the wormhole box to communicate with the outside air. One end of an electrical wire 5 is connected to a power supply, and the other end is connected to a ventilation fan 8, powering the ventilation fan 8. A thermometer and hygrometer 6, a humidifier 7, and a ventilation fan 8 are all provided on the removable acrylic upper panel 2. The thermometer and hygrometer 6 includes a thermometer and hygrometer indicator 601 and a thermometer and hygrometer sensor 602. The thermometer and hygrometer indicator 601 is located above the removable acrylic upper panel 2, allowing external viewing of the current temperature and humidity readings. The thermometer and hygrometer sensor 602 extends into the interior of the wormhole box to measure the temperature and humidity inside the wormhole box. The humidifier 7 is used to regularly water the soil to maintain soil moisture. The ventilation fan 8 is used to facilitate air circulation within the wormhole box. The upper panel screw holes 203 and the front panel screw holes 108 are connected and secured by upper panel screws 201. The upper wall of the rear portion 4 of the earthworm excavation box is provided with screw holes 402 for the earthworm excavation box. The screw holes 203 on the upper plate are connected to the screw holes 402 via screws 201. The filter membrane 9 is vertically positioned between the removable acrylic front plate 1 and the rear portion 4 of the earthworm excavation box. A rear groove 104 is cut into the rear side of the removable acrylic front plate 1 to facilitate contact between the pH plane photoelectrode membrane 106 and the DGT adsorption membrane 107 on the imaging membrane carrier plate 103 and the soil 3 for imaging. The rear groove 104 is a groove formed within the front plate and communicates with the soil inside the earthworm excavation box. By filling the groove with soil, each membrane has sufficient space to contact the soil. The membranes used for soil imaging are, from the outside in, the DGT adsorption membrane 107 and the pH plane photoelectrode membrane 106. The front plate screw holes 105 are secured to the front screw holes 401 of the earthworm excavation box using screws 101.

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

[0053] The second step is earthworm cultivation: Neutral, organic-rich soil 3 is selected as the earthworm culture medium. Before filling the earthworm box with soil, the soil must be air-dried and sieved through a 2mm sieve. To further provide a suitable soil environment for earthworm growth, the soil moisture is adjusted to 60% to 70% of its field capacity using a humidifier 7 during or after filling, as determined by thermometer and hygrometer 6. Mature and vigorous earthworms 302 are selected and cultured in the soil 3. After 7 to 14 days of cultivation, a stable and sufficient number of earthworm pores 301 are formed. During the cultivation process, the earthworm box is wrapped with aluminum film around the sides and on the top and bottom to simulate the dark environment of soil. The earthworm box is placed in a greenhouse at 25°C and 60% humidity.

[0054] Step 3: Solution preparation:

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

[0056] The fourth step involved constructing the imaging system: The filter membrane 9, DGT adsorption membrane 107, and pH planar optode membrane 106 were sequentially attached to the soil profile with earthworm pores. A plastic spacer was placed between the pH planar optode membrane and the imaging membrane carrier plate 103 to ensure a tight fit between the membrane assembly and the soil profile, thereby enhancing the reliability of solute imaging by the DGT high-resolution adsorption membrane and pH planar optode membrane. During the experimental period, the optimal placement time for the DGT and planar optode was determined. Images were processed using ImageJ software, and the spatial resolution of the DGT and pH planar optode images was confirmed. The filter membrane, which serves as part of the diffusion layer in the DGT device and reduces radial diffusion to maintain stable diffusion and adsorption of the leachate, 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. and is a precipitated zirconium oxide adsorption membrane (GDZR) with a thickness of 0.1 mm. pH planar optode membranes were purchased from Zhongke Zhigan (Nanjing) Environmental Technology Co., Ltd. After the reaction time, DGT membranes and pH planar optode membranes were imaged separately. (a) DGT membrane imaging: The DGT high-resolution adsorption membrane was attached to the soil profile for 12 hours, then removed. Soil particles adhering to the surface were rinsed with deionized water and immersed in molybdenum blue colorimetric solution. After reacting at 35°C for 30 minutes, the membrane was rinsed with deionized water and dried with dust-free paper. The membrane was then scanned on a scanner to obtain a color image. (b) pH optode membrane imaging: The pH optode membrane was attached to the soil profile. To prevent air bubbles from forming between the membrane and the soil profile, a small amount of deionized water was added between the membrane and the soil. The membrane was allowed to stand for 5 minutes to allow the soil system to fully stabilize. The earthworm digging box was placed in front of a planar UV light source with a wavelength of 355 nm. A camera was used to capture the fluorescence signal emitted by the pH optode membrane. A 370 nm bandpass filter was placed in front of the camera lens to filter out UV light and prevent interference with the fluorescence signal emitted by the pH optode membrane.

