Bacillus velezensis and application thereof in prevention and treatment of plant fungal diseases

Abstract

The invention belongs to the field of microorganisms, and discloses bacillus velezensis and application thereof in prevention and treatment of plant fungal diseases. The bacillus velezensis disclosed by the invention is bacillus velezensis YC46, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and has the preservation number of CGMCC No.33975. The bacillus velezensis can grow by taking leftovers in an aquatic product processing process as a unique nutrient source, and has the advantages of high yield, low cost and the like. The bacillus subtilis has the antibacterial activity on plant pathogenic fungi such as fusarium oxysporum, botrytis cinerea, alternaria alternata, colletotrichum gloeosporioides and penicillium, thalli or fermentation products of the thalli can be used as active ingredients for preparing biocontrol microbial agents or bacteriostatic agents for preventing and treating plant fungal diseases, and the bacillus subtilis has good application value and ecological environmental protection value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus belye and its application in the prevention and control of plant fungal diseases. Background Technology

[0002] Plant fungal diseases are a significant factor restricting agricultural production, leading to reduced crop yields, decreased quality, and even total crop failure. Common plant pathogenic fungi include Fusarium oxysporum (…). Fusarium oxysporum ), Botrytis cinerea ( Botrytis cinerea Alternaria ( Alternaria spp.), Colloidal anthracnose ( Colletotrichum gloeosporioides ) and Penicillium ( Penicillium These pathogens (such as spp.) can cause wilt, gray mold, black spot, anthracnose, and blue mold, respectively, seriously threatening the safe production of economic crops such as tomatoes, cucumbers, cabbages, and citrus.

[0003] Currently, the control of plant fungal diseases mainly relies on chemical fungicides, such as triazoles, methoxyacrylates, and benzimidazoles. However, these chemical control methods have problems such as environmental risks, pesticide residues, increased costs, and resistance. Biological control, as an environmentally friendly alternative strategy, has received widespread attention in recent years. Microbial biocontrol agents have become a research hotspot due to their high safety and good environmental compatibility. Currently, existing biopesticides on the market mainly rely on a few types of microorganisms, such as Bacillus (…). Bacillus ), Pseudomonas spp. Pseudomonas ), Bacillus belesi ( Bacillus velezensis These microbial strains, such as [list of strains], can produce a variety of active substances that inhibit plant pathogenic fungi. However, the existing antibacterial spectrum of these strains is limited, and some require high-cost synthetic culture media for fermentation, which restricts their large-scale industrial application.

[0004] There is an urgent need to develop new strains with inhibitory activity against more plant pathogenic fungi and to explore their adaptability on low-cost natural culture media, so as to promote their application in the prevention and control of plant fungal diseases. Summary of the Invention

[0005] To address the problems existing in the background art, this invention provides a Bacillus bellis and its application in the control of plant fungal diseases. This Bacillus bellis can grow using byproducts from aquatic product processing as its sole nutrient source and exhibits antibacterial effects against various plant pathogenic fungi.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a Bacillus belyssus, which is Bacillus belyssus ( Bacillusvelezensis YC46 was deposited on March 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33975.

[0007] The present invention further provides the application of the above-mentioned Bacillus belye in the preparation of antifungal agents for plant pathogenic fungi.

[0008] The plant pathogenic fungi include one or more of the following: Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium.

[0009] This invention provides the application of the above-mentioned Bacillus belye in the preparation of drugs for the prevention and control of plant fungal diseases.

[0010] The plant fungal diseases mentioned include one or more of the following: tomato wilt, cucumber gray mold, Chinese cabbage black spot, citrus brown spot, and citrus blue mold.

[0011] Secondly, the present invention provides a biocontrol agent comprising the aforementioned Bacillus berleis. Bacillus berleis is the active ingredient of the agent, used to inhibit plant pathogenic fungi.

[0012] According to the above scheme, the biocontrol agent is in the form of a liquid agent, a lyophilized powder, or a tablet or aqueous solution containing the lyophilized powder and optional nutrients obtained by culturing the Bacillus berberis in a liquid culture medium.

[0013] Furthermore, the liquid bacterial agent is a culture medium, concentrated culture medium, or culture suspension of Bacillus belyssus.

[0014] This invention also provides the application of the above-mentioned biocontrol agent in the preparation of antifungal agents for plant pathogenic fungi.

