Preparation method of pesticide fertilizer for preventing and treating soybean root rot

Abstract

The invention relates to the technical field of crop fertilizers, in particular to a preparation method of a pesticide fertilizer for preventing and treating soybean root rot, which comprises the following steps: S1, cultivating and pretreating biological bacteria; s2, preparing and storing a chemical agent; s3, preparing a fertilizer; s4, mixing and granulating the pesticide fertilizer; s5, post-treatment of the pesticide fertilizer; according to the scheme, chemical reagents and biological bacteria are combined, and the chemical reagents fludioxonil and metalaxyl are located on the outer layer of the pesticide fertilizer, can be rapidly dissolved and released after being applied into soil, respectively interfere signal transduction pathways of pathogenic bacteria and inhibit the activity of RNA polymerase, and synergistically reduce the cardinal number of the pathogenic bacteria and relieve the infection pressure; the biological bacteria bacillus subtilis and bacillus amyloliquefaciens are located on the inner layer of the pesticide fertilizer, after colonization in soil, growth of pathogenic bacteria is inhibited by generating extracellular enzyme, competing for nutrient space, secreting antibacterial substances, inducing plant resistance and the like, microecological balance of soybean root systems is maintained for a long time, and due to the multi-layer structural design of the pesticide fertilizer, all the components can play a role in order, so that the pesticide fertilizer has a good application prospect. The control effect on the soybean root rot is improved.

Description

Technical Field

[0001] The invention relates to the technical field of crop fertilizers, and in particular to a preparation method of a medicinal fertilizer for preventing and treating soybean root rot. Background Art

[0002] Soybean root rot is one of the most common and serious diseases in the soybean production process. It occurs widely in soybean-growing areas. Its pathogens are complex and diverse, including Pythium, Phytophthora, Fusarium and other pathogens. They can infect the roots of soybeans at various growth stages, causing root rot and poor plant growth. In severe cases, it can even cause the death of the entire plant, causing huge losses to soybean yield and quality, and greatly restricting the healthy development of the soybean industry.

[0003] Currently, soybean root rot is primarily controlled through chemical, biological, and agricultural measures. While commonly used chemical agents, such as fludioxonil and metalaxyl, have strong inhibitory and killing effects on pathogens in the short term, quickly controlling the spread of the disease, the long-term use of these agents alone can lead to numerous problems. Pathogens can easily develop resistance to these agents, resulting in a gradual decline in their effectiveness. Increasing dosages not only increase control costs but also exacerbate damage to the soil environment and ecosystems, leading to adverse consequences such as soil compaction, imbalanced microbial communities, and water pollution, posing a threat to sustainable agricultural development.

[0004] Pure biological control primarily utilizes beneficial microorganisms such as Bacillus subtilis and Bacillus amyloliquefaciens to inhibit the growth of pathogens. While biological control offers advantages such as environmental friendliness and minimal disturbance to soil ecosystems, its effectiveness is typically relatively slow, making it difficult to quickly control disease outbreaks. Furthermore, its effectiveness is easily affected by environmental factors, such as changes in soil temperature, humidity, and pH, which can affect the activity and colonization of beneficial microorganisms. This results in unstable control and an inability to provide sustained, reliable protection for soybean growth.

[0005] Although agricultural measures such as reasonable crop rotation and deep plowing and sun-drying can help reduce the accumulation of pathogens and improve the physical structure of the soil, these measures are complex to operate and have a long implementation cycle. In actual production, they are limited by factors such as land resources and planting habits, making them difficult to implement comprehensively and effectively. They often cannot solve the problem of soybean root rot alone.

[0006] Given the aforementioned shortcomings of existing control methods, a more comprehensive, efficient, and environmentally friendly approach is urgently needed. Therefore, we have developed a soybean root rot control fertilizer that combines the advantages of both chemical and biological control while providing comprehensive nutritional support for soybean growth. Summary of the Invention

[0007] In view of the deficiencies in the prior art, the present invention provides a method for preparing a medicinal fertilizer for preventing and treating soybean root rot, thereby solving the technical problems mentioned in the background technology.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] A method for preparing a medicinal fertilizer for preventing and treating soybean root rot, the method comprising the following steps:

[0010] S1. Cultivation and Pretreatment of Bio-Bacteria: Cultivation and pretreatment of bio-bacteria include operations on Bacillus subtilis and Bacillus amyloliquefaciens, both purchased from the China Center for Type Culture Collection. Both Bacillus subtilis and Bacillus amyloliquefaciens were prepared, inoculated, cultured, diluted, and stored refrigerated in their respective culture media.

[0011] S2. Preparation and Storage of Chemicals: Chemical reagents include fludioxonil and metalaxyl. When preparing the fludioxonil solution and metalaxyl solution, 10 g and 8 g, respectively, were weighed using an electronic balance and placed in a brown volumetric flask and beaker, respectively. Acetone and anhydrous ethanol were quantitatively added, respectively. Finally, the volumes were adjusted to 500 mL and 1000 mL, respectively, with distilled water, and transferred to brown bottles for storage in the dark.

[0012] S3. Fertilizer preparation: Fertilizers include organic-inorganic compound fertilizer and bio-organic fertilizer. The organic-inorganic compound fertilizer is prepared according to a predetermined ratio. 15% urea, 25% diammonium phosphate, 10% potassium sulfate, 0.2% borax, and 0.1% zinc sulfate are accurately weighed and thoroughly mixed with the previously mixed organic components. The bio-organic fertilizer is prepared by crushing plant straw and sawdust, adjusting the moisture content, inoculating EM bacteria, and regularly turning and mixing the compost.

[0013] S4. Mixing and granulating of medicinal fertilizers: The mixing and granulating of medicinal fertilizers comprises the following steps:

[0014] S41. The granulator is started and the fertilizer particles are formed: a small amount of about 5% of the weight of the fertilizer is added to the granulator to form a preliminary adhesion layer, which is slowly poured into the hopper of the granulator. The granulator is started and the speed is set to 30 rpm. The granulator drum is rotated to form spherical fertilizer particles attached to the bio-organic fertilizer;

[0015] S42. Addition of diluted Bacillus subtilis solution: The amount of diluted Bacillus subtilis solution to be added is determined by calculation and added to a metering device to precisely control the amount of solution added. The viable Bacillus subtilis count reaches 1×10 7 CFU / g. During the addition process, adjust the granulator speed to 35 rpm and continue stirring for 30 minutes. The bacterial solution is evenly attached to the fertilizer particles to form the prototype of biological bacterial fertilizer particles.

