A prothioconazole+flumetover+flufenerim seed treatment suspension concentrate and its preparation method and application

Abstract

The application discloses a propiconazole-azoxystrobin- clothianidin seed treatment suspending agent and a preparation method and application thereof, and belongs to the technical field of pesticide preparations; the agent takes propiconazole, azoxystrobin and clothianidin as active ingredients, takes beta-nicotinamide mononucleotide (NMN) as a functional antimicrobial component, and is supplemented with styrene-maleic anhydride copolymer sodium salt, fatty alcohol polyoxyethylene ether, xanthan gum, magnesium aluminum silicate, glycerol, polydimethylsiloxane defoaming agent, sodium benzoate, polyvinyl alcohol and deionized water to prepare the agent. The application is suitable for seed coating treatment for preventing and treating rice false smut, thrips and wheat sharp eyespot and aphids; NMN promotes the accumulation of phenolic compounds in the phloem of plants, and cooperates with clothianidin on the same insecticidal target, thereby significantly improving the prevention and treatment effect on aphids and thrips; the degradation product of NMN promotes the proliferation of beneficial microorganisms in the rhizosphere, improves soil enzyme activity, and realizes soil micro-ecological restoration.

Description

Technical Field

[0001] This invention relates to the field of pesticide formulation technology, specifically to a seed treatment suspension of prothioconazole oxychloride and its preparation method and application. Background Technology

[0002] Prothioconazole is a triazolylthion broad-spectrum systemic fungicide that binds to the active sites of key enzymes in fungal ergosterol biosynthesis, blocking the synthesis of key structural components of the fungal cell membrane. It is widely used to control important crop diseases such as wheat sheath blight and rice bakanae disease. Fludioxonil is a phenylpyrrole contact fungicide that targets the osmotic-sensitive mitogen-activated protein kinase cascade in the fungal osmolarity signaling pathway. Its target is completely different from that of prothioconazole. The combination of both has a synergistic effect on important pathogens such as Fusarium and Rhizoctonia, helping to delay the development of resistance under single-target treatment conditions. Thiamethoxam is a third-generation neonicotinoid insecticide that selectively acts on insect nicotinic acetylcholine receptors and has extremely low toxicity to mammals. When combined with the above two fungicides to form a seed treatment suspension, it can achieve simultaneous control of major seedling diseases and pests with a single coating, making it an important means of integrated pest management for wheat and rice seedlings.

[0003] Current ternary formulations rely solely on the dual-target combination of two fungicides to delay the development of pathogen resistance, failing to intervene in the resistance formation process. With long-term use, the accumulation of resistant strains is difficult to fundamentally prevent; prothioconazole-resistant strains have already been reported in the field, limiting the long-term lifespan of these formulations. Furthermore, current ternary formulations are purely chemical insecticides, lacking the ability to activate acquired resistance in crops. After the effective period of the active ingredient ends, they lack a sustained immune protection mechanism, resulting in a short overall effective protection period and insufficient disease control in the mid-to-late seedling stages. The synergistic insecticidal effect of current ternary formulations relies entirely on the single chemical component of thiamethoxam, lacking auxiliary mechanisms that can mobilize the plant's own natural active substances to synergistically enhance the effect of chemical insecticides. The insecticidal potential of these formulations needs further improvement. While controlling target pests and diseases, purely chemical formulations inhibit the normal metabolic activities of beneficial rhizosphere microorganisms to varying degrees, which is detrimental to maintaining soil microecological balance and does not align with the development direction of green agriculture and reducing pesticide application while increasing efficiency. At the formulation and process level, how to ensure the integrity of the activity of the novel functional auxiliary components and the dispersion stability of the formulation system through scientific process design while introducing novel bioactive functional auxiliary components is also a process challenge that needs to be overcome by existing preparation technologies.

[0004] Therefore, there is an urgent need for an improved seed treatment suspension that can achieve multiple technical goals, such as multi-target synergistic antibacterial activity, plant immune activation and sequential chemical control, field resistance overcoming, synergistic insecticidal effect and soil microecological restoration, by introducing novel components with multiple auxiliary functions and combining them with innovative preparation processes, while retaining the basic control of the three effective components. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides a seed treatment suspension of prothioconazole·fludioxonil·thiamethoxam, which, by weight, consists of the following components:

[0006] Prothioconazole 10-20 parts; fludioxonil 3-10 parts; thiamethoxam 60-100 parts; β-nicotinamide mononucleotide 0.5-1.5 parts; sodium salt of styrene-maleic anhydride copolymer 8-20 parts; fatty alcohol polyoxyethylene ether 4-15 parts; xanthan gum 0.8-2.5 parts; magnesium aluminum silicate 2.5-6.0 parts; glycerol 25-55 parts; polydimethylsiloxane defoamer 0.8-4.0 parts; sodium benzoate 0.8-2.5 parts; polyvinyl alcohol 4-15 parts; deionized water balance.

[0007] In the preferred embodiment, the weight parts of each component are as follows:

[0008] 15 parts of prothioconazole; 5 parts of fludioxonil; 80 parts of thiamethoxam; 0.8 parts of β-nicotinamide mononucleotide;

[0009] Sodium salt of styrene-maleic anhydride copolymer: 12 parts; fatty alcohol polyoxyethylene ether: 8 parts; xanthan gum: 1.6 parts;

[0010] Magnesium aluminum silicate 4.0 parts; glycerol 40 parts; polydimethylsiloxane defoamer 2.4 parts;

[0011] Sodium benzoate 1.6 parts; polyvinyl alcohol 8 parts; deionized water balance.

[0012] This invention also provides a method for seed treatment suspension, comprising the following steps:

[0013] S1, β-nicotinamide mononucleotide is pre-dissolved in deionized water to prepare an aqueous solution for later use;

[0014] S2, dispersant, wetting agent, suspending agent, antifreeze, defoamer, preservative, and three active ingredients are sequentially added to deionized water. After high-speed shear dispersion, the mixture is transferred to a sand mill for grinding. The material temperature is controlled to not exceed 35℃ during the grinding process until the formulation particle size D is reached. 90 No larger than 3 micrometers;

[0015] S3. After the temperature of the grinding suspension obtained in S2 drops below 25°C, slowly pump the β-nicotinamide mononucleotide solution obtained in S1 into the solution under low-speed stirring. After the pumping is completed, continue stirring to confirm that the absolute value of the Zeta potential of the formulation is not lower than 28mV.

[0016] S4, add film-forming agent solution and water, stir evenly to obtain the finished product.

[0017] In the preferred embodiment, the specific implementation process of S1 is as follows:

[0018] S11, weigh β-nicotinamide mononucleotide, add it to deionized water at 25℃ at a mass ratio of 1:49, stir until completely dissolved, and prepare an aqueous solution with a mass fraction of 2%;

[0019] S12, adjust the pH of the above solution to 5.5-6.0 with 0.1 mol / L citric acid solution, filter through a 0.45 micrometer microfiltration membrane, seal the filtrate in a brown bottle, and store it at 25°C in the dark for no more than 24 hours before use.

[0020] In the preferred embodiment, the specific implementation process of S2 is as follows:

[0021] S21. Add deionized water (65% of the total water volume) to a container, then add sodium salt of styrene-maleic anhydride copolymer and fatty alcohol polyoxyethylene ether in sequence, stirring until completely dissolved. Next, add magnesium aluminum silicate and xanthan gum in sequence, stirring until no lumps remain. Add glycerol, polydimethylsiloxane defoamer, and sodium benzoate, stirring until homogeneous. Add the three technical materials in the order of thiamethoxam, prothioconazole, and fludioxonil. After each material is added, stir at 500 rpm for 1 minute to wet it, then increase the speed to 3000-5000 rpm and shear for 3 minutes. After all three materials are added, continuously shear at the highest speed for 10 minutes to obtain the particle size D of the technical material. 90 Coarse dispersions no larger than 50 micrometers;

[0022] S22, the coarse dispersion obtained in S21 is transferred to a horizontal sand mill, and zirconia beads with a diameter of 0.8-1.0 mm and a purity of not less than 95% are added, with a zirconia bead volume filling rate of 60%-65%; the material temperature in the grinding chamber is controlled to not exceed 35℃ by jacket cooling water, and sand milling is performed in a circulating manner; the particle size D is measured every 30 minutes. 90 When D 90 Stop grinding when the particle size is no larger than 3 micrometers; clean the grinding chamber with 5% of the total water volume of deionized water, and add the cleaning solution to the grinding suspension.

