Bacillus belye ZF516 and its application in the control of various soil-borne diseases

Abstract

This invention belongs to the field of biotechnology. To improve the effectiveness of biological control of plant diseases, this invention discloses *Bacillus velezensis* ZF516 and its application in controlling various soil-borne diseases. The *Bacillus velezensis* strain number is ZF516, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30136. This *Bacillus velezensis* strain can prevent and / or treat tomato bacterial canker, tomato bacterial wilt, or cowpea root rot, or can be used to prepare products for the prevention and / or treatment of these diseases. This strain also exhibits good inhibitory effects against *Fusarium oxysporum*, *Diplostomum spp.*, *Rhizoctonia solani*, *Fusarium solanum*, *Corynebacterium multiflorum*, *Botrytis cinerea*, *Agrobacterium tumefaciens*, *Pectinobacter brasiliensis*, *Ralstonia solanacearum*, *Streptomyces spp.*, etc.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to Bacillus belye ZF516 and its application in the prevention and control of three soil-borne diseases: tomato bacterial canker, tomato bacterial wilt, and cowpea root rot. Background Technology

[0002] Bacterial canker of tomato is a systemic vascular disease caused by *Clavibacillus termiichiganensis* subsp. *michiganensis*. It can infect plants from seedlings to mature plants, with typical symptoms including stem cracking and browning of the vascular bundles, ultimately leading to wilting of the entire tomato plant. In recent years, with abnormal climate changes and the expansion of tomato cultivation, the severity of the disease has increased year by year. Currently, the main control measures are chemical control and field management, but there is still no rapid and effective method for controlling tomato bacterial canker, either domestically or internationally. Biological control, with its green, environmentally friendly, safe, and efficient characteristics, provides a new approach for controlling bacterial canker of tomato in agricultural production.

[0003] Tomato bacterial wilt is a systemic vascular disease caused by Ralstonia solanacearum, primarily affecting mature plants. In the early stages, the top leaves wilt and droop at midday on sunny days, followed by the middle and lower leaves, which recover by evening. This pattern continues for several days before ceasing to recover. Dissecting the stem reveals a hollow pith and browned vascular bundles. Under high humidity, pressing the cut surface will release a large amount of white liquid. In recent years, with the expansion of tomato cultivation, the severity and affected area of ​​the disease have increased. Currently, chemical control is the primary method, but its effectiveness is low and it easily leads to drug resistance in the pathogen. Biological control, with its environmental safety and stable efficacy, provides a new approach to controlling bacterial wilt in tomato production.

[0004] Cowpea root rot is a typical soil-borne disease of cowpea, mainly caused by Fusarium solani. The infection begins at the root tip and spreads upwards, forming brown lesions that extend from the lateral roots to the taproot, leading to necrosis of the entire root system and causing the plant to wilt. With the increasing number of years cowpeas have been cultivated, the area planted has expanded, and varieties have changed, this disease has become the most severe, damaging, and difficult to control disease of cowpeas. Current control measures mainly rely on field management and chemical control. There are currently no green and efficient control methods available domestically or internationally. Biological control, due to its safety and high efficiency, provides a new approach to controlling root rot in cowpea production.

[0005] Bacillus spp. possesses broad-spectrum antibacterial activity and stress resistance, inhibiting pathogen growth by producing proteases, heptaphiles, bacteriocins, lipopeptides, polyketides, and polypeptides. Studies by Bai, XF, et al. have shown that Bacillus velezensis HN-Q-8 produces fengycin and surfactant, causing hyphal distortion and inhibiting the growth of Rhizoctonia solani. Furthermore, B. velezensis can control plant pathogens due to its secreted antibiotic-like substances and siderophores. Simultaneously, genome-wide bioinformatics analysis by scientists both domestically and internationally has revealed numerous secondary metabolic gene clusters in the B. velezensis genome related to the synthesis of antibacterial compounds, laying the foundation for the development of safe and efficient bacterial-derived native antibacterial agents. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to improve the effectiveness of biological control of plant diseases.

[0007] To solve the above-mentioned technical problems, the present invention first provides a strain of Bacillus velezensis ZF516.

[0008] The *Bacilus velezensis* ZF516 provided in this invention has the registration number CGMCC No. 30136 at the China General Microbiological Culture Collection Center (CGMCC). This strain was deposited at CGMCC on March 26, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Hereinafter referred to as *Bacilus velezensis* ZF516.

[0009] Bacillus belye ZF516 initially grows as milky-white, oval or nearly circular colonies on LB agar plates, with a moist and smooth surface. Later, it transforms into off-white, oval or nearly circular colonies with ridge-like raised wrinkles on the surface, becoming relatively dry. Scanning electron microscopy reveals the strain to be rod-shaped with peritrichous flagella and a wrinkled surface, measuring (1.5-2.0) μm × (0.5-1.2) μm. This Gram-positive bacterium thrives in environments with a pH of 5-6 and can grow in NaCl concentrations of 1%-8%. Biolog results indicate that strain ZF516 can utilize dextran, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, and D-minobiose, but cannot utilize carbon sources such as stachyose, N-Acetyl-D-galactosamine, N-Acetyl-mannosamine pyruvate, D-galactose, 3-Methylglucose, and D-serine. The bacteria are motile. MR, VP, and gelatin liquefaction tests were positive, while citrate utilization, nitrate reduction, and starch hydrolysis tests were negative. *Bacillus belyssus* strain ZF516 possesses 16S rDNA containing the sequence shown in SEQ ID No. 1, the gyrB gene containing the sequence shown in SEQ ID No. 2, the rpoA gene containing the sequence shown in SEQ ID No. 3, the rpoB gene containing the sequence shown in SEQ ID No. 4, the atpD gene containing the sequence shown in SEQ ID No. 5, and the rho gene containing the sequence shown in SEQ ID No. 6.

[0010] Metabolites of Bacillus belyssus ZF516 and / or cultures of Bacillus belyssus ZF516 are also within the scope of protection of this invention.

[0011] In the above text, the metabolites of *Bacillus belyssus* ZF516 can be the fermentation broth of *Bacillus belyssus* ZF516. The fermentation broth of *Bacillus belyssus* ZF516 can be prepared as follows: *Bacillus belyssus* ZF516 is cultured in a liquid fermentation medium, and the fermentation broth (containing *Bacillus belyssus* ZF516 and substances secreted into the liquid medium) is collected. This fermentation broth is the metabolite of *Bacillus belyssus* ZF516.

