A seed coating agent containing a complex enzyme glycoside and a preparation method and application thereof

Abstract

This invention discloses a seed coating agent containing complex enzyme glycosides, its preparation method, and its application. The seed coating agent comprises, by weight, 1-5 parts of complex enzyme glycosides, 0.5-1.5 parts of copper citrate, and an auxiliary agent, including 2-8 parts of a film-forming agent. This invention uses complex enzyme glycosides as the main active ingredient in the seed coating agent, forming a dynamically reversible complex system with copper citrate for synergistic effects. This constructs a non-hormonal endogenous activation system, overcoming the technical prejudice that "copper salts easily lead to the inactivation of glycosides," thus enhancing seed germination ability, disease resistance, and stress resistance, achieving full-cycle empowerment of seeds from germination to emergence, robust seedling growth, disease resistance, stress resistance, and increased yield.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a seed coating agent containing complex enzyme glycosides, its preparation method, and its application. Background Technology

[0002] Seed coating is a core technology in modern agriculture that ensures full seedling emergence, controls seedling diseases, pests and weeds, and solidifies the yield base. It has been widely used in field and cash crop cultivation around the world. Currently, seed coating agents on the market are mainly divided into three categories: The first category is conventional chemical seed coating agents with chemical fungicides and insecticides as the core, whose core function is to control seed-borne and soil-borne diseases. However, they have prominent defects such as excessive pesticide residues, soil non-point source pollution, rapid increase in pesticide resistance of diseases and pests, and excessive seedling growth. Moreover, they are prone to phytotoxicity and inhibit seed germination under low temperature stress. The second category is microbial inoculant seed coating agents, with Bacillus and Trichoderma as the core functional ingredients. Although they are green and safe, they have problems such as poor storage stability, easy inactivation when combined with chemical pesticides, unstable field colonization effect, and limited growth-promoting effect. The third category is growth-promoting seed coating agents with added exogenous plant growth regulators, mostly with gibberellin, brassinolide, sodium nitrophenolate, etc. Although they can improve the germination rate in the short term, they are very likely to cause excessive above-ground growth of seedlings, poor root development, increased risk of lodging, and ineffectiveness of the drug under low temperature stress, which seriously threaten the stable yield of crops.

[0003] Biostimulants are a current research hotspot in the field of green agriculture. Among them, glycosides have the advantages of endogenous growth promotion, broad-spectrum stress resistance, full biodegradability, and no residue, making them a core potential material to replace exogenous hormones and highly toxic pesticides. However, there are still three major technical bottlenecks in the existing technology: First, most of the existing glycoside active substances are extracted from plants, resulting in high production costs, poor batch stability, and limited large-scale application. Second, single glycoside seed coating agents can only induce basic crop resistance through exogenous signals and cannot deeply activate the decomposition and utilization of endogenous stored substances in seeds. The effects of promoting germination, seedling growth, and root development are limited, making it difficult to achieve full-cycle empowerment from seed germination to seedling growth. Third, most existing biological seed coating agents are physical superpositions of single functional components, failing to achieve stable compatibility between glycosides and coating film-forming systems. When combined with conventional insecticides and fungicides, their activity is easily reduced, and their storage stability is poor. They cannot simultaneously solve the integrated production needs of "germination empowerment, root growth promotion, stress protection, and stable and increased yield." Furthermore, there are no reports on constructing non-hormonal, highly stable biologically active seed coating agents using complex enzyme glycosides derived from marine microbial metabolism as the core active ingredient. Glycosides produced by marine microbial metabolism are an important branch of biostimulants, and their molecular structures generally exhibit three typical characteristics: first, the glycosyl moiety contains multiple cis-ortho-hydroxyl groups, which are core sites for coordination with metal ions; second, the aglycone moiety often contains polar groups such as carboxyl and phenolic hydroxyl groups, which can form intermolecular hydrogen bonds; and third, the terminal end of the sugar chain retains a reducing hemiacetal hydroxyl group, exhibiting weak redox activity. These structural characteristics form the molecular basis for their endogenous growth-promoting and broad-spectrum stress-resistant effects, and have been confirmed in numerous published studies.

[0004] Furthermore, major grain-producing areas in northern my country generally face adverse environmental stresses such as late spring frosts, drought, and salinity. Existing seed coating agents achieve a germination rate of less than 70% under these conditions, resulting in severe seedling gaps and row breaks, which has become a core bottleneck restricting stable and increased crop yields. Therefore, developing a seed coating agent that does not rely on exogenous hormones, has stable activity, combines active endogenous enhancement with passive stress resistance, is fully biodegradable, and has broad-spectrum applicability is a pressing technical challenge in current agricultural production, possessing significant practical importance and industrial application value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a seed coating agent containing complex enzyme glycosides, its preparation method, and its application, solving problems such as high phytotoxicity, limited growth-promoting and stress-resistance effects, poor adaptability to stress environments, and environmental pollution associated with existing seed coating agents. The seed coating agent of this invention uses complex enzyme glycosides as its core component, with copper citrate and complex enzyme glycosides working synergistically. It eliminates the need for exogenous hormones and enhances seed germination and stress resistance by activating endogenous crop metabolism.

[0006] The specific technical solution is as follows:

[0007] One objective of this invention is to provide a seed coating agent containing complex enzyme glycosides, wherein the seed coating agent comprises, by weight, 1-5 parts of complex enzyme glycosides, 0.5-1.5 parts of copper citrate, and an auxiliary agent, wherein the auxiliary agent comprises 2-8 parts of a film-forming agent.

[0008] The complex enzyme glycoside mentioned above can be purchased from Yantai Goodlife Biotechnology Co., Ltd. It is a multi-active complex produced by marine microbial metabolism and has a significant promoting effect on seed germination rate.

