Method for relieving oxidative stress of citrus huanglongbing based on nano-enzyme

Abstract

This invention discloses a method for alleviating oxidative stress in citrus Huanglongbing (HLB) based on nanozymes. The method involves applying manganese-based nanozymes to HLB-infected citrus plants. By replacing some antioxidant enzymes, the nanozymes eliminate excess reactive oxygen species (ROS) produced by the oxidative stress response induced by the pathogen infection, thus alleviating oxidative stress and indirectly improving the plant's own antioxidant defense mechanism, thereby alleviating HLB symptoms. The manganese-based nanozymes used in this invention not only effectively remove ROS but also penetrate the citrus tissue through spraying and are slowly metabolized by the citrus plant itself without accumulation. This method is safe to use, does not affect citrus growth, and has good application value and promising prospects for promotion.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes. Background Technology

[0002] Citrus Huanglongbing (HLB), also known as "citrus AIDS," is caused by a bacterium called *Cassibacterium*. CandidatusLiberibacterspp, Clas Citrus Huanglongbing (HLB) is a plant disease caused by *Cassibiricum*. It is primarily transmitted by phytophagous insects such as the yellow orange aphid and psyllid. Once *Cassibiricum* enters the host plant cells, it colonizes the sieve tubes and parenchyma tissues, where the pathogen begins to grow and multiply, disrupting the plant's normal physiological processes. Infected citrus trees exhibit chlorosis, with leaves gradually turning yellow, usually starting from the base of the canopy and spreading upwards. Infected citrus trees also experience slow growth, reduced yields, and decreased fruit quality, including abnormal fruit development, deformities, rough peels, and bitter flesh. Reports indicate that HLB is currently the most destructive citrus disease, causing billions of dollars in economic losses globally each year and severely hindering the healthy development of the citrus industry.

[0003] However, there are currently no effective measures for the control of citrus Huanglongbing (HLB). To prevent the spread of HLB, agricultural departments typically take measures such as pruning and burning infected plants, controlling insect transmission, using disease-resistant varieties, or other technical means to reduce the impact of the disease. Removing and burning diseased trees not only results in significant losses but also causes environmental pollution. Controlling insect transmission requires the use of large amounts of fungicides or insecticides, which can cause food safety issues. Furthermore, the penetrants in pesticides increase cell membrane permeability, which can also harm plants. Developing disease-resistant varieties is costly and has a low success rate. RNAi (RNA interference)-based biopesticides are considered a disruptive technology in plant protection. This technology can silence important genes in the growth and development of pests, causing growth retardation or death, or specifically downregulate the expression of pesticide resistance genes in pests, thereby improving the efficiency of pesticide use and significantly changing the way humans control agricultural pests. However, it is undeniable that RNAi technology is also very expensive to develop, and siRNA has poor stability, easily leading to cytotoxicity and off-target risks.

[0004] Studies have found that when Clas infects citrus trees, it enters and multiplies within plant cells, simultaneously triggering oxidative stress in the citrus trees. This results in the production of excessive reactive oxygen species (ROS), including highly reactive oxides such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. These ROS attack cellular biomolecules such as lipids, proteins, and nucleic acids, leading to cell membrane oxidation, protein oxidation and damage, and DNA damage, thus affecting cell structure and function. High levels of ROS can also trigger apoptosis (programmed cell death) or necrosis, leading to cell death, affecting plant growth and development, and causing loss of tissue and organ function. Furthermore, excessive ROS accumulation can impair photosynthesis, affecting the production of photosynthetic products, reducing plant growth and yield, and contributing to the symptoms of Huanglongbing (HLB). Excessive ROS production also leads to oxidative stress in citrus, making cells unable to cope with normal biological and environmental stressors, affecting gene expression regulation, interfering with normal cell metabolism and nutrient absorption, and causing metabolic abnormalities such as a lack of essential nutrients in the plant. Therefore, plants need to maintain an appropriate antioxidant defense system to balance the production and clearance of reactive oxygen species (ROS) to ensure normal cell and tissue function. Wenxiu Ma et al. reported that citrus infection with Clas is similar to an autoimmune disease in humans. In their study, they used gibberellin to inhibit ROS-mediated cell death, alleviating the symptoms of HLB in citrus. Although gibberellin is a low-toxicity plant hormone, its aqueous solution gradually deactivates at room temperature or above 50°C and is unstable under neutral and alkaline conditions, requiring immediate preparation and use. In addition, excessively high concentrations of gibberellin can lead to malformation of stems, leaves, and fruits.

