Separation and identification of fumigating bacillus velezensis ZF525 and application of fumigating bacillus velezensis ZF525 in tomato bacterial wilt

Abstract

The invention discloses separation and identification of fumigation type bacillus velezensis ZF525 and application of the fumigation type bacillus velezensis ZF525 in tomato bacterial wilt. The invention belongs to the technical field of biology, and particularly relates to separation and identification of fumigation type bacillus velezensis ZF525 and application of the fumigation type bacillus velezensis ZF525 in tomato bacterial wilt. The bacillus velezensis provided by the invention is bacillus velezensis, the strain number of the bacillus velezensis is ZF525, and the preservation number of the bacillus velezensis in the China General Microbiological Culture Collection Center is CGMCC (China General Microbiological Culture Collection Center) No.32941. The bacillus velezensis ZF525 has bacteriostatic ability to various vegetable pathogenic bacteria, provides data support for development and application of the biocontrol strain, and provides a new strain resource for biological control work of tomato bacterial wilt.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the isolation and identification of a fumigation-type Bacillus belye ZF525 and its application in tomato bacterial wilt. Background Technology

[0002] Bacterial wilt of tomatoes is one of the most serious diseases affecting tomatoes, caused by Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum Bacterial wilt (Ralstonia solanacearum) is a soil-borne bacterial disease that often causes large-scale outbreaks after rain and under high temperatures. Its environmental adaptability and evolutionary capacity are extremely strong, allowing it to survive in soil and on non-host plants, making chemical control of it difficult. It has been reported that Ralstonia solanacearum can survive for 7 months in diseased plant debris, 2-3 years in soil or compost, and in some cases even 8-25 years. Furthermore, the pathogenic mechanism of Ralstonia solanacearum is very complex, involving many pathogenic genes, and the resistance mechanisms between Ralstonia solanacearum and its host plants are even more complex. Currently, the application of chemical fungicides is the main method for controlling bacterial wilt, but chemical control is not ideal, and long-term use of chemical fungicides leads to environmental pollution, harmful residues, and pathogen resistance. However, microbial fumigation, as an environmentally friendly soil fumigation technology, is a direct, rapid, and effective method for controlling soil-borne diseases, with broad research and application prospects.

[0003] In recent years, the study of the antibacterial properties of volatile substances from microorganisms has become a hot topic. The earliest reports of Bacillus subtilis (B. subtilis) since the 1990s... Bacillus subtilis NCIMB strains control Rhizoctonia solani by releasing antimicrobial volatile organic compounds. Rhizoctonia solani ) and ultimate pyridaben ( Pythium μLtimum Furthermore, researchers have discovered that gases produced by various Bacillus species can have antibacterial effects against different pathogens in plants. Bacillus also possesses strong environmental tolerance, making it a primary strain resource for commercial development and application. Current research utilizes headspace solid-phase microextraction and gas chromatography-mass spectrometry (GC-MS) to collect and analyze the volatile organic compounds produced by Bacillus. Researchers can use stored compounds in mass spectrometry databases for comparative analysis or search for relevant data on Bacillus volatile substances in the PubMed database. In recent years, Bacillus has been recognized as a major biocontrol bacterium, as its volatile organic compounds can achieve certain antibacterial effects. Among these, alcohols, ketones, esters, aldehydes, phenols, and alkenes show great potential application value in biocontrol. Summary of the Invention

[0004] The main problem to be solved by this invention is how to use biological methods to suppress bacterial wilt of tomatoes and obtain biocontrol strains that have significant preventive effects against bacterial wilt of tomatoes.

[0005] To address the above problems, the present invention provides a biocontrol bacterium, Bacillus belye.

[0006] The Bacillus belesiensis of the present invention ( Bacillus velezensis The strain number is ZF525, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 32941.

