Watermelon acidophilic bacteriophage and applications thereof

Abstract

The application discloses watermelon acidovorax bacteriophages and application thereof, and relates to the field of biotechnology. The application provides watermelon acidovorax bacteriophages (Acidovorax citrulli phage) ACP1 and ACP2, and the preservation numbers of the two bacteriophages in China Center for Type Culture Collection are CCTCC NO: M 20242161 and CCTCC NO: M 2025461 respectively. The watermelon acidovorax bacteriophages ACP1 and ACP2 provided by the application have strong lytic activity on watermelon acidovorax, and have good stability and high safety, and can be used for preparing watermelon acidovorax detection products, and can also be used as biological disinfectants or biological pesticides, so that bacterial diseases caused by watermelon acidovorax can be effectively prevented and treated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to watermelon acidophilic bacteriophage and its applications. Background Technology

[0002] *Acidovorax citrulli* (AC) is Gram-negative and does not produce spores. Its optimal growth temperature is 24°C–28°C, with a maximum tolerable growth temperature of 41°C and a minimum growth temperature of 4°C. It can grow within a salinity range of 1%–9%. On KB medium, *Acidovorax citrulli* forms smooth, pale yellow, round colonies with regular edges, a viscous texture, and slight elevation.

[0003] Acidophilus citrinum is the pathogen that causes bacterial fruit spot disease in cucurbits. Bacterial fruit spot disease in cucurbits seriously damages the leaves and fruits of various cucurbitaceous crops, such as watermelon, cantaloupe, cucumber, and pumpkin, and causes yield reduction. Due to its strong resistance, Acidophilus citrinum can survive on cucurbit seeds for 35 years. This disease has seriously affected the production of watermelon and cantaloupe (Li Leshu, Ge Yixin, Tian Yanli, et al. Development of biological seed dressing agent for the prevention and control of bacterial fruit spot disease (BFB) in cucurbits [J]. Journal of Agricultural Biotechnology, 2015, 23(12): 1649-1659.).

[0004] Bacteriophages are a class of viruses that infect bacteria. They are primarily composed of proteins and nucleic acids and are widely found in soil, air, water, and organisms, exhibiting strong specificity. Highly virulent bacteriophages can rapidly adsorb, invade, replicate, assemble, and lyse cells, achieving their bactericidal effect. Since Frederik Tword first discovered bacteriophages in 1915, increasing research has demonstrated their high antibacterial activity and specificity, showing no infectivity against humans, other mammals, plants, or non-target microorganisms. However, current research on bacteriophages largely focuses on screening and exploring their biological characteristics, with few reports on their applications. Therefore, screening and applying suitable highly virulent bacteriophages is an effective approach to developing novel antibacterial agents.

[0005] Currently, research on watermelon acidophilus bacteriophages is almost nonexistent, and there are few reports on the use of bacteriophage therapy to control bacterial fruit spot disease in cucurbits caused by watermelon acidophilus. Therefore, developing watermelon acidophilus bacteriophages for the prevention and control of related pathogens is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a watermelon acidophilus bacteriophage and its applications to solve the problems existing in the prior art. The watermelon acidophilus bacteriophage provided by this invention has strong lytic activity against watermelon acidophilus, and also has good stability and high safety. It can be used to prepare watermelon acidophilus detection products, and can also be used as a biological disinfectant or biological pesticide, thereby effectively preventing and controlling bacterial diseases caused by watermelon acidophilus.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a watermelon acidophilic bacteriophage ACP1, which was deposited on October 11, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242161.

[0009] The present invention also provides a watermelon acidophilic bacteriophage ACP2, which was deposited on March 13, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025461.

[0010] The present invention also provides a watermelon acidophilic bacteriophage combination, including the above-mentioned watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2.

[0011] The present invention also provides the application of the above-mentioned watermelon acidophilic bacteriophage ACP1, watermelon acidophilic bacteriophage ACP2 or watermelon acidophilic bacteriophage combination in the preparation of watermelon acidophilic detection products.

[0012] The present invention also provides the application of the above-mentioned watermelon acidophilic bacteriophage ACP1, watermelon acidophilic bacteriophage ACP2 or watermelon acidophilic bacteriophage combination in the preparation of biological pesticides for the prevention and control of bacterial diseases caused by watermelon acidophilic bacteria.

