A copper tungsten polyoxometalate, a preparation method thereof and application thereof in the field of agricultural fungicides

Abstract

The application belongs to the technical field of agricultural fungicidal active ingredients, and particularly relates to a copper-tungsten polyoxometalate and a preparation method and application thereof in the field of agricultural fungicides. The existing agricultural fungicidal POM compounds have defects such as insufficient targeted bacteriostatic activity and insufficient preparation stability. The application provides a copper-tungsten polyoxometalate compound Cu-W-POMs, which exhibits excellent fungicidal activity and can effectively inhibit the cucumber fusarium wilt pathogen. Meanwhile, the compound also has a good inhibitory effect on crop disease pathogenic bacteria such as soybean seed rot, pear black spot and apple anthracnose. In particular, the compound can be used for preparing an agricultural fungicide to prevent and control cucumber fusarium wilt and other crop diseases.

Description

Technical Field

[0001] This invention belongs to the technical field of agricultural bactericidal active ingredients, specifically relating to a copper-tungsten polyoxometalate, its preparation method, and its application in the field of agricultural bactericidal treatment. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] China is a major global producer and consumer of cucumbers. In recent years, the cucumber planting area has remained stable at over 1 million hectares, with a total output exceeding 60 million tons. Cucumbers are a crucial crop for ensuring domestic vegetable market supply and supporting the development of facility agriculture. However, diseases severely restrict the healthy development of the cucumber industry. Cucumber wilt is one of the most prominent soil-borne fungal diseases, caused by *Fusarium oxysporum* var. *cucumber*. In continuously cropped areas, the incidence rate can reach 30%–50%, and in severely affected areas, there can even be total crop failure. Infected plants exhibit vascular bundle browning, wilting, and death, resulting in yield losses exceeding 20% ​​and reducing the marketable quality of cucumbers, posing a significant threat to farmers' income and the stability of the vegetable supply chain.

[0004] Currently, the main control measures for cucumber wilt include agricultural control, physical control, and chemical control. Agricultural control (such as crop rotation and grafting disease-resistant rootstocks) is limited by planting patterns and site conditions, making it difficult to promote on a large scale in intensive facility cultivation. Physical control (such as soil disinfection) is costly, complex to operate, and easily damages the soil microecology. Chemical control is still the most relied-upon method in current production, with traditional fungicides such as carbendazim and hymexazol commonly used. However, long-term single use has led to a significant increase in pathogen resistance, and the field control effect has dropped from more than 80% in the early stage to less than 50%. At the same time, some agents pose a risk of exceeding the residue limit, which does not meet the requirements of green agriculture development.

[0005] Polyoxometalates (POMs), as a class of structurally tunable inorganic functional materials, have gradually gained attention in the field of agricultural fungicides due to their excellent redox activity and broad-spectrum antibacterial properties. However, existing agricultural POM compounds have shortcomings such as insufficient targeted antibacterial activity (poor specificity against cucumber wilt) and weak formulation stability (field residual effect of less than 7 days), making it difficult to meet the actual control needs of cucumber wilt. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide an active ingredient with stable structure, good environmental compatibility, and antibacterial activity. To achieve this technical objective, this invention provides a copper-tungsten polyoxometalate compound that exhibits good bactericidal activity, effectively inhibiting cucumber wilt, soybean stem rot, pear black spot, and apple anthracnose. This compound can be used to prepare agricultural fungicides for the control of plant diseases, and is of great significance for solving the problem of cucumber wilt control and promoting the development of green agriculture.

[0007] Based on the above-mentioned technical effects, the present invention provides the following technical solution:

[0008] Firstly, a copper-tungsten polyoxometalate compound with the molecular formula C is provided. 48 H 32 Cu2N8O 40 W 12 The compound is a monoclinic crystal with space group P21 / c and unit cell parameters of: a = 26.18881(12) Å, b = 11.83944(4) Å, c = 23.40596(11) Å, α = 90°, β = 113.7335°, γ = 90°.

[0009] The copper-tungsten polyoxometalate compounds mentioned in the first aspect above, abbreviated as Cu-W-POMs, have the Keggin-type tungsten-based polyoxometalate cluster W in their molecular formula. 12 O 40 As the basic building unit, it is related to Cu(C) 12 The H8N2)2 coordination units, after being bonded together by coordination bonds, further form a one-dimensional chain-like supramolecular structure along the α-axis through hydrogen bonding. Among them, the Keggin-type W... 12 O 40 Oxyacid clusters are the first active sites, possessing strong redox capabilities, and can disrupt pathogenic cell membranes and enzyme systems. Cu(C) 12 The H8N2)2 coordination unit is the second active site, which can realize the coordinated state of Cu. 2+ The slow release of the substance blocks the respiratory chain and nucleic acid synthesis of pathogens. The two active sites are covalently linked by coordination bonds, achieving a synergistic effect of oxidative antibacterial and metal ion antibacterial. The antibacterial efficiency is much higher than that of traditional antibacterial components with a single active site, and it can effectively reduce the probability of pathogens developing drug resistance.

[0010] Furthermore, the hydrogen bonds in the compound, being non-covalent bonds, ensure structural flexibility (adapting to the microenvironment of pathogen surfaces) while enhancing the overall chemical stability and dispersibility, preventing compound aggregation in aqueous solutions or non-specific reactions with environmental ions. The one-dimensional chain-like spatial arrangement provides the compound with a larger specific surface area, allowing for more thorough contact with pathogens and improving the targeting of its antibacterial effect. The supramolecular encapsulation also enhances the Cu... 2+ The active sites are not easily degraded by light and oxygen, which prolongs the field effect and adapts to the complex environment in agricultural production. It effectively overcomes the defects of poor stability and insufficient antibacterial activity of existing agricultural bactericides.

