A biocontrol agent composition and its application in improving growth obstacles caused by soil high temperature sterilization and preventing plant diseases

Abstract

This invention belongs to the field of microbial preparation technology, specifically relating to a biocontrol agent composition and its application in improving growth barriers caused by high-temperature soil sterilization and controlling plant diseases. The technical problem this invention aims to solve is to provide a new option for alleviating soil obstacles caused by high-temperature sterilization. The technical solution of this invention is a *Pseudomonas aeruginosa* 115-17, with the preservation number CCTCC No: M2026293. This invention provides a novel *Pseudomonas aeruginosa* 115-17 strain, which possesses nitrogen-fixing, inorganic phosphorus-solubilizing, and siderophore-producing abilities; it can also promote pepper growth, control plant diseases (mainly pepper blight), and improve plant growth inhibition caused by high-temperature soil sterilization. This invention also provides a combination of Bacillus velezensis Bv-6 (CCTCC No: M20191106) and newly isolated Pseudomonas aeruginosa 115-17 to form a microbial inoculum, thereby effectively alleviating plant growth obstacles caused by high-temperature sterilization and effectively preventing and controlling pepper blight.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, specifically relating to a biocontrol agent composition and its application in improving growth barriers caused by high-temperature sterilization of soil and controlling plant diseases. Background Technology

[0002] Continuous cropping obstacles are a common problem in greenhouse vegetable production. Due to long-term monoculture, pathogens such as Fusarium and Phytophthora, as well as autotoxins, accumulate in the soil, leading to poor root development, yellowing and wilting, and even widespread plant death, severely restricting yield and quality improvement (Ren Jianquan, 2023; Guo Chenxi et al., 2020). To address this problem, high-temperature soil sterilization is a traditional and effective control method. Through short-term high-temperature treatment (such as high-temperature fumigation), during the summer months of July and August when the greenhouse soil is idle, plastic film is used to simultaneously seal the greenhouse and soil surface. Under strong sunlight, the greenhouse and soil temperatures are rapidly increased to 60-70°C, using this high temperature to disinfect the greenhouse, thereby killing pathogens, pests, and weeds. The application of this technology can effectively improve soil physical and chemical properties, increase fertility, and kill and reduce the harm of pests, diseases, and weeds, especially soil-borne diseases, to vegetable production (Xu Guangming, 2012). However, while high-temperature sterilization eliminates harmful organisms, it also indiscriminately inactivates beneficial microorganisms in the soil (such as plant growth-promoting bacteria and antagonistic bacteria), leading to a severe imbalance in the structure and function of the soil microbial community (Dong Meilin et al., 2025). Studies have shown that microbial diversity in sterilized soil is significantly reduced, which not only weakens the soil's natural disease-suppressing ability but may also exacerbate secondary infections by pathogens due to niche vacancies. More seriously, repeated sterilization can cause problems such as microbial flora disorder, accelerated decomposition of organic matter, and accumulation of soluble salts, ultimately leading to hindered seedling root development and reduced nutrient absorption efficiency (Zheng Jiahui et al., 2017). Currently, methods for restoring soil ecological function after high-temperature sterilization mainly include inoculation with beneficial biocontrol bacteria and the addition of soil conditioners. However, existing technologies still have significant limitations. For example, although a single strain can promote plant growth through mechanisms such as secreting volatile organic compounds, its colonization capacity in the soil after sterilization is limited, and the effect is often not ideal. While applying soil conditioners can partially alleviate the side effects of sterilization, it is difficult to quickly rebuild the functional microbial network, making it difficult to fully repair the soil functional impairment caused by high-temperature sterilization. Moreover, in the past, soil improvement was mostly carried out using single inorganic conditioners, which still has many drawbacks such as soil compaction (Liu et al., 2025).

[0003] Phytophthora capsici is one of the most destructive soil-borne oomycetes worldwide, capable of systemically infecting over 300 species of crops from at least 20 families, including Solanaceae (pepper, tomato, tobacco, eggplant), Cucurbitaceae (cucumber, watermelon, pumpkin, melon), Malvaceae (cotton), and Fabaceae (bean). It can cause disease from seedling to mature plant stages, with typical symptoms including root rot, black rot at the stem base, damping-off, and wilting and death of the entire plant. It is characterized by rapid onset, short incubation period, rapid spread, and severe losses, often causing significant yield reductions or even total crop failure (Lamour et al., 2012). Phytophthora capsici occurs in many parts of my country, including Jiangxi, Chongqing, Anhui, and Qinghai, and exhibits rich genetic diversity, with its mating types showing an uneven distribution (Zhang et al., 2022). Currently, chemical control remains the primary method for controlling Phytophthora blight in peppers, with most treatments using chemical agents such as fluazinam, metalaxyl, cymoxanil, and azoxystrobin (Li Weishan et al., 2022). Long-term, large-scale use of chemical agents easily leads to environmental pollution, excessive pesticide residues, increased pathogen resistance, and increased planting costs. The technical limitations of chemical treatments are becoming increasingly prominent, and they will be gradually prohibited due to environmental legislation (Zhang Rui et al., 2022).

