Bacillus velezensis, biocontrol agent prepared from the bacillus and application thereof

Abstract

The present application relates to a bacillus velezensis, a biocontrol agent prepared from the bacillus velezensis and an application thereof, and belongs to the technical field of biological bacterial agents. The bacillus velezensis provided by the present application is bacillus velezensis B. velezensis, named JK1, the preservation unit is China General Microbiological Culture Collection Center, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation center number is CGMCC No. 31375, and the preservation time is July 19, 2024. The bacillus JK1 has excellent inhibition effect on different pathogenic bacteria whether it is a living bacterium, a supernatant or a cell lysate. The bacillus JK1 has strong living ability, and is suitable for a wide range of nutrition sources, temperature and humidity and pH. The bacillus JK1 has no pathogenicity to fruits and vegetables, and has good tolerance to a broad-spectrum bactericide, and has great application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol agents technology, specifically relating to a strain of Bacillus belye, a biocontrol agent made from the Bacillus belye, and its application in antibacterial and antimicrobial preservation of fruits and vegetables. Background Technology

[0002] Fruits and vegetables are susceptible to soil-borne pathogens during cultivation, frequently threatened by bacterial and fungal infections, with fungal diseases being the most common. Common symptoms include necrosis (leaf spot, leaf blight), rot (root rot, fruit rot), and wilting (infection of roots, stem base, and vascular tissue). Traditionally, disease control methods include chemical fungicides, breeding disease-resistant varieties, intercropping, and grafting, but each of these measures has its advantages and disadvantages.

[0003] On the other hand, fresh fruits and vegetables undergo changes in their physiological and biochemical characteristics after harvest and during long-term storage, making them susceptible to varying degrees of infectious diseases (pathogenic microbial infection). Postharvest infectious diseases in fruits and vegetables are caused by pathogenic microorganisms infecting the host plants during their growth and development in the field. Cross-contamination during transportation and storage is also a contributing factor. The presence of pores in the skin of most fruits and vegetables, as well as wounds caused by improper transportation, facilitates the invasion of pathogenic microorganisms into the internal flesh, exacerbating the severity of postharvest infectious diseases. Currently, the main methods for controlling postharvest infectious diseases in fruits and vegetables are pre-harvest pesticide spraying and post-harvest lowering of storage temperature. However, excessive pesticide use can lead to serious pesticide residue exceeding standards, endangering human health. Furthermore, long-term excessive pesticide use can cause pathogenic microorganisms to develop resistance, significantly reducing the effectiveness of disease control.

[0004] Biological control is a technique that uses live biocontrol bacteria and their metabolically active substances to control the occurrence of diseases. Biocontrol bacteria can colonize and grow in crop plants and rhizosphere, forming a biological barrier to protect crops from pathogens. Their metabolically active substances inhibit and kill pathogenic fungi on the one hand, and induce plants to improve their disease resistance on the other.

[0005] Biological control methods have significant advantages over chemical control methods. Besides the ease of production and use of biological agents, mixed inoculants made from biocontrol bacteria and their metabolites are more environmentally friendly, producing no pollution or pesticide residues, thus aligning with my country's sustainable development strategy. Furthermore, biocontrol agents are less sensitive to environmental changes and exhibit stable efficacy. Additionally, the nutrients decomposed and transformed by biocontrol bacteria can be utilized by crops, thereby improving crop quality and increasing yield.

[0006] However, while existing biocontrol strains and agents have shown some effectiveness, and some possess broad-spectrum antibacterial properties, the inhibition rates of existing Bacillus biocontrol agents are generally low, particularly lacking *Bacillus belyssiensis*. Furthermore, few biocontrol strains have been discovered against fruit and vegetable pathogens such as *Colletotrichum fioriniae* and *Colletotrichum citricola*, and their inhibition rates are not high. Additionally, no effective biocontrol strains have been reported against the four pathogens *Stemphylium eturmiunum*, *Clathrospora diplospora*, *Chaetomium cochliodes*, and *Galactomyces candidus*.

