Antibacterial nano-preparation, preparation method and application thereof

Abstract

The application discloses an antibacterial nano preparation and a preparation method thereof, which is obtained by loading organic-inorganic hybrid polyiodide crystal materials on a nano carrier POA modified by oleylamine; and further discloses application of the organic-inorganic hybrid polyiodide crystal materials or the antibacterial nano preparation in inhibiting plant pathogenic bacteria or preventing and treating plant diseases. The organic-inorganic hybrid polyiodide crystal materials are loaded on the POA nano carrier, and water solubility of the organic-inorganic hybrid polyiodide crystal materials is improved through nanocrystallization treatment; safety and penetrability of the organic-inorganic hybrid polyiodide crystal materials are significantly improved; and experiments prove that the organic-inorganic hybrid polyiodide crystal materials have a significant bactericidal effect in prevention and treatment of citrus Huanglongbing. The antibacterial nano preparation POA@I is used as a composite carrier to deliver antibacterial drugs for preventing and treating plant infectious diseases, thereby widening application of the POA in agricultural drug delivery, widening new application of the polyiodide crystal materials, and providing a new drug for prevention and treatment of citrus Huanglongbing.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease prevention and control, and in particular to an antibacterial nano preparation and a preparation method and application thereof. Background Art

[0002] Huanglongbing (HLB), a devastating citrus disease that has caused severe economic losses in citrus-producing areas worldwide, has been a major challenge for fruit growers and agricultural researchers since its discovery.

[0003] Huanglongbing is primarily caused by Candidatus Liberibacter, which can be divided into Asian species (Clas), African species (CLaf), and American species (CLam). This pathogen is spread between citrus trees by vector insects such as the citrus psyllid (Diaphorina citri). Once a citrus tree is infected with Huanglongbing, its initial symptoms often manifest as mottled yellowing of leaves and yellowing of branches, followed by gradual reduction in size, deformity, and abnormal coloring of the fruit, ultimately leading to the decline and death of the entire tree. Because the Huanglongbing pathogen cannot be cultured in vitro, the current prevention and control method used in production is still a three-pronged strategy. In the field of chemical control, the effect of existing fungicides against the Huanglongbing pathogen is not ideal. On the one hand, since the Huanglongbing pathogen grows in the phloem of citrus trees, its special living environment makes it difficult for many conventional fungicides to accurately reach the target and exert an effective fungicidal effect; on the other hand, the pathogen has developed a certain resistance to some existing fungicides, further reducing the application value of these fungicides in the prevention and control of Huanglongbing.

[0004] Compared with traditional methods, nano-delivery systems show many advantages, such as enhancing bioavailability and reducing losses, achieving efficient circulation of chemicals, achieving slow and stable release of drugs, and reducing phytotoxicity. Nanopesticides can be used to treat plant diseases. Most synthetic pesticides contain hydrophobic active ingredients (AIs), which can be encapsulated or attached to the hydrophobic groups of nanoparticles, thereby improving the performance of the original pesticide's active ingredients, targeted transport, pesticide protection, and pesticide release control functions. Compared with traditional pesticides, nanopesticides have the advantages of low dosage and high efficiency.

[0005] As a commercial bactericidal agent, elemental iodine has been widely used in the medical field, thanks to its high efficiency and broad-spectrum antibacterial properties, which can effectively fight bacteria, fungi and viruses. Compared with other antibacterial agents, iodine-containing antibacterial agents show significant advantages in reducing the generation and spread of drug-resistant bacteria. However, elemental iodine itself has some limitations, such as high volatility, poor chemical stability, and difficulty in modification. These shortcomings restrict its wider application. In order to overcome these limitations, combining active iodine with functional materials and developing new functional composite materials has become a hot research direction. The organic-inorganic hybrid polyiodine crystal materials reported in recent years have largely solved the problem of iodine stability, but their poor water solubility limits the possibility of direct application. Summary of the Invention

[0006] The purpose of the present invention is to provide an antibacterial nanoparticle preparation and its preparation method and application in response to the above problems.

[0007] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides an antibacterial nanoformulation, which is obtained by loading an organic-inorganic hybrid polyiodine crystal material onto a nanocarrier POA;

[0009] The nanocarrier POA is a modified polysuccinimide nanoparticle prepared by modifying polysuccinimide with oleylamine;

[0010] The molecular formula of the organic-inorganic hybrid polyiodine crystal material is [(C6 H 21 I 26 O3 S3)2] n , where n is a natural number greater than or equal to 1.

[0011] The nanocarrier POA is obtained by mixing polysuccinimide and oleylamine in an organic solvent A, reacting at 60-100° C. for 2-12 hours, and then washing to remove the organic solvent A.

[0012] Preferably, the organic solvent A is N,N-dimethylformamide, and the number average molecular weight of the polysuccinimide is 6000-10000.

[0013] The preparation method of the nanocarrier POA comprises the following steps: adding polysuccinimide and oleylamine to an organic solvent A, mixing them in a mass volume ratio of polysuccinimide to oleylamine of 1 g:0.6-1.4 ml (preferably 1 g:0.8-1.2 ml); stirring and reacting the mixture at 60-100° C. (preferably 60-80° C.) for 2-12 hours (preferably 6-12 hours); cooling the reaction product, washing it with an organic solvent B to remove the organic solvent A; and collecting the precipitate by centrifugation to obtain the nanocarrier POA, wherein the organic solvent B is methanol or ethanol.

[0014] Preferably, the precipitate collected by centrifugation after washing is freeze-dried in vacuum to form a freeze-dried powder.

[0015] The second aspect of the present invention provides a method for preparing any of the above-mentioned antibacterial nanoformulations, comprising the following steps:

[0016] The nanocarrier POA and the organic-inorganic hybrid polyiodine crystal material are added to an organic solvent C and mixed to obtain a polymer mixture; polyvinyl alcohol is added to the polymer mixture, mixed thoroughly, and then reacted under stirring. After the reaction is completed, the nanoparticles are collected by centrifugation and washed to obtain the antibacterial nanoformulation.

