Pseudomonas syringae phage as well as composition and application thereof

Abstract

The invention discloses a pseudomonas syringae phage as well as a composition and application thereof, and belongs to the technical field of biological prevention and control. The pseudomonas syringae bacteriophage PhiPs-1 which is short in incubation period, short in outbreak period, large in splitting quantity, high in titer, wide in host spectrum, acid-resistant, heat-resistant, chloroform-resistant and ultraviolet-resistant is obtained through screening, and the preservation number of the pseudomonas syringae bacteriophage PhiPs-1 is CCTCC NO: M 20252347; the composition can be developed into a medicinal preparation for preventing and treating the tomato bacterial leaf spot disease; according to the invention, the pseudomonas syringae phage phi Ps-1 and the biocontrol bacterium NBL-AP73 are combined for use, so that the control effect on the tomato bacterial leaf spot disease is synergistically improved.

Description

Technical Field

[0001] This invention relates to the field of biocontrol technology, specifically to a strain of Pseudomonas syringae bacteriophage, its composition, and its applications. Background Technology

[0002] Pseudomonas syringae ( Pseudomonas syringae *Pseudomonas syringae* is a Gram-negative, aerobic, and saprophytic bacterium belonging to the genus *Pseudomonas*. It ranks first among the top ten plant pathogens in molecular plant pathology, and is widely distributed in natural environments such as soil, water bodies, and plant leaves. It is a microbial community with strong ecological adaptability and significant phenotypic variation. *Pseudomonas syringae* can infect more than 300 economic crops, including those in the legume, cruciferous, solanaceous, and roseaceae families, causing diseases such as bean blight, stone fruit canker, peach canker, bacterial diseases of peas, and black spot disease in cruciferous plants, resulting in significant losses to agricultural production.

[0003] Tomatoes are one of the world's most important economic crops, and their production is often threatened by various pathogenic microorganisms. Among them, bacterial leaf spot caused by *Pseudomonas syringae* is one of the most important diseases in tomato cultivation. This pathogen infects leaves, stems, and fruits, forming characteristic necrotic spots, which leads to inhibited photosynthesis, premature aging of plants, and reduced commercial value of fruits. In severe cases, it can cause a yield loss of 20-50%, and the risk of epidemics is extremely high, especially in cool and humid regions (such as temperate greenhouses and early spring open-field planting areas).

[0004] For the control of bacterial leaf spot disease caused by *Pseudomonas syringae*, traditional agricultural measures such as crop rotation, deep plowing and sun-drying of soil, and breeding of disease-resistant and hybrid varieties have reduced the incidence rate. However, these measures are slow and subject to environmental constraints and the influence of the variety selection cycle. Chemical control mainly relies on copper preparations and antibiotics. Although these have a certain control effect on the disease, long-term single-use has led to increased drug resistance in pathogens (such as the frequent occurrence of multi-resistant strains to streptomycin and copper ions). Furthermore, excessively high concentrations of copper ions in fungicides can affect the normal growth and development of plants. Overuse can also lead to the accumulation of copper residues in the environment, disrupt the soil ecological balance, and cause environmental pollution problems.

[0005] Bacteriophages possess advantages such as precise targeting, environmental friendliness, and low drug resistance. They can specifically infect and kill specific pathogens without affecting beneficial microorganisms, thus reducing the use of chemical pesticides and lowering environmental pollution. In recent years, based on the high host specificity and environmental compatibility of bacteriophages, their biocontrol technology has become a research hotspot. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Pseudomonas syringae bacteriophage, its composition and application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Pseudomonas syringae* bacteriophage ΦPs-1 (… Pseudomonas syringae PhageΦPs-1 was deposited on October 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: M 20252347.

[0008] The *Pseudomonas syringae* phage ΦPs-1 of this invention belongs to the order Caudate Phages and family Brachypophagaceae; its nucleic acid type is DNA, belonging to the DNA virus group. The *Pseudomonas syringae* phage ΦPs-1 of this invention has the advantages of a short incubation period, a short outbreak period, and a large lysis rate; it has a broad lysis spectrum against *Pseudomonas* strains, and exhibits strong lysis against non-pathogenic *Pseudomonas koreanum* PK1 (ACCC60112), making it suitable for phage culture with high safety; moreover, the *Pseudomonas syringae* phage ΦPs-1 has a certain degree of tolerance to temperature, pH, chloroform, and ultraviolet light.

[0009] In a second aspect, the present invention provides a phage formulation, wherein the phage formulation uses the above-mentioned Pseudomonas syringae phage ΦPs-1 as the active ingredient.

[0010] In some preferred embodiments of the present invention, the bacteriophage preparation is prepared by the following method: Pseudomonas Koreanis PK1 was inoculated into fermentation medium and fermented until the OD of the bacterial culture reached a certain level. 590 When the concentration reaches 0.6-0.7, add *Pseudomonas syringae* phage ΦPs-1 and continue culturing until OD reaches 0.6-0.7. 590 The concentration was reduced to 0.2 to obtain the phage culture medium; Centrifuge the phage culture medium, discard the precipitate, collect the supernatant and add chloroform to prepare the phage preparation.

[0011] Preferably, the fermentation medium consists of: 30 g molasses, 15 g fish peptone, 7 g dipotassium hydrogen phosphate, 3 g potassium dihydrogen phosphate, 0.1 g magnesium sulfate, 1 g ammonium sulfate, 0.5 g sodium citrate, 0.3 g carvacrol, 1 g inulin, and 1 L distilled water, with a pH of 6.2-7.2, and sterilized at 121°C for 20 min.

[0012] This invention optimizes the composition of the fermentation medium by adding carvacrol and inulin, which synergistically increases the titer of the cultured bacteriophages.

[0013] Preferably, the fermentation conditions are: a culture temperature of 32℃, a stirring speed of 160 r / min, and an aeration rate of 1.0 vvm.

[0014] In a third aspect, the present invention provides the use of the above-mentioned *Pseudomonas syringae* bacteriophage ΦPs-1 or bacteriophage preparation in the prevention and control of plant diseases caused by *Pseudomonas syringae*.

[0015] Preferably, the plant disease is bacterial leaf spot of tomato.

[0016] In a fourth aspect, the present invention provides a composition for controlling bacterial leaf spot disease of tomatoes, the composition being formulated by combining the above-mentioned phage preparation and a bacterial suspension of biocontrol bacterium NBL-AP73 with preservation number CCTCC NO: M20241295.

[0017] In the composition, the bacteriophage preparation and the bacterial suspension of biocontrol bacteria NBL-AP73 can be diluted in different proportions according to actual needs, and then the diluted bacteriophage preparation and the diluted bacterial suspension of biocontrol bacteria NBL-AP73 can be compounded; or, the bacteriophage preparation and the bacterial suspension of biocontrol bacteria NBL-AP73 can be compounded and then diluted for use.

[0018] In some preferred embodiments of the present invention, the composition is prepared by mixing a phage preparation diluted 25 times with a bacterial suspension of biocontrol bacterium NBL-AP73 diluted 50 times at a volume ratio of 1:1.

[0019] A fifth aspect of the present invention provides a method for controlling bacterial leaf spot disease of tomatoes, comprising the following steps: Apply the above-mentioned phage preparation to tomato plants at risk of infection by *Pseudomonas syringae*, or apply the above-mentioned composition for controlling bacterial leaf spot disease in tomatoes.