[0007] Therefore, there is a need to provide a Bacillus beleilli biocontrol strain with a broader spectrum of antibacterial effects, a significantly high inhibition rate against fruit and vegetable pathogens such as Colletotrichum fioriniae and Colletotrichum citricola, and a significant inhibitory effect against four pathogens: Stemphylium eturmiunum, Clathrospora diplospora, Chaetomium cochliodes, and Galactomyces candidus. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a biocontrol strain applicable to the prevention and control of fruit and vegetable diseases and for antibacterial preservation. This invention provides a *Bacillus belyssiensis* strain that is highly effective in the prevention and control of fruit and vegetable diseases and for antibacterial preservation, exhibiting a 100% inhibition rate against pathogens such as *Colletotrichum fioriniae* and *Colletotrichum citricola*, and demonstrating significant antibacterial effects against four pathogens: *Stemphylium eturmiunum*, *Clathrospora diplospora*, *Chaetomium cochliodes*, and *Galactomyces candidus*. The invention also provides a biocontrol agent made from this *Bacillus* and its application in the antibacterial and antimicrobial preservation of fruits and vegetables.

[0009] Through extensive experimental research and exploration, the inventors of this invention obtained a strain of Bacillus velezensis, named JK1. The depositary institution is 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. The deposit number is CGMCC No. 31375, and the deposit date is July 19, 2024.

[0010] The gene sequence of Bacillus JK1 provided by the present invention is shown in SEQ NO.1.

[0011] The present invention further provides a biocontrol agent comprising the above-mentioned Bacillus JK1.

[0012] The present invention further provides the application of the biocontrol agent in the antibacterial activity against pathogens in fruits and vegetables, and the application of the Bacillus in the preparation of fruit and vegetable preservatives.

[0013] Specifically, the fruit and vegetable pathogenic bacteria include Colletotrichum fioriniae, Colletotrichumcitricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichumacutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergilluspuulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeriavariabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyceschartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccumparvum, Fusarium proliferatum, Rhizomucor variabilis, Phomaherbarum, Sclerotium hydrophilum, Fusariumincarnatum, Fusarium graminearum and Penicillium oxalicum.

[0014] The concentration of Bacillus JK1 in the biocontrol agent of this invention is 1×10⁻⁶. 6-8 cfu / mL.

[0015] The beneficial effects of this invention are as follows:

[0016] (1) The live Bacillus JK1 cells of the present invention have extremely high antibacterial activity against a variety of different pathogens, and their broad-spectrum antibacterial activity is excellent. Among them, the live Bacillus JK1 cells have the most significant antibacterial effect against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthe fusicola, and Pyriculariaoryzae, with an inhibition rate of 100%, and can completely inhibit the growth of pathogenic fungi. Among them, live JK1 bacteria showed an inhibition rate of over 90% against Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, and Fusarium decemcellulare, demonstrating good inhibitory effects; and against Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, and Fusarium The antibacterial effects of oxysporum, Galactomyces candidus, Neofuusicoccum parvum, Fusarium proliferatum, Rhizomucor variabilis, and Phona herbarum are generally moderate, with an antibacterial rate greater than 80%.

[0017] (2) The supernatant of Bacillus JK1 of the present invention has a certain antibacterial effect on different pathogenic fungi. Among them, the supernatant of Bacillus JK1 showed the best antibacterial effect against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthe fusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, and Clathrospora diplospora, with inhibition rates all greater than 90%. The supernatant of Bacillus JK1 showed certain antibacterial effects against Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum, and Rhizomucorvariabilis, with inhibition rates all greater than 80%.

[0018] (3) The Bacillus JK1 cell lysate of the present invention has a certain antibacterial effect against different pathogenic fungi, but the antibacterial effect is slightly lower than that of the supernatant of the JK1 strain. Among them, the Bacillus JK1 cell lysate has a better antibacterial effect against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, and Penicillium decumbens, with an inhibition rate of more than 90%. JK1 cell lysates exhibited antibacterial activity against Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, and Neofusicoccum parvum, with inhibition rates all exceeding 80%.

