Bacillus velezensis dispersible oil suspending agent and application thereof

Abstract

The invention discloses a bacillus velezensis dispersible oil suspending agent. The dispersible oil suspending agent is prepared from the following components in percentage by mass: 1%-5% of bacillus velezensis BJ-1 parent drug, 16%-20% of an emulsifier, 8%-12% of a wetting dispersant, 1%-2% of a thickening agent, 1%-2% of a stabilizer and the balance of peanut oil or soybean oil. Normal-temperature storage results show that the live bacterium rate of a parent drug can be increased by 10% or above when the preparation is stored for 6 months. According to the bacillus velezensis dispersible oil suspending agent disclosed by the invention, the viable bacterium rate of the bacillus velezensis during normal-temperature storage can be improved, the lasting period of effective components can be prolonged, and the prevention and treatment effects of the bacillus velezensis can be improved by frequently storing and screening different auxiliaries and dispersion media and selecting auxiliaries or dispersion media with good biocompatibility to a parent drug. An indoor potting result shows that the relative prevention and treatment effect on tomato gray mold is 78-88%. In addition, the bacillus velezensis dispersible oil suspending agent also has a good prevention and treatment effect on tomato fusarium wilt and cucumber powdery mildew, and can promote crop growth and increase the income of planters.

Description

Technical Field

[0001] This invention relates to a Bacillus vesiculosus dispersible oil suspension and its application. Background Technology

[0002] Bacillus belesiensis ( Bacillus velezensis It can produce a variety of enzymes and antibiotics, with broad-spectrum antibacterial activity, and can be used to prevent and control gray mold, wilt and powdery mildew in crops; at the same time, the enzymes it produces can promote the absorption of minerals by plants and produce plant growth-related hormones, which also have a good growth-promoting effect on plants.

[0003] Currently, the market for Bacillus vesiculus preparations is mainly dominated by wettable powders, but these suffer from poor formulation quality and low spore survival rates during processing and storage. Dispersible oil suspensions offer significant advantages such as environmental friendliness, good wetting and spreading properties, strong erosion resistance, and a marked synergistic effect. Encapsulating Bacillus vesiculus in an oil phase medium aims to improve its duration of action and extend the shelf life of the formulation.

[0004] Mineral oils have poor environmental compatibility and may affect the balance of soil microorganisms. While vegetable oils are more environmentally friendly, the free fatty acids they contain may accelerate the degradation of Bacillus. Therefore, it is crucial to select a biocompatible vegetable oil as the dispersion medium for Bacillus belyss dispersible oil suspensions. Summary of the Invention

[0005] Based on this, the present invention provides a Bacillus belye dispersible oil suspension and its preparation method.

[0006] The Bacillus belye dispersible oil suspension provided by this invention is composed of the following components by mass percentage: Bacillus vesiculosus technical grade: 1%–5%, Emulsifier: 16%–20%, Wetting and dispersing agent: 8%–12%, Thickener: 1%–2%, Stabilizer: 1%–2%, Dispersion medium: balance, wherein the dispersion medium is peanut oil or soybean oil.

[0007] The Bacillus berberis mother drug is Bacillus berberis ( Bacillus velezensis BJ-1 bacterial powder, with an effective live bacteria count of 1.0 × 10⁻⁶. 11 ~1.5×10 11 cfu / g. The Bacillus belesia ( Bacillus velezensis BJ-1 has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 24113.

[0008] The emulsifier is selected from one or more of EL-10, Tween-80, AEO-3, NP-4, OP-10, 6501, and Span-80; the wetting and dispersing agent is PEG400; the thickener is one or more of N20st, SK04, and CMC-Na. The stabilizer is one of BHT, dextrin, and ascorbic acid; the dispersion medium is peanut oil or soybean oil.

[0009] Preferably, the emulsifier is a combination of EL-10 and Tween-80, wherein EL-10 accounts for 80% to 90% of the total mass of the emulsifier and Tween-80 accounts for 10% to 20%; the thickener is a combination of SK04 and CMC-Na, wherein SK04 accounts for 75% to 90% of the total mass of the thickener and CMC-Na accounts for 10% to 25%; the stabilizer is dextrin; and the dispersion medium is peanut oil or soybean oil.

[0010] Preferably, the Bacillus berberis dispersible oil suspension is composed of the following ingredients in weight percentages: 1%–5% Bacillus berberis technical, 15%–17% EL-10, 1%–3% Tween-80, 8%–12% PEG400, 0.5%–1.5% SK04, 0.1%–0.5% CMC-Na, 1%–2% dextrin, and the balance being peanut oil or soybean oil.

[0011] More preferably, the Bacillus berberis dispersible oil suspension is composed of the following ingredients in weight percentage: 5% Bacillus berberis technical, 15%–17% EL-10, 1%–3% Tween-80, 10% PEG400, 1.5% SK04, 0.5% CMC-Na, 1%–2% dextrin, and the balance being peanut oil or soybean oil.

[0012] The *Bacillus belyceae* dispersible oil suspension provided by this invention, through screening the biocompatibility of different vegetable oils, determined the preferred order of dispersion media to be peanut oil > soybean oil > sunflower oil > rapeseed oil. This selection is based on the results of room temperature storage tests. The viable rate of *Bacillus belyceae* in peanut oil reached 87.34% ± 3.79%, and in soybean oil it was 86.1% ± 4.96%, both significantly higher than those in sunflower oil (84.64% ± 3.39%) and rapeseed oil (75.71% ± 3.83%), thus maximizing the protection of the activity of the active ingredients. Although sunflower oil and rapeseed oil are feasible to some extent, their viable cell retention rates are lower than those in peanut oil and soybean oil, and therefore they are not considered preferred embodiments.

[0013] The emulsifiers used are a combination of EL-10 and Tween-80. EL-10 mainly plays an emulsifying role, and its influence on the water dispersion behavior of the formulation is mainly reflected in its synergistic effect with Tween-80. The emulsion droplet size D after emulsification is...50 Only 1.010μm, D 90 The particle size is 2.187 μm, ensuring a uniform and stable system. When the two are mixed at 15%~17% + 1%~3%, filamentous dispersion can be achieved. It can be completely dispersed with slight stirring, without any floating paste or excessive precipitation.

[0014] The thickeners selected are a combination of SK04 and CMC-Na, and the system stability is controlled by adjusting the ratio. When SK04 is 1.5% and CMC-Na is 0.5%, the oil separation rate is only 4.2%; when SK04 is 1.5% and CMC-Na is 0.3%, the oil separation rate is 4.8%, both of which can form a stable system, prevent particle sedimentation, and avoid excessive viscosity affecting spray flowability.

[0015] The optimal addition amount of wetting and dispersing agent PEG400 is about 10%. At this level, the viscosity of the formulation is within a suitable range, which can not only fully coat the Bacillus berberis particles to reduce sedimentation, but also promote the rapid spread of the dispersible oil suspension to form a uniform drug film, thus facilitating the release of active ingredients.

[0016] While ascorbic acid and BHT are common stabilizers, they can lead to increased oil separation rate or decreased viable bacterial count in the system of this invention, making them unsuitable for the dispersible oil suspension system of this invention. Therefore, dextrin is the preferred stabilizer in this invention. The preparation method of the Bacillus belyss dispersible oil suspension also falls within the protection scope of this invention, and the specific steps include: 1) Weigh each component according to the stated mass percentage; 2) Without affecting the biological activity of Bacillus belysinus, mix the prepared materials except for the Bacillus belysinus mother drug, add zirconium beads at a mass ratio of 1:1.2 of the mixture to 2mm zirconium beads, and grind for 80 to 100 minutes at a grinding speed of 1800 to 2300 r / min. The particle size of the ground material is D50≤1.5μm and D90≤3μm. 3) Filter to remove zirconium beads, add Bacillus belysinogen mother drug to the filtrate, and stir at 600-720 r / min for 30 min to obtain Bacillus belysinogen dispersible oil suspension.

