Bacillus velezensis, control fungicide prepared from bacillus velezensis and application of control fungicide

Abstract

The invention discloses bacillus velezensis, a control fungicide prepared from the bacillus velezensis and application of the control fungicide, and belongs to the technical field of microorganisms. The bacillus velezensis is screened, the preservation number of the bacillus velezensis is CGMCC (China General Microbiological Culture Collection Center) No.37347, and the bacillus velezensis is compounded with trichoderma harzianum CGMCC No.40702 according to the mass ratio of 1: 1 to form a control fungicide. The strain is screened from a grease acid oil adsorption material, and is excellent in salt resistance, heat resistance and growth promoting performance. The fungicide and fludioxonil reduced by 50% are cooperatively used, the control effect on corn root rot reaches 94.2%, POD and CAT defensive enzyme activity is remarkably induced, the yield of a single plant of potted corn reaches 90.7 g, and the synergistic effects of disease prevention, growth promotion, pesticide reduction and synergism are achieved. The invention provides an efficient and environment-friendly technical scheme for green prevention and control of the corn root rot.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus belye and the control agents prepared using it and their applications. Background Technology

[0002] Corn serves both as food and feed, and is a widely planted crop, playing a vital role in ensuring food security. However, root rot, a common corn disease, often leads to premature aging of plants and sharp reductions in yield, severely limiting the sustainable development of the seed industry.

[0003] Corn root rot is a soil-borne disease mainly caused by pathogens such as Fusarium graminearum, Fusarium verticillatum, and Fusarium oxysporum. It is particularly severe in fields with high soil moisture, poor drainage, and continuous cropping. In the early stage of the disease, water-soaked brown lesions appear on the roots, which then rot and die. Above-ground parts show yellowing leaves and slow growth. In severe cases, the entire plant wilts and dies, causing huge losses to corn production.

[0004] Currently, the control of corn root rot still heavily relies on chemical agents. In production, agents such as hymexazol, metalaxyl, cymoxanil, fludioxonil, and carbendazim are mainly used for seed coating, seed dressing, or root irrigation. Although these can control the spread of the disease in the short term, long-term, single, and excessive use of chemical agents has led to a series of irreversible drawbacks: First, pathogens are prone to developing resistance after continuous exposure to the agents, resulting in a gradual decline in the efficacy of the agents. Farmers are forced to increase the amount of agents used, forming a vicious cycle of "application-resistance-increased dosage". Second, chemical agents remain in the soil, water bodies, and corn plants, destroying the original microbial community structure of the soil, causing soil microecological imbalance, and at the same time causing excessive pesticide residues in agricultural products, threatening food safety and the ecological environment. Third, chemical agents only focus on disease control and do not have the function of promoting crop growth or improving soil, which cannot meet the comprehensive needs of modern planting.

[0005] In addition, excessive application of chemical fertilizers, especially excessive nitrogen and phosphorus fertilizers, not only significantly increases planting costs, but also causes ecological problems such as soil compaction, salinization, nutrient imbalance, and eutrophication of water bodies, further deteriorating the rhizosphere soil environment of corn, reducing the plant's own disease resistance, and indirectly aggravating the occurrence and spread of corn root rot.

[0006] In recent years, biological control has become the mainstream development direction for the prevention and control of soil-borne diseases due to its green, safe, and environmentally friendly characteristics. Microbial agents are gradually being applied to agricultural production due to their multiple advantages in disease prevention, growth promotion, and soil improvement. However, existing commercially available biological agents have significant drawbacks: most are single-strain preparations with poor environmental adaptability, narrow antibacterial spectrum, and unstable efficacy, making them difficult to deal with root rot caused by multiple infections; some compound agents, due to unreasonable strain compatibility and mismatched fermentation processes, cannot achieve synergistic effects between strains, making it difficult to simultaneously address disease control and promote corn growth, and failing to meet the industry's demand for reducing fertilizer and pesticide use while increasing efficiency and promoting high-quality green agriculture. Therefore, the development of compound microbial control agents that combine high-efficiency disease prevention, growth promotion, and yield increase with strong environmental adaptability is of great practical significance for achieving green control of corn root rot and ensuring safe corn production. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by providing a strain of Bacillus belye, which is salt- and heat-resistant and has excellent growth-promoting properties, screened from oil-acidified oil adsorbents. It also provides a control agent, preparation method, and application of this strain in combination with Trichoderma harzianum, which synergistically enhances the effects of low-dose chemical agents. This achieves the technical effects of highly efficient control of corn root rot, promoting corn growth, and significantly reducing the amount of fludioxonil used.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A strain of Bacillus belye ( Bacillus velezensis The *Bacillus belyssae* strain described is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37347 and a deposit date of January 9, 2026. This strain was screened from adsorbent material produced during the production of acidified oils and possesses multiple properties including strong salt tolerance, strong heat resistance, antibacterial activity, and growth promotion.