[0007] The present invention provides a microbial agent containing the aforementioned Bacillus belye and / or metabolites of the Bacillus belye; The inoculant is a pathogen inhibitor or disease inhibitor, and the pathogen is *Ralstonia solanacearum* (…). Ralstonia solanacearum ), Corynebacterium multiflorum ( Corynespora cassiicola ) 、 Botrytis cinerea ( Botrytis cinerea Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum Fusarium solani ( ) Fusarium solani Anthrax bacillus (pepper) (Colletotrichum capsici) Eggplant stalk mold ( Stemphylium solani ).

[0008] The diseases mentioned are bacterial wilt of tomato, leaf spot of cucumber, anthracnose of pepper, gray mold of tomato, leaf spot of tomato, damping-off of rice, wilt of pea and / or root rot of melon.

[0009] The active ingredient of the above-mentioned microbial agent may be the above-mentioned Bacillus belyssus and / or the metabolites of the above-mentioned Bacillus belyssus. The active ingredient of the above-mentioned microbial agent may also contain other biological or non-biological components. Other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art based on their inhibitory effect on diseases.

[0010] In addition to the active ingredient, the above-mentioned microbial agent also contains a carrier. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0011] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0012] Depending on the needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the above-mentioned microbial agents.

[0013] As described above, the metabolites can be obtained from the fermentation broth of *Bacillus belyssiensis*. The metabolites can be sterile metabolites of *Bacillus belyssiensis* or bacterial metabolites of *Bacillus belyssiensis*. Specifically, the sterile metabolites of *Bacillus belyssiensis* (sterile fermentation filtrate) can be prepared by culturing *Bacillus belyssiensis* in a liquid culture medium and filtering to remove the *Bacillus belyssiensis* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of *Bacillus belyssiensis* can be prepared by culturing *Bacillus belyssiensis* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolites of *Bacillus belyssiensis*.

[0014] The present invention also provides the use of the above-mentioned Bacillus belyssus in the preparation of any of the following products: 1) Inoculants for the prevention and control of plant diseases, the diseases being tomato bacterial wilt, cucumber leaf spot, pepper anthracnose, tomato gray mold, tomato leaf spot, rice damping-off, pea wilt, and / or melon root rot; 2) Pathogen inhibitor, wherein the pathogen is Ralstonia solanacearum (… Ralstonia solanacearum ), Corynebacterium multiflorum ( Corynespora cassiicola ) 、 Botrytis cinerea ( Botrytis cinerea Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum Fusarium solani ( ) Fusarium solani Anthrax bacillus (pepper) (Colletotrichum capsici) Eggplant stalk mold ( Stemphylium solani ).

[0015] The present invention also provides a method for culturing the above-mentioned Bacillus belye, comprising the step of culturing the Bacillus belye described above in a culture medium for culturing Pseudomonas.

[0016] The present invention also provides a method for preparing the bacterial agent, comprising the following steps: using the aforementioned Bacillus belye as the active ingredient to obtain the bacterial agent.

[0017] The present invention also provides a bio-organic fertilizer containing the aforementioned Bacillus belye or the aforementioned microbial agent.

[0018] The present invention also provides the use of the aforementioned Bacillus berleis or the aforementioned bacterial agent as a biocontrol agent.

[0019] The present invention also provides the use of the aforementioned Bacillus berleis or the aforementioned bacterial agent as an antibacterial agent or in the preparation of volatile antibacterial agents.

[0020] This invention also provides the application of the aforementioned Bacillus belye in the prevention and control of plant diseases, wherein the diseases are any of the following: 1) Bacterial wilt of tomatoes; 2) Anthracnose of peppers; 3) Tomato gray mold; 4) Tomato leaf spot disease; 5) Rice damping-off; 6) Pea wilt; 7) Melon root rot; 8) Cucumber leaf spot disease caused by Corynebacterium tumefaciens.

[0021] In the above applications, the plant is a tomato, melon, cucumber, pepper, or pea.