[0023] In the preferred embodiment, the specific implementation process of S3 is as follows:

[0024] S31, transfer the grinding suspension into a jacketed cooling stainless steel mixing tank, turn on the jacket cooling water, and wait for the material temperature to drop below 25℃.

[0025] S32, with the impeller speed at 150-200 rpm, slowly pump the β-nicotinamide mononucleotide aqueous solution into the system using a peristaltic pump at a rate not exceeding 2% of the total system volume per minute, for a pumping time of not less than 30 minutes; after the pumping is completed, continue stirring for 20 minutes.

[0026] S33, sample and measure the Zeta potential of the system. If the absolute value of the Zeta potential is not less than 28mV, this step is complete. If the absolute value of the Zeta potential is less than 28mV, continue stirring at low speed for 10 minutes and then measure again until the absolute value of the Zeta potential is not less than 28mV.

[0027] In the preferred embodiment, the specific implementation process of S4 is as follows:

[0028] S41, slowly sprinkle polyvinyl alcohol powder into hot water at 85-90℃ to prepare a 10% polyvinyl alcohol solution by mass, and cool it to below 40℃ for later use.

[0029] S42, under the condition of stirring paddle speed of 150-200 rpm, slowly add the polyvinyl alcohol solution obtained in S41 to the material obtained in S3, and continue stirring for 15 minutes after the addition is complete; add deionized water to make up to the specified total weight, stir at 150 rpm for 5 minutes, mix evenly, and the finished product is obtained.

[0030] This invention also provides the application of seed treatment suspension in the prevention and control of diseases and pests in rice and wheat seedlings, by treating rice seeds or wheat seeds by seed coating to prevent rice bakanae disease and rice thrips, as well as wheat sheath blight and wheat aphids.

[0031] The beneficial effects achieved by this invention are as follows:

[0032] First, the seed treatment suspension prepared in this invention uses three active ingredients—prothioconazole, fludioxonil, and thiamethoxam—as the core control basis of the formulation. Prothioconazole exerts a broad-spectrum systemic fungicidal effect against important pathogens such as rice bakanae disease and wheat sheath blight by inhibiting the biosynthesis of ergosterol in pathogenic fungi and disrupting the integrity of fungal cell membranes. Fludioxonil exerts a contact-killing fungal inhibitory effect against the above pathogens by interfering with the fungal osmotic signal transduction pathway. Moreover, its target is completely different from that of prothioconazole. The combination of the two forms a dual-target synergistic attack, significantly improving the antifungal efficacy against Fusarium and Rhizoctonia species, and effectively delaying the effects of single-target treatment. The rate of resistance development under different pesticide conditions; thiamethoxam selectively acts on insect nicotinic acetylcholine receptors and has extremely low toxicity to mammals. It can control aphids, thrips, and other seedling pests while controlling diseases, achieving the goal of simultaneously controlling major seedling diseases and pests with a single seed coating treatment, reducing the labor costs of multiple applications, and meeting the production needs of reducing pesticide application and increasing efficiency; at the same time, β-nicotinamide mononucleotide (NMN) is introduced as a functional auxiliary antimicrobial component based on the ternary system. NMN competitively occupies the active site of nicotinamide mononucleotide adenylate transferase of pathogenic fungi in the rhizosphere microenvironment of seed germination, interfering with the NAD of pathogenic fungi. + The biosynthesis of NMN is disrupted, thus comprehensively inhibiting the energy metabolism of pathogens. The antimicrobial effect, combined with the chemical inhibition of ergosterol synthesis by prothioconazole and the interference of cycloheximedium on the osmotic signaling pathway, constitutes a triple-target attack. These targets three independent critical life nodes—pathogen cell membrane synthesis, energy metabolism, and osmotic pressure regulation—mutually reinforcing and synergistically enhancing each other. This results in a significantly higher overall combined toxicity coefficient compared to the ternary control formulation without NMN, producing an antibacterial efficacy enhancement that cannot be achieved by simple ternary chemical compounding. Simultaneously, after entering the plant, NMN is metabolized into NAD+. + It activates the salicylic acid immune signaling pathway, inducing systemic acquired resistance in plants. Its effects, combined with the rapid chemical killing effects of prothioconazole and fludioxonil, work over time. The chemical components rapidly kill pathogens in the early stages, while the NMN-induced immune protection continues to exert its effect after the chemical components' duration of action ends, significantly extending the overall effective protective period of the formulation. Furthermore, NMN also works through NAD... + The Sirtuin-dependent protein deacetylase pathway affects the epigenetic regulation of pathogens, downregulates the overexpression level of target genes in strains that have developed resistance to prothioconazole, and partially restores the sensitivity of resistant strains to prothioconazole. Thus, without changing the structure and dosage of prothioconazole, the field resistance can be overcome by the auxiliary component NMN.

[0033] Secondly, in the seed treatment suspension of the present invention, NMN increases NAD in plants. +After activating the phenylpropane metabolic pathway, the content of phenolic compounds in the phloem sap of plants increases significantly. When aphids and thrips suck plant sap, they ingest these phenolic compounds. Their weak agonistic activity on nicotinic acetylcholine receptors, combined with the selective inhibition of the same receptor by thiamethoxam, causes the receptor to become overexcited and eventually inactivated, accelerating the process of pest poisoning and death. This forms a new efficacy model in which plant-derived natural active substances and chemical insecticides work synergistically on the same receptor target, significantly improving the insecticidal efficacy of thiamethoxam against aphids and thrips. In field efficacy verification, the NMN-containing formulation showed significantly better control effect on wheat aphids than the NMN-free ternary formulation. After NMN is applied to the soil, it degrades into nicotinamide and ribose-5-phosphate, both of which are high-quality carbon and nitrogen sources for beneficial microorganisms in the rhizosphere. They significantly promote the proliferation of beneficial bacteria such as nitrogen-fixing bacteria and Bacillus. The proliferating beneficial microorganisms continuously inhibit the population of soil pathogenic fungi by secreting antagonists such as chitinase, forming an organic succession system of short-term chemical and long-term biological control with chemical control. At the same time, it enhances the activity of soil urease and phosphatase, improves the nitrogen and phosphorus cycle in the rhizosphere soil, and achieves the active restoration of the soil microecological environment.

[0034] Third, the formulation of this invention uses sodium salt of styrene-maleic anhydride copolymer as the main dispersant, which works synergistically with fatty alcohol polyoxyethylene ether wetting agent to ensure good surface wettability and dispersion stability of the three active ingredients. This effectively prevents the active ingredients from agglomerating during storage, maintaining the particle size of the formulation at the level required for uniform coating, and ensuring the uniformity of seed coating and the uniform distribution of active ingredients. Xanthan gum and magnesium aluminum silicate together form a composite suspension aid system. The two work synergistically to thicken the formulation, forming a weak gel network structure in the formulation system, effectively inhibiting the sedimentation of solid particles. This allows the formulation to maintain excellent physical stability under both hot and cold storage conditions, without crystallization or stratification. Glycerol acts as an antifreeze agent to ensure the stability of the formulation in low-temperature storage and transportation environments. Polyvinyl alcohol acts as a film-forming agent to form a uniform, continuous, and firmly adhered coating film on the seed surface, ensuring the stable and slow release of active ingredients during seed germination. Sodium benzoate acts as a preservative to effectively inhibit microbial contamination of the formulation during storage.