[0019] (4) The Bacillus JK1 provided by the present invention has a broad antibacterial spectrum, involving 38 kinds of fruit and vegetable pathogens. In contrast, the prior art (CN114369556B-A strain of Bacillus, a biocontrol agent made from the Bacillus and its application) also provides a strain of Bacillus biocontrol, but it involves only 32 kinds of fruit and vegetable pathogens. Attached Figure Description

[0020] Figure 1The antibacterial effect of live Bacillus JK1 cells against different pathogens is shown; in each group, the left side is the control (CK), and the right side is the experimental group containing JK1.

[0021] Figure 2 The turbidity of Bacillus JK1 cultured at different temperatures;

[0022] Figure 3 The turbidity of Bacillus JK1 cultured at different pH values. Detailed Implementation

[0023] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0024] Example 1

[0025] I. Source of the strain

[0026] In 2023, peach tree leaves were collected from a peach planting base in Dazhu County, Dazhou City, Sichuan Province, placed in sterile sealed bags, and brought back to the Institute of Agricultural Products of Chengdu Academy of Agricultural and Forestry Sciences to isolate and purify antagonistic bacterial strains.

[0027] II. Identification of the strain

[0028] Bacterial DNA was extracted and 16S rDNA sequenced by PCR. The gene sequence is shown in SEQ NO. 1 of the sequence listing. The obtained sequence was compared with the NCBI website, and it was classified and named Bacillus velezensis, and further named Bacillus JK1. The depositary institution is the China General Microbiological Culture Collection Center, the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number is CGMCC No. 31375, and the deposit date is July 19, 2024.

[0029] III. Antibacterial Tests of Strains

[0030] (I) Live cell antibacterial test of Bacillus JK1

[0031] The strain has a wide range of antibacterial properties and is effective in preventing and controlling pathogens on strawberries, blueberries, grapes, apples, mangoes, cherries, kiwifruit, jujubes, peppers, wax apples, eggplants, garlic, and loofahs.

[0032] 1. Experimental Design

[0033] A single colony of strain JK1 was inoculated into LB broth and cultured at 37°C with constant shaking at 150 rpm for 24 h. The cell suspension density was determined to be 1 × 10⁻⁶ using a hemocytometer. 8 cfu / mL. JK1 strain cell suspension was added to PDA medium, vortexed to mix, and plates were prepared. The cell density of JK1 strain on each plate was 1×10⁻⁶. 6 cfu / mL. Using a 6mm sterile punch, a mycelial cake of *Botrytis cinerea* was placed in the center of a PDA plate containing strain JK1. A PDA plate containing 100 mg / L carbendazim served as a pesticide control, and a PDA plate without JK1 bacteria served as a blank control. Each treatment was repeated in triplicate. The plates containing the mycelial cake were sealed in resealable bags and incubated at 26°C. The width of the inhibition zone was measured when the mycelium had fully grown on the control plate.

[0034] Calculate the inhibition rate R of mycelial growth.

[0035] R(%) = (R1 - R2) / R1 × 100%

[0036] Where R is the percentage of radial hyphal growth inhibited, R1 is the hyphal growth of the blank control, and R2 is the hyphal growth of the treatment group.

[0037] 2. Test Results

[0038] The experimental results are shown in Table 1 and Figure 1 As shown. By Figure 1As shown in Table 1, live Bacillus JK1 cells exhibit certain antibacterial effects against various pathogens. The most significant antibacterial effects of live JK1 cells were observed against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthe fusicola, and Pyricularia oryzae, with an inhibition rate of 100%, completely suppressing the growth of these pathogenic fungi. Among them, live JK1 bacteria showed an inhibition rate of over 90% against Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, and Fusarium decemcellulare, demonstrating good inhibitory effects; and against Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, and Fusarium Oxysporum, Galactomycescandidus, Neofuusicoccumparvum, Fusarium proliferatum, Rhizomucor variabilis, and Phona herbarum have certain antibacterial effects, with an antibacterial rate greater than 80%.