[0022] This chapter describes the isolation of Bacillus from the rhizosphere soil of healthy tomatoes in greenhouses in Pingluo County, Ningxia Hui Autonomous Region, Tongliao City, Inner Mongolia Autonomous Region, Shouguang City, Shandong Province, and Tianjin City using the dilution plating method. Antagonistic strains exhibiting significant inhibitory effects against Ralstonia solanacearum were initially screened using the plate-to-plate method. Subsequent screening using in vivo pot experiments with fumigation equipment identified biocontrol strains with significant control efficacy against tomato bacterial wilt. Based on the morphological characteristics, physiological and biochemical assays, Biolog assays, and molecular biological identification of the biocontrol strains, a multi-gene phylogenetic tree was constructed to determine their species. The inhibitory activity against various vegetable pathogens was also measured, providing data support for the development and application of these biocontrol strains and offering new strain resources for the biological control of tomato bacterial wilt.

[0023] Deposition description Bacterial strain name: Bacillus belesiensis Latin name: Bacillus velezensis Strain number: ZF525 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: December 6, 2024 Registered with the China National Collection Center (CGMCC) No.: 32941. Attached Figure Description

[0024] Figure 1 This study determined the antagonistic effect of three Bacillus strains against Ralstonia solanacearum on agar plates. Treatment 1: ZF525 fumigation; Treatment 2: NX02-5 fumigation; Treatment 3: NX01-1 fumigation; Treatment 4: sterile water treatment. Figure 2 Morphological characteristics of strain ZF506 were observed. Treatment 1: Colony morphology under visible light; Treatment 2: Single colony morphology; Treatment 3: Gram staining results; Treatment 4: Colony morphology under scanning electron microscopy; Treatment 5: Strain morphology under scanning electron microscopy.

[0025] Figure 3 Construction of a multigene phylogenetic tree for strain ZF525.

[0026] Figure 4 The effects of fumigation with different bacterial strains on the control of bacterial wilt in potted tomatoes were investigated. Treatment 1: fumigation with ZF525; Treatment 2: fumigation with NX02-5; Treatment 3: fumigation with NX01-1; Treatment 4: fumigation with water.

[0027] Figure 5 This study compares the effects of commercial inoculant fumigation and ZF525 fumigation on the control of bacterial wilt in potted tomatoes. Treatment 1: Guandiwang fumigation; Treatment 2: Nongwangxiang fumigation; Treatment 3: Aishanggen fumigation; Treatment 4: Zhongnong Lvkang fumigation; Treatment 5: Gengnongren fumigation; Treatment 6: Hugenling fumigation; Treatment 7: Calcium cyanide fumigation; Treatment 8: ZF525 fumigation; Treatment 9: Water fumigation.

[0028] Figure 6 The study investigated the inhibitory effect of strain ZF525 on the growth of six pathogens. Treatment 1: *Fusarium oxysporum*; Treatment 2: *Cyclocarya pallida*; Treatment 3: *Anthracnose capsulatum*; Treatment 4: *Botrytis cinerea*; Treatment 5: *Fusarium solani*; Treatment 6: *Pseudomonas solani*. Detailed Implementation

[0029] 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.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0032] The cucumber variety 'Zhongnong No. 6' used in the following examples was purchased from Beijing Zhongshu Seed Industry Technology Co., Ltd.

[0033] The tested pathogenic strain Ralstonia solanacearum in the following examples (Ralstonia solanacearum) Ralstonia solanacearum The biological material is preserved in the Vegetable Disease Integrated Prevention Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and has been recorded in: 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. DOI:10.16420 / j.issn. The public can obtain this biological material from the applicant. This biological material is only for the purpose of repeating the experiments of this invention and shall not be used for other purposes.

