A bacillus spizizenii and uses thereof

Abstract

The present application relates to a bacillus spizizenii. The bacillus spizizenii, its inactivator, its ferment and / or at least one metabolite not only have the ability of broad-spectrum inhibiting plant pathogenic fungi activity and preventing and treating plant insect pests, especially for preventing and treating root-knot nematode pests, thrips pests and the like, but also have the ability of nitrogen fixation and iron carrier production, and can promote plant growth.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a Bacillus sp. pizizenii and its uses. Background Technology

[0002] Root-knot nematodes are highly specialized, omnivorous plant pathogenic nematodes that can damage more than 130 crops from 39 families. Root-knot nematodes typically exist as eggs or second-instar larvae left in the soil or manure with plant debris. They overwinter or, when environmental conditions are suitable the following year, invade through tender roots as second-instar larvae, multiplying and causing damage. Root-knot nematode disease is a root disease caused by the invasion of root-knot nematodes into the plant's root system and the formation of root knots. Once a host plant is infected with root-knot nematodes, it not only inhibits plant growth but also reduces the plant's resistance to other pathogens, thus inducing other soil-borne diseases such as black shank and root rot, thereby significantly reducing crop yields.

[0003] Currently, there is a lack of practical, efficient, and sustainable control technologies for root-knot nematodes. Chemical nematicides can only control nematode populations in the short term, but they suffer from high costs, potential pollution, and short-lasting effects. There is a lack of highly efficient, easily cultured, industrially viable, and commercially viable biocontrol products for root-knot nematodes on the market.

[0004] The application of microorganisms to prevent and control root-knot nematode damage aligns with the requirements of green agricultural product production. Bacillus is a type of bacteria widely used in plant disease control. Patent CN202311011636.1 discloses the application of a strain of Bacillus spizizenii HMQ20YJ19 in drugs for controlling pathogenic fungi. This strain showed good control effects in pot experiments on tomato gray mold and pepper blight, and it can adapt to the soil ecological environment and climate conditions of Gansu, showing promising application prospects in the control of common diseases of summer vegetables in the highland areas of Gansu. Liu Ni et al. studied the extracellular products and antibacterial activity of Bacillus subsp. spizizenii Bspi2104, finding that the extracellular products of Bspi2104 had good physicochemical stability, and its 70% ammonium sulfate precipitate extract and ethyl acetate extract had the best antibacterial effects (Liu Ni et al., 2024). Existing technologies have focused on the antibacterial activity of Bacillus stearothermiae, but have paid less attention to its effectiveness in the biological control of nematodes or thrips. This invention provides a Bacillus stearothermiae that, in addition to having broad-spectrum antibacterial capabilities, also has insect-resistant capabilities, and has good application prospects in the development of microbial preparations that provide long-lasting resistance to insect pests and diseases and promote growth and increase yield. Summary of the Invention

[0005] This disclosure provides a strain of Bacillus spizizenii, which not only has the ability to broadly inhibit the activity of plant pathogenic fungi and control plant pests, especially for the control of root-knot nematodes and thrips, but also has nitrogen-fixing ability and siderophore production ability, which can promote plant growth.

[0006] Bacillus spizizenii was initially published as a subspecies of Bacillus subtilis, namely Bacillus subtilis subsp. spizizenii. In 2020, it was officially promoted to species status (Dunlap CA, Bowman MJ, Zeigler DR. Promotion of Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris to species status. Antonie Van Leeuwenhoek. 2020 Jan; 113(1):1-12. doi:10.1007 / s10482-019-01354-9. Epub 2019 Nov 12. PMID:31721032.). For clarity, the nomenclature of Bacillus spizizenii will be used in this application. It should be noted that the term Bacillus spizizenii is synonymous with, and is interchangeable with, Bacillus subtilis subsp. spizizenii.

[0007] According to one aspect of this disclosure, a Bacillus spizizenii is provided, said Bacillus spizizenii being any one of the following A1) to A4):

[0008] A1) Strains whose 16S rRNA has at least 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the sequence shown in SEQ ID NO: 1, and / or whose gyrB gene has at least 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the sequence shown in SEQ ID NO: 2;

[0009] A2) Strains with an average nucleotide identity (ANI) of at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% with the Bacillus spizizenii type strain;

[0010] A3) The strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 64116;

[0011] A4) Strains whose 16S rRNA gene has at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the gyrB gene sequence of the strain described in A3), and / or strains whose gyrB gene sequence has at least 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the gyrB gene sequence of the strain described in A3);

[0012] A5) Strains whose genomes are at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to the genomes of the strains described in A3);

[0013] Strains of strains A6), A1), A2), A4), and A5 that produce at least one metabolite of chrysophanol, cyclic proline-phenylalanine, or cyclic (L-proline-L-valine), and / or strains whose genome contains gene clusters that synthesize at least one secondary metabolite of bacilysin, pulcherriminic acid, mycosubtilin, surfactantin, subtilin, rhizocticin A, or bacillibactin.

[0014] According to another aspect of this disclosure, a culture of *Bacillus stearothermiae* as described herein is provided, said culture comprising any one of the following B1) to B5):

[0015] B1) The fermentation broth of Bacillus stearothermiae;

[0016] B2) Metabolites of Bacillus stearothermiae;

[0017] B3) The supernatant of the fermentation broth of Bacillus stearothermiae;

[0018] B4) is the concentrated or dried product of B1), B2) or B3).

[0019] In some embodiments, the culture medium used for the preparation of the culture is any one of LB medium, TSB medium, and TSA medium, and / or the culture temperature is 28-32°C, and / or the culture time is 2-10 days.

[0020] In some embodiments, the metabolites or fermentation broth supernatant include at least one of antibacterial compounds and insecticidal compounds.

[0021] In some embodiments, the antimicrobial compound is at least one of bacilysin, subtilin, fengycin, surfactantin, and mycosubtilin.

[0022] In some embodiments, the insecticidal active compound includes at least one of Chrysophanol, acetophenone, cyclo(L-proline-L-valine) (Cyclo(Pro-Val)), cyclo(L-proline-L-leucine) (Gancidin W), and cyclo(proline-phenylalanine) (Cyclo(Pro-Phe)).

[0023] According to another aspect of this disclosure, a microbial agent is provided, comprising Bacillus stearothermiae as described in this disclosure, its inactivated form, and / or the culture as described in this disclosure.

[0024] According to another aspect of this disclosure, a composition is provided comprising Bacillus stearothermii as described in this disclosure, an inactivated form thereof, or a culture as described in this disclosure.

[0025] According to another aspect of this disclosure, a composition is provided comprising an insecticidal active compound isolated from Bacillus stearothermiae.

[0026] In some embodiments, the insecticidal active compound includes at least one of Chrysophanol, acetophenone, cyclo(L-proline-L-valine) (Cyclo(Pro-Val)), cyclo(L-proline-L-leucine) (Gancidin W), and cyclo(proline-phenylalanine) (Cyclo(Pro-Phe)).

[0027] In some embodiments, the composition further comprises additional activators and / or excipients.

[0028] In some embodiments, the adjuvant activator is selected from one or more biological control agents, one or more chemical control agents, one or more fertilizers, one or more herbicides, one or more plant growth promoters, or any combination thereof.

[0029] In some embodiments, the biocontrol agent includes: bacterial, fungal, viral biocontrol agents, insect biocontrol agents, and nematode biocontrol agents.

[0030] In some embodiments, the biocontrol agent is selected from biocontrol bacteria of the genera *Burkholderia*, *Penicillium*, *Trichoderma*, or *Bacillus*, or any combination thereof, or from metabolites of biocontrol bacteria of the genera *Burkholderia*, *Penicillium*, *Trichoderma*, or *Bacillus*, or any combination thereof.

[0031] In some embodiments, the bacteria of the genus Bacillus are selected from: Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus coagulans, Paenibacillus polymyxa, Bacillus amyloliquefaciens, or any combination thereof.

[0032] In some embodiments, the first biocontrol agent is selected from at least one of Trichoderma harzianum, Purpureocillium lilacinum, Penicillium bilaiae, Bacillus subtilis, Burkholderiarinojensis, Bacillus pumilus, Bacillus velezensis, Methylobacterium rhodesianum, Methylobacterium extorquens, and Paenibacillus peoriae.

[0033] In some embodiments, the *Trichoderma harzianum* is *Trichoderma harzianum* with accession number CGMCC NO.15679.

[0034] In some embodiments, the *Paecilomyces lilacinus* is *Paecilomyces lilacinus* with accession number CGMCC NO.12773.

[0035] In some embodiments, the Penicillium baqara is Penicillium baqara with accession number CGMCC NO.12767.

