Bacillus belye AMC102 with antibacterial and plant protection functions, and its applications and products

Abstract

This invention relates to Bacillus bellis AMC102 with antibacterial and plant protection functions, as well as its applications and products, belonging to the field of microbial technology. This invention provides a Bacillus bellis AMC102 strain, with the preservation number CGMCC NO.29826. Based on this strain AMC102, this invention also provides its applications in the preparation of antibacterial agents, agricultural inoculants, plant growth promoters or biopesticides, and / or for non-disease-treatment purposes of antibacterial activity, and / or for promoting plant growth, and / or for controlling plant diseases, as well as its antibacterial agents, agricultural inoculants, plant growth promoters or biopesticides, and other products. The Bacillus bellis AMC102 provided by this invention can efficiently produce enzymes, effectively promote seed germination, control plant diseases, and has broad-spectrum antibacterial properties.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to Bacillus belye AMC102 with antibacterial and plant protection functions, as well as its applications and products. Background Technology

[0002] One of the major factors leading to yield reduction in agricultural crop production is plant disease. According to statistics from the Food and Agriculture Organization of the United Nations, plant diseases account for 14% of global crop production losses annually, including 42% of fungal diseases and 27% of bacterial diseases. Furthermore, most farmers lack sufficient understanding of disease control methods and rely primarily on the widespread application of chemical pesticides. However, the irrational overuse of pesticides not only affects the quality of fruits and vegetables but also leads to pesticide resistance in plant pathogens, ultimately resulting in reduced effectiveness of chemical control, serious environmental pollution, and threats to human health. Therefore, agricultural production needs alternative methods to chemical control of plant diseases. Biological control, which uses organisms to inhibit or eliminate harmful organisms, has gradually gained attention. Studies have shown that compared to chemical control, biological control is both effective in controlling diseases and environmentally friendly. Developing effective microorganisms with pathogen-killing properties is an important health and disease prevention measure for future agricultural development.

[0003] Bacillus is one of the most studied rhizosphere growth-promoting bacteria in plants, and has been proven to promote plant growth, improve plant resistance, and inhibit the growth of plant pathogens. Currently, the generally accepted mechanisms of action for Bacillus's antibacterial activity mainly include competitive action, production of antimicrobial substances, and induction of resistance. Furthermore, biocontrol bacteria typically exert their antibacterial capabilities through a combination of these mechanisms. Many Bacillus metabolites can cause pathogenic mycelial malformation, cell rupture, protoplasmic decomposition, and leakage, leading to loss of activity.

[0004] Bacillus velezensis, a biocontrol bacterium in the genus Bacillus, was first discovered in 1998. It possesses broad-spectrum antibacterial activity and is widely found in soil, food, the gut, and marine environments, exhibiting characteristics of being harmless to humans and animals and environmentally friendly. Bacillus velezensis can secrete various antibacterial substances, such as cell wall-degrading enzymes and lipopeptides, which directly kill plant pathogens by disrupting their cell walls and membranes, causing cytoplasmic leakage, or by causing abnormal development of spores and mycelia. Bacillus velezensis grows rapidly and preferentially occupies growth space and nutrients, competitively inhibiting the growth and development of pathogens through population dominance. Furthermore, Bacillus velezensis can inhibit the growth, development, infection, and colonization of pathogens by altering the plant microenvironment and secreting abundant secondary metabolites, such as surfactants, iturobacillusin, streptomycin, and bacteriocins, thus attracting significant attention in agricultural applications.

[0005] Rhizoctonia solani and Sclerotinia sclerotiorum, as soil-borne diseases, are widely distributed geographically and have a broad host range, causing various important crop diseases and leading to significant agricultural and economic losses. Symptoms of Rhizoctonia solani infection vary depending on the host plant, including seedling drop, stem canker, root rot, or stem rot. Sclerotinia sclerotiorum can infect from the seedling stage to the mature plant stage, mainly damaging leaves, stems, and fruits, causing stem soft rot and fruit rot. Cucumber damping-off and cucumber sclerotinia rot are caused by Rhizoctonia solani and Sclerotinia sclerotiorum, respectively, and are common soil-borne fungal diseases in cucumber production, leading to large-scale seedling death or yield reduction. However, there are few reports on the control of cucumber sclerotinia rot using Bacillus belyceae and its metabolites. Currently, there are many methods for disease control, including breeding resistant varieties, applying chemical pesticides, and biological control. The most common method is chemical control using pesticides, but chemical control causes serious problems such as environmental pollution and pesticide residues, and threatens human health. In contrast, biological control is safe and friendly, and has great potential to replace chemical control methods.

[0006] As an agricultural bacterium, Bacillus belye has been reported to have an antibacterial spectrum that is mostly concentrated on agricultural pathogens. Its inhibition spectrum against pathogens in other fields is quite limited. Furthermore, the existing agricultural performance of Bacillus belye is not comprehensive and still has room for optimization.

[0007] Therefore, there is a need in this field to develop more new strains of Bacillus belyss that possess both broad-spectrum antibacterial properties and excellent agricultural properties such as enzyme production, phosphorus solubilization, control of plant diseases, heat and salt tolerance, and promotion of seed germination. Summary of the Invention

[0008] To address the technical problems of limited inhibitory spectrum, incomplete agricultural performance, and the need for further optimization of Bacillus velezensis in the prior art, this invention provides a Bacillus velezensis strain AMC102 with broad-spectrum antibacterial activity, as well as its applications and products.

[0009] The technical solution of the present invention is as follows:

[0010] A strain of Bacillus velezensis, AMC102, with accession number CGMCCNO.29826.

[0011] Application of Bacillus velezensis strain AMC102 with accession number CGMCC NO.29826 in the preparation of antibacterial agents, agricultural inoculants, plant growth promoters or biopesticides, and / or for non-disease treatment purposes of antibacterial activity, and / or for promoting plant growth, and / or for the prevention and control of plant diseases.

[0012] Disease refers to a disease in humans or animals;

[0013] Preferably, the non-disease treatment-targeted antibacterial activity includes inhibiting pathogenic bacteria or inhibiting pathogenic bacteria through enzyme production;

[0014] Preferably, the pathogens include Rhizoctonia solani, Rice blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium wilt, Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile;

[0015] Preferably, the enzyme production refers to the production of protease, amylase, cellulase, chitinase, or manganese peroxidase.

[0016] Preferably, promoting plant growth refers to promoting cucumber seed germination;

[0017] Preferably, the plant diseases include: cucumber damping-off and cucumber sclerotinia stem rot.

[0018] An antibacterial agent comprising an antibacterial active ingredient, the antibacterial active ingredient comprising: a strain of Bacillus velezensis AMC102 with accession number CGMCC NO.29826.

[0019] The antibacterial agent further includes: excipients; preferably, the antibacterial spectrum of the antibacterial agent includes: Rhizoctonia solani, Rice blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium wilt of cabbage, Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile.

