Pseudomonas donghuensis and application thereof in preventing and treating bacterial soft rot of Chinese cabbage

Abstract

The application discloses a Pseudomonas donghuensis strain and application thereof in prevention and treatment of bacterial soft rot of Chinese cabbage. The application relates to the field of microorganisms, in particular to a Pseudomonas donghuensis strain and application thereof in prevention and treatment of bacterial soft rot of Chinese cabbage. The Pseudomonas donghuensis strain of the application is Pseudomonas donghuensis ZF510, and the preservation number of the strain in the China General Microbiological Culture Collection Center is CGMCC No. 27639. The strain ZF510 can produce protease, has the abilities of synthesizing IAA, dissolving phosphorus and being iron-loving, has broad-spectrum antagonistic effect, can effectively inhibit the growth of pathogenic bacteria and fungi, and the in-vivo potting prevention effect of the strain ZF510 on the bacterial soft rot of Chinese cabbage can reach 63.06%. In conclusion, the strain ZF510 has good biocontrol potential and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of *Pseudomonas aeruginosa* and its application in the prevention and control of bacterial soft rot in Chinese cabbage. Background Technology

[0002] Pseudomonas spp. bacteria are typical rhizosphere growth-promoting bacteria with a wide range of adaptability. Some biocontrol strains, represented by *Pseudomonas fluorescens*, can produce various antibacterial substances. The large-scale production of antibiotics is a key characteristic of Pseudomonas biocontrol bacteria; therefore, many Pseudomonas strains have been commercially used for plant disease control. Currently, Pseudomonas are mainly used to control root and stem diseases and for growth promotion. Researchers have successfully used Pseudomonas to control infectious diseases in various plants such as tomatoes, peppers, cucumbers, and melons. Numerous studies have also reported on the phosphorus-solubilizing ability and salt tolerance of Pseudomonas. Studies have found that *Pseudomonas donghuensis* can produce siderophores, hydrogen cyanide, and antibiotics to inhibit the infection of crops by pathogenic fungi, bacteria, and nematodes. However, research reports on the biological control applications of *Pseudomonas donghuensis* are relatively few. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to enhance the disease resistance of plants and inhibit or prevent plant diseases.

[0004] To solve the above-mentioned technical problems, the present invention first provides a strain of *Pseudomonas aeruginosa*.

[0005] The *Pseudomonas donghuensis* provided in this invention is *Pseudomonas donghuensis* CGMCC No. 27639, which has the accession number CGMCC No. 27639 at the China General Microbiological Culture Collection Center.

[0006] The present invention also provides a microbial agent containing the aforementioned *Pseudomonas aeruginosa* and / or metabolites of the aforementioned *Pseudomonas aeruginosa*.

[0007] The above-mentioned microbial agents can be pathogen inhibitors or disease inhibitors, and the pathogens can be pathogenic fungi or pathogenic bacteria.

[0008] In this invention, the pathogenic fungi may be Rhizoctonia solani, Ascochyta citrullina, and Stemphylium solani.

[0009] In this invention, the pathogenic bacteria may be Xanthomonas campestris pv. campestris, Pseudomonas syringaepv. tomato, Clavibacter michiganensis subsp. michiganensis, Acidovorax citrulli, Clavibacter michiganense subsp. sepedonicum, Pectobacterium brasiliense, and Pseudomonas syringaepv. lachrymans.

[0010] In this invention, the disease is soft rot of Chinese cabbage.

[0011] The present invention also provides a bio-organic fertilizer containing the aforementioned *Pseudomonas aeruginosa* or its metabolites.

[0012] This invention also provides the use of the aforementioned *Pseudomonas aeruginosa* in the preparation of any of the following products:

[0013] 1) A fungicide for the prevention and control of plant diseases, the disease being soft rot of Chinese cabbage;

[0014] 2) Pathogen inhibitors, wherein the pathogens are pathogenic fungi or pathogenic bacteria;

[0015] The pathogenic fungi may be *Rhizoctonia solani*, *Ascochytacitrullina*, and *Stemphylium solani*; the pathogenic bacteria may be *Xanthomonas campestris* pv. campestris, *Pseudomonas syringae* pv. tomato, *Clavibactermichiganensis* subsp. michiganensis, *Acidovorax citrulli*, *Clavibacter michiganense* subsp. sepedonicum, *Pectobacterium brasiliense*, and *Pseudomonas syringaepv. lachrymans*.

[0016] The present invention also provides a method for culturing the aforementioned *Pseudomonas donghuensis*, comprising the step of culturing the *Pseudomonas donghuensis* in a culture medium for culturing *Pseudomonas*.

[0017] The present invention also provides a method for preparing the bacterial agent described above, comprising the following steps: using the aforementioned *Pseudomonas aeruginosa* as the active ingredient to obtain the bacterial agent.

[0018] The active ingredient of the above-mentioned microbial agent may be the above-mentioned *Pseudomonas donghuensis* and / or the metabolites of the above-mentioned *Pseudomonas donghuensis*. The active ingredient of the above-mentioned microbial agent may also contain other biological or non-biological components. Other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art based on their inhibitory effect on diseases.

