A strain of *Bacillus polymyxa* and its application in biocontrol and growth promotion.

Abstract

This invention belongs to the field of microbial and biological control technology, specifically relating to a strain of *Bacillus polymyxa* and its application in biocontrol and growth promotion. Specifically, this invention screens and obtains a strain of *Bacillus polymyxa* (… Paenibacillus polymyxa GLZ219, as demonstrated in experiments, exhibits a high inhibition rate against the pathogen causing tomato neck and root rot; simultaneously, this strain also... Rhizoctonia solani It exhibits good antagonistic effects against nine common plant pathogens and can be widely applied to the green control of various crop diseases. Furthermore, *Bacillus polymyxa* GLZ219 possesses nitrogen-fixing and potassium-solubilizing abilities, and can secrete indoleacetic acid and siderophores, effectively promoting tomato plant growth. Tomato seedlings treated with its fermentation broth show a significant increase in plant height. It can control diseases, improve crop growth, and increase crop yield and quality, demonstrating broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial and biological control technology, specifically relating to a strain of polymyxa Bacillus and its application in biocontrol and growth promotion. Background Technology

[0002] tomato( Solanum tomato Tomatoes, as an important economic crop and vegetable widely cultivated globally, have yields and quality that directly affect agricultural economic benefits and market supply stability. However, during the winter and spring production seasons of greenhouse tomatoes, the low-temperature, high-humidity, enclosed environment leads to frequent outbreaks of soil-borne fungal diseases, among which Fusarium oxysporum tomato neck rot and root rot (… Fusarium oxysporum Tomato neck rot and root rot caused by *F. sp. radicis-lycopersici* (Forl.) has become one of the key diseases restricting the safe production of greenhouse tomatoes.

[0003] Currently, the control of tomato neck and root rot still relies primarily on traditional chemical pesticides, such as pyraclostrobin, for soil treatment or root irrigation. While chemical control can achieve some short-term efficacy, long-term and excessive use of chemical pesticides easily leads to the following problems: First, pathogens develop resistance, causing pesticide efficacy to decline year by year, necessitating increased dosage and frequency of application, creating a vicious cycle of "pesticide abuse - increased resistance - reduced efficacy"; second, the accumulation of chemical pesticide residues in the soil disrupts the soil microbial community structure, leading to soil degradation, and also pollutes water bodies, the atmosphere, and other ecological environments through food chain enrichment or surface runoff; third, excessive pesticide residues directly affect the quality and safety of tomato fruits, posing a potential threat to human health, which contradicts current consumer demand for green and safe agricultural products and the green development strategy of agriculture. Therefore, developing safe, efficient, and sustainable disease control technologies has become an urgent need for the development of the tomato industry. Biological control, due to its advantages such as environmental friendliness, low resistance rate, and safety for humans and animals, has become an important direction for replacing chemical control. Among them, the use of plant rhizosphere growth-promoting bacteria (PGPR) for biological control of diseases is a current research hotspot in the field of agricultural microbiology.

[0004] Polymyxin Bacillus ( Paenibacillus polymyxa As an important class of PGPR strains, *Polymyxobinus* has been reported to have antagonistic effects against some soil-borne pathogens. However, there are few highly effective antagonistic strains specifically targeting Tomato neck and root rot (Forl), and existing strains generally suffer from narrow antibacterial spectrum, unstable efficacy, and unclear biocontrol mechanisms, making it difficult to meet actual production needs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain of *Bacillus polymyxa* and its application in biocontrol and growth promotion. Specifically, experimental verification shows that this strain exhibits excellent control effects against tomato neck rot and root rot, with a broad antibacterial spectrum and strong growth-promoting ability. Furthermore, its biocontrol and growth-promoting mechanisms have been clarified, providing a new effective strain and theoretical basis for the green control of plant diseases such as tomato neck rot and root rot. Based on the above research results, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions:

[0007] In a first aspect, the present invention provides a strain of *Bacillus polymyxa* (…). Paenibacillus polymyxa GLZ219, this strain was deposited on April 24, 2022 at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 24766.