[0005] Nanoenzymes are artificially synthesized systems that utilize nanomaterials to mimic the catalytic function of natural enzymes (biocatalysts). Similar to natural enzymes, nanoenzymes can promote chemical reactions. Because nanoenzymes are generally more stable than natural enzymes, and their catalytic properties can be customized by adjusting the composition, structure, and shape of the nanomaterials to suit different catalytic reactions, they have attracted widespread attention. In recent years, nanoenzyme research has rapidly emerged, and its scope has gradually broadened, encompassing various fields such as materials science, physics, chemistry, biology, medicine, and the environment. The excellent ability of nanoenzymes to scavenge reactive oxygen species has been extensively reported in the treatment of various diseases, including tumors and inflammation. However, their application in plants, particularly in the control of citrus Huanglongbing (HLB), remains relatively unexplored. Therefore, using nanoenzymes to alleviate and control HLB symptoms is a novel research topic with significant research importance and value. Summary of the Invention

[0006] To address the aforementioned problems in the current control of citrus Huanglongbing (HLB), this invention proposes a method for alleviating oxidative stress in HLB-affected citrus plants based on nanozymes. This method involves applying nanozymes based on manganese and other metal cofactors to HLB-affected citrus plants, enhancing their antioxidant defense mechanisms, and thereby alleviating the oxidative stress caused by HLB. The specific technical solution is as follows: A method for alleviating oxidative stress caused by Huanglongbing (HLB) in citrus based on nanozymes involves directly applying nanozymes with reactive oxygen species (ROS) scavenging capabilities to citrus plants infected with HLB. The nanozymes remove excess reactive oxygen species produced by the HLB-infected plants due to oxidative stress caused by the corresponding pathogen infection, thereby alleviating the oxidative stress caused by HLB.

[0007] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes includes, but is not limited to, any one or more of nanozymes with manganese, iron, copper, zinc, and molybdenum as metal cofactors.

[0008] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes uses manganese-based nanozymes, which are prepared as follows: 1) By volume, mix 1 part oleic acid with 50 parts soluble manganese salt solution with a concentration of 10-50 mM thoroughly and stir vigorously to form a stable emulsion; 2) Continue stirring the obtained emulsion at room temperature for 4 hours to obtain a brownish-black block-shaped manganese-based nanozyme semi-finished product; 3) Wash the manganese-based nanozyme semi-finished product with ethanol several times and centrifuge at 5000-8000 rpm to remove residual reactants; 4) Vacuum dry the washed product to obtain the manganese-based nanozyme product, and store it at room temperature for later use.

[0009] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes includes, but is not limited to, any one or more of manganese carbonate, manganese chloride, potassium manganate, and potassium permanganate; and the vacuum drying includes, but is not limited to, any one of freeze-drying and low-temperature vacuum drying.

[0010] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes prepares manganese-based nanozymes that are flower-shaped manganese-based nanoparticles with a particle size of 150–200 nm.

[0011] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes involves dispersing the prepared manganese-based nanozymes in a solvent to form a manganese-based nanozyme suspension with a concentration of 0.005–1 mg / mL, and then applying it to citrus plants suffering from Huanglongbing through one or more of the following methods, including but not limited to spraying, injection, root application, and seedling infusion.

[0012] In the aforementioned method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes, the solvent is selected from deionized water, mineral water, or tap water; the spraying and injection both involve directly applying the manganese-based nanozyme suspension to the leaves of citrus plants suffering from Huanglongbing.