[0019] In addition to the active ingredient, the above-mentioned microbial agent also contains a carrier. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0020] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0021] Depending on the needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the above-mentioned microbial agents.

[0022] In the above text, the metabolites can be obtained from the fermentation broth of *Pseudomonas donghuensis*. The metabolites can be sterile metabolites of *Pseudomonas donghuensis* or bacterial metabolites of *Pseudomonas donghuensis*. Specifically, the sterile metabolites of *Pseudomonas donghuensis* (sterile fermentation filtrate) can be prepared by culturing *Pseudomonas donghuensis* in a liquid culture medium and filtering to remove the *Pseudomonas donghuensis* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of *Pseudomonas donghuensis* can be prepared by culturing *Pseudomonas donghuensis* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolite of *Pseudomonas donghuensis*.

[0023] This invention also provides the application of *Pseudomonas aeruginosa* or its inoculum as described above in the prevention and control of soft rot in Chinese cabbage and / or the increase of Chinese cabbage yield.

[0024] The present invention also provides the application of the aforementioned *Pseudomonas aeruginosa* or the aforementioned inoculum in the cultivation of Chinese cabbage.

[0025] This study used a dilution-spreading method to isolate and screen a biocontrol strain, ZF510, from the rhizosphere soil of Chinese cabbage in Guizhou Province. This strain exhibited significant inhibitory effects against *Pectobacterium brasiliense*, a bacterial soft rot pathogen. Based on morphological observation, physiological and biochemical characteristics, Biolog assays, and polygenic phylogenetic analysis, it was identified as *Pseudomonas donghuensis*. Preliminary studies on the biocontrol mechanism revealed that strain ZF510 produces proteases, possesses IAA synthesis capabilities, phosphate-solubilizing abilities, and iron-loving properties during its metabolism. Strain ZF510 exhibits broad-spectrum antagonistic activity, effectively inhibiting the growth of seven pathogenic bacteria. Its volatile substances showed significant inhibitory effects against *Rhizoctonia solani*, *Diplostomum spp.*, and *Pseudomonas solanum*. Under in vivo pot cultivation conditions, Chinese cabbage inoculated with strain ZF510 showed significantly reduced disease incidence and disease index, with a control efficacy of 63.06%. In conclusion, strain ZF510 has good biocontrol potential and application prospects. Attached Figure Description

[0026] Figure 1 Morphological characteristics of strain ZF510 observed on KB plates. A: colony front; B: colony back.

[0027] Figure 2 These are scanning electron microscope (SEM) images of strain ZF510. The left image shows the structure of multiple bacterial cells under a 10,000x microscope, and the right image shows the structure of a single bacterial cell under a 30,000x microscope.

[0028] Figure 3 Phylogenetic tree of strain ZF510 based on 16S rDNA, gyrB, and rpoB gene sequences.

[0029] Figure 4 This is a graph showing the growth curve of strain ZF510.

[0030] Figure 5 The inhibitory effect of ZF510 on pathogenic bacteria was investigated. A1–G1: ZF510 treatments; A2–G2: blank controls. A: *Pseudomonas syringae* (tomato pathogen); B: *Acidophilus citrinum* (watermelon pathogen); C: *Xanthomonas alopecuroides* (wild rapeseed pathogen); D: *Corynebacterium miltanense* (potato ring rot pathogen); E: *Pseudomonas syringae* (laziness pathogen); F: *Pectinobacter brasiliensis*; G: *Corynebacterium miltanense* (miltan subsp. *miltanense*).

[0031] Figure 6 The graph shows the inhibitory effects of ZF510 volatile substances on Rhizoctonia solani, Diplostomum cirrhifolium, and Pleurotus ostreatus. A: Rhizoctonia solani, B: Diplostomum cirrhifolium, C: Pleurotus ostreatus; A1-C1: ZF510 inoculation treatment, A2-C2: blank control.

[0032] Figure 7 The effects of ZF510 on protease, IAA synthesis, phosphate solubilization, and iron affinity are shown. A: ZF510 protease production capacity; B: ZF510 phosphate solubilization capacity; C: ZF510 iron affinity; D: ZF510 IAA synthesis capacity; E: IAA standard curve.

[0033] Figure 8 The safety assay results for strain ZF510 are shown. A represents the front of the plate; B represents the back of the plate.

[0034] Figure 9 The antibiotic resistance levels of strain ZF510 are shown below. A represents the resistance level of strain ZF510 to ampicillin; B represents the resistance level of strain ZF510 to spectinomycin; C represents the resistance level of strain ZF510 to rifampin; D represents the resistance level of strain ZF510 to tetracycline; E represents the resistance level of strain ZF510 to chloramphenicol; F represents the resistance level of strain ZF510 to streptomycin; G represents the resistance level of strain ZF510 to kanamycin; and H represents the resistance level of strain ZF510 to gentamicin.

[0035] Figure 10The efficacy of different dilutions of strain ZF510 against live potted plants was determined. A: 100-fold dilution of strain ZF510; B: 200-fold dilution of strain ZF510; C: 300-fold dilution of strain ZF510; D: 5% kasugamycin control; E: pathogenic bacteria control; F: healthy control.

