Acid response type nano preparation for inhibiting Candidatus Liberibacter asiaticum and preparation method thereof

Abstract

The invention belongs to the technical field of pesticide delivery, and particularly relates to an acid response type nano preparation for inhibiting Candidatus Liberibacter asiaticum and a preparation method of the acid response type nano preparation. Mesoporous nano silicon dioxide is used as a carrier, and a functional organic matter with amino is modified, so that the loading capacity of the mesoporous nano silicon dioxide is improved, and the acid response type nano preparation is used for inhibiting Candidatus Liberibacter asiaticum; and the chemical agent berberine can be loaded. Meanwhile, iron ions are added during loading, the loading rate of berberine can be further improved by utilizing the punctuality characteristic of the iron ions, finally, acid-responsive dopamine hydrochloride is used for packaging to prepare the nano medicament, and dopamine hydrochloride packaging improves the slow release capacity of the nano preparation and also improves the stability of the nano preparation. The nano preparation provided by the invention has a slow-release characteristic and a multi-component synergistic antibacterial effect, not only can significantly inhibit proliferation of CLas, but also can effectively improve the physiological status of diseased plants, and provides an innovative slow-release treatment strategy for comprehensive treatment of the citrus huanglongbing.

Description

Technical Field

[0001] This invention belongs to the field of pesticide delivery technology, and specifically relates to an acid-responsive nano-formulation for inhibiting Huanglongbing (HLB) of citrus and its preparation method. Background Technology

[0002] Citrus Huanglongbing (HLB), caused by *Candidatus Liberibacter asiaticus* (CLAs), is one of the most devastating diseases affecting the global citrus industry. The pathogen spreads through the field via its vector, the citrus psyllid (*Diaphorinacitri*). Control strategies for HLB primarily focus on using chemical agents (such as neonicotinoids) to kill the psyllid and employing antimicrobial agents (such as tetracycline antibiotics) to inhibit the reproduction of the HLB pathogen.

[0003] In recent years, innovative achievements have been made in the application research of nanodelivery systems in agricultural production. Nanodelivery systems can effectively improve the utilization efficiency of pesticides and enhance the control of pests and diseases.

[0004] In the prior art, Chinese Patent CN119655261A provides a method for preparing pH-responsive dopamine-modified tebuconazole layered double hydroxide nanocomposites and their application. The method prepares magnesium-aluminum layered double hydroxide nanocomposites by co-precipitation, uses SDS / PVP / CTAC to pillar-modify the layered double hydroxide nanocomposites, loads the antifungal drug tebuconazole, and then uses dopamine interface modification to modify the loaded tebuconazole layered double hydroxide nanocomposites, which can improve the antifungal bioactivity of tebuconazole. For example, Chinese patent CN119563634A discloses a porous chitosan-pyraclostrobin nano-slow-release agent, its preparation method and application. It prepares porous chitosan nanoparticles to carry pyraclostrobin, so as to achieve the purpose of nano-formulation and controlled release of pyraclostrobin. The porous chitosan nanoparticles with uniform morphology and particle size are prepared by interfacial polymerization at room temperature. They are used to carry the broad-spectrum fungicide pyraclostrobin. When used in the field, they can prolong the duration of action of the agent, reduce the number of applications, and reduce the amount of pesticide.

[0005] Therefore, developing a nano-formulation that utilizes a nano-delivery system to treat citrus Huanglongbing (HLB) and improve its therapeutic effect has become an urgent technical problem to be solved. Summary of the Invention To address the lack of nano-preparations for inhibiting Huanglongbing (HLB) in existing nanodelivery systems, this invention provides an acid-responsive nano-preparation for inhibiting HLB and its preparation method. Functionalized mesoporous silica nanoparticles are used as a carrier, loaded with berberine and iron ions, and then encapsulated in an acid-responsive manner to obtain the final nano-preparation. This nano-preparation can effectively inhibit HLB.

[0006] This invention provides a method for preparing an acid-responsive nano-formulation for inhibiting Huanglongbing (HLB) of citrus, the preparation method comprising the following steps: S1. Triethanolamine was added to deionized water, and after stirring, hexadecyltrimethylammonium bromide and sodium salicylate were added. After dispersion, the silicon source was injected, and the product was collected by centrifugation after heating and stirring. The product was refluxed in the mixed solution to remove hexadecyltrimethylammonium bromide and then extracted. The product was freeze-dried to obtain mesoporous silica nanoparticles DMSN. S2. The DMSN prepared in S1 is dispersed in an ethanol solution, a silane coupling agent is added and stirred, and then the excess coupling agent is removed by centrifugation to obtain functionalized mesoporous silica nanoparticles ND. The ND is dispersed in ethanol, berberine and an iron ion source are added, and after stirring, the mixture is centrifuged to obtain berberine-supported mesoporous silica nanoparticles NDBF. S3. Dissolve NDBF in ethanol, add HCl-PDA solution dropwise, and immediately after the HCl-PDA solution is added, add ammonia solution dropwise and continue stirring the reaction. After the reaction is completed, wash and dry to obtain acid-responsive silica nanoparticles NDBFP.

[0007] Furthermore, in step S1, the mixed solution is a mixture of concentrated hydrochloric acid and methanol in a volume ratio of 1:1; the DMSN particle size is 150-200 nm, and the silicon source is tetraethyl orthosilicate.

[0008] Furthermore, in step S2, the silane coupling agent includes 3-aminopropyltriethoxysilane, and the mass ratio of the silane coupling agent to DMSN is 10 to 30:1.

[0009] Furthermore, in step S2, the mass ratio of ND to berberine is 2 to 1:1, the iron ion source includes ferric chloride, and the iron ion concentration is 5 to 6 mM / L.

