Paenibacillus H31 and application thereof

Abstract

The invention discloses paenibacillus sp. H31, which is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20252669, and the preservation date is November 24, 2025. The invention further discloses a preparation method of the paenibacillus sp. The paenibacillus H31 disclosed by the invention not only has broad-spectrum disease-resistant activity on pathogenic fungi of various plants, but also has a remarkable growth-promoting effect on pepper, so that the paenibacillus H31 can be widely applied to prevention and treatment of agricultural diseases and insect pests, and sustainable development of green agricultural production is promoted. In addition, the paenibacillus H31 also has the effects of hydrolyzing proteinase and cellulose and decomposing ferritin, so that the paenibacillus H31 also can be applied to the scenes of industrial production and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to Bacillus subtilis H31 and its applications. Background Technology

[0002] With global agriculture facing severe challenges such as soil degradation, overuse of chemical pesticides, and increased pathogen resistance, developing green and sustainable crop disease control and growth promotion strategies has become an important direction for modern agriculture. Chili pepper (Capsicum annuum L.) holds a significant position among vegetable crops in my country and is cultivated globally. Phytophthora capsici L., caused by *Phytophthora capsici* L., and bacterial wilt, caused by *Ralstonia pseudosolanacearum*, are devastating soil-borne diseases affecting chili pepper production. These two diseases develop rapidly and spread quickly under suitable temperature and humidity conditions, causing large-scale death of chili peppers within a short period. With the expansion of chili pepper cultivation areas, the difficulty of disease control, especially Phytophthora capsici and bacterial wilt, has increased, becoming a major factor restricting the healthy development of the chili pepper industry. Current chemical control methods can achieve some control, but they cause serious damage to the ecological environment, necessitating the search for a safe, efficient, and environmentally friendly control method. The plant rhizosphere microbiome, as a core component of plant-soil-microbe interactions, plays a crucial role in plant nutrient absorption, stress adaptation, and disease defense. Among them, Paenibacillus, a group of Gram-positive, spore-forming beneficial rhizosphere bacteria, has received widespread attention in recent years due to its outstanding ability to promote plant growth and induce resistance.

[0003] Bacillus species are widely distributed in the rhizosphere soils of various plants and exhibit strong environmental adaptability and ecological plasticity. Several species in this genus, such as *Paenibacillus polymyxa*, *P. mucilaginosus*, *P. illinoisensis*, *P. azotofixans*, *P. kribbensis*, *P. edaphicus*, and *P. peoriae*, have been shown to promote plant growth through various mechanisms, including nitrogen fixation, phosphorus solubilization, production of plant hormones (such as indoleacetic acid, gibberellins, and cytokinins), secretion of siderophores, and induction of systemic resistance (ISR). In addition, Bacillus subtilis can produce a series of antibacterial active substances, such as antibiotics, lipopeptides, enzymes and volatile organic compounds, which can effectively inhibit the infection and spread of a variety of plant pathogens (such as Ralstonia solanacearum, Fusarium oxysporum, Phytophthora infestans, etc.).

[0004] Compared with other biocontrol bacteria, Bacillus subtilis exhibits stronger stress resistance and colonization ability, forming stable biofilm structures in plant roots and exerting long-term growth-promoting and disease-preventing effects. In recent years, with the development of omics technologies, the genomic characteristics, metabolic potential, and molecular mechanisms of interaction between Bacillus subtilis and plants have been gradually revealed. For example, studies have found that it can enhance plant resistance to pathogens by activating the jasmonic acid (JA) and salicylic acid (SA) signaling pathways; simultaneously, some strains can also induce root hair development and enhance nutrient absorption capacity, thereby promoting overall plant growth.

[0005] Although some Bacillus-like strains have been isolated and applied in agricultural production, the following problems still exist in the existing technology:

[0006] Single-function strains: Most reported strains only have one of the functions of promoting growth or resisting disease, and there is a lack of excellent strains that have both highly effective disease prevention and significant growth promotion effects.

[0007] Poor adaptability: Some strains have weak colonization ability under different soil types or climatic conditions, and their field application effects are unstable;

[0008] The mechanism of action is unclear: the lack of systematic research on the mechanism by which the strain induces plant resistance and its interaction with plants limits its precise application and product development;

[0009] Limited strain resources: Currently, there are relatively few Bacillus strains used in agricultural production, and there is still a need to explore new strain resources with higher activity and wider adaptability.