[0017] During the preparation process, the grinding parameters were set to ensure that all materials except the parent drug were ground to a particle size of D50≤1.5μm and D90≤3μm, guaranteeing uniform dispersion of emulsifiers, thickeners, and other additives, thus laying the foundation for subsequent mixing with the parent drug. The stirring parameters ensured thorough integration of the Bacillus belysse parent drug with the ground system, preventing uneven local concentrations from affecting activity. Through the above component selection and preparation parameter settings, the resulting formulation exhibited a high viable cell retention rate under room temperature storage conditions. Related experimental results showed that after 6 months of storage, the viable cell rate was more than 10% higher than the control sample, with a suspension rate ≥96.8% and good cold and hot storage stability.

[0018] This invention achieves a balance between storage stability, water dispersion performance, and application flowability of the formulation while ensuring the bioactivity of *Bacillus belyssiensis* through the synergistic effect of a specific emulsifier compound system, wetting and dispersing agents, and thickeners. This invention also provides the application of the aforementioned *Bacillus belyssiensis* dispersible oil suspension in the prevention and control of plant diseases and / or the promotion of plant growth. The plants may include tomatoes or cucumbers, etc.

[0019] Specifically, the plant disease can be one or more of tomato gray mold, tomato wilt, and cucumber powdery mildew; the Bacillus berberis dispersible oil suspension provided by this invention can be used to control tomato gray mold; this preparation can also be used to control common crop diseases such as cucumber and tomato wilt and cucumber powdery mildew. The application method is foliar spraying or irrigation, the application dosage is 200-600 mL per acre, diluted 100-500 times, and applied once by irrigation.

[0020] For those skilled in the art, without departing from the concept of this invention, conventional adjustments to the proportions of the components or process conditions can still yield dispersible oil suspensions with similar properties.

[0021] One of the advantages of this invention is that the selection of components is strongly correlated with biocompatibility. By screening the most biocompatible vegetable oils (peanut oil > soybean oil > sunflower oil > rapeseed oil) as the dispersion medium, and combining them with adjuvants that are compatible with Bacillus belyssus (EL-10+Tween-80 compound emulsifier, PEG400 wetting and dispersing agent, SK04+CMC-Na compound thickener), the effective ingredients are protected from the source, the shelf life is extended, and the environmental impact of mineral oil is avoided, making it more environmentally friendly.

[0022] The second advantage of this invention is that the optimized ratio is strongly correlated with system stability and spraying effect. The emulsifier compound ratio (EL-10 15%~17%, Tween-80 1%~3%) ensures rapid dispersion in water, the thickener compound ratio (SK04 0.5%~1.5%, CMC-Na 0.1%~0.5%) controls the oil separation rate to ≤4.8%, and the wetting and dispersing agent addition amount (PEG400 8%~12%) optimizes viscosity. The three factors work synergistically to improve the stability of the formulation and the spraying effect, reduce agent loss, and lower production costs.

[0023] The third advantage of this invention is that the preparation parameters are strongly correlated with the fusion effect of the components. Specific grinding and stirring parameters ensure that each component is fully mixed, avoiding uneven local concentrations, so that the various indicators of the preparation (suspension rate, fineness, stability) meet the standards. The viable bacteria rate increases by more than 10% after 6 months of storage at room temperature. The control effect on tomato gray mold is 78% to 88%, the control effect on tomato wilt is 70% to 72%, and the control effect on cucumber powdery mildew is 76% to 78%. It can also promote plant growth and increase growers' income. Attached Figure Description

[0024] Figure 1 Biocompatibility of Bacillus vesiculus BJ-1 in different dispersion media; a: viability of Bacillus vesiculus BJ-1 in different dispersion media; b: Bacillus vesiculus colonies on slabs in different dispersion media.

[0025] Figure 2 The biocompatibility of Bacillus vesiculus in emulsifiers and wetting and dispersing agents; a: viability of Bacillus vesiculus BJ-1 in emulsifiers and wetting and dispersing agents; b: Bacillus vesiculus colonies on slabs in emulsifiers and wetting and dispersing agents.

[0026] Figure 3 The biocompatibility of Bacillus vesiculus in the thickener; a: viability of Bacillus vesiculus BJ-1 in the thickener; b: slab colony count of Bacillus vesiculus BJ-1 in the thickener.

[0027] Figure 4 Viscosity curves of PEG400 with different addition amounts Figure 5 Rheological curves of dispersible oil suspensions with different amounts of SK04 Figure 6 The inhibitory effect of Bacillus belye BJ-1 on different Botrytis cinerea strains (left side is CK, right side is Bacillus belye treatment). Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1: Inhibitory effect of Bacillus belye BJ-1 on different Botrytis cinerea strains 1.1 Experimental Methods: The Bacillus berberis mother drug is Bacillus berberis ( Bacillus velezensis BJ-1 bacterial powder, with an effective live bacteria count of 1.0 × 10⁻⁶. 11 ~1.5×10 11 cfu / g, provided by the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences. The *Bacillus belye* (… Bacillus velezensis BJ-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24113, and is described in patent document CN116445357A. The *Bacillus belyssus* (…) Bacillus velezensis The preparation method of BJ-1 bacterial powder is as follows: (1) Material preparation and strain activation: The experimental strain BJ-1 was confirmed to be in good condition. LB, seed, counting and specific component fermentation media were prepared, and a protectant (a solution containing 5% skim milk powder, 5% trehalose and 5% glucose, where % is the mass percentage) and sterile consumables were prepared. In a clean bench, slant strains were streaked onto LB plates and activated by incubation at 37℃ for 24 hours. Subsequently, single colonies were inoculated into LB liquid medium and propagated by primary seed culture for 24 hours and secondary seed culture for 15 hours at 37℃ and 180 rpm.

[0031] (2) Shake flask fermentation: The fermentation medium (180 mL of liquid in a 500 mL Erlenmeyer flask) was sterilized at 121 °C and cooled. The secondary seed liquid was inoculated at a rate of 3% and cultured at 38 °C and 219 rpm for 79 hours.

[0032] (3) Treatment of bacterial culture and preparation of bacterial powder: After fermentation, the bacterial culture was centrifuged at 4℃ and 5000rpm to collect the bacterial cells, and washed twice with sterile physiological saline. The bacterial sludge and the protective agent (a solution containing 5% skim milk powder, 5% trehalose and 5% glucose, where % is the mass percentage) were mixed at a weight ratio of 1:1 and dispensed into freeze-drying bottles. After pre-freezing at -80℃, the samples were freeze-dried for 48 hours. The freeze-dried samples were ground, passed through an 80-mesh sieve to obtain a uniform powder, and finally vacuum-sealed in aluminum foil bags.

[0033] (4) Detection and analysis: The viable cell count was determined by the 10-fold dilution plating method; after killing vegetative cells by water bath treatment at 80℃ for 10 minutes, the count was performed, and the spore rate was calculated as (spore count / viable cell count) × 100%. The effective viable cell count was determined to be 1.0 × 10⁻⁶. 11 ~1.5×10 11 cfu / g.

