An in-situ detection method for the oxalic acid content in rhizosphere soil

Abstract

An in-situ detection method for the oxalic acid content in rhizosphere soil according to an embodiment of the present invention includes: preparing a substrate solution by using sulfuric acid, rhodamine, potassium dichromate / potassium bromate; mixing oxalic acid with the substrate solution to obtain fluorescent standard substances with different oxalic acid concentrations and soaking different fiber membrane media to obtain first fiber membrane media at different oxalic acid concentrations; excavating a soil profile for in-situ cultivation of root plants; after culturing for a period of time, covering the rhizosphere soil with another fiber membrane media soaked in the substrate solution and continuing in-situ cultivation to obtain a second fiber membrane media after in-situ cultivation; respectively performing fluorescence spectral imaging on each of the first fiber membrane media and the second fiber membrane media; fitting a linear regression equation by using the imaging photos of the first fiber membrane media; substituting the gray values of each pixel in the imaging photo of the second fiber membrane media into the linear regression equation to generate a two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of soil detection, and in particular to an in-situ detection method for the oxalic acid content in rhizosphere soil. Background Art

[0002] At present, the detection of the oxalic acid content in rhizosphere soil mainly combines the collection of field root plants or rhizosphere soil samples with indoor test analysis using equipment such as gas chromatographs, ion chromatographs, high-performance liquid chromatographs, and chromatograph-mass spectrometers in the laboratory. Among them, soil sample sampling usually adopts fixed-point sampling, layered sampling, or rhizosphere mixed sampling methods, which belong to destructive sampling, and then tests are carried out in the suspension prepared after grinding and sieving the samples in the laboratory. The above several equipment analysis and testing are expensive, and the soil samples need to be preserved, transported, and subjected to complex and time-consuming pretreatment in the laboratory before equipment analysis and testing. This series of processes usually causes a huge deviation between the detection results and the actual situation.

[0003] Patent CN117517507A discloses a method for in-situ identification and quantitative determination of organic acids in the rhizosphere hot spots of soil. It uses an enzyme spectrum image rhizosphere hot spot area precise identification device and technology, combined with a high-performance liquid chromatography method to test the detection method of 7 organic acids including oxalic acid, indirectly reflecting the quantitative and qualitative spatial information of rhizosphere soil organic acids in the hot spot area and non-hot spot area based on enzyme spectrum identification. However, it still requires the collection, preservation, transportation, and indoor treatment of rhizosphere soil samples and the combination with indoor high-performance liquid chromatography testing, and it is not known whether the enzyme activity rhizosphere hot spot area and non-hot spot area reflected by the enzyme spectrum image are consistent with the spatial distribution of rhizosphere organic acid content. Summary of the Invention

[0004] The embodiments of the present invention provide an in-situ detection method for the oxalic acid content in rhizosphere soil to solve the above technical problems.

[0005] The method includes:

[0006] Prepare a fluorescent substrate solution using sulfuric acid, rhodamine, and potassium dichromate / potassium bromate;

[0007] Mix oxalic acid with different amounts of the substrate solution to obtain fluorescent reference substances with different oxalic acid concentrations. Among them, oxalic acid is used to weaken the fluorescence of the substrate solution. The higher the oxalic acid concentration, the weaker the fluorescence of the fluorescent reference substance;

[0008] Soak different fiber membrane media uniformly with fluorescent reference substances with different oxalic acid concentrations to obtain the first fiber membrane media at different oxalic acid concentrations;

[0009] Excavate the soil profile and place it into a soil root culture device for in-situ cultivation of root plants, or cultivate plants using the original soil indoor soil root culture device;

[0010] After culturing for a period of time, cover the rhizosphere soil with another fiber membrane medium soaked in the substrate solution and continue the in-situ culture; after the oxalic acid in the rhizosphere soil weakens the fluorescence of the substrate solution in the other fiber membrane medium, use the other fiber membrane medium as the second fiber membrane medium.

[0011] Perform fluorescence spectral imaging on each first fiber membrane medium and the second fiber membrane medium respectively. Among them, the higher the fluorescence of the solution in the fiber membrane medium, the higher the imaging gray level.

[0012] Use the imaging photos of each first fiber membrane medium to fit a linear regression equation in which the imaging gray level decreases with the increase of oxalic acid concentration.

[0013] Substitute the gray level values of each pixel in the imaging photo of the second fiber membrane medium into the linear regression equation respectively to generate the two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil.