[0010] Therefore, developing a new strain of Bacillus subtilis with broad-spectrum disease-resistant activity and significant growth-promoting effects is of great significance for the prevention and control of diseases in chili peppers, as well as for promoting the development of green agriculture and the industrialization of microbial preparations. Summary of the Invention

[0011] In response to the shortcomings of existing technologies and practical needs, this invention provides a novel Bacillus subtilis, which can be applied to the prevention of diseases and promotion of growth in plants such as chili peppers, as well as to hydrolyze proteases, cellulose, and decompose iron.

[0012] Specifically, the present invention adopts the following technical solution:

[0013] In a first aspect, the present invention provides a Paenibacillus sp. H31, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252669 and deposit date November 24, 2025.

[0014] Secondly, the present invention provides a microbial preparation containing Bacillus subtilis H31 as described in the present invention, or a fermentation broth containing Bacillus subtilis H31, or a lyophilized powder containing Bacillus subtilis H31, or inactivated cells containing Bacillus subtilis H31, or lysates containing Bacillus subtilis H31, or an extract containing Bacillus subtilis H31.

[0015] It should be understood that microbial strains used for biocontrol generally produce certain special products during their growth or fermentation process, such as antibiotics, bacteriocins, proteins, or other antibacterial substances. Therefore, the scope of protection of this invention should also include the products of *Bacillus subtilis* H31, including but not limited to fermentation broth of *Bacillus subtilis* H31, lyophilized powder of *Bacillus subtilis* H31, inactivated cells of *Bacillus subtilis* H31, lysates of *Bacillus subtilis* H31, and extracts of *Bacillus subtilis* H31.

[0016] Thirdly, the present invention provides the use of the aforementioned Bacillus subtilis H31 or the aforementioned microbial preparation in any of the following:

[0017] (1) Application in the prevention and control of plant diseases;

[0018] (2) Application in inhibiting plant pathogenic fungi;

[0019] (3) Application in promoting the growth of chili pepper plants;

[0020] (4) Applications in hydrolyzing proteases, cellulose and decomposing iron.

[0021] In one or more embodiments, in the application (1) or (2), the plant is at least one of chili pepper, eggplant, tomato and cucumber.

[0022] In one or more embodiments, in the application (1), the plant disease is at least one of pepper blight, pepper bacterial wilt, pepper anthracnose, pepper wilt, pepper black spot, pepper root rot, eggplant wilt, tomato early blight, and cucumber wilt.

[0023] In one or more embodiments, in the application (2), the pathogenic fungus is at least one of Phytophthora capsici L., Ralstonia pseudosolanacearum, Colletotrichum capsici, Fusarium oxysporu, Alternaria alternate, Fusarium spp., Fusarium verticillioide, Alternaria solani (Ell. et Mart.) Sorauer, and Fusarium oxysporum (Schl.) F. sp cucumerinum Owen.

[0024] In one or more embodiments, the application (3) of the above-ground application includes promoting the growth of chili plants by increasing at least one of the following indicators: stem diameter, plant height, root length, above-ground dry and fresh weight, and below-ground dry and fresh weight.

[0025] Fourthly, the present invention provides a pesticide formulation comprising the Bacillus subtilis H31 or the microbial formulation described herein.

[0026] In one or more embodiments, the pesticide formulation further includes other microbial agents that have a synergistic effect with the Bacillus thuringiensis H31 described in this invention.

[0027] It should be understood that although the Bacillus thuringiensis H31 described in this invention can achieve the disease prevention and growth promotion effects described in this invention by using it alone, it is not excluded that it can be used in combination with other biocontrol bacteria with similar functions or other pathogen control functions, so that the pesticide of this invention has a stronger effect of inhibiting a variety of plant diseases and / or inhibiting a variety of plant pathogenic fungi and / or promoting the growth of pepper plants and / or hydrolyzing proteases, cellulose and decomposing iron, or even has a broader function of controlling other pathogens.

[0028] In one or more embodiments, the pesticide formulation is a suspension concentrate, an oil suspension concentrate, a powder, a wettable powder, or a granule.

