Composition for preventing and treating eggplant root rot

Abstract

The invention discloses a composition for preventing and treating eggplant root rot, and relates to the technical field of biological prevention and treatment. The composition consists of three parts, namely a double-stranded RNA (Ribonucleic Acid) delivery body controlled by a kaolinite nanotube-metal polyphenol network-boric acid ester, a nitric oxide / hydrogen sulfide double-pulse microcapsule and volatile organic compound particles of beta-cyclodextrin cross-linked aerogel, wherein the three parts are matched according to the total mass ratio of 1: (0.8-1.5): (0.8-1.2). Through structural design, time sequence release of non-overlapping peak values is realized, and a prevention and control mode for inhibiting environment enhancement host precise silencing is formed. The deliverer can be selectively disintegrated under the condition of pathogen-induced organic acid, iron chelating agent and hydrogen peroxide, so that the targeted release of the double-stranded RNA is realized; the double-pulse microcapsule respectively releases nitric oxide and hydrogen sulfide in a transplanting stress stage and an early disease stage, and induces host defensive gene expression; the aerogel particles continuously release 2, 3-butanediol and aromatic aldehyde to inhibit germination of pathogenic bacteria and serve as rhizosphere signals to enhance host resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological control, and particularly relates to a composition for preventing and treating eggplant root rot. BACKGROUND

[0002] Eggplant root rot is an important soil-borne disease caused by the infection of Fusarium oxysporum f. sp. melongenae in the rhizosphere. The disease has a high occurrence rate at the stages of seedling and transplanting, and the main symptoms are root cortex necrosis, stem base browning and plant wilting, which often leads to a reduction of 30% to 60% in yield, and even absolute yield in severe cases. It is one of the main diseases that restricts the efficient production of eggplants.

[0003] In the prior art, the measures for preventing and treating the disease mainly include chemical fungicides, disease-resistant variety breeding and biological control. Although chemical fungicides have a quick effect, long-term use may lead to the generation of pathogen resistance, drug residue exceeding the standard and imbalance of soil microecology; the breeding cycle of disease-resistant varieties is long and the resistance is easily degenerated; biological control mainly relies on antagonistic strains or a single inducer, and the action intensity is greatly affected by environmental conditions and has insufficient stability.

[0004] In recent years, the use of nano carriers to deliver double-stranded RNA for targeted silencing of key pathogenic genes, and the induction of host resistance by applying gaseous signaling molecules such as nitric oxide and hydrogen sulfide, have become emerging control methods. However, the existing technologies have the following limitations: Common layered double hydroxides (LDHs) or liposome carriers have a single release mode, which is difficult to match the timing of the host rhizosphere signals, and the release peak of double-stranded RNA is often ahead of or does not coincide with the pathogen infection window. Most existing NO or H2S donors are released in a single release, which cannot cover the key periods of transplanting stress and disease infection at the same time, and the induction effect is limited. Few technologies combine volatile organic compounds (VOCs) to regulate microbial communities, resulting in a difficult synergy between environmental antibacterial, host enhancement and pathogen silencing.

[0005] Therefore, there is an urgent need for a composition that can achieve multi-component time-release, form non-overlapping action peaks in the rhizosphere, and simultaneously have the functions of inhibiting pathogens, enhancing hosts and precise targeting, in order to improve the prevention and treatment effect of eggplant root rot and solve the problems of poor stability and short duration of the existing technologies. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art and provide a composition for preventing and treating eggplant root rot to solve the above problems.

[0007] The application aims at preventing and treating eggplant root rot by the following technical scheme: a composition for preventing and treating eggplant root rot, the composition is composed of three parts in a mass ratio of A:B:C=1:(1-1.5):(1-1.2) based on the total mass, and does not contain layered double hydroxide or hydrotalcite material: A: kaolinite nanotube-metal polyphenol network-borate ester gated double-stranded RNA delivery body, comprising: The kaolinite nanotube carrier has a volume distribution median particle size D50 of 100-300 nm and a lumen diameter of 20-70 nm, and the lumen is electrostatically loaded with double-stranded RNA through low-molecular-weight cationic polysaccharides or polycations, the low-molecular-weight cationic polysaccharides or polycations being selected from at least one of chitosan oligosaccharides, polylysine and polyethylenimine with a molecular weight of less than 10 kilodaltons; the double-stranded RNA has a length of 200-400 base pairs and targets a conserved functional region of Fusarium pathogenicity-related genes Fmk1 (mitogen-activated protein kinase) and / or ChsVb (chitin synthase); The metal polyphenol network shell layer coated on the outer surface of the kaolinite nanotube is formed by in-situ self-assembly of tannic acid and trivalent iron ions under the condition of pH 5.5-7.0, and has a thickness of 10-50 nm; The aryl boronic acid-carboxymethyl chitosan layer is arranged outside the metal polyphenol network shell layer, wherein the aryl boronic acid forms a borate ester bond with the ribose cis-diol of the double-stranded RNA, which can be cleaved by hydrogen peroxide; The metal polyphenol network shell layer is grafted with siderophore ligands on the outer surface, the siderophore ligands being selected from at least one of deferoxamine and hydroxamic acid ligands, and the grafting density is 0.1-1.0 micromole per gram of carrier; The delivery body disintegrates when there are organic acids or iron chelators in the rhizosphere, and the metal polyphenol network shell layer disintegrates by 50% or more within 2-6 hours under the condition of citric acid 0.5-5 millimoles per liter or deferoxamine 10-50 micromoles per liter, and the borate ester bond is broken to release the double-stranded RNA under the condition of hydrogen peroxide 10-100 micromoles per liter; B: nitric oxide / hydrogen sulfide double-pulse microcapsules, comprising: S-nitrosoglutathione liposomes are embedded in sodium alginate calcium ion cross-linked microbeads, the mass ratio of lecithin to cholesterol in the liposomes is 8:2, the particle size is 120-180 nm, and the microbeads are coated with a chitosan shell layer cross-linked by aryl boronic acid and dialdehyde, and the shell layer is broken in the presence of hydrogen peroxide to quickly release nitric oxide; A prodrug complex of GYY4137 and polysaccharides is used as a slow-release source of hydrogen sulfide, the polysaccharides being selected from at least one of alginic acid and hyaluronic acid, and the complex is coated with a chitosan-chitinase degradable layer; The microcapsules exhibit the following performance under the conditions of 25℃±5℃, pH 5.5-7.0, and soil moisture content of 15%-25%: the peak of nitric oxide release appears at 24-72 hours after application, and the apparent half-life is 1-2 days; the peak of hydrogen sulfide release appears at 7-14 days after application, and the apparent half-life is 7-12 days; C: a volatile organic compound particle of a beta-cyclodextrin crosslinked aerogel, which is composed of a porous framework formed by crosslinking beta-cyclodextrin with citric acid, has a specific surface area of not less than 150 square meters per gram, a pore size of 5-50 nanometers, loads 2,3-butanediol at 6-10% of the mass of the particle, and loads aromatic aldehyde at 0.3-0.8% of the mass of the particle, the aromatic aldehyde being at least one selected from benzaldehyde and phenylacetaldehyde, and the particle has a thin layer of at least one selected from pectin and calcium alginate on the outer surface thereof, the amount of the thin layer being 0.5-2% of the mass of the particle; under the conditions of 25℃±5℃, pH 5.5-7.0, and soil moisture content of 15%-25%, the particle releases volatile organic compounds at a volatile flux of 0.5-1.5 micrograms per square centimeter per hour in the first week after application; When A, B and C are applied to the root zone of eggplant in a mass ratio of A:B:C, the release rates of the active components are determined under the conditions of 25℃±5℃, pH 5.5-7.0, and soil moisture content of 15%-25%, to obtain the peak time (the time point at which the release rate reaches the maximum value) of each active component, and the peak times satisfy the order relationship of t VOC <t NO <t H2S <t dsRNA Under the conditions of pathogenic inoculation, the disease index of root rot is further reduced by 20% or more relative to a binary combination composed of any two of the components.