[0034] The *Cyclocarya multiflora* strain described in the following examples was preserved in the Vegetable Disease Integrated Prevention Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and is documented in: Wang Shaohua. The effect of droplet splashing on the spread of *Cyclocarya multiflora* leaf spot pathogen in soil [D]. Chinese Academy of Agricultural Sciences, 2024. DOI:10.27630 / d.cnki.gznky.2024.000361. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and shall not be used for any other purpose.

[0035] The *Anthracnose spp.* in the following examples was preserved in the Vegetable Disease Integrated Prevention Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and has been recorded in: Zhao Zixuan, Zeng Xianfeng, Qin Shiyang, et al. Identification of *Bacillus belyssus* strain ZF438 and its antibacterial effect of fermentation supernatant on anthracnose of pepper [J]. Journal of Agricultural Biotechnology, 2023, 31(10):2163-2175. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0036] The *Botrytis cinerea* in the following examples: preserved in the Vegetable Disease Integrated Prevention Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and described in: Shi Yanxia, ​​Tang Ming, Jin Zhiwen, et al. Evaluation of resistance of *Botrytis cinerea* to different types of fungicides in vegetable crops [J]. Chinese Vegetables, 2016, (03): 60-65. The public can obtain this biological material from the applicant. This biological material is only for repeating the experiments of this invention and shall not be used for other purposes.

[0037] The *Stemona japonica* species used in the following examples were preserved in the Vegetable Disease Integrated Prevention Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and have been documented in: Xie Xuewen, Liu Shicheng, Shi Yanxia, ​​et al. Establishment and application of rapid LAMP detection system for *Stemona japonica* [J]. Journal of Agricultural Biotechnology, 2022, 30(10):2045-2052. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating the experiments of this invention and may not be used for any other purpose.

[0038] The *Fusarium oxysporum* strains used in the following examples were preserved in the Integrated Disease Control Group of the Vegetable Disease Research Institute, Chinese Academy of Agricultural Sciences, and have been documented in: Liu Jialin, Shi Yanxia, ​​Xie Xuewen, et al. Evaluation of the efficacy of jinggangmycin combined with seed dressing agent in controlling cowpea wilt [J]. Pesticides, 2024, 63(10):760-764. DOI:10.16820 / j.nyzz.2024.1011. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and shall not be used for any other purpose.

[0039] The *Fusarium solani* strains described in the following examples were preserved by the Integrated Disease Control Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and have been documented in: Yan Wenxue, Shi Yanxia, ​​Chai Ali, et al. Identification of pathogens causing root rot of cowpea [J]. Journal of Plant Protection, 2019, 46(03):717-718. DOI:10.13802 / j.cnki.zwbhxb.2019.2018069. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and shall not be used for any other purpose.

[0040] The test media used in the following examples were NA medium (L8291), LB medium, and PDA medium (P8931), which were purchased from Beijing Solarbio Science & Technology Co., Ltd.; the test reagent was calcium cyanamide (Jinan Taihe Chemical Co., Ltd.).

[0041] The following examples used Excel 2010 software for data organization, statistics and analysis, SPSS 22.0 software for data significance testing, and MEGA 11 and other software for constructing multigene phylogenetic trees. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and Duncan's new multiple range method was used to analyze the significance of differences in the data (p<0.05). Different lowercase letters are used to indicate the significance of differences between different treatments.

[0042] Example 1: Isolation, purification and screening of Bacillus subtilis 1. Soil samples for testing Soil samples were collected from the rhizosphere soil of healthy tomatoes in greenhouses in Pingluo County, Ningxia Hui Autonomous Region, Tongliao City, Inner Mongolia Autonomous Region, Shouguang City, Shandong Province, and Tianjin Municipality, totaling 23 soil samples. Approximately 100 g of rhizosphere soil (10–20 cm below the soil surface) was collected from healthy tomato plants that had contracted bacterial wilt in the tomato fields. The soil samples were placed in sterile self-sealing bags and stored at 4℃ for later use.