[0013] The present invention also provides the application of the above-mentioned watermelon acidophilic bacteriophage ACP1, watermelon acidophilic bacteriophage ACP2 or watermelon acidophilic bacteriophage combination in the preparation of biological disinfectants of watermelon acidophilic bacteria.

[0014] The present invention also provides a detection product for watermelon acidophilus, comprising the above-mentioned watermelon acidophilus phage ACP1, watermelon acidophilus phage ACP2, or a combination of watermelon acidophilus phages.

[0015] The present invention also provides a biological pesticide for preventing and controlling bacterial diseases caused by watermelon acidophilus, the active ingredients of which include the above-mentioned watermelon acidophilus bacteriophage ACP1, watermelon acidophilus bacteriophage ACP2 or a combination of watermelon acidophilus bacteriophages.

[0016] Furthermore, the biopesticide also includes pesticide-acceptable excipients.

[0017] The present invention also provides a biological disinfectant of watermelon acidophilus, the active ingredients of which include the above-mentioned watermelon acidophilus phage ACP1, watermelon acidophilus phage ACP2 or a combination of watermelon acidophilus phages.

[0018] The present invention discloses the following technical effects:

[0019] (1) Watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2 are virulent bacteriophages isolated from nature. They do not contain virulence genes or harmful genes, and their DNA does not encode proteins that may cause potential health risks. They do not carry pathogen genes. This invention does not perform any genetic modification on the bacteriophages.

[0020] (2) Both watermelon acidophilus phage ACP1 and watermelon acidophilus phage ACP2 have high fermentation titers. During fermentation, the optimal MOI for watermelon acidophilus phage ACP1 to infect the host watermelon acidophilus is 0.0001. It is a virulent phage with high affinity and lytic ability. Fermentation at the optimal MOI can reach 6.35 × 10⁻⁶ within 12 h. 9 A titer of PFU / mL or higher; the optimal MOI for the ACP2 bacteriophage of *Acidophilus hygrophilus* to infect the host *Acidophilus hygrophilus* is 0.000001. It is a highly potent bacteriophage with strong affinity and lytic ability, and fermentation at the optimal MOI can reach 8.00 × 10⁸ PFU / mL within 12 h. 13 Potency above PFU / mL. Both ACP1 and ACP2 of watermelon acidophilus bacteriophage can specifically partially or completely inactivate watermelon acidophilus, requiring only a small amount of initial bacteriophage to complete large-scale proliferation, providing a high-quality source of bacteriophage strains for the industrial production of bacteriophage bactericides.

[0021] (3) Both watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2 of the present invention are strictly virulent bacteriophages with high specificity and lytic activity against host bacteria. Both watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2 can be used as effective ingredients in various products for environmental disinfection, including but not limited to disinfecting water distribution systems, irrigation facilities, public and private facilities, or other environmental surfaces by means of liquid immersion, spraying, or combined use with aqueous carriers, which can effectively control the growth and activity of target bacteria. Among them, liquid immersion and spraying include but are not limited to detergents, disinfectants, and stain removers; aqueous carriers include but are not limited to SM buffer, phosphate buffer, KB medium, LB medium, and chlorinated free water.

[0022] (4) Neither watermelon acidophilic bacteriophage ACP1 nor watermelon acidophilic bacteriophage ACP2 could interact with non-host bacteria and could not identify any of the 6 tested non-host bacteria, showing good specificity.

[0023] (5) Watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2 have good stability. Under conditions of -80 to 37℃, their titers do not change significantly within 24 hours. They also have good stability under pH conditions of 5 to 12, and can maintain good stability in weakly acidic to strongly alkaline environments.

[0024] (6) Watermelon acidophilus phage ACP1 and watermelon acidophilus phage ACP2 can be used to prepare compositions, reagents or kits, and can be applied to the rapid detection of watermelon acidophilus, including but not limited to the detection of watermelon acidophilus in target samples in the form of test strips, kits, etc., or the screening of target pathogens in samples, which can effectively ensure the sensitivity of detection.