[0017] Preferably, in the technical solution of the preparation method, the mass ratio of the organic-inorganic hybrid polyiodine crystalline material to POA in the polymer mixture is 1:5-15 (preferably 1:8-12); preferably, the concentrations of POA and the organic-inorganic hybrid polyiodine crystalline material in the polymer mixture are 10-30 mg / mL (preferably 15-25 mg / mL) and 1-5 mg / mL (preferably 1-3 mg / mL), respectively;

[0018] The polyvinyl alcohol is a 2-3 wt% polyvinyl alcohol solution, and the volume ratio of the polymer mixture to the polyvinyl alcohol solution is 1:7-13;

[0019] The organic solvent C is dimethyl sulfoxide or chloroform.

[0020] Preferably, in the technical solution of the preparation method, after adding polyvinyl alcohol to the polymer mixture, ultrasonic treatment is performed to fully mix the components, and the ultrasonic treatment conditions are: 160-200W, 1.5-3.0min; after ultrasonic treatment, the reaction is stirred for 2-16 hours (preferably 6-12 hours);

[0021] After the reaction is completed, the nanoparticles collected by centrifugation are washed with deionized water to remove the polyvinyl alcohol, thereby obtaining the antibacterial nanoparticle preparation.

[0022] The third aspect of the present invention provides an organic-inorganic hybrid polyiodine crystal material or any of the above-mentioned antibacterial nanoformulations for inhibiting plant pathogens or preventing and controlling plant diseases; the molecular formula of the organic-inorganic hybrid polyiodine crystal material is [(C6 H 21 I 26 O3 S3)2] n , where n is a natural number greater than or equal to 1.

[0023] In the application technology solution, the plant pathogens include pathogenic bacteria or fungi; and the plant diseases are plant diseases caused by bacterial or fungal pathogens.

[0024] In the application technology solution, the plant disease is Huanglongbing disease of Rutaceae plants, and the Rutaceae plants refer to Citrus, Poncirus, Fortunella or Murraya.

[0025] In the application technology scheme, the antibacterial nanoformulation is prepared into a solution and then administered by injection or spraying; the working concentration of the antibacterial nanoformulation is 150-1500 μg / mL, preferably 180-1000 μg / mL or 350-1000 μg / mL or 350-800 μg / mL.

[0026] The beneficial effects of the present invention are:

[0027] Organic-inorganic hybrid polyiodine crystalline materials are a new iodine material in recent years. They are fluid above 6°C and have high light and heat stability below 100°C, overcoming the volatilization of elemental iodine above 4°C. However, organic-inorganic hybrid polyiodine crystalline materials have the characteristics of self-assembly and easily polymerize into high polymers. Their large molecular size prevents them from entering plant cells, greatly limiting their application in plant disease control. The present invention loads them onto POA nanocarriers and enhances their water solubility through nano-processing, significantly improving their safety and penetrability. Experiments have confirmed that they have a significant bactericidal effect in the prevention and treatment of citrus Huanglongbing.

[0028] The present invention uses oleylamine to modify the surface of polysuccinimide to obtain a nanocarrier POA, which successfully loads an organic-inorganic hybrid polyiodine crystal material to obtain the antibacterial nanoformulation POA@I. This is applied to citrus Huanglongbing, achieving a three-dimensional control of citrus HLB, including inhibiting the growth of CLas, activating the plant antioxidant system, and activating the plant SAR. The antibacterial nanoformulation of the present invention exhibited strong antibacterial activity in in vitro and in vivo antibacterial tests. While not causing abiotic stress to infected citrus leaves, it increased antioxidant enzyme activity by upregulating antioxidant-related genes, alleviated oxidative stress in infected sweet orange leaves, and increased the chlorophyll content in the leaves. It also significantly upregulated defense marker genes, activated the SAR pathway, and successfully inhibited the growth of CLas.

[0029] The POA@I of the present invention is used as a composite carrier to deliver antibacterial drugs for the prevention and treatment of plant infectious diseases, which broadens the application of POA in agricultural drug delivery and also broadens the new application of organic-inorganic hybrid polyiodine crystal materials. At the same time, it provides a new drug for the prevention and treatment of citrus Huanglongbing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Characterization and dispersion of POA@I: (A)(B) TEM electron micrographs of PSI; (C)(D) TEM electron micrographs of POA@I; (E) SEM electron micrographs of POA@I; (F) images of PI (caged iodine) (left) and POA@I (right) dispersed in aqueous solution; (G) UV-visible absorption spectra of PSI, PI (caged iodine), POA and POA@I; (H) IR spectra of PI (caged iodine), POA and POA@I; (I) Zeta potential of PSI, PI (caged iodine), POA and POA@I.

[0031] Figure 2 A standard curve for caged iodine is shown.

[0032] Figure 3 The in vitro antibacterial activity of POA@I is shown: (A) Inhibition rate of different concentrations of POA@I against Gram-negative bacteria Sinorhizobium fredii; (B) Dilution coating plate of Sinorhizobium fredii.

[0033] Figure 4 The in vitro antibacterial activity of PI (caged iodine) is shown: (A) Inhibition rate of different concentrations of I against Gram-negative bacteria Sinorhizobium fredii; (B) Dilution coating plate of Sinorhizobium fredii.

[0034] Figure 5The in vitro antibacterial activity of POA is shown: (A) Inhibition rate of different concentrations of POA against Gram-negative bacteria Sinorhizobium fredii; (B) Dilution coating plate of Sinorhizobium fredii.

[0035] Figure 6 The relative changes in pigments in HLB-positive sweet orange leaves treated with POA@I within nine days, as well as the determination results of pigment contents in HLB-positive and healthy sweet orange leaves: (A) relative chlorophyll a content; (B) relative chlorophyll b content; (C) relative total chlorophyll content; (D) relative carotenoid content; (E) chlorophyll a content; (F) chlorophyll b content; (G) total chlorophyll content; (H) carotenoid content.