[0036] Preservation Instructions

[0037] Classification and nomenclature: *Pseudomonas donghuensis*

[0038] Latin name: Pseudomonas donghuensis

[0039] Strain number: ZF510

[0040] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0041] Collection institution abbreviation: CGMCC

[0042] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0043] Deposit date: June 15, 2023

[0044] Registration number at the Preservation Center: CGMCC No. 27639. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0048] The Chinese cabbage variety, Fast-Growing Xiulv, used in the following examples was purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd.

[0049] The pathogenic species of Xanthomonas campestris pv. campestris described in the following examples has been documented in: Zhang Yang, Li Jinping, Zhou Huimin, and Li Baoju. Occurrence patterns and control of bacterial black rot in cruciferous vegetables. China Vegetables, 2011, 17: 23-25. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating the experiments of this invention and shall not be used for any other purpose.

[0050] The pathogenic tomato species *Pseudomonas syringae* pv. tomato described in the following examples is described in: Chai Ali, Patiguli, Guo Weitao, Shi Yanxia, ​​Xie Xuewen, Xi Xianmei, and Li Baoju. Establishment and application of real-time fluorescence quantitative PCR detection method for bacterial spot disease of tomato. Journal of Horticulture, 2019, 46(01): 182-192. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of this invention and cannot be used for other purposes.

[0051] The Clavibacter michiganensis subsp. michiganensis described in the following examples is described in: Kang Huajun, Chai Ali, Shi Yanxia, ​​Xie Xuewen, Yuan Junhai, Li Baoju. Quadruple PCR detection method for bacterial spot, canker, bacterial wilt and scab of tomato. Journal of Horticulture 2018, 45(11):2254-2264. The biological material is available to the public from the applicant and is only for repeating the experiments of this invention and shall not be used for other purposes.

[0052] The watermelon acid-eating bacterium (Acidovorax citrulli) described in the following examples is described in: Tian Hui, Xie Xuewen, Li Huanling, Wei Songhong, and Li Baoju. Dr. Li Baoju's Diagnostic Notes (70): Occurrence and Control of Seed-borne Bacterial Fruit Spot Disease in Pumpkin Rootstock. China Vegetables 2014, (05): 65-66. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0053] The Clavibacter michiganense subsp. sepedonicum used in the following examples has been described in: Ma Haiyan, Zhang Jiasen, Wang Li, Gao Zhongqiang, Yu Hongjun, Jiang Weijie. Dr. Jiang Weijie: Focusing on the front line of production (XIV) High-efficiency cultivation technology of early spring greenhouse potatoes in Tengzhou City. China Vegetables 2015, (08):70-73. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of this invention and cannot be used for other purposes.

[0054] The *Pectobacterium brasiliense* used in the following examples has been described in: Li Lei, Zhao Yurong, Zheng Fei, Shi Yanxia, ​​Chai Ali, Xie Xuewen, and Li Baoju. Screening and control effects of antagonistic Bacillus against celery soft rot. Chinese Journal of Biological Control, 2020, 36(03): 388-395. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0055] The *Pseudomonas syringaepv. lachrymans* strain in the following examples has been described in: Chai Ali, Patiguli, Guo Weitao, Shi Yanxia, ​​Xie Xuewen, Xi Xianmei, Li Baoju. Establishment and application of real-time fluorescence quantitative PCR detection method for bacterial spot disease of tomato. Journal of Horticulture, 2019, 46(01): 182-192. The public can obtain this biological material from the applicant. This biological material is only for repeating the experiments of this invention and cannot be used for other purposes.

[0056] The Rhizoctonia solani described in the following examples is described in: Gao Wei, Li Baoju, Sun Junde, Shi Yanxia, ​​Jin Dan. Antagonistic effect of Trichoderma viride on Rhizoctonia solani and Fusarium oxysporum. China Vegetables, 2008, (6): 9-12. The biological material is available to the public from the applicant and is only for repeating the experiments of this invention and shall not be used for other purposes.

[0057] The watermelon shell spore (Ascochyta citrullina) described in the following examples is described in: Zhao Yanjie, Li Baoju, Shi Yanxia, ​​et al. Occurrence and control of cucurbit vine blight [J]. Chinese Vegetables, 2008(02):56-57+70. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0058] The *Stemphylium solani* described in the following examples is described in: Li Xinyu, Li Lei, Chen Lida, Shi Yanxia, ​​Chai Ali, Xie Xuewen, and Li Baoju. Screening and identification of antagonistic bacteria against leaf spot disease of *Stemphylium solani* in tomato. Journal of Horticulture, 2020, 47(04): 741-748. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of this invention and cannot be used for other purposes.

[0059] The culture medium information in the following examples is as follows:

[0060] King's B medium (KB medium): 20.0 g peptone, 10.0 mL glycerol, 1.5 g MgSO4·7H2O, 1.5 g K2HPO4, distilled water to a final volume of 1000 mL, pH 7.0±0.2.

[0061] Nutrient Broth medium (NB medium): 10.0g peptone, 3.0g beef meal, 5.0g NaCl, distilled water to 1000mL, pH 7.0±0.2.