[0011] The XRD diffraction of the above compounds is in 2 θ Characteristic peaks were observed at 8.2±0.2°, 25.7±0.2°, 35.7±0.2°, and 48.5±0.2°. Among them, 8.2±0.2° corresponds to the supramolecular stacking peak of the (002) crystal plane, 25.7±0.2° is the (211) skeleton diffraction peak corresponding to the organic ligand o-phenanthroline, 35.7±0.2° is the (310) WOW bridging oxygen characteristic peak corresponding to the Keggin-type tungsten oxide cluster, and 48.5±0.2° is the characteristic peak corresponding to the Cu-O coordination structure. These peak positions are completely matched with the structure of the target compound. Its microstructure is a cuboid structure with a particle size of 200~500 nm (average about 350 nm).

[0012] Secondly, a method for preparing the copper-tungsten polyoxometalate compound described in the first aspect is provided, comprising the following steps:

[0013] S1 - Preparation of dodecantungstooxide cluster precursor solution: Ammonium metatungstate hydrate (NH4)6 (H2W) 12 O 40 ) Dissolve 3H2O in water and stir at 60-70℃ for 10-20 minutes until the solid is completely dissolved. Slowly add acid to adjust the pH to 1.8-2.2 and continue stirring for 20-40 minutes to obtain a pale yellow precursor solution.

[0014] S2 - Introducing copper salt and organic ligand: Add copper salt, adipic acid and o-phenanthroline sequentially to the precursor solution obtained in S1, and continue stirring at 60~70℃ until a blue-green stable solution is obtained.

[0015] S3-Hydrothermal reaction: Adjust the pH of the blue-green solution obtained from S2 to 3.8~4.2, transfer it to a high-pressure reactor, heat it to 150~170℃ and react for 90~100h to obtain a suspension;

[0016] S4 - Purification treatment: Separate the precipitate from the suspension obtained in S3, wash and dry it to obtain a dark green rod-shaped single crystal product.

[0017] In S1 above, the dosage ratio of ammonium metatungstate hydrate to water is 0.2~0.4g:10~15ml; the stirring speed is 250~350r / min; the acid solution is preferably an inorganic acid, such as hydrochloric acid or sulfuric acid. In a more effective embodiment, hydrochloric acid is used with a concentration of 5~7mol / L.

[0018] In S2 above, the copper salt is preferably a copper-soluble inorganic salt, such as copper sulfate, copper chloride, copper nitrate, or copper bromide. The present invention can conventionally select the above inorganic salt based on factors such as cost and water solubility. In one embodiment verified by the present invention, copper chloride is used to reduce the introduction of impurity ions. In this embodiment, the molar ratio of adipic acid or o-phenanthroline to copper chloride is 1~3:1.

[0019] In the above S3, the filling degree of the solution in the high-pressure reactor is preferably 75-85%. During the reaction heating stage, the preferably heating rate is 4-6℃ / min, and after the reaction is completed, the preferably cooling rate is 8-12℃ / min.

[0020] In S4 above, the precipitate can be separated by methods such as filtration and centrifugation. The precipitate is then washed with water and ethanol in sequence and then dried. The drying method includes, but is not limited to, negative pressure drying, thermal radiation drying, or a combination of methods.

[0021] Thirdly, a composition is provided, the composition comprising an active dose of the copper-tungsten polyoxometalate compound of the first aspect, and further comprising a carrier acceptable in the field of pesticide science.

[0022] This invention provides corresponding safe and antibacterial concentrations for the copper-tungsten polyoxometalate compound. When the pesticide composition of this invention is used in agriculture, appropriate application amounts and concentrations can be set according to conditions including formulation type, frequency, location and method of application, pest species and degree of damage.

[0023] The pesticide-acceptable carriers include various solid and liquid carriers known in the art. Solid carriers may be, for example, fine powders or granules of clay materials such as kaolin, diatomaceous earth, synthetic hydrated silica, bentonite, Fubasami clay, and acid clay; fine powders or granules of various talc, ceramic, and other inorganic materials such as sericite, quartz, sulfur, activated carbon, calcium carbonate, and hydrated silica; and fine powders or granules of fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and ammonium chloride.

[0024] Liquid carriers may include, for example, water; alcohols such as methanol and ethanol; ketones such as acetone and methyl ethyl ketone; hydrocarbons such as hexane, cyclohexane, kerosene and light oil; and vegetable oils such as soybean oil and cottonseed oil.

[0025] The pesticide composition of the present invention may also contain surfactants, fixatives, dispersants, stabilizers, etc. The pesticide composition of the present invention can be prepared by mixing the various components in the pesticide composition of the present invention with each other.

[0026] The pesticide composition of the present invention, thus formulated, can be used directly or diluted with water. Furthermore, it can be mixed with other biocontrol agents, fertilizers, soil conditioners, and / or animal feed, or used simultaneously without mixing.

[0027] Fourthly, the application of the copper-tungsten polyoxometalate compound described in the first aspect and the composition described in the third aspect in the field of agricultural sterilization is provided.

[0028] The applications described in the fourth aspect above include at least one of the following aspects:

[0029] (1) A method for preventing and controlling crop diseases, comprising applying the copper-tungsten polyoxometalate compound or composition to the crop or soil to be treated;

[0030] (2) The copper-tungsten polyoxometalate compound or composition is used to prepare agricultural fungicides.

[0031] In aspect (1) above, the method of application is specifically such as spraying onto crop leaves or applying to the roots of crops in the soil.