[0008] A second aspect of the present invention provides a fermentation production method for the above-mentioned Bacillus polymyxa, the fermentation production method comprising: inoculating the Bacillus polymyxa into a fermentation medium for fermentation culture.

[0009] A third aspect of the present invention provides a microbial inoculant containing the aforementioned Bacillus polymyxa or its ferments or metabolites.

[0010] The aforementioned microbial agents can be pathogen inhibitors or disease inhibitors.

[0011] A fourth aspect of the present invention provides the use of the above-described Bacillus polymyxa GLZ219 and / or the above-described microbial agent in any one or more of the following (a)-(e):

[0012] (a) Inhibiting plant pathogens or preparing plant pathogen inhibitors;

[0013] (b) Suppressing plant diseases or preparing plant disease inhibitors;

[0014] (c) Promoting plant growth or preparing plant growth promoters;

[0015] (d) Nitrogen fixation and potassium solubilization;

[0016] (e) Production of indoleacetic acid (IAA) and iron carriers.

[0017] In a fifth aspect, the present invention provides a microbial fertilizer, wherein the active ingredients of the microbial fertilizer include the above-mentioned Bacillus polymyxa GLZ219 and / or the above-mentioned microbial agent.

[0018] A sixth aspect of the present invention provides a method for preventing and controlling plant diseases and / or promoting plant growth, the method comprising applying the above-mentioned Bacillus polymyxa GLZ219, the above-mentioned microbial inoculant and / or the above-mentioned microbial fertilizer to tomatoes (such as tomato roots) and / or tomato growing environment (such as tomato rhizosphere soil), thereby inhibiting the growth of plant pathogens and achieving prevention and control of plant diseases; and promoting the plant height of tomatoes.

[0019] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0020] The above-mentioned technical method isolated a plant rhizosphere growth-promoting bacterium from tomato rhizosphere soil, named GLZ219. Physiological and biochemical methods, as well as 16S rRNA sequencing alignment and phylogenetic tree construction, confirmed that strain GLZ219 is a *Bacillus polymyxa* (*Bacillus polymyxa*). Paenibacillus polymyxa Experiments have shown that this strain exhibits an inhibition rate of up to 78.91% against the pathogen causing tomato neck and root rot, with a potted plant control efficacy of 82.46%, superior to 30% pyraclostrobin suspension. Furthermore, this strain also demonstrates resistance to… Rhizoctonia solani, Botrytis cinerea It exhibits good antagonistic effects against nine common plant pathogens and can be widely applied to the green control of various crop diseases. Furthermore, *Bacillus polymyxa* GLZ219 possesses nitrogen-fixing and potassium-solubilizing abilities, and can secrete indoleacetic acid (IAA) and siderophores, effectively promoting tomato plant growth. Tomato seedlings treated with its fermentation broth show a significant increase in plant height. It can control diseases, improve crop growth, and increase crop yield and quality, demonstrating broad application prospects. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This invention demonstrates the inhibitory effect of strain GLZ219 on the mycelial growth of *Fusarium oxysporum* var. *tomatoides*, a specialized form of *Fusarium oxysporum*, on the neck and root rot of tomato. A shows the normal colony morphology of *Fusarium oxysporum* var. *tomatoides* on a PDA plate; B shows the colony morphology of *Fusarium oxysporum* var. *tomatoides* on a PDA plate inhibited by GLZ219; C shows the normal mycelial morphology at the colony edge (scale bar: 100 μm); D shows the inhibited mycelial morphology at the colony edge (scale bar: 100 μm); E shows a close-up of the inhibited mycelial morphology at the colony edge; F shows normal conidial germination and sporulation (scale bar: 20 μm); G shows the inhibited conidial germination state (scale bar: 20 μm).