[0036] Figure 7 The relative changes in H2O2 and antioxidant enzyme activities in HLB-positive sweet orange leaves treated with POA@I within nine days, as well as the determination results of H2O2 and antioxidant enzyme activities in Huanglongbing-positive and healthy sweet orange leaves: (A) relative change in H2O2; (B) relative change in CAT activity; (C) relative change in POD activity; (D) relative change in SOD activity; (E) H2O2 activity; (F) CAT activity; (G) POD activity; (H) SOD activity.

[0037] Figure 8 The results of POA@I regulation on antioxidant-related genes and defense marker genes in HLB-positive sweet orange leaves: (A) relative expression level of HLBas2; (B) relative expression level of SOD-3; (C) relative expression level of SOD-1; (D) relative expression level of SOD-2; (E) relative expression level of CAT-1; (F) relative expression level of CAT-2; (G) relative expression level of POD; (H) relative expression level of APX; (I) relative expression level of PAL; (J) relative expression level of RbohA; (K) relative expression level of PR-1; (L) relative expression level of PR-2. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0040] Example 1

[0041] 1 Experimental Materials

[0042] 1.1 Main experimental reagents

[0043]

[0044] The organic-inorganic hybrid polyiodine crystal material used in the present invention is a caged organic-inorganic hybrid polyiodine crystal material in Chinese Patent ZL202211219738.8 (Announcement No. CN 116254608B, Invention Name: A Caged Organic-Inorganic Hybrid Polyiodine Crystal Material and Its Preparation Method and Application), and its molecular formula is [(C6H 21 I 26 O3 S3)2] n , where n is a natural number greater than or equal to 1; it is a triclinic crystal system with a space group of With the following unit cell parameters: α=97.8884(125)°,β=97.4394(119)°,γ=109.5092(103)°,unit cell volume The number of molecules in the unit cell Z = 2. The preparation method of the organic-inorganic hybrid polyiodine crystal material refers to Chinese Patent ZL202211219738.8. In the embodiments of the present invention, the organic-inorganic hybrid polyiodine crystal material is referred to as caged iodine.

[0045] 1.2 Solutions and culture medium formulations used

[0046] 1×PBS: 0.01 mol / L PBS buffer: Phosphate buffer solid powder purchased from Seville Bioservices Co., Ltd. was dissolved in 2000 mL of distilled water at room temperature to obtain 0.01 mol / L PBS buffer.

[0047] SG medium (1 L): 1.0 g (NH4)2SO4, 3.0 g sodium D-gluconate, 0.35 g KH2PO4, 0.25 g K2HPO4, 0.15 g MgSO4·7H2O, 0.1 g NaCl, CaCl2·2H2O, 1.7 g yeast powder, 1000 mL deionized water; if SG solid medium is needed, add 15.0 g agar.

[0048] 10% trichloroacetic acid (TCA): 10.0 mL of TCA solution was added to 90.0 mL of deionized water in a 100.0 mL volumetric flask to obtain a 10% TCA solution.

[0049] 80% Ca(NO3)2 solution: Weigh 80.0 g of solid Ca(NO3)2 powder and add it to 20.0 g of deionized water. Ultrasonic dispersion is performed for 5 minutes to obtain 80% Ca(NO3)2 solution.

[0050] 0.5% I2-KI solution: Weigh 0.5g I2 and 1.5g KI solid, dissolve in 98.0g deionized water to obtain 5% I2-KI solution, and store in a brown bottle.

[0051] 1.3 Primer names and sequences used

[0052]

[0053] 2 Experimental methods

[0054] 2.1 Synthesis of polysuccinimide nanocarriers and preparation of caged iodine loading materials

[0055] (1) Preparation of POA

[0056] 1.0 g of polysuccinimide (PSI) (number-average molecular weight 6,000-10,000) was dissolved in 10 ml of N,N-dimethylformamide (DMF), followed by the addition of 995 μL of oleylamine (OA, i.e., cis-oleyl primary amine) and stirred at 500 rpm and 70°C overnight. The product after the overnight reaction was cooled at room temperature and washed three times with methanol to remove excess DMF. The resulting precipitate, after methanol washing, was centrifuged and vacuum freeze-dried to yield a pale pink oleylamine-modified polysuccinimide nanocarrier, designated POA. The vacuum freeze-drying process involved pre-freezing the product at a temperature below -20°C for 12 hours. The freezer was then turned on to pre-cool the equipment until the cold trap temperature dropped below -40°C. The POA was then placed on a pre-freeze rack and placed in the cold trap, covered with a cold lid, and maintained for approximately one hour to ensure sufficient pre-freezing. After pre-freezing, the pre-frozen POA was placed on a drying rack and dried for 24 hours under the conditions of a vacuum value less than 20 Pa, a cold trap temperature less than -40°C, and a sample temperature less than -20°C. After the water was completely sublimated, a light pink oleylamine-modified polysuccinimide nanocarrier was obtained.

[0057] (2) Preparation of POA@I

[0058] 10 mg of an organic-inorganic hybrid polyiodine crystalline material (referred to as caged iodine) was added to 1 mL of dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL caged iodine solution. 200 μL of the caged iodine solution, 20 mg of POA, and 800 μL of DMSO were added to a new test tube to create a polymer mixture with a total volume of 1 mL. 10 mL of a 2.5 wt% aqueous solution of polyvinyl alcohol (PVA) was then added to the polymer mixture, and the solution was sonicated for 2 minutes at 180 W for 2 minutes to uniformly disperse the components. After sonication, the solution was allowed to stand at room temperature and stirred overnight at 300 rpm using a 1 cm magnetic bar. After stirring overnight, the solution was centrifuged at 10,000 rpm for 10 minutes to obtain nanoparticles (NPs). The NPs were washed twice with deionized water to remove the PVA, resulting in POA loaded with caged iodine, designated POA@I. The POA@I was then redispersed in water to prepare solutions of varying concentrations for subsequent experiments. Polyvinyl alcohol (PVA) is used as a solvent, adsorbing onto the surface of the polymer particles during the reaction to form a protective film, thereby reducing interfacial tension and increasing the viscosity of the system, thereby improving the dispersion stability of the suspension polymerization system.