[0012] In the above text, the culture of Bacillus belyss ZF516 is a substance obtained by culturing Bacillus belyss ZF516 in a microbial culture medium (such as fermentation broth containing Bacillus belyss ZF516 and substances secreted into a liquid culture medium, or substances containing Bacillus belyss ZF516 and substances secreted into a solid culture medium).

[0013] The metabolites of Bacillus belyssus ZF516 and / or cultures of Bacillus belyssus ZF516 have at least one of the following functions: W1-W16

[0014] W1, inhibits bacterial activity;

[0015] W2 inhibits the activity of Gram-positive bacteria;

[0016] W3, inhibits the activity of Gram-negative bacteria;

[0017] W4. Inhibits fungal activity;

[0018] W5 inhibits the activity of Clavibactermichiganensis subsp. michiganensis;

[0019] W6 inhibits the activity of Ralstonia solanacearum;

[0020] W7 inhibits the activity of Fusarium solani, the causal agent of solanaceous diseases;

[0021] W8 inhibits the activity of Fusarium oxysporum;

[0022] W9 inhibits the activity of Ascochytacitrulina, a type of watermelon spore.

[0023] W10 inhibits the activity of Rhizoctonia solani;

[0024] W11 inhibits the activity of Corynesporacassiicola;

[0025] W12 inhibits the activity of Botrytiscinerea;

[0026] W13 inhibits the activity of Agrobacterium tumefaciens;

[0027] W14 inhibits the activity of Pectobacterium brasiliense;

[0028] W15 inhibits the activity of Acidovoraxcitruli, a bacterium found in watermelons;

[0029] W16 inhibits the activity of Streptomycescabies.

[0030] To address the above technical problems, the present invention also provides a product containing Bacillus belyceae ZF516 or / and metabolites of Bacillus belyceae ZF516 or / and cultures of Bacillus belyceae ZF516.

[0031] The product may be a microbial agent or a microecological preparation containing the microbial agent.

[0032] The product may specifically be any of the following products:

[0033] U1. Products for the prevention and / or treatment of tomato bacterial canker;

[0034] U2, Products for the prevention and / or treatment of bacterial wilt in tomatoes;

[0035] U3, Products for the prevention and / or treatment of cowpea root rot;

[0036] U4, Products that inhibit fungi;

[0037] U5, products that inhibit bacteria.

[0038] In the above products, the plant can be a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plant can be a plant of the Solanaceae family (e.g., a plant of the genus Solanum, specifically a tomato) or a plant of the Fabaceae family (e.g., a plant of the genus Cowpea, specifically a cowpea).

[0039] The active ingredient of the above-mentioned product may be Bacillus beleibrio ZF516 or / and Bacillus beleibrio ZF516 metabolites or / and Bacillus beleibrio ZF516 culture. The active ingredient of the above-mentioned product may also contain other biological or non-biological components. Other active ingredients of the above-mentioned product can be determined by those skilled in the art based on the product's effects.

[0040] The above products can be liquid or solid microbial agents.

[0041] The product may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material or a biological material; the mineral material may be at least one of peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material may be at least one of various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal manure; the liquid carrier may be water; in the product, Bacillus beleices ZF516 or / and Bacillus beleices ZF516 metabolites may be present in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0042] The product can be in various dosage forms, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0043] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the product.

[0044] The use of Bacillus vesicularis ZF516 or / and its metabolites or / and its cultures or / and the products in the prevention and / or treatment of tomato canker is within the scope of this invention.

[0045] The use of Bacillus vesiculosus ZF516 or / and its metabolites or / and its culture or / and the product in the prevention and / or treatment of bacterial wilt in tomatoes falls within the scope of protection of this invention.

[0046] The use of Bacillus vesiculosus ZF516 or / and its metabolites or / and its culture or / and the product in the prevention and / or treatment of cowpea root rot is within the scope of protection of this invention.

[0047] The following applications of Bacillus belyssus ZF516 or / and its metabolites or / and its cultures or / and the product thereof also fall within the scope of this invention:

[0048] V1. Application in inhibiting fungal activity;

[0049] V2. Application in inhibiting bacterial activity.

[0050] In the above applications, the plant can be a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plant can be a plant of the Solanaceae family (e.g., a plant of the genus Solanum, specifically tomato) or a plant of the Fabaceae family (e.g., a plant of the genus Cowpea, specifically cowpea).

[0051] The method for culturing the aforementioned Bacillus belyssus ZF516 is also within the scope of protection of this invention.

[0052] The method for culturing Bacillus belyssus ZF516 provided by the present invention includes the step of culturing Bacillus belyssus ZF516 in a culture medium.

[0053] The method for preparing the product is also within the scope of protection of this invention.

[0054] The method for preparing the product provided by the present invention includes the step of using Bacillus belyssus ZF516 or / and a culture of Bacillus belyssus ZF516 or / and a metabolite of Bacillus belyssus ZF516 as a component of the product, wherein the product is a liquid bacterial agent or a solid bacterial agent.

[0055] In the above method, the product can be a liquid bacterial agent. In the above method, the *Bacillus belye* ZF516 can be cultured in a fermentation medium to obtain a fermentation broth. The fermentation broth is then mixed with a carrier to obtain the liquid bacterial agent. The fermentation medium can consist of: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, and distilled water to a final volume of 1L, pH 7.0.

[0056] Experiments have demonstrated that the *Bacillus berberis* ZF516 of this invention possesses good antibacterial activity, inhibiting not only fungal activity but also bacterial activity. *Bacillus berberis* ZF516 of this invention exhibits excellent biocontrol effects against tomato bacterial canker, tomato bacterial wilt, and cowpea root rot. *Bacillus berberis* ZF516 poses no environmental pollution problems, has simple cultivation conditions, is easy to preserve and process, and is suitable for development and application.

[0057] Preservation Instructions

[0058] Classification and nomenclature of biological materials: Bacillus belesiensis

[0059] The Latin scientific name of the biological material is: Bacilus velezensis

[0060] Strain number of the biological material: ZF516

[0061] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0062] Abbreviation of depositary institution: CGMCC

[0063] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101

[0064] Deposit date: March 26, 2024

[0065] Accession number: CGMCC No. 30136 Attached Figure Description

[0066] Figure 1 This invention illustrates the inhibitory effect of biocontrol bacteria on tomato canker pathogens in Example 1. Wherein, A is ZF203; B is ZF210; C is ZF316; D is ZF516; E is ZF517; F is ZF534; G is ZF536; H is ZF547; I is ZF582; and J is ZF583.

