Method for detecting activity of earthworm enzyme in alkaline soil and differential application

Abstract

The present application relates to the technical field of soil improvement, and particularly relates to a method for detecting activity of earthworm enzyme in alkaline soil and differentiating application, comprising: obtaining hardness and oxygen content data of soil at different depths; monitoring enzyme activity data after application of earthworm enzyme, determining attenuation coefficient and active attenuation half-life; calculating correlation factors of attenuation coefficient, hardness and oxygen content, determining hardness mutation depth range and oxygen content mutation depth range; determining interaction coefficient according to the mutation depth range, and determining the action depth range based on the interaction coefficient, calculating the active retention time, and determining the action depth; applying earthworm enzyme by using the fertilization mode dominated by hardness or oxygen content according to the location of the action depth. The present application detects the activity attenuation law of earthworm enzyme in alkaline soil, identifies the dominant limiting factor at different depths and adopts a differentiated fertilization strategy, thereby significantly improving the utilization efficiency of earthworm enzyme and the soil improvement effect.

Description

Technical Field

[0001] This invention relates to the field of soil improvement technology, and in particular to a method for detecting and differentially applying earthworm enzyme activity in alkaline soil. Background Technology

[0002] Globally, soil salinization has become a major threat to agricultural production, food security, and sustainable development in arid and semi-arid regions. Statistics show that, to date, due to topographical and anthropogenic factors, over 424 million hectares of topsoil (0–30 cm depth) and 833 million hectares of subsoil (30–100 cm depth) have been affected by salinization. High concentrations of sodium ions disrupt soil aggregate structure, reduce soil microbial activity, and consequently significantly decrease soil water retention capacity and nutrient availability, severely negatively impacting the productivity of crops in saline-alkali soils. In Northwest my country, where soils are predominantly high in salinity and alkalinity, adopting environmentally friendly, efficient, and sustainable methods to improve soil properties is particularly important.

[0003] Earthworm enzyme, also known as lumbrokinase, is an active protease extracted from earthworms. It hydrolyzes fixed nutrients such as phosphorus, calcium, and magnesium in the soil, loosens the soil, and improves soil aggregate structure. Studies have shown that earthworm enzyme can activate soil enzyme activity, promote the reproduction and stability of beneficial microorganisms, reduce the content of heavy metals and toxins in the soil, and increase fertilizer utilization efficiency by more than 20%. Earthworm enzyme also shows promising application prospects in the improvement of saline-alkali land, effectively reducing soil pH and salinity, and promoting crop growth.

[0004] Chinese Patent Publication No. CN121379598A discloses a soil conditioner containing earthworm enzymatic hydrolysate and its preparation method. The soil conditioner comprises the following components: earthworm enzymatic hydrolysate microcapsules and auxiliary agent particles in a mass ratio of (2-4):1; the earthworm enzymatic hydrolysate is obtained by enzymatic hydrolysis of fresh earthworm homogenate with a complex enzyme, the complex enzyme including cellulase and protease; the wall material of the earthworm enzymatic hydrolysate microcapsules includes at least one selected from chitosan, calcium alginate, gelatin, and gum arabic; the auxiliary agent includes humic acid and inorganic natural minerals. The soil conditioner containing earthworm enzymatic hydrolysate, by encapsulating the earthworm enzymatic hydrolysate in microcapsules, can significantly improve the retention rate of active ingredients in the earthworm enzymatic hydrolysate. Combined with humic acid and inorganic natural minerals, it can further improve soil conditions.

[0005] In academic research, numerous studies have revealed the patterns of soil enzyme activity variation with depth. Research indicates that soil enzyme activity decreases sequentially from the surface to the deeper layers. For example, in forest soils, the activities of urease, β-glucosidase, cellulase, arylsulfatase, acid phosphatase, and alkaline phosphatase are highest in the surface layer, decreasing by an average of 50%, 81%, 71%, 71%, 59%, and 66% with increasing soil depth, respectively. In karst forest soils, the activities of alkaline phosphatase, polyphenol oxidase, and protease also decrease with increasing soil depth. Furthermore, studies have found a significant positive correlation between soil organic carbon and urease, alkaline phosphatase, and sucrase. These studies only reveal the objective laws governing the decline of enzyme activity with depth, but they all stop at describing these laws and have not translated them into engineered detection methods and fertilization decision-making schemes.

[0006] Therefore, the following problems exist in the existing technology: First, the focus is on product formulation, lacking application decision-making. Existing technologies mainly concentrate on compound products of earthworm enzymes with humic acid and inorganic natural minerals, using earthworm enzymes as a fixed component mixed into soil conditioners for one-time application, without addressing post-application activity testing and application decisions. Although microencapsulation technology is used to encapsulate earthworm enzyme hydrolysates to improve the retention rate of active ingredients, its purpose is to ensure product storage stability, not to test activity attenuation after application to the soil, and it does not correlate microencapsulation technology with environmental factors such as soil hardness and oxygen content.

[0007] Second, although there is an objective law that enzyme activity decreases with depth, no correlation model has been established between the decay coefficient and soil hardness and oxygen content, and no engineering method has been formed to guide differentiated fertilization.

[0008] Third, the application method is singular and lacks a differentiated strategy. Existing technologies use a one-time application method with a uniform dosage and depth, without considering the differentiated effects of environmental factors such as soil hardness and oxygen content at different depths on earthworm enzyme activity, resulting in low utilization rates of enzyme preparations in deep soil.

[0009] Fourth, the dominant influencing factors and their interactions have not been identified. Existing technologies have not analyzed whether hardness or oxygen content has a greater impact on earthworm enzyme activity at different depths, lack methods for judging and identifying the effects of hardness and oxygen content, and have not analyzed the interactive effects of the two on earthworm enzyme activity when they work together.

[0010] Therefore, there is an urgent need for a method that can systematically detect the activity decay patterns of earthworm enzymes at different depths and time points in alkaline soils, identify the degree of influence and interaction of hardness and oxygen content at different depths, and adopt differentiated fertilization strategies based on the detection results, so as to improve the utilization efficiency of earthworm enzymes and the soil improvement effect. Summary of the Invention

[0011] Therefore, this invention provides a method for detecting and differentially applying earthworm enzyme activity in alkaline soil, in order to overcome the problems of existing technologies that use a single method for earthworm enzyme application and do not consider the effects of soil depth, hardness and oxygen content on enzyme activity.