[0016] S43. Addition of diluted Bacillus amyloliquefaciens solution: Determine the amount of diluted Bacillus amyloliquefaciens solution to be added according to calculations, add it to the metering device, and accurately control the amount of solution added. The viable number of Bacillus amyloliquefaciens reaches 5×10 6 CFU / g, stirring was continued for 20 minutes during the addition process, and the Bacillus amyloliquefaciens was fully mixed with the previous spherical fertilizer to form complete biological fertilizer particles;

[0017] S44. Formation of a buffer layer: The organic - inorganic compound fertilizer is slowly poured into the hopper of the granulator, the granulator is started, the speed is set to 30 rpm, the organic - inorganic compound fertilizer is tumbled in the drum, and a buffer layer is formed on the outer surface of the complete bio-bacteria fertilizer particles;

[0018] S45. Addition and coating of fludioxonil solution: The 2% fludioxonil solution was loaded into a peristaltic pump container of a flow control device, the spray device of the granulator was turned on, and the fertilizer was evenly sprayed on the surface of the fertilizer particles in a mist at a mass ratio of 0.05% by weight of the fertilizer, and stirring was continued for 20 minutes to form a uniform preliminary chemical coating layer;

[0019] S46. Addition of metalaxyl solution and secondary coating: The 0.8% metalaxyl solution is loaded into the peristaltic pump container of the flow control device, the spray device of the granulator is turned on, and it is evenly sprayed on the surface of the fertilizer particles in a mist at a mass ratio of 0.03% by weight of the fertilizer, and stirred for 15 minutes to form a uniform and complete chemical coating layer;

[0020] S47. Adding the remaining bio-organic fertilizer: Finally, add the remaining bio-organic fertilizer (10% by weight of the total fertilizer) into the granulator and continue stirring for 40 minutes until the bio-organic fertilizer is evenly coated on the outside of the fertilizer granules.

[0021] S5. Post-processing of the fertilizer: After granulation, the fertilizer is naturally air-dried for 36 hours. After air-drying, the fertilizer is sieved with a vibrating screen to retain 2-4mm fertilizer particles. The fertilizer is then quality-tested. After the test is completed, the fertilizer is stored in a moisture-proof, ventilated and dark place.

[0022] In a possible implementation, the components and contents of the Bacillus subtilis culture medium are 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar, the solvent is 1000 mL of distilled water, and the pH value is 7.0-7.2.

[0023] In a possible implementation, the Bacillus amyloliquefaciens culture medium is prepared by adding 20 g / L of starch to the Bacillus subtilis culture medium, and other components and conditions are the same as those of the Bacillus subtilis culture medium.

[0024] In a possible implementation, during the post-processing of the fertilizer, the packaging is moisture-proof with a water vapor transmission rate of no more than 5g / m 2 For 24-hour plastic woven bags, the warehouse environment temperature should not be higher than 25℃, the relative humidity should not be higher than 70%, and the ventilation volume should not be less than 1000m 3 / h.

[0025] In a possible implementation, during the biological bacteria cultivation and pretreatment process, the inoculation amount of each strain is calculated and determined based on the number of viable bacteria in the inoculum to achieve a corresponding initial inoculation concentration.

[0026] Beneficial effects compared with existing technologies:

[0027] 1. In this scheme, chemical reagents and biological bacteria are combined to jointly prevent and control soybean root rot through different mechanisms of action. The chemical reagents fludioxonil and metalaxyl are in the outer layer of the fertilizer, and can be quickly dissolved and released after being applied to the soil, interfering with the pathogen signal transduction pathway and inhibiting the activity of RNA polymerase, respectively, synergistically reducing the pathogen base number and alleviating the infection pressure; the biological bacteria Bacillus subtilis and Bacillus amyloliquefaciens are located in the inner layer of the fertilizer. After colonization in the soil, they inhibit the growth of pathogens by producing extracellular enzymes, competing for nutrient space, secreting antibacterial substances, and inducing plant resistance, thereby maintaining the microecological balance of the soybean root system in the long term. The multi-layer structure design of the fertilizer enables each component to play its role in an orderly manner, improving the prevention and control effect of soybean root rot and providing full protection for soybean growth;

[0028] 2. In this program, the organic-inorganic compound fertilizer and bio-organic fertilizer in the medicated fertilizer provide macroelements such as nitrogen, phosphorus, and potassium, as well as trace elements such as boron and zinc, during soybean growth. These elements meet the nutritional needs of soybeans at different growth stages, participate in physiological processes such as photosynthesis, respiration, and protein synthesis, promote plant growth, and enhance resistance. The organic matter in the bio-organic fertilizer improves the physical structure of the soil, making it loose and breathable, which is conducive to root elongation and expansion, increasing the contact area between the root system and the soil, improving nutrient absorption efficiency, and at the same time enhancing the soil's ability to retain water and fertilizer, creating a good soil environment for soybeans.

[0029] 3. In this scheme, the effectiveness and stability of each component of the fertilizer are ensured through a series of steps such as the cultivation and pretreatment of biological bacteria, the preparation and preservation of chemical agents, the preparation of fertilizers, the mixing and granulation of fertilizers and post-processing. In actual application, comparative experiments have shown that the invented fertilizer can significantly reduce the incidence and disease index of soybean root rot, promote plant growth, and increase soybean yield. It has good economic benefits and has broad application prospects in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0031] Table 1 shows the incidence of soybeans at different growth stages;

[0032] Table 2 shows the disease index of soybean at different growth stages;

[0033] Table 3 shows the average plant height of soybean at different growth stages;

[0034] Table 4 shows the average stem diameter of soybean at different growth stages;

[0035] Table 5 shows the soybean yield per mu in different soybean growth groups;

[0036] Figure 1 This is a flow chart of the method for preparing medicinal fertilizer of the present invention;

[0037] Figure 2 The present invention is a flow chart of the mixed granulation process of the medicinal fertilizer. DETAILED DESCRIPTION

[0038] Preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention can be implemented in various forms, and therefore the present invention is not limited to the embodiments described below. In addition, in order to more clearly describe the present invention, components that are not related to the present invention will be omitted from the drawings.