[0008] The kaolinite nanotube is activated by acid washing, and the inner wall of the kaolinite nanotube is pre-coated with chitosan oligosaccharide, and the quaternary ammonium substitution degree of the chitosan oligosaccharide is 10%-20%; the loading efficiency of double-stranded RNA in the kaolinite nanotube is not less than 60%; the length of the double-stranded RNA is 220-350 base pairs, and the target sequence is located in the conserved fragment of the Fmk1 kinase domain and / or ChsVb catalytic core region.

[0009] The molar ratio of tannic acid to ferric ion in the metal polyphenol network shell layer is 8-15:1; the shell layer is disintegrated by 70% or more within 2-6 hours under the condition of 1-3 millimoles per liter of citric acid or 10-50 micromoles per liter of deferoxamine; and the grafting density of the siderophore ligand is 0.2-0.8 micromoles per gram.

[0010] The aryl boronic acid-carboxymethyl chitosan layer is prepared by a coupling reaction of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, the aryl boronic acid is p-boronic acid, and the substitution degree of the aryl boronic acid on the carboxymethyl chitosan is 5% to 15%; under the condition of 10 to 100 micromole / liter of hydrogen peroxide, the half-life of the boronic acid ester bond is 0.5 to 4 hours.

[0011] The S-nitrosoglutathione liposome is prepared by a film hydration and ultrasonic extrusion process, and the zeta potential is-10 to-25 millivolt; the cross-linking of sodium alginate is performed by using a 1% to 3% calcium chloride solution; and the cross-linking degree of the aryl boronic acid-dialdehyde cross-linked chitosan is 5% to 15%.

[0012] In the prodrug complex formed by GYY4137 and polysaccharide, the mass ratio of GYY4137 to polysaccharide is 1:3 to 1:8; the thickness of the outermost chitosan-chitinase degradable layer is 0.2 to 2 microns, and under the condition of chitinase activity not less than 10 units / milliliter, the degradation rate reaches 60% and above within 7 to 14 days.

[0013] The cross-linking degree of the β-cyclodextrin cross-linked aerogel is 5% to 12%; the molar ratio of 2,3-butanediol to aromatic aldehyde is 10 to 40:1; the pectin or calcium alginate coating outside the particles contains 0.5% to 1.0% of soluble starch, and under the conditions of 25℃±5℃, pH 5.5 to 7.0, and soil moisture content of 15% to 25%, the initial release start time of volatile organic compounds is delayed by 2 to 12 hours relative to the corresponding particles without soluble starch.

[0014] The method for preventing and treating eggplant root rot by the composition is to bury the particles of the C part in the root area on the day of transplanting, and the dosage per plant is 2 to 4 grams; simultaneously, the A part is applied to the root system, and the dipping application is performed by using a suspension with a mass concentration of 0.2 to 0.4 grams / liter, or the trench application is performed by using a solid dosage of 1 to 2 kilograms / mu; the B part is applied by drip irrigation or water flushing on the 7th day±2 days after transplanting, and the dosage is 0.3 to 0.6 kilograms / mu; under the conditions of 25℃±5℃, pH 5.5 to 7.0, and soil moisture content of 15% to 25%, the expression level of the eggplant PR1 gene and / or PR5 gene is increased by 2 times and above, and the disease index is reduced by 30% and above relative to the non-application or any one of the two control groups.

[0015] The use of the composition of in the preparation of an agricultural preparation for rhizosphere application to prevent and treat eggplant root rot caused by Fusarium is that the adjuvant of the agricultural preparation contains at least one substance selected from the group consisting of lignin sulfonate (with a dosage of 5% to 15%), at least one selected from the group consisting of montmorillonite and kaolin (with a dosage of 20% to 50%), and microcrystalline cellulose (with a dosage of 5% to 15%).

[0016] It is prepared as a triple combination application package, the application package is independently packaged with part A, part B and part C of claim 1 respectively, and is accompanied by an application instruction, the application instruction stipulates that part A, part B and part C are matched according to the mass ratio defined in claim 1; part C is shallowly buried on the day of transplanting, part B is applied on the 7th day ± 2 days after transplanting, and part A is applied on the day of transplanting; and stipulates that the apparent half-life of nitric oxide in part B is 1-2 days, the apparent half-life of hydrogen sulfide is 7-12 days, and the volatile organic compound release flux of part C in the first week after application is 0.5-1.5 micrograms per square centimeter per hour.

[0017] The beneficial effects of the present application are: The present application realizes the scheme for preventing and treating eggplant root rot by constructing a kaolinite nanotube-metallic polyphenol network-borate gate double-stranded RNA delivery body, a nitric oxide / hydrogen sulfide double-pulse microcapsule and a volatile organic compound particle of beta-cyclodextrin cross-linked aerogel three-part synergistic system. Compared with the prior art, the present application has at least the following beneficial effects: According to the order of t VOC <t NO <t H2S <t dsRNA Non-overlapping peak release windows are formed, mutual interference between different components is avoided, and the continuous timing prevention and control of the lead signal activation, pathogen pressure buffering and final gene silencing is realized.

[0018] A protective mode similar to natural immune rhythm is established in the rhizosphere environment, which is significantly better than the effect of any single agent or two-component combination.

[0019] Through the combination design of kaolinite nanotube loading, metallic polyphenol network shell and aryl borate-carboxymethyl chitosan gate structure, the double-stranded RNA is not easy to degrade in the conventional rhizosphere environment, and is only triggered to release under the conditions of pathogen infection rich in organic acids, iron chelators and hydrogen peroxide, thereby ensuring the timeliness and targeting of the action.

[0020] Compared with the layered double hydroxide (LDH) carrier, the delivery system of the present application can significantly delay the release peak of double-stranded RNA, making it the latest response event in the entire prevention and treatment process, ensuring that the pathogen expansion period can still obtain effective inhibition.

[0021] Nitric oxide forms an early pulse about 36 hours after application, rapidly inducing host defense response; hydrogen sulfide forms a secondary pulse in the 7th-14th day, prolonging the duration of rhizosphere defense effect.

[0022] This double-pulse design realizes the precursor-maintenance mode of host immunity, and enhances the stability of induced resistance (ISR).

[0023] The beta-cyclodextrin cross-linked aerogel particles provide sustained and stable 2,3-butanediol and aromatic aldehyde release, can effectively inhibit the growth of pathogenic fungal mycelium, and promote the expression of eggplant PR genes as a rhizosphere signal molecule.

[0024] The introduction of soluble starch in the outer pectin / calcium alginate thin layer can delay the initial release for 2-12 hours, avoiding the problems of waste and excessive stimulation caused by too fast release.

[0025] In field and greenhouse tests, the disease index of the triple combination of the present application is reduced by more than 60% compared with the blank control, and can still be reduced by an additional 20%-30% compared with any double combination, which is significantly higher than the expected additive effect.

[0026] Compared with the LDH-dsRNA control prepared according to the prior patent WO2015089543A1, the present application can still obtain an additional reduction of about 38% at the equivalent dose of dsRNA.

