Bacillus velezensis s2 and application thereof in prevention and treatment of tomato early blight

Abstract

This invention discloses a strain of *Bacillus belyssus* S2 and its application in the control of early blight in tomato, belonging to the field of microbial technology. *Bacillus belyssus* S2 is deposited at the China Center for Type Culture Collection (CCTCC) on March 30, 2026, with accession number CCTCC NO: M 2026551. The *Bacillus belyssus* S2 isolated and screened in this invention can secrete siderophores, cellulases, proteases, 1,2-β-glucanases, and lipases. Its soluble and volatile substances can inhibit the mycelial growth of tomato pathogens and cause mycelial malformation, reduce spore germination rate, delay the onset of early blight in tomato fruits, and enhance the fruit's resistance, providing a new microbial resource for the control of diseases such as early blight in tomato.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bacillus belye S2 and its application in the prevention and control of early blight in tomatoes. Background Technology

[0002] Early blight of tomato is one of the major diseases of tomatoes, caused by various Alternaria species. This disease is prevalent throughout the entire tomato growth cycle, causing seedling death, leaf drop, and fruit drop, leading to a 20%-30% yield reduction. It also severely impacts post-harvest storage, causing fruit rot and spoilage, resulting in significant economic losses. Numerous studies have shown that Alternaria fungi produce various Alternaria toxins during tomato infection, such as Alternaria alcohol (AOH), Alternaria alcohol monomethyl ether (AME), Alternene (ALT), Alternaria toxins I, II, and III (ATX-I, II, and III), tenozolic acid (TEA), isotenozolic acid (isoTEA), and tenozolin (TEN). Currently, limited by fruit and vegetable processing technology, the main way to reduce the risk of toxin and pathogen contamination in processed tomato products is by removing rotten parts. However, this cannot completely eliminate the potential risk of mycotoxin contamination in tomato products. Multiple Alternaria toxins have been detected in secondary processed tomato products. Among them, Alternaria alternataic acid (TeA) had the highest detection rate, followed by Alternaria alternataol (AOH) and Alternaria alternataol monomethyl ether (AME). Alternaria alternataol (AOH) and Alternaria alternataol monomethyl ether (AME) exhibit severe cytotoxicity and genotoxicity in humans and animals even at micromolar concentrations.

[0003] Currently, the control of early blight in tomatoes still relies primarily on chemical methods, with commonly used agents including mancozeb, difenoconazole, and cyprodinil. While chemical agents can rapidly control the occurrence and spread of the disease in the short term, long-term continuous application leads to pesticide residue problems, causing serious environmental pollution and food safety risks, while also resulting in a continuous increase in the risk of drug resistance in the tomato early blight pathogen. Studies by Shi Xiaojing et al. found that the tomato early blight pathogen in Shanxi Province has developed resistance to multiple agents such as difenoconazole and cyprodinil, with a resistance index to difenoconazole ranging from 0.295 to 0.447. Malandrakis et al. confirmed that the sensitivity of the tomato early blight pathogen to mancozeb was significantly reduced, with its half-maximal inhibitory concentration (EC50) decreasing to below 100%. 50 The effectiveness of chemical control is significantly improved at conventional dosages, while the control effect is greatly reduced. Therefore, developing green, environmentally friendly, safe, and efficient control technologies to reduce the application of chemical agents has become an urgent need in the field of tomato early blight control. Biological control, due to its significant advantages of environmental friendliness and high safety, has become a research hotspot in plant disease control. *Trichoderma* (*Trichoderma* genus) Trichoderma spp.), Bacillus spp. Bacillus spp.), Actinomycetes ( Actinomycetales spp.) and Pseudomonas spp. Pseudomonas Various microorganisms, including *Bacillus spp.*, have been shown to have significant antagonistic activity against early blight pathogens of tomato. Among them, *Bacillus* species are widely studied due to their ability to form highly resistant endospores, their combined aerobic and facultative anaerobic metabolic pathways, their rich variety of metabolites, their strong environmental adaptability, and their high biocompatibility. For example, *Bacillus subtilis* DB2203A, isolated by Chen Jiahui et al., showed an in vitro growth inhibition rate of 74.77% against tomato gray mold pathogens and produced lipopeptide antibacterial active substances. *Bacillus amyloliquefaciens* Y6-7, screened from healthy strawberry rhizosphere soil by He Haowen et al., achieved a field control effect of 80.16% against strawberry angular leaf spot and also showed significant inhibitory effects against various plant pathogens, exhibiting excellent broad-spectrum antibacterial properties. *Bacillus laterosporus* Bl13, screened and identified by Sun Yifan et al., can effectively control early blight of tomato by regulating soil microbial community structure, increasing the abundance of beneficial soil microorganisms, and inducing the enhancement of defensive enzyme activity in tomato leaves through multiple synergistic effects. It is worth noting that current research on biological control mainly focuses on disease control during the plant's growth period in the field, while research on biological control of diseases in fruits and vegetables during the post-harvest storage stage is relatively scarce. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Bacillus belyssus* S2 and its application in the control of early blight in tomato, thereby addressing the problems existing in the prior art. The *Bacillus belyssus* S2 isolated and screened by this invention can secrete siderophores, cellulases, proteases, and 1,2-β-glucanase. Its soluble and volatile substances can inhibit the mycelial growth of tomato pathogens and cause mycelial malformation, reduce spore germination rate, delay the onset of early blight in tomato fruits, and enhance the fruit's resistance to adverse conditions, providing a new microbial resource for the control of diseases such as early blight in tomato.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Bacillus belye ( Bacillus velezensis S2, deposited at the China Center for Type Culture Collection, on March 30, 2026, with accession number CCTCC NO: M 2026551.