[0034] The Bacillus berberis BJ-1 technical material was prepared with sterile distilled water to a concentration of 2.0 × 10⁻⁶. 9 Prepare a suspension of cfu / mL for later use.

[0035] Botrytis cinerea strains B.cinerea F2462FD1, B.cinerea F2462FB3 B.cinerea F2462LA3 were pathogenic bacterial strains isolated from tomato fruits, leaves, and stems in Shunyi District, Beijing, and were identified using standard methods. A 5mm diameter bacterial cake of the tested pathogen was inoculated into the center of a PDA plate. The plate was divided into four equal parts by marking "X". At four points 2.5cm from the center of the plate, Bacillus vesiculosus BJ-1 bacterial suspension (concentration 2.0 × 10⁻⁶) was added at the top, bottom, left, and right points. 9 Incubate 2 μL of the bacterial culture (cfu / mL) at 25℃ for 3–7 days, observe the growth of pathogenic hyphae, and measure the colony diameter using calipers.

[0036] 1.2 Experimental Results: Table 1. Inhibitory effect of Bacillus belye BJ-1 on different Botrytis cinerea strains Pathogenic fungi Control colony diameter Treatment of colony diameter Antibacterial rate Botrytis cinerea F2462FD1 75.47±1.20 36.43±0.93 51.73±1.23c Botrytis cinerea F2462FB3 74.37±0.80 32.24±2.20 56.65±3.10b Botrytis cinerea F2462LA3 74.67±1.77 28.12±1.58 62.34±2.11a Note: The letters a, b, and c following the data indicate the results of the significance analysis (significance level). P <0.05) As shown in Table 1, Bacillus belye BJ-1 exhibited inhibitory effects against different strains of Botrytis cinerea. The inhibition rate of Bacillus belye BJ-1 against different strains of Botrytis cinerea was greater than 50%. B.cinerea The F2462LA3 showed the best inhibition rate at 62.34%.

[0037] Example 2: Determination of the oil suspension formulation of the present invention 2.1 Main test materials and instruments (Table 2) Table 2.

[0038] The emulsifiers, wetting and dispersing agents and thickeners mentioned above can be commercially available products. Their specific models can be equivalently replaced according to the technical features described in this invention. As long as they can achieve the same or similar technical effects, they should be considered to fall within the protection scope of this invention.

[0039] Experimental instruments: Electronic balance with an accuracy of 0.0001g, purchased from Sartorius Technology Co., Ltd.; Thermal storage stability test chamber, purchased from Beijing Qincheng Yixin Technology Development Co., Ltd.; Laser particle size analyzer, purchased from Dandong Baite Instrument Co., Ltd.; Rheometer, purchased from Anton Paar (Shanghai) Trading Co., Ltd.

[0040] 2.2 Biocompatibility of Bacillus belye in different dispersion media Add 2% of Bacillus belysin BJ-1 technical material to soybean oil, peanut oil, sunflower oil, and rapeseed oil. After bottling, store at 30°C. Take samples every 7 days and count Bacillus belysin colonies using the dilution plating method. Set up 3 replicates for each group.

[0041] During 70 days of normal storage, different dispersion media had varying effects on the viability of Bacillus belyss BJ-1 spores. The viable percentage of Bacillus belyss in peanut oil (87.34±3.79)% was superior to that in soybean oil (86.1±4.96)% and sunflower oil (84.64±3.39)% and significantly superior to that in rapeseed oil (75.71±3.83)%. Figure 1 (a) The varying free fatty acid content in vegetable oils affects the stability of *Bacillus belyssiensis*. Peanut oil and soybean oil have suitable free fatty acid content, causing minimal damage to spore activity; after 70 days of storage at room temperature, the viable cell rate remains above 86%. Rapeseed oil, due to its higher free fatty acid content, has a viable cell rate of only about 75%, and is therefore excluded as a preferred dispersion medium. Therefore, the dispersion medium can be selected from peanut oil, soybean oil, or sunflower oil, with peanut oil or soybean oil being preferred. This selection directly relates to the duration of efficacy of the formulation, ensuring that the active ingredients maintain high activity during storage.

[0042] 2.3 Biocompatibility of Bacillus vesiculosus in emulsifiers and wetting / dispersing agents 2% of Bacillus vesiculus BJ-1 technical material (same as 2.2) was added to EL-10, Tween-80, and PEG400. The control was Bacillus vesiculus technical material (diluted 100-fold with sterile water before measurement). After bottling, the samples were stored at 30°C, and samples were taken every 7 days to count the viable bacteria using the dilution plating method. Each group was configured with 3 replicates.

[0043] In the presence of emulsifiers EL-10 and Tween-80, the viable cell rate of *Bacillus belyssiensis* BJ-1 stored at room temperature for 70 days was not significantly different from the control. In the presence of Tween-80, the viable cell rate of *Bacillus belyssiensis* BJ-1 was higher than the control at multiple time points. In the presence of EL-10, the viable cell rate of *Bacillus belyssiensis* BJ-1 spores remained the same as the control. Figure 2 (a) These results indicate that EL-10 and Tween-80 have good biocompatibility with Bacillus belyssae BJ-1 and can be selected as emulsifiers for this formulation. In the presence of wetting and dispersing agent PEG400, the viability rate of Bacillus belyssae BJ-1 after 70 days of normal storage was higher than the control, indicating good biocompatibility with Bacillus belyssae BJ-1 and its suitability as a wetting and dispersing agent for this formulation.

[0044] 2.4 Biocompatibility of Bacillus vesiculosus in Thickener Bacillus belyceta var. BJ-1 technical material (same as 2.2) was added at a concentration of 2% to a system containing 15%–17% EL-10, 1%–3% Tween-80, 8%–12% PEG400, 0.5%–3% SK04 or 0.5%–3% CMC-Na, with the remainder being soybean oil. After bottling, the mixture was stored at 30°C, and samples were taken at 0 and 24 hours for measurement using the dilution plating method. Three replicates were performed for each group.

[0045] In the presence of thickener SK04 and CMC-Na, the viable count of Bacillus belyss BJ-1 stored for 24 hours did not change significantly. Figure 3 (a, b) The results show that these two thickeners have good biocompatibility with Bacillus belyssus BJ-1 and can be used as thickeners for this formulation.

[0046] 2.5 Determination of Different Types of Emulsifiers and Their Addition Content Add 15% of EL-10, Tween-80, AEO-3, NP-4, OP-10, 6501, Span-80, and 1%–5% of Bacillus belyssus BJ-1 technical material to peanut oil, mix well, and then test. Add 100 mL of standard hard water (calculated as CaCO3, 342 mg / L) to a 100 mL stoppered graduated cylinder, add 0.5 mL of the prepared system, observe its dispersibility in water, and incubate at 30℃ for 1 hour to observe its emulsion state. Measure its OD using a laser particle size analyzer. 50 OD 90 .

[0047] Add 1% - 5% of the mother drug of Bacillus velezensis BJ - 1 and EL - 10 to peanut oil at an addition amount of 10% - 20%, mix well and wait for testing. Add 100 mL of standard hard water to a 100 mL stoppered graduated cylinder, add 0.5 mL of the prepared system, observe its water - in - oil dispersibility, keep it in a constant temperature water bath at 30 °C for 1 h, and observe its emulsion state.

[0048] Add 1% - 5% of the mother drug of Bacillus velezensis BJ - 1 to a system containing 15% EL - 10, and add 1% - 15% of Tween - 80 for compounding respectively. The remaining component is soybean oil. Mix well and wait for testing. Add 100 mL of standard hard water to a 100 mL stoppered graduated cylinder, add 0.5 mL of the prepared system, observe its water - in - oil dispersibility, keep it in a constant temperature water bath at 30 °C for 1 h, and observe its emulsion state.