[0015] The plant pathogenic fungi include one or more of the following: Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium.

[0016] This invention provides the application of the above-mentioned biocontrol agent in the preparation of drugs for controlling plant fungal diseases.

[0017] The plant fungal diseases mentioned include one or more of the following: tomato wilt, cucumber gray mold, Chinese cabbage black spot, citrus brown spot, and citrus blue mold.

[0018] Thirdly, the present invention provides an antibacterial agent comprising the fermentation product of the aforementioned Bacillus belye.

[0019] According to the above scheme, the antibacterial agent includes the fermentation broth or supernatant of the above-mentioned Bacillus belye inoculated with fermentation broth, and the fermentation broth or supernatant of fermentation broth contains the above-mentioned fermentation products.

[0020] The present invention further provides the application of the above-mentioned antibacterial agent in the preparation of antibacterial agents for plant pathogenic fungi.

[0021] According to the above scheme, the plant pathogenic fungi include one or more of the following: Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium.

[0022] This invention also provides the application of the above-mentioned antibacterial agent in the preparation of drugs for the prevention and control of plant fungal diseases.

[0023] According to the above scheme, the plant fungal diseases include one or more of the following: tomato wilt, cucumber gray mold, Chinese cabbage black spot, citrus brown spot, and citrus blue mold.

[0024] The present invention further provides a method for preparing the above-mentioned antibacterial agent, wherein Bacillus belye is inoculated into a fermentation medium for fermentation to obtain a fermentation broth containing fermentation products; or further, the fermentation broth is centrifuged and filtered to remove the bacterial cells to obtain the supernatant of the fermentation broth, which is the above-mentioned antibacterial agent; the fermentation medium contains by-products from the processing of cephalopod aquatic products, which are homogenized and then prepared into a fermentation medium.

[0025] Byproducts from the processing of cephalopod aquatic products include one or more of the following: viscera, head (eyes, mouth), skin, cartilage, and processed meat scraps. Viscera include, but are not limited to, squid viscera, octopus viscera, and cuttlefish viscera.

[0026] The Bacillus berleis of the present invention can grow using by-products from the processing of cephalopod aquatic products as the sole nutrient source.

[0027] Further preferred, each liter of fermentation medium includes: 12-18g of cephalopod aquatic processing by-products and 27-33g of glucose.

[0028] According to the above scheme, the fermentation conditions are: inoculum size of 1-5%, temperature of 27-47℃, pH of 6.0-11.0, and fermentation time of 2-7 days.

[0029] Preferably, the fermentation conditions are: inoculum size of 1-3%, temperature of 27-37℃, pH of 9.0-10.0, and fermentation time of 3 days.

[0030] The beneficial effects of this invention are: This invention screened and obtained a strain of Bacillus belye ( Bacillus velezensisYC46 exhibits broad-spectrum antibacterial activity against a variety of plant pathogens, including Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium. It provides a new microbial resource with broad-spectrum antibacterial activity for the green control of plant fungal diseases and the development of environmentally friendly biological pesticides, and has significant application value. Bacillus belesiensis ( Bacillus velezensis YC46 can be mass-produced through culture for the preparation of Bacillus belyssus (… Bacillus velezensis YC46 is a biocontrol agent with active substances, or an antibacterial agent with fermentation products of Bacillus belye as active ingredients. It is used for the control of plant fungal diseases. It is environmentally friendly, easy to apply, and has good application prospects and ecological and environmental protection value. The Bacillus belesiensis of the present invention ( Bacillus velezensis YC46 can grow using by-products from seafood processing, such as animal offal, as its sole nutrient source. By using inexpensive seafood processing by-products as a fermentation substrate, it can significantly reduce production costs and is suitable for large-scale production. Attached Figure Description

[0031] Figure 1 The colony morphology of the four dominant strains obtained in the initial screening in Example 1 of this invention; Figure 2 This is a colony morphology diagram of strain YC46 in Example 1 of the present invention; Figure 3 This is a Gram staining image of strain YC46 in Example 1 of the present invention; Figure 4 This is a phylogenetic tree of the 16S rDNA of Bacillus belyssus YC46 in Example 1 of the present invention; Figure 5 The antibacterial effect of fermentation broth fermented with different inoculum amounts of Bacillus vesiculosus YC46 in Example 2 of this invention; Figure 6 This invention demonstrates the antibacterial effect of fermentation broth from Bacillus vesiculosus YC46 fermented at different temperatures in Example 2 of this invention. Figure 7 The antibacterial effect of the fermentation broth of Bacillus vesiculosus YC46 fermented at different initial pH values ​​in Example 2 of this invention; Figure 8 This invention demonstrates the antibacterial effect of fermentation broth obtained from Bacillus vesiculosus YC46 at different fermentation times in Example 2 of this invention. Detailed Implementation