[0020] An agricultural microbial agent comprising: a strain of Bacillus velezensis, AMC102, with preservation number CGMCC NO.29826.

[0021] A plant growth promoter comprising an active ingredient; the active ingredient comprises: a strain of Bacillus velezensis, AMC102, with accession number CGMCC NO.29826.

[0022] The plant growth promoter also includes: excipients.

[0023] A biological pesticide, comprising a phytoactive ingredient; the phytoactive ingredient comprises: a strain of Bacillus velezensis, AMC102, with accession number CGMCCNO.29826.

[0024] The aforementioned biological pesticide also includes pharmaceutically acceptable excipients.

[0025] The present invention has the following outstanding beneficial effects:

[0026] The *Bacillus belyss* strain provided by this invention has a high viable cell count and spore count, can produce highly effective antibacterial substances, exhibits broad-spectrum antibacterial activity, and can tolerate high-salt, high-alkali, and high-temperature environments. Furthermore, this strain can produce manganese peroxidase with lignin-degrading capabilities, showing great application potential in agricultural disease control and the degradation of waste crop straw. Experiments have confirmed that the *Bacillus belyss* strain provided by this invention can inhibit pathogenic fungi and bacteria such as *Rhizoctonia solani*, *Bacillus oryzae*, *Sclerotinia sclerotiorum*, *Fusarium oxysporum*, *Fusarium wilt*, *Serratia marcescens*, *Streptococcus pseudopneumococcus*, *Streptococcus mutans*, *Bacteroides fragilis*, and *Clostridium difficile*, exhibiting broad-spectrum antibacterial activity. Simultaneously, this strain produces high levels of protease, amylase, cellulase, chitinase, and manganese peroxidase, and possesses excellent agricultural properties such as promoting seed germination, heat and salt tolerance, and highly effective control of cucumber damping-off and cucumber sclerotinia stem rot. Therefore, it has been named AMC102 and deposited for preservation, with the following preservation information:

[0027] Accession number: CGMCC NO.29826;

[0028] Classification and nomenclature: Bacillus velezensis;

[0029] Deposit date: January 31, 2024;

[0030] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0031] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0032] Figure 1 The growth curves of 10 strains of Bacillus belysium that underwent secondary screening are shown.

[0033] Figure 2 The inhibitory effects of Bacillus belyss AMC102 on five pathogenic fungi and five pathogenic bacteria are shown in the figure. The labels are listed below: (a) Rhizoctonia solani, (b) Rice blast fungus, (c) Sclerotinia sclerotiorum, (d) Fusarium oxysporum, (e) Fusarium wilt of cabbage, (f) Serratia marcescens, (g) Streptococcus pseudopneumococcus, (h) Streptococcus mutans, (i) Bacteroides fragilis, (j) Clostridium difficile. CK is the blank control group, and AMC102 is the Bacillus belyss AMC102 treatment group.

[0034] Figure 3 Figures show the colony morphology, cell morphology, spore morphology, and biofilm of Bacillus belysae AMC102. The labels in the figures are listed below: (a) colony morphology, (b) cell morphology, (c) spore morphology, and (d) biofilm.

[0035] Figure 4 Qualitative results of plate analysis of protease, amylase, cellulase, chitinase and manganese peroxidase produced by Bacillus belyssus AMC102; the labels in the figure are listed below: (a) protease, (b) amylase, (c) cellulase, (d) chitinase, (e) manganese peroxidase.

[0036] Figure 5 Qualitative plate plot of the inorganic phosphorus solubility of Bacillus belyssus AMC102.

[0037] Figure 6 The results of the inhibition of Rhizoctonia solani, Sclerotinia sclerotiorum and Bacillus oryzae by cell-free filtrate of Bacillus belyi AMC102 are shown in the figure. The labels in the figure are listed as follows: (a) Rhizoctonia solani, (b) Sclerotinia sclerotiorum, (c) Bacillus oryzae. CK is the blank control group, and AMC102 is the AMC102 treatment group.

[0038] Figure 7 The results of Bacillus belyssus AMC102 producing volatile substances that inhibit Rhizoctonia solani, Sclerotinia sclerotiorum and rice blast fungus are shown in the figure. The labels in the figure are as follows: (a) Rhizoctonia solani, (b) Sclerotinia sclerotiorum, (c) Rice blast fungus. CK is the blank control group, and AMC102 is the AMC102 treatment group. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments and experimental examples, but the implementation of the present invention is not limited thereto, nor is it intended to limit the scope of protection of the present invention.

[0040] I. Experimental Culture Media

[0041] PDA (Potato Dextrose Agar) medium: 200g peeled potatoes, 20g glucose, 15g agar, 1000mL distilled water, sterilized at 121℃ for 20min.

[0042] LB (Luria Broth) agar medium: 10g tryptone, 5g NaCl, 5g yeast extract, 15g agar powder, 1L distilled water, pH 7.2, autoclaved at 121℃ for 15min.

[0043] LB liquid medium: 10g tryptone, 5g NaCl, 5g yeast extract, 1L distilled water, pH 7.2, autoclaved at 121℃ for 15min.

[0044] BHI culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0045] Protease culture medium: 15g skim milk powder, 2g yeast powder, 10g NaCl, 20g agar, sterilized at 115℃ for 20min, pH 7.0.

[0046] Amylase medium: 5g yeast extract, 10g peptone, 5g NaCl, 18g agar, 10g starch, sterilized at 121℃ for 20min, pH 7.0.

[0047] Cellulase detection plate: 2-10g sodium carboxymethyl cellulose, 1.5g potassium dihydrogen phosphate, 2.5g peptone, 0.5g yeast extract, 2.5g disodium hydrogen phosphate, 1L water, 15g agar, pH 7.0, sterilized at 121℃ for 20min.

[0048] Chitinase assay plate: 10g colloidal chitin, 5g tryptone, 10g NaCl, 5g yeast extract, 15g agar powder, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 20min.

[0049] Manganese peroxidase assay plate: 10g tryptone, 5g NaCl, 5g yeast extract, 0.4g manganese chloride tetrahydrate, 15g agar powder, 1L distilled water, pH 7.2, sterilized at 121℃ for 20min.

[0050] Inorganic phosphorus qualitative culture medium: glucose 10g, ammonium sulfate 0.5g, NaCl 0.2g, KCl 0.2g, MgSO4·7H2O 0.1g, MnSO4·7H2O 0.005g, FeSO4·7H2O 0.005g, yeast powder 0.5g, Ca3(PO4) 5.0g, distilled water 1L, agar 18g, pH 7.0-7.2.

[0051] II. Sources of biological materials

[0052] (I) The pathogenic fungi Rhizoctonia solani, Sclerotinia sclerotiorum, and Fusarium oxysporum f.sp. conglutinans used in Experiment Example 2 were kindly provided by the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University and are currently stored in the applicant's laboratory. The applicant promises to release them to the public for verification of the technical effects of the present invention within 20 years from the date of application of this invention.