[0039] The technical solution in the embodiments of the present application is to solve the problems of the above-mentioned background technology, and the overall idea is as follows:

[0040] Example 1:

[0041] This embodiment introduces a preparation method of a medicinal fertilizer for preventing and treating soybean root rot, comprising the following steps:

[0042] S1. Cultivation and pretreatment of biological bacteria

[0043] S11. Bacillus subtilis YJ001 operation process

[0044] S111. Source Confirmation and Characterization: Bacillus subtilis YJ001, designated CCTCC No. M20211461, was purchased from the China Center for Type Culture Collection, a professional microbial culture collection. Previous studies have shown that this strain produces a variety of extracellular enzymes, such as proteases and cellulases, during its growth and reproduction. These enzymes not only help it obtain nutrients in complex soil environments but also improve the soil structure in the soybean rhizosphere, enhancing soil permeability and water retention. Testing revealed that the effective viable count of the purchased Bacillus subtilis inoculum was ≥1×10 10 CFU / g, providing a high activity basis for subsequent cultivation.

[0045] S112. Culture medium preparation and inoculation: According to the recipe, weigh 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar, dissolve them in 1000 mL of distilled water, and adjust the pH value to between 7.0 and 7.2 using a precision pH meter to ensure that the composition and pH of the culture medium fully meet the growth requirements of the strain. Then sterilize the culture medium with high temperature and high pressure (121°C, 1.05 kg / cm 2 , 20-30 minutes), cool and set aside. In a sterile operating environment, inoculate the purchased Bacillus subtilis inoculum onto the prepared culture medium. The inoculation amount is calculated based on the number of viable bacteria in the inoculum to make the initial inoculation concentration reach 1×10 6 CFU / mL or so.

[0046] S113. Cultivation and proliferation: Place the inoculated culture medium in a constant temperature incubator at 30°C for 36 hours. During this period, closely monitor the growth of the strain to ensure its normal reproduction. After the incubation period, count the bacterial solution. At this time, the number of viable Bacillus subtilis in the bacterial solution can reach 5×10 8 CFU / mL or so, achieving the proliferation of biological bacteria.

[0047] S114. Bacterial Liquid Processing and Storage: After the incubation period, transfer the bacterial liquid to a high-speed centrifuge and centrifuge at 8000 rpm for 10 minutes to allow the bacterial pellet to settle at the bottom of the centrifuge tube. Carefully discard the supernatant and gently wash the pellet three times with sterile saline to remove impurities. Finally, concentrate the pellet to a bacterial content of 5 × 10 9 CFU / mL high concentration bacterial solution, accurately measure 10mL bacterial solution, and dilute it with 90mL sterile water at a ratio of 1:10 (v / v). During the dilution process, place the container containing the bacterial solution and sterile water on a magnetic stirrer and stir at a slow and steady speed for 30 minutes to ensure that the bacterial solution is evenly dispersed, and obtain a bacterial content of 5×10 8The diluted bacterial solution with a CFU / mL was transferred to a sterile and well-sealed container and stored in a 4°C refrigerator for future use.

[0048] S12. Operational procedures for Bacillus amyloliquefaciens NBL-AP73

[0049] S121. Source and Characteristics: This strain was also purchased from the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens NBL-AP73, with a collection number of CCTCC NO: M20241295. Bacillus amyloliquefaciens NBL-AP73 possesses strong antimicrobial properties and can secrete antimicrobial peptides, iturin, and other antimicrobial substances. These antimicrobial substances have significant antagonistic effects on the pathogens that cause soybean root rot, making it an important biological resource for the prevention and treatment of soybean root rot. The effective viable count of its microbial agent is ≥5×10 9 CFU / g.

[0050] S122. Medium adjustment and preparation: Based on the Bacillus subtilis medium formula, add 20 g / L of starch to promote the growth of Bacillus amyloliquefaciens. Accurately weigh, dissolve, adjust the pH to between 7.0 and 7.2, and sterilize (121°C, 1.05 kg / cm 2 , 20-30 minutes) and other steps to prepare the culture medium, and then cool it for use.

[0051] S123. Inoculation, culture and processing: Under sterile conditions, according to the calculated inoculum volume (so that the initial inoculum concentration reaches 8×10 5 CFU / mL) was inoculated with Bacillus amyloliquefaciens inoculum and cultured in a 30°C constant temperature incubator for 36 hours. After the culture was completed, the culture solution was centrifuged, washed, and concentrated according to the bacterial solution treatment method of Bacillus subtilis to obtain a bacterial content of 3×10 9 CFU / mL of bacterial solution.

[0052] S124. Dilution and refrigeration: Dilute the bacterial solution with sterile water at a ratio of 1:15 (v / v) and stir it with a magnetic stirrer for 20 minutes to obtain a bacterial content of 2×10 8 The diluted bacterial solution with a CFU / mL should be sealed and stored in a 4°C refrigerator to ensure the stability of the strain activity and be available for subsequent use at any time.

[0053] S2. Preparation and storage of chemical agents

[0054] S21. Preparation of Fludioxonil Solution

[0055] S211. Raw material preparation: Select industrial-grade fludioxonil technical with a purity of more than 98%, prepare a 500mL brown volumetric flask, analytical grade acetone, distilled water, and an electronic balance with an accuracy of 0.0001g.

[0056] S212. Dissolution process: On a well-ventilated chemical laboratory workbench, accurately weigh 10.0000g of fludioxonil technical drug and carefully transfer it to a 500mL brown volumetric flask. Next, use a graduated cylinder to accurately measure 100mL of analytical grade acetone and slowly add it to the volumetric flask. Immediately secure the stopper on the flask and shake the volumetric flask using an oscillator or manually for 15 minutes to ensure that the technical drug is completely dissolved in the acetone to form a uniform solution.

[0057] S213. Volume adjustment and storage: After the original drug is completely dissolved, dilute the solution to the 500mL mark with distilled water. Gently shake the solution and transfer the prepared 2% (w / v) fludioxonil solution to a brown reagent bottle. Seal the bottle with sealing film or cap and store it in a dark, cool and dry reagent cabinet to prevent light and humidity from affecting the chemical properties of the drug and ensure its stability and effectiveness.