[0035] Fourth, the seed treatment suspension preparation process designed in this invention, which pre-dissolves NMN in deionized water and adjusts it to a weakly acidic environment for separate preparation, effectively protects the chemical structural stability of NMN under neutral to weakly acidic conditions. This avoids glycosidic bond hydrolysis and activity loss caused by high temperature or alkaline conditions, ensuring the integrity of NMN's biological activity in the final formulation. The process design, which strictly controls the material temperature during sand milling by using jacketed cooling water to prevent the heat-sensitive active ingredient from decomposing due to frictional heat accumulation during grinding, ensures the stability of the content of each active ingredient. The innovative operation of slowly pumping the NMN solution in at low speed using a peristaltic pump after sand milling and material cooling effectively avoids competitive ion interactions between high-concentration NMN and the anionic dispersant, preventing particle aggregation and suspension rate decrease caused by localized high concentrations of NMN in the dispersion system. The subsequent processes are further guaranteed by confirming system stability through online monitoring of the Zeta potential. The process of adding the film-forming agent after it has been dissolved separately in the final stage effectively prevents the mutual interference that may occur between polyvinyl alcohol and other components during the high-temperature dissolution process. This allows the film-forming agent to fully perform its function, and makes the formulation significantly superior to conventional preparation methods in terms of stability, uniformity, and retention of the activity of each component. Attached Figure Description

[0036] Figure 1 This is a line graph comparing the suspension rate and zeta potential of formulations with different NMN addition levels.

[0037] Figure 2 This is a comparison chart of the virulence-related indicators of Examples 1-3 and Comparative Examples 1-3 against Bakanae disease of rice. (a) is a virulence regression curve of the linear relationship between log concentration and probability value of each treatment group against Bakanae disease of rice, and (b) is a bar chart of the co-virulence coefficient values ​​of each treatment group with error bars.

[0038] Figure 3 This is a comparison chart of the germination-related indicators of rice and wheat seeds under different dosage treatments of the formulation in Example 1. Among them, (a) is a line graph of the germination rate of rice and wheat under each dosage treatment, (b) is a bar graph of the main root length of rice and wheat under each dosage treatment, and (c) is a bar graph of the fresh weight of seedlings of rice and wheat under each dosage treatment.

[0039] Figure 4 This is a comparison chart of the changes in the microecological indicators of rice rhizosphere soil over time between Example 1, Comparative Example 1, and the water control. (a) is a line graph showing the change in rhizosphere soil bacterial count over time for each treatment, (b) is a line graph showing the change in rhizosphere soil fungal count over time for each treatment, (c) is a line graph showing the change in rhizosphere soil respiration rate over time for each treatment, and (d) is a line graph showing the change in rhizosphere soil urease activity over time for each treatment.

[0040] Figure 5 This is a flowchart of a method for preparing a seed treatment suspension of prothioconazole nitrile thiamethoxam according to the present invention. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to illustrate the specific implementation of the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. In the following embodiments, all raw materials used are commercially available products that meet the corresponding quality standards. Unless otherwise specified, all percentages are weight percentages.

[0042] The active ingredients and auxiliary ingredients and their functions in the seed treatment suspension (FS formulation) of this invention are as follows: Prothioconazole (CAS No.: 178928-70-6) is a triazole thionine broad-spectrum systemic fungicide. It exerts its fungicidal activity by coordinating with the heme iron atom of sterol 14α-demethylase (CYP51, i.e., cytochrome P450 sterol demethylase), a key enzyme in fungal ergosterol biosynthesis, thereby inhibiting the conversion of lanosterol to ergosterol and disrupting the structural integrity of the fungal cell membrane; Fludioxonil (CAS No.: 131341-86-1) is a phenylpyrrole contact fungicide. Its target is the osmotic stress-activated mitogen-activated protein kinase (os-MAPK) cascade in the fungal osmolarity signaling pathway, which is completely different from the target of prothioconazole. The two together target Fusarium species. It has a synergistic effect with important pathogens such as *Rhizoctonia spp.* and *Rhizoctonia spp.*; Clothiadin (CAS No.: 210880-92-5) is a third-generation neonicotinoid insecticide that selectively acts on the α4β2 subtype of the insect's nicotinic acetylcholine receptor (nAChR), causing sustained excitation of the insect's postsynaptic membrane, paralysis, and death. It has extremely low toxicity to mammals.

[0043] β-Nicotinamide Mononucleotide (NMN, CAS No.: 1094-61-7, molecular formula C) 11 H 15 N2O8P (molecular weight 334.22 Da) is nicotinamide adenine dinucleotide (NAD). +NMN is a direct biosynthetic precursor of Nicotinamide Adenine Dinucleotide (NMN), belonging to a single compound with a well-defined structure. In the formulation of this invention, NMN acts as a functional auxiliary antimicrobial component, and its main mechanism of action is as follows: (1) In the rhizosphere soil microenvironment of seed germination, NMN competitively occupies the active site of Nicotinamide Mononucleotide Adenylyl Transferase (NMNAT, EC 2.7.7.1) of the pathogenic fungus at an exogenous concentration much higher than the steady-state concentration of endogenous NMN in the pathogenic fungus. This enzyme catalyzes the condensation of NMN with adenosine triphosphate (ATP) to generate NAD. + It is the pathogen NAD + The key rate-limiting step in biosynthesis; when NMNAT is competitively inhibited by exogenous NMN, intracellular NAD in pathogens... + The level drops sharply, energy metabolism (including glycolysis, tricarboxylic acid cycle, electron transport chain, etc.) is completely blocked, growth is inhibited or even death occurs, and it plays a direct role in disinfection and antimicrobial function (corresponding to IPC classification number A01P 1 / 00); (2) After NMN enters the plant, it is metabolized into NAD. + Intracellular NAD in plants + The increase in levels activates poly ADP-ribose polymerase (PARP), which in turn initiates the salicylic acid (SA) signaling pathway, upregulates disease resistance genes such as pathogenesis-related protein NPR1 (Nonexpresser of PR genes 1) and PR-1 (Pathogenesis-Related Protein 1), and induces systemic acquired resistance (SAR) in plants; (3) through NAD + The Sirtuins-dependent deacetylase pathway affects the epigenetic regulation of pathogens and delays the formation of field resistance to prothioconazole.

[0044] Auxiliary components: Styrene-Maleic Anhydride copolymer sodium salt (SMAS) is used as the main dispersant; Fatty Alcohol Polyoxyethylene Ether (AEO-7, with an average addition of 7 ethylene oxide units) is used as a wetting and spreading agent; Xanthan Gum and Magnesium Aluminum Silicate (montmorillonite) are used together as suspending agents, synergistically thickening; Glycerol (CAS No.: 56-81-5) is used as an antifreeze agent; Polydimethylsiloxane (PDMS) is used to suppress foaming during preparation and use; Sodium Benzoate (CAS No.: 532-32-1) is used as a preservative; Polyvinyl Alcohol (PVA) is used as a preservative. Alcohol (85%–90% degree of hydrolysis) is used as a film-forming agent to form a uniform and stable coating film on the seed surface; deionized water (conductivity ≤2 μS / cm) is used as a continuous phase dispersion medium.

[0045] Example 1: This example describes the preparation of a seed treatment suspension containing 1.5% prothioconazole, 0.5% fludioxonil, 10.0% thiamethoxam, and 0.10% NMN.

[0046] Based on a total weight of 800 g, the amounts of each component are as follows: 12.50 g of prothioconazole technical (96% purity); 4.12 g of fludioxonil technical (97% purity); 81.63 g of thiamethoxam technical (98% purity); 0.80 g of β-nicotinamide mononucleotide (NMN, purity ≥98%, pH (1% aqueous solution) 5.5); 12.00 g of sodium styrene-maleic anhydride copolymer (SMAS); 8.00 g of fatty alcohol polyoxyethylene ether (AEO-7); 1.28 g of xanthan gum; 3.20 g of magnesium aluminum silicate; 32.00 g of glycerol; 1.60 g of polydimethylsiloxane defoamer; 1.28 g of sodium benzoate; 6.40 g of polyvinyl alcohol (87% degree of hydrolysis, PVA-1788); and deionized water (conductivity ≤2 μS / cm) to bring the total to 800 g. g (approximately 635.19 g).

