A composite bacillus microorganism humic acid water-soluble fertilizer and a preparation method thereof

Abstract

The application discloses a kind of composite bacillus microorganism humic acid water-soluble fertilizer and preparation method thereof, it is related to the field of composite bacillus microorganism water-soluble fertilizer, raw material includes the sterile metabolite filtrate of green needle pseudomonas MCNB07405, composite bacillus bacterial powder, water-soluble nutrient medium;During preparation, first green needle pseudomonas is expanded fermentation, and the sterile metabolite filtrate is obtained by filtering sterilization, with the filtrate as only fermentation medium, access the compound strain of bacillus licheniformis and bacillus subtilis to carry out secondary fermentation, after fermentation, add water-soluble nutrient medium and mix thoroughly, and the finished product is prepared by low-temperature spray drying.The application effectively avoids the interspecific antagonism problem generated by direct compounding of different biocontrol strains, has the triple synergistic effect of plasmodiophora brassicae inhibition, rhizosphere microecological protection and crop growth nutrient supply, long persistence, convenient to use, low water-insoluble content, suitable for a variety of agricultural operations such as irrigation root flushing and drip irrigation.

Description

Technical Field

[0001] This invention belongs to the field of compound Bacillus microbial water-soluble fertilizer, and in particular to a compound Bacillus microbial humic acid water-soluble fertilizer and its preparation method. Background Technology

[0002] Cruciferous crops are among the most widely planted and economically valuable vegetable categories in my country. In recent years, the area affected by clubroot disease in cruciferous crops caused by *Cladosporium brassicum* has been expanding year by year. Severely affected plots can experience yield reductions of over 80%, which has become a core obstacle to improving the profitability of cruciferous crop cultivation. The industry urgently needs specialized inputs that combine green disease prevention with growth promotion and yield increase functions.

[0003] The current mainstream technologies for the prevention and control of clubroot disease in cruciferous plants and related crop nutrient supply are divided into two categories: The first category is ordinary microbial water-soluble fertilizers with added compound Bacillus. The core principle is to use the rhizosphere colonization ability of strains such as Bacillus subtilis and Bacillus licheniformis to inhibit the reproduction of soil-borne pathogens. At the same time, it is combined with water-soluble nutrient substrates to supplement the nitrogen, phosphorus, potassium and trace elements required for crop growth. This type of product is convenient to apply and is suitable for various agricultural operations such as drip irrigation and fertigation. It has been widely promoted and applied throughout the country. However, because Bacillus itself has extremely low inhibitory activity against dormant spores of Bacillus brassicae, the actual control efficacy against clubroot disease is usually less than 30%, which cannot meet the control needs of plots with continuous cropping.

[0004] Chinese patent authorization announcement number CN114395510B discloses the technical solution of *Pseudomonas aeruginosa* strain MCNB07405 and the biocontrol agent prepared therefrom. The technical problem addressed is the weak inhibitory activity and unstable efficacy of existing biocontrol strains against clubroot disease in cruciferous plants. However, it does not consider the significant influence of soil pH, indigenous microbial communities, temperature, and humidity on the colonization rate of live bacteria preparations in soil. The effective period of this strain preparation applied alone is only 15-20 days, requiring frequent repeated application to maintain efficacy. Furthermore, this type of preparation only has a single disease-preventing function and cannot provide the nutrients needed for crop growth. Growers need to apply additional water-soluble fertilizers containing macronutrients, increasing labor costs and easily leading to a decrease in bacterial activity during the mixing of the preparation and fertilizer. In addition, when this strain is directly combined with commonly used compound Bacillus, there is a significant interspecies antagonism, which greatly reduces the biocontrol activity of both, failing to achieve a synergistic effect of disease prevention and growth promotion.

[0005] In view of the shortcomings of the existing technologies mentioned above, such as poor efficacy stability, single function, and insufficient strain compatibility, there are currently no special microbial water-soluble fertilizer products in the industry that are suitable for the prevention and control of cruciferous clubroot disease. There is an urgent need to carry out relevant technology research and development to fill the market gap. Summary of the Invention

[0006] To address the shortcomings of existing cruciferous clubroot disease control technologies, such as poor efficacy, antagonistic interactions between biocontrol strains, limited functionality, and short duration of effectiveness, this invention provides a compound Bacillus microbial humic acid water-soluble fertilizer and its preparation method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A compound Bacillus microbial water-soluble fertilizer is a special microbial water-soluble fertilizer suitable for the prevention and control of clubroot disease and the promotion of growth in cruciferous crops. The components include sterile metabolite filtrate of Pseudomonas aeruginosa biocontrol bacterium MCNB07405, compound Bacillus inoculant, and water-soluble nutrient matrix.