[0049] Table 3 Water - in - oil dispersibility and emulsion stability of EL - 10 and Tween - 80 emulsifier Amount added (%) Dispersed state Emulsion stability <![CDATA[D 50 (μm)]]> <![CDATA[D 90 (μm)]]> EL-10 15 Stirring and dispersing No floating paste, small amount of sediment 1.010 2.187 Tween-80 15 Stirring and dispersing There is floating paste and a small amount of sediment. 3.922 11.39 AEO-3 15 Stirring and dispersing There is floating paste and a small amount of sediment. 3.991 9.958 NP-4 15 Stirring and dispersing There is floating paste and a small amount of sediment. 2.333 7.288 OP-10 15 Stirring and dispersing There is floating paste and a small amount of sediment. 4.336 12.75 6501 15 Not dispersed Oil floats, no sediment. —— —— Span-80 15 Not dispersed Oil floats, no sediment. —— —— Table 4 Emulsification results of different contents of EL - 10 EL-10 addition amount (%) Dispersed state Emulsion stability 10% Stirring and dispersing No floating paste, small amount of sediment 15%~17% When stirred, it disperses slightly upon contact with water. No floating paste, small amount of sediment 20% Stirring and dispersing No floating paste, small amount of sediment After stirring, emulsifier 6501 and Span - 80 cannot be dispersed and form floating oil on the water surface (Table 3). They cannot reduce the oil - water interfacial tension between peanut oil and water, and an emulsion cannot be formed between oil and water. The rest of the emulsifiers can be dispersed after stirring. The results show that after a constant temperature water bath at 30 °C, no floating paste appears for EL - 10, while floating paste appears for Tween - 80, AEO - 3, NP - 4, and OP - 10. This is because the hydrophilic - lipophilic balance of different emulsifiers is different. During the water bath, the rupture of the emulsion film causes the milk droplets to become larger, and further development makes the emulsion turn into an oil - water two - phase system, resulting in floating paste. The average particle size D 50 of EL - 10 < NP - 4 < AEO - 3 < Tween - 80 < OP - 10, D90 of EL - 10 < NP - 4 < AEO - 3 < Tween - 80 < OP - 10, and both D 50 and D 90 of EL - 10 are lower than those of other emulsifiers, indicating that the milk droplets after emulsification are smaller and more uniform, and the emulsification state is optimal.

[0050] Table 4 shows that when the addition amount of emulsifier EL-10 is 15%~17%, the aqueous dispersion state of the system changes, indicating that the oil-water interface is lowered at this point, and the interfacial film thickness formed by the emulsifier molecules at the oil-water interface is suitable. Therefore, 15%~17% is a suitable addition amount for EL-10. EL-10 is a nonionic surfactant with a moderate hydrophilic-lipophilic balance. When used alone, its spontaneous dispersion ability in water is limited, and it needs to be compounded with Tween-80, which has a high HLB value, to improve its aqueous dispersion behavior. Therefore, Tween-80 was chosen to compound with it to improve the dispersibility of the system.

[0051] Table 5. Emulsification results of 15% EL-10 and Tween-80 compounded. EL-10 addition amount (%) Tween-80 addition amount (%) Dispersed state Emulsion stability 15% 1~3 The filamentous dispersion disperses completely after slight stirring. No floating paste, small amount of sediment 15% 5~10 The filamentous dispersion disperses completely after slight stirring. Slight floating paste, small amount of sediment 15% 10~15 The filamentous dispersion disperses completely after slight stirring. Slight floating paste, but more sediment The amount of emulsifier added plays a crucial role in the emulsification of the dispersion medium. Insufficient addition fails to effectively stabilize the emulsion system, leading to the formation of a floating paste and precipitation. Excessive addition results in system instability, also causing floating paste and precipitation. As shown in Table 5, when the addition amount of EL-10 is 15% and the addition amount of Tween-80 is 1%~3%, no floating paste appears in the system, indicating a stable emulsion system and good emulsification effect. Therefore, nonionic surfactants such as EL-10 and Tween-80 are preferred emulsifiers, with a combination of EL-10 and Tween-80 being the preferred choice. EL-10 has a suitable hydrophilic-lipophilic balance value, enabling it to form a stable interfacial film at the oil-water interface, reducing interfacial tension and resulting in fine and uniform droplets. Tween-80 has a higher hydrophilic-lipophilic balance value, compensating for the insufficient dispersibility of EL-10 in water. The synergistic effect of the two ensures rapid emulsification of the formulation after entering water, forming a stable suspension system, reducing pesticide loss during spraying, and improving the control effect.

[0052] The optimal range, verified through multiple experiments, is recommended to be controlled at 15%~17% for EL-10 and 1%~3% for Tween-80. At this ratio, the emulsion system exhibits no floating paste and minimal sedimentation.

[0053] 2.6 Determination of the content of wetting and dispersing agent PEG400 was added at a concentration of 5%–30% to a system containing emulsifier, Bacillus vesiculus BJ-1, and peanut oil. After grinding, the specific amount of PEG400 added was determined using the viscosity curve method. Five replicates were set up for each group.

[0054] The viscosity of the Bacillus vesiculus BJ-1 dispersible oil suspension initially decreased and then increased with increasing dispersant concentration. When the dispersant concentration was less than 10%, the dispersant did not achieve saturated adsorption on the surface of Bacillus vesiculus BJ-1, and the pesticide particles could not be fully coated by the dispersant molecules. Under the influence of van der Waals forces and gravity, the particles aggregated and settled, resulting in increased viscosity. When the dispersant concentration was around 10%, the viscosity decreased to a relatively low value that met the requirements for pourability (160 mPa·s). Figure 4 Continuing to increase the amount of dispersant will cause excess dispersant molecules in the suspension to become entangled with the coating layer on the surface of the active ingredient particles, causing bridging between particles and leading to flocculation, which increases the viscosity of the formulation. Therefore, based on the viscosity curve trend, the recommended addition amount of wetting and dispersing agent PEG400 in actual production processes is 10%±2%, ranging from 8% to 12%.

[0055] 2.7 Screening of Thickener Types and Determination of Addition Amount 2% SK04, 2% CMC-Na, and 2% N20st were ground in a system containing emulsifier, wetting and dispersing agent, Bacillus vesiculus BJ-1, and peanut oil, and then stored at 54°C for 14 days. The precipitation and oil separation were observed.

[0056] Adding 0.5%–3% SK04 to a system containing emulsifiers, wetting and dispersing agents, Bacillus vesiculosus BJ-1, and peanut oil, followed by 14 days of heat storage and rheological studies, was conducted. After optimizing the content, to improve its performance, a certain amount of other thickeners was added for heat storage, and precipitation, oil separation, and other conditions were observed. The viscosity values ​​before and after heat storage were measured using a digital viscometer.

[0057] Table 6. Thermal storage results for different thickeners Thickener content(%) Layering Oil separation rate (%) Conclusion ointment SK04 2 Unlayered 9.7 none none CMC-Na 2 Unlayered 9.4 none have N20st 2 Layering —— —— —— Thickener SK04 utilizes the layered structure of organobentonite as a physical cross-linking point for polymers, resulting in a more stable spatial structure. CMC-Na can form colloidal or bentonite structures, encapsulating particulate matter and adsorbing onto the surface of particles to act as a bridging agent. N20st can improve system stability through adsorption, forming a network structure. SK04 exhibits good system stability at 2% addition and can be used as a thickener in this system (Table 6). CMC-Na showed paste-like formation at 2% addition, possibly due to excessive addition; excessive addition led to overly strong colloidal action, causing paste-like formation, requiring a reduction in its dosage. N20st exhibited stratification upon addition, indicating its inability to adsorb with substances in the system and form an effective structure.