[0067] Figure 2 The results show the morphological characteristics of strain ZF516 in Example 2 of this invention. Figure 2 A represents the colony morphology of strain ZF516; Figure 2 B represents the morphology of the colony on the reverse side of strain ZF516; Figure 2 C is a scanning electron microscope image of strain ZF516; Figure 2 D is a scanning electron microscope image of a single cell of strain ZF516.

[0068] Figure 3 This is a phylogenetic tree of strain ZF516 in Example 2 of the present invention, based on 16S rDNA and the gene sequences of rpoA, rpoB, gyrB, atpD, and rho.

[0069] Figure 4 This is the plate confrontation result of strain ZF516 against different pathogens in Example 3 of the present invention. Figure 4 A is Fusarium oxysporum; Figure 4 B is Ascochytacitrulina, a disporum podzolium. Figure 4 C is Rhizoctonia solani; Figure 4 D is Fusarium solani, the fungus that causes solanaceous diseases. Figure 4 E is Corynesporacassiicola; Figure 4 F is Botrytiscinerea; Figure 4 The G in the text refers to *Agrobacterium tumefaciens*. Figure 4 H stands for Pectobacterium brasiliense; Figure 4 The I-type is Ralstonia solanacearum; Figure 4 J is the watermelon acidophilic bacterium Acidovoraxcitruli; Figure 4 K represents Streptomyces cabiei; Figure 4 L represents Clavibactermichiganensis subsp. michiganensis.

[0070] Figure 5 The results show the activity of protease, heptaphilin, hydrocyanic acid, and phosphate solubility of strain ZF516 in Example 3 of this invention. Figure 5 A represents the result of the protease; Figure 5B represents the result of heptaphilin; Figure 5 The result is that C represents hydrogen cyanide. Figure 5 D represents the phosphorus solubility result.

[0071] Figure 6 The results are PCR amplification of antimicrobial lipopeptides and resistance-related genes of strain ZF516 in Example 3 of this invention. Wherein, M is DNA Marker 5000bp; CK is FZB42; 1 is sfrAA; 2 is pks2C; 3 is fenD; 4 is difA; 5 is dhbF; 6 is baeC; 7 is bacB; and 8 is ituC.

[0072] Figure 7 This invention demonstrates the efficacy of strain ZF516 in Example 3 of the present invention against live potted tomato canker. Figure 7 A is a ZF516 bacterial suspension treatment; Figure 7 B was treated with a 700-fold dilution of thiamethoxam zinc suspension; Figure 7 C is the treatment of ZF582 bacterial suspension; Figure 7 D is the treatment of ZF536 bacterial suspension; Figure 7 E represents the treatment of ZF210 bacterial suspension; Figure 7 F is the treatment of ZF583 bacterial suspension; Figure 7 G represents the treatment of Clavibactermichiganensis subsp. michiganensis strain (i.e., pathogen control); Figure 7 H represents the control treatment for healthy tomatoes.

[0073] Figure 8 This invention demonstrates the efficacy of strain ZF516 in Example 3 of the present invention against bacterial wilt in living potted tomato plants. Figure 8 A represents the root irrigation treatment with ZF516 bacterial suspension; Figure 8 B is the root irrigation treatment with ZF536 bacterial suspension; Figure 8 C represents root irrigation treatment with ZF210 bacterial suspension; Figure 8 D represents the root irrigation treatment with ZF582 bacterial suspension; Figure 8 The E is 20% thiamethoxam zinc suspension for root irrigation treatment; Figure 8 F represents the pathogen control treatment; Figure 8 G represents the healthy control group.

[0074] Figure 9 This invention demonstrates the efficacy of strain ZF516 in Example 3 against cowpea root rot in living potted plants. Figure 9 A represents the aboveground parts (left) and roots (right) treated with ZF516 bacterial suspension; Figure 9 B represents the aboveground parts (left) and roots (right) treated with ZF582 bacterial suspension; Figure 9C represents the aboveground parts (left) and roots (right) treated with ZF536 bacterial suspension; Figure 9 D represents the aboveground parts (left) and roots (right) treated with a 500-fold dilution of carbendazim wettable powder; Figure 9 E represents the aboveground parts (left) and roots (right) treated with ZF210 bacterial suspension; Figure 9 F represents the aboveground parts (left) and roots (right) of the pathogen control treatment; Figure 9 G represents the aboveground parts (left) and roots (right) of the healthy cowpea control treatment. Detailed Implementation

[0075] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0076] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Experimental data were statistically analyzed using Excel and SPSS software.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are conventional biochemical reagents and are commercially available.

[0078] The pathogens used in the following examples are as follows:

