A strain of Pseudomonas oryzae NF9-1 and its application

Abstract

This invention relates to a biocontrol agent, wherein the effective active ingredient is *Pseudomonas veronii* NF9-1. The biocontrol agent is characterized by being an aqueous solution, with the following composition and weight percentages: *Pseudomonas veronii* NF9-1: 89.5-99.8%; sodium dodecyl sulfate: 0.1-10%; potassium sorbate: 0.1-0.5%. In application, the aqueous solution is sprayed on the leaves to control strawberry anthracnose and powdery mildew, and applied as a root drench to control strawberry wilt, etc. Its advantages are: by introducing *Pseudomonas veronii* NF9-1, which is safe for humans and animals and has good control effects against various strawberry diseases, it achieves the goals of controlling plant diseases using biocontrol agents; reducing the amount of chemical pesticides used; and simultaneously improving the ecological environment and reducing the residues of chemical pesticides in agricultural products.

Description

Technical Field

[0001] This invention relates to a biocontrol agent and its application, particularly a strain of *Pseudomonas aeruginosa* NF9-1 and its application in controlling strawberry diseases. Background Technology

[0002] Plant diseases are a major factor affecting stable, high-yield, and high-quality agricultural production. Pathogens mainly include fungi, bacteria, nematodes, and viruses. Currently, plant disease control in production relies primarily on chemical pesticides, which not only causes serious environmental pollution but also directly increases the residues of toxic chemicals in agricultural products, posing a serious threat to human health. To effectively control plant diseases and reduce the use of chemical pesticides, the use of biological control techniques is receiving increasing attention from governments and people worldwide. Biological control techniques mainly utilize biodiversity by releasing or introducing beneficial microorganisms into nature to sustainably control plant diseases. Currently, the most widely used biocontrol bacteria both domestically and internationally include Bacillus and Pseudomonas.

[0003] Pseudomonas species are widely distributed in nature, including soil, water, food, and air. They possess capsules, flagella, and pili, have low nutritional requirements, and are diverse. The main species associated with human infection include *Pseudomonas aeruginosa* and *Pseudomonas pseudomallei*. Many species in the *Pseudomonas* genus have biocontrol effects, and the production of antimicrobial substances is one of the important factors in their biocontrol of plant diseases. It has been reported that the antimicrobial substances produced by *Pseudomonas* mainly include pyrrolnitrin (Pfender et al., 1993), pyrolurin (Howell et al., 1980), phenazines (Thomashow et al., 1988), and 2,4-diacetylphloroglucinol (2,4-DAPG) (Keel et al., 1992). Lee et al. (2003) isolated a strain of Pseudomonas GC-B26 from soil, which showed strong inhibitory activity against Phytophthora capsici. Zhang Liqun et al. (2004) found that a strain of Pseudomonas fluorescens 2P24, isolated from soil where wheat take-all disease had naturally subsided, exhibited good control efficacy against wheat take-all (Gaeumannomyces graminis), tomato bacterial wilt (Ralstonia solanacearum), and Fusarium spp. root rot.

[0004] The Nanjing Agricultural Sciences Research Institute in the hilly areas of Jiangsu Province collected soil samples from strawberry greenhouses in Xianlin Street, Qixia District, Nanjing City, Jiangsu Province, and obtained a novel biocontrol strain NF9-1 after isolation and purification. Previous research results showed that biocontrol strain NF9-1 has good control effects against several important diseases in strawberry production (such as anthracnose and powdery mildew). After testing and identification by the Institute of Microbiology, Chinese Academy of Sciences, based on comprehensive analysis of experimental data including cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence, strain NF9-1 was identified as *Pseudomonas veronii*. *Pseudomonas veronii* NF9-1 was registered with the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 26092.