[0013] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing (HLB) based on nanozymes involves spraying 2-3 times daily. To increase the transdermal penetration rate and residence time of the nanozymes, and to enhance their ability to scavenge reactive oxygen species through metabolic action, spraying should ideally be done on sunny, windless days without dew, avoiding the hottest times of day, before 10:00 AM and after 3:00 PM. Each spray consists of one even application or 2-3 repeated applications at a concentration of 0.1-0.2 mL / cm³. 2 Leaves should be moistened but not dripping wet; spray for one day or more.

[0014] The aforementioned method for alleviating oxidative stress in citrus Huanglongbing (HLB) based on nanozymes involves injecting a manganese-based nanozyme suspension into HLB-infected citrus leaves in multiple injections. The injection time should ideally be chosen to avoid the hottest times of day, such as before 10:00 AM and after 3:00 PM. Because citrus leaves have a thick waxy layer, gently make incisions on the underside of the diseased leaf before injection, avoiding piercing the leaf and the veins. Then, use a syringe without the needle to inject the manganese-based nanozyme suspension into the leaf tissue through the incisions. Treat 2-3 times daily, with 0.5 mL injected per leaf each time, for one day or more.

[0015] Preferably, the scratches are 1-2 mm in size, and at least one scratch is made on each leaf, with the scratches located at or near the lesion on the diseased leaf. This method can significantly increase the transdermal penetration and utilization rate of the nanoenzyme suspension, effectively reducing losses of nanoenzymes caused by factors such as rain erosion. Fertilization and water management after injection should follow the conventional management methods for citrus seedlings.

[0016] The beneficial effects of this invention are: 1) This invention is the first to propose applying nanozymes based on manganese and other metal cofactors to citrus plants infected with Huanglongbing (HLB). By using nanozymes instead of antioxidant enzymes, excess ROS accumulated in the diseased plants is eliminated, alleviating oxidative stress caused by HLB and indirectly improving the plants' own antioxidant defense mechanisms, thereby alleviating HLB symptoms. The nanozymes used in this invention not only effectively eliminate ROS, but also, after entering the citrus tissue, are slowly metabolized by the citrus plant itself, without causing accumulation in the plant. This makes them safe to use, does not affect citrus growth, and has good application value and promising prospects for promotion.

[0017] 2) The nanozyme used in this invention is a manganese-based nanozyme, which possesses various enzyme activities, giving it a broad-spectrum effect in scavenging reactive oxygen species (ROS) and a significant ability to scavenge multiple ROS. Furthermore, this enzyme can be prepared at room temperature under mild reaction conditions, requiring no complex chemical modifications, and can be completed within 4 hours. The preparation method is rapid, simple, and inexpensive. Simultaneously, the prepared nanozyme can be stored at room temperature for extended periods after vacuum drying, without requiring stringent storage conditions. It can be directly dispersed in solvents at room temperature for use, exhibiting stable properties and facilitating widespread application.

[0018] 3) The manganese-based nanoenzyme used in this invention can be applied in various ways, and can directly enter citrus cells by spraying without the use of a penetrant, with significant effects. Within 3-4 days of application, it can upregulate the activity of some reactive oxygen species scavenging enzymes in citrus, and at the same time, it can keep the hydrogen peroxide content in a downregulated state within 3 days, so as to alleviate Huanglongbing symptoms from the root and reduce the losses caused by the worsening of the disease, which has important economic value. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the use of the manganese-based nanozyme prepared in this embodiment of the invention to alleviate oxidative stress in Huanglongbing lesions; Figure 2 This is a schematic diagram illustrating the characterization of manganese-based nanozymes prepared in an embodiment of the present invention; Figure 3 This is an evaluation of the reactive oxygen species scavenging ability of manganese-based nanozymes in the embodiments of the present invention; Figure 4 This illustrates the distribution of manganese-based nanozymes in plant tissues after spraying and injection treatment in this embodiment of the invention. Figure 5 In this embodiment of the invention, NBT staining was used to observe the ROS level in plant leaf tissue after treatment with manganese-based nanozymes. Figure 6 The effect of injecting manganese-based nanozymes on plant enzyme activity in an embodiment of the present invention; Figure 7 This invention illustrates the effect of spraying manganese-based nanoenzymes on plant enzyme activity in an embodiment of the invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0021] Example 1 This embodiment describes a method for alleviating oxidative stress in citrus Huanglongbing (HLB) based on nanozymes. Figure 1As shown, nanozymes based on metal cofactors such as manganese were applied to citrus Huanglongbing (HLB) infected plants. By replacing part of the antioxidant enzymes, the nanozymes eliminated the excessive reactive oxygen species (ROS) produced by the oxidative stress response caused by the corresponding pathogen infection in HLB infected plants, thus alleviating the oxidative stress of HLB and indirectly improving the antioxidant defense function of the infected plants, thereby achieving the goal of alleviating HLB symptoms.