[0004] Rhizosphere growth-promoting bacteria (PGPRs), such as Bacillus spp. and Pseudomonas spp., promote plant growth through multiple mechanisms (Chen et al., 2025; Bai et al., 2025). Bacillus can fix nitrogen, dissolve insoluble phosphorus and potassium, and secrete siderophores and plant hormones (such as IAA), directly promoting plant nutrient absorption and growth. In addition, Bacillus can also inhibit the growth of soil-borne pathogens by producing antibiotics (such as surfactants and iturobrine) and inducing systemic resistance (ISR), thus indirectly promoting plant growth (Yang et al., 2024). In addition to possessing similar functions as phosphate solubilizers, siderophore producers, and plant hormone producers, *Pseudomonas* can also secrete ACC deaminase to reduce ethylene levels under plant stress and alleviate abiotic stress. Antibiotics produced by *Pseudomonas* (such as 2,4-diacetylphloroglucinol) and the induced ISR response can further enhance plant disease resistance (Zheng et al., 2012). These beneficial microorganisms effectively improve the rhizosphere microenvironment and enhance crop stress resistance and yield through the dual effects of direct nutrient promotion and indirect biological control.

[0005] Bacillus velezensis and Pseudomonas aeruginosa have attracted much attention in biocontrol due to their unique metabolic characteristics, but their synergistic application in alleviating the soil barrier of high-temperature sterilization and controlling pepper blight has not yet been reported. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a new option for alleviating the obstacles of high-temperature sterilization of soil.

[0007] The technical solution of the present invention is a Pseudomonas aeruginosa 115-17, whose preservation number is: CCTCC No:M2026293.

[0008] The present invention also provides the application of the aforementioned Pseudomonas aeruginosa 115-17 in improving growth barriers caused by high-temperature sterilization of soil.

[0009] Specifically, the growth obstacle mentioned is a growth obstacle in chili pepper cultivation caused by high-temperature sterilization of the soil.

[0010] The present invention also provides the application of the aforementioned Pseudomonas aeruginosa 115-17 in the prevention and control of plant blight.

[0011] Specifically, the plant in question is a chili pepper.

[0012] Furthermore, the pathogen of the disease is Phytophthora capsici.

[0013] The present invention also provides the application of the aforementioned Pseudomonas aeruginosa 115-17 in nitrogen fixation, inorganic phosphorus solubilization and / or siderogenic carriers.

[0014] The present invention also provides the application of the aforementioned Pseudomonas aeruginosa 115-17 in promoting plant growth.

[0015] Specifically, the plant in question is a chili pepper.

[0016] The present invention also provides the application of Bacillus velezensis Bv-6 in improving growth barriers caused by high-temperature sterilization of soil, wherein the preservation number of Bacillus velezensis Bv-6 is CCTCC No:M20191106.

[0017] Specifically, the growth obstacle mentioned is a growth obstacle in chili pepper cultivation caused by high-temperature sterilization of the soil.

[0018] The present invention also provides a biocontrol agent composition comprising *Pseudomonas aeruginosa* 115-17 and *Bacillus velezensis* Bv-6; the preservation number of *Pseudomonas aeruginosa* 115-17 is CCTCC No: M2026293, and the preservation number of *Bacillus velezensis* Bv-6 is CCTCC No: M20191106.

[0019] Specifically, in the biocontrol agent composition, the ratio of Pseudomonas aeruginosa 115-17 to Bacillus velezensis Bv-6 is 1:1.

[0020] Furthermore, the biocontrol agent composition comprises a suspension of Pseudomonas aeruginosa 115-17 and a suspension of Bacillus velezensis Bv-6.

[0021] Specifically, the bacterial suspension is obtained by centrifuging the fermentation broth of the bacterial strain, collecting the bacterial cells, and resuspending them in sterile water.

[0022] Preferably, the concentration of the *Pseudomonas aeruginosa* 115-17 bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0023] Preferably, the concentration of the Bacillus velezensis Bv-6 bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0024] Furthermore, the biocontrol agent composition comprises fermentation broth of Pseudomonas aeruginosa 115-17 and fermentation broth of Bacillus velezensis Bv-6.

[0025] The present invention also provides the application of the biocontrol agent composition in improving growth barriers caused by high-temperature sterilization of soil.

[0026] Specifically, the growth obstacle mentioned is a growth obstacle in chili pepper cultivation caused by high-temperature sterilization of the soil.

[0027] The present invention also provides the application of the biocontrol agent composition in the prevention and control of plant blight.

[0028] Specifically, the plant in question is a chili pepper.

[0029] Furthermore, the pathogen of the disease is Phytophthora capsici.

[0030] The present invention also provides a method for improving growth barriers caused by high-temperature sterilization of soil, comprising the following steps: treating plants grown on high-temperature sterilized soil with *Pseudomonas aeruginosa* 115-17 and / or *Bacillus velezensis* Bv-6; wherein the preservation number of *Pseudomonas aeruginosa* 115-17 is CCTCC No: M2026293, and the preservation number of *Bacillus velezensis* Bv-6 is CCTCC No: M20191106.

[0031] Specifically, the plant in question is a chili pepper.