[0029] It should be understood that the pesticide formulations described in this invention refer to compositions that utilize beneficial microorganisms to kill or suppress the number of pathogenic organisms to control the occurrence and development of plant diseases. The pesticide formulations described in this invention contain the aforementioned Bacillus subtilis H31 or the aforementioned microbial preparations, and can be prepared into formulations of different forms according to reagent requirements. Furthermore, the pesticide formulations of the present invention may, as needed, include appropriate carriers or excipients such as solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Of course, the addition of these carriers or excipients should not affect the original biocontrol effect of the Bacillus subtilis H31 or the microbial formulations.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The Bacillus thuringiensis H31 described in this invention not only exhibits broad-spectrum disease resistance against pathogenic fungi in various plants, but also demonstrates a significant growth-promoting effect on chili peppers. Therefore, it can be widely applied in agricultural pest and disease control, promoting the sustainable development of green agricultural production. Furthermore, the Bacillus thuringiensis H31 described in this invention also possesses the ability to hydrolyze proteases, cellulose, and decompose iron, thus it can also be applied in industrial production and other scenarios. Attached Figure Description

[0032] Figure 1 This is the colony morphology of strain H31 in Example 1 on a TSA plate.

[0033] Figure 2This is a genomic collinearity map of strain H31 and strain Paenibacillus polymyxa ATCC 842 in Example 1.

[0034] Figure 3 This refers to the antagonistic effect of strain H31 in Example 2 against Phytophthora capsici.

[0035] Figure 4 This refers to the antagonistic effect of strain H31 against Ralstonia solanacearum in Example 2.

[0036] Figure 5 This is a diagram showing the control effect of strain H31 on pepper blight and bacterial wilt in Example 3.

[0037] Figure 6 This describes the antagonistic effect of strain H31 in Example 4 against eight pathogens, where A and H are: Colletotrichum capsici, Fusarium oxysporu, Alternaria alternate, Fusarium spp., Sclerotium rolfsii Sacc., Fusarium verticillioide, Alternaria solani (Ell. et Mart.) Sorauer, and Fusarium oxysporum (Schl.) f. sp cucumerinum Owen.

[0038] Figure 7 This refers to the growth-promoting effect of strain H31 on pepper seedlings in Example 5.

[0039] Figure 8 This refers to the ability of strain H31 in Example 6 to hydrolyze proteases, cellulases, and decompose iron. Detailed Implementation

[0040] Bacillus species are widely distributed in the rhizosphere soils of various plants and exhibit strong environmental adaptability and ecological plasticity. Several species in this genus, such as *Paenibacillus polymyxa*, *P. mucilaginosus*, *P. illinoisensis*, *P. azotofixans*, *P. kribbensis*, *P. edaphicus*, and *P. peoriae*, have been shown to promote plant growth through various mechanisms, including nitrogen fixation, phosphorus solubilization, production of plant hormones (such as indoleacetic acid, gibberellins, and cytokinins), and effective inhibition of the infection and spread of various plant pathogens (such as *Ralstonia solanacearum*, *Fusarium oxysporum*, and *Phytophthora infestans*).

[0041] Although existing technologies disclose that Bacillus subtilis can be used to control a variety of crop diseases, there are no reports of its broad-spectrum inhibitory activity against a variety of plant pathogenic fungi, nor are there reports of its application in hydrolyzing proteases, cellulose, and decomposing iron.

[0042] It should be understood that in this invention, Bacillus subtilis H31, strain H31, or the abbreviated description "H31" are all different names for the same strain, and those skilled in the art would not consider these strains with different names to be different strains.

[0043] Example

[0044] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0045] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0046] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0048] The culture media involved in the following examples include TSA medium, TSB medium, PDA medium, CPG medium, etc., which are all common culture media in the art, and therefore are not described in detail here.

[0049] Example 1: Isolation, identification and preservation of Bacillus subtilis H31

[0050] 1.1 Isolation of strain H31 and preparation of its bacterial culture

[0051] Strain H31 was isolated and screened from the rhizosphere soil of chili peppers at the Yazhou Batou base and stored at -80℃ for later use. The stored strain H31 was activated by culturing on tryptic soy agar (TSA). Once single colonies appeared, their morphology was observed. Single colonies were then picked and cultured on tryptic soy liquid medium (TSB) at 28℃ with shaking at 200 rpm for 24 h. The culture temperature was adjusted to OD using deionized water (SDW). 600 =1.0 (ca. 2×10 7 The CFU / mL solution was prepared as the test bacterial solution and was ready for use.

[0052] 1.2 Identification of strain H31

[0053] (1) Morphological identification: After being cultured on TSA medium for 36 h, strain H31 exhibited a round, irregularly edged, milky-white colony morphology with a smooth, raised surface (e.g. Figure 1 (As shown).

