Antibacterial nano preparation as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial nano preparation and a preparation method thereof. The antibacterial nano preparation is obtained by loading an organic-inorganic hybrid polyiodine crystal material onto a nano carrier POA modified by oleylamine, the invention also discloses an application of the organic-inorganic hybrid polyiodine crystal material or the antibacterial nano preparation in inhibition of plant pathogenic bacteria or prevention and treatment of plant diseases. The organic-inorganic hybrid polyiodine crystal material is loaded on the POA nano-carrier, the water solubility of the POA nano-carrier is improved through nano-crystallization treatment, the safety and the penetrability of the POA nano-carrier are remarkably improved, and experiments prove that the POA nano-carrier has a remarkable sterilization effect in prevention and treatment of citrus huanglongbing. The antibacterial nano preparation POA (at) I serves as a composite carrier to deliver antibacterial drugs for preventing and treating plant infectious diseases, the application range of POA in the aspect of agricultural drug delivery is widened, the new application range of a polyiodine crystal material is widened, and meanwhile a new drug is provided for preventing and treating 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 fruit disease, has caused severe economic losses in citrus producing areas around the world. Since its discovery, it has been a major problem for fruit growers and agricultural researchers.

[0003] Huanglongbing is mainly caused by Candidatus Liberibacter. The pathogen 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 citrus psyllid (Diaphorina citri). Once a citrus tree is infected with Huanglongbing, its initial symptoms are often mottled yellowing of leaves and yellowing of branches, followed by gradual reduction, deformity, and abnormal coloring of fruits, eventually leading to the decline and death of the entire fruit tree. Since the Huanglongbing pathogen cannot be cultured in vitro, the current prevention and control method used in production is still the 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. For plant diseases, nano-pesticides can be used for treatment. Most synthetic pesticides contain hydrophobic active ingredients (AI), which can be encapsulated or attached to the hydrophobic groups of nanoparticles, thereby improving the performance of the active ingredients of the original pesticides, targeted transport, pesticide protection, and pesticide release control functions. Compared with traditional pesticides, nano-pesticides have the advantages of low dosage and high efficiency.

[0005] As a commercial bactericide, 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, iodine itself has some limitations, such as high volatility, poor chemical stability, and difficulty in modification, which 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 crystalline 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 nano preparation and a preparation method and application thereof in view of the above problems.

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

[0008] The first aspect of the present invention provides an antibacterial nano-preparation, which is obtained by loading an organic-inorganic hybrid polyiodine crystal material onto a nano-carrier 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 to 100° C. for 2 to 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 for mixing, wherein the mass volume ratio of polysuccinimide to oleylamine is 1 g:0.6-1.4 ml (preferably 1 g:0.8-1.2 ml); stirring and reacting at 60-100° C. (preferably 60-80° C.) for 2-12 hours (preferably 6-12 hours); after the reaction is completed, cooling the reaction product, washing 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 vacuum freeze-dried to a freeze-dried powder.

[0015] The second aspect of the present invention provides a method for preparing the antibacterial nanoformulation described in any one of the above, comprising the following steps:

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

[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 carried out under stirring conditions 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 antibacterial nanoformulations for use in 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 scheme, the plant pathogenic bacteria include pathogenic bacteria or fungi; the plant diseases are plant diseases caused by bacterial or fungal pathogens.

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

[0025] In the application technology scheme, the antibacterial nano preparation is prepared into a solution and then applied by injection or spraying; the working concentration of the antibacterial nano preparation 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 crystal material is a new iodine material in recent years. It is in a fluid state above 6°C and has high light and heat stability below 100°C, overcoming the characteristic that elemental iodine volatilizes above 4°C. However, organic-inorganic hybrid polyiodine crystal material has the characteristics of self-assembly, is easily polymerized into polymers, has a large molecular volume, and cannot enter plant cells, which greatly limits its application in plant disease prevention and control. The present invention loads it on a POA nanocarrier, improves its water solubility through nano-processing, and significantly improves its safety and penetration. It has been confirmed by experiments that it has a significant bactericidal effect in the prevention and control of citrus Huanglongbing.

