In-situ chemical imaging method and system applied to soil earthworm contact ring

Abstract

The invention discloses an in-situ chemical imaging method and system applied to a soil earthworm contact ring, and belongs to the technical field of agricultural tests. The method comprises the following steps: S1, preparing an earthworm digging box and filling soil; s2, proper earthworms are selected and placed in soil to be cultured, and enough earthworm pores are generated in the soil; s3, a filter membrane, a DGT adsorption membrane and a pH plane opto-electrode membrane are sequentially arranged on the soil profile of the earthworm contact ring needing to be imaged, so that the three membranes can be fully attached to the soil profile of the earthworm contact ring, ultraviolet light is applied under the dark condition, and culture is conducted for a period of time; capturing a fluorescence signal emitted by the pH planar opto-electrode film to obtain a pH fluorescence image; s4, carrying out color development on the DGT adsorption film; and S5, capturing a DGT adsorption film developing image to obtain a DGT image, and carrying out picture processing on the DGT image and the pH fluorescence image to obtain a spatial distribution image of available phosphorus and pH in the earthworm contact ring. The problem that in-situ detection of the biogeochemical cycle process driven by earthworm activity in soil is difficult can be solved.

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, playing a key role in increasing crop yields, maintaining terrestrial ecosystem functions, and improving the health of the earth. Soil phosphorus includes two forms of phosphorus: inorganic phosphorus and organic phosphorus. Due to improper agronomic practices and overplanting, the effective 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 been gradually 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. It is precisely because of their vital role in shaping the physical, chemical and biological properties of the soil that they are given 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 conducive to increasing crop yields while maintaining the basic functions of the ecosystem.

[0003] Earthworm-mediated phosphorus cycling has been a topic of extensive research. For example, Vos et al. studied the changes in available phosphorus levels in vermicompost of eight earthworm species and reference soils and found that earthworm activity increased soil phosphorus availability (Vos, HMJ, Koopmans, GF, Beezemer, L., deGoede, 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 the 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 earthworm-mediated soil phosphorus activation process has been widely studied by a large number of chemical methods, the role of earthworms as a driver of soil microbial activity is similar to the roots in the rhizosphere and the litter in the debris layer, forming a microbial hot zone defined as the "earthly 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 chemicals, have been used to study biogeochemical processes in the rhizosphere and debris layer, providing information on nutrient dynamics and spatial distribution of pH. For example, Fang et al. used high-resolution zirconium oxide 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 at the roots of wheat, thereby providing a certain theoretical basis for the migration and utilization of nutrients in 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] The 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, selecting suitable earthworms and placing them in the soil of the earthworm digging box, and cultivating the earthworms for a period of time to produce enough earthworm pores in the soil;

[0011] S3, sequentially set a filter membrane, a DGT adsorption membrane and a pH plane photoelectrode membrane 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 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 cultivate for a period of time; and use a camera to capture the fluorescence signal emitted by the pH plane photoelectrode 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 effective phosphorus in the earthworm tactile circle.

[0014] In addition, the in-situ chemical imaging method applied to soil earthworm contact circles 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 moisture of the soil in S1 is 60% to 70% of the field capacity.

[0017] In some of the 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 of the 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 of the embodiments, the camera device is preceded by a 370 nm bandpass filter to prevent interference of the excitation light on the fluorescent 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 of the 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 developing solution, reacted at 35°C for 30 minutes, and the DGT adsorption film after the reaction is washed with deionized water and the moisture is absorbed with dust-free paper.

[0023] In some embodiments, the molybdenum blue color 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, and stir evenly to obtain solution A;

[0025] Weigh a certain amount of ammonium molybdate and place it in 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 adjust the volume to a preset volume to obtain a 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 evenly to obtain the molybdenum blue developer solution.

[0029] In some embodiments, the length of the earthworms in S2 is 3 cm, the number is 2 to 3, and the culture time is 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 the embodiment of the present invention, the in-situ chemical imaging method applied to the earthworm contact circle in the soil is a technology combining the DGT technology and the pH plane photoelectrode technology to reveal the influence of earthworm activity on the spatial distribution characteristics of soil available phosphorus and pH.