[0032] In aspect (2) above, the agricultural fungicide is specifically one of the following: insecticide, nematicide, acaricide, fungicide, antifungal agent, herbicide, plant growth regulator or synergist.

[0033] Furthermore, this agricultural fungicide is a broad-spectrum fungicide, and the pathogens it inhibits include, but are not limited to, Fusarium wilt pathogen of cucumber (…). Fusarium oxysporum ), soybean stem rot pathogen ( Phomopsis longicolla Apple anthracnose bacteria ( Colletotrichum gloeosporioides ), pear black spot fungus ( Alternaria alternata One or more of the following:

[0034] Furthermore, the crop disease to which the agricultural fungicide is applicable is cucumber wilt.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] The copper-tungsten polyoxometalate compound of the present invention has a novel structure, a high degree of precision matching in crystal structure, excellent physicochemical stability, and a supramolecular chain structure that enhances the structural durability of the material.

[0037] The preparation process of the copper-tungsten polyoxometalate compound of the present invention is simple to operate and can be completed using conventional hydrothermal synthesis equipment. The reaction conditions are mild and easy to control, which facilitates large-scale preparation.

[0038] When the copper-tungsten polyoxometalate compound of the present invention is applied in the field of agricultural fungicides, it exhibits outstanding antibacterial activity against the pathogen of cucumber wilt, good environmental compatibility, and reduces the risk of pesticide residues. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Figure 1 The diagram shows the characterization and molecular structure model of the copper-tungsten polyoxometalate compound described in this invention.

[0041] in, Figure 1 Image A is a microscope photograph of a copper-tungsten polyoxometalate compound; Image B is a ball-and-stick model of the molecular structure of a copper-tungsten polyoxometalate compound.

[0042] Figure 2 The image shows the X-ray powder diffraction (XRD) pattern of the copper-tungsten polyoxometalate compound described in Example 1.

[0043] Figure 3 The Fourier transform infrared spectrum of the copper-tungsten polyoxometalate compound described in Example 1;

[0044] Figure 4 This is an X-ray photoelectron spectroscopy (XPS) analysis chromatogram of the copper-tungsten polyoxometalate compound described in Example 1;

[0045] Figure 4 A represents the full-spectrum analysis of the copper-tungsten polyoxometalate. Figure 4 B is the W 4f orbital analysis of the copper-tungsten polyoxometalate; Figure 4 C in the figure represents the O 1s orbital analysis of the copper-tungsten polyoxometalate. Figure 4 D in the middle represents the Cu 2p orbital of the copper-tungsten polyoxometalate.

[0046] Figure 5 The results of the safety concentration screening of the copper-tungsten polyoxometalate compound described in Example 4;

[0047] Figure 5In Figure A, the results of the determination of the toxicity threshold concentration of copper tungsten polyoxometalate to Arabidopsis thaliana are shown. Figure 5 In Figure B, the effect of copper-tungsten polyoxometalates on the fresh weight of Arabidopsis thaliana is shown. Figure 5 In the middle, C represents the effect of copper-tungsten polyoxometalates on the dry weight of Arabidopsis thaliana;

[0048] Figure 6 The results of the in vitro antibacterial activity evaluation of the copper-tungsten polyoxometalate described in Example 4;

[0049] Figure 6 From left to right, A represents the in vitro inhibition of cucumber wilt pathogens by copper tungsten polyoxometalates at concentrations of 0 μM, 50 μM, 100 μM, and 200 μM. Fusarium oxysporum Renderings;

[0050] Figure 6 From left to right, B represents the in vitro inhibition of soybean stem rot pathogens by copper tungsten polyoxometalates at concentrations of 0 μM, 50 μM, 100 μM, and 200 μM. Phomopsis longicolla Renderings;

[0051] Figure 6 The values ​​of C from left to right represent the in vitro inhibition of apple anthracnose by copper-tungsten polyoxometalates at concentrations of 0 μM, 50 μM, 100 μM, and 200 μM. Colletotrichum gloeosporioides Renderings;

[0052] Figure 6 From left to right, the concentrations of copper tungsten polyoxometalates (0 μM, 50 μM, 100 μM, and 200 μM) inhibited the in vitro growth of pear black spot pathogens. Alternaria alternata Renderings;

[0053] Figure 7 The image shows the effect of copper-tungsten polyoxometalate in controlling cucumber wilt disease as described in Example 5.

[0054] Figure 7 Figure A shows the phenotypic effect of copper-tungsten polyoxometalates on the control of cucumber wilt disease; Figure 7 In section B, the effect of copper-tungsten polyoxometalate on SOD enzyme activity is represented. Figure 7 In the figure, C represents the effect of copper-tungsten polyoxometalate on PPO enzyme activity. Detailed Implementation

[0055] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0057] In the context of this specification, the word "comprising" is considered to mean "especially including". It should not be interpreted as "consisting of only".

[0058] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0059] The reagents used in the following examples are all of analytical grade and require no further purification. Their specific specifications and sources are as follows:

[0060] Ammonium metatungstate hydrate (NH4)6(H2W) 12 O 40 ) 3H2O (purity ≥99.0%, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0061] Copper chloride dihydrate CuCl2 2H2O (purity ≥ 99.5%, Sinopharm Chemical Reagent Co., Ltd.);

[0062] o-phenanthroline C 12 H8N2 H2O (purity ≥ 99.0%, Shanghai Yuanye Biotechnology Co., Ltd.);

[0063] Adipic acid C6H 10 O4 (purity ≥ 99.0%, Sigma-Aldrich Trading Ltd.);

[0064] Hydrochloric acid (HCl) (concentration 36 ~ 38%, Sinopharm Chemical Reagent Co., Ltd.);

[0065] Sodium hydroxide (NaOH) (purity ≥ 96.0%, Sinopharm Chemical Reagent Co., Ltd.);

[0066] Deionized water: conductivity ≤ 10 μS / cm, prepared in the laboratory.