[0059] 2.2 Drawing of the caged iodine standard curve

[0060] Accurately weigh 10.0 mg of caged iodine (PI) and dissolve it in 1.0 mL of DMSO to obtain a 10.0 mg / mL caged iodine stock solution, which was then diluted with water to 200, 125, 100, 62.5, 50, 31.25, and 25 concentrations of the test solution. The absorbance of each concentration was measured at 351 nm.

[0061] 2.3 Characterization of POA@I

[0062] Preparation of material dispersion: 500 μg of PSI, caged iodine, POANPs and POA@I NPs were dispersed in 1.0 mL of deionized water by water bath ultrasonication for 3 min to obtain a material dispersion.

[0063] TEM: 10 μL of POANPs and POA@I NPs dispersion was dropped onto a copper grid and dried at room temperature for 24 h. The material morphology was observed under TEM.

[0064] HD test: 500 μL of POANPs and POA@I NPs were moved to the particle size test pool, and the hydrated particle size of the materials was analyzed using a nanoparticle size analyzer.

[0065] Zeta potential test: 500 μL of PSI, caged iodine, POANPs and POA@I NPs dispersions were transferred to the Zeta potential test cell, and the Zeta potential of the materials was analyzed using a nanoparticle size analyzer.

[0066] Infrared spectroscopy test: 5 mg of PSI, caged iodine, POANPs, and POA@I NPs solid samples were placed in an agate mortar and ground thoroughly. 200 mg of potassium bromide powder was added and thin slices were prepared using a tablet press. The infrared spectra were obtained by scanning with an infrared spectrometer.

[0067] UV spectroscopy test: 500 μL PSI, caged iodine, POANPs, and POA@I NPs were transferred to a micro-UV cuvette, and the UV spectra of the materials were tested in the wavelength range of 200 nm-800 nm.

[0068] 2.4 POA encapsulation efficiency and loading rate

[0069] After the mixture was tumbled, it was centrifuged at room temperature and 12,000 rpm for 5 minutes. 500 μL of the supernatant was taken and diluted 8-fold. The absorbance of the supernatant was measured at 351 nm and applied to the standard curve. The residual concentration of I in the supernatant was calculated by subtraction. The encapsulation efficiency and loading rate of POA were calculated according to formulas (1) and (2).

[0070]

[0071] 2.5 Bacterial strains and growth conditions

[0072] The bacterial strains used in this study to evaluate the in vitro antimicrobial activity included the Gram-negative bacterium Sinorhizobium fredii HH103. HH103 was grown in liquid SG medium at 27°C and 180 rpm in a thermostatic shaker for 24-48 hours.

[0073] 2.6 In vitro antibacterial test

[0074] The antibacterial activity of caged iodine, POANPs, and POA@I NPs was evaluated using the dilution plate method. The specific steps are as follows:

[0075] (1) Interaction between POA@I NPs and bacteria: After culturing bacteria in SG medium at 27°C for 1 day, POA@I NPs were diluted in a clean bench and then an equal volume of bacterial solution (10 6The final concentrations of POA@INPs were 1500 μg / mL, 750 μg / mL, 375 μg / mL, 187.5 μg / mL, and 93.75 μg / mL. The mixture was incubated in a biochemical incubator at 27°C for 4 h. Subsequently, 10 μL of the mixture diluted to the appropriate concentration was plated on SG solid medium. After 48 h, the number of colonies on the solid medium was counted. The in vitro antibacterial test procedures for caged iodine and POANPs were the same as above. The final concentrations of POANPs were 1500 μg / mL, 750 μg / mL, 375 μg / mL, 187.5 μg / mL and 93.75 μg / mL, and the final concentrations of caged iodine were 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL and 125 μg / mL. The antibacterial activity of the materials was evaluated by the bacterial survival rate, and the bacterial inhibition rate was calculated using formula (3).

[0076]

[0077] (2) Plate coating method: Pre-immerse the coating rod in a beaker containing 75% alcohol for disinfection, then place it over the flame of an alcohol burner to sterilize it, and use it for plate coating after cooling; open the cover of the pre-made plate in a clean bench and blow it for 15-30 minutes to keep the surface of the plate relatively dry to prevent excessive moisture from spreading the colony into a piece during coating; draw an appropriate amount of bacterial solution and add it dropwise to the surface of the culture medium, and evenly spread the bacterial solution on the surface of the plate; after the coating is completed, the coating rod needs to be burned and sterilized before the next sample coating operation is carried out.

[0078] 2.7 Treatment of plants and their samples

[0079] The plant material was 1.5-year-old Huanglongbing-infected Valencia orange plants grown in a greenhouse (temperature 27±2°C, relative humidity 50±5% and natural light duration). The leaves were injected with 750 μg / mL POA@I, and the leaves were sampled on the first, third, fifth, seventh and ninth days.

[0080] Sampling and processing: Use a leaf hole sampler with a diameter of 1 cm to take symmetrical samples with the midrib as the symmetry axis, and weigh the samples after sampling.

[0081] Sample processing: The leaves were collected and quickly frozen in liquid nitrogen, then ground into a uniform powder using a fully automatic sample rapid grinder, and the corresponding buffer was added according to different experiments.

[0082] 2.8 In vivo antibacterial test

[0083] Total DNA was extracted from treated leaves using a plant genomic extraction kit according to the manufacturer's instructions. After DNA purification, the DNA was eluted from the adsorption column with 50 μL of DEPC HO. Total DNA concentration was determined using an ultraviolet spectrometer, and the total DNA samples were stored in a 4°C freezer. The in vivo antibacterial activity of POA@I was evaluated by qPCR analysis of HLBas relative quantification (RQ) values ​​between the POA@I and CK groups, using Csactin-2 as an internal reference gene. The qPCR solution consisted of 5 μL of qPCRMIX, 1 μL of each upstream and downstream primer, 2 μL of RNase-free HO, and 1 μL of diluted template DNA, for a total of 10 μL. qPCR reactions were performed in a Quantistudio 3 using a standard rapid protocol: a 10-min initial denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 15 seconds. The survival rate of CLas was used to measure the antibacterial activity of POA@I against CLas in vivo, and the CLas survival rate was calculated using formula (4).