[0020] The beneficial effects of this invention are: (1) The present invention has isolated a phage ΦPs-1 of Pseudomonas syringae with a short incubation period, short outbreak period, large lysis volume, high titer, broad host spectrum, acid resistance, heat resistance, chloroform resistance and ultraviolet resistance, which can be developed into a drug preparation for the prevention and control of bacterial leaf spot disease of tomatoes.

[0021] (2) The present invention uses non-pathogenic Pseudomonas koreanum PK1 as a host bacteriophage for phage culture, which improves the safety of phage preparation preparation; the composition of the culture medium is optimized by adding carvacrol and inulin to the culture medium, thereby significantly improving the titer of phage.

[0022] (3) The present invention combines the phage ΦPs-1 of Pseudomonas syringae with the biocontrol bacterium NBL-AP73 to synergistically improve the control effect against bacterial leaf spot disease of tomato. Attached Figure Description

[0023] Figure 1 : The morphology of bacteriophage ΦPs-1 in culture medium.

[0024] Figure 2 Morphage ΦPs-1 under a transmission electron microscope.

[0025] Figure 3 The results of DNase I and RNase A digestion of phage ΦPs-1 nucleic acid are shown in the following: Lane 1 is the GeneRuler 1 kbPlus DNA molecular weight standard; Lane 2 is the phage ΦPs-1 nucleic acid digested with RNase A; and Lane 3 is the phage ΦPs-1 nucleic acid digested with DNase I.

[0026] Figure 4 : One-step growth curve of bacteriophage ΦPs-1.

[0027] Figure 5 The antibacterial effects of Bacillus amyloliquefaciens NBL-AP73 and Bacillus amyloliquefaciens 44 on Pseudomonas syringae Ps4.

[0028] Figure 6 pH stability of bacteriophage ΦPs-1.

[0029] Figure 7 Thermal stability of bacteriophage ΦPs-1.

[0030] Figure 8 UV stability of bacteriophage ΦPs-1.

[0031] Figure 9 Chloroform stability of bacteriophage ΦPs-1.

[0032] Figure 10 Effects of culture medium on biomass accumulation of Pseudomonas koreanum PK1.

[0033] Figure 11 Effect of culture medium on the lysis rate of bacteriophage ΦPs-1.

[0034] Figure 12 Effects of temperature on biomass accumulation of Pseudomonas koreanum PK1.

[0035] Figure 13 Effect of temperature on the lysis rate of bacteriophage ΦPs-1.

[0036] Figure 14 Effect of ventilation on biomass accumulation of Pseudomonas koreanum PK1.

[0037] Figure 15 Effect of ventilation rate on the lysis rate of bacteriophage ΦPs-1.

[0038] Figure 16 Effect of fermenter rotation speed on biomass accumulation of Pseudomonas koreanum PK1.

[0039] Figure 17 Effect of fermenter rotation speed on the lysis rate of phage ΦPs-1.

[0040] Figure 18 : Antagonistic effect of biocontrol bacteria against Korean Pseudomonas PK1. Detailed Implementation It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein: Nutrient broth medium (NB medium): 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L distilled water, natural pH, sterilized at 121℃ for 20 min.

[0042] NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 15 g agar, 1 L distilled water, pH at rest, sterilized at 121°C for 20 min.

[0043] Semi-solid NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 7 g agar, pH at rest, sterilized at 121°C for 20 min.

[0044] Fermentation medium 1: molasses 30g, fish peptone 15g, dipotassium hydrogen phosphate 7g, potassium dihydrogen phosphate 3g, magnesium sulfate 0.1g, ammonium sulfate 1g, sodium citrate 0.5g, distilled water 1L, pH 6.2-7.2, sterilized at 121℃ for 20 min.

[0045] Fermentation medium 2: 11g glycerol, 0.15g yeast extract, 6.5g disodium hydrogen phosphate, 1.6g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g ferric chloride, 1L distilled water, pH 6.2-7.2, sterilized at 121℃ for 20 min.

[0046] Fermentation medium 3: molasses 30 g, fish peptone 15 g, dipotassium hydrogen phosphate 7 g, potassium dihydrogen phosphate 3 g, magnesium sulfate 0.1 g, ammonium sulfate 1 g, sodium citrate 0.5 g, carvacrol 0.3 g, distilled water 1 L, pH 6.2-7.2, sterilized at 121℃ for 20 min.

[0047] Fermentation medium 4: molasses 30 g, fish peptone 15 g, dipotassium hydrogen phosphate 7 g, potassium dihydrogen phosphate 3 g, magnesium sulfate 0.1 g, ammonium sulfate 1 g, sodium citrate 0.5 g, inulin 1 g, distilled water 1 L, pH 6.2-7.2, sterilized at 121℃ for 20 min.

[0048] Fermentation medium 5: molasses 30 g, fish peptone 15 g, dipotassium hydrogen phosphate 7 g, potassium dihydrogen phosphate 3 g, magnesium sulfate 0.1 g, ammonium sulfate 1 g, sodium citrate 0.5 g, carvacrol 0.3 g, inulin 1 g, distilled water 1 L, pH 6.2-7.2, sterilized at 121℃ for 20 min.

[0049] Physiological saline: 0.9 g sodium chloride, 100 mL distilled water, sterilized at 121℃ for 20 min.

[0050] 2% Phosphotungstic acid (PTA): 0.2 g phosphotungstic acid, 9.8 mL distilled water, pH 7.0, filtered through a 0.22 μm aqueous filter membrane, stored at 4°C protected from light.

[0051] 0.5 M EDTA solution: 18.61 g of EDTA·2Na·2H2O, diluted to 100 mL with ultrapure water, pH 8.0, filtered through a 0.22 μm filter membrane, and stored at 4℃.

[0052] 16% PEG6000 solution: 16 g PEG6000, diluted to 100 mL with ultrapure water, filtered through a 0.22 μm filter membrane, and stored at 4℃.

[0053] 5 M NaCl solution: 29.22 g sodium chloride, diluted to 100 mL with ultrapure water, sterilized at 121℃ for 20 min.

[0054] The biocontrol bacterium NBL-AP73 is Bacillus amyloliquefaciens NBL-AP73, with accession number CCTCC NO: M20241295; it is described in patent CN 118931780B.

[0055] Bacillus amyloliquefaciens 44, with accession number CCTCC NO: M2018564, is described in patent CN 110205273B.

[0056] Pseudomonas syringae Ps4 was preserved and provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Bilan Biotechnology Co., Ltd. TE buffer, DNase I and RNase A, viral RNA / DNA miniprep kit, and GeneRuler 1 kb Plus DNA molecular weight standard were all purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0057] Example 1: Isolation, purification and identification of bacteriophages 1. Isolation and purification of bacteriophages 1.1 Test Methods: Preparation of *Pseudomonas syringae* Ps4 bacterial culture: *Pseudomonas syringae* Ps4 was inoculated into NB medium and cultured at 30℃ with shaking at 180 r / min for 12 h until the logarithmic growth phase (OD50). 590 =0.6-0.7), to prepare Ps4 bacterial culture of Pseudomonas syringae.