[0032] Specifically, the treatment involves preparing a suspension of Pseudomonas aeruginosa 115-17 and / or a suspension of Bacillus velezensis Bv-6 for root irrigation of the plants.

[0033] Specifically, the bacterial suspension is obtained by centrifuging the fermentation broth of the bacterial strain, collecting the bacterial cells, and resuspending them in sterile water.

[0034] Preferably, the concentration of the *Pseudomonas aeruginosa* 115-17 bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0035] Preferably, the concentration of the Bacillus velezensis Bv-6 bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0036] More preferably, the ratio of Pseudomonas aeruginosa 115-17 to Bacillus velezensis Bv-6 is 1:1.

[0037] The present invention also provides a method for preventing and controlling plant diseases, comprising the following steps: treating plants with Pseudomonas aeruginosa 115-17 and / or Bacillus velezensis Bv-6; wherein the preservation number of Pseudomonas aeruginosa 115-17 is CCTCC No:M2026293, and the preservation number of Bacillus velezensis Bv-6 is CCTCC No:M20191106.

[0038] Specifically, the plant in question is a chili pepper.

[0039] Furthermore, the pathogen of the disease is Phytophthora capsici.

[0040] Specifically, the treatment involves drenching the plants with a fermentation broth of Pseudomonas aeruginosa 115-17 or / and Bacillus velezensis Bv-6.

[0041] Preferably, the ratio of Pseudomonas aeruginosa 115-17 to Bacillus velezensis Bv-6 is 1:1.

[0042] The beneficial effects of this invention are as follows: This invention provides a novel *Pseudomonas aeruginosa* strain, *Pseudomonas aeruginosa* 115-17, which possesses nitrogen-fixing, inorganic phosphorus-solubilizing, and siderophore-producing abilities; it can also promote pepper growth, control plant blight (mainly pepper blight), and improve plant growth inhibition caused by high-temperature sterilization of soil. This invention also discovered that *Bacillus velezensis* Bv-6 possesses nitrogen-fixing, inorganic phosphorus-solubilizing, and siderophore-producing abilities; as well as the ability to control plant blight (mainly pepper blight) and improve plant growth inhibition caused by high-temperature sterilization of soil. Experiments combining the two strains show that the combined use of *Bacillus velezensis* Bv-6 and *Pseudomonas aeruginosa* 115-17 can significantly improve the growth of peppers in high-temperature treated soil, effectively alleviate the negative effects of high-temperature sterilization, and effectively control the occurrence of pepper blight. Compared to the control (CK) treatment of high-temperature sterilized soil, the Bv-6 treatment alone, the 115-17 treatment alone, and the Bv-6+115-17 mixed treatment increased the fresh weight of peppers by 35.63%, 24.71%, and 81.03%, respectively. Furthermore, compared to the CK control, the control efficacy of Bv-6 alone, the 115-17 treatment alone, and the Bv-6+115-17 mixed treatment against *Phytophthora capsici* was 20.83%, 70.83%, and 81.25%, respectively.

[0043] The Bacillus velezensis used in this invention is specifically Bacillus velezensis strain Bv-6, which was deposited on December 25, 2019, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number CCTCC No: M20191106, and has been disclosed in the patent application document with application number 201911381534.2.

[0044] The Pseudomonas aeruginosa used in this invention is specifically strain Pseudomonas aeruginosa 115-17, which was deposited at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on January 30, 2026, with accession number CCTCC No: M2026293. Attached Figure Description

[0045] Figure 1This is a schematic diagram of the morphological characteristics and phylogenetic tree of Pseudomonas aeruginosa 115-17. A: Culture morphology of 115-17 after 3 days of culture on CN medium; B: Single colony morphology of 115-17 after 3 days of culture on CN medium; C: Phylogenetic tree of 115-17 based on the gyrB sequence.

[0046] Figure 2 This is a schematic diagram illustrating the evaluation results of the growth-promoting properties of Bacillus velezensis Bv-6 and Pseudomonas aeruginosa 115-17 strains. A: Nitrogen fixation test; B: Siderophore production test; C: Inorganic phosphorus solubilization test; D: Ammonia production test; E: IAA production test.

[0047] Figure 3 This diagram illustrates the results of Bacillus velezensis Bv-6 and Pseudomonas aeruginosa 115-17 strains in alleviating the growth inhibition of peppers caused by high-temperature sterilization in the soil. Both single treatments with Bv-6 and 115-17, as well as the combined treatment of the two strains, significantly alleviated the growth inhibition caused by high-temperature sterilization. Compared to the control (CK) treatment of high-temperature sterilized soil, the Bv-6, 115-17, and Bv-6+115-17 treatments increased the fresh weight of peppers by 35.63%, 24.71%, and 81.03%, respectively.

[0048] Figure 4 The results (3 dpi) show the inhibition of mycelial growth of *Phytophthora capsici* by *Bacillus velezensis* Bv-6 and *Pseudomonas aeruginosa* 115-17 strains. Compared with the control (CK), 10% concentrations of Bv-6, 115-17, and Bv-6+115-17 inhibited mycelial growth of *Phytophthora capsici* by 27.91%, 48.33%, and 68.94%, respectively; while 20% concentrations of Bv-6, 115-17, and Bv-6+115-17 inhibited mycelial growth by 36.86%, 77.62%, and 85.39%, respectively.