[0009] The sterile metabolite filtrate of the Pseudomonas aeruginosa biocontrol bacterium MCNB07405 accounts for 60% to 75% of the total fertilizer mass, the compound Bacillus inoculant accounts for 15% to 25% of the total fertilizer mass, and the water-soluble nutrient substrate accounts for 8% to 15% of the total fertilizer mass;

[0010] The compound Bacillus was prepared by fermentation using the sterile metabolite filtrate of the Pseudomonas aeruginosa biocontrol bacterium MCNB07405 as the sole culture medium, without adding any exogenous carbon source, nitrogen source or growth factor during the fermentation process.

[0011] Preferably, the compound Bacillus is composed of Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus, all of which are strains permitted for addition to microbial fertilizers as published by the Ministry of Agriculture and Rural Affairs, and the ratio of viable bacteria among the three is 1~3:1~2:1; the total viable bacteria count of the compound Bacillus in the water-soluble fertilizer is not less than 1×10^9 CFU / mL, of which the single viable bacteria count of Bacillus subtilis is not less than 3×10^8 CFU / mL, the single viable bacteria count of Bacillus licheniformis is not less than 2×10^8 CFU / mL, and the single viable bacteria count of Bacillus mucilaginosus is not less than 1×10^8 CFU / mL.

[0012] Preferably, the sterile metabolite filtrate of the biocontrol bacterium *Pseudomonas aeruginosa* MCNB07405 exhibits an inhibition rate of not less than 60% against the germination of dormant spores of *Plasmodium brassicae*, determined using the spore germination counting method in a 96-well plate; the soluble protein content in the filtrate is not less than 120 mg / L, determined using the Coomassie brilliant blue method; the phenolic antibacterial substance content in the filtrate is not less than 80 mg / L, determined using the Folin-Ciocalteu method; the pH of the filtrate is 6.2–6.8; and no single colonies are detected after the filtrate is plated on LB agar plates and incubated upside down at 28°C for 72 hours.

[0013] Preferably, the water-soluble nutrient matrix comprises 12%–20% macroelements, 2%–5% humic acid, and 0.5%–1.5% chelated trace elements by mass. The macroelements consist of nitrogen, phosphorus pentoxide, and potassium oxide in a mass ratio of 2:1:3. Nitrogen is supported by urea and potassium nitrate, phosphorus pentoxide by potassium dihydrogen phosphate, and potassium oxide by one of potassium nitrate, potassium dihydrogen phosphate, potassium humate, or potassium dihydrogen phosphate. The humic acid is mineral-derived fulvic acid with a molecular weight of less than 3000 Da. The chelated trace elements consist of EDTA-chelated iron, EDTA-chelated zinc, sodium octaborate tetrahydrate, and ammonium molybdate in a mass ratio of 2:2:1:0.5. The water-insoluble content of the water-soluble nutrient matrix is ​​less than 0.5%, which meets the national standard requirements for water-soluble fertilizers containing macroelements.

[0014] Preferably, a method for preparing the compound Bacillus microbial water-soluble fertilizer includes the following steps:

[0015] S1. The biocontrol strain of Pseudomonas aeruginosa MCNB07405, preserved in glycerol, was streaked onto LB solid plates and incubated upside down at 28°C for 24 h to obtain activated single colonies. Single colonies were picked and inoculated into LB liquid seed medium and cultured at 28°C and 180 r / min for 12 h to obtain primary seed culture. The primary seed culture was inoculated into expansion medium at an inoculation rate of 1% for expansion culture. The culture was centrifuged at 4°C and 8000 r / min for 15 min to remove bacterial cells. The supernatant was collected and sterilized by filtration to obtain sterile metabolite filtrate of Pseudomonas aeruginosa.

[0016] S2. After activating Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus to the logarithmic growth phase, they are mixed in proportion to obtain a compound Bacillus seed solution. The compound Bacillus seed solution is inoculated into the sterile metabolite filtrate of Pseudomonas aeruginosa and aerobic fermentation is carried out in a mechanically stirred fermenter. No exogenous carbon or nitrogen source is added during the fermentation process. After the culture is completed, the compound Bacillus fermentation broth is obtained.

[0017] S3. After the water-soluble nutrient substrate is sterilized at high temperature and cooled to room temperature, it is added to the compound Bacillus fermentation broth and stirred evenly in a closed sterile mixing tank. After the mixture is filtered to remove impurities, the pH value is adjusted to 6.5~7.5 with 0.1mol / L citric acid solution or 0.1mol / L sodium hydroxide solution to obtain the compound Bacillus microbial water-soluble fertilizer.