[0062] WA medium: 5.0g agar, add to 1000mL distilled water.

[0063] NBRIP Inorganic Phosphorus Medium: Ca3(PO4)2 5g, glucose 10g, (NH4)2SO4 0.15g, KCl2·6H2O 0.5g, MgSO4·7H2O 0.25g; distilled water to 1000mL.

[0064] CAS medium: agar 15.0g, glucose 9.0g, peptone 4.5g, beef extract 2.7g, NaCl 4.5g, chromium azurite 121mg, hexadecyltrimethylammonium bromide 145.8mg, FeCl3 0.054g, HCl 0.167mL, distilled water to 1000mL.

[0065] The 5% kasugamycin wettable powder was purchased from Fujian Kaili Biological Products Co., Ltd., registration certificate number: PD20170764.

[0066] Ampicillin [Amp] 100mg / ml (Solebo, A8180), Kanamycine [Kan] 50mg / ml (Solebo, K8020), Chloramphenicol [Cm] 25mg / ml (Solebo, C8050), Streptomycin sulfate [Str] 50mg / ml (Coolbac, CS10481), Tetracycline [Tet] 10mg / ml (Maclean, T829835), Gentamicin sulfate [Gm] 50mg / ml (Coolbac, CG5551), Rifampicin [Rif] 50mg / ml (Coolbac, CR9551), Spectinomycin hydrochloride [Spec] 50mg / ml (Sigma, G1914).

[0067] The following examples used Excel 2019 software for statistical analysis and graphing, and SPSS 20 software for difference analysis. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, with P < 0.05 (*) indicating a significant difference, P < 0.01 (**) indicating a highly significant difference, and P < 0.001 (***) indicating a highly significant difference.

[0068] Example 1: Isolation, Screening, Identification and Preservation of Strains

[0069] 1. Strains isolation and screening

[0070] Soil samples were collected in 2022 from the rhizosphere soil of healthy Chinese cabbage plants in Guizhou. The soil samples were serially diluted using the dilution-spread plate method, with 100 μL of each sample representing a 10:10 concentration. -4 10 -5 10 -6 The suspension was spread onto KB solid medium and incubated at 28°C for 24 hours. Single colonies were purified by streak plating 3–5 times. After the colony morphology stabilized, the colonies were stored for later use. A total of 341 strains were isolated.

[0071] 2. Screening of antagonistic strains

[0072] The antibacterial spectrum of the above 341 strains was determined using a mixed culture method. 1×10⁻⁶ microparticles were pipetted into the culture. 8 Add 150 μL of CFU / mL *Bacillus brasiliensis* suspension to a 250 mL Erlenmeyer flask containing 150 mL of sterilized King's B medium at approximately 50 °C. Shake well, pour into a sterile petri dish, and cool to form a plate.

[0073] Sterile filter paper was placed in the center of the plate, and 5 μL of biocontrol solution was dropped onto the filter paper using a micropipette. Blank culture medium was added as a blank control. The plate was incubated at 28°C for 24 h. The size of the inhibition zone was observed and measured. Each strain was repeated 3 times. The diameter of the inhibition zone was measured using the cross-sectional method.

[0074] Twenty-four antagonistic bacteria with good inhibitory effects were obtained using the mixed bacterial method and numbered ZF490–ZF513. The experimental results (Table 1) show that seven antagonistic bacteria have a certain inhibitory effect on *Pectinobacter brasiliensis*, among which strain ZF510 has the best inhibitory effect, with an inhibition rate of 57.26% (Table 1).

[0075] Table 1. Determination of the antagonistic effect of 24 strains of Pseudomonas against pathogenic bacteria.

[0076] ZF490 Pseudomonas aeruginosa 52.16±2.10 ZF502 Pseudomonas aeruginosa 49.62±2.34 ZF491 Fluorescent Pseudomonas 43.29±1.62 ZF503 Korean Pseudomonas 45.34±1.65 ZF492 Pseudomonas aeruginosa 41.56±2.16 ZF504 Korean Pseudomonas 34.69±2.15 ZF493 Korean Pseudomonas 35.46±3.65 ZF505 Pseudomonas aeruginosa 50.64±1.68 ZF494 Pseudomonas aeruginosa 53.16±2.11 ZF506 Fluorescent Pseudomonas 52.39±1.25 ZF495 Pseudomonas aeruginosa 46.34±1.65 ZF507 Pseudomonas aeruginosa 49.52±2.14 ZF496 Fluorescent Pseudomonas 50.26±3.21 ZF508 Pseudomonas aeruginosa 47.89±2.91 ZF497 Pseudomonas aeruginosa 47.39±1.39 ZF509 Fluorescent Pseudomonas 53.29±1.64 ZF498 Fluorescent Pseudomonas 50.96±1.65 ZF510 Donghu Pseudomonas 57.26±1.33 ZF499 Fluorescent Pseudomonas 49.68±2.31 ZF511 Korean Pseudomonas 39.49±2.51 ZF500 Pseudomonas aeruginosa 48.26±2.61 ZF512 Fluorescent Pseudomonas 38.46±3.65 ZF501 Fluorescent Pseudomonas 54.23±1.65 ZF513 Pseudomonas aeruginosa 40.39±1.39

[0077] 3. Identification of strain ZF510

[0078] 1) Observation of strain morphology and determination of physiological and biochemical indicators

[0079] Single colonies of strain ZF510 were picked up using sterile toothpicks and inoculated onto KB solid medium, and incubated at 28°C for 24 hours. The morphological characteristics of the strain and the determination of its physiological and biochemical indicators were performed in accordance with the "Common Bacterial Systematic Identification Handbook" and "Bergey's Manual of Bacterial Identification".

