Application of malic acid and its preparations in alleviating or improving the inhibition of pesticide residues on soil

Abstract

The present invention discloses the application of malic acid as a soil remediation agent in alleviating or improving the inhibition of pesticide residues on soil, which belongs to the new application technology of biological secretions, especially the field of soil remediation technology. The present invention deeply studies the enzyme activity-microbiological mechanism, and uses in situ zymography to find that the application of malic acid can relieve the anti-competitive inhibition caused by imidacloprid, reduce the binding of imidacloprid and enzyme, and restore enzyme activity. Malic acid and root secretions can reduce the pH value of the soil, and at the same time effectively improve the problem of reduced phosphorus content, nitrate nitrogen content, and organic carbon content in the soil caused by imidacloprid. The present invention provides a practical and feasible remediation technology for pesticide-contaminated soil, providing a new way for the healthy development of agriculture.

Description

Technical Field

[0001] The present invention relates to a new application technology of malic acid, a biological secretion, and particularly to the field of soil improvement and remediation technology, specifically to the application of malic acid and its preparations as soil remediation agents in alleviating or improving the inhibition of pesticide residues on soil. Background Art

[0002] As early as the 1990s, China ranked first in the world in both apple production and cultivated area, making it a major apple producer. While apple trees, as an economic tree, boost local farmers' incomes and provide ecological benefits such as windbreak and sand fixation, air purification, soil erosion prevention, and water conservation, according to the "Chinese Fruit Tree Diseases and Insects," apples are susceptible to over 100 pests and diseases, leading to the frequent use of chemical pesticides.

[0003] Overuse and irrational pesticide application in orchards often lead to high detection rates and residue levels. These pesticides can affect pollutant transport and nutrient cycling within the soil-plant system by influencing soil physicochemical properties, enzyme activity, and microbial community dynamics. The effects of pesticides on soil enzyme activity are related to the type of pesticide and soil enzyme, soil physicochemical properties, and pesticide residue concentration. Current research on soil pollution focuses on inorganic pollution, such as drought stress, heavy metal stress, and salt stress. Research on organic pollution, such as pesticides, is lacking. Research focuses on source analysis of residual organic pesticide contamination after application, the effects of pesticides on soil enzyme activity and microbial activity, and the mechanisms of soil remediation. Soil enzyme activity, a key indicator used by researchers both domestically and internationally to assess soil environmental quality and safety, can be combined with metagenomics, a field widely used in recent years, to further elucidate the mechanisms by which pollutants affect soils. Current methods for measuring enzyme activity in research still rely on traditional spectrophotometry and fluorescence analysis. However, these methods only measure the maximum potential activity in the soil and fail to accurately reflect the true distribution of soil enzyme activity within the environment. In situ zymography is a recently developed technique that integrates fluorescent substrates to analyze the two-dimensional distribution of enzyme activity in soil (Spohn and Kuzyakov; 2013, Vandooren et al.; 2013). This technique is useful for a variety of hydrolytic enzymes, providing extensive information on their activity, including enzyme form and localization. Therefore, further integration of in situ zymography with metabolomics can effectively and intuitively interpret changes in soil ecology.

[0004] Imidacloprid (IMI), a widely used nicotinoid insecticide, is widely used in orchards due to its high efficiency in pest control. Imidacloprid residues can cause various stresses on plants, including oxidative stress. , metabolic disorders and changes in rhizosphere microbial communities These stress mechanisms not only affect plant growth and development, but also pose potential risks to ecosystem health. Therefore, rational use of pesticides is necessary, and effective remediation and degradation measures should be studied for residual pesticides. Summary of the Invention

[0005] The present invention aims to study the effect of imidacloprid on the nutrient cycle, root characteristics, soil enzyme kinetics and rhizosphere microorganisms of the soil-plant system, and in combination with the application of root secretions, to further study the enzyme activity-microbiological mechanism, and to find that the application of malic acid can relieve the anti-competitive inhibition caused by imidacloprid, reduce the binding of imidacloprid and enzymes, and restore enzyme activity using in situ zymography. Malic acid and root secretions can reduce the soil pH value, and effectively improve the problem of reduced phosphorus content, nitrate nitrogen content and organic carbon content in the soil caused by imidacloprid.

[0006] Firstly, the present invention provides the use of malic acid in alleviating or improving the inhibition of pesticide residues on soil.