[0025] (7) Watermelon acidophilus bacteriophage ACP1 and watermelon acidophilus bacteriophage ACP2 can be used to prepare biological disinfectants or biological pesticides, thereby effectively preventing and controlling bacterial diseases caused by watermelon acidophilus. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Images of phage plaques from two watermelon acidophilic bacteriophages;

[0028] Figure 2 Electron micrographs of two watermelon acidophilic bacteriophages;

[0029] Figure 3 Figure 1 shows the nucleic acid properties determination results of two watermelon acidophilic bacteriophage bacteriophages.

[0030] Figure 4 This is a pot experiment showing the preventive effect of watermelon acidophilic bacteriophage ACP1 on bacterial fruit spot disease in cucurbits; where A and C represent untreated seeds, disinfectant-treated seeds, and bacteriophage-treated seeds, respectively.

[0031] Figure 5 This is a pot experiment showing the preventive effect of watermelon acidophilic bacteriophage ACP2 on bacterial fruit spot disease in cucurbits; where A and C represent untreated seeds, disinfectant-treated seeds, and bacteriophage-treated seeds, respectively.

[0032] Figure 6 The figure shows the test results of the effect of applying bacteriophage-coated seeds on the control of bacterial fruit spot disease in cucurbits in the field.

[0033] Figure 7 This is a graph showing the test results of the therapeutic effect of bacteriophage on bacterial fruit spot disease in melons. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Biological Preservation: Acidovorax citrulli phage ACP1 was deposited at the China Center for Type Culture Collection (CCTCC) on October 11, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242161; Acidovorax citrulli phage ACP2 was deposited at the China Center for Type Culture Collection (CCTCC) on March 13, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025461.

[0040] The culture medium formulations involved in the following examples are as follows:

[0041] KB liquid medium: 20 g peptone, 10 mL glycerol, 1.5 g K2HPO4, 1.5 g MgSO4·7H2O, 1000 mL distilled water;

[0042] KB solid medium: 20 g peptone, 10 mL glycerol, 1.5 g K2HPO4, 1.5 g MgSO4·7H2O, 18 g agar, 1000 mL distilled water;

[0043] KB semi-solid agar medium: 20 g peptone, 10 mL glycerol, 1.5 g K2HPO4, 1.5 g MgSO4·7H2O, 8 g agar, 1000 mL distilled water.

[0044] Example 1: Isolation, preparation, purification, and culture of watermelon acidophilus bacteriophage ACP1 and ACP2

[0045] The source samples of watermelon acidophilic bacteriophage ACP1 in this invention were collected from melon fields in Liling City, Zhuzhou City, Hunan Province. The fruit peels were homogenized with water, filtered through double-layered filter paper, and the filtrate was centrifuged at low speed at room temperature. The supernatant was then filtered through a 0.22 μm filter membrane. The source samples of watermelon acidophilic bacteriophage ACP2 were collected from melon fields in Hainan Province. The soil from which the melons were grown was mixed with water, filtered through double-layered filter paper, and the filtrate was centrifuged at low speed at room temperature. The supernatant was then filtered through a 0.22 μm filter membrane.

[0046] Isolation of bacteriophages:

[0047] (1) Add 1 mL of the filtered sample to 50 mL of KB culture medium and 1 mL of logarithmic host bacterial culture. Incubate at 28°C and 200 rpm for 12 h to obtain the culture.

[0048] (2) Centrifuge the culture obtained in step (1) at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane, and store at 4℃ for later use.

[0049] (3) Take 0.5 mL of the host bacterial culture in the logarithmic phase, add it to 5 mL of semi-solid KB medium at a temperature not exceeding 45℃, mix well, pour it onto KB plates, and prepare a double-layer plate containing the host bacteria. Take 5 μL of the filtered supernatant and drop it onto the solidified double-layer plate. After air drying under aseptic conditions, place it at 28℃ for 12 h and observe the growth of phage plaques.

[0050] Phage purification:

[0051] (1) Use a sterile needle to puncture a single phage plaque into 2 mL of logarithmic host bacterial culture, culture at 200 rpm for 12 h at 28℃, filter with a 0.22 μm filter membrane, and then perform 10-fold serial dilutions of the filtrate;

[0052] (2) Add 0.5 mL of the host bacterial culture in the logarithmic phase to 5 mL of semi-solid KB medium at a temperature not higher than 45℃, mix well and immediately pour onto KB plates, wait for it to solidify, and use it for later use.