[0014] This method is based on the principle of fluorescence reaction-spectrum imaging. By using a fluorescence imaging device and an image processing method, it realizes the visualization expression of the distribution characteristics, spatial quantification and qualitative information of organic acids in rhizosphere soil in the case of in-situ soil, provides hardware for monitoring the spatial distribution of soil organic acids, and is particularly suitable for the in-situ detection of oxalic acid content in field rhizosphere soil. The whole detection system is convenient to use and very economical to obtain data results; the whole method does not change the original soil environmental conditions, the detection data is more accurate, and can accurately reflect the quantitative and qualitative spatial information of oxalic acid in rhizosphere soil. Description of the Drawings

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 is a schematic diagram of an in-situ detection system for the oxalic acid content in rhizosphere soil provided by an embodiment of the present invention;

[0017] Figure 2 is a flowchart of an in-situ detection method for the oxalic acid content in rhizosphere soil provided by an embodiment of the present invention;

[0018] Figure 3 is a flowchart of another in-situ detection method for the oxalic acid content in rhizosphere soil provided by an embodiment of the present invention;

[0019] Figure 4It is a schematic diagram of the key results of an in-situ detection method for the oxalic acid content in rhizosphere soil provided by an embodiment of the present invention;

[0020] Figure 5 It is a schematic diagram of a neural network structure model used for denoising provided by an embodiment of the present invention;

[0021] Reference numerals:

[0022] 1. Industrial control computer;

[0023] 2. High-resolution camera;

[0024] 3. Ultraviolet light source;

[0025] 4. Control valve;

[0026] 5. Mobile rack. Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the scope of protection of the present invention.

[0028] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be construed as a limitation of the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0029] In the description of the present invention, it should also be noted that unless otherwise clearly specified and defined, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0030] As described in the background art, the existing methods for detecting the oxalic acid content in rhizosphere soil have at least the following defects:

[0031] 1. The sample collection destroys the original soil environment, and the analysis results cannot reflect the native environment;

[0032] 2. The uncertainties of the series sample pretreatment process and the indoor instrument test analysis are superimposed, and the accuracy of the data results cannot be evaluated;

[0033] 3. It cannot accurately and truly reflect the two-dimensional spatial distribution of rhizosphere organic acids;

[0034] 4. The analysis period for quantitatively determining organic acids by the instrument is relatively long and costly.

[0035] In view of at least one of the above problems, the embodiments of the present invention provide an in-situ detection method for the oxalic acid content in rhizosphere soil. To illustrate this method, an in-situ detection system for the oxalic acid content in rhizosphere soil that supports the implementation of this method is introduced preferentially. Figure 1 is a schematic diagram of an in-situ detection system for the oxalic acid content in rhizosphere soil provided by the embodiments of the present invention. As Figure 1 shown, the system includes a soil root culture device and a fluorescence spectral imaging device.

[0036] Optionally, the size of the soil root culture device is 50 cm × 50 cm × 50 cm or 100 cm × 100 cm × 100 cm. The main part of the soil root culture device is a rhizobox, which is used for soil root culture of root plants. In front of the rhizobox is a movable baffle, which is used to place a fiber membrane closely attached to the side of the root soil during the detection process, for the detection experiment of the root longitudinal section, that is, for generating the two-dimensional spatial distribution of the oxalic acid concentration in the root longitudinal section; for root enrichment, below the rhizobox is a 45° inclined movable baffle, which is used to place a fiber membrane closely attached to the bottom of the root soil during the detection process, for the detection experiment of the root bottom section, that is, for generating the two-dimensional spatial distribution of the oxalic acid concentration in the root bottom section. Optionally, the movable baffle is a transparent glass baffle.

[0037] Optionally, the fluorescence spectral imaging device mainly includes a high-resolution camera 2, an ultraviolet light source 3, a lifting platform, and an industrial control computer 1. During the detection process, the imaged fiber membrane can be placed on the lifting platform. The industrial control computer 1 controls the ultraviolet light wavelength irradiation and the high-resolution camera 2 imaging respectively. The moving frame 5 and the regulating valve 4 of the lifting platform control the horizontal movement and vertical lifting of the fiber membrane respectively, so as to be better irradiated by the ultraviolet light source. After the irradiation, fluorescence imaging can be performed by the high-resolution camera 2 and the imaging photos can be output to the industrial control computer 1 or other electronic devices.

[0038] Based on the above system, Figure 2 is a flowchart of an in-situ detection method for the oxalic acid content in rhizosphere soil provided by the embodiments of the present invention. As Figure 2 shown, the method specifically includes:

[0039] S110. Prepare a fluorescent substrate solution using sulfuric acid, rhodamine, and potassium dichromate / potassium bromate; mix oxalic acid with different amounts of the substrate solution to obtain fluorescent reference substances with different oxalic acid concentrations. Here, oxalic acid is used to weaken the fluorescence of the substrate solution. The higher the oxalic acid concentration, the weaker the fluorescence of the fluorescent reference substance. Uniformly soak different fiber membrane media with the fluorescent reference substances of different oxalic acid concentrations to obtain the first fiber membrane media at different oxalic acid concentrations.

