Application of N-phenylmaleimide as copper-based bactericide synergist

Abstract

The invention discloses an application of N-phenylmaleimide as a synergist of a copper-based bactericide, and finds for the first time that the N-phenylmaleimide and the copper-based bactericide are mixed for use, so that the dosage of the copper-based bactericide can be reduced, and meanwhile, good bactericidal activity can be ensured; and verification is carried out on mango bacterial angular leaf spot bacteria and citrus canker bacteria, and the N-phenylmaleimide (NPM) shows a remarkable synergistic effect when being mixed with the copper-based bactericide. The use amount of the copper-based bactericide in unit area is reduced, the accumulation risk of heavy metals in soil is reduced, resistance evolution of pathogenic bacteria to copper is delayed, and the method has important significance for solving the increasingly prominent drug resistance problem of pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to a bactericide synergist, specifically to the application of N-phenylmaleimide as a copper-based bactericide synergist. Background Technology

[0002] Drug resistance and antimicrobial activity in plant pathogens is a major global problem. In recent years, the most commonly used methods for controlling bacterial diseases include the use of copper-based fungicides, agricultural antibiotics, and microbial preparations. Microbial preparations are not suitable for large-scale use due to their high cost. Copper-based fungicides, on the other hand, are widely used in disease control due to their broad-spectrum and long-lasting effects.

[0003] Copper is an essential trace element for both prokaryotes and eukaryotes, playing a crucial role in cellular metabolic processes such as respiration. However, excessive copper is toxic to organisms, and the extensive use of copper-based fungicides inevitably harms the environment, increases heavy metal pollution in soil, and allows pathogens to evolve resistance to copper. Reducing the amount of copper-based fungicides used will not achieve the goal of killing pathogens and controlling plant diseases.

[0004] In recent years, with the increasing attention people pay to food safety and environmental pollution, research on non-toxic, harmless, and low-residue new pesticides and new pesticide synergists that can reduce pesticide usage has become an urgent need and a promising research direction in plant disease control.

[0005] Mango bacterial angular leaf spot (also known as mango bacterial black spot or mango bacterial canker) is caused by the fungus *Hypericum mangoum*. Xanthomonas citri pv.mangiferaeindicae , Xcm Citrus canker is a common and important disease of mangoes, caused by the fungus *Citrus canker*, which seriously affects mango yield and the commercial value of the fruit worldwide. Xanthomonas citri pv. citri , Xac Citrus canker, caused by copper-based fungicides, is a common disease affecting citrus fruits and is a global disease, as well as a significant disease in China's citrus-producing regions. Therefore, there is a need to develop a synergist that can effectively reduce the dosage of copper-based fungicides while maintaining or enhancing their fungicidal effect, in order to address the environmental pollution and pathogen resistance issues associated with the use of copper-based fungicides in existing technologies.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide an application of N-phenylmaleimide as a synergist for copper-based fungicides, which solves the problems of environmental pollution and pathogen resistance caused by excessive use of copper-based fungicides in the prior art. This invention can reduce the amount of copper-based fungicides used while ensuring good fungicidal activity, providing a new and effective way for the green control of bacterial diseases in agricultural production.

[0008] To achieve the above objectives, the present invention provides the application of N-phenylmaleimide as a synergist for copper-based bactericides.

[0009] The N-phenylmaleimide used in this invention can be purchased directly or synthesized artificially, such as by reacting maleic anhydride with aniline.

[0010] Preferably, the applicable bacteria include: *Mango angularis* (a bacterium causing mango bacterial angular spot disease). Xanthomonas citri pv. Mangiferaeindicae , Xcm ) or / and citrus canker pathogen ( Xanthomonas axonopodis pv. citri , Xac ).

[0011] Preferably, the application involves mixing the copper-based bactericide with N-phenylmaleimide.

[0012] Preferably, the concentration of the N-phenylmaleimide is above 20 μM.

[0013] More preferably, the concentration of the N-phenylmaleimide is 20~400 μM.

[0014] Preferably, the copper-based bactericide is selected from inorganic copper and / or organic copper bactericides.

