Nanometer bactericide and preparation method and application thereof

Abstract

The application provides a nano bactericide and a preparation method and application thereof. The Mag@ZIF-8 nano composite material synthesized based on a competitive coordination strategy and containing two coordination structures of zinc ion-2-methyl imidazole and zinc ion-magnolol simultaneously, significantly increases the inhibition of ZIF-8 on pathogenic fungi. In addition, the results of the synergistic effect experiment show that there is a synergistic effect between Mag@ZIF-8 and the bactericide fludioxonil, which provides a new idea for the synergistic effect of the bactericide in a reduced amount.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a nano bactericide and a preparation method and application thereof. BACKGROUND

[0002] Metal-organic frameworks (MOFs) are a new type of porous crystalline material with periodic network structure formed by self-assembly of metal ions and organic ligands through coordination bonds. Due to the characteristics of large specific surface area, adjustable pore size and easy functionalization of the surface, MOFs are often used for the construction of nanomedicine delivery systems. In addition to being used as nanocarriers, the good antibacterial activity and unique antibacterial mechanism of MOFs have attracted the attention of many researchers. So far, a variety of MOFs (such as MOF-5, zeolite imidazole framework-8 (ZIF-8) and material institute lavoisier-100 (MIL-100)) have been developed as non-carrier type nano bactericides to inhibit the growth of pathogenic bacteria and improve the protection of hosts. ZIF-8, as a widely used MOF material in biomedicine, is connected by zinc ions and 2-methyl imidazole through coordination bonds, and can release zinc ions under acidic conditions to inhibit the growth and germination of pathogenic bacteria. ZIF-8 shows good inhibitory effect on pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and has great potential to be developed as a commercial bactericide. However, the biological activity of ZIF-8 on pathogenic fungi is low, which limits its application in the prevention and control of fungal diseases. Magnolol is a natural product extracted from the bark of Chinese magnolia, which shows broad-spectrum antibacterial activity and has the potential to increase the inhibitory effect of ZIF-8 on pathogenic fungi.

[0003] At present, due to the much lower sensitivity of pathogenic fungi to metal ion release mediated antibacterial effect than pathogenic bacteria, the research and application of antibacterial MOFs are more focused on the field of bacterial diseases. To solve this problem, researchers mainly introduce another antibacterial metal ion into MOFs through the strategy of constructing bimetallic MOFs to enhance their antibacterial activity, but this method does not essentially change the mode of action of antibacterial MOFs on pathogenic bacteria. SUMMARY

[0004] The purpose of the present application is to provide a new type of nano bactericide and a preparation method and application thereof.

[0005] In order to achieve the purpose of the present application, in a first aspect, the present application provides a nano bactericide, which is prepared by adding magnolol in the preparation process of metal-organic framework ZIF-8.

[0006] In a second aspect, the present application provides a preparation method of the nano-antiseptic, which is synthesized by one-pot method: adding magnolol to a mixed aqueous solution of zinc acetate dihydrate and 2-methylimidazole, collecting the precipitate by magnetic stirring and centrifugation, then washing the precipitate with anhydrous ethanol and deionized water in sequence to remove unreacted reactants, and finally drying and grinding to obtain the nano-antiseptic.

[0007] In the present application, the zinc acetate dihydrate can be replaced by zinc nitrate hexahydrate.

[0008] The preparation method comprises the following steps:

[0009] (1) respectively preparing 60-80 mg / mL zinc acetate dihydrate aqueous solution 25 mL, 200-300 mg / mL 2-methylimidazole aqueous solution 25 mL and 50-80 mg / mL magnolol ethanol solution 5 mL;

[0010] (2) adding the zinc acetate dihydrate aqueous solution dropwise into the 2-methylimidazole aqueous solution, when the addition is 10-20 mL, adding the magnolol ethanol solution dropwise into the mixed solution of zinc acetate dihydrate and 2-methylimidazole, and then adding the remaining zinc acetate dihydrate aqueous solution dropwise into the mixed solution, and stirring the reaction system by a magnetic stirrer during the whole addition process;

[0011] (3) after all the reactants are added, continuously stirring the reaction system by the magnetic stirrer for 1-2 hours;

[0012] (4) after the stirring is completed, collecting the precipitate by centrifugation, and washing the precipitate with anhydrous ethanol and deionized water in sequence, for 3-5 times;

[0013] (5) after the washed precipitate is dried in an oven, grinding the precipitate by a mortar to obtain nano-antiseptic powder (Mag@ZIF-8), which is stored in a 1.5 mL centrifuge tube for use.

