Seed coating agent as well as preparation method and application thereof

Abstract

The invention provides a seed coating agent as well as a preparation method and application thereof, and belongs to the technical field of crop growth promotion. The seed coating agent comprises a microbial agent and a film-forming agent; the microbial agent comprises any two or three of bacillus velezensis, bacillus subtilis and trichoderma longibrachiatum. The seed coating agent is obtained by combining the microbial agent provided by the invention with a film-forming agent. The seed coating agent is stable in physicochemical property, the pH value is 7-8, the coating uniformity is larger than or equal to 90%, the coating falling rate is smaller than or equal to 8%, and the seed coating agent has high-temperature and low-temperature stability. After seeds are coated with the seed coating agent, the germination rate of the seeds can be remarkably increased, and the germination period of the seeds can be shortened; the growth of the seedlings is obviously promoted, the overground part length and the underground part length of the seedlings are increased, the dry weight and the fresh weight of the seedlings are increased, and the root-shoot ratio of the seedlings is increased. In conclusion, the seed coating agent provided by the invention has high germination rate, stability and growth promoting ability, and a theoretical basis is provided for development and application of the microbial seed coating agent.

Description

Technical Field

[0001] This invention belongs to the field of crop growth promotion technology, specifically relating to a seed coating agent, its preparation method, and its application. Background Technology

[0002] Wheat, as a major food crop, plays a vital role in national economic development. However, wheat is susceptible to various biological stresses during its germination stage. On the one hand, pathogens (such as Fusarium head blight)... Fusarium graminearum Root rot pathogens Bipolaris sorokiniana Infection by pests such as grubs and larvae hinders crop germination; on the other hand, underground pests (such as grubs) also contribute to this problem. Holotrichia oblita Wireworm Agriotes fuscicollis The damage caused by the grazing of animals (such as larvae) seriously affects the seedling survival rate.

[0003] Traditional seed treatment in agriculture often employs soaking and coating techniques, but both suffer from drawbacks such as short-lasting efficacy, high environmental pollution risks, unsuitability of seed sizes for mechanized sowing, and low operational efficiency. To address these issues, seed coating technology was developed and rapidly adopted. Today, this technology, with its high efficiency and practicality, has become a widely used advanced seed treatment method in agriculture. Seed coating technology is a modern seed treatment technique developed based on traditional seed soaking and coating processes through the deep integration and innovation of knowledge from multiple disciplines. Based on the colloidal chemical stability mechanism, this technology has successfully developed a functional membrane layer that combines slow-release properties, air permeability, and high biocompatibility. By precisely coating the seed surface with a composite coating material integrating nutrients, plant growth regulators, and pest and disease control components, a multi-functional protective barrier is constructed. While ensuring normal seed germination, it achieves the continuous and slow release of active ingredients within the membrane, providing long-term protection and nutrient supply for crop growth.

[0004] Related studies show that seed coating technology can serve as an important technical approach to optimize crop growth and improve crop yield. It provides multi-dimensional protection for crops in the early stages of growth, ultimately achieving the dual goals of improved growth quality and increased yield. In the field of alfalfa seed coating agents, experimental data show that seed coating significantly promotes the optimization of plant growth indicators, specifically manifested in increased plant height, biomass accumulation, number of root nodules, and overall improvement in nitrogenase catalytic activity, thereby enhancing the plant's nitrogen conversion efficiency and overall growth competitiveness. In maize planting trials, compared with the uncoated control group, the seed-coated group showed significant increases in both stem and root length, with stem length increasing by 18% and root length by an even higher 21.45%. Currently, mainstream seed coating agents on the market still rely on chemical pesticides such as imidacloprid and furans as core ingredients, primarily depending on the active components of traditional chemical pesticides to construct their formulation systems. While these agents can effectively suppress crop diseases such as root rot and leaf spot, they pose significant safety risks. On the one hand, chemical residues may accumulate through the food chain, potentially harming the health of humans and livestock. On the other hand, their application can easily lead to pollution of soil, water, and other ecological environments. Long-term use may also cause target organisms to develop resistance, further exacerbating the ecological pressure on agricultural production. Therefore, the development and preparation processes of safe and environmentally friendly microbial seed coating agents have become a current research hotspot. However, some existing microbial seed coating agents often suffer from unstable efficacy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a seed coating agent with stable physicochemical properties and effects, which can effectively promote wheat seed germination and seedling growth.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a seed coating agent, comprising: a microbial inoculant and a film-forming agent; The microbial agent includes any two or three of Bacillus vesiculosus, Bacillus subtilis, and Trichoderma longifolia.

[0007] Preferably, the Bacillus berleis includes Bacillus berleis SSF6; the Bacillus subtilis includes Bacillus subtilis LU5; and the Trichoderma longifolia includes Trichoderma longifolia C1, strain number CICC 40136.

[0008] Preferably, when the microbial agent includes any two of Bacillus belyssioides, Bacillus subtilis, and Trichoderma longifolia, the volume ratio of the two microbial agents is (1~2):(1~2); the viability of the seed liquid of Bacillus belyssioides is 1×10⁻⁶. 10CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of *Trichoderma longifolia* C1 was 1×10⁻⁶. 7 CFU / mL.

[0009] Preferably, when the microbial agent comprises Bacillus belyssioides, Bacillus subtilis, and Trichoderma longifolia, the volume ratio of the three microbial agents is 1:(1~2):(1~2); the viability of the seed liquid of Bacillus belyssioides is 1×10⁻⁶. 10 CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of the *Trichoderma longifolia* was 1×10⁻⁶. 7 CFU / mL.