[0033] In the embodiment of the present invention, the in-situ chemical imaging method applied to the earthworm contact circle in the soil is provided to realize the in-situ monitoring of the nutrient cycle process caused by the earthworm activity in the soil; different from the conventional chemical inspection method (the change of the mineral composition or content of the soil before and after the addition of earthworms), the present invention carries out in-situ monitoring of the earthworm activity micro-area (the range of 2mm near the earthworm pore, also known as the earthworm contact circle), that is, the nutrients and pH of the key areas of the soil are directly imaged and detected during the earthworm activity process;

[0034] In the embodiment of the present invention, the provided in-situ chemical imaging method applied to the earthworm haptic circle in the soil realizes the visualization of the biochemical reaction process of the earthworm haptic circle; the visualization methods used for earthworm haptic circles in the past mainly reflect the degree of biochemical reactions that may exist, but 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 effective phosphorus content and pH value during the earthworm activity process, intuitively display the degree of biochemical reactions in the area, and conduct quantitative analysis;

[0035] In an embodiment of the present invention, an in situ chemical imaging method applied to the soil earthworm haptic circle is provided, which realizes the synchronous monitoring of multiple substances during the mineral-solution interface reaction process; through the combination of DGT and pH planar photoelectrode technology, the synchronous 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 given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0040] Figure 4A schematic diagram of a digging box filled with soil and introduced with earthworms 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 The following is a result diagram of low-phosphorus earthworm contact circle (earthworm pore) soil imaging disclosed in one embodiment of the present invention; wherein (a) is a soil profile diagram, 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 contact circle (earthworm pore) in the middle phosphorus according to an 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 another embodiment of the present invention that discloses the soil imaging result of the earthworm pores in the medium phosphorus earthworm contact circle; 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 plate; 101. Front plate screws; 102. Front plate side holes; 103. Imaging film carrier plate; 104. Front plate rear groove; 105. Front plate screw holes; 106. pH plane photoelectrode membrane; 107. DGT adsorption membrane; 108. Front plate screw holes; 2. Removable acrylic upper plate; 201. Upper plate screws; 202. Air holes; 203. Upper plate screw holes; 3. Soil; 301. earthworm pores; 302. earthworms; 4. rear part of earthworm digging box; 401. front screw holes of earthworm digging box; 402. upper screw holes of earthworm digging box; 5. electric wires; 6. thermometer and hygrometer; 601. thermometer and hygrometer indicator; 602. thermometer and hygrometer sensor; 7. humidifier; 701. water pipe; 702. shower head; 8. ventilating fan; 801. ventilating fan shaft; 802. ventilating fan blades; 9. filter membrane. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are 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 in conjunction with 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 technique (DGT) and pH plane photoelectrode technology to achieve in-situ detection of soil available phosphorus (labile-P) and pH in the earthworm contact circle (soil micro-area within the earthworm activity range) to achieve in-situ analysis of soil biogeochemical processes mediated by earthworm activity. Different from the traditional potted soil sampling method, the design of the present invention overcomes the changes in soil physical and chemical properties that may be caused by soil destructive sampling, and greatly shortens the detection time and reduces the detection cost in terms of operation and economic benefits. 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) earthworm cultivation; (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 that is sensitive to pH changes. Under ultraviolet light excitation, H + Induced membrane fluorescence intensity changes. The combination of the above two imaging technologies can realize the visualization and quantitative analysis of the spatial distribution of available phosphorus and pH in the soil. The specific scheme of this technology is described in detail below.