[0080] The results are as follows: Strain ZF510 appeared pale yellow on the front and yellow on the back of KB agar plates. Figure 1 (A and B), has a distinctive odor; under scanning electron microscopy, strain ZF510 appears as a short rod, approximately 1.05 μm in length. Figure 2 ).

[0081] 2) Biolog measurement

[0082] A single colony of ZF510 was picked and inoculated onto a slant of KB medium in a test tube and incubated at 28°C for 24 hours. The unique carbon source utilization was determined using the BIOLOGGEN III kit (operated according to the kit instructions).

[0083] The results of the biolog assay are as follows: strain ZF510 can utilize carbon sources such as α-D-glucose, D-fructose, and D-serine, but cannot utilize carbon sources such as D-merbiose, D-maltose, D-trehalose, and D-cellobiose (Table 2).

[0084] Table 2. Determination of the unique carbon source utilization of strain ZF510 using BIOLOG GEN III reagent strips

[0085]

[0086]

[0087]

[0088] Note: + indicates positive; - indicates negative; w indicates weak positive.

[0089] 3) Construction of a multi-gene phylogenetic tree for strain ZF510

[0090] Using the TIANGEN bacterial genomic DNA extraction kit, a small amount of genomic DNA was extracted from strain ZF510 as a template. Conserved genes such as 16S rDNA, gyrB, and rpoB were selected for PCR amplification and sequencing. The PCR products were sequenced by Beijing Bomaide Biotechnology Co., Ltd. The sequencing results are shown in Table 3.

[0091] Strain ZF510 possesses a 16S rDNA gene with the nucleotide sequence of sequence 1 in the sequence listing, a gyrB gene with the nucleotide sequence of sequence 2 in the sequence listing, and an rpoB gene with the nucleotide sequence of sequence 3 in the sequence listing. Sequencing results were analyzed using software such as MEGA 6.0, Sequence Matrix, and Seaview4. A maximum likelihood phylogenetic tree was constructed to analyze its taxonomic position.

[0092] Table 3. Primer information for constructing a multigene phylogenetic tree

[0093]

[0094] After PCR amplification, the 16S rDNA, gyrB, and rpoB gene sequences of strain ZF510 were obtained. A multi-gene phylogenetic tree of ZF510 was constructed, placing it on the same branch as *Pseudomonas donghuensis* HYS. Based on these taxonomic characteristics, the taxonomic position of strain ZF510 was determined to be *Pseudomonas donghuensis*. Figure 3 ).

[0095] 4) Growth curve determination of strain ZF510

[0096] Single colonies of the purified strain ZF510 were picked and transferred to 50 mL of KB(+kan) liquid medium for incubation. The seed culture of the test bacteria was diluted to a bacterial suspension concentration of 1×10⁻⁶. 8 After obtaining cfu / mL, transfer the inoculum at a rate of 1% to a sterile 250mL Erlenmeyer flask containing 100mL of the culture medium to be tested. Incubate at 28℃ and 180rpm for 48 hours, measuring OD every 4 hours. 600 The bacterial count and colony count were determined, and the lost KB medium in the fermentation flask was replenished. Three replicates were designed. Sampling began 4 hours after transfer, with 100 μL of bacterial solution taken each time. When counting on plates, colonies that emitted fluorescence were selected under UV light. The data were integrated to obtain the growth curve of strain ZF510.

[0097] OD 600 Values ​​were determined using a turbidimetric method. The absorbance of the fermentation broth at 600 nm was measured using the corresponding blank culture medium as a control. Each treatment was repeated in triplicate.

[0098] Colony count: determined using a serial dilution method. The fermentation broth was serially diluted to 10⁻⁶ using the corresponding blank culture medium. -6 10 -7 10 -8 To determine the concentration, take 100 μL of bacterial suspension and spread it evenly on a KB plate. Incubate at 28°C for 1 day, then count the colonies. Each treatment was repeated three times.

[0099] The results showed that strain ZF510 experienced a lag phase of growth from 0-4 hours after inoculation; it entered the logarithmic phase from 4-24 hours, with cell numbers increasing logarithmically; and the strain activity decreased after 28 hours. Therefore, the mid-exponential phase was the period of most vigorous growth for the strain, and the optimal culture time for the seed culture was determined to be 12 hours. Figure 4 ).

[0100] 4. Preservation of strain ZF510

[0101] Pseudomonas donghuensis ZF510 was deposited on June 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27639. It will be referred to as Pseudomonas donghuensis CGMCC No. 27639 or strain ZF510.