[0005] Existing literature reports that *Pseudomonas oryzae* is mostly used to produce proteases and catalases. *Pseudomonas oryzae* also exhibits phosphate-solubilizing activity, producing distinct phosphate-solubilizing zones on inorganic phosphorus plates, indicating its potential application value in the bioremediation of saline-alkali soils (Liu Ximin et al., 2019, *Journal of Tianjin Agricultural College*). Furthermore, there are reports that *Pseudomonas oryzae* can degrade triphenylmethane dyes such as malachite green and volatile harmful compounds such as α-pinene (Wang Yani et al., 2019, *Biotechnology Bulletin*; Zhou Dandan, 2020, Master's Thesis, Zhejiang University of Technology). The *Pseudomonas oryzae* strains with existing patents are mainly used for wastewater treatment, degrading nitrogenous compounds in wastewater (patent application numbers: CN202011509694.3; CN202010673897.X; CN201911299027.4); highly efficient phosphorus solubilization (patent application number: CN201410174982.6); and degradation of triphenylmethane dyes and α-pinene compounds, especially showing excellent removal rates for malachite green, crystal violet, or gentian violet (patent application numbers: CN201910314629.6; CN201610345493.1). Furthermore, *Pseudomonas oryzae* produces the CIP104663 protein, which can serve as a catalyst for the synthesis of L-2-piperidinecarboxylic acid (CN201610196783). Currently, there are no reports, either domestically or internationally, on the biocontrol applications of Pseudomonas veronii against strawberry anthracnose, powdery mildew, and wilt. Summary of the Invention

[0006] The purpose of this invention is to provide a biocontrol strain with good application prospects - Pseudomonas veronii NF9-1. This strain was registered with the China General Microbiological Culture Collection Center on November 14, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 26092.

[0007] This invention provides a biocontrol agent, wherein the effective active ingredient is composed of Pseudomonas aeruginosa NF9-1.

[0008] The biocontrol agent of the present invention contains three antibacterial-related substances, namely protease, hepatophilia and chitinase, in the metabolites secreted by Pseudomonas aeruginosa NF9-1.

[0009] The biocontrol agent described in this invention is an aqueous solution.

[0010] The aqueous agent of the present invention is characterized in that: the components and their weight percentages are as follows: Pseudomonas aeruginosa NF9-1: 89.5-99.8%; sodium dodecyl sulfonate: 0.1-10%; potassium sorbate: 0.1-0.5%; pH value: 6.5-7.0.

[0011] The aqueous solution described in this invention can be sprayed on plant leaves to prevent and control strawberry anthracnose and strawberry powdery mildew, and used for root irrigation to prevent and control various plant fungal diseases such as strawberry wilt.

[0012] The advantages of this invention are: a biocontrol strain, *Pseudomonas aeruginosa* NF9-1, which is safe for humans and animals and has good application prospects, has been developed, enriching biocontrol resources; this biocontrol agent can be used to control a variety of plant diseases, reduce the amount of chemical agents used in production, and at the same time improve the ecological environment and reduce the amount of chemical agents residues in agricultural products. Attached Figure Description

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0014] Figure 1 The inhibitory effect of Pseudomonas aeruginosa NF9-1 on bacteria.

[0015] Figure 2 This refers to the inhibitory effect of Pseudomonas aeruginosa NF9-1 on pathogenic fungi.

[0016] Figure 3 It is the protease produced by Pseudomonas oryzae NF9-1.

[0017] Figure 4 It is Pseudomonas rosenbergii NF9-1 that produces ferrophiles.

[0018] Figure 5 It is chitinase produced by Pseudomonas oryzae NF9-1.

[0019] In the diagram: A: *Pseudomonas aeruginosa* NF9-1; B: *Bacillus megaterium*; C: *Erwinia*; D: *Rhizoctonia solani* (bacterial wilt of tomato); E: *Rhizoctonia solani* (bacterial leaf streak of rice); F: *Fusarium wilt* (bacterial wilt of tomato); G: *Anthracnose* (bacterial rot of strawberry); H: *Rhizoctonia solani* (sheath blight of rice); I: *Black spot* (bacterial rot of cabbage); J: *Sclerotinia sclerotiorum* (bacterial rot of rapeseed); K: *Gray mold* (bacterial rot of strawberry). Detailed implementation method:

[0020] Example 1

[0021] Inhibitory effect of Pseudomonas aeruginosa NF9-1 on bacteria

[0022] A. Test pathogenic bacteria

[0023] The tested bacteria included *Bacillus megaterium*, *Erwinia*, *Ralstonia solanacearum*, and *Bacterium tumefaciens*, which were provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. They were cultured in LB broth (10.0 g peptone, 5.0 g yeast extract, 10 g NaCl, 1000 mL distilled water) at 28°C with shaking at 150 rpm for 2 days, and then diluted with sterile water to a final concentration of 10. 8 cfu / mL, for later use.

[0024] B. Test methods:

[0025] The *Pseudomonas aeruginosa* NF9-1 strain, screened by the Nanjing Agricultural Science Research Institute in the hilly areas of Jiangsu Province, was activated on LB medium, then transferred to 50 mL of LB medium and cultured at 28 °C with shaking at 150 rpm for 2 days. The culture was then diluted with sterile water to a final concentration of 10. 8 cfu / mL, for later use.