[0047] Reference Figure 5The preparation process of the seed treatment suspension in this embodiment is as follows: S1: Pre-preparation of β-nicotinamide mononucleotide (NMN) solution. S11: Accurately weigh 0.80 g of NMN and add it to 39.20 g of deionized water at 25℃ at a mass ratio of 1:49. Stir at 300 r / min for about 10 min on a magnetic stirrer until NMN is completely dissolved to prepare a 2% (w / w) NMN aqueous solution. S12: Adjust the pH of the above NMN aqueous solution to 5.5-6.0 with 0.1 mol / L citric acid solution (detected with a precision pH meter, accuracy 0.01). Filter through a 0.45 μm microfiltration membrane (water-based, polyethersulfone material). Collect the filtrate in a brown sealed bottle and store it at 25℃ in the dark for no more than 24 h for later use. The entire process should be carried out at room temperature and should not be heated, to prevent NMN from undergoing glycosidic bond hydrolysis at high temperatures (>60℃), generating nicotinamide (NAM) and ribose-5-phosphate (R5P), which would lead to a loss of activity.

[0048] S2: Mixing, shearing and grinding. S21: Add 65% (approximately 412.9 g) of deionized water to a stainless steel container, then add 12.00 g of SMAS and 8.00 g of AEO-7 sequentially, stirring at 200 r / min until completely dissolved; then slowly add 3.20 g of magnesium aluminum silicate and 1.28 g of xanthan gum sequentially, stirring at 300 r / min until no lumps remain; add 32.00 g of glycerol, 1.60 g of polydimethylsiloxane defoamer, and 1.28 g of sodium benzoate, stirring until homogeneous; add the three technical materials in the following order: thiamethoxam (81.63 g), prothioconazole (12.50 g), and fludioxonil (4.12 g). After each technical material is added, stir at 500 r / min for 1 min to fully wet the material, then increase the speed to 3000–5000 r / min and shear for 3 min; after all three technical materials are added, stir at the highest speed (5000 r / min)... Continuous high-speed shearing at r / min for 10 min yields the original drug particle size D. 90 A coarse dispersion with a particle size not exceeding 50 μm. S22: Transfer the coarse dispersion obtained in S21 into a horizontal sand mill, add zirconia beads (ZrO2) with a diameter of 0.8–1.0 mm and a purity of not less than 95%, with a zirconium bead volume filling rate of 60%–65%; control the material temperature in the grinding chamber to not exceed 35℃ using jacket cooling water (water temperature set at 10℃), and perform circulating sand milling; take samples every 30 minutes to test the particle size D. 90 (Laser diffractometer, Malvern Mastersizer 3000 or equivalent instrument), when D 90Stop grinding when the particle size is no larger than 3 μm (in this embodiment, grinding takes about 2 hours and is repeated 3 times); clean the grinding chamber with 5% (about 25.8 g) of deionized water, and add the cleaning solution to the grinding suspension.

[0049] S3: Add NMN solution. S31: Transfer the ground suspension to a jacketed, cooled stainless steel mixing tank (effective volume 10L), turn on the jacket cooling water (cold water temperature set to 10℃), and wait for the material temperature to drop below 25℃ (approximately 30 minutes). S32: With the impeller speed at 150–200 r / min, slowly pump the NMN aqueous solution obtained in S12 (approximately 40.00 g, equivalent to a final concentration of 0.10%) using a peristaltic pump at a rate not exceeding 2% / min (approximately 16 mL / min) of the total system volume, for at least 30 minutes. After pumping, continue low-speed stirring for 20 minutes to ensure NMN is uniformly dispersed in the system, preventing competitive ion interactions between locally high concentrations of NMN and the anionic dispersant SMAS, which could lead to particle aggregation and a decrease in suspension rate in the dispersion system. S33: The Zeta potential (ζ) of the system is measured using a dynamic light scattering Zeta potential meter. If |ζ| ≥ 28 mV, this step is complete; if |ζ| < 28 mV, continue stirring at low speed for 10 min and measure again until |ζ| ≥ 28 mV. An absolute value of the Zeta potential |ζ| ≥ 28 mV indicates that the electrostatic repulsion between colloidal particles is sufficient to overcome van der Waals forces, and the formulation system is in a stable dispersion state.

[0050] S4: Add film-forming agent and replenish water. S41: Slowly sprinkle 6.40g of polyvinyl alcohol (PVA, degree of hydrolysis 87%) powder into hot water at 85-90℃ (approximately 57.6g of water is used to make the final PVA solution concentration 10%). Dissolve for about 30 minutes under stirring at 300r / min until completely transparent, then cool to below 40℃ for later use. S42: Under the condition of stirring paddle speed of 150-200r / min, slowly add the polyvinyl alcohol solution obtained in S41 (approximately 64g) to the material obtained in S3. After the addition is complete, continue stirring for 15 minutes; replenish with deionized water to the specified total weight of 800g, stir at 150r / min for 5 minutes until uniformly mixed, and the finished product is obtained.

[0051] The obtained products were subjected to quality testing, and the test results are shown in Table 1.

[0052] Table 1. Product quality inspection results of Example 1

[0053]

[0054] Example 2: In this example, a seed treatment suspension containing 1.0% prothioconazole, 0.3% fludioxonil, 8.0% thiamethoxam, and 0.05% NMN was prepared, and the formulation performance was investigated when the content of each active ingredient and NMN was at the lower limit of the claims.

[0055] Formula composition (based on a total weight of 800g): Prothioconazole technical (96% purity) 8.33g; Fludioxonil technical (97% purity) 2.47g; Thiamethoxam technical (98% purity) 65.31g; NMN (≥98% purity) 0.40g; SMAS 8.00g; AEO-7 4.00g; Xanthan gum 0.64g; Magnesium aluminum silicate 2.00g; Glycerol 20.00g; Polydimethylsiloxane defoamer 0.64g; Sodium benzoate 0.64g; PVA-1788 3.20g; Deionized water to 800g.

[0056] The preparation process was carried out according to steps S1 to S4, the same as in Example 1. Specifically, in S12, the volume of the NMN aqueous solution (2%) was 20.00 g, corresponding to a final concentration of 0.05%; in S21, the high-speed shearing speed was 3500 r / min, and the shearing time was 10 min; in S22, the sand milling was carried out for approximately 1.5 h. 90 The acceptable test result is ≤3μm; the peristaltic pumping rate in S32 shall not exceed 2% / min of the total system volume (approximately 13mL / min), and the pumping time shall be ≥30min.

[0057] Quality test results: Prothioconazole content 1.01%, fludioxonil content 0.301%, thiamethoxam content 8.06%, NMN content 0.049%; pH 5.6; suspension rate 93.8%, suspension rate after heat storage (54℃ / 14d) 92.0%; D 90 2.9μm; |ζ|=28.5mV; decomposition rate of each effective component after thermal storage <3%; no crystallization or stratification during cold storage (0℃ / 7d); Zeta potential meets stability requirements.

[0058] Example 3: In this example, a seed treatment suspension with 3.0% prothioconazole, 1.0% fludioxonil, 15.0% thiamethoxam, and 0.20% NMN was prepared, and the formulation performance was investigated when the content of each active ingredient and NMN was at the upper limit of the claims.

[0059] Formula composition (based on a total weight of 800g): Prothioconazole technical (purity 96%) 25.00g; Fludioxonil technical (purity 97%) 8.25g; Thiamethoxam technical (purity 98%) 122.45g; NMN (purity ≥98%) 1.60g; SMAS 20.00g; AEO-7 12.00g; Xanthan gum 1.60g; Magnesium aluminum silicate 4.80g; Glycerol 40.00g; Polydimethylsiloxane defoamer 2.40g; Sodium benzoate 1.60g; PVA-1788 10.40g; Deionized water to 800g.