[0028] The present invention obtains nanocarrier POA by surface modification of polysuccinimide with oleylamine, realizes successful loading of organic-inorganic hybrid polyiodine crystal material to obtain antibacterial nanoformulation POA@I, and applies it to citrus Huanglongbing, achieving triple-dimensional control of citrus HLB including inhibiting CLas growth, activating plant antioxidant system and activating plant SAR. The antibacterial nanoformulation of the present invention exhibits strong antibacterial activity in in vitro and in vivo antibacterial tests. While not generating abiotic stress on the infected citrus leaves, it increases antioxidant enzyme activity by up-regulating antioxidant-related genes, alleviates oxidative stress in the infected sweet orange leaves, and increases the chlorophyll content in the leaves; it significantly up-regulates defense marker genes, can activate the SAR pathway, and successfully inhibits the growth of CLas.

[0029] The POA@I of the present invention is used as a composite carrier to deliver antibacterial drugs for preventing and controlling 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, while providing a new drug for the prevention and control of citrus Huanglongbing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Characterization and dispersion of POA@I: (A)(B) TEM electron microscope images of PSI; (C)(D) TEM electron microscope images of POA@I; (E) SEM electron microscope images 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) The inhibition rate of different concentrations of POA@I against the 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) The inhibition rate of different concentrations of POA against the Gram-negative bacteria Sinorhizobium fredii; (B) Dilution coating plate of Sinorhizobium fredii.

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

[0036] Figure 7 The relative changes of 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 of H2O2; (B) relative change of CAT activity; (C) relative change of POD activity; (D) relative change of 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 in conjunction with 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 crystalline material used in the present invention is a caged organic-inorganic hybrid polyiodine crystalline material in Chinese Patent ZL202211219738.8 (Announcement No. CN 116254608B, Invention Name: A caged organic-inorganic hybrid polyiodine crystalline 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 system, and the space group is 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 embodiment 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: Dissolve phosphate buffer solid powder purchased from Sewell Bioservices Co., Ltd. in 2000 mL of distilled water at room temperature to obtain 0.01 mol / L PBS buffer.

[0047] SG medium (1L): 1.0g (NH4)2SO4, 3.0g sodium D-gluconate, 0.35g KH2PO4, 0.25g K2HPO4, 0.15g MgSO4·7H2O, 0.1g NaCl, CaCl2·2H2O, 1.7g yeast powder, 1000mL deionized water; if SG solid medium is needed, add 15.0g 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 80.0g of solid Ca(NO3)2 powder and add it to 20.0g of deionized water, and ultrasonically disperse it 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 a 5% I2-KI solution, which is stored 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] 1.0g polysuccinimide (PSI) (number average molecular weight is 6000-10000) was dissolved in 10ml N,N-dimethylformamide (DMF), and then 995μL oleylamine (OA, i.e. cis-oleyl primary amine) was added and stirred at 500rpm and 70℃ for overnight reaction. The product after overnight reaction was cooled at room temperature and washed three times with methanol to remove excess DMF. The precipitate obtained by centrifugation after methanol washing was vacuum freeze-dried to obtain a light pink oleylamine-modified polysuccinimide nanocarrier, which was recorded as POA. The specific steps of vacuum freeze-drying are: first pre-freeze at a temperature below -20℃ for 12h, then turn on the refrigerator to pre-cool the equipment until the temperature of the cold trap drops below -40℃, put POA on the pre-freeze rack and put it into the cold trap together, and cover it with a cold cover for about an 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 organic-inorganic hybrid polyiodine crystalline material (referred to as caged iodine) was added to 1 mL of dimethyl sulfoxide (DMSO) to prepare a caged iodine solution with a concentration of 10 mg / mL. 200 μL of caged iodine solution, 20 mg of POA, and 800 μL of DMSO were added to a new test tube to prepare a polymer mixture with a total volume of 1 mL. Then 10 mL of polyvinyl alcohol (PVA) aqueous solution (concentration of 2.5 wt%) was added to the polymer mixture, and the solution was ultrasonically treated for 2 minutes to uniformly disperse the components. The ultrasonic treatment conditions were: 180 W, 2 min. After ultrasonic treatment, the solution was placed at room temperature and stirred overnight at a stirring speed of 300 rpm using a 1 cm magnetic bar. The solution stirred overnight was centrifuged at 10,000 rpm for 10 minutes to obtain nanoparticles (NPs). The NPs were washed twice with deionized water to remove PVA, and POA loaded with caged iodine was obtained, denoted as POA@I. POA@I was redispersed in water to prepare solutions of different concentrations for subsequent experiments. Polyvinyl alcohol can be adsorbed on the surface of polymer particles during the reaction as a solvent to form a protective film, thereby reducing the interfacial tension and increasing the viscosity of the system, thereby improving the dispersion stability of the suspension polymerization system.