[0026] Take 5 μL of *Pseudomonas aeruginosa* NF9-1 bacterial suspension and spot-inoculate it in the center of an LB agar plate. After incubating at 28°C for 48 h, spray with a diluted pathogenic bacterial solution and incubate overnight at 28°C. Observe the diameter of the inhibition zone. Each treatment is repeated 3 times, and the average value is taken. A water control is included.

[0027] C. Experimental Results:

[0028] The test results are shown in Figure 1 The bacterial suspension of *Pseudomonas aeruginosa* NF9-1 showed some inhibitory effect on all four tested bacteria. The inhibition zone diameter was 42.7 mm against *Bacillus megaterium*, 39.0 mm against *Erwinia*, and 42.2 mm against *Bacillus streakus*, the causal agent of rice bacterial leaf streak. The inhibitory effect was weakest against *Ralstonia solanacearum*, with an inhibition zone diameter of only 9.64 mm.

[0029] like Figure 1 As shown.

[0030] Example 2

[0031] Inhibitory effect of Pseudomonas aeruginosa NF9-1 against 6 plant pathogenic fungi

[0032] A. Test pathogenic fungi:

[0033] The pathogenic fungi included *Fusarium wilt*, *Anthracnose*, *Rhizoctonia solani*, *Black spot*, *Sclerotinia sclerotiorum*, and *Gray mold*, all provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. They were cultured on PDA medium (200g potato, 20g glucose, 20g agar, and 1000mL distilled water, pH 6.5-7.0) at 28℃ for 5 days for later use.

[0034] B. Test methods:

[0035] The preparation of Pseudomonas oryzae NF9-1 bacterial suspension was the same as in Example 1.

[0036] A 6 mm diameter fungal disc was placed on one side of a blank PDA plate, 30 mm from the edge. On the other side of the plate, 30 mm from the edge, 5 μL of *Pseudomonas aeruginosa* NF9-1 bacterial suspension was inoculated. The plates were incubated at 28 °C with a water control. When the control plate showed confluent pathogen colonies, the diameter of the pathogens in each treatment was measured, and the inhibition rate was calculated. Each treatment was repeated three times, and the average value was taken.

[0037] Inhibition rate of pathogenic fungi = [(Diameter of growth of control pathogens - diameter of growth of treated pathogens) / diameter of growth of control pathogens] × 100%.

[0038] C. Experimental Results:

[0039] The test results are shown in Table 1 and Figure 2 The results showed that *Pseudomonas aeruginosa* NF9-1 bacterial suspension had a strong inhibitory effect on all six tested pathogenic fungi in the laboratory. Among them, the inhibitory effect on the mycelial growth of *Fusarium wilt* of strawberry was the strongest, with an inhibition rate of 65%. This was followed by *Sclerotinia sclerotinia* of rapeseed and *Botrytis cinerea* of strawberry, with inhibition rates of 63.67% and 60.11%, respectively.

[0040] Table 1. Inhibitory effect of *Pseudomonas aeruginosa* NF9-1 on tested pathogenic fungi.

[0041]

[0042]

[0043] like Figure 2 As shown.

[0044] Example 3

[0045] Production of antimicrobial-related substances from the metabolic secretions of *Pseudomonas aeruginosa* NF9-1

[0046] Test materials: Protease detection medium: 100g skim milk powder, 20g agar, adjusted to 1000mL. Ferrophilic acid detection medium: 60.5mg CAS, 10mL ferric solution (1mmol·L⁻¹). -1 FeCl3·6H2O), HDTMA 72.9mg, agar 20g, bring to 1000mL, pH 7.0. Chitinase detection medium: chitin 15g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, K2HPO4 0.7g, KH2PO4 0.3g, agar 20g, bring to 1000mL, pH 7.0-7.2. Cellulase detection medium: peptone 10g, yeast extract 10g, sodium carboxymethyl cellulose 10g, NaCl 5g, KH2PO4 1g, agar 20g, bring to 1000mL, pH 7.0.

[0047] The preparation of *Pseudomonas aeruginosa* NF9-1 bacterial suspension was the same as in Example 1. 5 μL of *Pseudomonas aeruginosa* NF9-1 bacterial suspension was inoculated at the center of each test medium plate, with each treatment repeated three times. The plates were incubated at 28°C for 3-7 days, and then observed.