[0023] Figure 2Morphological characteristics of strain GLZ219 in this embodiment of the invention: A: Colony morphology; B: Gram staining observation; C: Scanning electron microscopy observation.

[0024] Figure 3 This is a phylogenetic tree constructed using the neighbor-joining method based on 16S RNA sequences in an embodiment of the present invention.

[0025] Figure 4 This invention illustrates the inhibitory effect of strain GLZ219 on pathogenic fungi in an embodiment of the invention, where A represents... Pythium aphaniderm B is Rhizoctonia solani C is Colletorichum lagenerium D is Fusarium chlamydospore E is Fusarium gramineae F is Fusarium pseudogramineum G is Corynespora cassicola H is Colletotrichum acutatum I is Botrytis cinerea .

[0026] Figure 5 The following describes the extracellular enzyme production of strain GLZ219 in this embodiment of the invention, where A is protease, B is amylase, C is cellulase, and D is β-1,3-glucanase.

[0027] Figure 6 This invention relates to the growth-promoting effects and characteristics of strain GLZ219 in this embodiment. Specifically, A represents the growth-promoting effect of strain GLZ219 on tomato; B represents GLZ219 on potassium-rich bacteria solid culture medium; C represents the growth status of GLZ219 on Assumption nitrogen-fixing medium; and D represents the IAA production capacity assay of GLZ219, with P representing the positive control.

[0028] Figure 7 This invention illustrates the biocontrol effect of strain GLZ219 against tomato neck and root rot in this embodiment. Group A represents treatment 1; Group B represents treatment 2; Group C represents treatment 3; and Group D represents treatment 4. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0031] In a typical embodiment of the present invention, a strain of *Bacillus polymyxa* (…) is provided. Paenibacillus polygamous GLZ219, this strain was deposited on April 24, 2022 at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 24766.

[0032] In another specific embodiment of the present invention, a fermentation production method for the above-mentioned Bacillus polymyxa GLZ219 is provided, the fermentation production method comprising: inoculating the Bacillus polymyxa GLZ219 into a fermentation medium for fermentation culture.

[0033] In this invention, no specific limitation is made to the fermentation production method; any conventional bacterial fermentation culture method can be used for cultivation.

[0034] The fermentation medium can be any common bacterial culture medium, such as LB medium in one specific embodiment of the present invention.

[0035] In another specific embodiment of the present invention, a microbial inoculant is provided, which contains the aforementioned Bacillus polymyxa GLZ219 or its fermentation product or its metabolites.

[0036] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the fermentation culture of *Bacillus polymyxa* GLZ219, and therefore can also be called fermentation broth; the liquid may contain bacteria (cells), but does not necessarily need to contain bacteria. Preferably, the liquid contains metabolites produced by the *Bacillus polymyxa* GLZ219 of this invention.

[0037] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant." In the present invention, the supernatant contains extracellular metabolites of Bacillus polymyxa GLZ219. In embodiments of the present invention, the bacterial agent may also contain this supernatant.

[0038] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is centrifuged, filtered, settled, or otherwise known in the art to separate the bacterial cells grown in the fermentation broth or culture medium from the liquid to obtain bacterial cells. The bacterial cells can be broken up to obtain bacterial fragments. The breaking method can be ultrasound (e.g., ice bath ultrasound to break up cells) or other methods known in the art. Alternatively, the bacterial fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In the present invention, the bacterial fragments or cell-free extract contain intracellular metabolites of Bacillus polymyxa GLZ219. In embodiments of the present invention, the bacterial agent may also contain the bacterial fragments or cell-free extract.

[0039] Furthermore, in embodiments of the present invention, for ease of storage and transportation, and to improve the survival rate of the bacterial strain, the bacterial agent may also be a solid, and more preferably a freeze-dried powder. That is, it is obtained by further freeze-drying the aforementioned *Bacillus polymyxa* GLZ219 or its fermentation product or its metabolites. The freeze-drying technology (including vacuum freeze-drying technology) can be carried out using conventional methods, and will not be elaborated further here.