[0079] The strain of Clavibacillus termichiganensis subsp. michiganensis, the pathogen of tomato bacterial canker, has been published in the literature "Li Huanling, Shi Yanxia, ​​Xie Xuewen, Li Baoju. Occurrence patterns and control techniques of tomato bacterial canker. China Vegetables, 2011, 23:24-27." and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0080] The strain of Ralstonia solanacearum, the pathogen of bacterial wilt of tomato, has been published in the literature "Kang Huajun, Chai Ali, Shi Yanxia, ​​et al. Quadruple PCR detection method for bacterial spot, canker, bacterial wilt and scab of tomato[J]. Journal of Horticulture, 2018, 45(11):2254-2264." and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0081] The strain of *Fusarium solani*, the pathogen causing root rot of cowpea, has been published in the literature “Chai Ali, Han Yun, Wu Jun, Shi Yanxia, ​​Xie Xuewen, Li Baoju. Detection of spore activity of *Fusarium solani* based on FDA-PI double fluorescence counterstaining method. Chinese Journal of Agricultural Science, 2015, 48(14):2757-2766.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0082] Fusarium oxysporum has been published in the literature “Shi Yanxia, ​​Zhang Xiaohui, Xu Yufang, Xie Xuewen, Chai Ali, Li Baoju. Induction of resistance to Fusarium wilt in cucumber by pyrazolopyrimidine derivative BDO-1 [J]. Journal of Horticulture, 2019, 46(05):877-890.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0083] The spore species *Ascochytacitrulina* has been published in the literature “Li Wei, Zhang Aixiang, Jiang Jiao, Yang Xingping, Chen Huaigu. Identification and biological characteristics of the pathogen of melon vine blight [J]. Jiangsu Journal of Agricultural Sciences, 2008, (02): 148-152.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0084] Rhizoctonia solani has been published in the literature “Huang Yishuo, Xie Xuewen, Shi Yanxia, ​​Chai Ali, Li Lei, Li Baoju. Control effect of Bacillus polymyxa ZF197 on stem base rot of Chinese cabbage. Journal of Horticulture: 1-13.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0085] Corynesporacassiicola has been published in the literature “Gao Wei, Li Baoju, Shi Yanxia, ​​Xie Xuewen. Differentiation of pathogenicity of Corynesporacassiicola on cucumber, tomato and eggplant hosts. Journal of Horticulture, 2011, 38(3): 465-470”, and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0086] Botrytiscinerea has been published in the literature “Shi Yanxia, ​​Tang Ming, Jin Zhiwen, Xie Xuewen, Chai Ali, Li Baoju. Evaluation of resistance of Botrytiscinerea to different types of fungicides in vegetable crops. China Vegetables, 2016(03):60-65.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0087] The agrobacterium tumefaciens species has been published in the literature “Li Yujia, Li Qian, Zhang Zhixiang, et al. Screening and identification of peach endophytic bacteria antagonizing agrobacterium tumefaciens [J]. Chinese Agricultural Science, 2017, 50(20):3918-3929”, and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0088] Pectobacterium brasiliense has been published in the literature “Zhao Yurong, Xie Xuewen, Xu Shuai, Xie Hua, Shi Yanxia, ​​Chai Ali, Li Lei, Li Baoju. Control effect of Pectobacterium brasiliense ZF390 on bacterial soft rot of cucumber. Chinese Journal of Biological Control, 2022, 38(02):476-486.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0089] The acidophilus citruli of watermelon has been published in the literature “Zhao Zixuan, Li Junhui, Du Gongfu, et al. Isolation and identification of the antipyretic ZF509 and its control effect on bacterial fruit spot disease of melon [J]. Chinese Journal of Biological Control, 2024, 40(03):690-700.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0090] Streptomyces scabies has been published in the literature “Chen Lida, Xie Xuewen, Shi Yanxia, ​​Chai Ali, Pan Haoqin, Li Lei, Li Baoju. Construction and application of real-time fluorescence quantitative PCR detection system for Streptomyces scabies. Journal of Agricultural Biotechnology, 2020, 28(07):1314-1321.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0091] The strain of *Bacilus velezensis* FZB42 has been published in the literature “Wu LM, Wu HJ, Chen L N, Yu XF, Borriss R, Gao X W. 2015. Difficidin and bacilysin from *Bacilus amyloliquefaciens* FZB42 have antibacterial activity against *Xanthomonasoryzae* rice pathogens. Scientific Reports, 5: 12975.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0092] The tomato variety “Hongza 33” (variety registration number: GPD tomato (2019) 110202) used in the following examples was purchased from China Vegetable Seed Industry Technology (Beijing) Co., Ltd.

[0093] The cowpea variety “Yujiang” used in the following examples was purchased from China Vegetable Seed Industry Technology (Beijing) Co., Ltd.

[0094] The culture media used in the following examples are as follows:

[0095] Tryptone medium (LB): 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, distilled water to a final volume of 1000mL, pH 7.0, used for activating and shaking culture of biocontrol bacteria.

[0096] Beef Peptone Liquid Culture Medium (NA): 3g beef powder, 10g peptone, 5g sodium chloride, 15g agar, and distilled water to a final volume of 1000mL.

[0097] PDA medium: 200g potato, 20g D+ anhydrous glucose, 15g agar, and distilled water to a final volume of 1000mL.

[0098] WA medium: 4.5g agar, diluted with distilled water to 1000mL.

[0099] PVK medium: yeast extract 0.50g, glucose 10.00g, Ca3(PO4)2 5.00g, (NH4)2SO4 0.50g, KCl 0.20g, MgCl2 0.10g, MnSO4 0.10mg, FeSO4 0.10mg, agar 15.00g, distilled water to a final volume of 1000mL.

[0100] Culture medium for protease detection: 5.00 g tryptone, 3.00 g yeast extract, 1.00 g glucose, 15 g agar, add distilled water to 1000 mL, pH 7.0, autoclave at 121℃ for 20 min, cool to about 50℃ and add 10% skim milk by volume.

[0101] CAS medium: Dissolve 121 mg of chromaine in 50 mL of 1 mmol / L Fe. 3+ Mix the solution thoroughly, add 145.8 mg HDTMA, adjust the pH to 7.0, and bring the volume to 100 mL. Finally, sterilize and mix with 900 mL of solution a. (Note: The last part, "1 mmol / L Fe," appears to be an unrelated fragment and is omitted from the translation.) 3+The solutes and their concentrations in the solution are: 1 mmol / L FeCl3·6H2O, 10 mmol / L HCl. Solution a: 15 g agar, 9 g glucose, 4.5 g peptone, 2.7 g beef extract, 4.5 g NaCl, with distilled water added to 1000 mL.

[0102] The 20% thiazole zinc suspension used in the following examples was purchased from Zhejiang Xinong Chemical Co., Ltd. (Registration No.: PD20096932).

[0103] Example 1: Isolation and Screening of Strains

[0104] 1. Isolation and purification of bacterial strains

[0105] In June 2023, soil samples were collected from the rhizosphere of healthy tomato plants in an area affected by tomato canker in Chifeng, Inner Mongolia, by collector Li Lei (contact number: 15711155718, email: lilei01@caas.cn). Antagonistic bacteria were isolated using a serial dilution method. 10g of the soil was mixed thoroughly and weighed, dissolved in 90ml of sterile water, and placed on a shaker at 28℃ and 180r / min for 0.5h. The fully suspended soil suspension was then serially diluted, with each dilution factor of 10... -3 10 -4 10 -5 10 -6 10 -7 The diluted solution was evenly spread on LB solid medium and placed in an incubator at 28°C for 48 hours. After incubation, single colonies of bacteria with different morphologies were picked using a sterile inoculation loop for secondary purification. After stable single colonies were formed, they were stored for later use.