[0039] Compared with the antibacterial rate of 100 mg / L carbendazim, JK1 viable bacteria were effective against Alternaria alternata, Alternariaporri, Alternaria angustiovoidea, Aspergilluspuulaauensis, Aspergillustubingensis, Colletotrichum acutatum, Colletotrichum citricola, Colletotrichumgloeosporioides, Cladosporium bruhnei, Clathrospora diplospora, Chaetomiumcochliodes, Curvularia lunata, Didymosphaeria variabile, Diaporthefusicola, Fusarium decemcellulare, Fusarium proliferatum, Galactomycescandidus, Lasiodiplodia theobromae, Neofusicoccumparvum, Nigrospora oryzae, Penicillium decumbens, Penicillium Oxalicum,Phytophthora The antibacterial effects of *Capsici*, *Pithomyces chartarum*, *Rhizoctonia solani*, *Rhizomucor variabilis*, *Stemphylium eturmiunum*, and *Talaromyces verruculosus* were more pronounced, with inhibition rates significantly higher than those of treatment with 100 mg / L carbendazim. Treatment with 100 mg / L carbendazim showed even better antibacterial effects against *Colletotrichum siamense*, *Fusarium solani*, *Fusarium graminearum*, *Fusarium incarnatum*, *Phoma herbarum*, and *Sclerotium hydrophilum*, with inhibition rates all higher than those of live Bacillus JK1 cells. The antibacterial effects of treatment with live JK1 cells and 100 mg / L carbendazim were similar against *Colletotrichum fioriniae*, *Fusarium oxysporum*, *Pyricularia oryzae*, and *Botryosphaeria dothidea*.

[0040] Table 1. Antibacterial effects of Bacillus JK1 live cells and carbendazim against different pathogens.

[0041]

[0042]

[0043] (II) Antibacterial test of Bacillus JK1 supernatant

[0044] The supernatant antibacterial test mainly tests the antibacterial effect of extracellular antibacterial substances. This method mainly eliminates nutrient competition factors and is an important indicator for evaluating the biocontrol effect of biocontrol bacteria.

[0045] 1. Experimental Design

[0046] After 24 hours of culture, Bacillus JK1 bacteria were repeatedly centrifuged and filtered to remove viable cells, resulting in JK1 supernatant. 200 μL of this supernatant was evenly spread onto a PDA plate. Different pathogenic bacterial pellets were placed in the center of the PDA plate using a 6 mm sterile punch, with an equal volume of sterile water as a control. Each treatment was repeated three times. The prepared plates were incubated at 26°C. The width of the inhibition zone was measured when the control plate reached full mycelial growth.

[0047] Calculate the inhibition rate R of mycelial growth.

[0048] R(%) = (R1 - R2) / R1 × 100%

[0049] Where R is the percentage of radial hyphal growth inhibited, R1 is the hyphal growth of the blank control, and R2 is the hyphal growth of the treatment group.

[0050] 2. Test Results

[0051] The antibacterial effects of Bacillus JK1 supernatant against different pathogenic fungi are shown in Table 2. Among them, the supernatant of JK1 showed the best antibacterial effect against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, and Clathrospora diplospora, with inhibition rates all greater than 90%. The supernatant of Bacillus JK1 showed certain antibacterial activity against Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum, and Rhizomucor variabilis, with inhibition rates all greater than 80%. The supernatant of Bacillus JK1 showed moderate antibacterial activity against Phona herbarum, Sclerotium hydrophilum, and Fusarium incarnatum, with inhibition rates greater than 70%.

[0052] Table 2. Antibacterial effects of Bacillus JK1 supernatant against different pathogenic fungi.