[0040] Fluorescent substances emit light under ultraviolet light irradiation. This property can be used to detect the content of fluorescent substances. The stronger the light, the more the substance content. Oxalic acid does not have fluorescence. Therefore, there is no method or technology for detecting the oxalic acid content using fluorescence spectral imaging. In this embodiment, the interaction and reaction law of oxalic acid catalyzing the oxidation of rhodamine by a strong oxidant potassium dichromate under acidic conditions will be utilized, and the fluorescence kinetic characteristics of rhodamine fluorescence quenching will be used to prepare a fiber membrane medium and perform spectral imaging on the fiber membrane medium to achieve two-dimensional distribution characteristics and quantitative expression of rhizosphere soil organic acid - oxalic acid. Specifically, in this step, two types of fiber membrane media are first prepared through relevant chemical reagents. One type is soaked with the substrate mixture at different oxalic acid concentrations, and the other type is soaked with the substrate solution without oxalic acid.

[0041] In a specific embodiment, the preparation process of each fiber membrane medium may include the following steps:

[0042] Step 1. Prepare the substrate solution. Optionally, slowly add 1.12 mL of concentrated sulfuric acid to approximately 300 mL of a large amount of ultrapure water, and continuously stir. Cool to room temperature; add 134 mg of rhodamine B (or rhodamine 6G), stir to dissolve, then add 353 mg of potassium dichromate (or 1002 mg of potassium bromate), stir to dissolve, and then make up the volume to 500 mL with ultrapure water in a volumetric flask to prepare the substrate solution. It should be noted that all chemical reagents used in this embodiment are required to be of analytical pure grade, and the ultrapure water is not less than the secondary requirement of experimental water.

[0043] Step 2: Mix oxalic acid with different amounts of substrate solution to obtain fluorescent reference materials with different oxalic acid concentrations. Optionally, weigh 100 mg of oxalic acid, add 40 mL of substrate solution, stir to dissolve, and then make up the volume to 100 mL in a volumetric flask or colorimetric tube with substrate solution to prepare a stock standard solution of 1000 mg / L. Respectively pipette 12.5 mL, 7.5 mL, 5 mL, 2.5 mL, 1.5 mL, 1.0 mL, 0.25 mL, and 0 mL of the stock solution into 25-mL volumetric flasks or colorimetric tubes, and make up the volume to 25 mL with substrate solution to prepare standard solutions with gradient concentrations of 500 mg / L, 300 mg / L, 200 mg / L, 100 mg / L, 60 mg / L, 40 mg / L, 10 mg / L, and 0 mg / L. Among them, the concentrations of the above standard solutions can be adjusted in different gradients according to the possible concentration range of the organic acids in the soil to be measured, and the number of gradient standards can also be adjusted.

[0044] Step 3: Prepare fluorescent reference material-fiber membrane medium. Optionally, use the mixed solution prepared by dissolving oxalic acid, rhodamine B (or rhodamine 6G), potassium dichromate, and concentrated sulfuric acid in ultrapure water in Step 2 as the fluorescent reference material. The membrane material is a polyester fiber membrane. Immerse the fiber membrane in the fluorescent reference material with different concentration gradients for 10 - 15 minutes, preferably 12 minutes; then take it out and let it stand on an inclined tin foil (or aluminum foil) for 2 minutes to prepare fluorescent reference material-fiber membrane media with different gradient concentrations.

[0045] Meanwhile, prepare substrate-fiber membrane medium. Optionally, immerse the fiber membrane in the substrate solution prepared in Step 1 for 10 - 15 minutes, preferably 12 minutes, then take it out and let it stand on an inclined tin foil (or aluminum foil) for 2 minutes to prepare substrate-fiber membrane medium for later use. Specifically, when immersing, multiple polyester fiber membranes or nylon fiber membranes with a specification model of 12 cm × 5 cm and 0.45 μm can be respectively placed in a glass petri dish with a diameter of 15 cm, and different concentration gradient standard substance solutions and substrate solutions can be respectively poured in for immersion.

[0046] It can be understood that the dosages of the chemical reagents concentrated sulfuric acid, rhodamine B (or rhodamine 6G), and potassium dichromate (or potassium bromate) for preparing the substrate solution in the process of preparing the fiber membrane medium can be slightly adjusted, but the adhesion effect and imaging effect in the fiber membrane medium need to be ensured.