[0040] In another specific embodiment of the present invention, the microbial agent may further include excipients acceptable to the agent.

[0041] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

[0042] In another specific embodiment of the present invention, the above-mentioned microbial agent may specifically be a plant pathogen inhibitor or a plant disease inhibitor.

[0043] The active ingredient of the above-mentioned plant pathogen inhibitor may be Bacillus polymyxa GLZ219 or its fermentation product or its metabolites. The active ingredient of the above-mentioned plant pathogen inhibitor may also contain other biological or non-biological components. Other active ingredients of the above-mentioned plant pathogen inhibitor can be determined by those skilled in the art based on the inhibitory effect on plant pathogens.

[0044] The active ingredient of the above-mentioned plant disease inhibitor may be Bacillus polymyxa GLZ219 or its fermentation product or its metabolites. The active ingredient of the above-mentioned plant disease inhibitor may also contain other biological or non-biological components. Other active ingredients of the above-mentioned plant disease inhibitor can be determined by those skilled in the art based on their inhibitory effect on plant diseases.

[0045] In another specific embodiment of the present invention, the above-mentioned Bacillus polymyxa GLZ219 and / or microbial inoculant are used in any one or more of the following (a)-(e):

[0046] (a) Inhibiting plant pathogens or preparing plant pathogen inhibitors;

[0047] (b) Suppressing plant diseases or preparing plant disease inhibitors;

[0048] (c) Promoting plant growth or preparing plant growth promoters;

[0049] (d) Nitrogen fixation and potassium solubilization;

[0050] (e) Production of indoleacetic acid (IAA) and iron carriers.

[0051] The plant pathogens include at least the following: Fusarium oxysporum f. sp.radicis-lycopersici, Forl、 Rhizoctonia solani , Botrytis cinerea , Pythium aphanidermatum , Fusarium chlamydosporum , Fusarium gramineae , Corynespora cassicola , Colletorichum lagenerium , Colletotrichum acutatum and Fusarium pseudogramineum .

[0052] The plant diseases mentioned include, but are not limited to, plant diseases caused by the aforementioned plant pathogens, such as tomato neck rot and root rot, tomato damping-off, tomato gray mold, tomato seedling blight, root rot of cruciferous vegetables, wheat scab, cucumber leaf spot, cucurbit anthracnose, strawberry root rot, and wheat stem base rot.

[0053] The plant may be a crop, specifically a food crop or a cash crop, such as wheat, tomato, cucumber, strawberry, etc.

[0054] The promotion of plant growth specifically manifests as promoting plant height.

[0055] In another specific embodiment of the present invention, a microbial fertilizer is provided, wherein the active ingredients of the microbial fertilizer include the above-mentioned Bacillus polymyxa GLZ219 and / or the above-mentioned microbial agent.

[0056] In another specific embodiment of the present invention, a method for preventing and controlling plant diseases and / or promoting plant growth is provided. The method includes applying the above-mentioned Bacillus polymyxa GLZ219, the above-mentioned microbial inoculant and / or the above-mentioned microbial fertilizer to tomatoes (such as tomato roots) and / or tomato growing environment (such as tomato rhizosphere soil), thereby inhibiting the growth of plant pathogens and achieving prevention and control of plant diseases; and promoting the plant height of tomatoes.

[0057] The plant pathogens include Fusarium oxysporum, a specialized strain of Tomato neck and root rot, and the plant disease is Tomato neck and root rot.