[0053] (ii) The pathogenic fungi Magnaphalthe oryzae and Fusarium oxysporum used in Experiment Example 2 were kindly donated by the Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, and are currently stored in the applicant's laboratory. The applicant promises to release them to the public for verification of the technical effects of the present invention within 20 years from the date of application of this invention.

[0054] (III) The *Streptococcus mutans* ATCC25175, *Bacteroides fragilis* ATCC25285, and *Clostridium difficile* ATCC43593 used in Experiment Example 2 were all purchased from the Guangdong Provincial Microbial Culture Collection Center; *Serratia marcescens* was kindly donated by the Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences; and *Streptococcus pseudopneumococcus* was isolated by our laboratory. All of the above strains are currently preserved in the applicant's laboratory, and the applicant undertakes to release them to the public for verification of the technical effects of this invention within 20 years from the date of this application.

[0055] Group 1 Examples, Strain AMC102 of the present invention

[0056] This set of examples provides a Bacillus velezensis strain AMC102, with the accession number CGMCC NO.29826.

[0057] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, altering, selling, or offering for sale a strain of Bacillus velezensis AMC102 with accession number CGMCC NO.29826, and / or the act of using a strain of Bacillus velezensis AMC102 with accession number CGMCC NO.29826 in combination with other probiotics, and / or the use of Bacillus velezensis AMC102 with accession number CGMCC NO.29826 to antagonize various pathogens, and / or the preparation of antibacterial products, and / or the preparation of drugs to improve constipation or regulate intestinal flora, and / or the preparation of health foods with laxative or intestinal flora regulating functions, falls within the scope of protection of this invention.

[0058] Other probiotics include, but are not limited to: *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus delbrueckii* subsp. lactis, *Lactobacillus helveticus*, *Lactobacillus casei*, *Lactobacillus brevis*, *Lactobacillus johnsonii*, *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus salivarius*, *Saccharomyces cerevisiae*, *C. delbrueckii*, *C. candida*, *Wickham's yeast*, *Pichia pastoris*, *Blanctomyces boulardii*, *Pichia pastoris*, *Blanctomyces leucosus*, *Saccharomyces cerevisiae*, and *Schizosaccharomyces cerevisiae*. *Bacillus boulardii*, *Bacillus thuringiensis*, *Bacillus brevis*, *Bacillus megaterium*, *Bacillus mucilaginosus*, *Bacillus azoosus*, *Bacillus spheroides*, *Clostridium butyricum*, *Bifidobacterium adolescentis*, *Bifidobacterium angularis*, *Bifidobacterium animalis*, *Bifidobacterium astrum*, *Bifidobacterium bifidum*, *Bifidobacterium bovis*, *Bifidobacterium brevis*, *Bifidobacterium densiflorum*, *Bifidobacterium infantis* (i.e., *Bifidobacterium longum* subsp. *infantii*), *Bifidobacterium lactis* (i.e., *Bifidobacterium animalis* subsp. *milk fat*), *Bifidobacterium longum*, *Bifidobacterium pseudosporidis*, as well as *Bifidobacterium thermophilum* and *Bifidobacterium acidophilus*.

[0059] Those skilled in the art can, based on actual production needs and in conjunction with conventional technical means or basic common sense in pharmaceutical manufacturing processes (e.g., the Encyclopedia of Pharmaceutical Technology, Pharmaceutical Preparation Technology, etc.), routinely select or adjust pharmaceutical excipients, thereby producing products with different dosage forms, storage conditions, and shelf lives from a strain of Bacillus velezensis AMC102 with accession number CGMCC NO.29826. This is not a technical obstacle for those skilled in the art and is feasible and easy to do.

[0060] Group 2 Examples, Application of the present invention strain AMC102

[0061] This set of embodiments provides the application of Bacillus velezensis strain AMC102 with accession number CGMCC NO.29826 in the preparation of antibacterial agents, agricultural microbial agents, plant growth promoters or biopesticides, and / or antibacterial agents for non-disease treatment purposes, and / or plant growth promotion and / or prevention and control of plant diseases.

[0062] In a specific implementation, "disease" refers to a disease in a human or animal;

[0063] Preferably, the non-disease treatment-targeted antibacterial activity includes inhibiting pathogenic bacteria or inhibiting pathogenic bacteria through enzyme production;

[0064] Preferably, the pathogens include Rhizoctonia solani, Rice blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium wilt, Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile;

[0065] Preferably, the enzyme production refers to the production of protease, amylase, cellulase, chitinase, or manganese peroxidase.

[0066] Preferably, promoting plant growth refers to promoting cucumber seed germination;

[0067] Preferably, the plant diseases include: cucumber damping-off and cucumber sclerotinia stem rot.

[0068] Group 3 Examples, the antibacterial agent of the present invention

[0069] This set of embodiments provides an antibacterial agent. All embodiments in this set share the following common feature: the antibacterial agent includes an antibacterial active ingredient, which includes: a Bacillus velezensis strain AMC102 with accession number CGMCC NO.29826.

[0070] In a further embodiment, the antibacterial agent further includes: excipients;

[0071] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0072] According to the present invention, and considering the different needs in actual production applications, combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Bacillus velezensis AMC102 with accession number CGMCC NO.29826 into different dosage forms, such as powders, tablets, suppositories, gels, patches, sprays, lotions, granules, etc.

[0073] In a specific embodiment, the dosage form of the product is selected from one or more of the following: powder, tablet, and liquid.

[0074] Preferably, the antibacterial spectrum of the antibacterial agent includes: Rhizoctonia solani, blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium wilt of cabbage, Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile.

[0075] Group 4 Examples, Agricultural Microbial Agents of the Present Invention

[0076] This set of embodiments provides an agricultural microbial agent. All embodiments in this set share the following common feature: the agricultural microbial agent comprises a strain of Bacillus velezensis, AMC102, with preservation number CGMCC NO.29826.

[0077] In a further embodiment, the agricultural microbial agent further includes: auxiliary materials.

[0078] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0079] According to the present invention, and considering the different needs in actual production applications, combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Bacillus velezensis AMC102 with accession number CGMCC NO.29826 into different dosage forms, such as powders, tablets, suppositories, gels, patches, sprays, lotions, granules, etc.

[0080] In a specific embodiment, the dosage form of the product is selected from one or more of the following: powder, tablet, and liquid.

[0081] Group 5 Examples, Plant Growth Promoter of the Present Invention

[0082] This set of embodiments provides a plant growth promoter. All embodiments in this set share the following common feature: the plant growth promoter includes an active ingredient; the active ingredient includes: a strain of Bacillus velezensis, AMC102, with accession number CGMCC NO.29826.

[0083] In a further embodiment, the plant growth promoter further includes: excipients.