[0010] Preferably, the film-forming agent includes: NNO, ethylene glycol, Permanent Red F4R, xanthan gum, magnesium aluminum silicate, and sodium benzoate.

[0011] Preferably, the seed coating agent comprises, by mass-volume percentage, 3%~5% NNO, 4%~5% ethylene glycol, 0.2%~0.3% Permanent Red F4R, 0.3%~0.5% xanthan gum, 0.5%~1% magnesium aluminum silicate, and 0.07%~0.1% sodium benzoate.

[0012] This invention provides a method for preparing the seed coating agent described in the above technical solution, comprising: The seed liquids of any two or three of the Bacillus vesiculosus, Bacillus subtilis and Trichoderma longifolia are mixed to obtain the microbial inoculant. Adding a film-forming agent to a microbial inoculant yields a seed coating agent.

[0013] This invention provides an application of the seed coating agent described in the above technical solution or the seed coating agent prepared by the preparation method described in the above technical solution in promoting wheat growth.

[0014] Preferably, the method of promoting wheat growth includes at least one of the following: (1) Improve the germination rate of wheat seeds; (2) Increase the length of the aboveground part and / or the underground part of the seedling; (3) Increase the fresh weight and / or dry weight of seedlings; (4) Increase the root-to-shoot ratio of seedlings.

[0015] This invention provides a method for promoting wheat growth, comprising: Seeds are coated with the seed coating agent prepared by the above-described technical solution or the preparation method described above; the ratio of seed coating agent to seed is 1 mL: (50~75) g.

[0016] The beneficial effects of this invention are: This invention provides a seed coating agent, comprising: a microbial inoculant and a film-forming agent; the microbial inoculant includes any two or three of Bacillus belye, Bacillus subtilis, and Trichoderma longifolia. The seed coating agent is prepared by combining the microbial inoculant with the film-forming agent. The seed coating agent exhibits stable physicochemical properties, with a pH of 7-8, a coating uniformity ≥90%, and a coating shedding rate ≤8%, and demonstrates high-temperature and low-temperature stability. Furthermore, coating seeds with the seed coating agent significantly improves seed germination rate and shortens the germination cycle; it also significantly promotes seedling growth, increasing the length of the aboveground and underground parts of the seedlings, increasing the dry and fresh weight of the seedlings, and increasing the root-to-shoot ratio. In summary, the seed coating agent provided by this invention combines high germination rate, stability, and growth-promoting ability, providing a theoretical basis for the development and application of microbial seed coating agents.

[0017] Biological Preservation Information Bacillus subtilis LU5, Latin scientific name: Bacillus subtilis It was deposited on June 15, 2023 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC No: M20231029.

[0018] Bacillus belyssus SSF6, Latin scientific name: Bacillus velezensis It was deposited on July 14, 2022 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25298. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Image of the seed after coating; Figure 2 The graph shows the coating peeling rate of the samples from treatments 1 to 3 corresponding to formulation 1 in Example 3. Figure 3 This is a graph showing the effect of different seed coating agents and different seed ratios on wheat germination rate in Example 3; Figure 4 This is a graph showing the effect of the microbial seed coating agent on the aboveground parts and length of wheat in Example 4. Detailed Implementation

[0021] The present invention provides a seed coating agent, comprising: a microbial agent and a film-forming agent; wherein the microbial agent comprises any two or three of Bacillus belye, Bacillus subtilis and Trichoderma longifolia.

[0022] As an optional embodiment of the present invention, the microbial agent is any two of Bacillus bellis, Bacillus subtilis, and Trichoderma longicornis; the microbial agent can be Bacillus bellis and Bacillus subtilis, or Bacillus subtilis and Trichoderma longicornis, or Bacillus bellis and Trichoderma longicornis. As an optional embodiment of the present invention, when the microbial agent includes any two of Bacillus bellis, Bacillus subtilis, and Trichoderma longicornis, the volume ratio of the two microbial agents can be (1~2):(1~2), or 1:1, 1:2, or 2:1. As an optional embodiment of the present invention, the viability of the seed liquid of Bacillus bellis is 1×10⁻⁶. 10 CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of the *Trichoderma longifolia* was 1×10⁻⁶. 7 CFU / mL.

[0023] As an optional embodiment of the present invention, when the microbial agent is *Bacillus belyssiensis*, *Bacillus subtilis*, and *Trichoderma longicornis*, the volume ratio of *Bacillus belyssiensis*, *Bacillus subtilis*, and *Trichoderma longicornis* in the microbial agent can be 1:(1~2):(1~2), or 1:1:1, 1:2:2, 1:2:1, or 1:1:2; the viability of the seed liquid of *Bacillus belyssiensis* is 1×10⁻⁶. 10 CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of the *Trichoderma longifolia* was 1×10⁻⁶. 7 CFU / mL.

[0024] As an optional embodiment of the present invention, the Bacillus berleis includes Bacillus berleis SSF6; the Bacillus subtilis includes Bacillus subtilis LU5; and the Trichoderma longifolia includes Trichoderma longifolia C1, strain number CICC40136.