[0014] Preferably, the magnetic stirring conditions in steps (2) and (3) are 20-25°C and 600-800 rpm.

[0015] Preferably, the centrifugation conditions in step (4) are 10000-12000 rpm and 3-5 minutes.

[0016] Preferably, the drying conditions in step (5) are 60-70°C and 12-16 hours.

[0017] In a specific embodiment of the present application, the preparation method of the nano-antiseptic comprises the following steps:

[0018] (1) respectively prepare 60 mg / mL zinc acetate dihydrate aqueous solution 25 mL, 224 mg / mL 2-methylimidazole aqueous solution 25 mL and 60 mg / mL magnolol ethanol solution 5 mL;

[0019] (2) add the zinc acetate dihydrate aqueous solution dropwise to the 2-methylimidazole aqueous solution, when 12.5 mL is added, add the magnolol ethanol solution dropwise to the mixture of the zinc acetate dihydrate and 2-methylimidazole, then add the remaining 12.5 mL of the zinc acetate dihydrate aqueous solution dropwise, and stir the reaction system through a magnetic stirrer during the whole dropwise adding process;

[0020] (3) after all the reactants are added, continue to stir the reaction system through the magnetic stirrer for 2 hours;

[0021] (4) after the stirring is completed, centrifugally collect the precipitate, and wash the precipitate with anhydrous ethanol and deionized water in sequence, for a total of 3 times;

[0022] (5) after the washed precipitate is dried, grind it, and the nano bactericide powder is obtained.

[0023] In a third aspect, the present application provides a compound bactericide, wherein the active ingredient is the nano bactericide and fludioxonil.

[0024] Preferably, the mass ratio of the nano bactericide and fludioxonil is 1:1.

[0025] In a fourth aspect, the present application provides any one of the following applications of the nano bactericide or the compound bactericide or a material containing the nano bactericide or the compound bactericide:

[0026] 1) for preparing a broad-spectrum antibacterial agent;

[0027] 2) for inhibiting pathogenic fungi;

[0028] 3) for fungal disease prevention and control;

[0029] 4) for developing a new type of bacteriostatic material.

[0030] In the present application, the fungi include but are not limited to Botrytis cinerea (Botrytis cinerea Botrytis cinerea ), Fusarium graminearum (Fusarium graminearum Fusarium graminearum ), Rhizoctonia solani (Rhizoctonia solani Rhizoctonia solani ), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum Sclerotinia sclerotiorum ), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides Colletotrichum gloeosporioides ), and Phoma exigua (Phoma exigua Colletotrichum acutatum ).

[0031] Through the above technical solution, the present application has at least the following advantages and beneficial effects:

[0032] The magnolol / ZIF-8 nanocomposite (Mag@ZIF-8) synthesized based on the competitive coordination strategy contains two coordination structures of zinc ion-2-methyl imidazole and zinc ion-magnolol, and the inhibition of ZIF-8 on the pathogenic fungi is significantly increased. The bioassay experiment results show that the median effective concentration (EC 50 ) of ZIF-8 on the mycelium growth of Botrytis cinerea is 886.1 μg / mL, and the EC 50 of Mag@ZIF-8 on the mycelium growth of Botrytis cinerea is 68.3 μg / mL. It is shown that under the same experimental conditions, the inhibition activity of Mag@ZIF-8 on the pathogenic fungi is higher than that of ZIF-8, and it is also shown that the addition of magnolol significantly improves the inhibition of ZIF-8 on the pathogenic fungi.

[0033] In addition, the compounding and synergistic experiment results show that the actual inhibition rate (67.8%) of the binary mixture (1:1) of Mag@ZIF-8 and the fungicide fludioxonil on the mycelium growth of Botrytis cinerea is higher than the theoretical inhibition rate (57.1%), and it is shown that there is a synergistic effect between Mag@ZIF-8 and fludioxonil, which provides a new idea for the reduction and synergistic effect of fungicides. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is the characterization of ZIF-8 and Mag@ZIF-8 in the preferred embodiment of the present application. (A) Transmission electron microscope images of ZIF-8 and Mag@ZIF-8. (B) Particle size distribution of ZIF-8 and Mag@ZIF-8. (C) Macroscopic morphology of ZIF-8 and Mag@ZIF-8 suspensions.