[0058] S22. Preparation of Metalaxyl Solution

[0059] S221. Reagent and instrument preparation: Prepare metalaxyl technical with a purity of more than 95%, a 250mL beaker, a small amount of anhydrous ethanol (analytical grade), a 1000mL volumetric flask, distilled water, an electronic balance, and a graduated cylinder.

[0060] S222. Preliminary dissolution: In a chemical fume hood, accurately weigh 8.0000g of metalaxyl technical stock and place it in a 250mL beaker. Use a graduated cylinder to measure 5mL of anhydrous ethanol and add it to the beaker. Stir with a glass rod to completely dissolve the technical stock to form a clear solution.

[0061] S223. Transfer and adjust volume: Drain the solution in the beaker through a glass rod and slowly transfer it to a 1000mL volumetric flask. Wash the inner wall of the beaker with distilled water at least 3 times to ensure that all the original drug is transferred to the volumetric flask. Then adjust the volume to the 1000mL mark with distilled water. Shake gently to obtain a 0.8% (w / v) metalaxyl solution.

[0062] S224. Protect from light: Transfer the prepared metalaxyl solution into a brown reagent bottle, seal it and store it in a light-proof reagent cabinet to prevent light from causing decomposition of the agent and affecting its bactericidal effect.

[0063] S3. Fertilizer preparation

[0064] S31. Preparation of organic-inorganic compound fertilizer

[0065] S311. Raw material collection and processing: Collect decomposed chicken manure and soybean meal, ensure that the chicken manure has been fully treated with high-temperature composting, and the composting temperature reaches 55-65℃ for at least 15 days to kill pathogens, insect eggs and weed seeds. Mix the chicken manure and soybean meal in a ratio of 30% and 20% and set aside. At the same time, prepare the raw materials of urea, diammonium phosphate, potassium sulfate and trace elements boron and zinc to ensure their purity and quality meet the requirements.

[0066] S312. Fertilizer preparation: According to the predetermined proportions, accurately weigh 15% urea, 25% diammonium phosphate, 10% potassium sulfate, 0.2% boron (added in the form of borax) and 0.1% zinc (added in the form of zinc sulfate), and mix them thoroughly with the previously mixed organic ingredients. Use a blender to mix all the ingredients evenly to ensure that the various nutrients in the fertilizer are uniformly distributed to form a preliminary product of organic-inorganic compound fertilizer. The stirring time should be no less than 30 minutes to ensure mixing uniformity.

[0067] S313. Quality inspection and adjustment: Sample inspection is carried out on the prepared fertilizer, mainly to test whether its nitrogen, phosphorus, potassium content and organic matter content are in line with the expected ratio. Its nitrogen-phosphorus-potassium (NPK) ratio is precisely adjusted to 15-15-15, which can meet the needs of soybeans for macroelements at different growth stages. It also contains appropriate amounts of trace elements, such as calcium, magnesium, zinc, boron, etc. Although these trace elements are used in small amounts, they play an indispensable role in the physiological metabolism and disease resistance of soybeans. Conventional chemical analysis methods are used for testing. If content deviations are found, timely adjustments are made to ensure that the quality of the fertilizer is stable and reliable and can meet the nutritional needs of soybean growth.

[0068] S32. Preparation of bio-organic fertilizer

[0069] S321. Raw material pretreatment: Crush plant straw and wood chips to a particle size suitable for fermentation, generally 2-5 mm. Then, adjust the moisture content of the crushed raw materials to control the moisture content between 50% and 60% to create a good moisture environment for microbial fermentation. When adjusting the moisture, spray or gradually add water and stir to ensure that the moisture is evenly distributed.

[0070] S322. Inoculation and fermentation: The raw materials with adjusted moisture content are piled into a fermentation pile, and EM bacteria are introduced with an inoculation amount of 0.5%-1% of the weight of the raw materials. The inoculation amount is evenly spread on the surface of the fermentation pile according to the inoculation amount, and then the pile is turned and mixed to ensure that the microbial flora is in full contact with the raw materials. During the fermentation process, the temperature of the fermentation pile is monitored regularly, measured at least twice a day, the humidity is tested with a moisture meter, and the pH value is tested once every three days. The fermentation conditions are controlled by turning the pile once every three to five days, so that the fermentation process proceeds smoothly. The fermentation cycle is generally 2-3 months. When the temperature of the fermentation pile drops to close to the ambient temperature, the color of the material turns black, the texture is loose and there is no odor, it indicates that the fermentation and composting are complete.

[0071] S323. Composting and post-processing: The fermentation products are screened to remove incompletely decomposed impurities and larger particles, and a bio-organic fertilizer with an organic matter content of more than 40% is obtained. In addition to being rich in organic matter, it is also rich in a variety of beneficial microbial metabolites, such as polysaccharides and vitamins. On the one hand, these metabolites can provide additional carbon sources, nitrogen sources and other nutrients for beneficial microorganisms, promoting their colonization and reproduction in the soil; on the other hand, they can stimulate the growth of soybean roots, enhance root vitality, and improve the efficiency of soybean nutrient absorption, thereby enhancing the overall stress resistance of soybeans, including resistance to root rot.

[0072] S4. Mixing and granulation of pesticides and fertilizers

[0073] S41. Start the granulator and form fertilizer-attached particles: Check the equipment status of the drum granulator to ensure that the equipment is clean and free of residual materials, and that the motor, transmission device, agitator and other components are operating normally. First, add a small amount of bio-organic fertilizer (about 5% of the fertilizer weight) to the granulator to form a preliminary attachment layer, and slowly pour it into the hopper of the granulator. Start the granulator and set the speed to 30 rpm. Allow the bio-organic fertilizer to slowly roll in the drum to form spherical fertilizer-attached particles, preparing for the subsequent addition of agents and bacterial solution.