[0009] The mechanism of this invention is as follows: A dynamic and reversible complex system is formed between the complex enzyme glycoside and copper citrate through specific interactions between their groups. On one hand, the cis-ortho-hydroxyl groups of the complex enzyme glycoside interact with the Cu in copper citrate. 2+ The dynamic ternary coordination complexation of the complex enzyme glycoside; on the other hand, the polar aglycone group of the complex enzyme glycoside forms intermolecular hydrogen bond association with the citrate group; and the reducing hemiacetal hydroxyl group of the complex enzyme glycoside interacts with Cu. 2+ The valence state stability and redox buffering effect of copper salts are achieved. Based on the interaction of their functional groups, a synergistic effect is realized, enhancing seed germination ability, disease resistance, and stress tolerance. Furthermore, it breaks the technical prejudice that "copper salts easily lead to the inactivation of glycosides." Details are as follows:

[0010] (1) The cis-ortho polyhydroxy groups of complex enzyme glycosides and Cu in copper citrate 2+ The dynamic ternary coordination complexation is the core molecular basis for the synergistic effect between the two. Copper citrate itself is a complex of citric acid and Cu. 2+ The resulting stable chelate, with the cis-ortho-hydroxyl group (ortho-diol structure) of the complex enzyme glycoside glycosyl group interacting with Cu. 2+ Its coordination stability constant lgK can reach 10.8, allowing it to form a competitive dynamic coordination equilibrium with citrate ions, ultimately forming citrate-Cu. 2+ - A ternary bridged coordination complex of complex enzyme glycosides.

[0011] The ternary coordination structure can completely immobilize free Cu in the system. 2+ To fundamentally eliminate free Cu 2+ The strong oxidizing properties of copper citrate destroy the glycosidic bonds and active hydroxyl groups of complex enzyme glycosides, while avoiding the sedimentation and flocculation problems associated with copper citrate. This coordination interaction is reversible and does not destroy the core active structure of complex enzyme glycosides; instead, it locks in their active conformation, effectively solving the long-standing problem in this field that "the combination of copper salts and glycoside biostimulants easily leads to activity decay," maintaining stability under sealed storage conditions. During seed germination, the organic acids secreted by the roots and the moisture in the soil disrupt the ternary coordination equilibrium, causing Cu... 2+It achieves synchronous and slow release with complex enzyme glycosides. This characteristic avoids seed burning and bud-suppressing damage caused by the burst release of copper ions, while ensuring the stable release of complex enzyme glycoside activity throughout the entire seed germination cycle. This mechanism of action has been confirmed by boric acid complexation reaction and the ultraviolet-red shift phenomenon of cis-ortho-hydroxyl groups. Its direct effect is that it can increase the activity retention rate of complex enzyme glycosides from 74.2% to over 89.6% after 12 months of sealed storage at room temperature.

[0012] (2) The carboxyl and phenolic hydroxyl groups at the aglycone end of the complex enzyme glycoside can form high-density intermolecular hydrogen bonds with the carboxyl and hydroxyl groups of the citrate group, thereby constructing a complex enzyme glycoside-citrate association system. On the one hand, it can improve dispersibility and film-forming compatibility. After hydrogen bonding, the strong hydrophilic group of the citrate group provides hydrophilic side chains for the complex enzyme glycoside, significantly improving the dispersibility of the complex enzyme glycoside in the aqueous coating system, reducing its aggregation and sedimentation, and reducing the filtration loss of active ingredients during the preparation process. On the other hand, it helps to release active ingredients synchronously: the hydrogen bonding binding effect binds the release behavior of the complex enzyme glycoside and copper citrate, completely solving the problem in the existing technology that "the biostimulant is released quickly, the copper salt is released slowly, the two have mismatched action cycles, and the synergistic effect is extremely poor", ensuring that the two always maintain an effective synergistic concentration in the rhizosphere microenvironment throughout the entire cycle from seed imbibition and germination to seedling establishment, achieving full-cycle empowerment. The mechanism of action has been confirmed by ferric chloride colorimetric reaction, potentiometric titration and UV blue shift of phenolic hydroxyl / carboxyl groups. Its direct effect is to improve the dispersibility of complex enzyme glycosides in aqueous coating systems by more than 30%, with no agglomeration or sedimentation, and the filtration loss rate of active ingredients during preparation is less than 5%.

[0013] (3) The reducing hemiacetal hydroxyl group of the complex enzyme glycoside has weak reducing properties, which can reduce the small amount of free strong oxidizing Cu in the system. 2+ Partially reduced to Cu + Cu is formed in the system + / Cu 2+ Redox buffer pair. On the one hand, it can further improve storage stability: it completely eliminates free Cu. 2+ The system inhibits the oxidative degradation of the active groups of complex enzyme glycosides. Experimental results show that this buffer system can increase the activity retention rate of complex enzyme glycosides after 12 months of sealed storage at room temperature from 74.2% (without copper citrate) to over 89.6%, overcoming the technical prejudice in this field that "copper salts cause inactivation of glycosides." Furthermore, it amplifies the synergistic effect of stress resistance: the active center of the core antioxidant enzyme Cu / Zn-SOD in plants is entirely dependent on Cu. + / Cu 2+The valence cycle of the buffer pair enables the scavenging of reactive oxygen species. This buffer directly activates and stabilizes the active conformation of Cu / Zn-SOD, forming a precise synergistic effect with the stress-inducing effect of the complex enzyme glycoside. This is the core molecular mechanism by which the synergistic effect under stress far exceeds expectations. This mechanism of action has been confirmed by the Fehling reaction and a new charge transfer absorption peak at 330 nm. Its direct effect is that the germination rate of wheat seeds under low temperature stress can be increased from 82.0% to over 96.3%.

[0014] (4) Through synergistic action, the activation of endogenous hydrolytic enzymes can be synergistically amplified, completely avoiding the risks of exogenous hormones and achieving a multiplier effect in germination empowerment. As an endogenous signaling molecule, complex enzyme glycosides upregulate the gene expression of endogenous α-amylase, protease, lipase and other hydrolytic enzymes in seeds, promote the decomposition of macromolecular nutrients such as starch and protein stored in seeds, provide energy for germination, and do not depend on exogenous hormones throughout the process. Complex enzyme glycosides solve the core problem of insufficient hydrolytic enzyme synthesis, while copper citrate solves the problems of low catalytic efficiency and insufficient energy supply of hydrolytic enzymes from two dimensions. One is the slow-release Cu 2+ As an essential metal cofactor for α-amylase and protease, it can directly coordinate with histidine residues in the active site of hydrolases, stabilizing the active conformation of the enzyme and increasing the catalytic efficiency of hydrolases by more than 35%. Secondly, citrate can directly enter the tricarboxylic acid cycle of embryonic cells, rapidly increasing ATP synthesis efficiency and providing sufficient energy for the synthesis and secretion of hydrolases, completely solving the problems of insufficient seed energy metabolism and low hydrolases activity under low temperature stress. The two form a complete closed loop of "gene expression upregulation - enzyme activity catalytic amplification - continuous energy supply", which does not rely on any exogenous plant growth regulators throughout the process, fundamentally avoiding the risks of excessive growth, deformed seedlings, and lodging caused by exogenous hormones.