[0015] More preferably, the copper-based bactericide is selected from any one or more of copper sulfate, copper hydroxide, cuprous oxide, basic copper sulfate, copper oxychloride, quinoline copper, thiamethoxam copper, thiamethoxam copper, amino acid copper, copper succinate, copper acetate, and copper rosinate.

[0016] More preferably, the copper-based bactericide is selected from copper sulfate, and the concentration of the copper sulfate is greater than 0 and less than or equal to 0.7 mM.

[0017] More preferably, the copper-based bactericide is selected from any one or more of 46% copper hydroxide, quinoline copper, and 30% copper oxychloride.

[0018] More preferably, the copper-based bactericide and N-phenylmaleimide are mixed, and the concentration of the copper-based bactericide in the mixture is diluted by less than 10,000 times.

[0019] The application of N-phenylmaleimide of the present invention as a synergist for copper-based fungicides solves the problems of environmental pollution and pathogen resistance caused by excessive use of copper-based fungicides in the prior art, and has the following advantages: (1) This invention first discovered that by mixing N-phenylmaleimide (NPM) with copper-based fungicides, it is possible to reduce the amount of copper-based fungicides used while ensuring good fungicidal activity. This was verified on mango bacterial angular leaf spot and citrus canker. The mixture of NPM and copper-based fungicides showed a significant synergistic effect. (2) In this invention, NPM is mixed with different types of copper-based fungicides (such as copper sulfate, copper hydroxide, quinoline copper, etc.) in a certain proportion. The inhibitory effect on mango bacterial angular leaf spot and citrus canker is significantly better than that of copper-based fungicides alone, and the amount of copper-based fungicide used is reduced. (3) This invention not only reduces the amount of copper-based fungicide used per unit area and reduces the risk of heavy metal accumulation in the soil, but also helps to delay the evolution of resistance to copper by pathogens. It is of great significance to solve the increasingly prominent problem of drug resistance of pathogens. This invention meets the current development needs of green prevention and control in agriculture and ecological environmental protection, and provides a practical and feasible technical solution for the sustainable management of bacterial diseases in crops such as mango and citrus. Attached Figure Description

[0020] Figure 1 This refers to the effect of different concentrations of NPM and copper sulfate mixed on bacterial angular leaf spot of mango in Example 2 of the present invention. Xcm GXBS06 sterilization results; A: Coating results; B: Spotting results.

[0021] Figure 2 The bacterial angular leaf spot pathogen of mango in Example 2 of this invention Xcm Growth of GXBS06 in liquid culture medium containing different concentrations of NPM and copper sulfate.

[0022] Figure 3 The effects of different concentrations of NPM and copper sulfate mixed on citrus canker pathogens in Example 3 of this invention. Xac 306 sterilization results; A: Coating results; B: Spot test results.

[0023] Figure 4 The citrus canker pathogen in Example 3 of this invention Xac Growth of 306 in liquid culture media containing different concentrations of NPM and copper sulfate.

[0024] Figure 5 The citrus canker pathogen in Example 4 of this invention Xac 306 Pathogenicity of mixtures of copper sulfate and NPM at different concentrations; A: XacPathogenicity of plants under 306 and PBS conditions; B: Xac Pathogenicity of 306 plants under 0 μM NPM and 0.2 mM (left) / 0.3 mM (right) copper sulfate; C: Xac Pathogenicity of 306 plants under 20 μM NPM and 0.2 mM (left) / 0.3 mM (right) copper sulfate; D: Xac Pathogenicity of 306 plants under 40 μM NPM and 0.2 mM (left) / 0.3 mM (right) copper sulfate; D: Xac Pathogenicity of 306 plants under 60 μM NPM and 0.2 mM (left) / 0.3 mM (right) copper sulfate.

[0025] Figure 6 The bacterial angular leaf spot pathogen of mango in Example 5 of this invention Xcm The pathogenicity of GXBS06 at 0.2 mM and 0.3 mM copper sulfate concentrations and at different small molecule compound concentrations; A: 0 μM NPM, 20 μM NPM; B: 40 μM NPM, 60 μM NPM.