[0102] Example 2: Study on the antibacterial ability of strain ZF510

[0103] 1. Determination of the antibacterial spectrum of strain ZF510

[0104] The inhibitory effect of strain ZF510 on seven pathogenic bacteria was determined using a double-layer culture method. 5 μL of a 1×10⁻⁶ concentration was used. 8 A bacterial suspension of strain ZF510 at cfu / mL was inoculated into the center of KB plates and incubated at 28°C for 48 h. The bacteria were then inactivated by fumigation with 3 mL of chloroform. 5 mL of WA medium containing the pathogen was used as the top layer. After incubation at 28°C for 48 h, the diameter of the inhibition zone was measured, and the inhibition rate was calculated. Each treatment was repeated three times. Inhibition rate (%) = (diameter of inhibition zone / diameter of control colony) × 100.

[0105] The results showed that strain ZF510 could effectively inhibit *Xanthomonas spp.* (a pathogenic strain of *Xanthomonas spp.*), *Pseudomonas syringae* (a pathogenic strain of *P. spp.*), *Corynebacterium miltanense* (a pathogenic strain of *P. miltanense*), *Pectobacterium brasiliensis*, *Cypericum cirrhosa* (a pathogenic strain of *P. spp.*), *Corynebacterium miltanense* (a pathogenic strain of *P. spp.*), and *P. syringae* (a pathogenic strain of *P. spp.*), with inhibition rates greater than 50% for all seven bacteria (Table 4). Figure 5 (A1-G1 and A2-G2).

[0106] Table 4. Antibacterial effect of strain ZF510 against pathogens

[0107]

[0108] 2. Determination of the antibacterial activity of volatile substances in bacterial agent ZF510

[0109] A single colony of strain ZF510 was inoculated into liquid KB medium and incubated at 28°C for 24 hours with shaking. The concentration was then adjusted to 1×10⁻⁶. 8CFU / mL was used to obtain ZF510 bacterial agent for later use.

[0110] Add KB medium and PDA medium to two separate agar plates. Using a sterile toothpick, streak ZF510 inoculant onto the side containing KB medium, and add a 6mm pathogenic fungal disc to the side containing PDA medium. Incubate inverted positions at 28℃. Use sterile water without ZF510 inoculant as a blank control. When the pathogenic fungus in the blank control group grows to the edge of the plate, observe its diameter and calculate the inhibition rate. Each treatment is repeated three times. Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100.

[0111] The results of the inhibitory effect of the volatile substances of fungal agent ZF510 on pathogenic fungi showed that the volatile substances of fungal agent ZF510 were effective against Rhizoctonia solani, Diplostomum cirrhosa, and Pseudomonas spp., with inhibition rates of 75.73%, 61.96%, and 62.30%, respectively (Table 5). Figure 6 (A1, A2, B1, B2, C1, and C2).

[0112] Table 5. Inhibition effect of volatile substances by bacterial agent ZF510

[0113]

[0114] Example 3: Study on the biocontrol mechanism of strain ZF510

[0115] Preparation of bacterial agent ZF510: A single colony of *Pseudomonas aeruginosa* CGMCC No. 27639 was inoculated into liquid KB medium and shaken at 180 r / min for 24 h at 28℃. The bacterial suspension concentration was then adjusted to 1 × 10⁻⁶. 8 ZF510 bacterial agent was obtained by using cfu / mL (with liquid KB medium as a blank control).

[0116] 1. Protease activity assay

[0117] Inoculate 5 μL of a 1×10⁻⁶ solution at the center of the skim milk culture medium. 8 The bacterial culture was incubated with ZF510 at CFU / mL at 28°C for 24 hours, and the digestion zone was observed. Each treatment was repeated three times.

[0118] 2. Phosphorus solubility test

[0119] Using the clear zone method, 5 μL of a 1×10⁻⁶ solution was inoculated at the center of NBRIP inorganic phosphorus medium. 8 The bacterial agent ZF510 at cfu / mL was used in three replicates per group and cultured at 28℃ for 7 days. The diameter of the phosphate-solubilizing clear zone and the colony diameter were measured, and the ratio of the clear zone diameter to the colony diameter, HD / CD, was calculated.

[0120] 3. Iron absorption capacity test

[0121] Inoculate 5 μL of a 1×10⁻⁶ solution at the center of the CAS medium. 8 Inoculate with ZF510 at cfu / mL, repeating three times per group, and incubate upside down at 28°C for 7 days. If the strain produces a distinct orange-yellow clear zone on CAS plates, it indicates the strain's ability to secrete siderophores. The size and color of the clear zone indicate the strain's siderophoretic production capacity; a larger clear zone and a deeper color indicate a stronger siderophoretic production capacity. Measure the diameter of the phosphate-solubilizing clear zone and the colony diameter, and calculate the ratio of clear zone diameter to colony diameter, HD / CD.