[0053] (3) Take 5 μL of each of the above gradient dilutions and drop them onto a double-layer plate. After air drying under sterile conditions, place the plate in a 28℃ incubator for 12 h and observe. This will yield a double-layer plate containing a single phage plaque.

[0054] (4) Puncture a single plaque into 2 mL of logarithmic host bacterial culture and purify it at least 3 times in the above manner to finally form plaques of uniform shape and size on the plate where the plaques are formed.

[0055] (5) Single phage plaques of uniform shape and size were placed in a bacterial culture containing 2 mL of logarithmic host bacteria and cultured at 200 rpm for 12 h at 28°C. The mixed culture was then added to 50 mL of KB medium and cultured at 200 rpm for 12 h at 28°C.

[0056] (6) Centrifuge the mixed culture at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the purified phage solution. Both ACP1 and ACP2 phages of *Acidophilus hygrophilus* produce single, circular plaques on the *Acidophilus hygrophilus* mycelium, with a clear center and a diameter of 4-5 mm (see [link to relevant documentation]). Figure 1 ).

[0057] Example 2: Electron microscopic observation of bacteriophages

[0058] The purified watermelon acidophilic bacteriophage ACP1 and ACP2 solutions prepared in Example 1 were observed under electron microscopy: 20 μL of sample was dropped onto a copper grid and allowed to precipitate naturally for 15 min. Excess liquid was absorbed from the side with filter paper, 1 drop of 2% phosphotungstic acid (PTA) was added to the copper grid, and the staining was carried out for 10 min. The staining solution was then absorbed from the side with filter paper, and the sample was dried before observation under a transmission electron microscope.

[0059] The results are as follows Figure 2 As shown, bacteriophages ACP1 and ACP2 exhibit a polyhedral, three-dimensionally symmetrical head and a short, non-retractable tail under an electron microscope. Based on their unique size and morphology, bacteriophages ACP1 and ACP2 are systematically classified into the family Podoviridae according to the definition of the International Committee on Taxonomy of Viruses (ICTV).

[0060] Example 3: Extraction and sequencing of bacteriophage genomes

[0061] Take 200 μL of the purified watermelon acidophilus phage ACP1 and ACP2 solutions prepared in Example 1, and extract the phage nucleic acid using the FastPure Viral DNA / RNA Mini Kit (Vazyme). The extraction procedure follows the kit instructions. Add DNase, RNase, and water to the extracted watermelon acidophilus phage ACP1 and ACP2 nucleic acids, respectively. After reacting for 30 minutes, take 5 μL for agarose gel electrophoresis. The results are shown in the figure. Figure 3 .

[0062] The results showed that the nucleic acids of bacteriophages ACP1 and ACP2 could be degraded by DNase, indicating that they are DNA bacteriophages. After gel electrophoresis to detect the bacteriophage DNA, its concentration and purity were determined using a NanoDrop ND-1000, and the products were sent to Shanghai Paisenno Biotechnology Co., Ltd. for sequencing. The genome length of *Acidophilus hygrophilus* bacteriophage ACP1 is 42426 bp, and its accession number in the National Genome Data Center (NGDC) is C_AA120265.1. The genome length of *Acidophilus hygrophilus* bacteriophage ACP2 is 41594 bp, and its accession number in the NGDC is C_AA120266.1.

[0063] Example 4: Detection of virulence genes in bacteriophages

[0064] This invention selects 15 virulence genes (Table 1) that have been identified as originating from lysogenic bacteriophages in pathogenic bacteria. By comparing the whole genomes of watermelon acidophilus bacteriophage ACP1 and watermelon acidophilus bacteriophage ACP2 and performing bioinformatics analysis, it is determined whether they contain the above-mentioned virulence genes.

[0065] Table 1. Major known virulence genes of lysogenic bacteriophages within pathogenic bacteria.

[0066]

[0067] The results showed that neither bacteriophages ACP1 nor ACP2 contained the aforementioned virulence genes.