[0036] In some embodiments, the Bacillus subtilis is Bacillus subtilis with accession number CGMCC NO.12908.

[0037] In some embodiments, the *Rhinoceros johnsonii* is *Rhinoceros johnsonii* with accession number GDMCC NO.61156.

[0038] In some embodiments, the *Bacillus pumilus* is the *Bacillus pumilus* with accession number GDMCC NO.61962.

[0039] In some embodiments, the Bacillus belyssus is Bacillus belyssus with accession number GDMCC NO.61434.

[0040] In some embodiments, the *Methylobacterium roximatee* is *Methylobacterium roximatee* with accession number GDMCC NO.60729.

[0041] In some embodiments, the *Tylomycinus* is *Tylomycinus* with accession number GDMCC NO.62943.

[0042] In some embodiments, the *Bacillus pilaris* is *Bacillus pilaris* with accession number GDMCC NO.60482.

[0043] In some embodiments, the chemical control agent is a chemical control agent whose target does not include bacteria.

[0044] In some embodiments, the chemical control agent is a fungicide, insecticide, acaricide, nematicide, rodenticide, herbicide, or ablative.

[0045] In some embodiments, the plant growth promoter includes plant growth regulators, medium-level nutrients, micronutrients, humates, fulvic acids, or any combination thereof.

[0046] In some embodiments, the medium nutrients include calcium salts, magnesium salts, sulfates, sulfites, or any combination thereof, and the micronutrients include iron salts, borates, manganese salts, copper salts, zinc salts, molybdenum salts, chloride salts, or any combination thereof.

[0047] In some embodiments, the excipients are selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film-forming agents, spontaneous accelerators, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof.

[0048] In some embodiments, the composition is in solid form, liquid form, powder form, or any combination thereof.

[0049] In some embodiments, the composition is selected from at least one of solutions, powders, granules, emulsions, emulsions, suspensions, tablets, microparticles, and wettable powders.

[0050] According to another aspect of the present invention, a plant growth regulator, seed coating agent, root drenching agent, seed soaking agent, microbial insecticide, microbial fertilizer, microbial pesticide or biostimulant is provided, comprising Bacillus stearothermii, its inactivated form, the culture, the microbial agent, or the composition thereof as disclosed herein.

[0051] According to another aspect of the present invention, the use of the Bacillus sp. pizizenii described herein, the culture, the microbial agent, or the composition thereof in the preparation of products having the function of preventing and controlling plant diseases and pests or having the function of promoting plant growth is provided.

[0052] In some embodiments, the pests and diseases include fungal diseases, bacterial diseases, nematode pests, and / or insect pests.

[0053] In some embodiments, the plant pathogenic fungi include Rhizoctonia, Colletotrichum, Fusarium, or Magnaporthe.

[0054] In some embodiments, the *Rhizoctonia* genus includes *Rhizoctonia solani*; the *Anthracnose* genus includes *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, *Glomerella cingulata*, and *Colletotrichum capsici*; the *Fusarium* genus includes *Fusarium graminearum*, *Fusarium commune*, and *Fusarium oxysporum*; and the *Magnaportheoryzae* genus includes *Magnaportheoryzae*.

[0055] In some embodiments, the pathogens of the nematode pest include root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp.), and root-rot nematodes (Pratylenchus spp.).

[0056] In some embodiments, the root-knot nematodes include, but are not limited to, one or more of the following: southern root-knot nematode (Meloidogyne incognita), northern root-knot nematode (Meloidogyne hapla), Javan root-knot nematode (Meloidogyne javanica), and peanut root-knot nematode (Meloidogyne arenaria).

[0057] In some embodiments, the pathogens of the insect pests include Thysanoptera or Arachnida.

[0058] In some embodiments, the thrips pests include at least one of the following: *Thrips flevas Schrank*, *Thrips alliorum*, *Stenchaetothrips biformis*, *Frankliniella occidentalis*, *Frankliniella intonsa*, *Thrips palmmi Karny*, *Megalurothrips usitatus*, and *Scirtothrips dorsalis*.

[0059] In some embodiments, the arachnid pests include pests from the superfamily Tardiformes, Tetranychus, and Erythroformes.

[0060] In some embodiments, the arachnid pests include at least one of the following: Cyclamen stenoptera, Cyclamen stearothermii, Cyclamen lateralis, alfalfa moth, wheat mite, fruit moth, jujube leaf mite, cotton leaf mite, two-spotted leaf mite, hawthorn leaf mite, cinnabar leaf mite, truncated leaf mite, citrus pterocaryon, apple pterocaryon, citrus leaf mite, six-spotted leaf mite, cypress leaf mite, rice leaf mite, bamboo leaf mite, millet gall mite, and tulip mite.

[0061] According to another aspect of this disclosure, the use of the Bacillus stearothermii, the culture, the microbial agent, the composition, or the plant growth regulator, seed coating agent, root drenching agent, seed soaking agent, microbial insecticide, microbial fertilizer, microbial pesticide or biostimulant described herein in the prevention and control of plant diseases and pests or in the promotion of plant growth is provided.

[0062] In some embodiments, the pests and diseases include fungal diseases, bacterial diseases, nematode pests, and / or insect pests.

[0063] In some embodiments, the plant pathogenic fungi include Rhizoctonia, Colletotrichum, Fusarium, or Magnaporthe.

[0064] In some embodiments, the *Rhizoctonia* genus includes *Rhizoctonia solani*; the *Anthracnose* genus includes *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, *Glomerella cingulata*, and *Colletotrichum capsici*; the *Fusarium* genus includes *Fusarium graminearum*, *Fusarium commune*, and *Fusarium oxysporum*; and the *Magnaportheoryzae* genus includes *Magnaportheoryzae*.

[0065] In some embodiments, the pathogens of the nematode pest include root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp.), and root-rot nematodes (Pratylenchus spp.).

[0066] In some embodiments, the root-knot nematodes include, but are not limited to, one or more of the following: southern root-knot nematode (Meloidogyne incognita), northern root-knot nematode (Meloidogyne hapla), Javan root-knot nematode (Meloidogyne javanica), and peanut root-knot nematode (Meloidogyne arenaria).

[0067] In some embodiments, the pathogens of the insect pests include Thysanoptera or Arachnida.

[0068] In some embodiments, the thrips family pests include at least one of the following: *Thrips flevas Schrank*, *Thrips alliorum*, *Stenchaetothrips biformis*, *Frankliniella occidentalis*, *Frankliniella intonsa*, *Thrips palmmi Karny*, *Megalurothrips usitatus*, and *Scirtothrips dorsalis*.

[0069] Preferably, the arachnid pests include pests from the superfamily Tardiformes, Tetranychus, and Erythroformes.

[0070] In some embodiments, promoting plant growth includes promoting the absorption of nutrients by the plant.

[0071] In some embodiments, promoting plant growth includes promoting the absorption of macroelements and / or microelements by the plant. Macroelements include, but are not limited to: carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P), sulfur (S), potassium (K), magnesium (Mg), calcium (Ca), silicon (Si), etc.; microelements include, but are not limited to: iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), boron (B), molybdenum (Mo), chlorine (Cl), sodium (Na), nickel (Ni), etc.

[0072] In some embodiments, promoting plant growth includes providing a nitrogen source to the plant.

[0073] In some embodiments, promoting plant growth includes promoting the absorption of nitrogen and / or iron ions by the plant.

[0074] In some embodiments, promoting plant growth includes reducing root knots, increasing the dry weight of individual plants, increasing leaf spread, increasing stem diameter, and / or promoting leaf greening.

[0075] According to another aspect of this disclosure, the use of the Bacillus stearothermii, the culture, the microbial agent, and the composition described herein is provided in soil remediation or soil improvement or nitrogen fixation.

[0076] In some embodiments, the soil remediation or soil improvement is to enhance the availability of soil nutrients.

[0077] In some embodiments, the soil remediation or soil improvement includes improving nitrogen supply and / or generating iron carriers.

[0078] The disclosed Bacillus steggii strain MNA0003 not only has the ability to broadly inhibit the activity of plant pathogenic fungi and control plant pests, especially for the control of root-knot nematodes and thrips, but also has nitrogen-fixing and siderophore-producing capabilities, which can promote plant growth and have broad application prospects.

[0079] Biological Preservation

[0080] The disclosed Bacillus spizizenii strain MNA0003 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) under the accession name Bacillus spizizenii MN223761, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on December 6, 2023, with accession number GDMCC NO.64116. The proposed taxonomic name is Bacillus spizizenii. Attached Figure Description

[0081] Figure 1 The phylogenetic tree of strain MNA0003 in Example 1 of this disclosure is shown.