[0026] Figure 7 The bacterial angular leaf spot pathogen of mango in Example 6 of this invention Xcm GXBS06 and Citrus Canker Xac 306 The bactericidal synergistic effect of different dilution ratios of 46% copper hydroxide, quinoline copper, and 30% copper oxychloride NPM mixtures; A, B: respectively Xcm GXBS06 Xac Growth of 306 in 46% copper hydroxide at different dilutions under 0 μM NPM and 60 μM NPM conditions; C and D: respectively Xcm GXBS06 Xac Growth of quinoline copper at different dilutions in 306 under 0 μM NPM and 60 μM NPM conditions; E and F: respectively Xcm GXBS06 Xac Growth of 306 in 30% copper oxychloride at different dilutions under conditions of 0 μM NPM and 60 μM NPM. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0029] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0030] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0031] In the description of this invention, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0032] This invention provides the application of N-phenylmaleimide as a synergist for copper-based fungicides. The study found that the combination of the small molecule compound N-phenylmaleimide (NPM) with copper-based fungicides can significantly improve the efficacy of the fungicide, and has a certain synergistic fungicidal effect on the prevention and control of bacterial angular leaf spot in mangoes and citrus canker.

[0033] The molecular structure of N-phenylmaleimide is as follows: Targeting copper resistance-related targets, this method inhibits bacterial resistance to copper-based pesticides. Compared to traditional pesticide synergists, which reduce pesticide dosage and extend pesticide shelf life by delaying pesticide metabolism and enhancing the wetting, penetration, and anti-drift capabilities of pesticide solutions, this synergist screened for bacterial copper resistance targets is safer, greener, more targeted, and more efficient, thus having a broader application prospect.

[0034] Furthermore, copper-based fungicides can be selected from any one or more of copper sulfate, copper hydroxide, cuprous oxide, basic copper sulfate, copper oxychloride, quinoline copper, thiamethoxam copper, thiabendazole copper, amino acid copper, copper succinate, copper acetate, and copper rosinate, but are not limited to these. Among them, copper sulfate, as a broad-spectrum inorganic copper fungicide, is widely used in agricultural production. Its mechanism of action is through the binding of copper ions to proteins on the cell membrane of pathogens, causing protein denaturation and coagulation, thereby destroying the cell structure of pathogens and inhibiting their growth and reproduction.

[0035] This invention mixes N-phenylmaleimide with the above-mentioned copper-based fungicide in a certain ratio. The specific ratio can be adjusted according to different types of copper-based fungicides and target diseases. Within this ratio range, the synergistic effect is significant, which can effectively reduce the amount of copper-based fungicide used and improve the control effect on target diseases.

[0036] In practical applications, the mixed composition can be prepared into different formulations such as wettable powder, suspension, and water-in-oil emulsion to meet the needs of different crops and application methods. For example, wettable powder can be applied to the surface of crop leaves by spraying with water, while suspension has better dispersibility and stability and is suitable for a variety of application equipment.

[0037] The following examples illustrate in detail the application of N-phenylmaleimide provided by the present invention as a synergist for copper-based bactericides.

[0038] Example 1: Formulation of NPM and copper-based fungicide Accurately weigh 0.035 g of NPM powder using a balance, dissolve the weighed powder in 20 mL of anhydrous ethanol to prepare a 10 mM NPM stock solution for subsequent experiments; accurately weigh 1.25 g of copper sulfate pentahydrate powder, dissolve the accurately weighed powder in 50 mL of deionized water to prepare a 10 mM copper sulfate stock solution for subsequent experiments.