[0025] As an optional embodiment of the present invention, the film-forming agent comprises: NNO, ethylene glycol, Permanent Red F4R, xanthan gum, magnesium aluminum silicate, and sodium benzoate. In the present invention, by mass-volume percentage, the seed coating agent comprises 3%~5% NNO, 4%~5% ethylene glycol, 0.2%~0.3% Permanent Red F4R, 0.3%~0.5% xanthan gum, 0.5%~1% magnesium aluminum silicate, and 0.07%~0.1% sodium benzoate. As an optional embodiment of the present invention, the film-forming agent includes 3%~5% NNO, or 5%; the humectant includes 4%~5% ethylene glycol, or 5%; the film-forming agent includes 0.2%~0.3% Permanent Red F4R, or 0.3%; the film-forming agent includes 0.3%~0.5% xanthan gum, or 0.3%; the film-forming agent includes 0.5%~1% magnesium aluminum silicate, or 1%; the film-forming agent includes 0.07%~0.1% sodium benzoate, or 0.1%. The film-forming agent provided by the present invention will not adversely affect the microbial agent, and may even enhance the effect of the microbial agent, enabling the seed coating agent to effectively promote wheat growth.

[0026] This invention provides a method for preparing the seed coating agent described in the above technical solution, comprising: The seed liquids of any two or three of the Bacillus vesiculosus, Bacillus subtilis and Trichoderma longifolia are mixed to obtain the microbial inoculant. Adding a film-forming agent to a microbial inoculant yields a seed coating agent.

[0027] This invention does not impose any particular limitation on the preparation method of the seed culture of each bacterial agent; any conventional preparation method in the art can be used. As an optional embodiment of this invention, the bacterial activity of the *Bacillus belyssus* seed culture is 1 × 10⁻⁶. 10 CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of the *Trichoderma longifolia* was 1×10⁻⁶. 7 CFU / mL. In this invention, various microbial agents are mixed according to the volume ratio described in the above technical solution to obtain microbial agents.

[0028] After obtaining the microbial inoculant, the present invention adds a film-forming agent to the microbial inoculant. The present invention does not specifically limit the method of addition; any conventional method in the art can be used.

[0029] The seed coating agent prepared according to this invention has a simple process, and the resulting seed coating agent has stable physicochemical properties, a pH value of 7-8, a coating uniformity of ≥90%, a coating shedding rate of ≤8%, and exhibits high and low temperature stability. The seed coating agent provided by this invention combines high germination rate, stability, and growth-promoting ability.

[0030] This invention provides an application of the seed coating agent described in the above-described technical solution or the seed coating agent prepared by the preparation method described in the above-described technical solution in promoting wheat growth. As an optional embodiment of this invention, promoting wheat growth includes at least one of the following: (1) increasing wheat seed germination rate; (2) increasing the aboveground length and / or underground length of seedlings; (3) increasing the fresh weight and / or dry weight of seedlings; (4) increasing the root-to-shoot ratio of seedlings. The results of the embodiments of this invention show that coating seeds with the seed coating agent can significantly improve the seed germination rate and shorten the seed germination cycle; it can significantly promote seedling growth, increase the aboveground and underground length of seedlings, increase the dry and fresh weight of seedlings, and increase the root-to-shoot ratio of seedlings.

[0031] This invention provides a method for promoting wheat growth, comprising: Seeds are coated with the seed coating agent described in the above technical solution or the seed coating agent prepared by the preparation method described in the above technical solution; the ratio of seed coating agent to seed can be 1 mL: (50~75) g. As an optional embodiment of the present invention, the present invention can coat sterilized seeds. The present invention does not specifically limit the sterilization method; any conventional seed sterilization method in the art can be used. As an optional embodiment of the present invention, the ratio of seed coating agent to seed can be 1 mL: 50 g, 1 mL: 60 g, 1 mL: 65 g, 1 mL: 70 g, or 1 mL: 75 g.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] The experimental materials used in the following technical solutions and their sources are as follows: 1. Microbial materials: Test microbial strain: Bacillus belyssus SSF6 ( Bacillus velezensis The accession number is: CGMCC No. 25298; Bacillus subtilis LU5 ( Bacillus subtilis The accession number is: CCTCC No: M20231029. *Trichoderma longifolia* (CICC 40136) Trichoderma longibrachiatum Purchased from the China Industrial Microbial Culture Collection Center, it was named Trichoderma longifolia C1 for easy recording in the laboratory.

[0034] Bacillus belyssus SSF6 has been described in published patent CN115725447A; Bacillus subtilis LU5 has been described in published patent CN119286706A.

[0035] 2. Test culture medium: LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, distilled water to 1L, pH 7.0, 121℃ for 20 min.

[0036] LB solid medium: beef extract 3 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar powder 20 g / L, pH 7.0, 121℃ for 20 min.

[0037] PDA medium: 15 g / L potato starch, 20 g / L agar powder, pH 6.0, 121℃ for 20 min. 3. Additives and materials: dispersant NNO (Zhejiang Lianhui New Materials Co., Ltd.), xanthan gum (Shandong Fufeng Group Co., Ltd.), magnesium aluminum silicate (Henan Gongfa New Materials Technology Co., Ltd.), permanent red F4R (Shandong Sunshine Pigment Co., Ltd.), ethylene glycol (China Petroleum & Chemical Corporation), sodium benzoate (Hebei Bailingwei Ultrafine Materials Co., Ltd.).