[0049] Figure 5The in vivo control efficacy (5 dpi) of Bacillus velezensis Bv-6 and Pseudomonas aeruginosa 115-17 strains against Phytophthora capsici was determined. Compared with the control (CK), the control efficiencies of Bv-6, 115-17, and Bv-6+115-17 against Phytophthora capsici were 20.83%, 70.83%, and 81.25%, respectively. Detailed Implementation

[0050] Bacillus velezensis and Pseudomonas aeruginosa are both widely used biocontrol bacteria in agricultural production, but research on their combined use to alleviate soil barriers caused by high-temperature sterilization and synergistically control pepper blight has not been reported. Based on the previously disclosed Bacillus velezensis Bv-6 (CCTCC No: M20191106), the applicant has for the first time combined it with the newly isolated Pseudomonas aeruginosa 115-17 to create a microbial inoculum combination, thereby effectively alleviating plant growth barriers caused by high-temperature sterilization and providing effective control of pepper blight.

[0051] To better explain the present invention, the main contents of the present invention are further illustrated below with reference to specific embodiments, but the contents of the present invention are not limited to the following embodiments. Unless otherwise specified, the technical solutions involved in the embodiments of the present invention are conventional solutions in the art; the reagents or materials mentioned, unless otherwise specified, are all from commercial sources.

[0052] Example 1: Isolation, identification, and biological characteristics analysis of Pseudomonas aeruginosa 115-17 1.1 Isolation and Screening of Strain 115-17 In the vegetable greenhouse of the Wuhan Academy of Agricultural Sciences, Hubei Province, pepper plants treated with CanL-30 powder for two months were selected. Soil samples were taken from the rhizosphere and placed in sterile sampling bags, then immediately stored in ice packs for later use. In the laboratory, Pseudomonas bacteria were isolated from the rhizosphere soil of the pepper plants. Soil that could be naturally air-dried was crushed in a sterile mortar and passed through a 20-mesh sieve. 5 g of soil sample was placed in a conical flask containing 45 mL of sterile water, with a few glass beads added. The flask was then incubated on a shaker at 150 rpm for 2 h and allowed to stand for 1 h. 1 mL of the incubation was then transferred to a centrifuge tube containing 9 mL of sterile water and thoroughly shaken to prepare 10... -2 Soil dilution solution, and so on, to make 10 -3 10 -4 and 10 -5The soil suspension is then prepared for use.

[0053] Using a sterile pipette, 200 μL of each suspension at different concentrations was taken and added to CN medium (formulation per liter: 16 g gelatin peptone, 10 g tryptone, 10 g potassium sulfate, 1.4 g magnesium chloride, 15 g agar, with 0.2 g hexadecyltrimethylamine bromide and 0.015 g naphthiocarboxylic acid as selective inhibitors, and 10 mL glycerol as a carbon source; each component was dissolved in 1 L distilled water, the pH was adjusted to 7.1±0.2, and the medium was autoclaved at 121 °C for 15 min, cooled to 45-50 °C, and poured onto plates for later use). The suspensions were spread evenly using a sterile spreader, and the medium was then inverted and incubated at 30 °C for 48 h. After incubation, seven single colonies with typical morphology and color were picked, and these single colonies were streaked for purification, then transferred to LB slant medium, incubated at 30 °C for 24 h, and then stored at 4 °C for later use. To screen for strains with growth-promoting functions, each isolated strain was inoculated into LB medium and cultured at 28°C with shaking at 180 rpm for 48 h; subsequently, the bacterial cells were collected by centrifugation at 12000 rpm and resuspended in sterile water to a final volume of 1×10⁻⁶. 8 CFU / mL. The obtained bacterial suspension was used to treat pepper plants by root irrigation, and the fresh weight was measured after 14 days. After two rounds of screening, the strain with the most significant growth-promoting effect was obtained and named 115-17.

[0054] 1.2 Morphological characteristics of strain 115-17: On CN (Cetrimide-Nalidixic acid) plates, this strain grows rapidly. After 24 hours of growth, *Pseudomonas aeruginosa* forms bright green to blue-green colonies with a diameter of 2-4 mm, round, raised surfaces, and neat edges. The surface is moist, smooth, and opaque (as shown in the image). Figure 1 (A and B).

[0055] 1.3 Identification of the taxonomic attributes of strain 115-17: Strains 115-17 were inoculated into LB medium and cultured with shaking at 180 rpm for 24 h. After centrifugation to remove the filtrate, the 115-17 cells were washed twice with physiological saline. Genomic DNA of 115-17 was extracted using the CTAB method (Liu Binghui et al., 2008). PCR amplification was performed using universal gyrB amplification primers (SEQ ID No. 1, gyrB-F: 5′-GAAGGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′; SEQ ID No. 2, gyrB-R: 5′-AGCAGAGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′).