[0022] In this embodiment, the nanozyme with reactive oxygen species (ROS) scavenging function includes, but is not limited to, any one or more of nanozymes using manganese, iron, copper, zinc, and molybdenum as metal cofactors, preferably manganese-based nanozymes. This is because research has shown that manganese-based nanozymes possess a variety of enzyme activities, exhibit a broad spectrum of ROS scavenging effects, and demonstrate significant scavenging capabilities against multiple ROS. Furthermore, this enzyme can be prepared at room temperature under mild reaction conditions, requiring no complex chemical modifications, and can be prepared within 4 hours, resulting in low preparation cost and a rapid and simple preparation method. The prepared nanozyme can be vacuum-dried and stored at room temperature for extended periods without requiring stringent storage conditions. It can be directly dispersed in solvents at room temperature for use, exhibiting stable properties and facilitating widespread application.

[0023] In this embodiment, the preparation method of the manganese-based nanozyme is as follows: 1) By volume, mix 1 part oleic acid with 50 parts soluble manganese salt solution with a concentration of 10-50 mM thoroughly and stir vigorously to form a stable emulsion; 2) Continue stirring the obtained emulsion at room temperature for 4 hours to obtain a brownish-black block-shaped manganese-based nanozyme semi-finished product; 3) Wash the manganese-based nanozyme semi-finished product with ethanol several times and centrifuge at 5000-8000 rpm to remove residual reactants; 4) Vacuum dry the washed product to obtain the manganese-based nanozyme product, and store it at room temperature for later use.

[0024] The soluble manganese salt described in this embodiment includes, but is not limited to, any one or more of manganese carbonate, manganese chloride, potassium manganate, and potassium permanganate, with potassium permanganate being preferred. The vacuum drying includes, but is not limited to, any one of freeze-drying and low-temperature vacuum drying, with low-temperature vacuum drying being preferred, and the drying temperature being 40–60°C. The prepared manganese-based nanozyme consists of flower-shaped manganese-based nanoparticles with a particle size of 150–200 nm.

[0025] The application involves dispersing the prepared manganese-based nanozyme in a solvent to form a manganese-based nanozyme suspension with a concentration of 0.005–1 mg / mL. This suspension is then applied to citrus plants infected with Huanglongbing (HLB) via one or more of the following methods: spraying, injection, root application, and seedling infusion. Spraying and injection are preferred. The solvent is selected from deionized water, mineral water, or tap water. Both spraying and injection methods involve directly applying the manganese-based nanozyme suspension to the leaves of citrus plants infected with HLB.

[0026] The spraying process is as follows: Spray on a windless, sunny day, avoiding periods of dew and midday high temperatures; spray 2-3 times daily, each time applying a single, even coat or repeating the spray 2-3 times, at a concentration of 0.1-0.2 mL / cm³. 2 Leaves should be moist but not dripping wet; spray for one day or more. To increase the transdermal penetration and retention time of the nanoenzyme, and enhance its ability to scavenge reactive oxygen species through metabolism, spraying should ideally be done on a sunny, windless day without dew, avoiding the hottest times of day, before 10:00 AM and after 3:00 PM. Fertilization and water management should follow standard management methods for citrus seedlings.