[0032] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0033] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0035] In this invention, the inventors screened a bacterium, YC46, from fish sauce. This bacterium can grow using byproducts from seafood processing, such as animal viscera, as its sole nutrient source and exhibits antibacterial effects against various plant pathogenic fungi. Morphological and molecular biological identification confirmed that this bacterium is *Bacillus belye* (…). Bacillus velezensis YC46 was deposited on March 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33975.

[0036] The present invention also relates to a biocontrol agent with the above-mentioned Bacillus berleis as an active ingredient.

[0037] The biocontrol agent is in the form of a liquid agent, a lyophilized powder, or a tablet or aqueous solution containing the lyophilized powder and optional nutrients, obtained by culturing the Bacillus berberis in a liquid culture medium.

[0038] Preferably, the liquid bacterial agent is a culture medium, concentrated culture medium, or culture suspension of Bacillus belesiensis.

[0039] The above-mentioned biocontrol agents can be used to control plant pathogenic fungi, specifically such as Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium.

[0040] The above-mentioned biocontrol agents can be used to control plant fungal diseases. Specifically, they can be applied to the control of one or more of the following diseases: tomato wilt, cucumber gray mold, cabbage black spot, citrus brown spot, and citrus blue mold.

[0041] Specifically, biocontrol agents can be applied by spraying or root drenching.

[0042] The present invention also relates to the use of Bacillus belye fermentation products as antibacterial agents with active ingredients.

[0043] In some specific embodiments of the present invention, the antibacterial agent comprises the fermentation broth or supernatant of the above-mentioned Bacillus belye, and the fermentation broth or supernatant contains the above-mentioned fermentation product.

[0044] All of the above-mentioned antibacterial agents can be used to control plant fungal diseases. Specifically, the antibacterial agents can be sprayed on the fruit, leaves, or drenched in water.

[0045] The present invention further provides a method for preparing the above-mentioned antibacterial agent, wherein the above-mentioned Bacillus belye is inoculated into a fermentation medium for fermentation to obtain a fermentation broth containing fermentation products. Optionally, the fermentation broth is centrifuged and filtered to remove the bacterial cells to obtain the supernatant of the fermentation broth, which is the antibacterial agent.

[0046] The fermentation medium for the aforementioned Bacillus belye contains by-products from cephalopod seafood processing, which are homogenized and then formulated into the fermentation medium. Cephalopod seafood processing by-products include one or more of the following: viscera, head (eyes, mouth), skin, cartilage, and processed meat scraps. Viscera include, but are not limited to, squid viscera, octopus viscera, and cuttlefish viscera.

[0047] The *Bacillus belye* strain of the present invention can grow using by-products from cephalopod seafood processing as the sole nutrient source. More preferably, the fermentation medium for *Bacillus belye* contains by-products from cephalopod seafood processing and glucose. Specifically, each liter of fermentation medium contains: 12-18 g of by-products from cephalopod seafood processing and 27-33 g of glucose.

[0048] In one specific embodiment, each liter of fermentation medium comprises: 15g of cephalopod aquatic processing by-products and 30g of glucose.

[0049] The fermentation conditions for the above-mentioned Bacillus belyssus are: inoculum size of 1-5%, temperature of 27-47℃, pH of 6.0-11.0, and fermentation time of 2-7 days. Preferably, the fermentation speed is 170-210 r / min.

[0050] Preferably, the fermentation conditions for the above-mentioned Bacillus belyss are: inoculum size of 1-3%, temperature of 27-37℃, pH of 9.0-10.0, speed of 170-190 r / min, and fermentation time of 3 days.

[0051] In one specific embodiment of the present invention, Bacillus bellis was fermented in a liquid culture medium, and the bacterial cells were removed by centrifugation and filtration to obtain the supernatant of the fermentation broth. Using plant pathogenic fungi Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium as antibacterial activity indicator bacteria, the antibacterial activity of the Bacillus bellis fermentation broth was determined by the mycelial growth inhibition method. The inhibition rates against Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium were 93.35%, 95.12%, 96.37%, 96%, and 87.85%, respectively.