[0058] Table 7. Thermal storage results of SK04 with different contents SK04 content (%) Oil separation rate (%) Conclusion Liquidity Dispersed state 0.5 78.2 bottom excellent The filamentous dispersion disperses completely after slight stirring. 1~2 9.7~15.4 slight good The filamentous dispersion disperses completely after slight stirring. 2.5 9.4 none good The filamentous dispersion disperses completely after slight stirring. 3 4.0 none Difference The filamentous dispersion disperses completely after slight stirring. Table 8. Rheological parameters of SK04 dispersible oil suspensions with the same content content(%) τ0(Pa) K N <![CDATA[R 2 ]]> 0.5 0.0829 0.0694 0.9242 0.999 1 0.2699 0.1156 0.9172 0.999 1.5 0.3761 0.1574 0.9016 0.999 2 0.418 0.1193 0.8674 0.999 2.5 0.8443 0.149 0.8784 0.999 3 0.9929 0.1475 0.8726 0.999 Note: τ0 is the yield value, and its magnitude is related to the stability of the system. K is the consistency index, N is the fluidity index, and R... 2 The correlation coefficient is the one used for fitting.

[0059] The results of thermal storage with different contents of SK04 showed that as the content increased, the oil separation rate gradually decreased, and the bottoming condition improved, but the fluidity deteriorated (Table 7), and the system viscosity increased, which is consistent with the rheological investigation. However, only when the addition amount was 3% did the oil separation rate of the system fall below 5%. At this point, the system had poor fluidity and high viscosity, which was not conducive to pouring. When the addition amount was less than 2%, the fluidity index of the system was relatively high (Table 8). Therefore, it is advisable to choose an addition amount of less than 2% to compound with CMC-Na.

[0060] Table 9. Results of thermal storage using SK04 and CMC-Na composite materials

[0061] As shown in Table 9, the Bacillus vesiculosus BJ-1 dispersible oil suspension system, after adding 1.5% SK04 and 0.5% CMC-Na, exhibited an oil separation rate of only 4.2%, the lowest among all qualified combinations. Furthermore, the viscosity change before and after heat storage was gradual, with a viscosity of 518.22±4.65 mPa·s before heat storage and 549.42±5.53 mPa·s after heat storage. This ensured system stability while avoiding excessive viscosity that could affect spray flowability. In contrast, the oil separation rate was slightly higher (4.8%) after adding 1.5% SK04 and 0.3% CMC-Na, indicating a slightly weaker resistance to stratification. While the oil separation rate met the standard (5.2%) after adding 1% SK04 and 1% CMC-Na, the viscosity value (575.16±5.01 mPa·s) was significantly higher, posing a slight risk of paste formation during long-term storage and potentially causing excessively large droplets and uneven adhesion during spraying. For other addition amounts, the oil separation rate increased, and the viscosity of the thickener system also increased after heat storage, but the viscosity change was not significant. This may be due to the expansion and dispersion of SK04 during heat storage, forming a thixotropic gel with a cardboard roof structure.

[0062] Therefore, the preferred thickener is a combination of SK04 and CMC-Na, which synergistically form a stable system with the dispersant. SK04 uses the organic bentonite lamellar structure as a physical cross-linking point, while CMC-Na forms a colloidal encapsulation of particles. The combination significantly reduces oil separation. When the SK04 addition is below 1%, the system shows obvious sedimentation; above 2%, the fluidity deteriorates; and when the CMC-Na addition exceeds 0.5%, it is prone to paste formation. Therefore, the optimal combination ratio is determined to be 1.5% SK04 and 0.5% CMC-Na. This ratio directly determines the storage stability of the system, avoids particle sedimentation and oil separation, and extends the shelf life of the formulation.

[0063] 2.8 Screening of Stabilizer Types Add 1%–2% stabilizer to 1%–5% Bacillus belyssioides technical material, 15%–17% EL-10, 1%–3% Tween-80, 8%–12% PEG400, 1%–1.5% SK04, 0%–0.5% CMC-Na, 1%–2% stabilizer, and the balance is peanut oil or soybean oil. After grinding, heat-store and observe the state of the formulation.

[0064] Table 10 Thermal storage results with different stabilizers stabilizer content(%) Layering Oil separation rate Conclusion Liquidity in conclusion ascorbic acid 1 Unlayered More none good Unqualified ascorbic acid 1.5 Unlayered More none good Unqualified ascorbic acid 2 Unlayered More none good Unqualified dextrin 1 Unlayered normal none good qualified dextrin 1.5 Unlayered normal none good qualified dextrin 2 Unlayered normal none Difference Unqualified BHT 1 Unlayered More slight good Unqualified BHT 1.5 Unlayered More slight good Unqualified BHT 2 Unlayered More slight good Unqualified As shown in Table 10, although ascorbic acid and BHT can be used as common stabilizers, they will lead to an increase in oil separation rate in the system of the present invention and are not suitable for the dispersible oil suspension system of the present invention. Therefore, the stabilizer in the present invention is preferably dextrin, and the suitable addition amount range is 1% to 1.5%, and the system has good fluidity.

[0065] To obtain a *Bacillus vesiculosus* dispersible oil suspension with good dispersibility, storage stability, and bioactivity, this invention first systematically screened and optimized the emulsifier system, wetting and dispersing agents, types and amounts of thickeners, and dispersion media. While maintaining a consistent viable count of the *Bacillus vesiculosus* parent drug, we formulated seven *Bacillus vesiculosus* BJ-1 dispersible oil suspension formulations to screen suitable formulation systems. In formulations 1-6, the emulsifiers were all nonionic surfactants EL-10 and Tween-80, the wetting and dispersing agent was PEG400, the thickeners were selected from SK04 and CMC-Na, the stabilizer was dextrin, and the dispersion media were peanut oil or soybean oil. Formulations 1-6 were used to investigate the effect of different emulsifier ratios, wetting and dispersing agent contents, and dispersion media differences on system stability within a reasonable range. In formulation 7, the addition amount of CMC-Na was significantly higher than in the other formulations, used to verify the adverse effects of excessive thickener on system performance, serving as a comparative example.

[0066] Formula 1: 5% Bacillus belyssus BJ-1 mother drug, 17% EL-10, 3% Tween-80, 8% PEG400, 1.5% SK04, 0.5% CMC-Na, 1% dextrin, and the balance is peanut oil.

[0067] Formula 2: 5% Bacillus belyssus BJ-1 mother drug, 15% EL-10, 1% Tween-80, 10% PEG400, 1.5% SK04, 0.5% CMC-Na, 2% dextrin, and the balance is peanut oil.

[0068] Formula 3: 5% Bacillus vesiculus BJ-1 mother drug, 15% EL-10, 3% Tween-80, 10% PEG400, 1.5% SK04, 0.5% CMC-Na, 1.5% dextrin, and the balance is peanut oil.

[0069] Formula 4: 5% Bacillus belyssus BJ-1 mother drug, 15% EL-10, 2% Tween-80, 12% PEG400, 1% SK04, 0.5% CMC-Na, 1% dextrin, and the balance is soybean oil.