[0027] Under the treatment of the triple combination, the expression levels of eggplant PR1 and PR5 genes are significantly increased by 2-3 times at 72 hours, indicating that the present application not only relies on exogenous disease resistance factors, but also can induce the systemic resistance of the host itself.

[0028] The ITS copy number detection of rhizosphere samples shows that on the 14th day and the 21st day, the pathogen copy number of the triple combination of the present application is significantly lower than that of the double combination and the control, indicating that the present application can effectively inhibit the continuous expansion of pathogenic fungi in the rhizosphere.

[0029] The triple combination composition increases the yield of single plant by more than 26% in field tests, and still has about 8% yield increase compared with the best double combination, which has high input-output ratio and good popularization and application prospect. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0031] Example 1 This embodiment prepares a composition for preventing and treating eggplant root rot, which is composed of three parts with a mass ratio of A:B:C=1:1.2:1.1 based on the total mass, the whole preparation process is completed in a clean environment at 25℃±2℃, all aqueous phases use ultrapure water without RNase treatment, and the composition does not contain layered double hydroxide or hydrotalcite materials.

[0032] Preparation of kaolinite nanotube-metal polyphenol network-borate ester gated double-stranded RNA delivery vehicle. Commercial grade kaolinite nanotubes were acid washed with 3.0 mol / L hydrochloric acid at 60°C for 2 hours, washed with deionized water to pH about 6.5, and vacuum dried at 60°C. The treated kaolinite nanotubes were dispersed in 0.02 mol / L acetic acid buffer (pH 5.8), and exfoliated with an ultrasonic probe at 200W power for 10 minutes to obtain a stable dispersion with a median particle size D50 of 180 nanometers and an average inner cavity diameter of 40 nanometers. Chitosan oligosaccharide with a quaternary ammonium substitution degree of 14% was dissolved in acetic acid buffer to prepare a 1.0 mg / mL solution, and the kaolinite nanotube dispersion was added with a chitosan oligosaccharide to kaolinite nanotube mass ratio of 0.08:1. An inner cavity cationization pre-coating layer was formed by slow stirring at room temperature for 30 minutes.

[0033] A double-stranded RNA loading solution was prepared by selecting a double-stranded RNA with a length of 280 base pairs, which targets the conserved fragments of the Fusarium Fmk1 mitogen-activated protein kinase functional region and the ChsVb chitin synthase catalytic core region, and preparing a concentration of 0.5 mg / mL. The double-stranded RNA solution was slowly added to the pre-coating system with a double-stranded RNA to kaolinite nanotube mass ratio of 0.06:1, and allowed to stand for 30 minutes for adsorption. Free double-stranded RNA was removed by centrifugation at 10,000 g for 10 minutes, and the loading efficiency was measured to be 64%.

[0034] A metal polyphenol network shell layer was prepared by dispersing the above-mentioned loaded body in a 1.0 mg / mL tannic acid solution at pH 6.3, slowly adding a 0.1 mg / mL trivalent iron ion solution, controlling the molar ratio of tannic acid to trivalent iron ions to be 10:1, and adding the solution for a total of 20 minutes. The reaction was continued for 20 minutes to form a metal polyphenol network shell layer in situ. Transmission electron microscopy showed that the shell layer was about 22 nanometers thick.

[0035] A layer of arylboronic acid-carboxymethyl chitosan was prepared. A 0.5 mg / mL carboxymethyl chitosan solution (amine group equivalent 0.8 mmol / L) was prepared. A p-boronic acid was activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and the molar ratio of the three was 1:1.2:1.2. After 30 minutes of activation, the activated p-boronic acid was mixed with the carboxymethyl chitosan for 2 hours to obtain a reaction solution with a substitution degree of arylboronic acid on carboxymethyl chitosan of 9%. The reaction solution was coated on the outside of the metal polyphenol network shell layer to form a layer of arylboronic acid-carboxymethyl chitosan. After standing at room temperature for 30 minutes, the small molecules were removed by dialysis. The residual carboxyl groups on the outer surface were grafted with deferoxamine under the action of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide to introduce siderophore ligands, and the grafting density was 0.45 micromole per gram of carrier (μmol / g).

[0036] The dispersion was spray dried to obtain the Part A powder. A laboratory spray dryer was used, with an inlet temperature of 140±5°C, an outlet temperature of 70±5°C, a feed flow rate of 5-10 mL / min, an atomizing gas flow rate of 0.5-0.7 m³ / h, and a nozzle diameter of 0.7 mm. The resulting powder had a concentrated particle size distribution and no free double-stranded RNA. The delivery body was shaken in a system containing 2.0 mmol / L citric acid at pH 6.0 for 2 hours, and the disintegration ratio of the metal polyphenol network shell layer reached 72%. In the presence of 30 μmol / L deferoxamine, the disintegration ratio reached 75% after 2 hours. In the presence of 50 μmol / L hydrogen peroxide, the half-life of the borate ester bond was about 1.6 hours. Under the condition of no RNase, it was confirmed that the double-stranded RNA was released from the carrier and remained intact.

[0037] Part B nitric oxide / hydrogen sulfide double-pulse microcapsules were prepared. Egg phospholipid and cholesterol were dissolved in chloroform at a mass ratio of 8:2, and a film was prepared by rotary evaporation. The film was hydrated with 0.5 mg / mL S-nitrosoglutathione aqueous solution at 4°C, and ultrasonically treated with an ultrasonic probe at a power of 180 W for a total of 10 minutes of intermittent operation. The resulting particles were successively extruded through 100, 80, and 50 nanometer polycarbonate membranes, and the particle size was 145 nanometers and the zeta potential was -18 millivolts as measured by dynamic light scattering. The liposomes were dispersed in a 1.5% sodium alginate solution, and were added dropwise to a 2.0% calcium chloride coagulation bath at a rate of 300 drops per minute through a 0.25 mm nozzle to crosslink into sodium alginate calcium ion crosslinked microbeads, with a bead diameter of about 0.8 millimeters.

[0038] A 1.0 mg / mL chitosan solution was prepared and p-boronic acid and glutaraldehyde were added, with an amine group to dialdehyde molar ratio of 10:1 and an aryl boronic acid to chitosan monosaccharide unit molar ratio of 0.08, to form an aryl boronic acid and dialdehyde cross-linked chitosan shell. Sodium alginate calcium ion cross-linked microbeads were immersed in the chitosan solution for 20 minutes, with slight shaking at room temperature to allow the microbeads to fully contact the solution, washed with deionized water until no free aldehyde coloration was observed, and dried to a moisture content of about 20%, with a cross-linking degree of about 9% measured by titration. In a 50 μmol / L aqueous hydrogen peroxide solution, the shell layer reversibly cleaved to release S-nitrosoglutathione, with a nitric oxide release peak at about 36 hours after administration and an apparent half-life of 1.4 days measured at 25℃±5℃ and pH 6.0.

[0039] To construct a slow pulse of hydrogen sulfide, the carboxyl group was activated with alginate and N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and GYY4137 was added to form a prodrug complex with a hydrolysable covalent bond, with a GYY4137 to alginate mass ratio of 1:5. After 4 hours of reaction, small molecules were removed by dialysis, and the powder was obtained by spray drying. The powder was redispersed and immersed in a chitosan solution for 15-30 minutes to form a chitosan-chitinase degradable layer. By adjusting the chitosan solution concentration (0.5-2.0%) and the number of immersions (1-3 times), the coating thickness was about 0.8 microns. The prodrug complex was degraded at a rate of about 68% in a chitinase activity of 10 units / mL buffer for 7-14 days, with a hydrogen sulfide release peak at day 9 after administration and an apparent half-life of 9.3 days measured at 25℃±5℃ and pH 6.5. The nitric oxide module and the hydrogen sulfide module were uniformly mixed in a mass ratio of 1:1.2 to obtain Part B.