[0082] Figure 2 The results of siderophore production detection by strain MNA0003 in Example 4 of this disclosure are shown.

[0083] Figure 3 The results of plate antagonism of strain MNA0003 against pathogenic fungi in Example 5 of this disclosure are shown.

[0084] Figure 4 This demonstrates that the MNA0003 strain in Example 6 of this disclosure can grow on Ashby medium without a nitrogen source.

[0085] Figure 5 This demonstrates that the MNA0003 strain in Example 7 of this disclosure is a strong biofilm-forming strain. Detailed Implementation

[0086] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are provided to better understand the invention and are not intended to limit the scope of the invention. They do not constitute a limitation on the content or scope of protection of this invention. Any product identical or similar to this invention, derived by any person under the guidance of this invention or by combining features of this invention with other prior art, falls within the protection scope of this invention.

[0087] As used herein, the term "progeny" refers to daughter cells or subcultured progeny cells produced by the growth and culture of a microorganism (e.g., growth in a culture medium). During the growth and culture of microorganisms, particularly bacteria, the genetic material may undergo certain changes (e.g., mutations of one or more bases). Therefore, in this document, the term "progeny of Bacillus stearothermiae" is intended to encompass both progeny whose genetic material has not changed and those whose genetic material has changed compared to the Bacillus stearothermiae of this invention. Of course, said progeny still retain the functions of the strain from which it is derived, such as the control of plant diseases and pests, and the promotion of plant growth.

[0088] As used herein, “identity” of nucleic acid sequences refers to the degree to which two nucleic acid sequences have the same nucleotide residues at the same positions when compared to achieve the maximum level of identity, expressed as a percentage. The “identity” between the sequences of two nucleic acid molecules can be determined using known computer algorithms, such as at least one of the GCG package, BLASTN, or FASTA, and other commercially or publicly available programs such as the DNAStar-“MegAlign” program.

[0089] Furthermore, with the rapid development of second- and third-generation sequencing technologies, bacterial species identification based on whole-genome sequencing has become possible, leading to more accurate identification results. Average nucleotide identity (ANI) refers to the similarity of homologous genes between two bacterial genomes. Currently, various mature tools exist for calculating ANI values, such as the local software Jspecies ( / jspecies) and Gegenees ( / documentation.html), and online tools like ANI calculator (http: / / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani), and ANItools. Therefore, ANI values ​​can be calculated using any of the aforementioned local software, online tools, or other tools or methods known in the art.

[0090] As used herein, “metabolite,” “culture,” “fermentation broth,” and “extract” include any component, compound, substance, or byproduct (including, but not limited to, small molecule secondary metabolites) produced by the *Bacillus stearothermiae* strain described herein that has any of the beneficial effects described in this disclosure. These beneficial effects include nitrogen fixation, secretion of ammonium nitrogen, siderophore production, and promotion of plant growth.

[0091] In some embodiments, the adjuvant active agent described herein may be phytonutrients, plant growth promoters (growth stimulants), biostimulants (micronutrients), fertilizers, seed coating agents, biological control agents, microorganisms, chemical agents, herbicides, etc.

[0092] Preferably, the fertilizer includes, but is not limited to, at least one of amino acid fertilizers, humic acid fertilizers, seaweed fertilizers, humic acid fertilizers, fertilizers containing pollen polysaccharides, polypeptide fertilizers, and microbial fertilizers. As used herein, the term "microbial fertilizer" refers to a product containing specific microorganisms that, when applied in agricultural production, can achieve specific fertilizer effects. These effects include not only the supply of nutrients to the soil, environment, and plants, but also the beneficial effects of its metabolites on plants.

[0093] In some embodiments, the adjuvant active agent described herein may include one or more chemical agents and one or more microbial preparations. In some embodiments, the chemical agent includes, but is not limited to, insecticides, bactericides, fungicides, viricides, nematicides, insecticides, acaricides, gastropod toxicants, herbicides, etc., or combinations thereof. In preferred embodiments, the chemical agent described herein includes one or more of bactericides, fungicides, and nematicides. Preferably, the bactericides described herein include, but are not limited to, one or more of, kasugamycin, thiabendazole, thiabendazole, allicin, copper hydroxide, kasugamycin, cymoxanil, copper oxychloride, copper oxychloride, chlorobromoisocyanuric acid, trichloroisocyanuric acid, copper acetate, or copper succinate. Preferably, the fungicides described herein include, but are not limited to, one or more of, carbendazim, mancozeb, aluminum fosetyl-aluminum, metalaxyl, metalaxyl, cymoxanil, dimethomorph, flumorph, silver oxychloride, benzalkonium chloride, cymoxanil, tebuconazole, or propiconazole. Preferably, the nematicides described herein include, but are not limited to, one or more of halogenated hydrocarbons, methyl thioisothiocyanate, organophosphates, carbamates, or avermectin.

[0094] In some embodiments, the adjuvant active agent described herein may include one or more biocontrol agents. Preferably, the biocontrol agent includes, but is not limited to, microbial control agents, bacteria, fungi (e.g., yeast), viral biocontrol agents, insect biocontrol agents, nematode biocontrol agents, or any combination thereof. Preferably, the bacteria herein may include, for example, bacteria selected from the genera *Bacillus* and *Burkholderia*, or any combination thereof.

[0095] In a preferred embodiment, the adjuvant described herein may include a combination of two or more chemical agents and biological control agents described herein.

[0096] The terms "biofertilizer" and "plant growth promoter" used in this article are interchangeable, referring to preparations that use organisms or their derivatives as the main active ingredient and have the effect of promoting plant growth. The terms "biopesticide" and "bioinsecticide" used in this article are interchangeable, referring to preparations that utilize organisms or their derivatives to kill or inhibit agricultural pests such as plant pathogens that cause plant diseases and pests. The biofertilizers and biopesticides described in this article can also be called microbial fertilizers or microbial pesticides. Specifically, the organisms used include Bacillus stearothermiae as mentioned above.

[0097] The compositions described herein may be provided in the form of solids, liquids, powders, or any combination thereof. For example, the compositions described herein may be formulated into at least one of the following dosage forms: solutions, powders, granules, emulsions, emulsions, suspensions, tablets, microparticles, and wettable powders.

[0098] This invention may also include a dried product obtained by drying the bacterial strain or its culture medium, and a biopesticide containing the product. The product may be selected from hydrating agents (WP), curing agents (GM), granular hydrating agents (WG), granules (GR), powders (DP), and seed treatment hydrating agents (WS). Compared to conventional liquid formulations, these biopesticide formulations exhibit excellent stability and physicochemical properties, and can be used for the control of plant diseases.

[0099] Microbial preparations can be liquid wetting agents (SC), liquid suspensions (SM), adsorbed granules (adsorbed GR), granular formulations (granular GR), or powdered hydrating agents (WP). Powdered hydrating agents are preferred. In biopesticides, wettable powders refer to pesticide formulations that hydrate after dilution in water, while solid formulations refer to formulations obtained by mixing or adsorbing microbial culture broth into a solid substance, which is not the same as powders. Granular wettable powders. Furthermore, granular wettable powders are pesticide formulations used by diluting them in water in granular form, granules are pesticide formulations used in their original granular form, and powders are formulations of the pesticide used. Seed treatment suspensions, in their original powder form, refer to pesticide formulations that are prepared into a suspension through hydration before being used as powder treatment of seeds.

[0100] The plants mentioned in this document include those used in agriculture, forestry, and other fields. Preferably, these plants include, but are not limited to, food crops, cash crops, or root crops.

[0101] In some embodiments, the aforementioned food crops include, but are not limited to, at least one of cereal crops, tuber crops, and legume crops. In other embodiments, the cereal crops include, but are not limited to, at least one of the following grasses: rice, wheat, corn, barley, oats, rye, sorghum, millet, foxtail millet, barnyard millet, and buckwheat. In still other embodiments, the tuber crops include, but are not limited to, at least one of the following: sweet potato, yam, and potato. In some embodiments, the legume crops include, but are not limited to, at least one of the following: soybean, broad bean, pea, mung bean, and peanut.

[0102] In other embodiments, the economic crops described herein include, but are not limited to, at least one of the following economic crops: plants of the Solanaceae, Rosaceae, Rutaceae, Musaceae, Cucurbitaceae, Brassicaceae, Orchidaceae, Fabaceae, Asteraceae, Liliaceae, Zingiberaceae, Passifloraceae, Bromeliaceae, Araliaceae, and Cactaceae families.

