Pseudomonas protegens pflp and its application in preventing and treating bacterial fruit blotch of melon

Abstract

The present application discloses a strain of Pseudomonas protegens and its application in preventing and treating bacterial fruit blotch of melon. The present application relates to the technical field of microbial pesticides, in particular to a strain of Pseudomonas protegens and its application in preventing and treating bacterial fruit blotch of melon. The Pseudomonas protegens of the present application is Pseudomonas protegens ZF509, and its preservation number in China General Microbiological Culture Collection Center is CGMCC No. 27638. The strain ZF509 is identified through morphological observation, physiological and biochemical characteristic determination and multi-gene phylogenetic tree construction analysis, and it is proved that the strain has broad-spectrum bacteriostatic property and prevention and treatment effect on melon in vitro leaf and living body potting, and provides a strain resource for developing a biocontrol agent applicable to bacterial fruit blotch of melon, and has application potential in biological prevention and treatment of bacterial fruit blotch of melon.

Description

Technical Field

[0001] This invention relates to the field of microbial pesticide technology, specifically to a strain of Pseudomonas aeruginosa and its application in controlling bacterial fruit spot disease in melons. Background Technology

[0002] Fruit spot disease is a common disease in melon production, primarily infecting melon leaves and fruits. It spreads rapidly after onset, causing significant economic losses globally. The causative agent of bacterial fruit spot disease in melons is *Acidovorax citrulli*, which can infect various cucurbitaceous crops. Currently, there are no commercially available resistant varieties for the control of bacterial fruit spot disease in cucurbits; control measures mainly rely on seed disinfection and quarantine.

[0003] Biological control has become a hot research area in recent years. Biocontrol agents, as a low-toxicity biological control technology with specific target control properties, have been widely applied in crop disease research. Regarding the development of biocontrol strains for bacterial fruit spot disease in cucurbits, several reports on spore-forming strains have been published, indicating that the application of biocontrol strains in the control of bacterial fruit spot disease in cucurbits has certain research space and development potential. As a class of highly efficient biocontrol bacteria, *Pseudomonas* strains can secrete a variety of antibacterial active substances and have demonstrated application value in crop disease control research. For the control of bacterial fruit spot disease in melons, *Pseudomonas* strains show certain development potential. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the disease resistance of plants and inhibit or prevent infectious diseases of plants.

[0005] To address the aforementioned technical problems, the present invention first provides a strain of Pseudomonas aeruginosa for defense.

[0006] The defensive pseudomonad provided by this invention is Pseudomonas protegens CGMCC No. 27638, which has the accession number CGMCC No. 27638 at the China General Microbiological Culture Collection Center.

[0007] The present invention also provides a microbial agent containing the above-mentioned anti-pseudomonas and / or metabolites of the anti-pseudomonas.

[0008] The above-mentioned microbial agents can be pathogen inhibitors or disease inhibitors, and the pathogens can be pathogenic fungi or pathogenic bacteria.

[0009] The pathogenic fungi mentioned above are Rhizoctonia solani, Ascochytacitrullina, Stemphylium solani, Colletotrichum acutatum, Corynespora cassiicola, Fusarium oxysporum, and Alternaria solani Sorauer.

[0010] The pathogenic bacteria mentioned above are *Acidovorax citrulli*, *Xanthomonas campestris* pv. campestris, *Clavibacter michiganensis* subsp. michiganensis, and *Pectobacterium carotovorum*.

[0011] The disease mentioned above is melon fruit spot disease.

[0012] 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.

[0013] The active ingredient of the above-mentioned microbial agent may be the above-mentioned Pseudomonas aeruginosa and / or the above-mentioned Pseudomonas aeruginosa metabolites. 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.

[0014] In addition to the active ingredients, the above-mentioned microbial agents also contain a carrier. The carrier can be a biologically inert carrier commonly used in the pesticide or fertilizer field. The carrier can be a solid carrier or a 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.

[0015] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0016] 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.

[0017] In the above text, the metabolites can be obtained from the fermentation broth of the *Pseudomonas aeruginosa*. The metabolites can be sterile metabolites of the *Pseudomonas aeruginosa* or bacterial metabolites of the *Pseudomonas aeruginosa*. Specifically, the sterile metabolites of the *Pseudomonas aeruginosa* (sterile fermentation filtrate) can be prepared by culturing the *Pseudomonas aeruginosa* in a liquid culture medium and filtering to remove the *Pseudomonas aeruginosa* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of the *Pseudomonas aeruginosa* can be prepared by culturing the *Pseudomonas aeruginosa* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolites of the *Pseudomonas aeruginosa*.

[0018] The present invention also provides a bio-organic fertilizer containing the aforementioned agent for defending against Pseudomonas or metabolites of the aforementioned Pseudomonas.

[0019] This invention also provides the use of the foregoing defense against Pseudomonas aeruginosa in the preparation of any of the following products:

[0020] 1) A fungicide for the prevention and control of plant diseases, the disease being melon fruit spot disease;

[0021] 2) Pathogen inhibitor, wherein the pathogen is a pathogenic fungus or a pathogenic bacterium.

[0022] The above-mentioned microbial agents can be pathogen inhibitors or disease inhibitors, and the pathogens can be pathogenic fungi or pathogenic bacteria.

[0023] In the above applications, the pathogenic fungi are Rhizoctonia solani, Ascochyta citrullina, Stemphylium solani, Colletotrichumacutatum, Corynespora cassiicola, Fusarium oxysporum, and Alternaria solani Sorauer.

[0024] In the above applications, the pathogenic bacteria are *Acidovorax citrulli*, *Xanthomonas campestris pv. campestris*, *Clavibacter michiganensis subsp. michiganensis*, and *Pectobacterium carotovorum*.

[0025] The present invention also provides a method for culturing the aforementioned defensive Pseudomonas, comprising the step of culturing the defensive Pseudomonas in a culture medium for culturing the defensive Pseudomonas.

[0026] The present invention also provides the application of the aforementioned defense against Pseudomonas aeruginosa or the aforementioned inoculum in the cultivation of melons.