[0084] Group 6 Examples, Biological Pesticides of the Present Invention

[0085] This set of embodiments provides a biological pesticide. All embodiments in this set share the following common feature: the biological pesticide includes a phytoactive ingredient; the phytoactive ingredient includes: a strain of Bacillus velezensis, AMC102, with accession number CGMCC NO.29826.

[0086] In a further embodiment, the biological pesticide further includes pharmaceutically acceptable excipients.

[0087] In a more specific embodiment, the pharmaceutically acceptable excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0088] According to the present invention, and considering the different needs in actual production applications, combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned pharmaceutically acceptable excipients, and prepare Bacillus velezensis AMC102 with accession number CGMCC NO.29826 into different dosage forms, such as powders, tablets, suppositories, gels, patches, sprays, lotions, granules, etc.

[0089] In a specific embodiment, the dosage form of the drug is selected from one or more of the following: powder, tablet, and liquid.

[0090] Experimental Example 1: Isolation, Identification and Preservation of Bacillus belye

[0091] (1) Weigh 5g of soil sample and add it to 45ml of sterile water. Incubate in a constant temperature shaker at 30℃ and 200r / min for 30min. After standing for 20min, obtain the sample suspension.

[0092] (2) Take 1.8 mL of the supernatant of the soil suspension obtained in step (1) and transfer it to a 2 mL cryovial. Treat it in a water bath at 80°C for 10 min.

[0093] (3) Take 100 μL of the water bath sample suspension obtained in step (2), and dilute it in a 10-fold gradient to obtain 10 μL of the water bath sample suspension. -1 10 -2 10-3 A sample dilution of 10 times;

[0094] (4) Take the 10 obtained in step (3) respectively -2 10 -3 100 μL of a sample dilution was spread onto a nitrogen-free solid medium plate, and the plate was incubated upside down in a 30°C incubator for 48 h; colonies grew on the LB solid medium plate after incubation.

[0095] (5) Pick a single colony grown on the LB solid medium plate from step (4), and transfer it to a new LB solid medium plate by streaking in three zones. Place it in a constant temperature incubator at 30°C for 48 hours. If only one colony morphology is observed in the LB solid medium plate after culture, and pure colonies are obtained, it indicates that the isolation and purification are complete.

[0096] (6) The purified monoclonal strain from step (5) was cultured in liquid, the bacterial cells were collected, and genomic DNA was extracted. The 16S rDNA fragment was amplified using the universal primers 27F and 1492R described in paragraph 58 of Chinese Invention Patent ZL202210478937.4. The PCR amplification products were detected by agarose gel electrophoresis and sequenced. The PCR reaction system consisted of: 10×buffer 10 μL, 10 mM dNTP 2 μL, upper and lower primers 1 μL each, DNA template 2 μL, Taq enzyme 0.5 μL, and ddH2O 34 μL. Pre-denaturation was performed at 95℃ for 10 min; then 35 cycles were performed at 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 1 min, followed by an extension at 72℃ for 5 min. The PCR products were detected by gel electrophoresis and then sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The identified gene sequences were compared with the NCBI database using the BLAST tool.

[0097] A total of 120 strains of Bacillus belyssus were obtained through PCR molecular identification, and the strains were preserved in ultra-low temperature glycerol tubes in the Microcon Probiotics Resource Bank.

[0098] Experiment Example 2: Initial screening of Bacillus belysin's antagonistic ability, and secondary screening of its high efficiency and antibacterial ability.

[0099] (1) The antagonistic effect was determined by the plate confrontation method for initial screening of strains. 120 strains of Bacillus belysiniana were activated on LB solid medium plates and incubated at 30℃ for 24 h to grow single colonies. Four different Bacillus belysiniana colonies were taken from each plate and inoculated into PDA solid plates. Then, the activated Rhizoctonia solani PDA plates were taken out, and circular mycelial discs were punched with a 5 mm diameter punch and transferred to the center of the PDA medium plate. The Rhizoctonia solani and each Bacillus belysiniana were equidistant and served as the Bacillus belysiniana AMC102 treatment group. Plates without Bacillus belysiniana were used as blank control group (CK). The inoculated PDA plates were incubated upside down in a 28℃ incubator. After the blank control plates were fully colonized, the results were observed. The presence of inhibition zones was observed, the strain numbers were recorded, and the size of the inhibition zones was measured. The 10 strains with the highest inhibition rates were screened from the 120 strains for further screening of antagonistic strains.

[0100] Inhibition rate (%) = (Diameter of control pathogen - Diameter of treated pathogen) / Diameter of control pathogen x 100%

[0101] (2) Based on the screening results of step (1) in Experiment Example 2, 10 strains of *Bacillus belyssiensis* were selected for further screening based on their high efficiency and antibacterial ability. Ten activated plates containing *Bacillus belyssiensis* were taken, and single colonies were transferred to LB liquid medium for expansion. The volume of the medium was 50 mL / 250 mL, and the medium was cultured at 30℃ and 220 r / min for 16 h to obtain a seed culture. The seed culture was then transferred to LB liquid medium at a 2% inoculum volume, with a volume of 50 mL / 250 mL, and cultured at 30℃ and 220 r / min. At 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, appropriate amounts of the bacterial culture were taken and the OD value was measured using a spectrophotometer at a wavelength of 600 nm. Samples were then examined under a microscope. The experiment was repeated three times, and the growth rate and spore formation of the 10 strains were compared as reference indicators for further screening. The growth curve of *Bacillus belyssiensis* is shown below. Figure 1 As shown, the results indicate that strain AMC102 grows faster than the other strains.

[0102] (3) The antagonistic effect of 10 initially screened Bacillus belyi strains was rescreened using the plate confrontation method, with the experiment repeated three times. The antibacterial results are shown in Table 1 and... Figure 2 As shown, *Bacillus belyssioides* AMC102 exhibits broad-spectrum antibacterial activity, with inhibition rates exceeding 60% against all five pathogenic fungi. Specifically, the inhibition rates against *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, *Magnapordia oryzae*, *Fusarium oxysporum*, and *Fusarium wilt* were 64.24±1.35%, 86.08±1.67%, 69.41±1.12%, 71.29±2.67%, and 69.15±1.33%, respectively.

[0103] Table 1. Antifungal effects of Bacillus belye AMC102 on five pathogenic fungi

[0104] Pathogens Rhizoctonia solani Sclerotium Rice blast fungus Fusarium oxysporum Fusarium wilt of cabbage Antibacterial rate (%) 64.24±1.35 86.08±1.67 69.41±1.12 71.29±2.67 69.15±1.33

[0105] (4) The Oxford cup method was used to detect the ability of Bacillus belyssus AMC102 strain to inhibit five pathogenic bacteria. The antibacterial test method is as follows:

[0106] Preparation of *Serratia marcescens* bacterial suspension: *Serratia marcescens* was inoculated into LB liquid medium at a 2% inoculum and cultured at 37°C and 250 rpm for 24 h on a shaker. The bacterial concentration was then adjusted to 10. 8 CFU / mL.