[0038] Example 1 Confirmation of microbial agent combinations (1) Activation of microbial strains: Microbial strains (Bacillus belye SSF6 and Bacillus subtilis LU5) stored at -80℃ were streaked on LB solid medium for activation. Single colonies were then picked and transferred to 250 mL Erlenmeyer flasks containing LB liquid medium and cultured at 30℃ and 180 r / min on a shaker until the viable count reached 1×10⁻⁶. 10 CFU / mL was used to prepare Bacillus belye SSF6 seed culture and Bacillus subtilis LU5 seed culture. Trichoderma longifolia mycelium was inoculated onto PDA medium and cultured at 30℃ for 4 days. After sporulation, the spores were washed with sterile physiological saline, and the spore concentration was adjusted to 1×10⁻⁶. 7 A suspension of Trichoderma spores was prepared using CFU / mL.

[0039] (2) Strain antagonism experiment Cross-cross method: The cross-cross method is used for bacterial antagonism experiments. An inoculation loop is dipped into bacterial suspension and streaked across the culture medium in a cross pattern, ensuring the streaks intersect. Three parallel experiments are set up, and the cultures are incubated upside down for 1 day. Whether antagonism occurs during bacterial growth is observed.

[0040] Plate confrontation method: The antagonistic effect between bacteria and fungi is detected using the plate confrontation method. A bacterial suspension is streaked in the center of an agar plate using an inoculation loop. Fungal spores are then collected using a sterile toothpick and spotted onto the plate. The plate is sealed with film and incubated at 28°C. Simultaneously, spores are inoculated onto the plate using a sterile toothpick, and a blank control is established to monitor strain growth and interaction. If two adjacent strains grow well and fuse at the interface, they are considered compatible; if one strain's growth slows and there is a gap at the interface, it is considered partially compatible; if one strain significantly inhibits the other and the interface is clear, it is considered incompatible. Strains exhibiting significant antagonistic behavior are not used for constructing compound bacterial systems.

[0041] The antagonistic effects of Bacillus subtilis LU5, Bacillus bereaves SSF6, and Trichoderma longifolia C1 were determined using the cross-cross method and the plate confrontation method. The results are shown in Table 1.

[0042] Table 1. Antagonistic effects among the three strains

[0043] Note: "+" indicates that the strains can be co-cultured without antagonism; " / " indicates that the antagonism between the strains is not obvious.

[0044] Observe the growth of each strain on the plate. B. subtilis LU5 and B. velzensis SSF6 and T. longifolia There was no growth inhibition among them, and all of them grew well; therefore, they were selected. B. subtilis LU5 B. velzensis SSF6 and T. longifolia The work of constructing a complex bacterial system was carried out.

[0045] (3) Screening the ratio of Bacillus subtilis LU5, Bacillus belye SSF6 and Trichoderma longifolia C1 Seed culture of Bacillus subtilis LU5, Bacillus belye SSF6, and Trichoderma longicornis C1 spore suspension were inoculated into PDA medium and cultured at 30℃ and 170 rpm for 24 h, either separately or in a specific volume ratio (see Table 2). After culture, the biomass of each treatment was measured by weighing to determine the ratio of Bacillus subtilis LU5, Bacillus belye SSF6, and Trichoderma longicornis. Biomass is the weight of the dry matter after centrifugation of the culture medium and drying of the mycelial sludge.

[0046] Table 2 Construction of the composite microbial system and its biomass

[0047] From Table 2, we can see that B. subtilisLU5 B. velzensis SSF6 and T. longifolia Combination D in C1 mixed fermentation showed higher biomass than other combinations, and combination D4 ( B. subtilis LU5 + B. velzensis SSF6 + T. longifolia Combination C1 had the highest biomass, followed by combination A1. Therefore, combinations A1 and D4 were used for subsequent studies.

[0048] Example 2: Preparation of different seed coating agents 1. Preparation of seed coating agents Mix the additives according to Formula 1: 5% NNO, 5% ethylene glycol, 0.3% Permanent Red F4R, 0.3% xanthan gum, 0.5% magnesium aluminum silicate, and 0.1% sodium benzoate; Additive 1; Formula 2: NNO: 4%, Xanthan gum: 0.45%, Ethylene glycol: 4%, Permanent Red F4R: 0.25%, Sodium benzoate: 0.1%; Additive 2; Formula 3: NNO: 4%, Magnesium aluminum silicate: 1.3%, Permanent Red F4R: 0.25%, Sodium benzoate: 0.1%; Additive 3.

[0049] 2. Preparation of Bacillus belyss SSF6 and Bacillus subtilis LU5 seed coating agents The Bacillus berberis SSF6 seed culture prepared in Example 1 and Bacillus subtilis LU5 seed culture were mixed at a volume ratio of 1:1 to obtain a composite microbial agent system. Adding the substances according to the composition of Formula 1 to the compound microbial agent system, we prepared Bacillus belyss SSF6 + Bacillus subtilis LU5 + seed coating agent 1; adding the substances according to the composition of Formula 2 to the compound microbial agent system, we prepared Bacillus belyss SSF6 + Bacillus subtilis LU5 + seed coating agent 2; adding the substances according to the composition of Formula 3 to the compound microbial agent system, we prepared Bacillus belyss SSF6 + Bacillus subtilis LU5 + seed coating agent 3.