[0054] (2) Physiological and biochemical identification: The physiological and biochemical indicators of strain H31 were evaluated using the EasyID bacterial biochemical identification kit (Huankai Biotechnology). The results showed that strain H31 could utilize mannitol, glucose, starch hydrolysis and citrate, but could not utilize lysozyme, lysozyme-induced reaction and nitrate reduction reaction. In addition, the motility assay, VP assay, gelatin liquefaction and 3% hydrogen peroxide were all positive (as shown in Table 1).

[0055] Table 1 Physiological and biochemical characteristics of strain H31

[0056]

[0057] Note: "+" indicates positive; "−" indicates negative.

[0058] (3) Average Nucleic Acid Similarity (ANI) Identification

[0059] Total DNA was extracted from H31 using the FastPure Bacteria DNA Isolation Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.). Whole-genome sequencing was performed by Guangdong Megagene Technology Co., Ltd., and the sequenced sequences were compared using an average similarity (ANI). An ANI > 95% indicated that the two genomes belonged to the same species. The target genome and reference genome were compared using MUMmer software (Version 3.23). Figure 2 This is a map showing the collinearity of the genomes of strain H31 and strain Paenibacilluspolymyxa ATCC 842.

[0060] The experimental results (as shown in Table 2) show that the ANI of strain H31 and the model strain of Paenibacillus polymyxa ATCC 842 is 93.5%, which is less than 95%, indicating that the two genomes do not belong to the same species. Therefore, it is named Paenibacillus sp. H31.

[0061] Table 2. H31 Average Calculated Similarity Analysis

[0062]

[0063] 1.3 Identification of strain H31

[0064] The *Paenibacillus* sp. H31 isolated above was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252669 and deposit date November 24, 2025.

[0065] Example 2: Antagonistic effect of strain H31 against Phytophthora capsici and Ralstonia solanacearum

[0066] 2.1 Antagonistic effect against Phytophthora capsici

[0067] Experimental Methods: The plate confrontation method was used for screening. The indicator fungus Phytophthora capsici Leonian LT1534 (provided by Professor Chen Qinghe's team from the College of Tropical Agriculture and Forestry, Hainan University) was activated in potato dextrose agar (PDA). The indicator fungus block was taken with an 8 mm punch and placed in the center of the PDA culture medium (90 mm Petri dish). 0.5 μL of the test bacterial solution was inoculated at the four sides 20 mm away from the center. An equal volume of deionized water (SDW) was inoculated as a control. Each test strain was repeated three times. The culture was carried out at 28℃ until the control indicator fungus covered the entire Petri dish. The diameter of the indicator colony was measured and the inhibition rate was calculated as (control colony radius - treated colony radius) / control colony radius × 100%.

[0068] Experimental results are as follows Figure 3 As shown: When the mycelium of the control Phytophthora infestans had completely covered the petri dish (mycelial radius = 4.5 cm), Figure 3 In treatments A and C, the mycelial radius of *Phytophthora infestans* treated with H31 was 1.2 cm. Figure 3 The inhibition rates of H31 (B and C) were significantly lower than those of the control treatment, reaching 72.8%, indicating that H31 has a significant antagonistic effect on Phytophthora capsici.

[0069] 2.2 Antagonistic effect against Ralstonia solanacearum

[0070] Experimental Methods: A co-culture method was used, with 600 μL of the indicator bacterium Ralstonia pseusolanacearum strain RS03 (isolated and preserved by the research group) containing OD200. 600 =1.0, ca. 1×10 9 Add CFU / mL to 100 mL of CPG (Casamino Acids-Peptone-Glucose) medium, mix thoroughly, and pour the unsolidified medium evenly into 9 cm petri dishes, about 15 mL per dish. Use a 7 mm diameter punch to make holes, add 70 μL of H31 bacterial suspension to the holes, and use 70 μL of deionized water (SDW) as a blank control. Incubate at 30℃ for 48 h, and then observe the inhibition zone. The experiment was repeated 3 times.

[0071] The results are as follows Figure 4 As shown, a clear transparent halo appeared around the wells inoculated with H31 bacterial solution, while no halo was observed in the control treatment, indicating that strain H31 has an antagonistic effect on Ralstonia solanacearum.