[0106] A total of 483 bacterial strains were isolated and purified.

[0107] 2. Screening of antagonistic strains against bacterial canker in tomatoes

[0108] Using *Clavibacillus termichiganensis* subsp. *michiganensis*, the causal agent of tomato bacterial canker, as the target strain, a double-layer culture method was employed to screen for antagonistic bacteria (see reference "Zhao Yurong, Xie Xuewen, Xu Shuai, et al. Control effect of *Bacillus pilaris* ZF390 on bacterial soft rot of cucumber [J]. Chinese Journal of Biological Control. 2022, 38(02):476-486."). Details are as follows:

[0109] Use a micropipette to take 5 μL of the bacterial suspension (OD) of the strain to be screened. 600=1.0) was added to the center of LB medium, allowed to air dry, and then incubated at 28°C for 36 hours. 3 mL of chloroform was added to the lid of the glass culture dish, and the dish was inverted and placed in a fume hood for 12 hours of fumigation. OD was then measured using a micropipette. 600 150 μL of a bacterial suspension of *Clavibacillus termichiganensis* subsp. *michiganensis*, the causal agent of tomato bacterial canker, with a concentration of 1.0, was added to 5 mL of WA medium cooled to approximately 50°C. After thorough shaking and mixing, the mixture was poured onto the top layer of LB medium and incubated at 28°C for 36 h. If a transparent inhibition zone appeared around the colony, it indicated that the strain had an antagonistic effect against *Clavibacillus termichiganensis*. The size of the inhibition zone was measured, and the inhibition rate was calculated using Formula 1.

[0110]

[0111] Ten bacterial strains exhibiting significant antagonistic effects against *Tomato bacterial canker* were screened from 483 bacterial strains using a double-layer culture method. The results are shown below. Figure 1 And Table 1:

[0112] Table 1. Determination of antagonistic activity of biocontrol bacteria against Tomato bacterial canker pathogen.

[0113]

[0114] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data indicate significant differences (P < 0.05).

[0115] Among them, strains ZF516 and ZF582 had inhibition zones larger than 55.00 mm and inhibition rates larger than 60.00%. Strain ZF516 showed significantly greater antibacterial effect than other strains, with an inhibition zone diameter of 69.00 mm and an inhibition rate of 76.67%. In contrast, strain ZF518 had the smallest inhibition zone at 31.78 mm and an inhibition rate of 35.31%.

[0116] The following section describes the identification of the selected strain ZF516.

[0117] Example 2: Identification of strain ZF516

[0118] Morphological characteristics identification of strain ZF516

[0119] Antagonistic strain ZF516 was cultured on LB solid medium. After 36 hours of culture, its colony morphology was observed, and its cell morphology was examined using electron microscopy. Electron microscopy samples were fixed with 2.5% glutaraldehyde and dehydrated seven times with 20%-100% ethanol for 10 minutes each time. The samples were then observed using transmission electron microscopy.

[0120] See results Figure 2In the early stages of growth on LB agar, strain ZF516 forms milky-white, oval or nearly circular colonies with a moist and smooth surface. In the later stages of growth, it forms off-white, oval or nearly circular colonies with ridge-like raised wrinkles on the surface, and becomes relatively dry. Figure 2 A, Figure 2 (B); Scanning electron microscopy revealed that the strain was rod-shaped with peritrichous flagella and wrinkled cell surface. Figure 2 The size of D is (1.5-2.0) μm × (0.5-1.2) μm. Figure 2 (C).

[0121] Physiological and biochemical assays of strain ZF516 were performed according to Bergey's Manual of Bacterial Identification, 8th Edition, and the Manual of Systematic Identification of Common Bacteria. The unique carbon source utilization of strain ZF516 was determined by the China Agricultural Microbial Culture Collection Center using the BIOLOG GEN III kit. The results are shown in Table 2.

[0122] Table 2 Physiological and biochemical reactions of strain ZF516

[0123]

[0124] Note: + indicates that the experimental result is positive; - indicates that the experimental result is negative.

[0125] The results showed that strain ZF516 is a Gram-positive bacterium, with an optimal growth environment of pH 5-6, and can grow in NaCl concentrations of 1%-8%. Biolog results indicated that strain ZF516 can utilize dextran, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, and D-minobiose, but cannot utilize carbon sources such as stachyose, N-Acetyl-D-galactosamine, N-Acetyl-mannosamine pyruvate, D-galactose, 3-Methylglucose, and D-serine. The bacteria are motile, and the MR, VP, and gelatin liquefaction tests were positive, while the citrate utilization, nitrate reduction, and starch hydrolysis tests were negative.

[0126] Multigene amplification and sequence analysis of strain ZF516

[0127] Genomic DNA was extracted from strain ZF516 using a bacterial genomic DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.). Six genes—16S rDNA and gyrB, rpoA, rpoB, atpD, and rho—were amplified. Universal primer 27F / 1492R was used for 16S rDNA amplification, along with primers specifically targeting the genes gyrB, rpoA, rpoB, atpD, and rho: BF / BR, 72F / 328R, 1698f / 2014r, atpD-F / atpD-R, and OF / OR (primer sequences are shown in Table 3). These primers were used to specifically amplify strain ZF516.

[0128] Table 3 Primer sequences for strain identification and genes related to antimicrobial lipopeptide synthesis

[0129]

[0130]

[0131] The total reaction volume was 50 μL. The reaction conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, with a program of 34 cycles, followed by a full extension at 72℃ for 10 min. The reaction product was sent to Beijing Biomed Biotechnology Co., Ltd. for sequencing.

[0132] Bacillus belyssus strain ZF516 contains 16S rDNA containing the sequence shown in SEQ ID No. 1, the gyrB gene containing the sequence shown in SEQ ID No. 2, the rpoA gene containing the sequence shown in SEQ ID No. 3, the rpoB gene containing the sequence shown in SEQ ID No. 4, the atpD gene containing the sequence shown in SEQ ID No. 5, and the rho gene containing the sequence shown in SEQ ID No. 6.

[0133] The 16S rDNA and gyrB, rpoA, rpoB, atpD, and rho genes of strain ZF516 were compared with those of strains in the NCBI database. The sequencing results were analyzed using software such as MEGA 7.0, a phylogenetic tree was constructed, and the kinship of strain ZF516 was analyzed.