[0103] In some embodiments, the aforementioned Solanaceae plants include, but are not limited to, tomatoes, peppers, potatoes, eggplants, black nightshade, belladonna, and goji berries. In some embodiments, Rosaceae plants include, but are not limited to, strawberries and papayas. Preferably, Rutaceae plants include, but are not limited to, citrus fruits. Preferably, Musaceae plants include, but are not limited to, bananas. Preferably, Cucurbitaceae plants include, but are not limited to, cucumbers, gourds, winter melons, pumpkins, bitter melons, loofahs, watermelons, monk fruit, and bottle gourds. Preferably, Brassicaceae plants include, but are not limited to, cabbage, rapeseed, kale, radishes, and cauliflower. Preferably, Orchidaceae plants include, but are not limited to, orchids. Preferably, Fabaceae plants include, but are not limited to, soybeans. Preferably, Asteraceae plants include, but are not limited to, lettuce. Preferably, Liliaceae plants include, but are not limited to, garlic. Preferably, Zingiberaceae plants include, but are not limited to, ginger. Preferably, Passifloraceae plants include, but are not limited to, passion fruit. Preferably, Bromeliaceae plants include, but are not limited to, pineapples. Preferably, Araliaceae plants include, but are not limited to, Panax notoginseng. Preferably, Cactaceae plants include, but are not limited to, dragon fruit.

[0104] In some embodiments, the root and tuber crops described herein include, but are not limited to, potatoes, carrots, leafy vegetables, solanaceous vegetables, strawberries, grapes, citrus fruits, bananas, kiwifruit, dragon fruit, tomatoes, peppers, beans, ginger, Panax notoginseng, ginseng, etc.

[0105] The plants described herein include plants at various stages, including those in the seed germination stage, seedling growth stage, robust seedling development stage, mature seedling flowering stage, and seed formation stage. Therefore, the plants described herein include both seedlings and fully grown plants. Furthermore, the plants described herein also include plant tissues and plant organs. Preferably, the plant tissues include, but are not limited to, meristematic tissues, protective tissues, ground tissues, and vascular tissues. Preferably, the plant organs include, but are not limited to, roots, stems, leaves, flowers, fruits, and seeds.

[0106] The term "culture" refers to a group of microbial cells or their growth within a specific time and space, such as slant cultures or shake flask cultures of microorganisms. Specifically, in this invention, it refers to the product obtained by culturing isolated bacterial strains in a fermentation medium. The fermentation medium can be selected from natural or artificial media, such as solid or liquid media. In some embodiments, the fermentation medium includes at least one of peptones, such as meat peptone, casein peptone, whey peptone, or soybean peptone. Additionally, it may include other inorganic salts or organic substances used to promote bacterial growth.

[0107] In one embodiment, this document relates to a method for preparing a culture of Bacillus stenosum strain, comprising the steps of: first activating the Bacillus stenosum strain, and then fermenting it. Preferably, the activation culture is carried out on a solid culture medium. Preferably, the fermentation culture is carried out in a liquid culture medium.

[0108] In some implementations, the solid culture medium used for activation culture is any conventional culture medium, such as TSB solid medium, LB medium, NA (nutrient agar) medium, MSA medium, AMS medium, AGPS medium, etc.

[0109] In some embodiments, the liquid culture medium used for fermentation culture is TSB liquid medium or any of the above-mentioned solid culture media in liquid form.

[0110] In some embodiments, this disclosure also relates to a method for promoting plant growth, comprising the step of applying a strain of Bacillus spp. of this disclosure or its metabolites, cultures, fermentation broths, extracts, agents, or compositions to plants (including plant tissues, plant organs) or seeds. Preferably, the method comprises applying a fermentation broth of a Bacillus spp. strain to plants, plant tissues, plant organs, or seeds.

[0111] Preferably, the application methods include root soaking, foliar spraying, spraying, composting, seed soaking, coating, flood irrigation, drip irrigation of plants or plant organs, application of plant or plant organs, and drip application of plant or plant organs.

[0112] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0113] Example 1. Isolation, Identification and Screening of Strains

[0114] 1. Isolation of strains

[0115] Bacillus spizizenii MNA0003 was isolated from tomato rhizosphere soil samples in Beijing. The specific process is as follows:

[0116] Weigh 5g of soil, add 45mL of sterile water, vortex for 5 minutes, and then serially dilute to 10⁻⁶. -1 ~10 - 6 Take 10 g / mL of soil dilution. -4 g / mL, 10 -5 g / mL and 10 -6Soil dilutions at three different g / mL levels were plated onto LB agar plates (formulation: 5g yeast extract, 10g casein peptone, 10g sodium chloride, 15g agar, 1L water, pH=7). Each plate was replicated three times. The plates were incubated upside down at 30℃ for 4 days. After 4 days, single colonies of the strain were picked, streaked onto new LB plates, and incubated at 30℃ for 1 day to obtain strain MNA0003 and its pure culture. The culture was stored in glycerol tubes at -80℃.

[0117] 2. Identification of strains

[0118] Morphological characteristics: When strain MNA0003 was inoculated onto TSB solid agar plates (17g tryptone, 3g soybean peptone, 5g sodium chloride, 2.5g anhydrous dipotassium hydrogen phosphate, 2.5g glucose, 5g agar powder, diluted to 1000mL, autoclaved at 121℃ for 20 minutes), and incubated aerobically at 30℃ for 24 hours, visible colonies formed on the plates. The colonies were round, flat, dull, rough-surfaced, milky white, and had intact edges. The bacteria were rod-shaped, with a few arranged in chains. The strain was Gram-positive. Spore staining showed the presence of spores, which were columnar, central, or apical.

[0119] 3. Genetic identification

[0120] 16S rDNA and gyrB gene sequence analysis: Genomic DNA was extracted from bacterial cells, amplified using primers, and the PCR products were sequenced. The 16S rDNA amplification primers and sequencing primers were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:3) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:4). The gyrB gene amplification primers were UP-1: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' (SEQ ID NO:5) and UP-2r: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3' (SEQ ID NO:6).

[0121] After proofreading and splicing, the sequenced data was compared with sequences of related species in the GenBank database using BLAST. The results showed that the 16S rDNA sequence (SEQ ID NO:1) of this strain had 99% homology with the sequence of Bacillus spizizenii, and the gyrB gene sequence (SEQ ID NO:2) had the highest homology with the sequence of Bacillus spizizenii, at 97%.

[0122] Based on bacterial morphology, 16S rDNA, and gyrB gene sequence analysis, this strain was identified as *Bacillus spizizenii* and named *Bacillus spizizenii* MNA0003. Multiple sequence alignment was performed between the gyrB gene sequence of MNA0003 and the gyrB gene sequences of closely related *Bacillus* strains, and a phylogenetic tree was constructed using MEGA5 software. The results are as follows. Figure 1 As shown.

[0123] Bacillus spizizenii MNA0003 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) under the name Bacillus spizizenii MN223761, accession number GDMCC64116, on December 6, 2023. The address is Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. The proposed taxonomic name is Bacillus spizizenii.

[0124] The sequence of SEQ ID NO:1 is as follows:

[0125]

[0126] The sequence of SEQ ID NO:2 is as follows:

[0127]

[0128] 4. Genome analysis

[0129] Whole-genome sequencing of strain MNA0003 yielded 1.30 Gbp of data, with Q20 accounting for 96.705%. Genome assembly revealed a total gene length of 3.99 Mbp, an N50 length of 324.8 kbp, and a GC content of 43.83%. A total of 3962 coding sequences (CDS) were predicted, with an average CDS sequence length of 894 bp.

[0130] First, Prokka (Rapid prokaryotic genome annotation) was used to predict genes in the strain and obtain gene coding sequences. Then, the coding genes were annotated based on multiple databases (COG, KEGG, Uniprot). Finally, the secondary metabolite synthesis gene clusters (BGCs) predicted by Antismash 7.0 and deepBGC were combined to discover metabolite gene clusters in the strain.

[0131] The gene cluster analysis of potential secondary metabolites is shown in Table 1 below:

[0132] Table 1: MNA0003 potential secondary metabolite gene clusters

[0133]

[0134]

[0135] The results showed that *Bacillus spp.* MNA0003 possesses multiple gene clusters for the synthesis of secondary metabolites, enabling the production of various secondary metabolites, including bacilysin, pulcherriminic acid, mycosubtilin, bacillaene, surfactantin, bacillibactin, subtilin, and rhizocticin A. Among these, bacilysin, pulcherriminic acid, mycosubtilin, surfactantin, subtilin, and rhizocticin A are all substances with antibacterial activity, while bacillibactin is a siderophore. Therefore, *Bacillus spp.* MNA0003 can produce a variety of antibacterial and growth-promoting substances.