[0056] The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 1 min, 56℃ annealing for 1.5 min, 72℃ extension for 30 s, 35 cycles, followed by a 72℃ extension for 10 min. PCR products were detected by 1% agarose gel electrophoresis. PCR product recovery and purification were performed using an Axygen DNA recovery kit. Sequencing results were submitted to the NCBI nucleic acid database for BLAST alignment. The gyrB sequence of the relevant Pseudomonas aeruginosa and an outgroup strain, Bacillus velezensis Hao 2023, were selected. Multiple sequence alignment was performed using MEGA 7.0 software, and a phylogenetic tree was constructed using the neighbor-joining method to determine the taxonomic position of the strains. The phylogenetic tree results showed that strain 115-17 clustered with Pseudomonas aeruginosa Jade-X in one branch (…). Figure 1 Based on the morphological characteristics of strain 115-17 (C), strain 115-17 was finally identified as Pseudomonas aeruginosa.

[0057] SEQ ID No. 3 Pseudomonas aeruginosa 115-17 gyrB sequence:

[0058] Example 2: Evaluation of the growth-promoting properties of Bacillus belyssus Bv-6 and Pseudomonas aeruginosa 115-17 2.1 Determination of nitrogen fixation capacity of strains Single colonies of *Bacillus belyssioides* Bv-6 and *Pseudomonas aeruginosa* 115-17 grown on LA medium were picked and transferred to LB liquid medium and cultured with shaking at 28°C and 180 rpm for 48 hours. The fermentation broth was collected, centrifuged at 12000 rpm to obtain bacterial cells, resuspended in sterile water, and the OD value of the suspension was adjusted to 0.6. 3 μL of each of the Bv-6 and 115-17 bacterial suspensions were added dropwise to the surface of Ashby nitrogen-free medium (10.0 g glucose, 0.2 g KH₂PO₄, 0.2 g MgSO₄·7H₂O, 0.2 g NaCl, 0.1 g K₂SO₄, 5.0 g CaCO₃, 15.0 g agar powder, 1000 mL distilled water, sterilized at 121°C for 20 min), with three replicates for each strain. The inoculated plates were incubated at 28°C for 2 days, and the growth of the strains was observed. If there is obvious colony growth at the inoculation site, the strain is considered to have nitrogen-fixing ability.

[0059] 2.2 Determination of the strain's ability to produce siderophores Take 10 μL of pretreated (see Section 2.1) Bacillus belyssus Bv-6 and Pseudomonas aeruginosa 115-17 fermentation broth supernatant, respectively, and use a sterile 6 mm diameter punch to make evenly distributed wells on CAS detection medium. Carefully remove the agar block from the wells, and then add an equal volume of fermentation broth supernatant to the corresponding wells. Each strain was replicated in triplicate. The CAS medium was prepared as follows: 60.5 mg of Chrome Azurol S was dissolved in 50 mL of a solution containing 1 mmol / L FeCl3·6H2O and 10 mmol / L HCl. After mixing, 40 mL of an aqueous solution containing 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA) was slowly added under continuous stirring to adjust the pH to 7.0, and the volume was brought to 100 mL to obtain the CAS colorimetric solution. Separately, 900 mL of basal medium was prepared, containing 20 g agar, 4.5 g peptone, 9 g glucose, 2.7 g beef extract, and 4.5 g NaCl, with the pH adjusted to 7.0. Both solutions were sterilized at 115℃ for 20 minutes, then mixed thoroughly under aseptic conditions and poured into plates for later use. The inoculated plates were incubated at 28℃ for 2 days. If a distinct orange-yellow halo appeared around the colony, it was considered a positive result for siderophore secretion.

[0060] 2.3 Determination of the strain's ability to solubilize inorganic phosphorus Take 3 μL of pretreated (as described in "2.1") Bacillus belyssus Bv-6 and Pseudomonas aeruginosa 115-17 bacterial suspensions and spot them onto the surface of NBRIP medium (10 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 5 g Ca3(PO4)2, 0.3 g MgSO4, 0.3 g K2SO4, 0.03 g MnSO4, 0.03 g FeSO4, 15 g agar, 1 L distilled water, sterilized at 121℃ for 20 min). Each strain was replicated in triplicate. After incubation at 28℃ for 2 days, observe the results. The appearance of a lysing zone indicates a positive result.

[0061] 2.4 Determination of the strain's ability to produce ammonia Bv-6 and 115-17 strains were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of peptone water (10 g / mL) and cultured at 28℃ and 180 rpm for 2 days. 2 mL of each strain was then transferred to a 5 mL centrifuge tube, 0.5 mL of Nessler's reagent was added, and the mixture was shaken well before observation. Peptone water was used as a control. A reddish-brown color was considered positive.