[0010] Furthermore, in step S2, the stirring time continues for 12–24 hours.

[0011] Furthermore, in step S3, the concentrations of the HCl-PDA solution and the NDBF ethanol solution are the same, the volume ratio of the HCl-PDA solution to the NDBF ethanol solution is 1:1, and the volume ratio of the ammonia water added to the previous solution is 1 to 2:5000.

[0012] The present invention also provides an acid-responsive nano-formulation for inhibiting Huanglongbing (HLB) of citrus, comprising a functionalized nanocarrier, berberine, iron ions and an acid-responsive encapsulating agent, wherein the functionalized nanocarrier is an amino-modified DMSN and the acid-responsive encapsulating agent is dopamine hydrochloride.

[0013] The beneficial effects of this invention are as follows: This invention utilizes mesoporous nano-silica as a carrier and enhances its loading capacity by modifying it with amino-functionalized organic compounds, enabling the loading of the chemical agent berberine. Simultaneously, iron ions are added to the loading process; the positive polarity of iron ions further increases the loading rate of berberine. Finally, acid-responsive dopamine hydrochloride is used for encapsulation to prepare the nano-drug. Dopamine hydrochloride encapsulation improves the sustained-release capability and stability of the nano-formulation. The nano-formulation provided by this invention possesses sustained-release properties and multi-component synergistic antibacterial activity. It not only significantly inhibits CLas proliferation but also effectively improves the physiological state of diseased plants, providing an innovative sustained-release treatment strategy for the integrated management of citrus Huanglongbing (HLB). Attached Figure Description

[0014] Figure 1 TEM image of the DMSN prepared in the embodiments of the present invention ( Figure 1 A), TEM image of NDBFP ( Figure 1 B), Particle size distribution chart ( Figure 1 C), zeta potential statistics ( Figure 1 D); Figure 2 The standard curve of BBR in Experimental Example 1 of this invention ( Figure 2 A, Figure 2 C) and the relationship between NDBF and NDBFP release rates and time ( Figure 2 B); Figure 3 This is the antibacterial growth curve of BBR in Experimental Example 2 of this invention. Figure 3 A), NDB antibacterial growth curve ( Figure 3 B), NDBF antibacterial growth curve ( Figure 3 C), NDBFP antibacterial growth curve ( Figure 3 D), NDBP antibacterial growth curve ( Figure 3 E); Figure 4 This refers to the NDBFP processing in Experimental Example 2 of this invention. HH103 Survival rate statistics of bacteria ( Figure 4 A), after NDBFP processing Xcc Survival rate statistics of bacteria ( Figure 4 B), after NDBFP processing HH103 Figure of dilution plating of bacteria ( Figure 4C), after NDBFP processing HH103 Figure of dilution plating of bacteria ( Figure 4 D); Figure 5 This is a gel electrophoresis image from Experiment 3 of this invention. Figure 5 A), CLas Survival rate statistics chart ( Figure 5 B); Figure 6 This is a statistical chart showing the catalytic decomposition effect of different components on hydrogen peroxide in Experimental Example 4 of this invention; Figure 7 This is a graph showing the change in H2O2 content in HLB-positive sweet orange leaves after NDBFP treatment in Experimental Example 4 of this invention. Figure 7 A), Graph showing changes in H2O2 content in diseased sweet oranges treated with NDBFP, healthy groups, and blank control group ( Figure 7 B); Figure 8 This is a statistical graph showing the changes in antioxidant enzyme activity after NDBFP treatment of HLB citrus in Experimental Example 4 of this invention. Figure 8 A represents CAT activity. Figure 8 B represents POD activity. Figure 8 C represents SOD activity; Figure 9 This is a statistical graph showing the changes in chlorophyll content in Experimental Example 5 of this invention; Figure 10 This is a statistical comparison chart of the changes in total chlorophyll content and the changes in endogenous carotenoid content in Experimental Example 5 of this invention; Figure 11 The figures show statistical comparisons of changes in malondialdehyde content and soluble sugar content in HLB-positive sweet orange leaves after NDBFP treatment in Experimental Example 5 of this invention. Detailed Implementation

[0015] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be considered in isolation, but can be combined with each other to achieve better technical effects. Example

[0016] This embodiment provides a method for preparing an acid-responsive nano-formulation for inhibiting Huanglongbing (HLB) of citrus, comprising the following steps: S1. Triethanolamine was added to deionized water, and after stirring, hexadecyltrimethylammonium bromide and sodium salicylate were added. After dispersion, the silicon source was injected, and the product was collected by centrifugation after heating and stirring. The product was refluxed in the mixed solution to remove hexadecyltrimethylammonium bromide and then extracted. The product was freeze-dried to obtain mesoporous silica nanoparticles DMSN. Specifically, 68.0 mg of triethanolamine (TEA) was added to 25 mL of water, and the mixture was stirred continuously at 80°C for 0.5 h. Next, 380 mg of hexadecyltrimethylammonium bromide (CTAB) and 168 mg of sodium salicylate were added sequentially to the well-stirred mixture, and the mixture was stirred for another 1 h. Then, 4 mL of tetraethyl orthosilicate (TEOS) was slowly added to the mixture, and the mixture was stirred gently at 80°C for 2 h. After the reaction was complete, the mixture was centrifuged at 8500 rpm for 10 min to collect the product. The collected product was washed three times with anhydrous ethanol. After washing, the product was redispersed in a mixture of 30 mL of 37% concentrated HCl and 30 mL of methanol. The mixture containing the dispersed product was refluxed at 60°C for 6 h to remove the CTAB template. After this step, the extraction process was repeated. Finally, the obtained DMSN was redispersed in 100 mL of ethanol for later use.