[0027] This invention also provides a method for cultivating melons, including...

[0028] 1) Spray the melon leaves with the anti-pseudomonas bacteria or the aforementioned inoculant;

[0029] 2) Spray the cultivated melon seedlings with the anti-pseudomonas bacteria or the bacterial agent described above.

[0030] This invention also provides the application of the aforementioned defense against Pseudomonas aeruginosa in the prevention and control of melon fruit spot disease and / or the increase of melon yield.

[0031] In this article, the prevention and control of melon fruit spot disease specifically refers to the prevention and control of fruit spot disease on detached melon leaves and / or fruit spot disease in potted melons.

[0032] This study isolated a strain of *Pseudomonas* ZF509 from potato rhizosphere soil in Harbin, Heilongjiang Province. The strain was identified through morphological observation, physiological and biochemical characterization, and polygenic phylogenetic tree construction. The antibacterial spectrum of the strain was also determined, confirming its broad-spectrum antibacterial activity. The control efficacy of the fermentation broth of this strain on detached leaves and live potted plants of watermelon and melon was evaluated. This study provides a strain resource for developing biocontrol agents applicable to bacterial fruit spot disease in melons and shows potential for application in the biological control of bacterial fruit spot disease in cucurbits. However, its practical application still requires field validation. Attached Figure Description

[0033] Figure 1 The inhibitory effect of strain ZF509 on bacterial fruit spot pathogen of melon was investigated. A. ZF509 strain on a plate containing the pathogen, with strain ZF509 in the center (scale bar: 10 mm). B. ZF509 strain on a plate not inoculated with bacterial fruit spot pathogen of melon (scale bar: 10 mm).

[0034] Figure 2 Colony morphology and ultrastructure of strain ZF509 were observed. A. Strain ZF509 was cultured on KB medium for 2 and 5 days (scale bar: 10 mm). C. Gram staining observation. DF. Scanning electron microscopy observation (scale bars: 10, 2.0, and 1.0 μm, respectively).

[0035] Figure 3 This is a phylogenetic tree of the ZF509 strain constructed based on multiple genes.

[0036] Figure 4 The results show the 48-hour drug resistance of strain ZF509. A. Resistance to kanamycin (48 hours) of strain ZF509; B. Resistance to gentamicin sulfate (48 hours) of strain ZF509; C. Resistance to chloramphenicol (48 hours) of strain ZF509; D. Resistance to ampicillin (48 hours) of strain ZF509; E. Resistance to tetracycline (48 hours) of strain ZF509; F. Resistance to spectinomycin hydrochloride (48 hours) of strain ZF509; G. Resistance to rifampin (48 hours) of strain ZF509; H. Resistance to streptomycin (48 hours) of strain ZF509; I. Resistance to erythromycin (48 hours) of strain ZF509.

[0037] Figure 5 The enzyme activity, biofilm formation, and hydrogen cyanide synthesis capabilities of strain ZF509 were determined. Specifically: A. Protease synthesis assay of strain ZF509 (scale bar: 10 mm); B. Phosphoesterase synthesis assay of strain ZF509 (scale bar: 10 mm); C. Cellulase synthesis assay of strain ZF509 (scale bar: 10 mm); D. Ferrophilic synthesis assay of strain ZF509 (scale bar: 10 mm); E. Biofilm formation assay of strain ZF509; F. HCN synthesis assay of strain ZF509.

[0038] Figure 6 Electrophoresis diagram of the antibacterial substance of strain ZF509.

[0039] Figure 7 This is a schematic diagram of the growth curve of strain ZF509.

[0040] Figure 8The inhibitory effect of strain ZF509 on the growth of four pathogenic bacteria: A. Xanthomonas campestris pv. campestris; B. Clavibacter michiganensis subsp. michiganensis; C. Pectobacterium carotovorum; and D. Acidovorax citrulli. (Scale bar: 10 mm).

[0041] Figure 9 The inhibitory effects of volatile substances from strain ZF509 on seven pathogenic fungi. These fungi include: A. *Rhizoctonia solani*; B. *Ascochyta citrullina*; C. *Colletotrichumacutatum*; D. *Corynespora cassiicola*; E. *Fusarium oxysporum*; F. *Stemphylium solani*; and G. *Alternaria solani* Sorauer. (Scale bar: 10 mm).

[0042] Figure 10 The study assesses the control efficacy of ZF509 against detached leaves of melon fruit spot disease. A. Disease incidence of bacterial fruit spot disease in cucurbits; B. Straw strain ZF509; C. Chemical agent thiamethoxam.

[0043] Figure 11 The study investigated the preventive effect of ZF509 on live potted melon fruit spot disease. The results included: AD. Seedling status three days after inoculation; EH. Seedling status five days after inoculation; A and E. Healthy controls; B and F. Disease incidence of bacterial fruit spot disease in cucurbits; C and G. Strain ZF509; D and H. The chemical agent thiamethoxam.

[0044] Figure 12 The therapeutic effect of ZF509 on live potted melon fruit spot disease is shown. AD represents seedling growth three days after inoculation; EH represents seedling growth five days after inoculation; A and E represent healthy controls; B and F represent the incidence of bacterial fruit spot disease in cucurbits; C and G represent strain ZF509; and D and H represent the chemical agent thiamethoxam.

[0045] Figure 13The therapeutic effect of ZF509 on live potted plants with watermelon fruit spot disease is shown. AD represents seedling status three days after inoculation; EH represents seedling status five days after inoculation; A and E represent healthy controls; B and F represent the incidence of bacterial fruit spot disease in cucurbits; C and G represent strain ZF509; and D and H represent the chemical agent thiamethoxam.

[0046] Preservation Instructions

[0047] Bacterial species name: *Pseudomonas aeruginosa*

[0048] Latin name: Pseudomonas protegens

[0049] Strain number: ZF509

[0050] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0051] Collection institution abbreviation: CGMCC

[0052] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0053] Deposit date: June 15, 2023

[0054] Registration number at the Preservation Center: CGMCC No. 27638. Detailed Implementation

[0055] 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.