[0068] Example 5: Determination of the optimal multiple of infection (MOI) of watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2.

[0069] Single colonies of *Acidophilus citrinum* host bacteria were picked and inoculated into Erlenmeyer flasks containing 50 mL of KB culture medium. The flasks were incubated at 28°C with shaking at 200 rpm for 48 h to obtain a host bacterial suspension. Pure cultures of *Acidophilus citrinum* phages ACP1 and ACP2 (prepared in Example 1) and host bacteria (MOI = number of phages / number of host bacteria) were added in proportions of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001, respectively. KB liquid medium was added to ensure the total volume of each tube was the same, and the tubes were incubated at 28°C with shaking at 200 rpm for 12 h. After incubation, the tubes were centrifuged at 12000 g for 10 min, and the supernatant was collected. The titer of each phage was determined using the double-layer plate method. Each dilution should be performed in triplicate. The average of the three replicates for each dilution is used for counting. The MOI that produces the highest phage titer is considered the optimal multiple of infection. The experiment is repeated three times.

[0070] Table 2. Titer of ACP1 from *Acidophilus hygrophilus* phage at different multiplicity of infection

[0071]

[0072] As shown in Table 2, the titer of watermelon acidophilic bacteriophage ACP1 reached its highest level (6.35 × 10⁻⁶) when the MOI was 0.0001. 9 (PFU / mL). The optimal MOI for watermelon acidophilic bacteriophage ACP1 is 0.0001. This indicates that watermelon acidophilic bacteriophage ACP1 requires only a very small amount of initial bacteriophage to rapidly multiply and produce a large number of bacteriophages. This provides a high-quality source of bacteriophage strains for the industrial production of bacteriophage preparations for controlling bacterial fruit spot disease in cucurbits, and also provides the important production parameter of the multiplicity of infection (MOI) of watermelon acidophilic bacteriophage ACP1.

[0073] Table 3. Titer of ACP2 phage from *Acidophilus hygrophilus* at different multiplicity of infection

[0074]

[0075] As shown in Table 3, the titer of watermelon acidophilic bacteriophage ACP2 reached its highest level (8.00 × 10⁻⁶) when the MOI was 0.000001. 13 (PFU / mL). The optimal MOI for watermelon acidophilic bacteriophage ACP2 was 0.000001. This indicates that watermelon acidophilic bacteriophage ACP2 requires only a very small amount of initial bacteriophage to rapidly multiply into a large number of bacteriophages. This provides a high-quality source of bacteriophage strains for the industrial production of bacteriophage preparations for controlling bacterial fruit spot disease in cucurbits, and also provides the important production parameter of the multiplicity of infection (MOI) of watermelon acidophilic bacteriophage ACP2.

[0076] Example 6: Determination of the thermal stability of watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2

[0077] Take 1 mL of 1×10 10 PFU / mL Watermelon acidophilus bacteriophage ACP1 and 1×10 12 PFU / mL pure culture medium of *Acidophilus hygrophilus* phage ACP2 (prepared in Example 1) was placed in sterile centrifuge tubes and incubated at -80℃, -20℃, 4℃, 28℃, 37℃, and 55℃ for 3 h, 6 h, 12 h, and 24 h, respectively. After the incubation period, the centrifuge tubes were removed and immediately placed at room temperature for equilibration. After appropriate dilution, the phage efficacy was determined using the double-layer plate method. The experiment was repeated three times.

[0078] Table 4. Stability of ACP1 bacteriophage from watermelon acidophilus at different temperatures

[0079]

[0080] As shown in Table 4, the titer of watermelon acidophilic bacteriophage ACP1 decreased by only one order of magnitude under conditions of -80 to 37℃, which indicates that watermelon acidophilic bacteriophage ACP1 has good room temperature and low temperature preservation characteristics.

[0081] Table 5. Stability of ACP2 bacteriophage from watermelon acidophilus at different temperatures

[0082]

[0083] As shown in Table 5, the titer of watermelon acidophilic bacteriophage ACP2 did not change significantly under conditions of -80 to 37℃, indicating that watermelon acidophilic bacteriophage ACP2 has good room temperature and low temperature preservation characteristics.