[0107] Preparation of *Streptococcus pseudopneumococcus* bacterial suspension: *Streptococcus pseudopneumococcus* was inoculated into BHI blood plates containing 5% defibrinated sheep blood and incubated at 37°C for 16–20 h. The bacterial concentration was then adjusted to 10⁻⁶. 8 CFU / mL.

[0108] Preparation of Streptococcus mutans bacterial suspension: Streptococcus mutans was inoculated into BHI liquid medium at an inoculum of 2%, and cultured at 37°C and 250 rpm for 24 h on a shaker. The bacterial concentration was then adjusted to 10. 8 CFU / mL.

[0109] Preparation of Bacteroides fragilis suspension: Bacteroides fragilis was inoculated into BHI blood plates containing 5% defibrinated sheep blood and incubated at 37°C for 16–20 h. The bacterial concentration was then adjusted to 10. 8 CFU / mL.

[0110] Preparation of Clostridium difficile bacterial suspension: Clostridium difficile was inoculated into BHI blood plates containing 5% defibrinated sheep blood and incubated at 37°C for 16–20 h. The bacterial concentration was then adjusted to 10. 8 CFU / mL.

[0111] Cool the BHI solid medium containing 5% defibrinated sheep blood (LB solid medium is used for Serratia marcescens) to about 55°C, then mix the pathogenic bacteria into the medium in a certain proportion to ensure that the viable count of the pathogenic bacteria is above 10. 6 The concentration was on the order of CFU / mL. The medium was then quickly poured into plates pre-placed with Oxford cups. After the medium cooled and solidified, the Oxford cups were removed, and 150 μL of Bacillus belysae AMC102 fermentation broth was added to the wells. After overnight incubation at 37°C, the diameter of the inhibition zone was measured.

[0112] The antibacterial results are shown in Table 2 and Figure 2 As shown, Bacillus belye AMC102 has a certain inhibitory effect on all five pathogenic bacteria.

[0113] Table 2. Antibacterial effect of Bacillus belyceta var. AMC102 against five pathogenic bacteria

[0114] Pathogens Diameter of the inhibition zone (mm) Serratia marcescens 15.70±0.08 Pseudomonas pneumoniae 17.26±0.17 Streptococcus mutans 30.67±0.47 Bacteroides fragilis 11.63±0.05 Clostridium difficile 11.46±0.05

[0115] Based on a comprehensive comparison of the results of experiments on the antibacterial ability, growth rate, and sporulation of 120 strains, *Bacillus belyssae* with a fast growth rate and significant antagonistic effect was screened out and named AMC102, which was then deposited for preservation. Its preservation information is as follows:

[0116] Accession number: CGMCC NO.29826;

[0117] Classification and nomenclature: Bacillus velezensis;

[0118] Deposit date: January 31, 2024;

[0119] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0120] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0121] When cultured on LB agar plates, the colonies were observed to be pale yellow, raised, with irregular edges, and entirely convex with a rough, opaque surface and obvious wrinkles. The longer the culture time, the drier the colony surface became. During static culture in liquid medium, a biofilm formed on the surface of the medium. Biofilm formation can reduce the severity of plant diseases and improve the disease control ability of the strain. Microscopic observation showed that the strain's cells were short rod-shaped, produced elliptical spores, and were Gram-positive. The colony morphology, cell morphology, spore morphology, and biofilm of AMC102 are as follows: Figure 3 As shown.

[0122] Experimental Example 3: Determination of Enzyme Production Capacity of Bacillus belyssus AMC102

[0123] (1) Detection of the protease production ability of Bacillus belyssus AMC102

[0124] A small amount of *Bacillus berberis* AMC102 colonies was aspirated onto a protease medium using a toothpick and incubated at 30°C for 4 days. Three replicates were performed, and the appearance of a clear zone was observed. A larger clear zone indicates a stronger protease production capacity. Results are as follows: Figure 4 As shown in (a), Bacillus belye AMC102 can produce a distinct clear zone with a diameter of up to 49.83±2.02 mm, and has a strong protease production capacity.

[0125] (2) Detection of the amylase production capacity of Bacillus belyssus AMC102

[0126] A small amount of *Bacillus belye* AMC102 colonies was aspirated onto an amylase medium using a toothpick and incubated at 30°C for 4 days. Triple replicates were performed. The medium was then stained with dilute iodine solution, rinsed with water, and the presence of a clear zone was observed. A larger clear zone indicates a stronger amylase-producing capacity. Results are as follows: Figure 4 As shown in (b), Bacillus belyssus AMC102 can produce a distinct clear zone with a diameter of up to 40.67±0.58 mm, and has a good ability to produce amylase.

[0127] (3) Detection of the cellulase production capacity of Bacillus belyssus AMC102

[0128] A small amount of *Bacillus belye* AMC102 colonies was aspirated onto cellulase medium using a toothpick and incubated at 30°C for 4 days. Three replicates were performed. After incubation, the plates were stained with 1 g / L Congo red solution for 1 hour, then destained twice with 1 M NaCl solution for 30 minutes each time. The presence of a cellulose hydrolysis zone around the colonies was observed; a larger hydrolysis zone indicated a stronger cellulase production capacity. Results are as follows: Figure 4 As shown in (c), Bacillus belye AMC102 can produce a distinct clear zone with a diameter of up to 40.16±0.76 mm, and has a good ability to produce cellulase.

[0129] (4) Detection of chitinase production capacity of Bacillus belyssus AMC102

[0130] A small amount of *Bacillus belye* AMC102 colonies was aspirated onto chitinase medium using a toothpick and incubated at 30°C for 4 days. Three replicates were performed, and the appearance of a clear zone was observed. Results are as follows: Figure 4 As shown in (d), the plate qualitative method did not detect the ability of Bacillus belyss AMC102 to produce chitinase.

[0131] (5) Detection of the ability of Bacillus belyssus AMC102 to produce manganese peroxidase

[0132] A small amount of *Bacillus belye* AMC102 colonies was aspirated onto manganese peroxidase medium using a toothpick and incubated at 30°C for 7 days. Triple replicates were performed to observe for the appearance of a clear zone. Results are as follows: Figure 4 As shown in (e), the plate qualitative method detected that Bacillus belye AMC102 produced a brownish color zone with a diameter of up to 25.89±0.09 mm, indicating that it has the ability to produce manganese peroxidase.

[0133] Enzyme production experiments showed that *Bacillus belye* AMC102 possesses the ability to synthesize proteases and cellulases. Studies have reported that these activities contribute to plant pathogen antagonism and the biological control of plant diseases. Furthermore, this strain has the ability to produce manganese peroxidase, demonstrating potential applications in the treatment of field crop waste and improving feed utilization.