[0134] A multigene phylogenetic tree constructed using 16S rDNA and the rpoA, rpoB, rho, gyrB, and atpD gene sequences showed that strain ZF516 clustered with Bacillus velezensis ZF2 in the same branch (see [link to phylogenetic tree]). Figure 3 ).

[0135] Based on the above morphological characteristics analysis, physiological and biochemical indicators, BIOLOG unique carbon source utilization characteristics, 16S rDNA and rpoA, rpoB, rho, gyrB and atpD gene sequence analysis results, strain ZF516 was identified as Bacillus velezensis.

[0136] Strain ZF516 is *Bacilus velezensis*, which was deposited on March 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Its registration number at CGMCC is CGMCC No. 30136. Hereinafter referred to as *Bacilus velezensis* ZF516.

[0137] Example 3: Biocontrol effect of Bacillus belyssus ZF516

[0138] 1. Determination of the antibacterial spectrum of strain ZF516

[0139] The plate confrontation method (reference "Huang Yishuo, Xie Xuewen, Shi Yanxia, ​​et al. Control effect of Bacillus polymyxa ZF197 on stem base rot of Chinese cabbage [J]. Journal of Horticulture. 2020, 47(6):1059-1071.") was used to determine the inhibitory effect of strain ZF516 on six pathogenic fungi: Fusarium oxysporum, Ascochytacitrulina, Rhizoctonia solani, Fusarium solani, Corynesporacassiicola, and Botrytiscinerea. The results are as follows:

[0140] Use a sterile punch to collect fungal cakes (5 mm in diameter) and inoculate them into the center of PDA medium. Place sterile filter paper discs (5 mm in diameter) at the four corners, 2.5 cm from the center of the plate, forming a cross shape. Add 5 μL of ZF516 bacterial suspension to the filter paper discs. Set up a sterile water control. After the control mycelia have covered the plate, conduct an investigation and measure the colony diameter. Calculate the inhibition rate using Formula 1 above.

[0141] The inhibitory effects of strain ZF516 on six pathogenic bacteria were determined using a double-layer culture method (same as the double-layer culture method in Example 1): *Agrobacterium tumefaciens*, *Pectobacterium brasiliense*, *Ralstonia solanacearum*, *Acidovorax citruli*, *Streptomyces cabie*, and *Clavibacillus termichiganensis subsp. michiganensis*.

[0142] See results Figure 4 And Table 4:

[0143] Table 4. Antibacterial effect of Bacillus belye ZF516 against pathogens

[0144]

[0145]

[0146] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data indicate significant differences (P < 0.05).

[0147] The results showed that the inhibition zone for *Fusarium oxysporum* was 39.70 mm with an inhibition rate of 53.57%; for *Ascochytacitrulina*, the inhibition zone was 41.32 mm with an inhibition rate of 57.06%; for *Rhizoctonia solani*, the inhibition zone was 59.52 mm with an inhibition rate of 72.97%; for *Fusarium solani*, the inhibition zone was 32.12 mm with an inhibition rate of 50.33%; for *Corynesporacassicola*, the inhibition zone was 42.25 mm with an inhibition rate of 65.76%; for *Botrytiscinerea*, the inhibition zone was 55.77 mm with an inhibition rate of 69.71%; and for *Agrobacterium tumefaclor*, the inhibition rate was significantly higher. The inhibition zone of *Ralstonia solanacearum* was 42.24 mm, with an inhibition rate of 48.55%; the inhibition zone of *Pectobacterium brasiliense* was 53.84 mm, with an inhibition rate of 61.88%; the inhibition zone of *Ralstonia solanacearum* was 36.71 mm, with an inhibition rate of 42.20%; the inhibition zone of *Acidovorax citruli* was 30.15 mm, with an inhibition rate of 34.66%; the inhibition zone of *Streptomyces cabie* was 37.71 mm, with an inhibition rate of 42.86%; and the inhibition zone of *Clavibacillus termichiganensis subsp. michiganensis* was 60.15 mm, with an inhibition rate of 66.83%.

[0148] 2. Detection of biocontrol factors in strain ZF516

[0149] Phosphate-solubilizing capacity test: Take 5 μL of ZF516 bacterial suspension (OD) 600 =0.8) was added dropwise to the center of PVK medium, placed in an incubator at 28℃, and incubated for 48 hours. Then, it was observed whether a hydrolysis zone was formed.

[0150] HCN synthesis detection: The puncture inoculation method was used to inoculate strain ZF516 into LB medium. The sensitive test strip for HCN detection was taken and suspended and sealed in a centrifuge tube inoculated with strain ZF516. ZF509 was used as the control strain. The tube was placed in an incubator at 28℃ and incubated for 24 hours. After incubation, the test strip was observed to see if it turned blue.

[0151] Ferrophilin assay: Take 5 μL of ZF516 bacterial suspension (OD) 600=0.8) was added to the center of the heptaphilic medium, and after air drying, it was incubated in a 28℃ incubator. The colonies were observed daily to see if a yellow halo was formed around them.

[0152] Protease detection: Take 5 μL of ZF516 bacterial suspension (OD) 600 =0.8) was added to the center of the protease detection medium, placed in a 28℃ incubator, and observed after 24 hours of incubation.

[0153] See results Figure 5 The enzyme activity assay results showed that strain ZF516 produced a clear hydrolysis zone on the protease detection medium. Figure 5 The presence of A indicates that strain ZF516 can produce a protease. A yellow-green halo is produced on CAS medium. Figure 5 The result of B indicates that strain ZF516 can produce heptaphilin. The HCN test strip did not turn blue. Figure 5 The presence of C indicates that strain ZF516 does not produce HCN. A distinct transparent hydrolysis zone is produced on inorganic phosphorus-containing plates. Figure 5 The D) indicates that strain ZF516 has phosphate-solubilizing ability.

[0154] 3. Detection of antibiotic synthesis-related genes in strain ZF516

[0155] To detect the genes for the synthesis of lipopeptide antibiotics, namely Surfactin, Macrolactin, Fengycin, Difficidin, Bacillibactin, Bacillaene, Bacilysin, and Iturin, in the genome of Bacillus (genes and primer pairs are shown in Table 3), Bacillus velezensis strain FZB42 was used as a positive control strain, and genomic DNA of ZF516 was used as a template for PCR amplification.