[0006] The present invention also provides the application of the above-mentioned Bacillus berberis S2 in the preparation of microbial agents.

[0007] The present invention also provides a microbial inoculant containing the above-mentioned Bacillus belye S2, its metabolites and / or its volatile substances.

[0008] This invention also provides the application of the above-mentioned Bacillus berberis S2 or the above-mentioned microbial inoculant in inhibiting the growth of plant pathogens, wherein the plant pathogens include Alternaria alternata (… Alternaria alternata ), strawberry gray mold ( Botrytis cinerea Pers. Alternaria alternifolia ( ) Alternaria solani Fusarium graminearum ( ), Fusarium grasses ), corn leaf blight pathogen ( Turkish sedge ), corn leaf blight fungus ( Exerohyl Turkish ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Haematonectria haematococci ), Fusarium graminearum ( Fusarium pseudogramineum Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani-tobacco-specific type ( Rhizoctonia solani ) and Rhizoctonia solani-soybean-specific ( Rhizoctonia solani ).

[0009] The present invention also provides a product for inhibiting the growth of plant pathogens, the product comprising the above-mentioned Bacillus berberis S2 or the above-mentioned microbial agent.

[0010] Furthermore, the plant pathogens include Alternaria alternata (…). Alternaria alternata ), strawberry gray mold ( Botrytis cinerea Pers. Alternaria alternifolia ( ), Alternaria alternifolia Alternaria solani Fusarium graminearum ( ), Fusarium gramineae ), corn leaf blight pathogen ( Turkish sedge ), corn leaf blight fungus ( Turkish sedge ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Haematonectria haematococci ), Fusarium graminearum ( Fusarium pseudogramineum Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani-tobacco-specific type ( Rhizoctonia solani ) and Rhizoctonia solani-soybean-specific ( Rhizoctonia solani ).

[0011] The present invention also provides a method for preventing and controlling early blight in tomatoes, comprising the step of applying the above-mentioned Bacillus berberis S2, the above-mentioned microbial agent, or the above-mentioned product to tomato plants.

[0012] The present invention also provides a method for preventing and controlling postharvest early blight of tomatoes, comprising the step of applying the above-mentioned Bacillus berberis S2, the above-mentioned microbial agent or the above-mentioned product to postharvest tomato fruits.

[0013] The present invention also provides the application of the above-mentioned Bacillus berberis S2 or the above-mentioned microbial agent in the production of enzyme preparations, wherein the enzyme preparations include cellulase preparations, protease preparations, 1,2-β-glucanase preparations and lipase preparations.

[0014] The present invention also provides the application of the above-mentioned Bacillus vesiculosus S2 or the above-mentioned microbial agent in the production of iron carriers.