[0052] In one specific embodiment of the present invention, Bacillus belye was fermented in a liquid culture medium, and the bacterial cells were removed by filtration to obtain the supernatant of the fermentation broth. The control effect of the supernatant on plant fungal diseases such as tomato wilt, cucumber gray mold, Chinese cabbage black spot, citrus brown spot, and citrus blue mold was tested. The results showed that the control effects on tomato wilt, cucumber gray mold, Chinese cabbage black spot, citrus brown spot, and citrus blue mold were 83.22%, 85.83%, 89.38%, 89.15%, and 87.34%, respectively.

[0053] The following are specific examples.

[0054] Example 1: Isolation, purification and identification of Bacillus belye (1) Separation and purification a. Sample Collection and Pretreatment: Ten samples of naturally fermented fish sauce were collected from Lanshan District, Rizhao City, Shandong Province. They were placed in sterile sampling bottles, stored at 4℃, and transported to the laboratory. 5g of the naturally fermented fish sauce sample was added to 45mL of sterile physiological saline, shaken to mix, and allowed to stand for 10min. The supernatant was then serially diluted (10⁻¹⁰). -3 ~10 -6 ).

[0055] b. Initial screening of strains Bacterial strains were isolated from fish sauce samples using the conventional dilution plate method in the primary screening medium.

[0056] The primary screening medium is a medium containing squid processing waste. In this embodiment, the specific waste is squid processing waste such as squid viscera. The squid viscera are washed and homogenized. The specific components of the primary screening medium are: 25g of squid viscera, distilled water to a final volume of 1L, 25g of agar, and natural pH.

[0057] Take 100µL of each concentration of sample solution and spread it evenly on the primary screening medium plate. Incubate at 37℃ for 2 days. Pick single colonies with different morphologies and transfer them to commercial LB liquid medium. Incubate at 37±1℃ and 180r / min for 1 day for later use.

[0058] Initial screening results are as follows Figure 1As shown, four dominant bacteria strains that use squid processing waste such as squid viscera as their sole nutrient source were isolated from the fish sauce sample and named strain I, strain II, strain III, and strain IV, respectively.

[0059] c. Preparation of fermentation broth from primary screening of dominant strains Prepare a fermentation medium consisting of 15g squid viscera, 30g glucose, and distilled water to a final volume of 1L, adjusting the pH to 8.0.

[0060] The four dominant bacterial strains obtained from the initial screening were inoculated into the fermentation medium at an inoculum rate of 3% (V / V) and fermented at 37℃ for 3 days at 180 rpm. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4℃, the supernatant was collected, and the bacterial cells were removed by filtering through a 0.45 μm filter membrane. The filtrate was stored at 4℃ for later use.

[0061] d. Antibacterial activity of fermentation broth from preliminarily screened dominant strains The antibacterial activity of the fermentation broth of the pre-screened dominant strains against *Fusarium oxysporum*, *Botrytis cinerea*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, and *Penicillium* was determined using the mycelial growth inhibition method. The specific method is as follows: 6 mL of the dominant strain's fermentation broth was added to 44 mL of sterile commercial potato dextrose agar (PDA) medium at 50–60 °C. After mixing, the agar plates were poured to prepare PDA plates containing the dominant strain's fermentation broth. *Fusarium oxysporum*, *Botrytis cinerea*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, and *Penicillium* were used as indicator bacteria for antibacterial activity. Mature indicator bacterial discs (6 mm in diameter) were prepared and placed on fresh PDA plates containing the fermentation broth. The discs were incubated in the dark at 25 °C. Sterile water was used as a blank control group instead of the fermentation broth. When the control group colonies reached approximately 2 / 3 of the petri dish, the diameter of the treated group colonies was measured using the cross-crossing method. The mycelial growth inhibition rate (%) was calculated for each PDA plate containing fermentation broth according to the formula: Mycelial growth inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / (Coronary diameter of control group - Diameter of mycelial cake) × 100%. Each treatment was performed in triplicate, and the inhibition rate was taken as the mean ± standard deviation.

[0062] The results of the antibacterial activity are shown in Table 1 below.