[0040] Preparation of volatile organic compound particles of the β-cyclodextrin crosslinked aerogel of Part C. An aqueous solution of β-cyclodextrin and citric acid with a mass ratio of 10:1 was prepared, 1.0% of the total mass of β-cyclodextrin and citric acid was added as a catalyst, and after vacuum freeze-drying, a crosslinked porous framework was formed by heat curing at 140°C for 2 hours. The specific surface area was about 165 m² / g, the pore size distribution was concentrated in 5-40 nm, and the crosslinking degree was about 8%. A mixture of 2,3-butanediol and benzaldehyde with a mass ratio of 20:1 was used as the loading liquid, and after impregnation at 20°C and 20 kPa for 2 hours, the 2,3-butanediol loading was 8.2% and the benzaldehyde loading was 0.45%. The loaded framework was immersed in a 1.0% pectin solution for 3 minutes, then transferred to a 50 mmol / L calcium chloride solution for crosslinking for 10 minutes, and a pectin-calcium thin layer was formed on the surface. The water content was dried to 10%, and the amount of the thin layer was 1.2% of the mass of the particles. Under the conditions of 25°C±5°C, pH 6.0, and soil water content 20% (artificially prepared sandy loam soil), the volatile organic compound release flux was 0.9 μg·cm⁻²·h⁻¹ in the first week after application, as determined by gas chromatography in a closed diffusion cell.

[0041] The Part A powder, Part B microcapsules, and Part C particles were mixed in a mass ratio of 1:1.2:1.1 to form a composition. A plastic pot (25 cm in diameter) was filled with sandy loam soil sterilized by high-pressure steam in a greenhouse, and the pH was adjusted to 6.3 with a water content of 20%. The eggplant seedlings were transplanted at the 3-leaf stage. The Part C particles were shallowly buried on both sides of the root zone in a hole application manner, and the application amount was 2.5 g / plant. The Part A was made into a 0.3 g / L suspension and dipped into the roots for 30 seconds before planting. The Part B was added with water at 0.4 kg / acre on the 7th day after planting. 6 The pathogen inoculation used a Fusarium oxysporum f. sp. melongenae suspension with a concentration of 10

[0042] The release and action timing was monitored from 0 to 28 days after application: the volatile organic compound flux of Part C was determined by electronic nose and gas chromatography, the peak time appeared at about 12 hours after application; the micro-zone nitric oxide concentration was determined by chemiluminescence probe, the peak time appeared at about 36 hours after application; the hydrogen sulfide concentration was cross-verified by electrochemical sensor and methylene blue colorimetry, the peak time appeared on the 9th day after application; the uptake of double-stranded RNA in pathogens was detected by fluorescently labeled double-stranded RNA and qPCR, and the target gene transcription level was taken as a representation, the peak time appeared on the 16th day after application. The peak times of the four meet the order relationship of t VOC <t NO <t H2S <t dsRNA .

[0043] The addition of citric acid in rhizosphere solution verified the disintegration of the iron-robbed metal polyphenol network shell, and both the 2-hour absorbance change and the transmission electron microscope image showed that the shell structure was obviously loose; the kinetics of double-stranded RNA release triggered by borate ester bond breakage was monitored in a 50 μmol / L hydrogen peroxide system, and the half-life was consistent with the previous in vitro results. The "peak time" in the present application refers to the time point at which the release rate per unit time reaches the maximum under the specified environmental conditions (25℃±5℃, pH 5.5-7.0, soil moisture content 15%-25%).

[0044] The "non-overlapping peak" of each active component is determined by different limiting steps: the volatile organic compounds of β-cyclodextrin cross-linked aerogel are controlled by pore size and surface layer diffusion to form a leading peak; nitric oxide and hydrogen sulfide are determined by the response of S-nitrosoglutathione and GYY4137 prodrug outer coating and the prodrug hydrolysis / enzymatic degradation rate to form fast / slow pulses; the release peak of double-stranded RNA is limited by a series of gates, the first limiting step is that rhizosphere organic acids (represented by citric acid) and / or iron chelators gradually promote the disintegration of the metal polyphenol network shell, and the second limiting step is that the hydrogen peroxide triggered by pathogen infection triggers the breakage of borate ester bond. The formation and accumulation of the above triggers in the rhizosphere have a timing, resulting in the overall release peak of double-stranded RNA appearing on the 10th to 28th day after application, thereby meeting the order relationship of t VOC <t NO <t H2S <t dsRNA .

[0045] In the rhizosphere simulation system at 25℃, pH 6.2, and soil moisture content of 20%, the volatile organic compound flux peaked at 9-18 hours after application, the nitric oxide release peaked at 30-48 hours after application with an apparent half-life of 1-2 days, the hydrogen sulfide peaked at 8-12 days after application with an apparent half-life of 7-12 days, and the double-stranded RNA peaked at 16-20 days after application. The fitting of the double-stranded RNA release showed that the accumulation of the gate trigger rather than the borate ester cleavage reaction itself was the rate-limiting step, which explained why the overall release peak was still delayed to the weekly level despite the completion of chemical bond cleavage in hours.

[0046] During the work process, part C quickly establishes the root zone gas phase concentration of volatile organic compounds on the day of transplanting, inhibits conidial germination and early mycelial extension, and at the same time induces resistance background to the root of eggplant; in part B, S-nitrosoglutathione liposomes trigger the cross-linking of the shell layer of aryl boronic acid and dialdehyde cross-linked chitosan to form a nitric oxide "fast pulse" in 24-72 hours, which coincides with the transplanting stress window and promotes the expression of defense-related genes such as PR1 and PR5; then the GYY4137-polysaccharide prodrug complex gradually degrades in the chitosan-chitinase degradable layer Example 2 This example prepares a composition for preventing and treating eggplant root rot, which is composed of three parts with a mass ratio of A:B:C = 1:1.5:1.0 based on the total mass, and focuses on process optimization and release kinetics verification of volatile organic compound particles of part B nitric oxide / hydrogen sulfide double-pulse microcapsules and part C β-cyclodextrin cross-linked aerogel. The entire preparation process is completed in a clean environment at 25℃±2℃, all aqueous phases use RNase-free ultrapure water, and the composition does not contain layered double hydroxide or hydrotalcite materials.

[0047] Preparation of Part A kaolinite nanotube-metallic polyphenol network-borate ester gated double-stranded RNA delivery vehicle. Part A was prepared by the same procedure as Example 1, including acid washing activation of kaolinite nanotubes (3.0 mol / L hydrochloric acid, 60°C, 2 hours), intracavum pre-coating of quaternary ammonium chitosan oligosaccharide (degree of substitution 14%, mass ratio 0.08:1), double-stranded RNA loading (280 base pairs, targeting Fmk1 / ChsVb, loading efficiency 64%), tannic acid-trivalent iron ion metallic polyphenol network shell (molar ratio 10:1, shell thickness 22 nanometers), aryl boronic acid-carboxymethyl chitosan layer (p-boronic acid benzene acid substitution degree 9%), and deferoxamine grafting (0.45 micromole / gram carrier). Part A powder was prepared by spray drying, and the disintegration ratio reached 72% in a 2.0 mmol / L citric acid system for 2 hours, and the borate ester bond half-life was 1.6 hours under 50 micromol / L hydrogen peroxide conditions.