[0015] The present invention discloses the following technical effects: This invention isolated and screened a strain of *Bacillus belyssus* S2, and explored its antibacterial mechanism through microbial culture and fruit wound infection experiments. The results showed that *Bacillus belyssus* S2 can secrete siderophores, cellulases, proteases, and 1,2-β-glucanase. Its soluble and volatile substances can inhibit the mycelial growth of tomato pathogens and cause mycelial malformation, reducing spore germination rate. It can also delay the onset of early blight in tomato fruits, reduce malondialdehyde content by 12.05%-45.78%, increase CAT enzyme activity by 4-7 times, increase POD enzyme activity by 36.0%-63.0%, and while SOD enzyme activity slightly decreased but remained relatively stable, indicating that this strain can enhance the fruit's resistance to adverse conditions. This invention provides a theoretical basis and usable microbial resources for further research on the control of postharvest early blight in tomatoes. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results of screening and identification of Bacillus belyss S2 are shown below; where A is the Gram staining image of Bacillus belyss S2, B is the plate colony morphology of Bacillus belyss S2, C is the result of the plate confrontation experiment of the control group (without inoculation with antibacterial agents), D is the result of the plate confrontation experiment of Bacillus belyss S2, and E is the statistical result of the lesion diameter of Bacillus belyss S2 in the plate confrontation experiment. Figure 2 Phylogenetic tree of Bacillus belyssus S2; Figure 3 The results of the detection of the effect of Bacillus belyss S2 on the mycelial growth of early blight of tomato (magnified 400 times); where A is the control group and B is the Bacillus belyss S2 treatment group; Figure 4 The results show the effect of Bacillus belyss S2 on the germination of tomato early blight spores; where A represents the statistical results of spore germination inhibition rate, and B represents the spore germination under a microscope. Figure 5 The antibacterial effect of volatile substances from Bacillus belyss S2 is shown; the left figure shows the Bacillus belyss S2 treatment group, and the right figure shows the control group. Figure 6 The results are for the extracellular enzyme assays of Bacillus belyssus S2; where A represents 1,2-β-glucanase secretion, B represents siderophore secretion, C represents lipase secretion, D represents protease secretion, and E represents cellulase secretion. Figure 7 The results show the inhibitory effect of Bacillus belyss S2 on early blight of tomato fruit; where A represents the statistical results of incidence rate; and B represents the statistical results of lesion diameter. Figure 8 The image shows the inhibitory effect of Bacillus berleis S2 on early blight of tomato fruit; the concentrations of Bacillus berleis S2 in the AE treatment were 0 (CK) and 10, respectively. 6 cfu / mL, 10 7 cfu / mL, 10 8 cfu / mL, 10 9 cfu / mL; Figure 9 The results show the effects of Bacillus belyssus S2 on the antioxidant capacity of tomato fruit cells; where A represents the MDA content detection result; B represents the CAT enzyme activity detection result; C represents the POD enzyme activity detection result; and D represents the SOD enzyme activity detection result. Figure 10 This is the result of whole genome analysis of Bacillus belyssus S2; the diagram is arranged from the outside to the inside: First circle: CDS, rRNA, tRNA, tmRNA on the positive strand; Second circle: GC content; Third circle: GC skew; Fourth circle: CDS on the negative strand; Fifth circle: Genomic sequence position coordinates. Figure 11 Gene annotation results based on the CAZy database; Figure 12 Gene annotation results based on the NR database; Figure 13 The results of enrichment analysis based on the GO database; Figure 14 Gene annotation results based on the COG database; Figure 15 The results are based on enrichment analysis using the KEGG database. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0024] Example 1 1. Materials and Methods 1.1 Test Materials Nutrient agar, sorghum grain culture medium: 5 g sorghum grains, 20 mL distilled water, natural pH; PDA medium: 200 g potato, 20 g glucose, 20 g agar, 1000 mL distilled water, natural pH.

[0025] CAS medium: CAS 0.060g, FeCl3·6H2O 0.1g, CTAB 0.020g, sucrose 10g, yeast extract 1.0g, K2HPO4 0.5g, MgSO4·7H2O 0.2g, agar 15g, distilled water 1000mL, pH 7.0-7.2.

[0026] Congo red cellulose medium: CMCC-Na 2.0 g, (NH4)2SO4 2.0 g, K2HPO4 1.0 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, agar 20.0 g, 5 mL of 0.1 g / L Congo red staining solution dissolved in 1000 mL of distilled water, and the pH was adjusted to 7.2.

[0027] Chitin medium: 20 g chitin, 1.0 g (NH4)2SO4, 0.5 g KH2PO4, 0.5 g K2HPO4, 1.0 g yeast extract, 0.3 g MgSO4·7H2O, 0.01 g FeSO4, and 20.0 g agar, dissolved in 1000 mL distilled water, pH 7.0.

[0028] Protease screening medium: 15 g skim milk powder and 20 g agar dissolved in 1000 mL distilled water.

[0029] Chitinase medium: 20 g chitin, 1.0 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g KH2PO4, 1.0 g yeast extract, 0.3 g MgSO4·7H2O, 0.01 g FeSO4, and 20.0 g agar, dissolved in 1000 mL distilled water, pH 7.0.

[0030] Soil source: Tomato planting area of ​​the Agricultural College of Bayi Agricultural Reclamation University, Heilongjiang Province.

[0031] Early blight of tomato (Alternaria alternifolia, Alternaria alternata It is separated from the tomato fruit.

[0032] Cherry tomatoes were purchased from Huachen Supermarket in Daqing City, Heilongjiang Province. Fruits of similar size, consistent color, undamaged skin, and free from pests and diseases were selected. They were disinfected by soaking in 75% ethanol solution for 2 minutes, washed with sterile water, and dried for later use.

[0033] Preparation of spores of *Early Blight*, the pathogen of *Early Blight*, was activated on PDA medium and then prepared into 9 mm diameter fungal discs. These discs were placed in sorghum grain medium, with 3-5 discs per bottle. After incubation at 27°C for 5 days, the fungal concentrators were washed with sterile water and filtered through four layers of gauze to remove mycelia, thus preparing a spore suspension. The concentration was adjusted to 10%. 5 CFU / mL is prepared for later use.

[0034] 1.2 Test Methods 1.2.1 Isolation and Identification of Biocontrol Bacteria Healthy tomato root soil samples were collected from the tomato growing area of ​​Daqing, Heilongjiang Province. After natural air drying, 10 g of the tomato root soil sample was weighed and placed in a conical flask containing 100 mL of sterile water and a small amount of glass beads. The flask was then thoroughly shaken, and a 10% concentration was prepared using a gradient dilution method. 4 10 5 10 6 Diluted solutions were prepared, with 100 μL of each concentration evenly spread onto NA nutrient agar plates and incubated at 27℃ for 48 h. Single colonies with different morphology, color, gloss, and size were then picked, purified, and preserved. Using tomato early blight as an indicator bacterium, a cross-cross test was conducted, with each group replicated three times. The differences in antibacterial effects among different strains were calculated, and the strain with the strongest antibacterial effect was retained for preservation.