[0136] Example 2. Preparation of fermentation culture of Bacillus stearothermiae strain MNA0003

[0137] The preparation of fermentation culture of Bacillus stearothermia MNA0003 includes the following steps:

[0138] 1. Activation culture of the strain: The isolated and preserved Bacillus stearotherm MNA0003 was inoculated onto TSB medium plates (17g tryptone, 3g soybean peptone, 5g sodium chloride, 2.5g anhydrous dipotassium hydrogen phosphate, 2.5g glucose, 5g agar powder, and the volume was adjusted to 1000mL, and the plates were autoclaved at 121℃ for 20 minutes) and incubated at 30℃ for 2 days.

[0139] 2. Fermentation culture of the strain: The activated Bacillus steganus MNA0003 obtained from the TSB medium plate was transferred to TSB liquid medium (200mL medium / 500mL Erlenmeyer flask, TSB liquid medium formula: 17g tryptone, 3g soybean peptone, 5g sodium chloride, 2.5g anhydrous dipotassium hydrogen phosphate, 2.5g glucose, adjusted to 1000mL, autoclaved at 121℃ for 20 minutes), and placed in a shaker for 48h to obtain the fermentation broth of Bacillus steganus MNA0003. The culture conditions were 30℃ and 200r / min.

[0140] Example 3. Test of nematicidal ability of Bacillus stearothermiae MNA0003 strain

[0141] 1. In vitro necrotic activity

[0142] Obtaining a suspension of second instar larvae (J2s) of the southern root-knot nematode (Mi, Meloidogyne incongnita).

[0143] Wash the water spinach roots used for propagating root-knot nematodes with clean water, and collect the egg masses with tweezers. Disinfect the collected egg masses with a 0.5% NaClO solution, then rinse three times with sterile water. Place them in a 90mm petri dish and incubate in the dark. After 48 hours, collect the second-instar larvae of the root-knot nematodes and prepare a suspension for later use.

[0144] The fermentation broth of the strain obtained in Example 2 was prepared into treatment solutions with five-fold and ten-fold dilutions. 450 μL of the treatment solution and 50 μL of second-instar larval suspension (2000 larvae / mL) were added to a 24-well cell culture plate, the plate was capped, and sealed with sealing film. The plate was incubated at 28°C in the dark for 24 hours. The number of rigid and dead second-instar larvae was observed and recorded under a stereomicroscope, and the rigidity rate and mortality rate were calculated. Judgment criteria: larvae that remained rigid were considered rigid, while those that moved or curled were considered live; nematodes that remained rigid after stimulation with 1 mol / L NaOH solution were considered dead. Sterile water was used as a blank control, and a 1500-fold dilution of 5% avermectin emulsifiable concentrate (Xingbake nematode) was used as a positive control. Each treatment was repeated in five replicates.

[0145] Wherein: Rigidity rate (%) = (Number of rigid linear worms / Number of linear worms) × 100%

[0146] Mortality rate (%) = (Number of dead nematodes / Number of dead nematodes) × 100%

[0147] The results of the in vitro contact nematode killing test are shown in Table 2. From the results in Table 2, it can be seen that the MNA0003 fermentation broth can cause nematodes to become rigid and die.

[0148] Table 2: Results of in vitro contact nematode killing test of MNA0003

[0149]

[0150] 2. Centrifuge tube protection

[0151] Cucumber seeds were sown in petri dishes. After germination, one seed was transplanted into each 50mL centrifuge tube and placed in a light-controlled culture chamber. The photoperiod and temperature were set as follows: 16 hours of light at 28°C, followed by 8 hours of darkness at 20°C. One week later, each cucumber plant was irrigated with 10mL of the treatment solution. One day later, each cucumber plant was inoculated with 1mL of a suspension of second-instar larvae (200 larvae / mL). Treatment solution preparation: 2mL of the fermentation broth obtained from Example 2 was applied to each cucumber plant, and water was added to make up to 10mL. After 14 days, the number of root knots and the aboveground dry / fresh weight were counted, and the root knot reduction rate and dry weight growth rate were calculated. Water was used as a blank control, and 1500-fold diluted abamectin emulsifiable concentrate was used as a positive control. Each treatment consisted of 5 seedlings, replicated 3 times.

[0152] Wherein: Root knot reduction rate (%) = [(Number of root knots in control group - Number of root knots in treatment group) / Number of root knots in control group] × 100

[0153] Dry weight growth rate (%) = [(Dry weight of control group - Dry weight of treatment group) / Dry weight of control group] × 100

[0154] The results are shown in Table 3 below. The results indicate that the fermentation broth of Bacillus stearothermii MNA0003 can reduce the occurrence of root knots and promote plant growth.

[0155] Table 3: Results of MNA0003 centrifuge tube control of nematodes

[0156] deal with Number of root nodes / Root knot reduction rate Fresh weight of aboveground parts / g aboveground dry weight / g Dry weight growth rate water 70.60 - 0.682 0.085 - Avermectin dilution 0.00 100.00％ 0.833 0.091 15.63％ MNA0003 40.67 42.40％ 1.475 0.110 28.91％

[0157] 3. Potted plant experiment

[0158] When cucumber plants reached the 2-leaf-1-heart stage, they were irrigated with MNA0003 treatment solution and placed in a light-treated culture chamber. The photoperiod and temperature were set to 16 hours of light at 28°C and 8 hours of darkness at 20°C. Treatment solution preparation: 15 mL of the fermentation broth obtained from Example 2 was applied to each cucumber seedling, and water was added to bring the volume to 60 mL. After 24 hours, a suspension of second-instar root-knot nematode larvae (300 larvae / mL) was inoculated into each plant at 1 mL. After 15 days, the stem diameter was measured 2 cm below the cotyledon using calipers with the outer measuring claw perpendicular to the cotyledon growth direction. The leaf spread was measured by unfolding the first and second true leaves of the cucumber seedling and measuring the distance between the leaf tips with a ruler. After 30 days, the number of root knots was observed and counted to evaluate the root knot reduction rate of the tested treatment on root-knot nematodes. Water was used as a blank control, and 1500-fold diluted abamectin emulsifiable concentrate was used as a positive control. Each treatment consisted of 8 seedlings, and the experiment was repeated 3 times.

[0159] The root knot reduction rate (%) is calculated as follows: [(Number of root knots in the control group - Number of root knots in the treatment group) / Number of root knots in the control group] × 100

[0160] The results are shown in Table 4 below. The results indicate that the fermentation broth of Bacillus stearothermii MNA0003 can reduce the occurrence of root knots and promote plant growth.

[0161] Table 4: Results of pot experiment on nematode control using MNA0003

[0162] Stem diameter / mm Surface unfolding / cm Number of root nodes / Root knot reduction rate / % water 2.03 5.69 31.23 - Avermectin dilution 2.11 5.54 0.00 100.00 MNA0003 4.14 24.50 10.79 65.44

[0163] 4. Natural metabolites—Metabolomics analysis

[0164] To investigate the active substances with nematicidal activity in the fermentation broth of strain MNA0003, further metabolomics analysis was performed on the fermentation broth.

[0165] 1. Experimental Methods

[0166] a. Sample processing

[0167] Take 400 μL of the bacterial fermentation broth sample, add 1600 μL of acetonitrile (chromatographic or mass spectrometry grade), vortex for 30 s, centrifuge at 14000 rpm for 5 min, take 500 μL of the supernatant, filter it through a 0.22 μm membrane, add it to the test bottle, label it, and send it for testing.

[0168] b. Instruments and consumables

[0169] Mass spectrometry system: Xevo G2-XS Q-Tof mass spectrometer

[0170] Liquid chromatography system: ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph

[0171] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (1.7μm, 2.1×50mm)

[0172] c. Chromatographic conditions

[0173] Injection volume 5 μL, column temperature 40℃, sample chamber temperature 10℃; mobile phase B-acetonitrile, A-water (containing 0.1% formic acid), flow rate 0.30 mL / min, 0-10 min, 1-100% B phase (acetonitrile); 10-15 min, 100-100% B phase (acetonitrile); 15-20 min, 1% B phase (acetonitrile).

[0174] d. Mass spectrometry conditions

[0175] Positive and negative ion ionization mode ESI + / ESI - Capillary voltage: 2.0KV, cone voltage: 40.0V, cone backflush gas: 50L / h, ion source temperature: 140℃, desolvation gas temperature (N2): 500℃, desolvation gas flow rate: 800L / h, acquisition mode: DDA, low collision energy: 6eV, high collision energy: 10-50eV, acquisition quality range: 50-2000m / z.