[0035] Figure 2 It is the inhibition of ZIF-8 (A) and Mag@ZIF-8 (B) on the mycelium growth of Botrytis cinerea in the preferred embodiment of the present application.

[0036] Figure 3 It is the antibacterial activity of Mag@ZIF-8 on pathogenic fungi such as Fusarium graminearum (A), Pellicularia sasakii (B), Sclerotinia sclerotiorum (C), Colletotrichum gloeosporioides (D) and Phaeosphaeria nodorum (E) in the preferred embodiment of the present application.

[0037] Figure 4 It is the compounding and synergistic effect between Mag@ZIF-8 and common fungicides in the preferred embodiment of the present application. DETAILED DESCRIPTION

[0038] The present application aims to provide a kind of nano fungicide and its synthesis method, and its application in fungal disease prevention and control.

[0039] The application adopts the following technical solutions:

[0040] The application synthesizes the nanometer bactericide Mag@ZIF-8 by one-pot method. Specifically, by adding magnolol to a mixed aqueous solution of zinc acetate dihydrate and 2-methylimidazole, a precipitate is obtained by magnetic stirring and centrifugation, and then the unreacted reactants are removed by washing with anhydrous ethanol and deionized water. Finally, Mag@ZIF-8 powder is obtained by drying and grinding. Mag@ZIF-8 is a nanosphere with an average particle size of 148.7 nm. The water suspension of Mag@ZIF-8 is tan.

[0041] The Mag@ZIF-8 of the application has better inhibitory effect on the growth of Botrytis cinerea than ZIF-8, and can be compounded with the bactericide fludioxonil to enhance the effect.

[0042] The following examples are used to illustrate the application, but are not used to limit the scope of the application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0043] The Botrytis cinerea B05.10 used in the following examples was provided by Mr. Hou Yiping of the College of Plant Protection, Nanjing Agricultural University. The strain B05.10 can be referred to Bian, C., Duan, Y., Wang, J., Xiu, Q., Wang, J., Hou, Y.,... & Zhou, M. (2020). Validamycin A induces broad-spectrum resistance involving salicylic acid and jasmonic acid / ethylene signaling pathways. Molecular Plant-Microbe Interactions, 33(12), 1424-1437.

[0044] Fusarium graminearum (PH-1), Sclerotinia sclerotiorum (YD5), Colletotrichum gloeosporioides (LN-48) and Colletotrichum acutatum (HTTJ1) were provided by Mr. Liu Feng of the College of Plant Protection, Shandong Agricultural University, and Rhizoctonia solani (AG4) was provided by Mr. Liu Wende of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Fusarium graminearum can be referred to Ma, D., Wang, G., Zhu, J., Mu, W., Dou, D., & Liu, F. (2022). Green leaf volatile trans-2-hexenal inhibits the growth of Fusarium graminearumby inducing membrane damage, ROS accumulation, and cell dysfunction. Journal of Agricultural and Food Chemistry, 70(18), 5646-5657. Rhynchosporium can be found in Zhang, Z., Xia, X., Du, Q., Xia, L., Ma, X., Li, Q., & Liu, W. (2021). Genome sequence of Rhynchosporium oryzae from different hosts in Shandong, China. Plant Disease, 103(1), 34-43. Rhizoctonia solani anastomosis group 4 strain Rhs4ca, a widespread pathomycete in field crops. Molecular Plant-Microbe Interactions, 34(7), 826-829. Sclerotinia can be found in Huang, X., Luo, J., Li, B, Song, Y, Mu, W., & Liu, F. (2019). Bioactivity, physiological characteristics and efficacy of the SDHI fungicide pydiflumetofen against Sclerotinia sclerotiorum. Pest Management Science, 75(10), 2570-2577. Sclerotinia sclerotiorum . Pesticide Biochemistry and Physiology, 160, 70-78. Colletotrichum gloeosporioides and Colletotrichum acutatum can be found in He, L., Li, X., Gao, Y., Li, B, Mu, W., & Liu, F. (2019). Characterization and fungicide sensitivity of Colletotrichum gloeosporioides and C. acutatum from different hosts in Shandong, China. Plant Disease, 103(1), 34-43. Colletotrichum

[0045] The honokiol used in the following examples was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. with the item number M813634, and fludioxonil was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. with the item number F980645.