[0084]

[0085] 2.9 Determination of chlorophyll and carotenoids

[0086] After treatment, 1 mL of anhydrous ethanol was added to the tissue sample and shaken at 250 r / min at room temperature for 15 minutes to extract the pigment. Subsequently, the sample was centrifuged at 6000 r / min for 5 minutes, and the supernatant was collected. 1 mL of anhydrous ethanol was then added to the precipitate, and the above steps were repeated twice. After combining 3 mL of the supernatant, the absorbance of the supernatant was measured at wavelengths of 470 nm, 649 nm, and 665 nm. Chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, and their relative contents were calculated using formulas (5), (6), (7), (8), and (9), respectively.

[0087] Chlorophyll a concentration C a (mg / L)=13.95A 665- 6.88A 649 Formula (5)

[0088] Chlorophyll b concentration C b (mg / L)=24.96A 649- 7.32A 665 Formula (6)

[0089] Total chlorophyll concentration (mg / L) = chlorophyll a concentration + chlorophyll b concentration Formula (7)

[0090]

[0091] 2.10 Determination of MDA and soluble sugar content

[0092] 1.0 mL of 10% TCA was added to homogenized leaf samples that had been quick-frozen in liquid nitrogen. Malondialdehyde (MDA) and soluble sugars were extracted from the tissue at room temperature under shaking conditions of 250 rpm for 5 minutes. The mixed solution was centrifuged at 6000 rpm for 5 minutes, and the supernatant was collected. 500 μL of the supernatant was mixed with an equal volume of 0.6% thiobarbituric acid and reacted in a boiling water bath for 10 minutes. After the reaction, the mixed solution was placed on ice to cool and the reaction solution was centrifuged at 3500 rpm for 10 minutes. The supernatant was then collected and its ultraviolet absorbance was measured at 450 nm, 532 nm, and 600 nm. The soluble sugars, MDA, and their relative contents were calculated using equations (10) and (11).

[0093] Soluble sugar concentration (μμmol / L) = 11.71A 450 Formula (10)

[0094] The concentration of malondialdehyde (μμmol / L) = 6.45 (A 532 -A 600 )-0.56A 450 Formula (11)

[0095] 2.11 Determination of H2O2 content

[0096] The corresponding 0.1 mol / L PBS buffer was added to the tissue samples at a ratio of 1:9 sample mass (mg): PBS volume (μL). The H₂O₂ content in the sweet orange leaves was determined and calculated according to the manufacturer's instructions for the hydrogen peroxide detection kit. The relative H₂O₂ content was calculated using Equation (9).

[0097] 2.12 Antioxidant enzyme activity assay

[0098] The corresponding 0.1 mol / L PBS buffer was added to the tissue samples at a ratio of 1:9 sample mass (mg): PBS (μL) to obtain extracts of antioxidant-related enzymes. The activities of the corresponding antioxidant enzymes in sweet orange leaves were measured and calculated according to the manufacturer's instructions for the CAT, POD, and SOD kits. The relative activities of the antioxidant-related enzymes were calculated using Equation (12).

[0099]

[0100] 2.13 Analysis of relative gene expression levels

[0101] Total RNA was extracted from homogenized leaf samples using the Huiling RNA extraction kit according to the manufacturer's instructions. After total RNA extraction, RNA concentration was measured using an ultra-micro-UV instrument, and 10 μL of the diluted RNA was used to synthesize cDNA using a reverse transcription cDNA kit. The RT-qPCR reaction system was then prepared. A 10 μL reaction solution contained 1 μL of cDNA, 1 μL of each upstream and downstream primer, 5 μL of qPCR mix, and 2 μL of DEPC HO. RT-qPCR reactions were performed in a Quantistudio 3 using a standard rapid protocol: 10 min of initial denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 15 seconds. Primers for PAL, PR-1, and PR-2, which have been previously reported (Huang et al. 2021), were designed using NCBI.

[0102] 3 Results and Analysis

[0103] 3.1 Synthesis and characterization of materials

[0104] In this study, polysuccinimide was functionalized with oleylamine and then POA@I NPs were obtained by electrostatic adsorption of caged iodine. Caged iodine has low water solubility and rapid metabolism. Therefore, loading caged iodine into POANPs can not only solve these limitations but also target disease sites, such as Figure 1 As shown in F, caged iodine has poor water solubility ( Figure 1 F (glass bottle on the left), but after the synthesis of POA@I NPs, the dispersion in aqueous solution was greatly improved Figure 1 F glass bottle on the right).

[0105] like Figure 1 As shown in A and B, TEM characterization of PSI was performed. Figure 1 The TEM characterization results of POA@I NPs (C and D) show that the particle size increases, which more intuitively proves the successful synthesis of POA@I NPs. Figure 1 E) further illustrates the change in material size, which is consistent with the TEM data. After PSI was modified with oleylamine, the Zeta potential of POA was -39.97mV. The partial hydrolysis of the functional groups at the end of the polymer chain (i.e., aspartic acid) or the succinimide groups on the surface of NPs caused aspartic acid to cause the negative charge of the NPs. After the caged iodine was loaded, the potential became +20.77mV ( Figure 1 I). The UV-visible absorption spectrum of POA@INPs showed absorption peaks at 287nm and 351nm ( Figure 1H), while the fluorescence intensity of POA@I NPs is between that of PI and POA ( Figure 1 H), also confirmed the synthesis of the material.

[0106] The encapsulation efficiency and loading rate of caged iodine were determined by the standard curve method, and the standard curve was y = 0.00691x-0.2177 ( Figure 2 ), the encapsulation efficiency of drug cage iodine was calculated to be 44.27%, and the loading rate was 59.03%.

[0107] 3.2 In vitro antibacterial activity

[0108] After confirming the successful synthesis of POA@I NPs, in vitro antibacterial tests were performed to evaluate the antibacterial activity of POA@I NPs. Given that CLas was originally a branch of rhizobia and cannot be cultured in vitro, the Gram-negative bacterium Sinorhizobium fredii strain HH103 was selected for in vitro antibacterial activity evaluation.