[0059] 2.2 Drawing of 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, and dilute it with water to 200, 125, 100, 62.5, 50, 31.25 and 25 test solutions, and measure the absorbance of each concentration at 351 nm.

[0061] 2.3 Characterization of POA@I

[0062] Preparation of material dispersion: 500 μg PSI, caged iodine, POANPs and POA@I NPs were dispersed in 1.0 mL deionized water by water bath ultrasound 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 POANPs and POA@I NPs were moved to the particle size test pool, and the hydrated particle size of the material was analyzed by 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 material was analyzed by a nanoparticle size analyzer.

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

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

[0068] 2.4 Encapsulation rate and loading rate of POA

[0069] After the above mixed solution was turned over, it was centrifuged at room temperature and 12000r / min for 5 minutes, and 500μL of the supernatant was taken and diluted 8 times. The absorbance of the supernatant solution was measured at 351nm, and the absorbance was inserted into the standard curve. The residual concentration of I in the supernatant solution 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 h.

[0073] 2.6 In vitro antibacterial test

[0074] The antibacterial activities of caged iodine, POANPs, and POA@I NPs were 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 graded 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 cultured in a biochemical incubator at 27 °C for 4 h, and then 10 μL of the mixture diluted to an appropriate concentration was plated on SG solid culture medium. After 48 h, the number of colonies on the solid culture medium was counted. The in vitro antibacterial test steps 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 by formula (3).

[0076]

[0077] (2) Plate coating method: Pre-soak 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 it cools down; open the cover of the pre-made plate in an ultra-clean workbench and blow it for 15-30 minutes to keep the surface of the plate relatively dry to prevent excessive moisture during coating from spreading the colonies; draw an appropriate amount of bacterial solution and add it dropwise to the surface of the culture medium, and evenly spread the added bacterial solution on the surface of the plate; after the coating is completed, the coating rod needs to be sterilized by burning before coating the next sample.

[0078] 2.7 Treatment of plants and their samples

[0079] The plant material was 1.5-year-old Huanglongbing-infected Valencia sweet orange plants grown in a greenhouse (temperature 27±2°C, relative humidity 50±5% and natural light hours). 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 process: Use a leaf hole sampler with a diameter of 1 cm to take symmetrical samples with the midrib as the axis of symmetry, and weigh the samples after sampling.

[0081] Sample processing: The leaves were collected and quickly frozen in liquid nitrogen, then ground into 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] According to the instructions provided by the manufacturer, the total DNA of the treated leaves was extracted using a plant genome extraction kit. After DNA purification, the DNA on the adsorption column was eluted with 50 μL DEPC H2O. The total DNA concentration was measured by an ultra-micro UV instrument, and the total DNA samples were stored in an ultra-low temperature refrigerator at 4°C. Csactin-2 was used as an internal reference gene, and the RQ value of HLBas between POA@I and CK groups was analyzed by qPCR to evaluate the antibacterial activity of POA@I in vivo. The qPCR solution system consisted of 5 μL qPCRMIX, 1 μL of upstream and downstream primers, 2 μL RNase-free H2O, and 1 μL diluted template DNA, totaling 10 μL. The qPCR reaction was performed in Quantistudio3, and the reaction was carried out according to the standard rapid protocol: 10 min 95°C pre-denaturation, followed by 15 sec 95°C denaturation, 20 sec 60°C annealing, and 15 sec 72°C extension as one cycle, and a total of 40 cycles. 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 the treatment, 1 mL of anhydrous ethanol was added to the tissue sample, and the sample was shaken in a shaker at 250 r / min and room temperature for 15 min to extract the pigment. Subsequently, the sample was centrifuged at 6000 r / min for 5 min, and the supernatant was collected. Subsequently, 1 mL of anhydrous ethanol was added to the precipitate, and the above steps were repeated twice. After 3 mL of the supernatant was combined, 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 by formula (5), formula (6), formula (7), formula (8), and formula (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 the homogenized leaf sample after quick freezing in liquid nitrogen, and malondialdehyde (MDA) and soluble sugar in the tissue were extracted at room temperature under shaking conditions of 250 r / min for 5 min. The mixed solution was centrifuged at 6000 r / min for 5 min to collect the supernatant. 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 min. After the reaction, the mixed solution was placed on ice to cool, and the reaction solution was centrifuged at 3500 r / min for 10 min. The supernatant was then taken and its ultraviolet absorbance was measured at 450 nm, 532 nm and 600 nm. The soluble sugar, MDA and the relative content of the two were calculated by formula (10) and formula (11).