[0067] Example 1

[0068] In this embodiment, a copper-tungsten polyoxometalate C is provided. 48 H 32 Cu2N8O40 W 12 The preparation method of the copper-tungsten polyoxometalate specifically includes the following:

[0069] (I) Preparation of copper-tungsten polyoxometalates

[0070] S1 - Preparation of dodecantungstenoxate cluster precursor solution: Accurately weigh 0.30 g of ammonium metatungstate hydrate, add 12 mL of deionized water to a 250 mL three-necked flask, set up a constant temperature stirring device, heat to 65 ℃, and magnetically stir at a constant temperature (300 r / min) for 15 min until the solid is completely dissolved, obtaining a clear and transparent solution. Slowly add 6 mol / L hydrochloric acid solution dropwise while stirring, monitor the pH in real time with a precision pH meter, adjust the pH of the system to 2.0, and continue stirring at a constant temperature for 30 min to allow the dodecantungstenoxate cluster to fully dissociate and stabilize, obtaining a pale yellow polyacid precursor solution.

[0071] S2 - Introduction of copper salt and organic ligand: Add 0.34 g of copper chloride dihydrate to the aforementioned precursor solution, and stir at 65 ℃ and 300 r / min for 10 min until the copper salt is completely dissolved and the system turns pale blue. Then add 0.72 g of o-phenanthroline and 0.58 g of adipic acid (molar ratio of o-phenanthroline, adipic acid and copper chloride dihydrate is 2:2:1), and continue stirring at a constant temperature for 20 min. During this period, the color of the system gradually deepens to blue-green. After stirring, a homogeneous and stable mixed solution is obtained, with no obvious precipitation or layering.

[0072] S3-Hydrothermal Reaction Crystallization: The pH of the mixed solution was adjusted to 4.0 with 1 mol / L sodium hydroxide solution. After stirring evenly, the solution was transferred to a 50 mL polytetrafluoroethylene-lined high-pressure reactor with a filling degree of 80%. The reactor was sealed and placed in a constant temperature drying oven, and the temperature was programmed to rise to 160 °C (heating rate 5 °C / min) and reacted at this temperature for 96 h. After the reaction was completed, the heating device was turned off, and the mixture was slowly cooled to room temperature at a rate of 10 °C / h (approximately 16 h). The reactor was then opened to obtain a suspension containing dark green crystals.

[0073] S4 - Post-treatment purification: The suspension was filtered using a Buchner funnel, and the precipitate was collected. The precipitate was first washed three times with deionized water (10 mL each time) to remove residual unreacted salts and impurities, and then washed once with anhydrous ethanol (10 mL) to remove surface-adsorbed organic impurities. The washed crystals were placed in a vacuum drying oven and dried at 60 ℃ and -0.09 MPa for 12 h. After drying, a dark green rod-shaped single crystal product was obtained, named Cu-W-POMs, with a yield of 72.3%.

[0074] (II) Product Characterization and Verification

[0075] 1. Morphological and structural characterization

[0076] The morphology and structure of the copper-tungsten polyoxometalate prepared in this embodiment were observed using an optical microscope. As shown in Figure 1A, the copper-tungsten polyoxometalate is a dark green to emerald green rod-shaped single crystal with complete crystal form and uniform surface luster. Although some crystals are fragmented, they still maintain a rod-shaped morphology, indicating that the crystal growth was sufficient during the hydrothermal synthesis process, and that it possesses good morphological regularity and mechanical stability. This indicates that the target copper-tungsten polyoxometalate (named Cu-W-POM) was successfully prepared in this invention. X-ray single-crystal diffraction data were analyzed, as shown... Figure 1 As shown in Figure B, the ball-and-stick model clearly reveals the supramolecular structural features of the compound: the core is a Keggin-type tungsten oxide cluster (blue W atoms, red O atoms), forming a stable inorganic framework; orange Cu ions serve as connecting sites, coordinating with the organic ligand o-phenanthroline (gray C atoms, light blue N atoms); the organic ligand assembles with the inorganic cluster through π-π stacking and hydrogen bonding to form a one-dimensional chain-like supramolecular structure, with a compact and symmetrical overall structure. In summary, this study successfully prepared a well-formed copper-tungsten polyoxometalate single crystal, whose molecular structure verifies the ternary coordination design of "inorganic tungsten oxide cluster - copper ion - organic ligand," and the supramolecular chain-like stacking mode is also completely consistent with expectations, providing a solid structural foundation for subsequent research on antibacterial activity and material properties.

[0077] 2. Crystalline characterization based on XRD diffraction

[0078] The crystal structure data of the copper-tungsten polyoxometalates prepared by the above method by X-ray single-crystal diffraction (XRD) are shown in Table 1 below:

[0079] Table 1 Crystal structure data of copper-tungsten polyoxometalates

[0080]

[0081] X-ray diffraction (XRD) of the copper-tungsten polyoxometalate Cu-W-POMs is as follows: Figure 2 As shown in the XRD pattern, the copper-tungsten polyoxometalate exhibits extremely high crystallinity, with sharp diffraction peaks, stable baselines, and no obvious amorphous background. The characteristic peaks in the spectrum perfectly match the supramolecular packing, organic ligand binding, and inorganic cluster framework structure of the target compound, confirming the successful assembly of the ternary supramolecular structure of "Keggin-type tungsten-oxygen cluster-copper ion-o-phenanthroline ligand," providing a reliable structural basis for subsequent performance studies.