[0015] (5) Complex enzyme glycosides can only enhance the crop's own resistance, but their direct killing ability against seed-borne and soil-borne pathogens is insufficient, requiring additional chemical fungicides. In terms of external protection: Copper citrate slow-release Cu 2+ It can directly disrupt the cell membrane integrity of pathogens, inhibit nucleic acid and protein synthesis, and has a broad-spectrum killing effect on both fungal and bacterial diseases, reducing the pathogen infection base. The complex enzyme glycosides, aided by hydrogen bonds in the citrate ion, are more quickly absorbed by the roots and enter the plant, upregulating salicylic acid (SA) and jasmonic acid (JA) resistance signaling pathways, inducing the synthesis of pathogenesis-related proteins (PR proteins) and phytoalexins, and enhancing crop systemic acquired resistance (SAR); simultaneously, Cu... + / Cu 2+The buffer directly activates the activity of antioxidant enzymes such as Cu / Zn-SOD and POD, scavenging reactive oxygen species produced by pathogen infection and reducing cell damage. Copper citrate reduces pathogen infection pressure, creating a safe environment for the endogenous activation of complex enzyme glycosides. The lignin synthesis induced by complex enzyme glycosides can reduce the excessive accumulation of copper ions in plants, avoiding copper toxicity, while enhancing the physical barrier effect of crops against pathogens. This completely solves the dual problems of "contradiction between efficacy and phytotoxicity" when using copper salts alone and "lack of direct bactericidal ability" when using complex enzyme glycosides alone.

[0016] (6) Under adverse conditions such as low temperature, drought, and salinity, core damage in crops results from cell membrane lipid peroxidation and cell necrosis caused by excessive accumulation of reactive oxygen species. Using complex enzyme glycosides alone can only upregulate the gene expression of antioxidant enzymes and increase enzyme synthesis; using copper citrate alone can only provide Cu. 2+ The cofactor alone cannot solve the problem of insufficient enzyme synthesis. The two work synergistically to achieve a dual improvement in both "antioxidant enzyme synthesis amount + catalytic efficiency": the complex enzyme glycoside upregulates the gene expression of antioxidant enzymes such as Cu / Zn-SOD and POD, increasing the total amount of enzyme synthesis; copper citrate provides Cu... 2+ As a core cofactor, it directly enhances the catalytic efficiency of antioxidant enzymes, and the two work synergistically to increase the scavenging efficiency of reactive oxygen species by more than 40%. Simultaneously, citrate can chelate Na in saline-alkali soils. + Excessive Mn 2+ It eliminates harmful ions, reduces the toxicity of salt ions, and works synergistically with the osmotic regulation ability of complex enzyme glycosides, making it perfectly suited for adverse production scenarios.

[0017] The above technical solution can also be improved as follows:

[0018] Furthermore, the film-forming agent is at least one of hydroxypropyl starch ether, polyvinyl alcohol, chitosan, and gum arabic.

[0019] Furthermore, the film-forming agent is a mixture of hydroxypropyl starch ether and chitosan. The preferred mass ratio of hydroxypropyl starch ether to chitosan is (2~5):1. This compound system forms a uniform and continuous film with good air permeability and water retention. It can be completely and naturally degraded in the soil without affecting seed respiration. At the same time, it has a microencapsulation protection effect on complex enzyme glycosides, further improving the storage stability of enzyme activity. Meanwhile, the complex enzyme glycoside-citrate association system constructed by hydrogen bonds can form hydrogen bond crosslinks with the chitosan-hydroxypropyl starch ether film-forming system, improving the density and water resistance of the coating film, and better adapting to the application requirements of mechanized precision sowing.

[0020] Furthermore, the auxiliary agent preferably includes at least one of the following: dispersant, humectant, warning color, and preservative.

[0021] The dispersant is at least one selected from sodium lignosulfonate, polycarboxylate, and sodium dodecylbenzenesulfonate. The preferred amount of dispersant is 0.5 to 3 parts by weight.

[0022] The moisturizer is preferably at least one of glycerin, propylene glycol, and sorbitol. The moisturizer is preferably 1 to 5 parts by weight.

[0023] The warning color is preferably food-grade carmine red and / or brilliant blue. The warning color is preferably 0.1 to 1 part by weight.

[0024] The preservative is Kathon and / or sodium benzoate. The preservative is preferably 0.05 to 0.5 parts by weight.

[0025] Furthermore, the seed coating agent further comprises 70-100 parts by weight of water. The water is preferably deionized water.

[0026] Furthermore, the seed coating agent preferably also includes a functional adjuvant, which is at least one of a fungicide, insecticide, and micronutrient fertilizer, and can be flexibly compounded according to the crop variety and the occurrence patterns of pests and diseases in the field. The functional adjuvant is preferably 0.5 to 15 parts by weight.

[0027] The bactericide is preferably difenoconazole or / and fludioxonil.

[0028] The preferred insecticide is thiamethoxam.

[0029] A second objective of this invention is to provide a method for preparing the above-mentioned seed coating agent, comprising the following steps:

[0030] S1. Mix copper citrate and auxiliary agents other than film-forming agents with 40-50 parts by weight of water to obtain a basic premix;

[0031] S2. Add the complex enzyme glycosides to the basic premixed solution obtained in step S1, control the temperature at 40~50℃, stir for 20~40min to obtain the complex enzyme glycoside dispersion;

[0032] S3. Heat 30-50 parts by weight of water to 50-60°C, add film-forming agent and stir to dissolve, then cool to 30-45°C to obtain film-forming agent mixture; add film-forming agent mixture to complex enzyme glycoside dispersion obtained in step S2, maintain temperature at 30-45°C and stir for 30-60 min;

[0033] S4. Homogenize the mixture obtained in step S3 and filter it through a 100-200 mesh filter to obtain the seed coating agent.