[0062] 2.5 IAA production capacity Bacillus velezensis Bv-6 and Pseudomonas aeruginosa 115-17 strains were inoculated into 100 mL LB liquid medium (supplemented with L-tryptophan to a final concentration of 100 mg·L⁻¹). -1 The culture medium was placed in a 250 mL Erlenmeyer flask and incubated at 28 °C and 180 rpm with shaking for 48 hours. After incubation, the culture medium was centrifuged, and the supernatant was collected. 100 μL of the supernatant was added to each well of a 96-well plate, along with an equal volume of Salkowski colorimetric solution (made by mixing 50 mL of 35% perchloric acid solution with 1 mL of 0.5 mol·L⁻¹). -1 The solution was prepared using ferric chloride solution, shaken to mix, and then allowed to stand for reaction. A negative control was a mixture of uninoculated LB liquid medium (containing an equal amount of L-tryptophan) and an equal volume of colorimetric solution, using 500 μg / mL... -1 A mixture of the IAA standard solution and an equal volume of colorimetric solution was used as a positive control, with three replicates for each treatment. If the reaction system turned red, the strain was considered to have the ability to produce indole-3-acetic acid (IAA).

[0063] Evaluation of the growth-promoting properties of *Bacillus belyssae* Bv-6 and *Pseudomonas aeruginosa* 115-17 showed that *Pseudomonas aeruginosa* 115-17 possesses nitrogen fixation, inorganic phosphorus hydrolysis, and siderophore production capabilities, but lacks the ability to produce IAA; *Bacillus belyssae* Bv-6 possesses nitrogen fixation and siderophore production capabilities, but lacks the ability to hydrolyze inorganic phosphorus and produce IAA. Specific results are detailed in [link to relevant documentation]. Figure 2 As shown in the figure. Among them: Figure A shows the nitrogen fixation ability of the strain; Figure B shows the siderophore production ability of the strain; Figure C shows the inorganic phosphorus solubilization ability of the strain; Figure D shows the ammonia production ability of the strain; Figure E shows the IAA production ability of the strain.

[0064] Table 1. Evaluation of the growth-promoting properties of Bacillus belyssus Bv-6 and Pseudomonas aeruginosa 1115-17 .

[0065] Example 3: Evaluation of the remedial effect of combined application of Bacillus vesiculosus Bv-6 and Pseudomonas aeruginosa 115-17 on pepper growth impairment induced by high-temperature sterilization soil. The combined application of *Bacillus berleis* Bv-6 and *Pseudomonas aeruginosa* 115-17 to repair the growth impairment of peppers induced by high-temperature sterilization of soil was investigated. Single colonies of *Bacillus berleis* Bv-6 and *Pseudomonas aeruginosa* 115-17 grown on LA medium were transferred to LB liquid medium and cultured at 28℃ and 180 rpm for 48 hours with shaking. The fermentation broth was collected, centrifuged at 12000 rpm to obtain bacterial cells, and resuspended in sterile water to obtain a bacterial suspension of 1×10⁻⁶. 8 CFU / mL is available for use. The experiment is divided into the following five treatments: 1. CK+: Peppers are planted in soil without high-temperature sterilization. After the peppers are two weeks old, each pepper plant is irrigated with 20 mL of sterile water. 2. CK-: After the soil is sterilized at high temperature (121℃, 30 min), chili peppers are planted. Two weeks after the chili peppers are two weeks old, each chili pepper plant is irrigated with 20 mL of sterile water. 3. Bv-6: After the soil is sterilized at high temperature (121℃, 30 min), peppers are planted. Two weeks after the peppers are grown, 10 mL of Bacillus vesicle Bv-6 suspension and 10 mL of sterile water are mixed and applied to the roots. 4. 115-17: After the soil is sterilized at high temperature (121℃, 30 min), plant peppers. Two weeks after the peppers are grown, mix 10 mL of Pseudomonas aeruginosa suspension and 10 mL of sterile water and drench the roots. 5. Bv-6+115-17: After the soil is sterilized at high temperature (121℃, 30 min), peppers are planted. Two weeks after the peppers are grown, 10 mL of Bacillus vesiculosus Bv-6 and 10 mL of Pseudomonas aeruginosa 115-17 are mixed and applied to the roots.

[0066] Plants of each treatment were cultured in a 24℃ incubator (light:dark = 16 hours:8 hours). After 14 days of root irrigation for each treatment, the fresh weight of the pepper plants was counted. Each treatment group consisted of 8 pepper plants replicated. All data are expressed as mean ± standard deviation.

[0067] The results are as follows Figure 3 As shown in Table 2, after 14 days of treatment, compared with unsterilized healthy soil (CK+), high-temperature sterilized soil (CK-) significantly inhibited the growth of chili seedlings, reducing their fresh weight by 22.41%. In the high-temperature sterilized soil, both Bv-6 and 115-17 single-agent treatments significantly alleviated the growth inhibition caused by high-temperature sterilization. Specifically, the Bv-6 treatment increased the fresh weight of chili peppers by 35.63% compared to CK-, while the 115-17 treatment increased the fresh weight by 24.71%. The combined application of Bv-6 and 115-17 showed a significant synergistic effect. In the high-temperature sterilized soil, the fresh weight of the mixed-agent treatment was significantly higher than that of the 115-17 single-agent treatment, and significantly better than that of the Bv-6 and 115-17 single-agent treatments, with a fresh weight increase of 81.03% compared to CK-.

[0068] Table 2. Statistics on plant height and fresh weight of chili peppers in each treatment (14 days) .