[0043] 2. Isolation, purification, and preservation of Bacillus in soil Weigh 10 g of each soil sample and add it to an Erlenmeyer flask containing 90 mL of sterile water. Shake and incubate at 170 r / min and 28℃ for 30 min to obtain a soil suspension. Then place the suspension in an 80℃ water bath for 15 min. Dilute the soil suspension using the dilution plating method, selecting four gradients: 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶. Take 100 μL of each gradient and spread it onto LB agar plates. Repeat each dilution and each culture medium three times. Incubate the plates at 28℃ for 1–2 days. Select single colonies with inconsistent morphology for purification and re-culturing, and store them at -80℃ using 40% glycerol.

[0044] The results are as follows: 329 bacterial strains were initially screened from the rhizosphere soil of healthy tomato plants in tomato-growing areas of Pingluo County, Ningxia Hui Autonomous Region.

[0045] 3. Screening of antagonistic strains producing volatile substances 1) Preparation of pathogen suspension Ralstonia solanacearum stored at -40℃ was activated with NA solid medium and cultured in a dark environment at 28℃ for 2 days. Single colonies that had grown well were picked and transferred to NB medium, then activated at 180 rpm for 24 hours in a constant temperature shaker at 28℃. The bacterial concentration was adjusted to 102. 8 cfu / mL.

[0046] 2) Preparation of biocontrol bacteria suspension Bacillus spores stored at -80℃ were activated using LB agar plates and cultured at 28℃. After single colonies grew, they were picked up with sterile toothpicks and placed on LB liquid medium. The culture was then incubated for 36 h at 28℃ and 170 r / min on a shaker. The bacterial suspension concentration was adjusted to 1×10⁻⁶ using sterile water. 8 The biocontrol bacterial solution was prepared at cfu / mL and used for later use.

[0047] 3) Screening of antagonistic bacterial strains producing volatile substances Antagonistic bacterial strains producing volatile substances were screened using the plate-on-plate method. The specific steps are as follows: 100 μL of tomato was inoculated onto one side of an NA medium plate. R.solanacearumThe bacterial suspension was evenly spread. On the other side of the NA medium plate, 100 μL of the biocontrol bacterial suspension prepared in step 2) was inoculated and evenly spread. A treatment plate with an equal volume of sterile physiological saline was used as a control. The plates were sealed with plastic wrap and incubated at 30°C for 1-2 days. The tomato content in the treatment and control groups was recorded using the dilution plate technique. R.solanacearum To determine the effect of volatile substances on tomatoes, the number of colonies is used. R.solanacearum The antibacterial rate was determined, and the effect of volatile substances on tomatoes was calculated using the following formula. R.solanacearum Inhibition rate of bacteria: Inhibition rate (%) = [(number of control colonies - number of treated colonies) / number of control colonies] × 100. The Bacillus strain with an inhibition rate of up to 85% was selected as the target strain.

[0048] Through the above-mentioned plate-to-plate screening method, three strains with control efficacy of over 80% against R. solanacearum were obtained, among which strain ZF525 had the highest antagonistic efficiency of 93.93%. Figure 1 (and Table 1).

[0049] Table 1. Determination of the plate antagonistic effect of three Bacillus strains against Ralstonia solanacearum.

[0050] Example 2: Classification and identification of ZF525, a volatile substance antagonistic bacterial strain. 1. Morphological observation and determination of physiological and biochemical characteristics The target strain ZF525 was identified by morphology and physiological and biochemical characteristics according to Bergey's Manual of Identification, and its carbon source utilization (BIOLOG) was also measured.