[0084] Example 7 pH stability test of watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2

[0085] Add 900 μL of KB medium at different pH values ​​(pH=2-12) to each sterile centrifuge tube. Place the centrifuge tubes in a constant temperature water bath at 28℃. After temperature equilibration, add 100 μL of pure culture medium of watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2 (prepared in Example 1) to each tube, so that the initial titer of watermelon acidophilic bacteriophage ACP1 is 1×10⁻⁶. 10 PFU / mL, the initial titer of ACP1 phage from *Watermelon acidophilus* was 1×10⁻⁶ PFU / mL. 12 PFU / mL, incubated at 28℃ for 2 h, and then sampled to determine the potency. The experiment was repeated 3 times.

[0086] Table 6. Stability of ACP1 bacteriophage from watermelon acidophilus under different pH conditions

[0087]

[0088] As shown in Table 6, the titer of watermelon acidophilic bacteriophage ACP1 decreased by only one order of magnitude or even showed no significant change under pH conditions of 5 to 12, indicating that the bacteriophage has good stability under neutral, weakly acidic and alkaline conditions. Therefore, if watermelon acidophilic bacteriophage ACP1 is applied in the field, it will not be easily affected by the pH of the adjuvant.

[0089] Table 7. Stability of watermelon acidophilic bacteriophage ACP2 under different pH conditions

[0090]

[0091] As shown in Table 7, the titer of watermelon acidophilic bacteriophage ACP2 decreased by only one order of magnitude or even showed no significant change under pH conditions of 5 to 12, indicating that the bacteriophage has good stability under neutral, weakly acidic and alkaline conditions. Therefore, if watermelon acidophilic bacteriophage ACP2 is applied in the field, it will not be easily affected by the pH of the adjuvant.

[0092] Example 8. UV stability determination of watermelon acidophilic bacteriophage ACP1 and watermelon acidophilic bacteriophage ACP2

[0093] Take 5 mL of 1×10 10 PFU / mL Watermelon acidophilus bacteriophage ACP1 and 1×10 12 PFU / mL pure culture medium of *Acidophilus hygrophilus* phage ACP2 (prepared in Example 1) was spread evenly in a sterile petri dish with a diameter of 90 mm and placed in a laminar flow hood under ultraviolet light. Samples were taken at 0 min, 5 min, 10 min, 15 min, 25 min, and 50 min, respectively, and incubated in the dark for 30 min before the phage titer was determined using the double-layer plate method.

[0094] Table 8. UV stability determination of ACP1 bacteriophage from watermelon acidophilus.

[0095]

[0096] As shown in Table 8, the titer of watermelon acidophilic bacteriophage ACP1 did not change significantly after 50 min of ultraviolet irradiation. Therefore, watermelon acidophilic bacteriophage ACP1 has strong tolerance to ultraviolet radiation and can be used in the field without being affected by ultraviolet radiation.

[0097] Table 9. UV stability determination of ACP2 bacteriophage from watermelon acidophilus.

[0098]

[0099] As shown in Table 9, the titer of watermelon acidophilic bacteriophage ACP2 did not change significantly after 50 minutes of ultraviolet irradiation. Therefore, watermelon acidophilic bacteriophage ACP2 has strong tolerance to ultraviolet radiation and can be used in the field without being affected by ultraviolet radiation.

[0100] Example 9: Lysis test of watermelon acidophilic bacteriophage ACP1 and ACP2 on non-host pathogenic bacteria.

[0101] Six single colonies of non-host bacteria, including *Xanthomonas oryzae* and *Bacillus*, were selected and inoculated into Erlenmeyer flasks containing 50 mL of KB liquid medium. The flasks were incubated at 28°C with shaking at 200 rpm for 48 h to obtain bacterial suspensions for each strain. One mL of each bacterial suspension was mixed with 5 mL of KB semi-solid medium and plated onto KB agar plates. Ten μL of *Acidophilus citrinum* phage ACP1 and ACP2 phage solutions were spotted onto the plates, and after air-drying, incubated at 28°C for 12 h. The results were then observed. The experiment was repeated three times.

[0102] Table 10. Lysis test of watermelon acidophilic bacteriophages ACP1 and ACP2 on non-host bacteria.

[0103]

[0104] Note: "-" indicates no cleavage.

