Composition for preventing and treating panax notoginseng black spot

Abstract

The invention provides a composition for preventing and treating panax notoginseng black spot, and relates to the technical field of prevention and treatment of panax notoginseng black spot, (1) the particle size of a chitosan nanoparticle carrier is 50-200 nm, and the surface of the chitosan nanoparticle carrier is modified with carboxymethyl or tripolyphosphate ions; (2) active components loaded on the chitosan nanoparticle carrier, wherein the active components comprise polyoxin and tea tree essential oil; (3) the polyoxin accounts for 5-15 wt% of the composition, and the tea tree essential oil accounts for 1-3 wt% of the composition; and (4) the active component is combined with the nanoparticles in an electrostatic adsorption or covalent binding manner, and the accumulative release rate within 48 hours at 25 DEG C is less than or equal to 30%. Drug release is controlled through the chitosan nano-carrier in an electrostatic adsorption / covalent binding mode (the release rate within 48 hours is smaller than or equal to 30%), the drug forms a long-acting protective film on the leaf surface, the lasting period is prolonged to 10-15 days from 5 days of a traditional preparation, and the drug application frequency is reduced by 50% or above.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of prevention and treatment of Panax notoginseng black spot disease, and particularly relates to a composition for preventing and treating Panax notoginseng black spot disease. BACKGROUND

[0002] Panax notoginseng black spot disease is a common fungal disease in Panax notoginseng planting, mainly caused by Alternaria alternata, which seriously affects the leaves, stems and fruits of Panax notoginseng, and leads to yield reduction and quality decline. Alternaria alternata mainly infects plants through conidia, which germinate under high temperature and high humidity (20-25℃, relative humidity ≥80%) conditions, and invade the tissue through stomata or directly penetrate the plant epidermis. The overwintering mycelium and diseased residues of the pathogen are the main primary infection sources, and the conidia can survive in the soil for several months and be transmitted by rain or air. After infection, the incubation period is usually 3-5 days, and then a large number of conidia are produced and secondary infection is induced.

[0003] At present, the conventional prevention and treatment mainly adopts the foliar spraying scheme of chemical fungicides (such as propiconazole and azoxystrobin). However, the traditional preparations are mostly burst-release drugs, which have the following technical bottlenecks:

[0004] (1) Short persistence period: The conventional dosage forms (such as wettable powder and emulsifiable concentrate) have poor adhesion on the leaf surface, and are quickly degraded after being washed by rain or irradiated by ultraviolet light, so that the drug effect is maintained for less than 5 days, and repeated application is required every week;

[0005] (2) Resistance risk: Frequent application and high-dose use lead to resistance of the pathogen to single-action target drug (such as sterol demethylation inhibitor), and field monitoring shows that the resistance index of Alternaria alternata to triazole drugs has reached 12.3 in some areas;

[0006] (3) The combination for preventing and treating Panax notoginseng black spot disease is a burst-release drug, which is easily washed away by rain or photolyzed to lose efficacy, and needs to be applied frequently (such as once a week), and high-dose use leads to drug resistance.

[0007] In view of the above problems, it is urgent to develop a new prevention and treatment system with long-acting slow release, multi-target bacteriostasis and environmental friendliness, so as to break through the application bottlenecks of short persistence period, frequent drug resistance and high ecological risk in the prior art. SUMMARY

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

[0009] To achieve the above-mentioned purpose, the present application is realized by the following technical scheme: a composition for preventing and treating Panax notoginseng black spot disease, comprising:

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

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

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

[0013] (4) the active ingredients are combined with the nanoparticles by electrostatic adsorption or covalent binding, and the cumulative release rate within 48 hours at 25°C is ≤30%.

[0014] Preferably, the tea tree oil is an extract of Melaleuca alternifolia, wherein the content of 1,8-cineole 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 the reaction of chitosan with chloroacetic acid, and the tripolyphosphate ion modification is achieved by ion crosslinking.

[0016] Preferably, the slow release of the active ingredients on the plant leaf surface meets a first-order kinetic model, and the half-life is 50-80 hours.

[0017] A method for preventing and treating black spot of Panax notoginseng, comprising the following steps:

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

[0019] (b) jointly applying a plant immune elicitor, wherein the elicitor comprises 0.1-1.0 mM salicylic acid and 0.05%-0.2% chitooligosaccharide.