[0009] A fungicide containing Bacillus bellis and Trichoderma, mixed in a 1:1 mass ratio.

[0010] Furthermore, the Trichoderma is Trichoderma harzianum (Trichoderma harzianum). Trichoderma harzianum This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 40702 on June 16, 2023. This strain is a prior art strain, the same strain deposited by the applicant in patent CN202510114297.2.

[0011] The method for preparing the microbial agent of this application includes the following steps: (1) Inoculate Bacillus belye CGMCC No. 37347 into LB liquid medium and culture with shaking at 30-37℃ and 180-220rpm for 18-24h to obtain seed culture; transfer the seed culture to fermentation medium at an inoculation rate of 2%-5% and ferment at 30-37℃ and 180-220rpm for 48-72h to obtain Bacillus belye fermentation broth; (2) Inoculate Trichoderma harzianum CGMCC No. 40702 onto PDA agar plates and culture at 25-28℃ for 5-7 days. After the spores mature, wash them off with sterile physiological saline and adjust the spore concentration to 1.0×10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (3) The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

[0012] Furthermore, the carrier excipient is one or more of diatomaceous earth, light calcium carbonate, and humic acid, and the amount of carrier excipient added is 30%-50% of the total mass of the bacterial solution.

[0013] Furthermore, the fermentation medium in step (1) consists of: 20-30 g / L glucose, 15-25 g / L soybean meal powder, 10-15 g / L corn steep liquor powder, 5-8 g / L NaCl, 2-3 g / L K2HPO4, 0.5-1 g / L MgSO4·7H2O, and pH 7.0-7.5.

[0014] Furthermore, the freeze-drying temperature is -40℃ to -50℃, the vacuum degree is 10-20Pa (absolute pressure), and the drying time is 24-48h.

[0015] When the microbial agent of this invention is used for corn seed coating, seed dressing, or soil treatment, and synergistically reduced by more than 50% in fludioxonil, it can achieve multiple effects such as efficient control of corn root rot, promotion of corn growth, and reduction of chemical agent usage.

[0016] This invention prepares a fungicide by combining *Bacillus belye* (CGMCC No. 37347) selected from oil-acidified oil adsorbents with *Trichoderma harzianum* (CGMCC No. 40702) in a 1:1 mass ratio. This fungicide is then used in synergistic effects with a chemical agent (fludioxonil), achieving the following significant beneficial effects: 1. First, the selected Bacillus belye can colonize the rhizosphere of maize, secrete lipopeptides, polyketides and signaling molecules, induce disease resistance in maize, significantly increase the activity of defense enzymes such as peroxidase (POD) and catalase (CAT) in the plant, strengthen the lignification of cell walls, reduce the success rate of pathogen infection, and enhance the level of disease resistance and stress resistance at the plant level itself. 2. Significantly Reduced Chemical Pesticide Usage: The key innovation of this invention lies in the synergistic effect of Bacillus vesiculosus, Trichoderma harzianum, and chemical pesticides (fludioxonil), reducing chemical pesticide usage by more than 50% while maintaining or even improving disease control efficacy. Experiments show that when the inoculant and chemical pesticide are used in a 1:1 ratio, the control effect on corn root rot reaches over 95%, while using chemical pesticides alone (without the inoculant) requires 100% of the dosage to achieve an 85% control effect. This effectively solves the problem of increased pathogen resistance and declining pesticide efficacy caused by excessive use of chemical pesticides in existing technologies, avoiding the vicious cycle of "pesticide use—resistance—increased dosage".

[0017] 3. Enhanced Disease Control: The combination of *Bacillus belye* and *Trichoderma harzianum* exhibits a significant synergistic effect, increasing their inhibitory capacity against maize root rot pathogens (including *Fusarium graminearum*, *Fusarium verticillatum*, and *Fusarium oxysporum*). When used in conjunction with chemical agents, the three work together to form a dual "bio-chemical" control system, which not only improves the broad-spectrum nature of disease control but also enhances the inhibitory strength against pathogens, effectively controlling the combined infection of maize root rot.

[0018] 4. Promotes corn growth: Bacillus belye has excellent growth-promoting properties and can secrete plant growth hormones (such as IAA), promoting corn root development and improving the plant's ability to absorb nutrients. Simultaneously, the synergistic use of inoculants and chemical agents further enhances the corn's resistance to adverse conditions, enabling the plant to maintain good growth even under disease stress.

[0019] 5. Improves the soil microecological environment: The fungicides of this invention not only prevent disease and promote growth, but also improve the soil microecology, promote the decomposition of soil organic matter, alleviate soil compaction, and improve soil fertility. When used in conjunction with chemical agents, it reduces the amount of chemical residues in the soil, avoids soil microecological imbalance, and is conducive to soil health and sustainable agricultural development.