[0072] Example 3: Evaluation of H31's resistance to Phytophthora blight and bacterial wilt in peppers

[0073] 3.1 Preparation of chili seedlings

[0074] One-year-old chili pepper seedlings of CA181 (Capsicum annuum L.) and drooping chili pepper CB81 (Capsicum baccatum L., provided by the chili pepper research group of Hainan University) at the 4-5 leaf stage were used as experimental materials. CA181 chili peppers were used for infection with Ralstonia solanacearum, and CB81 chili peppers were used for infection with Phytophthora. Chili pepper seeds were disinfected with 75% ethanol solution for 1 min, followed by disinfection with 2% sodium hypochlorite for 3 min, and then rinsed 5-6 times with sterile deionized water (SDW). The seeds were germinated at room temperature, and after the seeds showed white sprouts, they were sown in 50-cell seedling trays and placed in a seedling room with normal water and fertilizer management until the chili peppers grew 4-5 true leaves.

[0075] 3.2 Preparation of bacterial culture

[0076] (1) Preparation of Phytophthora inoculum: The preserved Phytophthora (same as Phytophthora capsici in Example 2) was propagated using PDA medium. After the Phytophthora had filled the entire dish, it was cultured under light for 4-5 days. The sporangia were scraped off and placed in deionized water (SDW) and then placed in a refrigerator at 4 ℃ for 40 min. After that, it was placed under a greenhouse and filtered through three layers of gauze to obtain the Phytophthora inoculum inoculum. The concentration was adjusted to 1.0 × 10⁻⁶ using a hemocytometer counting method. 5 CFU / mL.

[0077] (2) Preparation of Ralstonia solanacearum culture: RS03 (same as Ralstonia solanacearum in Example 2) was cultured on CPG agar plates for 48 h. Single colonies were picked and transferred to CPG liquid medium, and cultured with shaking at 200 rpm and 28±2℃ for 24 h. The fermentation broth was centrifuged twice at 8000 rpm and diluted with deionized water (SDW) to adjust the concentration to ca. 1×10⁻⁶. 9 CFU / mL available for use.

[0078] 3.3 Inoculation Trial

[0079] (1) Phytophthora seedling inoculation experiment: CB81 pepper seedlings were transferred to 10×10cm seedling cups and placed in an artificial incubator (26±2℃, 14 h light / 10 h dark) for 2 days. The roots were then wounded 2cm away from the roots of the pepper seedlings with a knife and 10mL of H31 bacterial solution was applied to each seedling. Deionized water (SDW) was used as a control and the seedlings were placed in an incubator under the same conditions for 3 days before Phytophthora inoculation. 5mL of pathogenic bacterial solution was injected into the wounded roots and the seedlings were placed in an artificial incubator for cultivation. The disease incidence of the experimental seedlings was observed and recorded. 10 seedlings were treated in each group and the experiment was repeated three times. The disease severity grading standard referenced the method in existing technology (Li Ye. Preliminary study on the biocontrol effect and antibacterial mechanism of Bacillus licheniformis BL06 against pepper blight [D]. Nanjing Agricultural University, 2022), ranging from asymptomatic (level 0) to plant death (level 5). The disease severity index was calculated based on the severity grading: Disease Severity Index = ∑(Number of diseased plants at each level × Disease Severity value) / Total number of plants surveyed × Highest Disease Severity value) × 100%. Control Efficacy = (Control Disease Severity Index - Treatment Disease Severity Index) / Control Disease Severity Index × 100%.

[0080] (2) Bacterial wilt seedling inoculation test: CA181 pepper seedlings were transplanted into 10×10cm seedling cups (bottom layer 150ml substrate, middle layer 20g river sand, top layer 150ml substrate), and then H31 bacterial solution was applied, 10mL per plant. The control group was treated with the same amount of SDW. 3 days later, 100mL / plant of RS03 bacterial solution (concentration approximately 1×10) was applied from the bottom. 7 CFU / g soil), the treated pepper seedlings were placed in an incubator at 28℃, 14 / 10h day / night, and 70% humidity for observation and recording of disease incidence. The disease severity grading standard for bacterial wilt was based on the existing technology (Gao Yulan, Gao Lianbao, Fu Huizhen, et al. Study on sugar screening and resistance mechanism of pepper bacterial wilt [J]. Journal of Southern Agriculture, 2024, 55(06):1653-1661), from asymptomatic (level 0) to plant death (level 5). The disease index was calculated according to the disease severity grading, and the disease index = ∑(number of diseased plants at each level × representative value at each level) / total number of plants investigated × highest representative value) × 100%. The control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%. The experiment was repeated three times, with 10 seedlings in each treatment.