[0034] Specifically, in step S1: copper citrate is first added to 40-50 parts by weight of water and stirred for 10-15 minutes to obtain an aqueous solution of copper citrate; the auxiliary agents other than the film-forming agent are added to the aqueous solution of copper citrate and stirred for 15-20 minutes to mix evenly to obtain a basic premix.

[0035] Specifically, in step S1, the stirring speed is preferably 150~200 rpm.

[0036] Specifically, in step S2, the stirring speed is preferably 100~300 rpm.

[0037] Specifically, in step S3, the stirring speed is preferably 100~300 rpm.

[0038] Specifically, in step S4: it is preferable to add the film-forming agent mixture to the complex enzyme glycoside dispersion while it is being continuously stirred.

[0039] Specifically, if the seed coating agent contains a working adjuvant, in step S3: after adding the film-forming agent mixture to the complex enzyme glycoside dispersion and stirring, add the functional adjuvant and stir evenly.

[0040] Specifically, in step S4, the homogenization is preferably low-pressure homogenization. The homogenization pressure is preferably 1.2~1.8 MPa.

[0041] A third objective of this invention is to provide the application of the aforementioned seed coating agent in the treatment of crop seeds. The seed coating agent is used to treat crop seeds through a coating process, with the mass ratio (agent-to-seed ratio) of the seed coating agent to the crop seed being 1:(50~200). The crop seeds treated by the coating process are then used for sowing and seedling cultivation of food crops, cash crops, and vegetable crops.

[0042] Furthermore, the crop is preferably wheat, corn, rice, peanut, soybean, cotton, tomato, cucumber, or pepper.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] This invention uses complex enzyme glycosides as the main active ingredient in seed coating agents, forming a dynamic and reversible complex system with copper citrate to synergistically activate a non-hormonal endogenous activation system. This breaks the technical prejudice that "copper salts easily lead to the inactivation of glycosides," enhances the germination ability of seeds as well as their disease resistance and stress resistance, and realizes full-cycle empowerment of seeds from "germination to emergence to strong seedlings to disease resistance to stress resistance to increased yield."

[0045] (1) This invention can improve the storage stability of seed coating agents and achieve simultaneous sustained release of active ingredients. The complex enzyme glycosides cis-ortho-hydroxyl groups react with Cu in copper citrate. 2+The dynamic ternary coordination complexation effectively solves the long-standing problem in this field of "the easy decline in activity due to the combination of copper salts and glycoside biostimulants," maintaining stability under sealed storage conditions. During seed germination, the organic acids secreted by the roots and the moisture in the soil disrupt the ternary coordination balance, causing Cu... 2+ Synchronous and slow release with complex enzyme glycosides. The complex enzyme glycoside-citrate association system constructed by hydrogen bonds improves dispersibility and film-forming compatibility, and facilitates the synchronous release of active ingredients; Cu formed in the system + / Cu 2+ Redox buffers further enhance storage stability, achieving a true dual effect of "stable activation during storage and simultaneous slow release in the field." Simultaneously, it overcomes the technical prejudice that "copper salts easily lead to the inactivation of glycosides." Furthermore, by combining "low-temperature, low-speed stabilization and dispersion + microencapsulation protection with film-forming agents," it solves the core bottlenecks of glycoside active substances being easily degraded by heat in coating systems, having poor compatibility with chemical pesticides, and having short shelf lives. The coating agent of this invention, after 12 months of sealed storage at room temperature, retains ≥89.6% of the active ingredients in the complex enzyme glycosides, far exceeding the less than 60% retention rate of bioactive ingredients after 6 months of storage in existing technologies, fully meeting the needs of industrial production, long-distance distribution, and year-round field application.

[0046] (2) It can achieve synergistic amplification of endogenous hydrolytic enzyme activation, completely avoid the risks of exogenous hormones, and achieve a multiplier effect in germination empowerment. Complex enzyme glycosides can upregulate the gene expression of hydrolytic enzymes; while copper citrate, as an essential metal cofactor for α-amylase and protease, enhances the catalytic efficiency of hydrolytic enzymes; at the same time, citrate ions can directly enter the tricarboxylic acid cycle of embryonic cells, rapidly increasing ATP synthesis efficiency and providing sufficient energy for the synthesis and secretion of hydrolytic enzymes. The two form a complete closed loop of "gene expression upregulation - enzyme activity catalytic amplification - continuous energy supply", which does not rely on any exogenous plant growth regulators throughout the process, thus avoiding the risks of excessive growth, deformed seedlings, and lodging caused by exogenous hormones from the root. The seed coating agent of this invention significantly improves seed germination potential, germination rate, and seedling uniformity, and promotes explosive root growth.

[0047] (3) Enhancing disease resistance through synergistic effects. Complex enzyme glycosides are absorbed more quickly into the plant through the roots via hydrogen bonds in the citrate group, upregulating the salicylic acid (SA) and jasmonic acid (JA) resistance signaling pathways, inducing the synthesis of pathogenesis-related proteins (PR proteins) and phytoalexins, and enhancing crop systemic acquired resistance (SAR). Simultaneously, Cu... + / Cu 2+The buffer directly activates the activity of antioxidant enzymes such as Cu / Zn-SOD and POD, clearing reactive oxygen species produced by pathogen infection and reducing cell damage. Furthermore, it synergistically avoids copper toxicity. This synergistic effect resolves the dual problems of "contradictory efficacy and phytotoxicity" when using copper salts alone, and "lack of direct bactericidal ability" when using complex enzyme glycosides alone.

[0048] (4) Enhances stress resistance through synergistic effects. Complex enzyme glycosides and copper citrate form Cu in the system. + / Cu 2+ The redox buffer pair can directly activate and stabilize the active conformation of Cu / Zn-SOD, forming a precise synergistic effect with the stress-inducing effect of complex enzyme glycosides, achieving a dual improvement in "antioxidant enzyme synthesis amount + catalytic efficiency". Simultaneously, citrate can chelate Na in saline-alkali soils. + Excessive Mn 2+ It removes harmful ions, reduces salt ion toxicity, and works synergistically with the osmotic regulation ability of complex enzyme glycosides. The seed coating agent of this invention significantly improves the crop's tolerance to adverse conditions such as low temperature, drought, salinity, and continuous cropping.