[0051] Strain ZF525 was cultured on LB medium for 48 h. The colonies were round, pale yellow, opaque, with irregular edges and ridge-like raised wrinkles on the surface. Figure 2It was identified as a Gram-positive bacterium that can grow at temperatures ranging from 28 to 37 °C; it was positive for starch hydrolysis, VP reaction, catalase and motility tests, and positive for gelatin liquefaction and nitrate reduction (Table 2). Regarding carbon source utilization, strain ZF525 can utilize D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, D-raffinose, D-mannitol, and L-rhamnose; however, it cannot utilize D-serine, minocycline, lincomycin, niaproof 4, stachyose, or α-D-lactose (Table 3). Table 2. Physiological and biochemical characteristics of strain ZF525

[0052] Note: +: positive; -: negative; W: weak positive.

[0053] Table 3. Carbon source utilization rate of strain ZF525

[0054] Note: +: positive; -: negative.

[0055] 2. Construction of a multi-gene phylogenetic tree Genomic DNA was extracted from strain ZF525 using a rapid DNA extraction and detection kit (KG203, Tiangen Biotech Co., Ltd., Beijing). Primers targeting 16S rDNA, gyrA, purH, and rpoB were synthesized according to primers designed by HUANG et al. and ZHAO et al. (Table 4). PCR amplification was performed, and the amplified products were sequenced by Beijing Bomeide Biotechnology Co., Ltd. A multi-gene (16S rDNA-gyrA-purH-rpoB) phylogenetic tree was constructed based on the sequencing results to analyze the taxonomic position of strain ZF525.

[0056] Table 4. Primer information for constructing the multigene phylogenetic tree of strain ZF525

[0057] Four sequences from strain ZF525, targeting 16S rDNA (nucleotide sequence 1), gyrA (nucleotide sequence 2), purH (nucleotide sequence 3), and rpoB (nucleotide sequence 4) genes obtained by PCR amplification, were uploaded to NCBI. NCBI BLAST comparison revealed that all five gene sequences of this strain shared ≥97% homology with their corresponding gene sequences from *Bacillus velezensis*.

[0058] In addition, a multigene phylogenetic tree was constructed based on 16S rDNA, gyrA, purH, and rpoB ( Figure 3 The results showed that strain ZF525 clustered in the same group as the type strains *B. velezensis* FZB42, AP46, and 12Y, among other previously reported strains. Therefore, strain ZF525 was identified as *Bacillus belyesense*. Bacillus velezensis ).

[0059] 3. Bacillus belye ( Bacillus velezensis Preservation of strain ZF525 Bacillus belesiensis ( Bacillus velezensis ZF525 was deposited on December 6, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32941. It will be referred to as Bacillus belyssus CGMCC No. 32941 or strain ZF525.

[0060] Example 3: Pot control efficacy of antagonistic bacteria against bacterial wilt of tomato 1. The effect of strain ZF525 on greenhouse pot cultivation under greenhouse conditions R.solanacearum The efficacy of preventing bacterial wilt in tomatoes The two-dish confrontation method was used to screen and obtain the pair R.solanacearum The B. velezensis strain ZF525, which exhibits high antagonistic activity, was further investigated using a simulated soil fumigation method to determine its antagonistic effect on plants grown in greenhouse pots under these conditions. R.solanacearum The efficacy in preventing bacterial wilt of tomatoes. The specific steps are as follows: Microbial fumigation treatment was simulated using a dryer. Tomatoes that had been shaken for 2 days were... R.solanacearum bacterial suspension (1×10) 8 CFU·mL -1 )according to R.solanacearum The bacterial solution was prepared by mixing bacterial culture and soil at a ratio of 1:10, and then the bacterial soil was placed on top of the desiccator; the bacterial culture of strain ZF525 (1×10⁻⁶) was added. 8 CFU·mL -1Add ZF525 bacteria to soil at a ratio of 1:10, mix well, and then place at the bottom of a desiccator. Seal the desiccator and leave it at room temperature for 7 days. Then, remove the tomato-containing product fumigated with ZF525 bacteria from the top of the desiccator. R.solanacearum The inoculum was placed in seedling trays, and tomato seedlings at the two-leaf-one-heart stage were transplanted into the inoculum. Three replicates were set up, with 18 tomato seedlings in each replicate. Sterile water mixed with soil was used as a negative control, and calcium cyanamide fumigation treatment was used as a chemical control.