[0176] 2. Data Processing

[0177] Data processing was performed using Progenesis QI software, with Tag Filter: Anova p-value ≤ 0.05; Maxfold change ≥ 10; sorting method: abundance from largest to smallest. The NPatlas and HMDB databases were used for identification, and results were sorted by abundance. The collected metabolites were then uploaded to the MetaboAnalyst library ("https: / / www.metaboanalyst.ca / MetaboAnalyst / Secure / pathway / PathResultView.xhtml"). The HMDB ID input type was selected in the pathway analysis section, and the metabolite encoding information (HMDB ID) detected by the QTOF-MS mass spectrometry system was uploaded. Combined with the metabolic pathway analysis, the secondary metabolites produced by the complete metabolic pathway can be viewed.

[0178] 3. Experimental Results

[0179] Secondary metabolites that the strain may produce:

[0180] Using Progenesis QI software in conjunction with the NPatlas and HMDB databases, 7777 secondary metabolites were identified from this strain. The most abundant metabolites included phospholipids, terpenes, spironolactones, long-chain fatty acids, medium-chain fatty acids, and ceramides. Phospholipids can serve as effective nutrients, providing crops with phosphorus and fatty acids, promoting crop growth and development. Terpenes typically possess antibacterial properties, helping plants fight pathogenic microorganisms.

[0181] In addition, three nematicidal compounds were identified, including Chrysophanol (102.59 mg / L). –1 The mortality rate against *L. elegans* can reach 50%; acetophenone has a strong attractant effect on nematodes, causing them to feed and die after entering the nematode's intestines; cyclic proline-phenylalanine, in controlling root-knot nematodes, showed a 48-hour mortality rate of 38.52±1.33% at 10 mg / mL and 60.45±2.59% at 20 mg / mL; in controlling *C. elegans*, the 48-hour mortality rate was 42.79±1.47% at 10 mg / mL and 67.37±4.85% at 20 mg / mL. Cyclic (L-proline-L-valine) compounds are present. Therefore, it is speculated that the active substances in this strain are these types of compounds.

[0182] Example 4. Siderophore production capacity test of Bacillus stearothermiae strain MNA0003

[0183] 1. Iron starvation treatment

[0184] Using a sterile toothpick, scrape *Bacillus stearothermiae* MNA0003 from the plate in step 1 of Example 2 and inoculate it into a 50 mL centrifuge tube containing 10 mL of MKB iron-free medium (MKB iron-free medium: 5 g casein amino acids, 15 mL glycerol, 2.5 g magnesium sulfate heptahydrate, 1000 mL ultrapure water, pH 7.2, autoclaved at 121°C for 20 minutes). Incubate at 30°C with shaking at 200 rpm for 24 hours. Before inoculation, add 10 mL of dipotassium hydrogen phosphate solution (2.5 g dipotassium hydrogen phosphate, 50 mL ultrapure water, autoclaved at 121°C for 20 minutes) to every 200 mL of medium. After bacterial cell formation (the medium becomes turbid), centrifuge at 8000 rpm for 5 minutes and discard the supernatant. Wash the bacterial cells twice with 5 mL of sterile ultrapure water by centrifugation at 8000 rpm for 5 minutes, and then dilute 10 times with sterile ultrapure water to obtain a bacterial suspension.

[0185] 2. CAS plate cover method for detecting siderophores

[0186] Siderophores are low-molecular-weight substances that can specifically bind iron ions and supply them to microbial cells. Iron is an essential element for microbial growth. In iron-deficient environments, microorganisms secrete siderophores to chelate iron ions to meet their own growth needs and those of plants, playing a crucial role in plant photosynthesis and respiration.

[0187] Take 10 μL of bacterial suspension and inoculate it in 3 drops onto iron-free MKB plates (MKB iron-free liquid medium with 15 g agar added), with 3 replicates for each strain; incubate at 30℃ for 1 day; after clear single colonies have grown on the plates, pour a layer of CAS medium (Qingdao Haibo, weigh 4.35 g of medium powder, add 400 mL of ultrapure water, autoclave at 116℃ for 30 minutes) onto the plates with colonies, and incubate at 30℃ for 24 hours. Observe the color change of each plate, and measure the diameter of the siderophore chelation halo and the colony diameter, and calculate the siderophore solubility index (iron).

[0188] Siderophore solubility index = diameter of siderophore chelation halo / colony diameter

[0189] See results Figure 2 Bacillus stearothermiae MNA0003 produced a distinct orange-yellow siderophore chelating halo, with a siderophore solubility index of 2.41, indicating strong affinity for Fe. 3+ It has strong chelating power.

[0190] Example 5. Test on the antifungal activity of Bacillus stearothermiae MNA0003 strain against pathogenic fungi.

[0191] (1) Preparation of pathogens

[0192] Pathogenic fungi: Colletotrichum siamense (Cs), Fusarium graminearum (Fg), and Magnaphorthe oryzae (Mo).

[0193] The pathogenic fungus was activated on PDA plates (200g potato, 20g glucose, 15-20g agar, 1L distilled water, sterilized at 121℃ for 20min), and cultured at 30℃ for 5 days for later use.

[0194] (2) Preparation of antagonistic bacterial cake

[0195] The MNA0003 bacterial plate from step 1 of Example 2 was used to make bacterial cakes with a diameter of 6 mm using a sterilized pipette tip.

[0196] (3) Plate antagonism: Plate antagonism against pathogenic fungi

[0197] After 5 days of cultivation, 8 mm-sized bacterial discs were formed along the edge of the colony of the pathogen. These bacterial discs were then inoculated into the center of an LB agar plate (formula: 5 g yeast extract, 10 g casein peptone, 10 g sodium chloride, 15 g agar, 1 L water, pH = 7). Antagonistic bacterial discs were then inoculated around the same plate with an equal radius. A blank control was prepared by inoculating only the pathogen bacterial discs. Three replicates were performed. The plates were incubated at 30°C for 5 days. The results were observed, photographed, and the growth radius of the pathogen was measured using the cross-cross method.

[0198] Wherein, the inhibition rate (%) = (radius of control group - radius of treatment group) / radius of control group × 100%.

[0199] The results are shown in Table 5 below. (See illustration below.) Figure 3 The results showed that strain MNA0003 exhibited significant antagonistic activity against three different pathogenic fungi (anthrax fungus, Fusarium graminearum, and rice blast fungus), demonstrating broad-spectrum antifungal activity.

[0200] Table 5: Antimicrobial activity of MNA0003 against pathogenic fungi

[0201]

[0202] Example 6. Nitrogen fixation capacity test of Bacillus stearothermiae MNA0003 strain

[0203] Using a sterile inoculation loop, scrape an appropriate amount of MNA0003 cells from the plate in step 1 of Example 2, resuspend them in 1 mL of sterile water, and inoculate them evenly onto Ashby plates (formulation: 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5.0 g calcium carbonate, 10.0 g mannitol, 0.1 g calcium sulfate, 15.0 g agar, 1 L water, pH = 7.0 ± 0.1). Perform 3 replicates on each plate. After incubation in the dark at 30°C for 4–6 days, observe the growth of the strain on the Ashby plates and select rapidly growing strains to measure the colony diameter.

[0204] The results are as follows Figure 4 As shown, MNA0003 can grow on Ashby medium without nitrogen source, and after 5 days of culture, the colony diameter is greater than 4 mm, indicating nitrogen fixation potential.

[0205] Example 7. Biofilm experiment of Bacillus stearothermiae strain MNA0003

[0206] The MNA0003 strain from Example 2 was inoculated into TSB liquid medium and cultured for 24 hours in a clean bench at 30°C and 200 rpm. The OD of the bacterial culture was measured using a spectrophotometer. 600 The value was adjusted to OD using TSB medium. 600=1. Pipette 100 μL of bacterial culture adjusted to OD600 = 1 into a 96-well plate and incubate for 24 hours. Wash once with 200 μL of PBS solution, then place the 96-well plate in a 60°C oven for 1 hour to fix the biofilm. Next, add 50 μL of 0.4% crystal violet solution to the wells for staining for 15 minutes, then wash twice with 200 μL of PBS. Dry at 37°C for 10 minutes until the liquid is completely dry, add 200 μL of 70% ethanol, and then measure the absorbance at 600 nm using a microplate reader.

[0207] The average of three replicates of the measured absorbance (D) was taken, and twice the average value of the negative control Ck was used as the cutoff value (Dc). When D > 2 × Dc, the strain was determined to be a strong biofilm-forming strain; when Dc < D ≤ 2 × Dc, it was a weak biofilm-forming strain; and when D ≤ Dc, it was a non-biofilm-forming strain.