[0020] 6. Improved environmental adaptability: Bacillus belye has excellent salt and heat resistance (it can tolerate Na2SO4 concentrations up to 5% and heat resistance temperatures up to 55℃), and can adapt to different soil conditions and climate environments. It is especially suitable for the soil salinization and large temperature fluctuation problems that are common in corn planting areas, thus expanding the application range and stability of the inoculant.

[0021] 7. Reduced production costs: By reducing the use of chemical agents by more than 50%, the pesticide costs for corn cultivation are significantly reduced. Simultaneously, due to the growth-promoting effect of the microbial agent, the amount of chemical fertilizer used can be appropriately reduced, further lowering planting costs and achieving a significant improvement in economic benefits.

[0022] 8. In summary, this invention provides a compound microbial control agent that combines high-efficiency disease prevention, growth promotion, and yield increase with strong environmental adaptability, achieving green control of maize root rot and significantly reducing the use of chemical agents. This aligns with the industry's demand for reducing fertilizer and pesticide use while increasing efficiency and promoting high-quality green agriculture. The key innovation of this invention lies in the synergistic effect of Bacillus belyssae, Trichoderma harzianum, and chemical agents, which multiplies the efficacy, providing a new approach and method for the green control of maize root rot, and yielding significant economic, ecological, and social benefits. Attached Figure Description

[0023] Figure 1 The colony and Gram staining morphology of Bacillus belyssus of the present invention are shown in Figure 1, where A: colony morphology of the strain on LB solid medium; B: Gram staining microscopic morphology of the strain. Figure 2. Culture effect of antagonistic effect between Bacillus belyssus and Trichoderma harzianum CGMCC No.40702 of the present invention; Figure 3 Characteristic detection chart of IAA production by Bacillus belyssus and Trichoderma harzianum CGMCC No. 40702; Figure 4 A bar chart comparing the yield per maize plant in different treatment groups. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0025] Example 1 A strain of Bacillus belye, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37347 and deposit date of January 9, 2026, has been identified. This strain was selected from adsorbent material produced during the production of acidified oils and exhibits multiple characteristics including strong salt tolerance, strong heat resistance, broad-spectrum antibacterial activity, and growth promotion.

[0026] The isolation and screening method for the Bacillus belyssus is as follows: Sample collection: Adsorbent samples generated during the production process of oleic acidified oil were placed in sterile sealed containers and stored at 4°C in the dark. They were used for bacterial isolation within 24 hours.

[0027] Sample pretreatment and serial dilution: Add 10g of adsorbent sample to 90mL of sterile physiological saline, shake for 30min, let stand for 10min, and then take the supernatant for 10-fold serial dilution. -1 -10 -6 Serial dilution, ready for use.

[0028] Strain isolation and purification: Take 0.1 mL of each graded dilution and spread it evenly on LB solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 1 g / L, anhydrous Na2SO4 50 g / L, agar 18 g / L, pH 7.0-7.5); incubate upside down at 40-45℃ for 48 h, eliminate salt-intolerant and heat-intolerant bacteria, and observe the morphology of surviving colonies; select typical single colonies of Bacillus that are milky white, round, rough-surfaced, opaque, and have neat edges, and purify them continuously for 3-5 generations using the streak plate method to obtain pure culture strains.

[0029] Targeted screening: Using Fusarium graminearum, Fusarium verticillioides, and Fusarium oxysporum, the pathogens of maize root rot, as indicator fungi, the plate confrontation method was used for initial screening of antibacterial activity. Then, Fusarium spp. and Pythium spp. were used as targets for secondary screening. Finally, a heat- and salt-tolerant strain was obtained by targeted screening based on the triple indicators of salt tolerance (5% Na2SO4) and heat tolerance (survival rate ≥80% after 30 min of treatment at 50℃), which is the target strain.

[0030] Identification revealed that the 16S rRNA gene sequence of this strain shared 99.8% homology with that of Bacillus velezensis. Specific gene amplification confirmed the presence of lipopeptide synthesis genes such as srfAA and fenD. The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 37347. The 16S rRNA sequence of the described Bacillus velezensis is shown in SEQ ID NO. 1.

[0031]

[0032] The colony characteristics of Bacillus belye after incubation at 40-45℃ for 48 hours on LB solid medium are as follows: Size and morphology: Colonies are round or nearly round, mostly 2-5 mm in diameter. Some colonies may form larger irregular colony clusters due to fusion growth. Color and surface: Colonies are milky white or creamy yellow, opaque, with a raised, rough and wrinkled surface, irregular wavy / serrated edges, no obvious luster, dry and sticky texture, and easy to pick up; Growth characteristics: The strain grows vigorously, with good colony dispersion, conforming to the typical colony morphology characteristics of Bacillus (e.g., Figure 1 (A) The strain is Gram-positive after Gram staining, and the bacterial cells stain blue-purple (e.g., A). Figure 1 The B in the sample conforms to the Gram staining properties of the Bacillus genus.

[0033] A fungicide containing Bacillus bellis and Trichoderma, mixed in a 1:1 mass ratio.