[0012] To achieve the above objectives, this invention provides a method for detecting and differentially applying earthworm enzyme activity in alkaline soil, comprising: Step S1: Obtain hardness and oxygen content data of soil samples at different depths of the target alkaline soil; Step S2: Apply earthworm enzyme preparation to each soil sample. At several time points after the application of earthworm enzyme preparation, monitor the enzyme activity data of soil samples at each depth to determine the decay coefficient and activity decay half-life of earthworm enzyme at the corresponding depth. Step S3: Calculate the attenuation coefficient at each depth and the hardness correlation factor corresponding to several hardness data, and the attenuation coefficient at each depth and the oxygen content correlation factor corresponding to several oxygen content data. Determine the hardness mutation depth range based on each hardness correlation factor, and determine the oxygen content mutation depth range based on each oxygen content correlation factor. Step S4: Determine the interaction coefficient based on the hardness mutation depth range and the oxygen content mutation depth range; Step S5: Determine the range of action depth based on the interaction coefficient, calculate the activity retention time at each depth within the range of action depth according to the activity decay half-life and decay coefficient, and determine the action depth based on the activity retention time.

[0013] Furthermore, the steps for determining the decay coefficient and activity decay half-life of earthworm enzymes at the corresponding depth include: Obtain initial values ​​of enzyme activity data for soil samples at any depth at the initial moment of application of earthworm enzyme preparation; Enzyme activity data in the soil were collected at several time points in a time sequence and compared with the initial values ​​of enzyme activity data. The activity decay half-life is determined based on the time required for enzyme activity in the soil to decay to 50% of its initial value. Based on the same time point, enzyme activity data of soil samples at different depths are determined, and the decay coefficient is determined based on the average decrease rate of enzyme activity data within a unit depth.

[0014] Further, in step S3, the steps of determining the hardness mutation depth range based on each hardness-related factor and determining the oxygen content mutation depth range based on each oxygen content-related factor include: The range of hardness mutation depth is determined based on the depth range corresponding to the first time that the hardness-related factor exceeds the preset hardness threshold; The depth range of oxygen content mutation is determined based on the depth range corresponding to the first time that oxygen-related factors exceed a preset oxygen content threshold.

[0015] Further, in step S4, determining the interaction coefficient based on the hardness abrupt change depth range and the oxygen content abrupt change depth range includes: Within the range of hardness mutation depth, a first representative depth is determined for oxygenation treatment. The residual enzyme activity data after oxygenation treatment is measured and recorded as the first residual data. The first rate of change is calculated based on the first residual data and the enzyme activity data corresponding to the first representative depth in step S2. A second representative depth is determined within the range of oxygen content mutation depth for hardening treatment. The residual enzyme activity data after hardening treatment is measured and recorded as the second residual data. The second rate of change is calculated based on the second residual data and the enzyme activity data corresponding to the second representative depth in step S2. The interaction coefficient is determined based on the absolute value of the ratio of the first rate of change to the second rate of change.

[0016] Further, in step S4, the step of determining the first representative depth and the second representative depth includes: The first representative depth is determined based on the median depth of the hardness abrupt change depth range; The second representative depth is determined based on the median depth of the oxygen content mutation depth range.

[0017] Furthermore, the oxygenation treatment includes injecting a first effective amount of calcium peroxide into a first representative depth, and the hardening treatment includes injecting a second effective amount of clay slurry into a second representative depth.

[0018] Furthermore, the determination of the interaction depth range based on the interaction coefficient in step S5 includes: If the interaction coefficient is greater than or equal to the reference value, then the oxygen content mutation depth range is used as the reference action depth range, and the reference action depth range is extended in combination with the hardness mutation depth range direction to determine the action depth range. If the interaction coefficient is less than the reference value, the range of hardness change depth is used as the range of action depth, and the reference range of action depth is extended in the direction of oxygen content change depth to determine the range of action depth.

[0019] Further, determining the depth of action includes: Several alternative points of action are determined at different depths within the range of the stated depth of action. The activity retention time of each candidate site of action is determined based on the activity decay half-life and decay coefficient. The depth of action is determined based on the minimum duration of activity retention for each candidate site of action.

[0020] Further, the step of determining the duration of activity retention includes: The duration of activity retention is determined based on the activity decay half-life at the depth of each candidate action site, the decay coefficient at the corresponding depth, and the interaction coefficient.

[0021] Furthermore, it also includes step S6, applying earthworm enzymes according to the location of the depth of action, using a hardness-dominant fertilization method, an oxygen content-dominant fertilization method, or a combined fertilization method. The hardness-dominant fertilization method involves deep injection of earthworm enzyme preparations to the depth of action. The oxygen content-driven fertilization method involves implementing oxygenation measures at the specified depth of application.

[0022] Compared with the prior art, the beneficial effects of the present invention are that by detecting the activity decay law of earthworm enzyme in alkaline soil, analyzing the correlation and interaction between the decay coefficient and soil hardness and oxygen content, the present invention can accurately determine the optimal application depth and adopt differentiated fertilization strategies, thereby overcoming the shortcomings of the prior art in that the application method is singular and does not consider the influence of depth and environmental factors, thus improving the utilization efficiency of earthworm enzyme and the soil improvement effect.

[0023] Furthermore, by monitoring the residual activity data of earthworm enzymes at different time points and depths, the decay coefficient and activity decay half-life of earthworm enzymes at the corresponding depths were determined. This systematically detected the activity decay pattern of earthworm enzymes after they were applied to the soil, overcoming the shortcomings of existing technologies that only focus on product formulations and do not involve activity decay detection, and providing a data basis for subsequent differentiated fertilization.

[0024] Furthermore, by calculating the attenuation coefficient at each depth and the hardness correlation factor of the hardness data, as well as the oxygen correlation factor of the oxygen content data, the depth range of hardness mutation and oxygen content mutation is determined. This transforms the objective law of enzyme activity attenuation with depth in academic research into quantifiable engineering parameters, overcoming the deficiency of existing technologies that have not established a correlation model between the attenuation coefficient and soil hardness and oxygen content, and providing a basis for identifying the dominant limiting factors at different depths.

[0025] Furthermore, by calculating the first rate of change through oxygenation treatment within the range of hardness mutation depth and the second rate of change through hardening treatment within the range of oxygen content mutation depth, the interaction coefficient is obtained by dividing the absolute value of the first rate of change by the second rate of change. This quantifies the degree of interaction between hardness and oxygen content on earthworm enzyme activity, overcoming the deficiency of existing technologies that do not analyze the interaction when the two work together, and providing a corrective basis for determining the range of action depth.