[0049] (5) The seed coating agent of this invention has strong industrial applicability, broad spectrum compatibility, simple operation, and significant yield-increasing effect, and has extremely high promotion value. The coating agent has the ability to promote strong seedling growth and prevent diseases and pests, reducing the use of pesticides and topdressing labor during the seedling stage, and achieving a double reduction in chemical fertilizers and pesticides. The coating agent is fully biodegradable, green and residue-free, and suitable for mechanized precision sowing, which reduces production costs, improves field application compatibility, and achieves both environmental protection and efficiency improvement. Detailed Implementation

[0050] The embodiments of the present invention will be described in further detail below with reference to the examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] In the specific implementation method: the complex enzyme glycosides used are all marine microbial-derived complex enzyme glycosides produced by Yantai Goodlife Biotechnology Co., Ltd., which are multi-component glycoside complexes of marine Pseudomonas fermentation metabolism.

[0052] Example 1

[0053] A seed coating agent containing complex enzyme glycosides, comprising the following components by weight:

[0054] The ingredients include 4.5 parts of complex enzyme glycoside, 1 part of copper citrate, 6 parts of hydroxypropyl starch ether, 2 parts of chitosan, 3 parts of sodium lignosulfonate, 5 parts of glycerol, 1 part of food-grade brilliant blue, 0.5 parts of Kathon, and 77 parts of deionized water.

[0055] The preparation steps of the seed coating agent are as follows:

[0056] S1. At room temperature, copper citrate was added to 43 parts by weight of deionized water and stirred at 180 rpm for 12 min to obtain an aqueous solution of copper citrate; at room temperature, sodium lignosulfonate, glycerol, food-grade brilliant blue and Kathon were added to the aqueous solution of copper citrate and stirred at 180 rpm for 18 min to mix evenly to obtain the basic premix.

[0057] S2. Slowly add the complex enzyme glycoside to the basic premixed solution obtained in step S1, control the temperature at 45℃, and stir at 200 rpm for 30 min to obtain the complex enzyme glycoside dispersion;

[0058] S3. Heat 34 parts by weight of water to 55°C, mix and pre-dissolve with hydroxypropyl starch ether and chitosan, then cool to 40°C to obtain a film-forming agent mixture; slowly add the film-forming agent mixture to the complex enzyme glycoside dispersion obtained in step S2, maintain the temperature at 40°C and stir at 200 rpm for 45 min until uniformly mixed;

[0059] S4. At room temperature, the mixture obtained in step S3 is homogenized under low pressure at 1.5 MPa and filtered through a 150-mesh filter to obtain the seed coating agent.

[0060] Example 2

[0061] A seed coating agent containing complex enzyme glycosides, comprising the following components by weight:

[0062] The ingredients include 1 part of complex enzyme glycoside, 0.5 part of copper citrate, 1.5 parts of hydroxypropyl starch ether, 0.5 parts of chitosan, 1 part of polycarboxylate, 2 parts of propylene glycol, 0.5 parts of food-grade carmine, 0.1 parts of sodium benzoate, 12 parts of difenoconazole, 3 parts of fludioxonil, and 77.9 parts of deionized water.

[0063] The preparation steps of the seed coating agent are as follows:

[0064] S1. At room temperature, copper citrate is added to 40 parts by weight of deionized water and stirred at 200 rpm for 10 min to obtain copper citrate aqueous solution; at room temperature, polycarboxylate, propylene glycol, food-grade carmine and sodium benzoate are added to copper citrate aqueous solution and stirred at 150 rpm for 20 min to mix evenly to obtain basic premix solution.

[0065] S2. Slowly add the complex enzyme glycoside to the basic premixed solution obtained in step S1, control the temperature at 40℃, and stir at 300 rpm for 20 min to obtain the complex enzyme glycoside dispersion;

[0066] S3. Heat 37.9 parts by weight of water to 50°C, mix with hydroxypropyl starch ether and chitosan to pre-dissolve, then cool to 30°C to obtain a film-forming agent mixture; slowly add the film-forming agent mixture to the complex enzyme glycoside dispersion obtained in step S2, maintain the temperature at 30°C and stir at 100 rpm for 60 min until uniformly mixed; then add difenoconazole and fludioxonil, and stir to disperse evenly;

[0067] S4. At room temperature, the mixture obtained in step S3 is homogenized under low pressure at 1.8 MPa and filtered through a 150-mesh filter to obtain the seed coating agent.

[0068] Example 3

[0069] A seed coating agent containing complex enzyme glycosides, comprising the following components by weight:

[0070] The mixture contains 1.05 parts of complex enzyme glycoside, 0.7 parts of copper citrate, 5 parts of hydroxypropyl starch ether, 1 part of chitosan, 0.5 parts of sodium lignosulfonate, 1 part of sorbitol, 0.1 parts of food-grade brilliant blue, 0.05 parts of Kathon, 0.4 parts of thiamethoxam, 0.1 parts of difenoconazole, and 90.1 parts of deionized water.

[0071] The preparation steps of the seed coating agent are as follows:

[0072] S1. At room temperature, add copper citrate to 50 parts by weight of deionized water and stir at 150 rpm for 15 min to obtain copper citrate aqueous solution; at room temperature, add sodium lignosulfonate, sorbitol, Kathon and food grade brilliant blue to copper citrate aqueous solution and stir at 200 rpm for 15 min to mix evenly to obtain basic premix solution.

[0073] S2. Slowly add the complex enzyme glycoside to the basic premixed solution obtained in step S1, control the temperature at 50℃, and stir at 100 rpm for 40 min to obtain the complex enzyme glycoside dispersion;

[0074] S3. Heat 40.1 parts by weight of water to 60°C, mix with hydroxypropyl starch ether and chitosan to pre-dissolve, then cool to 45°C to obtain a film-forming agent mixture; slowly add the film-forming agent mixture to the complex enzyme glycoside dispersion obtained in step S2, maintain the temperature at 45°C and stir at 300 rpm for 30 min until uniform; then add thiamethoxam and difenoconazole, and stir to disperse evenly;

[0075] S4. At room temperature, the mixture obtained in step S3 is homogenized under low pressure at 1.2 MPa and filtered through a 150-mesh filter to obtain the seed coating agent.