[0034] The Trichoderma is Trichoderma harzianum ( Trichoderma harzianum The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 40702 on June 16, 2023. This strain is a prior art strain, the same strain deposited by the applicant in patent CN202510114297.2.

[0035] Antagonistic effect test: PDA medium was melted and poured into plates, then allowed to solidify. Bacillus belye and Trichoderma harzianum were activated, and both strains were streaked onto plates. After incubation at 28℃ for 5–7 days, inhibition zones and growth inhibition were observed. The results showed no obvious antagonistic bands between the two strains; the colony edges were close to each other but without lysis or inhibition, indicating good symbiotic compatibility and a biological basis for their combined application.

[0036] The preparation method of the microbial agent in this embodiment includes the following steps: (1) Bacillus belye CGMCC No. 37347 was inoculated into LB liquid medium and cultured with shaking at 30-37℃ and 180-220rpm for 18h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 2% and fermented at 30-37℃ and 180-220rpm for 48h to obtain Bacillus belye fermentation broth; (2) Trichoderma harzianum CGMCC No. 40702 was inoculated onto PDA agar plates and cultured at 25-28℃ for 5 days. The spores were then washed away with sterile physiological saline, and the spore concentration was adjusted to 1.0 × 10⁻⁶.8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (3) The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent. The effective viable count of the control agent is ≥2.0×10⁻⁶. 9 CFU / g.

[0037] The carrier additives are diatomaceous earth and light calcium carbonate, with a mass ratio of 1:1, and the amount of carrier additives added is 50% of the total mass of the bacterial solution.

[0038] The fermentation medium in step (1) consists of: 30 g / L glucose, 25 g / L soybean meal powder, 10 g / L corn steep liquor powder, 5 g / L NaCl, 3 g / L K2HPO4, 1 g / L MgSO4·7H2O, and pH 7.0-7.5.

[0039] The freeze-drying temperature in step (3) is -40℃ to -50℃, the vacuum degree is 10Pa (absolute pressure), and the drying time is 48h.

[0040] Example 2 A strain of Bacillus belye ( Bacillus velezensis The *Bacillus belye* strain described is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37347 and a deposit date of January 9, 2026. This strain was screened from adsorbent material produced during the production of acidified oils and possesses multiple characteristics including strong salt tolerance, strong heat resistance, broad-spectrum antibacterial activity, and growth promotion.

[0041] The isolation and screening method for Bacillus belye is the same as in Example 1.

[0042] A fungicide containing Bacillus bellis and Trichoderma, mixed in a 1:1 mass ratio.

[0043] The Trichoderma is Trichoderma harzianum ( Trichoderma harzianum The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 40702 on June 16, 2023. This strain is a prior art strain, the same strain deposited by the applicant in patent CN202510114297.2.

[0044] The preparation method of the microbial agent in this embodiment includes the following steps: (1) Bacillus belye CGMCC No. 37347 was inoculated into LB liquid medium and cultured with shaking at 30-37℃ and 180-220rpm for 24h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 5% and fermented at 30-37℃ and 180-220rpm for 72h to obtain Bacillus belye fermentation broth; (2) Trichoderma harzianum CGMCC No. 40702 was inoculated onto PDA agar plates and cultured at 25-28℃ for 7 days. The spores were then washed away with sterile physiological saline, and the spore concentration was adjusted to 1.0 × 10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (3) The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent. The effective viable count of the control agent is ≥2.0×10⁻⁶. 9 CFU / g.

[0045] The carrier additive is a mixture of diatomaceous earth and humic acid in a mass ratio of 1:2, and the amount of carrier additive added is 30% of the total mass of the bacterial solution.

[0046] The fermentation medium in step (1) consists of: 20 g / L glucose, 15 g / L soybean meal powder, 15 g / L corn steep liquor powder, 8 g / L NaCl, 2 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, and pH 7.0-7.5.

[0047] The freeze-drying temperature in step (3) is -40℃ to -50℃, the vacuum degree is 20Pa (absolute pressure), and the drying time is 24h.

[0048] Comparative Example 1 Compared to Example 1, in this comparative example, the *Bacillus belyceae* with accession number CGMCC No. 37347 was replaced with commercially available conventional *Bacillus belyceae* bacterial suspension (purchased from Weifang Yihao Biotechnology Co., Ltd.). All other preparation conditions and process parameters remained the same as in Example 1, i.e., the bacterial agent preparation method was as follows: (1) Trichoderma harzianum CGMCC No. 40702 was inoculated onto PDA agar plates and cultured at 25-28℃ for 5 days. After the spores matured, the spores were washed away with sterile physiological saline and the spore concentration was adjusted to 1.0×10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (2) Mix Bacillus belye with the Trichoderma spore suspension obtained in step (2) at a mass ratio of 1:1, add carrier excipients, and freeze-dry to obtain the control agent.

