Preparation method of nano bamboo charcoal for treating betel nut liberobacter asiaticum

Abstract

The invention discloses a preparation method of nano bamboo charcoal for treating betel nut yellow shoot, and relates to the technical field of polymer modified materials. Innovation is achieved through the following steps that firstly, 50-100 nm nano bamboo charcoal dispersion liquid is prepared through a ball milling method; (2) polyvinyl alcohol and a modifier are used for synergistically modifying the nano bamboo charcoal, so that the surface activity and stability are enhanced; 3) tetracycline antibiotics are loaded, and the targeting property is improved through a pH response release mechanism; the preparation method has the advantages that the nano bamboo charcoal has antibacterial, antioxidant and slow-release functions, the inhibition rate of the nano bamboo charcoal to the Candidatus Liberibacter asiaticum reaches 96%, the lasting period is prolonged to 20 days, the environmental residue is reduced to be less than 5 ppm, and an efficient solution is provided for green prevention and control of betel nut diseases.

Description

Technical Field

[0001] This invention relates to the field of polymer modified materials technology, specifically a method for preparing nano-bamboo charcoal for treating Huanglongbing in areca palm. Background Technology

[0002] Huanglongbing (HLB) is a highly destructive disease in areca nut cultivation, caused by a phloem-restricting Gram-negative bacterium. Candidatus Liberibacter asiaticus Caused by CLas, this disease is transmitted by psyllids. It leads to asymmetrical yellowing of areca leaves, branch dieback, root degeneration, and fruit deformities, and in severe cases, plant death. Traditional control methods primarily rely on antibiotic application (such as tetracyclines) and vector insect control; however, long-term use can induce antibiotic resistance in pathogens, and the environmental residues of chemical agents are a significant concern. In recent years, nanotechnology has gradually become a research hotspot in the field of plant disease control due to its advantages in targeted delivery, slow-release synergistic effects, and biosafety.

[0003] Huanglongbing Pathogenesis and Limitations of Existing Control Techniques: After infecting areca palms, clas primarily colonize the phloem sieve tubes, interfering with plant immune signaling pathways by secreting effector proteins, inducing reactive oxygen species (ROS) bursts and abnormal callose deposition, and blocking the transport of photosynthetic assimilates. Studies have shown that malondialdehyde (MDA) content is significantly increased and chlorophyll and carotenoid levels are decreased in the leaves of diseased plants, indicating that oxidative stress is a key link in the pathological process. Traditional control methods have the following drawbacks: poor antibiotic penetration: the phloem tissue where clas reside is dense, making it difficult for conventional agents to penetrate effectively; for example, direct injection of oxytetracycline can only achieve local inhibitory concentrations. Environmental toxicity risks: continuous control of disease vectors by chemical pesticides leads to ecological imbalance and poses a risk of pesticide residues. Insufficient induction of systemic resistance: existing technologies mostly target the direct killing of pathogens, lacking regulation of the plant's own antioxidant and immune response capabilities.

[0004] The technological advantages of nanomaterials in plant disease control: Nanocarriers can achieve targeted drug delivery through size effects and surface modification, improving control efficiency. Studies show that particles with a diameter of less than 100 nm can penetrate the waxy layer of the plant epidermis and enter the phloem via plasmodesmata. For example: Manganese oxide nanoenzymes (MONPs): By mimicking the activities of superoxide dismutase (SOD) and catalase (CAT), they directly scavenge ROS, reducing ROS levels in diseased leaves by 60% and upregulating the expression of antioxidant genes; Water-in-oil nanoemulsion systems: Using polyoxyethylene castor oil / Span80 as an emulsifier, nanoemulsions loaded with ampicillin have a 3.3-fold increased transdermal efficiency and significantly increased drug concentration in the phloem; Carbon quantum dot-oxytetracycline complex: Utilizing the fluorescent tracer properties of carbon quantum dots, it has been confirmed that they can migrate across vascular bundles, reducing CLas loading by 70%.

[0005] The technological potential and application prospects of bamboo charcoal nanofibers: After high-temperature carbonization, bamboo charcoal forms a porous structure with a specific surface area of ​​300-2000 m² / g, exhibiting excellent adsorption performance and biocompatibility. Nano-bamboo charcoal (NBC) can enhance the control effect of Huanglongbing (HLB) through the following mechanisms: Drug loading and sustained release: The mesoporous structure (2-50 nm) of NBC can efficiently load antibiotics or plant immune inducers (such as Flg22 peptide), extending the action period through pH-responsive release; ROS scavenging and oxidation balance: The abundant oxygen-containing functional groups (-COOH, -OH) on the surface can capture free radicals, synergistically reducing oxidative damage in diseased plants; Microbial community regulation: NBC improves the soil environment, promotes the colonization of beneficial bacteria (such as Bacillus subtilis), and inhibits the proliferation of Clas. Experiments have shown that nano-preparations based on bamboo charcoal have demonstrated application potential in the control of citrus HLB. For example, after trunk injection of carbon quantum dot-oxytetracycline nanocomposites, the drug concentration in the sieve tubes of diseased plants increased by 2.7 times compared to traditional methods, and the effective period was extended to 20 days. In addition, field trials of bamboo charcoal nanofiber and MnO2 composite materials showed that they could increase leaf chlorophyll content by 15% and reduce callose deposition by 40%.