[0039] Example 2: Detection of the bactericidal effect of different concentrations of NPM mixed with copper sulfate on bacterial angular leaf spot pathogens of mango. The small molecule compound NPM was dissolved in anhydrous ethanol as a solvent and then treated with a certain OD value by mixing with a 10 mM copper sulfate mother liquor. 600 The bacterial suspension of *N. mongolica*, a fungicide for mango bacterial angular leaf spot, was analyzed using the dilution-spread method. The number of bacterial colonies on the plate was used to determine the synergistic effect of different concentrations of the small molecule compound NPM on the bactericidal efficacy of copper sulfate. The specific procedures are as follows:

[0040] In the laminar flow hood, pour NB medium into the small white bottle to one-third full, and use the sterile plate tip to pick up an appropriate amount of the activated strain from the plate ( XcmThe test strain was cultured overnight in a shaker at 200 rpm and 28 ℃ using GXBS06. The OD of the strain was then measured. 600 Adjust the value to 1.0, then dilute the bacterial solution to 10. -5 The concentrations of NPM (0, 20, 40, 60, 100, 200, and 400 μM) and copper sulfate (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mM) were mixed and added to NA medium according to the corresponding mixed concentrations. Copper sulfate at gradient concentrations was added to NA medium alone as a positive control. Each concentration was added to three replicate culture plates. 200 μL of bacterial culture was taken and spread on NA medium containing different copper ion and inhibitor concentrations. After the bacterial culture was dried, it was incubated at 28 ℃ for three days, and the bacterial growth was observed.

[0041] The results are as follows Figure 1 As shown, the results indicate that when the pathogen is dispersed on a solid culture medium in the absence of NPM, Xcm GXBS06 can resist copper ions smaller than 0.6 mM; under NPM-only conditions, a concentration of 400 μM can completely inhibit [copper ions]. Xcm The growth of GXBS06. And... Xcm GXBS06 showed antibacterial effects at low concentrations (20–60 μM) of NPM and low concentrations of copper ions (0.1–0.3 mM). It exhibited more significant antibacterial effects, even complete inhibition, at copper ion concentrations of 0.4–0.5 mM. Xcm Growth of GXBS06. At higher concentrations (100–400 μM) of NPM, it can achieve antibacterial effects with lower concentrations (0–0.2 mM) of copper ions.

[0042] In addition, the OD strain in the above experiment 600 Adjust the value to 0.2, then dilute the bacterial solution to 10. -5 Experiments were conducted using double-spot plate assays, with NPM concentrations adjusted to 0, 25, 50, 75, 100, and 125 μM. The results are as follows... Figure 1 As shown in B, it can be seen that Xcm GXBS06 showed significant antibacterial effects at 100 μM NPM and copper ion concentrations of 0.1–0.2 mM, and even at a copper ion concentration of 0.3 mM. Xcm The growth of GXBS06 was completely inhibited at a concentration of 200 μM NPM and a copper ion concentration of 0.1 mM. Xcm The growth of GXBS06 was essentially inhibited.

[0043] Furthermore, overnight cultivation XcmFor strain GXBS06, the OD value was adjusted to 1.0. 90 μL of liquid culture medium containing different concentrations of NPM (0, 25, 50, 100, 200, and 400 μM) and copper sulfate (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM) was added to each well of a 96-well plate. The plate was then incubated at 600 r / min for 24 h, and the OD value was measured using a microplate reader. 600 Values. The experiment was conducted three times, with three parallel groups each time. Results are shown in [reference needed]. Figure 2 .

[0044] like Figure 2 As shown, the antibacterial effect of copper sulfate gradually increases with the concentration of NPM, and the antibacterial effect of copper sulfate is significant when the NPM concentration is 25 μM.

[0045] Combination Figure 1 B and Figure 2 The result, with Figure 1 The A shown Xcm The antibacterial effect of GXBS06 is basically the same at different NPM concentrations.