[0070] Formula 5: 5% Bacillus belyssus BJ-1 mother drug, 15% EL-10, 3% Tween-80, 10% PEG400, 1.5% SK04, 0.5% CMC-Na, 1% dextrin, and the balance is soybean oil.

[0071] Formula 6: 5% Bacillus vesiculus BJ-1 mother drug, 15% EL-10, 3% Tween-80, 12% PEG400, 1.5% SK04, 0.5% CMC-Na, 1.5% dextrin, and the balance is soybean oil.

[0072] Formula 7: 5% Bacillus vesiculus BJ-1 mother drug, 15% EL-10, 3% Tween-80, 10% PEG400, 1% SK04, 1% CMC-Na, 2% dextrin, and the balance is peanut oil.

[0073] The preparation method is as follows: 1) Weigh each component according to the mass percentages described in claim 1 or 5; 2) Without affecting the bioactivity of Bacillus beryl, mix the prepared materials except for the Bacillus beryl BJ-1 mother drug, add zirconium beads at a mass ratio of 1:1.2 between the mixture and 2mm zirconium beads, and grind for 80-100 minutes at a grinding speed of 1800-2300 r / min. The particle size of the ground material is D50≤1.5μm and D90≤3μm; 3) Filter to remove the zirconium beads, add the Bacillus beryl mother drug to the filtrate, and stir at 600-720 r / min for 30 minutes to obtain the Bacillus beryl dispersible oil suspension. This preparation parameter and component ratio work synergistically to achieve the technical effect of meeting the suspension index and good cold and hot storage stability.

[0074] Example 3: Detection of Indicators of Bacillus belesiensis BJ-1 Dispersible Oil Suspension 3.1 Experimental methods: After processing formulations 1-7, the following indicators were tested: spore content was determined by dilution plating method; suspension rate was determined according to GB / T14825-2006; fineness was determined by wet sieving method according to GB / T16150; pH was determined according to GB / T1601; dispersibility was determined according to GB / T1603; and storage stability was determined according to GB / T21459.1-2008.

[0075] 3.2 Experimental Results: Table 11 Detection results of indicators for Bacillus belyssibirica BJ-1 dispersible oil suspension. formula Suspension rate % pH Moisture% Fineness% Residue W1 (%) after dumping Residue W2 (%) after washing in conclusion 1 97.5 7.1 0.38 99.72 5.02 0.42 qualified 2 99.8 6.99 0.44 99.85 4.75 0.29 qualified 3 99.6 7.12 0.41 98.96 4.86 0.32 qualified 4 96.8 6.86 0.52 99.74 5.04 0.56 qualified 5 98.9 6.92 0.48 98.86 4.68 0.38 qualified 6 99.1 6.84 0.49 97.48 4.95 0.43 qualified 7 98.8 6.94 0.36 98.42 4.58 0.45 qualified The seven formulations obtained from the screening were tested for relevant indicators of dispersible oil suspensions, as shown in Table 11. All relevant indicators met the relevant standards or guidelines.

[0076] Example 4: Cold and hot storage test of Bacillus vesiculosus dispersible oil suspension 4.1 Experimental Methods: Formulas 1 to 7 of Bacillus belyss BJ-1 dispersible oil suspension were placed in constant temperature storage boxes at 54℃ and 0℃ for hot storage and cold storage, respectively. The hot storage time was 14 days and the cold storage time was 7 days. Samples were taken at the corresponding time points, diluted and coated, and the colony count was counted to calculate the viability rate (%).

[0077] 4.2 Experimental Results: Table 12 Results of Cold and Heat Storage of Bacillus belye Dispersible Oil Suspension

[0078] After heat storage, the viability of Bacillus vesiculus BJ-1 in formulations 2, 3, 5, and 6 was >80%, and after cold storage, the viability of Bacillus vesiculus BJ-1 was >90% (Table 12), indicating that the formulations have good stability and can meet the storage requirements.

[0079] Example 5: Storage test of Bacillus belysin dispersible oil suspension at room temperature (30°C) 5.1 Experimental Methods: Formulas 1-7 of Bacillus belyss BJ-1 dispersible oil suspension and Bacillus belyss BJ-1 mother powder were packaged into reagent bottles and stored in a 30℃ constant temperature storage box. Samples were taken at 0 and 6 months, diluted and coated, and the number of colonies was counted to calculate the viability rate (%).

[0080] 5.2 Experimental Results: Table 13 Results of normal storage of Bacillus belysin dispersible oil suspension

[0081] As shown in Table 13, the results of room temperature storage show that, compared with the Bacillus vesiculus mother powder of the present invention (70.6% viability after 6 months of room temperature storage), the dispersible oil suspension of the present invention (such as formulation 3) can achieve a viability of 88.0% after 6 months of storage, a significant increase of 17.4%, indicating that the formulation has good stability and can meet the storage requirements to a certain extent, improve the viability, and solve the problem of shelf life of microbial preparations. Although formulations 1 and 4 can also form a stable oil suspension system, their viability retention rate and long-term storage stability are relatively lower, indicating that their formulation parameters are on the edge of the feasible range. In contrast, when the CMC-Na addition is increased to 1% (formulation 7), the viscosity of the system increases significantly, the fluidity of the preparation deteriorates, and the stability and viability retention rate decrease during long-term storage, indicating that the amount of thickener needs to be controlled within a reasonable range to ensure storage stability while taking into account the performance of the preparation.

[0082] Example 6: Inhibitory effect of Bacillus belye against Botrytis cinerea 6.1 Experimental Methods: The inhibitory effect of Bacillus belyceta var. BJ-1 dispersible oil suspension on gray mold was detected using the mycelial growth rate method. While dissolving PDA medium, Bacillus belyceta var. BJ-1 spore suspension and Bacillus belyceta var. BJ-1 water-dispersible granules (final concentration of Bacillus belyceta var. BJ-1 was 2.0 × 10⁻⁶) were dissolved in sterile water. 10Polyvinyl alcohol 6.0%; sodium dodecyl sulfate 4.0%; ammonium sulfate 4%; polyethylene glycol 4%; talc to bring the total to 100% (the percentages are by mass percentages) and Bacillus bereaves BJ-1 dispersible oil suspension were diluted to 1,500,000, 300,000, 60,000, 12,000, 2,400, and 480 CFU / mL. 1 mL of the diluted suspension was added to 99 mL of melted culture medium to prepare agar plates containing 15,000, 3,000, 600, 120, 24, and 4.8 CFU / mL. Plates without sample solution were used as blank controls (CK). After the culture medium solidified, 5 mm diameter mycelial discs were punched along the edge of the colony using a sterile punch. The mycelial side was placed in the center of each plate, one disc per plate, for a total of 5 plates. After incubation at 25℃ for 3-7 days, the growth of *Botrytis cinerea* colonies on tomatoes was observed. Colony diameters were measured using calipers, and the average value was calculated. The data were then transformed and subjected to regression analysis to determine the toxicity regression equations for each agent and the effective inhibitory concentration (EC50) of each agent. 50 .