[0109] like Figure 3 As shown, the inhibition rate of HH103 in SG medium increased with increasing POA@I NPs concentration, demonstrating the concentration-dependent antibacterial activity of POA@I NPs. Furthermore, the inhibition rate of HH103 in the POA@I NPs group reached 96.82% at a concentration of 750 μg / mL and 100% at a concentration of 1500 μg / mL. POA@I NPs also exhibited a moderate antibacterial effect at other concentrations.

[0110] In addition to evaluating the antibacterial effect of POA@I NPs on HH103, the antibacterial effect of carrier POA on HH103 was also evaluated, e.g. Figure 4 As shown, the results showed that POA had a certain antibacterial effect compared with CK, but it was significantly lower than the antibacterial effect of POA@INPs, and the inhibition rate of HH103 in SG medium was not concentration-dependent with the concentration of POA.

[0111] In addition, the antibacterial effect of caged iodine on HH103 was evaluated, such as Figure 5 As shown in the results, the inhibition rate of caged iodine in aqueous solution against HH103 can reach 100% at a concentration of 2000 μg / mL, the inhibition rate against HH103 at a concentration of 1000 μg / mL is 80.53%, and the inhibition rate at a concentration of 500 μg / mL can only reach 52.26%. This result shows that the antibacterial effect of pure drug caged iodine dissolved in aqueous solution is worse than that of POA@I NPs, which reflects the improvement of the antibacterial activity of POA@INPs compared with caged iodine.

[0112] 3.3 Increase in pigment content

[0113] Chlorophyll content is correlated with photosynthetic activity and the severity of biotic and abiotic stresses to which plants are exposed. When plants experience severe stress, their chlorophyll content decreases, and consequently, their photosynthetic activity gradually weakens. Although combined nutrient deficiencies cause chlorosis in HLB-positive citrus leaves, the direct cause is the decreased chlorophyll content in Huanglongbing-positive leaves, which leads to reduced photosynthetic activity. Therefore, chlorophyll content in HLB-positive sweet orange leaves treated with POA@I NPs is an important criterion for evaluating the photosynthetic toxicity of POA@I NPs and their ability to mitigate the negative effects of HLB on sweet orange leaves.

[0114] like Figure 6 As shown in A, after treatment with POA@I NPs, the chlorophyll a content in HLB-positive sweet orange leaves increased to 107.44%, 106.66%, 118.27%, 129.91% and 117.47% compared with the CK group ( Figure 6 A). The chlorophyll b content increased compared with CK on the first, third, fifth, seventh and ninth days after treatment, increasing by 1.86%, 18.48%, 24.02%, 18.79% and 2.10% respectively ( Figure 6 B). As the contents of chlorophyll a and chlorophyll b increased, the total chlorophyll contents on the first, third, fifth, seventh and ninth days after treatment were 105.34%, 111.54%, 127.46%, 117.98% and 104.19% of that in CK, respectively ( Figure 6 C). In addition, it was observed that the carotene content increased to varying degrees on the third, fifth, seventh, and ninth days after POA@I NPs treatment, increasing by 21.80%, 27.19%, 7.21%, and 8.68%, respectively ( Figure 6 D).

[0115] By comparing and analyzing the chlorophyll and carotenoid contents of HLB-positive sweet oranges in the CK group, the POA@I NPs-treated group and the healthy sweet oranges, the degree to which POA@I NPs treatment alleviates the adverse stress of HLB-positive sweet orange leaves was determined.

[0116] like Figure 6 E, the chlorophyll a content of the POA@I NPs group was 523.87 μg / g, which was significantly higher than the chlorophyll a content of the control group (365.75 μg / g). The chlorophyll b content of the leaves of the POA@I NPs-treated group was 62.52 μg / g higher than that of the CK group ( Figure 6 F), the content was lower than that of WT group (87.39 μg / g). Figure 6As shown in G, the total chlorophyll content of HLB-positive sweet orange leaves was 797.91 μg / g in the POA@I NPs group and 626.02 μg / g in the CK group. The increase in carotenoids after POA@INPs treatment was greater than that of chlorophyll ( Figure 6 H), the carotenoid content of the POA@I NPs group reached 84.42 μg / g, which was significantly increased compared with 66.37 μg / g in the CK group and approached the carotenoid content of the WT group (148.26 μg / g).

[0117] The relative increase in chlorophyll and carotenoid content suggests that POA@I NPs promote nitrogen assimilation in plants. Carotenoids also act as a protective shield for chlorophyll, and their increased content effectively protects chlorophyll. Chlorophyll absorbs light energy and converts it into easily storable chemical energy within the body, making it an essential pigment for photosynthesis. Increased chlorophyll content enhances light absorption, thereby enhancing photosynthesis and increasing photosynthetic product production (Curran et al. 1990). Overall, the results demonstrate that POA@I NPs are non-toxic to photosynthesis in HLB-positive leaves. The increased chlorophyll and carotenoid content compared to the CK group suggests that POA@I NPs treatment can protect and even increase chlorophyll content in HLB-positive leaves.

[0118] 3.4 Changes in H2O2 and Antioxidant Enzyme Activities

[0119] CLas-induced excessive H2O2 production and abnormal starch accumulation are significant metabolic abnormalities in HLB-positive leaves (Pourreza et al. 2015) and are important indicators for evaluating the potential of materials to control HLB.

[0120] The H2O2 content in plants will show an increasing trend under adverse stress. Therefore, the effect of POA@I NPs on the H2O2 concentration in HLB-positive leaves was studied ( Figure 7 A, E). H2O2 concentrations decreased by 5.68%, 7.77%, 17.62%, and 26.54% in the first 7 days, respectively. However, on the 9th day, H2O2 concentrations rebounded and were closer to those in the CK group ( Figure 7 A). On the 7th day, the H2O2 content dropped to a minimum of 1094.53 μmol / g ( Figure 7E), closer to the WT group than the CK group. The decrease in H2O2 concentration indicates that the oxidative stress experienced by HLB-positive leaves was alleviated. This consistency suggests that POA@INPs NPs, in addition to their excellent antimicrobial properties against CLas, can also mitigate the deleterious effects of CLas on HLB-positive leaves. The decrease in H2O2 concentration may be due to a decrease in the number of CLas in HLB-positive sweet orange leaves, thereby slowing the continuous induction of H2O2 production. It is also possible that POA@INPs NPs activated their antioxidant system.