[0074] S42. Addition of diluted Bacillus subtilis solution: Remove the refrigerated diluted Bacillus subtilis solution, gently shake it, and place it into a container equipped with a metering device (metering accuracy of ±0.5 mL). Connect the container to the feed port of the granulator. Then, slowly add the diluted Bacillus subtilis solution, accurately controlling the amount of solution added using the metering device, so that the number of viable Bacillus subtilis in the final fertilizer product reaches 1×10 7 CFU / g. During the addition process, appropriately adjust the granulator speed to 35rpm and continue stirring for 30 minutes to ensure that the bacterial solution is evenly attached to the fertilizer particles, so that the biological bacteria and fertilizer are initially combined to form the prototype of biological bacteria fertilizer particles.

[0075] S43. Addition of diluted Bacillus amyloliquefaciens solution: Following the same operating procedures as for adding diluted Bacillus subtilis solution, add diluted Bacillus amyloliquefaciens solution to the granulator to increase the number of viable Bacillus amyloliquefaciens bacteria in the fertilizer to 5 × 10 6 CFU / g, and stirring was continued for 20 minutes during the addition process to fully mix the Bacillus amyloliquefaciens with the previously added ingredients, further improve the biological bacteria composition of the fertilizer granules, form biological bacteria fertilizer granules, and enhance its comprehensive prevention and control capabilities against soybean root rot.

[0076] S44. Formation of a buffer layer: Slowly pour the prepared organic-inorganic compound fertilizer into the hopper of the granulator, start the granulator, set the speed to 30 rpm, and allow the organic-inorganic compound fertilizer to slowly tumble in the drum, forming a buffer layer around the formed bio-fertilizer particles to prevent subsequent chemical reagents from directly contacting the bio-bacteria and affecting their activity.

[0077] S45. Addition and coating of fludioxonil solution: The prepared 2% fludioxonil solution is placed in a peristaltic pump container of a flow control device (flow accuracy of ±0.1 mL / min), and the infusion tube is connected to ensure that the end of the infusion tube is located above the granulator drum and can evenly cover the fertilizer particles. The peristaltic pump is started and the fludioxonil solution is slowly added dropwise at a ratio of 0.05% (w / w) of the fertilizer weight. At the same time, the spray device of the granulator is turned on (the spray pressure is 0.2-0.3 MPa) to spray the solution evenly on the surface of the fertilizer particles in the form of mist. During the addition process, the wetness of the fertilizer particles and the distribution of the solution are closely observed. Stirring is continued for 20 minutes to ensure that the fludioxonil solution fully coats each fertilizer particle to form a uniform preliminary chemical coating layer.

[0078] S46. Addition of metalaxyl solution and secondary coating: After the coating of the fludioxonil solution is completed, stop the peristaltic pump and the spray device, and slowly add 0.8% metalaxyl solution into the granulator through the peristaltic pump at a ratio of 0.03% (w / w) of the weight of the fertilizer in the same way. Turn on the spray device (the spray pressure is 0.2-0.3 MPa) and the stirring device again, and continue stirring for 15 minutes to evenly distribute the metalaxyl solution on the surface of the fertilizer particles coated with fludioxonil, further thicken the coating layer, and enhance the protection and bactericidal function of the agent.

[0079] S47. Addition of the remaining bio-organic fertilizer: Finally, slowly add the remaining bio-organic fertilizer (about 10% of the total fertilizer weight) into the granulator and continue stirring for 40 minutes to fully mix the bio-organic fertilizer and the medicinal fertilizer particles. The addition of the outermost layer of bio-organic fertilizer can not only supplement the organic matter content in the medicinal fertilizer, but also provide a more suitable living environment for the biological bacteria, promote their stable survival in the medicinal fertilizer and subsequent growth and reproduction in the soil, and at the same time help to improve the physical properties of the medicinal fertilizer particles, making them easier to store and apply.

[0080] S5. Post-processing of pesticides and fertilizers

[0081] S51. Natural air drying and preliminary screening: After the fertilizer and drug mixture is granulated, stop the granulator and carefully take out the fertilizer and drug particles from the granulator. Spread them evenly in a well-ventilated area (ventilation volume of not less than 500m 3 / h), in a dry and clean environment, away from direct sunlight and dust contamination. Air dry for 36 hours, turning the fertilizer granules every 6 hours to ensure even surface drying and prevent adhesion between particles. After air drying, use a vibrating screen with a pore size of 2-4mm to perform preliminary screening of the fertilizer granules to remove oversized or undersized particles. Retain fertilizer granules with a particle size between 2-4mm as the preliminary finished product, ready for the next step of quality testing.

[0082] S52. Quality Inspection and Adjustment: After preliminary screening, the finished product of the medicinal fertilizer undergoes comprehensive quality inspection, including but not limited to indicators such as active ingredient content (such as chemical agent content and viable bacterial count), nutrient content (nitrogen, phosphorus, potassium, and trace elements), moisture content, granule strength, and pH value. Chemical agent content is determined using high-performance liquid chromatography (HPLC), viable bacterial counts are determined using the plate count method, nutrient content is determined using conventional chemical analysis methods, moisture content is determined using the drying method (drying the sample in a 105°C oven to constant weight), granule strength is determined using a pressure testing machine (applying pressure until the granules break and recording the maximum pressure value), and pH is determined using a pH meter. If any indicator is found to not meet quality standards during testing, the cause will be promptly analyzed and adjustments will be made, such as supplementing missing ingredients, adjusting moisture content, or remixing, to ensure that the quality of the finished medicinal fertilizer fully meets requirements.

[0083] S53. Packaging and storage: The finished product of the fertilizer that has passed the quality inspection shall be stored in a moisture-proof container (the moisture-proof performance shall be such that the water vapor transmission rate is not higher than 5g / m 2The finished product of the fertilizer should be stored in a cool (temperature not higher than 25℃) and dry (relative humidity not higher than 70%) warehouse. The warehouse floor should be treated with moisture-proof treatment and covered with moisture-proof mats with a thickness of not less than 5mm. The warehouse should also have good ventilation facilities (ventilation volume not less than 1000m 3 / h), avoid direct sunlight and high temperature and humidity environments. During storage, regularly check the storage status of the fertilizer once a month to prevent moisture, deterioration, or other contamination to ensure the stability of the fertilizer's quality and a shelf life of more than 12 months.