[0082] 3. Structural characterization based on infrared spectroscopy

[0083] The structure of the copper-tungsten polyoxometalates Cu-W-POMs prepared in this embodiment was characterized using Fourier transform infrared spectroscopy. Figure 3 As shown, the prepared Cu-W-POMs were obtained at 980 cm⁻¹. - The characteristic absorption at ¹ corresponds to the stretching vibration of the W=O bond in a Keggin-type tungsten oxide cluster; 1450 cm⁻¹ - ¹ and 1590 cm - The characteristic absorption at ¹ is induced by the vibration of the benzene ring in the ligand; in addition, at 580 cm⁻¹ - The absorption peak near ¹ corresponds to the vibration of the Cu-O bond. It can be seen that the Cu-W-POMs structure contains the characteristic structure of a Keggin-type tungsten oxide cluster and a copper-ligand coordination unit. In summary, the FT-IR results confirm that its structure includes a Keggin-type tungsten oxide cluster, an organic o-phenanthroline ligand, and a copper-oxygen coordination unit, which is completely consistent with the designed structure of the target compound, providing a reliable material basis for subsequent research on antibacterial activity and applications.

[0084] 4. Characterization of elemental composition and chemical valence state

[0085] The elemental composition and chemical valence state of the copper-tungsten polyoxometalate were characterized by X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 4 In the full spectrum of compound A, characteristic signals of Cu 2p, O 1s, C 1s, N 1s, W 4f, and Cl 2p were detected. Cu, W, and O are the core elements of the inorganic cluster framework; C and N are derived from the organic ligand o-phenanthroline; Cl is a residual element from the preparation raw material (CuCl2). The overall elemental composition is similar to that of the target compound C. 48 H 32 Cu2N8O 40 W 12 The molecular formulas match perfectly. Figure 4 B-4D is an analysis of elemental chemical valence states and chemical environments, with W in the inorganic cluster framework. 6+ Mainly, with a small amount of W 5+ Cu 2+ Mainly composed of Cu, with a small amount present. + Oxygen comprises three chemical environments: inorganic cluster metal-oxygen bonds, surface hydroxyl groups, and organic ligand CO bonds. The organic ligand o-phenanthroline was successfully incorporated into the compound, and the overall structure matched the design expectations.

[0086] The above investigation results show that the copper-tungsten polyoxometalate Cu-W-POMs successfully prepared by hydrothermal synthesis in Example 1 is a single crystal with good crystallinity, complete structure, high yield, and purity that meets the requirements of subsequent activity evaluation tests.

[0087] Example 2

[0088] This embodiment provides another method for preparing copper-tungsten polyoxometalate Cu-W-POMs, which specifically includes the following steps:

[0089] S1 - Preparation of dodecantungstenoxate cluster precursor solution: Accurately weigh 0.30 g of ammonium metatungstate hydrate, add 12 mL of deionized water to a 250 mL three-necked flask, set up a constant temperature stirring device, heat to 60 ℃, and magnetically stir at a constant temperature (250 r / min) for 20 min until the solid is completely dissolved, obtaining a clear and transparent solution. Slowly add 5 mol / L hydrochloric acid solution dropwise while stirring, monitor the pH in real time with a precision pH meter, adjust the pH of the system to 1.8, and continue stirring at a constant temperature for 40 min to allow the dodecantungstenoxate cluster to fully dissociate and stabilize, obtaining a pale yellow polyacid precursor solution.

[0090] S2 - Introduction of copper salt and organic ligand: Add 0.34 g of copper chloride dihydrate to the aforementioned precursor solution, and stir at 60 ℃ and 300 r / min for 10 min until the copper salt is completely dissolved and the system turns pale blue. Then add 0.36 g of o-phenanthroline and 0.29 g of adipic acid (molar ratio of o-phenanthroline, adipic acid and copper chloride dihydrate is 2:1:1), and continue stirring at a constant temperature for 20 min. During this period, the color of the system gradually deepens to blue-green. After stirring, a homogeneous and stable mixed solution is obtained, with no obvious precipitation or layering.

[0091] S3-Hydrothermal Crystallization: The pH of the mixed solution was adjusted to 3.8 with 1 mol / L sodium hydroxide solution. After stirring evenly, the solution was transferred to a 50 mL polytetrafluoroethylene-lined high-pressure reactor with a filling degree of 75%. The reactor was sealed and placed in a constant temperature drying oven, and the temperature was programmed to rise to 170℃ (heating rate 4℃ / min) and reacted at this temperature for 90 h. After the reaction was completed, the heating device was turned off, and the mixture was slowly cooled to room temperature at a rate of 8℃ / h (approximately 16 h). The reactor was then opened to obtain a suspension containing dark green crystals.

[0092] S4 - Post-treatment purification: The suspension was filtered using a Buchner funnel, and the precipitate was collected. The precipitate was first washed three times with deionized water (15 mL each time) to remove residual unreacted salts and impurities, and then washed once with anhydrous ethanol (15 mL) to remove surface-adsorbed organic impurities. The washed crystals were placed in a vacuum drying oven and dried at 65 °C and -0.09 MPa for 12 h, yielding a dark green rod-shaped single crystal product.