[0208] The test results are shown in Table 6, and the images are shown in [images not provided]. Figure 5 This indicates that MNA0003 is a strain with strong biofilm formation. Strong biofilm formation ability enhances the adhesion of microorganisms to complex environments and promotes their growth. Strains with strong biofilm formation ability are more likely to survive in different environments and effectively control nematodes.

[0209] Table 6: Biomembrane Experiments with MNA0003

[0210] strain D Standard error Dc 2XDc determination MNA0003 0.777 0.103 0.286 0.572 Strong biofilm-forming strains

[0211] Example 8. Colonization experiment of Bacillus stearothermiae strain MNA0003

[0212] Soak corn seeds in 75% alcohol for 3 minutes, rinse twice with sterile water, and repeat twice. Then soak in 5% sodium hypochlorite for 5 minutes, and rinse three times with sterile water. After washing, the seeds were placed on 1 / 2 MS medium plates (containing: potassium nitrate 0.95 g / L, ammonium nitrate 0.825 g / L, potassium dihydrogen phosphate 0.085 g / L, magnesium sulfate 0.09 g / L, calcium chloride 0.165 g / L, potassium iodide 0.00083 mg / L, boric acid 0.0062 mg / L, manganese sulfate 0.0169 mg / L, zinc sulfate 0.0086 mg / L, sodium molybdate 0.00025 mg / L, copper sulfate 0.000025 mg / L, inositol 0.1 g / L, cobalt chloride 0.000025 mg / L, glycine 0.002 mg / L, sodium EDTA 0.03726 mg / L, vitamin B1 0.0001 mg / L, ferrous sulfate 0.0152 mg / L, niacin 0.0005 mg / L, vitamin B6). Incubate at 22°C for 5 days on 0.0005 mg / L agar (10 g / L).

[0213] The MNA0003 fermentation broth prepared in Example 2 was diluted to OD. 600 =1. Select uncontaminated, uniformly growing seeds and soak them in bacterial solution with adjusted OD for 1 hour. Dry the seeds and transplant them into glass tubes containing 1 / 2 MS medium, one tube at the top and one at the bottom. Seal the tubes and incubate at 22℃ for 14 days. Each strain has 3 replicates.

[0214] Samples were taken at 7 and 14 days. For each sample, 1 mL of 1‰ Tween water and 3 sterile glass beads were added to a 2 mL EP tube. Using sterile scissors, 1 cm sections of corn seedling roots were cut and placed into the EP tubes. The tubes were shaken for 15-30 seconds, then sonicated for 1 minute. This process was repeated twice. After shaking, the samples were serially diluted and plated. The plates were incubated overnight at 30°C. After a clear colony count appeared, the colony counts were counted at each concentration gradient, and the colony forming units (CFU) of the samples were calculated.

[0215] The experimental data are shown in Table 7. The results show that MNA0003 can colonize the roots of corn, thereby effectively protecting the plant and preventing the invasion of pests and diseases.

[0216] Table 7: Colonization experiment of strain MNA0003

[0217]

[0218] Example 9. Thrips pathogenicity test of Bacillus steggii strain MNA0003

[0219] Western flower thrips: In the laboratory, clean insect sources were continuously reared for more than 15 generations using peanut seedlings. The rearing conditions were: 25±2℃, relative humidity 55±1%, and a light cycle of 12h:12h. Adult western flower thrips with uniform growth were selected as the insect source for the experiment.

[0220] Treatment method: The immersion method was used. The test fermentation broth prepared in Example 2 was diluted 5 times with ddH2O. Adult western flower thrips of uniform growth were placed in a 5 mL pipette tip, and sealed with absorbent cotton wrapped in 200-mesh gauze (sterilized). 20 thrips were placed per device, with 3 replicates, and the actual count was used as the standard. 0.025% Tween-80 sterile solution was used as a negative control (CK). The diluted fermentation broth or control solution was drawn into the pipette tip and immersed in adult western flower thrips for 5 seconds. The devices containing thrips after treatment were placed in an insect chamber at 25±2℃, 55% relative humidity, and a photoperiod of 12h:12h. The survival of the thrips was observed daily, and they were fed 25% honey water every other day. The number of dead thrips was counted after 3 days.

[0221] Calculate the mortality rate and adjusted mortality rate using the following formulas:

[0222] Mortality rate (%) = (Number of dead insects / Number of tested insects) × 100%;

[0223] Corrected mortality rate (%) = [(treatment mortality rate - negative control mortality rate) / (1 - negative control mortality rate)] × 100%.

[0224] The experimental results are shown in Table 8 below. The results indicate that the MNA0003 fermentation broth has a certain pathogenicity towards thrips.

[0225] Table 8: Thrips pathogenicity of strain MNA0003

[0226] deal with OD600 Number of test insects Number of dead insects in 4 days mortality rate / % CK / 30 3 10.0 MNA0003 0.94 30 12 40.0

[0227] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of Bacillus spizizenii or its progeny, characterized in that, It is any one of the following A1) to A5): A1) Strains whose 16S rRNA has at least 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the sequence shown in SEQ ID NO: 1, and / or whose gyrB gene has at least 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the sequence shown in SEQ ID NO: 2; A2) Strains with an average nucleotide identity (ANI) of at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% with the Bacillus spizizenii type strain; A3) The strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 64116; A4) Strains whose 16S rRNA gene has at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the 16S rRNA gene of the strain described in A3), and / or whose gyrB gene sequence has at least 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identity with the gyrB gene sequence of the strain described in A3); A5) Strains whose genomes are at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to the genomes of the strains described in A3); Strains of strains A6), A1), A2), A4), and A5 that produce at least one metabolite of chrysophanol, cyclic proline-phenylalanine, or cyclic (L-proline-L-valine), and / or strains whose genome contains gene clusters that synthesize at least one secondary metabolite of bacilysin, pulcherriminic acid, mycosubtilin, surfactantin, subtilin, rhizocticin A, or bacillibactin.

2. The culture of Bacillus stearothermiae according to claim 1, characterized in that, Includes any one of the following: B1) to B5) B1) The fermentation broth of Bacillus stearothermiae; B2) Metabolites of Bacillus stearothermiae; B3) The supernatant of the fermentation broth of Bacillus stearothermiae; B4) The concentrated or dried products of B1), B2) or B3); Preferably, the culture medium used for the preparation of the culture is any one of LB medium, TSB medium, and TSA medium, and / or the culture temperature is 28-32°C, and / or the culture time is 2-10 days; Preferably, the metabolites or fermentation broth supernatant include at least one of antibacterial compounds and insecticidal active compounds; Preferably, the antibacterial compound is selected from at least one of bacilysin, subtilin, fengycin, surfactantin, and mycosubtilin. Preferably, the insecticidal active compound includes at least one of Chrysophanol, acetophenone, cyclo(L-proline-L-valine) (Cyclo(Pro-Val)), cyclo(L-proline-L-leucine) (Gancidin W), and cyclo(proline-phenylalanine) (Cyclo(Pro-Phe)).

3. A microbial inoculant, characterized in that, The microbial agent includes Bacillus stearothermii as described in claim 1, its inactivated form, and / or the culture as described in claim 2.

4. A composition, characterized in that, The composition comprises Bacillus stearothermiae of claim 1, its inactivated form, or the culture of claim 2.

5. A composition, characterized in that, The composition comprises an insecticidal active compound isolated from Bacillus stearothermiae, preferably, the insecticidal active compound comprising at least one of Chrysophanol, acetophenone, cyclo(L-proline-L-valine) (Cyclo(Pro-Val)), cyclo(L-proline-L-leucine) (Gancidin W), and cyclo(proline-phenylalanine) (Cyclo(Pro-Phe)).