[0035] Physiological and biochemical identification was performed using the Common Bacterial Systematics Manual and Berger's Manual. For molecular identification, total DNA of the strain was prepared using a bacterial genomic DNA extraction kit. Universal primers 27F / 1492R were selected for amplification of the 16S rRNA gene. The PCR reaction system (25 µL) consisted of: 12.5 µL of 2× Taq PCR MasterMix; 1.0 µL each of forward and reverse primers (10 µmol / L); 1.0 µL of template DNA (approximately 50 ng); and ddH2O to a final volume of 25 µL. The 16S rRNA amplification program was: 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 1.5 min; and a final extension at 72 ℃ for 10 min. The amplified products were purified and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for Sanger bidirectional sequencing. The obtained sequences were compared with the type strains using the 16S-based-ID in the EzBioCloud database (EzBioCloud.net | Search about Bacteria or Archaea). High homology reference sequences were selected, and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with a bootstrap value set to 1000 using MEGA11.0 software (v10.2.6). In addition, ANI (Average Nucleotide Identity) and DDH (DNA-DNA hybridization) analyses were performed based on the whole genome sequencing results using the NCBI database. The taxonomic position was clarified by combining morphological, physiological and biochemical characteristics and molecular biological data.

[0036] 1.2.2 Determination of the antibacterial spectrum of biocontrol bacteria The plate confrontation method was used to test strawberry gray mold with a diameter of 6 mm. Botrytis cinerea Early blight of tomatoes ( Alternaria solani Fusarium graminearum ( ), Fusarium gramineae ), corn leaf blight pathogen ( Exerohyl Turkish ), corn leaf blight fungus ( Turkish sedge ), Sclerotinia sclerotiorum ( Sclerotinia sclerotia Fusarium solani () Haematonectria haematococci ), Fusarium graminearum ( Fusarium pseudograsses Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia nightshade Place the bacterial culture disc on a PDA plate and spot-inoculate 5 μL (C=10) at a distance of 2.5 cm from the center of the pathogenic bacterial disc. 8 A biocontrol bacterium (cfu / mL) was used, with an uninoculated control group. After incubation at 27℃ for 5 days, the inhibitory activity of the biocontrol bacterium against other pathogens was determined. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (colon radius of control group - colony radius of treatment group) / colony radius of control group.

[0037] 1.2.3 Determination of the antagonistic ability of Bacillus belyss S2 against early blight pathogen of tomato Place a tomato early blight pathogen inoculation dish (Φ=6 mm) in the center of a PDA medium, and spot-inoculate 10 μL of Bacillus belye bacterial suspension (C=10) at four points 2.5 cm from the edge of the pathogen. 8 (cfu / mL), repeated three times, incubated at 27℃ for 5-7 days. Culture was stopped when the mycelium of the pathogen in the control group almost completely covered the surface. The colony radius of the control group and the *Bacillus belyssus* S2 treatment group was recorded, and the inhibition rate was calculated using the following formula: Antibacterial rate = (colon radius of control group - colony radius of treatment group) / colony radius of control group × 100%.

[0038] Hyphae from the edges of different treatments were examined under an optical microscope to observe their morphology, integrity, and any abnormal features (such as deformity, breakage, swelling, etc.).

[0039] 1.2.4 Effects of Bacillus belyss S2 on spore germination of early blight pathogen of tomato 100 mL of NB culture medium was prepared in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 20 min. S2 was inoculated into the culture medium at a 1% inoculum rate and incubated at 150 r / min for 48 h. The bacterial suspension was diluted 10, 20, 30, 40, and 50 times, respectively, with sterile water as the control group. 20 μL of the diluted solution and 20 μL of spores of the tomato early blight pathogen (C=10) were taken from each flask. 5 The mixture (cfu / mL) was prepared at a 1:1 volume ratio and incubated in a concave glass slide at a constant temperature and humidity for 12 h. After 12 h, 200 spores were randomly collected, and the number of germinations was recorded. The effect of S2 on the spore germination of *Pseudomonas aeruginosa* was calculated. The inhibition rate was calculated using the following formula: Inhibition rate = (Total number of spores - Total number of germinations) / Total number of spores × 100%.

[0040] 1.2.5 Effects of VOCs from Bacillus belyssus S2 on the growth of early blight pathogen in tomato Single colonies of the biocontrol bacteria were picked and incubated in NB medium at 27°C and 120 r / min for 24 h. The bacterial suspension was then diluted to 10⁻⁶. 6 After achieving cfu / mL concentration, 100 μL of the bacterial suspension was spread onto NA medium. After incubation at 27℃ for 24 h, the culture was inverted onto PDA medium newly inoculated with *Tomato Early Blight*. The control group consisted of uninoculated blank NA medium. After incubation at 27℃, once the *Tomato Early Blight* bacteria from the control group had completely covered the plates, the growth status of the *Tomato Early Blight* bacteria in the treatment groups was observed, the colony diameter was measured, and the antibacterial effect of volatile substances was detected.