[0058] Tomato plant samples infected with bacterial leaf spot were washed, chopped, and added to the logarithmic growth phase (OD50). 590 In a Pseudomonas syringae Ps4 bacterial culture with a concentration of 0.6-0.7 μg / mL, the culture was incubated overnight at 30°C and 180 r / min with shaking. After centrifugation at 8000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm microporous filter to obtain the phage stock solution. The phage was isolated and purified using the double-layer agar plate method. The phage stock solution was diluted 10-fold serially, and 100 μL of each of the 10-fold serial dilutions was taken. 2 10 3 10 4 10 5 Mix the diluted solution with 100 μL of Pseudomonas syringae Ps4 bacterial suspension, add 5 mL of semi-solid NA medium, mix well and immediately pour onto a pre-prepared NA medium plate. Set up 3 replicates for each dilution, and incubate in an inverted 30℃ incubator for 12 h to observe whether plaques are formed.

[0059] After observing plaques, select a single plaque and inoculate it during the logarithmic growth phase (OD). 590 The phage culture was incubated overnight at 30°C and 180 rpm with shaking in a Ps4 culture medium containing 0.6-0.7 μm of bacteria. After centrifugation at 8000 rpm for 5 min, the supernatant was collected, filtered through a 0.22 μm microporous filter, and then cultured in an inverted double-layer plate incubator at 30°C for 12 h using the double-layer plate method. This process was repeated 5 times until the phage plaques were clear and of similar diameter. The plaques at this stage were then inoculated into a logarithmic-phase Ps4 culture medium and cultured at 30°C and 180 rpm with shaking for 3 h. After centrifugation at 8000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm microporous filter. The resulting filtrate was the pure phage culture medium.

[0060] 1.2 Test Results: Using *Pseudomonas syringae* Ps4 as the host bacterium, three bacteriophages were isolated from tomato plants infected with bacterial leaf spot disease and named ΦPs-1, ΦPs-4, and ΦPs-11, respectively. The bacteriophage ΦPs-1, which exhibited the best lysis performance, was selected for further identification.

[0061] 2. Identification of bacteriophages (1) Phage morphology identification Bacteriophage ΦPs-1 can produce clear, well-defined plaques with a diameter of 3–5 mm on the above-mentioned NA medium, without a surrounding halo (see...). Figure 1 ).

[0062] Take 10 μL (10 9 Phage ΦPs-1 culture medium (PFU / mL) was dropped onto a copper grid and allowed to adsorb for 20 min. After absorbing excess liquid with filter paper, 15 μL of 2% phosphotungstic acid (PTA) was added to the copper grid for staining for 5 min. After absorbing residual liquid with filter paper, the morphology of phage ΦPs-1 was observed using a transmission electron microscope after drying at room temperature.

[0063] The results are as follows Figure 2 As shown, the head of bacteriophage ΦPs-1 is a regular polyhedron with a diameter of 50-55 nm, and the tail is a short conical protrusion (14-15 nm in length). The tail lacks a basal plate structure. According to the classification system of the International Committee on Taxonomy of Viruses, bacteriophage ΦPs-1 belongs to the order Tailed Bacteriophages (…). Caudovirales Short-tailed bacteriophages ( Podoviridae ).

[0064] (2) Identification of bacteriophage nucleic acid types To extract nucleic acid from bacteriophage ΦPs-1, the ΦPs-1 culture medium was first filtered through a 0.22 μm microfiltration filter, followed by centrifugation at 35000×g for 2 h. The supernatant was discarded, and the precipitate was dissolved in TE buffer. DNase I and RNase A were then added to the solution, and the mixture was incubated at 37°C for 30 min. 0.5 M EDTA was then added, and the mixture was treated at 65°C for 10 min. Next, 0.4 volumes of 16% PEG6000 solution and 5 M NaCl solution were added, and the mixture was incubated on ice for 30 min. The mixture was then centrifuged at 13000×g for 30 min at 4°C, and the precipitate was collected. Finally, nucleic acid was extracted using a viral RNA / DNA miniprep kit. The extracted ΦPs-1 nucleic acid was digested according to the DNase I and RNase A instructions, and the digestion results were detected by 0.9% agarose gel electrophoresis.

[0065] Agarose gel electrophoresis results showed that the nucleic acid of bacteriophage ΦPs-1 could be degraded by DNase I, but was not affected by RNase A (see...). Figure 3Therefore, it can be determined that its nucleic acid type is DNA, and bacteriophage ΦPs-1 is a DNA virus group.

[0066] The bacteriophage ΦPs-1 was biopreserved using a patented procedure. The preservation information is as follows: The culture requested for preservation is: Pseudomonas syringae phage ΦPs-1 ( Pseudomonas syringae phageΦPs-1); Accession number: CCTCC NO: M 20252347; Preservation date: October 27, 2025.

[0067] Example 2: Performance evaluation of bacteriophage ΦPs-1 1. Phage titer and optimal multiple of infection (MOI) assessment: The optimal multiplicity of infection (MOI) is the ratio of the number of infected cells to the total number of cells.

[0068] Pseudomonas syringae Ps4 was cultured to the logarithmic growth phase (OD). 590 =0.6-0.7), with multiplicity of infection (MOI) of 0.001, 0.01, 0.1, 1, 10, and 100, phage ΦPs-1 culture medium was added. After standing at room temperature for 20 min, the culture was incubated at 30℃ and 180 rpm for 2 h with shaking. After centrifugation at 8000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm microporous filter. The filtrate was serially diluted, and 100 μL of the diluted solution was mixed with 100 μL of *Pseudomonas syringae* Ps4 culture medium. 5 mL of semi-solid NA medium was added, mixed well, and immediately poured onto a pre-prepared NA medium plate. The plate was incubated upside down at 30℃ for 12 h. The number of phage plaques was counted. The experiment was repeated 3 times. Phage titer calculation method: Phage titer (PFU / mL) = Average number of phage plaques × Dilution factor × 10 The phage titer results corresponding to different infection multiples are shown in Table 1.

[0069] Table 1: Results of Multiplicity of Infection (Multiple Infection Count) assay for phage ΦPs-1 The results showed that when the multiplicity of infection of phage ΦPs-1 was 1, the highest phage titer was (2.93±0.19)×10⁻⁶. 11 PFU / mL, meaning the optimal multiplicity of infection for phage ΦPs-1 is 1.

[0070] 2. Examination of the one-step growth curve of bacteriophage: Pseudomonas syringae Ps4 was cultured to the logarithmic growth phase (OD).590 =0.6-0.7), add bacteriophage ΦPs-1 with MOI=1, incubate at room temperature for 20 min, then centrifuge at 8000 rpm for 5 min at 4℃, and discard the supernatant. Wash the precipitate twice with NB medium, discard the supernatant, add 30 mL of NB medium, mix thoroughly, and immediately place in a 30℃ constant temperature shaker for oscillation at 180 r / min. Take 500 μL samples every 15 min, centrifuge the samples at 8000 rpm for 5 min, filter the supernatant through a 0.22 μm microporous filter, serially dilute the filtrate, and determine the bacteriophage titer using the double-layer plate method. Repeat the experiment 3 times. Plot the one-step growth curve of bacteriophage ΦPs-1 with the culture time as the x-axis and the logarithm of the bacteriophage titer as the y-axis to analyze the latency period, outbreak period, and lysis rate of the bacteriophage.