[0156] PCR reaction system: 12.5 μL of 2×Taq PCR Mix, 1 μL each of 10 μmol / L forward and reverse primers, 1 μL (50 ng) of genomic DNA as template, and ultrapure water to a final volume of 25 μL.

[0157] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 54℃ annealing for 40 s, 72℃ extension for 50 s, 34 cycles; 72℃ extension for 10 min, and storage at 4℃.

[0158] After the PCR reaction was completed, 5 μL of the amplification product was electrophoresed in a 1.5% agarose gel, observed and photographed on a BIO-RAD gel imaging system, and compared with the expected size of the PCR amplification product for comparative analysis.

[0159] See results Figure 6 The results showed that bright and single bands were amplified in each lane, and the size of the amplified bands was consistent with the expected results. This demonstrates that the genome of strain ZF516 contains genes related to the synthesis of lipopeptides such as Surfactin, Macrolactin, Fengycin, Difficidin, Bacillibactin, Bacillaene, Bacilysin, and Iturin, suggesting that secretion of lipopeptides may be one of the antibacterial pathways of strain ZF516.

[0160] 4. Test on the control efficacy of strain ZF516 against bacterial canker in tomato plants (pot cultivation)

[0161] The topping method will be selected for inoculation (refer to "Ji Yuwei, Li Ziyan, Deng Shuozhen, et al. Comparison of pathogenicity of tomato bacterial canker pathogen under different inoculation methods [J]. Journal of China Agricultural University. 2022, 27(9):109-116."), as follows:

[0162] At the 3-4 true leaf stage of the tomato plant, dip the tip 1cm of a 14cm surgical scissor (model: PTJ-3) into a suspension of *Clavibacillus termichiganensis* subsp. *michiganensis* (concentration 1×10⁻⁶). 8 The main stem was cut off at the new leaf tip (cfu / mL). A control was prepared by inoculating with sterile water. After inoculation, the cells were placed in a humidifier and cultured for 24 hours at 30℃ and 80%-90% humidity. A biocontrol bacterial suspension (concentration 1×10⁻⁶) was then inoculated using a spray inoculation method. 8 The control group was inoculated with 20% zinc thiazo SC (cfu / mL) as a control agent. The pathogen control was inoculated with only tomato canker pathogen, and the healthy control was inoculated with only sterile water. After the water was drained, the control group was placed in a constant temperature incubator (28℃, RH 80%). After the pathogen control group developed full disease, the severity of the disease was investigated and the disease index was calculated according to the tomato canker grading standard (see Formula 2), and the control effect of the agent was calculated (see Formula 3).

[0163] Disease index = ∑(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100 Formula 2 Control effect (%) = ((disease index of disease control - disease index of each treatment) / disease index of disease control) × 100 Formula 3 Table 5 Control efficacy of strain ZF516 against tomato bacterial canker in potted plants

[0164] Serial Number deal with Disease index Relative efficacy % 1 ZF516 18.40±4.93d 78.70±5.71a 2 20% Thiazole Zinc 24.80±2.16cd 71.30±2.50ab 3 ZF582 33.08±7.19c 61.72±8.32b 4 ZF536 60.00±7.06b 30.56±8.17c 5 ZF210 78.40±4.93a 9.26±5.71d 6 ZF583 79.20±4.78a 8.33±5.53d 7 Pathogen control 86.40±3.24a / 8 Health comparison / /

[0165] Note: Data are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0166] The results are shown in Table 5 and Figure 7 Seven days after inoculation, the control group showed full disease development, exhibiting typical symptoms such as blackening of the inoculation site, stem ulceration, and wilting of the entire plant, with a disease index reaching 86.40. The incidence of tomato ulceration was significantly reduced in tomatoes treated with fermentation broth of strain ZF516. Figure 7 The disease index was only 18.40, and the control efficacy reached 78.70%, which is better than the control efficacy of thiazolium zinc suspension against tomato bacterial canker (Table 5).

[0167] 5. Control effect of strain ZF516 on bacterial wilt of tomato

[0168] This experiment used 5-6 leaf stage tomato seedlings (Zhongza 201) to determine the effects of different treatments on the control of tomato bacterial wilt by biocontrol bacteria. For the pathogen-mixed soil method: except for the control (CK) without pathogens, all other treatments had tomato bacterial wilt bacteria suspension pre-mixed evenly into the soil to a final concentration of 10%. 6 CFU / g soil. After treating the soil with the pathogen by mixing it with soil for 1 day, the soil was inoculated with a biocontrol bacterial suspension (concentration 1×10⁻⁶) via root drenching. 8 The control group was inoculated with 20% zinc thiazo SC (cfu / mL) as a control. The pathogen control was inoculated with only the tomato bacterial wilt pathogen, while the healthy control was inoculated with only sterile water. The control groups were placed in a glass room (28℃, RH 80%). After the pathogen control group had fully developed the disease, the severity of the disease was investigated and the disease index was calculated according to the tomato bacterial wilt grading standard (see Formula 2), and the control effect of the pesticide was calculated (see Formula 3).

[0169] Table 6. Control efficacy of strain ZF516 against bacterial wilt in live potted tomato plants.

[0170]

[0171]

[0172] Note: Data are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0173] The results of the indoor potted plant experiment are shown in Table 6 and Figure 8 Five days after inoculation, the control group showed full disease development, exhibiting typical symptoms of bacterial wilt in tomatoes, with the entire plant wilting and a disease index reaching 81.94. The incidence of bacterial wilt in tomatoes treated with fermentation broth of strain ZF516 was significantly reduced. Figure 8 The disease index was only 27.78, and the control efficacy reached 66.10%, which was better than the 23.72% control efficacy of thiamethoxam zinc suspension against tomato bacterial wilt (Table 6).