[0046] Example 1 Synthesis of ZIF-8, Mag@ZIF-8

[0047] ​Mag@ZIF-8 was synthesized by one-pot method, and the specific steps were as follows:

[0048] (1) Prepare zinc acetate dihydrate aqueous solution (25 mL, 60 mg / mL), 2-methylimidazole aqueous solution (25 mL, 224 mg / mL) and magnolol ethanol solution (5 mL, 60 mg / mL) respectively;

[0049] (2) Add zinc acetate dihydrate aqueous solution dropwise to 2-methylimidazole aqueous solution through a rubber bulb dropper, and when half (12.5 mL) is added, add magnolol ethanol solution dropwise to the mixture of zinc acetate dihydrate and 2-methylimidazole, then add the remaining zinc acetate dihydrate aqueous solution (12.5 mL) dropwise, and stir the reaction system through a magnetic stirrer (25°C, 600 rpm) during the whole process;

[0050] (3) After all the reactants are added, continue to stir the reaction system through a magnetic stirrer (25°C, 600 rpm) for 2 hours;

[0051] (4) After stirring, centrifuge the reaction product through a centrifuge (10000 rpm, 3 minutes), and wash the obtained precipitate with anhydrous ethanol and deionized water to remove unreacted reactants, a total of three times;

[0052] (5) After drying the washed precipitate in an oven (60°C, 12 hours), grind the precipitate thoroughly through a mortar, and finally obtain Mag@ZIF-8 powder, which is stored in a 1.5 mL centrifuge tube for use.

[0053] The synthesis steps of ZIF-8 are the same as those of Mag@ZIF-8, except that magnolol is not added.

[0054] Example 2 Characterization of ZIF-8 and Mag@ZIF-8

[0055] An appropriate amount of Mag@ZIF-8 powder was added to a centrifuge tube containing deionized water to prepare a 250 μg / mL suspension. Then, the suspension was placed in an ultrasonic disrupter (power 500W, 30 minutes) for ultrasonic treatment to uniformly disperse Mag@ZIF-8 in deionized water. The preparation of ZIF-8 suspension also refers to the above method.

[0056] 5 μL of the Mag@ZIF-8 suspension was dropped on the grid with a pipette, and the nanoparticles were fixed on the grid by natural air-drying at room temperature for 12 hours. The prepared sample was stored in a desiccator to prevent the sample from being damp or contaminated. The micro-morphology of the Mag@ZIF-8 was observed and photographed by transmission electron microscopy (model: Tecnai G2 F30, FEI Company, USA). The particle size of the nanoparticles in the transmission electron microscopy photos was counted by ImageJ software (version: 1.52a, National Institutes of Health, USA), and the particle size distribution of the nanoparticles was analyzed by GraphPad software (version: 8.0.2, GraphPad Software, USA). ZIF-8 was used as a control. The results showed that the ZIF-8 nanoparticles were polyhedron, with an average particle size of 209.1 nm; the Mag@ZIF-8 nanoparticles were nearly spherical, with an average particle size of 148.7 nm Figure 1 ).

[0057] 1.5 mL of the Mag@ZIF-8 suspension was taken into a sample injection vial with a pipette, and the macroscopic state of the suspension was observed and photographed by a digital camera (model: EOS R100, Canon, Japan). ZIF-8 suspension was used as a control. The results showed that the ZIF-8 suspension was white, while the Mag@ZIF-8 suspension was tan Figure 1 ).

[0058] Example 3 Inhibition of Botrytis cinerea mycelial growth by ZIF-8 and Mag@ZIF-8

[0059] The inhibition of Botrytis cinerea mycelial growth by ZIF-8 and Mag@ZIF-8 was determined by the mycelial growth rate method, and the specific steps were as follows:

[0060] (1) A proper amount of Mag@ZIF-8 powder was added to a centrifuge tube containing sterilized deionized water to prepare a 5000 μg / mL stock solution, and the nanoparticles were dispersed by ultrasonic for 30 minutes. The stock solution was diluted to 2500, 1250, 625 and 312.5 μg / mL with deionized water. The preparation of ZIF-8 test solution also referred to the above method;

[0061] (2) A proper amount of Mag@ZIF-8 suspension was added to the pre-cooled potato dextrose agar (PDA) medium at about 50°C to make the final concentration of 0, 31.25, 62.5, 125, 250 and 500 μg / mL, and then vortexed and poured into disposable plastic culture dishes (15 mL / dish). After drying, the mycelium was inoculated with Botrytis cinerea (strain B05.10) fungus cake (diameter 7 mm) with the mycelium facing down. ZIF-8 with the same concentration was used as a control.