[0026] Furthermore, by determining the range of action depth based on the interaction coefficient, calculating the activity retention time at each depth within the action depth range, and selecting the depth with the shortest activity retention time as the action depth, the optimal location for earthworm enzyme application can be precisely located. This overcomes the shortcomings of existing technologies that require uniform depth and dosage application, and provides a targeted location for differentiated fertilization.

[0027] Furthermore, by applying earthworm enzymes according to the depth of application, either hardness-based or oxygen-based fertilization methods are used, thus achieving differentiated and precise fertilization targeting the dominant limiting factors at different depths. This overcomes the shortcomings of existing technologies with their single application methods and further improves the utilization efficiency of earthworm enzymes and the soil improvement effect. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to an embodiment of the present invention. Figure 2 This is a flowchart of step S2 of the method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to an embodiment of the present invention; Figure 3 This is a flowchart of step S3 of the method for detecting and differentially applying earthworm enzyme in alkaline soil according to an embodiment of the present invention; Figure 4 This is a flowchart of step S4 of the method for detecting and differentially applying earthworm enzyme in alkaline soil according to an embodiment of the present invention. Detailed Implementation

[0029] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0030] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0031] In this embodiment, for illustrative purposes, the conventional procedures such as soil sample collection, air drying, sieving, and soil column filling are all performed according to known process parameters and equipment conditions in the art, and will not be described in detail here. In this embodiment, soil columns of the target alkaline soil have been collected, and earthworm enzyme application effect data are obtained through the method of this embodiment. After verification, the data are then applied to field fertilization.

[0032] Please see Figure 1 The diagram shows a flowchart of the method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to an embodiment of the present invention. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to this embodiment includes: Step S1: Obtain hardness and oxygen content data of soil samples at different depths of the target alkaline soil; Step S2: Apply earthworm enzyme preparation to each soil sample. At several time points after the application of earthworm enzyme preparation, monitor the enzyme activity data of soil samples at each depth to determine the decay coefficient and activity decay half-life of earthworm enzyme at the corresponding depth. Step S3: Calculate the attenuation coefficient at each depth and the hardness correlation factor corresponding to several hardness data, and the attenuation coefficient at each depth and the oxygen content correlation factor corresponding to several oxygen content data. Determine the hardness mutation depth range based on each hardness correlation factor, and determine the oxygen content mutation depth range based on each oxygen content correlation factor. Step S4: Determine the interaction coefficient based on the hardness mutation depth range and the oxygen content mutation depth range; Step S5: Determine the range of action depth based on the interaction coefficient, calculate the activity retention time at each depth within the range of action depth according to the activity decay half-life and decay coefficient, and determine the action depth based on the activity retention time.

[0033] Step S6: Apply earthworm enzyme preparations according to the location of the depth of action, using either a hardness-based fertilization method or an oxygen-based fertilization method. The hardness-dominant fertilization method involves deep injection of earthworm enzyme preparations to the depth of action. The oxygen content-driven fertilization method involves implementing oxygenation measures at the specified depth of application.

[0034] Specifically, in step S1, hardness data and oxygen content data of soil samples at different depths of the target alkaline soil are obtained.

[0035] In this embodiment, hardness data is measured using a soil hardness meter, and oxygen content data is measured using a soil oxygen content meter. The test results will serve as the input for determining the mutation depth range in step S3. The range of soil samples at different depths should at least cover the planting depth range of crops grown in the target alkaline soil, that is, the depth range from the soil surface to the extension of the crop root system. By measuring the oxygen content and hardness of soil samples at each depth, soil quality data of the target alkaline soil is obtained to accurately match the action site of earthworm enzymes and leverage the soil-improving effect of earthworm enzyme preparations to improve soil quality.

[0036] Alternatively, hardness data can be measured using an electronic soil hardness tester or a handheld penetrant, and oxygen content data can be measured using gas chromatography or the oxygen electrode method. These are all conventional techniques in the field and will not be elaborated upon here.

[0037] Please see Figure 2As shown, it is a flowchart of step S2 of the method for detecting and differentially applying earthworm enzyme in alkaline soil according to an embodiment of the present invention.

[0038] Specifically, the steps for determining the decay coefficient and activity decay half-life of earthworm enzymes at a corresponding depth include: Step S21: Apply the same dose of earthworm enzyme preparation to the sampling depth corresponding to each soil sample depth, record the time after the earthworm enzyme preparation is applied as the start time, collect each soil sample at several preset time points, and measure the residual enzyme activity concentration at each time point. Specifically, the steps for applying earthworm enzyme preparations include: The sampling depths corresponding to soil samples at each depth from which hardness and oxygen content data were obtained were determined as the corresponding application depths. Soil samples at each depth were placed in sealed containers. The soil moisture content was kept constant by supplementing with humid air, thereby maintaining its natural hardness conditions. The natural oxygen content conditions were maintained by periodically supplementing with oxygen or introducing oxygen-containing gas. After the hardness and oxygen content of soil samples at each depth have stabilized, the same dose of earthworm enzyme preparation is applied at each application depth.

[0039] It is understandable that maintaining soil samples at each depth under their natural hardness and oxygen content before applying earthworm enzyme preparations ensures that subsequent differences in enzyme activity decay only reflect the inherent differences in soil environmental factors at different depths. Applying the same dose of earthworm enzyme preparations to each depth ensures that the initial enzyme activity of soil samples at each depth is consistent at the beginning, thereby eliminating the influence of dosage differences on enzyme activity decay and making subsequent correlation analysis more accurate.

[0040] Step S22: Based on the enzyme activity residual concentration at each time point, plot the first decay curve of the activity residual rate at each depth as a function of time, and read the time corresponding to the activity residual rate decaying to 50% from the first decay curve as the activity decay half-life at that depth. Understandably, the first decay curve is a curve constructed with time on the x-axis and residual activity rate on the y-axis, used to determine the decay pattern of earthworm enzyme activity over time at the same depth. For each depth, the enzyme activity at the initial moment is used as the initial value, and the residual activity rate at each time point is calculated. Connecting these points gives the first decay curve for that depth. By constructing the first decay curve, discrete enzyme activity measurement data can be made continuous and visualized, intuitively reflecting the rate of decay of earthworm enzyme in the soil at the corresponding depth. The time corresponding to the residual activity rate decaying to 50% from this curve is the activity decay half-life. Using 50% as the criterion for determining the decay half-life is because in the first-order exponential decay process, the half-life is inversely proportional to the decay rate constant. This value is not affected by the initial concentration, facilitating horizontal comparisons between different depths. The activity decay half-life is the core indicator characterizing the rate of decay of earthworm enzyme in the soil at the corresponding depth. The shorter the half-life, the faster the enzyme activity decays at that depth, requiring more frequent replenishment.