[0007] In the aforementioned application, preferably, the inhibitory effect of the pesticide residue on the soil includes at least one of reduced soil enzyme activity, reduced phosphorus content, reduced nitrate nitrogen content, and reduced organic carbon content. Preferably, the soil enzyme is BG (β-glucosidase), and the pesticide is imidacloprid. For the application described herein, a preferred method involves slowly releasing malic acid into the soil containing pesticide residues to establish an effective malic acid concentration gradient within the soil. If malic acid fails to react with the pesticide in a timely manner, it will reach excessive concentration, potentially causing soil damage such as acidification. This can be achieved by slowly releasing malic acid into the soil using a Rhizon sampler and incubating the solution to establish a gradient throughout the soil surrounding the Rhizon sampler (artificial root). The malic acid concentration is adjusted appropriately based on the level of pesticide residue and can generally range from 100 to 400 micromol carbon / ml. Preferably, malic acid at a concentration of 200 micromol carbon / ml can be added to the root box via the Rhizon sampler (artificial root) at a rate of 1.0 ml per day and incubated for 14 days to ensure the gradient is established throughout the artificial root.

[0008] Secondly, the present invention also provides the use of plant root exudates containing malic acid to alleviate or improve the inhibitory effects of pesticide residues on soil. Preferably, the inhibitory effects of pesticide residues on soil include at least one of reduced soil enzyme activity, reduced phosphorus content, reduced nitrate nitrogen content, and reduced organic carbon content. The plants can be fruit trees such as apples, pears, peaches, etc. The soil enzyme is BG enzyme (β-glucosidase). The pesticide is imidacloprid.

[0009] Thirdly, the present invention also provides the use of a malic acid preparation to alleviate or improve the inhibitory effects of pesticide residues on soil. The malic acid preparation can be a malic acid-added agent, such as a compound formulation made by adding malic acid to some agricultural bio-infectants or fertilizers, which can then be applied to the soil. The inhibitory effects of pesticide residues on soil include at least one of reduced soil enzyme activity, reduced phosphorus content, reduced nitrate nitrogen content, and reduced organic carbon content. The soil enzyme is BG enzyme (β-glucosidase). The pesticide is imidacloprid.

[0010] The present invention has found that the application of malic acid can relieve the uncompetitive inhibition caused by imidacloprid. Malic acid and imidacloprid are combined through hydrogen bonds and charge transfer, thereby reducing the binding of imidacloprid and enzymes and restoring enzyme activity. Malic acid and root secretions can reduce the pH value of the soil, while effectively improving the problem of reduced phosphorus content, nitrate nitrogen content, and organic carbon content in the soil caused by imidacloprid. Therefore, it can be used as a soil remediation agent for imidacloprid contamination. The present invention provides a practical and feasible repair technology for soil contaminated by the pesticide imidacloprid, which provides new ideas and feasible methods for the green, sustainable and healthy development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figures show validation experiments in a rhizobox. (a) A real-time photograph of the rhizobox after the Rhizon® sample was placed before the experiment began. The white line in the middle represents the artificial root, and the dark shaded area represents the soil sampling range, which is determined by the range of enzyme activity in the zymogram. (b) A zymogram captured in a darkroom under ultraviolet (UV) light at an excitation wavelength of 355 nm. (c) An example zymogram of the response of malic acid to β-glucosidase activity in the presence of imidacloprid. The legend on the right is proportional to the enzyme activity (nmol cm - 2 h -1 ).

[0012] Figure 2 Soil zymogram analysis; (a): Response of malic acid to β-glucosidase activity under different treatments. Imidacloprid addition reduced BG enzyme activity. (b): Malic acid addition increased BG enzyme activity. (c), (d), and (e): Simulated secretion of malic acid using rhizoctonia increased the BG enzyme hotspot area and enzyme extension range. This indicates that malic acid can repair the enzyme activity reduction caused by imidacloprid residues.

[0013] Figure 3 This is a soil enzyme kinetic analysis diagram.

[0014] Figure 4 This is an analysis chart of the content of imidacloprid and malic acid in soil.

[0015] Figure 5 This is the Fourier infrared spectrum analysis of rhizosphere soil.

[0016] Figure 6 The following is a graph showing the changes in soil parameters before and after the addition of malic acid. (a) Soil pH under different treatments. After the addition of malic acid, the soil pH decreased. (b) Available phosphorus content in the soil under different treatments. After the addition of malic acid, the available phosphorus content in the soil increased. (c) Nitrate nitrogen content in the soil under different treatments. After the addition of malic acid, the nitrate nitrogen content in the soil increased significantly. (d) Changes in dissolved organic carbon under different treatments. After the addition of malic acid, the dissolved organic carbon content in the soil increased significantly.