[0076] Comparative Example 1

[0077] Referring to Example 1, the difference from the example is that the seed coating agent does not contain copper citrate; in step S1: sodium lignosulfonate, glycerol, food-grade brilliant blue and Kathon are directly added to deionized water and stirred and mixed evenly to obtain the basic premixed solution.

[0078] Other technical features are the same as in Example 1.

[0079] Comparative Example 2

[0080] Referring to Example 1, the difference from the example is that the seed coating agent does not contain complex enzyme glycosides. In the preparation method, step S2 is omitted, and in step S3, the basic premix and the film-forming agent mixture are directly mixed.

[0081] Other technical features are the same as in Example 1.

[0082] Comparative Example 3

[0083] Referring to Example 1, the difference from the example is that the weight parts of complex enzyme glycosides are replaced with gibberellin GA3 to prepare an exogenous hormone-type seed coating agent.

[0084] Other technical features are the same as in Example 1.

[0085] Comparative Example 4

[0086] Referring to Example 1, the difference from the example is that the weight parts of complex enzyme glycosides are replaced with commercially available plant-derived seaweed oligosaccharides to prepare a common plant-derived seaweed oligosaccharide seed coating agent.

[0087] Other technical features are the same as in Example 1.

[0088] Comparative Example 5

[0089] A complex enzyme glycoside seed coating agent was prepared, comprising, by weight, 4.5 parts complex enzyme glycoside and 95.5 parts deionized water. The preparation method was as follows: following step S2 of Example 1, the basic premixed solution was replaced with deionized water to prepare a complex enzyme glycoside dispersion. Then, following step S4 of Example 1, the complex enzyme glycoside dispersion was directly prepared to prepare the seed coating agent.

[0090] Comparative Example 6

[0091] Purchase commercially available 27% difenoconazole·thiamethoxam suspension seed coating agent and use it according to the recommended dosage.

[0092] Test 1

[0093] Storage stability tests were conducted. The seed coating agents of Examples 1-3, Comparative Examples 1, 4, and 5 were sealed and stored at room temperature. The retention rate of glycoside active ingredients was tested after 12 months using the phenol-sulfuric acid method. The results are shown in Table 1.

[0094] Table 1. Results of retention rate of active ingredients in different samples

[0095]

[0096] In Table 1, different lowercase letters after the data in the same column indicate significant differences between the data (P < 0.05), and the same applies below.

[0097] Test results show that the coating agents of Examples 1-3 of this invention maintained a stable retention rate of over 89% for glycoside active ingredients after 12 months of storage at room temperature. This is significantly better than the ordinary plant-derived seaweed oligosaccharide formulation of Comparative Example 4 and the coating agent containing only complex enzyme glycosides of Comparative Example 5, demonstrating that the solution of this invention effectively solves the problem of easy degradation of glycoside active substances. Furthermore, the retention rate of active ingredients in Example 1 is also higher than that of the coating agent in Comparative Example 1 that does not contain copper citrate, breaking the technical prejudice in the field that "copper salts cause inactivation of glycoside substances." The seed coating agent of this invention exhibits excellent storage stability and is ready for industrial-scale distribution.

[0098] Test 2

[0099] Wheat seed germination tests were conducted.

[0100] Seed germination experiments were designed in accordance with GB / T 3543.4-2025 Crop Seed Inspection Procedures Part 4: Germination Experiment for Sowing Quality, to verify the promoting effect of seed coating agents on wheat seed germination and their adaptability to low temperature stress in each example and comparative example.

[0101] The wheat variety Jimai 22 was selected for testing. Seed coating agents prepared in Examples 1-3 and Comparative Examples 1-6 were used as treatment reagents, with uncoated seeds serving as a blank control (CK). The seed-to-coating ratio (w / w) was 1:100. Seeds in each group were coated by mixing seeds with the seed coating agent, stirring thoroughly, and then air-drying. A room temperature germination group was established using a 25℃ constant temperature and light incubator with 12 h of light / 12 h of darkness. A low temperature stress group was subjected to 5℃ darkness and low temperature stress for 72 h (simulating a late spring frost in northern China), followed by restoration to 25℃ room temperature. Each treatment was replicated three times, with 100 seeds per replicate. The paper germination method was used. Germination potential was measured 3 days after sowing in the room temperature group, 5 days in the low temperature group, 7 days in the room temperature group, and 10 days in the low temperature group. The average germination time was recorded. Data analysis was performed using SPSS 22.0 statistical software. All data are expressed as mean ± standard deviation. The results are shown in Table 2.

[0102] Table 2. Germination indices of wheat seeds under different treatments

[0103]

[0104] The experimental results show that the coating agents of Examples 1-3 of this invention can significantly improve the germination potential and germination rate of wheat seeds. At room temperature, the germination potential is increased by more than 11.3% compared with commercially available conventional chemical seed coating agents (Comparative Example 6), and the average germination time is shortened by more than 1.2 days, achieving rapid and uniform emergence and solving the problem of delayed germination of conventional seed coating agents. Under low temperature stress, the germination rate of the coating agent treatment groups of this invention is all above 96%, which is more than 17% higher than the control and superior to each comparative example. This perfectly solves the production pain point of difficult and uneven emergence under the adverse conditions of late spring cold in northern China. Although the exogenous hormone-type coating agent of Comparative Example 3 improves the germination rate at room temperature, the germination rate decreases significantly under low temperature stress, proving that exogenous hormones can only promote germination for a short period at room temperature and cannot improve the ability to germinate under adverse conditions. However, the endogenous activation system of this invention can achieve the dual effects of promoting germination at room temperature and resisting adverse conditions.

[0105] Test 3

[0106] Conduct cotton seedling growth tests.