[0056] 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.

[0057] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0058] The following examples use the honeydew melon (Cucumis melo L.) horned honey (variety registration number: GPD honeydew melon (2017) 110042), which can be obtained by the public from China Vegetable Seed Industry Technology (Beijing) Co., Ltd.

[0059] The pathogenic bacteria tested in the following examples (four pathogenic bacteria) were all preserved by the Vegetable Disease Integrated Prevention Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The four pathogenic bacteria include: *Acidovorax citrulli*, *Xanthomonas campestris pv. campestris*, *Clavibacter michiganensis subsp. michiganensis*, and *Pectobacterium carotovorum*. The seven pathogenic fungi include: Rhizoctonia solani, Ascochyta citrullina, Stemphylium solani, Colletotrichum acutatum, Corynespora cassiicola, Fusarium oxysporum, and Alternaria solani Sorauer.

[0060] The *Pectobacterium brasiliense* in the following examples has been described in: Zhao Zixuan, Zeng Xianfeng, Qin Shiyang, et al. Identification of *Bacillus belyssus* strain ZF438 and its antibacterial effect of fermentation supernatant on anthracnose of pepper [J]. Journal of Agricultural Biotechnology, 2023, 31(10):2163-2175. It is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, for the purpose of repeating the experiments in this application, but should not be used for other purposes.

[0061] The *Acidovorax citrulli* and *Stemphylium solani* mentioned in the following examples have been described in: Huang Yishuo, Xie Xuewen, Shi Yanxia, ​​et al. Effect of *Bacillus polymyxa* ZF197 on the control of stem base rot in Chinese cabbage [J]. Journal of Horticulture, 2020, 47(06):1059-1071. DOI:10.16420 / j.iss n.0513-353x.2019-0915. They are available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, for the purpose of repeating the experiments in this application, but should not be used for other purposes.

[0062] The following examples include Xanthomonas campestris pv. campestris, Clavibacter michiganensis subsp. michiganensis, Rhizoctonia solani, Colletotrichuma cutatum, Corynespora cassiicola, Fusarium oxysporum, and Alternaria solani. Sorauer has been documented in: Zhao Yurong, Li Lei, Xie Xuewen, et al. Control effect of Bacillus bereaves ZF2 against Pleurotus erythrorhizon [J]. Chinese Journal of Biological Control, 2019, 35(02):217-225. DOI:10.16409 / j.cnki.2095-039x.2019.02.018. It can be obtained by the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiments of this application, and shall not be used for other purposes.

[0063] The Ascochyta citrullina in the following examples has been 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. DOI:10.19928 / j.cnki.1000-6346.2008.02.023. It can be obtained by the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, for the purpose of repeating the experiments of this application, but should not be used for other purposes.

[0064] The Pseudomonas protegens pf5 in the following examples were provided by the Department of Plant and Plant Pathology, Oregon State University, and are described in: Henkels MD, Kidarsa TA, Shaffer BT, et al. Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites. Molecular Plant-microbe Interactions: MPMI. 2014 Jul; 27(7):733-746. DOI:10.1094 / mpmi-10-13-0311-r.PMID:24742073. It is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, for the purpose of repeating the experiments in this application, but should not be used for other purposes.

[0065] The preparation method of KB (King's B) liquid culture medium in the following examples is as follows: 20.0g peptone, 1.5g K2HPO4, 1.5g MgSO4·7H2O, 10mL glycerol, and 1000mL distilled water; KB solid culture medium: 13g agar was added to the KB liquid culture medium.

[0066] The preparation method for NB (Nutrient Broth) liquid culture medium is as follows: 10g peptone, 3g beef extract, 5g NaCl, 20g agar, and 1000mL distilled water; NA (Nutrient Agar) solid culture medium: add 13g agar to the NB liquid culture medium.

[0067] The preparation method for WA (Water Agar) culture medium is as follows: 4.5g of agar and 1000mL of distilled water are dispensed into 5mL glass test tubes.

[0068] The PDA (Potato Dextrose Agar) medium is prepared as follows: 200.0g potato, 20.0g glucose, 13.0g agar, and 1000mL distilled water.

[0069] The preparation method for protease selective culture medium is as follows: 5g tryptone, 3g yeast extract, 1g glucose, 15g agar, and distilled water to a final volume of 1000mL. After inoculation, add 100mL skim milk.

[0070] The preparation method of inorganic phosphorus (PVK) solid culture medium is as follows: 0.5g yeast extract, 10g glucose, 5g Ca3(PO4)2, 0.5g (NH4)2SO4, 0.2g KCl, 0.1g MgCl2 (Beijing Bailingwei, GBW(E)084284), 0.1mg MnSO4, 0.1mg FeSO4, 15g agar, and distilled water to a final volume of 1000mL.

[0071] The culture medium for cellulase detection is prepared as follows: 0.25g MgSO4·7H2O, 0.50g K2HPO4, 1.88g Carboxymethylcellulose sodium CMC (Soleb, C8621), Congo red, 15.0g agar (Shanghai Yuanye, B67416), and distilled water to a final volume of 1000mL.

[0072] The culture medium for ferrophosphate detection was prepared as follows: 0.02% 121 mg of Chromium Azuril S indicator (Aladdin, C299259), 0.0135 g of FeCl3·6H2O, and 50 mL of 10 mmol / L HCl were mixed. Then, 145.8 mg of 99% Hexadecyltrimethylammonium bromide (HDTMA) (Shanghai Yuanye, S15001) was added, and the mixture was brought to a final volume of 100 mL with distilled water. After sterilization, the mixture was mixed with 900 mL of solution a. Solution a: 9 g glucose, 4.5 g peptone, 2.7 g beef extract, 4.5 g NaCl, and 15 g agar.

[0073] Reagents required for detecting biofilm synthesis: anhydrous ethanol (China National Pharmaceutical Group, 100092008), crystal violet (Sigma, C0775) 0.01 g, distilled water 10 mL.