[0060] The preparation process was carried out according to steps S1 to S4, the same as in Example 1. In S12, the volume of the NMN aqueous solution (2%) was 80.00 g, corresponding to a final concentration of 0.20%. In S22, the milling time was approximately 2.5 h, repeated 4 times, until D... 90 ≤3μm; the peristaltic pumping rate in S32 does not exceed 2% / min of the total system volume (approximately 16mL / min). Due to the large amount of NMN, the pumping time is appropriately extended to ≥40min.

[0061] Quality test results: Prothioconazole content 3.02%, fludioxonil content 1.003%, thiamethoxam content 15.1%, NMN content 0.199%; pH 5.7; suspension rate 92.5%, suspension rate after heat storage (54℃ / 14d) 90.8%; D 90 3.0μm; |ζ|=27.8mV (in this example, |ζ| is near the warning value of 28mV, verifying that 0.20% is the reasonable upper limit of NMN dosage. When the dosage is exceeded, the stability of the anionic system is slightly affected); the decomposition rate of each effective component after heat storage is <4%; no crystallization or stratification occurs during cold storage.

[0062] Example 4: This example prepares a seed treatment suspension containing 2.0% prothioconazole, 0.5% fludioxonil, 12.0% thiamethoxam, and 0.15% NMN. The weight ratio of prothioconazole to fludioxonil is 4:1, and the weight ratio of NMN to the total amount of the three active ingredients is approximately 1:96. The overall performance of the formulation when the ratio is moderate is investigated.

[0063] Formula composition (based on a total weight of 800g): Prothioconazole technical (purity 96%) 16.67g; Fludioxonil technical (purity 97%) 4.12g; Thiamethoxam technical (purity 98%) 97.96g; NMN (purity ≥98%) 1.20g; SMAS 14.00g; AEO-7 9.00g; Xanthan gum 1.00g; Magnesium aluminum silicate 3.20g; Glycerol 35.00g; Polydimethylsiloxane defoamer 2.00g; Sodium benzoate 1.20g; PVA-1788 8.00g; Deionized water to 800g.

[0064] The preparation process was carried out according to steps S1 to S4, the same as in Example 1, except that in S12, the volume of the NMN aqueous solution (2%) was 60.00 g, corresponding to a final concentration of 0.15%; and in S32, the pumping time was ≥35 min. Quality test results: prothioconazole content 2.01%, fludioxonil content 0.502%, thiamethoxam content 12.05%, NMN content 0.149%; pH 5.7; suspension rate 94.8%, suspension rate after heat storage 92.5%; D 90 2.8μm; |ζ|=29.0mV; decomposition rate of each component in thermal storage <3%; no crystallization or stratification in cold storage.

[0065] Comparative Example 1 is a ternary seed treatment suspension without NMN. The formulation is the same as that of Example 1, but the NMN component is removed and made up with an equal amount of deionized water. It is used to directly compare the contribution of NMN to the physicochemical properties and biological activity of the formulation.

[0066] Formula composition (based on a total weight of 800g): Prothioconazole technical (96% purity) 12.50g; Fludioxonil technical (97% purity) 4.12g; Thiamethoxam technical (98% purity) 81.63g; SMAS 12.00g; AEO-7 8.00g; Xanthan gum 1.28g; Magnesium aluminum silicate 3.20g; Glycerol 32.00g; Polydimethylsiloxane defoamer 1.60g; Sodium benzoate 1.28g; PVA-178 86.40g; Deionized water to 800g.

[0067] The preparation process was carried out according to steps S2 and S4, with the remaining process parameters being the same as in Example 1. Quality test results: prothioconazole content 1.50%, fludioxonil content 0.500%, thiamethoxam content 10.00%; pH 5.9; suspension rate 91.2%, suspension rate after heat storage (54℃ / 14d) 88.6%; D 90 3.4 μm; |ζ| = 27.1 mV; decomposition rate of each component after thermal storage <3%; no crystallization or stratification during cold storage. Compared with Example 1, the suspension rate of Comparative Example 1 is 4.4 percentage points lower, and the suspension rate after thermal storage is 4.5 percentage points lower. D 90 The value is relatively large (indicating that NMN makes a positive contribution to the dispersion stability of the system), while |ζ| is slightly low.

[0068] Comparative Example 2 uses chitosan oligosaccharide instead of NMN. Chitosan oligosaccharide (COS, degree of deacetylation DD≥85%, average molecular weight Mw≤3200Da) is a known natural polysaccharide degradation product that can induce plant defense responses, but it does not have the competitive inhibition function of NMNAT, nor does it have an epigenetic intervention mechanism.

[0069] Formula composition (based on a total weight of 800g): Prothioconazole technical (96% purity) 12.50g; Fludioxonil technical (97% purity) 4.12g; Thiamethoxam technical (98% purity) 81.63g; Chitosan oligosaccharide (DD≥85%, Mw≤3200Da) 0.80g (equal replacement for NMN); SMAS 12.00g; AEO-7 8.00g; Xanthan gum 1.28g; Magnesium aluminum silicate 3.20g; Glycerol 32.00g; Polydimethylsiloxane defoamer 1.60g; Sodium benzoate 1.28g; PVA-1788 6.40g; Deionized water to 800g.

[0070] The preparation process was the same as in Example 1. Chitosan oligosaccharides were dissolved in deionized water and added using the same pump-in process (step S3). Quality test results: suspension rate 90.5%, suspension rate 87.1% after heat storage (54℃ / 14d) (chitosan oligosaccharides carry positively charged amino groups, which neutralize the charge of the anionic dispersant SMAS, resulting in significantly lower suspension stability than in Example 1); D 90 3.3μm; |ζ|=26.8mV; decomposition rate of each component in thermal storage <4%; no crystallization in cold storage.

[0071] Comparative Example 3 is a ternary formulation in which salicylic acid (SA, CAS No.: 69-72-7) replaces NMN. Salicylic acid is a known plant SAR activator.

[0072] Formula composition (based on a total weight of 800g): 12.50g of prothioconazole technical (96% purity); 4.12g of fludioxonil technical (97% purity); 81.63g of thiamethoxam technical (98% purity); 0.80g of salicylic acid (equal amount to replace NMN); other adjuvants are the same as in Example 1; deionized water is added to bring the total to 800g. The preparation process is the same as in Example 1, with SA directly dissolved in deionized water and then slowly pumped in. Quality test results: suspension rate 91.8%, suspension rate after heat storage 89.5%; D 90 3.2 μm; |ζ|=27.5 mV; SA has a simple chemical structure and has little impact on anionic systems. Its formulation stability is slightly better than Comparative Example 2, but slightly lower than Example 1.

[0073] Example 1: Effect of NMN addition on suspension rate and zeta potential of the formulation; This example uses the preparation process of Example 1, and sets up 6 NMN addition amounts (0%, 0.05%, 0.10%, 0.15%, 0.20%, 0.30%) to prepare formulation samples, corresponding to the numbers E1 (containing 0% NMN, i.e., Comparative Example 1), E2 (containing 0.05% NMN, i.e., Example 2), E3 (containing 0.10% NMN, i.e., Example 1), E4 (containing 0.15% NMN, i.e., Example 4), E5 (containing 0.20% NMN, i.e., Example 3), and E6 (containing 0.30% NMN, exceeding the upper limit of the claims, reference group).

[0074] The initial suspension rate and the suspension rate after heat storage (54℃, 14d) of each sample were determined according to GB / T 14825-2006 "Determination of Pesticide Suspension Rate". |ζ| (mV) of each sample was measured using a dynamic light scattering Zeta potential meter (Malvern Zetasizer Nano ZS). Each treatment was repeated three times, and the average value was taken. The experimental results are as follows: Figure 1 As shown. Figure 1 Line graph showing the effect of NMN addition on the suspension rate (initial and after heat storage) and Zeta potential. The horizontal axis is NMN concentration (%), the left vertical axis is suspension rate (%), and the right vertical axis is |ζ| (mV). The mean ± standard deviation of each data point is marked (n=3).