[0006] Despite the promising prospects of nano-bamboo charcoal formulations, the following bottlenecks still need to be overcome: Large-scale preparation process: The physical / chemical parameters of bamboo charcoal nano-processing (such as ball milling and acid hydrolysis) need to be optimized to balance yield and particle size uniformity; Environmental behavior assessment: The metabolic pathways and long-term ecological safety of NBC in areca nut tissue need to be clarified to avoid the bioaccumulation of nanoparticles; Multi-mechanism synergistic design: Develop composite formulations with antibacterial, antioxidant and immune-activating functions, such as NBC carriers loaded with MONPs and Flg22 peptide.

[0007] In summary, nano-bamboo charcoal formulations offer an innovative solution for the green control of Huanglongbing (HLB) in Areca catechu by integrating multiple mechanisms, including targeted delivery, oxidative stress relief, and microecological regulation. Future research should focus on the material-pathogen-host interaction mechanisms to promote the translation of this technology from the laboratory to field applications. Summary of the Invention

[0008] This invention aims to address the following key problems in the prevention and control of Huanglongbing (HLB) in existing technologies: poor antibiotic penetration: traditional drugs are difficult to penetrate effectively due to the dense structure of the phloem, resulting in insufficient local drug concentration (e.g., oxytetracycline injection only provides localized bacteriostasis); environmental toxicity and drug resistance: long-term use of chemical pesticides leads to ecological imbalance and pesticide residues, and pathogens (CLas) are prone to developing drug resistance (literature shows that the inhibition rate of a single antibiotic is only 70%); systemic defense deficiencies: existing technologies focus on the direct killing of pathogens, neglecting the plant's own antioxidant and immune response regulation (ROS accumulation in HLB-infected plants leads to oxidative damage); short duration of effectiveness: conventional formulations degrade quickly (e.g., water-in-oil nanoemulsions have an effective period of only 15 days), requiring frequent application.

[0009] The innovative solutions of this invention are as follows: Nano-targeted delivery system: 50-100nm nano-bamboo charcoal is prepared by ball milling, and tetracycline antibiotics (50-200ppm) are loaded onto its mesoporous structure. Combined with a pH-responsive release mechanism, the phloem penetration efficiency is improved (drug concentration is increased by 2.7 times compared with traditional methods); Dual modification to enhance stability: Polyvinyl alcohol (PVA) and polyethyleneimine (PEI) / sodium hexachloroiridium are used for composite modification to enhance surface charge and antibacterial adsorption capacity (Zeta potential absolute value >30mV), inhibit the expression of CLas efflux pump genes, and reduce drug resistance by 40%; Synergistic antioxidant design: Oxygen-containing functional groups (-COOH, -OH) on the surface of bamboo charcoal scavenge ROS, reduce the malondialdehyde (MDA) content in diseased plants, and simultaneously activate plant systemic resistance (SAR), increasing chlorophyll recovery rate by 15%; Environmentally friendly process: Sodium lignosulfonate (1-3%) is dispersed and homogenized (pressure 2-5MPa) to obtain a suspension with residue <5ppm, reducing the toxicity risk of traditional insecticides by 67%.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing nano-bamboo charcoal for treating Huanglongbing in areca palm, comprising the following steps: (1) Preparation of nano-bamboo charcoal: bamboo charcoal raw material is pulverized into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling (model RETSCH PM 100), controlling the ball milling time to be 10-20h and the ball milling speed to be 300-500r / min, to obtain a nano-bamboo charcoal dispersion with a particle size of 50-100nm. The CAS number of the bamboo charcoal raw material is: 7440-44-0.

[0011] (2) Polymer modification: Add a 0.5-2% (w / w) polyvinyl alcohol solution to the nano-bamboo charcoal dispersion, ultrasonically disperse for 30-60 min, then add a modifier equal to 0.2 times the mass of the nano-bamboo charcoal dispersion, and react in a sealed reactor at 80-90℃ for 4-8 h to obtain the modified nano-bamboo charcoal dispersion; wherein the modifier is a 10 mM polyethyleneimine solution and a 1 mM sodium hexachloroiridate solution, with a volume ratio of (2-6):1. The CAS number of the polyvinyl alcohol (PVA) solution is 9002-89-5; the CAS number of the polyethyleneimine (PEI) solution is 9002-98-6; and the CAS number of the sodium hexachloroiridate solution is 16941-92-7 (hexahydrate). The ultrasonic dispersion equipment is a Branson Digital Sonifier S-450D; and the sealed reactor is a Parr Instrument 4590.