[0027] The injection involves injecting a manganese-based nanoenzyme suspension into the leaves of citrus plants infected with Huanglongbing (HLB) in multiple injections. The specific procedure is as follows: Injections are administered before 10:00 AM and after 3:00 PM, avoiding the hottest times of day. Before injection, gently make incisions on the underside of the diseased leaf, avoiding piercing the leaf and the veins. Then, using a syringe without the needle, inject the manganese-based nanoenzyme suspension into the leaf tissue through the incisions. This is done 2-3 times daily, with 0.5 mL injected per leaf each time, for at least one day. The incisions should be 1-2 mm in size, with at least one incision made on each leaf, located at or near the lesion. This method significantly increases the transdermal penetration and utilization rate of the nanoenzyme suspension, effectively reducing losses due to rain erosion and other factors. Post-injection fertilization and water management follow standard citrus seedling management methods.

[0028] Example 2 This embodiment examines the morphological characteristics of the manganese-based nanozyme prepared by the method described in Example 1. According to the method described in the example, 1 part by volume of oleic acid and 50 parts by volume of potassium permanganate solution (concentration 35 mM) were thoroughly mixed and vigorously stirred with a magnetic stirrer to form a stable emulsion. The mixture was stirred continuously at room temperature for 4 hours to obtain a brownish-black block, which was the crude manganese-based nanozyme product. The crude manganese-based nanozyme product was then washed three times with ethanol and centrifuged at 8000 rpm to remove as much residual reactant as possible. Finally, the washed product was vacuum dried at low temperature (drying temperature 40–60 °C) to obtain the manganese-based nanozyme, which could then be stored at room temperature.

[0029] The prepared manganese-based nanozyme was characterized by TEM (transmission electron microscopy) and TEM-mapping (elemental analysis), and the results are as follows: Figure 2 As shown. Among them, Figure 2 Figure A shows a schematic diagram of TEM (transmission electron microscopy) characterization of manganese-based nanozymes. As can be seen from the figure, the synthesized manganese-based nanozymes have good dispersibility, with a particle size of 150-200 nm, uniform size, and a uniform flower-like structure, making them easily identifiable. Figure 2 B represents TEM-mapping (elemental analysis) of the nanozyme. As shown in the figure, the main elements of the manganese-based nanozyme synthesized in this embodiment are Mn and O, with uniform elemental distribution and good active centers.

[0030] Example 3 This embodiment examines the ability of the manganese-based nanozyme prepared in Example 2 to scavenge reactive oxygen species.

[0031] S01. Testing the activity of manganese-based nanozymes (POD enzymes, peroxidases). The following method was adopted: The manganese-based nanozyme prepared in Example 2 was weighed to prepare material dispersions with concentrations ranging from 0.005 to 1 mg / mL (0.005, 0.0075, 0.01, 0.015, 0.02, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.75, and 1 mg / mL). A 50 mM hydrogen peroxide solution and 2 mg / mL TMB (3,3',5,5'-tetramethylbenzidine) were prepared for the reaction. 0.3 mL of each concentration of nanozyme dispersion was added to a centrifuge tube, followed by 0.5 mL of TMB and 0.5 mL of hydrogen peroxide using a multi-channel pipette. The reaction was allowed to proceed for 10 min. After color development and stabilization, the absorbance of each well at 652 nm was measured using a microplate reader.

[0032] The results are as follows Figure 3 As shown in Figure A, as the concentration of manganese-based nanozymes increases, the absorbance increases, and the ability of manganese-based nanozymes to scavenge peroxides increases. Furthermore, the concentration of nanozymes exhibits a certain concentration dependence in the range of 0–0.5 mg / mL.

[0033] S02, Testing the ability of manganese-based nanozymes to scavenge hydroxyl radicals The method employed involved utilizing the Fenton reaction to generate a large number of hydroxyl radicals, followed by colorimetric analysis with salicylic acid to reflect the concentration level of these radicals. Specifically, nanozyme dispersions were prepared according to the procedure in S01. 0.5 mL of each concentration of nanozyme dispersion was mixed with 0.5 mL of 10 mM ferrous sulfate solution in centrifuge tubes, followed by the addition of 0.5 mL of 10 mM hydrogen peroxide solution. The reaction proceeded for 30 min, using the nanozyme dispersion as a blank control. Finally, 2 mL of salicylic acid solution was added, and the mixture was incubated at 37°C for 15 min for color development. After colorimetric stability, the supernatant was centrifuged, and its absorbance was measured at 510 nm.