[0047] S120. Excavate the soil profile and place it into the soil root culture device for in-situ cultivation of root plants, or cultivate plants using the original soil in the indoor soil root culture device; after cultivating for a period of time, cover the rhizosphere soil with another fiber membrane medium soaked in the substrate solution and continue in-situ cultivation; after the oxalic acid in the rhizosphere soil weakens the fluorescence of the substrate solution in the other fiber membrane medium, use the other fiber membrane medium as the second fiber membrane medium.

[0048] This step conducts in-situ monitoring of the rhizosphere soil. When detecting wild soil, the excavated soil profile is the wild soil profile, and the roots are wild root plants.

[0049] In a specific embodiment, soil profiles of different sizes can be excavated for different types of plants, place the root box closely against the soil profile, and then backfill the soil. After 1 - 3 months of in-situ cultivation, dig open the soil profile, open the soil root culture device, cover the substrate-fiber membrane medium on the soil profile where the roots are distributed, and make the substrate-fiber membrane medium closely fit with the rhizosphere soil for in-situ cultivation for 30 - 60 minutes.

[0050] Specifically, combined with Figure 2 , the movable baffles on the sides and bottom of the root box can be inserted and removed. After 1 - 3 months of in-situ cultivation as above, the movable baffles on the sides and bottom can be taken out respectively, and then take two substrate-fiber membrane media prepared in S110 and place them on the two movable baffles respectively; then place the two movable baffles with the substrate-fiber membrane media back to the sides and bottom of the root box respectively, so that the two substrate-fiber membrane media are closely attached to the sides and bottom of the rhizosphere soil respectively, and continue in-situ cultivation.

[0051] After the above in-situ cultivation is completed, the substrate solution in the fiber membrane medium has undergone sufficient chemical reactions (catalysis is also a chemical reaction) with the oxalic acid in the rhizosphere soil, which will be used as the basis for detecting the oxalic acid content in the subsequent rhizosphere soil. For the convenience of distinction and description, in this embodiment, the fluorescence standard substance-fiber membrane medium prepared in S110 is called the first fiber membrane medium, and the fiber membrane medium that has fully undergone chemical reactions with the rhizosphere soil substances is called the second fiber membrane medium.

[0052] S130. Perform fluorescence spectral imaging on each of the first fiber membrane media and the second fiber membrane medium respectively. Among them, the higher the fluorescence of the solution in the fiber membrane medium, the higher the imaging gray level; and use the imaging photos of each first fiber membrane medium to fit a linear regression equation in which the imaging gray level decreases with the increase of oxalic acid concentration.

[0053] In this step, the imaging methods for each fiber membrane medium are similar. Taking the second fiber membrane medium as an example, first, move the second fiber membrane medium into the reaction darkroom and adjust it to a suitable position through the horizontal movement of the moving rack 5 and the up-and-down movement of the regulating valve 4; then, rely on the industrial control computer 1 to control the excitation of the ultraviolet light source 3, the emission wavelength, and the irradiation duration, and irradiate the substrate-fiber medium with ultraviolet light; finally, perform fluorescence imaging through the high-resolution camera 2 and output the imaging photo to other electronic devices (such as other PC terminals). Among them, the ultraviolet light irradiation is 1-5 minutes, preferably 3 minutes; and before putting the second fiber membrane medium into the reaction darkroom, first remove the soil particles on its surface with a brush.

[0054] According to the same method, put each first fiber membrane medium into the reaction darkroom in turn, and the imaging photos (also called standard substance images) of each first fiber membrane medium can be obtained respectively. Based on these standard substance images, a linear regression equation between the oxalic acid concentration and the imaging gray level can be fitted. In a specific embodiment, this fitting process can be implemented by the image processing module of the other electronic device, and specifically includes the following steps:

[0055] Step 1: Convert the color pixel values of each imaging photo into gray values. Optionally, using image preprocessing technology, the captured color image can be converted into a gray image, that is, the red (R), green (G), and blue (B) color components of each pixel are combined into a single gray value Gray, so that each pixel in the image is represented by only one intensity value (usually between 0 and 255), 0 usually represents black, 255 represents white, and the intermediate value represents different degrees of gray. According to different camera imaging modes, the calculation formula can be:

[0056] Gray = 0.299×R + 0.587×G + 0.114×B

[0057] Or Gray = (R + G + B) / 3 (1)

[0058] Step 2: Using the gray average value of the imaging photos of each first fiber membrane medium and the corresponding oxalic acid concentration as samples, fit a linear regression equation between the oxalic acid concentration and the imaging gray level. Optionally, average the gray values of all pixels of each standard substance image to obtain the gray value Gray of the standard substance s ; and combine the different oxalic acid concentrations C configured in S110 s to perform linear regression calculation, and the obtained linear regression equation is:

[0059] Gray s = Intercept + Slope*C s (2)

[0060] S140. Substitute the gray values of each pixel in the imaging photo of the second fiber membrane medium into the linear regression equation respectively to generate the two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil.