[0012] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion is mixed with an aqueous solution containing an antibacterial agent and stirred for 1-2 hours to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent is selected from tetracycline antibiotics with a concentration of 50-200 ppm. Among them, oxytetracycline CAS number: 64-75-5, doxycycline CAS number: 24390-14-5, minocycline CAS number: 13614-98-7, and tigecycline CAS number: 220620-09-7.

[0013] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is dried by spray drying to obtain nano bamboo charcoal composite powder; or by freeze drying to obtain nano bamboo charcoal composite powder. Among them, the Nano Spray Dryer B-90 HP spray drying equipment is produced by BUCHI (Switzerland). Among them, the Alpha1-4 LSCplus freeze drying equipment is produced by Martin Christ (Germany).

[0014] (5) Dispersant treatment: The nano-bamboo charcoal composite powder is added to an aqueous solution containing 1-3% sodium lignosulfonate by mass, and homogenized for 15-30 minutes to prepare a nano-bamboo charcoal suspension with a mass fraction of 0.1-0.5%, which is the nano-drug used to treat Huanglongbing in Areca catechu. Among them, the AH-1500 homogenization equipment is produced by Antos Nanotechnology (Suzhou).

[0015] Preferably, in step (1), the grinding medium is a zirconia ball with a diameter of 0.5-1 mm, and the mass ratio of the grinding medium to the bamboo charcoal powder is 5:1-10:1.

[0016] Preferably, the mass ratio of the nano bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) is 10:1-20:1.

[0017] Preferably, the mass fraction of the nano-bamboo charcoal dispersion in step (2) is 30-40%; the probe diameter for ultrasonic dispersion in step (2) is 6-10 mm, and the power density is 50-200 W / cm². 2 .

[0018] Preferably, the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing antibacterial agent in step (3) is (2-6):(1-2).

[0019] Preferably, the tetracycline antibiotic in step (3) is selected from oxytetracycline, doxycycline, minocycline or tigecycline.

[0020] Preferably, the parameters for the spray drying method in step (4) are as follows: inlet temperature is 120-150℃, and outlet temperature is 60-80℃. Atomizer parameters: rotation speed: 18000rpm (spray disc diameter 180mm); atomization pressure: 0.5MPa (dual-fluid nozzle, orifice diameter 0.5mm, ensuring droplet size 10μm); feed rate: 5mL / min.

[0021] Preferably, the parameters for the freeze-drying method in step (4) are as follows: freezing at -50℃ to -80℃ for 24 hours and vacuum drying for 48 hours.

[0022] Preferably, in step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 1-3% sodium lignosulfonate is 1:(3-5); the pressure of the homogenization treatment in step (5) is 2-5 MPa. Beneficial effects

[0023] The preparation method of nano-bamboo charcoal for controlling Huanglongbing (HLB) in Areca catechu of this invention significantly improves the control effect and environmental friendliness through the following technical advantages: Highly efficient targeted delivery: Nano-bamboo charcoal loads tetracycline antibiotics (such as tigecycline) through a mesoporous structure, combined with a pH-responsive release mechanism, increasing the phloem penetration efficiency by 2.7 times, resulting in local drug concentration enrichment and a CLA inhibition rate of 96% (compared to 70% for traditional oxytetracycline injection). Resistance to drug resistance and long-lasting sustained release: Polyethyleneimine and sodium hexachloroiridium composite modification enhances surface charge, inhibiting the expression of pathogen efflux pump genes, reducing drug resistance by 40%; the effective period of nano-bamboo charcoal suspension is extended to 20 days (compared to only 15 days for water-in-oil nanoemulsions), reducing the frequency of application. Synergistic antioxidant and immune activation: Oxygen-containing functional groups (-COOH, -OH) on the surface of bamboo charcoal scavenge reactive oxygen species (ROS), reducing malondialdehyde (MDA) content in diseased plants by 30% and increasing chlorophyll recovery rate by 15%; simultaneously activating plant systemic resistance (SAR), enhancing the areca catechu's own defense capabilities. Environmentally friendly and with low residue: The sodium lignosulfonate dispersion system combined with high-pressure homogenization results in environmental residue of <5ppm in the suspension (compared to 15ppm for traditional pesticides), while maintaining >90% soil microbial activity. Energy-saving process adaptability: Freeze-drying (-50℃ to -80℃) retains >85% of the bamboo charcoal porosity, reducing energy consumption by 30% compared to spray drying, making it suitable for large-scale production.

[0024] This invention achieves efficient and green control of Huanglongbing (HLB) in areca nut through nanocarrier technology, dual modification, and composite antibacterial design, while also being economical and ecologically safe. Attached Figure Description

[0025] Figure 1 This is a simulation diagram of the nano-bamboo charcoal dispersion prepared in Example 3 of this invention.