[0081] The experimental results are shown in Table 3 and Figure 5 As shown, on day 21 after inoculation with Phytophthora capsici, the disease severity of the control treatment peppers reached 90%, while the disease severity of the H31 treatment peppers was only 21.3%, with a control effect of 76.3%. On day 14 after inoculation with Ralstonia solanacearum, the disease severity of the control treatment peppers reached 90%, while the disease severity of the H31 treatment peppers was only 21.3%, with a control effect of 76.3%.

[0082] Table 3. Control efficacy of strain H31 against Phytophthora blight and bacterial wilt of pepper.

[0083]

[0084] Note: * indicates that P < 0.05 is significant.

[0085] The above research results indicate that strain H31 has a significant indoor control effect on pepper blight and bacterial wilt.

[0086] Example 4: Broad-spectrum antibacterial activity of strain H31

[0087] The plate confrontation method was used to detect the antagonistic effect of strain H31 against various pathogenic fungi of different vegetables, including *Colletotrichum capsici* (anthracnose pathogen of pepper), *Fusarium oxysporu* (fusarium wilt pathogen of pepper), *Alternaria alternate* (black spot pathogen of pepper), *Fusarium spp.* (root rot pathogen of pepper), *Sclerotium rolfsii* Sacc. (white mold pathogen of pepper), *Fusarium verticillioide* (fusarium verticillioide) (fusarium wilt pathogen of eggplant), *Alternaria solani (Ell. et Mart.) Sorauer* (early blight pathogen of tomato), and *Fusarium oxysporum (Schl.) F. sp cucumerinum Owen* (fusarium oxysporum cucumber-specific strain of cucumber wilt pathogen). All pathogenic fungal strains were provided by the Hunan Academy of Agricultural Sciences. The above-mentioned pathogenic fungi were activated on PDA medium, punched with an 8 mm punch, and transferred to fresh PDA medium. The H31 bacterial culture was cultured on TAB medium, centrifuged, and the concentration was adjusted to OD. 600 =1.0 (ca. 1×10 7 (CFU / mL) 0.5 μL was inoculated around the pathogen block at a distance of 2 mm from the surrounding area, with sterile distilled water used as a blank control. The mixture was incubated at 28°C until the control completely covered the entire culture dish, and the experimental results were recorded by photographing. The experiment was repeated three times.

[0088] The results are as follows Figure 6 As shown, strain H31 exhibits significant antagonism against pathogens such as *Polygonum aviculare*, *Fusarium oxysporum*, *Alternaria alternata*, *Fusarium*, *Fusarium verticillatum*, *Alternaria alternata*, and *Fusarium oxysporum* cucumber-specific strain.

[0089] Example 5: Growth-promoting effect of strain H31 on peppers

[0090] One-year-old CA181 pepper seedlings with two leaves and one bud (same as CA181 pepper in Example 3) were transplanted into seedling bags, and inoculated with H31 bacterial solution (OD) 3 days later. 600 =1), 10 mL per plant, with sterile water as a control, for a total of 3 inoculations, one inoculation every 7 days, and physiological indicators such as stem diameter, plant height, and root length were measured after 28 days (7 days after the third inoculation). The experiment was repeated 3 times, with 10 seedlings each time.

[0091] The results are shown in Table 4 and Figure 7 As shown, after applying H31 bacterial solution, the pepper plants grew significantly. Compared with the control, the pepper plants treated with H31 strain had significantly higher stem diameter, plant height, root length, above-ground dry and fresh weight, and underground dry and fresh weight than the control group, thus achieving a good growth-promoting effect.

[0092] Table 4. Growth-promoting effect of H31 on pepper seedlings

[0093]

[0094] Note: * This indicates that the result is significant at the P < 0.05 level.

[0095] Example 6: The ability of strain H31 to hydrolyze proteases, cellulose and decompose iron

[0096] 6.1 Detection of proteases

[0097] Pour approximately 10 mL of protease medium (Solution A: 4 g peptone, 3 g yeast extract, and 18 g agar / 1000 mL; Solution B: 24 g skim milk powder / 1000 mL, pH 7.5, Chinook brand) into a 90 mm Petri dish. After cooling to room temperature, punch holes in the center of the medium using a 7 mm diameter punch. Then, inoculate 5 μL of H31 bacterial culture into each hole and incubate at 30°C for 72 h. Observe the formation of clear zones in the medium. The appearance of clear zones indicates that the strain has the ability to hydrolyze proteases; otherwise, no clear zone is observed. The experimental treatment was performed in triplicate.