[0051] Step 1: Construction of earthworm digging box: To allow for the free movement of earthworms and the attachment of the test film to the soil profile, please refer to Figure 1-Figure 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 plate 1, a detachable acrylic upper plate 2, a rear part 4 of the earthworm digging box, an electric wire 5, a thermometer and hygrometer 6, a humidifier 7 and a ventilation fan 8. On the detachable acrylic front plate 1, a front plate screw 101 is used to fix the detachable acrylic front plate 1 and the rear part 4 of the earthworm digging box. A front plate side hole 102 is provided on the side of the detachable acrylic front plate 1, and the front plate 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 plate side hole 102. On the detachable acrylic upper plate 2, the upper plate screw 201 is used to fix the detachable acrylic upper plate 2 with the detachable acrylic front plate 1 and the rear part 4 of the earthworm digging box. A number of air holes 202 are provided on the detachable acrylic upper plate 2, for connecting the air in the earthworm digging box 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, for supplying power to the ventilation fan 8. Thermohygrometer 6, humidifier 7 and ventilation fan 8 are all provided on the detachable acrylic upper plate 2. Thermohygrometer 6 includes a thermohygrometer indicator 601 and a thermohygrometer sensor 602. The thermohygrometer indicator 601 is provided above the detachable acrylic upper plate 2, for external viewing of the current temperature and humidity measurement indication. The thermohygrometer sensor 602 extends into the internal space of the earthworm digging box, for measuring the temperature and humidity inside the earthworm digging box; the humidifier 7 is used to regularly sprinkle water on the soil to ensure soil humidity; the ventilation fan 8 is used to accelerate the air circulation in the earthworm digging box. The upper plate screw hole 203 and the screw hole 108 on the front plate are connected and fixed by the upper plate screw 201. The upper wall of the rear part 4 of the earthworm digging box is provided with an upper screw hole 402 of the earthworm digging box, and the upper plate screw hole 203 and the upper screw hole 402 of the earthworm digging box 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 earthworm digging box. The rear side of the detachable acrylic front plate 1 is cut with a front plate rear groove 104, which is convenient for the pH plane photoelectrode membrane 106 and the DGT adsorption membrane 107 on the imaging film carrier plate 103 to contact with the soil 3 for imaging. The front plate rear groove 104 is a groove opened inside the front plate, and is connected to the soil inside the earthworm digging box. By filling the groove with soil, each membrane has enough space to contact the soil. The order of the membranes used for soil imaging from outside to inside is DGT adsorption membrane 107 and pH plane photoelectrode membrane 106. The front plate screw hole 105 and the front screw hole 401 of the earthworm digging box are fixed by the front plate screw 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 absorb deionized water and transport it to the shower head 702. The shower head 702 is used to evenly spray the deionized water onto the surface of the soil 3. The ventilating fan 8 includes a rotating shaft 801 and fan blades 802, which are driven by the electricity transmitted by the wire 5 to ensure the oxygen content inside the earthworm digging box.

[0053] The second step is earthworm cultivation: a neutral soil 3 rich in organic matter is selected as the substrate for earthworm cultivation. Before the earthworm digging box is filled with soil, the soil used needs to be air-dried in advance and sieved through a 2mm sieve. To further provide a soil environment suitable for earthworm growth, the soil moisture needs to be adjusted to 60% to 70% of the field water holding capacity by a humidifier 7 during or after filling, and is determined by measuring with a thermometer and hygrometer 6. Mature and vigorous earthworms 302 are selected and cultured in the soil 3. After 7 to 14 days of cultivation, stable and sufficient earthworm pores 301 are formed. During the cultivation process, aluminum film is used to wrap the earthworm digging box around and on both sides to simulate the dark environment in the soil. The earthworm digging box is placed in a greenhouse at 25°C and 60% air humidity for cultivation.

[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, dissolve it in 50mL of deionized water, and 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 to avoid light and store at 4°C. This solution is used as a 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 is to construct the imaging system: the filter membrane 9, the DGT adsorption membrane 107 and the pH plane photoelectrode membrane 106 are attached to the soil profile with earthworm pores in sequence, and a plastic partition is filled between the pH plane photoelectrode membrane and the imaging membrane carrier plate 3 to ensure that the above-mentioned combined membrane is closely attached to the soil profile, so as to improve the reliability of the DGT high-resolution adsorption membrane and the pH plane photoelectrode membrane for imaging the solute. During the experimental period, the optimal placement time of the DGT and the plane photoelectrode needs to be determined. The captured images are processed using ImageJ software, and the spatial resolution of the DGT and pH plane photoelectrode images is confirmed at the same time. The filter membrane is part of the diffusion layer in the DGT device, and at the same time reduces radial diffusion to maintain the stable diffusion and adsorption of the dissolved substance. It is purchased from Whatman (Nuclepore Track-Etch Membrane, pore size 0.2μm). The DGT high-resolution adsorption membrane is used for the enrichment of effective phosphorus, purchased from Nanjing Weishen Environmental Protection Technology Co., Ltd., the model is a precipitated zirconium oxide adsorption membrane (GDZR), and the thickness is 0.1mm. The pH plane photoelectrode membrane was purchased from Zhongke Zhigan (Nanjing) Environmental Technology Co., Ltd. After the reaction time, the DGT membrane and the pH plane photoelectrode membrane were imaged separately. (a) DGT membrane imaging: The DGT high-resolution adsorption membrane was attached to the soil profile for 12 hours and then peeled off. The soil particles attached to the surface were rinsed with deionized water and immersed in molybdenum blue colorimetric solution. After reacting at 35°C for 30 minutes, it was washed with deionized water and dried with dust-free paper. The color image was scanned on a scanner. (b) pH photoelectrode membrane imaging: The pH photoelectrode membrane was attached to the soil profile. In order to avoid bubbles between the membrane and the soil profile, a small amount of deionized water can be added between the membrane and the soil, and the soil system was allowed to stand for 5 minutes to achieve complete stability. The earthworm digging box was placed in front of a flat ultraviolet light source with a wavelength of 355nm. The camera was used to capture the fluorescence signal emitted by the pH photoelectrode membrane, and a 370nm bandpass filter was placed in front of the camera lens to filter ultraviolet light to prevent it from interfering with the fluorescence signal emitted by the pH photoelectrode membrane.