[0134] Experiment Example 4: Determination of Phosphate Solubility of Bacillus belyssus AMC102

[0135] A small amount of *Bacillus belye* AMC102 colonies was aspirated onto an inorganic phosphorus qualitative plate using a toothpick and incubated at 30°C for 7 days. Three replicates were performed, and the appearance of a clear zone was observed. Results are as follows: Figure 5 As shown, the presence of a transparent halo around the Bacillus belye AMC102 colony indicates that the strain has phosphate-solubilizing ability and potential to promote plant growth.

[0136] Experiment Example 5: Effects of AMC102 cell-free filtrate on the hyphal growth of three pathogenic fungi

[0137] (1) Take out the Bacillus belysae AMC102 activated plate, pick a single colony on the plate and transfer it to LB liquid medium for expansion culture, with a liquid volume of 50mL / 250mL, and culture it in a shaker at 30℃ and 220r / min for 16h to obtain seed liquid; transfer the seed liquid to LB liquid medium at an inoculation rate of 2% and culture for 48h to obtain fermentation broth.

[0138] (2) The 48-hour fermentation broth of the strain was centrifuged at 8000 rpm for 10 min at 4℃ to remove the bacterial cells and obtain the supernatant. The supernatant was filtered through a 0.22 μm filter to obtain cell-free filtrate. The cell-free filtrate was inoculated at a ratio of 10% into melted but not hot PDA medium, thoroughly mixed, poured into plates, and allowed to solidify. Then, 5 mm diameter Rhizoctonia solani, Sclerotinia sclerotiorum, and Blastomyces oryzae were inoculated into the plates as the AMC102 treatment group. The three pathogenic fungal blocks inoculated into PDA plates without cell-free filtrate were used as the blank control group (CK). Each group was replicated three times. The inoculated PDA plates were incubated upside down in a 28℃ incubator. After the CK group had fully grown on the plates, the results were photographed and the growth inhibition rate was calculated.

[0139] The results are as follows Figure 6 As shown, the growth diameters of the three pathogenic fungi in the AMC102 treatment group were significantly smaller than those in the CK group, indicating that the cell-free filtrate of AMC102 had a significant inhibitory effect on Rhizoctonia solani, Sclerotinia sclerotiorum and rice blast fungus, with inhibition rates of 49.06±0.52%, 100%±0.00% and 100%±0.00%, respectively.

[0140] Experiment Example 6: Effects of volatile substances produced by AMC102 on the mycelial growth of three pathogenic fungi.

[0141] (1) Prepare AMC102 fermentation broth according to the method in step (1) of Example 5.

[0142] (2) Spread 100 μL of fermentation broth evenly on an LB agar plate. Simultaneously, inoculate 5 mm diameter *Rhizoctonia solani*, *Sclerotinia sclerotiorum*, and *Blastomyces oryzae* blocks onto the center of a PDA agar plate. Remove the caps from both plates. Tightly place the LB agar plate containing AMC102 and the PDA agar plate inoculated with the three pathogenic fungi onto each other, with the PDA agar plate on top and the LB agar plate on the bottom, and seal with tape. This serves as the AMC102 treatment group. LB agar plates containing blank culture medium and PDA agar plates inoculated with the three pathogenic fungi are placed together as a blank control group (CK group). Each group is replicated three times. The treated plates are incubated upside down in a 28°C incubator. After the CK group plates have fully grown, photographs are taken, and the growth inhibition rate is calculated.

[0143] like Figure 7 As shown, the growth diameters of the three pathogenic fungi in the AMC102 treatment group were significantly smaller than those in the CK group, indicating that the volatile substances produced by the growth and metabolism of AMC102 have a significant inhibitory effect on Rhizoctonia solani, Sclerotinia sclerotiorum, and rice blast fungus, with inhibition rates of 71.77±0.67%, 100%±0.00%, and 39.62%±0.35%, respectively.

[0144] Experimental Example 7: Preparation of Bacillus belye AMC102 bacterial powder

[0145] (1) Seed culture medium formula: 10g tryptone, 5g NaCl, 5g yeast powder, 1L distilled water, pH 7.2, autoclave at 121℃ for 15min.

[0146] (2) Fermentation medium formula: 15g sucrose, 0.2g K2HPO4, 0.5g MgSO4·7H2O, 0.2g NaCl, 0.2g K2SO4, 1g yeast powder, 1g ammonium sulfate, 1L distilled water, pH 7.0-7.2, sterilized at 121℃ for 20min for later use.

[0147] (3) Prepare the primary seed culture of Bacillus belysae AMC102 according to the method in step (2) of Example 2. Transfer the primary seed culture to a 5L fermenter at an inoculation rate of 5% to prepare the secondary seed culture. Transfer the secondary seed culture to a 100L fermenter containing fermentation medium for fermentation culture. Stop fermentation at 30℃ for 30-32h to obtain fermentation broth.

[0148] (4) The fermentation broth obtained in step (3) is centrifuged at 12000 rpm to collect the bacterial sludge, and then the bacterial powder is obtained by spray drying. The inlet air temperature of the spray drying tower is 158-162℃, the outlet air temperature is 80-84℃, and the vacuum degree inside the spray drying tower is -0.01Mpa~-0.02MPa.

[0149] (5) First, measure 99 mL of sterile physiological saline containing 0.1% Tween 80 and add it to a 250 mL Erlenmeyer flask with glass beads. Then, weigh 1 g of Bacillus berberis AMC102 bacterial powder obtained in step (4) and add it to the Erlenmeyer flask. Then, place it on a shaker at 20℃ and 250 r / min for 30 min to homogenize and obtain the initial bacterial suspension. Take 2 mL of the homogenized initial bacterial suspension into a cryovial and immerse it in an 80℃ water bath for 10 min. Remove the bacterial solution after the water bath and cool it to room temperature with cold water to obtain the spore count test solution. The initial bacterial suspension without water bath is the viable count test solution. Dilute the two test solutions tenfold with sterile physiological saline, select an appropriate dilution gradient for counting, repeat 3 times for each group, and finally calculate the spore rate of the bacterial powder.

[0150] Table 3. Viable cell count and spore count of Bacillus belyss AMC102 powder

[0151] strains <![CDATA[Number of viable bacteria (*10 11 CFU / g)]]> <![CDATA[Number of spores (*10 11 CFU / g)]]> Spore formation rate (%) AMC102 4.34±0.08 4.00±0.09 92.17％

[0152] The results are shown in Table 3. The viable count of Bacillus vesiculosus AMC102 powder was 4.34 × 10⁻⁶. 11 CFU / g, spore count 4.00×10 11 CFU / g, with a spore rate of up to 92.17%.

[0153] (6) Take 2 mL of the initial bacterial suspension after homogenization in step (5) and treat it in an 80℃ water bath and at a 90℃ high temperature. The treatment at 80℃ is 2 h and 5 h, respectively, and the treatment at 90℃ is 10 min. Take the treated solution and dilute it appropriately, and determine the spore count. Use the untreated Bacillus belyss suspension as a blank control group. Each group is repeated 3 times, and the spore survival rate of Bacillus belyss powder is calculated.