[0018] Preferably, the expansion culture in S1 uses a modified basic culture medium, the culture temperature is 28℃, the culture time is 48h, and the OD600 value is measured every 12h during the culture process. The culture is terminated when the OD600 stabilizes at 2.8~3.2. The components of the modified basic culture medium are 10.5g / L K2HPO4, 4.5g / L KH2PO4, 0.5g / L sodium citrate dihydrate, 1.0g / L yeast extract, 0.25g / L magnesium sulfate heptahydrate, and 2.0g / L sucrose. After the culture medium is prepared, it is autoclaved at 121℃ for 20min and then cooled before use. The sterilization filtration adopts a two-stage filtration process. First, it is pre-filtered through a 0.45μm microporous membrane to remove residual bacterial fragments, and then finally filtered through a 0.22μm polyethersulfone membrane. After filtration, 10mL of the filtrate is spread on LB plates and incubated at 28℃ for 72h. If no colony growth is observed, it is considered qualified.

[0019] Preferably, the total viable count of the compound Bacillus seed liquid in S2 is not less than 1×10^10 CFU / mL, and the total inoculum is 3%~5% of the volume of the sterile metabolite filtrate of Pseudomonas aeruginosa; the fermentation culture temperature is 28℃~32℃, the fermentation time is 24h~36h, the dissolved oxygen is maintained above 30% during the fermentation process, the stirring speed is 150~200r / min, the aeration rate is 1:0.8~1.2vvm, the dissolved oxygen is adjusted and controlled by the linkage between the stirring speed and the aeration rate, the viable count is measured every 6h during the fermentation process, and the fermentation is terminated when the total viable count reaches 2×10^9 CFU / mL or more.

[0020] Preferably, in the composite Bacillus fermentation broth prepared in S2, the total viable number of composite Bacillus is not less than 2×10^9 CFU / mL, and the spore formation rate is not less than 90%; the detection of miscellaneous bacteria is carried out by dilution plating method, and selective culture media corresponding to bacteria, fungi and actinomycetes are cultured for 72h respectively, and the detection rate of miscellaneous bacteria is 0; the pH value of the fermentation broth is 6.4~7.0, and the retention rate of phenolic antibacterial substances in the supernatant after fermentation is not less than 90%, and no significant decrease in antibacterial activity is observed.

[0021] Preferably, the mass ratio of the water-soluble nutrient substrate to the compound Bacillus fermentation broth added in S3 is 1:4~6. The water-soluble nutrient substrate is sterilized at 120℃ for 10 minutes before being added, and then cooled to room temperature to avoid killing the compound Bacillus at high temperature. The stirring speed is 100~120 r / min, and the stirring time is 20~30 minutes. Microfiltration is performed using a 5μm polypropylene organic filter membrane. The water-insoluble content of the water-soluble fertilizer obtained after filtration is less than 0.2%, which is suitable for various application methods such as fertigation, drip irrigation, and root irrigation.

[0022] The present invention has the following beneficial effects:

[0023] This invention employs a technique that uses the sterile metabolite filtrate of *Pseudomonas aeruginosa* biocontrol strain MCNB07405 as the sole fermentation medium for compound Bacillus. This method not only fully preserves the highly inhibitory active components against *Cladosporium brassicae* in the metabolites but also completely avoids the interspecies antagonism problem that occurs when live *Pseudomonas aeruginosa* bacteria are directly combined with compound Bacillus. At the same time, the natural nutrients in the metabolites can fully support the growth and reproduction of compound Bacillus. Highly active compound Bacillus inoculants can be obtained without the need for additional exogenous carbon and nitrogen sources. This effectively solves the defects of existing technologies, such as the significant decrease in activity when biocontrol strains are directly combined and the insufficient inhibitory activity of ordinary Bacillus water-soluble fertilizers against clubroot.

[0024] This invention constructs a synergistic system of "rapid disease suppression + long-term protection + nutrient supply" by optimizing the combination of antibacterial active components, compound Bacillus inoculants, and water-soluble nutrient substrates. The metabolites can quickly act on the dormant spores of Pseudomonas aeruginosa in the soil to reduce their germination rate. The compound Bacillus inoculants can stably colonize the rhizosphere of crops to form a long-term rhizosphere microecological protective barrier. The water-soluble nutrient substrate can be directly absorbed and utilized by the crop roots to meet their growth needs without the need for additional application of conventional water-soluble fertilizers. This effectively solves the shortcomings of existing Pseudomonas aeruginosa live bacteria preparations, such as short duration of action, single function, need for high-frequency application, and increased labor input due to the need for additional fertilizers.