[0063] Table 1

[0064] The fermentation broths of the four dominant strains all showed inhibitory activity against the indicator bacteria Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium. Among them, the fermentation broth of the dominant strain IV showed the highest inhibition rate against the above five indicator bacteria, with inhibition rates of 42.03±0.15%, 45.15±1.22%, 39.17±0.53%, 38.42±1.07%, and 33.86±1.42%, respectively.

[0065] e. Purification of the primary screening dominant strain IV The strain IV with the highest antibacterial activity was purified by the LB solid medium streak plate method, named YC46, and stored at -80℃.

[0066] (2) Growth characteristics and species identification of strain YC46 a. Strain morphological characteristics The selected YC46 cells were inoculated onto LB solid medium plates and incubated at 37°C for 1 day. The morphological characteristics of the colonies on the solid medium were observed, including their size, color, and raised shape, and Gram staining was performed.

[0067] The results are as follows Figure 2 and Figure 3 As shown, strain YC46 colonies on LB plates are milky white, smooth and moist, with neat edges, viscous texture, and easy to pick up; they are Gram-positive short rods.

[0068] b. Physiological and biochemical identification of strains The physiological and biochemical identification results of strain YC46 are shown in Table 2. The VP test, propionate, D-xylose, and L-arabinose utilization tests were negative; the citrate, gelatin liquefaction, nitrate reduction, and starch hydrolysis tests were positive. NaCl and pH tolerance tests showed that strain YC46 cannot grow under 7% NaCl conditions but can grow under pH=5.7 conditions. It is a facultative anaerobic bacterium, capable of growing in both aerobic and anaerobic environments. Based on morphological characteristics, strain YC46 is preliminarily identified as belonging to the genus *Bacillus*.

[0069] Table 2

[0070] c. Identification of 16S rDNA and construction of phylogenetic tree for strain YC46 The 16S rDNA gene was amplified using universal primers 27F / 1492R. The amplified product was recovered and sequenced. The 16S rDNA nucleotide sequence of strain YC46 is shown in SEQ ID NO:1. The 16S rDNA sequence of strain YC46 has a total nucleotide length of 1454 bp. Sequencing results were submitted to the NCBI database for comparison, and the results showed that strain C46 is similar to the type strain. Bacillus velezensis NRRL B-41580 showed only a one-base difference, with a similarity of 99.99%. Sequences closely related to the sequencing results were selected, and a phylogenetic tree was constructed using MEGA 11 software. The phylogenetic tree construction results are as follows: Figure 4 As shown.

[0071] Based on the above morphological and molecular biological identification results and phylogenetic tree, strain YC46 was identified as Bacillus belyesii (B. belyesii). Bacillus velezensis The strain was named *Bacillus belyssus* YC46. It was deposited on March 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33975, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0072] Example 2: Antibacterial effect of Bacillus vesiculosus YC46 bacterial suspension Bacillus belye YC46 bacterial culture was inoculated into LB liquid medium and cultured at 37 ℃ and 180 r / min for 1 day to obtain Bacillus belye YC46 seed culture. The seed culture was then inoculated into fermentation medium, and the antibacterial effect of Bacillus belye YC46 under different fermentation conditions (inoculum size, temperature, pH, and fermentation time) was determined.

[0073] The fermentation medium consisted of 15g of squid viscera, 30g of glucose, and distilled water to a final volume of 1L, with the pH adjusted to 8.0.

[0074] a. Inoculation amount for Bacillus belye ( Bacillus velezensis Influence of YC46 bacterial solution on antibacterial effect The above-mentioned *Bacillus belyssioides* YC46 seed culture was inoculated into the fermentation medium at inoculum rates of 1%, 2%, 3%, 4%, and 5%, respectively. The initial pH of the fermentation broth was 10.0, and fermentation was carried out at 180 r / min and 27℃ for 3 days. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4℃, and the supernatant was collected. The bacterial cells were removed by filtering through a 0.45 μm filter membrane, and the fermentation filtrate was stored at 4℃ for later use. Further, the antibacterial activity of the fermentation filtrate was investigated using the mycelial growth inhibition method described in Example 1. The antibacterial results of the fermentation broth of strain YC46 against *Fusarium oxysporum*, *Botrytis cinerea*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, and *Penicillium* are as follows: Figure 5 As shown, Bacillus belye ( Bacillus velezensisThe optimal inoculum size for YC46 fermentation is 1%. When the inoculum size is 1%, the inhibition rates of the fermentation broth of strain YC46 against Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum anthracis, and Penicillium are 94.88%, 96.77%, 99.58%, 95.96%, and 84.64%, respectively.