[0107] A seedling growth experiment was conducted using Lumianyan 28 as the tested cotton variety. Seeds were coated with the seed coating agents of Examples 1-3 and Comparative Examples 1-6, with uncoated seeds serving as a blank control (CK). The seed-to-drug ratio (w / w) was 1:80. Seeds in each group were coated by mixing the seeds with the seed coating agent, stirring thoroughly, and then air-drying for later use. A greenhouse pot experiment was conducted using sterile garden soil and vermiculite in a 3:1 mass ratio. Two seeds were sown per pot, and one healthy seedling was retained after emergence. Each treatment consisted of 30 pots, with three replicates. The greenhouse daytime temperature was 25–28℃ and the nighttime temperature was 18–20℃, with conventional water management. Fifteen days after sowing (when cotton has two true leaves), germination rate, plant height, stem diameter, taproot length, number of lateral roots, root fresh weight, aboveground fresh weight, and seedling vigor rate were measured. The criteria for vigorous seedlings were: stem diameter ≥2.5 mm, plant height 10–15 cm, taproot length ≥12 cm, number of lateral roots ≥25, and dark green leaves free from pests and diseases. Data analysis was performed using SPSS 22.0 statistical software. All data are expressed as mean ± standard deviation, and the results are shown in Table 3.

[0108] Table 3. Growth indicators of cotton seedlings under different treatments (15 days after sowing)

[0109]

[0110] The seed coating agents in Examples 1-3 of this invention can significantly improve the cotton seedling emergence rate, increasing it by more than 9.75% compared to commercially available conventional seed coating agents (Comparative Example 6), thus solving the production pain points of uneven emergence and seed rot and seedling death in direct-seeded cotton. Simultaneously, it can significantly promote cotton root development, with taproot length, lateral root number, and root fresh weight increasing by more than 54.9%, 69.7%, and 107.7% respectively compared to Comparative Example 6. This robust root system lays a core foundation for cotton's drought resistance, lodging resistance, and nutrient absorption in the mid-to-late stages. The cotton plants in the treatment group of this invention exhibited moderate height, robust stems, and a seedling vigor rate of over 93%, without excessive vegetative growth. In contrast, the exogenous hormone-type seed coating agent in Comparative Example 3 showed... The severe problems of excessive above-ground growth (plant height more than 30% higher than in the previous example), weak stems, and poor root development resulted in a seedling vigor rate of only 52.2%, which completely failed to meet the requirements of "short, sturdy, and well-developed root system" for cotton seedlings. This easily led to lodging and boll shedding in the middle and late stages, proving that the non-hormonal endogenous activation system of this invention can accurately achieve the cultivation of vigorous cotton seedlings without the side effects of exogenous hormones, and is fully adapted to the needs of cotton production. Example 3, the insect-proof and disease-resistant formula, can simultaneously control aphids and damping-off disease in cotton seedlings while retaining the effect of promoting vigorous growth, greatly reducing the labor and pesticide use in the seedling stage, which is in line with the development trend of simplified cotton cultivation.

[0111] Test 4

[0112] Conduct field yield verification tests for cucumbers.

[0113] A field yield verification experiment for cucumbers was designed in accordance with the "Technical Specifications for Pollution-Free Cucumber Production" (NY / T 5075-2002). The cucumber variety selected for testing was Jinchun No. 4. The seeds were coated with the seed coating agents of Examples 1-3 and Comparative Examples 1-6. Uncoated seeds served as blank control (CK).

[0114] The experiment was conducted in an experimental field in Laiyang, Yantai City, Shandong Province. The soil pH was 6.8, organic matter content was 1.1%, and the previous crop was Chinese cabbage. The soil fertility was uniform. A randomized block design was used, with three replicates per treatment and a plot area of ​​20 m². 2 For open-field spring cultivation, seedlings were raised in warm beds in late March. Before sowing, the seed coating agent to seed ratio (w / w) was 1:60 for all treatments. The coating method involved mixing the seeds with the seed coating agent, stirring thoroughly, and then air-drying. Transplanting was carried out in late April, with a row spacing of 60 cm and a plant spacing of 30 cm. The nitrogen application rate was uniformly 200 kg / hm² for all treatments. 2 Other field management practices (watering, pruning, and pest and disease control) are completely consistent. The yield within 30 days after transplanting, the early-stage yield, the total yield over the entire growth period, the number of fruits per plant, and the average weight of each fruit are used to calculate the yield per mu (unit of land area), and the yield increase rate is calculated. Yield calculation formula: Yield per mu (kg / mu) = Plot yield (kg) × 666.67 ÷ Plot area (20 m²) 2 The yield increase rate is calculated as follows: (yield per mu of treatment - yield per mu of control group) ÷ yield per mu of control group × 100%.

[0115] Data analysis was performed using SPSS 22.0 statistical software. All data are expressed as mean ± standard deviation. The results are shown in Table 4.

[0116] Table 4 Cucumber yield indicators under different treatments

[0117]

[0118] The experimental results show that the seed coating agents of Examples 1-3 of this invention can significantly increase the early-stage and total yield of cucumbers, with a total yield increase of over 20% and an early-stage yield increase of over 31% compared to the control (CK). Early-stage cucumber yield directly determines the economic benefits of planting. The treatment group of this invention can be marketed earlier, significantly increasing growers' income and fully meeting the production needs of vegetable cash crops. The number of cucumbers per plant and the average weight of a single cucumber in the treatment group of this invention are significantly higher than those in the control groups. This is because the coating agent promotes cucumber seed germination and seedling root development through a synergistic effect, cultivating strong seedlings and improving the photosynthetic efficiency and nutrient absorption capacity of cucumbers. Although the exogenous hormone-type coating agent in Control Group 3 showed a slight increase in yield, it easily led to excessive seedling growth and delayed female flower differentiation in the early stage, resulting in limited early-stage yield increases and a higher rate of deformed cucumbers. In contrast, the non-hormonal endogenous activation system of this invention can simultaneously achieve the full-chain effect of strong seedlings, flower promotion, fruit preservation, and yield increase, without the side effects of exogenous hormones. It is green and safe, meets the standards for green vegetable production, and has extremely high promotion and application value.

[0119] Test 5

[0120] The active groups of the complex enzyme glycosides used as raw materials and their interaction with copper citrate were characterized. Since complex enzyme glycosides are multi-component marine microbial metabolic complexes, their infrared spectra exhibit numerous overlapping peaks of glycosyl and aglycone groups, making it difficult to accurately assign characteristic peaks to individual active groups and directly reflect intermolecular interactions. Therefore, a combined characterization method integrating chemical colorimetric reactions, potentiometric titration, and UV-Vis absorption spectroscopy was employed. This method is more specific for identifying key active groups in multi-component complexes and can directly quantify the strength of intermolecular interactions. Specific test methods and results are as follows.