[0083] 6.2 Results: Table 14 Inhibitory effect of Bacillus belye dispersible oil suspension on Botrytis cinerea

[0084] As shown in Table 14, compared with its spore suspension, the various formulations of Bacillus belye BJ-1 dispersible oil suspension effectively inhibited medium-concentration EC. 50 There was a decrease in EC levels in formulas 1, 2, 3, 5, and 6. 50 The lower efficacy compared to water-dispersible granules indicates that dispersible oil suspensions can, to some extent, enhance the inhibition rate of *Bacillus belye* BJ-1 against *Botrytis cinerea*. Formulations 2, 3, 5, and 6 showed the most significant improvement in the inhibition rate against *Botrytis cinerea*, which is related to their enhanced dispersibility and spore activity. Furthermore, formulations 3 and 6 exhibited particularly outstanding inhibitory effects, demonstrating that *Bacillus belye* maintained high biological activity under the aforementioned emulsifier system, thickener combination, and dispersion medium conditions. While the inhibitory efficacy of formulation 7 was lower than the aforementioned preferred formulations, this further indicates that excessive thickener is detrimental to the maintenance of formulation performance and biological activity.

[0085] In summary, by rationally selecting the types and controlling the amounts of emulsifiers, wetting and dispersing agents, and thickeners, stable Bacillus belye dispersible oil suspensions can be prepared in different vegetable oil dispersion media. Preferred examples show that when the emulsifiers, thickeners, and stabilizers are within suitable ratios, the resulting formulation exhibits good storage stability and biocontrol efficacy.

[0086] Example 7: Effect of Bacillus vesiculosus dispersible oil suspension on gray mold in potted tomatoes.

[0087] 7.1 Test Methods: Protective and therapeutic effects: The test reagents included BJ-1 dispersible oil suspension (5×10⁻⁶). 9 (CFU / mL), formulations 2, 3, 5, and 6 are all used at a dosage of 400 mL / mu, diluted 200 times, with a spray concentration of 2.5 × 10⁻⁶. 7 CFU / mL; Commercially available Bacillus subtilis wettable powder (100 billion spores / g), dosage 70g / acre, diluted 500 times, spray concentration 200 million spores / g; Bacillus vesiculosus BJ-1 water-dispersible granules (Bacillus vesiculosus BJ-1 final concentration 2.0×10⁻⁶). 10 Polyvinyl alcohol 6.0%; Sodium dodecyl sulfate 4.0%; Ammonium sulfate 4%; Polyethylene glycol 4%; Talc to bring the total to 100%, the percentages are by weight (2×10). 10 (CFU / g), the dosage of the formulation is 225 g / mu, diluted 200 times, and the concentration of the spray solution is 1×10. 8 cfu / g; with water as a blank control (CK). The concentration prepared before the experiment was 1×10⁻⁶. 6 cfu / mL B.cinerea F2462FD1 bacterial suspension was prepared for use. The test plants were healthy tomato seedlings (Pink Crown No. 1, early flowering stage) grown in a greenhouse, and plants with uniform growth were selected for the experiment.

[0088] The experiment was divided into two parts: protective effect (preventive effect) and therapeutic effect (therapeutic effect).

[0089] 1) Protective effect group: First, spray different treatment solutions until the tomato leaves are completely soaked. 24 hours later, inoculate with pathogen suspension. Each treatment has 15 tomato seedlings and is repeated 3 times.

[0090] 2) Treatment group: Inoculate with pathogen suspension first, spray with treatment solution 24 hours later, repeat 3 times for every 10 tomato seedlings treated.

[0091] All treatment groups received a second treatment (using the same treatment solution) 7 days after the first treatment, and disease indices were assessed 14 days after the second treatment.

[0092] The disease severity index grading standard is as follows: Grade 0: No lesions; Grade 1: Lesion area ≤ 5%; Grade 3: 5% < lesion area ≤ 15%; Grade 5: 15% < Lesion area ≤ 25%; Grade 7: 25% < lesion area ≤ 50%; Grade 9: Lesion area > 50%.

[0093] The calculation formula is: Disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value) Disease control effect = (Disease index in control area - Disease index in control area) / Disease index in control area × 100 The control effect was calculated by comparing the disease index of the treatment group and the blank control group. During the experiment, the greenhouse environment should be controlled at a temperature of 25±2℃ and a humidity of 75-90%. The bacterial suspension should be prepared and used immediately, and the front and back of the leaves should be covered when spraying.

[0094] 7.2 Test Results: Table 15. Effects of Bacillus vesiculosus dispersible oil suspension on gray mold control in tomatoes. deal with Mode of action Disease index Prevention and control effect (%) Pathogen control Therapeutic effects 58.64±2.05 — Pathogen control Protective effect 61.20±1.54 — Bacillus vesiculosus water dispersible granules Therapeutic effects 14.40±0.60 75.30±2.00b Formula 2 Therapeutic effects 13.00±0.60 78.00±1.90b Formula 3 Therapeutic effects 10.30±0.70 82.50±2.20a Formula 5 Therapeutic effects 11.90±0.50 79.50±2.33b Formula 6 Therapeutic effects 9.30±0.90 84.12±2.90a Commercially available Bacillus subtilis Therapeutic effects 20.80±1.50 64.48±4.82c Bacillus vesiculosus water dispersible granules Protective effect 12.40±0.80 77.73±2.52b Formula 2 Protective effect 10.70±0.70 82.40±2.45b Formula 3 Protective effect 7.10±0.90 88.44±3.14a Formula 5 Protective effect 11.80±0.60 79.81±2.14b Formula 6 Protective effect 8.20±1.10 86.82±3.83a Commercially available Bacillus subtilis Protective effect 19.30±1.80 68.40±6.30c Note: The letters following the data indicate the results of the significance analysis (significance level). P <0.05) The results of the indoor potted plant experiment show that: the test reagent 5×10 9 The average control efficacy of various formulations of BJ-1 CFU / mL dispersible oil suspension (400 mL / acre) against tomato gray mold was 78%~84%. The preventive effect of each formulation was significantly higher than that of commercially available Bacillus subtilis wettable powder (Table 15). Among them, the therapeutic effects of formulations 3 and 6 (>82%) were significantly higher than those of Bacillus belyss water dispersible granules (75.30%).

[0095] Test reagent 5×10 9 The average control efficacy of BJ-1 CFU / mL dispersible oil suspension (400 mL / acre) against tomato gray mold was 79%~88% for each formulation. The control efficacy of each formulation was significantly higher than that of commercially available Bacillus subtilis wettable powder (Table 15). Among them, the control efficacy of formulations 3 and 6 (>85%) was significantly higher than that of Bacillus belyss water dispersible granules (77.73%).

[0096] Therefore, a formulation dosage of 400 mL / mu can be used as the recommended field dosage for formulations 2, 3, 5, and 6 of BJ-1 dispersible oil suspension.

[0097] Example 8: Control effect of Bacillus belye dispersible oil suspension on crop wilt and powdery mildew. 8.1 Experimental Methods Test pathogen: Fusarium wilt of tomato ( Fusarium oxysporum f. sp. lycopersici ), cucumber powdery mildew ( Sphaerotheca fuliginea All samples were provided by the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences, and were identified using conventional methods before being stored for future use.

[0098] Test reagents: Bacillus belyssin dispersible oil suspensions (concentration 5×10⁻⁶) of formulations 3 (peanut oil system) and 6 (soybean oil system). 9 The concentrations were 100 cfu / mL, and the concentrations were 50% commercially available carbendazim wettable powder and water was used as a blank control (CK).

[0099] Inhibition effect determination (mycelial growth rate method): The pathogen was inoculated onto a PDA plate and incubated at 25°C for 3 days. A 5mm diameter mycelial cake was then taken using a sterile punch and placed in the center of a new PDA plate. The test reagent was diluted to 1×10⁻⁶. 7 Add 2 μL of the agent at four points (2.5 cm away from the bacterial cake) on the plate at cfu / mL, and incubate at 25℃ for 5-7 days. Measure the colony diameter and calculate the inhibition rate.

