A composition for preventing and treating black spot disease in Panax notoginseng

Abstract

This invention provides a composition for preventing and controlling black spot disease in Panax notoginseng, relating to the field of Panax notoginseng black spot disease control technology. (1) Chitosan nanoparticle carrier with a particle size of 50-200 nm, and surface modified with carboxymethyl or tripolyphosphate ions; (2) Active ingredients loaded on the chitosan nanoparticle carrier, including polyoxin and tea tree oil; (3) The content of polyoxin in the composition is 5-15 wt%, and the content of tea tree oil is 1-3 wt%; (4) The active ingredients are bound to the nanoparticles by electrostatic adsorption or covalent bonding, and the cumulative release rate is ≤30% within 48 hours at 25°C. This invention controls drug release (48-hour release rate ≤30%) through electrostatic adsorption / covalent bonding of chitosan nanoparticle carrier, so that the drug forms a long-lasting protective film on the leaf surface, extending the effective period from 5 days of traditional preparations to 10-15 days, and reducing the frequency of application by more than 50%.

Description

Technical Field

[0001] This invention belongs to the field of prevention and control technology of Panax notoginseng black spot disease, specifically, it relates to a composition for preventing and controlling Panax notoginseng black spot disease. Background Technology

[0002] Black spot disease of Panax notoginseng is a common fungal disease in Panax notoginseng cultivation, mainly caused by Alternaria alternata. It severely affects the leaves, stems, and fruits of Panax notoginseng, leading to reduced yield and quality. Alternaria alternata mainly infects plants through conidia. Conidia germinate under high temperature and humidity conditions (20-25℃, relative humidity ≥80%), invading tissues through stomata or directly penetrating the plant epidermis. Overwintering mycelium and diseased plant debris are the main sources of primary infection. Conidia can survive in the soil for several months and are spread by rainwater or air currents. After infection, the incubation period is usually 3-5 days, after which a large number of conidia are produced, triggering secondary infections.

[0003] Currently, conventional control methods mostly employ foliar spraying of chemical fungicides (such as difenoconazole and azoxystrobin), but traditional formulations are mostly burst-release drugs, which present the following technical bottlenecks:

[0004] (1) Short duration of effect: Conventional formulations (such as wettable powders and emulsifiable concentrates) have poor adhesion to the leaf surface and are rapidly degraded after being washed away by rain or exposed to ultraviolet radiation. The efficacy lasts for less than 5 days and requires repeated application every week.

[0005] (2) Risk of drug resistance: Frequent application and high doses of drugs have led to resistance of pathogens to single-target agents (such as sterol demethylation inhibitors). Field monitoring shows that the resistance index of Alternaria alternata to triazole agents has reached 12.3 in some areas.

[0006] (3) The combination for preventing and treating black spot disease of Panax notoginseng is a burst-release drug, which is easily washed away by rainwater and photodegraded and becomes ineffective. Frequent application is required (e.g., once a week), and high doses can induce drug resistance.

[0007] To address the aforementioned issues, there is an urgent need to develop a novel prevention and control system that combines long-lasting sustained release, multi-target antibacterial activity, and environmental friendliness, in order to overcome the application bottlenecks of existing technologies, such as short duration of effectiveness, frequent drug resistance, and high ecological risks. Summary of the Invention

[0008] In order to overcome the problems existing in the prior art, the present invention proposes a composition for preventing and treating black spot disease of Panax notoginseng.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: a composition for preventing and treating black spot disease in Panax notoginseng, comprising:

[0010] (1) Chitosan nanoparticle carrier with a particle size of 50-200 nm and surface modified with carboxymethyl or tripolyphosphate ions;

[0011] (2) The active ingredients loaded on the chitosan nanoparticle carrier include polyoxin and tea tree oil;

[0012] (3) The polyoxin content in the composition is 5-15 wt%, and the tea tree oil content is 1-3 wt%;

[0013] (4) The active ingredient is bound to the nanoparticles by electrostatic adsorption or covalent bonding, and the cumulative release rate is ≤30% within 48 hours at 25°C.

[0014] Preferably, the tea tree oil is a Melaleuca alternifolia extract, wherein the 1,8-cineole content is ≥40%, and the mass ratio of polyoxin to tea tree oil is 1:5-1:15.

[0015] Preferably, the carboxymethyl modification is prepared by reacting chitosan with chloroacetic acid, and the tripolyphosphate ion modification is achieved by ion crosslinking.

[0016] Preferably, the sustained release of the active ingredient on the plant leaf surface satisfies a first-order kinetic model, with a half-life of 50-80 hours.

[0017] A method for preventing and treating black spot disease in Panax notoginseng includes the following steps:

[0018] (a) Applying the chitosan nanoparticle carrier delivery composition according to any one of claims 1-4;

[0019] (b) Combined application of a plant immune inducer, the inducer comprising 0.1-1.0 mM salicylic acid and 0.05%-0.2% chitosan oligosaccharide.