[0122] The results showed that strain ZF510 produced a clear zone in skim milk medium, with a diameter of 2.35 ± 0.05 cm. Figure 7 (A); a clear zone is produced in NBRIP inorganic phosphorus medium ( Figure 7 (B) The diameter of the transparent zone was 1.15 ± 0.08 cm; a pale yellow halo was formed in CAS medium. Figure 7 In C), the halo diameter is 0.66 ± 0.04 cm; in DF + Trace amounts of IAA can be produced in the culture medium. Figure 7 (D); According to the IAA standard curve y = 0.0396x - 0.0381(R) 2 =0.9972)( Figure 7 The concentration of IAA synthesized by strain ZF510 was calculated to be 11.94 μg / mL. This demonstrates that the metabolites of bacterial agent ZF510 contain proteases and possess the ability to synthesize IAA, solubilize phosphorus, and exhibit iron affinity.

[0123] 4. Safety Testing

[0124] Inoculate 5 μL of ZF510 bacterial agent into the center of a blood agar plate, air dry, and incubate at 28°C for 1 day. Repeat each treatment three times. Observe whether a clear zone forms. If no clear zone forms, it indicates that the bacterial agent ZF510 has good safety for organisms.

[0125] The results showed that no clear zone was formed in the hemolytic medium with bacterial agent ZF510, indicating that ZF510 has a certain degree of safety. Figure 8 (A and B in the middle).

[0126] 5. Antibiotic resistance level of strain ZF510

[0127] Eight antibiotics were selected: ampicillin, kanamycin, chloramphenicol, streptomycin, tetracycline, gentamicin, rifampin, and spectinomycin (Table 6). Gradient solutions of the eight antibiotics at concentrations of 50-600 μg / mL were prepared, and the growth was observed by inoculating with bacterial agent ZF510. The results were repeated three times.

[0128] Table 6. Types and Concentrations of Antibiotics

[0129]

[0130] The results are shown in Table 7 and Figure 9 As shown: Strain ZF510 for ampicillin (Amp) ( Figure 9 (A) Tolerance exceeds 600 μg / mL, spectinomycin (Spec) Figure 9 Rifampin (Rif) (B) Figure 9 C), tetracycline (Tet) Figure 9 The tolerance levels for chloramphenicol (Cm) were 350 μg / mL, 150 μg / mL, and 100 μg / mL, respectively, to chloramphenicol (Cm) ( Figure 9 (E), streptomycin (Str) Figure 9 The tolerance level for (F) was 50 μg / mL, and the tolerance level for kanamycin (Kan) was [missing information]. Figure 9 (G) Gentamicin (Gm) Figure 9 It is more sensitive to H).

[0131] Table 7. Analysis of antibiotic resistance level of strain ZF510

[0132]

[0133] Note: "+" indicates tolerance, and "-" indicates intolerance.

[0134] Example 4: Determination of the control effect of different dilution ratios of the fungal agent ZF510 on live potted plants in disease prevention.

[0135] 1. Preparation of bacterial agent ZF510: *Pseudomonas aeruginosa* CGMCC No. 27639, stored in cryovials at -80℃, was inoculated onto KB plates using an inoculation loop via streak. The plates were activated at 28℃ for 48 hours. Single colonies of the activated strain were then picked and inoculated into liquid KB medium. The medium was incubated at 28℃ with shaking at 180 rpm for 24 hours. The bacterial suspension was collected, and the bacterial concentration was adjusted to 1 × 10⁻⁶ with sterile water. 8 The bacterial suspension of agent ZF510 was obtained by measuring cfu / mL (with sterile water as a blank control). The bacterial suspension of agent ZF510 was diluted 100-fold with sterile water to obtain a 100-fold diluted solution; the bacterial suspension of agent ZF510 was diluted 200-fold with sterile water to obtain a 200-fold diluted solution; and the bacterial suspension of agent ZF510 was diluted 300-fold with sterile water to obtain a 300-fold diluted solution.

[0136] 2. Preparation of *Bacillus brasiliensis*, the pathogen causing soft rot in Chinese cabbage: *Bacillus brasiliensis*, stored in cryovials at -80℃, was inoculated onto KB plates using an inoculation loop. The plates were activated at 28℃ for 48 hours. Single colonies of the activated pathogen were then picked and inoculated into liquid KB medium. The medium was incubated at 28℃ with shaking at 180 rpm for 24 hours. The bacterial suspension was collected and the bacterial concentration was adjusted to 1 × 10⁻⁶ with sterile water. 8 CFU / mL (with sterile water as a blank control), yielded 1×10⁻⁶. 8 CFU / mL suspension of *Braziliana spp.* (a bacterium that causes soft rot in Chinese cabbage) is available for use.

[0137] 3. Effect of fungicide ZF510 on the control of soft rot in live potted plants of Chinese cabbage

[0138] Using the spray inoculation method, once the fast-growing Xiulv cabbage variety (China Vegetable Seed Industry Technology (Beijing) Co., Ltd.) has grown to an appropriate plant height (two leaves and two buds), a concentration of 1×10⁻⁶ is applied. 8 A suspension of *Bacillus brasiliensis*, the pathogen causing soft rot in Chinese cabbage, was sprayed onto the leaves of two true-leaf Chinese cabbage plants, 10 mL per plant, and cultured under moist conditions. One day after inoculation, 10 mL / plant of *ZF510* bacterial suspension at different dilutions were sprayed according to Table 8. Each treatment was repeated three times, with 18 seedlings per replicate, and the seedlings were placed in a greenhouse. When the disease index of the pathogen control treatment exceeded 50, the disease index and control effect were calculated. A 5% kasugamycin wettable powder was used as a positive control, a pathogen-only treatment was used as a negative control, and Chinese cabbage without added pathogens or bacterial agents was used as a healthy control.