[0046] Example 3: Detection of the bactericidal effect of different concentrations of NPM mixed with copper sulfate on citrus canker pathogens. The small molecule compound NPM was dissolved in anhydrous ethanol as a solvent and then treated with a certain OD value by mixing with a 10 mM copper sulfate mother liquor. 600 The bacterial suspension of *Bacillus cankerii*, the pathogen causing citrus canker, was analyzed using the dilution-spread method. The number of bacterial colonies on the plates was used to determine the synergistic effect of different concentrations of the small molecule compound NPM on the bactericidal efficacy of copper sulfate. The specific procedures are as follows: In the laminar flow hood, pour NB medium into the small white bottle to one-third full, and use the sterile plate tip to pick up an appropriate amount of the activated strain from the plate ( Xac The test strain was cultured overnight on a shaker at 200 rpm and 28 ℃ (306), and the OD of the strain was measured. 600 Adjust the value to 1.0, then dilute the bacterial solution to 10. -5 Different concentrations (0, 20, 40, 60, 100, 200, and 400 μM) of NPM and gradient concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM) of copper sulfate were added to NA medium. Copper sulfate at gradient concentrations was added to NA medium alone as a positive control. Each concentration was added to three replicate culture plates. 200 μL of each solution was spread on NA medium containing different concentrations of copper ions and inhibitors. After the bacterial culture was dried, it was incubated at 28 °C for three days, and the bacterial growth was observed.

[0047] like Figure 3As shown in Figure A, the results indicate that when the pathogen is dispersed on a solid culture medium in the absence of NPM, Xac 306 can resist copper ions smaller than 0.5 mM; under NPM-only conditions, a concentration of 400 μM can completely inhibit [the ion concentration]. Xac The growth of 306. And... Xac 306 showed significant antibacterial effects at low concentrations (20–60 μM) of NPM and low concentrations of copper ions (0.1–0.3 mM), and almost completely inhibited bacterial growth at concentrations of 0.4–0.5 mM. Xac The growth of 306. (From...) Figure 3 As can be seen from A, Xac 306, at 100 μM NPM, already showed a relatively significant antibacterial effect at copper ion concentrations of 0.1–0.2 mM, and even at a copper ion concentration of 0.3 mM... Xac The growth of 306 was completely inhibited at a concentration of 200 μM NPM and a copper ion concentration of 0.1 mM. Xac The growth of 306 was almost completely inhibited.

[0048] Furthermore, overnight cultivation Xac 306. Adjust the OD value of the strain to 1.0. Pipette 90 μL of liquid culture medium containing different concentrations of NPM (0, 25, 50, 100, 200, and 400 μM) and copper sulfate (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM) into each well of a 96-well plate. Add 10 μL of the test strain to each medium. Incubate at 600 r / min for 24 h. Measure the OD value using a microplate reader. 600 nm value. The experiment was conducted three times, with three parallel groups each time. The results are shown in [reference]. Figure 4 .

[0049] like Figure 4 As shown, the antibacterial effect of copper sulfate gradually increases with the concentration of NPM, and the antibacterial effect of copper sulfate is significant when the NPM concentration is 25 μM.

[0050] Combination Figure 3 B and Figure 4 The result, with Figure 3 The A shown Xac The antibacterial effect of 306 is basically the same at different NPM concentrations.

[0051] Example 4: Test on the pathogenicity of NPM mixed with copper sulfate in detached leaves for the control of citrus canker pathogen. Preparation of a suspension of citrus canker pathogen bacteria: ... XacAfter streaking 306 onto NA medium for 2 days, a single colony was picked and inoculated into NB liquid medium, and cultured overnight at 28°C. 600 =0.2, ready for inoculation.

[0052] Pathogenicity tests were conducted in an indoor incubator. Potted citrus varieties susceptible to the disease for over two years were purchased as experimental materials. Young leaves of similar size, color, and growth were selected as inoculation subjects. Before inoculation, the young leaves were disinfected with 0.8% sodium hypochlorite and washed three times with sterile water. Using a syringe needle, a bacterial solution containing NPM at certain concentrations (0, 20, 40, and 60 μM) and copper sulfate at certain concentrations (0.2 and 0.3 mM) was used to pierce the leaf from the underside, creating three evenly spaced perforations on both sides of the abaxial surface. 600 The bacterial suspension with a concentration of 0.2 was used as a positive control. Each treatment was repeated three times. After inoculation, the bacterial suspension was incubated at 28°C in the dark. The results were photographed and saved 14 days after inoculation.