[0034] The results are as follows Figure 3 As shown in Figure B, the absorbance of the reaction system decreased with increasing manganese-based nanozyme concentration, indicating that the ability of nanozyme to scavenge hydroxyl radicals increased with increasing nanozyme concentration, showing a certain concentration dependence in the range of 0–0.5 mg / mL.

[0035] S03, Testing the ability of manganese-based nanozymes (CAT enzymes, catalases) The method employed involves using the reaction of hydrogen peroxide with molybdate to form a complex, and measuring the absorbance at 405 nm to reflect the amount of hydrogen peroxide. Specifically, the nanozyme dispersions were prepared according to S01. 0.5 mL of each concentration of nanozyme dispersion was mixed with 0.5 mL of 10M hydrogen peroxide solution, and the mixture was allowed to react for 10 min. Then, 1 mL of ammonium molybdate solution was added for color development, which was allowed to proceed for 10 min. After the color stabilized, the absorbance was measured at 405 nm.

[0036] The results are as follows Figure 3 As shown in Figure C, the absorbance of the reaction system decreased with increasing manganese-based nanozyme concentration, indicating that the ability of nanozyme to remove hydrogen peroxide increased with increasing concentration, and this effect was concentration-dependent in the range of 0–0.5 mg / mL.

[0037] S04. Testing the ability of manganese-based nanozymes (SOD enzymes, superoxide dismutases) The method employed involves generating superoxide anion radicals through the reaction of xanthine and xanthine oxidase, followed by testing the amount of superoxide anion radicals using a DCFH reactive oxygen species fluorescent probe. Specifically, the manganese-based nanozyme prepared in Example 2 was weighed to prepare material dispersions with concentrations ranging from 0.01 to 2 mg / mL (0.01, 0.05, 0.1, 0.5, 1, and 2 mg / mL); xanthine (0.01 mM) and xanthine oxidase (0.1 U / mL) were prepared using phosphate buffer (50 mM, pH 7.4). -1A solution of xanthine and xanthine oxidase of equal volume was prepared and incubated at 37°C for 30 min to obtain a superoxide anion radical stock solution. Then, 0.2 mL of manganese-based nanozymes of different concentrations were mixed with 0.4 mL of the superoxide anion radical stock solution, and the mixture was reacted for 30 min. Finally, a DCFH reactive oxygen species fluorescent probe was added, and the fluorescence intensity was measured immediately after centrifugation. The DCFH reactive oxygen species fluorescent probe was excited at 488 nm and emitted at 525 nm.

[0038] The results are as follows Figure 3 As shown in Figure D, the fluorescence intensity of the reaction system decreases with increasing manganese-based nanozyme concentration, indicating that the ability of nanozymes to scavenge superoxide anion free radicals continuously increases with increasing nanozyme concentration.

[0039] Example 4 This embodiment investigates the distribution of the manganese-based nanozyme prepared in Example 2 within plant tissues. Specifically, it examines the distribution of the manganese-based nanozyme in citrus leaf tissues under two application methods: spraying and injection. Details are as follows: Weigh out the manganese-based nanozyme prepared in Example 2 to prepare a manganese-based nanozyme suspension with a concentration of 0.05 mg / mL, for later use. The plant material in this example is sweet orange (…) preserved in a greenhouse. Citrussinensis Citrus Huanglongbing (HLB) was inoculated through grafting. Fertilization and water management after spraying followed standard citrus seedling management methods. The infection status of the plants was observed and tested regularly, with branches from plants testing positive for HLB PCR serving as the experimental treatment group. Three plants testing positive for HLB PCR were selected for the experiment. One plant was sprayed three times a day using the spraying device according to the spraying protocol described in Example 1; one plant was injected three times a day using a syringe according to the injection protocol described in Example 1; and one plant received no treatment as the control group. After treatment, citrus leaves were removed, fixed with glutaraldehyde, and sectioned for tissue analysis. The distribution of manganese-based nanozymes in the leaf tissue was observed using transmission electron microscopy.