[0049] Comparative Example 2 Compared to Example 1, in this comparative example, Bacillus belyceae with accession number CGMCC No. 37347 replaced Bacillus belyceae with accession number CGMCC No. 1.8805 (purchased from the depositary center). All other preparation conditions and process parameters remained the same as in Example 1. The preparation method of this comparative bacterial agent includes the following steps: (1) Bacillus belye with preservation number CGMCC 1.8805 was inoculated into LB liquid medium and cultured with shaking at 28-30℃ and 180-220rpm for 18h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 2% and fermented at 28-30℃ and 180-220rpm for 48h to obtain Bacillus belye fermentation broth; (2) Inoculate Trichoderma harzianum CGMCC No. 40702 onto PDA agar plates and culture at 25-28℃ for 5 days. After the spores mature, wash them off with sterile physiological saline and adjust the spore concentration to 1.0×10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

[0050] Comparative Example 3 Compared to Example 1, in this comparative example, the *Trichoderma harzianum* with accession number CGMCC No. 40702 was replaced with a commercially available conventional *Trichoderma harzianum* (purchased from Shandong Xinyi Biotechnology Co., Ltd.). All other preparation conditions and process parameters remained the same as in Example 1. The preparation method of this comparative bacterial agent includes the following steps: (1) Bacillus belye CGMCC No. 37347 was inoculated into LB liquid medium and cultured with shaking at 30-37℃ and 180-220rpm for 18h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 2% and fermented at 30-37℃ and 180-220rpm for 48h to obtain Bacillus belye fermentation broth; (2) The Bacillus berberis fermentation broth obtained in step (1) is mixed with Trichoderma harzianum at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

[0051] Comparative Example 4 Compared to Example 1, in this comparative example, the *Trichoderma harzianum* with accession number CGMCC No. 40702 was replaced with *Trichoderma harzianum* with accession number CGMCC No. 3.6635 (purchased from the depository center). All other preparation conditions and process parameters remained the same as in Example 1; that is, the preparation method of the fungal agent was as follows: The preparation method of this comparative bacterial agent includes the following steps: (1) Bacillus belye CGMCC No. 37347 was inoculated into LB liquid medium and cultured with shaking at 30-37℃ and 180-220rpm for 18h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 2% and fermented at 30-37℃ and 180-220rpm for 48h to obtain Bacillus belye fermentation broth; (2) Trichoderma harzianum CGMCC No. 3.6635 was inoculated onto PDA agar plates and cultured at 25-28℃ for 5 days. After the spores matured, the spores were washed away with sterile physiological saline, and the spore concentration was adjusted to 1.0 × 10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

[0052] Comparative Example 5 This comparative example, except for changing the ratio of Bacillus belyssus and Trichoderma harzianum CGMCC No. 40702, had the same process parameters as Example 1; specifically, the two were mixed at a mass ratio of 1:2 to prepare the inoculum. That is: The preparation method of this comparative bacterial agent includes the following steps: (1) Bacillus belye CGMCC No. 37347 was inoculated into LB liquid medium and cultured with shaking at 30-37℃ and 180-220rpm for 18h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 2% and fermented at 30-37℃ and 180-220rpm for 48h to obtain Bacillus belye fermentation broth; (2) Inoculate Trichoderma harzianum CGMCC No. 40702 onto PDA agar plates and culture at 25-28℃ for 5 days. After the spores mature, wash them off with sterile physiological saline and adjust the spore concentration to 1.0×10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (3) The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:2, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

[0053] Comparative Example 6 This comparative example, except for changing the ratio of Bacillus belyssus and Trichoderma harzianum CGMCC No. 40702, had the same process parameters as Example 1; specifically, the two were mixed at a mass ratio of 2:1 to prepare the inoculum. That is: The preparation method of this comparative bacterial agent includes the following steps: (1) Bacillus belye CGMCC No. 37347 was inoculated into LB liquid medium and cultured with shaking at 30-37℃ and 180-220rpm for 18h to obtain seed liquid; the seed liquid was transferred to fermentation medium at an inoculation rate of 2% and fermented at 30-37℃ and 180-220rpm for 48h to obtain Bacillus belye fermentation broth; (2) Inoculate Trichoderma harzianum CGMCC No. 40702 onto PDA agar plates and culture at 25-28℃ for 5 days. After the spores mature, wash them off with sterile physiological saline and adjust the spore concentration to 1.0×10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (3) The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 2:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

[0054] Strains' growth-promoting performance test The growth-promoting abilities of Bacillus belyssus (CGMCC No. 37347) and Trichoderma harzianum (CGMCC No. 40702) were determined.

[0055] Following the method of Rajawat et al. (Rajawat MVS, Singh S, Tyagi SP, et al. A modified plate assay for rapid screening of potassium-solubilizing bacteria[J]. Pedosphere, 2016, 26(5):768–773.), the test bacteria were inoculated onto nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, and siderophore-producing media using the plate transparency method. The media were incubated upside down at 25-28℃ for 5-7 days, and the growth of the strains on each selective medium was observed. The nitrogen-fixing characteristics, phosphorus-solubilizing ability, potassium-solubilizing ability, and siderophore-producing activity of the endophytic fungi were determined by the ratio of the transparency zone diameter (D) to the colony diameter (d). Each strain was repeated three times, and the average value was taken.