[0053]

[0054]

[0055] (III) Antibacterial test of Bacillus JK1 cell lysate

[0056] 1. Experimental Design

[0057] Add 10 ml of sterile physiological saline to live Bacillus JK1 cells and shake well. Transfer the solution to a 50 ml centrifuge tube and lyse the JK1 cells using an ultrasonic cell disruptor. Filter the lysate once through a 0.22 μm microporous membrane filter to obtain the Bacillus JK1 cell lysate, which should be stored at 4°C for later use.

[0058] 200 μL of cell lysis buffer was evenly spread onto a PDA plate. Using a 6 mm sterile punch, different pathogenic fungal mold cakes were placed in the center of the PDA plate surface. An equal volume of sterile water was used as a control. Each treatment was repeated three times. The prepared plates were incubated at 26℃. The width of the inhibition zone was measured when the mycelium had fully grown on the control plate. The inhibition rate R of mycelial growth was calculated using the same formula as above.

[0059] 2. Test Results

[0060] Table 3 shows that Bacillus JK1 cell lysates exhibited certain antibacterial effects against various pathogenic fungi, but the antibacterial effects were slightly lower than those of the JK1 strain supernatant. Among these, Bacillus JK1 cell lysates showed the best antibacterial effects against Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, and Penicillium decumbens, with inhibition rates exceeding 90%. JK1 cell lysate exhibits antibacterial activity against Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyces chartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, and Neofusicoccum parvum, with inhibition rates exceeding 80%. JK1 cell lysate shows moderate antibacterial activity against Fusarium proliferatum, Rhizomucorvariabilis, and Phona herbarum, with inhibition rates exceeding 70%.

[0061] Table 3. Antibacterial effects of Bacillus JK1 cell lysate against different pathogenic fungi.

[0062]

[0063]

[0064] IV. Life Characteristics of Biocontrol Bacteria

[0065] Bacillus JK1 has a strong survival ability, mainly due to its wide adaptability to temperature, pH, field nutrient sources and humidity.

[0066] 1. Temperature adaptability

[0067] (1) Experimental Design

[0068] The JK1 strain was inoculated into LB broth and cultured at 4℃, 16℃, 28℃, 37℃, and 60℃ in a shaker at 150 rpm, with each treatment repeated three times. After 18 hours, the absorbance and transmittance were measured at 600 nm using a UV-Vis spectrophotometer. Uninoculated LB broth served as a blank control, and each treatment was repeated three times. Turbidity = (100 - transmittance) × 100%.

[0069] (2) Test Results

[0070] The test results are shown in Table 4 and Figure 2 As shown, Bacillus JK1 exhibits a wide temperature adaptability, demonstrating good survival ability within the range of 16-37℃, with the turbidity of the culture medium exceeding 65% after 18 hours. Bacillus JK1 can grow at 45℃, but growth is relatively slow, with the turbidity of the culture medium reaching 48.92% after 18 hours. At 4℃ or 60℃, Bacillus JK1 struggles to reproduce viable cells, with the turbidity of the culture medium reaching 0% after 18 hours.

[0071] Table 4. Determination of the adaptability of Bacillus JK1 to different temperature cultures.

[0072]

[0073] 2. pH adaptability

[0074] (1) Experimental Design

[0075] The pH of the culture medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using 1 mol / L HCl or 1 mol / L NaOH, respectively. After sterilization, 1 mL of Bacillus JK1 seed culture was inoculated into 100 mL of LB liquid medium adjusted to different pH values. The medium was incubated at 37°C and 150 rpm in a shaker. After 18 h, the absorbance and transmittance were measured at 600 nm using a UV-Vis spectrophotometer. Uninoculated LB liquid medium served as a blank control. Each treatment was repeated in triplicate. Turbidity was calculated as (100 - transmittance) × 100%.