[0075] Table 2. Suspension rate and zeta potential of formulations with different NMN additions

[0076]

[0077] From Table 2 and Figure 1 It can be seen that within the NMN addition range of 0.05% to 0.15%, the initial suspension rate and the suspension rate after heat storage of the formulation are both higher than those of the control group 1 without NMN, and |ζ| also remains in the stable range above 28 mV. This indicates that an appropriate amount of NMN has a positive contribution to the dispersion stability of the formulation system, which is speculated to be related to the phosphate group (-OPO3) in the NMN molecule. 2- The NMN content is related to the formation of the charge layer on the particle surface. When the NMN content exceeds 0.20%, both the suspension rate and |ζ| decrease significantly (|ζ| drops to 25.6 mV when the NMN content is 0.30%, which is lower than the critical value of 28 mV for colloidal stability). This is presumably due to the competitive ionic interaction between excess NMN and the anionic dispersant SMAS, which disrupts the stability of the colloidal double layer. Considering both suspension stability and bioactivity, 0.10% is determined to be the optimal addition amount of NMN, and 0.20% is the safe upper limit as claimed in the claims.

[0078] Experiment 2: Indoor Co-Toxicity Coefficient (CTC) Calculation; This experiment was conducted in accordance with NY / T 1154.7-2006 "Guidelines for Indoor Bioassays of Pesticides - Fungicides Part 7: Determination of Co-Toxicity Effects of Mixtures". The mycelial growth rate method was used to determine the inhibitory effects of Example 1 (containing 0.10% NMN), Example 2 (containing 0.05% NMN), Example 3 (containing 0.20% NMN), and Comparative Example 1 (without NMN) on Fusarium moniliforme ATCC 38932 (i.e., Fusarium moniliforme), the pathogen of rice seedling blight. The Wadley method was used to calculate the Co-Toxicity Coefficient (CTC).

[0079] The CTC calculation method is as follows: First, the CTC of each single dose is measured separately. (Half-maximum effective concentration, i.e., the concentration of the agent required to inhibit the mycelial growth of the tested pathogen by 50%, mg / L), then determine the mixture. Then calculate the theoretical value according to the Wadley formula. (Theoretical value) ,in , , , These represent the proportions of each component in the mixture (expressed as decimals, with the sum of the proportions of each component being 1). Equal to each single dose ; Final theoretical value and actual measured value CTC>120 indicates synergistic effect, 80≤CTC≤120 indicates additive effect, and CTC<80 indicates antagonistic effect.

[0080] Test strains: One susceptible strain each of *F. moniliforme*, the causal agent of rice seedling blight, and *Rhizoctonia cerealis*, the causal agent of wheat sheath blight. Culture medium: Potato dextrose agar (PDA, containing 200 g potato extract, 20 g glucose, and 15 g agar per liter). The test solutions were prepared by mixing acetone and water (9:1, v / v) with the formulations from each example to create a series of concentration gradients (5-7 concentrations in a geometric progression, with a common ratio of 2). Each treatment was repeated in triplicate. After culturing at 28°C for 5 days, the colony diameter was measured using the cross-section method with vernier calipers, and the inhibition rate was calculated. Probit analysis was performed using the DPS data processing system (or SPSS) to obtain the virulence regression equation and EC50. 50 Value. Experimental results are as follows: Figure 2 As shown in Table 3. Figure 2 (a) Regression curves of virulence of F. moniliforme for each treatment group (log concentration-probability value linear relationship). Figure 2(b) A bar chart of CTC values ​​for each treatment group (including error bars, n=3, with the horizontal dashed line marking the CTC=120 baseline).

[0081] Table 3. Results of indoor toxicity assays of *F. moniliforme* in each example and comparative example.

[0082]

[0083] From Table 3 and Figure 2 It can be seen that the EC of Example 1 (NMN 0.10%) 50 The concentration of NMN (0.142 mg / L) was significantly lower than that of Comparative Example 1 (0.185 mg / L), and the CTC increased from 130 to 148, indicating that the introduction of NMN significantly enhanced the combined inhibitory effect of the ternary main components on F. moniliforme. Compared with Comparative Example 2 (chitosan oligosaccharide, CTC=138) and Comparative Example 3 (SA, CTC=140), the CTC of Example 1 containing NMN was the highest, indicating that the synergistic effect of NMN in the ternary formulation is better than that of similar functional auxiliary components. This is a comprehensive result of the direct competitive inhibition of pathogenic NMNAT enzyme (A01P1 / 00 antimicrobial function) by NMN and the dual bactericidal synergy of triazole + pyrrole.

[0084] Experiment 3, Seed germination safety test; This experiment evaluates the safety of the formulation in Example 1 on the germination of rice and wheat seeds based on the basic principles of the national standard GB / T17980.68-2004 "Guidelines for Field Efficacy Tests of Pesticides (II) Part 68: Safety of Seed Treatment Agents for Crops".

[0085] Test seeds: rice (variety: Wuyunjing 27) and wheat (variety: Yangmai 25), with a seed purity ≥99% and a germination rate ≥95% (pre-calibrated). Five treatment doses were set: (1) water control (CK); (2) 0.5 times the recommended dose (0.5×); (3) 1 times the recommended dose (1×, 833mL / 100kg rice seeds, 860mL / 100kg wheat seeds); (4) 2 times the recommended dose (2×); (5) 5 times the recommended dose (5×). The preparation and seeds were mixed evenly in a seed coating machine (5g seeds / batch simulation) according to the specified ratio, dried, and placed in a petri dish (9cm in diameter) lined with double-layer filter paper, 50 seeds per dish, 3 dishes per treatment (replicated), and cultured in a constant temperature germination chamber at 25℃ (relative humidity 80%±5%), with an appropriate amount of distilled water added daily to keep the filter paper moist. Germination was counted on days 3, 5, and 7 (germination was defined as radicle length ≥ half the seed length), and germination rate (%) and germination potential (%, calculated as: number of germinated seeds on day 3 / total number of seeds tested × 100). On day 7, taproot length (mm, accuracy 1 mm) and seedling fresh weight (mg, accuracy 0.1 mg) were measured. The experimental results are as follows: Figure 3 As shown in Table 4. Figure 3 A comprehensive comparison chart of germination rate (line graph), taproot length and fresh weight (bar graph) of rice and wheat under different dosage treatments (2-row × 3-column sub-chart format, the first row is rice, the second row is wheat; the first column is germination rate, the second column is taproot length, and the third column is fresh weight).

[0086] Table 4. Effects of the formulation of Example 1 on the germination safety of rice and wheat at different dosages (day 7)

[0087]

[0088] From Table 4 and Figure 3 It can be seen that, at the recommended dose (1×), the germination rate (≥96%), taproot length, and fresh weight of the formulation in Example 1 were not lower than those of the water control in rice and wheat. Furthermore, at doses of 0.5× and 1×, the taproot length and fresh weight were slightly higher than those of the water control (rice taproot length was 47.8 and 48.5 mm vs. CK 45.2 mm, an increase of 5.8% and 7.3%, respectively), indicating that an appropriate amount of NMN (0.10%) has a certain effect on promoting seed germination and root growth. At 5 times the recommended dose, the germination rate decreased slightly but remained above 90%, meeting the pesticide formulation safety evaluation standard, i.e., the germination rate at 1 times the recommended dose is not lower than 90% of that of the water control. The formulation is safe for both rice and wheat without phytotoxicity.

[0089] Experiment 4: Dynamic monitoring of soil microorganisms; This experiment was conducted in accordance with the relevant principles of the national standard GB / T17993-2014 "Methods for determination of soil microbial biomass". A pot simulation experiment was used to evaluate the effects of the NMN-containing quaternary preparation of Example 1 and the ternary preparation of Comparative Example 1 on rhizosphere soil microorganisms and soil enzyme activity, and to verify the soil microecological restoration function of NMN (corresponding to the disinfection / antimicrobial and environmental remediation functions of IPCA01P1 / 00).