[0121] 3.5HLB inhibition and upregulation of antioxidant-related genes and defense marker genes

[0122] Antioxidant genes play a key role in encoding a series of enzymes, including SOD, POD, CAT, and APX, which work together to eliminate excess ROS and convert them into less toxic substances (Li et al. 2018a). Defense marker genes act as sentinels in plants, sensing and combating pathogen threats, similar to the immune system of animals (Jones et al. 2006).

[0123] Defense marker genes play a crucial role in plant defense against pathogens. SA and phenylpropionic acid are two key pathways of plant disease resistance. PR and PAL are the corresponding marker genes for SA and PAL, respectively. Upregulation of these genes indicates increased SA and PAL levels, inducing SAR in plants and enhancing resistance to infectious pathogens. Therefore, we used RT-qPCR to analyze PR-1, PR-2, PAL-1, and PAL-2 in diseased sweet orange leaves. By comparing gene expression levels between the POA@I treatment and the CK group on day three, we determined whether POA@I could enhance plant disease resistance.

[0124] Based on the results that POA@I NPs treatment decreased ROS levels and increased antioxidant enzyme activities in HLB-positive sweet orange leaves, it was hypothesized that POA@I NPs would affect the relative expression levels of genes in HLB-positive sweet orange leaves. The presence of POA@I NPs significantly upregulated the expression levels of antioxidant-related genes in leaf cells and activated defense marker genes. With the increase in the relative expression levels of antioxidant-related genes, antioxidant enzyme activities were enhanced, and the growth of CLas, which induced excessive ROS accumulation, was inhibited, ultimately reducing ROS levels.

[0125] To explore the multifaceted effects of POA@I NPs on HLB-positive sweet orange leaves, including its antibacterial activity and its complex regulation on antioxidant-related genes and defense marker genes in HLB-positive sweet orange, RT-qPCR experiments were performed; e.g. Figure 8As shown in A, the pot experiment showed that the relative expression level of the internal reference HLBas2 gene of Huanglongbing bacteria changed, reaching the lowest value on the 5th day, and the relative expression value was 32.86% of the control group, indicating that the titer of Huanglongbing bacteria decreased significantly.

[0126] The expression of genes related to antioxidant enzyme activity was quantified within 9 days after treatment with POA@I NPs, including CAT-1, CAT-2, SOD-1, SOD-2, SOD-3, POD, APX and RbohA. Among them, the relative expression level of RbohA can reflect the severity of oxidative stress in plants and is often used as a key marker to verify the alleviation of oxidative stress. The three defense marker genes are PR-1, PR-2 and PAL, which are the response genes of SA and PAL respectively. The increase in their relative expression levels indicates that the disease resistance of plants has been enhanced. Figure 8 As shown in E, the relative expression of CAT-1 in HLB-positive sweet orange leaves showed no significant increase except on the first day, but increased by 2.16 times, 4.22 times, 2.66 times and 1.86 times on the third, fifth, seventh and ninth days, respectively; while the relative expression level of CAT-2 also showed an increasing trend compared with the CK group, increasing to 181.28%, 334.33% and 236.94% of that in the CK group on the third, fifth and seventh days, respectively ( Figure 8 F) The increasing trend of the relative expression levels of CAT-1 and CAT-2 is consistent with the changing trend of CAT enzyme activity.

[0127] The results of RT-qPCR experiments showed that spraying of POA@I NPs could change the expression levels of CAT-1 and CAT-2 in leaves, so we continued to detect the expression levels of related genes encoding SOD, POD, and APX. Figure 8 As shown in C, the relative expression of SOD-1 within 9 days was 1.43 times, 2.83 times, 3.10 times, 2.43 times and 1.59 times that of CK, respectively; the relative expression of SOD-2 was 176.72%, 316.54%, 373.92%, 272.44% and 164.33% of that in the control group ( Figure 8 D); The relative expression level of SOD-3 also showed an upward trend compared with the CK group, increasing to 206.85%, 284.97% and 169.36% of the CK group on the 3rd, 5th and 7th days respectively ( Figure 8 B)

[0128] The relative expression level of POD reached its maximum on the 5th day, which was 3.54 times that of the CK group. The relative expression levels of POD on the remaining days were 1.63 times, 1.88 times, 2.76 times and 1.88 times that of the CK group respectively. Figure 8G), the changes in the relative expression level of POD were less volatile than those of the first four genes, which was consistent with the experimental results of the gentle changes in POD enzyme activity. Figure 8 As shown in Figure H, the relative expression level of the gene APX follows a similar trajectory to that of the POD, but with smaller fluctuations. Its relative expression levels on each day were 1.08, 1.33, 1.69, 1.28, and 1.18 times higher than those in the CK group, respectively. In summary, 9 days after POA@I NPs treatment, the changes in the relative expression levels of antioxidant-related genes corresponded to fluctuations in antioxidant enzyme activity, indicating that POA@I NPs activate the antioxidant system by regulating antioxidant-related genes.

[0129] The expression level of gene RbohA increased with the severity of plant oxidative stress. RT-qPCR results showed that the relative expression level of RbohA was completely opposite to the change trend of antioxidant-related genes. Its relative expression level on the third day was only 46.17% of that in CK ( Figure 8 J), demonstrating that oxidative stress in HLB-positive sweet orange leaves is alleviated by upregulating the expression of antioxidant-related genes. This phenomenon also confirms that the activation of the antioxidant system by POA@I NPs is, in part, due to the upregulation of these antioxidant genes. In addition to the significant upregulation of antioxidant-related gene expression levels after POA@I NPs treatment, the expression levels of defense marker genes also increased compared to the CK group.