[0069] Example 4: Evaluation of the plate antagonistic activity of Bacillus belyssus Bv-6 and Pseudomonas aeruginosa 115-17 against Phytophthora. Single colonies of *Bacillus belyssioides* Bv-6 and *Pseudomonas aeruginosa* 115-17 were picked and inoculated into liquid LB medium, and cultured with shaking at 28°C and 180 rpm for 48 hours. The fermentation broth was centrifuged at 4°C and 12000 rpm for 10 minutes, and the supernatant was collected and aseptically filtered through a 0.22 μm microporous membrane. The resulting sterile fermentation supernatant was stored at 4°C for later use.

[0070] The above fermentation supernatant was treated as follows and mixed with melted PDA medium to prepare a poisoned plate: 1. CK control: Use sterile water equal to the highest added volume of the experimental group (i.e., the volume corresponding to 20% concentration) to mix with PDA culture medium.

[0071] 2. Bv-6 treatment: Add Bv-6 fermentation supernatant to make the final volume fraction in the plates 10% and 20%, respectively.

[0072] 3. 115-17 treatment: Add 115-17 fermentation supernatant to make their final volume fraction in the plates 10% and 20%, respectively.

[0073] 4. Bv-6 + 115-17 treatment: Bv-6 and 115-17 fermentation supernatant were premixed at a ratio of 1:1 (v / v), and then added to PDA medium at a total volume fraction of 10% and 20%, respectively.

[0074] A 5 mm diameter mycelial cake, taken from the edge of a vigorous *Phytophthora capsici* colony, was inoculated in the center of each treatment plate. Three replicates were set up for each concentration of each treatment. All plates were incubated in the dark at 20°C for 3 days. After 3 days, the diameter of the pathogen colony in each plate was measured using the cross-hatching method. The inhibition rate of *Phytophthora capsici* mycelial growth by the fermentation supernatant was calculated using the following formula: Mycelial growth inhibition rate (%) = 100% × (Control lesion diameter - Treatment lesion diameter) / Control lesion diameter.

[0075] The results are as follows Figure 4 As shown in Table 3, based on the plate confrontation test results, the fermentation supernatants of *Bacillus belye* Bv-6 and *Pseudomonas aeruginosa* 115-17 both showed significant inhibitory effects on *Phytophthora capsici*, and their combined use exhibited a synergistic effect. Specifically, compared to the control (CK) lesion diameter of 67.33 mm, the 10% and 20% concentrations of Bv-6 treatments inhibited the lesion diameter to 48.53 mm and 42.52 mm, respectively, with inhibition rates of 27.91% and 36.86%. The inhibitory effect of 115-17 was even more significant; the lesion diameters in the 10% and 20% concentration treatments were 34.78 mm and 15.07 mm, respectively, with inhibition rates reaching 48.33% and 77.62%. When the fermentation broths of the two strains were mixed 1:1, the antibacterial activity was significantly enhanced: the lesion diameter in the 10% mixed treatment group was 20.90 mm, and the inhibition rate increased to 68.94%; while the 20% mixed treatment had the most outstanding effect, with the lesion diameter being only 9.95 mm and the inhibition rate as high as 85.39%, which was significantly better than the single strain treatment.

[0076] Table 3. Antagonistic activity of Bv-6 and 115-17 against Phytophthora capsici (3 days) on plate. .

[0077] Example 5: Evaluation of the control efficacy of Bacillus belye Bv-6 and Pseudomonas aeruginosa 115-17 against pepper blight. One-month-old pepper seedlings were transplanted into plastic pots filled with culture soil (nutrient soil: substrate soil: vermiculite = 8:4:1). After culturing in the dark for 7 days, pepper seedlings of uniform size were selected for the experiment. Preparation of *Phytophthora capsici* spore suspension: *Phytophthora capsici* spores that had fully colonized a PDA plate were added to 15 mL of 10% V8 culture medium and incubated in the dark at 25 °C for 3 days. The culture medium was discarded, and 10 mL of sterile water was added to cover the mycelium. Incubation continued, with the sterile water changed every 12 hours for a total of 3 times. The plate was then placed in a 4 °C refrigerator for 15 min, followed by a 25 °C refrigerator for 15 min, alternating hot and cold temperatures to stimulate the release of zoospores from the sporangia. The spore suspension was diluted to 10% with sterile water. 5 One spore / mL for use.

[0078] The experiment consisted of four treatments, all of which involved pricking the base of the chili pepper stems: 1. CK control: After drenching the roots with 20 mL of sterile water for 24 h, inoculate with 5 mL of zoospores of Phytophthora capsici. 2. Bv-6 root drenching treatment: After drenching the roots with 20 mL of Bv-6 fermentation broth for 24 h, inoculate with 5 mL of zoospores of Phytophthora capsici. 3. Root drenching treatment with Pseudomonas aeruginosa 115-17: After drenching the roots with 20 mL of Pseudomonas aeruginosa 115-17 fermentation broth for 24 h, inoculate with 5 mL of zoospores of Phytophthora capsici. 4. Root irrigation treatment with a mixture of Pseudomonas aeruginosa 115-17 and Bv-6: After irrigating the roots with 20 mL of a 1:1 mixture of Pseudomonas aeruginosa 115-17 and Bv-6 fermentation broth, inoculate with 5 mL of zoospores of Phytophthora capsici for 24 h.