[0048] Preparation of Part B nitric oxide / hydrogen sulfide double-pulse microcapsules. S-nitrosoglutathione liposomes were prepared by thin film hydration and ultrasonic extrusion. Egg phospholipid and cholesterol were dissolved in chloroform at a mass ratio of 8:2, rotary evaporation was performed to form a film, and 0.5 mg / mL S-nitrosoglutathione aqueous solution was hydrated at 4°C. The ultrasonic probe was intermittently operated at a power of 180 W for a total of 10 minutes, and then extruded through 100 and 80 nanometer polycarbonate membranes in sequence. Dynamic light scattering showed that the particle size distribution was concentrated in 140-165 nanometers, and the zeta potential was -17 to -22 millivolts. The liposomes were mixed with 1.8% sodium alginate solution at a volume ratio of 1:4, and then dropped into a 2.5% calcium chloride coagulation bath through a 0.25 mm nozzle at a rate of 300 drops per minute to crosslink into sodium alginate calcium ion crosslinked microbeads. The bead diameter was 0.6-1.0 millimeters after screening.

[0049] A chitosan solution containing p-boronic acid benzene acid and glutaraldehyde was prepared, with an amine group to dialdehyde molar ratio of 10:1 and an aryl boronic acid to chitosan monosaccharide unit molar ratio of 0.08. The microbeads were immersed in the solution for 20 minutes to construct an aryl boronic acid-dialdehyde crosslinked chitosan shell, washed with deionized water until no free aldehyde was developed, and dried to a water content of 15%-25%. The crosslinking degree was 7%-12% as measured by titration. Under the trigger of 50 micromol / L hydrogen peroxide, the release rate peak of gaseous nitric oxide was observed at 30-48 hours after administration in a 25°C, pH 6.2 buffer system, and the apparent half-life was 1.2-1.8 days as monitored by chemiluminescence.

[0050] To prepare the hydrogen sulfide slow pulse module, hyaluronic acid was selected as the polysaccharide carrier to prepare the GYY4137 prodrug complex. The carboxyl group of hyaluronic acid was activated in the system of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and GYY4137 was reacted with hyaluronic acid at a mass ratio of 1:4 at 25°C for 4 hours. After dialysis to remove small molecules, a light yellow powder was obtained by spray drying. The prodrug complex was redispersed in water, coated with a chitosan solution to form a chitosan-chitinase degradable layer, and the coating thickness was adjusted to 0.5-1.5 microns by adjusting the coating solid content and spin coating speed. In a chitinase activity of 10 units / mL phosphate buffer at 25°C, the degradation rate reached not less than 60% within 7-14 days. The release of hydrogen sulfide was cross-verified by the methylene blue method and electrochemical sensor, and the peak time was located at 8-12 days after administration, with an apparent half-life of 7.5-10.8 days. The nitric oxide module and the hydrogen sulfide module were mixed at a mass ratio of 1:1.1 to obtain Part B, which had no obvious breakage under a load of 50 N, ensuring the mechanical stability of drip irrigation and water flushing.

[0051] The volatile organic compound particles of the β-cyclodextrin cross-linked aerogel of Part C were prepared. A water solution of β-cyclodextrin and citric acid was prepared at a mass ratio of 10:1, 1.0% sodium metaphosphate was added as a catalyst, and vacuum freeze-drying was followed by heat curing at 140°C for 2 hours to form a cross-linked porous framework. The mercury intrusion method and nitrogen adsorption-desorption showed that the specific surface area was 150-175 m² / g, the pore size distribution was concentrated in 5-45 nanometers, and the cross-linking degree was 6%-10%. The 2,3-butanediol and aromatic aldehyde mixture was loaded by vacuum immersion method, the 2,3-butanediol loading amount was 6%-10%, the aromatic aldehyde was selected from at least one of benzaldehyde and phenylacetaldehyde, and the total loading amount was 0.3%-0.7%, ensuring that the total molar ratio of 2,3-butanediol and aromatic aldehyde was 10-40:1. The surface was regulated by a thin layer of pectin or calcium alginate, the thin layer accounted for 0.5%-2% of the mass of the particles, and soluble starch 0.5%-1.0% was added in the thin layer for the delayed demand batch. Gas chromatography was used to compare the volatile organic compound release curves of samples with and without soluble starch, and the initial release start time was defined as the time when the flux reached 0.2 micrograms·square centimeter⁻¹·hour⁻¹, and the initial release start time of the sample containing soluble starch was delayed by 2-10 hours compared with the control.

[0052] In the standard environment of 25°C±5°C, pH 6.0-6.8, and water content 15%-25% in the sandy loam soil column system, the volatile organic compound release flux in the first week after application was 0.6-1.3 micrograms·square centimeter⁻¹·hour⁻¹, which was measured by the diffusion cell and electronic nose combined method.

[0053] The Part A powder, Part B microcapsule, and Part C particle are mixed in a mass ratio of 1:1.5:1.0 to prepare a composition. A plastic pot (25 cm in diameter) is filled with sand loam soil that has been sterilized by high-pressure steam, and the pH is adjusted to 6.5 and the water content is adjusted to 20%. Eggplant seedlings at the 3-leaf stage are transplanted. The Part C particle is buried on both sides of the root zone in a hole application manner, and the application amount is 3.0 g / plant. The Part A is prepared into a 0.3 g / L suspension and used to dip the roots for 30 seconds before planting. The Part B is added with water at a dosage of 0.5 kg / mu on the 7th day after planting. The pathogen inoculation is performed using a Fusarium oxysporum f. sp. melongenae suspension with a concentration of 10 6 spores / mL, and 20 mL is poured into the rhizosphere of each plant under the condition of 25°C±2°C and sunlight.

[0054] To verify the peak time sequence of the three types of active components, a rhizosphere simulation device is built, and the application is completed in the same pot in the above manner and monitored online. The volatile organic compounds are sampled every 3 hours by gas chromatography, and the peak time is located at 9-18 hours after application. Nitric oxide is recorded at a resolution of 12 hours by chemiluminescence analysis, and the peak time is located at 30-48 hours after application. Hydrogen sulfide is cross-verified by an electrochemical sensor and a methylene blue colorimetric method, and the peak time is located at 8-12 days after application. The peak time of double-stranded RNA is quantified by qPCR and fluorescence tracing, and is located in the interval of 14-20 days after application, thereby confirming that the order of t VOC < t NO < t H2S < t dsRNA is stable and established.

[0055] The non-overlapping peaks of the active components reflect different release control mechanisms. The volatile organic compounds of the β-cyclodextrin cross-linked aerogel form a leading peak controlled by pore size and surface diffusion. Nitric oxide is triggered by hydrogen peroxide to form a fast pulse by the arylboronic acid-dialdehyde cross-linked chitosan shell of S-nitrosoglutathione liposomes. Hydrogen sulfide is controlled by the enzymatic degradation rate of the chitosan-chitinase degradable layer of the GYY4137-polysaccharide prodrug complex to form a slow pulse. The release of double-stranded RNA is limited by a series of gates. The first limiting step is the disintegration of the metal polyphenol network shell promoted by the rhizosphere organic acid / iron chelator, and the second limiting step is the hydrogen peroxide-triggered borate ester bond breakage induced by pathogen infection.