[0040] The results are as follows Figure 4 As shown, in untreated citrus tissue sections, a distinct cell wall structure is visible; in the sprayed and injected tissue sections, distinct nanofloral structures are distributed in the intercellular spaces, closely adjacent to the cell wall periphery. These nanofloral structures are similar to the TEM electron microscopy morphology of the manganese-based nanozyme prepared in Example 2 (see...). Figure 2 Similar to [other examples]. This indicates that manganese-based nanozymes can enter plant tissues through injection and spraying and distribute in the intercellular spaces.

[0041] Example 5 This example aims to investigate the ROS levels in the leaves of citrus plants infected with Huanglongbing after treatment with the manganese-based nanozyme prepared in Example 2. Details are as follows: Weigh out the manganese-based nanozyme prepared in Example 2 to prepare a manganese-based nanozyme suspension with a concentration of 0.05 mg / mL. Treat the leaves of citrus plants infected with Huanglongbing (HLB) with the manganese-based nanozyme suspension by injection. Specifically, draw 1 mL of the material dispersion into a syringe, remove air bubbles, remove the needle, and inject the solution into the infected citrus leaves from the underside of the leaf. Repeat this treatment three times a day, with 0.5 mL injected each time. Untreated diseased leaves served as disease controls, and untreated healthy leaves served as healthy controls.

[0042] After treatment, histochemical staining was performed using NBT (nitrotetrazole blue chloride). The leaves were washed and soaked in NBT solution (50 mM PBS, pH 7.8). Due to the waxy layer on citrus leaves, vacuum permeation was required for 20 minutes, followed by incubation in the dark at room temperature for 12 hours. Chlorophyll was then removed using 75% ethanol in a boiling water bath before photography.

[0043] Shooting results as follows Figure 5 As shown in Figure A; the percentage of NBT-stained area to the total leaf area was analyzed using Image-J image analysis software, and the results are as follows. Figure 5 As shown in Figure B, the NBT staining intensity of leaves treated with manganese-based nanozymes was lower than that of the untreated disease control group, indicating that the ROS accumulation level in the treated group was significantly lower than that in the untreated group.

[0044] Example 6 This example investigates how manganese-based nanozymes enhance the antioxidant enzyme activity of citrus plants infected with Huanglongbing (HLB). Details are as follows: Weigh out the manganese-based nanozyme prepared in Example 2 to prepare a manganese-based nanozyme suspension with a concentration of 0.05 mg / mL. By regularly observing and testing the infection status of the plants, three plants that tested positive for Huanglongbing (HLB) by PCR were selected as the spray treatment group, three plants that tested positive for HLB by PCR were selected as the injection treatment group, three plants that tested positive for HLB by PCR were selected as the untreated control group, and three healthy citrus plants that were not grafted with HLB-infected branches were selected as the healthy control group. The spray treatment group was sprayed three times a day using a spraying device according to the spraying protocol described in Example 1. The injection treatment group was injected three times a day using a syringe according to the injection protocol described in Example 1. Leaf samples were taken on days 1, 3, and 5 after treatment to test leaf enzyme activity. Fertilization and water management for both the treatment and control groups were carried out according to the conventional management methods for citrus seedlings.

[0045] Before the test, the tissue was pretreated. The plant tissue was accurately weighed and nine times the volume of homogenizing medium (0.1MPBS, pH 7.2) was added at a weight (g) : volume (mL) ratio of 1 : 9. The tissue homogenate was prepared under ice-water bath conditions to a 10% concentration. After centrifugation for 10 min, the supernatant was collected for analysis.