[0100] Potted plant control efficacy test: Tomato seedlings (Fen Guan No. 1) were selected for wilt disease control, and cucumber seedlings (Bi Chun) were selected for powdery mildew control. Each group consisted of 10 plants, with 3 replicates. Wilt disease treatment group: The agent was diluted 200 times and applied to the soil before transplanting (100 mL per plant). 24 hours later, the plants were inoculated with a suspension of *Fusarium wilt* fungus (1×10⁻⁶). 6 cfu / mL); Powdery mildew treatment group: Inoculated first with cucumber powdery mildew spore suspension (1×10⁻⁶ cfu / mL); 5 (Spores / mL), foliar spraying with a 200-fold diluted agent 24 hours later, followed by a second application (same treatment solution) 7 days after the first application, and disease index assessed 14 days after the second application. The control group was sprayed / irrigated with an equal volume of water; disease index was assessed after disease onset. Refer to Example 7 to calculate the control effect.

[0101] 8.2 Experimental Results Table 16. Antibacterial effect of Bacillus vesiculosus dispersible oil suspension on Fusarium wilt of tomato and powdery mildew of cucumber.

[0102] Table 17. Effects of Bacillus vesiculosus dispersible oil suspension on the control of tomato wilt and cucumber powdery mildew in pot plants.

[0103] Note: The letters following the data indicate the results of the significance analysis (significance level). P <0.05) As shown in Tables 16 and 17, the *Bacillus vesiculosus* dispersible oil suspension of this invention has a significant inhibitory effect on *Fusarium wilt* of tomato and *Powdery mildew* of cucumber, with an inhibition rate of 54.9%–61.0%. The control effect in potted plants reaches 70.1%–78.4%, and there is no significant difference in control effect compared with commercially available carbendazim wettable powder. These results indicate that *Bacillus vesiculosus* BJ-1 has broad-spectrum antibacterial activity, and the formulation of this invention can not only effectively control tomato gray mold but also effectively inhibit *Fusarium wilt* and powdery mildew.

[0104] Example 9: Control effect of Bacillus vesiculosus dispersible oil suspension on gray mold in potted tomatoes. 9.1 Experimental Methods Test materials: Tomato seedlings (Pink Crown No. 1) grown in a solar greenhouse. Select plants with uniform growth, 15 plants per group, and repeat 3 times.

[0105] Test reagents: Bacillus belyssin dispersible oil suspensions (concentration 5×10⁻⁶) of formulations 3 (peanut oil system) and 6 (soybean oil system). 9 (cfu / mL), diluted 200 times, the spray concentration is 2.5×10⁻⁶. 7 cfu / mL; commercially available Bacillus subtilis wettable powder (100 billion spores / g), formulation dosage 70g / acre, diluted 500 times, spray concentration 200 million spores / g; water as blank control (CK).

[0106] Treatment method: Foliar spraying was carried out 7 days and 14 days after transplanting, with a spraying amount of 100mL / plant, ensuring that both sides of the leaves were completely soaked; the blank control group was sprayed with an equal amount of water.

[0107] Measurement indicators: Tomato plant height, stem diameter (2cm from the base), and fresh weight (above ground) were measured 30 days after transplanting.

[0108] 9.2 Experimental Results Table 18. Growth-promoting effect of Bacillus belyssus dispersible oil suspension on tomatoes (30 days after transplanting) deal with Plant height (cm) Stem diameter (mm) Fresh weight of above-ground parts (g) Blank control (CK) 32.5±1.8c 5.2±0.3c 88.6±2.1c Commercially available Bacillus subtilis 36.8±2.0b 5.6±0.3b 96.8±2.5b Formula 3 37.6±2.2b 5.7±0.2b 97.2±2.4b Formula 6 41.2±2.3a 6.3±0.3a 103.5±2.8a Note: The letters following the data indicate the results of the significance analysis (significance level). P <0.05) As shown in Table 18, all formulations of the Bacillus vesiculosus dispersible oil suspension of the present invention significantly improved the plant height, stem diameter, and fresh weight of tomatoes. Among them, formulation 6 showed the best growth-promoting effect, significantly better than other treatments, while formulation 3 showed a significantly better growth-promoting effect than the blank control and no significant difference from commercially available Bacillus subtilis. After applying formulations 3 and 6, the plant height 30 days after transplanting increased by 15.7% and 26.8% respectively compared to the blank control, the stem diameter increased by 9.6% and 21.2% respectively, and the above-ground fresh weight increased by 9.7% and 16.8% respectively. These results show that the formulation of the present invention not only has excellent disease prevention effects but also promotes the growth and development of tomato plants, further improving planting efficiency.

Claims

1. A Bacillus belesiensis dispersible oil suspension, comprising the following components in weight percentage: Bacillus vesiculosus technical grade: 1%–5%, Emulsifier: 16%–20%, Wetting and dispersing agent: 8%–12%, Thickener: 1%–2%, Stabilizer: 1%–2%, Dispersion medium: Balance; The Bacillus berberis mother drug is Bacillus berberis ( Bacillus velezensis BJ-1 bacterial powder, with an effective live bacteria count of 1.0 × 10⁻⁶. 11 ~1.5×10 11 cfu / g; The emulsifier is selected from one or a combination of EL-10 and Tween-80; the wetting and dispersing agent is PEG400; the thickener is one or a combination of SK04 and CMC-Na; and the stabilizer is dextrin. The dispersion medium is peanut oil or soybean oil.

2. The Bacillus belesi dispersible oil suspension according to claim 1, characterized in that, The Bacillus belesi ( Bacillus velezensis BJ-1 has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 24113.

3. The Bacillus belesi dispersible oil suspension according to claim 1, characterized in that, The emulsifier is a combination of EL-10 and Tween-80, wherein EL-10 accounts for 80%–90% of the total mass of the emulsifier and Tween-80 accounts for 10%–20%; the wetting and dispersing agent is PEG400; the thickener is a combination of SK04 and CMC-Na, wherein SK04 accounts for 75%–90% of the total mass of the thickener and CMC-Na accounts for 10%–25%; the stabilizer is dextrin; and the dispersion medium is peanut oil or soybean oil.

4. The Bacillus belye dispersible oil suspension according to claim 1, characterized in that, It is composed of the following components in weight percentage: 1%–5% Bacillus belye technical, 15%–17% EL-10, 1%–3% Tween-80, 8%–12% PEG400, 0.5%–1.5% SK04, 0.1%–0.5% CMC-Na, 1%–2% dextrin, and the balance being a dispersion medium; wherein the dispersion medium is peanut oil or soybean oil.

5. The method for preparing the Bacillus belye dispersible oil suspension according to any one of claims 1-4, characterized in that, It includes the following steps: 1) Weigh each component according to its content; 2) After mixing the prepared materials except for the Bacillus vesiculus parent material, add grinding media and perform wet grinding to make the particle size of the resulting system D50≤1.5μm and D90≤3μm; 3) After removing the grinding media, add Bacillus belysinogen mother drug to the obtained system, stir and mix evenly to obtain Bacillus belysinogen dispersible oil suspension.

6. The use of the Bacillus belye dispersible oil suspension according to any one of claims 1-4 in the prevention and control of plant diseases and / or the promotion of plant growth.

7. The application according to claim 6, characterized in that, The plant diseases mentioned include one or more of the following: gray mold, wilt, or powdery mildew. The crops mentioned include tomatoes or cucumbers. The application method is foliar spraying or irrigation. The dosage per acre is 200-600 mL, diluted 100-500 times before use.

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