[0075] b. The effect of temperature on Bacillus belesiensis ( Bacillus velezensis Influence of YC46 bacterial solution on antibacterial effect The above-mentioned *Bacillus belyssioides* YC46 seed culture was inoculated into the fermentation medium at an inoculum rate of 1% (V / V). The initial pH of the fermentation broth was 10.0. Fermentation was carried out at 180 r / min for 3 days at 27℃, 32℃, 37℃, 42℃, and 47℃, respectively. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4℃, and the supernatant was collected. The bacterial cells were removed by filtering through a 0.45 μm filter membrane, and the fermentation filtrate was stored at 4℃ for later use. Furthermore, the antibacterial activity of the fermentation filtrate was investigated according to the mycelial growth inhibition method in Example 1. The antibacterial results of the fermentation broth of strain YC46 against *Fusarium oxysporum*, *Botrytis cinerea*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, and *Penicillium* are as follows: Figure 6 As shown, Bacillus belye ( Bacillus velezensis The optimal fermentation temperature for YC46 is 27℃. When the fermentation temperature is 27℃, the inhibition rates of the fermentation broth of strain YC46 against Fusarium oxysporum, Staphylococcus aureus, Alternaria alternata, Colletotrichum anthracis, and Penicillium are 91.51%, 94.63%, 92.70%, 91.80%, and 88.39%, respectively.

[0076] c. pH value on Bacillus belesi ( Bacillus velezensis Influence of YC46 bacterial solution on antibacterial effect The above-mentioned *Bacillus belye* YC46 seed culture was inoculated into the fermentation medium at an inoculum rate of 1% (V / V). The initial pH values ​​of the fermentation broth were 6, 7, 8, 9, 10, and 11, respectively. Fermentation was carried out at 180 r / min and 27℃ for 3 days. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4℃, and the supernatant was collected. The bacterial cells were removed by filtering through a 0.45 μm filter membrane, and the fermentation filtrate was stored at 4℃ for later use. Further, the antibacterial activity of the fermentation filtrate was investigated according to the mycelial growth inhibition method in Example 1. The antibacterial results of the fermentation broth of strain YC46 against *Fusarium oxysporum*, *Botrytis cinerea*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, and *Penicillium* are as follows. Figure 7 As shown, Bacillus belye ( Bacillus velezensis The optimal initial pH for fermentation of YC46 is 10. When the initial pH of the fermentation broth is 10, the inhibition rates of the fermentation broth of strain YC46 against Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum anthracis, and Penicillium are approximately 91.32%, 92.31%, 96.15%, 97.35%, and 88.42%, respectively.

[0077] d. Fermentation time for Bacillus belesiensis ( Bacillus velezensis Influence of YC46 bacterial solution on antibacterial effect The above-mentioned *Bacillus belyssioides* YC46 seed culture was inoculated into the fermentation medium at an inoculum rate of 1% (V / V). The initial pH of the fermentation broth was 10. Fermentation was carried out at 180 r / min and 27℃ for 2, 3, 4, 5, 6, and 7 days, respectively. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4℃, and the supernatant was collected. The bacterial cells were removed by filtering through a 0.45 μm filter membrane, and the fermentation filtrate was stored at 4℃ for later use. Further, the antibacterial activity of the fermentation filtrate was investigated according to the mycelial growth inhibition method in Example 1. The antibacterial results of the fermentation broth of strain YC46 against *Fusarium oxysporum*, *Botrytis cinerea*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, and *Penicillium* are as follows: Figure 8 As shown, Bacillus belye ( Bacillus velezensis The optimal fermentation time for YC46 is 3 days. When the fermentation time is 3 days, the inhibition rates of the fermentation broth of strain YC46 against Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum anthracis and Penicillium are approximately 95.69%, 96.75%, 97.05%, 98.86% and 89.94%, respectively.

[0078] Bacillus belesiensis ( Bacillus velezensis YC46 fermentation broth has good antibacterial properties, with a high inhibition rate against fungi such as Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum anthracnose, and Penicillium. It can be used as a safe biological pesticide or antifungal agent for application to plants or their growth environment.