[0074] The reagents required for the sensitive test strip for HCN detection are: 4,4′-tetramethyldiaminodiphenylmethane Methane Base 98% (Shanghai Yuanye, S32035) 25mg, Copper(II) Ethylacetoacetate 97% (Shanghai Yuanye, T21050) 25mg, and chloroform (China National Pharmaceutical Group, HW049401) 10mL.

[0075] Antibiotics: Ampicillin [Amp] 100 mg / mL (Solebo, A8180), Kanamycine [Kan] 50 mg / mL (Solebo, K8020), Chloramphenicol [Cm] 25 mg / mL (Solebo, C8050), Streptomycin Sulfate [Str] 50 mg / mL (Coolbac, CS10481), Tetracycline [Tet] 10 mg / mL (Maclean, T829835), Gentamicin Sulfate [Gm] 50 mg / mL (Coolbac, CG5551), Rifampicin [Rif] 50 mg / mL (Coolbac, CR9551), Spectinomycin Hydrochloride sulfate [Spec] 50 mg / mL (Sigma, G1914), erythromycin [Em] 50 mg / mL (Coolbac, CE5091).

[0076] Example 1: Isolation and identification of Pseudomonas protegens strain ZF509

[0077] 1. Strains isolation and purification

[0078] Soil samples were collected from Harbin, Heilongjiang Province, and grouped and numbered. 10g of each sample was weighed and added to 90mL of sterile water. The mixture was then incubated at 28℃ in a shaker for 30min. The resulting soil suspension was then serially diluted, with a dilution factor of 10⁻⁶. 4 10 5 10 6 10 7 Soil suspensions were spread on KB plates and incubated at 28°C for 36 hours. Once single colonies grew on the plates, single colonies of different morphologies were picked up with sterile toothpicks and streaked onto KB solid plates for purification. The plates were then incubated at 28°C until single colonies with uniform morphology grew. Finally, the plates were stored at 4°C for later use.

[0079] 2. Screening of antagonistic strains against watermelon acidophilic bacteria

[0080] The inhibition rate of the isolated strains against *Acidovorax citrulli* was determined using a mixed culture method. 4 mL of the shake-cultured *Acidovorax citrulli* suspension was added to 200 mL of sterile KB solid medium (melted at 50°C) using a micropipette. After mixing and shaking, the mixture was poured into sterile petri dishes and allowed to solidify at room temperature. Sterilized filter paper discs of uniform size were affixed to the center and sides of the plates. 5 μL of the shake-cultured soil sample suspension was then added to the filter paper discs using a micropipette. The plates were incubated at 28°C for 48 h, with each treatment repeated three times. The diameters of the control and treated colonies were measured using a cross-hatching method to calculate the inhibition rate and obtain the antagonistic strains.

[0081] Inhibition rate (%) = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100%

[0082] The results are as follows Figure 1 As shown: A antagonistic bacterium with good inhibitory effect on bacterial fruit spot disease of melon was obtained by the mixed culture method and was numbered ZF509. The inhibition rate of strain ZF509 against bacterial fruit spot disease of melon was determined again by the plate confrontation method. The center of the plate is strain ZF509. The bacterial fruit spot disease solution of melon was mixed into the plate culture medium. Figure 1 In the study, strain 509 was found to have an inhibition rate of 71.41%, with plates containing no bacterial fruit spot pathogens of melon used as a control. Figure 1 (B)

[0083] 2. Physicochemical properties and molecular biological identification of strain ZF509

[0084] 1) Morphological observation

[0085] Morphological characteristics of strain ZF509 were observed, including colony size, color, shape, colony edge morphology, and whether the colony surface was glossy, raised or flat, and smooth or rough in texture. The streak plate method was used; activated single colonies were picked and streaked onto KB solid agar plates, incubated at 28°C for 48 hours, and then observed, referring to the methods in *Common Bacterial Systematic Identification Handbook* (Dong Xiuzhu et al., 2001) and *Bergey's Manual of Bacterial Identification* (Buchanan et al., 1984).

[0086] 2) Biolog measurement

[0087] Single colonies of strain ZF509 were picked and inoculated onto KB medium slant tubes and incubated at 28°C for 48 hours. The unique carbon source utilization of strain ZF509 was determined by the China Agricultural Microbial Culture Collection Center using the BIOLOG GEN III kit (operated according to the kit instructions).

[0088] The results are as follows Figure 2 As shown: After culturing strain ZF509 on KB plates for 48 hours, the colonies were slightly raised, round or nearly round, light white, with smooth edges and a moist surface. Figure 2 (A), cultured for 5 days, the colonies turned orange ( Figure 2 (B) Gram staining is negative. Figure 2 (C) Under scanning electron microscopy, the colonies are rod-shaped and approximately 1.5 μm in length. Figure 2 (ZF509). Physiological and biochemical identification results showed that strain ZF509 was negative in gelatin liquefaction test, tolerated salt concentrations of 1-4 g / mL (NaCl), and was suitable for a slightly acidic environment (Table 1).

[0089] Table 1. Identification of physiological and biochemical characteristics of strain ZF509

[0090]

[0091]

[0092] Note: +: Positive reaction or growth is possible; -: Negative reaction or no growth; W: Weak positive reaction or growth is possible.

[0093] 3) Multi-gene amplification and sequence analysis

[0094] Genomic DNA was extracted from strain ZF509 using a bacterial genomic DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.). Conserved genes (Table 2) that can be used to identify Pseudomonas species, namely 16S rDNA, DNA gyrase subunit B (gyrB), RNA polymerase subunit β (rpoB), and RNA polymerase sigma-70 factor (rpoD), were selected for PCR amplification of strain ZF509.

[0095] The amplification system consisted of 50 μL of components, including 1 μL of the front primer, 1 μL of the back primer, 1 μL of template DNA, 25 μL of T-Taq Mix, and 22 μL of ddH2O. The PCR amplification program was set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 29 cycles; and a final extension at 72℃ for 10 min. The PCR products were sequenced by Beijing Bomeide Biotechnology Co., Ltd.