[0050] 3. Preparation of seed coating agents for Bacillus belyssus SSF6, Bacillus subtilis LU5, and Trichoderma longifolia C1 The Bacillus berberis SSF6 seed solution, Bacillus subtilis LU5 seed solution and Trichoderma longicornis spore suspension prepared in Example 1 were mixed in a volume ratio of 1:2:2 to obtain a composite microbial agent system. Adding the substances according to the composition of Formula 1 to the compound microbial agent system yields Bacillus belye SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed coating agent 1; adding the substances according to the composition of Formula 2 to the compound microbial agent system yields Bacillus belye SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed coating agent 2; adding the substances according to the composition of Formula 3 to the compound microbial agent system yields Bacillus belye SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed coating agent 3.

[0051] Example 3: Determination of the ratio of different coating agents 1) Seed pretreatment: Soak the seeds in a 5% sodium hypochlorite solution for 5 min to sterilize them, then rinse them twice with sterile distilled water and air dry them in a clean bench.

[0052] 2) The properties and viable microbial counts of the six coating agents prepared in Example 2 were tested.

[0053] 3) The coating peeling rate of the six coating agents prepared in Example 2 was tested.

[0054] 4) Effects of seed coating agents on seed germination rate The experiment included five gradient ratios (volume-to-weight ratio, V / W), where different seed coating agents were applied to seeds at ratios of 1:10, 1:25, 1:50, 1:75, and 1:100 (mL / g). The resulting seed coatings are shown in the image below. Figure 1 As shown, the left image shows seeds coated with a mixture of *Bacillus vesalis* SSF6, *Bacillus subtilis* LU5, *Trichoderma longicornis*, and seed coating agent 1 at a ratio of 1:50 (mL / g); the right image shows seeds coated with a mixture of *Bacillus vesalis* SSF6, *Bacillus subtilis* LU5, *Trichoderma longicornis*, and seed coating agent 1 at a ratio of 1:75 (mL / g). The germination rate of seeds in each treatment group was measured to investigate the effect of the optimal seed coating agent dosage on seed vigor. The water-treated seed group served as the control group (CK); the seed coating agent (adjuvant) treated seeds served as treatment 1; the *Bacillus vesalis* SSF6 + *Bacillus subtilis* LU5 + seed coating agent treated seeds served as treatment 2; and the *Bacillus vesalis* SSF6 + *Bacillus subtilis* LU5 + *Trichoderma longicornis* + seed coating agent treated seeds served as treatment 3. After mixing the seed coating agents with the seeds, the mixture was stirred with a glass rod until the seed surface was evenly covered with the seed coating agent, and then air-dried in a clean bench for 3 hours.

[0055] The germination rate of coated seeds was determined using the paper bed germination method. Two hundred coated seeds were arranged face down on moist filter paper, with each treatment performed in triplicate to ensure the reliability of the experimental data. The seed-containing petri dishes were placed in a constant temperature and humidity incubator at 20 ± 0.5℃ and 20% light intensity to simulate the natural germination environment. Germination was determined by the emergence of fresh seed buds through the seed coat (the "white sprouting" phenomenon). The number of germinated seeds in each petri dish was counted manually every 12 hours, and the germination rate was calculated.

[0056] The two groups with the highest germination rates of the treated medicinal herbs were identified, and the growth and physiological indicators of the crop seedlings were measured.

[0057] 5) Test Results (1) The properties and viable microbial counts of different seed coating agents in formulations 1, 2, and 3 are shown in the table below: Table 3 Properties and viable microbial counts of different seed coating agents

[0058] Note: "-, +, ++, +++" represent the degree of goodness or badness of the property, where "-" indicates poor; "+" indicates good effect; "++" indicates good effect; "+++" indicates the best effect. Taking suspension stability as an example, the corresponding explanations are as follows: "-" indicates poor suspension stability; "+" indicates good suspension stability; "++" indicates good suspension stability; "+++" indicates the best suspension stability, which can form a stable mixed system.

[0059] As shown in Table 3, xanthan gum in Formulation 1 provides high viscosity and strong suspending power, preventing sedimentation; magnesium aluminum silicate provides a three-dimensional network structure and excellent thixotropy, preventing stratification during storage. For high concentrations of microbial agents or long shelf-life requirements, this formulation provides the most robust physical protection. 5% ethylene glycol provides antifreeze and moisture-retaining properties for the seed coating agent, broadening its adaptability and applicability, improving shelf life, microbial activity, and stability, and showing high application potential in cool northern regions. Therefore, Formulation 1 was selected as the seed coating agent adjuvant formulation for further research.

[0060] (2) The coating peeling rate of the three coating samples corresponding to treatment 1 to treatment 3 of formulation 1 is shown in the figure below. Figure 2 As shown, A represents the drop rate test results of a single coating agent sample under different drug ratios; B represents the drop rate test results of two coating agent samples under different drug ratios; and C represents the drop rate test results of three coating agent samples under different drug ratios.

[0061] The test tubes contain three groups of suspension seed coating samples treated with different drug-to-seed ratios. Figure 2It was found that there were no significant differences in the color of samples with different drug-to-seed ratios among the treatments, and the different drug-to-seed ratio treatment groups could not be effectively distinguished by appearance alone. Based on this, wheat seeds were coated with suspension seed coating agents with different drug-to-seed ratios, and germination rate was measured to explore the influence of the drug-to-seed ratio gradient on seed germination rate.

[0062] (3) An experiment was conducted using wheat germination rate as the evaluation index. The germination rate was calculated by counting the number of germinated wheat germinations 24 hours after germination. The results are as follows: Figure 3 As shown in Table 4. Figure 3 Different lowercase letters in the text indicate significant differences between treatments (P<0.05).