[0048] Protease detection: Observe whether a clear zone is produced. If a clear zone is produced, it indicates that the antagonistic bacteria can secrete proteases.

[0049] Ferrophilic assay: After incubation at 28℃ for 7-10 days, observe for the formation of a yellow halo. If a yellow halo appears, it indicates that the antagonistic bacteria can secrete ferophilic substances.

[0050] Chitinase assay: Observe whether a clear zone is formed. If a clear zone is formed, it indicates that the antagonistic bacteria can secrete chitinase to degrade chitin.

[0051] Cellulase detection: Observe whether a clear zone is formed around the colony. If a clear zone is formed, it indicates that the antagonistic bacterium can secrete cellulase.

[0052] The experimental results showed that *Pseudomonas aeruginosa* NF9-1 cannot secrete cellulase; however, it can secrete protease, which forms a clear zone around the colony when used to detect proteins on a plate. Figure 3 It can secrete hematophilic acid, which interacts with Fe in the detection plate. 3+ Combined, an orange-yellow halo forms around the colony. Figure 4 It can secrete chitinase, which, when used to detect chitin in a plate, forms a clear zone around the colony. Figure 5 ).

[0053] like Figure 3 , Figure 4and Figure 5 As shown.

[0054] Example 4

[0055] Obtaining Pseudomonas aeruginosa NF9-1 aqueous solution

[0056] A. Preparation and activation of bacterial strains.

[0057] LB medium (same as in Example 1) was sterilized after adding agar using standard methods and made into slant agar. Pseudomonas oryzae NF9-1 was inoculated and cultured at 28°C for 2 days.

[0058] B. Liquid fermentation.

[0059] Primary fermentation: Transfer one loopful of the cultured bacterial growth from the test tube slant into 300 mL of LB broth using an inoculation loop (same as in Example 1). Incubate at 28°C and 150 rpm with shaking for 2 days, then set aside.

[0060] Secondary fermentation: Culture medium formula: 1% soybean meal, 0.5% soybean flour, 0.15% yeast extract, 1% corn flour, 0.5% corn steep liquor, 0.5% sucrose, 0.05% NaCl, 0.18% CaCO3. The pH value before sterilization is 7.0. 1 ton of material is added to a 2-ton fermenter. Sterilize at 121℃ for 30 minutes. When cooled to approximately 30℃, inoculate with 300mL of the above primary fermentation inoculum. Liquid fermentation temperature is 32℃, stirring speed is 300rpm. Aeration rate is 1:1.5. The fermentation cycle is 18 hours.

[0061] Tertiary fermentation: The culture medium formula is the same as that for secondary fermentation. 10 tons of material are added to a 15-ton fermenter. Sterilize at 121℃ for 30 minutes. When cooled to approximately 30℃, inoculate with 1 ton of the aforementioned secondary fermentation inoculum. Liquid fermentation temperature is 32℃, stirring speed is 300 rpm. Aeration ratio is 1:1.5. The fermentation cycle is approximately 24 hours; then remove from the fermenter.

[0062] C. Preparation of aqueous solution.

[0063] The fermentation broth was placed in a storage tank, and 0.1-10% sodium dodecyl sulfate and 0.1-0.5% potassium sorbate were added and stirred thoroughly. The pH of the fermentation broth was measured to be 6.5-7.0, and the viable cell count was 10. 9 -2×10 9 cfu / mL. Bottled into an aqueous solution of *Pseudomonas aeruginosa* NF9-1.

[0064] Example 5

[0065] A small-scale trial of the efficacy of Pseudomonas aeruginosa NF9-1 aqueous solution against strawberry anthracnose.

[0066] Experiment location: Strawberry plantation at the Lishui Experimental Base of Jiangsu Academy of Agricultural Sciences.

[0067] The experimental variety was Red Cheek, provided by the Fruit Tree Research Institute of Jiangsu Academy of Agricultural Sciences.

[0068] Test and control agents: *Pseudomonas aeruginosa* NF9-1 aqueous solution was provided in Example 4. The control agent was 25% prochloraz, a commonly used chemical agent for controlling strawberry anthracnose in production, provided by Jiangsu Huifeng Agrochemical Co., Ltd., and used according to its recommended field dosage.