[0096] The three genes of the other control strains were downloaded from the NCBI website. The gene sequences of 16S rDNA, gyrB, and rpoB were aligned in tandem using MEGA 7.0, SequenceMatrix, and Seaview 4 software. A phylogenetic tree was constructed using the maximum likelihood method (bootstrapping number of 1000) to determine the taxonomic position of ZF509.

[0097] Table 2. Primer Information

[0098]

[0099] Strain ZF509 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, an rpoB gene with the nucleotide sequence of sequence 3 in the sequence listing, and an rpoD gene with the nucleotide sequence of sequence 4 in the sequence listing. Based on the 16S rDNA, gyrB, and rpoB gene sequences of strain ZF509, a multigene phylogenetic tree of ZF509 was constructed. The results are as follows: Figure 3 As shown, strain ZF509 clustered in one branch with the Pseudomonas protegens model strain. Based on morphological observation, physiological and biochemical characteristic detection, and phylogenetic tree construction, strain ZF509 was identified as belonging to Pseudomonas protegens.

[0100] 3. Preservation of ZF509 strain

[0101] The defensive pseudomonad ZF509 strain was deposited on June 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27638. It will be referred to as defensive pseudomonad CGMCC No. 27638 or strain ZF509.

[0102] 4. Determination of antibiotic tolerance levels in defensive pseudomonocyte ZF509

[0103] The results of 24-hour and 48-hour shake-flask culture on eight antibiotic-containing culture media inoculated with ZF509 bacterial suspension are shown in the figure. Figure 4The results are shown in Table 3. Within 48 hours, strain ZF509 showed strong resistance to tetracycline, rifampin, ampicillin, spectinomycin hydrochloride, streptomycin, and erythromycin, with tolerance levels ranging from 50 μg / mL to 500 μg / mL. Its tolerance level to chloramphenicol was 50 μg / mL to 300 μg / mL. It showed no resistance to kanamycin and gentamicin sulfate.

[0104] Table 3. Drug resistance of strain ZF509 over 24 hours

[0105]

[0106] +: resistant or able to grow; -: no resistance or unable to grow; W: weakly resistant or able to grow.

[0107] 5. Enzyme activity detection, biofilm synthesis assay, and HCN synthesis assay of strain ZF509

[0108] The activated ZF509 strain was inoculated at the center of protease-selective medium, inorganic phosphorus-selective medium, cellulase detection medium, and hematophilic acid detection medium. Each treatment was repeated three times. After incubation at 28°C for 24 hours, the presence of a transparent digestion zone at the inoculation site was observed. The results are as follows: Figure 5 As shown, strain ZF509 has the ability to produce proteases and dissolve inorganic phosphorus. Figure 5 (A and B) do not produce cellulase and heptaphilin (A and B). Figure 5 (C and D in the middle).

[0109] The next day, dilute the cultured strain ZF509 to OD200. 600 =0.5, then transfer 30 μL of bacterial culture to a sterile tube containing 3 mL of KB medium (1:100). Incubate at 28°C for 24 h. Pour off the bacterial culture from the tube, rinse once with deionized water, and add 4 mL of 0.1% (w / v) crystal violet for staining at room temperature for 15 min. Rinse the tube twice more with deionized water and observe for the appearance of purple rings on the tube wall. Results are as follows... Figure 5 As shown in Figure E, strain ZF509 has the ability to produce biofilms.

[0110] Filter paper strips were soaked in 10 mL of chloroform containing 25 mg of 4,4′-tetramethyldiaminodiphenylmethane and 25 mg of copper(II) ethylacetoacetate to prepare sensitive test strips for HCN detection. These strips were suspended and sealed on KB solid medium inoculated with ZF509 strain. Each treatment was repeated three times. After incubation at 28°C for 24 h, the test strips were observed to see if they turned blue. Results are as follows: Figure 5 As shown in Figure F, strain ZF509 can produce hydrogen cyanide.

[0111] 6. Determination of antibacterial substances in strain ZF509

[0112] By comparing the amplified products of specific primers (primer sequence information is shown in Table 4) with the synthetic sequences of antimicrobial substances, and referring to the known antimicrobial substances contained in the positive control strain P. protegens pf5, the results are as follows: Figure 6 As shown, strain ZF509 was found to contain four classes of antibacterial substances: 2,4-DAPG, nitropyrrolizin, gambogeysin, and 2-hydroxyphenazine.

[0113] Table 4. Antibiotic gene and primer sequence information

[0114]

[0115] 7. Growth curve determination of strain ZF509

[0116] A single colony of purified ZF509 was transferred to 3 mL of KB medium and incubated for 12 h to obtain a seed culture. The seed culture was then diluted to OD200. 600 After reaching 0.5 μL, the culture was transferred to a sterile 250 mL Erlenmeyer flask containing 50 mL of KB(+kan) medium and incubated at 28 °C and 200 rpm on a shaker. Three replicates were designed. Samples were taken every 4 hours after transfer, with 100 μL of bacterial culture dissolved in 900 μL of sterile water as a primary dilution gradient (sampling points are shown in Table 5). 10... 4 -10 7 Bacterial suspensions within the dilution range were plate-spread and counted. The results are as follows: Figure 7 As shown, strain ZF509 was in the logarithmic growth phase from 12 to 20 hours, and tended to stabilize after 20 hours, with a viable cell concentration of 1×10⁻⁶. 9 Within the CFU / mL range, the activity of the strain began to decline after 28 hours.

[0117] Table 5. Sampling nodes for the growth curve of ZF509

[0118]

[0119] Example 2: Detection of the antibacterial spectrum of strain ZF509

[0120] 1. Antibacterial spectrum

[0121] The inhibitory effect of the ZF509 strain against pathogenic bacteria was determined using a double-layer culture method. The ZF509 strain was cultured in KB liquid medium until the bacterial concentration reached 1×10⁻⁶. 8CFU / mL, using a 10μL pipette, pipette 5μL of the test bacterial culture and spot it in the center of KB medium (glass dish), incubate at 28℃ for 48h; invert the culture dish, use a dropper to add 5mL of chloroform along the slits of the glass dish, and fumigate in a fume hood for at least 9h. Blank control group: Spot 5μL of KB medium in the center of the KB medium as a control. Treatment groups were set up according to the pathogen species, with three replicates per group.