[0071] The results are as follows Figure 4 As shown, the incubation period of bacteriophage ΦPs-1 is about 30 min, the outbreak period is about 45 min, and the lysis rate is 190.06 PFU / cell (the calculation method of lysis rate is referenced in: Li Jinmin, Gao Xuna, Sang Ruixin, et al. Isolation and bactericidal effect evaluation of a strain of avian Escherichia coli bacteriophage [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(02):783-790.). This bacteriophage has the advantages of short incubation period, short outbreak period and large lysis rate.

[0072] 3. Phage host profile determination: The lytic activity of bacteriophage ΦPs-1 against seven tested bacterial strains was determined, including five Pseudomonas strains: *Pseudomonas syringae* Ps4, *Pseudomonas syringae* BIM B-266, *Pseudomonas syringae* BIM B-267, *Pseudomonas syringae* BIM B-268, and *Pseudomonas amyloliquefaciens* PK1, and two biocontrol Bacillus strains (*Bacillus amyloliquefaciens* NBL-AP73 and *Bacillus amyloliquefaciens* 44). *Pseudomonas amyloliquefaciens* PK1 was purchased from the China Agricultural Microbial Culture Collection Center (strain number ACCC60112), while the other strains were provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Bilan Biotechnology Co., Ltd. *Pseudomonas amyloliquefaciens* PK1 was non-pathogenic. *Bacillus amyloliquefaciens* NBL-AP73 and *Bacillus amyloliquefaciens* 44 showed significant inhibitory effects against *Pseudomonas syringae* Ps4 (see [link to study]). Figure 5 ).

[0073] Single colonies of the seven bacterial strains were picked and inoculated into 50 mL of NB medium. The culture was then incubated overnight at 30℃ with shaking at 180 r / min. 100 μL of the seven bacterial cultures were spread evenly onto NA medium plates using a spreader. 3 μL of phage ΦPs-1 culture was added to each plate, while 3 μL of NB medium was added to the control group. Three parallel addition points were set for each plate. After air drying, the plates were incubated upside down in a 30℃ incubator for 12 h to observe whether phage plaques were formed. The results are shown in Table 2.

[0074] Table 2: Host profile determination results of bacteriophage ΦPs-1 Note: "+" indicates the presence of plaques; "-" indicates the absence of plaques.

[0075] Post-culturing observations revealed that, in addition to Pseudomonas syringae Ps4, phage ΦPs-1 could also successfully lyse Pseudomonas syringae BIM B-266 and BIM B-267 and Pseudomonas koreanum PK1 (this strain is non-pathogenic); however, it had no lysing effect on Pseudomonas syringae BIMB-268 and two strains of Bacillus amyloliquefaciens (NBL-AP73 and 44).

[0076] The above results indicate that the host range of bacteriophage ΦPs-1 is concentrated in the genus *Pseudomonas*, exhibiting a broad lytic spectrum against *Pseudomonas* strains, but showing no cross-genus lytic ability against the two *Bacillus* strains. Bacteriophage ΦPs-1 shows strong lytic activity against *Pseudomonas koreanum* PK1, and since PK1 is non-pathogenic, it can be used for subsequent culture of bacteriophage ΦPs-1. *Bacillus amyloliquefaciens* NBL-AP73 and 44 exhibit excellent antibacterial activity against *Pseudomonas syringae* Ps4. Specifically, the inhibition zone diameter of *Bacillus amyloliquefaciens* NBL-AP73 against *Pseudomonas syringae* Ps4 was 3.46 ± 0.14 cm; the inhibition zone diameter of *Bacillus amyloliquefaciens* 44 against *Pseudomonas syringae* Ps4 was 3.19 ± 0.12 cm; and both were not lysed by bacteriophage ΦPs-1, thus synergistically inhibiting *Pseudomonas syringae* Ps4 with bacteriophage ΦPs-1.

[0077] 4. Phage pH stability study: The pH of NB medium was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 using 1 mol / L HCl and NaOH, respectively. Phage ΦPs-1 culture was then added to NB medium at different pH values, resulting in a final phage concentration of 10. 10 PFU / mL. After incubation at 30℃ for 1 h, take 200 μL of phage culture, serially dilute it, and determine the phage titer using the double-layer agar plate method, with 3 replicates for each pH.

[0078] The results are as follows Figure 6As shown, the titer of bacteriophage ΦPs-1 remained stable at pH 5-9. At pH 4, 10, and 11, the titer decreased significantly, and at pH ≤ 3 or pH ≥ 12, the titer was 0, indicating complete inactivation of the bacteriophage. These results demonstrate that ΦPs-1 has a wide pH adaptation range (pH = 5-9) and exhibits good titer stability within this range.

[0079] 5. Study on the thermal stability of bacteriophages: Add 2 mL of phage ΦPs-1 culture medium to 40 mL of NB medium to achieve a final phage concentration of 10. 10 PFU / mL was incubated in water baths at 4℃, 30℃, 40℃, and 50℃, with samples taken every 30 min (200 μL). After serial dilution, the phage titer was determined using the double-layer agar plate method to evaluate the thermal stability of phage ΦPs-1 at different temperatures. Each temperature was repeated three times.

[0080] The results are as follows Figure 7 As shown, the titer of bacteriophage ΦPs-1 remained stable within the temperature range of 4-30℃. At 40℃, bacteriophage ΦPs-1 was relatively stable, with the titer decreasing slowly over time. At 50℃, bacteriophage ΦPs-1 was completely inactivated after 2 hours.

[0081] 6. Study on the ultraviolet stability of bacteriophages: Take 5 mL of phage ΦPs-1 (9.78 × 10⁻⁶) 9 PFU / mL phage culture medium was placed in a 90 mm sterile petri dish and placed in a clean bench. The petri dish was opened and placed under a 254 nm UV lamp at a distance of 20 cm for irradiation. After irradiation for 0, 3, 5, 15, 30, 45, and 60 min, 200 μL of phage culture medium was taken each time and incubated in the dark for 30 min. After serial dilution, the phage ΦPs-1 titer was determined by the double-layer agar plate method. The experiment was set up in 3 replicates, and the curve of phage titer affected by ultraviolet light was plotted.

[0082] The results are as follows Figure 8 As shown, bacteriophage ΦPs-1 exhibits a certain degree of tolerance to ultraviolet radiation; however, the titer of bacteriophage ΦPs-1 decreases with increasing ultraviolet irradiation time, decreasing from 9.78 × 10⁻⁶ after 1 hour of ultraviolet irradiation. 9 PFU / mL decreased to 8.48 × 10⁻⁶ 6 PFU / mL.

[0083] 7. Chloroform stability study of bacteriophage: Take 5 mL of phage ΦPs-1 culture medium (10 10PFU / mL), and different volumes of chloroform were added to make the final chloroform concentrations 5%, 8% and 10% (v / v), respectively. Each concentration was repeated 3 times. After thorough mixing, 200 μL samples were taken at 0, 30, 60, 90 and 120 min respectively. After serial dilution, the phage titer was determined by the double-layer agar plate method, and the curve of the effect of chloroform on the phage titer was plotted.