[0079] Example 3: Evaluation of the control effect of Bacillus vesiculosus YC46 fermentation broth on plant fungal diseases Plant seedlings (tomato, cucumber, cabbage, citrus, and mandarin orange) with similar growth were selected for a pot experiment to evaluate the control effect of Bacillus vesiculosus YC46 fermentation broth on plant fungal diseases. One day before transplanting, the seedlings were drenched with 50 mL of pathogen spore suspension (Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, Penicillium, 10...) for the control group (CK). 5 CFU·mL -1 ) and 50 mL of distilled water; the treatment group consisted of 50 mL of pathogenic spore solution (10) 5 CFU·mL -1 The treatments were administered with 50 mL of Bacillus belyss YC46 fermentation broth. Each group was replicated three times, with eight seedlings per group. All treatments were cultured at room temperature under a 16 / 8 h day / night light / dark cycle for 30 days, and the disease incidence was recorded for each group.

[0080] The control effect is calculated based on the disease index. Disease index = ∑(number of diseased plants at each level × disease level value) / (total number of plants × highest level value) × 100; Control effect = (CK disease index - treatment disease index) / CK disease index × 100%.

[0081] The disease index and control efficacy are shown in Table 3. The disease indexes of the blank control group were 90.25%, 92.34%, 85.18%, 80.64%, and 79.28%, respectively. In contrast, the plants treated with Bacillus vesiliflorus YC46 fermentation broth showed good growth, with disease indices decreasing to 15.14%, 13.08%, 9.05%, 8.75%, and 10.04, respectively, and control efficacy reaching approximately 83.22%, 85.83%, 89.38%, 89.15%, and 87.34%, respectively. These results indicate that Bacillus vesiliflorus YC46 fermentation broth has a significant inhibitory effect on plant fungal diseases, and its application in the control of plant fungal diseases shows promising prospects.

[0082] Table 3. Control efficacy of Bacillus belye YC46 against plant fungal diseases.

[0083] The above indicates that the selected Bacillus belyss ( Bacillus velezensis YC46 exhibits excellent antifungal effects against plant pathogenic fungi, including Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium. It also provides good control over plant fungal diseases such as tomato wilt, cucumber gray mold, cabbage black spot, citrus brown spot, and citrus penicillium.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus belesii, characterized in that, Bacillus belesiensis ( Bacillus velezensis YC46 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33975.

2. A biocontrol agent, characterized in that, It includes Bacillus belesiensis as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, It is obtained by culturing the Bacillus berberis in a liquid culture medium in the form of a liquid inoculum, a lyophilized powder, or a tablet or aqueous solution containing the lyophilized powder and optional nutrients.

4. An antibacterial agent, characterized in that, Fermentation products containing Bacillus belye as described in claim 1.

5. The antibacterial agent according to claim 4, characterized in that, The fermentation broth or supernatant of the Bacillus berberis as described in claim 1 contains the fermentation product.

6. The method for preparing the antibacterial agent as described in claim 4 or 5, characterized in that, The Bacillus berberis described in claim 1 is inoculated into a fermentation medium for fermentation to obtain a fermentation broth containing fermentation products; or further, the fermentation broth is centrifuged and filtered to remove the bacterial cells to obtain the supernatant of the fermentation broth, which is the antibacterial agent; the fermentation medium contains by-products from the processing of cephalopod aquatic products, which are homogenized and then prepared into a fermentation medium.

7. The method for preparing the antibacterial agent according to claim 6, characterized in that, Each liter of fermentation medium includes: 12-18g of by-products from cephalopod aquatic product processing, and 27-33g of glucose.

8. The method for preparing the antibacterial agent according to claim 6, characterized in that, Fermentation conditions are as follows: inoculum size 1-5%, temperature 27-47℃, pH 6.0-11.0, and fermentation time 2-7 days.

9. The application of Bacillus vesiculosus as described in claim 1, the biocontrol agent as described in claim 2 or 3, and the antibacterial agent as described in claim 4 or 5 in the preparation of antibacterial agents for plant pathogenic fungi, wherein the plant pathogenic fungi include one or more of Fusarium oxysporum, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium.

10. The use of *Bacillus belye* as described in claim 1, the biocontrol agent as described in claim 2 or 3, and the antibacterial agent as described in claim 4 or 5 in the preparation of drugs for controlling plant fungal diseases, wherein the plant fungal diseases include: One or more of the following: tomato wilt, cucumber gray mold, cabbage black spot, citrus brown spot, and citrus blue mold.

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