[0098] 6.2 Detection of decomposed cellulose

[0099] Pour approximately 10 mL of cellulose Congo red medium (Qingdao Haibo Biotechnology) into a 90 mm Petri dish. After cooling to room temperature, punch holes in the center of the medium using a 7 mm diameter punch. Then, inoculate 5 μL of H31 bacterial suspension into each hole and incubate at 30 °C. After 48 h, first add 1 mg / mL Congo red solution for 1 h, then add 1 mol / L NaCl for 1 h. Observe the formation of clear zones in the medium. The appearance of clear zones indicates that this strain has some hydrolytic effect on cellulose; otherwise, no effect is observed. The experiment was performed in triplicate.

[0100] 6.3 Detection of hydrolyzed ferrophosphate

[0101] Pour approximately 10 mL of heptaphilite detection medium (CAS agar, Qingdao Haibo Biotechnology) into a 90 mm Petri dish. After cooling to room temperature, punch a hole in the center of the medium using a 7 mm diameter punch. Then, inoculate H31 bacterial suspension into the hole and incubate at 30°C. After 5 days, observe the color change in the medium. A change in medium color from blue to orange-yellow indicates that the strain can hydrolyze heptaphilite; otherwise, it cannot. The experiment was performed in triplicate.

[0102] The test results are as follows Figure 8 As shown, transparent halos appeared around the protease medium, cellulose medium, and iron-loving medium after inoculation with H31, indicating that strain H31 has the ability to hydrolyze proteases, cellulose, and decompose iron.

[0103] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A Paenibacillus sp. H31, characterized in that, The Paenibacillus sp. H31 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20252669, and the preservation date is November 24, 2025.

2. A microbial preparation, characterized in that, The microbial preparation contains the Paenibacillus sp. H31 of claim 1, or contains the fermentation broth of the Paenibacillus sp. H31, or contains the freeze-dried powder of the Paenibacillus sp. H31, or contains the inactivated bacterial body of the Paenibacillus sp. H31, or contains the lysate of the Paenibacillus sp. H31, or contains the extract of the Paenibacillus sp. H31.

3. The Paenibacillus sp. H31 of claim 1 or the microbial preparation of claim 2 is used in any one of the following: (1) application in preventing and treating plant diseases; (2) application in inhibiting plant pathogenic fungi; (3) application in promoting the growth of pepper plants; (4) application in hydrolyzing protease, cellulose and decomposing ferrite.

4. The use according to claim 3, wherein the compound is ###0002### The plant in (1) or (2) is at least one of pepper, eggplant, tomato and cucumber.

5. The use according to claim 3, wherein the compound is ###0002### The plant disease in (1) is at least one of pepper blight, pepper bacterial wilt, pepper anthracnose, pepper wilt, pepper black spot, pepper root rot, eggplant wilt, tomato early blight and cucumber wilt.

6. The use according to claim 3, wherein the compound is ###0002### The pathogenic fungi in (2) is at least one of pepper Phytophthora capsici L., Ralstonia pseudosolanacearum, Colletotrichum capsici, Fusarium oxysporu, Alternaria alternate, Fusarium spp., Fusarium verticillioide, Alternaria solani (Ell.et Mart.) Sorauer and Fusarium oxysporum (Schl.) F. sp cucumerinum Owen.

7. The use according to claim 3, wherein the compound is ###0002### The promotion of the growth of pepper plants in (3) includes the promotion of the increase of at least one of stem diameter, plant height, root length, aboveground dry fresh weight and underground dry fresh weight of pepper plants.

8. A pesticidal formulation, characterized by, The pesticide preparation contains the Paenibacillus sp. H31 of claim 1 or the microbial preparation of claim 2.

9. The pesticide formulation of claim 8, wherein The pesticide preparation further comprises other microbial agents having synergistic effect with the Paenibacillus sp. H31 of claim 1.

10. The agricultural chemical preparation according to claim 8, wherein The dosage form of the pesticide preparation is one of suspension, oil suspension, powder, wettable powder and granules.

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