[0084] In summary, the outermost layer of bio-organic fertilizer modifies and fertilizes the applied soil, creating conditions for the subsequent growth and reproduction of biological bacteria. The second layer of chemical agents, fludioxonil and metalaxyl, then form the first line of defense against pathogens. When applied to the soil, the moisture in the soil rapidly dissolves and releases the outer layer of chemicals. Fludioxonil, with its unique chemical structure, precisely interferes with the pathogen's signaling pathways. Pathogens rely on complex signaling systems to regulate physiological processes such as growth, reproduction, and pathogenicity. Fludioxonil's intervention blocks these signaling pathways, preventing them from properly sensing and responding to environmental signals, thereby inhibiting their growth and reproduction. Metalaxyl, on the other hand, targets the pathogen's RNA polymerase, which plays a key catalytic role in protein synthesis. By specifically inhibiting this enzyme, metalaxyl prevents the pathogen from synthesizing proteins, thereby curbing its proliferation at its source. These two chemical agents work synergistically to form a powerful inhibitory force, rapidly reducing the base number of root rot bacteria in the soil in a short period of time, significantly reducing the initial infection pressure of pathogens on soybean roots, and providing key protection for the early growth of soybeans.

[0085] Afterward, the organic-inorganic compound fertilizer and some bio-organic fertilizers, located in the middle layer of the fertilizer, play a crucial role in the mid-stage after the fertilizer is applied to the soil. The organic-inorganic compound fertilizers contain macronutrients such as nitrogen, phosphorus, and potassium, as well as trace elements such as boron and zinc, which are essential nutrients for soybean growth. At different stages of soybean growth, these nutrients are absorbed and utilized by the roots and participate in physiological processes such as photosynthesis, respiration, and protein synthesis. This provides an adequate material foundation for the growth and development of the soybean plant, promoting healthy growth and enhancing its resistance. Furthermore, the organic matter in the bio-organic fertilizer improves the soil's physical structure, making it more porous and breathable, which facilitates root elongation and expansion, increases root-soil contact area, and improves nutrient absorption efficiency. Organic matter also enhances the soil's ability to retain water and nutrients, reduces nutrient loss, and creates a stable and fertile soil environment for soybean growth.

[0086] Finally, the Bacillus subtilis and Bacillus amyloliquefaciens bacteria, located in the innermost layer of the fertilizer, are key to long-term control. As the fertilizer gradually decomposes in the soil, the inner bacterial communities are released and colonize the soybean rhizosphere. Extracellular enzymes such as proteases and cellulases produced by Bacillus subtilis play a multifaceted role. Firstly, these enzymes break down residual organic macromolecules in the soil, converting them into smaller nutrients such as amino acids and glucose, which are readily absorbed and utilized by soybean roots, improving nutrient availability. Secondly, enzymatic reactions help improve soil aggregate structure, increase soil porosity, and further optimize soil aeration and water permeability, creating more favorable conditions for root growth and microbial activity. Furthermore, during its growth and reproduction, Bacillus subtilis indirectly and directly inhibits the growth of root rot pathogens through various means, including competing for nutrient space, secreting antimicrobial substances, and inducing plant resistance. Antimicrobial peptides and iturin, among other antimicrobial substances secreted by Bacillus amyloliquefaciens, possess strong antibacterial activity. They can directly act on the cell membranes, cell walls, or metabolic processes of pathogens, disrupting their cellular structure and physiological functions, thereby effectively inhibiting their growth and reproduction. The synergistic effect of these two agents maintains the long-term microecological balance surrounding soybean roots, preventing a resurgence of pathogens and continuously safeguarding the health of soybean roots, providing stable protection throughout the soybean growth cycle.

[0087] Example 2:

[0088] On the basis of Example 1, this example further explores the preventive and control effects of the prepared fertilizer on soybean root rot and its comprehensive impact on soybean growth by setting up a control group for comparative experiments.

[0089] 1. Experimental Grouping and Preparation

[0090] 1. Setting up a control group: According to the method for preparing the medicated fertilizer in Example 1, the formula was modified to prepare a chemical group containing only the chemical reagents fludioxonil and metalaxyl, a biocontrol group containing only the biological bacteria Bacillus subtilis and Bacillus amyloliquefaciens, and a blank group without using any medicated fertilizer.

[0091] 2. Soybean Variety Selection: The "Heihe 43" soybean variety, which is widely cultivated locally and is sensitive to root rot, was selected. This variety has strong adaptability and stable yields. Its growth characteristics and response to root rot are representative of the local area and can better reflect the effectiveness of pesticide and fertilizer application in actual production.

[0092] 3. Experimental plot selection: A plot with black loam, medium fertility, flat terrain and corn as the previous crop was selected as the experimental site. The plot was divided into four areas, which were used as the experimental group (medicine fertilizer prepared in Example 1), the biocontrol group (containing no chemical agents, only containing Bacillus subtilis and Bacillus amyloliquefaciens in Example 1), the chemical group (containing no biological bacteria, only containing fludioxonil and metalaxyl in Example 1) and the blank control group (no medicine fertilizer was applied). Three replicate plots were set in each area, with a plot area of 30 square meters, and a 0.5 meter wide isolation belt was set between the plots to avoid mutual interference between the treatments.

[0093] 4. Sowing time: Based on local climate conditions and soybean growth habits, the sowing time was determined to be May 10. At this time, the soil temperature and humidity are suitable for soybean seed germination and seedling growth, which can provide relatively consistent starting conditions for the experiment.

[0094] 2. Planting and Management

[0095] 1. Fertilization: Before sowing, deep plowing (depth of 20-25 cm) and leveling were performed on the soil of each plot to ensure that the soil was loose and finely broken, which was conducive to soybean seed germination and root growth. The experimental group applied fertilizer according to the standard of 40 kg of the fertilizer prepared by Example 1 per mu; the biocontrol group adjusted the amount of fertilizer applied according to the number of viable bacteria of the biological bacteria so that it was equivalent to the biological bacteria content in the experimental group, and applied 35 kg per mu; the chemical group adjusted the amount of fertilizer applied according to the content of the chemical agent so that it was identical to the content of the effective ingredient of the chemical agent in the experimental group, and applied 30 kg per mu; the blank control group did not apply any fertilizer. The fertilization method was strip application, and fertilizer was evenly applied in the sowing furrow, and then covered with a thin layer of soil (thickness of 3-5 cm) before sowing.