[0017] S2. The DMSN prepared in S1 is dispersed in an ethanol solution, a silane coupling agent is added and stirred, and then the excess coupling agent is removed by centrifugation to obtain aminated dendritic mesoporous silica ND. The ND is dispersed in ethanol, berberine and an iron ion source are added, and after stirring, it is centrifuged to obtain berberine-supported mesoporous silica nanoparticles NDBF. Specifically, 100 mg of DMSN and 2 mL of 97% pure APTES were added to 98 mL of anhydrous ethanol, and then stirred at room temperature for 2 h. To remove unreacted APTES, the mixture was centrifuged at 12000 r / min for 5 min, followed by washing three times with anhydrous ethanol for 10 min each time, thus obtaining ND. The ND was dried and stored for later use.

[0018] The dried ND was redispersed in anhydrous ethanol and diluted to a concentration of 1 mg / mL. Then, 70 mg of BBR and FeCl3 were added to the solution. . H2O was added and stirred overnight in the dark for 12 h. After stirring, the mixture was centrifuged three times with deionized water at 12000 r / min for 10 min each time to obtain NDBF.

[0019] S3. Dissolve NDBF in ethanol, add HCl-PDA solution dropwise, and immediately after the HCl-PDA solution is added, add ammonia solution dropwise and continue stirring the reaction. After the reaction is completed, wash and dry to obtain acid-responsive silica nanoparticles NDBFP.

[0020] Specifically, 10 mg of NDBF was accurately weighed and dissolved thoroughly in 5 mL of anhydrous ethanol. A 2 mg / mL HCl-PDA solution was then slowly added dropwise to this solution. Immediately after the HCl-PDA addition was complete, 1 μL of ammonia solution was added dropwise to the mixture. The mixture was stirred continuously overnight. After the reaction was complete, the product was washed multiple times with anhydrous ethanol to remove unreacted impurities. Subsequently, the washed product was processed using freeze-drying technology to finally obtain NDBFP.

[0021] This embodiment also provides an acid-responsive nano-formulation for inhibiting Huanglongbing (HLB) of citrus, comprising a functionalized nanocarrier, berberine, iron ions, and an acid-responsive encapsulating agent. The functionalized nanocarrier is an amino-modified DMSN, and the acid-responsive encapsulating agent is dopamine hydrochloride.

[0022] The prepared NDBFP and DMSN were characterized, and the results are attached. Figure 1 As shown in the figures, A is a TEM image of DMSN; B is a TEM image of NDBFP; C is a statistical graph of the particle size of nanoparticles at each stage; and D is a statistical graph of the zeta potential of nanoparticles at each stage. Observations revealed that NDBFP consists of uniformly sized spheres with excellent dispersibility. The edges of NDBFP changed from the dendritic structure of the original DMSN to a spherical morphology after being coated with HCl-PDA. The average size of the loaded NDBFP increased to 190 nm.

[0023] Experimental Example 1: Berberine Loading Rate and Release Experiment Loading rate experimental method: The loading rate of berberine (BBR) was calculated using the differential method. The NDBF prepared in the examples was used as the experimental group. The supernatant of the NDBF preparation process and its washing liquid was accurately collected, diluted by a certain factor, and the absorbance was measured and substituted into the BBR standard curve to calculate the loading rate of BBR in the NDBF.

[0024] Release rate experimental method: Accurately weigh 1 mg of NDBF and NDBFP samples and add them separately to 1 mL of pH 6.0 buffer solution. Place the solutions in a completely dark environment and transfer them to a shaker. Set the shaker speed to 60 rpm and perform sampling at specific time points: 0 min, 15 min, 30 min, 60 min, 120 min, and 24 min. At each time point, accurately transfer 200 μL of solution sample. To maintain a constant total volume, immediately replenish with an equal volume of pH 6 buffer solution after each sampling. Dilute the sample solutions according to a pre-determined appropriate ratio and measure their absorbance at 264 nm using a high-resolution spectrophotometer. Substitute the measured absorbance data into a pre-constructed and validated standard curve equation to accurately calculate the cumulative release of berberine at each time point.

[0025] Construction of BBR standard curves: Accurately weigh 10.00 mg of BBR powder (using a 0.01 g analytical balance), dissolve it separately in appropriate amounts of deionized water and ethanol, sonicate for 10 min, transfer to a 10 mL volumetric flask and dilute to volume to obtain a 1.00 mg / mL BBR stock solution. The solution should be filtered through a 0.22 μm filter membrane and stored at 4°C protected from light for later use. Further dilute to 10.0 μg / mL, 5.0 μg / mL, 2.5 μg / mL, 1.25 μg / mL and 0.625 μg / mL, 0.3125 μg / mL, 0.156 μg / mL test solutions, and measure the absorbance at 264 nm to plot the BBR standard curves. This yields standard curves for both the ethanol and aqueous phases of BBR. The aqueous phase standard curve is used to test the release rate, while the ethanol phase standard curve is used to test the loading rate.

[0026] The experimental results are attached. Figure 2 As shown, A is the standard curve of BBR in the ethanol phase; B is the relationship between the release rates of NDBF and NDBFP and time; and C is the standard curve of BBR in the H2O phase.

[0027] Experimental results analysis: Calculations show that the loading rate of BBR in NDBF is 64.62%. It should be noted that the addition of positively charged iron ions in this invention can increase the loading rate of BBR, from 23.44% to 64.62%.