[0041] Step S23: At the same time point, obtain the residual enzyme activity concentration at different depths and plot the second decay curve of the residual activity rate as a function of depth. Step S24: Fit the second attenuation curve to obtain a fitted curve, and calculate the slope of the fitted curve as the attenuation coefficient for that depth.

[0042] Understandably, the second decay curve is constructed with depth on the x-axis and residual activity rate on the y-axis, used to characterize the decay pattern of earthworm enzyme activity with depth at the same time point. The enzyme activity at the sampling depth closest to the application surface is used as the baseline value, as this depth is relatively less affected by diffusion distance and decay rate, and best represents the initial activity level. Setting the activity at this depth to 100%, the relative residual activity rate at each depth is calculated, and connecting these points yields the second decay curve, which is used to make the enzyme activity data at different depths continuous and visual. Fitting the second decay curve can eliminate measurement errors and obtain a more accurate decay pattern. The slope of the fitted curve is the decay coefficient, which characterizes the average rate of decrease in earthworm enzyme activity per unit depth with increasing depth. The larger the decay coefficient, the more significant the effect of depth on enzyme activity decay. It should be noted that the time point for plotting the second decay curve is selected when the activity differences between depths are fully apparent, while the enzyme activity is still within the detectable range.

[0043] Understandably, according to the principles of enzyme reaction kinetics, the decay of enzyme activity in soil follows a first-order exponential decay law, and the decay rate constant is closely related to soil environmental factors such as hardness, oxygen content, and pH. In alkaline soils, hardness and oxygen content are two key environmental factors affecting earthworm enzyme activity. Higher hardness means denser soil particles, stronger binding of enzyme molecules to soil particles, and a greater degree of obscuring of active sites; lower oxygen content means weaker microbial activity and faster oxidative inactivation of enzyme molecules. The half-life and decay coefficient obtained through the above steps can quantify the influence of hardness and oxygen content on earthworm enzyme activity, providing a basis for identifying the dominant limiting factors at different depths in subsequent step S3.

[0044] In this embodiment, enzyme activity data were determined using the fluorescein diacetate hydrolysis method. As a preferred embodiment, sampling time points were set at day 1, day 3, day 7, day 14, day 21, and day 28 to cover the main decay phase of earthworm enzyme activity, with the initial enzyme activity measured on day 1 as the baseline.

[0045] Specifically, please refer to Figure 3 As shown, it is a flowchart of step S3 of the method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to an embodiment of the present invention. The steps for determining the hardness mutation depth range based on various hardness-related factors and the oxygen content mutation depth range based on various oxygen content-related factors include: The range of hardness mutation depth is determined based on the depth range corresponding to the first time that the hardness-related factor exceeds the preset hardness threshold; The depth range of the oxygen content mutation is determined based on the depth range corresponding to the first time that the oxygen-related factors exceed the preset oxygen content threshold. Among them, the hardness correlation factor is the correlation coefficient between the attenuation coefficient and the hardness data, and the oxygen content correlation factor is the correlation coefficient between the attenuation coefficient and the oxygen content data, which are used to quantify the degree of influence of hardness and oxygen content on the attenuation of earthworm enzyme activity.

[0046] Understandably, a higher absolute value of the correlation coefficient indicates a stronger correlation between the two variables; conversely, a lower absolute value indicates a weaker correlation. When the absolute value of the correlation coefficient first exceeds a corresponding preset threshold, it indicates that the factor begins to play a dominant role in the attenuation of earthworm enzyme activity, and this depth range is defined as the mutation depth range. In this embodiment, both the preset hardness threshold and the preset oxygen content threshold are determined based on the statistical standard for determining strong correlation. When the absolute value of the correlation coefficient is greater than this threshold, it indicates a strong correlation between the two variables.

[0047] Alternatively, the correlation coefficient can be calculated using Spearman's rank correlation coefficient or Kendall's rank correlation coefficient, and the range of mutation depth can be determined using the sliding window method or piecewise regression method.

[0048] Specifically, in step S4, the interaction coefficient is determined based on the hardness abrupt change depth range and the oxygen content abrupt change depth range, including: Specifically, the interaction coefficients are determined based on the hardness abrupt change depth range and the oxygen content abrupt change depth range, including: Step S41: Determine the first representative depth within the range of hardness abrupt change depth and perform oxygenation treatment; Step S42: Measure the residual data of earthworm enzyme activity after oxygenation treatment and record it as the first residual data. Step S43: Calculate the first rate of change based on the first residual data and the first enzyme activity data representing the first depth in step S2; Step S44: Determine the second representative depth within the range of oxygen content mutation depth and perform hardening treatment; Step S45: Measure the residual data of earthworm enzyme activity after hardening treatment and record it as the second residual data; Step S46: Calculate the second rate of change based on the second residual data and the enzyme activity data representing the second depth in step S2; Step S47: Determine the interaction coefficient based on the ratio of the absolute values ​​of the first rate of change and the second rate of change; Understandably, the interaction coefficient is used to quantify the degree of interaction between hardness and oxygen content on earthworm enzyme activity. The first rate of change reflects the effect of oxygenation treatment on earthworm enzyme activity, i.e., the degree of influence of the oxygen content limiting factor. The larger the first rate of change, the stronger the limiting effect of oxygen content on enzyme activity, and the more significant the increase in enzyme activity after improving oxygen content. The second rate of change reflects the effect of hardening treatment on earthworm enzyme activity, i.e., the degree of influence of hardness limiting factor. The larger the second rate of change, the stronger the limiting effect of hardness on enzyme activity, and the more significant the increase in enzyme activity after improving hardness. Taking the ratio of the first rate of change to the second rate of change as the interaction coefficient can eliminate the influence of the difference in enzyme activity itself on the absolute value of the rate of change, and obtain the relative magnitude of the influence of the two factors on enzyme activity. Through the interaction coefficient, the degree of interaction between two environmental factors on earthworm enzyme activity can be quantified, thereby identifying the dominant limiting factors in different depth ranges, and providing a basis for the subsequent formulation of differentiated fertilization strategies.