[0057] Step 5: Establish a standard curve for quantitative analysis of the previous image processing results;

[0058] The imaging displays the two-dimensional distribution of available phosphorus and pH at all pixels within the membrane, not just the average concentration within the area or the concentration change in a certain dimension. Without a standard curve, the data displayed is only the grayscale value of the image recognized by the software. The test results obtained after correction and analysis using a standard curve are of practical significance.

[0059] See also Figure 5 As shown in Figure 2, (a) is the effective phosphorus-gray value standard curve. Phosphorus standard substances were used to prepare phosphorus concentrations of 0, 20, 50, 100, 200, 500, 750, 1000, and 2000 μg L -1A standard phosphorus solution was prepared. A disc-shaped DGT membrane was placed in the phosphorus solution and allowed to adsorb for 12 hours before developing the color in a molybdenum blue colorimetric solution. The colored DGT membrane was immediately rinsed several times with 4°C deionized water to remove any residual developer from the surface. The membrane was then immersed in deionized water for another 5 minutes (to stop the color development reaction). The membrane was removed, its surface wiped clean with filter paper, and placed face down on a scanner at a resolution of 1200 dpi. After scanning, the image was converted to grayscale using ImageJ software. The exponential function with the highest correlation was used to fit the P accumulation per unit area on the membrane and its corresponding grayscale intensity on the membrane surface. This formed the standard curve (G(f)) of the analyte accumulation per unit area (f) versus 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 optode standard film in a cuvette and submerge the optode film in deionized water until it adheres completely to the cuvette. Then, pour standard solutions representing different pH values ​​into the cuvette in sequence. Expose the sample under 355nm UV light for color development. Record the light intensity using a camera equipped with a prefilter. Use ImageJ software to determine concentrations and plot a standard curve. (c) Select the Calibration bar option in ImageJ software to insert the effective phosphorus concentration and pH scale into the image.

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

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

[0062] (2) Material preparation: Cut a 2.5 cm diameter DGT high-resolution adsorption membrane to soak the adsorbed phosphorus standard solution and draw a standard curve of effective phosphorus concentration-grayscale value. At the same time, cut several 5 cm × 5 cm DGT membranes to attach to the earthworm tactile circle section to image the effective phosphorus spatial distribution. Prepare a filter membrane slightly larger than the imaging membrane to maintain the stability of the soil-solution interface. Prepare a certain concentration gradient of phosphorus standard solution and molybdenum blue color development solution, which will be used for the reaction and color development of the phosphorus standard adsorption membrane and the imaging membrane.

[0063] (3) Imaging operation: After a period of incubation, remove the detachable acrylic front plate of the earthworm digging box, paste the filter membrane on the soil profile to be imaged, and place the DGT high-resolution adsorption membrane on the imaging carrier plate. Cover it with the acrylic front plate and insert the imaging membrane carrier plate. To ensure that the acrylic plate and the DGT membrane fit tightly, insert a plastic sheet between the DGT membrane and the imaging membrane carrier plate so that the DGT membrane and the filter membrane are fully attached to the soil profile. After incubating in the dark for 12 hours, remove the imaging membrane carrier plate, peel off the DGT membrane, and wash the DGT membrane surface with deionized water. Place it in molybdenum blue color developing solution for 30 minutes. After rinsing the residual color developing solution on the DGT membrane surface with deionized water, use a flatbed scanner to capture the color image. Standard phosphorus adsorption membrane imaging is carried out in the same way as above.