[0090] Test soil: Topsoil (0-20cm) from paddy fields in the suburbs of a city was taken, air-dried and sieved through a 2mm sieve. Organic matter content was 22.3g / kg, pH 6.5, and total nitrogen was 1.85g / kg. Test plant: Rice (Wuyunjing 27). Three treatments were set up: A (water-coated control), B (Comparative Example 1, ternary formulation coating), and C (Example 1, NMN-containing quaternary formulation coating). Six pots (25cm diameter, 1.5kg soil / pot) were used for each treatment and cultured in a 25℃ / 20℃ (day / night) light incubator, with water added as needed to 70% of field capacity. Rhizosphere soil samples were collected from each treatment at 7, 14, 21, and 30 days after sowing (the soil adhering to the roots within 1–2 mm after shaking off loose soil from the root surface). Three soil samples from each treatment were combined and divided into two aliquots: one aliquot for microbial counting (bacterial counting was performed using the dilution plating method on beef extract peptone medium, 28℃ / 48h; fungal counting was performed using the dilution plating method on Martin agar, 28℃ / 5d), with results expressed as CFU / g dry soil; the other aliquot was used for soil enzyme activity determination. Urease activity was determined using the indophenol blue colorimetric method (phenol-sodium hypochlorite method), with results expressed as μgNH3-N / (g·h); phosphatase activity was determined using the p-nitrophenyl phosphate colorimetric method, with results expressed as μgp-NPP / (g·h). Soil respiration rate was measured using the alkaline absorption method (NaOH sealed culture), with results expressed as mgCO2 / (100g soil·24h). Experimental results are as follows Figure 4 As shown in Table 5. Figure 4 Line graphs showing the changes in rhizosphere soil bacterial count, fungal count, soil respiration rate, and urease activity over time for each treatment (2-row × 2-column sub-graph format, with groups A, B, and C distinguished by different colored lines), with each sub-graph labeled (a), (b), (c), and (d).

[0091] Table 5 Effects of Example 1 and Comparative Example 1 on rhizosphere soil microorganisms and enzyme activity (30 days after sowing)

[0092]

[0093] From Table 5 and Figure 4It was found that, compared with Comparative Example 1 (ternary formulation group B), the rhizosphere soil bacterial count of Example 1 (group C) containing NMN increased by 75% on day 30 after sowing (5.46 vs 3.12 × 10⁻⁶). 6 The CFU / g of beneficial bacteria was associated with the proliferation of beneficial microorganisms promoted by NMN degradation products nicotinamide (NAM) and ribose-5-phosphate (R5P) as high-quality carbon and nitrogen sources; soil fungal counts decreased by 45% (12.6 vs 22.8 × 10⁻⁶). 3 The concentration of NMN (CFU / g) indicates that the fungi were mainly pathogenic fungi, while beneficial bacteria, after proliferation, inhibited pathogenic fungi by secreting antagonists such as chitinase, demonstrating the sustained effect of NMN's direct antimicrobial disinfection function at the soil level. Soil respiration rates (C>A>B) show that the overall metabolic activity of soil microorganisms was significantly enhanced after treatment with the NMN-containing preparation, while chemical pesticides alone (Group B) slightly inhibited soil respiration. Urease and phosphatase activities were significantly higher than in Comparative Example 1 (increased by 42.7% and 38.1%, respectively), demonstrating the environmental remediation function of NMN degradation products in participating in nitrogen and phosphorus cycling and enhancing soil enzyme activity. These results prove that Example 1, containing NMN, achieved chemical control, NMN supplementation of microbial carbon and nitrogen sources, beneficial microbial proliferation, biological antagonism of pathogens, and soil environmental remediation, exhibiting significant effects in rhizosphere soil microecological restoration and environmental governance.

[0094] Experiment 5: Verification of Prothioconazole Sensitivity Recovery in Resistant Strains; This experiment, based on the relevant principles of NY / T 1156.14-2014 "Guidelines for Indoor Bioassay Testing of Pesticides - Fungicides Part 14: Risk Assessment of Resistance", verifies the effect of NMN in partially restoring the susceptibility of prothioconazole-resistant strains to prothioconazole through epigenetic pathways.

[0095] Tested resistant strains: F. moniliforme prothioconazole field-resistant strains collected from rice-growing areas of Jiangsu Province (resistance index RI>5, RI=EC of resistant strain). 50 / Sensitive reference strain EC 50 The resistant strain was screened by continuous subculturing on PDA medium containing prothioconazole (concentration gradient gradually increasing, 0.5-1.0-2.0 mg / L, 5 days per generation). After 5 generations of screening, the RI was confirmed to be 8.3. Three treatments were set up: T1 (prothioconazole only, without NMN); T2 (prothioconazole + 0.10% NMN pretreatment, i.e., the resistant strain was inoculated on PDA medium containing 0.10% NMN and cultured for 72 h, then transferred to medium containing prothioconazole for EC measurement). 50 T3 (prothiophanate-methyl + equal volume of solvent, negative control). The EC50 of prothiophanate-methyl for each treatment on resistant strains was determined. 50The differences between T1 and T2 were compared. Total RNA was extracted from mycelia after each treatment culture, and the relative expression level of the CYP51 gene was determined by real-time quantitative PCR (RT-qPCR, referring to the relevant methods in NY / T 3589-2020 "Plant Quarantine"). The experiment was repeated three times, and the data were analyzed using SPSS 22.0 for one-way ANOVA and LSD multiple comparisons (significance level p<0.05). The experimental results are shown in Table 6.

[0096] Table 6. Effects of NMN pretreatment on prothioconazole-resistant strain EC 50 Effects on CYP51 gene expression

[0097]

[0098] Note: * indicates a significant difference (p<0.05); ns indicates a non-significant difference (p>0.05).

[0099] Table 6 shows that NMN pretreatment for 72 hours (T2) reduced the EC50 of resistant strains to prothioconazole. 50 The concentration decreased significantly from 1.534 mg / L to 1.106 mg / L (a decrease of 27.9%, p<0.05), while the relative expression level of the CYP51 gene was downregulated by approximately 2.4-fold (log2 change -1.28), which was significantly different from T1 (only prothioconazole was used, and CYP51 overexpression remained unchanged); the T3 solvent control showed no significant difference from T1, ruling out the solvent effect. These results are from EC... 50 Confirmed from both gene expression and NAD, NMN works through NAD. + The Sirtuin pathway downregulated the overexpression of the CYP51 gene in resistant strains, partially restoring their sensitivity to prothioconazole.

[0100] Experiment 6, Field Efficacy Verification, Taking Wheat Sheath Blight / Aphids as an Example; This experiment was conducted in Hanjiang District, Yangzhou City, Jiangsu Province, according to the agricultural industry standards NY / T1464.4-2007 "Field Efficacy Test Guidelines for Pesticides - Fungicides for the Control of Wheat Sheath Blight" and NY / T1464.34-2010 "Field Efficacy Test Guidelines for Pesticides - Insecticides for the Control of Wheat Aphids". The field efficacy test was carried out to evaluate the control effect of the NMN-containing preparation in Example 1 on wheat sheath blight and wheat aphids.

[0101] Test crop: Wheat (Yangmai 25). Treatment design: (1) Water control; (2) Comparative Example 1 formulation (ternary, NMN-free), dosage 860 mL / 100 kg seeds; (3) Example 1 formulation (containing 0.10% NMN), dosage 860 mL / 100 kg seeds; (4) Commercially available control drug (Hailier Pharmaceutical, propoxur·fludioxonil·thiamethoxam FS, 3%+1.5%+25%), dosage at the lower limit of the recommended dose. Each treatment plot area was 30 m². 2 Repeat 3 times, randomized block design, mechanically coat seeds before sowing, and sow after drying.