[0062] (3) After inoculation, the culture dish was placed in a 25℃, dark condition for culture;

[0063] (4) After 2 days of culture, the colony expansion diameter was measured by the cross method to calculate the inhibition rate, and the DPS software (version: v9.05, Hangzhou Ruifeng Information Technology Co., Ltd.) was used to convert the test concentration and the inhibition rate to calculate the EC 50 value.

[0064] The results show that the EC 50 value of ZIF-8 on the mycelial growth of Botrytis cinerea is 886.1 μg / mL, and the EC 50 value of Mag@ZIF-8 on the mycelial growth of Botrytis cinerea is 68.3 μg / mL, indicating that the inhibition activity of Mag@ZIF-8 on Botrytis cinerea is obviously higher than that of ZIF-8 (P < 0.05). Figure 2

[0065] Example 4 Inhibition activity of Mag@ZIF-8 on Fusarium graminearum and other pathogenic fungi

[0066] The inhibition effect of Mag@ZIF-8 on the mycelial growth of Fusarium graminearum and other five kinds of pathogenic fungi was determined by the mycelial growth rate method, and the specific steps were as follows:

[0067] (1) Mag@ZIF-8 suspensions with concentrations of 312.5, 625, 1250, 2500 and 5000 μg / mL were prepared with deionized water;

[0068] (2) An appropriate amount of Mag@ZIF-8 suspension was added to the PDA medium pre-cooled to about 50℃ to make the final concentration 0, 31.25, 62.5, 125, 250 and 500 μg / mL, and then mixed and poured into a plastic culture dish;

[0069] (3) The pathogenic fungi (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides or Phoma exigua var. fischeri) were inoculated in the dried culture dish with a diameter of 7 mm, and the inoculated culture dish was transferred to a 25℃, dark condition for culture;

[0070] (4) After 2-5 days of culture, the colony expansion diameter was measured by the cross method to calculate the EC 50 value of Mag@ZIF-8 on the mycelial growth.

[0071] The results show that Mag@ZIF-8 exhibits good inhibition activity on Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides and Phoma exigua var. fischeri, and the EC 50 ​The values are 113.6, 148.1, 103.1, 107.5 and 207.8 μg / mL, respectively Figure 3 ).

[0072] Example 5 Synergistic effect evaluation of Mag@ZIF-8 and fungicides

[0073] The synergistic effect of Mag@ZIF-8 and 8 commonly used fungicides was evaluated by simple mixing method, and the specific steps were as follows:

[0074] (1) Mag@ZIF-8 (2500 μg / mL), carbendazim (40 μg / mL), boscalid (25 μg / mL), pyraclostrobin (2 μg / mL), propiconazole (0.5 μg / mL), procymidone (2.5 μg / mL), fludioxonil (0.05 μg / mL), dimethirimol (1 μg / mL) and probenazole (0.5 μg / mL) test solutions were prepared with sterilized deionized water;

[0075] (2) Mag@ZIF-8 (1.5 mL) and single fungicide (1.5 mL) test solutions were added to PDA medium (15 mL) pre-cooled to about 50°C at the same time, and then vortexed and poured into a plastic culture dish. PDA plates containing only deionized water, Mag@ZIF-8 or fungicides were used as controls;

[0076] (3) A piece of B. cinerea (strain B05.10) mycelium cake (7 mm in diameter) was inoculated into the dried culture dish, and the inoculated culture dish was transferred to 25°C in the dark for incubation;

[0077] (4) After 2 days of incubation, the colony expansion diameter was measured by cross method to calculate the actual joint inhibition rate of Mag@ZIF-8 and fungicide binary mixture. The theoretical joint inhibition rate of Mag@ZIF-8 and fungicide binary mixture = inhibition rate of Mag@ZIF-8 + inhibition rate of fungicide. If the actual joint inhibition rate of Mag@ZIF-8 and fungicide binary mixture is higher than / equal to / lower than its theoretical joint inhibition rate, it indicates that there is synergistic / additive / antagonistic effect between them.