[0057] Step 5: 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 range, and does not just obtain the average concentration in the area or the concentration change in a certain dimension; the data displayed without a standard curve is only the image grayscale value recognized by the software, and the test results obtained after correction and analysis by the standard curve are of practical significance;

[0059] See also Figure 5 As shown in the figure, (a) is the effective phosphorus-gray value standard curve. The phosphorus concentrations of 0, 20, 50, 100, 200, 500, 750, 1000 and 2000 μg L were prepared using phosphorus standard substances. -1The DGT membrane was placed in the P solution and adsorbed for 12 hours before coloring in a molybdenum blue color developing solution. The colored DGT membrane was immediately washed several times with 4°C deionized water to clean the residual color developer on the surface, and then immersed in deionized water for 5 minutes (to stop the color development reaction). The DGT membrane was taken out, the surface water was wiped off with filter paper, and it was placed on the scanner (face down), and the resolution was set to 1200dpi. After scanning and obtaining the image, the image was converted into grayscale using ImageJ software, and the exponential function with the highest correlation was selected to fit the P accumulation per unit area on the membrane and its corresponding grayscale intensity on the membrane surface, which was the standard curve (G(f)) of the analyte accumulation per unit area (f)-grayscale value (G). (b) pH-grayscale value standard curve: prepare pH standard solution with sodium acetate-acetic acid solution (pH 4-6) and potassium dihydrogen phosphate-borax solution (pH 6-9), place the long strip pH photoelectrode standard film in the cuvette, and immerse the photoelectrode film with deionized water to make it completely fit the cuvette. Then pour in standard solutions representing different pH values ​​in turn, place under 355nm ultraviolet light for color development, and use a camera equipped with a front filter to record the light intensity, use ImageJ software to determine the concentration value and draw a standard curve. (c) Select the Calibration bar function option of ImageJ software to insert the effective phosphorus concentration and pH value scale into the image.

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

[0061] (1) Earthworm culture: Choose low-phosphorus soil (effective phosphorus content of 8.75 mg kg -1 ) were filled into the designed earthworm digging box, and 3 Eisenia fetida with a length of 3 cm and a weight of 0.5 g were selected and cultured in it for 7 days to produce enough earthworm pores. During the culture process, the earthworm digging box was wrapped with aluminum film to simulate the dark environment in the soil. The earthworm digging box was placed in a greenhouse at 25°C and 60% air humidity for culture. During the culture process, an appropriate amount of water was sprinkled on the top regularly to maintain soil moisture.

[0062] (2) Material preparation: Cut a DGT high-resolution adsorption membrane with a diameter of 2.5 cm to soak the adsorbed phosphorus standard solution and draw a standard curve of effective phosphorus concentration-grayscale value. At the same time, cut several DGT membranes with a size of 5 cm × 5 cm to attach to the earthworm touch circle profile for effective phosphorus spatial distribution imaging. 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 reaction and color development of the phosphorus standard adsorption membrane and the imaging membrane.