[0063] Table 4 Germination rate data for different treatments

[0064] Depend on Figure 2 It can be seen that the germination rate of seeds treated with any of the three methods was significantly improved compared with that treated with water (CK). When the ratio of drug to seed was 1:10, the germination rate of seeds in each treatment increased by 32.52%, 34.23%, and 32.52%, respectively; when the ratio was 1:25, the germination rate of seeds in each treatment increased by 39.35%, 32.95%, and 32.95%, respectively; when the ratio was 1:50, the germination rate of seeds in each treatment increased by 49.57%, 47.44%, and 56.39%, respectively; when the ratio was 1:75, the germination rate of seeds in each treatment increased by 48.29%, 52.98%, and 41.05%, respectively; and when the ratio was 1:100, the germination rate of seeds in each treatment increased by 44.03%, 34.23%, and 48.29%, respectively.

[0065] Using seed dressing agent-treated wheat seeds (treatment 1) as a control, at a seed-dressing agent ratio of 1:10, there was no significant difference in germination rate among the three treatments. At a seed-dressing agent ratio of 1:25, the germination rates of treatments 2 and 3 were both lower than that of treatment 1, decreasing by 4.59% compared to treatment 1. At a seed-dressing agent ratio of 1:50, there was no significant difference between treatments 1 and 2, but treatment 3 had the highest germination rate, increasing by 4.56% compared to treatment 1. At a seed-dressing agent ratio of 1:75, there was no significant difference between treatments 1 and 2, but both had higher germination rates than treatment 3. Compared to treatment 1, treatment 2 had a 3.16% higher germination rate, while treatment 3 had a 5.13% lower germination rate. At a seed-dressing agent ratio of 1:100, there was no significant difference between treatments 1 and 3, but treatment 2 had the lowest germination rate. Compared to treatment 1, treatment 3 had a 2.96% higher germination rate, while treatment 2 had a 6.8% lower germination rate.

[0066] Based on the above results, wheat seeds treated with two seed dressing agents—Treatment 2 (Bacillus belye SSF6 + Bacillus subtilis LU5 + seed dressing agent) and Treatment 3 (Bacillus belye SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed dressing agent)—achieved the best germination rates. Among these, wheat seeds with a herb-to-seed ratio of 1:75 in Treatment 2 and 1:50 in Treatment 3 showed the highest germination rates. Subsequently, the physicochemical properties of these two treatments were measured, and a wheat pot experiment was conducted to determine the physiological indicators of the wheat.

[0067] Example 4: Physicochemical properties of *Bacillus belyssus* SSF6 + *Bacillus subtilis* LU5 + seed dressing agent and *Bacillus belyssus* SSF6 + *Bacillus subtilis* LU5 + *Trichoderma longifolia* + seed dressing agent obtained from Example 3 were determined. 1. Quality determination of microbial seed coating agents Determination of pH value of seed coating agent: Weigh 0.5 g of microbial seed coating agent into a clean beaker, add 50 mL of distilled water to dilute it thoroughly, and use a pH meter to measure the acidity and alkalinity of the solution. Each sample is measured 3 times.

[0068] The pH values ​​of Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + seed dressing agent and Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed dressing agent are 7.39 and 7.38, respectively, which meet the requirements of GB 20287-2006 "Agricultural Microbial Agents" and NY 621-2002 "Duo·Fu·Ke Suspension Seed Dressing Agent".

[0069] 2. Determination of the uniformity of microbial seed coating agent coating Prepare 20 10 mL capped shaking tubes. Using a pipette, accurately measure 2 mL of 95% ethanol solution and add it to each tube sequentially. Then, randomly select 20 seeds coated with the test seed dressing agent and place them into each of the ethanol-coated shaking tubes. Seal the tubes and start the shaking device, continuously shaking for 15 minutes. After standing until a clear red solution layer appears in the tubes, use 95% ethanol as a blank control solution. Measure and record the absorbance (A) of each sample at a wavelength of 550 nm using a spectrometer.

[0070] The 20 measured absorbance data points were sorted in ascending order of value, and their average absorbance was calculated and denoted as Aa. Then, the coating uniformity X1 (%) of the sample was determined according to the following formula: X1=(n / 20)×100 (1) In the formula: n represents the total number of coated seeds with absorbance values ​​in the range of 0.7 to 1.3.

[0071] The uniformity of seed coating agent coverage directly reflects its distribution on the seed surface and is closely related to the overall control efficacy of the product. Uniform coating avoids localized insufficient application leading to ineffective pest and disease control or damage caused by excessive application. The average absorbance (Aa) of the Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + seed coating agent group (1:75 ratio) was 0.9935, and the coating uniformity (Xa) was 90%. The average absorbance (Ab) of the Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia C1 + seed coating agent group (1:50 ratio) was 0.9735, and the coating uniformity (Xb) was 95%. The results indicate that the seed coating uniformity is not less than 90%, meeting the standard requirements. The average absorbance (Aa) of the seed coating agent group without added microorganisms (1:50) was 0.991, and the coating uniformity (Xa) was 85%.