[0025] The microbial water-soluble fertilizer prepared by this invention has an extremely low content of water-insoluble matter, making it suitable for various conventional agricultural operations such as root irrigation, fertigation, and drip irrigation. It can be widely applied to different planting scenarios such as open field and facility planting for various cruciferous crops, and can be applied to plots with different disease severity without adjusting existing agricultural operation procedures. It has high promotion and application value and effectively solves the defects of existing similar products in terms of limited application scenarios and poor adaptability. Attached Figure Description

[0026] Figure 1 The flowchart of the overall preparation process of the microbial water-soluble fertilizer proposed in this invention is shown.

[0027] Figure 2 This is a sub-flowchart of Pseudomonas aeruginosa fermentation and aseptic filtrate preparation proposed in this invention.

[0028] Figure 3 This is a key logic diagram of the secondary fermentation of compound Bacillus proposed in this invention.

[0029] Figure 4 This is a diagram illustrating the finished product formulation and low-temperature spray drying process proposed in this invention.

[0030] Figure 5 This is a product performance testing and defect verification evaluation diagram proposed in this invention. Detailed Implementation

[0031] The following will refer to the appendices in the embodiments of the present invention. Figure 1-5 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] This embodiment addresses the shortcomings of existing technologies, such as interspecific antagonism caused by direct compounding of biocontrol strains, poor efficacy against clubroot disease, short duration of effectiveness, single function, and high content of water-insoluble matter, which makes them unsuitable for refined agricultural operations. In conjunction with the technical solution defined in the claims of this patent, four sets of examples with different parameters and one set of comparative examples using existing technologies are set up. All strains and reagents are commercially available and publicly available conventional products, and the detection methods all adopt national or industry standard methods. Those skilled in the art can repeat the experiments according to the description to obtain the corresponding results.

[0033] The *Pseudomonas aeruginosa* MCNB07405 used in all the following examples is a publicly available standard strain collected by the Microbial Fertilizer and Edible Fungus Strains Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs. The compound Bacillus is a seed liquid of a 1:1 ratio of live bacteria of the standard strain of *Bacillus licheniformis* (accession number CGMCC 1.813) and the standard strain of *Bacillus subtilis* (accession number CGMCC 1.3358). The water-soluble nutrient substrate is a compound of agricultural water-soluble urea, food-grade potassium dihydrogen phosphate, and mineral-derived potassium humate in corresponding proportions.

[0034] Example 1

[0035] S1 Pseudomonas aeruginosa extended fermentation: Activated Pseudomonas aeruginosa MCNB07405 was inoculated into sterilized LB liquid medium at an inoculation rate of 1%. The shaker speed was controlled at 180 rpm, the fermentation temperature at 28℃, and the fermentation time at 24 h. After fermentation, the mixture was sterilized by pressure filtration using a 0.22 μm microporous membrane to obtain sterile metabolite filtrate. After testing the filtrate and finding no viable bacteria, the mixture proceeded to the next step.

[0036] S2 compound Bacillus secondary fermentation: A seed culture of Bacillus licheniformis and Bacillus subtilis with a live count ratio of 1:1 was inoculated into the above sterile metabolite filtrate at an inoculation rate of 1%. The shaking speed was controlled at 200 rpm, the fermentation temperature at 28℃, and the fermentation time at 24 h. After the fermentation was completed, the live count of compound Bacillus in the fermentation broth was detected to be ≥500 million / mL.

[0037] S3 Product Preparation: Add water-soluble nutrient substrate to the fermented bacterial solution. The ratio of water-soluble nutrient substrate is urea: potassium dihydrogen phosphate: potassium humate = 3:2:5. Stir for 30 minutes until completely mixed, then send to a low-temperature spray drying device. Control the inlet air temperature to 110℃ and the outlet air temperature to 60℃. After spray drying, collect the powdered product and test the total viable bacteria count in the product to be ≥200 million / g.

[0038] This embodiment employs a low inoculum size and short fermentation time process to verify the effectiveness of this technical solution in addressing strain antagonism under low parameter conditions, while also verifying the product's basic disease prevention and nutrient supply performance.

[0039] Example 2

[0040] S1 Pseudomonas aeruginosa extended fermentation: Activated Pseudomonas aeruginosa MCNB07405 was inoculated into sterilized LB liquid medium at an inoculation rate of 1%. The shaker speed was controlled at 180 rpm, the fermentation temperature at 28℃, and the fermentation time at 48 h. After fermentation, the mixture was sterilized by pressure filtration using a 0.22 μm microporous membrane to obtain sterile metabolite filtrate. After testing the filtrate and finding no viable bacteria, the mixture proceeded to the next step.

[0041] S2 compound Bacillus secondary fermentation: A seed culture of Bacillus licheniformis and Bacillus subtilis with a live count ratio of 1:1 was inoculated into the above sterile metabolite filtrate at an inoculation rate of 5%. The shaking speed was controlled at 200 rpm, the fermentation temperature at 32℃, and the fermentation time at 48 h. After the fermentation was completed, the live count of compound Bacillus in the fermentation broth was detected to be ≥800 million / mL.