[0079] Five days after vaccination, the disease index was statistically analyzed. The disease index grading standard for Phytophthora capsici is as follows: Level 0: No obvious symptoms Grade 1: A small number of brown lesions appear at the base of the seedlings. Grade 2: The basal lesions extend to the heart leaves or encircle the entire stem, causing the plant to slightly droop. Level 3: The plant is lodged, and most leaves are noticeably wilted or yellowed. Level 4: Plants are lodged and wilted. Level 5: Plant withered and dead like Figure 5As shown in Table 4, both *Bacillus belye* Bv-6 and *Pseudomonas aeruginosa* 115-17, when applied alone, significantly inhibited the occurrence of *Phytophthora capsici* and effectively reduced the disease index. Compared with the blank control (CK) with a disease index of 96, the disease index of the Bv-6 treatment group was 76, with a control efficacy of 20.83%; the disease index of the 115-17 treatment group was 28, with a control efficacy of 70.83%. When the two strains were applied synergistically, the control effect was further enhanced, the disease index further decreased to 18, and the control efficacy reached 81.25%, significantly better than either single-strain treatment, indicating that the two strains have a synergistic effect in inhibiting *Phytophthora capsici*.

[0080] Table 4. Efficacy determination of Bv-6 and 115-17 against Phytophthora capsici (5 days) .

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A biocontrol agent composition, characterized in that: This includes *Pseudomonas aeruginosa* 115-17 and *Bacillus velezensis* Bv-6; the preservation number of *Pseudomonas aeruginosa* 115-17 is CCTCC No: M2026293, and the preservation number of *Bacillus velezensis* Bv-6 is CCTCC No: M20191106.

2. The biocontrol agent composition according to claim 1, characterized in that: In the biocontrol agent composition, the ratio of Pseudomonas aeruginosa 115-17 to Bacillus velezensis Bv-6 is 1:

1.

3. The biocontrol agent composition according to claim 1, characterized in that: The biocontrol agent composition includes a suspension of Pseudomonas aeruginosa 115-17 and a suspension of Bacillus velezensis Bv-6. Alternatively, the biocontrol agent composition may comprise fermentation broth of Pseudomonas aeruginosa 115-17 and Bacillus velezensis Bv-6.

4. The biocontrol agent composition according to claim 3, characterized in that: The bacterial suspension is obtained by centrifuging the fermentation broth of the strain, collecting the bacterial cells, and resuspending them in sterile water. Preferably, the concentration of the *Pseudomonas aeruginosa* 115-17 bacterial suspension is 1×10⁻⁶. 8 CFU / mL; Preferably, the concentration of the Bacillus velezensis Bv-6 bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

5. The use of the biocontrol agent composition according to any one of claims 1 to 4 in improving growth barriers caused by high-temperature sterilization of soil or in controlling plant diseases.

6. The application according to claim 5, characterized in that: The growth obstacle is a growth obstacle in chili pepper cultivation caused by high-temperature sterilization of the soil; or, the plant is chili pepper; or, the pathogen of the disease is Phytophthora capsici.

7. A method for improving growth barriers caused by high-temperature sterilization of soil, characterized in that: The procedure includes the following steps: treating plants grown in high-temperature sterilized soil with *Pseudomonas aeruginosa* 115-17 and / or *Bacillus velezensis* Bv-6; the preservation number of *Pseudomonas aeruginosa* 115-17 is CCTCC No: M2026293, and the preservation number of *Bacillus velezensis* Bv-6 is CCTCC No: M20191106; Preferably, the plant is a chili pepper; Preferably, the treatment involves preparing a suspension of Pseudomonas aeruginosa 115-17 and / or a suspension of Bacillus velezensis Bv-6 for root irrigation of the plants.

8. The method according to claim 7, characterized in that: The bacterial suspension is obtained by centrifuging the fermentation broth of the strain, collecting the bacterial cells, and resuspending them in sterile water. Preferably, the concentration of the *Pseudomonas aeruginosa* 115-17 bacterial suspension is 1×10⁻⁶. 8 CFU / mL; Preferably, the concentration of the Bacillus velezensis Bv-6 bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

9. The method according to claim 7, characterized in that: The ratio of Pseudomonas aeruginosa 115-17 to Bacillus velezensis Bv-6 is 1:

1.

10. A method for preventing and controlling plant diseases, characterized in that: The procedure includes the following steps: treating plants with *Pseudomonas aeruginosa* 115-17 and / or *Bacillus velezensis* Bv-6; the *Pseudomonas aeruginosa* 115-17 has the accession number CCTCC No: M2026293, and the *Bacillus velezensis* Bv-6 has the accession number CCTCC No: M20191106; Preferably, the plant is a chili pepper; the pathogen of the blight is *Phytophthora capsici*. Preferably, the treatment method is to prepare a fermentation broth of Pseudomonas aeruginosa 115-17 and / or Bacillus velezensis Bv-6 and apply it to the roots of the plant for irrigation. Preferably, the ratio of Pseudomonas aeruginosa 115-17 to Bacillus velezensis Bv-6 is 1:1.

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