[0122] Using sterilized glass test tubes, dispense 5 mL of WA medium into each tube and incubate at 50°C in a water bath. Incubate the pathogenic bacterial suspension in KB medium for 24 hours prior to the test. Using a 200 μL pipette, add 150-200 μL of the pathogenic bacterial suspension to each glass test tube and pour it onto the top layer of the bacterial layer to be screened. Incubate at 28°C for 48 hours and observe the inhibition zone. Measure the diameter of the control and treated colonies using the cross-hatching method to calculate the inhibition rate and obtain the antagonistic strain.

[0123] Inhibition rate (%) = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100%

[0124] The results are as follows Figure 8 As shown in Tables AD and 6, ZF509 can effectively inhibit *Acidovoraxcitrulli*, *Xanthomonas campestris* pv. campestris, *Clavibacter michiganensis* subsp. michiganensis, and *Pectobacterium carotovorum*, with inhibition diameters greater than 45 mm.

[0125] Table 6. Inhibitory effects of ZF509 strain on four pathogenic bacteria

[0126]

[0127]

[0128] 2. Determination of the antibacterial activity of volatile substances against Pseudomonas ZF509

[0129] The ZF509 bacterial culture, frozen at -80℃, was thawed at 4℃. 500 μL of the culture was then added to a test tube containing 5 mL of liquid KB medium for activation. The culture was incubated at 28℃ and 150 rpm for 12 hours using a constant temperature shaker. The activated culture was then streaked onto a plate and incubated at 28℃ for 24 hours. Single colonies were picked and streaked again for purification. Finally, single colonies were incubated in KB liquid medium for 48 hours to obtain the ZF509 anti-pseudomonas agent at a concentration of 1.0 × 10⁻⁶. 8 CFU / mL.

[0130] Pour PDA and KB solid culture media onto both sides of a bipartite dish, respectively, and allow to air dry before use. Using a sterile toothpick, pick up a single colony of biocontrol strain ZF509 and streak it onto one side of the KB solid culture medium. Incubate at 28°C for 24 hours. Perform three replicates for each target treatment group. For bipartite dishes inoculated with biocontrol strain ZF509 and incubated for 24 hours, add a 5.0 mm diameter pathogenic fungal disc 2.25 cm from the middle partition on the PDA side, and incubate upside down at 28°C. Observe the growth of the pathogenic fungus to the edge of the plate in the blank control, and determine the diameter of the pathogenic fungus using the cross-cross method to calculate the inhibition rate.

[0131] Inhibition rate (%) = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100%

[0132] The blank control group was set up as follows: KB medium was not inoculated with ZF509.

[0133] The results are as follows Figure 9 As shown in Table 7, the volatile substances of ZF509 have significant inhibitory effects on Rhizoctonia solani, Diplospermae citrinum, Crestis spp., and Alternariae spp., with inhibition rates exceeding 55%; they also have some inhibitory effects on Colletotrichum spp. and Colletotrichum spp., with inhibition rates of 45.3% and 36.4%, respectively; and they have a slight inhibitory effect on Fusarium oxysporum, with an inhibition rate of only 22.5%.

[0134] Table 7. Inhibitory effects of volatile substances from strain ZF509 on seven pathogenic fungi.

[0135]

[0136]

[0137] Example 3: Determination of the protective efficacy of Pseudomonas ZF509 against fruit spot disease in melons and watermelons.

[0138] 1. Preparation of antibacterial agent against Pseudomonas ZF509

[0139] The ZF509 bacterial culture, frozen at -80℃, was thawed at 4℃. 50 μL of the culture was then added to a test tube containing 5 mL of liquid KB medium for activation. The culture was incubated at 28℃ and 150 rpm for 12 hours using a constant temperature shaker. The activated culture was then streaked onto a plate and incubated at 28℃ for 24 hours. Single colonies were picked and streaked again for purification. Finally, single colonies were incubated in KB liquid medium for 48 hours to obtain the ZF509 anti-pseudomonas agent at a concentration of 1.0 × 10⁻⁶. 8 CFU / mL.

[0140] 2. Efficacy of Pseudomonas ZF509 in preventing fruit spot disease from detached melon leaves.

[0141] Select healthy melon seedlings with two leaves and one bud at the same height. Wrap the roots of the seedlings with sterile cotton cloth soaked in water and incubate them in a sterile glass dish with humidity control. Activate the watermelon acid-eating bacteria preserved in glycerol tubes on KB plates. Use a sterile toothpick or inoculation loop to pick up single colonies and inoculate them into shake tubes containing 3 mL of liquid KB medium. Incubate at 28°C with shaking for 24 hours. The entire process is performed in a sterile operating room. Adjust the concentration of the watermelon acid-eating bacteria suspension to 1×10⁻⁶. 8 CFU / mL was added to the leaves using a sterile pipette tip, ensuring that the bacterial solution adhered to both sides of each leaf. 24 hours after inoculation, the ZF509 bacterial agent obtained in step 1 of this example was sprayed on both sides of the leaves, and the control efficacy was investigated according to the grading standards in Table 8. References for disease grading are: Li Leshu. Development of biological seed dressing agent for controlling bacterial fruit spot disease in cucurbits [D]. Nanjing Agricultural University, 2017; Fenoa, Chen Huamin, Yang Yuwen, et al. New progress in foreign research on bacterial fruit spot disease in cucurbits [J]. China Cucurbits and Vegetables, 2022, 35(07):1-5. The control group (CK) consisted of leaves sprayed with water 24 hours after inoculation, and the disease incidence was observed as a control.