[0053] like Figure 5 As shown, A is Xac Pathogenicity of plants under 306 and PBS conditions, B being... Xac Pathogenicity of 306 plants at copper sulfate concentrations of 0 μM NPM and 0.2 mM (left) / 0.3 mM (right), where C represents... Xac Pathogenicity of Xac 306 at 20 μM NPM and 0.2 mM (left) / 0.3 mM (right) copper sulfate concentrations, D represents the pathogenicity of Xac 306 at 40 μM NPM and 0.2 mM (left) / 0.3 mM (right) copper sulfate concentrations, E represents... Xac The pathogenicity of 306 in plants at copper sulfate concentrations of 60 μM NPM and 0.2 mM (left) / 0.3 mM (right). On citrus leaves, the disease manifests as grayish-brown spot-like lesions surrounded by a faint yellow halo. PBS was used as a negative control, eliminating the need for pathogen inoculation. Figure 5 In Figure A, the PBS on the right side is merely a small puncture wound. As the NPM concentration increases, the severity of the disease decreases at a 0.2 mM copper sulfate concentration, reaching its weakest point at an NPM concentration of 60 μM.

[0054] Example 5: Test of NPM and copper sulfate mixture on the control of bacterial angular leaf pathogens in mangoes. Preparation of a suspension of bacterial angular leaf spot fungus from mangoes: ... Xcm After streaking GXBS06 onto NA medium for 2 days, a single colony was picked and inoculated into NB liquid medium, and cultured overnight at 28°C. 600 =0.2, ready for inoculation.

[0055] Pathogenicity tests were conducted in an indoor incubator. Potted mango varieties susceptible to the disease for over two years were purchased as experimental materials. Young leaves of similar size, color, and growth were selected as inoculation subjects. Before inoculation, the young leaves were disinfected with 0.8% sodium hypochlorite and washed three times with sterile water. Using a syringe needle, a bacterial solution containing NPM at certain concentrations (0, 20, 40, and 60 μM) and copper sulfate at certain concentrations (0.2 and 0.3 mM) was used to puncture the leaf from the underside, creating three evenly spaced perforations on both sides of the abaxial surface. 600 The bacterial suspension with a concentration of 0.2 was used as a positive control. Each treatment was repeated three times. After inoculation, the bacterial suspension was incubated at 28°C in the dark. The results were photographed and saved 14 days after inoculation.

[0056] See results Figure 6 On mango leaves, dark brown raised lesions appear. With increasing NPM concentration, the severity of the disease decreases at a 0.2 mM copper sulfate concentration, but at an NPM concentration of 20 μM, the severity is almost identical to that of PBS. Figure 6 (A); As NPM concentration increases, the severity of disease decreases at a concentration of 0.3 mM copper sulfate, and at a concentration of 40 μM, it is almost identical to that of PBS ( ). Figure 6 B).

[0057] Example 6: Detection of the bactericidal effects of different dilutions of 46% copper hydroxide, quinoline copper, and 30% copper oxychloride mixed with NPM on mango bacterial angular leaf spot and citrus canker. The activated test strain was inoculated into NB medium containing the corresponding antibiotic and cultured in a shaker at 28°C and 200 rpm until the mid-to-late logarithmic growth phase. The bacterial culture concentration was then diluted to OD0.05. 600 =0.2, take 2 μL of bacterial suspension and spot it onto copper-based bactericides (diluted with water) at different dilution ratios and NPM at different concentrations. Figure 7 The samples were plated on NA plates (all concentrations and dilutions are after mixing), then incubated upside down at 28°C for 3 days. The growth of each sample was observed, and the influence of different concentrations of stress factors on the bacterial growth of the samples was determined based on the size and morphology of the colonies and their growth on plates under different stress concentrations. Photos were taken and saved, and three parallel samples were prepared for each sample, with three replicates.