[0017] Figure 7 This is the result of apple root exudate extraction and analysis. DETAILED DESCRIPTION

[0018] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0019] Example 1

[0020] Effect of malic acid on enzyme activities at the root-soil interface contaminated by the pesticide imidacloprid:

[0021] (1) Collecting uncontaminated soil

[0022] Before the experiment, soil samples were collected and basic physical and chemical properties (organic matter, total nitrogen, ammonium nitrate nitrogen, available phosphorus, available potassium, and pH) were measured. Soil samples were collected from the surface layer (0-10 cm depth) at the Weibei Dryland Experimental Station of Northwest Agriculture and Forestry University in Baishui County, Shaanxi Province, China (109°56′E, 35°21′N, 838 m above sea level) in 2022. To avoid interference from malic acid and the use of pesticides, soil was collected from fallow land 20 meters away from apple trees, where neither crops nor fruit trees had been cultivated for more than five years. The soil was mixed thoroughly by hand, and tree roots and rocks were removed. The soil was then sealed in sealed bags and stored at -4°C. Before incubation, the soil was passed through a 2 mm sieve. Residual imidacloprid concentrations in the soil were measured before treatment and were below the detection limit (0.01 mg / kg).

[0023] (2) Make a root box

[0024] The entire experiment was conducted in a rhizobox (8 × 10 × 2 cm). The rhizobox is a transparent plastic box with a removable front panel that can be opened without disturbing the soil surface and artificial roots (Rhizon) ( Figure 1 ). A 100 mm long Rhizon® sampler (Rhizosphere Research Products, Wageningen) was buried along the longitudinal center with its upper surface at the same level as the soil. When filling the soil, first place the root box horizontally, open one side, and then slowly add the sieved soil. After pouring a small amount of soil into the root box, close the open side, re-place the sample vertically, and then gently shake it to make it uniform. Repeat the above steps until the root box is filled with stable soil. The specific operation is to divide 1.2 kg of soil into three equal parts and pass it through a 2 mm sieve. Each soil sample is evenly transferred to two root boxes. Then use a pipette to drop imidacloprid (IM) solution on the soil surface. In actual application, the dosage of pesticides is often higher than the recommended dosage. To account for this, three imidacloprid concentrations were set up in root boxes (8 × 10 × 2 cm): 0 mg / kg (IM0), 1.0 mg / kg (IM1, recommended concentration), and 5.0 mg / kg (IM5, 5 times the recommended concentration). Soil samples with a concentration of 0 mg / kg were treated with an equal volume of distilled water.

[0025] (3) Simulating the process of roots secreting malic acid into the soil

[0026] Paired rhizoboxes containing the same imidacloprid concentration were compared using a Rhizon® sampler (artificial root) to treat malic acid (MA) or distilled water (CK). Malic acid (MA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., China. Four replicates were prepared for each treatment, resulting in a total of 24 rhizoboxes for the experiment. The prepared rhizoboxes were first incubated for 6 days. On the 7th day, malic acid at a concentration of 200 μmol C / mL was added to the rhizoboxes via a Rhizon® sampler (artificial root) at a rate of 1.0 mL per day. Incubation was continued for 14 days to ensure the establishment of a gradient around the artificial root. Throughout the incubation period, the rhizoboxes were maintained in a climate chamber at a stable temperature of 25 ± 1°C.

[0027] like Figure 1 For verification and results, the white line in the center of the rhizobox represents artificial roots. (a) is a real-time photograph of the rhizobox after the Rhizon® sample was placed before the experiment began. The dark shaded area represents the soil sampling range, which is determined by the range of enzyme activity in the zymogram. (b) is a zymogram captured in a darkroom under ultraviolet (UV) light at an excitation wavelength of 355 nm. (c) is an example of a zymogram of the response of malic acid to β-glucosidase activity in the presence of imidacloprid. The legend on the right is proportional to the enzyme activity (nmol·cm -2 h -1(d) Compared with the control, the soil enzyme activity after adding imidacloprid decreased significantly on the third day. On the seventh day, the difference was not obvious. On the 30th day, the soil enzyme activity after adding imidacloprid was not significant compared with the control.

[0028] Soil zymogram analysis: performed after 28 days of incubation. Soil zymogram analysis was used to determine the spatial distribution of soil enzymes around Rhizon® after addition of malic acid in the presence of imidacloprid. Figure 2 .