[0063] (3) Imaging operation: After a period of cultivation, remove the detachable acrylic front plate of the earthworm digging box, paste the filter membrane at the soil profile to be imaged, and place the DGT high-resolution adsorption membrane on the imaging carrier plate, cover the acrylic front plate, and insert the imaging membrane carrier plate; to ensure the close fit between the acrylic plate and the DGT membrane, insert the 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 culturing in the dark for 12 hours, take out the imaging membrane carrier plate, peel off the DGT membrane and wash the surface of the DGT membrane with deionized water, place it in molybdenum blue color developing solution for 30 minutes, rinse the residual color developing solution on the surface of the DGT membrane with deionized water, and capture the color image with a flat scanner. 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 correspond the effective phosphorus concentration value to the pseudo-color displayed on the image, and then you can get the spatial distribution image of the effective phosphorus in the earthworm tactile circle. Please refer to 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: Choose medium phosphorus soil (effective phosphorus content 40 mg kg -1 ) were filled into the designed earthworm digging box, and 3 Eisenia fetida with a length of 3 cm and a weight of 0.5 g were selected and cultured in it for 7 days to produce enough earthworm pores. During the culture process, the earthworm digging box was wrapped with aluminum film to simulate the dark environment in the soil. The earthworm digging box was placed in a greenhouse at 25°C and 60% air humidity for culture. During the culture process, an appropriate amount of water was sprinkled on the top regularly to maintain soil moisture.

[0067] (2) Material preparation: Cut a DGT high-resolution adsorption membrane with a diameter of 2.5 cm to soak the standard solution for adsorption of phosphorus and draw a standard curve of effective phosphorus concentration-grayscale value. At the same time, cut several DGT high-resolution adsorption membranes with a size of 5 cm × 5 cm to attach to the earthworm touch circle profile for imaging of 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 reaction and color development of the phosphorus standard adsorption membrane and the imaging membrane.

[0068] (3) Imaging operation: After a period of cultivation, remove the detachable acrylic front plate of the earthworm digging box, paste the filter membrane at the soil profile to be imaged, and place the DGT high-resolution adsorption membrane on the imaging carrier plate, cover the acrylic front plate, and insert the imaging membrane carrier plate; to ensure the close fit between the acrylic plate and the DGT membrane, insert the 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 culturing in the dark for 12 hours, take out the imaging membrane carrier plate, peel off the DGT membrane and wash the surface of the DGT membrane with deionized water, place it in molybdenum blue color developing solution for 30 minutes, rinse the residual color developing solution on the surface of the DGT membrane with deionized water, and capture the color image with a flat scanner. Standard phosphorus adsorption membrane imaging is carried out in the same way as above.

[0069] (4) Image processing: The two images taken 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 ​​one by one. Through the calibration curve, the concentration of fluorescent substances and the concentration value of effective phosphorus were matched to the pseudo colors displayed on the images one by one, and the distribution images of phosphatase and effective phosphorus at the interface were obtained, as shown in Fig. Figure 7 shown.

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

[0071] (1) Earthworm culture: Choose medium phosphorus soil (effective phosphorus content 40 mg kg -1 ) were filled into the designed earthworm digging box, and 3 Eisenia fetida with a length of 3 cm and a weight of 0.5 g were selected and cultured in it for 7 days to produce enough earthworm pores. During the culture process, the earthworm digging box was wrapped with aluminum film to simulate the dark environment in the soil. The earthworm digging box was placed in a greenhouse at 25°C and 60% air humidity for culture. During the culture process, an appropriate amount of water was sprinkled on the top regularly to maintain soil moisture.

[0072] (2) Material preparation: (a) Cut a standard pH photoelectrode membrane with a size of 1cm×3cm for immersion imaging in a standard pH solution, and prepare a pH standard solution with a certain concentration gradient. After the pH photoelectrode membrane is immersed in the standard solution for 5 minutes, it is excited and imaged under 355nm ultraviolet light, and the fluorescence image is captured by a camera with a 370nm bandpass filter in front of the lens. In addition, a 5cm×5cm pH photoelectrode membrane is cut and attached to the imaging membrane carrier plate and fully contacted with the soil profile for in-situ measurement of the pH spatial distribution of the soil profile; (b) A DGT disc membrane with a diameter of 2.5cm is cut and used to immerse the adsorbed phosphorus standard solution and draw a standard curve of effective phosphorus concentration-grayscale value. At the same time, several DGT square membranes with a size of 5cm×5cm are cut and used to attach to the earthworm touch circle profile for imaging of 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 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 phosphorus standard adsorption film and imaging film.