[0041] 1.2.6 Determination of the ability of Bacillus belyssus S2 to secrete siderophores and produce hydrolytic enzymes Prepare CAS, protease, lipase, chitinase, cellulase and 1,2-β-glucanase media, and inoculate 10 μL (c=10) in the center of each solid medium. 8 Incubate Bacillus berberis S2 culture (cfu / mL) for 3-7 days and observe the strain's ability to produce related enzymes.

[0042] 1.2.7 Tests on the control of early blight of tomato by Bacillus belye S2 (1) The effect of S2 on the control of early blight of tomato fruit After activating S2, inoculate it into NB medium at a rate of 1%, and incubate at 27°C and 120 rpm for 24 hours. Then, dilute the bacterial culture to a concentration of 10. 6 -10 9 CFU / mL (A6-A9) is prepared for use. Select cherry tomatoes of uniform size and ripeness with no skin damage. Rinse them thoroughly with clean water, then soak them in 75% ethanol for 3 minutes. Rinse with sterile water and air dry.

[0043] A 3mm diameter and 3mm deep wound was created on the tomato skin at the equator using a disposable syringe. 10μL of different concentrations of diluted solutions were added, while the control group received an equal volume of sterile water. After air drying, 10μL of each solution was added. 5 A suspension of *E. erythropoietin* spores (cfu / mL) was administered, with 20 spores per dose, and each group was repeated three times. The mixture was kept at 27℃ and humidified for 5 days. The incidence rate and lesion size at the initial stage of disease in tomatoes treated with different concentrations were recorded, as well as the incidence rate and lesion diameter after 5 days.

[0044] (2) Effect of S2 on the activity of tomato-related resistance enzymes Following the method of Cao Jiankang et al., the pulp of the treated tomatoes was sampled to detect the activity of disease-resistant defense enzymes and the content of malondialdehyde (MDA), and to investigate the effect of S2 on the stress resistance of tomatoes. The content of MDA, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were determined by the thiobarbituric acid method, the nitroblue tetrazolium method, the guaiacol method, and ultraviolet spectrophotometry, respectively.

[0045] 1.2.8 Analysis of the S2 gene of Bacillus belyssus After activating single colonies in NB medium, inoculate 1% of the culture into fresh NB medium and incubate at 27°C and 120 r / min for 24 h. Collect the bacterial culture and centrifuge at 5000 r / min for 10 min. Discard the supernatant and wash the precipitate 2-3 times with pre-cooled PBS buffer, centrifuging at 5000 r / min for 5 min after each wash. Remove the supernatant completely and collect the bacterial cells in 1.5 mL centrifuge tubes. Quick-freeze in liquid nitrogen for 15 min and send the samples to Novogene Beijing for whole genome sequencing and metabolomics analysis.

[0046] Whole-genome sequencing was performed using the Illumina Novaseq platform. After removing low-quality base sequences, the sequences were assembled using SPAdes, Abyss, and CISA software. Gene prediction was performed using GeneMarkS (Version 4.17), and the genome was visualized using Proksee gene loop mapping. Secondary metabolite synthesis gene clusters of strain S2 were predicted using antiSMASH (Antibiotics and Secondary Metabolite Analysis Shell, https: / / antismash.secondarymetabolites.org / ). Gene ontology was analyzed using GO (Gene Ontology, http: / / geneontology.org / ), KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg), COG (Cluster of Orthologous Groups of proteins, http: / / www.ncbi.nlm.nih.gov / COG / ), NR (Non-Redundant Protein Database), and CAZy (Carbohydrate-Active Enzymes). Functional gene annotation is performed using databases such as Databases.

[0047] 1.2.9 Data Analysis Analysis of variance was performed using SPSS, and plotting was done using Origin.

[0048] 2. Results and Analysis 2.1 Screening and Identification of Biocontrol Bacteria Eleven strains exhibiting antifungal activity against early blight pathogens of tomato were isolated from the rhizosphere soil of healthy tomatoes. Among them, strain S2 showed significant antifungal activity, with an inhibition rate of up to 72.41% against the mycelial growth of the pathogen measured using the plate confrontation method (see...). Figure 1 (C, D, and E).

[0049] On NA solid medium, S2 colonies are white, round, with a smooth, moist surface, a viscous texture, and wrinkles and bulges (see...). Figure 1 Microscopic observation combined with Gram staining results indicated that this strain was Gram-positive (G). + ) bacillus, producing spores (see Figure 1(A). Physiological and biochemical identification results showed (see Table 1) that S2 has a wide range of carbon source metabolism capabilities, and can utilize glucose, mannitol, and starch. It was positive for gelatin liquefaction and VP tests, negative for MR tests, and could hydrolyze casein.