[0061] This step can also be implemented by the image processing module in the other electronic devices. In a specific embodiment, the color pixel values at each pixel position in the imaging photo of the second fiber membrane medium can be converted into gray values at each pixel position by the same method; then, substitute the gray values at each pixel position into the linear regression equation to obtain the oxalic acid concentration C at each pixel position:

[0062] C = (Gray – Intercept) / Slope (3)

[0063] Arrange the oxalic acid concentrations according to the pixel positions, and the two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil can be obtained.

[0064] Furthermore, the oxalic acid concentration in the two-dimensional spatial distribution can be normalized to the image gray space of [0 255]; perform color rendering on the normalized two-dimensional data to realize the visual representation of the oxalic acid concentration in the rhizosphere soil. The normalization formula is:

[0065] Gray’ = (C - C min ) × 255 / (C max - C min ) (4)

[0066] Figure 3 It is the flowchart of another in-situ detection method for the oxalic acid content in the rhizosphere soil provided by the embodiment of the present invention, which shows the key steps and device modules used in the method of this embodiment from another perspective. Referring to Figure 3 the method of this embodiment can be better understood.

[0067] In order to verify the reliability of the method of this embodiment, the following uses this method to conduct an experiment on a soil root culture root box of Stipa grandis vegetation planted indoors for 7 months. The whole process can include the following steps:

[0068] Step 1. Prepare the reference material - polyester fiber membrane medium. Select a mixed solution prepared by dissolving oxalic acid, rhodamine B (or rhodamine 6G), potassium dichromate, and concentrated sulfuric acid in ultrapure water as the fluorescent reference material. Set 8 concentration gradients of 500, 300, 200, 100, 60, 40, 10, and 0 mg / L. Place a 12 cm × 5 cm 0.45 μm polyester fiber membrane in a petri dish with a diameter of 15 cm, pour in the reference material solutions with different concentration gradients, soak for 12 minutes, take out and let it stand on an inclined tin foil (or aluminum foil) for 2 minutes to obtain the reference material - polyester fiber membrane media with different concentration gradients, and accurately label each reference material - polyester fiber membrane medium.

[0069] Step 2. Fit the linear regression equation between the oxalic acid concentration and the imaging gray level. Use a fluorescence imaging device to image each reference material - polyester fiber membrane medium, perform quantitative processing on the gray values of the images, and fit the gray values of the images with the reference material concentrations to obtain the linear regression equation between the oxalic acid concentration and the imaging gray level.

[0070] Step 3. In-situ monitor the rhizosphere soil. Select a mixed solution prepared by dissolving rhodamine B, potassium dichromate, and concentrated sulfuric acid in ultrapure water as the substrate solution. Place a 12 cm × 5 cm 0.45 μm polyester fiber membrane in the substrate solution in a glass petri dish with a diameter of 15 cm, soak for 12 minutes, take out and let it stand on an inclined tin foil (or aluminum foil) for 2 minutes to obtain the substrate - polyester fiber membrane medium. Select Stipa grandis planted as a representative of herbaceous plants for in-situ detection experiments. Remove the movable baffle of the rhizosphere soil monitoring root box to make the substrate - polyester fiber membrane medium closely fit with the rhizosphere soil, and incubate in-situ for 30 minutes, then take out and remove the soil particles on its surface with a soft brush, and use a fluorescence imaging device to image it.

[0071] Step 4. Visual expression of the rhizosphere soil image. Perform quantitative processing on the gray values of the rhizosphere soil image, substitute the fluorescence imaging gray values into the linear equation, convert the gray values into organic acid concentrations, and thus quantitatively express the distribution characteristics of the content of organic acids in the two-dimensional rhizosphere soil.