[0020] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0021] (1) Extended duration of effect: Chitosan nanocarriers control drug release through electrostatic adsorption / covalent bonding (48-hour release rate ≤30%), so that the drug forms a long-lasting protective film on the leaf surface, extending the duration of effect from 5 days of traditional formulations to 10-15 days, and reducing the frequency of application by more than 50%.

[0022] (2) Resistance to environmental interference: The stability of the nanocarrier modified with carboxymethyl or tripolyphosphate is improved by 60% in the pH range of 5-8, and the drug residue rate after rainwater washing is ≥85% (≤30% for traditional formulations), which significantly improves the applicability in the field.

[0023] (3) Multi-target antibacterial activity: The combined use of polyoxin (which inhibits fungal cell wall synthesis) and high-purity tea tree oil (1,8-cineole disrupts the cell membrane structure) increases antibacterial activity by 40%–60% (experimental data: EC). 50(Decreased from 15.2 μg / mL to 6.8 μg / mL).

[0024] (4) Reduced risk of resistance: The synergistic effect of the two components makes it difficult for pathogens to develop resistance through single gene mutation. Field trials show that after three consecutive seasons of application, the resistance index of Alternaria is only 1 / 3 of that of traditional agents.

[0025] (5) Immune activation assistance: When sustained-release drugs are used in combination with plant immune inducers (such as salicylic acid and chitosan oligosaccharide), the peroxidase (POD) activity of Panax notoginseng leaves is increased, and the systemic resistance is enhanced. Detailed Implementation

[0026] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0027] Example 1: Preparation and Performance Testing of Chitosan Nanocarrier Delivery Composition

[0028] Objective: To verify the preparation, active ingredient loading, and sustained-release properties of chitosan nanoparticles.

[0029] Materials and Methods:

[0030] 1. Preparation of carboxymethylated chitosan nanoparticles:

[0031] Take 1.0 g of chitosan with a degree of deacetylation ≥90%, dissolve it in 1% acetic acid solution (pH 4.5), add 0.5 g of chloroacetic acid, and react at 50℃ for 4 hours. During the reaction, maintain the pH at 4.5±0.2 with 1 M NaOH.

[0032] After dialysis purification (molecular weight cutoff 10 kDa), the chitosan was freeze-dried to obtain carboxymethylated chitosan.

[0033] Carboxymethyl chitosan and sodium tripolyphosphate (TPP) were mixed at a mass ratio of 5:1 and formed into nanoparticles under magnetic stirring (800 rpm). The average particle size was determined to be 85±15 nm by dynamic light scattering (DLS), with a polydispersity index (PDI) of 0.21 and a zeta potential of -32.5 mV.

[0034] 2. Active ingredient loading:

[0035] Polyoxin (total input 100 mg) and tea tree oil (total input 20 mg, GC-MS detected 1,8-cineole content 45%) were mixed at a mass ratio of 1:10, dissolved in ethanol, and then added to the nanocarrier suspension. The mixture was stirred at 30°C for 6 hours.

[0036] Centrifugation (12000 rpm × 20 min) removes unloaded drug. The encapsulation efficiency is calculated using the following formula:

[0037]

[0038] The loading amount is 92.5 mg of polyoxin and 18.3 mg of tea tree oil per gram of nanoparticles.

[0039] 3. Sustained-release performance test:

[0040] In phosphate buffer at 25°C and pH 6.5, the cumulative release rate over 48 hours was 27.9% (HPLC determination), which meets the requirement of ≤30%.

[0041] (1) Dynamic model fitting: based on the first-order dynamic model Nonlinear regression analysis was performed on the released data, where:

[0042] Q∞ represents the theoretical maximum cumulative release (%).

[0043] k is the release rate constant (h⁻¹);

[0044] t represents the release time (in hours).

[0045] (2) Fitting results:

[0046] Optimal parameters: Q∞=30.2%, k=0.012 h−1;

[0047] Fitting equation: Goodness of fit R 2 =0.975;

[0048] half life: .

[0049] (3) Verification Analysis

[0050] t=24h: , measured value (7.1%);

[0051] t=48h: , measured value (27.8%);

[0052] t=72h: , Measured value (29.5%).

[0053] Residual analysis showed that the data were evenly distributed on both sides of the fitted curve, with no systematic bias.

[0054] Example 2: Validation of the antibacterial activity and field efficacy of the composition

[0055] Objective: To evaluate the inhibitory effect of the composition on Alternaria alterniflora and its practical application effect.

[0056] Experimental Design:

[0057] 1. Antibacterial test:

[0058] The mycelial growth rate method (strain: Alternaria, culture medium: PDA, culture time: 72 hours) was used to test the EC50 of the composition against Alternaria. 50 The value was 6.5 μg / mL.