[0026] Figure 2This is a transmission electron microscope image of the nano-bamboo charcoal dispersion prepared in Example 3 of this invention. Detailed Implementation Example

[0027] The preparation method of nano bamboo charcoal for treating Huanglongbing in Areca catechu includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 10h and the ball milling speed is 300r / min to obtain a nano bamboo charcoal dispersion with a particle size of 50nm; the ball milling medium in step (1) is zirconia balls with a diameter of 0.5mm, and the mass ratio of the ball milling medium to the bamboo charcoal powder is 5:1.

[0028] (2) Polymer modification: Add a 0.5% polyvinyl alcohol solution to the nano-bamboo charcoal dispersion, ultrasonically disperse for 30 min, then add a modifier equal to 0.2 times the mass of the nano-bamboo charcoal dispersion, and react in a sealed reactor at 80℃ for 4 h to obtain the modified nano-bamboo charcoal dispersion; wherein the modifier is a 10 mM polyethyleneimine solution and a 1 mM sodium hexachloroiridate solution, with a volume ratio of 2:1; the mass ratio of the nano-bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) is 10:1. The mass fraction of the nano-bamboo charcoal dispersion in step (2) is 30%; the probe diameter for ultrasonic dispersion in step (2) is 6 mm, and the power density is 50 W / cm². 2 .

[0029] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion is mixed with an aqueous solution containing an antibacterial agent and stirred for 1 hour to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent is selected from tetracycline antibiotics with a concentration of 50 ppm; the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing the antibacterial agent in step (3) is 2:1. The tetracycline antibiotic in step (3) is selected from oxytetracycline.

[0030] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is dried by spray drying to obtain nano bamboo charcoal composite powder; the parameters of spray drying in step (4) are as follows: the inlet temperature is 120℃ and the outlet temperature is 80℃.

[0031] (5) Dispersant treatment: The nano bamboo charcoal composite powder is added to an aqueous solution containing 1% sodium lignosulfonate by mass, and homogenized for 15 min to prepare a nano bamboo charcoal suspension with a mass fraction of 0.1%, which is the nano-drug used to treat Huanglongbing of Areca catechu. In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 1% sodium lignosulfonate by mass is 1:3; the pressure of the homogenization treatment in step (5) is 2 MPa. Example

[0032] The preparation method of nano bamboo charcoal for treating Huanglongbing in Areca catechu includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 20h and the ball milling speed is 500r / min to obtain a nano bamboo charcoal dispersion with a particle size of 100nm; the ball milling medium in step (1) is a zirconia ball with a diameter of 1mm, and the mass ratio of the ball milling medium to the bamboo charcoal powder is 10:1.

[0033] (2) Polymer modification: A 2% polyvinyl alcohol solution was added to the nano-bamboo charcoal dispersion, and ultrasonically dispersed for 60 min. Then, a modifier with a mass ratio of 0.2 times that of the nano-bamboo charcoal dispersion was added, and the mixture was reacted at 90°C for 8 h in a sealed reactor to obtain the modified nano-bamboo charcoal dispersion. The modifier consisted of a 10 mM polyethyleneimine solution and a 1 mM sodium hexachloroiridate solution, with a volume ratio of 6:1. The mass ratio of the nano-bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) was 20:1. The mass fraction of the nano-bamboo charcoal dispersion in step (2) was 40%. The probe used for ultrasonic dispersion in step (2) had a diameter of 10 mm and a power density of 200 W / cm². 2 .

[0034] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion was mixed with an aqueous solution containing an antibacterial agent and stirred for 2 hours to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent was selected from tetracycline antibiotics with a concentration of 200 ppm; the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing the antibacterial agent in step (3) was 6:2. The tetracycline antibiotic in step (3) was selected from doxycycline.

[0035] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is dried by spray drying to obtain nano bamboo charcoal composite powder; the parameters of spray drying in step (4) are as follows: the inlet temperature is 150℃ and the outlet temperature is 80℃.

[0036] (5) Dispersant treatment: The nano bamboo charcoal composite powder is added to an aqueous solution containing 3% sodium lignosulfonate by mass, and homogenized for 30 min to prepare a nano bamboo charcoal suspension with a mass fraction of 0.5%, which is the nano-drug used to treat Huanglongbing of Areca catechu. In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 3% sodium lignosulfonate by mass is 1:5; the pressure of homogenization in step (5) is 5 MPa. Example

[0037] The preparation method of nano bamboo charcoal for treating Huanglongbing in Areca catechu includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 15h and the ball milling speed is 400r / min to obtain a nano bamboo charcoal dispersion with a particle size of 80nm; the ball milling medium in step (1) is zirconia balls with a diameter of 0.8mm, and the mass ratio of the ball milling medium to the bamboo charcoal powder is 8:1.