[0078] The results showed that there was synergistic effect between Mag@ZIF-8 and fludioxonil (actual joint inhibition rate 67.8%> theoretical joint inhibition rate 57.1%), additive effect between Mag@ZIF-8 and carbendazim (actual joint inhibition rate 30.8%=theoretical joint inhibition rate 30.8%), antagonistic effect between Mag@ZIF-8 and boscalid (actual joint inhibition rate 50.9%<theoretical joint inhibition rate 69.6%), azoxystrobin (actual joint inhibition rate 38.8%<theoretical joint inhibition rate 54.5%), propiconazole (actual joint inhibition rate 49.1%<theoretical joint inhibition rate 60.3%), procymidone (actual joint inhibition rate 32.1%<theoretical joint inhibition rate 50.4%), dimethirimol (actual joint inhibition rate 32.1%<theoretical joint inhibition rate 50.4%) and pyrimethanil (actual joint inhibition rate 33.5%<theoretical joint inhibition rate 39.7%) Figure 4

[0079] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.​

Claims

1. A nano-bactericide, characterized in that, It is a magnolol / ZIF-8 nanocomposite material prepared by adding magnolol during the preparation of metal-organic framework ZIF-8. The nanocomposite material contains two coordination structures: zinc ion-2-methylimidazole and zinc ion-magnolol. The nano-bactericide is synthesized using a one-pot method, comprising the following steps: (1) Prepare 25 mL of 60-100 mg / mL zinc acetate dihydrate aqueous solution, 25 mL of 200-300 mg / mL 2-methylimidazole aqueous solution and 5 mL of 50-80 mg / mL magnolol ethanol solution respectively; (2) Add zinc acetate dihydrate aqueous solution dropwise to 2-methylimidazole aqueous solution. When the solution is 10-20 mL, add magnolol ethanol solution dropwise to the mixture of zinc acetate dihydrate and 2-methylimidazole. Then add the remaining zinc acetate dihydrate aqueous solution dropwise. Stir the reaction system with a magnetic stirrer throughout the dropwise addition process. (3) After all reactants have been added, continue to stir the reaction system continuously with a magnetic stirrer for 1-2 hours. (4) After stirring, centrifuge to collect the precipitate, and wash the precipitate with anhydrous ethanol and deionized water in sequence, for a total of 3-5 times; (5) After washing, the precipitate is dried and then ground to obtain nano-bacterial agent powder.

2. The preparation method of the nano-bactericide according to claim 1, characterized in that, The nano-bactericide is synthesized using a one-pot method, comprising the following steps: (1) Prepare 25 mL of 60-100 mg / mL zinc acetate dihydrate aqueous solution, 25 mL of 200-300 mg / mL 2-methylimidazole aqueous solution and 5 mL of 50-80 mg / mL magnolol ethanol solution respectively; (2) Add zinc acetate dihydrate aqueous solution dropwise to 2-methylimidazole aqueous solution. When the solution is 10-20 mL, add magnolol ethanol solution dropwise to the mixture of zinc acetate dihydrate and 2-methylimidazole. Then add the remaining zinc acetate dihydrate aqueous solution dropwise. Stir the reaction system with a magnetic stirrer throughout the dropwise addition process. (3) After all reactants have been added, continue to stir the reaction system with a magnetic stirrer for 1-2 hours. (4) After stirring, centrifuge to collect the precipitate, and wash the precipitate with anhydrous ethanol and deionized water in sequence, for a total of 3-5 times; (5) After washing, the precipitate is dried and then ground to obtain nano-bacterial agent powder.

3. The method according to claim 2, characterized in that, The conditions for magnetic stirring in steps (2) and (3) are: 20-25℃, 600-800rpm.

4. The method according to claim 2, characterized in that, The centrifugation conditions in step (4) are: 10000-12000 rpm, 3-5 minutes.

5. The method according to claim 2, characterized in that, The drying conditions in step (5) are: 60-70℃, 12-16 hours.

6. A compound bactericide, characterized in that, The active ingredients are the nano-bactericide described in claim 1 and fludioxonil.

7. The compound bactericide according to claim 6, characterized in that, The mass ratio of the nano-bactericide to fludioxonil is 1:

1.

8. Any of the following applications of the nano-bactericide of claim 1, the compound bactericide of claim 6 or 7, or a material containing the nano-bactericide of claim 1 or the compound bactericide of claim 6 or 7: A) Used for the prevention and control of fungal diseases; B) Used for developing novel antibacterial materials.

9. Any of the following applications of the nano-bactericide of claim 1, the compound bactericide of claim 6 or 7, or a material containing the nano-bactericide of claim 1 or the compound bactericide of claim 6 or 7: a) Used to inhibit pathogenic fungi; b) Used in the preparation of broad-spectrum antibacterial agents.

10. The application according to claim 8 or 9, characterized in that, The fungi mentioned include Botrytiscinerea, Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides, and Colletotrichum acutatum.

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