[0096] 2. Sowing: Use manual sowing, with row spacing of 60 cm and plant spacing of 10 cm. Sow 3-4 seeds per hole at a sowing depth of 3-5 cm. Cover with soil immediately after sowing to ensure close contact between the seeds and the soil, reduce soil voids, maintain soil moisture, and create good conditions for seed germination.

[0097] 3. Irrigation: Irrigate appropriately based on soybean growth needs. During the seedling stage, maintain soil moisture at 60%-70%. During the flowering and pod-setting stages, water requirements are higher, so ensure soil moisture is between 70% and 80%. Each irrigation should soak the soil to a depth of 20-30 cm. Avoid waterlogging in the field to prevent diseases caused by excessive humidity.

[0098] 3. Weeding: Use a combination of manual and chemical weeding. In the early stages of soybean growth, combine manual weeding with tillage 2-3 times at a depth of 3-5 cm. This not only removes weeds but also loosens the soil and promotes root growth. In the middle and late stages of soybean growth, select a highly effective, low-toxic, and low-residue herbicide for chemical weed control, depending on weed growth. Strictly follow the instructions for use to control the dosage and application schedule to avoid pesticide damage.

[0099] 3. Data Recording

[0100] 1. Morbidity Survey: Root rot incidence surveys were conducted in each plot during the seedling stage (June 15), flowering stage (July 20), and podding stage (August 25) of soybean growth. A five-point sampling method was used in each plot. Fifteen soybean plants were randomly selected at each point. The roots of the plants were carefully dug out and washed with clean water. The root disease was carefully observed, the number of affected plants was recorded, and the incidence was calculated. The incidence calculation formula is:

[0101] Table 1: Incidence of soybean disease at different growth stages

[0102] Seedling stage (%) Flowering period (%) Pod-setting period (%) Experimental group 3.3 8.9 13.3 Chemistry Group 6.7 13.3 20 Biocontrol Group 8.9 17.8 26.7 Blank group 15.6 26.7 37.8

[0103] The results showed that throughout the growth cycle, the incidence of the experimental group was significantly lower than that of the other three groups, and the increase in incidence was relatively small as the soybean growth progressed. This shows that the medicated fertilizer of the present invention has obvious advantages in preventing soybean root rot and can effectively reduce the risk of pathogen infection.

[0104] 2. Disease index assessment: At the same time as the morbidity survey, the disease index of the diseased plants is assessed. The disease grading standards are as follows: Level 0, no obvious symptoms in the soybean root system; Level 1, partial discoloration of the soybean main root or lateral root, and the lesion area is less than 10% of the total root area; Level 2, obvious discoloration of the soybean main root or lateral root, and the lesion area accounts for 10%-25% of the total root area; Level 3, most of the soybean main root or lateral root is discolored, and the lesion area accounts for 25%-50% of the total root area; Level 4, severe discoloration and rot of the soybean main root or lateral root, and the lesion area is greater than 50% of the total root area. The disease index calculation formula is:

[0105] Table 2: Disease index of soybean at different growth stages

[0106] Seedling stage Flowering period pod-setting period Experimental group 1.7 4.4 6.7 Chemistry Group 3.3 6.7 10.0 Biocontrol Group 4.4 8.9 13.3 Blank group 7.8 13.3 18.9

[0107] The changing trend of the disease index is consistent with the disease incidence rate, which further confirms that the medicated fertilizer of the present invention has a strong inhibitory effect on the development of soybean root rot and can effectively reduce the degree of damage caused by the disease to soybean plants.

[0108] 3. Plant height and stem diameter measurement: During the seedling stage (June 15), flowering stage (July 20), and podding stage (August 25) of soybean growth, 10 soybean plants were randomly selected from each plot. The plant height (the distance from the ground to the top growth point of the plant) was measured using a ruler, and the stem diameter (the diameter of the stem at 5 cm from the ground) was measured using a vernier caliper. The measurement data were recorded and the average value was calculated.

[0109] Table 3: Average plant height of soybean at different growth stages

[0110] Seedling stage (cm) Flowering period (cm) Pod-setting period (cm) Experimental group 15.2 45.6 70.5 Chemistry Group 14.0 42.0 68.0 Biocontrol Group 13.5 40.5 65.0 Blank group 12.0 35.0 58.0

[0111] Table 4: Average stem diameter of soybean at different growth stages

[0112] Seedling stage (cm) Flowering period (cm) Pod-setting period (cm) Experimental group 0.35 0.65 0.85 Chemistry Group 0.33 0.60 0.80 Biocontrol Group 0.32 0.58 0.75 Blank group 0.30 0.52 0.70

[0113] The data showed that at all stages of soybean growth, the experimental group had superior plant height and stem diameter compared to the other three groups. This indicates that the medicated fertilizer of the present invention not only has a preventive and therapeutic effect on root rot, but also promotes the growth and development of soybean plants, making them stronger and enhancing their adaptability to the environment and resistance to stress.

[0114] 4. Yield Determination: After soybeans matured (September 20), each plot was harvested manually. The soybean plants were cut from the roots and air-dried in the field for 3-5 days to allow the pods to fully dry before threshing. The actual yield of each plot was measured using an electronic scale and converted to yield per mu.

[0115] Table 5: Soybean yield per mu in different soybean growth groups

[0116] Experimental group Chemistry Group Biocontrol Group Blank group Yield per mu (kg) 280 240 220 180

[0117] The yield of the experimental group was significantly higher than that of the other three groups, which was consistent with the results of the incidence rate and disease index. This shows that the medicated fertilizer of the present invention effectively prevents and treats root rot, improves soybean growth, and thus significantly increases soybean yield, with good economic benefits.

[0118] In summary, through comparative experiments with the biological control group, chemical group and blank group, the soybean root rot prevention and treatment fertilizer prepared by the present invention showed significant advantages in terms of morbidity control, disease index reduction, plant growth promotion and yield increase. The main reason is the synergistic effect of biological bacteria and chemical agents in the fertilizer, as well as the comprehensive support of fertilizer components for soybean growth. After the biological bacteria colonize in the soil, they inhibit the growth of pathogens through various mechanisms and improve the soil microecological environment; chemical agents quickly exert a bactericidal effect in the early stage and reduce the base number of pathogens; fertilizers provide sufficient nutrients for soybean growth, promote plant growth and development, and enhance its stress resistance.