[0076] (2) Test Results

[0077] The test results are shown in Table 5 and Figure 3 From Table 5 and Figure 2 It can be seen that the turbidity of the culture medium of Bacillus JK1 was greater than 50% after 18 hours under pH conditions of 5-8, indicating that Bacillus JK1 exhibited good survival ability under these conditions. Under pH conditions of 9 and 10, the turbidity of the culture medium after 18 hours was 40.87% and 21.25%, respectively, indicating that Bacillus JK1 can grow under slightly alkaline conditions, but the growth is relatively slow. When pH = 4, the turbidity of the culture medium after 18 hours was less than 10%, indicating that Bacillus JK1 has very little chance of growing in a strongly acidic environment.

[0078] Table 5. Determination of Bacillus JK1's adaptability to different pH cultures.

[0079]

[0080]

[0081] 3. Adaptability to field nutrient sources

[0082] (1) Experimental Design

[0083] Based on the nutrient classification standards of the Second National Soil Survey and the existing soil conditions in Sichuan Province, the following soil nutrient conditions were proposed: total nitrogen 2 g / kg, total phosphorus 1 g / kg, total potassium 20 g / kg, and field capacity 70%. Based on these conditions, the following single-factor variable design was conducted for N, P, and K nutrients, with five different gradients for each factor (see Table 6):

[0084] (2) Test Results

[0085] Table 7 shows that Bacillus JK1 exhibited strong survival ability after 60 days of cultivation under different nutrient source conditions. Under the nutrient source conditions of 2 g / kg total nitrogen, 1 g / kg total phosphorus, and 20 g / kg total potassium, Bacillus JK1 could survive well for more than 60 days. The JK1 strain can survive under low nitrogen, phosphorus, and potassium content. Even when nitrogen, phosphorus, and potassium are deficient respectively, Bacillus JK1 can survive in soil for more than 60 days, indicating that the JK1 strain does not depend on any specific nutrient element for survival and can provide itself with nutrients from other elements.

[0086] Table 6. Single-factor levels of field nutrient sources for Bacillus JK1

[0087]

[0088] Note: Among the individual factors, the non-variable factors maintain the formula for the highest soil nutrient conditions.

[0089] Table 7. Nutrient source adaptability of Bacillus JK1 after 60 days

[0090]

[0091]

[0092] Note: "++" indicates a viable count ≥10 8 cfu / L, "+" indicates a viable count ≥10 6 cfu / L, "-" indicates no viable bacteria.

[0093] 4. Field humidity adaptability

[0094] (1) Experimental Design

[0095] In sterile sand tubes, a sterile nutrient solution containing 2 g / kg total nitrogen, 1 g / kg total phosphorus, and 20 g / kg total potassium was added, with moisture content controlled at 10%, 30%, 50%, and 70%. 1 mL of JK1 seed culture solution was inoculated into each tube, and the tubes were sealed with a sealing film and incubated at 37°C. Each treatment was repeated three times. After 60 days, soil samples were taken and dissolved in physiological saline. A small amount of sand was spread on LB agar plates to observe the presence and quantity of viable bacteria.

[0096] (2) Test Results

[0097] Table 8 shows the survival of Bacillus JK1 after 60 days under different field moisture contents. The results indicate that Bacillus JK1 can survive for 60 days under soil moisture contents ≥10%. When the soil moisture content is 50–70%, Bacillus JK1 exhibits stronger survival ability.

[0098] This strain is highly tolerant of high temperature and humidity environments, but not adapted to extremely low temperatures. However, most plants remain dormant at 4°C, therefore, this strain is viable in most crop soils. When air humidity is high, this strain can be mixed with inorganic nutrient solution and sprayed on the crop surface as a foliar fertilizer and protectant.

[0099] Table 8. Adaptability of Bacillus JK1 to different field water contents after 60 days

[0100]

[0101] Note: "++" indicates a viable count ≥10 8 cfu / L, "+" indicates a viable count ≥10 6 cfu / L, "-" indicates no viable bacteria.