[0072] 3. Determination of coating shedding rate Two coated seed samples were precisely measured, each weighing strictly controlled to 10.00 ± 0.02 g, and transferred to cleaned conical flasks. 100 mL of 95% ethanol solution was accurately added to one of the flasks, and the flask opening was sealed with sealing film. The flask was then placed in an ultrasonic cleaner and run in vibration mode for 10 minutes to ensure complete dissolution and release of the seed coating agent adhering to the seed surface. The flask was removed and allowed to stand for 10 minutes. 10 mL of the supernatant was collected and transferred to a 50 mL volumetric flask, diluted to the mark with ethanol, and shaken well; this is solution A.

[0073] Take another seed sample and place it in a shaker for 10 min. Transfer the shaken seeds to a clean conical flask and prepare the test solution B using the standard procedure for solution A.

[0074] Using ethanol as a blank reference, the absorbance of solution B was measured using a spectrophotometer at a wavelength of 550 nm. The coating shedding rate X2 (%) was calculated using the following formula: X2 (2) Where: m0—the mass of seeds to be coated in solution A; m1 — the mass of seeds to be coated in solution B; A0—The absorbance of the measured solution A; A1—The absorbance of the measured solution B.

[0075] Determining the coating shedding rate of microbial seed coating agents can evaluate the stability of the drug film, the loss of active ingredients, and the product quality, so as to ensure the control effect and guide production applications. For the determination of the shedding rate of the seed coating agent samples, the measured data of the Bacillus velezensis SSF6 + Bacillus subtilis LU5 + seed coating agent group (drug-to-seed ratio 1:75) are m0 = 10.0002 g, m1 = 10.0003 g, A0 = 0.4, and A1 = 0.37. The calculated coating shedding rate is 7.5%; for the Bacillus velezensis SSF6 + Bacillus subtilis LU5 + Trichoderma longibrachiatum + seed coating agent group (drug-to-seed ratio 1:50), the measured data are m0 = 10.0002 g, m1 = 10.0001 g, A0 = 0.5, and A1 = 0.4618. The coating shedding rate is determined to be 7.65%. According to the results, the coating shedding rates of the two treatment preparations after being applied to wheat seeds are both not more than 8%. This meets the standard requirements.

[0076] 4. Determination of the low-temperature stability of microbial seed coating agents Put the microbial seed coating agent sample into a 20 mL stoppered graduated cylinder and store it in a 4°C refrigerator. After 7 days, observe whether there are any precipitates, stratifications, crystallizations, etc. If the test results meet the standard requirements, it is considered qualified. The same applies hereinafter.

[0077] The setting of this index aims to ensure the stability of the seed coating agent during low-temperature storage or transportation, prevent stratification, caking, or solidification under low-temperature conditions, avoid the inactivation of active ingredients and the destruction of the dosage form structure. After the seed coating agent samples are stored at 4°C for 7 days in the test, the results show that both samples have a small amount of stratification, no caking, and can return to their original state after shaking. This meets the standard.

[0078] 5. Determination of the hot storage stability of microbial seed coating agents Put the microbial seed coating agent sample into a 20 mL stoppered graduated cylinder and store it in a 54°C constant-temperature oven. After 7 days, observe whether there are any precipitates, stratifications, crystallizations, etc.

[0079] This index is formulated to ensure the ability of the seed coating agent to maintain the stability of active ingredients and performance under high-temperature storage conditions. Avoid the decomposition and inactivation of active ingredients (microbial agents, active substances) due to high temperature, and ensure the continuous发挥 of the effects of preventing and controlling pests and diseases and promoting crop growth after sowing. Prevent the generation of harmful by-products or changes in physical properties (such as stratification, caking) during hot storage. After the seed coating agent samples are stored at 54°C for 7 days in the test, the results show that both samples have a small amount of stratification, no caking, and can return to their original state after shaking. This meets the standard.

[0080] The determination results of the above physicochemical property indexes of the seed coating agent are shown in Table 5.

[0081] Table 5 Determination results of the physicochemical property indexes of the seed coating agent

[0082] Example 4: Effects of microbial seed coating agents on crop growth The treatments described below, namely Treatment 2 (Bacillus belye SSF6 + Bacillus subtilis LU5 + seed dressing agent) and Treatment 3 (Bacillus belye SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed dressing agent), are the Bacillus belye SSF6 + Bacillus subtilis LU5 + seed dressing agent and Treatment 3 (Bacillus belye SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed dressing agent) obtained from the screening in Example 3. The drug-to-species ratio is also the drug-to-species ratio obtained from the screening in Example 3.

[0083] The pot experiment consisted of one control group and three treatment groups: Treatment 1: Seeds treated with water as a blank control group; Treatment 2: Seeds treated with a mixture of Bacillus vesalis SSF6 + Bacillus subtilis LU5 + seed coating agent; Treatment 3: Seeds treated with a combination of Bacillus vesalis SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed coating agent; Treatment 4: Seeds irrigated with a mixed bacterial solution of Bacillus vesalis SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia. The nutrient substrate and ordinary soil were thoroughly mixed at a 1:1 (v / v) ratio and evenly packed into 15 cm × 15 cm × 20 cm plastic culture boxes. 20 coated seeds were sown evenly in each box. Three parallel biological experiments were conducted in both the control and treatment groups. Ten days after sowing, nine uniformly growing plants were randomly selected from each treatment group, carefully dug up, and gently separated into root and above-ground parts. The length of each part was measured, and then the fresh weight was weighed using an electronic balance. The separated plants were placed in an electric heating drying oven, first blanched at 105℃ for 30 minutes, and then dried at 80℃ until constant weight was achieved. The dry weight of the roots and aboveground parts was measured separately, and the root-to-shoot ratio (dry weight of underground parts / dry weight of aboveground parts) was calculated according to the formula.