[0084] The results are as follows Figure 9 As shown, phage ΦPs-1 exhibits high tolerance to chloroform, and its titer remained stable after treatment with 5%, 8%, and 10% chloroform for 2 h.

[0085] 8. Investigation into bacteriophage preservation methods: The culture medium of phage ΦPs-1 was filtered through a 0.22 μm microporous filter, and the phage titer was determined by the double-layer agar plate method. Then, it was stored under low-temperature cryopreservation, freeze-drying, and refrigeration at 4°C. The storage methods are as follows.

[0086] Low-temperature cryopreservation: Take 500 μL of phage ΦPs-1 culture medium into a sterile cryotube, add 500 μL of 20% glycerol (final glycerol concentration is 10%), mix well, and then place at 4℃ for 15-20 min, and then freeze to -20℃ and -70℃ for storage.

[0087] Freeze-drying preservation: Phage ΦPs-1 culture medium was mixed with a cryoprotectant (sterile skim milk) to a final concentration of 10%. 200 μL of the mixture was then transferred to ampoules. Freeze-drying was performed in two stages: first, at -55°C and a vacuum of 8 Pa for 5 hours; second, at room temperature and a vacuum of 8 Pa for 2 hours. Afterward, the ampoules were sealed with an alcohol torch and stored at 4°C.

[0088] Store at 4°C: Add 1 mL of chloroform to 99 mL of phage ΦPs-1 culture medium, mix well, and store in a refrigerator at 4°C.

[0089] Each sample was serially diluted with sterile water, and the phage titer was determined using the double-layer agar plate method. The samples were incubated upside down in a 30℃ incubator for 12 h. Each sample was repeated three times. The number of phage plaques was counted, and the phage titer (PFU / mL) was calculated. The results are shown in Table 3.

[0090] Table 3: Titer changes of bacteriophage ΦPs-1 after long-term storage (1 year) The results showed that the titer of phage ΦPs-1 decreased after one year of storage under different preservation methods compared to the initial titer. The best results were achieved by cryopreservation at -70℃ and freeze-drying. After cryopreservation at -70℃, the phage titer was (2.02±0.32)×10⁻⁶. 11 PFU / mL; after freeze-drying, the titer was (1.14±0.26)×10⁻⁶. 11 PFU / mL. The titer decreased significantly under both methods, and the phage activity remained high. After one year of cryopreservation at -20℃, the phage titer decreased to (2.16±0.13)×10⁻⁶. 10 The PFU / mL titer decreased significantly compared to the initial titer, indicating a substantial loss of phage activity. Storage at 4°C showed the worst effect, with the titer dropping to only (7.45±1.32)×10⁻⁶ after one year. 9 PFU / mL was the most significant titer decrease among all preservation methods, indicating severely impaired phage activity.

[0091] In summary, freezing at -70℃ and freeze-drying are the two methods that best preserve the activity of phage ΦPs-1 and are the optimal methods for preserving phage ΦPs-1.

[0092] Example 3: Optimization of culture conditions for bacteriophage ΦPs-1 1. Culture medium optimization: (1) Selection of basal culture medium: Preparation of seed culture of Pseudomonas Koreanis PK1: Pseudomonas Koreanis PK1 was inoculated into NB medium and cultured at 30℃ and 180r / min for 12 h with shaking to obtain the seed culture.

[0093] In this study, *Pseudomonas kwangsiensis* PK1 seed culture was inoculated into three different culture media (fermentation medium 1, fermentation medium 2, and NB medium) at an inoculation rate of 10% (v / v). OD was measured at 1 mL per 30 min. 590 Dynamically monitor bacterial growth until the bacterial solution OD 590 When the value reaches 0.6-0.7, subsequent experiments will be conducted.

[0094] The inoculation titers for the three different culture media were (1.83±0.32)×10⁻⁶ at a 1% (v / v) inoculation rate. 10 Gently mix PFU / mL of phage ΦPs-1 culture medium, incubate at room temperature for 20 min, then incubate at 30℃ with shaking at 180 r / min. Take a 1 mL sample every 30 min to measure OD. 590The growth of the bacteria was dynamically monitored, and the titer was determined using the double-layer agar plate method with Pseudomonas syringae Ps4 as the host bacterium. The lysis effect of bacteriophage ΦPs-1 on Pseudomonas koreanum PK1 was also studied. The experiment was repeated in three replicates.

[0095] The effects of different culture media on the growth of Pseudomonas Koreanis PK1, such as Figure 10 As shown, *Pseudomonas koreanum* PK1 reached the logarithmic growth phase (OD50) in three different culture media. 590 The time required to reach the logarithmic phase (=0.6-0.7) varied significantly; the shortest time to reach the logarithmic phase was 2 h in fermentation medium 1, 2.5 h in NB medium, and the longest time was 2.75 h in fermentation medium 2.

[0096] The effects of different culture media on the titer of phage ΦPs-1 are shown in Table 4.

[0097] Table 4: Effect of culture medium on phage ΦPs-1 titer Different culture media significantly affected the titer of bacteriophage ΦPs-1. The highest titer of bacteriophage ΦPs-1 was observed when cultured in fermentation medium 1, reaching (4.71 ± 0.31) × 10⁻⁶. 11 The PFU / mL was significantly higher than that of the other two culture media.

[0098] The lytic effects of bacteriophage ΦPs-1 on Pseudomonas Koreanis PK1 under different culture media are as follows: Figure 11 As shown, the OD values ​​of Pseudomonas Koreanis PK1 bacterial suspensions cultured in different culture media increased after the addition of bacteriophage ΦPs-1. 590 All showed a decrease, when OD 590 When the concentration dropped to 0.2 and the bacterial culture became clear, it indicated that *Pseudomonas kwangsiensis* PK1 was completely lysed by bacteriophage ΦPs-1. Comparatively, in fermentation medium 1, complete lysis was achieved in just 2 hours after the addition of bacteriophage ΦPs-1; while in fermentation medium 2 and NB medium, it took 2.5 hours to achieve the same lysis effect. Figure 11 ).

[0099] In summary, in fermentation medium 1, phage ΦPs-1 exhibited the best lytic activity and the highest titer against Pseudomonas koreanum PK1, therefore fermentation medium 1 was selected as the basal medium.

[0100] (2) Optimization of basal culture medium: Based on fermentation medium 1, carvacrol (0.3 g / L) and inulin (1 g / L) were added separately to form fermentation medium 3 and fermentation medium 4, respectively; simultaneously, carvacrol (0.3 g / L) and inulin (1 g / L) were added to form fermentation medium 5. 10% (v / v) of *Pseudomonas kwangsiensis* PK1 seed culture was added to each medium, and cell growth was dynamically monitored until the OD of the bacterial culture was reached. 590 After reaching a titer of 0.6-0.7, add 1% (v / v) at a titer of (1.67±0.21)×10. 10 Phage ΦPs-1 culture medium of PFU / mL was gently mixed, incubated at room temperature for 20 min, and then cultured at 30℃ with shaking at 180 r / min. After 2 h, a 200 μL sample was taken, and the titer of phage ΦPs-1 was determined using the double-layer agar plate method. The experiment was repeated in triplicate. The results are shown in Table 5.