[0064] (4) Image processing: Import the captured image into ImageJ and convert it into an 8-bit grayscale image. Select appropriate pseudo-color to assign to the grayscale image. The pseudo-color displayed on the image corresponds to the grayscale value. Use the calibration curve to match the effective phosphorus concentration value with the pseudo-color displayed on the image. The spatial distribution image of the effective phosphorus in the earthworm's tactile circle can be obtained. Figure 6 shown.

[0065] Example 2: Visualization of the spatial distribution of available phosphorus in earthworm diatoms in medium-phosphorus soils

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

[0067] (2) Material preparation: Cut a 2.5 cm diameter DGT high-resolution adsorption membrane to soak the adsorbed phosphorus standard solution and draw a standard curve of effective phosphorus concentration-grayscale value. At the same time, cut several 5 cm × 5 cm DGT high-resolution adsorption membranes to attach to the earthworm tactile circle profile to image 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 a phosphorus standard solution with a certain concentration gradient and a molybdenum blue color developing solution, which will be used for the subsequent reaction and color development of the phosphorus standard adsorption membrane and the imaging membrane.

[0068] (3) Imaging operation: After a period of incubation, remove the detachable acrylic front plate of the earthworm digging box, paste the filter membrane on the soil profile to be imaged, and place the DGT high-resolution adsorption membrane on the imaging carrier plate. Cover it with the acrylic front plate and insert the imaging membrane carrier plate. To ensure that the acrylic plate and the DGT membrane fit tightly, insert a plastic sheet between the DGT membrane and the imaging membrane carrier plate so that the DGT membrane and the filter membrane are fully attached to the soil profile. After incubating in the dark for 12 hours, remove the imaging membrane carrier plate, peel off the DGT membrane, and wash the DGT membrane surface with deionized water. Place it in molybdenum blue color developing solution for 30 minutes. After rinsing the residual color developing solution on the DGT membrane surface with deionized water, use a flatbed scanner to capture the color image. Standard phosphorus adsorption membrane imaging is carried out in the same way as above.

[0069] (4) Image processing: The two images were imported into ImageJ and converted into 8-bit grayscale images. Appropriate pseudo colors were selected to assign to the grayscale images. The pseudo colors displayed on the images corresponded to the grayscale values. Through the calibration curve, the concentration of fluorescent substances and the effective phosphorus concentration values ​​were matched to the pseudo colors displayed on the images. The distribution images of phosphatase and effective phosphorus at the interface were obtained, as shown in Figure 2. Figure 7 shown.

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

[0071] (1) Earthworm culture: Select medium phosphorus soil (available phosphorus content of 40 mg kg -1 ) were placed in a designed earthworm-boring box and three Eisenia fetida (Eisenia fetida) earthworms, each 3 cm long and weighing 0.5 g, were cultured for 7 days to create sufficient earthworm pores. During the culture process, the earthworm-boring box was wrapped with aluminum film to simulate the dark environment of soil. The earthworm-boring box was placed in a greenhouse at 25°C and 60% humidity. Water was regularly sprinkled on the top of the box to maintain soil moisture.

[0072] (2) Material preparation: (a) Cut a standard pH photoelectrode membrane of size 1cm×3cm for immersion imaging in standard pH solution, and prepare a pH standard solution with a certain concentration gradient. After soaking the above pH photoelectrode membrane in the standard solution for 5 minutes, excite the imaging under 355nm ultraviolet light, 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 photoelectrode membrane and attach it to the imaging membrane carrier plate, and make full contact with the soil profile for in situ measurement of the pH spatial distribution of the soil profile; (b) Cut a DGT disc membrane with a diameter of 2.5cm and use it to soak the adsorbed phosphorus standard solution to draw the effective phosphorus concentration-grayscale value standard curve. At the same time, cut several DGT square membranes of size 5cm×5cm and use them to attach to the earthworm touch circle profile for imaging the effective phosphorus spatial distribution. Prepare a filter membrane slightly larger than the imaging membrane size to maintain the stability of the soil-solution interface. Prepare phosphorus standard solutions and molybdenum blue color developing solution with a certain concentration gradient, which are subsequently used for the reaction and color development of the phosphorus standard adsorption film and the imaging film.