[0028] The cumulative release rate of BBR in NDBF rapidly reached approximately 94.85% within 60 minutes and approached 100% after 100 minutes, exhibiting a rapid release characteristic. In contrast, the cumulative release rate of NDBFP increased significantly and gradually, reaching only about 35.33% at 60 minutes and only 46.1% at 250 minutes. Compared to NDBF, the release process of NDBFP was slower and more sustained, without any burst release. This data difference clearly indicates that the HCl-PDA encapsulation of the nanomaterials significantly inhibited the release of BBR, resulting in a reduced release rate and prolonged release time. This confirms that the HCl-PDA encapsulation structure of NDBFP effectively regulates the release kinetics of BBR, achieving a sustained-release function and fully demonstrating the crucial role of HCl-PDA in delaying drug release.

[0029] Experiment Example 2: In vitro antibacterial effect experiment Experimental methods: Inhibition rate test: Considering the inherent difficulties in the pure in vitro culture of HLB pathogens, this study carefully selected several bacterial strains to evaluate the in vitro antibacterial efficacy of NDBFP. The selected strains included *Rhizobium fischeri*, a close relative of CLAs. Sinorhizobium fredii HH103 ), closely related fungi of citrus Huanglongbing fungus RM1021 and Xanthomonas citrus subsp. citrus ( Xanthomonas citri subsp. citri, Xcc This experiment used the 96-well plate method to determine... RM1021 Growth curves of the bacteria were obtained to investigate the effects of different materials on their growth. BBR at concentrations of 62.5 μg / mL, 125 μg / mL, 250 μg / mL, and 500 μg / mL, NDB loaded with BBR, and Fe were added. 3+ NDBF containing ions, NDBP and NDBFP nanomaterial solutions encapsulated with HCl-PDA were added respectively to solutions containing ions RM1021 In 96-well plates containing bacterial culture, a blank control group was also included. The 96-well plates were placed in a constant temperature incubator, and the OD values ​​of each well were measured using a microplate reader at intervals between 0 and 36°C. 600 Before measurement, gently shake the well plate to ensure even distribution of the bacterial culture; record the data and remove outliers, then plot the values ​​on the x-axis as time and OD. 600 The value is the vertical axis, and a growth curve is plotted using professional software.

[0030] Survival test: Cultured in SG medium and NA medium at 27°C respectively. HH103 bacteria and Xcc After 5 days of bacterial culture, NDBFP was serially diluted in a clean bench. Equal volumes of the diluted NDBFP solutions were then added to the solutions. HH103 bacterial solution (concentration of 10)6 The NDBFP concentrations were adjusted to 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 900 μg / mL by shaking (or vortexing, etc.). Similarly, an equal volume of CFU / mL was added to the diluted NDBFP solution. Xcc bacterial solution (concentration of 10) 6 The NDBFP concentrations were adjusted to 56.25 μg / mL, 112.5 μg / mL, 225 μg / mL, 450 μg / mL, and 900 μg / mL by shaking (or vortexing, etc.). The mixtures were then incubated at 27°C for 4 hours, followed by dilution to appropriate concentrations for plating.

[0031] The experimental results are attached. Figure 3 and attached Figure 4 As shown, the attached Figure 3 A represents the inhibition of different concentrations of BBR. RM1021 B is the growth curve of the bacteria; B represents the inhibition of different concentrations of NDB. RM1021 Growth curve of bacteria; C represents the inhibition of different concentrations of NDBF. RM1021 Growth curves; D represents the inhibition of different concentrations of NDBFP. RM1021 Growth curve of bacteria; E represents the inhibition of different concentrations of NDBP. RM1021 Growth curve of the bacteria. (Attached) Figure 4 In the middle, A represents the results after treatment with different concentrations of NDBFP. HH103 B is a statistical chart of bacterial survival rates; B represents the survival rates of bacteria after treatment with different concentrations of NDBFP. Xcc Survival rate statistics of bacteria; C represents the survival rate of bacteria after treatment with different concentrations of NDBFP. HH103 Diluted plating of bacteria; D represents different concentrations of NDBFP after treatment. HH103 Image of diluted bacterial plating.

[0032] Analysis of Experimental Results: (Appendix) Figure 3 In group A, after 12 hours of incubation, at a concentration of 250 μg / mL, the inhibition rate of BBR against bacteria was 9.5%, lower than that of NDB (74.07%), NDBF (78.10%), NDBP (85.27%), and NDBFP (94.88%). At other concentrations, NDB, NDBF, NDBP, and NDBFP showed relatively low inhibition rates against bacteria. RM1021 The antibacterial activity of these bacteria was also stronger than that of BBR. BBR, NDB, NDBF, NDBP, and NDBFP were respectively comparable to... RM1021 After co-incubation with the bacteria for 20 days, the inhibition rates were 18.77%, 48.63%, 76.91%, 70.86%, and 69.85%, respectively (see appendix). Figure 3(AE). With increasing time, NDBFP showed higher antibacterial efficiency than NDBF, likely due to NDBFP's sustained-release properties. The structure of NDBFP allows for the slow release of its antibacterial components, prolonging the effective ingredient's duration of action in the system and providing sustained inhibition. RM1021 While NDBF does not possess this long-lasting sustained-release mechanism, it inhibits bacterial growth, thus NDBFP exhibits higher antibacterial efficiency after the same time interval.

[0033] Based on the combined experimental data and phenomena, NDBFP... HH103 and Xcc Both citrus model bacteria exhibited significant antibacterial activity. (Observation) HH103 strains (attached) Figure 4 (A) When the NDBFP material concentration was 62.5 μg / mL, the bacterial survival rate was 80.65%. As the concentration increased sequentially to 125 μg / mL, 250 μg / mL, and 500 μg / mL, the survival rate decreased to 70.23%, 65.41%, and 63.78%, respectively, and dropped to 0% at a concentration of 900 μg / mL. The corresponding dilution plating results are attached. Figure 4 C) shows that as the treatment concentration increases, the number of colonies on the plate gradually decreases, from a dense distribution at the initial concentration to no colony growth at 900 μg / mL, clearly demonstrating the effect of NDBFP on... HH103 The antibacterial effect of the strain is concentration-dependent.