[0073] (3) Imaging operation: After a period of cultivation, the detachable acrylic front plate of the earthworm digging box is removed, and the filter membrane is pasted at the soil profile to be imaged, and the pH photoelectrode membrane and the DGT square membrane are placed on the imaging membrane carrier plate in turn, and a plastic sheet is filled between the pH photoelectrode membrane and the imaging membrane carrier plate, and 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 culturing in the dark for 12 hours, the earthworm digging box is first placed directly under ultraviolet light for in-situ pH imaging, and then the imaging membrane carrier plate is taken out, the pH photoelectrode membrane and the 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 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 flat scanner. The imaging of the standard phosphorus adsorption membrane is carried out in the same way as above. The flat photoelectrode membrane is used to capture the fluorescence image under 355nm ultraviolet light with a camera with a pre-lens filter.

[0074] (4) Image processing: Import the two images taken 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 correspond the fluorescent substance concentration and the effective phosphorus concentration value to the pseudo colors displayed on the images. Then, 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 planar optode combined with DGT technology is widely used in situ detection of the spatial distribution of solutes (heavy metals) in rhizosphere soil, there are essential differences between the rhizosphere environment and the earthworm environment, such as the difference in the growth environment of plants and earthworms, the difference in soil nutrient turnover by plant roots and earthworms, and the micro-area differences formed by root growth and earthworm movement. Specifically:

[0076] 1) Plant growth depends on a large amount of soil moisture to supply plant metabolism, especially rice plants are more dependent on flooded soil environment, and the soil moisture reaches or exceeds 100%. However, earthworm activity generally requires soil moisture of 60-70% or even lower. It is difficult for earthworms to survive in wet soil.

[0077] 2) The rhizosphere plays an important role in plant growth, such as absorbing mineral nutrients from the soil, and the nutrient content in the rhizosphere is often lower than that in undisturbed soil. Earthworm activities (such as secreting coelomic fluid and excreting earthworm feces) directly increase the nutrient content in the earthworm contact circle soil on the one hand, and promote soil nutrient activation by supplying soil microbial nutrient resources on the other hand.

[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 configured in a semi-open manner 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 the 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. In order to further simulate the soil environment suitable for earthworm growth, the device of the present invention installs a thermometer / hygrometer and a humidifying 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) Different from the traditional soil in situ imaging device, this experimental device is designed to facilitate operation by cutting a rear groove on the detachable acrylic front plate and placing a movable imaging film carrier plate to facilitate timely replacement of the pH plane photoelectrode membrane and DGT membrane.

[0083] The parts of the present invention that are not described in detail may refer to the prior art in the art or are 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 modes, which are merely illustrative rather than 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 within the protection of the present invention.

Claims

1. An in-situ chemical imaging method for earthworm contact circles 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; S2, selecting suitable earthworms and placing them in the soil of the earthworm digging box, and cultivating the earthworms for a period of time to produce enough earthworm pores in the soil; S3, sequentially set a filter membrane, a DGT adsorption membrane and a pH plane photoelectrode membrane 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 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 cultivate for a period of time; and use a camera to capture the fluorescence signal emitted by the pH plane photoelectrode membrane to obtain a pH fluorescence image; 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 contact circles in soil according to claim 1, characterized in that: The soils in S1 were low and medium in available phosphorus content.

3. The in-situ chemical imaging method for earthworm contact circles in soil according to claim 1, characterized in that: The soil moisture in S1 was 60%~70% of the field capacity.

4. The in-situ chemical imaging method for earthworm contact circles in soil according to claim 1, characterized in that: 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.

5. The in-situ chemical imaging method for earthworm contact circles in soil according to claim 4, characterized in that: After adding deionized water between the pH plane photoelectrode membrane and the earthworm contact circle soil profile, the soil system was allowed to stand for more than 5 minutes to achieve complete stability.

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

7. The in-situ chemical imaging method for earthworm contact circles 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.

8. The in-situ chemical imaging method for earthworm contact circles in soil according to claim 1, characterized in that: The color development of 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 adhering to the surface of the DGT adsorption film are rinsed with deionized water, and then immersed in molybdenum blue color developing solution, reacted at 35°C for 30 minutes, and the DGT adsorption film after the reaction is washed with deionized water and dried with dust-free paper.

9. The in-situ chemical imaging method for earthworm contact circles in soil according to claim 8, characterized in that: The preparation method of the molybdenum blue color developing solution is: Measure a certain amount of concentrated sulfuric acid and pour it into a certain amount of deionized water, and stir evenly to obtain solution A; Weigh a certain amount of ammonium molybdate and place it in 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 adjust the volume to a preset volume to obtain a 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 evenly to obtain the molybdenum blue developer solution.

10. 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 haptosphere as described in any one of claims 1 to 9.

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