[0029] like Figure 3 , the addition of imidacloprid reduced the activity of BG enzyme. After malic acid was used to simulate secretion by rhizoctonia, it was found that the hot spot area of ​​BG enzyme and the extension range of enzyme increased. This shows that malic acid can repair the decrease in enzyme activity caused by imidacloprid residue. Enzyme kinetic analysis showed that the application of imidacloprid led to a decrease in soil Vmax (maximum reaction rate) and Km (Michaelis constant), which produced an uncompetitive inhibition on soil BG enzyme activity. After the application of malic acid, Vmax increased, Km increased, enzyme activity increased, and to a certain extent, the uncompetitive inhibition caused by imidacloprid was relieved. Malic acid may repair soil enzyme activity through the following mechanisms: (1) Competitive displacement: Malic acid may compete with imidacloprid to bind to the enzyme-substrate complex, thereby displacing imidacloprid and restoring enzyme activity. (2) Hydrogen bond, charge transfer: Because malic acid has a carboxyl group, it can bind to imidacloprid through hydrogen bond and charge transfer, making imidacloprid less bound to the enzyme-substrate complex, thus restoring enzyme activity. To verify this idea, we also measured the content of imidacloprid and malic acid in the soil, and the results are as follows: Figure 4 The concentrations of imidacloprid and malic acid decreased in the soil with malic acid added, indicating that malic acid combined with imidacloprid through hydrogen bonding and charge transfer, thereby reducing the binding between imidacloprid and the enzyme and restoring enzyme activity.

[0030] like Figure 5 Further Fourier transform infrared spectroscopy analysis of rhizosphere soil showed that the characteristic absorption peak in the Fourier transform infrared spectrum (FTIR) changed at approximately 1700-1730 cm⁻¹ after the application of malic acid, indicating the C=O stretching vibration of the carboxyl group. This shows that the carboxyl group (–COOH) and hydroxyl group (–OH) in malic acid can indeed form coordination bonds or hydrogen bonds with the nitrogen atoms in imidacloprid, which is also the fundamental reason why malic acid can repair imidacloprid residual soil.

[0031] Figure 6This is a graph showing the changes in soil-related parameters before and after the addition of malic acid. (a): Soil pH under different treatments. After adding malic acid, soil pH decreased. (b): Available phosphorus content in soil under different treatments. After adding malic acid, available phosphorus content in soil increased. (c): Nitrate nitrogen content in soil under different treatments. After adding malic acid, nitrate nitrogen content in soil increased significantly. (d): Changes in dissolved organic carbon under different treatments. After adding malic acid, dissolved organic carbon content in soil increased significantly. Different lowercase letters indicate significant differences. The differences decrease from a to d.

[0032] Example 2

[0033] Extraction and analysis of root exudates

[0034] (1) Collection of basic soil physical and chemical properties samples

[0035] Soil samples were collected before the experiment to determine the basic physical and chemical properties of the soil (organic matter, total nitrogen, ammonium nitrate nitrogen, available phosphorus, available potassium content, pH, etc.).

[0036] (2) Extraction and analysis of apple root exudates

[0037] The root exudates were collected using an in situ collection method. Root exudates were collected from three apple trees of similar diameter at breast height (DBH) at the Weibei Dryland Experimental Station of Northwest Agriculture and Forestry University (109°56′E, 35°21′N, 838 m above sea level) in Baishui County, Shaanxi Province, China. The collection mainly focused on finding fine roots buried in the 0-10 cm soil layer along the exposed roots of each selected tree. The roots were completely dug out and the soil particles on the surface of the fine roots were rinsed with deionized water. Other polymers on the surface were removed with tweezers. The clean and complete roots were then placed in a sterile 50 ml syringe, filled with about 1 mm of sterile quartz sand, and sealed with a syringe rubber stopper. The sterile syringe containing the selected fine roots was wrapped with tin foil and buried back in the original location (following the original growth direction of the roots and fixing the syringe with the original soil to restore the original growth environment of the roots as much as possible). After a stabilization period of about 2 days, 20 ml of a pre-prepared carbon-free nutrient solution (mainly containing 0.5 mM NH4NO3, 0.1 mM KH2PO4, 0.2 mM K2SO4, 0.2 mM MgSO4, and 0.3 mM CaCl2) was added to each sterile syringe to eliminate the interference of soluble carbon. At the same time, a control was set up in each sample plot. After 24 hours, use a sterile syringe with a 0.22 μm filter tip to collect the nutrient solution in each syringe containing the root exudates and promptly refrigerate the collected solution. Filter the solution using a 0.22 μm syringe filter within 24 hours of collection to obtain the apple root exudates.