[0034] As attached Figure 4 As shown in Figure B, during the process of increasing the concentration from 56.25 μg / mL to 900 μg / mL, Xcc The survival rate decreased from 53.61% to 30.88%. Notably, at a concentration of 225 μg / mL, the antibacterial effect stabilized, and the survival rate decreased to 30.82%, with subsequent increases in concentration not causing significant fluctuations in the survival rate. (See attached dilution plating results). Figure 4(D) The number of colonies decreased significantly with increasing concentration, becoming sparse from an initial dense state to a high concentration, further confirming the inhibitory effect of NDBFP on this strain. The antibacterial mechanism of NDBFP may be as follows: First, BBR, as an alkaloid antibacterial component, can embed into the bacterial cell membrane, disrupting the membrane structure and causing leakage of contents; second, iron ions can induce ROS accumulation in bacteria through the Fenton reaction, triggering oxidative stress, damaging bacterial nucleic acids, proteins, and other biomolecules, and inhibiting their metabolic activities; finally, the aminated silicon nanocarrier and HCl-PDA encapsulation structure not only improve the dispersibility and biocompatibility of the antibacterial component, but also, due to the nanoscale size effect, enable it to act more precisely on specific target sites on the bacterial surface, enhancing the targeting effect on bacteria. Multiple mechanisms synergistically amplify the antibacterial effect, achieving a 1+1 greater than 2 effect. The phenomenon that the bacterial survival rate decreases with increasing concentration and the number of colonies decreases in the experiment is a manifestation of the synergistic inhibition of bacterial proliferation by multiple antibacterial mechanisms of NDBFP, fully demonstrating its clear antibacterial efficacy against the citrus model bacteria.

[0035] Experiment Example 3: Antibacterial Effect of Citrus Fruit in Vivo Experimental Methods: Valencia sweet orange trees aged 2-3 years and positive for HLB infection were selected as experimental materials. They were cultivated in a greenhouse (temperature 27℃±2°C, relative humidity 50%±5%, and natural light duration). 100 μL of water (CK) and 225 μg / mL NDBFP were sprayed onto leaves of similar phenotypes on the same branch. Samples were taken on days 3, 7, 12, and 16 after spraying. A 1 cm diameter perforator was used to make mirror images of the leaves, with the veins as the dividing line. The total sample size was approximately 100 mg. The samples were then placed in 2 mL reinforced centrifuge tubes containing three 0.5 cm diameter steel balls. The collected leaf samples were immediately frozen in liquid nitrogen for 5 min, then transferred to an automated rapid grinder (160 Hz, 5 min) for thorough homogenization. The appropriate extraction solvent was then added according to the downstream experimental requirements.

[0036] Immediately reverse transcribe the DNA into cDNA using a DNA reverse transcription kit. Perform PCR using the cDNA; the solution composition is 1 μL cDNA, forward and reverse primers. actin-2 or HLBas2 1 μL and 5 μL of PCR mix and 2 μL of RNase-free H2O were added. The PCR program consisted of 10 min of pre-denaturation at 95 °C, followed by 15 s of denaturation at 95 °C, 20 s of annealing at 60 °C, and 15 s of extension at 72 °C, for a total of 40 cycles. After the reaction, 3 μL of the PCR reaction solution was subjected to gel electrophoresis on a 2% agarose gel.

[0037] The content of CLAs in confirmed infected sweet oranges was detected by qPCR using the SYBR Green dye method. The qPCR reaction system was prepared using total DNA extracted by the CTAB method, consisting of 1 μL gDNA, 1 μL each of forward and reverse primers, 5 μL qPCR MIX, and 2 μL RNase-free H2O in 10 μL reaction solution. The qPCR program was 10 min 95 °C pre-denaturation, followed by 15 s 95 °C denaturation, 20 s 60 °C annealing, and 15 s 72 °C extension as one cycle, for a total of 32 cycles. Fluorescence signal was read at the end of each cycle at 72 °C. Three biological replicates and three technical replicates were set up for each sample. actit-2 The CT value was used as an internal reference, and 2 -ΔΔCT Results were analyzed, with the gDNA template of sweet oranges suffering from HLB as the control and the gDNA template of sweet oranges after drug administration as the experimental group.

[0038] The experimental results are attached. Figure 5 As shown, A is a gel electrophoresis image; B is a statistical graph of the survival rate of CLas in HLB-positive sweet orange leaves after NDBFP treatment.

[0039] Experimental Results Analysis: PCR detection was performed on sweet oranges exhibiting HLB symptoms in a greenhouse (see attached). Figure 5 A), Lane 1-3 Manager actin-2 Stable amplification of the HLB-specific gene (approximately 150 bp) and the gene (approximately 110 bp) in lanes 4-6 confirmed the presence of CLAs, the pathogen of citrus Huanglongbing (HLB), in sweet orange leaves, laying the foundation for subsequent antibacterial experiments. Sweet orange trees were treated with 225 μg / mL NDBFP, and qPCR analysis was performed (see attached). Figure 5 B) shows that on day 3 after application, the survival rate of CLas decreased to 25.01%; on day 7, it further decreased to 6.8%, close to the 1.27% of the WT group; although the survival rate fluctuated slightly on day 12 (9.5%) and day 16 (13.34%), it was significantly lower than that of the control group (CK). This dynamic change directly proves that NDBFP has an inhibitory effect on CLas and can effectively reduce the survival level of pathogens. This antibacterial effect is due to the synergistic effect of the materials: BBR can destroy the cell membrane integrity of CLas and interfere with their metabolic processes; iron ions induce the accumulation of reactive oxygen species in CLas through oxidative stress, damaging nucleic acids and proteins; the amino-coated silicon nanocarrier and HCl-PDA encapsulation structure enhance the targeted delivery efficiency of antibacterial components, and the multi-mechanism synergistic inhibition of CLas proliferation is ultimately reflected in the significant decrease in the survival rate of pathogens in the experiment, which clearly verifies the antibacterial efficacy of NDBFP against the pathogen of citrus Huanglongbing.