[0050] Table 1. Physiological and Biochemical Test Results To further clarify their taxonomic position, the 16S rRNA sequences of the screened strains were determined and analyzed. A phylogenetic tree constructed based on the 16S rRNA gene sequences is shown below. Figure 2 The results showed that S2 was associated with Bacillus belye. Bacillus velezensis The closest kinship is preliminarily determined to be... Bacillus velezensis According to ANI and GGDC and multiple strains Bacillus from Velez Far exceeding the species definition thresholds (ANI ≥ 95%, dDDH ≥ 70%), and beyond that... Bacillus velezensis ANI value is higher than Bacillus amyloliquefaciens The strain and GC offset were relatively low (see Table 2), thus identifying S2 as Bacillus belesii.

[0051] The Bacillus berlesi ( Bacillus velezensis S2 was deposited on March 30, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026551.

[0052] Table 2 Comparison results of strain S2 and the model strain 2.2 Effects of S2 on the growth of early blight mycelium in tomato S2 inhibited the mycelial growth of *Alternaria alternata*, the causal agent of early blight of tomato. Compared with the control group, after co-culturing S2 with *Alternaria alternata*, the pathogenic mycelia exhibited severe swelling and enlargement, with severely affected areas displaying a beaded appearance and severe mycelial deformities (see...). Figure 3 ).

[0053] 2.3 Effects of S2 on the germination of early blight spores in tomato Experiments showed that S2 bacterial solution caused changes in the morphology and structure of pathogenic fungal spores, with obvious swelling and deformity of the spore heads, significantly inhibiting the germination of tomato early blight fungal spores. With increasing bacterial solution concentration, the germination rate of pathogenic fungal spores decreased significantly; when the bacterial solution was diluted 10 times, the germination rate of pathogenic fungal spores was only 4.0% (see...). Figure 4 ).

[0054] 2.4 Antibacterial effect of S2 volatile substances Compared to the untreated control group, the growth of early blight pathogens in tomatoes almost stopped after treatment with VOCs generated by S2, the diameter of the pathogen colonies was significantly smaller than that in the control group, and the edges were solidified (see...). Figure 5 The results indicate that the VOCs produced by this strain have a significant inhibitory effect on the growth of its mycelium. The measured inhibition rate of S2 volatile substances on the growth of pathogens was 74.65% ± 2.97%.

[0055] 2.5 Extracellular enzyme assay of strain S2 Plate culture results (see) Figure 6 The study found that strain S2 exhibited distinct clear zones on CAS medium, sodium carboxymethyl cellulose medium, skim milk powder medium, 1,2-β-glucanase medium, and lipase medium, but no clear zone on chitin medium. This indicates that S2 possesses the ability to produce siderophores, cellulases, proteases, 1,2-β-glucanases, and lipases, but not chitinases. Furthermore, the relative siderophore activity of S2, determined by the CAS method (chrome azurite S colorimetric method), was 70.26%, classifying it as a strong siderophore-producing strain.

[0056] 2.6 Prevention and control of early blight in tomatoes 2.6.1 The impact of S2 on early blight disease in tomatoes like Figure 7 and Figure 8 As shown, with prolonged storage time, the incidence and lesion diameter of early blight in tomatoes of all treatment groups increased. The disease rate and lesion diameter growth rate of the control group were both higher than those of the treatment groups. The S2 treatment group showed significant control of early blight in tomatoes during the early transfection stage (1-3 days), with a significantly lower incidence rate than the control group. However, with prolonged storage time (4-6 days) and differences in biocontrol bacteria concentrations among the treatment groups, the incidence rate of tomatoes in the treatment groups increased. Low concentration treatment (C=10) 6 Although the incidence rate of tomato treated with high concentrations (cfu / mL) was similar to that of the control group, the diameter of lesions was still significantly smaller than that of the control group (P<0.05). Furthermore, by day 5 of co-cultivation, the incidence rate of tomatoes treated with high concentrations was significantly lower than that of the control group, indicating that S2 has a certain control ability against postharvest early blight in tomatoes, and the strength of the control effect is dependent on the concentration of the biocontrol bacteria.

[0057] 2.6.2 Effects of S2 on the antioxidant capacity of tomato fruit cells Malondialdehyde (MDA) content is a core indicator reflecting the degree of oxidative damage to fruit cell membranes; its level directly corresponds to the degree of cell damage caused by ROS attack. For example... Figure 9 As shown, the MDA content in tomato cells of the control group (CK) was 3.16 × 10⁻⁶. -5The S2 strain had a concentration of μmol / g, significantly higher than the MDA content in tomatoes in all treatment groups (P<0.05), indicating a higher degree of oxidative damage in the fruit. The MDA content in tomatoes treated with S2 decreased by 12.18%-45.83% compared to the control group. These results indicate that as early blight pathogens invade tomato fruit cells, the degree of oxidative damage increases and the fruit's resistance decreases. The addition of strain S2 significantly alleviated the degree of oxidative damage in tomato fruits, helping the fruit defend against the continuous invasion of pathogens and thus slowing down the disease progression. Furthermore, the study found that S2 significantly enhanced the activity of antioxidant enzymes in tomato fruits, helping fruit cells process intracellular ROS, reducing oxidative damage to fruit cells, and enhancing their resistance. The activities of CAT and POD enzymes increased by 400%-700% and 36.0%-63.0% respectively compared to the control group, while the activity of SOD enzyme decreased slightly compared to the control group, but remained relatively stable among the treatment groups.