[0058] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0059] Example

[0060] I. Material Preparation

[0061] 1. Test strain: *Tomato neck rot and root rot pathogen* ( Fusarium oxysporum f. sp. root - tomatoes Forl), the pathogen of tomato damping-off ( Rhizoctonia solani ), tomato gray mold pathogen ( Botrytis cinerea ), tomato damping-off pathogen ( Pythium aphanidermatum ), the pathogen of root rot in cruciferous vegetables ( Fusarium chlamydosporum Anthracnose pathogen of melons ( Colletotrichum lager Strawberry root rot pathogen ( Colletotrichum acutatum ), wheat scab pathogen ( Fusarium gramineae ), the pathogen of cucumber leaf spot disease ( ) Corynespora cassicola ) was isolated and preserved in this laboratory; the pathogen of wheat stem rot ( Fusarium pseudogramineum (This was kindly donated by the Institute of Plant Protection, Shandong Academy of Agricultural Sciences)

[0062] 2. Tomato variety tested: “Heinz1706” tomato variety, provided by the Solanaceous Eggplant Breeding and Cultivation Team of the Vegetable Research Institute of Shandong Academy of Agricultural Sciences.

[0063] 3. Culture media: Potato dextrose agar (PDA), potato dextrose broth (PDB), LB solid medium, LB liquid medium; protease detection medium, cellulase detection medium, starch hydrolysis medium, chitinase medium; iron ferrite detection solid medium, Assumption solid medium, silicate medium (potassium bacteria solid medium), Monkina organic phosphorus medium and Monkina inorganic phosphorus medium were purchased from Beijing Coollife Technology Co., Ltd.; R2A medium and YEPD medium containing tryptophan were prepared according to conventional formulas.

[0064] II. Strains Screening and Purification

[0065] Collect 10g of rhizosphere soil sample from tomatoes, place it in a sterile conical flask containing 90mL of sterile water, shake at 200r / min and 28℃ for 30min, and let stand for 5min to obtain soil suspension.

[0066] Take 100 μL of the supernatant from the soil suspension and dilute it sequentially with sterile water to a concentration of 10. -2 10 -3 10 -4 10 -5 10 -6 For each concentration, take 100 μL of the diluted solution and spread it evenly on LB solid medium plates. Set up 3 replicates for each dilution and incubate in an inverted incubator at 28°C for 3-5 days.

[0067] After single colonies appear on the plate, different single colonies are picked according to their morphology (color, size, edge, surface condition) and streaked on LB solid medium for purification. The streaking is repeated 3 times to obtain purified bacterial strains.

[0068] Activated Fusarium oxysporum tomato neck and root rot specific strain (Forl): Inoculate the pathogen onto PDA medium and incubate at 28°C for 7 days. Use a sterile punch (5 mm in diameter) to collect mycelial cakes from the edge of the colony.

[0069] Plate contrast screening: One Forl bacterial disc was inoculated in the center of PDA medium, and four purified bacterial strains (5 μL of bacterial suspension for each strain) were inoculated at a cross position 2.5 cm away from the bacterial disc. PDA plates inoculated only with Forl bacterial discs were used as controls. Each treatment was set up in 3 replicates and incubated at 28°C until the control colonies covered the plate.

[0070] The diameter of the control colony (D) and the diameter of the pathogen colony (d) in the treatment group were measured. The inhibition rate was calculated using the formula "inhibition rate = (D - d) / D × 100%". The strain with the highest inhibition rate was selected and named GLZ219, with an inhibition rate of 78.91%. The inhibited colonies became darker in color. Figure 1 (A to B); the hyphae growing normally at the edge of the colony are of uniform thickness and smooth. Figure 1 In the middle (C), the inhibited hyphae are dense, with significantly increased branching, swelling, twisting and deformity, and disordered growth direction. Figure 1 (D to E); GLZ219 treatment resulted in localized swelling or shrinkage of hyphae, and cell wall damage ( Figure 1 In contrast, the hyphae in the control group, which was not treated with antagonistic bacteria, were round and plump, and were able to produce conidia (F). Figure 1 (G). The above results indicate that GLZ219 can produce antagonistic active substances that inhibit the growth of Fusarium oxysporum, interfere with the branching pattern of hyphae, and disrupt the integrity of hyphal cell walls.