[0040] Experiment Example 4: Antioxidant Performance Experiment TMB colorimetric experiment: TMB colorimetric solution and 50 mmol / L H2O2 were mixed at a volume ratio of 1:1, and then an appropriate amount of NDBFP was added. After incubation at room temperature in the dark for 30 min, the absorbance was measured at 652 nm immediately.

[0041] Hydrogen peroxide content and antioxidant enzyme activity determination: After accurately weighing the plant tissue, it was frozen in liquid nitrogen and ground in an automated sample homogenizer. 0.1 mol / L PBS buffer (0.1 mol / L pH 7.0) was added to the tissue sample at a sample mass (mg):PBS volume ratio of 1:9 to obtain the extract of antioxidant-related enzymes, which was then stored at low temperature. The homogenate was prepared mechanically according to the kit instructions. The homogenate was centrifuged at 3000-10000 r / min at 4℃ for 10 min (specific speed as per the instructions), and the supernatant was collected for determining the activities of H2O2, SOD, CAT, and POD in citrus tissue. The relevant detection kits were used according to the kit instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu Province, China: A064-1 for H2O2 detection, A084-3 for POD detection, A001-1 for SOD detection, and A007-1 for CAT detection).

[0042] The experimental results are attached. Figure 6-8 As shown, the attached Figure 7 Table A shows the change in H2O2 content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; Table B shows the change in H2O2 content in diseased sweet oranges, healthy groups, and the blank control group after foliar spraying with 225 μg / mL NDBFP for 16 days. (See attached table.) Figure 8 The graph in Figure AC represents the statistical changes in antioxidant enzyme activity after NDBFP treatment of HLB citrus, where A represents CAT activity, B represents POD activity, and C represents SOD activity. Figure 8 The DF values ​​represent the antioxidant enzyme activities of diseased sweet oranges, healthy groups, and blank control groups after foliar spraying with 225 μg / mL NDBFP for 16 days. D represents CAT activity, E represents POD activity, and F represents SOD activity.

[0043] Analysis of Experimental Results: (Appendix) Figure 6In the experiment, NDBFP exhibited highly efficient catalytic activity for the decomposition of H2O2. A blue radical intermediate (TMB⁺) was generated, exhibiting a characteristic maximum absorption peak at 652 nm. With increasing NDBFP concentration gradient, TMB gradually changed from colorless to blue-black. This is because the amino group in the TMB molecule undergoes single-electron oxidation to form a cationic radical, which then associates with another TMB molecule through charge transfer to form a dimerized charge-transfer complex. During this process, NDBFP participates in the reaction, inhibiting the oxidation of TMB and the resulting color change, confirming its antioxidant properties and its ability to effectively promote the decomposition of H2O2.

[0044] In the TMB colorimetric experimental system, the antioxidant capacity of NDBFP can be characterized by the relationship between absorbance and concentration. As the material concentration increases from 7.81 μg / mL to 1000 μg / mL, the absorbance increases significantly. This phenomenon is consistent with its antioxidant mechanism: after the addition of NDBFP, its phenolic hydroxyl groups and other antioxidant active groups play a core role. The o-dihydroxyl groups of surface dopamine hydrochloride act as "electron donors," preferentially reducing the loaded Fe³⁺ to Fe²⁺. The generated Fe²⁺ immediately undergoes a Fenton reaction with H₂O₂, forming an orderly cycle of "Fe³⁺→Fe²⁺→・OH". Simultaneously, the nitrogen-containing groups of berberine chelate Fe³⁺, regulating the cycle rate and preventing a sudden increase in ・OH that could lead to excessive oxidation of TMB, making the colorimetric process smoother and more controllable. Furthermore, the antioxidant effect of the phenolic hydroxyl groups inhibits excessive TMB color development. This dual effect jointly confirms its antioxidant activity and catalytic efficiency.

[0045] Appendix Figure 7 In this study, infection by the citrus Huanglongbing pathogen (CLas) significantly affected the H2O2 content in sweet oranges. Compared with data from healthy plants (WT group) at the same time point, the H2O2 content in diseased sweet oranges (CK group) increased by 135.28%. Compared with the initial level in the CK group before application, the application of NDBFP reduced the H2O2 content in diseased sweet oranges. At 3, 7, 12, and 16 days after application, the H2O2 content in diseased sweet oranges decreased by 14.98%, 34.64%, 30.77%, and 20.33%, respectively. NDBFP had a sustained scavenging effect on H2O2 in diseased sweet oranges within 7 days after application. As a type of reactive oxygen species (ROS), reducing H2O2 content helps alleviate oxidative damage to cell structures and biomolecules, thereby effectively alleviating oxidative stress levels in plants, maintaining intracellular environmental stability, and having a positive effect on improving the physiological state of diseased sweet oranges.

[0046] Appendix Figure 8 In, such as Figure 8As shown in Figure A, compared with the CK group, CAT activity in the NDBFP group showed no significant fluctuation from day 3 to day 12. On day 16, the CAT activity in the NDBFP group reached 363.66 U / g, an increase of 32.50 U / g compared to the CK group (331.16 U / g), similar to the level in the healthy WT group. The trend of POD activity change was similar to that of CAT, with an increase of 81.12% relative to the CK group on day 12. Figure 8 B), 1395.71 U / g in the near-healthy WT group on day 16.