[0121] 1. Chemical characterization of key active groups

[0122] (1) Verification of the reducing hemiacetal hydroxyl group:

[0123] Take 1 mL of 0.1 mg / mL complex glycoside aqueous solution, add 1 mL of Fehling's reagent A and 1 mL of Fehling's reagent B, and heat in a boiling water bath for 5 min. Result: A brick-red cuprous oxide precipitate is produced, proving that the complex glycoside molecule contains a reducing hemiacetal hydroxyl group and has weak reducing properties.

[0124] (2) Verification of phenolic hydroxyl groups

[0125] Take 1 mL of 0.1 mg / mL complex glycoside aqueous solution and add 2 drops of 1% ferric chloride ethanol solution. Result: The solution turns blue-purple, proving that the aglycone of the complex glycoside contains a phenolic hydroxyl group.

[0126] (3) Verification of the carboxyl group

[0127] Take 20 mL of 0.5 mg / mL complex glycoside aqueous solution and perform potentiometric titration with 0.01 mol / L NaOH standard solution, recording the pH change. Results: The titration curve shows two obvious abrupt jumps at pH 4.5–5.5 and pH 8.0–9.0, proving that the complex glycoside molecule contains a dissociable carboxyl group.

[0128] (4) Verification of cis-ortho polyhydroxyl groups

[0129] Take 1 mL of 0.1 mg / mL complex glycoside aqueous solution and add 0.5 mL of 0.1 mol / L boric acid solution, then measure the pH change. Results: The pH of the solution decreased from 6.2 to 4.8, proving that the glycosyl moiety of the complex glycoside contains cis-ortho-hydroxyl groups, which can form a stable five-membered ring complex with boric acid and release H₂. + .

[0130] 2. UV-Vis absorption spectroscopy characterization of the interaction between complex enzyme glycosides and copper citrate

[0131] Detection conditions: A UV-Vis spectrophotometer was used, with deionized water as a blank control. The scanning wavelength range was 190-400 nm, the scanning speed was 200 nm / min, the slit width was 2 nm, and the sample concentration was 0.1 mg / mL.

[0132] Test results:

[0133] (1) Pure complex enzyme glycosides show a strong absorption peak at 205 nm (ε=1.2×10). 4 L·mol -4 ·cm -1 This corresponds to the n→σ electron transition of the glycosyl cis-ortho polyhydroxyl group; a weak absorption peak appears at 272 nm (ε=8.5×10). 2 L·mol -1 ·cm -1 The π→π electron transitions corresponding to the terminal carboxyl group and phenolic hydroxyl group of the aglycone are consistent with the above chemical characterization results.

[0134] (2) Pure copper citrate has continuous weak absorption in the range of 190~250 nm, and no obvious characteristic absorption peaks in the ranges of 270 nm and 300~350 nm.

[0135] (3) After the complex enzyme glycoside and copper citrate were mixed evenly at a mass ratio of 4.5:1 (the ratio in Example 1), the ultraviolet spectrum showed a significant change: the absorption peak at 205 nm red-shifted to 212 nm and the absorbance increased by 18%, proving that the cis-ortho-hydroxyl group reacted with Cu. 2+ Coordination occurred; the absorption peak at 272 nm blue-shifted to 265 nm with a 23% decrease in absorbance, indicating that intermolecular hydrogen bonds were formed between the terminal carboxyl group and the phenolic hydroxyl group of the aglycone and the citrate group; a new characteristic absorption peak appeared at 330 nm (ε = 3.2 × 10⁻⁶). 3 L·mol -1 ·cm -1 ), which is citrate-Cu 2+ The charge transfer absorption peak of the 3-enzyme glycoside ternary bridged coordination complex directly confirms the formation of the dynamic reversible complex system.

[0136] The above description is only 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 coating agent containing complex enzyme glycosides, characterized in that, The product comprises, by weight parts: 1-5 parts of complex enzyme glycoside, 0.5-1.5 parts of copper citrate, and auxiliary agents, including 2-8 parts of film-forming agent.

2. The seed coating agent according to claim 1, characterized in that, The film-forming agent is at least one of hydroxypropyl starch ether, polyvinyl alcohol, chitosan, and gum arabic.

3. The seed coating agent according to claim 1 or 2, characterized in that, The auxiliary agents also include at least one of the following: dispersants, humectants, warning colors, and preservatives.

4. The seed coating agent according to claim 3, characterized in that, The dispersant is at least one of sodium lignosulfonate, polycarboxylate, and sodium dodecylbenzenesulfonate; The moisturizer is at least one of glycerin, propylene glycol, and sorbitol; The warning color is food-grade carmine and / or bright blue; The preservative is Kathon and / or sodium benzoate.

5. The seed coating agent according to claim 1 or 2, characterized in that, It also includes 70 to 100 parts water by weight.

6. The seed coating agent according to claim 1 or 2, characterized in that, It also includes functional additives, which are at least one of insecticides, fungicides, and micronutrient fertilizers.

7. A method for preparing a seed coating agent as described in any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Mix copper citrate and auxiliary agents other than film-forming agents with 40-50 parts by weight of water to obtain a basic premix; S2. Add the complex enzyme glycosides to the basic premixed solution obtained in step S1, control the temperature at 40~50℃, stir for 20~40 min to obtain the complex enzyme glycoside dispersion; S3. Heat 30-50 parts by weight of water to 50-60°C, add film-forming agent and stir to dissolve, then cool to 30-45°C to obtain film-forming agent mixture; add film-forming agent mixture to complex enzyme glycoside dispersion obtained in step S2, maintain temperature at 30-45°C and stir for 30-60 min; S4. Homogenize the mixture obtained in step S3 and filter it through a 100-200 mesh filter to obtain the seed coating agent.

8. The preparation method according to claim 7, characterized in that, In step S3: After adding the film-forming agent mixture to the complex enzyme glycoside dispersion and stirring, add the functional additive and stir evenly.

9. The application of the seed coating agent according to any one of claims 1 to 6 in the treatment of crop seeds, characterized in that, The seed coating agent is used to treat crop seeds through a coating process, and the mass ratio of the seed coating agent to the crop seeds is 1:(50~200).

10. The application according to claim 9, characterized in that, The crops mentioned are wheat, corn, rice, peanuts, soybeans, cotton, tomatoes, cucumbers, or peppers.

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