[0102] VI. Tolerance to broad-spectrum fungicides

[0103] 1. Experimental Design

[0104] Seventeen chemical fungicides, including pyraclostrobin, tebuconazole, flutriafol, carbendazim, azoxystrobin, propiconazole, pyraclostrobin-mancozeb, cyazofamid, mancozeb, difenoconazole, boscalid, fluopyram, iprodione, pyraclostrobin, pyrimidine nucleotide antibiotics, prochloraz, and flusilazole, were added to LB liquid medium to achieve pesticide concentrations equal to or 10 times the normal field application doses of the aforementioned fungicides. LB liquid medium without fungicides or biocontrol bacteria served as a control, inoculated with the same concentration of strain JK1. The cultures were incubated at 37℃ and 120 rpm in a shaker. After 24 hours, LB liquid medium was streaked onto LB plates using an inoculation loop, and the growth of strain JK1 was observed. Each treatment was replicated three times.

[0105] 2. Test Results

[0106] The experimental results are shown in Table 9. Bacillus JK1 exhibited varying resistance to different fungicides. Specifically, after co-culturing Bacillus JK1 with normal doses and 10 times the concentration of pyraclostrobin, tebuconazole, flutriafol, carbendazim, azoxystrobin, propiconazole, cyazofamid, mancozeb, difenoconazole, boscalid, fluopyram, iprodione, azoxystrobin, and flusilazole for 24 hours, Bacillus JK1 successfully grew on LB agar plates. This indicates that Bacillus JK1 showed good resistance to these 10 commonly used fungicides at low concentrations and could survive under the conditions of these 10 pesticide spraying. Bacillus JK1 could also grow viable bacteria after co-culturing with normal doses of pyraclostrobin, pyrimidine nucleotide antibiotics, and prochloraz for 24 hours, but could not survive after co-culturing with 10 times the concentration for 24 hours.

[0107] Table 9. Tolerance of Bacillus JK1 to common fungicides

[0108]

[0109] Note: "+" indicates the presence of live bacteria, and "-" indicates the absence of live bacteria.

Claims

1. A strain of Bacillus, characterized in that, The Bacillus species described is *Bacillus velezensis*, named JK1. It is deposited 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 deposit number CGMCC No. 31375, on July 19, 2024. The gene sequence of the Bacillus species is shown in SEQ NO.

1.

2. A biocontrol agent, characterized in that, The biocontrol agent comprises Bacillus as described in claim 1.

3. The application of the biocontrol agent according to claim 2 in the antibacterial activity against pathogens in fruits and vegetables.

4. The application of the Bacillus subtilis according to claim 1 in the preparation of fruit and vegetable preservatives.

5. The application according to claim 3, characterized in that, The fruit and vegetable pathogens include Colletotrichum fioriniae, Colletotrichum citricola, Colletotrichum gloeosporioides, Diaporthefusicola, Pyricularia oryzae, Cladosporium bruhnei, Alternaria alternata, Colletotrichum acutatum, Alternaria angustiovoidea, Talaromyces verruculosus, Aspergillus puulaauensis, Stemphylium eturmiunum, Botryosphaeria dothidea, Nigrospora oryzae, Alternaria porri, Penicillium decumbens, Didymosphaeria variabile, Chaetomium cochliodes, Colletotrichum siamense, Curvularia lunata, Aspergillus tubingensis, Clathrospora diplospora, Fusarium decemcellulare, Rhizoctonia solani, Lasiodiplodia theobromae, Phytophthora capsici, Pithomyceschartarum, Fusarium solani, Fusarium oxysporum, Galactomyces candidus, Neofusicoccum parvum, Fusarium proliferatum, Rhizomucor variabilis, Phomaherbarum, Sclerotium hydrophilum, Fusarium incarnatum, Fusarium graminearum and Penicillium oxalicum.

6. The application according to claim 3, characterized in that, The concentration of Bacillus JK1 in the biocontrol agent is 1×10⁻⁶. 6 -10 8 cfu / L.

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