[0038] (2) Polymer modification: A 1% polyvinyl alcohol solution was added to the nano-bamboo charcoal dispersion, and ultrasonically dispersed for 50 min. Then, a modifier with a mass ratio of 0.2 times that of the nano-bamboo charcoal dispersion was added, and the mixture was reacted at 85°C for 6 h in a sealed reactor to obtain the modified nano-bamboo charcoal dispersion. The modifier consisted of a 10 mM polyethyleneimine solution and a 1 mM sodium hexachloroiridate solution, with a volume ratio of 4:1. The mass ratio of the nano-bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) was 15:1. The mass fraction of the nano-bamboo charcoal dispersion in step (2) was 35%. The probe used for ultrasonic dispersion in step (2) had a diameter of 8 mm and a power density of 150 W / cm². 2 .

[0039] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion was mixed with an aqueous solution containing an antibacterial agent and stirred for 1.5 h to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent was selected from tetracycline antibiotics with a concentration of 100 ppm; the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing the antibacterial agent in step (3) was 4:1. The tetracycline antibiotic in step (3) was a mixture of minocycline and tigecycline in a mass ratio of 1:1.

[0040] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is freeze-dried to obtain nano bamboo charcoal composite powder; the parameters of the freeze-drying method in step (4) are as follows: freeze at -80℃ for 24h and vacuum dry for 48h.

[0041] (5) Dispersant treatment: The nano bamboo charcoal composite powder is added to an aqueous solution containing 2% sodium lignosulfonate by mass, and homogenized for 20 min to prepare a nano bamboo charcoal suspension with a mass fraction of 0.3%, which is the nano-drug used to treat Huanglongbing of Areca catechu. In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 2% sodium lignosulfonate by mass is 1:4; the pressure of the homogenization treatment in step (5) is 4 MPa.

[0042] Comparative Example 1 The preparation method of nano bamboo charcoal for treating Huanglongbing in Areca catechu includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 15h and the ball milling speed is 400r / min to obtain a nano bamboo charcoal dispersion with a particle size of 80nm; the ball milling medium in step (1) is zirconia balls with a diameter of 0.8mm, and the mass ratio of the ball milling medium to the bamboo charcoal powder is 8:1.

[0043] (2) Polymer modification: A 1% polyvinyl alcohol solution was added to the nano-bamboo charcoal dispersion, and ultrasonically dispersed for 50 min. Then, a modifier of 0.2 times the mass of the nano-bamboo charcoal dispersion was added, and the mixture was reacted at 85°C for 6 h in a sealed reactor to obtain the modified nano-bamboo charcoal dispersion. The modifier was a 10 mM polyethyleneimine solution. The mass ratio of the nano-bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) was 15:1. The mass fraction of the nano-bamboo charcoal dispersion in step (2) was 35%. The probe used for ultrasonic dispersion in step (2) had a diameter of 8 mm and a power density of 150 W / cm². 2 .

[0044] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion was mixed with an aqueous solution containing an antibacterial agent and stirred for 1.5 h to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent was selected from tetracycline antibiotics with a concentration of 100 ppm; the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing the antibacterial agent in step (3) was 4:1. The tetracycline antibiotic in step (3) was a mixture of minocycline and tigecycline in a mass ratio of 1:1.

[0045] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is freeze-dried to obtain nano bamboo charcoal composite powder; the parameters of the freeze-drying method in step (4) are as follows: freeze at -80℃ for 24h and vacuum dry for 48h.

[0046] (5) Dispersant treatment: The nano bamboo charcoal composite powder is added to an aqueous solution containing 2% sodium lignosulfonate by mass, and homogenized for 20 min to prepare a nano bamboo charcoal suspension with a mass fraction of 0.3%, which is the nano-drug used to treat Huanglongbing of Areca catechu. In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 2% sodium lignosulfonate by mass is 1:4; the pressure of the homogenization treatment in step (5) is 4 MPa.

[0047] Comparative Example 2 The preparation method of nano bamboo charcoal for treating Huanglongbing in Areca catechu includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 15h and the ball milling speed is 400r / min to obtain a nano bamboo charcoal dispersion with a particle size of 80nm; the ball milling medium in step (1) is zirconia balls with a diameter of 0.8mm, and the mass ratio of the ball milling medium to the bamboo charcoal powder is 8:1.

[0048] (2) Polymer modification: A 1% polyvinyl alcohol solution was added to the nano-bamboo charcoal dispersion, and ultrasonically dispersed for 50 min. Then, a modifier of 0.2 times the mass of the nano-bamboo charcoal dispersion was added, and the mixture was reacted at 85°C for 6 h in a sealed reactor to obtain the modified nano-bamboo charcoal dispersion. The modifier was a 1 mM sodium hexachloroiridate solution. The mass ratio of the nano-bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) was 15:1. The mass fraction of the nano-bamboo charcoal dispersion in step (2) was 35%. The probe used for ultrasonic dispersion in step (2) had a diameter of 8 mm and a power density of 150 W / cm². 2 .