[0154] Table 4. Heat resistance of Bacillus belyssus AMC102 bacterial powder

[0155]

[0156] As shown in Table 4, the results indicate that Bacillus belye AMC102 spores have strong heat resistance. After treatment in a water bath at 80℃ for 2 hours, the spore survival rate was 98.20%; after treatment in a water bath at 80℃ for 5 hours, the spore survival rate was 95.10%; and after treatment at 90℃ for 10 minutes, the spore survival rate was 90.98%.

[0157] Experiment Example 8: Salt and Alkali Tolerance Experiment of Bacillus belyssus AMC102 Fermentation Broth

[0158] (1) Adjust the salt concentration of LB medium to 1%, 2.5%, 5%, 7.5%, and 10% using NaCl. Prepare AMC102 seed culture according to step (2) of Example 2. Inoculate the seed culture into LB medium with different NaCl concentrations at an inoculation rate of 2%, with a liquid volume of 50 mL / 250 mL. Culture in a shaker at 30℃ and 220 r / min for 48 h. Repeat each group 3 times. The NaCl concentration of the LB medium without salt adjustment is 0.5%. Observe the growth of the strain.

[0159] Table 5. Salt tolerance of Bacillus belyssus AMC102 strain

[0160]

[0161] "+" indicates that growth is possible; "-" indicates that growth is not possible.

[0162] As shown in Table 5, the results indicate that Bacillus belye AMC102 can grow under six different salinities, and the strain has strong salt tolerance.

[0163] (2) The initial pH of the LB medium was adjusted to 4, 5, 6, 7, 8, 9 and 10 respectively using 1M HCl and 1M NaOH. AMC102 seed culture was prepared according to the method in step (2) of Example 2. The seed culture was inoculated into LB medium with different pH values ​​at an inoculation rate of 2%, with a liquid volume of 50mL / 250mL. The culture was carried out at 30℃ and 220r / min for 48h. Each group was repeated 3 times, and the growth of the strain was observed.

[0164] Table 6. Acid and alkali resistance of Bacillus belyssus AMC102 strain

[0165]

[0166] "+" indicates that growth is possible; "-" indicates that growth is not possible.

[0167] As shown in Table 6, the results indicate that the strain hardly grows at pH 4 and 10; however, it can grow at pH 5 to 9, demonstrating a certain degree of acid and alkali tolerance.

[0168] In summary, the AMC102 strain exhibits certain tolerance to alkali and salt, and its powder demonstrates strong resistance to high temperatures. This strain possesses strong ecological adaptability and stability, enabling it to adapt to various application environments and demonstrating significant commercial potential.

[0169] Experiment Example 9: Cucumber Seed Growth Promotion Experiment

[0170] Referring to GB / T 3543.4-1995, the Procedure for Testing Crop Seeds (Germination Test), plump cucumber seeds were selected and first soaked in 75% alcohol for 30 seconds, then rinsed with sterile water until no alcohol residue remained. Next, they were soaked in 1% sodium hypochlorite solution for 20 minutes, followed by rinsing several times with sterile water to complete seed disinfection. Sterile filter paper was placed in petri dishes (9cm in diameter). In a clean bench, 25 disinfected cucumber seeds (per dish) were evenly sprinkled onto the filter paper. Two different treatment solutions were added: sterile water (control group) and AMC102 fermentation broth cultured for 48 hours (treatment group). The filter paper was kept moist, and each group was repeated three times. The plants were placed in a 30℃ constant temperature incubator for germination. Seed germination was recorded after 7 days.

[0171] Seed germination rate (%) = Number of germinated seeds / Number of seeds sown × 100

[0172] Table 7. Effects of Bacillus belyssus AMC102 fermentation broth on cucumber seed germination

[0173] Group Germination rate (%) sterile water <![CDATA[83.33±2.08 a ]]> AMC102 <![CDATA[91.33±1.15 b ]]>

[0174] Note: Data in the table are mean ± standard error. Lowercase letters indicate significant differences in germination rate between the control group and the treatment group (P<0.05).

[0175] As shown in Table 7, cucumber seeds treated with 48h fermentation broth of strain AMC102 had a germination rate of 91.33±1.15%, while the germination rate of the sterile water control group was 83.33±2.08%. The significant difference between the groups indicates that strain AMC102 can promote seed germination.

[0176] Experiment Example 10: Potted Plant Control Experiment for Cucumber Damping-off Disease

[0177] (1) Preparation of pathogens: Take out the Rhizoctonia solani PDA plate stored at 4℃, use a 5mm diameter punch to make a circular mycelial cake in a clean bench, transfer it to the center of the PDA medium plate, and incubate the inoculated PDA plate upside down in a 28℃ incubator for 7 days for later use.

[0178] (2) Fermentation broth preparation: Prepare AMC102 fermentation broth according to the method in step (1) of Example 5, and adjust the viable cell count of the fermentation broth to contain 1 x 10⁻⁶ cells / mL. 8 CFU / mL available for use.

[0179] (3) Sowing: Select cucumber seeds from Experiment 9 that have germinated and grown evenly. Transfer them to pots (11cm in diameter, 9cm in height, and 7.5cm in bottom diameter) containing nutrient soil (containing a certain proportion of peat moss, vermiculite, perlite, etc.) that have been sterilized by high-pressure steam at 121℃ for 1 hour. Inoculate 3 cucumber seeds into each pot and then cover the surface with 1-2cm of sterilized nutrient soil. When the cucumber seedlings grow their first true leaves, use a small garden shovel to damage the root system of the 3 cucumber seedlings near the base. Irrigate each pot with 50mL of AMC102 fermentation liquid. Use an equal volume of sterile water as a blank control group and use an equal volume of 2000 times diluted 98% oxadixyl soluble powder as a positive control group. There are 6 pots in each treatment group and 18 cucumber seedlings in each group.

[0180] (5) Transplanting: Eight hours after inoculation with AMC102 fermentation broth, holes were dug at the base of each seedling to damage the roots, and 5mm diameter Rhizoctonia solani mycelium blocks were inoculated, with 3 blocks per pot. The seedlings were then placed in a greenhouse at 27±1℃, 55%±5% relative humidity, and a photoperiod of 14 hours light / 10 hours darkness to cultivate and develop disease. After 7 days, the soil around the cucumber seedling roots was cleaned, and the disease incidence in different treatment groups was recorded to assess the severity of the disease (Table 8). The disease index and relative control efficacy were calculated using the following formula.