6. The composition according to claim 4 or 5, characterized in that, The composition further comprises additional activators and / or excipients; Preferably, the adjuvant activator is selected from one or more biological control agents, one or more chemical control agents, one or more fertilizers, one or more herbicides, one or more plant growth promoters, or any combination thereof; Preferably, the biological control agent includes: bacterial, fungal, viral biological control agents, insect biological control agents, and nematode biological control agents; Preferably, the biocontrol agent is selected from biocontrol bacteria of the genera *Burkholderia*, *Penicillium*, *Trichoderma*, or *Bacillus*, or any combination thereof, or is selected from metabolites of biocontrol bacteria of the genera *Burkholderia*, *Penicillium*, *Trichoderma*, or *Bacillus*, or any combination thereof. More preferably, the bacteria of the genus Bacillus are selected from: Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus coagulans, Paenibacillus polymyxa, Bacillus myloliquefaciens, or any combination thereof; More preferably, the first biocontrol agent is selected from at least one of Trichoderma harzianum, Purpureocillium lilacinum, Penicillium bilaiae, Bacillus subtilis, Burkholderia rinojensis, Bacillus pumilus, Bacillus velezensis, Methylobacterium rhodesianum, Methylobacterium extorquens, and Paenibacillus peoriae; More preferably, the *Trichoderma harzianum* is *Trichoderma harzianum* with accession number CGMCC NO.15679; More preferably, the *Paecilomyces lilacinus* is *Paecilomyces lilacinus* with accession number CGMCC NO.12773; More preferably, the Penicillium baqensis is Penicillium baqensis with accession number CGMCC NO.12767; More preferably, the Bacillus subtilis is Bacillus subtilis with accession number CGMCC NO.12908; More preferably, the *Burkholderia renjohnsonii* is *Burkholderia renjohnsonii* with accession number GDMCC NO.61156; More preferably, the *Bacillus pumilus* is the *Bacillus pumilus* with accession number GDMCC NO.61962; More preferably, the Bacillus belyssus is Bacillus belyssus with accession number GDMCC NO.61434; More preferably, the *Methylobacterium roximatee* is *Methylobacterium roximatee* with accession number GDMCC NO.60729; More preferably, the *Methylobacterium tumefaciens* is *Methylobacterium tumefaciens* with accession number GDMCC NO.62943; More preferably, the *Bacillus pilaris* is *Bacillus pilaris* with accession number GDMCC NO.60482; Preferably, the chemical control agent is a chemical control agent whose target does not include bacteria; Preferably, the chemical control agent is a fungicide, insecticide, acaricide, nematicide, rodenticide, herbicide, or ablative agent; Preferably, the plant growth promoter includes plant growth regulators, medium-quantity nutrients, micronutrients, humates, fulvic acids, or any combination thereof; Preferably, the medium-nutrient elements include calcium salts, magnesium salts, sulfates, sulfites, or any combination thereof, and the micronutrient elements include iron salts, borates, manganese salts, copper salts, zinc salts, molybdenum salts, chloride salts, or any combination thereof. Preferably, the excipients are selected from agriculturally or horticulturally acceptable carriers, diluents, stabilizers, fillers, wetting agents, colorants, solvents, co-solvents, film-forming agents, spontaneous growth promoters, emulsifiers, dispersants, preservatives, antifreeze agents, thickeners, adjuvants, or any combination thereof; Preferably, the composition is in solid form, liquid form, powder form, or any combination thereof; Preferably, the composition is selected from at least one of solutions, powders, granules, emulsions, emulsions, suspensions, tablets, microparticles, and wettable powders.

7. A plant growth regulator, seed coating agent, root drenching agent, seed soaking agent, microbial insecticide, microbial fertilizer, microbial pesticide, or biostimulant, characterized in that, It includes Bacillus stearothermiae as described in claim 1, its inactivated form, the culture as described in claim 2, the microbial agent as described in claim 3, or the composition as described in any one of claims 4-6.

8. The use of Bacillus spizizenii as described in claim 1, the culture as described in claim 2, the microbial agent as described in claim 3, or the composition as described in any one of claims 4-6 in the preparation of products with the function of preventing and controlling plant diseases and pests or products with the function of promoting plant growth; Preferably, the pests and diseases include fungal diseases, bacterial diseases, nematode pests, and / or insect pests. More preferably, the plant pathogenic fungi include Rhizoctonia, Colletotrichum, Fusarium, or Magnaporthe. Preferably, the *Rhizoctonia* genus includes *Rhizoctonia solani*; the *Anthracnose* genus includes *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, *Glomerella cingulata*, and *Colletotrichum capsici*; the *Fusarium* genus includes *Fusarium graminearum*, *Fusarium commune*, and *Fusarium oxysporum*; and the *Magnaporthe* genus includes *Magnaporthe oryzae*. Preferably, the pathogens of the nematode pests include root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp.), and root-rot nematodes (Pratylenchus spp.); Preferably, the root-knot nematodes include, but are not limited to, one or more of the following: southern root-knot nematode (Meloidogyne incognita), northern root-knot nematode (Meloidogyne hapla), Javan root-knot nematode (Meloidogyne javanica), and peanut root-knot nematode (Meloidogyne arenaria). Preferably, the pathogens of the insect pests include Thysanoptera pests or Arachnida pests; Preferably, the thrips pests of the order Thysanoptera include at least one of the following: *Thrips flevas Schrank*, *Thrips alliorum*, *Stenchaetothrips biformis*, *Frankliniella occidentalis*, *Frankliniella intonsa*, *Thrips palmi Karny*, *Megalurothrips usitatus*, and *Scirtothrips dorsalis*. Preferably, the arachnid pests include pests from the superfamily Tardiformes, Tetranychus, and Erythromorpha; Preferably, the arachnid pests include at least one of the following: Cyclamen tarsiforme, Cyclamen schoenleinii, Cyclamen lateralis, alfalfa moth, wheat moth, fruit moth, jujube leaf mite, cotton leaf mite, two-spotted leaf mite, hawthorn leaf mite, cinnabar leaf mite, truncated leaf mite, citrus leaf mite, apple leaf mite, citrus leaf mite, six-spotted leaf mite, cypress leaf mite, rice leaf mite, bamboo leaf mite, millet gall mite, and tulip mite.

9. The use of Bacillus stearothermii of claim 1, the culture of claim 2, the microbial agent of claim 3, the composition of any one of claims 4-6, or the plant growth regulator, seed coating agent, root irrigation agent, seed soaking agent, microbial insecticide, microbial fertilizer, microbial pesticide or biostimulant of claim 7 in the prevention and control of plant diseases and pests or in promoting plant growth. Preferably, the pests and diseases include fungal diseases, bacterial diseases, nematode pests, and / or insect pests. More preferably, the plant pathogenic fungi include Rhizoctonia, Colletotrichum, Fusarium, or Magnaporthe. Preferably, the *Rhizoctonia* genus includes *Rhizoctonia solani*; the *Anthracnose* genus includes *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, *Glomerella cingulata*, and *Colletotrichum capsici*; the *Fusarium* genus includes *Fusarium graminearum*, *Fusarium commune*, and *Fusarium oxysporum*; and the *Magnaporthe* genus includes *Magnaporthe oryzae*. Preferably, the pathogens of the nematode pests include root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp.), and root-rot nematodes (Pratylenchus spp.); Preferably, the root-knot nematodes include, but are not limited to, one or more of the following: southern root-knot nematode (Meloidogyne incognita), northern root-knot nematode (Meloidogyne hapla), Javan root-knot nematode (Meloidogyne javanica), and peanut root-knot nematode (Meloidogyne arenaria). Preferably, the pathogens of the insect pests include Thysanoptera pests or Arachnida pests; Preferably, the thrips pests of the order Thysanoptera include at least one of the following: *Thrips flevas Schrank*, *Thrips alliorum*, *Stenchaetothrips biformis*, *Frankliniella occidentalis*, *Frankliniella intonsa*, *Thrips palmi Karny*, *Megalurothrips usitatus*, and *Scirtothrips dorsalis*. Preferably, the arachnid pests include pests from the superfamily Tardiformes, Tetranychus, and Erythromorpha; Preferably, the arachnid pests include at least one of the following: Cyclamen stenoptera, Cyclamen stenoptera, Cyclamen lateralis, alfalfa moth, wheat mite, fruit moth, jujube leaf mite, cotton leaf mite, two-spotted leaf mite, hawthorn leaf mite, carmine leaf mite, truncate leaf mite, citrus pterocaryon, apple pterocaryon, citrus leaf mite, six-spotted leaf mite, cypress leaf mite, rice leaf mite, bamboo leaf mite, millet gall mite, and tulip mite. Preferably, promoting plant growth includes promoting the absorption of nutrients by the plant; Preferably, promoting plant growth includes promoting the absorption of macroelements and / or microelements by plants; Preferably, promoting plant growth includes providing a nitrogen source to the plant and promoting the plant's absorption of nitrogen and / or iron ions; Preferably, promoting plant growth includes reducing root knots, increasing the dry weight of individual plants, increasing leaf spread, increasing stem diameter, and / or promoting leaf greening.

10. The use of the Bacillus stearotherm of claim 1, the culture of claim 2, the microbial agent of claim 3, and the composition of any one of claims 4-6 in soil remediation, soil improvement, or nitrogen fixation; Preferably, the soil remediation or soil improvement aims to enhance the availability of soil nutrients, and preferably includes improving the supply of nitrogen and / or generating iron carriers.

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