[0058] 2.7 Inhibitory effect of biocontrol bacteria on other pathogens As shown in Table 3, the experimental results indicate that S2 is effective against strawberry gray mold (… Botrytis cinerea Pers. Early blight of tomatoes ( Alternaria solani Fusarium graminearum ( ), Fusarium gramineae ), corn leaf blight pathogen ( Turkish sedge ), corn leaf blight fungus ( Turkish sedge ), Sclerotinia sclerotiorum ( Sclerotinia sclerotia Fusarium solani () Haematonectria haematococci ), Fusarium graminearum ( Fusarium pseudograsses Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani-tobacco-specific type ( Rhizoctonia solani Rhizoctonia solani-soybean-specific strain ( Rhizoctonia solani All of them have significant inhibitory effects, with an inhibition rate of over 80% against Sclerotinia sclerotiorum.

[0059] Table 3 S2 Antibacterial Spectrum Note: Different letters after the data in the same column indicate significant differences (P<0.05), and the same letter indicates no significant differences (P>0.05).

[0060] 2.8 S2 whole genome analysis like Figure 10As shown, the S2 genome consists of one chromosome with a total length of 3,906,291 bp and an average GC content of 46.45%. Gene prediction results show that the S2 genome encodes a total of 4,022 genes with a total length of 3,506,610 bp, accounting for 89.77% of the total genome length; among them, there are 82 tRNAs, 9 5S rRNAs, 1 16S rRNA, 5 23S rRNAs, 14 sRNAs, and 1 tmRNA.

[0061] like Figure 11 As shown, the CAZy database (carbohydrate-active enzymes database) annotates S2 as containing 76 glycoside hydrolases (GHs), 60 glycosyltransferases (GTs), 3 polysaccharide lyases (PLs), 14 carbohydrate esterases (CEs), 3 auxiliary redox enzymes (AAs), and 43 non-catalytic carbohydrate-binding modules (CBMs). Further analysis revealed that S2 contains several key antagonistic enzymes, including β-glucosidase, maltose-6-phosphate glucosidase, endo-β-1,4-glucanase, GH43 (β-xylosidase), GH53 (endo-β-1,4-galactosidase), CE1 (carboxylesterase), CE3 (pectin esterase), and AA3 (glucose oxidase). These enzymes catalyze the hydrolysis of polysaccharides, components of the pathogen's cell wall, inhibiting the pathogen's respiratory chain and disrupting its secretory toxin structure. This indicates that S2 can degrade the pathogen's cell wall, interfere with its energy metabolism, and thus inhibit its growth.

[0062] like Figure 12 As shown, the NR (Non-Redundant Protein Database) annotates 3936 S2 genes, of which... Bacillus There are 3042 homologous genes, accounting for the largest proportion, at 77.29%; B. velezensis There are a total of 672 homologous genes, accounting for 17.07%; B.amyloliuquefaciens This accounts for 5.64% of the annotation results. S2 strain and... Bacillus Genus, especially B. velezensis They are highly homologous.

[0063] like Figure 13As shown, the amino acid sequence of strain S2 was compared with the GO database. 63.51% of the genes in the genome were annotated to GO functions, totaling 2553. Based on gene function, they can be classified into: molecular function, biological process, and cellular component, with 13, 19, and 3 functional branches respectively. 3845 genes were annotated to molecular functions, with the most genes involved in binding, catalytic activity, and transport activity (1169, 1412, and 325 respectively). 6887 genes were annotated to biological processes, with the most genes involved in cellular processes and metabolic processes (1422 and 1356 respectively). 2621 genes were annotated to cellular components, with the most genes involved in cellular anatomical entities (792), corresponding to core structures such as cell parts, cells, and membranes.

[0064] like Figure 14 As shown, COG data annotated 2875 genes in S2, divided into 24 categories, accounting for 71.52% of the total coding genes. Among these, amino acid transport and metabolism (E, 303 genes), transcription (K, 290 genes), and carbohydrate transport and metabolism (G, 255 genes) accounted for the highest proportions, at 10.54%, 10.09%, and 8.87% of the annotated genes, respectively. Translation, ribosome structure and biogenesis (J, 224 genes), and signal transduction mechanisms (T, 206 genes) followed, accounting for 7.79% and 7.16%, respectively. This indicates that the strain possesses a highly efficient material and energy metabolism base and exhibits excellent characteristics such as strong environmental adaptability and stress resistance. In addition, cell wall / membrane / enveloping biogenesis (M, 190 genes) and inorganic ion transport and metabolism (P, 175 genes) are also relatively abundant, accounting for 6.61% and 6.09% of the annotated genes, respectively. It is speculated that S2 can inhibit the growth of pathogens through a combination of multiple mechanisms, including competing for nutrients, occupying space sites, and secreting antibacterial substances.