[0042] S3 Product Preparation: Add water-soluble nutrient substrate to the fermented bacterial solution. The ratio of water-soluble nutrient substrate is urea: potassium dihydrogen phosphate: potassium humate = 4:3:3. Stir for 30 minutes until completely mixed, then send to a low-temperature spray drying device. Control the inlet air temperature at 130℃ and the outlet air temperature at 60℃. After spray drying, collect the powdered product and test the total viable bacteria count in the product to be ≥400 million / g.

[0043] This embodiment employs a high inoculum size and long fermentation time process to verify the fermentation stability of this technical solution under high parameter conditions, and at the same time to verify the improved disease prevention performance of the product under high active metabolite content.

[0044] Example 3

[0045] S1 Pseudomonas aeruginosa extended fermentation: Activated Pseudomonas aeruginosa MCNB07405 was inoculated into sterilized LB liquid medium at an inoculation rate of 1%. The shaker speed was controlled at 180 rpm, the fermentation temperature at 28℃, and the fermentation time at 36 h. After fermentation, the mixture was sterilized by pressure filtration using a 0.22 μm microporous membrane to obtain sterile metabolite filtrate. After testing the filtrate and finding no viable bacteria, the mixture proceeded to the next step.

[0046] S2 compound Bacillus secondary fermentation: A seed culture of Bacillus licheniformis and Bacillus subtilis with a live count ratio of 1:1 was inoculated into the above sterile metabolite filtrate at an inoculation rate of 3%. The shaking speed was controlled at 200 rpm, the fermentation temperature at 30℃, and the fermentation time at 36 h. After the fermentation was completed, the live count of compound Bacillus in the fermentation broth was detected to be ≥1 billion / mL.

[0047] S3 Product Preparation: Add water-soluble nutrient substrate to the fermented bacterial solution. The ratio of water-soluble nutrient substrate is urea: potassium dihydrogen phosphate: potassium humate = 3.5: 2.5: 4. Stir for 30 minutes until completely mixed, then send to a low-temperature spray drying device. Control the inlet air temperature at 120℃ and the outlet air temperature at 60℃. After spray drying, collect the powdered product and test the total viable bacteria count in the product to be ≥500 million / g.

[0048] This embodiment employs the intermediate optimal parameter process, which is the preferred implementation method of this patent, and verifies the comprehensive solution effect of this technical solution on all existing defects under the optimal parameters.

[0049] Example 4

[0050] S1 Pseudomonas aeruginosa extended fermentation: Activated Pseudomonas aeruginosa MCNB07405 was inoculated into sterilized LB liquid medium at an inoculation rate of 1%. The shaker speed was controlled at 180 rpm, the fermentation temperature at 28℃, and the fermentation time at 42 h. After fermentation, the mixture was sterilized by pressure filtration using a 0.22 μm microporous membrane to obtain sterile metabolite filtrate. After testing the filtrate and finding no viable bacteria, the mixture proceeded to the next step.

[0051] S2 compound Bacillus secondary fermentation: A seed culture of Bacillus licheniformis and Bacillus subtilis with a live count ratio of 1:1 was inoculated into the above sterile metabolite filtrate at an inoculation rate of 4%. The shaking speed was controlled at 200 rpm, the fermentation temperature was 31℃, and the fermentation time was 42 h. After the fermentation was completed, the live count of compound Bacillus in the fermentation broth was detected to be ≥900 million / mL.

[0052] S3 Product Preparation: Add water-soluble nutrient substrate to the fermented bacterial solution. The ratio of water-soluble nutrient substrate is urea: potassium dihydrogen phosphate: potassium humate = 3.2:2.2:4.6. Stir for 30 minutes until completely mixed, then send to a low-temperature spray drying device. Control the inlet air temperature at 125℃ and the outlet air temperature at 60℃. After spray drying, collect the powdered product and test the total viable bacteria count in the product to be ≥450 million / g.

[0053] This embodiment uses suboptimal parameter processes to verify the performance stability of the technical solution under small parameter fluctuations and to confirm the adaptability range of the process.

[0054] Comparative Example

[0055] This comparative example employs a conventional direct compounding process for biocontrol bacteria, with the following specific steps: S1. *Pseudomonas aeruginosa*, *Bacillus licheniformis*, and *Bacillus subtilis* are fermented separately using their respective optimal fermentation processes. After cultivation, each is spray-dried to obtain single-strain powders, with viable cell counts ≥1 billion / g for all three powders. S2. The three single-strain powders are mixed uniformly at a 1:1:1 mass ratio, and a water-soluble nutrient substrate with the same proportions as in Example 3 is added. After stirring for 30 minutes until completely mixed, the final product is obtained. The total viable cell count in the final product is ≥300 million / g. This comparative example completely utilizes existing technical solutions and is used to verify the improved effects of the technical solution of this invention.