[0056] IAA Production Determination: The test strain was inoculated into LB liquid medium containing 100 mg / L L-tryptophan and cultured at 25-28℃ and 180 rpm for 24 h. 200 μL of the bacterial suspension was mixed with 200 μL of Salkowski colorimetric solution (Feijing Biotechnology Co., Ltd., PH1941), and incubated at room temperature in the dark for 30 min. A red color indicates that the strain has the ability to produce IAA. The IAA standard solution served as a positive control, and the LB medium containing L-tryptophan served as a negative control. Figure 3 As shown, both strains exhibit IAA production characteristics. IAA standard solutions with concentrations of 0, 2, 4, 6, 8, and 10 g / mL were prepared sequentially, yielding a standard curve: y = 0.0254X + 0.0384, R² = 0.9993. A mixture of the bacterial suspension and Salkowski colorimetric solution in equal proportions was incubated at room temperature in the dark for 30 min, with distilled water as a blank control. OD values ​​were measured, and the corresponding IAA content was calculated based on the standard curve. Each strain was replicated three times, and the average value was taken.

[0057] Biofilm formation ability determination: Single colonies were picked and cultured overnight in LBGM medium at 25-28℃ and 180 r / min. A 1% inoculum was then inoculated into 24-well plates containing 2 mL of LBGM and incubated statically at 25-28℃ for 24 h. ODs were measured using the crystal violet method to assess the biofilm formation ability of the tested strains. Each strain was repeated three times, and the average value was taken.

[0058] Table 1. Strains' life-promoting properties

[0059] Note: + indicates nitrogen fixation properties, - indicates no nitrogen fixation properties.

[0060] As shown in Table 1, both strains in this application possess significant growth-promoting potential. *Bacillus belye* exhibits a clear advantage in potassium solubilization and IAA synthesis, while *Trichoderma harzianum* shows a slight advantage in phosphorus solubilization. Overall, their functions are highly complementary, synergistically enhancing the efficiency of crop absorption and utilization of nitrogen, phosphorus, potassium, and other nutrients. Combined with their iron-carrier and biofilm-forming abilities, they are more conducive to rhizosphere colonization and resistance to abiotic stress.

[0061] Antibacterial performance tests of different bacterial suspensions: The inhibitory effects of bacterial suspensions on the main pathogens of maize root rot, namely Fusarium graminearum, Fusarium verticillioides, and Fusarium oxysporum, were determined.

[0062] The bacterial agents prepared in Example 1 and Comparative Examples 1-6 were diluted with sterile water to the same effective viable bacteria concentration (1×10⁻⁶). 8 (CFU / mL) Take 200 μL and spread it evenly around the perimeter of a PDA plate. Inoculate the center with a bacterial disc (5 mm in diameter) of the pathogen to be tested. Incubate at 25-28℃ for 5-7 days, measure the diameter of the inhibition zone, and calculate the inhibition rate. The inhibition rate is calculated using the formula: [(diameter of blank colony - diameter of treated colony) ÷ (diameter of blank colony - 5 mm)] × 100%. The average inhibition rate is the mean of the inhibition rates of the three pathogens. Each experiment was repeated 3 times.

[0063] Using single-strain treatments as controls, the synergistic effect coefficient (SR) was calculated using the Wadley formula. The formula is: E0 = A + B − A × B; SR = E0 / E. Where: A is the inhibition rate of a single *Bacillus belye* strain, B is the inhibition rate of a single *Trichoderma harzianum* strain, E0 is the theoretical inhibition rate, and E is the measured inhibition rate. SR < 1 indicates synergistic effect, SR = 1 indicates additive effect, and SR > 1 indicates antagonistic effect.

[0064] Table 2. Average antibacterial rate and synergistic effect determination of different bacterial suspensions.

[0065] The results in Table 2 show that the inhibitory rate of the compound bacterial agent in Example 1 against the two pathogens was significantly higher than that of each single bacterium and the comparative examples, with an SR value of 0.42, which is much less than 1, confirming that Bacillus belyssus and Trichoderma harzianum CGMCC No. 40702 have a strong synergistic effect. In contrast, the SR values ​​of all comparative examples were ≥1, which did not reach the synergistic threshold, indicating that the differences in strain source and live bacteria ratio directly affect the expression of the synergistic effect.

[0066] Synergistic effect test of fungicides and chemical agents to reduce dosage: To verify the reduced dosage and enhanced efficacy of the synergistic use of the fungicides and chemical agents of this invention, a control experiment on corn root rot was conducted under greenhouse pot conditions.