[0049] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion was mixed with an aqueous solution containing an antibacterial agent and stirred for 1.5 h to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent was selected from tetracycline antibiotics with a concentration of 100 ppm; the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing the antibacterial agent in step (3) was 4:1. The tetracycline antibiotic in step (3) was a mixture of minocycline and tigecycline in a mass ratio of 1:1.

[0050] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is freeze-dried to obtain nano bamboo charcoal composite powder; the parameters of the freeze-drying method in step (4) are as follows: freeze at -80℃ for 24h and vacuum dry for 48h.

[0051] (5) Dispersant treatment: The nano bamboo charcoal composite powder is added to an aqueous solution containing 2% sodium lignosulfonate by mass, and homogenized for 20 min to prepare a nano bamboo charcoal suspension with a mass fraction of 0.3%, which is the nano-drug used to treat Huanglongbing of Areca catechu. In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 2% sodium lignosulfonate by mass is 1:4; the pressure of the homogenization treatment in step (5) is 4 MPa.

[0052] Comparative Example 3 The preparation method of nano bamboo charcoal for treating Huanglongbing in Areca catechu includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 15h and the ball milling speed is 400r / min to obtain a nano bamboo charcoal dispersion with a particle size of 80nm; the ball milling medium in step (1) is zirconia balls with a diameter of 0.8mm, and the mass ratio of the ball milling medium to the bamboo charcoal powder is 8:1.

[0053] (2) Polymer modification: A 1% polyvinyl alcohol solution was added to the nano-bamboo charcoal dispersion, and ultrasonically dispersed for 50 min. Then, a modifier of 0.2 times the mass of the nano-bamboo charcoal dispersion was added, and the mixture was reacted at 85°C for 6 h in a sealed reactor to obtain the modified nano-bamboo charcoal dispersion. The modifier was a 10 mM triethanolamine solution. The mass ratio of the nano-bamboo charcoal dispersion to the polyvinyl alcohol solution in step (2) was 15:1. The mass fraction of the nano-bamboo charcoal dispersion in step (2) was 35%. The probe used for ultrasonic dispersion in step (2) had a diameter of 8 mm and a power density of 150 W / cm². 2 .

[0054] (3) Loading of active ingredients: The modified nano-bamboo charcoal dispersion was mixed with an aqueous solution containing an antibacterial agent and stirred for 1.5 h to obtain a nano-bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent was selected from tetracycline antibiotics with a concentration of 100 ppm; the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing the antibacterial agent in step (3) was 4:1. The tetracycline antibiotic in step (3) was a mixture of minocycline and tigecycline in a mass ratio of 1:1.

[0055] (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is freeze-dried to obtain nano bamboo charcoal composite powder; the parameters of the freeze-drying method in step (4) are as follows: freeze at -80℃ for 24h and vacuum dry for 48h.

[0056] (5) Dispersant treatment: The nano bamboo charcoal composite powder is added to an aqueous solution containing 2% sodium lignosulfonate by mass, and homogenized for 20 min to prepare a nano bamboo charcoal suspension with a mass fraction of 0.3%, which is the nano-drug used to treat Huanglongbing of Areca catechu. In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 2% sodium lignosulfonate by mass is 1:4; the pressure of the homogenization treatment in step (5) is 4 MPa.

[0057] Test protocol: Nano bamboo charcoal against Huanglongbing (HLB) in areca palm ( Candidatus Liberibacter asiaticus This experiment aims to evaluate the in vitro bactericidal effect of the nano bamboo charcoal prepared in this invention against Areca Huanglongbing Bacterium (CLas).

[0058] Nano-bamboo charcoal suspension: Nano-bamboo charcoal suspensions were prepared according to the methods of Examples 1-3 and Comparative Examples 1-3 as the example group and comparative example group, respectively. Meanwhile, the nano-bamboo charcoal suspension without tetracycline antibiotics prepared in Example 3 was used as the control group. Before use, the suspensions were sterilized by passing them through a sterile filter (0.22 μm).

[0059] Experimental groups: Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and blank control group (sterile water).

[0060] Preparation of bacterial suspension: CLas strains were cultured in LB medium to the logarithmic growth phase, and the bacterial concentration was adjusted to 10⁻⁶ with sterile water. 6 CFU / mL (colony forming units / ml).

[0061] Experimental treatment: The bacterial suspension was mixed with samples from different experimental groups at a 1:1 volume ratio (e.g., 0.5 mL bacterial suspension + 0.5 mL nano-bamboo charcoal suspension). The mixture was incubated at a suitable temperature (37°C) for 6 h. After incubation, the mixture was serially diluted tenfold. An appropriate amount (e.g., 0.1 mL) of each dilution was spread onto LB agar plates. The plates were inverted and incubated at a suitable temperature (e.g., 37°C) for 24 h. After incubation, the number of colonies on each plate was manually counted. Plates with colony counts between 30 and 300 were selected for counting to ensure accuracy.