[0047] SOD activity was higher in the NDBFP group than in the CK group. Over time, on day 3, the SOD activity in the NDBFP group was 146.38% of that in the CK group; on day 7, although it decreased slightly, it remained at 79.33% of the CK group; on day 12, it increased significantly, reaching 233.35% of the CK group; and on day 16, it was 699.33 U / g, which was 140.75% of the CK group and quite close to the 571.21 U / g of the healthy WT group.

[0048] From the perspective of plant stress response mechanisms, under normal circumstances, when ROS increases within a certain range, the activity of antioxidant enzymes will increase synchronously to maintain homeostasis; however, under long-term biotic or abiotic stress, the ability of the antioxidant system to regulate ROS will be impaired. In HLB-positive sweet orange leaves, the antioxidant enzyme activity in the CK group was significantly lower than that in the WT group (…). Figure 8 The presence of DF indicates that the antioxidant system is unable to cope with the excessive accumulation of ROS. However, the antioxidant enzyme activity in the NDBFP group was significantly higher than that in the CK group, confirming that NDBFP can activate the antioxidant defense system in HLB-positive sweet oranges, enabling them to regain the ability to regulate ROS, and providing important physiological mechanism support for alleviating the damage of citrus Huanglongbing.

[0049] Experiment Example 5: Citrus Growth Experiment Pigment content determination experiment: 1 mL of 75% ethanol was added to the homogenized tissue sample, and pigments were extracted for 15 min at room temperature in a shaker at 250 r / min in the dark. After extraction, the mixture was centrifuged at 6000 r / min for 10 min to separate the supernatant and precipitate. This operation was repeated twice to ensure complete pigment extraction. Finally, 3 mL of the supernatant was combined, diluted according to the preset dilution ratio, and added to a cuvette with a 1 cm optical path. Using 75% ethanol as a blank, the absorbance was measured using a UV-Vis spectrophotometer. The absorbance values ​​were read at wavelengths of 490 nm, 649 nm, and 665 nm to obtain the chlorophyll a concentration (C). a ), chlorophyll b concentration (C b ) concentration, total chlorophyll (C t ) concentration and endogenous carotene concentration (C carThe formula is as follows: C a =13.95A 665 -6.88A 649 ; C b =24.96A 649 -7.32A 665 ; C t =C a +C b ; C car =100*(1000A) 490 -3.27C a -104C b ) / 229.

[0050] Finally, the chlorophyll content in plant tissue can be further calculated using the following formula: Chlorophyll content (mg / g FW) = (Chlorophyll concentration × Extract volume × Dilution factor) / Sample fresh weight. Experimental results are attached. Figure 9-10 As shown, the attached Figure 9 A is a statistical graph showing the change in chlorophyll a content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; B is a statistical graph showing the change in chlorophyll b content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; C is a statistical graph showing the chlorophyll a content in diseased sweet oranges, healthy groups, and blank control groups after 16 days of foliar spraying with 225 μg / mL NDBFP; D is a statistical graph showing the chlorophyll b content in diseased sweet oranges, healthy groups, and blank control groups after 16 days of foliar spraying with 225 μg / mL NDBFP. Figure 10 Figure A shows the statistical changes in total chlorophyll content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; Figure B shows the statistical changes in carotenoid content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; Figure C shows the comparison of total chlorophyll content in diseased sweet oranges, healthy groups, and blank control group after 16 days of foliar spraying with 225 μg / mL NDBFP; Figure D shows the comparison of carotenoid content in diseased sweet oranges, healthy groups, and blank control group after 16 days of foliar spraying with 225 μg / mL NDBFP.

[0051] Analysis of experimental results: such as Figure 9 As shown in Figure A, after NDBFP treatment, the chlorophyll a content in HLB-positive sweet orange leaves increased by 33.13%, 36.91%, 47.23%, and 41.19% compared to the control group at days 3, 7, 12, and 16, respectively, with the most significant increase observed on day 7, reaching 1.16 mg / g. The chlorophyll b content increased by 106.36%, 59.77%, 64.79%, and 57.03% compared to the control group at each corresponding day. Figure 9 B). Meanwhile, the carotene content, starting from day 7 after NDBFP treatment, increased by 0.38%, 14.64%, and 14.74% compared to the CK group, respectively. Figure 10 B, Figure 10 D).

[0052] Chlorophyll, a key pigment in photosynthesis, contributes to enhanced photosynthetic capacity of sweet orange leaves by increasing its content, providing more energy for plant growth. Carotene, an antioxidant, enhances the plant's antioxidant capacity by increasing its content, alleviating oxidative stress caused by HLB pathogen infection. Although chlorophyll b content decreased at some time points after NDBFP treatment, total chlorophyll and chlorophyll a content generally showed an upward trend, and carotene content also increased. These changes collectively reflect the positive effect of NDBFP treatment on improving the physiological state of HLB-positive sweet oranges.

[0053] Determination of malondialdehyde (MDA) and soluble sugar content: After freezing the leaves with liquid nitrogen and thoroughly grinding them, approximately 100 mg of the treated sample was dispersed in 1 mL of 10% TCA and shaken at 250 r / min in a shaker at room temperature for 5 min. Subsequently, the tissue mixture was centrifuged at 10000 r / min for 10 min. 500 μL of the supernatant was reacted with 500 μL of 0.6% TBA in a boiling water bath for 10 min. After the reaction, the mixture was rapidly cooled on ice and then centrifuged at 10000 r / min at 4 ℃ for 5 min. The sample was zeroed with deionized water, and the absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm. The formulas for calculating MDA and soluble sugar content are as follows: C Sugar (μmol / L) = 11.71A 450 C MDA (μmol / L) = 6.45 (A) 532 -A 600 -0.56A 450 The MDA and soluble sugar content in the plant tissue can then be calculated using the following formula: Soluble sugar / MDA content (μmol / g FW) = (concentration × extract volume × dilution factor) / fresh weight of sample.