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

[0094] MDA concentration (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 above tissue samples at a ratio of sample mass (mg): PBS volume (μL) of 1:9. The H2O2 content in the sweet orange leaves was measured and calculated according to the instructions provided by the manufacturer in the hydrogen peroxide detection kit. The relative content of H2O2 was calculated using formula (9).

[0097] 2.12 Antioxidant enzyme activity assay

[0098] According to the ratio of sample mass (mg): PBS (μL) volume of 1:9, the corresponding 0.1 mol / L PBS buffer was added to the above tissue samples to obtain the extract of antioxidant-related enzymes. The corresponding antioxidant enzyme activities in sweet orange leaves were measured and calculated according to the instructions provided by the manufacturers of CAT, POD and SOD kits. The relative activities of antioxidant-related enzymes were calculated by formula (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, and the steps provided in the manufacturer's instructions were followed. After the total RNA extraction was completed, the RNA concentration was measured using an ultra-micro UV instrument, and 10 μL of the diluted RNA was taken to synthesize cDNA using a reverse transcription cDNA kit. Subsequently, the RT-qPCR reaction system was prepared, and the 10 μL reaction solution included 1 μL cDNA, 1 μL of upstream and downstream primers, 5 μL qPCR MIX, and 2 μL DEPC H2O. The RT-qPCR reaction was performed in Quantistudio3, and the reaction was performed according to the standard rapid protocol: 10 min 95 ° C pre-denaturation, followed by 15 sec 95 ° C denaturation, 20 sec 60 ° C annealing, and 15 sec 72 ° C extension as one cycle, and the cycle was repeated 40 times. Except for the primers of PAL, PR-1, and PR-2, which have been reported (Huang et al. 2021), the remaining primers were designed at NCBI.

[0102] 3 Results and analysis

[0103] 3.1 Synthesis and characterization of materials

[0104] In this study, polysuccinimide was functionalized by 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 (left glass bottle), 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 C and D show that the particle size of POA@I NPs 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), the synthesis of the material was also confirmed.

[0106] The encapsulation rate 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. Considering that CLas was one of the branches of rhizobia in the early days and could not be cultured in vitro, we selected the Gram-negative bacterium Sinorhizobiumfredii strain HH103 for in vitro antibacterial activity evaluation test.

[0109] like Figure 3 As shown in the figure, the inhibition rate of HH103 in SG medium increased with the increase of POA@I NPs concentration, which indicated that POA@I NPs had concentration-dependence of antibacterial activity. In addition, the inhibition rate of HH103 in the POA@I NPs group could reach 96.82% at a concentration of 750 μg / mL, and 100% at a concentration of 1500 μg / mL. At other concentrations, POA@I NPs also had a certain antibacterial effect.

[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 had no concentration dependence on 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 figure, the inhibition rate of caged iodine in aqueous solution for HH103 can reach 100% at a concentration of 2000 μg / mL, the inhibition rate for HH103 at a concentration of 1000 μg / mL is 80.53%, and the inhibition rate can only reach 52.26% at a concentration of 500 μg / mL. 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 related to photosynthetic activity and the severity of biotic and abiotic stresses suffered by plants. When plants are subjected to severe adverse stress, chlorophyll content decreases, and photosynthetic activity gradually weakens. Although compound nutrient deficiency causes yellowing of HLB-positive citrus leaves, the direct cause is the decrease in chlorophyll content in Huanglongbing-positive leaves, which leads to weakened photosynthetic activity. Therefore, after HLB-positive sweet orange leaves are treated with POA@I NPs, the chlorophyll content is also one of the important criteria for evaluating whether POA@I NPs have photosynthetic toxicity and whether they can alleviate the negative impact of HLB on sweet orange leaves.

[0114] like Figure 6 As shown in A, after POA@I NPs treatment, 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 on the first, third, fifth, seventh and ninth days after treatment compared with CK, increasing by 1.86%, 18.48%, 24.02%, 18.79% and 2.10% respectively ( Figure 6 B). With the increase of chlorophyll a and chlorophyll b contents, 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 of 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 orange and healthy sweet orange in the CK group, the POA@I NPs-treated group, and the effect of POA@I NPs treatment on the relief of adverse stress in HLB-positive sweet orange leaves, the authors determined the extent to which POA@I NPs treatment could alleviate adverse stress in HLB-positive sweet orange leaves.