[0071] III. Strain Identification

[0072] 1. Morphological identification: GLZ219 was inoculated onto LB solid medium and incubated at 28°C for 4 days. Colonies were observed to be nearly round, viscous, milky white, with a moist and smooth surface and irregular edges. Figure 2 (A); Gram staining result was positive ( Figure 2 (B) Scanning electron microscopy revealed that the cells were rod-shaped and possessed peritrichous flagella ( Figure 2 (C)

[0073] 2. Physiological and biochemical characteristics determination: The determination was carried out in accordance with the "Handbook of Systematic Identification of Common Bacteria". The results showed (see Table 1): GLZ219 was positive for VP test, gelatin liquefaction, indole reaction, citrate utilization, starch hydrolysis and catalase reaction, and negative for methyl red test; it can tolerate 5% NaCl concentration; it can grow in an environment with pH 10, indicating that the strain has a certain degree of salt and alkali tolerance; at the same time, it can utilize glucose, sucrose, maltose, lactose and glycerol as carbon sources.

[0074] 3. Molecular biological identification:

[0075] Genomic DNA was extracted from GLZ219 and PCR amplification of the 16S rRNA gene was performed using primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.1) and primer 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO.2). The target fragment of approximately 1500 bp was obtained and detected by 1.0% agarose gel electrophoresis, showing clear and uniform bands.

[0076] After sequencing the PCR product, a BLAST comparison was performed in the NCBI database. The results showed that the sequence matched the accession number NR_114810. Paenibacillus polymyxa The similarity to strain DSM 36 was 99.03%.

[0077] A phylogenetic tree was constructed using MEGA 7.0 software, and the results are as follows: Figure 3 As shown, the results indicate that strain GLZ219 and Paenibacillus polymyxa strain CF05 (accession number JX997944) Paenibacillus polygamous strain SQR-21 (accession number FJ600406) Paenibacillus polymyxa strain DSM 36 (accession number NR_114810) Paenibacillus polymyxa strain IAM 13419 (accession number NR_112117) clusters into a single branch, with a high confidence level.

[0078] Based on the comprehensive analysis of morphological, physiological and biochemical characteristics, and 16S rRNA gene sequence (nucleotide sequence as shown in SEQ ID NO.3), strain GLZ219 was identified as a polymyxinobacterium (Bacillus polymyxa). Paenibacillus polymyxa This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24766.

[0079] Table 1. Physiological and biochemical characteristics of GLZ219

[0080]

[0081] In Table 1, "+" indicates a positive reaction or that GLZ219 has this characteristic, and "-" indicates a negative reaction or that GLZ219 does not have this characteristic.

[0082] IV. Functional Verification of Strains

[0083] 1. Antibacterial spectrum determination:

[0084] Nine tested pathogenic fungi were inoculated into the center of PDA medium, and GLZ219 bacterial suspension (5 μL / point) was inoculated at a cross position 2.5 cm away from the fungal block. Each treatment was set up in 3 replicates, and cultured at 28°C until the control colonies covered the plate.

[0085] The results are as follows Figure 4 As shown in A to I, strain GLZ219 is effective against... Pythium aphanidermatum , Rhizoctonia nightshade , Colletotrichum lagenerium , Fusarium chlamydosporum , Fusarium graminearum , Fusarium pseudograminearum , Corynespora cassiicola , Colletotrichum Acutatum and Botrytis cinerea All nine common plant pathogens showed good inhibitory effects, with clearly visible inhibition zones, indicating a broad spectrum of inhibition.