[0142] Table 8. Grading Standards for Fruit Spot Disease

[0143]

[0144]

[0145] Disease index = 100 × ∑(number of diseased leaves at each level × representative value of the relative level) / (total number of leaves × representative value of the highest level)

[0146] Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100%

[0147] The results are as follows Figure 10As shown in Tables AC and 9, the pathogen control group inoculated only with *Pseudomonas sylvestris* exhibited severe disease, with sunken, dark brown lesions on the upper surface of leaves surrounded by yellow halos, and water-soaked lesions on the lower surface. The ZF509 treatment group showed significantly milder disease, with scattered pale yellow lesions on the upper surface of the leaves, and the overall leaf color remained green. The thiamethoxam treatment group also showed milder disease, with fewer pale yellow lesions, and the overall leaf color remained green. Disease index analysis based on the percentage of lesion area on diseased leaves showed that the control efficacy against *Pseudomonas sylvestris* strain ZF509 was 74.1%, while the control agent thiamethoxam showed a control efficacy of 82.8%, indicating similar control efficacy.

[0148] Table 9. Control efficacy of ZF509 against bacterial fruit spot disease in melons

[0149] ZF509 16.7 74.1 Thiamethoxam 10.9 82.8 CK 63.3 ——

[0150] 3. Efficacy of Pseudomonas ZF509 in preventing fruit spot disease in potted melons.

[0151] Select plump melon seeds, soak them in a 50℃ water bath for 10 minutes, then wrap the seeds in sterilized gauze and place them in a humidity-controlled box. Place them in a 28℃ constant temperature incubator to germinate. When the radicle grows to 0.3cm-0.5cm, prepare for sowing. Try to select germinated seeds with uniform root growth and sow them in 72-cell trays to ensure uniform germination. Select melon seedlings with two leaves and one bud (when the first true leaf has just unfolded) for inoculation.

[0152] Vaccination is divided into two methods: prevention and treatment. 1) Treatment group: First, administer a suspension of *Hydroxyphae pilosa* (bacterial suspension concentration of 1.0 × 10⁻⁶). 8 The bacterial suspension (CFU / mL) was sprayed onto the leaves, ensuring that both sides of each leaf were covered with the bacterial solution. After 36 hours, the ZF509 bacterial agent (bacterial suspension concentration 1.0 × 10⁻⁶) obtained in step 1 of this embodiment was sprayed. 8 The control group was treated with a mixture of ZF509 (CFU / mL) and thiamethoxam (1.6% active ingredient diluted 600 times). The prevention group was treated by spraying melon seedlings with the same concentration of biocontrol agent ZF509 and the control agent thiamethoxam. After a 36-hour interval, the pathogenic agent *Alternaria hygrophila* was sprayed. Both were inoculated by spraying, with an average of 5 mL of *Alternaria hygrophila* suspension and the control agent per seedling. Disease incidence was observed 3 and 5 days after inoculation with *Alternaria hygrophila* suspension. The disease index was calculated based on the area of ​​lesions on diseased leaves to evaluate the in vivo control efficacy of ZF509.

[0153] Disease index = 100 × ∑(number of diseased plants at each level × representative value of relative level) / (total number of plants × representative value of the highest level)

[0154] Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100%

[0155] 1) The preventive effect of Pseudomonas ZF509 on fruit spot disease in potted melons

[0156] Prevention and treatment group: First, spray the same concentration of biocontrol bacterial solution ZF509 and control agent thiamethoxam, and then spray the pathogen solution after an interval of 36 hours.

[0157] The results are as follows Figure 11 As shown in Table 10 and the AH, the pathogen control group inoculated only with *Acidobacterium hygrophilum* exhibited severe disease, with large, obvious water-soaked lesions and multiple evenly distributed pale yellow spots on the leaves. In severe cases, the lesions merged into dark brown patches, and the leaf edges turned yellowish-green. The melon leaves treated with strain ZF509 showed no large water-soaked lesions on the upper surface, but had scattered small, pale yellow lesions, and the leaves remained mostly green. The leaves in the thiamethoxam-treated group showed milder disease, with fewer small, pale yellow lesions and water-soaked lesions, and the leaves remained generally green. On the third day after inoculation, strain ZF509 showed an 83.0% preventive effect against bacterial fruit spot disease in melons, while thiamethoxam showed an 87.1% preventive effect. On the fifth day after inoculation, strain ZF509 showed a 75.3% preventive effect, while thiamethoxam showed a 69.3% preventive effect. The results showed that the control efficacy of strain ZF509 was relatively stable and did not decrease significantly over time, while the control efficacy of the chemical agent thiamethoxam was less stable and decreased over time.

[0158] Table 10. Preventive effect of ZF509 on bacterial fruit spot disease in melon.

[0159]

[0160] 2) The therapeutic effect of Pseudomonas ZF509 on fruit spot disease in potted melons

[0161] Treatment: First, inoculate with pathogenic bacterial solution, and after an interval of 36 hours, spray with biocontrol bacterial solution ZF509 and control agent thiamethoxam.

[0162] The results are as follows Figure 12As shown in Tables A and H and Table 11, the pathogen control group inoculated only with *Acidobacterium hygrophilum* exhibited severe disease, with large, obvious water-soaked lesions and multiple evenly distributed pale yellow spots on the leaves. In severe cases, these lesions merged into dark brown patches. The melon leaves treated with strain ZF509 showed no large water-soaked lesions on the upper surface, but had small, pale yellow lesions distributed on the upper surface, and the leaves remained largely green. The leaves in the thiamethoxam-treated group showed milder disease, with fewer small, pale yellow lesions and water-soaked lesions, and the leaves remained generally green. Overall, the disease severity in the treatment group was higher than that in the prevention group. On the third day after inoculation, strain ZF509 showed a 50.4% efficacy rate against bacterial fruit spot disease in melons, while thiamethoxam showed a 48.6% efficacy rate. On the fifth day after inoculation, the efficacy rate of strain ZF509 was 43.4%, and that of thiamethoxam was 40.1%. These results indicate that strain ZF509 has a more stable control effect and is superior to the control agent thiamethoxam. Over time, the therapeutic and preventive efficacy of strain ZF509 and thiamethoxam both decreased.