[0056] Experimental data and tables

[0057] All test indicators in this section were tested using standard methods: spore survival rate was determined according to the method in GB 20287-2006 "Agricultural Microbial Agents"; the inhibition rate of *Platycodon brassicae* was determined using the plate confrontation method, with the inhibition rate of dormant spore germination of *Platycodon brassicae* as the statistical index; the water-insoluble matter content was determined according to the method in NY 1107-2020 "Water-soluble Fertilizers with Macroelements"; the control efficacy against clubroot disease in potted plants was tested using Chinese cabbage as the test crop, with 2g of the corresponding fertilizer applied per plant at transplanting, and the incidence of clubroot disease investigated 30 days after transplanting to calculate the relative control efficacy; the duration of efficacy was determined using field plot trials, with 5kg of the corresponding fertilizer applied per acre, and the incidence of clubroot disease investigated monthly, with the duration of efficacy defined as the number of days when the control efficacy decreased to below 50%.

[0058] Table 1. Preparation process parameters for each experimental group

[0059] Group Fermentation time of green needle mushrooms (h) Compound spore inoculation amount (%) Secondary fermentation temperature (°C) Secondary fermentation time (h) Water-soluble matrix ratio (urea: potassium dihydrogen phosphate: potassium humate) Inlet air temperature for spray drying (°C) Example 1 24 1 28 24 3:02:05 110 Example 2 48 5 32 48 4:03:03 130 Example 3 36 3 30 36 3.5:2.5:4 120 Example 4 42 4 31 42 3.2:2.2:4.6 125 Comparative Example / / / / 3.5:2.5:4 /

[0060] Table 1 Explanation: This table clarifies the process parameter gradients for the four embodiments, covering reasonable value ranges for fermentation time, inoculum size, fermentation temperature, and substrate ratio. It can verify the stability of product performance under different process conditions. The comparative examples do not involve secondary fermentation steps and adopt the process of direct mixing of strains in the existing technology, which is consistent with the preparation method of mainstream similar products on the market. It can be effectively used as a control group to verify the improvement effect of the process of this invention.

[0061] Table 2 Performance test results of each test group

[0062] Group Spore survival rate (%) Inhibition rate of *Cladosporium brassicum* (%) Water-insoluble matter content (%) Prevention efficacy against clubroot disease in potted plants (%) Shelf life (d) Example 1 92.3 72.1 0.21 68.4 92 Example 2 90.7 85.3 0.18 81.2 118 Example 3 95.6 89.7 0.12 86.5 127 Example 4 94.1 87.5 0.15 84.3 121 Comparative Example 61.2 52.4 0.87 47.6 45

[0063] Table 2 Explanation: The data in this table show that the spore survival rate, *Bacillus brassicae* inhibition rate, potted plant efficacy, and duration of effectiveness of all embodiments are significantly higher than those of the comparative example, while the water-insoluble content is much lower than that of the comparative example. This indicates that the secondary fermentation process using sterile metabolic filtrate as the culture medium in this invention effectively solves the interspecies antagonism problem caused by the direct combination of different biocontrol strains in the prior art, greatly improves the survival rate and disease control effect of the strains, and extends the duration of effectiveness. At the same time, the use of fully water-soluble raw materials ensures that the water-insoluble content of the product meets the requirements of refined agricultural operations such as drip irrigation and sprinkler irrigation. The performance of the embodiments with different parameters is stable, verifying that the technical solution of this invention has a wide range of applicability and high reliability.

[0064] refer to Figure 1 This diagram illustrates the entire lifecycle preparation path of the product described in this invention. The process begins with the expanded fermentation of *Pseudomonas aeruginosa*. Through a crucial pressure filtration step, live bacteria are separated from their metabolites, yielding a sterile filtrate containing bioactive substances. A secondary fermentation stage then begins, using this filtrate as a nutrient base to inoculate a compound of *Bacillus* for deep culture. This step cleverly solves the interspecies antagonism problem when different biocontrol bacteria coexist. Finally, the system mixes the fermentation broth with a high-purity water-soluble nutrient matrix (urea, potassium dihydrogen phosphate, etc.) and converts it into a highly active solid powder using low-temperature spray drying technology. This overall process embodies the scientific logic from "strain metabolic utilization" to "nutrient compounding" and then to "formulation transformation," establishing the foundation for its dual functions of disease prevention and fertilization.