[0105] As shown in Table 10, neither the ACP1 nor ACP2 bacteriophages of *Acidophilus hygrophilus* could recognize any of the six tested non-host bacteria. This indicates that the tested bacteriophages have extremely strong host specificity and do not damage the remaining microbial communities.

[0106] Example 10: Control of bacterial fruit spot disease in cucurbits by watermelon acidophilus bacteriophages ACP1 and ACP2

[0107] Preparation of disinfected seeds: Wash the melon seeds purchased from the agricultural supply store with 1% sodium hypochlorite for 20 minutes to remove bacterial contamination on the seed surface, and then rinse them three times with sterile water to obtain disinfected seeds.

[0108] Preparation of phage-coated seeds: A concentration of 1×10⁻⁶ seeds was prepared. 9 ACP1 phage at a concentration of 1×10 PFU / mL and a concentration of 1×10 9 ACP2 phage at PFU / mL was diluted 100-fold to obtain two dilutions. Sterilized seeds were then mixed and cultured in both dilutions for 2 h, followed by drying for 20 minutes to obtain either ACP1-coated or ACP2-coated seeds.

[0109] The experiment used two groups of phage-coated seeds, sterilized seeds, and untreated seeds as control groups. The experimental treatment groups included one group of ACP1-coated seeds and one group of ACP2-coated seeds, two groups of sterilized seeds, and two groups of untreated seeds. Each experimental group contained 40 seeds, sown in a culture pot containing 200g of autoclaved soil. The six experimental treatments were sown in six different pots. Diluted *A. citrulli* bacteria were added after sowing. The culture pots were covered with plastic covers, and the lids were periodically opened to spray sterile water to maintain humidity. Seven days after inoculation, the disease index of each melon leaf was recorded and photographed.

[0110] like Figure 4 As shown, bacteriophage ACP1 has a significant effect on the prevention of bacterial fruit spot disease in cucurbits. Seeds treated with bacteriophage were significantly better than untreated seeds and disinfected seeds. Treatment with bacteriophage ACP1 achieved a control efficacy of 85% against bacterial fruit spot disease in cucurbits.

[0111] like Figure 5 As shown, bacteriophage ACP2 has a significant effect on the prevention of bacterial fruit spot in cucurbits. Seeds treated with bacteriophage were significantly better than untreated seeds and seeds treated with disinfectants. Treatment with bacteriophage ACP2 achieved a control efficacy of 80% against bacterial fruit spot disease in cucurbits.

[0112] Example 11: Evaluation of the field control efficacy of bacteriophages ACP1 and ACP2 against bacterial fruit spot disease in cucurbits.

[0113] Control group (H2O): Melon seeds purchased from agricultural supply stores were washed with 1% sodium hypochlorite for 20 minutes to remove bacterial contamination on the seed surface, then rinsed 3 times with sterile water, and then placed in 100 mL of sterile water and cultured on a shaker at 100 rpm for 2 hours. After draining the seeds for 20 minutes, they were germinated with clean water.

[0114] Experimental group: Melon seeds purchased from an agricultural supply store were washed with 1% sodium hypochlorite for 20 minutes to remove bacterial contamination from the seed surface, and then rinsed three times with sterile water to obtain disinfected seeds. A concentration of 1×10⁻⁶ sodium hypochlorite was used... 9 ACP1 phage at a concentration of 1×10 PFU / mL 9 Mix 1 mL of each of PFU / mL ACP2 phages thoroughly, then dilute 100 times, place the sterilized seeds in the mixture, and incubate on a shaker at 100 rpm for 2 h to obtain phage-coated seeds. After draining the seeds for 20 minutes, germinate them with water.

[0115] After the seeds in each group germinated, the control group seeds were planted in two rows at the Hunan Agricultural University base, with 16 seedlings per row. The experimental group seeds were also planted in two rows, with 16 seedlings per row. Ten days later, the seedlings were inoculated with *Acidophilus hygrophilus* and the disease incidence was observed.