[0139] According to the disease grading standards in the "Guidelines for Field Efficacy Trials of Pesticides", the disease severity is graded according to the proportion of lesion area to leaf area: Grade 0 is 0%; Grade 1 is 1%–5%; Grade 2 is 6%–10%; Grade 3 is 11%–25%; Grade 4 is 26%–50%; and Grade 5 is above 50%. Disease index = ∑(number of diseased leaves at each level × disease grade) / (total number of leaves surveyed × highest disease grade) × 100, and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0140] Table 8. Efficacy of ZF510 inoculant against soft rot in potted Chinese cabbage at different inoculation concentrations.

[0141]

[0142] The results are shown in Table 9 and Figure 10 As shown: Three days after inoculation with *Bacillus brasiliensis*, the control treatment showed obvious lesions on the leaves; the 200-fold dilution of the ZF510 bacterial suspension sprayed on cabbage showed a control efficacy of 63.06% against bacterial soft rot, higher than the 62.87% control efficacy of the 5% kasugamycin control treatment (P < 0.05) (Table 9). Figure 10 (AF).

[0143] Table 9. Efficacy test of fungicide ZF510 on live potted plants

[0144]

[0145] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. *Pseudomonas aeruginosa*, characterized by: The *Pseudomonas aeruginosa* mentioned is *Pseudomonas aeruginosa* (…). Pseudomonas donghuensis ZF510, its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 27639.

2. A microbial agent, characterized in that: The bacterial agent contains *Pseudomonas aeruginosa* as described in claim 1; The microbial agent is a pathogen inhibitor or disease inhibitor, and the pathogen is a pathogenic fungus or pathogenic bacteria; The pathogenic fungus is Rhizoctonia solani ( ). Rhizoctonia solani ), watermelon shell dispore ( Ascochyta citrullina ) and stalk mold ( Stemphylium solani ); The pathogenic bacteria is Xanthomonas spp., a pathogenic strain of *Xanthomonas spp.* (also known as *Xanthomonas spp.*). Xanthomonas campestris pv. campestris ), syringomorphic tomato disease strain ( Pseudomonas syringae pv . tomato ), Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp . michiganensis ), watermelon acidophilic bacteria ( Acidovorax citrulli ), Corynebacterium micranthae, a pathogenic species of potato ring rot ( Clavibacter michiganense subsp .sepedonicum ), Brazilian pectinobacterium ( Pectobacterium brasiliense ) and Pseudomonas syringae var. lacrimae ( Pseudomonas syringae pv .lachrymans ); The disease in question is soft rot of Chinese cabbage.

3. A bio-organic fertilizer, characterized by: The bio-organic fertilizer contains the *Pseudomonas aeruginosa* of claim 1 or the microbial agent of claim 2.

4. The use of the *Pseudomonas aeruginosa* according to claim 1 in the preparation of any of the following products: 1) A fungicide for the prevention and control of plant diseases, the disease being soft rot of Chinese cabbage; 2) Pathogen inhibitors, wherein the pathogens are pathogenic fungi or pathogenic bacteria; The pathogenic fungus is Rhizoctonia solani ( ). Rhizoctonia solani ), watermelon shell dispore ( Ascochyta citrullina ) and stalk mold ( Stemphylium solani ); The pathogenic bacteria is Xanthomonas spp., a pathogenic strain of *Xanthomonas spp.* (also known as *Xanthomonas spp.*). Xanthomonas campestris pv .campestris ), syringomorphic tomato disease strain ( Pseudomonas syringae pv. tomato ), Corynebacterium micranthae subsp. micranthae ( Clavibacter michiganensis subsp . michiganensis ), watermelon acidophilic bacteria ( Acidovorax citrulli ), Corynebacterium micranthae, a pathogenic species of potato ring rot ( Clavibacter michiganense subsp .sepedonicum ), Brazilian pectinobacterium ( Pectobacterium brasiliense ) and Pseudomonas syringae var. lacrimae ( Pseudomonas syringae pv .lachrymans ).

5. A method for culturing *Pseudomonas donghuensis* according to claim 1, comprising the step of culturing the *Pseudomonas donghuensis* in a culture medium for culturing *Pseudomonas*.

6. The method for preparing the bacterial agent according to claim 2 includes the following steps: using the *Pseudomonas aeruginosa* according to claim 1 as the active ingredient to obtain the bacterial agent.

7. The application of *Pseudomonas aeruginosa* as described in claim 1 in the prevention and control of soft rot in Chinese cabbage.

8. The application of the *Pseudomonas aeruginosa* of claim 1 or the inoculum of claim 2 in the cultivation of Chinese cabbage.

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