[0069] Experimental Methods: Strawberry mother plants were transplanted on April 12, 2022. The experiment included five treatments: 10x dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution, 100x dilution of NF9-1 aqueous solution, 750x dilution of 25% prochloraz, 1500x dilution of 25% prochloraz, and a water control. The experiment was replicated three times, with a total of 15 plots, each 40 m², randomly arranged. The first application of the pesticide was made on June 10, 2022, evenly spraying the strawberry plants, both sides of the leaves, and the runners. A second application was made 7 days later, for a total of four applications. Ten and twenty days after the last application, 50 runners were randomly sampled from each plot to investigate disease incidence, calculate the disease index, and determine the control efficacy. The severity grading criteria for strawberry anthracnose are as follows:

[0070] Grade 0: No lesions;

[0071] Grade 1: The lesion area covers less than 5% of the entire stolon;

[0072] Grade 3: The lesions cover 6%-10% of the entire stolon;

[0073] Grade 5: The lesion area covers 11%-25% of the entire stolon;

[0074] Grade 7: The lesion area covers 26%-50% of the entire stolon;

[0075] Level 9: The lesion covers more than 50% of the entire stolon or the seedlings die.

[0076] Disease index = [(Number of cases at each level × Representative value at each level) / (Total number of plants surveyed × Highest representative value)] × 100

[0077] Prevention and control efficacy (%) = [(Disease index of control - Disease index of prevention and control) / Disease index of control] × 100%

[0078] The experimental results are shown in Table 2. Ten days after the last application, the control efficacy of a 10-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution was 70.11%, and that of a 100-fold dilution was 49.43%. The control efficacy of a conventional chemical agent, 25% prochloraz, diluted 750 times, was 75.86%. Twenty days after application, the disease index of the water control increased significantly. The control efficacy of the 10-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution and the 750-fold dilution of 25% prochloraz was comparable, both greater than 75%. The control efficacy of the 100-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution and the 1500-fold dilution of 25% prochloraz was approximately 60%. *Pseudomonas aeruginosa* NF9-1 can significantly replace the chemical agent prochloraz in the control of strawberry anthracnose.

[0079] Table 2. Field control efficacy of Pseudomonas aeruginosa NF9-1 aqueous solution against strawberry anthracnose.

[0080]

[0081] Example 6

[0082] A small-scale trial of the efficacy of Pseudomonas aeruginosa NF9-1 aqueous solution against strawberry powdery mildew.

[0083] Test location: Baitu Town, Jurong City, Jiangsu Province.

[0084] The experimental variety was Red Cheek, provided by the Fruit Tree Research Institute of Jiangsu Academy of Agricultural Sciences.

[0085] Test and control agents: *Pseudomonas aeruginosa* NF9-1 aqueous solution was provided in Example 4. The control agent was 10% difenoconazole water-dispersible granules, a commonly used chemical agent for controlling strawberry powdery mildew in production, provided by Zhejiang Shijia Technology Co., Ltd., and used according to its recommended field dosage.

[0086] Experimental Methods: The experimental greenhouse had a history of severe powdery mildew. Five treatments were included: 10x dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution, 100x dilution of NF9-1 aqueous solution, 600x dilution of 10% difenoconazole, 1200x dilution of 10% difenoconazole, and a water control. The experiment was repeated three times, with a total of 15 plots, each plot measuring 50 m². 2 The plants were randomly arranged. The first application of the pesticide was performed on November 20, 2022, evenly spraying both sides of the strawberry leaves and the surface of young fruits. A second application was made 7 days later, for a total of three applications. Seven and fourteen days after the last application, five plants were randomly selected from each plot to investigate the disease incidence on all leaves of each plant, and the disease index and control efficacy were calculated. The grading criteria for strawberry powdery mildew are as follows:

[0087] Grade 0: No disease spots on the leaves;

[0088] Grade 1: The lesion area accounts for less than 5% of the total leaf area;

[0089] Grade 3: Lesions cover 6%-15% of the total leaf area;

[0090] Level 5: Lesions cover 16%-25% of the total leaf area;

[0091] Level 7: Lesions cover 26%-50% of the total leaf area;

[0092] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0093] Disease index = [(Number of cases at each level × Representative value at each level) / (Total number of plants surveyed × Highest representative value)] × 100

[0094]

[0095] Where: CK0 - disease index before drug application in the blank control area; CK1 - disease index after drug application in the blank control area; PT0 - disease index before drug application in the drug-treated area; PT1 - disease index after drug application in the drug-treated area.