[0056] Under the conditions of transplanting stress and pathogen infection, the root system and rhizosphere microorganisms of eggplant gradually produce time-sequential triggers: the concentration of citric acid in the rhizosphere solution gradually increases from 0.2-0.6 mmol / L to 1.4-4.2 mmol / L within 7-18 days; the effective concentration of chelating agents such as siderophores reaches 15-42 μmol / L in the same period, which promotes the selective disintegration of the metal polyphenol network shell; the oxidative burst induced by pathogen infection causes the transient concentration of H2O2 in the root surface microdomain to reach 18-72 μmol / L (for 30-90 minutes / episode) within 10-18 days, triggering the borate ester bond breakage. The gradual accumulation and burst of triggers determine the peak release of double-stranded RNA to be delayed to 10-28 days after application.

[0057] During the work process, Part C rapidly establishes the root zone gas phase concentration of volatile organic compounds on the day of transplanting, inhibiting the germination of conidia and the early extension of mycelium; in Part B, S-nitrosoglutathione liposomes form a nitric oxide pulse within 24-72 hours, promoting the expression of defense-related genes such as PR1 and PR5; GYY4137 prodrug complexes form a slow hydrogen sulfide pulse within 7-14 days, maintaining root system antioxidant and cell wall reinforcement; Part A achieves delayed release of double-stranded RNA and is taken up by pathogens, targeting down-regulation of the transcription levels of Fmk1 and ChsVb, forming a time-sequential synergy of first inhibiting the environment, then enhancing the host, and finally precise silencing.

[0058] In greenhouse verification, the disease index of the triple-application group is 33-40 percentage points lower than that of the blank control and 20-23 percentage points lower than that of the B+C two-application group; within 1-3 days after application, the expression levels of PR1 and PR5 genes in root tissues increase by 2-3 times, the lignin staining area increases, the activities of peroxidase and superoxide dismutase significantly increase, and the content of malondialdehyde decreases; within 8-14 days, the hydrogen sulfide signal is maintained, and the TTC reduction rate of root system activity increases by 15-20 percentage points; within 14-20 days, the expression of target sequence-related genes is down-regulated, and the re-isolation rate in the rhizosphere decreases.

[0059] Storage stability evaluation shows that, in a sealed aluminum foil bag, the S-nitrosoglutathione content retention rate is higher than 85% at 4°C in the dark for 90 days, and the peak position of nitric oxide release changes by no more than 6 hours; the effective content retention rate of GYY4137 is higher than 88%, and the peak position of hydrogen sulfide release changes by no more than 1 day; the loading loss of 2,3-butanediol and aromatic aldehyde in β-cyclodextrin cross-linked aerogel is less than 15%, and the flux decreases by less than 20% in the first week. At room temperature of 25°C, the content retention rates of S-nitrosoglutathione and GYY4137 are higher than 72% and 80%, respectively.

[0060] The process parameters used in this example all fall within the scope defined by the invention, and the calcium alginate ion-crosslinked microbeads can be produced by multi-nozzle dripping or coaxial spraying for capacity expansion, and the beta-cyclodextrin cross-linked aerogel can be continuously produced by tray-type freeze-drying and tunnel-type heat curing, with particle size distribution, shell cross-linking degree, loading rate and release curve as the core quality indicators. Based on this, those skilled in the art can stably reproduce and realize field application.

[0061] To verify the consistency of the molar ratio of 2,3-butanediol to aromatic aldehyde and the loading mass fraction, taking the parameters of this example as an example: when the loading amount of 2,3-butanediol is 7.5% (w / w) and the loading amount of benzaldehyde is 0.4% (w / w), according to the molecular weight 90.12 and 106.12 g / mol, the molar ratio is about (7.5 / 90.12) / (0.4 / 106.12)≈22:1, which falls within the range of 10~40∶1.

[0062] This example is prepared and added according to the total mass ratio of 1∶1.5∶1.0, and under the current process conditions, the active loading rates of parts A, B and C are 64%, 47% and 8.9% respectively, which is equivalent to a ratio of about 1∶1.6∶1.0 according to the effective ingredient, and all fall within the scope defined by the invention.

[0063] Example 3 This example discloses a method for controlling eggplant root rot using a composition, and also relates to the preparation of an agricultural preparation and the design of a triple application package.

[0064] The composition is composed of part A kaolinite nanotube-metal polyphenol network-borate ester gated double-stranded RNA delivery body, part B nitric oxide / hydrogen sulfide double-pulse microcapsule and part C volatile organic compound particles of beta-cyclodextrin cross-linked aerogel, with a total mass ratio of 1∶1.5∶1.0. The preparation method is the same as that of examples 1 and 2, which will not be repeated here.

[0065] On the day of transplanting, part C particles are shallowly buried 3-5 cm on both sides of the root zone at a dosage of 2-4 g / plant; at the same time, part A is applied to the eggplant root system, a suspension with a mass concentration of 0.3 g / L is prepared, the roots are dipped uniformly for 30 seconds, and then planted immediately, or the solid agent is evenly applied to the bottom of the planting trench at a dose of 1.5 kg / acre after covering the soil; on the 7th day±2 days after transplanting, part B microcapsules are applied by drip irrigation at a dose of 0.5 kg / acre.

[0066] To verify the timing effect of the method, rhizosphere dynamic monitoring was carried out under greenhouse conditions (25℃±2℃, relative humidity 65%-75%). Gas chromatography / electronic nose was used to monitor the release of 2,3-butanediol and aromatic aldehyde released by β-cyclodextrin cross-linked aerogel. The release flux reached a peak at 12 hours after application and was stably maintained at 0.5-1.4 μg·cm⁻²·h⁻¹ in the first week. Chemiluminescence and electrochemical probe cross verification was used to verify the release of nitric oxide and hydrogen sulfide. The peak of nitric oxide appeared at 8-10 days after transplanting, with a half-life of 1.3-1.7 days. The peak of hydrogen sulfide appeared at 14-18 days after transplanting, with a half-life of 7.5-11 days. The release of double-stranded RNA was quantitatively analyzed by fluorescence tracing and qPCR, and the peak appeared at 16-22 days after transplanting. The above monitoring results confirmed that the peak time sequence of the four types of active components met the requirements of t VOC <t NO <t H2S <t dsRNA .

[0067] Field application verification was carried out in a naturally infected test field. The soil was sandy loam, pH 6.4, water content 20%, and the background infection concentration of Fusarium was about 10 5 spores / g dry soil. The test design was randomized block with 4 replicates, 30 plants per block. Three-component combination group, A+B, A+C, B+C two-component combination group, single agent group and blank control group were set. The disease index of the three-component combination group was reduced by 34%-38% compared with the blank control group, and further reduced by 21%-26% compared with each two-component combination group, and the difference was significant (p<0.05) by ANOVA analysis.

[0068] Molecular level detection showed that the expression levels of PR1 and PR5 genes in the three-component combination group increased by 2.5-3.2 times at 3 days after transplanting, the activities of SOD and POD increased by 1.4-1.7 times at 10 days after transplanting, the MDA content decreased by 28%-33%, and the transcription levels of Fusarium Fmk1 and ChsVb were down-regulated by 55%-63% at 20 days after transplanting.

[0069] Part C provides an early barrier of volatile organic compounds to inhibit pathogen germination, Part B releases nitric oxide and hydrogen sulfide at the initial infection stage of the pathogen, rapidly induces host defense response and maintains medium-term resistance, and Part A delivers double-stranded RNA to precisely silence key pathogenic genes at the extension stage of the pathogen. The timing complementarity of the three types of components makes the prevention and control effect significantly better than single or two-component combination.