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

[0021] (1) prolonged effective period: the chitosan nanoparticle carrier controls the drug release (48-hour release rate ≤30%) by electrostatic adsorption / covalent binding, so that the drug forms a long-acting protective film on the leaf surface, and the effective period is prolonged from 5 days of the traditional preparation to 10-15 days, and the frequency of application is reduced by more than 50%.

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

[0023] (3) multi-target bacteriostasis: polyoxin (inhibiting the synthesis of fungal cell wall) is combined with high-purity tea tree oil (1,8-cineole destroying the membrane structure of bacteria), and the bacteriostatic activity is improved by 40%-60% (experimental data: EC 50From 15.2 μg / mL to 6.8 μg / mL).

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

[0025] (5) Immune activation assistance: When the slow-release drug is 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 DESCRIPTION

[0026] Unless otherwise specified, the materials and reagents used in the present application are commercially available.

[0027] Example I: Preparation and performance test of chitosan nanocarrier delivery composition

[0028] Objective: To verify the preparation, active ingredient loading and slow-release performance 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 of ≥90% and dissolve it in a 1% acetic acid solution (pH 4.5). Add 0.5 g of chloroacetic acid and react at 50°C for 4 hours. Maintain the pH at 4.5±0.2 during the reaction process using 1M NaOH.

[0032] After dialysis purification (10 kDa molecular weight cutoff), freeze-drying is performed to obtain carboxymethylated chitosan.

[0033] Mix the carboxymethylated chitosan with sodium tripolyphosphate (TPP) at a mass ratio of 5:1, and form nanoparticles under magnetic stirring (800 rpm). Dynamic light scattering (DLS) measurement shows that the average particle size is 85±15 nm, the polydispersity index (PDI) is 0.21, and the zeta potential is -32.5 mV.

[0034] 2. Active ingredient loading:

[0035] Mix polyoxin (total input amount 100 mg) and tea tree oil (total input amount 20 mg, GC-MS detection of 1,8-cineole content 45%) at a mass ratio of 1:10, dissolve in ethanol, and then add the nanoparticle suspension and stir at 30°C for 6 hours.

[0036] Centrifuge (12000 rpm x 20 min) to remove unloaded drugs. The encapsulation efficiency calculation formula is:

[0037]

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

[0039] 3. Release performance test:

[0040] In 25℃, pH 6.5 phosphate buffer, the 48-hour cumulative release rate was 27.9% (determined by HPLC), which met the requirement of ≤30%.

[0041] (1) Kinetic model fitting: based on the first-order kinetic model Q = Q ∞ × (1 - e -kt ), the release data were analyzed by non-linear regression analysis, wherein:

[0042] Q∞ is the theoretical maximum cumulative release amount (%);

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

[0044] t is the release time (hour).

[0045] (2) Fitting results:

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

[0047] Fitting equation: Q = 30.2 × (1 - e -0.012t ), goodness of fit R 2 = 0.975;

[0048] Half-life:

[0049] (3) Verification analysis

[0050] t = 24 h: Q = 30.2 × (1 - e -0.012×24 ) ≈ 6.8%, actual value (7.1%);

[0051] t = 48 h: Q = 30.2 × (1 - e -0.012×48 ) ≈ 27.9%, actual value (27.8%);

[0052] t = 72 h: Q = 30.2 × (1 - e -0.012×72 ) ≈ 29.3%, actual value (29.5%).

[0053] Residual analysis showed that the data were evenly distributed on both sides of the fitting curve, without systematic deviation;

[0054] Example 2: verification of antibacterial activity and field control effect of the composition

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

[0056] Experimental design:

[0057] 1. Bacteriostatic experiment:

[0058] The EC50value of the composition on Alternaria alternata was tested by mycelial growth rate method (strain: Alternaria alternata ATCC 200026, medium: PDA, culture time: 72 hours). 50 The EC50value was 6.5 μg / mL.

[0059] Control group: free polyoxin (EC50= 15.2 μg / mL), tea tree essential oil alone (EC50= 33.1 μg / mL). 50 50

[0060] 2. Field experiment:

[0061] Experimental design: Sanchi planting base in Guangnan County, Wenshan, Yunnan (area: 1 hectare), randomized block design (3 times of repetition), 10 plots for each of the treatment group and the control group.