[0163] Table 11. Therapeutic effect of ZF509 on bacterial fruit spot disease in melon.

[0164]

[0165] 4. Efficacy of Pseudomonas ZF509 in preventing fruit spot disease in potted watermelons

[0166] Select plump watermelon seeds, soak them in a 50℃ water bath for 10 minutes, then wrap the seeds in sterilized gauze and place them in a humidity-controlled box. Place them in a 28℃ constant temperature incubator to germinate. When the radicle grows to 0.3cm-0.5cm, prepare for sowing. Try to select germinated seeds with uniform root growth and sow them in 72-cell trays to ensure uniform germination. Select watermelon seedlings with two leaves and one bud (when the first true leaf has just unfolded) for inoculation.

[0167] Inoculation is a method of prevention and treatment: first, spray the watermelon seedlings with the ZF509 inoculant obtained in step 1 of this embodiment (concentration of 1.0 × 10⁻⁶). 8 CFU / mL) and the control agent thiamethoxam (active ingredient content 1.6% diluted 600 times, sprayed at a concentration of 1.0×10 after 72 hours) 8 A CFU / mL suspension of *Acidobacterium hygrophilum* was applied to each leaf, ensuring that both sides of the leaf were covered with the bacterial suspension. After 72 hours, the same concentration of ZF509 inoculant and the control agent thiamethoxam were sprayed. The disease incidence was observed 5 and 7 days after inoculation with the *Acidobacterium hygrophilum* suspension. The disease index was calculated based on the area of ​​lesions on diseased leaves to evaluate the in vivo control efficacy of ZF509.

[0168] Disease index = 100 × ∑(number of diseased plants at each level × representative value of relative level) / (total number of plants × representative value of the highest level)

[0169] Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100%

[0170] The results are as follows Figure 13 As shown in Tables A and H and Table 12, the pathogen control group inoculated only with *Acidobacterium hygrophilum* exhibited severe disease, with large, water-soaked "V"-shaped lesions. In severe cases, these lesions merged into dark brown patches, causing leaf edges to turn yellowish-green and resulting in wilting. Melon leaves treated with strain ZF509 showed no large water-soaked lesions on the leaf surface, but had scattered small, pale yellow lesions on the upper surface, and the leaves remained largely green. The thiamethoxam-treated group showed less disease than the pathogen control group, but more severe than the ZF509-treated group, with patches of lesions and water-soaked lesions, slight wilting, and smaller leaves than the healthy control group. Five days after inoculation, strain ZF509 showed an 83.0% preventative effect against bacterial fruit spot disease in watermelon, while thiamethoxam showed an 87.1% preventative effect. Seven days after inoculation, the preventative effect of strain ZF509 was 75.3%, and that of thiamethoxam was 69.3%. The results showed that the control efficacy of strain ZF509 was relatively stable and did not decrease significantly over time, which was superior to that of the chemical agent thiamethoxam.

[0171] Table 12. Control efficacy of ZF509 against bacterial fruit spot disease in watermelon

[0172]

[0173] 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. Defense against Pseudomonas ( ) Pseudomonas protegens ZF509, characterized in that: The preservation number for the defense against Pseudomonas is CGMCC No. 27638.

2. A microbial agent, characterized in that: The bacterial agent contains the anti-pseudomonas bacteria 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 ), creeping mold ( Stemphylium solani ), Colchicum aquiferum ( Colletotrichum acutatum ), Corynebacterium multiflorum ( Corynespora cassiicola Fusarium oxysporum ( Fusarium oxysporum ) and Alternaria solanacearum ( Alternaria solani Sorauer ); The pathogenic bacteria is *Liquidambar formosana* (a type of citrus acidophilus). Acidovorax citrulli ), Xanthomonas aurea, a pathogenic species of wild rapeseed ( Xanthomonas campestris pv. campestris ), Corynebacterium micranthae subsp. micranthae Clavibacter michiganensis subsp. michiganensis ) and Carotene soft rot pectinobacter ( Pectobacterium carotovorum ); The disease in question is melon fruit spot disease.

3. The method for preparing the bacterial agent according to claim 2 includes the following steps: using the anti-pseudomonas bacteria according to claim 1 as the active ingredient to obtain the bacterial agent.

4. Bio-organic fertilizer, characterized by: The bio-organic fertilizer contains the anti-pseudomonas bacteria agent as described in claim 1 or the microbial agent as described in claim 2.

5. The use of the anti-pseudomonas bacteria as described in claim 1 in the preparation of the product described in 1) or 2) below: 1) A fungicide for the prevention and control of plant diseases, the disease being melon fruit spot disease; 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 ), creeping mold ( Stemphylium solani ), Colchicum aquiferum ( Colletotrichum acutatum ), Corynebacterium multiflorum ( Corynespora cassiicola Fusarium oxysporum ( Fusarium oxysporum ) and Alternaria solanacearum ( Alternaria solani Sorauer ); The pathogenic bacteria is *Liquidambar formosana* (a type of citrus acidophilus). Acidovorax citrulli ), Xanthomonas aurea, a pathogenic species of wild rapeseed ( Xanthomonas campestris pv. campestris ), Corynebacterium micranthae subsp. micranthae Clavibacter michiganensis subsp. michiganensis ) and Carotene soft rot pectinobacter ( Pectobacterium carotovorum ).

6. A method for culturing the defensive Pseudomonas as claimed in claim 1, comprising the step of culturing the defensive Pseudomonas in a culture medium for culturing the defensive Pseudomonas.

7. The application of the anti-pseudomonas bacteria agent of claim 1 or the inoculant of claim 2 in the cultivation of melons.

8. Methods for cultivating melons include the following: 1) Spray the leaves of cultivated melons with the anti-pseudomonas agent as described in claim 1 or the inoculant as described in claim 2; 2) Spray the cultivated melon seedlings with the anti-pseudomonas bacteria agent as described in claim 1 or the bacterial agent as described in claim 2.

9. The application of the anti-pseudomonas species as described in claim 1 in the prevention and control of melon fruit spot disease.

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