[0061] Tomato bacterial wilt is classified according to the disease grading standards in the "Guidelines for Field Efficacy Trials of Pesticides": The classification is based on the degree of wilting of the plant and leaves. Five levels are assigned based on the severity of the disease: Level 0: Healthy plant, no wilting symptoms; Level 1: 1 / 4 of the leaves are wilted; Level 2: 1 / 2 of the leaves are wilted; Level 3: 2 / 3 of the leaves are wilted; Level 4: The entire plant wilts and dies.

[0062] The results showed that the control group (treated with sterile water instead of ZF525 strain for fumigation) had a more severe incidence of bacterial wilt in tomatoes, with a disease index of 75.00. In contrast, the group treated with ZF525 microbial fumigation had a milder disease incidence, with a disease index of only 41.67. This indicates that ZF525 microbial fumigation treatment achieved a control efficacy of 43.69% against bacterial wilt in tomatoes, which was higher than the control efficacy of chemical pesticide (calcium cyanide) treatment (41.24%). Figure 4 This indicates that the biocontrol strain ZF525 can be used to effectively control tomato bacterial wilt via microbial soil fumigation. 2. Comparison of the effects of fumigation with six commercially available microbial agents and ZF525 on the control of bacterial wilt in potted tomatoes. Six commercially available Bacillus preparations were selected, and the efficacy of ZF525 against tomato bacterial wilt was compared with these six preparations using a live potted plant method (Table 5). The efficacy against tomato bacterial wilt under greenhouse potted plant conditions was determined using a simulated soil fumigation method, following the same procedure as in step 1.

[0063] Table 5. Control efficacy of commercial microbial fumigation against bacterial wilt in potted tomatoes.

[0064] The results showed that the antagonistic strain ZF525 had the highest control efficacy against bacterial wilt of tomato, reaching 56.25%, while the control efficacy of calcium cyanamide fumigation against bacterial wilt of tomato was 50.00% (Table 6). The control efficacy of strain ZF525 was significantly higher than that of commercially available microbial agents. Among the six selected products, Nongwangxiang (Nongduole Agricultural Technology Co., Ltd.) had the best control efficacy, reaching 37.50%. Figure 5 ) Table 6. Comparison of control efficacy of commercial inoculant fumigation and ZF525 fumigation against bacterial wilt in potted tomatoes.

[0065] Example 4: Determination of the antibacterial spectrum of strain ZF525 1. Antagonistic effect of strain ZF525 against six common pathogenic fungi The inhibitory effect of strain ZF525 on six common pathogenic fungi (provided by the Vegetable Disease Integrated Management Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences) was determined using the dichotomous plate confrontation method (Table 7). Fungal discs (3 mm in diameter) were inoculated into the center of the left-hand PDA medium, and 100 µL of ZF515 bacterial suspension (1×10⁻⁶) was added. 8 CFU·mL -1 The culture medium was evenly spread on the right side of the same petri dish and incubated at 28 °C for 4 days. The diameter of the inhibition zone was measured, and the inhibition rate was calculated. Each treatment was repeated three times. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100.

[0066] Table 7. Information on tested strains

[0067] 2. Analysis of the antagonistic effects of volatile substances from strain ZF525 on different pathogenic fungi. The antagonistic effects of volatile substances from the screened ZF525 strain against different plant pathogenic fungi were determined by the dichotomous dish method (Table 8). Figure 6 ).