[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 365.75 μg / g in the control group. The chlorophyll b content of the leaves of the POA@I NPs 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. After being treated with POA@INPs, the increase in carotenoids 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 content and carotenoid content indicates that POA@I NPs can promote the assimilation of nitrogen sources in plants. Carotenoids are also the "protective umbrella" of chlorophyll. The increase in their content can effectively protect chlorophyll. Chlorophyll can absorb light energy and convert it into chemical energy that is easy to store in the body. It is the basic pigment for photosynthesis. The increase in chlorophyll content is conducive to improving light absorption, thereby enhancing photosynthesis and increasing the production of photosynthetic products (Curran et al. 1990). In short, the results show that POA@I NPs are not toxic to photosynthesis in HLB-positive leaves. Compared with the CK group, the content of chlorophyll and carotenoids has increased to varying degrees, which means that POA@I NPs treatment of HLB-positive leaves can protect chlorophyll and even increase its content.

[0118] 3.4 Changes in H2O2 and antioxidant enzyme activities

[0119] CLas-induced excessive H2O2 production and abnormal starch accumulation are significant abnormal metabolism 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 upward 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), which is closer to the WT group than the CK group. The decrease in H2O2 concentration indicates that the oxidative stress experienced by HLB-positive leaves is alleviated. This consistency means that in addition to having excellent antibacterial properties against CLas, POA@INPs NPs can also alleviate the harmful effects of CLas on HLB-positive leaves. The decrease in H2O2 concentration may be due to the reduction in the number of CLas in the leaves of HLB-positive sweet oranges, thereby slowing down the process of continuous induction of H2O2 production. There is also the possibility that POA@I NPs NPs activated its antioxidant system.

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

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

[0123] Defense marker genes play an important role in plant defense against pathogen infection, among which SA and phenylpropionic acid are two main pathways of plant disease resistance. PR and PAL are the corresponding marker genes of SA and PAL, respectively. The upregulation of the two genes means that the content of SA and PAL increases, thereby inducing the SAR of plants and improving resistance to infectious pathogens. Therefore, we analyzed PR-1, PR-2, PAL-1 and PAL-2 in the leaves of infected sweet oranges by RT-qPCR, and compared the differences in gene expression levels between the POA@I treatment and the CK group on the third day to determine whether POA@I can improve plant disease resistance.

[0124] Based on the results that the ROS level in HLB-positive sweet orange leaves decreased after treatment with POA@I NPs and the activity of antioxidant-related enzymes increased, it is speculated that POA@I NPs will affect the relative expression level of genes in HLB-positive sweet orange leaves. The presence of POA@INPs will significantly upregulate the expression level of antioxidant-related genes in leaf cells and activate defense marker genes. With the increase in the relative expression level of antioxidant-related genes, the activity of antioxidant enzymes is enhanced, and the growth of CLas that induces excessive accumulation of ROS is inhibited, and the ROS content eventually decreases.

[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, and the lowest value appeared on the 5th day. 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 within 9 days after POA@I NPs treatment was quantified, 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 relative to the CK group, increasing to 181.28%, 334.33% and 236.94% of 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 was consistent with the changing trend of CAT enzyme activity.

[0127] The results of RT-qPCR experiments showed that the 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 of 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, 1.88, 2.76 and 1.88 times that of CK, respectively. Figure 8G), the change in the relative expression level of POD was less volatile than that of the first four genes, which was consistent with the experimental results of the gentle change of POD enzyme activity. Figure 8 As shown in Figure H, the relative expression level change trajectory of gene APX is similar to the change trend of POD, but the fluctuation is smaller. Its relative expression levels on each day were 1.08, 1.33, 1.69, 1.28 and 1.18 times that of the CK group. In summary, at 9 days after POA@I NPs treatment, the changes in the relative expression levels of antioxidant-related genes can correspond to the 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. The results of RT-qPCR 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 of CK ( Figure 8 J), which proves that with the upregulation of the expression level of antioxidant-related genes, the oxidative stress suffered by HLB-positive sweet orange leaves is alleviated. This phenomenon also confirms that the phenomenon of POA@I NPs activating the antioxidant system is to some extent caused by the upregulation of these antioxidant genes. In addition to the significant upregulation of the expression level of antioxidant-related genes after POA@I NPs treatment, the expression level of defense marker genes also increased relative to the CK group.