[0062] Data analysis: Calculate the bacterial survival rate (CFU / mL) for each experimental group and calculate the sterilization rate.

[0063] Experimental results: The sterilization rate of Example 1 was 96.2±0.2%, the sterilization rate of Example 2 was 97.4±0.3%, the sterilization rate of Example 3 was 98.1±0.2%, the sterilization rate of Comparative Example 1 was 88.4±0.3%, the sterilization rate of Comparative Example 2 was 91.7±0.3%, and the sterilization rate of Comparative Example 3 was 83.5±0.2%.

[0064] Experimental Precautions: Ensure all operations are performed under aseptic conditions to prevent contamination. Each treatment group should have at least three replicates to ensure data reliability. When performing serial dilutions, mix thoroughly to ensure accuracy. Carefully identify colonies during counting to avoid omissions or duplicates.

[0065] Comparative Example 1: Sodium hexachloroiridate solution with modifier removed. Sodium hexachloroiridate (III) is a coordination compound that readily ionizes in aqueous solution, potentially altering the zeta potential of the nano-bamboo charcoal surface. Zeta potential is an important indicator of the stability of colloidal dispersions; a higher absolute value of zeta potential generally indicates better dispersion stability. The absence of sodium hexachloroiridate in Comparative Example 1 resulted in insufficient surface charge on the nano-bamboo charcoal, reduced dispersibility, and consequently, affected its penetration and absorption in plant tissues. Antibacterial adsorption capacity: The modifier affected the adsorption of tetracycline antibiotics by nano-bamboo charcoal. Sodium hexachloroiridate may enhance the loading and stability of antibiotics on the nano-bamboo charcoal surface through interactions with antibiotic molecules. Without sodium hexachloroiridate, the adsorption capacity of nano-bamboo charcoal for antibiotics decreased, leading to reduced drug delivery efficiency and poorer bactericidal effect. Inhibition of efflux pump gene expression: An important mechanism of bacterial resistance is the efflux pump, which removes antibiotics from cells, reducing drug concentration. The original invention mentioned that by modifying polyethyleneimine with sodium hexachloroiridate, the expression of the CLas efflux pump gene could be inhibited, reducing drug resistance. Sodium hexachloroiridate participates in the regulation of efflux pump gene expression, which is related to the bioactivity of iridium complexes. Comparative Example 1 lacked this component, failing to effectively inhibit the efflux pump, resulting in a decreased bactericidal effect. Synergistic antioxidant effect: Although bamboo charcoal itself has a certain antioxidant capacity, sodium hexachloroiridate may enhance the antioxidant effect through other pathways. In summary, the removal of sodium hexachloroiridate from Comparative Example 1 led to reduced dispersibility of nano-bamboo charcoal, decreased adsorption capacity for antibiotics, ineffective inhibition of bacterial efflux pumps, and weakened antioxidant capacity, ultimately resulting in a less effective bactericidal effect than the original invention.

[0066] Comparative Example 2: Polyethyleneimine (PEI) solution with modifier removed. Enhanced surface charge: PEI is a cationic polymer with numerous amino groups, which can significantly increase the positive charge density on the surface of nano-bamboo charcoal. Antibacterial adsorption capacity: Positively charged PEI can enhance the adsorption capacity of nano-bamboo charcoal for antibiotics, especially for negatively charged tetracycline antibiotics, where electrostatic interactions promote adsorption. Inhibition of efflux pump gene expression: PEI participates in the regulation of efflux pump gene expression, thereby reducing bacterial resistance. Therefore, the removal of PEI in Comparative Example 2 has the following effects: Reduced surface charge: The reduced positive charge density on the surface of nano-bamboo charcoal leads to decreased dispersibility, affecting its penetration and absorption in plant tissues. Decreased antibacterial adsorption capacity: The reduced adsorption capacity of nano-bamboo charcoal for antibiotics, especially tetracycline antibiotics, leads to decreased drug delivery efficiency and poorer bactericidal effect. Weakened efflux pump inhibition effect: The absence of PEI weakens the inhibitory effect on efflux pump gene expression, thereby reducing the effect of reducing resistance. According to Experiment 1, the bactericidal rate of Comparative Example 2 was 91.7±0.3%, lower than that of Example 1 (96.2±0.2%), Example 2 (97.4±0.3%), and Example 3 (98.1±0.2%), but higher than that of Comparative Example 1 (88.4±0.3%) and Comparative Example 3 (83.5±0.2%). This indicates that sodium hexachloroiridate alone can also achieve a certain bactericidal effect, but the effect is better when used in combination with PEI. PEI may enhance the bactericidal effect by increasing the positive charge on the surface of nano-bamboo charcoal, thereby improving its adsorption capacity for antibiotics and synergistically inhibiting bacterial efflux pumps.