[0174] 6. The control effect of strain ZF516 on cowpea root rot

[0175] Inoculation was carried out using the root drenching method. At the two-leaf-one-heart stage of cowpeas, each cowpea plant was treated with 10 mL of a Fusarium solani suspension (spore concentration 1×10⁻⁶) that had been shaken for 7 days. 8 Biocontrol bacteria suspension (concentration 1×10⁻⁶ / mL) was inoculated using sterile water as a control. After inoculation, the bacteria were placed in a glass room and cultured for 24 hours at 30℃ and 80%-90% humidity. Then, a root drenching inoculation method was used to inoculate the bacteria with a suspension of biocontrol bacteria (concentration 1×10⁻⁶ / mL). 8 The control group was inoculated with 50% carbendazim wettable powder (cfu / mL) as a control agent. The pathogen control was inoculated with cowpea root rot pathogen only, and the healthy control was inoculated with sterile water only. After the moisture was drained, the control group was placed in a glass room (28℃, RH 80%). After the pathogen control group was fully infected, the severity of the disease was investigated and the disease index was calculated according to the cowpea root rot grading standard (see formula 2), and the control effect of the agent was calculated (see formula 3).

[0176] Table 7. Control efficacy of strain ZF516 against cowpea root rot in live potted plants.

[0177] Serial Number deal with Disease index Relative efficacy % 1 ZF516 36.67±2.36d 56.00±2.83a 2 ZF582 43.33±2.36cd 48.00±2.83ab 3 ZF536 46.25±1.77bcd 44.50±2.12abc 4 50% Carbendazim 50.00±4.08bc 40.00±4.90bc 5 ZF210 56.67±8.50b 32.00±10.20c 6 Pathogen control 83.33±2.36a / 7 Health comparison / /

[0178] Note: Data are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0179] The results of the indoor potted plant experiment are shown in Table 7 and Figure 9 Seven days after inoculation, the control group showed full disease development, exhibiting typical symptoms such as reddening and browning of the roots, rotting of the root base, and loss of lateral roots, with a disease index reaching 83.33. The incidence of cowpea root rot was significantly reduced in cowpeas treated with fermentation broth of strain ZF516. Figure 9 The disease index was only 36.67, and the control efficacy reached 56.00%, which is better than the control efficacy of carbendazim wettable powder against cowpea root rot (Table 7).

[0180] This study isolated a strain of Bacillus belye ZF516 from the rhizosphere soil of tomatoes in Chifeng, Inner Mongolia. This strain exhibits good antagonistic effects against the tomato canker pathogen. It has broad-spectrum antibacterial activity and can inhibit various pathogenic fungi and bacteria. It can produce lipopeptides such as Surfactin, Macrolactin, Fengycin, Difficidin, Bacillibactin, Bacillaene, Bacilysin, and Iturin, as well as proteases and heparin. It also has good phosphorus-solubilizing ability. The fermentation broth of strain ZF516 showed a 78.70% control effect on tomato bacterial canker in potted plants, a 66.10% control effect on tomato bacterial wilt in potted plants, and a 56.00% control effect on cowpea root rot in potted plants, which was superior to the control agent carbendazim. This invention provides an efficient and safe biocontrol resource for the biological control of tomato bacterial canker, tomato bacterial wilt, and cowpea root rot. The antibacterial mechanism and field control effect of this strain still need further verification.

[0181] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Bacillus belye ( Bacillus velezensis ), characterized by: The specimen is Bacillus belye ZF516, which was deposited on March 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30136. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

2. Metabolites or cultures of Bacillus belyssus ZF516, characterized in that: The metabolite is the fermentation broth of Bacillus belyssus ZF516 as described in claim 1; the culture is a substance obtained by culturing Bacillus belyssus ZF516 as described in claim 1 in a microbial culture medium.

3. A medicine, characterized in that: Metabolites or cultures containing Bacillus belyssus ZF516 as described in claim 1 and / or Bacillus belyssus ZF516 as described in claim 2.

4. The medicine according to claim 3, characterized in that: It is any one of the following drugs: U1, medicines for the prevention and / or treatment of tomato scab; U2, medications for the prevention and / or treatment of bacterial wilt in tomatoes; U3. Medicines for the prevention and / or treatment of cowpea root rot; U4, a drug that inhibits fungi; the fungus being Fusarium oxysporum (…). Fusarium oxysporum ), watermelon shell dispore ( Ascochyta citrullina Rhizoctonia solani ( ) Rhizoctonia solani Fusarium solani ( ) Fusarium solani ), Corynebacterium multiflorum ( Corynespora cassiicola ) and Botrytis cinerea ( Botrytis cinerea Any one of the following; U5, a drug that inhibits bacteria; the bacteria being *Agrobacterium tumefaciens* (…). Agrobacterium tumefaciens ), Brazilian pectinobacterium ( Pectobacterium brasiliense Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum ), watermelon acidophilus ( Acidovorax citrulli ), Streptomyces scabbingus ( Streptomyces scabies ) and Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp. michiganensis Any one of them.

5. The use of the metabolites or cultures of Bacillus belyssus ZF516 as described in claim 1 and / or Bacillus belyssus ZF516 as described in claim 2 in the preparation of a medicine for the prevention and / or treatment of tomato canker.

6. The use of the metabolites or cultures of Bacillus vesiculosus ZF516 as described in claim 1 and / or Bacillus vesiculosus ZF516 as described in claim 2 in the preparation of a medicine for the prevention and / or treatment of bacterial wilt of tomato.

7. The use of the metabolites or cultures of Bacillus belyi ZF516 as described in claim 1 and / or Bacillus belyi ZF516 as described in claim 2 in the preparation of a medicine for the prevention and / or treatment of cowpea root rot.

8. Any of the following applications of the metabolites or cultures of Bacillus belyssus ZF516 of claim 1 and / or Bacillus belyssus ZF516 of claim 2: V1. Application in inhibiting fungal activity; the fungus is Fusarium oxysporum (…). Fusarium oxysporum ), watermelon shell dispore ( Ascochyta citrullina Rhizoctonia solani ( ) Rhizoctonia solani Fusarium solani ( ) Fusarium solani ), Corynebacterium multiflorum ( Corynespora cassiicola ) and Botrytis cinerea ( Botrytis cinerea Any one of the following; V2. Application in inhibiting bacterial activity; the bacteria are *Agrobacterium tumefaciens* (…). Agrobacterium tumefaciens ), Brazilian pectinobacterium ( Pectobacterium brasiliense Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum ), watermelon acidophilus ( Acidovorax citrulli ), Streptomyces scabbingus ( Streptomyces scabies ) and Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp. michiganensis Any one of them.

9. A method for culturing Bacillus belye ZF516 as described in claim 1, characterized in that: The step includes culturing the Bacillus belye ZF516 of claim 1 in a culture medium.

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