[0101] Table 5: Effects of four culture media on the titer of bacteriophage ΦPs-1 Compared to fermentation medium 1, the titer of bacteriophage ΦPs-1 was increased when using fermentation media 3, 4, and 5. The highest titer of bacteriophage ΦPs-1 was achieved using fermentation medium 5, which simultaneously added carvacrol and inulin, reaching (9.95±0.32)×10⁻⁶. 11 PFU / mL; Compared with fermentation medium 3 with carvacrol alone and fermentation medium 4 with inulin alone, the combination of carvacrol and inulin can synergistically increase the titer of cultured bacteriophages.

[0102] 2. Optimization of culture temperature: A 10% inoculum of *Pseudomonas kwangsiensis* PK1 seed culture was inoculated into fermentation medium 5 and cultured on a constant-temperature shaker at 180 r / min. Five temperature gradients were set: 20℃, 24℃, 28℃, 32℃, and 36℃, with three replicates for each temperature. OD was measured using 1 mL samples every 30 min. 590 Dynamically monitor bacterial growth until the bacterial solution OD 590 When the titer reaches 0.6-0.7, further experiments are conducted. A titer of (1.78±0.19)×10⁻⁶ is added at a 1% inoculum. 10 PFU / mL phage ΦPs-1 culture medium was gently mixed and incubated at room temperature for 20 min. The mixture was then transferred to a shaker and cultured at 180 r / min at different temperatures as required for the experiment. A 1 mL sample was taken every 30 min, and the OD was measured using a UV spectrophotometer. 590 The dynamic changes in bacterial growth and lysis throughout the entire process were recorded.

[0103] The effect of culture temperature on the growth of Pseudomonas Koreanis PK1 Figure 12 As shown, temperature significantly affects the growth of *Pseudomonas kwangsiensis* PK1. Specifically, *Pseudomonas kwangsiensis* PK1 showed the most active growth at 28℃ and 32℃, with rapid biomass accumulation and high OD values ​​in the bacterial culture. 590 The growth rate reached 0.6 within 2 hours; in contrast, the growth rate of the strain was slower at other temperatures; indicating that 28-32℃ is the optimal temperature range for the growth of Pseudomonas koreanum PK1.

[0104] The effect of temperature on the lysis efficiency of bacteriophage ΦPs-1 is as follows: Figure 13 As shown, the lysis efficiency of bacteriophage ΦPs-1 against Pseudomonas Koreanis PK1 varied significantly at different temperatures. At 32℃, bacteriophage ΦPs-1 exhibited the fastest lysis rate, with the highest OD value of the bacterial culture. 590 The OD of the bacterial culture decreased to 0.2 within 2 hours; under conditions of 28℃ and 36℃, the OD of the bacterial culture decreased. 590 The OD value decreased to 0.2 within 2.5 h; however, the lysis rate of phage ΦPs-1 was significantly reduced at 24℃, and phage ΦPs-1 failed to completely lyse Pseudomonas kwangsiensis PK1 after 2.5 h. 590 It is still higher than 0.2.

[0105] In summary, 32℃ is the optimal temperature for the growth of Pseudomonas Koreanis PK1 and the lysis of bacteriophage ΦPs-1. Therefore, 32℃ is selected as the optimal temperature parameter.

[0106] 3. Optimization of ventilation conditions: A 10 L automated fermenter (working volume 6 L) was used, with fermentation medium 5 as the fermentation medium. *Pseudomonas kwangsiensis* PK1 seed culture was inoculated into the fermenter at a 5% inoculum. The temperature was set at 32℃, and the stirring speed at 160 r / min. Aeration rates of 0.5, 1.0, and 1.5 vvm were set, with three replicates for each aeration rate. 1 mL samples were taken every 30 min to determine OD. 590 Dynamically monitor bacterial growth until the bacterial solution OD 590 When the titer reaches 0.6-0.7, add a titer of (1.61±0.41)×10 at a 1% inoculum. 10 The phage ΦPs-1 culture medium containing PFU / mL was cultured and incubated for an extended period. A 1 mL sample was taken every 30 min, and the OD was measured using a UV spectrophotometer. 590 The dynamic changes in bacterial growth and lysis throughout the entire process were recorded.

[0107] The results are as follows Figure 14 and Figure 15As shown in the figure, the results indicate that the aeration rate significantly affects the growth status of Pseudomonas Koreanis PK1. At aeration rates of 1.0 and 1.5 vvm, Pseudomonas Koreanis PK1 exhibits active growth and rapid biomass accumulation. There are no significant differences in the lysis rate and final lysis degree of bacteriophage ΦPs-1 under different aeration rates.

[0108] Based on a comprehensive evaluation of experimental data and economic benefits, 1.0 vvm was ultimately selected as the optimal ventilation rate.

[0109] 4. Optimized stirring speed: A 10 L automated fermenter was used, with fermentation medium 5 as the fermentation medium. A 5% inoculum of *Pseudomonas kwangsiensis* PK1 seed culture was inoculated into the fermenter. The temperature was set at 32℃, the aeration rate at 1.0 vvm, and the stirring speeds at 120, 160, and 200 r / min, with three replicates for each speed. OD was measured using 1 mL samples every 30 min. 590 Dynamically monitor bacterial growth until the bacterial solution OD 590 When the titer reaches 0.6-0.7, add a titer of (2.12±0.11)×10 at a 1% inoculum. 10 The phage ΦPs-1 culture medium containing PFU / mL was cultured and incubated for an extended period. A 1 mL sample was taken every 30 min, and the OD was measured using a UV spectrophotometer. 590 The dynamic changes in bacterial growth and lysis throughout the entire process were recorded.

[0110] The effect of stirring speed on the growth of Pseudomonas koreanum PK1 is as follows: Figure 16 As shown, stirring speed significantly affected the time for *Pseudomonas kwangsiensis* PK1 to enter the logarithmic phase and the biomass accumulation rate. At a stirring speed of 120 r / min, *Pseudomonas kwangsiensis* PK1 required 3 h to enter the logarithmic phase; however, increasing the stirring speed to 160 and 200 r / min significantly increased the biomass accumulation rate, with OD at 2 h. 590 Reaching 0.6 ( Figure 16 However, when the stirring speed is 200 r / min, a large amount of foam is generated in the fermentation broth, which causes the foam to adhere to the inner wall of the fermenter and the stirring blades, resulting in biomass loss and increasing the risk of contamination.

[0111] The effect of stirring speed on the lysis efficiency of bacteriophage ΦPs-1 is as follows: Figure 17 As shown, different stirring speeds significantly affect the lysis efficiency of phage ΦPs-1 against Pseudomonas koreanum PK1. Phage ΦPs-1 lyses fastest at 160 r / min, and the required lysis time (2 h) is much lower than that required at 120 r / min and 200 r / min (>2.5 h).

[0112] Experiments showed that at a stirring speed of 160 r / min, *Pseudomonas Koreanis* PK1 grew rapidly with minimal biomass loss, and phage ΦPs-1 exhibited the highest lysis efficiency. Considering all factors, 160 r / min was selected as the optimal stirring speed.