[0130] like Figure 8 As shown in Figure K, the treatment with POA@I NPs significantly increased the expression level of PR-1 in the infected sweet orange leaves, which was increased to 2.28 times compared with the CK group. The expression level of PR-2 was also observed to be 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 increase in the expression level of PAL-related genes was similar 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 the expression levels of PR-1, PR-2, and PAL means that POA@I NPs entering HLB-positive sweet orange leaves can significantly upregulate defense marker genes, and the significant upregulation of SA and PAL response genes corresponds to the increase in SA and PAL content to a certain extent. In short, POA@I NPs upregulate defense marker genes, activate plant SAR, and inhibit the growth of CLas.

[0132] In summary, the present invention studies the surface modification of PSI by oleylamine to form an amphiphilic nanocarrier POA, realizes the loading of hydrophobic drug caged iodine, realizes the inhibition of CLas in HLB-positive leaves by loading caged iodine, and activates the antioxidant system in the leaves by up-regulating the expression level of antioxidant-related genes, thereby reducing 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 the expression level of defense marker genes indicates that POA@I NPs can activate plant SAR, and the synergy with the antioxidant system can enhance the disease resistance of plants. The present invention studies and develops a multi-faceted strategy for the treatment of HLB, designs POA@I NPs nanomaterials that take into account anti-CLas, activation of plant antioxidant defense system and plant acquired immunity, and fully utilizes the good biocompatibility of POA to successfully load antibacterial drugs through surface modification, thereby realizing 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 nano 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 at 60 to 100° C. for 2 to 12 hours, and then washing to remove the organic solvent A. Preferably, the organic solvent A is N,N-dimethylformamide, and the number average molecular weight of the polysuccinimide is 6000-10000.

3. The antibacterial nano preparation according to claim 2, characterized in that: The preparation method of the nanocarrier POA comprises the following steps: taking polysuccinimide and oleylamine, adding them into an organic solvent A and mixing them, wherein the mass volume ratio of polysuccinimide to oleylamine is 1g:0.6-1.4ml, stirring and reacting them at 60-100°C for 2-12 hours, and after the reaction is finished, washing the reaction product with an organic solvent B to remove the organic solvent A, and collecting the precipitate by centrifugation after washing to obtain the nanocarrier POA, wherein the organic solvent B is methanol or ethanol; Preferably, the precipitate collected by centrifugation after washing is vacuum freeze-dried to a freeze-dried powder.

4. A method for preparing the antibacterial nano preparation according to any one of claims 1 to 3, characterized in that: The steps include: The nanocarrier POA and the organic-inorganic hybrid polyiodine crystal material are added to the organic solvent C and mixed to obtain a polymer mixture; polyvinyl alcohol is added to the polymer mixture, mixed thoroughly and evenly, and then reacted fully under stirring conditions, and after the reaction is completed, the nanoparticles are collected by centrifugation and washed to obtain the antibacterial nano preparation.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the organic-inorganic hybrid polyiodine crystalline material to POA in the polymer mixture is 1:5-15; preferably, 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.

6. The preparation method according to claim 4, 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, 1.5-3.0min; after the ultrasonic treatment, the mixture is reacted under stirring conditions 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.

7. Use of an organic-inorganic hybrid polyiodine crystalline material or the antibacterial nanoformulation according to any one of claims 1 to 3 in inhibiting plant pathogenic bacteria or preventing and controlling plant diseases; the molecular formula of the organic-inorganic hybrid polyiodine crystalline material is [(C6 H 21 I 26 O3 S3)2] n , where n is a natural number greater than or equal to 1.

8. The use according to claim 7, characterized in that: The plant pathogens include pathogenic bacteria or fungi; the plant diseases are plant diseases caused by bacterial or fungal pathogens.

9. The use according to claim 8, characterized in that: The plant disease is Huanglongbing of Rutaceae plants, and the Rutaceae plants refer to Citrus, Poncirus, Fortunella or Murraya.

10. The use according to claim 7, 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 nano preparation is 150-1500 μg / mL, preferably 180-1000 μg / mL or 350-1000 μg / mL or 350-800 μg / mL.

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