[0072] The whole experimental process and key results are as Figure 4As shown, the multiple imaging photos in the upper left part of the figure are the imaging photos of multiple first fiber membrane media, and the corresponding curves are the fitted linear regression equations. It can be seen that the method of this embodiment obtains data in the original soil environment, which has better representativeness and accuracy. At the same time, it can be understood through this application example that the execution order of S110 - S140 can be flexibly adjusted according to the actual situation and can also be interspersed. This embodiment does not make specific restrictions. To sum up, in order to realize the in-situ monitoring of the oxalic acid content in rhizosphere soil and overcome the deficiency that current sampling and analysis are difficult to reflect the spatial heterogeneity of oxalic acid, the embodiment of the present invention utilizes the interaction and reaction law of oxalic acid catalyzing the oxidation of rhodamine by strong oxidant potassium dichromate under acidic conditions, and the fluorescence kinetic characteristics of promoting the fluorescence quenching of rhodamine to prepare the fiber membrane medium. Then, the fluorescence image is output to the computer terminal through the fluorescence spectral imaging device, and a program is compiled to process and calculate the image, realizing the visualization expression of the distribution characteristics, spatial quantification and qualitative information of organic acids in rhizosphere soil in the case of in-situ soil, which can provide hardware for monitoring the spatial distribution of soil organic acids and is particularly suitable for the in-situ detection of the oxalic acid content in field rhizosphere soil. The entire detection system is convenient to use, and the obtained data results are very economical. The entire method does not change the original soil environmental conditions, the detection data is more accurate, and it can accurately reflect the quantitative and qualitative spatial information of oxalic acid in rhizosphere soil, providing a basis for systematically understanding microorganisms and their influencing factors in highly heterogeneous soil and rhizosphere environment.

[0073] Furthermore, in any of the above embodiments, the fluorescence imaging photos taken by the high-resolution camera, the two-dimensional distribution of oxalic acid concentration, and the final visualization image all contain image noise, and the noise includes at least one of the following: noise in fluorescence imaging, noise in camera shooting, and noise caused by uneven attachment of liquid in the fiber membrane medium, etc. In order to remove the noise, denoising processing can be performed after obtaining the fluorescence imaging photo, or after obtaining the two-dimensional distribution of oxalic acid concentration, or after obtaining the final visualization image. Optionally, this embodiment uses a neural network method for denoising. However, since there is currently no ground truth image of the two-dimensional distribution of oxalic acid in plant soil rhizosphere, in the process of detecting the two-dimensional distribution of oxalic acid in rhizosphere soil using the above method, this embodiment adjusts the distance and angle between the high-resolution camera and the fiber membrane medium to be photographed to continuously accumulate image samples of the oxalic acid distribution in rhizosphere soil.

[0074] In a specific embodiment, denoising can be performed after obtaining the fluorescence imaging photo, and the neural network structure model used for denoising is as Figure 5As shown, the model includes multiple groups of encoders and decoders. Each group (or each pair) of encoder and decoder corresponds to a combination of shooting angle and shooting distance. The encoder is used to encode the fluorescence imaging photos taken under this combination, and extract the structural depth features regarding the oxalic acid distribution in the soil roots. The decoder is used to decode the depth features and restore the fluorescence imaging photo corresponding to the input decoder. Among them, the decoder and encoder of the same group (such as encoder 1 and decoder 1) are inverse operations of each other. A CNN structure or a self-attention network structure can be adopted, and the number of internal layers and parameters can be flexibly set according to needs.

[0075] The training process and denoising mechanism of the model are described below. First, determine multiple fixed combinations of shooting angles and shooting distances, including combinations of different shooting distances and the forward shooting angle. The forward shooting angle refers to the shooting angle perpendicular to the placement plane of the fiber membrane medium. Then, use the method of S110 - S140 to detect the oxalic acid content in the root soil multiple times. During the detection process, when taking pictures of each fiber membrane medium, through the cooperation of the regulating valve, the moving rack, and the industrial control computer, the camera and the fiber membrane respectively meet the shooting angle and shooting distance in the above multiple combinations, take pictures respectively under each combination, and the fluorescence imaging photos of the same second fiber membrane medium under all combinations together constitute a denoising sample.

[0076] After the denoising samples reach a certain number, use multiple denoising samples to train the neural network model. Combining Figure 5 , during the training process, input the fluorescence imaging photos of each combination in the same denoising sample into the corresponding encoder respectively to obtain their respective depth features; then input their respective depth features into the corresponding decoder respectively to obtain their respective restored images. At the same time, construct the following loss function:

[0077]

[0078] Among them, M represents the number of denoising samples input into the neural network model in the same batch, N represents the number of combinations of shooting angles and distances; i ≠ j, F i，k and F j，k respectively represent the depth features of the i-th combination and the j-th combination in the k-th denoising sample, that is, the depth features under any two different combinations, P i，k and respectively represent the fluorescence imaging photo and the restored photo of the i-th combination in the k-th denoising sample; α and β are respectively the weight ratios of the loss terms.