[0086] 2. Research on antibacterial mechanisms:

[0087] Extracellular enzyme secretion capacity: GLZ219 cells were inoculated into protease detection medium, starch hydrolysis medium, cellulase detection medium, β-1,3-glucanase detection medium, and chitinase medium, respectively, and cultured at 28°C for 5 days. Results are as follows: Figure 5 As shown, on the protease, starch hydrolysis, cellulase, and β-1,3-glucanase detection media, a clear halo appeared around the colony, while no clear halo was observed on the chitinase detection media; on the CAS detection media, a faint orange-yellow halo appeared around the colony, indicating that GLZ219 can secrete proteases (…). Figure 5 (A) amylase ( Figure 5 (B) Cellulase ( Figure 5 (C), β-1,3-glucanase ( Figure 5 (Middle D), has a weak ability to produce heptaphiles, but does not produce chitinase.

[0088] Effects on pathogenic mycelial growth: Microscopic observation revealed that Forl hyphae treated with GLZ219 exhibited denser growth, significantly increased branching, swelling, and twisting deformities, with disordered growth direction; some hyphae showed localized swelling or wrinkling and depression, and cell wall damage; while the control group hyphae were uniform in thickness, smooth, round and plump, and could produce conidia, indicating that GLZ219 can destroy the mycelial structure of pathogens and inhibit their growth.

[0089] 3. Measurement of fertility-promoting capacity:

[0090] Nitrogen fixation ability: After GLZ219 was inoculated into Assumption nitrogen-fixing medium and cultured at 30°C for 3 days, the strain could grow normally, indicating that it has nitrogen fixation ability. Figure 6 (C)

[0091] Potassium solubilization ability: When strain GLZ219 was inoculated onto a potassium-rich bacteria solid culture medium and cultured at 28°C for 3 days, the strain grew normally, indicating that it possesses potassium solubilization ability. Figure 6 (B)

[0092] Siderophore production capacity: When strain GLZ219 was inoculated into CAS detection medium and cultured at 30°C for 6 days, an orange-yellow halo appeared around the colony, indicating that it could produce siderophores.

[0093] Phosphorus solubility: When strain GLZ219 was inoculated into Mongkina organic phosphorus medium and inorganic phosphorus medium and cultured at 30°C for 5 days, no transparent zone appeared on either medium, indicating that it does not have phosphorus solubility.

[0094] Indoleacetic acid (IAA) secretion capacity: After the GLZ219 bacterial suspension reacted with the colorimetric solution, the color turned pink. The colorimetric solution with added indoleacetic acid in the control group also turned pink, while the sterile water control group was colorless, indicating that GLZ219 can secrete indoleacetic acid (IAA). Figure 6 (D).

[0095] 4. Tomato growth-promoting effect experiment:

[0096] "Heinz1706" tomato seeds were soaked in 55℃ warm water for 30 minutes, kept moist at 28℃ for germination, and then sown. When the seedlings had grown to 2 to 3 true leaves, seedlings with uniform growth were selected and divided into treatment groups (5 mL root drenching at a concentration of 1×10⁻⁶). 8 The fermentation broth of strain GLZ219 (CFU / mL) and the control group (root irrigation with 5 mL of sterile water) were used, with 10 strains in each group and repeated 3 times.

[0097] Plant height was measured after 21 days of cultivation, and the results are as follows: Figure 6 As shown in Figure A, the average plant height of tomatoes in the treatment group increased by 24.4% compared with that in the control group, which is a significant difference, indicating that GLZ219 has a significant growth-promoting effect on tomatoes.

[0098] 5. Potted plant efficacy test for controlling tomato neck and root rot:

[0099] When the tomato seedlings have grown to 2 to 3 true leaves, four treatment groups were set up:

[0100] Treatment 1: Root irrigation with 5 mL of 1×10⁻⁵ mL solution only. 8 GLZ219 fermentation broth at CFU / mL;

[0101] Treatment 2: First, drench the roots with 5 mL of a 1×10 solution. 8 GLZ219 fermentation broth at CFU / mL, drenched with 1×10⁻⁶ CFU / mL for root irrigation after 1 day. 7 A suspension of Forl conidia;

[0102] Treatment 3: First spray with 30% pyraclostrobin suspension (diluted 1000 times), then drench the roots with 1×10⁶ spores one day later. 7 A suspension of Forl conidia;

[0103] Treatment 4: Root irrigation only 1×10 7 A suspension of Forl conidia.