[0046] For specific measurement procedures, please refer to the instruction manual of the reagent kit from Nanjing Jiancheng Bioengineering Institute (A084-3-1 is for peroxidase assay, A007-1-1 is for catalase assay, A064-1-1 is for hydrogen peroxide assay, and A001-1 is for superoxide dismutase assay).

[0047] Test results are as follows Figure 6 and Figure 7 As shown in the test results, it can be seen that through injection ( Figure 6 ) and spraying ( Figure 7 Manganese-based nanozymes can enhance the activity of CAT (catalase), POD (peroxidase), and SOD (superoxide dismutase) within 1-3 days. The most significant effect is the enhancement of catalase activity. Furthermore, both spraying and injecting manganese-based nanozymes can enhance the activity of enzymes related to oxidative defense in diseased citrus.

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for alleviating oxidative stress in citrus Huanglongbing (HLB) based on nanozymes, characterized in that: Nanoenzymes with the function of scavenging reactive oxygen species are directly applied to citrus plants suffering from Huanglongbing (HLB). The nanoenzymes remove the excessive reactive oxygen species produced by the oxidative stress response caused by the corresponding pathogen infection in the HLB plants, thereby alleviating the oxidative stress caused by HLB. The nanoenzyme with reactive oxygen species scavenging function is a manganese-based nanoenzyme, and its preparation method is as follows: 1) By volume, mix 1 part oleic acid with 50 parts soluble manganese salt solution with a concentration of 10-50 mM thoroughly and stir vigorously to form a stable emulsion; 2) Continue stirring the obtained emulsion at room temperature for 4 hours to obtain a brownish-black block-shaped manganese-based nanozyme semi-finished product; 3) Wash the manganese-based nanozyme semi-finished product with ethanol several times and centrifuge at 5000-8000 rpm to remove residual reactants; 4) Vacuum dry the washed product to obtain the manganese-based nanozyme product, and store it at room temperature for later use.

2. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to claim 1, characterized in that: The soluble manganese salt is selected from any one or more of manganese carbonate, manganese chloride, potassium manganate, and potassium permanganate. The vacuum drying is selected from either freeze-drying or low-temperature vacuum drying.

3. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to claim 2, characterized in that: The prepared manganese-based nanozymes are manganese-based nanoparticles with a particle size of 150-200 nm that exhibit a flower-like appearance.

4. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to any one of claims 1-3, characterized in that: The application involves dispersing the prepared manganese-based nanozyme in a solvent to form a manganese-based nanozyme suspension with a concentration of 0.005–1 mg / mL, and then applying it to citrus plants suffering from Huanglongbing by any one or more of the following methods: spraying, injection, root application, and seedling infusion.

5. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to claim 4, characterized in that: The solvent is selected from deionized water, mineral water or tap water; the spraying and injection are both methods of applying the manganese-based nanoenzyme suspension directly to the leaves of citrus plants suffering from Huanglongbing.

6. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to claim 5, characterized in that: The spraying is performed 2-3 times per day, and the specific operation is as follows: Choose a windless, sunny day for spraying, and avoid spraying during periods of dew and midday high temperatures; Each application involves spraying evenly once or repeating the spraying 2-3 times, with a spraying rate of 0.1-0.2 mL / cm³. 2 The leaves are moist but not dripping wet; Spray for 1 day or more.

7. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to claim 5, characterized in that: The injection involves injecting a manganese-based nanozyme suspension into the diseased leaves of citrus trees infected with Huanglongbing (HLB) in multiple injections. The specific procedure is as follows: Avoid the midday heat. Before injection, gently make a scratch on the back of the diseased leaf to avoid piercing the leaf and avoiding the leaf veins. Then, use a syringe with the needle removed to inject the manganese-based nanoenzyme suspension into the leaf tissue at the scratch. Treat 2-3 times a day, injecting 0.5 mL per leaf each time; Processing time is one day or more.

8. The method for alleviating oxidative stress in citrus Huanglongbing based on nanozymes according to claim 7, characterized in that: The scratches are 1-2 mm in size, and each leaf has at least one scratch. The scratches are located at or near the lesion on the diseased leaf.

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