[0116] Onset of illness, such as Figure 6 As shown, seeds coated with bacteriophages did not exhibit obvious symptoms of bacterial fruit spot disease in cucurbits, while seeds in the control group showed obvious disease after inoculation with *Acidophilus hygrophila*. This indicates that the combined use of bacteriophages ACP1 and ACP2 has a significant effect on the prevention of bacterial fruit spot disease in cucurbits in the field. Bacteriophage treatment of seeds is significantly superior to disinfectant treatment. Bacteriophages ACP1 and ACP2 show good control efficacy against bacterial fruit spot disease in cucurbits in the field.

[0117] Example 12: Evaluation of the therapeutic effects of bacteriophages ACP1 and ACP2 on bacterial fruit spot disease in melons.

[0118] After surface disinfecting the melons with 75% alcohol, the melon skin was punctured with a needle tip, and the melons were sprayed with *Acidophilus hygrophilus* for watermelon inoculation. The melons were then kept moist in a greenhouse to promote disease development. Once the fruit showed signs of disease on the 6th day after inoculation, a 1×10⁻⁶ dose was administered. 9 PFU / mL phage ACP1, 1×10 9 PFU / mL phage ACP2 and a mixture of the two (0.5 × 10⁻⁶) 9 PFU / mL ACP1+0.5×10 9 Spraying with PFU / mL ACP2 (2 mL) was performed, and the disease incidence was observed. A solvent control (using KB medium as the solvent) and a blank control (using water instead of water for inoculation with *Acidophilus hygrophilus*) were also set up.

[0119] Onset of illness, such as Figure 7 As shown in Table 11, the results indicate that the spread of bacterial fruit spot disease in melons treated with bacteriophages was better controlled than that in melons treated with water. The combined use of ACP1 and ACP2 showed better treatment results.

[0120] Table 11 Statistical results of lesion diameter

[0121]

[0122] In summary, the watermelon acidophilus phages ACP1 and ACP2 of the present invention have good stability and high safety. They can not only be used to prepare watermelon acidophilus detection kits, but also as biological disinfectants or biological pesticides to effectively prevent and control bacterial diseases caused by watermelon acidophilus.

[0123] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A watermelon acidophilic bacteriophage ( Acidovorax citrulli Phage) ACP1, characterized in that, The watermelon acidophilic bacteriophage ACP1 was deposited at the China Center for Type Culture Collection on October 11, 2024, with accession number CCTCCNO: M 20242161.

2. A strain of Acidophilic Citrullus bacteriophage ACP2, characterized by, The watermelon acidophilic bacteriophage ACP2 was deposited at the China Center for Type Culture Collection on March 13, 2025, with accession number CCTCC NO: M 2025461.

3. A watermelon acidophilic bacteriophage assemblage, characterized in that, It includes the watermelon acidophilic bacteriophage ACP1 as described in claim 1 and the watermelon acidophilic bacteriophage ACP2 as described in claim 2.

4. The use of the watermelon acidophilic bacteriophage ACP1 as described in claim 1, the watermelon acidophilic bacteriophage ACP2 as described in claim 2, or the combination of watermelon acidophilic bacteriophages as described in claim 3 in the preparation of a biological pesticide for the prevention and control of bacterial fruit spot disease in cucurbits caused by watermelon acidophilic bacteria.

5. The application of the watermelon acidophilic bacteriophage ACP1 as described in claim 1, the watermelon acidophilic bacteriophage ACP2 as described in claim 2, or the combination of watermelon acidophilic bacteriophages as described in claim 3 in the preparation of a biological disinfectant that inhibits watermelon acidophilic bacteria.

6. A biological pesticide for controlling bacterial fruit spot disease in cucurbits caused by *Acidophilus hygrophilus*, characterized in that, The active ingredients include the watermelon acidophilic bacteriophage ACP1 as described in claim 1, the watermelon acidophilic bacteriophage ACP2 as described in claim 2, or the combination of watermelon acidophilic bacteriophages as described in claim 3.

7. The biological pesticide according to claim 6, characterized in that, The biopesticide also includes pesticide-acceptable excipients.

8. A biological disinfectant for inhibiting watermelon acidophilus, characterized in that, The active ingredients include the watermelon acidophilic bacteriophage ACP1 as described in claim 1, the watermelon acidophilic bacteriophage ACP2 as described in claim 2, or the combination of watermelon acidophilic bacteriophages as described in claim 3.

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