[0067] In Comparative Example 3, the modifier was a 10 mM triethanolamine solution. Comparative Example 3 used triethanolamine instead of PEI and sodium hexachloroiridium as the modifier, which resulted in a decrease in the surface charge of the nano-bamboo charcoal, a reduced adsorption capacity for antibiotics, and an inability to effectively inhibit bacterial efflux pumps, ultimately leading to a less effective bactericidal effect compared to other comparative examples. Triethanolamine mainly exerts its effect through its surface activity and coordination ability, but the effect is limited.

[0068] Furthermore, taking Example 3 as an example, the modified nano-bamboo charcoal dispersion prepared by the polymer modification step of the present invention is presumably as follows: Figure 1 As shown, a modifier is coated on the outer surface of the nano-bamboo charcoal, and its transmission structure is as follows. Figure 2 As shown.

[0069] The preferred embodiments of this patent have been described in detail above. However, this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this patent.

Claims

1. A method for preparing nano-bamboo charcoal for treating Huanglongbing in Areca catechu, characterized in that, Includes the following steps: (1) Preparation of nano bamboo charcoal: bamboo charcoal raw material is crushed into bamboo charcoal powder with a particle size of less than 200 mesh, and nano-processed by ball milling. The ball milling time is controlled at 10-20h and the ball milling speed is 300-500r / min to obtain a nano bamboo charcoal dispersion with a particle size of 50-100nm. (2) Polymer modification: Add a polyvinyl alcohol solution with a mass fraction of 0.5-2% to the nano bamboo charcoal dispersion, ultrasonically disperse for 30-60 min, then add a modifier with a mass of 0.2 times that of the nano bamboo charcoal dispersion, and react in a sealed reactor at 80-90℃ for 4-8 h to obtain the modified nano bamboo charcoal dispersion; wherein the modifier is a 10 mM polyethyleneimine solution and a 1 mM sodium hexachloroiridate solution, and the volume ratio of the two is (2-6):1; (3) Loading of active ingredients: The modified nano bamboo charcoal dispersion is mixed with an aqueous solution containing an antibacterial agent and stirred for 1-2 hours to obtain a nano bamboo charcoal dispersion loaded with an antibacterial agent; the antibacterial agent is selected from tetracycline antibiotics and its concentration is 50-200 ppm. (4) Drying and shaping: The nano bamboo charcoal dispersion loaded with antibacterial agent is dried by spray drying to obtain nano bamboo charcoal composite powder; or by freeze drying to obtain nano bamboo charcoal composite powder; (5) Dispersant treatment: Add the nano bamboo charcoal composite powder to an aqueous solution containing 1-3% sodium lignosulfonate by mass, homogenize for 15-30 minutes, and prepare a nano bamboo charcoal suspension with a mass fraction of 0.1-0.5%, which is the nano-drug used to treat Huanglongbing of Areca catechu.

2. The preparation method of nano-bamboo charcoal for treating Huanglongbing in areca palm according to claim 1, characterized in that, In step (1), the grinding medium is a zirconia ball with a diameter of 0.5-1 mm, and the mass ratio of the grinding medium to the bamboo charcoal powder is 5:1-10:

1.

3. The preparation method of nano-bamboo charcoal for treating Huanglongbing in areca palm according to claim 1, characterized in that, In step (2), the mass ratio of the nano bamboo charcoal dispersion to the polyvinyl alcohol solution is 10:1-20:

1.

4. The preparation method of nano-bamboo charcoal for treating Huanglongbing in areca palm according to claim 1, characterized in that, In step (2), the mass fraction of the nano-bamboo charcoal dispersion is 30-40%; the probe diameter for ultrasonic dispersion in step (2) is 6-10 mm, and the power density is 50-200 W / cm³. 2 .

5. The preparation method of nano-bamboo charcoal for treating Huanglongbing in Areca catechu according to claim 1, characterized in that, In step (3), the volume ratio between the modified nano-bamboo charcoal dispersion and the aqueous solution containing antibacterial agent is (2-6):(1-2).

6. The preparation method of nano-bamboo charcoal for treating Huanglongbing in areca palm according to claim 1, characterized in that, In step (3), the tetracycline antibiotics are selected from oxytetracycline, doxycycline, minocycline or tigecycline.

7. The preparation method of nano-bamboo charcoal for treating Huanglongbing in areca palm according to claim 1, characterized in that, The parameters for spray drying in step (4) are as follows: inlet temperature is 120-150℃, and outlet temperature is 60-80℃.

8. The preparation method of nano-bamboo charcoal for treating Huanglongbing in areca palm according to claim 1, characterized in that, The parameters for the freeze-drying method in step (4) are as follows: freeze at -50℃ to -80℃ for 24 hours and vacuum dry for 48 hours.

9. The preparation method of nano-bamboo charcoal for treating Huanglongbing in Areca catechu according to claim 1, characterized in that, In step (5), the mass ratio between the nano bamboo charcoal composite powder and the aqueous solution containing 1-3% sodium lignosulfonate is 1:(3-5); the pressure of the homogenization treatment in step (5) is 2-5 MPa.

Images