[0065] refer to Figure 2 This diagram details the meticulous operation of the first step (S1) of the process. First, the activated *Pseudomonas aeruginosa* MCNB07405 is introduced into LB broth and cultured at high speed in a constant-temperature shaker. After fermentation, the bacterial culture is not used directly; instead, it is filtered under pressure using a 0.22-micron microporous membrane. The core technology of this step lies in "physical isolation": the extremely fine pores completely intercept live *Pseudomonas aeruginosa*, retaining only its secreted secondary metabolites with antibacterial effects. The subsequent live bacteria detection test is crucial to ensuring that the subsequent compound Bacillus can grow in an environment free from interspecies competition, thus avoiding the risk of reduced activity caused by direct compounding of biocontrol strains at the source.

[0066] refer to Figure 3This diagram illustrates the core "secondary fermentation" (S2) logic of this invention. In this stage, the sterile filtrate obtained in the previous step is no longer merely an extract, but serves as a special biological culture medium, supporting the growth of *Bacillus licheniformis* and *Bacillus subtilis*. The inoculum amount is dynamically adjusted between 1% and 5% to adapt to different implementation requirements. Under a temperature-controlled environment of 28 to 32 degrees Celsius, the *Bacillus* utilize the active ingredients in the filtrate to proliferate, ensuring a consistently high viable count at the end of the fermentation broth. This process not only enhances the activity of the *Bacillus* but also achieves deep integration of metabolites and viable bacteria, providing the product with strong biological resistance and a longer duration of effectiveness against clubroot disease.

[0067] refer to Figure 4 This figure illustrates the technical details of the conversion from liquid microbial agents to highly water-soluble solid fertilizer (S3). During the preparation process, the system strictly adheres to a specific ratio of urea, potassium dihydrogen phosphate, and potassium humate to formulate the water-soluble matrix. The selection of these raw materials aims to ensure that the finished product achieves extremely low water-insoluble content, thus adapting to refined irrigation systems. In the drying stage after mixing, low-temperature spray technology is employed. By controlling the inlet air temperature to 110-130 degrees Celsius and maintaining a constant outlet air temperature, rapid moisture removal is achieved while maximizing the protection of the thermal stability of microbial spores. The final collected dry powder not only has balanced nutrients but also an extremely high total viable bacteria count, overcoming the process defects of traditional compound fertilizers, such as easy strain inactivation and excessive water-insoluble matter during drying.

[0068] refer to Figure 5 This diagram summarizes the logical architecture for performance hedging verification of the embodiments and comparative examples. The systematic evaluation covers biological indicators (spore survival rate, antibacterial rate), physicochemical indicators (water-insoluble matter content), and field application indicators (clumproot control efficacy, duration of effect). By comparing the "secondary fermentation process" in the embodiments with the "direct compounding process" in the comparative examples, the significant advantages of this invention in addressing antagonistic effects can be intuitively observed. The diagram clearly illustrates how the technical solution addresses the pain points of existing technologies, such as single function and short duration of effect. The final evaluation results confirm that the fertilizer produced by this process has stronger applicability in refined agricultural operations, achieving dual efficacy in disease prevention and growth promotion.

[0069] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound Bacillus microbial water-soluble fertilizer, characterized in that, This fertilizer is a special microbial water-soluble fertilizer suitable for the prevention and control of clubroot disease and the promotion of growth in cruciferous crops. Its components include sterile metabolite filtrate of Pseudomonas aeruginosa biocontrol bacterium MCNB07405, compound Bacillus inoculant, and water-soluble nutrient matrix. The sterile metabolite filtrate of the Pseudomonas aeruginosa biocontrol bacterium MCNB07405 accounts for 60% to 75% of the total fertilizer mass, the compound Bacillus inoculant accounts for 15% to 25% of the total fertilizer mass, and the water-soluble nutrient substrate accounts for 8% to 15% of the total fertilizer mass; The composite Bacillus was prepared by fermentation using the sterile metabolite filtrate of the Pseudomonas aeruginosa biocontrol bacterium MCNB07405 as the sole culture medium.

2. The compound Bacillus microbial water-soluble fertilizer according to claim 1, characterized in that, The compound Bacillus is composed of Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus, all of which are strains permitted for addition to microbial fertilizers as published by the Ministry of Agriculture and Rural Affairs. The ratio of viable bacteria among the three is 1~3:1~2:

1. The total viable bacteria count of the compound Bacillus in the water-soluble fertilizer is not less than 1×10^9 CFU / mL, of which the viable single-strain count of Bacillus subtilis is not less than 3×10^8 CFU / mL, the viable single-strain count of Bacillus licheniformis is not less than 2×10^8 CFU / mL, and the viable single-strain count of Bacillus mucilaginosus is not less than 1×10^8 CFU / mL.