[0181] Incidence rate = (Number of infected plants / Total number of plants) × 100%

[0182] Disease index = 100% × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × 4)

[0183] Relative prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0184] Table 8. Grading Criteria for Cucumber Damping-off Disease

[0185] Disease level Cucumber seedlings with diseased stem circumference 0 No disease spots at the base of cucumber seedling stems 1 The proportion of diseased spots on the stem circumference of cucumber seedlings should be ≤25%. 2 The percentage of diseased spots on cucumber seedlings that account for 26% or less of the stem circumference is ≤50%. 3 The percentage of diseased spots on cucumber seedlings that cover 75% of the stem circumference is ≤51%. 4 76% or less of the percentage of stem circumference occupied by diseased spots in cucumber seedlings

[0186] Table 9. Control effect of Bacillus vesicle AMC102 fermentation broth on cucumber damping-off disease.

[0187] deal with Incidence rate (%) Disease index Preventive efficacy (%) sterile water 88.89 78.67 / AMC102 44.44 15.67 80.08 98% Hymexazol 50 17.33 77.97

[0188] The control effects in potted plants are shown in Table 9. The results showed that the disease index of both treatment groups decreased significantly compared with the sterile water control group. The disease index of the Bacillus vesalis AMC102 fermentation broth treatment group was 15.67%, with a control efficacy as high as 80.08%; while the disease index of the 98% oxadixyl treatment group was 17.33%, with a control efficacy of 77.97%. Bacillus vesalis AMC102 showed slightly better control efficacy against cucumber damping-off caused by Rhizoctonia solani than 98% oxadixyl.

[0189] Experiment Example 11: Potted Plant Control Experiment for Cucumber Sclerotinia Disease

[0190] (1) Preparation of pathogens: Take out the PDA plate of Sclerotinia sclerotiorum stored at 4℃, use a 5mm diameter punch to make a circular mycelial cake in the ultra-clean workbench, transfer it to the center of the PDA medium plate, and incubate the inoculated PDA plate upside down in a 28℃ incubator for 7 days for later use.

[0191] (2) Preparation of fermentation broth: Same as step (2) in Experiment Example 10.

[0192] (3) Sowing: Select cucumber seeds from Experiment Example 9 that have germinated and grown evenly. Transfer them to pots (11cm in diameter, 9cm in height, and 7.5cm in bottom diameter) containing nutrient soil (containing a certain proportion of peat moss, vermiculite, perlite, etc.) that have been sterilized by high-pressure steam at 121℃ for 1 hour. Inoculate 3 cucumber seeds into each pot, and then cover the surface with 1-2cm of sterilized nutrient soil. The experiment begins when the cucumber seedlings have grown four true leaves.

[0193] (4) Inoculation: Sclerotinia sclerotiorum was inoculated at the base of the cucumber plant stem and wrapped with absorbent cotton soaked in sterile water as a negative control group; Sclerotinia sclerotiorum was inoculated at the base of the cucumber plant stem and wrapped with absorbent cotton soaked in 1000 times diluted 50% iprodione wettable powder as a positive control group; Sclerotinia sclerotiorum was inoculated at the base of the cucumber plant stem and wrapped with absorbent cotton soaked in AMC102 fermentation broth as an experimental group; 6 pots were used for each treatment group, and 18 cucumber seedlings were used in each group.

[0194] (5) Culture: The plants were then placed in a greenhouse with a temperature of 27±1℃, a relative humidity of 55%±5%, and a photoperiod of 14 hours of light / 10 hours of darkness to cultivate for disease development. After 7 days, the disease incidence of plants in different treatment groups was counted, the severity of the disease was assessed (Table 10), and the disease index and relative control efficacy were calculated using the following formula.

[0195] Incidence rate = (Number of infected plants / Total number of plants) × 100%

[0196] Disease index = 100% × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × 4)

[0197] Relative prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0198] Table 10. Grading Criteria for Cucumber Sclerotinia Disease

[0199]

[0200]

[0201] Table 11. Control effect of Bacillus belye AMC102 fermentation broth on cucumber sclerotinia stem rot

[0202] deal with Incidence rate (%) Disease index Preventive efficacy (%) sterile water 94.44 91.67 / AMC102 50.00 14.25 84.46 50% iprodione 61.11 17.33 81.10

[0203] The control effects in potted plants are shown in Table 11. The results showed that the disease index of both treatment groups decreased significantly compared with the sterile water control group. The disease index of the Bacillus vesalivarius AMC102 fermentation broth treatment group was 14.25%, with a control efficacy as high as 84.46%; while the disease index of the 98% oxydemeton-methyl treatment group was 17.33%, with a control efficacy of 81.10%. Bacillus vesalivarius AMC102 showed slightly better control efficacy against cucumber sclerotinia disease caused by Sclerotinia sclerotiorum than 50% iprodione.

Claims

1. A type of Bacillus belesii ( Bacillus velezensis strain AMC102, characterized in that, Its accession number is CGMCC NO. 29826.

2. A species of Bacillus belye with accession number CGMCC NO. 29826 ( Bacillus velezensis The application of strain AMC102 in the preparation of antibacterial agents or plant growth promoters is characterized by, The antibacterial agent is used to inhibit pathogens; the plant growth promoter is used to promote cucumber seed germination; the pathogens include: Rhizoctonia solani, rice blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium wilt of cabbage, Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile.

3. A species of Bacillus belye with accession number CGMCC NO. 29826 ( Bacillus velezensis The application of strain AMC102 for non-disease-treatment purposes of inhibiting fungi, and / or promoting plant growth, and / or controlling plant diseases, is characterized by, The non-disease treatment target antibacterial agents include Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile; the plant disease pathogens include Rhizoctonia solani, Rice blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, and Fusarium wilt of cabbage. The plant diseases mentioned include: cucumber damping-off and cucumber sclerotinia stem rot; the promotion of plant growth refers to promoting cucumber seed germination.

4. An antibacterial agent comprising an antibacterial active ingredient, characterized in that, The antibacterial active ingredient includes: a type of Bacillus belyssus with preservation number CGMCC NO. 29826. Bacillus velezensis ) strain AMC102.

5. The antibacterial agent according to claim 4, characterized in that, Also includes: auxiliary materials; And / or, the antibacterial spectrum of the antibacterial agent includes: Rhizoctonia solani, blast fungus, Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium wilt of cabbage, Serratia marcescens, Streptococcus pseudopneumococcus, Streptococcus mutans, Bacteroides fragilis, and Clostridium difficile.

6. An agricultural microbial agent, characterized in that, include: A species of Bacillus belye with accession number CGMCC NO. 29826 ( Bacillus velezensis ) strain AMC102.

7. A plant growth promoter, comprising an active ingredient; characterized in that, The active ingredients include: a type of Bacillus belyssus with accession number CGMCC NO. 29826. Bacillus velezensis ) strain AMC102.

8. A plant growth promoter according to claim 7, characterized in that, Also includes: Auxiliary materials.

9. A biological pesticide, comprising a phytoactive ingredient; characterized in that, The active pharmaceutical ingredient includes: a type of Bacillus belyssus with accession number CGMCC NO. 29826. Bacillus velezensis ) strain AMC102.

10. A biological pesticide according to claim 9, characterized in that, Also includes: Pharmaceutically acceptable excipients.

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