[0058] The results are as follows Figure 7 As shown, A is Xcm Growth of GXBS06 under different dilutions of 46% copper hydroxide and in mixtures of 0 μM NPM and 60 μM NPM, B being... XacGrowth of 306 under different dilutions of 46% copper hydroxide and in mixtures of 0 μM NPM and 60 μM NPM, where C is... Xcm Growth of GXBS06 at different dilutions of quinoline copper and in mixtures of 0 μM NPM and 60 μM NPM, where D is... Xac Growth of 306 under different dilutions of quinoline copper and mixtures of 0 μM NPM and 60 μM NPM, where E represents... Xcm Growth of GXBS06 under different dilutions of 30% copper oxychloride and mixtures of 0 μM NPM and 60 μM NPM, F = Xac The growth of 306 under different dilutions of 30% copper hydroxide and in mixtures of 0 μM NPM and 60 μM NPM was investigated. The results showed that at different dilutions of 46% copper hydroxide, at a dilution of 10000, 60 μM NPM had the best effect on growth. Xcm GXBS06 and Xac 306 has a significant synergistic effect. Figure 7 A and Figure 7 (B) is much lower than the usage concentration of 46% copper hydroxide (1000~1500 times dilution); at different dilution ratios of quinoline copper concentration, at a dilution ratio of 50000 times, 60 μM NPM has a significant effect on... Xcm GXBS06 has a significant synergistic effect. Figure 7 (C); at different dilutions of quinoline copper, at a dilution of 2000 times, 60 μM NPM... Xac 306 has a synergistic effect ( Figure 7 (D); at different dilution factors, the effect of 60 μM NPM on 46% copper hydroxide concentration at a dilution factor of 10000: Xcm GXBS06 and Xac 306 has a significant synergistic effect. Figure 7 E and Figure 7 The concentration of F is much lower than that of 30% copper oxychloride (600~800 times dilution).

[0059] In summary, this invention utilizes N-phenylmaleimide as a synergist for copper-based fungicides. When mixed with copper-based fungicides such as copper sulfate, it significantly enhances the inhibitory effect against *Citrus canker* and *Citrus medicamentosa*, the pathogen causing bacterial angular leaf spot in mangoes. Both in in vitro antibacterial tests and in detached leaf pathogenicity tests, NPM exhibits a good synergistic effect. This provides strong experimental evidence for reducing the amount of copper-based fungicides used and mitigating potential environmental risks, demonstrating the significant application prospects of NPM in the field of green control of agricultural bacterial diseases.

[0060] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of N-phenylmaleimide as a synergist for copper-based bactericides.

2. The application according to claim 1, characterized in that, The applicable plant pathogens include: *Mango angularis* (a bacterium causing mango angular leaf spot). Xanthomonas citri pv. mangiferaeindicae ) or / and citrus canker pathogen ( Xanthomonas citri pv. citri ).

3. The application according to claim 1, characterized in that, The application involves mixing the copper-based bactericide with N-phenylmaleimide.

4. The application according to claim 1, characterized in that, The concentration of the N-phenylmaleimide is above 20 μM.

5. The application according to claim 4, characterized in that, The concentration of the N-phenylmaleimide is 20~400 μM.

6. The application according to any one of claims 1 to 5, characterized in that, The copper-based bactericide is selected from inorganic copper and / or organic copper bactericides.

7. The application according to claim 6, characterized in that, The copper-based bactericide is selected from any one or more of copper sulfate, copper hydroxide, cuprous oxide, basic copper sulfate, copper oxychloride, quinoline copper, thiamethoxam copper, thiamethoxam copper, amino acid copper, copper succinate, copper acetate, and copper rosinate.

8. The application according to claim 7, characterized in that, The copper-based bactericide is selected from copper sulfate, and the concentration of the copper sulfate is greater than 0 and less than or equal to 0.7 mM.

9. The application according to claim 7, characterized in that, The copper-based bactericide is selected from any one or more of 46% copper hydroxide, quinoline copper, and 30% copper oxychloride.

10. The application according to claim 9, characterized in that, The copper-based bactericide and N-phenylmaleimide are mixed, and the concentration of the copper-based bactericide in the mixture is diluted by less than 10,000 times.

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