[0038] Using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) analysis, we found that among a total of 8743 mass features detected by positive (51.3%) or negative ions (48.7%), the following were reported using the database (http: / / www.mzcloud.org) (Korenblum et al., 2020, Wright-Muelas et al., 2020). These included fatty acyl groups, carboxylic acids and their derivatives, organic oxygen compounds, steroids and steroid derivatives, organic nitrogen compounds, phenols, benzene and substituted derivatives, prenol lipids, pyridine and its derivatives, isoflavones, organophosphates and their derivatives, and purine nucleotides. Figure 7Figure a shows principal component analysis (PCA) of annotated metabolites strongly clustered according to the amount of imidacloprid added, with PC 1 and PC 2 accounting for 43.28% and 25.77% of the variance, respectively (pANOSIM < 0.05) (Figure a). A total of 56 differential metabolic signatures were modulated in the IM group compared to the control group (CK), including 22 downregulated genes and 34 upregulated genes (log2 (fold change) > 1.5, q < 0.05, Figure b). Using a 2-fold change threshold, 27 metabolites were found to be significantly different in the IM group compared to the CK group (p < 0.05, log2 (fold change) > |1|). In particular, L-malate and L-2-hydroxyglutarate were specifically enriched in the IM group compared to the CK group (fold change > 3) (Figure c), which are related to the tricarboxylic acid cycle (TCA cycle). Therefore, imidacloprid addition enriched the apple rhizosphere with specific organic acids, L-malic acid and its related product, L-2-hydroxyglutaric acid, with malic acid being the highest. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we found that the tricarboxylic acid cycle, carbon metabolism, glyoxylate and dicarboxylic acid metabolism, secondary metabolite biosynthesis, lysine degradation, sulfur metabolism, amino acid biosynthesis, alanine, aspartate, and glutamate metabolism, pyruvate metabolism, and carbon fixation in photosynthetic organisms were among the top 10 KEGG pathways. Compared with CK, IM addition significantly enriched these pathways (p < 0.05, t-test, Figure d). We focused on the TCA cycle and secondary metabolite biosynthesis pathways because they had high enrichment factors and P values, respectively.

[0039] The present invention discovered that apple root exudates, particularly malic acid, can relieve the uncompetitive inhibition caused by imidacloprid, restore soil BG enzyme activity, and lower soil pH. This, in turn, effectively alleviates the reduction in available phosphorus, nitrate nitrogen, and organic carbon in the soil caused by imidacloprid. Therefore, the present invention provides a practical and feasible technology for remediating pesticide-contaminated soil, offering new insights and feasible approaches for the green, sustainable, and healthy development of agriculture.

[0040] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

Claims

1. Use of malic acid in alleviating or improving the inhibition of pesticide residues on soil, wherein the inhibition of pesticide residues on soil includes reducing the activity of soil enzymes, and the soil enzymes are BG enzymes.

2. The use according to claim 1, characterized in that The pesticide residues inhibit soil from at least one of reducing phosphorus content, reducing nitrate nitrogen content, and reducing organic carbon content.

3. The use according to claim 1, wherein The pesticide is imidacloprid.

4. The use according to any one of claims 1 to 3, characterized in that Malic acid was slowly released into the soil through the Rhizon sampler, allowing a gradient to be established around the Rhizon sampler.

5. Use of plant root exudates containing malic acid in alleviating or improving the inhibition of pesticide residues on soil, wherein the inhibition of pesticide residues on soil includes a reduction in soil enzyme activity, and the soil enzyme is BG enzyme.

6. The use according to claim 5, characterized in that The pesticide residues inhibit soil from at least one of reducing phosphorus content, reducing nitrate nitrogen content, and reducing organic carbon content, and the soil enzyme is BG enzyme.

7. The use according to claim 5, characterized in that The pesticide is imidacloprid.

8. Use of a malic acid preparation in alleviating or improving the inhibition of pesticide residues on soil, wherein the inhibition of pesticide residues on soil includes a reduction in soil enzyme activity, wherein the soil enzyme is BG enzyme.

9. The use according to claim 8, characterized in that The pesticide residues inhibit soil from at least one of reducing phosphorus content, reducing nitrate nitrogen content, and reducing organic carbon content, and the soil enzyme is BG enzyme.

10. The use according to claim 8, characterized in that The pesticide is imidacloprid.

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