[0073] (3) Imaging operation: After a period of incubation, the detachable acrylic front plate of the earthworm digging box is removed, and the filter membrane is pasted on the soil profile to be imaged. The pH photoelectrode membrane and the DGT square membrane are placed on the imaging membrane carrier plate in sequence. A plastic sheet is filled between the pH photoelectrode membrane and the imaging membrane carrier plate. The acrylic front plate is covered and the imaging membrane carrier plate is inserted into the acrylic front plate so that the pH photoelectrode membrane, DGT membrane and filter membrane are fully attached to the soil profile. After incubation in the dark for 12 hours, the earthworm digging box is first placed directly under ultraviolet light for in situ pH imaging. Then the imaging membrane carrier plate is removed, the pH photoelectrode membrane and DGT membrane are peeled off, the surface of the DGT membrane is cleaned with deionized water, and placed in molybdenum blue color developing solution for reaction for 30 minutes. After the residual color developing solution on the surface of the DGT membrane is rinsed with deionized water, the color image is captured with a flatbed scanner. The imaging of the standard phosphorus adsorption membrane is carried out in the same manner as above. The flatbed photoelectrode membrane is used to capture the fluorescence image under 355nm ultraviolet light using a camera with a pre-lens filter.

[0074] (4) Image processing: Import the two images captured into ImageJ and convert them into 8-bit grayscale images. Select appropriate pseudo colors to assign to the grayscale images. The pseudo colors displayed on the images correspond one to one with the grayscale values. Use the calibration curve to match the fluorescent substance concentration and the effective phosphorus concentration value with the pseudo colors displayed on the image. The pH value and effective phosphorus distribution image at the interface can be obtained, as shown in Figure 2. Figure 8 shown.

[0075] Although the combination of planar optodes and DGT is currently widely used to detect the spatial distribution of solutes (heavy metals) in rhizosphere soil in situ, there are essential differences between the rhizosphere environment and the earthworm environment, such as the differences in the growth environments of plants and earthworms, the differences in soil nutrient turnover by plant roots and earthworms, and the micro-region differences formed by root growth and earthworm movement. Specifically:

[0076] 1) Plant growth relies on large amounts of soil moisture to fuel plant metabolism. Rice plants, in particular, rely on flooded soils, where soil moisture reaches or exceeds 100%. However, earthworm activity generally requires soil moisture of 60-70% or even lower; earthworms struggle to survive in wet soil.

[0077] 2) The plant rhizosphere plays a crucial role in plant growth, absorbing mineral nutrients from the soil. Consequently, nutrient content in the rhizosphere is often lower than in undisturbed soil. Earthworm activity (such as coelomic fluid secretion and vermicompost excretion) directly increases nutrient content in the earthworm sphere and promotes nutrient activation by providing nutrient resources 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 characteristics of the present invention are embodied in the following aspects:

[0080] 1) The device used in traditional soil in situ imaging technology is applied to the soil-plant system and is often set to a semi-open type to facilitate plant growth. However, this method is not suitable for the earthworm contact circle soil system where earthworms are active, which causes earthworms to escape and thus reduces the number of stable earthworm gaps in the device. Therefore, the soil in situ imaging technology device used in the present invention is different from the root box in that the detachable acrylic upper plate is provided to prevent earthworms from escaping during soil cultivation.

[0081] 2) The device used in traditional soil in situ imaging technology is applied to a soil-plant system in a flooded state. The soil moisture content often reaches or exceeds 100%, but this growth environment is not suitable for earthworm cultivation. To further simulate the soil environment suitable for earthworm growth, the device of the present invention installs a thermometer / hygrometer and a humidification device in the 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 on the detachable acrylic front plate to further facilitate operation, and a movable imaging film carrier plate is placed to facilitate timely replacement of the pH plane photoelectrode membrane and DGT membrane.

[0083] Parts of the present invention that are not described in detail may refer to the existing technology in the field or are well known to those skilled in the art, and will not be described in detail here.