[0059] Control group: Free polyoxin (EC) 50 =15.2 μg / mL), tea tree oil alone (EC) 50 =33.1 μg / mL).

[0060] 2. Field trials:

[0061] Experimental design: A randomized block design (3 replicates) was used at a Panax notoginseng planting base in Guangnan County, Wenshan City, Yunnan Province (1 hectare). The treatment group and the control group each had 10 plots.

[0062] Treatment group: Spray the composition prepared in Example 1 (diluted 200 times) once every 10 days for a total of 3 times.

[0063] Control group: Sprayed with 80% mancozeb wettable powder (conventional agent) once every 7 days, for a total of 4 times.

[0064] 3. Results: The incidence of melasma in the treatment group was 12.3% (standard deviation ± 2.1%), while it was 51.7% in the control group (standard deviation ± 4.5%). The relative efficacy was: The difference was significant (*p<0.05, t-test).

[0065] Example 3: Synergistic effect of combined plant immune inducers

[0066] Objective: To verify the synergistic effect of the composition in combination with the salicylic acid-chitosan oligosaccharide antagonist.

[0067] 1. Formulation and application:

[0068] Antidote preparation:

[0069] Salicylic acid 0.5 mM, chitosan oligosaccharide (degree of polymerization 8) 0.1%, Tween-80 0.05% (v / v), adjust pH to 6.0 with 1 mM NaOH, add 0.1% glycerol as a stabilizer, dilute 100 times before use.

[0070] Combined solution:

[0071] After spraying the composition of Example 1, the inducer was sprayed at 7-day intervals, and the application was repeated 3 times (total cycle 21 days).

[0072] 2. Effect Analysis:

[0073] Immunological indicators: The β-1,3-glucanase activity of Panax notoginseng leaves in the combined treatment group was 35.2 U / g FW (compared to 10.1 U / g FW in the control group), the peroxidase (POD) activity increased by 2.8 times, and the superoxide dismutase (SOD) activity increased by 2.3 times.

[0074] Disease control: The incidence of black spot disease was reduced to 8.7% (12.3% when using the combination alone), and the relative control efficacy was improved by 29.3%.

[0075] Example 4: Environmental stability and ecological safety test of the composition

[0076] 1. Objective: To verify the stability of the nanocarrier in a field environment and its safety to non-target organisms. Test content:

[0077] 2. Rainwater erosion test:

[0078] Simulated rainfall intensity of 20 mm / h lasting for 1 hour, and sampling and testing 24 hours after spraying, showed that the residue rate of the composition on the leaves was 83.5% (compared to 26.8% for traditional emulsifiable concentrate formulations).

[0079] 3. Soil degradation test:

[0080] Chitosan nanocarriers showed a degradation rate of 92.3% in soil after 30 days (morphological disintegration observed by SEM) and complete degradation after 90 days (residual rate <5%).

[0081] Microbial community analysis showed that the abundance of actinomycetes in the soil of the treatment group increased by 45%, and the fungal / bacterial ratio decreased by 60%.

[0082] 4. Ecotoxicity test:

[0083] Acute toxicity of bees: LD50 50 >1000 mg / kg (low toxicity level, OECD standard).

[0084] Earthworm acute toxicity: LC 50 >1000 mg / kg (48-hour exposure, no deaths).

[0085] Soil microbial respiration: The change rate of CO2 release in the treatment group was <8% (control group ±5%), with no significant inhibition.

[0086] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A composition for controlling black spot of Panax notoginseng, characterized by, include: (1) Chitosan nanoparticle carrier with a particle size of 50-200 nm, and surface modified with carboxymethyl and tripolyphosphate ions; (2) Active ingredients loaded on the chitosan nanoparticle carrier, including polyoxin and tea tree oil; (3) The content of polyoxin in the composition is 5-15 wt%, and the content of tea tree oil is 1-3 wt%; (4) The active ingredients are bound to the nanoparticles by electrostatic adsorption or covalent bonding. The tea tree essential oil is Melaleuca alternifolia essential oil, in which the 1,8-cineole content is ≥40%, and the mass ratio of polyoxin to tea tree essential oil is 1:5-1:

15.

2. The composition for preventing and treating black spot disease of Panax notoginseng according to claim 1, characterized in that, The carboxymethyl modification was prepared by reacting chitosan with chloroacetic acid, and the tripolyphosphate ion modification was achieved by ion crosslinking.

3. A method for preventing and controlling black spot disease in Panax notoginseng, characterized in that, The method comprises the following steps: (a) applying the composition according to any one of claims 1-2; and (b) applying in combination with a plant immune inducer, said inducer comprising 0.1-1.0 mM salicylic acid and 0.05%-0.2% chitosan oligosaccharide.