[0113] In summary, the optimal culture conditions for bacteriophage ΦPs-1 are as follows: A 10 L automated fermenter was used with fermentation medium 5, and the inoculum of *Pseudomonas kwangsiensis* PK1 seed culture was 5%. The temperature was set at 32℃, the aeration rate at 1.0 vvm, and the stirring speed at 160 r / min. OD was measured using 1 mL samples every 30 min. 590 Dynamically monitor bacterial growth until the bacterial solution OD 590 When the concentration reaches 0.6-0.7, add phage ΦPs-1 culture medium at a 1% inoculum rate. The concentration of phage ΦPs-1 is (1.54±0.24)×10⁻⁶. 10 PFU / mL, continue culturing until OD 590 The concentration was reduced to 0.2 to obtain the culture medium of phage ΦPs-1.

[0114] The culture medium was serially diluted, and the titer of the phage ΦPs-1 culture medium prepared under optimal culture conditions was determined using the double-layer agar plate method. The result was (4.81±1.82)×10⁻⁶. 12 PFU / mL.

[0115] Example 4: Preparation of phage formulation Large-scale culture of bacteriophage ΦPs-1 was carried out under the optimal culture conditions determined in Example 3 to obtain bacteriophage ΦPs-1 culture medium. Details are as follows: Pseudomonas Koreanis PK1 was inoculated into NB medium and cultured at 30℃ with shaking at 180 r / min for 12 h to obtain the seed culture. A 10 L automated fermenter was used with fermentation medium 5, the seed culture inoculation volume was 5%, the temperature was set at 32℃, the aeration rate was 1.0 vvm, and the stirring speed was 160 r / min. OD was measured by taking 1 mL samples every 30 min. 590 Dynamically monitor bacterial growth until the bacterial solution OD 590 When the concentration reaches 0.6-0.7, add phage ΦPs-1 culture medium at a 1% inoculum rate. The concentration of phage ΦPs-1 is (1.54±0.24)×10⁻⁶. 10 PFU / mL, continue culturing until OD 590 The titer was reduced to 0.2, yielding phage ΦPs-1 culture medium with a phage titer of (4.81±1.82)×10⁻⁶. 12 PFU / mL.

[0116] Then, the phage ΦPs-1 culture medium was centrifuged at high speed (8000 rpm, 5 min), the precipitate was discarded, and the supernatant was collected. After adding 1% volume of chloroform to the supernatant, the phage preparation was prepared and stored in a refrigerator at 4℃ for later use.

[0117] Example 5: Preparation of a composition for controlling bacterial leaf spot disease in tomatoes 1. Compatibility study of biocontrol bacteria with Pseudomonas aeruginosa: Both *Bacillus amyloliquefaciens* NBL-AP73 and *Bacillus amyloliquefaciens* 44 exhibit excellent antibacterial activity against *Pseudomonas syringae* Ps4 and are not lysed by bacteriophage ΦPs-1. Therefore, a combination of biocontrol bacteria and bacteriophage ΦPs-1 was considered. *Bacillus amyloliquefaciens* NBL-AP73 showed better antibacterial activity against *Pseudomonas syringae* Ps4 than *Bacillus amyloliquefaciens* 44; therefore, a combination of *Bacillus amyloliquefaciens* NBL-AP73 and bacteriophage ΦPs-1 was chosen.

[0118] The phage ΦPs-1 formulation was prepared using *Pseudomonas aeruginosa* PK1 as the host bacterium. To further investigate the feasibility of combining *Bacillus amyloliquefaciens* NBL-AP73 with phage ΦPs-1, the compatibility between *Bacillus amyloliquefaciens* NBL-AP73 and *Pseudomonas aeruginosa* PK1 was determined, as detailed below: The antagonistic effect between biocontrol bacteria NBL-AP73 and *Pseudomonas kwangsiensis* PK1 was verified using the streak method. Single colonies of both biocontrol bacteria NBL-AP73 and *Pseudomonas kwangsiensis* PK1 were inoculated into NB medium and cultured overnight at 30°C and 180 rpm with shaking. Four parallel horizontal streaks of *Pseudomonas kwangsiensis* PK1 were then drawn on NA medium, with biocontrol bacteria NBL-AP73 inoculated three times at equal intervals along the top horizontal line. The medium was then incubated upside down at 30°C. After 24 hours, the growth at the intersection of the streaks was observed. Normal growth at the intersection indicated good compatibility between the strains; no growth at the intersection indicated antagonistic activity.

[0119] The results are as follows Figure 18 As shown, there is no antagonistic effect between Korean Pseudomonas PK1 and biocontrol bacterium NBL-AP73.

[0120] 2. Preparation of the composition: The biocontrol bacterium NBL-AP73 was inoculated into NB medium and cultured at 37℃ with shaking at 180 r / min for 12 h. The viable count in the bacterial culture was then adjusted to 102. 8 The bacterial suspension of biocontrol bacterium NBL-AP73 was prepared by using cfu / mL.

[0121] The bacterial suspension of biocontrol bacterium NBL-AP73 was diluted 50 times with sterile water to obtain the diluted biocontrol bacterial suspension; the phage preparation prepared in Example 4 was diluted 25 times with sterile water to obtain the diluted phage preparation.

[0122] The diluted biocontrol bacterial solution was then mixed with the diluted bacteriophage preparation at a volume ratio of 1:1 to prepare the composition. The composition contained 2% bacteriophage preparation, and the viable count of the biocontrol bacteria was 10⁻⁶. 6 cfu / mL.

[0123] Experimental Example 1: Evaluation of the Application Effect of Bacteriophage Preparations 1. Test materials: 1% phage preparation: Dilute the phage preparation prepared in Example 4 100 times with sterile water.

[0124] 2% phage preparation: Dilute the phage preparation prepared in Example 4 with sterile water 50 times.

[0125] 2. Test methods: Tomato plants with uniform growth at the four-leaf-one-heart stage and free from pests and diseases were selected and three treatments were established. Treatment 1 (control group): Inoculated with *Pseudomonas syringae* Ps4 bacterial solution, followed by foliar spraying and root irrigation with a 100-fold diluted fermentation medium 5 (containing 1% chloroform). Treatment 2: Inoculated with *Pseudomonas syringae* Ps4 bacterial solution, followed by foliar spraying and root irrigation with a 1% bacteriophage preparation. Treatment 3: Inoculated with *Pseudomonas syringae* Ps4 bacterial solution, followed by foliar spraying and root irrigation with a 2% bacteriophage preparation.

[0126] *Pseudomonas syringae* Ps4 bacterial suspension was inoculated onto the leaves of all treated tomato plants using the wound inoculation method. After inoculation, treatments 2 and 3 were treated with foliar spraying and root irrigation with 1% and 2% bacteriophage preparations, respectively; treatment 1 was treated with foliar spraying and root irrigation with fermentation medium 5 diluted 100 times (containing 1% chloroform). For all treatments, 5 mL was sprayed on the leaves and 20 mL was irrigated the roots per tomato plant, for a total of 25 mL per plant. Treatments were administered once at 0, 12, and 24 days, for a total of three treatments. Each treatment contained 10 tomato plants, and each treatment was replicated three times. The disease index was calculated every 12 days, and the control efficacy was assessed to evaluate the control effect of the bacteriophage preparations against bacterial leaf spot disease in tomatoes.