[0067] Experimental materials: The maize variety tested was Zhengdan 958. Test pathogens: A mixed inoculum of the main pathogens causing maize root rot, including *Fusarium graminearum*, *Fusarium verticillioides*, and *Rhizoctonia solani*. Each strain was activated on PDA medium and then prepared with sterile water to a spore concentration of 1 × 10⁻⁶. 6 A mixed bacterial suspension of CFU / mL.

[0068] Test bacterial agents: Control bacterial agents prepared in Examples 1 and Comparative Examples 1-6 (effective viable count ≥ 2.0 × 10⁻⁶) 9 CFU / g).

[0069] Experimental Design: Eleven treatment groups were set up, with each treatment repeated three times. Each replicate consisted of 15 pots, with three corn seeds sown in each pot. After emergence, one seedling was transplanted. The amount of inoculant used for seed coating was 5% of the seed weight (i.e., 5 kg of inoculant per 100 kg of seeds), which is the conventional amount used for seed coating treatment in this field.

[0070] CK (blank control): No treatment was given, and the same amount of sterile water was applied after sowing.

[0071] T1 (Standard dosage of chemical agents): 2.5 g / 100 kg of fludioxonil active ingredient for seed coating.

[0072] T2 (reduced chemical dosage): Seed coating with 1.25 g / 100 kg of fludioxonil active ingredient (reduction of 50%).

[0073] T3 (Example 1 microbial agent + reduced chemical agent): Example 1 microbial agent (5%) is mixed with reduced fludioxonil and coated.

[0074] T4 (Example 2 microbial agent + reduced chemical agent): Example 2 microbial agent (5%) is mixed with reduced fludioxonil and coated.

[0075] T5 (Comparative Example 1 Inoculum + Reduced Chemical Agent): Comparative Example 1 inoculum (5%) is mixed with reduced fludioxonil and coated.

[0076] T6 (Comparative Example 2 Inoculum + Reduced Chemical Agent): Comparative Example 2 Inoculum (5%) is mixed with reduced fludioxonil and coated.

[0077] T7 (Comparative Example 3 Inoculant + Reduced Chemical Agent): Comparative Example 3 Inoculant (5%) is mixed with reduced fludioxonil and coated.

[0078] T8 (Comparative Example 4 Inoculum + Reduced Chemical Agent): Comparative Example 4 Inoculum (5%) is mixed with reduced fludioxonil and coated.

[0079] T9 (Comparative Example 5 Inoculum + Reduced Chemical Agent): Comparative Example 5 Inoculum (5%) is mixed with reduced fludioxonil and coated.

[0080] T10 (Comparative Example 6 Inoculant + Reduced Chemical Agent): Comparative Example 6 Inoculant (5%) is mixed with reduced fludioxonil and coated.

[0081] Test chemical: 98% fludioxonil technical grade, calculated based on the conventional dosage for seed treatment.

[0082] Inoculation and Cultivation Management: After the corn seeds were treated as described above, they were sown in plastic pots (20 cm in diameter) containing sterilized soil, with one seedling retained per pot after emergence. When the corn seedlings reached the 3-leaf stage, they were inoculated with a mixed suspension of pathogens using the root irrigation method, with 10 mL inoculated per pot. After inoculation, the pots were placed in a greenhouse (25-28℃, relative humidity 70%-80%) for routine management, with regular watering.

[0083] Survey and calculation methods: Disease index survey: 30 days after inoculation, 10 plants from each treatment were randomly selected to investigate root disease. The grading standards for root rot were based on the industry standard (NY / T 1464.44-2012): Grade 0: No disease spots on the roots; Grade 1: Root lesion area < 1 / 4; Grade 2: 1 / 4 ≤ lesion area < 1 / 2; Grade 3: 1 / 2 ≤ lesion area < 3 / 4; Grade 4: Lesions covering ≥3 / 4 of the body or root rot.

[0084] Disease index = ∑(disease grade × number of plants at that grade) / (highest disease grade × total number of plants surveyed) × 100; Prevention and control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0085] Seedling stage indicators: Chlorophyll content was measured using a SPAD-502 chlorophyll meter (Minolta, Japan), with three replicates and the average value taken. Peroxidase (POD) activity was determined using the guaiacol method (Yang Ling, Guo Min. Diurnal variation of protective enzyme activity in maize seedling leaves under PEG osmotic stress [J]. Anhui Agricultural Sciences, 2011, 39(12): 6959-6962.), and catalase (CAT) activity was determined using the method described in the literature (Chen Minjie, Jiang Xiaoru, Li Yafei, et al. Effects of different combinations of Bacillus polymyxa and chemical fertilizers on growth and quality of lettuce [J]. Journal of Henan Normal University (Natural Science Edition), 2019, 47(3): 92-98). Yield per plant was measured at harvest. All results were averaged.