3. The compound Bacillus microbial water-soluble fertilizer according to claim 1, characterized in that, The sterile metabolite filtrate of the biocontrol bacterium *Pseudomonas aeruginosa* MCNB07405 inhibited the germination of dormant spores of *Plasmodium brassicae* by no less than 60%, and the inhibition rate was determined by the spore germination counting method in a 96-well plate.

4. The compound Bacillus microbial water-soluble fertilizer according to claim 1, characterized in that, The water-soluble nutrient matrix comprises 12% to 20% macroelements, 2% to 5% humic acid, and 0.5% to 1.5% chelated trace elements by mass. The macroelements consist of nitrogen, phosphorus pentoxide, and potassium oxide in a mass ratio of 2:1:

3. Nitrogen is supported by urea and potassium nitrate, phosphorus pentoxide by potassium dihydrogen phosphate, and potassium oxide by one of potassium nitrate, potassium dihydrogen phosphate, potassium humate, or potassium dihydrogen phosphate. The humic acid is mineral-derived fulvic acid with a molecular weight of less than 3000 Da. The chelated trace elements consist of EDTA-chelated iron, EDTA-chelated zinc, sodium octaborate tetrahydrate, and ammonium molybdate in a mass ratio of 2:2:1:0.

5.

5. A method for preparing a compound Bacillus microbial water-soluble fertilizer, used to prepare the compound Bacillus microbial water-soluble fertilizer according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. The biocontrol strain MCNB07405 of Pseudomonas aeruginosa was activated and cultured on a large scale. The culture medium was centrifuged and sterilized and filtered to obtain the metabolite filtrate of Pseudomonas aeruginosa. S2. Inoculate the compound Bacillus strain into the filtrate of Pseudomonas aeruginosa metabolites and carry out aerobic fermentation culture to obtain the compound Bacillus fermentation broth. S3. Add water-soluble nutrient matrix to the compound Bacillus fermentation broth, stir and mix well, remove impurities by microfiltration, and adjust the pH value to 6.5~7.5 to obtain the compound Bacillus microbial water-soluble fertilizer.

6. The preparation method according to claim 5, characterized in that, The expansion culture in S1 uses a modified basic culture medium, the culture temperature is 28℃, the culture time is 48h, and the OD600 value is measured every 12h during the culture process. The culture is terminated when the OD600 stabilizes at 2.8~3.

2. The components of the modified basic culture medium are 10.5g / L K2HPO4, 4.5g / L KH2PO4, 0.5g / L sodium citrate dihydrate, 1.0g / L yeast extract, 0.25g / L magnesium sulfate heptahydrate, and 2.0g / L sucrose. After the culture medium is prepared, it is autoclaved at 121℃ for 20min and then cooled before use.

7. The preparation method according to claim 5, characterized in that, The total viable count of the compound Bacillus seed liquid in S2 is not less than 1×10^10 CFU / mL, and the total inoculum is 3%~5% of the volume of the sterile metabolite filtrate of Pseudomonas aeruginosa. The fermentation culture temperature is 28℃~32℃, the fermentation time is 24h~36h, the dissolved oxygen is maintained above 30% during the fermentation process, the stirring speed is 150~200r / min, the aeration rate is 1:0.8~1.2vvm, and the dissolved oxygen is adjusted and controlled by the linkage between the stirring speed and the aeration rate. The viable count is measured every 6 hours during the fermentation process, and the fermentation is terminated when the total viable count reaches 2×10^9 CFU / mL or more.

8. The preparation method according to claim 5, characterized in that, The total viable count of the composite Bacillus fermentation broth prepared by S2 is not less than 2×10^9 CFU / mL, and the spore formation rate is not less than 90%. The detection of miscellaneous bacteria is carried out by dilution plating method, and selective culture media corresponding to bacteria, fungi and actinomycetes are cultured for 72h respectively, and the miscellaneous bacteria detection rate is 0. The pH value of the fermentation broth is 6.4~7.0, and the retention rate of phenolic antibacterial substances in the supernatant after fermentation is not less than 90%.

9. The preparation method according to claim 5, characterized in that, The mass ratio of the water-soluble nutrient substrate to the compound Bacillus fermentation broth added in S3 is 1:4~6. The water-soluble nutrient substrate is sterilized at 120℃ for 10 minutes before being added, and then cooled to room temperature before being added. The stirring speed is 100~120 r / min and the stirring time is 20~30 minutes. Microfiltration is performed using a 5μm polypropylene organic filter membrane, and the water-insoluble matter content of the water-soluble fertilizer obtained after filtration is less than 0.2%.

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