[0102] Wheat sheath blight survey: Surveys were conducted twice, once at the jointing stage (approximately 60 days after sowing) and once at the booting stage (approximately 90 days after sowing). Five points were sampled diagonally across each plot, with 20 plants surveyed at each point (totaling 100 plants). The number of infected plants and the disease severity (0–5) were recorded. The disease index (DI) and control efficacy (FE) were calculated: FE (%) = (1 - DI of treatment area / DI of control area) × 100. Aphid survey: Surveys were conducted three times, at 30, 45, and 60 days after sowing. Five points were sampled per plot, with 10 plants at each point. The number of aphids per plant was counted, and the aphid population reduction rate was calculated. The experimental results are shown in Table 7.

[0103] Table 7. Field efficacy comparison between Example 1 and Comparative Example 1 (wheat, surveyed 60 days after sowing).

[0104]

[0105] As shown in Table 7, Example 1 (containing NMN, 0.10%) showed significantly better efficacy against wheat sheath blight (68.8%) than Comparative Example 1 (55.9%, difference 12.9 percentage points, p<0.05), and was also better than commercially available drugs of the same type (57.5%). Its efficacy against wheat aphids (90.1%) was also better than Comparative Example 1 (84.6%). The improved efficacy of Example 1 is related to the following combined effects of NMN: (1) NMN directly inhibits soil pathogens (NMNAT competition, A01P1 / 00 function) to reduce the amount of initial infecting pathogens; (2) NMN activates plant SA-SAR, and continues to provide biological immune protection after the efficacy of chemical components declines (the effective period of prothioconazole ends about 25 days after sowing), thus prolonging the effective protection period; (3) NMN and thiamethoxam have a synergistic effect, which further improves the efficacy against aphids.

[0106] In summary, the seed treatment suspension containing β-nicotinamide mononucleotide designed in this invention, namely, prothioconazole·fludioxonil·thiamethoxam, achieves the comprehensive effects of optimized and improved suspension stability of the formulation, enhanced direct antimicrobial efficacy against pathogens, synergistic effect of plant SAR immune activation and chemical control, overcoming prothioconazole resistance, and restoration of rhizosphere soil microecology.

[0107] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A seed treatment suspension of prothioconazole·fludioxonil·thiamethoxam, characterized in that, It consists of the following components in parts by weight: 10-20 parts of prothioconazole; 3-10 parts of fludioxonil; Thiamethoxam 60-100 parts; 0.5–1.5 parts of β-nicotinamide mononucleotide; 8-20 parts of sodium salt of styrene-maleic anhydride copolymer; 4-15 parts of fatty alcohol polyoxyethylene ether; Xanthan gum 0.8–2.5 parts; Magnesium aluminum silicate 2.5–6.0 parts; Glycerol 25-55 parts; Polydimethylsiloxane defoamer, 0.8–4.0 parts; Sodium benzoate 0.8–2.5 parts; 4-15 parts of polyvinyl alcohol; Deionized water balance.

2. The seed treatment suspension according to claim 1, characterized in that, The weight parts of each component are as follows: 15 parts of prothioconazole; 5 parts of fludioxonil; 80 parts of thiamethoxam; 0.8 parts of β-nicotinamide mononucleotide; Sodium salt of styrene-maleic anhydride copolymer: 12 parts; fatty alcohol polyoxyethylene ether: 8 parts; xanthan gum: 1.6 parts; Magnesium aluminum silicate 4.0 parts; glycerol 40 parts; polydimethylsiloxane defoamer 2.4 parts; Sodium benzoate 1.6 parts; polyvinyl alcohol 8 parts; deionized water balance.

3. A method for preparing the seed treatment suspension according to any one of claims 1 or 2, characterized in that, Includes the following steps: S1, β-nicotinamide mononucleotide is pre-dissolved in deionized water to prepare an aqueous solution for later use; S2, dispersant, wetting agent, suspending agent, antifreeze, defoamer, preservative, and three active ingredients are sequentially added to deionized water. After high-speed shear dispersion, the mixture is transferred to a sand mill for grinding. The material temperature is controlled to not exceed 35℃ during the grinding process until the formulation particle size D is reached. 90 No larger than 3 micrometers; S3. After the temperature of the grinding suspension obtained in S2 drops below 25°C, slowly pump the β-nicotinamide mononucleotide solution obtained in S1 into the solution under low-speed stirring. After the pumping is completed, continue stirring to confirm that the absolute value of the Zeta potential of the formulation is not lower than 28mV. S4, add film-forming agent solution and water, stir evenly to obtain the finished product.

4. The method according to claim 3, characterized in that, The specific implementation process of S1 is as follows: S11, weigh β-nicotinamide mononucleotide, add it to deionized water at 25℃ at a mass ratio of 1:49, stir until completely dissolved, and prepare an aqueous solution with a mass fraction of 2%; S12, adjust the pH of the above solution to 5.5-6.0 with 0.1 mol / L citric acid solution, filter through a 0.45 micrometer microfiltration membrane, seal the filtrate in a brown bottle, and store it at 25°C in the dark for no more than 24 hours before use.

5. The method according to claim 4, characterized in that, The specific implementation process of S2 is as follows: S21. Add deionized water (65% of the total water volume) to a container, then add sodium salt of styrene-maleic anhydride copolymer and fatty alcohol polyoxyethylene ether in sequence, stirring until completely dissolved. Next, add magnesium aluminum silicate and xanthan gum in sequence, stirring until no lumps remain. Add glycerol, polydimethylsiloxane defoamer, and sodium benzoate, stirring until homogeneous. Add the three technical materials in the order of thiamethoxam, prothioconazole, and fludioxonil. After each material is added, stir at 500 rpm for 1 minute to wet it, then increase the speed to 3000-5000 rpm and shear for 3 minutes. After all three materials are added, continuously shear at the highest speed for 10 minutes to obtain the particle size D of the technical material. 90 Coarse dispersions no larger than 50 micrometers; S22, the coarse dispersion obtained in S21 is transferred to a horizontal sand mill, and zirconia beads with a diameter of 0.8-1.0 mm and a purity of not less than 95% are added, with a zirconia bead volume filling rate of 60%-65%; the material temperature in the grinding chamber is controlled to not exceed 35℃ by jacket cooling water, and sand milling is performed in a circulating manner; the particle size D is measured every 30 minutes. 90 When D 90 Stop grinding when the particle size is no larger than 3 micrometers; clean the grinding chamber with 5% of the total water volume of deionized water, and add the cleaning solution to the grinding suspension.

6. The method according to claim 4, characterized in that, The specific implementation process of S3 is as follows: S31, transfer the grinding suspension into a jacketed cooling stainless steel mixing tank, turn on the jacket cooling water, and wait for the material temperature to drop below 25℃. S32, with the impeller speed at 150-200 rpm, slowly pump the β-nicotinamide mononucleotide aqueous solution into the system using a peristaltic pump at a rate not exceeding 2% of the total system volume per minute, for a pumping time of not less than 30 minutes; after the pumping is completed, continue stirring for 20 minutes. S33, sample and measure the Zeta potential of the system. If the absolute value of the Zeta potential is not less than 28mV, this step is complete. If the absolute value of the Zeta potential is less than 28mV, continue stirring at low speed for 10 minutes and then measure again until the absolute value of the Zeta potential is not less than 28mV.

7. The method according to claim 4, characterized in that, The specific implementation process of S4 is as follows: S41, slowly sprinkle polyvinyl alcohol powder into hot water at 85-90℃ to prepare a 10% polyvinyl alcohol solution by mass, and cool it to below 40℃ for later use. S42, under the condition of stirring paddle speed of 150-200 rpm, slowly add the polyvinyl alcohol solution obtained in S41 to the material obtained in S3, and continue stirring for 15 minutes after the addition is complete; add deionized water to make up to the specified total weight, stir at 150 rpm for 5 minutes, mix evenly, and the finished product is obtained.

8. The application of the seed treatment suspension as described in any one of claims 1-7 in the prevention and control of seedling diseases and pests in rice and wheat, characterized in that, Seed coating is used to treat rice or wheat seeds to prevent rice bakanae disease and rice thrips, as well as wheat sheath blight and wheat aphids.

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