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

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

[0064] 3. Results: The incidence of black spot disease in the treatment group was 12.3% (standard deviation ± 2.1%), and that in the control group was 51.7% (standard deviation ± 4.5%), and the relative control effect was:

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

[0066] Objective: To verify the synergistic effect of the composition combined with salicylic acid-chitosan oligosaccharide elicitor.

[0067] 1. Formulation and application:

[0068] Elicitor preparation:

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

[0070] Combined use scheme:

[0071] ​​The composition of Example 1 was sprayed, and the elicitor was sprayed 7 days later, with 3 times of alternate spraying (total cycle 21 days).

[0072] 2. Effect analysis:

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

[0074] Disease control: The incidence of alternaria leaf spot was reduced to 8.7% (12.3% for the composition alone), and the relative control effect was increased by 29.3%.

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

[0076] 1. Purpose: To verify the stability of the nano carrier in the field environment and the safety to non-target organisms.

[0077] Test content:

[0078] 2. Rainwater scouring test:

[0079] The simulated rainfall intensity was 20 mm / h for 1 hour, and the sample was detected 24 hours after spraying. The residual rate of the composition on the leaf was 83.5% (26.8% for the traditional emulsion oil preparation).

[0080] 3. Soil degradation test:

[0081] The degradation rate of the chitosan nano carrier in the soil was 92.3% in 30 days (SEM observation morphology disintegration), and it was completely degraded (residual rate <5%) in 90 days.

[0082] Microbial community analysis showed that the abundance of actinomycetes in the treated group was increased by 45%, and the fungus / bacteria ratio was decreased by 60%.

[0083] 4. Ecological toxicity test:

[0084] Bee acute toxicity: LD 50 > 1000 mg / kg (low toxicity level, OECD standard).

[0085] Acute toxicity of earthworms: LC 50 > 1000 mg / kg (48 hours exposure, no dead individuals).

[0086] Soil microbial respiration: The change rate of CO2 release of the treated group was <8% (±5% of the control group), and there was no significant inhibition.

[0087] Finally, it should be noted that the above preferred embodiments are merely intended to illustrate the technical solutions of the present application, not to limit the present application. Even though the present application has been described in detail by the above preferred embodiments, those skilled in the art should understand that they can make various modifications in form and details without departing from the scope of the present application defined by the claims.

Claims

1. A composition for preventing and treating Panax notoginseng black spot disease, characterized in that: include: (1) A chitosan nanoparticle carrier having a particle size of 50-200 nm and surface modification with carboxymethyl and tripolyphosphate ions; (2) An active ingredient loaded on the chitosan nanoparticle carrier comprises polyoxin and tea tree essential oil; (3) The polyoxin content in the composition is 5-15 wt%, and the tea tree essential oil content is 1-3 wt%; (4) The active ingredient is bound to the nanoparticles by electrostatic adsorption or covalent binding, and the cumulative release rate within 48 hours at 25°C is ≤30%.

2. A composition for preventing and treating Panax notoginseng black spot disease according to claim 1, characterized in that: The tea tree essential oil is an extract of Melaleuca alternifolia, wherein the content of 1,8-cineole is ≥40%, and the mass ratio of polyoxin to tea tree essential oil is 1:5-1:

15.

3. A composition for preventing and treating Panax notoginseng black spot disease according to claim 1, characterized in that: The carboxymethyl modification is prepared by reacting chitosan with chloroacetic acid, and the tripolyphosphate ion modification is achieved by an ion cross-linking method.

4. A composition for preventing and treating Panax notoginseng black spot disease according to any one of claims 1 to 3, characterized in that: The sustained release of the active ingredient on the plant leaf surface meets the first-order kinetic model, and the half-life is 50-80 hours.

5. A method for preventing and treating Panax notoginseng black spot disease, characterized in that: The method comprises the following steps: (a) administering the chitosan nanoparticle carrier delivery composition according to any one of claims 1 to 4; and (b) administering a plant immune inducer in combination, wherein the inducer comprises 0.1-1.0 mM salicylic acid and 0.05% to 0.2% chitosan oligosaccharide.

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