[0070] To realize large-scale promotion, the composition is prepared into an agricultural preparation. The adjuvants are selected from lignosulfonate (10% by weight), montmorillonite (30% by weight), and microcrystalline cellulose (10% by weight), and the wettable powder is prepared by high-speed mixing and granulation. The content retention rates of the active components of the obtained preparation are all not less than 80% after 12 months of storage.

[0071] Further, a triple administration package is designed, in which the first A part powder, the second B part microcapsule, and the third C part granule are independently packaged, and an administration instruction is attached to the outer package. The instruction clearly indicates that the first A part is administered on the day of transplanting, the second B part is administered 7 days ± 2 days after transplanting, and the third C part is shallowly buried on the day of transplanting. Technical indexes are specified: the apparent half-life of the second B part is 1-2 days, the apparent half-life of hydrogen sulfide is 7-12 days, and the volatile organic compound release flux of the third C part in the first week after administration is 0.5-1.5 μg·cm⁻²·h⁻¹.

[0072] In summary, the method and the administration package can realize a replicable three-stage time-release in the field, significantly inhibit the occurrence and development of eggplant root rot, and improve yield and input-output ratio.

[0073] Example 4 In this example, a composition for preventing and treating eggplant root rot is prepared, which is composed of three parts with a mass ratio of A:B:C = 1:1.3:1.1 based on the total mass. The technical advantages of the present application are verified by systematic comparison with a control group. The entire preparation process is completed in a clean environment at 25°C ± 2°C. All aqueous phases are prepared using ultrapure water treated with RNase. The composition does not contain layered double hydroxides or hydrotalcite materials.

[0074] The first A part is a kaolinite nanotube-metallic polyphenol network-borate ester gated double-stranded RNA delivery body, which is prepared by the same process as in Example 1, has a loading efficiency of 64%, and has a disintegration ratio of 72% and 75% under citric acid / deferoxamine conditions for 2 hours after spray drying. The half-life of the borate ester bond under hydrogen peroxide conditions is about 1.6 hours.

[0075] The second B part is a nitric oxide / hydrogen sulfide double-pulse microcapsule, which is prepared by the same process as in Example 2. S-nitrosoglutathione liposomes are encapsulated in calcium alginate ion cross-linked microbeads and coated with an aryl boronic acid-dialdehyde cross-linked chitosan shell layer. GYY4137-hyaluronic acid prodrug complexes are coated with a chitosan-chitinase degradable layer. The peak release time of nitric oxide is 36 ± 5 hours after administration, and the apparent half-life is 1.2-1.8 days. The peak release time of hydrogen sulfide is 9.0 ± 1.0 days after administration, and the apparent half-life is 7.5-10.8 days.

[0076] Part C is a β-cyclodextrin crosslinked aerogel with volatile organic compound particles, a skeletal specific surface area of 150-175 m2 / g, a pore size of 5-45 nm, a crosslinking degree of 6%-10%, a loading of 2,3-butanediol of 6%-10% and aromatic aldehyde of 0.3%-0.7%, a total molar ratio of 10-40:1, and a pectin-calcium coating. The release flux in the first week after application is 0.5-1.5 μg·cm⁻²·h⁻¹.

[0077] The three parts are mixed in proportion to form a composition. The test is simultaneously carried out in a greenhouse and in a field, the substrate is sandy loam soil, and the pH is adjusted to 5.5-7.0, the soil moisture content is 15%-25%, and the environment is maintained at 25°C±5°C. The test is carried out on eggplant seedlings at the 3-leaf stage, and 24 hours after transplanting, the seedlings are inoculated with a Fusarium spore suspension (10 6 spores / mL) of 20 mL / plant. Randomized block design with four replicates, 40 plants per plot. Part C is shallowly buried at 3.3 g per plant on the day of transplanting; Part A is dipped in a 0.3 g / L suspension for 30 seconds; and Part B is applied by drip irrigation with water at 0.65 kg / acre at 7 days±2 days after transplanting.

[0078] The test treatments include: blank control, A, B, C, A+B, A+C, B+C, patent control (LDH-dsRNA, Patent No. WO2015089543A1), and A+B+C. The triple combination of the present application is the same as in the previous examples. The LDH-dsRNA control is prepared and applied strictly in accordance with the previous patent disclosure, the carrier is a magnesium-aluminum system LDH, the loading mechanism is surface adsorption and intercalation, the loading ratio is dsRNA:LDH=1:5 (mass ratio), the loading rate is 82%, D50=185 nm, the zeta potential is +22 mV, and the dose is uniformly converted to "effective double-stranded RNA mass per plant".

[0079] The comparative test results are as follows: Table 1 Disease index and related indicators (mean±SD, four replicates)

[0080] The ablation experiment is as follows: Table 2 Ablation experiment results (mean±SD, four replicates)

[0081] The process parameters used in this example all fall within the scope defined by the present application, and through three-stage time sequence coordination, significant and stable technical progress is achieved relative to any two combinations and single agents; under equivalent conditions, additional effects are also exhibited relative to the layered double hydroxide carrier scheme (WO2015089543A1).

[0082] To verify the consistency of the molar ratio of 2,3-butanediol to aromatic aldehyde and the loading mass fraction: when the loading amount of 2,3-butanediol is 7.8% (w / w) and the loading amount of benzaldehyde is 0.45% (w / w), the ratio of the total molar amount of 2,3-butanediol to aromatic aldehyde is about 20.4:1 according to the molecular weight of 90.12 and 106.12 g / mol, which falls within the range of 10-40:1.

[0083] The present example is prepared and added according to the total mass ratio of 1:1.3:1.1, and the active load rates of the first A, B and C parts are 64%, 46% and 8.8% respectively under the current process conditions, which is converted to the ratio of about 1:1.4:1.1 according to the effective ingredient, and all fall within the range defined by the present application.

[0084] The above description is only the preferred embodiment of the present application, and it should be understood that the present application is not limited to the form disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein by the above teaching or related art or knowledge. Any modification and change made by the person skilled in the art without departing from the spirit and scope of the present application shall fall within the protection scope of the claims of the present application.