[0127] The disease classification standards are as follows (classification standards reference: Wang Lin, Zhang Hui. Screening of chemical control agents for bacterial leaf spot disease of pepper in Heilongjiang Province [J]. China Forestry and Special Products, 2025, (02): 9-12.): Grade 0: No obvious lesions on the leaves; Grade 1: Leaf lesions cover less than 25% of the total leaf area; Grade 2: Leaf lesions cover 26%-50% of the leaf area; Grade 3: Leaf spots cover 51%-75% of the leaf area; Level 4: Leaf lesions cover more than 75% of the leaf area.

[0128] Disease index = [Σ(disease grade value × number of plants at that disease grade) / (highest disease grade value × number of plants surveyed)] × 100.

[0129] Prevention and control effect = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.

[0130] 3. Test Results: The results are shown in Table 6.

[0131] Table 6: Control efficacy of bacteriophage preparations against bacterial leaf spot disease in tomato The results showed that the bacteriophage preparation effectively inhibited *Pseudomonas syringae*. With increasing treatment frequency, the control effect of the bacteriophage preparation against tomato bacterial leaf spot continuously increased. Different concentrations of the bacteriophage preparation showed significant differences in their inhibitory effects on *Pseudomonas syringae* Ps4, with higher concentrations (2%) exhibiting stronger inhibitory effects. The 2% bacteriophage preparation, after three applications, showed the highest control efficacy against tomato bacterial leaf spot, reaching 54.82%.

[0132] Experiment Example 2: Field Trial of Synergistic Control of Bacterial Leaf Spot Disease in Tomatoes by Bacteriophages and Biocontrol Bacteria 1. Test materials: Diluted biocontrol agent: The preparation method of the biocontrol agent NBL-AP73 bacterial solution is the same as in Example 5, diluted with sterile water to a viable count of 10. 6 cfu / mL.

[0133] 2% phage preparation: Dilute the phage preparation prepared in Example 4 with sterile water 50 times.

[0134] The composition prepared in Example 5 contains 2% bacteriophage preparation and has a viable count of 10 for the biocontrol bacterium NBL-AP73. 6 cfu / mL.

[0135] 2. Test methods: Tomato plants with uniform growth at the four-leaf-one-heart stage and free from pests and diseases were selected, and four treatments were established. Treatment 1: Inoculated with *Pseudomonas syringae* Ps4, and foliar sprayed and root-drenched with an equal volume mixture of 50-fold diluted fermentation medium 5 (containing 1% chloroform) and 50-fold diluted NB medium. Treatment 2: After inoculation with *Pseudomonas syringae* Ps4, foliar sprayed and root-drenched with a diluted biocontrol agent. Treatment 3: After inoculation with *Pseudomonas syringae* Ps4, foliar sprayed and root-drenched with a 2% bacteriophage preparation. Treatment 4: After inoculation with *Pseudomonas syringae* Ps4, foliar sprayed and root-drenched with the composition prepared in Example 5.

[0136] *Pseudomonas syringae* Ps4 was inoculated onto the leaves of all treated tomatoes using the wound inoculation method. The treatment amounts were: 5 mL for foliar spraying and 20 mL for root irrigation per plant, for a total of 25 mL per plant. Treatments were administered once at 0, 12, and 24 days, for a total of three treatments. The disease index was calculated 36 days after inoculation, and the control efficacy was assessed to evaluate the effectiveness of different treatments in controlling bacterial leaf spot disease of tomatoes.

[0137] The disease grading standards, disease index, and control effectiveness calculations are the same as in Experiment 1.

[0138] The Q-value was calculated using the Jin Zhengjun method to evaluate the interaction between the biocontrol bacterium NBL-AP73 and the bacteriophage ΦPs-1.

[0139] Q = Ea + b / (Ea + Eb - Ea × Eb) Where Ea+b represents the inhibition rate of combined drug use; Ea and Eb represent the inhibition rates of drug A and drug B used alone, respectively.

[0140] In the formula, the numerator represents the "measured merging effect", the denominator is the "expected merging effect", and Q is the ratio of the two. Q < 0.85 indicates antagonism, 0.85 ≤ Q < 1.15 indicates addition, and Q ≥ 1.15 indicates synergy.

[0141] 3. Test Results: The results are shown in Table 7.

[0142] Table 7: Control effects of different treatments on bacterial leaf spot disease of tomato at 36 days The results showed that biocontrol bacteria, bacteriophage preparations, and the combination of the two were all effective in controlling bacterial leaf spot disease of tomato. Among them, the control efficacy of biocontrol bacteria + bacteriophage preparations was significantly better than that of either alone, and the Q value was greater than 1.15, which confirmed that bacteriophages and biocontrol bacteria had a synergistic control effect on bacterial leaf spot disease of tomato under field conditions.

[0143] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of Pseudomonas syringae phage ΦPs-1, with the accession number CCTCC NO: M 20252347.

2. A phage preparation, characterized in that, The phage preparation uses the eugenol phage ΦPs-1 described in claim 1 as its active ingredient.

3. The phage preparation according to claim 2, characterized in that, The bacteriophage preparation is prepared by the following method: Pseudomonas Koreanis PK1 was inoculated into fermentation medium and fermented until the OD of the bacterial culture reached a certain level. 590 When the concentration reaches 0.6-0.7, add *Pseudomonas syringae* phage ΦPs-1 and continue culturing until OD reaches 0.6-0.

7. 590 The concentration was reduced to 0.2 to obtain the phage culture medium; Centrifuge the phage culture medium, discard the precipitate, collect the supernatant and add chloroform to prepare the phage preparation.

4. The phage preparation according to claim 3, characterized in that, The fermentation medium consisted of: 30g molasses, 15g fish peptone, 7g dipotassium hydrogen phosphate, 3g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 1g ammonium sulfate, 0.5g sodium citrate, 0.3g carvacrol, 1g inulin, and 1L distilled water, with a pH of 6.2-7.2; sterilized at 121℃ for 20 min.

5. The phage preparation according to claim 3, characterized in that, The fermentation conditions were: a culture temperature of 32℃, a stirring speed of 160 r / min, and an aeration rate of 1.0 vvm.

6. The use of the silymarinus phage ΦPs-1 according to claim 1 or the phage preparation according to any one of claims 2-5 in the prevention and control of plant diseases caused by silymarinus.

7. The application according to claim 6, characterized in that, The plant disease in question is bacterial leaf spot of tomato.

8. A composition for controlling bacterial leaf spot disease of tomatoes, characterized in that, The composition is prepared by combining the phage preparation according to any one of claims 2-5 and the bacterial suspension of biocontrol bacterium NBL-AP73 with accession number CCTCC NO: M20241295.

9. The composition according to claim 8, characterized in that, The composition is prepared by mixing a phage preparation diluted 25 times with a bacterial suspension of biocontrol bacterium NBL-AP73 diluted 50 times at a volume ratio of 1:

1.

10. A method for controlling bacterial leaf spot disease of tomatoes, characterized in that, Includes the following steps: Apply the bacteriophage preparation according to any one of claims 2-5, or the composition for controlling bacterial leaf spot of tomatoes according to claim 8 or 9, to tomato plants at risk of infection by *Pseudomonas syringae*.

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