[0093] Example 3

[0094] This embodiment provides another method for preparing copper-tungsten polyoxometalate Cu-W-POMs, which specifically includes the following steps:

[0095] S1 - Preparation of the dodecantungstenoxate cluster precursor solution: Accurately weigh 0.30 g of ammonium metatungstate hydrate, add 12 mL of deionized water to a 250 mL three-necked flask, set up a constant temperature stirring device, heat to 70℃, and magnetically stir at a constant temperature (350 r / min) for 10 min until the solid is completely dissolved, obtaining a clear and transparent solution. Slowly add 7 mol / L hydrochloric acid solution dropwise while stirring, monitor the pH in real time with a precision pH meter, adjust the pH of the system to 2.2, and continue stirring at a constant temperature for 20 min to allow the dodecantungstenoxate cluster to fully dissociate and stabilize, obtaining a pale yellow polyacid precursor solution.

[0096] S2 - Introduction of copper salt and organic ligand: Add 0.34 g of copper chloride dihydrate to the aforementioned precursor solution, and stir at 70℃ and 300 r / min for 10 min until the copper salt is completely dissolved and the system turns pale blue. Then add 1.08 g of o-phenanthroline and 0.87 g of adipic acid (molar ratio of o-phenanthroline, adipic acid and copper chloride dihydrate is 3:3:1), and continue stirring at a constant temperature for 20 min. During this period, the color of the system gradually deepens to blue-green. After stirring, a homogeneous and stable mixed solution is obtained, with no obvious precipitation or layering.

[0097] S3-Hydrothermal Crystallization: The pH of the mixed solution was adjusted to 4.2 with 1 mol / L sodium hydroxide solution. After stirring evenly, the solution was transferred to a 50 mL polytetrafluoroethylene-lined high-pressure reactor with a filling degree of 85%. The reactor was sealed and placed in a constant temperature drying oven, and the temperature was programmed to rise to 150℃ (heating rate 6℃ / min) and reacted at this temperature for 100 h. After the reaction was completed, the heating device was turned off, and the mixture was slowly cooled to room temperature at a rate of 12℃ / h. The reactor was then opened to obtain a suspension containing dark green crystals.

[0098] S4 - Post-treatment purification: The suspension was filtered using a Buchner funnel, and the precipitate was collected. The precipitate was first washed three times with deionized water (10 mL each time) to remove residual unreacted salts and impurities, and then washed once with anhydrous ethanol (10 mL) to remove surface-adsorbed organic impurities. The washed crystals were placed in a vacuum drying oven and dried at 60 °C and -0.09 MPa for 12 h, yielding a dark green rod-shaped single crystal product.

[0099] Example 4

[0100] This embodiment provides the application of the copper-tungsten polyoxometalate Cu-W-POMs prepared in Example 1 in the field of preparing agricultural antibacterial agents:

[0101] (I) Safety Concentration Screening of Copper-W-POMs

[0102] This part of the study aims to evaluate the effects of different concentrations of copper-tungsten polyoxometalates on the growth of Arabidopsis thaliana seedlings, clarify the safe application concentration range, and provide a reference for subsequent field crop application.

[0103] 1. Test materials

[0104] Arabidopsis thaliana seeds were of the Columbia ecotype (Col-0) and were prepared for use after surface sterilization; the test reagent was copper tungsten polyoxometalate (C... 48 H 32 Cu2N8O 40 W 12 Treatment solutions with concentrations of 200, 400, and 600 μM were prepared using sterile water, with water as a blank control (CK).

[0105] 2. Test Methods

[0106] Arabidopsis seeds were sown in seedling pots filled with nutrient soil (or a mixture of vermiculite and peat moss) and regularly watered with 1 / 2 MS nutrient solution. The seedlings were then placed in an artificial climate chamber (22 ℃, 16 h light / 8 h dark) until the cotyledons were fully expanded. Rhizosphere drenching was used to treat the seedlings, with 10 mL of the corresponding concentration applied to each pot. Each treatment was repeated three times, with each replicate containing 12 seedlings. After treatment, the seedlings were cultured for another 21 days. Seedling phenotypes were observed, and the aboveground fresh and dry weights (sterilized at 105 ℃ and dried at 70 ℃ to constant weight) were measured. Data were analyzed using one-way ANOVA for significance testing; a significant difference compared to the control was indicated by P < 0.05.

[0107] 3. Experimental Results

[0108] Phenotypic observation showed that Arabidopsis seedlings in the blank control (CK), 200 μM and 400 μM treatment groups grew vigorously with bright green leaves and uniform plant shape; seedlings in the 600 μM treatment group showed obvious yellowing and wilting symptoms, and some plants died. Figure 5 A). Fresh weight determination results showed no significant difference in seedling fresh weight among the CK group, the 200 μM group, and the 400 μM group (fresh weights were 0.29 g, 0.28 g, and 0.26 g, respectively); the fresh weight of the 600 μM treatment group was significantly reduced to 0.09 g (P<0.05), which was only 31.0% of the control. Figure 5B). Dry weight measurements showed no significant difference in seedling dry weight among the CK group, the 200 μM group, and the 400 μM group (dry weights were 0.019 g, 0.020 g, and 0.017 g, respectively); the dry weight of the 600 μM treatment group significantly decreased to 0.015 g (P<0.05), indicating that high concentration treatment significantly inhibited the accumulation of plant substances. Figure 5 C).

[0109] 4. Conclusion

[0110] In summary, the present invention has demonstrated through the above embodiments that copper tungsten polyoxometalate has no significant adverse effects on the growth (including fresh weight, dry weight and phenotype) of the model plant Arabidopsis thaliana at an application concentration of ≤ 400 μM, thus clarifying that its safe application threshold for Arabidopsis thaliana is 400 μM.