[0130] like Figure 8 As shown in Figure K, POA@I NPs treatment significantly increased the expression level of PR-1 in the infected sweet orange leaves, which was increased by 2.28 times compared with the CK group. The expression level of PR-2 was also significantly increased in the POA@I NPs treatment group, which was 2.20 times the expression level of the CK group ( Figure 8 In addition, the expression level of PAL-related genes increased similarly to that of PR genes. After POA@I NPs treatment, PAL was 2.78 times that of the CK group ( Figure 8 I).

[0131] The significant increase in PR-1, PR-2, and PAL expression levels indicates that POA@I NPs can significantly upregulate defense marker genes in HLB-positive sweet orange leaves. The significant upregulation of SA and PAL response genes also corresponds, to some extent, to the increased SA and PAL content. In summary, POA@I NPs upregulate defense marker genes, activate plant SAR, and inhibit CLas growth.

[0132] In summary, the present invention studies the formation of an amphiphilic nanocarrier POA by surface modification of PSI with oleylamine, enabling the loading of the hydrophobic drug caged iodine. By loading caged iodine, CLas inhibition is achieved in HLB-positive leaves. By upregulating the expression levels of antioxidant-related genes, the antioxidant system in the leaves is activated, thereby alleviating the adverse effects of HLB on sweet orange leaves, such as starch deposition, decreased pigment content, H2O2 accumulation, and increased MDA. The extremely significant upregulation of defense marker gene expression levels indicates that POA@I NPs can activate plant SAR, and synergistically with the antioxidant system can enhance the plant's disease resistance. The present invention studies and develops a multi-faceted strategy for the treatment of HLB, designing POA@I NPs nanomaterials that take into account anti-CLas, activation of the plant antioxidant defense system, and plant acquired immunity. The excellent biocompatibility of POA is fully utilized to successfully load antimicrobial drugs through surface modification, achieving a multi-dimensional treatment of HLB. POA@I NPs are used as composite carriers to deliver antimicrobial drugs for the prevention and control of plant infectious diseases, broadening the application of POA@INPs in agricultural drug delivery.

Claims

1. An antibacterial nanoparticle preparation, characterized in that: It is obtained by loading organic-inorganic hybrid polyiodine crystal material onto nanocarrier POA; The nanocarrier POA is a modified polysuccinimide nanoparticle prepared by modifying polysuccinimide with oleylamine; The molecular formula of the organic-inorganic hybrid polyiodine crystal material is [(C6 H 21 I 26 O3 S3 )2 ] n , where n is a natural number greater than or equal to 1.

2. The antibacterial nanoparticle preparation according to claim 1, characterized in that: The nanocarrier POA is obtained by mixing polysuccinimide and oleylamine in an organic solvent A, reacting the mixture at 60-100° C. for 2-12 hours, and then washing to remove the organic solvent A.

3. The antibacterial nanoparticle preparation according to claim 2, characterized in that: The organic solvent A is N,N-dimethylformamide, and the number average molecular weight of the polysuccinimide is 6000-10000.

4. The antibacterial nanoparticle preparation according to claim 2, characterized in that: The preparation method of the nanocarrier POA comprises the following steps: adding polysuccinimide and oleylamine to an organic solvent A and mixing them, wherein the mass volume ratio of polysuccinimide to oleylamine is 1 g: 0.6-1.4 ml; stirring and reacting the mixture at 60-100° C. for 2-12 hours; cooling the reaction product, washing it with an organic solvent B to remove the organic solvent A; and collecting the precipitate by centrifugation to obtain the nanocarrier POA. The organic solvent B is methanol or ethanol.

5. The antibacterial nanoparticle preparation according to claim 4, characterized in that: The washed precipitate collected by centrifugation is vacuum freeze-dried to obtain freeze-dried powder.

6. A method for preparing the antibacterial nanoformulation according to any one of claims 1 to 5, characterized in that: The steps include: The nanocarrier POA and the organic-inorganic hybrid polyiodine crystal material are added to an organic solvent C and mixed to obtain a polymer mixture; polyvinyl alcohol is added to the polymer mixture, mixed thoroughly, and then reacted under stirring. After the reaction is completed, the nanoparticles are collected by centrifugation and washed to obtain the antibacterial nanoformulation.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the organic-inorganic hybrid polyiodine crystalline material to POA in the polymer mixture is 1:5-15; the concentrations of POA and the organic-inorganic hybrid polyiodine crystalline material in the polymer mixture are 10-30 mg / mL and 1-5 mg / mL, respectively; The polyvinyl alcohol is a 2-3 wt% polyvinyl alcohol solution, and the volume ratio of the polymer mixture to the polyvinyl alcohol solution is 1:7-13; The organic solvent C is dimethyl sulfoxide or chloroform.

8. The preparation method according to claim 6, characterized in that: After adding polyvinyl alcohol to the polymer mixture, ultrasonic treatment is performed to fully mix the components. The ultrasonic treatment conditions are: 160-200W, 1.5-3.0 minutes; after ultrasonic treatment, the mixture is stirred for 2-16 hours; After the reaction is completed, the nanoparticles collected by centrifugation are washed with deionized water to remove the polyvinyl alcohol, thereby obtaining the antibacterial nanoparticle preparation.

9. Use of the antibacterial nanoformulation according to any one of claims 1 to 5 in inhibiting plant pathogens or preventing and controlling plant diseases.

10. The use according to claim 9, characterized in that: The plant pathogens include pathogenic bacteria or fungi; the plant diseases are plant diseases caused by bacterial or fungal pathogens.

11. The use according to claim 10, characterized in that: The plant disease is Huanglongbing disease of Rutaceae plants, and the Rutaceae plants refer to Citrus, Poncirus, Fortunella or Murraya.

12. The use according to claim 9, characterized in that: The antibacterial nanoparticle preparation is prepared into a solution and then administered by injection or spraying; the working concentration of the antibacterial nanoparticle preparation is 150-1500 μg / mL.

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