[0096] The experimental results are shown in Table 3. Before treatment, the disease index of each plot was basically consistent, ranging from 8.82 to 9.15. Seven days after three applications, the disease index of the control plot was 26.89, and the control efficacy of a 10-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution was 54.31%, comparable to the control efficacy of a 600-fold dilution of the conventional chemical agent 10% difenoconazole (56.62%). The control efficacy of a 100-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution was 46.76%, and the control efficacy of a 1200-fold dilution of 10% difenoconazole was 43.84%. Fourteen days after treatment, the disease index of the water control plot continued to increase to 35.42, and the control efficacy of both the 10-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution and the 600-fold dilution of 10% difenoconazole was greater than 60%. The results showed that *Pseudomonas aeruginosa* NF9-1 can replace the chemical agent difenoconazole in the control of strawberry powdery mildew.

[0097] Table 3. Field control efficacy of Pseudomonas aeruginosa NF9-1 aqueous solution against strawberry powdery mildew.

[0098]

[0099] Example 7

[0100] Field trial of the efficacy of Pseudomonas aeruginosa NF9-1 aqueous solution against strawberry wilt disease

[0101] Experiment location: Lishui Plant Base, Jiangsu Academy of Agricultural Sciences.

[0102] The experimental varieties were "Hongjia" and "Ningyu", provided by the Fruit Tree Research Institute of Jiangsu Academy of Agricultural Sciences.

[0103] Test and control agents: *Pseudomonas aeruginosa* NF9-1 aqueous solution was provided in Example 4. The control agent was 50% carbendazim, purchased from Jiangsu Huifeng Agrochemical Co., Ltd., a commonly used chemical agent for controlling strawberry wilt in production, applied according to the recommended field dosage.

[0104] Experimental Methods: Five treatments were set up: 10-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution, 100-fold dilution of NF9-1 aqueous solution, 800-fold dilution of 50% carbendazim, 1600-fold dilution of 50% carbendazim, and a water control. The experiment included two varieties, with three replicates, for a total of 30 plots, each plot being 100 m². 2 The plants were randomly arranged. The first root drenching was conducted on August 30, 2022, with 300 mL applied to each seedling. A second drenching was performed 10 days later, for a total of two treatments. At the end of September, the incidence of Fusarium wilt in the strawberry plants within the plot was investigated, and the control efficacy was calculated.

[0105] Control efficacy (%) = [(Number of diseased plants in control plot - Number of diseased plants in treatment plot) / Number of diseased plants in control plot] × 100%

[0106] The experimental results are shown in Table 4. The disease index of the control variety "Ningyu" was 15.80, while that of the "Hongjia" variety reached 18.90, indicating that "Ningyu" was more resistant to Fusarium wilt than "Hongjia". After treatment with a 10-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution, the control efficacy of "Ningyu" and "Hongjia" varieties was 77.72% and 71.27%, respectively, which was better than the control efficacy of an 800-fold dilution of 50% carbendazim (65.57% and 60.48%, respectively). A 100-fold dilution of *Pseudomonas aeruginosa* NF9-1 aqueous solution showed control efficacy of 56.46% and 57.72% against Fusarium wilt in both "Ningyu" and "Hongjia" varieties, while the control efficacy of a 1600-fold dilution of 50% carbendazim was poor, at 47.47% and 38.99%, respectively. The experimental results show that Pseudomonas aeruginosa NF9-1 aqueous solution can effectively control strawberry wilt disease.

[0107] Table 4. Field control efficacy of Pseudomonas aeruginosa NF9-1 aqueous solution against strawberry Fusarium wilt.

[0108]

Claims

1. A biocontrol agent, characterized in that: The effective active ingredient in the biocontrol agent is *Pseudomonas aeruginosa* (…). Pseudomonas veronii The composition of NF9-1 CGMCC No.26092; the metabolites secreted by the *Pseudomonas NF9-1* include three antibacterial-related substances: protease, heptaphilin, and chitinase; the biocontrol agent is an aqueous solution.

2. The biocontrol agent according to claim 1, characterized in that: The components and weight percentage are as follows: Pseudomonas veronii NF9-1: 89.5-99.8%; sodium dodecyl sulfonate: 0.1-10%; Potassium sorbate: 0.1-0.5%; pH value: 6.5-7.

0.

3. The use of biocontrol agent according to claim 1, characterized in that: The strawberry leaf surface is evenly sprayed to prevent and control strawberry anthracnose and strawberry powdery mildew, and the roots are irrigated to prevent and control strawberry wilt.

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