[0049] In this embodiment, the first representative depth is the median depth of the hardness mutation depth range, and the second representative depth is the median depth of the oxygen content mutation depth range. Alternatively, the first representative depth can also be selected from the depth corresponding to the peak value of the hardness-related factor, and the second representative depth can also be selected from the depth corresponding to the peak value of the oxygen content-related factor. The oxygenation treatment is achieved by injecting a first effective amount of calcium peroxide into the first representative depth, and the hardening treatment is achieved by injecting a second effective amount of clay slurry into the second representative depth. The first effective amount is the amount of calcium peroxide required to increase the oxygen content at the corresponding first representative depth to a preset standard oxygen content value, which is determined by measuring the oxygen content at the corresponding depth of normal soil in the region. The second effective amount is the amount of clay slurry required to increase the hardness at the corresponding second representative depth to a preset standard hardness value, which is determined by measuring the hardness at the corresponding depth of normal soil in the region.

[0050] Alternatively, oxygenation treatment can be achieved by injecting magnesium peroxide solution or introducing oxygen-enriched air, and hardening treatment can be achieved by injecting bentonite slurry or high-pressure grouting.

[0051] Specifically, in step S5, the step of determining the range of interaction depth based on the interaction coefficient includes: If the interaction coefficient is greater than or equal to the reference value, the oxygen content mutation depth range is used as the reference action depth range, and the reference action depth range is extended in combination with the hardness mutation depth range direction to determine the action depth range; if the interaction coefficient is less than the reference value, the hardness mutation depth range is used as the reference action depth range, and the reference action depth range is extended in combination with the oxygen content mutation depth range direction to determine the action depth range, wherein the reference value is the critical value when the oxygen content limiting effect and the hardness limiting effect are equal.

[0052] Understandably, the interaction coefficient is used to determine the relative limiting effect of hardness and oxygen content on earthworm enzyme activity. When the interaction coefficient is greater than or equal to the baseline value, it indicates that the limiting effect of oxygen content is stronger than or equal to that of hardness. Therefore, the range of oxygen content mutation depths is taken as the main range of action, and it is extended towards the range of hardness mutation depths to ensure coverage of the area jointly affected by both factors. When the interaction coefficient is less than the baseline value, it indicates that the limiting effect of hardness is stronger than that of oxygen content. Therefore, the range of hardness mutation depths is taken as the main range of action, and it is extended towards the range of oxygen content mutation depths.

[0053] Specifically, the steps for determining the depth of action include: Several alternative points of action are determined at different depths within the range of the stated depth of action. The activity retention time of each candidate site of action is determined based on the activity decay half-life and decay coefficient. The depth of action is determined based on the minimum duration of activity retention for each candidate site of action.

[0054] Understandably, the activity retention time is the duration for which earthworm enzymes can maintain effective activity at a corresponding depth. It is determined based on the activity decay half-life, decay coefficient, and interaction coefficient at each depth within the effective depth range. A shorter activity retention time indicates faster enzyme activity decay at that depth, requiring more focused intervention. The effective depth is the depth with the shortest activity retention time, i.e., the depth where earthworm enzyme activity decays most rapidly, and this depth is used as the target location for differentiated fertilization.

[0055] In this embodiment, the activity retention time is the ratio of the activity decay half-life at each depth to the decay coefficient at the corresponding depth, multiplied by the reciprocal of the interaction coefficient. The ratio of the activity decay half-life to the decay coefficient reflects the decay effect of depth on enzyme activity; multiplying by the reciprocal of the interaction coefficient introduces the interaction effect of hardness and oxygen content on the activity retention time. When the interaction coefficient is greater than the baseline value, the reciprocal of the interaction coefficient is less than 1, and the activity retention time is shortened, indicating that the oxygen content limiting factor has a greater influence. When the interaction coefficient is less than the baseline value, the reciprocal of the interaction coefficient is greater than 1, and the activity retention time is extended, indicating that the hardness limiting factor has a greater influence.

[0056] Alternatively, the duration of activity retention can also be calculated using a weighted summation method or a nonlinear combination method.

[0057] Specifically, in step S6, depending on the location of the depth of action, either a hardness-based fertilization method or an oxygen content-based fertilization method is adopted. The hardness-dominant fertilization method involves deep injection of earthworm enzyme preparations to the depth of action. The oxygen content-driven fertilization method involves implementing oxygenation measures at the specified depth of application.

[0058] Understandably, the core of hardness-driven fertilization lies in overcoming physical barriers. When the application depth is within the range of abrupt changes in hardness, the dense particles in high-hardness soil increase the diffusion resistance of enzyme molecules, making it difficult for conventional application rates to deliver sufficient earthworm enzymes to the desired depth. Therefore, deep injection bypasses the high-hardness surface layer and delivers the enzymes directly to the desired depth, ensuring fertilization effectiveness.

[0059] The core of oxygen-based fertilization lies in compensating for chemical decay. When the fertilization depth falls within the range of abrupt changes in oxygen content, the anaerobic environment causes earthworm enzyme activity to decay rapidly, making it difficult to maintain enzyme activity at the fertilization depth for an extended period. Therefore, oxygenation measures can be applied at the fertilization depth to improve the oxygen content environment, slow down the rate of enzyme activity decay, and prolong the effective fertilization time.

[0060] Alternatively, oxygenation measures can be implemented by introducing oxygen-enriched air or adding oxygen-releasing materials, and deep injection methods can be implemented by burying slow-release devices or high-pressure grouting.

[0061] Example 1:

[0062] This embodiment uses a typical saline-alkali farmland soil in Northwest China as the test object, and conducts earthworm enzyme activity detection and differentiated application according to the above method. The earthworm enzyme preparation used in this embodiment is a commercially available product, specifically a soil conditioner containing lumbrokinase active ingredients, wherein the earthworm enzyme content, calculated as lumbrokinase, is 0.3% to 1.0% of the total mass of the preparation. Alternatively, other commercially available products containing earthworm protease active ingredients can be used, such as water-soluble fertilizers or earthworm enzyme compound soil conditioners, and the earthworm enzyme content should be based on the enzyme activity units or mass percentage indicated in the product instructions.