[0054] The experimental results are attached. Figure 11As shown, A is a statistical graph showing the change in malondialdehyde (MDA) content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; B is a statistical graph showing the change in soluble sugar content in HLB-positive sweet orange leaves after 16 days of NDBFP treatment; C is a statistical graph showing the MDA content in diseased sweet oranges, healthy groups, and the blank control group after 16 days of foliar spraying with 225 μg / mL NDBFP; and D is a statistical graph showing the soluble sugar content in diseased sweet oranges, healthy groups, and the blank control group after 16 days of foliar spraying with 225 μg / mL NDBFP.

[0055] Analysis of experimental results: such as Figure 11 As shown in Figure A, NDBFP treatment reduced MDA content. The highest MDA content in the leaves of positive sweet oranges was 68.90 nmol / g. After NDBFP treatment, it decreased to 55.64 nmol / g on day 3, 26.27 nmol / g on day 7, and reached its lowest point on day 12, a reduction of 68.0% compared to the control group, gradually approaching the level of the total wheat harvester (WT) group. Figure 11 C). When plants are subjected to oxidative stress, they respond by increasing the synthesis and accumulation of soluble sugars. After treatment with NDBFP, the soluble sugar content of HLB citrus decreased because NDBFP alleviated oxidative stress, eliminating the need for plants to maintain high levels of soluble sugar synthesis. The soluble sugar concentrations on days 3, 7, 12, and 16 were 145.43 nmol / g, 67.99 nmol / g, 70.07 nmol / g, and 56.99 nmol / g, respectively. The lowest soluble sugar content was observed on day 16, close to the 44.93 nmol / g of the WT group. Figure 11 D).

[0056] The decrease in MDA and soluble sugar content indicates that these nanoparticles alleviated the abiotic stress faced by HLB-positive leaves. When CLas-induced ROS accumulates excessively to a critical level, it may lead to the death of companion cells and sieve cells. This oxidative stress phenomenon is considered one of the factors contributing to the higher MDA concentration in HLB-positive leaves compared to healthy leaves.

[0057] While several embodiments of the present invention have been provided herein, those skilled in the art should understand that modifications can be made to these embodiments without departing from the spirit of the invention. The above embodiments are merely exemplary and should not be construed as limiting the scope of the invention.

Claims

1. A method for preparing an acid-responsive nano-formulation for inhibiting Huanglongbing (HLB) of citrus, characterized in that, The preparation method includes the following steps: S1. Triethanolamine was added to deionized water, and after stirring, hexadecyltrimethylammonium bromide and sodium salicylate were added. After dispersion, the silicon source was injected, and the product was collected by centrifugation after heating and stirring. The product was refluxed in the mixed solution to remove hexadecyltrimethylammonium bromide and then extracted. The product was freeze-dried to obtain mesoporous silica nanoparticles DMSN. S2. The DMSN prepared in S1 is dispersed in an ethanol solution, a silane coupling agent is added and stirred, and then the excess coupling agent is removed by centrifugation to obtain functionalized mesoporous silica nanoparticles ND. The ND is dispersed in ethanol, berberine and an iron ion source are added, and after stirring, the mixture is centrifuged to obtain berberine-supported mesoporous silica nanoparticles NDBF. S3. Dissolve NDBF in ethanol, add HCl-PDA solution dropwise, and immediately after the HCl-PDA solution is added, add ammonia solution dropwise and continue stirring the reaction. After the reaction is completed, wash and dry to obtain acid-responsive silica nanoparticles NDBFP.

2. The method for preparing an acid-responsive nano-formulation for inhibiting citrus Huanglongbing (HLB) as described in claim 1, characterized in that, In step S1, the mixed solution is synthesized by mixing concentrated hydrochloric acid and methanol at a volume ratio of 1:1; the DMSN particle size is 150-200 nm.

3. The method for preparing an acid-responsive nano-formulation for inhibiting citrus Huanglongbing (HLB) as described in claim 1, characterized in that, In step S2, the silane coupling agent includes 3-aminopropyltriethoxysilane, and the mass ratio of the silane coupling agent to DMSN is 10 to 30:

1.

4. The method for preparing an acid-responsive nano-formulation for inhibiting citrus Huanglongbing (HLB) as described in claim 3, characterized in that, In step S2, the mass ratio of ND to berberine is 2 to 1:1, the iron ion source includes ferric chloride, and the iron ion concentration is 5 to 6 mM / L.

5. The method for preparing an acid-responsive nano-formulation for inhibiting citrus Huanglongbing (HLB) as described in claim 4, characterized in that, In step S2, the stirring time continues for 12 to 24 hours.

6. The method for preparing an acid-responsive nano-formulation for inhibiting citrus Huanglongbing (HLB) as described in claim 1, characterized in that, In step S3, the HCl-PDA solution and the NDBF ethanol solution have the same concentration, the volume ratio of HCl-PDA solution to NDBF ethanol solution is 1:1, and the volume ratio of ammonia added to the previous solution is 1-2:5000.

7. The acid-responsive nano-formulation for inhibiting citrus Huanglongbing (HLB) prepared by the preparation method according to any one of claims 1-6, characterized in that, It includes a functionalized nanocarrier, berberine, iron ions, and an acid-responsive encapsulating agent, wherein the functionalized nanocarrier is an amino-modified DMSN, and the acid-responsive encapsulating agent is dopamine hydrochloride.

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