[0104] Investigate disease incidence 30 days after inoculation and classify the disease according to the following standards: Grade 0: No disease in the plant; Grade 1: A few brown spots at the base of the stem, no root rot; Grade 2: The brown spots at the base of the stem enlarge, some roots rot, and the leaves wilt slightly; Grade 3: Brown lesions surround the base of the stem, most of the roots rot, and the leaves wilt significantly; Grade 4: The plant withers and dies.

[0105] The disease index and preventive efficacy were calculated, and the results showed (see Table 2 and...). Figure 7 (A to D): Treatment 4 (blank control) showed the highest disease index, indicating severe neck and root rot; Treatment 2 (GLZ219) achieved a control efficacy of 82.46%; Treatment 3 (chemical agent) had a control efficacy of 79.83%, indicating that GLZ219 has excellent control effects against tomato neck and root rot, and its efficacy is superior to that of 30% pyraclostrobin suspension. The results show that root irrigation with GLZ219 fermentation broth has good control efficacy against tomato neck and root rot.

[0106] Table 2. Control efficacy of GLZ219 against tomato neck rot and root rot in potted plants.

[0107]

[0108] V. Whole Genome Sequence Analysis

[0109] 1. Basic genomic information: The genome of strain GLZ219 is 5,669,264 bp in size, with a GC content of 45.53%, containing 5,018 protein-coding genes, accounting for 85.45% of the total genome length; in addition, the genome also contains 81 tRNAs, 10 rRNAs, 108 simple repeat sequences, 39 scattered repeat sequences, 5 short palindromic repeat sequences, and 2 gene islands.

[0110] 2. Prediction of Secondary Metabolite Synthesis Gene Clusters: Analysis using the antiSMASH online software revealed 16 secondary metabolite synthesis gene clusters in the GLZ219 genome. These included nine clusters of known-function secondary metabolites such as paenicidin B, paenibacillin, fusaricidin B, tridecaptin, and polymyxin; and seven NPRS-type gene clusters with unknown functions. This indicates the presence of gene clusters encoding unknown secondary metabolites in the GLZ219 genome, suggesting that the strain may antagonize pathogens by synthesizing novel antibacterial substances. These findings suggest that strain GLZ219 may exert its antifungal antagonistic effect by producing secondary metabolites with antibacterial activity.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Polymyxin Bacillus ( Paenibacillus polymyxa Applications of GLZ219 in the following (a)-(e): (a) Inhibiting plant pathogens or preparing plant pathogen inhibitors; the plant pathogens are Fusarium oxysporum f. sp.radicis-lycopersici、 Rhizoctonia solani , Botrytis cinerea , Pythium aphanidermatum , Fusarium chlamydosporum , Fusarium graminearum , Corynespora cassiicola , Colletorichum lagenerium , Colletotrichum Acutatum and Fusarium pseudograminearum ; (b) Suppressing plant diseases or preparing plant disease inhibitors; said plant disease is caused by said plant pathogen. Fusarium oxysporum The plant disease caused by f. sp. radicis-lycopersici is tomato neck rot and root rot; (c) Promoting plant growth or preparing plant growth promoters; the promotion of plant growth specifically manifests as promoting plant height; the plant is tomato; (d) Nitrogen fixation and potassium solubilization; And, (e) to produce indoleacetic acid and iron carrier; The polymyxa GLZ219 is resistant to salt and alkali. The polymyxa bacteria GLZ219 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2022, with the accession number CGMCC No. 24766.

2. The application as described in claim 1, characterized in that, The fermentation production method of the polymyxin Bacillus GLZ219 is as follows: the polymyxin Bacillus GLZ219 is inoculated into a fermentation medium for fermentation culture.

3. The application as described in claim 2, characterized in that, The fermentation medium used is LB medium.

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