[0063] Step S1: Obtain hardness and oxygen content data of soil samples at different depths of the target alkaline soil.

[0064] Topsoil samples were collected from a typical saline-alkali farmland in Northwest China at depths ranging from 0 to 60 cm. After removing stones and plant debris, samples were taken from each 10 cm layer at depths of 0–10 cm, 10–20 cm, 20–30 cm, 30–40 cm, 40–50 cm, and 50–60 cm. The soil samples were then air-dried, ground, and sieved through a 2 mm sieve before use.

[0065] Soil column preparation: Soil columns were prepared using plexiglass columns with an inner diameter of 15 cm and a height of 70 cm. Soil samples from each depth were sequentially placed into the plexiglass column according to the bulk density and depth of the undisturbed soil. After each layer was filled, it was gently compacted until the bulk density of the undisturbed soil was 1.3 g / cm³. Layers were separated by gauze to maintain clear stratification. After filling, the total height of the soil column was 60 cm, simulating a field soil profile.

[0066] Hardness data measurement: A WY-2 needle-type soil hardness tester with a range of 0–5000 kPa and an accuracy of 10 kPa was used. The probe of the hardness tester was vertically inserted into the soil column at depths of 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, and 60 cm. The penetration resistance value was read, and the average value was taken for each depth. The hardness data measured at each depth in this embodiment are shown in Table 1.

[0067] Oxygen content data determination: An OX-09 soil oxygen meter with a range of 0–30% and an accuracy of 0.1% was used. The oxygen probe was inserted into the soil column at various depths, and the readings were recorded after stabilization. Measurements were taken three times at each depth, and the average value was taken. The instrument was calibrated at two points before measurement. The oxygen content data at each depth obtained in this embodiment are shown in Table 1.

[0068] Table 1. Soil hardness and oxygen content data at various depths

[0069] Step S2: Apply earthworm enzyme preparation to each soil sample. At several time points after the application of earthworm enzyme preparation, monitor the enzyme activity data of soil samples at each depth to determine the decay coefficient and activity decay half-life of earthworm enzyme at the corresponding depth.

[0070] Earthworm enzyme preparation was evenly sprayed into the soil at various depths using a sprayer, with a spray volume of 50 ml per column, equivalent to approximately 2 kg of effective enzyme per acre. The application time was recorded as time 0. The soil columns were placed in a constant temperature incubator set at 25℃. Soil moisture content was controlled by weighing to maintain it at approximately 70% of field capacity. The moisture content measurements of soil samples at various depths are shown in Table 2.

[0071] Table 2 Soil moisture content at different depths

[0072] On days 1, 3, 7, 14, 21, and 28 after application, soil samples were taken from depths of 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, and 60 cm from the soil column. A 1 cm diameter sampling drill was used for sampling, and three points were taken from each depth and mixed together to form a representative sample for that depth.

[0073] Soil enzyme activity was determined using the fluorescein diacetate hydrolysis method. The specific procedure was as follows: 5g of fresh soil sample was weighed, 15ml of 60mM, pH 7.6 phosphate buffer was added, and the mixture was shaken for 10min. Then, 0.2ml of fluorescein diacetate working solution was added, and the mixture was incubated at 30℃ with shaking for 2 hours. The reaction was terminated by adding 15ml of acetone. After filtration, the absorbance was measured at 490nm. Enzyme activity was calculated based on the fluorescein standard curve and corrected to the unit dry soil enzyme activity.

[0074] Based on enzyme activity data at various time points, decay curves of residual activity at each depth were plotted as a function of time. A first-order exponential decay model was used for fitting, and the decay rate constant k and the activity decay half-life were calculated. In this embodiment, the decay rate constant k and the active decay half-life at each depth are... As shown in Table 3.

[0075] Table 3. Decay rate constants and active decay half-lives at different depths

[0076] On day 7, enzyme activity data at different depths were acquired. Using the enzyme activity at a depth of 10 cm from the surface as a baseline, the relative activity residual rate at each depth was calculated, and then the average activity decrease rate per unit depth was calculated as the decay coefficient for that depth. The decay coefficients for each depth in this embodiment are shown in Table 4.

[0077] Table 4 Attenuation coefficients at different depths

[0078] Step S3: Calculate the attenuation coefficient at each depth and the hardness correlation factor corresponding to several hardness data, and the attenuation coefficient at each depth and the oxygen content correlation factor corresponding to several oxygen content data. Determine the hardness mutation depth range based on each hardness correlation factor, and determine the oxygen content mutation depth range based on each oxygen content correlation factor.

[0079] In this embodiment, both the preset hardness threshold and the preset oxygen content threshold are set to 0.7. A layer-by-layer progressive method is used, starting from the surface and gradually increasing the depth. The correlation coefficients of the attenuation coefficient and hardness data, as well as the correlation coefficients of the attenuation coefficient and oxygen content data, are calculated sequentially within the interval from the surface to the current depth point. The absolute values ​​of the hardness correlation factor |rh(hi)| and the absolute values ​​of the oxygen content correlation factor |ro(hi)| for each depth interval are shown in Table 5.

[0080] Table 5. Absolute values ​​of hardness-related factors and oxygen content-related factors for each depth range.

[0081] The calculated depth range where the hardness correlation factor first exceeds 0.7 is the surface layer to 40cm. By calculating the increment of the correlation coefficient between adjacent intervals, the abrupt change is determined to occur between 30 and 40cm. Therefore, the 30-40cm interval is defined as the depth range for hardness abrupt changes. The depth range where the oxygen content correlation factor first exceeds 0.7 is the surface layer to 50cm. By calculating the increment of the correlation coefficient between adjacent intervals, the abrupt change is determined to occur between 40 and 50cm. Therefore, the 40-50cm interval is defined as the depth range for oxygen content abrupt changes.

[0082] Step S4: Determine the interaction coefficient based on the hardness mutation depth range and the oxygen content mutation depth range.

[0083] Within the hardness abrupt change depth range of 30–40 cm, the median depth of 35 cm was selected as the first representative depth. A 5% calcium peroxide solution (30 ml) was injected into this depth for oxygenation treatment, raising the oxygen content to the preset standard oxygen content value of 16.5%. The preset standard oxygen content value was obtained by measuring the oxygen content at a depth of 35 cm in normal soil samples from the same area.