[0084] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. An in situ chemical imaging method for earthworm haptosphere in soil, characterized in that: The steps of the method include: S1. Prepare an earthworm digging box and fill it with soil that meets the requirements; the soil moisture in S1 is 60% to 70% of the field water holding capacity; S2. Select suitable earthworms and place them in the soil of the earthworm digging box. Cultivate the earthworms for a period of time to create enough pores for earthworms in the soil. The earthworm digging box is a square box structure, comprising a detachable acrylic front panel (1), a detachable acrylic upper panel (2) and an earthworm digging box rear portion (4); on the detachable acrylic front panel (1), a front panel screw (101) is used to fix the detachable acrylic front panel (1) and the earthworm digging box rear portion (4); on the detachable acrylic upper panel (2), an upper panel screw (201) is used to fix the detachable acrylic upper panel (2) and the detachable acrylic front panel (1) and the earthworm digging box rear portion (4); an earthworm digging box upper screw hole (402) is provided on the upper wall of the earthworm digging box rear portion (4), and the upper panel screw hole (203) and the earthworm digging box upper screw hole (402) are connected and fixed by the upper panel screw (201); Wrap the sides and upper and lower sides of the earthworm digging box with aluminum film; S3. A filter membrane, a DGT adsorption membrane and a pH plane photoelectrode membrane are sequentially arranged at the soil profile of the earthworm contact circle to be imaged, so that the filter membrane, the DGT adsorption membrane and the pH plane photoelectrode membrane can all be fully attached to the soil profile of the earthworm contact circle, and ultraviolet light is applied to the earthworm digging box under light-proof conditions and cultured for a period of time; and a camera device is used to capture the fluorescence signal emitted by the pH plane photoelectrode membrane to obtain a pH fluorescence image; a certain amount of deionized water is added between the pH plane photoelectrode membrane and the earthworm contact circle soil profile to ensure that there are no bubbles between the pH plane photoelectrode membrane and the earthworm contact circle soil profile; after adding deionized water between the pH plane photoelectrode membrane and the earthworm contact circle soil profile, the mixture is allowed to stand for more than 5 minutes to allow the soil system to reach complete stability; S4, performing color reaction on the DGT adsorption film; S5. Use a flatbed scanner to capture the color image of the DGT adsorption film after color development to obtain a DGT image, and perform image processing on the DGT image and the pH fluorescence image obtained in S3 to obtain a spatial distribution image of available phosphorus and pH in the earthworm tactile circle.

2. The in-situ chemical imaging method for earthworm haptosphere in soil according to claim 1, characterized in that: The soils in S1 were low to medium available phosphorus content soils.

3. The in-situ chemical imaging method for soil earthworm contact circles according to claim 1, characterized in that: The camera device is pre-installed with a 370 nm bandpass filter to prevent ultraviolet light from interfering with the fluorescence signal.

4. The in-situ chemical imaging method for earthworm haptosphere in soil according to claim 1, characterized in that: The method further comprises the steps of: S6. Establishing an effective phosphorus-grayscale value calibration curve and / or a pH-grayscale value calibration curve, and using the established calibration curve to perform quantitative analysis on the imaging analysis results.

5. The in-situ chemical imaging method for earthworm haptosphere in soil according to claim 1, characterized in that: The color development of the DGT adsorption film includes the following steps: the DGT adsorption film is attached to the soil profile of the earthworm's tactile circle for 12 h and then peeled off; the soil particles adhering to the surface of the DGT adsorption film are rinsed with deionized water; the film is then immersed in molybdenum blue color developing solution and reacted at 35°C for 30 min; the DGT adsorption film after the reaction is washed with deionized water and dried with dust-free paper.

6. The in-situ chemical imaging method for soil earthworm haptosphere according to claim 5, characterized in that: The preparation method of the molybdenum blue developing solution is: Measure a certain amount of concentrated sulfuric acid and pour it into a certain amount of deionized water, stir evenly to obtain solution A; Weigh a certain amount of ammonium molybdate and add it to a certain amount of deionized water, heat and stir to obtain solution B; Weigh a certain amount of potassium antimony tartrate and dissolve it in a certain amount of deionized water to obtain solution C; Mix solution A, solution B and solution C evenly, and then dilute to the preset volume to obtain a color developer stock solution; A certain amount of ascorbic acid is weighed and dissolved in a certain amount of the developer stock solution, and then a certain amount of deionized water is added and mixed uniformly to obtain the molybdenum blue developer solution.

7. An in-situ chemical imaging system for soil earthworm contact circles, characterized in that: The system is used to implement the in-situ chemical imaging method applied to soil earthworm haptospheres as described in any one of claims 1 to 6.

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