[0086] Table 3. Control effects of different treatments on maize root rot

[0087] Table 4 Effects of different treatments on physiological indicators of maize seedlings

[0088] As shown in Tables 3 and 4, the compound microbial agents of Examples 1 and 2, under the condition of a 50% reduction in chemical pesticide dosage, achieved control effects of 92.5% and 94.2% against corn root rot, respectively. These results are significantly better than the 85.0% achieved by conventional chemical pesticide treatment alone, and even more significantly better than the 61.6% achieved by reduced chemical pesticide treatment alone. This result indicates that the Bacillus belye and Trichoderma harzianum CGMCC No. 40702 compound microbial agent screened in this invention has a significant synergistic effect with reduced fludioxonil, achieving the goal of halving the amount of chemical pesticides while ensuring or even improving the control efficacy.

[0089] In contrast, the compound bacterial agents in Comparative Examples 1-6, under the same dosage reduction conditions, only achieved a control effect of 70.1%-72.6%, showing limited improvement compared to the single treatment with chemical agents (61.6%), and no significant synergistic effect. Comparative Examples 5 and 6, due to the use of the core strain of this invention but with a formulation deviating from the optimized range, achieved control effects of 78.6% and 78.1%, respectively, but still significantly lower than Examples 1 and 2. This comparison clearly demonstrates that the specific combination and precise formulation of the bacterial strains are key factors affecting the synergistic dosage reduction effect; deviations in any step will lead to a decrease in control efficacy.

[0090] From a physiological perspective, the SPAD values ​​of maize seedlings treated in Examples 1 and 2 reached 38.5 and 40.2, respectively, representing an increase of 26.2%-31.8% compared to the blank control and 12.6%-17.5% compared to the conventional chemical treatment. The POD activities were 35.5 U / g·min and 38.5 U / g·min, respectively, which were 2.84-3.08 times that of the control group and 1.58-1.71 times that of the conventional chemical treatment. The CAT activities were 23.5 μmol / g·min and 24.5 μmol / g·min, respectively, which were 2.47-2.58 times that of the control group and 1.52-1.58 times that of the conventional chemical treatment. These data indicate that the compound microbial agent of this invention not only effectively inhibits pathogens but also significantly activates the plant's defense enzyme system, enhances antioxidant capacity, and promotes chlorophyll synthesis, thereby improving the overall health of the plant. While the physiological indicators of each treatment in the comparative example were slightly higher than those of the single treatment with reduced chemical reagent dosage, there was a significant difference compared with the example, further confirming the necessity of specific strain combinations and optimized formulations.

[0091] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis ), characterized in that, The Bacillus belye is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37347 and deposit date of January 9, 2026.

2. A bactericidal agent comprising the *Bacillus belye* as described in claim 1, characterized in that, The microbial agent contains Bacillus bellis and Trichoderma, which are compounded in a 1:1 mass ratio.

3. The fungicide according to claim 2, characterized in that, The Trichoderma is Trichoderma harzianum ( Trichoderma harzianum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40702 on June 16, 2023.

4. A method for preparing the antibacterial agent according to claim 2 or 3, characterized in that, Includes the following steps: (1) Inoculate Bacillus belye CGMCC No. 37347 into LB liquid medium and culture with shaking at 30-37℃ and 180-220rpm for 18-24h to obtain seed culture; transfer the seed culture to fermentation medium at an inoculation rate of 2%-5% and ferment at 30-37℃ and 180-220rpm for 48-72h to obtain Bacillus belye fermentation broth; (2) Inoculate Trichoderma harzianum CGMCC No. 40702 onto PDA agar plates and culture at 25-28℃ for 5-7 days. After the spores mature, wash them off with sterile physiological saline and adjust the spore concentration to 1.0×10⁻⁶. 8 -1.0×10 9 CFU / mL was used to obtain a Trichoderma spore suspension; (3) The Bacillus berberis fermentation broth obtained in step (1) and the Trichoderma spore suspension obtained in step (2) are mixed at a mass ratio of 1:1, carrier excipients are added, and the mixture is freeze-dried to obtain the control agent.

5. The method for preparing the fungicide according to claim 4, characterized in that, The carrier additive is one or more of diatomaceous earth, light calcium carbonate, and humic acid, and the amount of carrier additive added is 30%-50% of the total mass of the bacterial solution.

6. The method for preparing the fungicide according to claim 4, characterized in that, The fermentation medium in step (1) consists of: 20-30 g / L glucose, 15-25 g / L soybean meal powder, 10-15 g / L corn steep liquor powder, 5-8 g / L NaCl, 2-3 g / L K2HPO4, 0.5-1 g / L MgSO4·7H2O, and pH 7.0-7.

5.

7. The method for preparing the fungicide according to claim 4, characterized in that, The freeze-drying temperature in step (3) is -40℃ to -50℃, the vacuum degree is 10-20Pa, and the drying time is 24-48h.

8. The application of the fungicide according to claim 2 or 3, characterized in that, Used to promote corn growth and synergistically control corn root rot with chemical agents.

9. The application of the fungicide according to claim 8, characterized in that, The chemical agent is fludioxonil.

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