[0068] The results showed that strain ZF525 was effective against the six common plant pathogenic fungi tested (Fusarium oxyporum, Corynespora cassiicola, Colletotrichum caps). ici ; Botrytis cinerea ; Fusarium solani ; Stemphylium solani All of them showed antibacterial effects, with the highest inhibition rate against *Pseudomonas cremastoides* (91.11%). This indicates that the volatile substances produced by strain ZF525 have broad-spectrum antagonistic properties, and can be used not only to prevent and control *Pseudomonas cremastoides* but also to inhibit the growth of bacteria caused by *Pseudomonas cremastoides*. R.solanacearum Besides the bacterial wilt of tomatoes caused by [unspecified disease], for [unspecified disease] caused by [unspecified disease] R. solani It also has great potential for development and application in the prevention and control of various soil-borne diseases of vegetables.

[0069] Table 8. Inhibitory effect of strain ZF525 on six pathogens

[0070] Note: Experimental data are presented as mean ± standard deviation, with lowercase letters indicating variability. P <0.05).

[0071] The present invention has been described in detail above. Those skilled in the art will recognize that 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. While specific embodiments have been provided, 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.

Claims

1. Bacillus belye, characterized in that: The *Bacillus belesii* is *Bacillus belesii* (… Bacillus velezensis The strain number is ZF525, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 32941.

2. A microbial agent, characterized in that: The bacterial agent contains the Bacillus berleis as described in claim 1 and / or the metabolites of the Bacillus berleis; The inoculant is a pathogen inhibitor or disease inhibitor, and the pathogen is *Ralstonia solanacearum* (…). Ralstonia solanacearum ), Corynebacterium multiflorum ( Corynespora cassiicola ) 、 Botrytis cinerea ( Botrytis cinerea Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum Fusarium solani ( ) Fusarium solani Anthrax bacillus (pepper) (Colletotrichum capsici) Eggplant stalk mold ( Stemphylium solani The diseases mentioned are bacterial wilt of tomato, leaf spot of cucumber, anthracnose of pepper, gray mold of tomato, leaf spot of tomato, damping-off of rice, wilt of cowpea and / or root rot of cowpea.

3. The use of Bacillus belye as described in claim 1 in the preparation of any of the following products: 1) Inoculants for the prevention and control of plant diseases, the diseases being tomato bacterial wilt, cucumber leaf spot, pepper anthracnose, tomato gray mold, tomato leaf spot, rice damping-off, pea wilt, and / or melon root rot; 2) Pathogen inhibitor, wherein the pathogen is Ralstonia solanacearum (… Ralstonia solanacearum ), Corynebacterium multiflorum ( Corynespora cassiicola ) 、 Botrytis cinerea ( Botrytis cinerea Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), Fusarium oxysporum ( Fusarium oxysporum Fusarium solani ( ) Fusarium solani Anthrax bacillus (pepper) (Colletotrichum capsici) Eggplant stalk mold ( Stemphylium solani ).

4. A method for culturing Bacillus belye of claim 1, comprising the step of culturing the Bacillus belye in a culture medium for culturing Pseudomonas.

5. The method for preparing the bacterial agent according to claim 2 includes the following steps: using Bacillus belye as the active ingredient to obtain the bacterial agent.

6. Bio-organic fertilizer, characterized by: The bio-organic fertilizer contains the Bacillus berberis described in claim 1 or the microbial agent described in claim 2.

7. The use of Bacillus belye as claimed in claim 1 or the bacterial agent as claimed in claim 2 as a biocontrol agent.

8. The use of Bacillus belyssus of claim 1 or the bacterial agent of claim 2 as a volatile antibacterial agent in the preparation of such an agent.

9. The application of Bacillus belye as described in claim 1 in the prevention and control of plant diseases, wherein the disease is any one of the following: 1) Bacterial wilt of tomatoes; 2) Anthracnose of peppers; 3) Tomato gray mold; 4) Tomato leaf spot disease; 5) Rice damping-off; 6) Cowpea wilt; 7) Cowpea root rot; 8) Cucumber leaf spot disease caused by Corynebacterium tumefaciens.

10. The application according to claim 9, wherein the plant is a tomato, melon, cucumber, pepper, or cowpea.

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