[0084] The results are shown in Table 6 and Figure 4 As shown.

[0085] Table 6 Effects of seed coating agents on wheat dry weight and fresh weight

[0086] Note: Different lowercase letters indicate significant differences between treatments. P <0.05).

[0087] Depend on Figure 3It was found that, after treatment 2 (Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + seed coating agent), the wheat plant height and root length were significantly increased by 17.6% and 17.13% respectively compared with the water treatment. Treatment 3 (Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed coating agent) significantly increased the wheat plant height and root length by 25.92% and 26.42% respectively compared with the water treatment. Treatment 4 (Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia compound agent) irrigated wheat seeds significantly increased the plant height and root length by 24.33% and 23.51% respectively compared with the water treatment. Treatments 2 and 3 both significantly promoted wheat growth; therefore, the optimal ratio of herbicide to seed will be further discussed later.

[0088] When the ratio of herbicide to seed dressing agent in treatment 3 (Bacillus belyss SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed dressing agent) was 1:50, the average fresh weight of the coated wheat was 0.246 g, the average dry weight was 0.041 g, and the average root-to-shoot ratio was 49.15%, which were significantly higher than those in the water treatment by 23%, 73.91%, and 41%, respectively. When the ratio of herbicide to seed dressing agent in treatment 2 (Bacillus belyss SSF6 + Bacillus subtilis LU5 + seed dressing agent) was 1:75, the average fresh weight of the wheat was 0.241 g, the average dry weight was 0.033 g, and the average root-to-shoot ratio was 43.077%, which were significantly higher than those in the water treatment by 20%, 39.13%, and 14.08%, respectively. The herbicide-to-seed dressing agent ratio of 1:50 had a more significant growth-promoting effect compared to other ratios. In summary, the seed coatings prepared by this invention, consisting of Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + Trichoderma longifolia + seed coating agent and Bacillus vesiculosus SSF6 + Bacillus subtilis LU5 + seed coating agent, meet the standards in terms of pH value, coating uniformity, coating shedding rate, low-temperature stability, and thermal storage stability. The seed-to-drug ratio of the two microbial seed coating agents significantly increased the wheat germination rate, and both microbial seed coating agents significantly promoted wheat growth.

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A seed coating agent, characterized in that, include: Microbial agents and film-forming agents; The microbial agent includes any two or three of Bacillus vesiculosus, Bacillus subtilis, and Trichoderma longifolia.

2. The seed coating agent according to claim 1, characterized in that, The *Bacillus belyssus* includes *Bacillus belyssus* SSF6; the *Bacillus subtilis* includes *Bacillus subtilis* LU5; the *Trichoderma longifolia* includes *Trichoderma longifolia* C1, strain number CICC 40136.

3. The seed coating agent according to claim 1 or 2, characterized in that, When the microbial agent includes any two of Bacillus vesiculosus, Bacillus subtilis, and Trichoderma longifolia, the volume ratio of the two microbial agents is (1~2):(1~2); the bacterial activity of the seed liquid of Bacillus vesiculosus is 1×10⁻⁶. 10 CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of the *Trichoderma longifolia* was 1×10⁻⁶. 7 CFU / mL.

4. The seed coating agent according to claim 1 or 2, characterized in that, When the microbial inoculant includes Bacillus bellis, Bacillus subtilis, and Trichoderma longifolia, the volume ratio of the three microbial inoculants is 1:(1~2):(1~2); the viability of the seed liquid of Bacillus bellis is 1×10⁻⁶. 10 CFU / mL; the bacterial activity of the Bacillus subtilis was 1×10⁻⁶. 10 CFU / mL; the viability of the *Trichoderma longifolia* was 1×10⁻⁶. 7 CFU / mL.

5. The seed coating agent according to claim 1, characterized in that, The film-forming agents include: NNO, ethylene glycol, Permanent Red F4R, xanthan gum, magnesium aluminum silicate, and sodium benzoate.

6. The seed coating agent according to claim 5, characterized in that, The seed coating agent comprises, by mass-volume percentage, 3%~5% NNO, 4%~5% ethylene glycol, 0.2%~0.3% Permanent Red F4R, 0.3%~0.5% xanthan gum, 0.5%~1% magnesium aluminum silicate, and 0.07%~0.1% sodium benzoate.

7. A method for preparing the seed coating agent according to any one of claims 1 to 6, characterized in that, include: The seed liquids of any two or three of the Bacillus vesiculosus, Bacillus subtilis and Trichoderma longifolia are mixed to obtain microbial inoculants; Adding a film-forming agent to a microbial inoculant yields a seed coating agent.

8. The application of a seed coating agent according to any one of claims 1 to 6 or a seed coating agent prepared by the preparation method according to claim 7 in promoting wheat growth.

9. The application according to claim 8, characterized in that, The promotion of wheat growth includes at least one of the following: (1) Improve the germination rate of wheat seeds; (2) Increase the length of the aboveground part and / or the underground part of the seedling; (3) Increase the fresh weight and / or dry weight of seedlings; (4) Increase the root-to-shoot ratio of seedlings.

10. A method for promoting wheat growth, characterized in that, include: Seeds are coated with the seed coating agent prepared by any one of claims 1 to 6 or by the preparation method of claim 7; the ratio of seed coating agent to seed is 1 mL: (50~75) g.

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