[0079] After processing each batch of denoising samples in the model, update the model parameters by minimizing L. Among them, the loss term When minimized, the depth features of the same second fiber membrane medium under different combinations tend to be consistent. At this time, this feature represents the relatively fixed essential structural feature in the second fiber membrane medium, that is, the structural feature in the true two-dimensional oxalic acid distribution in the rhizosphere soil. This feature will not be changed due to uncertain disturbances during shooting (i.e., various noises mentioned above), thus achieving the purpose of denoising; the restored image obtained according to this feature That is, the image after denoising. Minimization can ensure that the restored image is similar enough to the original image, avoiding the loss of important information of the original image P i，k α and β can be set as needed. They can be used to balance the magnitude differences of the two loss terms and can also be used to balance the degree of denoising and the degree of information retention.

[0080] After the above model is trained, each group of encoders and decoders can denoise the fluorescence imaging photos under the corresponding combinations. The output of the decoder is the image after denoising. Optionally, combinations of different shooting distances and forward shooting angles are used as multiple preferred combinations, and the encoders and decoders corresponding to each preferred combination in the trained neural network model are used to denoise the fluorescence imaging photos of each first fiber membrane medium under each preferred combination respectively. That is, in this embodiment, the forward shooting angle is used as the preferred angle. After denoising, the method of fitting the linear regression equation in which the imaging gray level decreases with the increase of oxalic acid concentration in S130 is adopted. The denoised fluorescence imaging photos of each first fiber membrane medium under each preferred combination are used to refit the linear regression equation respectively, thereby obtaining the linear regression equation under each preferred combination. Optionally, the least squares method is used in this fitting process. If the average imaging gray level of the denoised fluorescence imaging photos of each first fiber membrane medium is used as each actual value, and the gray level values calculated by substituting each oxalic acid concentration into the linear regression equation are used as each predicted value, then the sum of the squared errors between each actual value and the predicted value after fitting is the smallest (also known as the least squares error). In this embodiment, this least squares error is used as the fitting error, and the fitting errors of each linear regression equation are compared. The equation with the smallest fitting error is used as the optimal linear regression equation, and the corresponding shooting distance is the best shooting distance. During subsequent detection, the fluorescence imaging photo of the second fiber membrane medium can be taken according to the best shooting distance and the forward shooting angle, the trained encoder and decoder corresponding to the combination of the best shooting distance and the forward shooting angle are used to denoise the fluorescence imaging photo, and the two-dimensional spatial distribution of the rhizosphere oxalic acid concentration is generated by using the optimal linear regression equation.

[0081] In view of the current technical situation that there is no ground truth image of the two-dimensional distribution of oxalic acid in the rhizosphere of plant soil, multiple captured images of the same soil oxalic acid distribution are used as a large sample to jointly train multiple groups of encoders and decoders, and the common features in the images are extracted to eliminate uncertain interference for the purpose of denoising. At the same time, the linear regression equation is corrected according to the denoised image, and the optimal shooting distance is determined to further improve the detection accuracy.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features. However, such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. An in-situ detection method for the oxalic acid content in rhizosphere soil, characterized in that, Comprising: Preparing a fluorescent substrate solution using sulfuric acid, rhodamine, and potassium dichromate / potassium bromate; Mixing oxalic acid with different amounts of the substrate solution to obtain fluorescent reference materials with different oxalic acid concentrations; Uniformly soaking different fiber membrane media with the fluorescent reference materials with different oxalic acid concentrations respectively to obtain first fiber membrane media at different oxalic acid concentrations; Excavating a soil profile and placing it into a soil root culture device for in-situ cultivation of root plants, or cultivating plants using a soil root culture device indoors with the original soil; After culturing for a period of time, covering the rhizosphere soil with another fiber membrane medium soaked with the substrate solution and continuing in-situ cultivation; after the oxalic acid in the rhizosphere soil weakens the fluorescence of the substrate solution in the other fiber membrane medium, taking the other fiber membrane medium as the second fiber membrane medium; Moving the second fiber membrane medium into a reaction darkroom; irradiating the second fiber membrane medium with ultraviolet light using an ultraviolet light source in the reaction darkroom; taking a fluorescence imaging photo of the second fiber membrane medium using a camera in the reaction darkroom; performing fluorescence spectral imaging on each first fiber membrane medium using the same operation; Using the imaging photos of each first fiber membrane medium to fit a linear regression equation where the imaging gray level decreases as the oxalic acid concentration increases; Substituting the gray level values of each pixel in the imaging photo of the second fiber membrane medium into the linear regression equation respectively to generate a two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil; Wherein, when performing fluorescence spectral imaging, the camera takes photos of the same fiber membrane medium from different angles and different distances; among them, the fluorescence imaging photos taken by the camera of the same second fiber membrane medium from different angles and different distances together constitute a denoising sample; Training a neural network model using multiple denoising samples so that the trained neural network model can denoise each fluorescence imaging photo; wherein, each combination of shooting angle and shooting distance in the neural network model corresponds to a pair of encoder and decoder, the encoder and decoder are inverse operations to each other, the encoder is used to extract the depth features of the fluorescence imaging photo under the corresponding combination, and the decoder is used to restore the fluorescence imaging photo according to the depth features; during the training process, the depth features of the fluorescence imaging photos under different combinations in the same denoising sample are constrained to be consistent, and the difference between each restored image and each fluorescence imaging photo is minimized to achieve denoising.