[0111] (II) Evaluation of the in vitro antibacterial activity of copper-tungsten polyoxometalates Cu-W-POMs

[0112] This section aims to evaluate the in vitro antibacterial activity of copper-tungsten polyoxometalates (Cu-W-POMs) against various important crop harmful fungi, clarify the relationship between their antibacterial spectrum and concentration effect, and provide data support for their application as broad-spectrum agricultural fungicides.

[0113] 1. Test materials

[0114] Test pathogen: Fusarium wilt of cucumber ( Fusarium oxysporum ), soybean stem rot pathogen ( Phomopsis longicolla Apple anthracnose bacteria ( Colletotrichum gloeosporioides ), pear black spot fungus ( Alternaria alternata All were activated and cultured to the logarithmic growth phase for later use.

[0115] Test reagent: Copper-tungsten polyoxometalate (C 48 H 32 Cu2N8O 40 W 12 Treatment solutions with concentrations of 50, 100, and 200 μM were prepared using sterile water, with sterile water containing 0.1% DMSO serving as a blank control (0 μM).

[0116] 2. Test Methods

[0117] Antibacterial activity was determined using the mycelial growth rate method: Different concentrations of copper-tungsten polyoxometalate solutions were mixed with PDA medium cooled to 45-50℃ to prepare drug-containing plates. Mycelial cakes from the edge of pathogen colonies were taken using a 5 mm diameter punch and inoculated into the center of the drug-containing plate, with three replicates per treatment. The plates were incubated at 25℃ for 5-7 days (adjusted according to the pathogen growth rate). Colony morphology was observed and colony diameter was measured to calculate the inhibition rate.

[0118] Inhibition rate (%) = [(Control colony diameter - Treated colony diameter) / (Control colony diameter – 0.5 cm)] × 100%

[0119] 3. Experimental results (see...) Figure 6 )

[0120] Figure 6 A represents the fungus that causes cucumber wilt (…). Fusarium oxysporum The colony diameter of the 0 μM group reached 8.2 cm, with dense and white flocculent hyphae; the colony diameter of the 200 μM group was only 1.9 cm, with an inhibition rate of 80.7%, and the colonies were significantly shrunken and lighter in color, showing the most significant inhibitory effect.

[0121] Figure 6 B represents the soybean stem rot pathogen ( ). Phomopsis longicolla The colony diameter of the 0 μM group was 6.1 cm, and it appeared as a yellow velvet; the colony diameter of the 200 μM group was 1.7 cm, with an inhibition rate of 78.5%, and mycelial growth was significantly inhibited.

[0122] Figure 6 C represents apple anthracnose bacteria ( Colletotrichum gloeosporioides The colony diameter of the 0 μM group was 5.9 cm, and it was white and dense; the colony diameter of the 200 μM group was 1.7 cm, with an inhibition rate of 77.8% and colony expansion was significantly restricted.

[0123] Figure 6 D in the middle represents the pear black spot pathogen ( Alternaria alternata The colony diameter of the 0 μM group was 5.8 cm, and it appeared as white fluff; the colony diameter of the 200 μM group was 1.6 cm, with an inhibition rate of 79.2% and a significant decrease in mycelial density.

[0124] Overall results showed that the antibacterial activity of copper tungsten polyoxometalates against the tested pathogens increased in a concentration-dependent manner. At a concentration of 200 μM, it showed significant inhibitory effects on all tested fungi, with the most prominent inhibitory activity against Cucumber Fusarium wilt.

[0125] 4. Conclusion

[0126] This embodiment confirms that copper-tungsten polyoxometalates (Cu-W-POMs) possess broad-spectrum and highly efficient in vitro antifungal activity against various harmful fungi in crops, with the activity increasing with increasing concentration. At a concentration of 200 μM, this compound exhibits an inhibition rate exceeding 75% against important pathogens such as *Fusarium wilt* of cucumber and *Pseudomonas aeruginosa* of soybean, demonstrating its core potential as a broad-spectrum agricultural fungicide and providing crucial evidence for its subsequent field application.

[0127] Example 5

[0128] In this embodiment, the field simulation control effect of copper-tungsten polyoxometalate Cu-W-POMs on cucumber wilt is provided to clarify its control efficacy in practical applications and its impact on crop growth.

[0129] 1. Test materials

[0130] Test plants: Healthy seedlings of cucumber variety 'Jinchun No. 4' cultivated to the three-leaf-one-heart stage;

[0131] Pathogen: Fusarium wilt of cucumber ( Fusarium oxysporum f. sp. cucumerinum (Foc), prepared at a concentration of 1×10 6 A spore suspension of 1 spore per mL;

[0132] Test reagent: Copper-tungsten polyoxometalate (C 48 H 32 Cu2N8O 40 W 12 ), to prepare a 200 μM treatment solution.

[0133] 2. Test Methods

[0134] Three treatment groups were set up, with three replicates per group and 12 seedlings per replicate:

[0135] POM group: 200 μM copper tungsten polyoxometalate solution was applied only as a root drench;

[0136] Foc group: root irrigation with Foc spore suspension only;

[0137] POM+Foc group: The roots were first drenched with copper tungsten polyoxometalate solution, and then inoculated with Foc spore suspension 24 h later.

[0138] After treatment, the plants were placed in an artificial climate chamber (25-28 ℃, 85% relative humidity) for 14 days. The plant phenotype was observed, and the activities of superoxide dismutase (SOD) and polyphenol oxidase (PPO) in the leaves were measured (using the nitro blue tetrazol method and the catechol colorimetric method).