[0084] Within the oxygen content mutation depth range of 40cm to 50cm, a median depth of 45cm was selected as the second representative depth. Clay slurry was injected into this depth for hardening treatment, raising the hardness to a preset standard hardness value of 520kPa. The clay and water were mixed at a ratio of 1:2.5, and 8% cement by dry weight was added. The preset standard hardness value was obtained by measuring the hardness of normal soil samples from the same area at a depth of 45cm.

[0085] After the treated soil column was cultured at 25℃ for 7 days, the enzyme activity at depths of 35 cm and 45 cm was measured. The enzyme activity at each depth on day 28, obtained from step S2, was used as the baseline value before treatment, as shown in Table 6.

[0086] Table 6 Enzyme activities at different depths on day 28

[0087] In this embodiment, the enzyme activity at a depth of 35 cm after oxygenation treatment was 15.8 μg / g dry soil·h, and the enzyme activity at a depth of 45 cm after hardening treatment was 9.5 μg / g dry soil·h.

[0088] The first rate of change is calculated to be 28.5%, and the second rate of change is 9.2%. The absolute value of the ratio of the first rate of change to the second rate of change is used as the interaction coefficient, which is 3.1 in this embodiment.

[0089] Step S5: Determine the range of action depth based on the interaction coefficient, calculate the activity retention time at each depth within the range of action depth based on the activity decay half-life and decay coefficient, and determine the action depth based on the activity retention time.

[0090] In this embodiment, the interaction coefficient 3.1 ≥ the reference value 1. Therefore, the range of oxygen content mutation depth 40-50cm is taken as the action depth range, and it is extended in the direction of hardness mutation depth range, with the final action depth range being 40-50cm.

[0091] The activity retention time is determined based on the activity decay half-life at each depth, the decay coefficient at the corresponding depth, and the interaction coefficient. In this embodiment, the activity retention time is the ratio of the activity decay half-life at each depth to the decay coefficient at the corresponding depth, multiplied by the reciprocal of the interaction coefficient. The calculated activity retention time at each depth is shown in Table 7.

[0092] Table 7. Duration of activity retention at different depths within the oxygen-containing mutation depth.

[0093] The depth of action is the depth at which the activity is maintained for the shortest duration within the depth range of action; in this embodiment, it is 50 cm.

[0094] Step S6: Apply earthworm enzymes according to the location of the depth of action, using an oxygen-dominant fertilization method.

[0095] In this embodiment, the depth of action is 50cm, which is within the range of oxygen content mutation depth. Therefore, the oxygen content-driven fertilization method is adopted: at the same time as each application of earthworm enzyme, a 5% concentration and 30ml volume of calcium peroxide solution is injected into the depth of 50cm to maintain the oxygen content at this depth at a preset standard oxygen content value of 16.5% or higher.

[0096] Example 2:

[0097] This embodiment uses the same test plot and method as Embodiment 1. The difference from Embodiment 1 is that the soil properties are different in this embodiment; the initial soil hardness is lower and the oxygen content is moderate, resulting in an interaction coefficient of 0.65 < the baseline value of 1.

[0098] Steps S1 to S4 are the same as in Example 1, wherein the hardness mutation depth range determined in step S3 is 30cm to 40cm, and the oxygen content mutation depth range is 40cm to 50cm; the interaction coefficient calculated in step S4 is 0.65.

[0099] In step S5, since the interaction coefficient is <1, the range of hardness abrupt change depth (30cm–40cm) is used as the effective depth range, and this range is extended towards the oxygen content abrupt change depth range. The calculated activity retention time at each depth within the effective depth range is shown in Table 8. Table 8. Duration of activity retention at various depths within the hardness mutation depth range.

[0100] The depth of action is 40 cm, which is the depth at which the activity is maintained for the shortest time.

[0101] In step S6, the depth of action of 40cm is within the range of hardness mutation depth, so the hardness-dominant fertilization method is adopted: the earthworm enzyme preparation is applied to a depth of 40cm by deep injection, and the application interval remains unchanged at 14 days.

[0102] Comparative Example 1: Conventional fertilization methods were used, without mutation depth detection or differentiated application. The specific procedures are as follows: Spray a 0.5% earthworm enzyme solution (60 ml) evenly onto the surface of the soil column. Apply once every 14 days (day 0, day 14, and day 28), for a total of three applications. Do not perform oxygenation treatment, deep injection, or change the application frequency or dosage.

[0103] Comparative Example 2: This comparative study only considered stratified fertilization based on soil depth, without calculating hardness-related factors and oxygen content-related factors, and therefore did not determine the range of mutation depths. The soil depth was divided into three layers: 0cm–20cm, 20cm–40cm, and 40cm–60cm. At the median depth of each layer (10cm, 30cm, and 50cm), a 0.5% concentration earthworm enzyme solution (20ml / layer, totaling 60ml) was injected via perforation. This was done every 14 days (days 0, 14, and 28), for a total of three applications. No oxygenation treatment was performed.

[0104] The above Examples 1, 2, Comparative Example 1, and Comparative Example 2 were tested for the following indicators, and the results are shown in Table 9: (1) Earthworm enzyme utilization rate: the average value of the activity residual rate at each depth within the depth range where the activity residual rate is greater than 30%.

[0105] (2) Average hardness reduction rate: the average value of the hardness reduction at each depth before and after treatment.

[0106] (3) Average oxygen content increase rate: the average value of the increase in oxygen content at each depth before and after treatment.

[0107] (4) Increase in available phosphorus: The average value of the increase in available phosphorus content at each depth before and after treatment.

[0108] Table 9 Comparison of the improvement effects of different treatment methods

[0109] (1) Compared with Comparative Example 1, the earthworm enzyme utilization rates of Examples 1 and 2 increased by 35.9% and 39.7%, respectively, representing increases of 84.3% and 93.2%; the hardness reduction rate increased by 17.1% and 20.3%, respectively; the oxygen content increase rate increased by 22.4% and 15.9%, respectively; and the available phosphorus increase increased by 13.4 mg / kg and 17.2 mg / kg, respectively. This indicates that the present invention achieves differentiated and precise fertilization through mutation depth range identification and interaction coefficient calculation, significantly improving the utilization efficiency of earthworm enzymes and the soil improvement effect.