[0119] Finally, it should be noted that the above embodiments are merely examples for the purpose of illustrating the present invention and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above description are possible. It is not necessary and impossible to provide an exhaustive list of all embodiments. However, obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing a medicinal fertilizer for preventing and treating soybean root rot, characterized in that: The method comprises the following steps: S1. Cultivation and Pretreatment of Bio-Bacteria: Cultivation and pretreatment of bio-bacteria include operations on Bacillus subtilis and Bacillus amyloliquefaciens, both purchased from the China Center for Type Culture Collection. Both Bacillus subtilis and Bacillus amyloliquefaciens were prepared, inoculated, cultured, diluted, and stored refrigerated in their respective culture media. S2. Preparation and Storage of Chemicals: Chemical reagents include fludioxonil and metalaxyl. When preparing the fludioxonil solution and metalaxyl solution, 10 g and 8 g, respectively, were weighed using an electronic balance and placed in a brown volumetric flask and beaker, respectively. Acetone and anhydrous ethanol were quantitatively added, respectively. Finally, the volumes were adjusted to 500 mL and 1000 mL, respectively, with distilled water, and transferred to brown bottles for storage in the dark. S3. Fertilizer preparation: Fertilizers include organic-inorganic compound fertilizers and bio-organic fertilizers. The organic-inorganic compound fertilizer is prepared by accurately weighing 15% urea, 25% diammonium phosphate, 10% potassium sulfate, 0.2% borax, and 0.1% zinc sulfate according to a predetermined ratio and thoroughly mixing them with the previously mixed organic components. The bio-organic fertilizer is prepared by crushing plant straw and sawdust, adjusting the moisture content, inoculating EM bacteria, and regularly turning and mixing the compost. S4. Mixing and granulating of medicinal fertilizers: The mixing and granulating of medicinal fertilizers comprises the following steps: S41. The granulator is started and the fertilizer particles are formed: a small amount of about 5% of the weight of the fertilizer is added to the granulator to form a preliminary adhesion layer, which is slowly poured into the hopper of the granulator. The granulator is started and the speed is set to 30 rpm. The granulator drum is rotated to form spherical fertilizer particles attached to the bio-organic fertilizer; S42. Addition of diluted Bacillus subtilis solution: The amount of diluted Bacillus subtilis solution to be added is determined by calculation and added to a metering device to precisely control the amount of solution added. The viable Bacillus subtilis count reaches 1×10 7 CFU / g. During the addition process, adjust the granulator speed to 35 rpm and continue stirring for 30 minutes. The bacterial solution is evenly attached to the fertilizer particles to form the prototype of biological bacterial fertilizer particles. S43. Addition of diluted Bacillus amyloliquefaciens solution: Determine the amount of diluted Bacillus amyloliquefaciens solution to be added according to calculations, add it to the metering device, and accurately control the amount of solution added. The viable number of Bacillus amyloliquefaciens reaches 5×10 6 CFU / g, stirring was continued for 20 minutes during the addition process, and the Bacillus amyloliquefaciens was fully mixed with the previous spherical fertilizer to form complete biological fertilizer particles; S44. Formation of a buffer layer: The organic - inorganic compound fertilizer is slowly poured into the hopper of the granulator, the granulator is started, the speed is set to 30 rpm, the organic - inorganic compound fertilizer is tumbled in the drum, and a buffer layer is formed on the outer surface of the complete bio-bacteria fertilizer particles; S45. Addition and coating of fludioxonil solution: The 2% fludioxonil solution was loaded into a peristaltic pump container of a flow control device, the spray device of the granulator was turned on, and the fertilizer was evenly sprayed on the surface of the fertilizer particles in a mist at a mass ratio of 0.05% by weight of the fertilizer, and stirring was continued for 20 minutes to form a uniform preliminary chemical coating layer; S46. Addition of metalaxyl solution and secondary coating: The 0.8% metalaxyl solution is loaded into the peristaltic pump container of the flow control device, the spray device of the granulator is turned on, and it is evenly sprayed on the surface of the fertilizer particles in a mist at a mass ratio of 0.03% by weight of the fertilizer, and stirred for 15 minutes to form a uniform and complete chemical coating layer; S47. Adding the remaining bio-organic fertilizer: Finally, add the remaining bio-organic fertilizer (10% by weight of the total fertilizer) into the granulator and continue stirring for 40 minutes until the bio-organic fertilizer is evenly coated on the outside of the fertilizer granules. S5. Post-processing of the fertilizer: After granulation, the fertilizer is naturally air-dried for 36 hours. After air-drying, the fertilizer is sieved with a vibrating screen to retain 2-4mm fertilizer particles. The fertilizer is then quality-tested. After the test is completed, the fertilizer is stored in a moisture-proof, ventilated and dark place.

2. The method for preparing a soybean root rot prevention and treatment medicinal fertilizer according to claim 1, wherein: The components and contents of the Bacillus subtilis culture medium are 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar, the solvent is 1000 mL of distilled water, and the pH value is 7.0-7.

2.

3. The method for preparing a soybean root rot prevention and treatment medicinal fertilizer according to claim 1, wherein: The Bacillus amyloliquefaciens culture medium is prepared by adding 20 g / L of starch to the Bacillus subtilis culture medium, and other components and conditions are the same as those of the Bacillus subtilis culture medium.

4. The method for preparing a soybean root rot prevention and treatment medicinal fertilizer according to claim 1, wherein: During the post-processing of the fertilizer, the packaging is moisture-proof and has a water vapor transmission rate of no more than 5g / m 2 For 24-hour plastic woven bags, the warehouse environment temperature should not be higher than 25℃, the relative humidity should not be higher than 70%, and the ventilation volume should not be less than 1000m 3 / h.

5. The method for preparing a soybean root rot prevention and treatment medicinal fertilizer according to claim 1, wherein: During the biological bacteria cultivation and pretreatment process, the inoculation amount of each strain is calculated and determined based on the number of viable bacteria in the inoculum to achieve the corresponding initial inoculation concentration.

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