[0139] 3. Experimental results (see...) Figure 7 )

[0140] Phenotypic observation (Figure 7A): Foc group seedlings showed obvious wilting and yellowing of leaves; POM+Foc group seedlings grew vigorously with bright green leaves and no significant difference in plant height compared with POM group, indicating that copper tungsten polyoxometalate effectively alleviated the damage of Fusarium wilt to plants.

[0141] SOD activity (Figure 7B): The SOD activity of seedlings in the Foc group was significantly increased to 208 U / g FW (98.1% higher than that in the POM group), reflecting the strong oxidative stress caused by the pathogen; the SOD activity of the POM+Foc group was 112 U / g FW, which was significantly lower than that in the Foc group (P<0.01), indicating that copper tungsten polyoxometalate effectively reduced the oxidative damage of the plants.

[0142] PPO activity (Figure 7, C): PPO activity in the Foc group seedlings was significantly increased to 521 U. g -1 min -1 (160.5% higher than the POM group); PPO activity in the POM+Foc group was 214 U. g - ¹ min - ¹, significantly lower than the Foc group (P<0.01), further confirming that copper tungsten polyoxometalates alleviate pathogen-induced oxidative stress.

[0143] In summary, copper tungsten polyoxometalate exhibits excellent control of cucumber wilt at 200 μM (safe threshold concentration) without adversely affecting cucumber growth and yield, demonstrating good potential for field application.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a copper-tungsten polyoxometalate compound or a composition thereof in the field of agricultural sterilization, characterized in that, The copper-tungsten polyoxometalate compound or composition is used to prepare agricultural fungicides; The molecular formula of the copper-tungsten polyoxometalate compound is C 48 H 32 Cu2N8O 40 W 12 The compound is a monoclinic crystal with space group P21 / c. Its unit cell parameters are: a = 26.18881(12) Å, b = 11.83944(4) Å, c = 23.40596(11) Å, α = 90°, β = 113.7335°, γ = 90°. The compound exhibits XRD diffraction patterns in 2... θ It has characteristic peaks at 8.2±0.2°, 25.7±0.2°, 35.7±0.2°, and 48.5±0.2°; its microstructure is a cuboid structure with a particle size of 200-500 nm; The composition comprises an active dose of the copper-tungsten polyoxometalate compound, and also includes a carrier acceptable in the field of pesticide science; The carriers accepted in the field of pesticide science are selected from solid carriers and liquid carriers; the solid carriers are selected from fine powders or granules of kaolin, diatomaceous earth, synthetic hydrated silica, bentonite, Fubasami clay, acid clay, talc, ceramics, sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea or ammonium chloride. The liquid carrier is selected from water, methanol, ethanol, acetone, methyl ethyl ketone, hexane, cyclohexane, kerosene, light oil, soybean oil, or cottonseed oil.

2. The application of the copper-tungsten polyoxometalate compound or its composition as described in claim 1 in the field of agricultural sterilization, characterized in that, The agricultural fungicide inhibits pathogens selected from one or more of the following: Fusarium wilt of cucumber, Pseudomonas stolonifera of soybean, Anthracnose of apple, and Black spot of pear.

3. The application of the copper-tungsten polyoxometalate compound or its composition as described in claim 1 in the field of agricultural sterilization, characterized in that, The preparation method of the copper-tungsten polyoxometalate compound includes the following steps: S1 - Preparation of dodecatungstic acid oxychloride cluster precursor solution: Ammonium metatungstate hydrate is dissolved in water and stirred at 60-70℃ for 10-20 min until the solid is completely dissolved. Acid solution is slowly added to adjust the pH to 1.8-2.2, and stirring is continued for 20-40 min to obtain a light yellow precursor solution. The dosage ratio of ammonium metatungstate hydrate to water is 0.2-0.4 g: 10-15 ml. S2 - Introducing copper salt and organic ligand: Add copper salt, o-phenanthroline and adipic acid sequentially to the precursor solution obtained in S1, and continue stirring at 60~70℃ until a blue-green stable solution is obtained. The molar ratio of o-phenanthroline, adipic acid and copper salt is 1~3:1~3:

1. S3-Hydrothermal reaction: Adjust the pH of the blue-green solution obtained from S2 to 3.8~4.2, transfer it to a high-pressure reactor, heat it to 150~170℃ and react for 90~100h to obtain a suspension; S4 - Purification treatment: Separate the precipitate from the suspension obtained in S3, wash and dry it to obtain a dark green rod-shaped single crystal product.

4. The application of the copper-tungsten polyoxometalate compound or its composition as described in claim 3 in the field of agricultural sterilization, characterized in that, In S1, the stirring speed is 250~350 r / min; the acid solution is hydrochloric acid with a concentration of 5~7 mol / L.

5. The application of the copper-tungsten polyoxometalate compound or its composition as described in claim 3 in the field of agricultural sterilization, characterized in that, Its features are, In S2, the copper salt is copper chloride.

6. The application of the copper-tungsten polyoxometalate compound or its composition as described in claim 3 in the field of agricultural sterilization, characterized in that, In S3, the filling degree of the solution in the high-pressure reactor is 75-85%; During the heating phase of the reaction, the heating rate is 4~6℃ / min, and after the reaction is completed, the cooling rate is 8~12℃ / min.

7. The application of the copper-tungsten polyoxometalate compound or its composition as described in claim 3 in the field of agricultural sterilization, characterized in that, Its features are, In S4, the separation method of the precipitate is filtration or centrifugation. The obtained precipitate is washed with water and ethanol in sequence and then dried. The drying method includes, but is not limited to, negative pressure drying, thermal radiation drying or a combination of methods.

Images