2. The method according to claim 1, wherein The step of preparing a fluorescent substrate solution using sulfuric acid, rhodamine, and potassium dichromate / potassium bromate comprises: Adding 1.12 mL of concentrated sulfuric acid to 300 mL of ultrapure water, continuously stirring, and cooling to room temperature; Adding 134 mg of rhodamine B or rhodamine 6G, stirring to dissolve, and then adding 353 mg of potassium dichromate or 1002 mg of potassium bromate, stirring to dissolve; Making up the volume to 500 mL with ultrapure water in a volumetric flask to prepare the substrate solution.

3. The method according to claim 1, wherein The step of mixing oxalic acid with different amounts of the substrate solution to obtain fluorescent reference materials with different oxalic acid concentrations comprises: Weigh 100 mg of oxalic acid, add 40 mL of the substrate solution, stir to dissolve, and then make up the volume to 100 mL in a volumetric flask or colorimetric tube with the substrate solution to prepare a standard stock solution of 1000 mg / L. Respectively pipette 12.5 mL, 7.5 mL, 5 mL, 2.5 mL, 1.5 mL, 1.0 mL, 0.25 mL and 0 mL of the stock solution into 25-mL volumetric flasks or colorimetric tubes, and make up the volume to 25 mL with the substrate solution to prepare fluorescent standard substances with oxalic acid concentrations of 500 mg / L, 300 mg / L, 200 mg / L, 100 mg / L, 60 mg / L, 40 mg / L, 10 mg / L and 0 mg / L respectively.

4. The method according to claim 1, wherein The soil root culture device includes a root box, and a movable baffle is arranged on the side of the root box, and another movable baffle is placed obliquely at the bottom of the root box. After culturing for a period of time, cover the rhizosphere soil with another fiber membrane medium soaked in the substrate solution, and continue in-situ culture, including: After culturing for a period of time, take out the two movable baffles respectively, and place the other two fiber membrane media soaked in the substrate solution on the two movable baffles respectively. Put the two movable baffles with the fiber membrane media on them back to the side and bottom of the root box respectively, so that the other two fiber membrane media are respectively attached to the side and bottom of the rhizosphere soil, wherein the other two fiber membrane media are respectively used to generate the two-dimensional spatial distribution of the oxalic acid concentration of the root longitudinal section and the bottom section.

5. The method according to claim 1, wherein The relative positions of each first fiber medium with respect to the ultraviolet light source and the camera in the reaction darkroom are the same as the relative positions of the second fiber membrane medium with respect to the ultraviolet light source and the camera in the reaction darkroom.

6. The method according to claim 1, wherein The method of fitting a linear regression equation in which the imaging gray level decreases with the increase of the oxalic acid concentration by using the imaging photos of each first fiber membrane medium includes: Convert the color pixel values of each imaging photo into gray level values. Taking the gray average value of the imaging photos of each first fiber membrane medium and the corresponding oxalic acid concentration as samples, fitting the imaging gray scale Gray s and the oxalic acid concentration C s for the linear regression equation between them: Gray s = Intercept + Slope * C s Among them, Intercept represents the intercept, and Slope represents the slope less than 0.

7. The method according to claim 1, wherein The method of substituting the gray level values of each pixel in the imaging photo of the second fiber membrane medium into the linear regression equation to generate the two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil includes: Convert the color pixel values at each pixel position in the imaging photo of the second fiber membrane medium into the gray level values at each pixel position respectively. Substitute the gray level values at each pixel position into the linear regression equation to obtain the oxalic acid concentration at each pixel position. Arrange the oxalic acid concentrations according to the pixel positions to obtain the two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil.

8. The method according to claim 1, wherein After generating the two-dimensional spatial distribution of the oxalic acid concentration in the rhizosphere soil, it further includes: Normalize the oxalic acid concentration in the two-dimensional spatial distribution to the image gray level space. Perform color rendering on the normalized two-dimensional data to realize the visual representation of the spatial distribution of the oxalic acid concentration in the rhizosphere soil.

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