[0065] like Figure 15As shown, 1951 genes of strain S2 were annotated in the KEGG database and mapped to 40 metabolic-related biological pathways. Significant enrichment analysis of metabolic pathways revealed that the core functional modules are mainly concentrated in two key areas: global metabolic network topology, carbohydrate conversion pathways (249, 12.76%), and amino acid synthesis and catabolism (207, 10.61%). These data reveal the significant characteristics of this strain at the level of material and energy metabolism, with its metabolic network architecture exhibiting a functional distribution pattern dominated by basic substance cycling. Furthermore, pathways such as cofactor and vitamin metabolism (167, 8.56%), signal transduction (147, 7.53%), transmembrane transport systems (153, 7.84%), energy metabolism (113, 5.79%), and prokaryotic cell community regulation (52, 2.67%) showed significant associations with the genomic functional characteristics of *Bacillus belyssae* strain S2. The high enrichment of genes related to cofactor and vitamin metabolism pathways and transmembrane transport systems suggests that this strain may possess efficient metabolite uptake and energy conversion mechanisms in complex environmental adaptations. The active annotation of signal transduction-related genes indicates a dynamic response to environmental signals. Furthermore, the annotation results of exogenous biodegradation and metabolic pathways (37, 1.90%) further suggest that this strain has potential functions in environmental pollutant degradation and stress adaptation.

[0066] The biosynthetic gene clusters (BGCs) of strain S2 were predicted using antiSMASH. The results are shown in Table 4. S2 contains 15 biosynthetic gene clusters, 8 of which are known. Among these, 7 are highly similar: macrolide H, bacillaene, fengycin, bacillibactin, bacilysin, difficidin, and surfactant. These metabolites encompass a variety of antibacterial active substances and can synergistically inhibit pathogen growth through different mechanisms, indicating that strain S2 possesses broad-spectrum antibacterial activity. Surfactin and bacillibactin are important secondary metabolites regulating microbial rhizosphere colonization, enhancing environmental adaptability, and promoting plant growth. This suggests that in addition to biocontrol, this strain also has potential applications in promoting plant growth and inducing disease resistance. In addition, six secondary metabolite synthesis gene clusters in the strain have not been functionally annotated, suggesting that the S2 strain may have the potential to synthesize new natural products.

[0067] Table 4. Prediction results of secondary metabolite synthesis gene clusters in strain S2 The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis S2, characterized in that, It is deposited at the China Center for Type Culture Collection on March 30, 2026, with accession number CCTCC NO: M 2026551.

2. The application of Bacillus belyssus S2 as described in claim 1 in the preparation of microbial inoculants.

3. A microbial inoculant, characterized in that, The microbial agent contains Bacillus belyssus S2 as described in claim 1, its metabolites and / or its volatile substances.

4. The application of Bacillus berberis S2 as described in claim 1 or the microbial agent as described in claim 3 in inhibiting the growth of plant pathogens, characterized in that, The plant pathogens include Alternaria alternifolia (… Alternaria alternata ), strawberry gray mold ( Botrytis cinerea Pers Alternaria alternifolia ( ), Alternaria alternifolia Alternaria solani Fusarium graminearum ( ), Fusarium graminearum ), corn leaf blight pathogen ( Exserohilum turcicum ), corn leaf blight fungus ( Exserohilum turcicum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Haematonectria haematococca ), Fusarium graminearum ( Fusarium pseudograminearum Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani-tobacco-specific type ( Rhizoctonia solani ) and Rhizoctonia solani-soybean-specific ( Rhizoctonia solani ).

5. A product that inhibits the growth of plant pathogens, characterized in that, The product contains either the Bacillus berberis S2 of claim 1 or the microbial agent of claim 3.

6. The product according to claim 5, characterized in that, The plant pathogens include Alternaria alternifolia (… Alternaria alternata ), strawberry gray mold ( Botrytis cinerea Pers Alternaria alternifolia ( ), Alternaria alternifolia Alternaria solani Fusarium graminearum ( ), Fusarium graminearum ), corn leaf blight pathogen ( Exserohilum turcicum ), corn leaf blight fungus ( Exserohilum turcicum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Fusarium solani () Haematonectria haematococca ), Fusarium graminearum ( Fusarium pseudograminearum Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani-tobacco-specific type ( Rhizoctonia solani ) and Rhizoctonia solani-soybean-specific ( Rhizoctonia solani ).

7. A method for preventing and controlling early blight in tomatoes, characterized in that, The method includes the step of applying the Bacillus berberis S2 of claim 1, the microbial agent of claim 3, or the product of claim 5 or 6 to tomato plants.

8. A method for preventing and controlling postharvest early blight in tomatoes, characterized in that, The method includes the step of applying the Bacillus berberis S2 of claim 1, the microbial agent of claim 3, or the product of claim 5 or 6 to postharvest tomato fruit.

9. The application of Bacillus belyssus S2 according to claim 1 or the microbial agent according to claim 3 in the production of enzyme preparations, characterized in that, The enzyme preparations include cellulase preparations, protease preparations, 1,2-β-glucanase preparations, and lipase preparations.

10. The use of Bacillus belyssus S2 of claim 1 or the microbial agent of claim 3 in the production of siderophores.

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