Claims

1. A composition for controlling eggplant root rot, characterized by, The composition is composed of three parts in a mass ratio of A:B:C = 1:(1-1.5):(1-1.2) by total mass, and does not contain layered double hydroxide or hydrotalcite-like materials: A: Kaolinite nanotube-metal polyphenol network-borate ester gated double-stranded RNA delivery vehicle, comprising: A kaolinite nanotube carrier with a volume distribution median particle size D50 of 100-300 nm and a lumen diameter of 20-70 nm, the lumen being electrostatically loaded with double-stranded RNA through low-molecular cationic polysaccharides or polycations selected from at least one of chitosan oligosaccharides, polylysine and polyethylenimine with a molecular weight less than 10 kilodaltons; the double-stranded RNA has a length of 200-400 base pairs and targets a conserved functional region of Fusarium pathogenicity-related genes Fmk1 (mitogen-activated protein kinase) and / or ChsVb (chitin synthase); A metal polyphenol network shell layer coated on the outer surface of the kaolinite nanotube, formed by in-situ self-assembly of tannic acid and ferric ions at pH 5.5-7.0, with a shell thickness of 10-50 nm; An aryl boronic acid-carboxymethyl chitosan layer arranged outside the metal polyphenol network shell layer, wherein the aryl boronic acid forms a borate ester bond with the ribo cis diol of the double-stranded RNA, which can be cleaved by hydrogen peroxide; Iron carrier ligands are grafted on the outer surface of the metal polyphenol network shell layer, the iron carrier ligands being selected from at least one of deferoxamine and hydroxamic acid ligands, with a grafting density of 0.1-1.0 micromole per gram of carrier; The delivery vehicle undergoes shell disintegration in the presence of organic acids or iron chelators in the rhizosphere, with the metal polyphenol network shell disintegrating by 50% or more within 2-6 hours in the presence of citric acid at 0.5-5 millimoles per liter or deferoxamine at 10-50 micromoles per liter, and the borate ester bond being cleaved to release the double-stranded RNA in the presence of hydrogen peroxide at 10-100 micromoles per liter; B: Nitric oxide / hydrogen sulfide double-pulse microcapsules, comprising: S-nitrosoglutathione liposomes are embedded in sodium alginate calcium ion cross-linked microbeads, the mass ratio of lecithin to cholesterol in the liposomes is 8:2, the particle size is 120-180 nm, and the microbeads are coated with a chitosan shell layer cross-linked by aryl boronic acid and dialdehyde, which is broken in the presence of hydrogen peroxide to rapidly release nitric oxide; A prodrug complex of GYY4137 and polysaccharides forming hydrolysable covalent bonds is used as a slow-release source of hydrogen sulfide, the polysaccharides being selected from at least one of alginic acid and hyaluronic acid, and the complex being coated with a chitosan-chitinase degradable layer; The microcapsules exhibit the following properties under the conditions of 25℃±5℃, pH 5.5-7.0 and soil moisture content of 15%-25%: the nitric oxide release peak appears at 24-72 hours after application, and the apparent half-life is 1-2 days; the hydrogen sulfide release peak appears at 7-14 days after application, and the apparent half-life is 7-12 days; C: a volatile organic compound particle of a beta-cyclodextrin crosslinked aerogel, comprising a porous framework formed by crosslinking beta-cyclodextrin with citric acid, having a specific surface area of not less than 150 square meters per gram, a pore size of 5-50 nanometers, loaded with 2,3-butanediol at 6-10% by mass of the particle, and loaded with aromatic aldehyde at 0.3-0.8% by mass of the particle, the aromatic aldehyde being at least one selected from benzaldehyde and phenylacetaldehyde, and an outer surface of the particle being coated with a thin layer of at least one selected from pectin and calcium alginate, the thin layer being at 0.5-2% by mass of the particle; the particle releases volatile organic compound at a volatile flux of 0.5-1.5 micrograms per square centimeter per hour in the first week after application under conditions of 25 DEG C + / - 5 DEG C, pH 5.5-7.0, and soil moisture content of 15-25%; When applied to the root zone of eggplant in the mass ratio of A, B and C as described, the release rate of each active component is determined under the conditions of 25℃±5℃, pH 5.5-7.0 and soil moisture content 15%-25%, to obtain the peak time (the time point at which the release rate reaches the maximum value) of each active component, and the peak time satisfies the order relationship of t VOC <t NO <t H2S <t dsRNA Under the condition of pathogenic inoculation, the disease index of root rot is further reduced by 20% or more relative to the double combination composed of any two parts.

2. The composition of claim 1, wherein: The high aspect ratio kaolinite nanotube is activated by acid washing, and the inner wall of the high aspect ratio kaolinite nanotube is pre-coated with chitosan oligosaccharide, the quaternary ammonium substitution degree of the chitosan oligosaccharide is 10% to 20%; the loading efficiency of double-stranded RNA in the high aspect ratio kaolinite nanotube is not less than 60%; the length of the double-stranded RNA is 220 to 350 base pairs, and the target sequence is located in the conserved fragment of the Fmk1 kinase domain and / or ChsVb catalytic core region.

3. The composition of claim 1, wherein: The molar ratio of tannic acid to ferric ion in the metal polyphenol network shell layer is 8-15:1; the shell layer disintegration ratio reaches 70% and above within 2-6 hours under the condition of 1-3 millimoles per liter of citric acid or 10-50 micromoles per liter of deferoxamine; and the grafting density of siderophore ligand is 0.2-0.8 micromoles per gram.

4. The composition of claim 1, wherein: The aryl boronic acid-carboxymethyl chitosan layer is prepared by a coupling reaction of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, the aryl boronic acid is p-boronic acid, and the substitution degree of the aryl boronic acid on the carboxymethyl chitosan is 5%-15%; under the condition of 10-100 micromoles per liter of hydrogen peroxide, the half-life of the boronic acid ester bond is 0.5-4 hours.

5. The composition of claim 1, wherein: The S-nitrosoglutathione liposome is prepared by a thin film hydration and ultrasonic extrusion process, and the zeta potential is-10 to-25 millivolts; the crosslinking of sodium alginate uses a 1%-3% calcium chloride solution; and the crosslinking degree of aryl boronic acid-dialdehyde crosslinked chitosan is 5%-15%.

6. The composition of claim 1, wherein: In the prodrug complex formed by the GYY4137 and the polysaccharide, the mass ratio of GYY4137 to the polysaccharide is 1:3-1:8; the thickness of the outermost chitosan-chitinase degradable layer is 0.2-2 micrometers, and the degradation rate reaches 60% and above within 7-14 days under the condition of chitinase activity of not less than 10 units per milliliter.

7. The composition of claim 1, wherein: The crosslinking degree of the beta-cyclodextrin crosslinked aerogel is 5%-12%; the molar ratio of 2,3-butanediol to the aromatic aldehyde is 10-40:1; and the pectin or calcium alginate coating on the particle contains 0.5%-1.0% of soluble starch, and the initial release of volatile organic compound is delayed by 2-12 hours relative to the corresponding particle without the soluble starch under the conditions of 25 DEG C + / - 5 DEG C, pH 5.5-7.0, and soil moisture content of 15%-25%.

8. The composition of claim 1, wherein: The method for preventing and treating eggplant root rot by the composition is to shallowly bury the granules of the C part in the root area on the day of transplanting, with a dosage of 2-4 grams per plant; simultaneously, the A part is applied to the root system, using a suspension with a mass concentration of 0.2-0.4 grams per liter for dipping application, or a solid dosage of 1-2 kilograms per mu for trench application; the B part is applied by drip irrigation or water flushing at 7 days ± 2 days after transplanting, with a dosage of 0.3-0.6 kilograms per mu; under the conditions of 25℃ ± 5℃, pH 5.5-7.0, and soil moisture content of 15%-25%, the expression level of the PR1 gene and / or the PR5 gene of eggplant is increased by 2 times or more, and the disease index is reduced by 30% or more relative to no application or any of the two control groups.

9. The composition of claim 1, wherein: The use of the composition in the preparation of an agricultural preparation for rhizosphere application to prevent and treat eggplant root rot caused by Fusarium is that the adjuvant of the agricultural preparation comprises at least one substance selected from the following: lignin sulfonate (with a dosage of 5%-15%), at least one selected from montmorillonite and kaolin (with a dosage of 20%-50%), and microcrystalline cellulose (with a dosage of 5%-15%).

10. The composition of claim 1, wherein, It is prepared as a triple application package, wherein the A part, the B part and the C part of claim 1 are independently packaged, and an application instruction is attached, which stipulates that the A part, the B part and the C part are matched in the mass ratio as defined in claim 1; the C part is shallowly buried on the day of transplanting, the B part is applied at 7 days ± 2 days after transplanting, and the A part is applied on the day of transplanting; and it is stipulated that the apparent half-life of the B part is 1-2 days for nitric oxide and 7-12 days for hydrogen sulfide, and the technical index of the C part is a volatile organic compound release flux of 0.5-1.5 micrograms per square centimeter per hour in the first week after application.

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