[0110] (2) Compared with Comparative Example 2, the earthworm enzyme utilization rate of Examples 1 and 2 increased by 26.7% and 30.5%, respectively; the hardness reduction rate increased by 12.3% and 15.5%, respectively; the oxygen content increase rate increased by 16.7% and 10.2%, respectively; and the available phosphorus increase increased by 9.7 mg / kg and 13.5 mg / kg, respectively. This indicates that the present invention, through mutation depth range identification, can more accurately locate the depth range that needs to be treated, and its effect is better than fixed-depth stratified fertilization.

[0111] (3) Comparing Example 1 and Example 2, when the interaction coefficient is ≥1, the oxygen content-dominant fertilization method increases the oxygen content by 35.2%, which is better than the 28.7% of Example 2; when the interaction coefficient is <1, the hardness-dominant fertilization method reduces the hardness by 35.6%, which is better than the 32.4% of Example 1. This shows that selecting the corresponding fertilization method according to the interaction coefficient can accurately improve the main limiting factors.

[0112] In summary, Examples 1 and 2 of this invention are significantly superior to Comparative Examples 1 and 2 in terms of earthworm enzyme utilization rate, hardness reduction rate, oxygen content increase rate, and available phosphorus increase. Comparative Example 1 shows that the earthworm enzyme utilization rate and soil improvement effect are poor when there is no differentiated strategy; Comparative Example 2 shows that although fixed-depth stratified fertilization is better than surface application, it still cannot accurately identify the mutation depth range and dominant factors. This invention, by systematically detecting the earthworm enzyme activity decay law, identifying the mutation depth range of hardness and oxygen content, calculating the interaction coefficient, and adopting a differentiated fertilization strategy, significantly improves the earthworm enzyme utilization efficiency and soil improvement effect, overcoming the various defects of the prior art.

[0113] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for detecting and differentially applying earthworm enzyme activity in alkaline soil, characterized in that, include: Step S1: Obtain hardness and oxygen content data of soil samples at different depths of the target alkaline soil; Step S2: Apply earthworm enzyme preparation to each soil sample. At several time points after the application of earthworm enzyme preparation, monitor the enzyme activity data of soil samples at each depth to determine the decay coefficient and activity decay half-life of earthworm enzyme at the corresponding depth. Step S3: Calculate the attenuation coefficient at each depth and the hardness correlation factor corresponding to several hardness data, and the attenuation coefficient at each depth and the oxygen content correlation factor corresponding to several oxygen content data. Determine the hardness mutation depth range based on each hardness correlation factor, and determine the oxygen content mutation depth range based on each oxygen content correlation factor. Step S4: Determine the interaction coefficient based on the hardness mutation depth range and the oxygen content mutation depth range; Step S5: Determine the range of action depth based on the interaction coefficient, calculate the activity retention time at each depth within the range of action depth according to the activity decay half-life and decay coefficient, and determine the action depth based on the activity retention time.

2. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 1, characterized in that, The steps for determining the decay coefficient and activity decay half-life of earthworm enzymes at a corresponding depth include: Obtain initial values ​​of enzyme activity data for soil samples at any depth at the initial moment of application of earthworm enzyme preparation; Enzyme activity data in the soil were collected at several time points in a time sequence and compared with the initial values ​​of enzyme activity data. The activity decay half-life is determined based on the time required for enzyme activity in the soil to decay to 50% of its initial value. Based on the same time point, enzyme activity data of soil samples at different depths are determined, and the decay coefficient is determined based on the average decrease rate of enzyme activity data within a unit depth.

3. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 2, characterized in that, In step S3, the steps of determining the hardness mutation depth range based on each hardness-related factor and determining the oxygen content mutation depth range based on each oxygen content-related factor include: The range of hardness mutation depth is determined based on the depth range corresponding to the first time that the hardness-related factor exceeds the preset hardness threshold; The depth range of oxygen content mutation is determined based on the depth range corresponding to the first time that oxygen-related factors exceed a preset oxygen content threshold.

4. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 1 or 3, characterized in that, In step S4, determining the interaction coefficient based on the hardness mutation depth range and the oxygen content mutation depth range includes: Within the range of hardness mutation depth, a first representative depth is determined for oxygenation treatment. The residual enzyme activity data after oxygenation treatment is measured and recorded as the first residual data. The first rate of change is calculated based on the first residual data and the enzyme activity data corresponding to the first representative depth in step S2. A second representative depth is determined within the range of oxygen content mutation depth for hardening treatment. The residual enzyme activity data after hardening treatment is measured and recorded as the second residual data. The second rate of change is calculated based on the second residual data and the enzyme activity data corresponding to the second representative depth in step S2. The interaction coefficient is determined based on the absolute value of the ratio of the first rate of change to the second rate of change.

5. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 4, characterized in that, In step S4, the steps of determining the first representative depth and the second representative depth include: The first representative depth is determined based on the median depth of the hardness abrupt change depth range; The second representative depth is determined based on the median depth of the oxygen content mutation depth range.

6. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 5, characterized in that, The oxygenation treatment includes: injecting a first effective amount of calcium peroxide into the first representative depth; The hardening treatment includes injecting a second effective amount of clay slurry into the second representative depth.

7. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 1, characterized in that, Step S5, which involves determining the depth range based on the interaction coefficient, includes: If the interaction coefficient is greater than or equal to the reference value, then the oxygen content mutation depth range is used as the reference action depth range, and the reference action depth range is extended in combination with the hardness mutation depth range direction to determine the action depth range. If the interaction coefficient is less than the reference value, the range of hardness change depth is used as the range of action depth, and the reference range of action depth is extended in the direction of oxygen content change depth to determine the range of action depth.

8. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 1, characterized in that, In step S5, determining the depth of action includes: Several alternative points of action are determined at different depths within the range of the stated depth of action. The activity retention time of each candidate site of action is determined based on the activity decay half-life and decay coefficient. The depth of action is determined based on the minimum duration of activity retention for each candidate site of action.

9. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 8, characterized in that, The steps for determining the duration of activity retention include: The duration of activity retention is determined based on the activity decay half-life at the depth of each candidate action site, the decay coefficient at the corresponding depth, and the interaction coefficient.

10. The method for detecting and differentially applying earthworm enzyme activity in alkaline soil according to claim 1, characterized in that, It also includes step S6, which involves applying either a hardness-based fertilization method or an oxygen content-based fertilization method based on the location of the depth of action. The hardness-dominant fertilization method involves deep injection of earthworm enzyme preparations to the depth of action. The oxygen content-driven fertilization method involves implementing oxygenation measures at the specified depth of application.

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