Cracking pantoea agglomerans phage and application thereof

Abstract

The invention relates to a lytic pantoea agglomerans phage and application thereof, and belongs to the technical field of bioengineering. The lytic pantoea agglomerans phage is preserved in the China Center for Type Culture Collection on October 30, 2025, the preservation address is Wuhan, China, and the preservation number is CCTCC NO: M 20252393; the strain is named as Pantoea agglomerans phage vBPanPgDPHH-6 in a classified manner, and the strain is named as Pantoea agglomerans phage The bacteriophage provides a brand new way for solving the problem of drug resistance of pantoea agglomerans, even if pathogenic bacteria generate resistance to conventional chemical pesticides or antibiotics, as long as the characteristics of the pathogenic bacteria serving as bacteriophage hosts are not changed, the bacteriophage can still efficiently split the pathogenic bacteria, and reliable guarantee is provided for preventing and controlling infection caused by drug-resistant strains.

Description

Technical Field

[0001] This invention relates to a lytic, clustering pan-bacterial phage and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] Pantothecinia clumps Pantoea agglomerans Pantotheca acuminata is a Gram-negative rod-shaped bacterium widely distributed on plant surfaces, in soil, and in water. Numerous studies have shown that Pantotheca acuminata can cause diseases in a variety of economic crops, including cotton, rice, corn, sorghum, walnuts, peaches, dragon fruit, jujubes, sweet onions, and Chinese cabbage. Some pathogenic strains are tumorigenic, inducing gall formation in edible beets, wisteria, Douglas fir, and cranberries. Furthermore, Pantotheca acuminata is also an opportunistic pathogen, causing hospital-acquired infections in immunocompromised individuals, leading to suppurative arthritis, synovitis, endocarditis, and osteomyelitis. Therefore, the control and treatment of pathogenic Pantotheca acuminata is an important issue in agricultural economic crops and public health.

[0003] Fungicides are an effective treatment for pantothenic infections. However, pantothenic bacteria, found in various environments (plants, soil, water), have ample opportunity to acquire additional drug-resistant genes from other bacteria through plasmid exchange. Furthermore, the widespread use of antibiotics in agricultural and hospital settings exerts strong selective pressure on pantothenic populations, screening for and enriching strains carrying multidrug-resistant genes. These factors significantly reduce the effectiveness of fungicides against pantothenic infections, necessitating the development of safe and effective treatments.

[0004] Bacteriophages are a class of viruses capable of infecting bacteria and other microorganisms. They lyse the host by multiplying within it and releasing a large number of progeny. This highly specific predator-prey relationship makes bacteriophages ubiquitous in nature, and they have co-evolved with bacteria, forming the most numerous biological entities on Earth. Based on this precise targeting and efficient bactericidal mechanism, using bacteriophages to control crop diseases or human infections caused by pan-microbe swarms demonstrates significant advantages over traditional fungicides. Furthermore, bacteriophages themselves are biodegradable organic matter; they naturally die after completing their mission and do not remain in crops, which is crucial for developing green organic agriculture, ensuring food safety, and reducing environmental pollution. Therefore, bacteriophages, with their unique advantages of high specificity, self-replication, and environmental friendliness, open up a promising new path for the sustainable control of bacterial diseases caused by pan-microbe swarms. Summary of the Invention

[0005] The purpose of this invention is to provide a lytic, clustering pan-bacterial phage and its application to solve the technical problems existing in the prior art as described above.

[0006] The technical solution provided by this invention is as follows: This invention discloses a lytic, clustering pan-bacterial phage named vB_PanPg_DPHH-6, which has been submitted for biodepository.

[0007] [Instructions for Biological Preservation] Depository: China Center for Type Culture Collection; Location of collection: Wuhan, China; Deposit date: October 30, 2025; Accession number: CCTCC NO: M 20252393; Category Naming: Pantoea agglomerans phage vB_PanPg_DPHH-6 。

[0008] Furthermore, the complete genome sequence of the bacteriophage is shown in SEQ ID NO.1.

[0009] The present invention also discloses the application of the lytic clump-forming pantothenic phage described above in the preparation of products that inhibit and / or kill clump-forming pantothenic bacteria.

[0010] The present invention also discloses the application of the lytic clustering pantothenic bacteriophage described above in the prevention and control of pantothenic infection in economic crops.

[0011] The present invention also discloses an environmental biological disinfectant, comprising the lytic clustering pan-bacteriophage as described above.

[0012] Furthermore, the environment includes agricultural product planting and storage environments, agricultural product and feed processing spaces, and hospital environments.

[0013] The technical solution provided by this invention has the following advantages compared with the prior art: The optimal multiplicity of infection (MOF) of the lytic pantothenic phage of this invention is 0.01, with a burst capacity of 278 PFU / mL, and it exhibits good stability under conditions of 4-40°C and pH 6-9. Furthermore, the genome of this phage, vB_PanPg_DPHH-6, does not contain lysogenic or drug-resistance genes, making it safe for the prevention and control of pantothenic agglomerates.

[0014] The bacteriophage of this invention provides a novel approach to solving the problem of drug resistance in clustered pantothenia. Even if the pathogen develops resistance to conventional chemical pesticides or antibiotics, as long as its characteristics as a bacteriophage host remain unchanged, this bacteriophage can still efficiently lyse it, providing a reliable guarantee for controlling infections caused by drug-resistant strains. Attached Figure Description

[0015] Figure 1Image of a plaque from bacteriophage vB_PanPg_DPHH-6.

[0016] Figure 2 Transmission electron microscopy image of bacteriophage vB_PanPg_DPHH-6.

[0017] Figure 3 The results are for the multiplicity of infection assay of bacteriophage vB_PanPg_DPHH-6.

[0018] Figure 4 The results show the one-step growth curve of bacteriophage vB_PanPg_DPHH-6.

[0019] Figure 5 The results show the stability of bacteriophage vB_PanPg_DPHH-6 at different temperatures.

[0020] Figure 6 The results show the stability of bacteriophage vB_PanPg_DPHH-6 at different pH values.

[0021] Figure 7 This is a genome annotation diagram for bacteriophage vB_PanPg_DPHH-6.

[0022] Figure 8 The figure shows the results of the determination of the bactericidal effect of bacteriophage vB_PanPg_DPHH-6 on the surface of Chinese cabbage to kill pan-bacteria. Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0024] This invention discloses a lytic, clumping pantothenic phage, named vB_PanPg_DPHH-6. This phage was isolated from wastewater from a farmers' market in Yicheng District, Zhumadian City, Henan Province, and was deposited on October 30, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20252393. Its classification is as follows: Pantoea agglomerans phage vB_PanPg_DPHH-6.

[0025] In the following embodiments, the culture medium used includes: LB (Luria broth) liquid medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, add distilled water to 1L, autoclave at 121℃ for 20min.

[0026] LB semi-solid medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, 7.5g agar powder, add distilled water to 1L, autoclave at 121℃ for 20min, then cool to 48℃ before use.

[0027] LB solid medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar powder, add distilled water to 1L, autoclave at 121℃ for 20min, cool to 50℃, pour into plates, let solidify, and then invert for later use.

[0028] Example 1: Preparation of Pantotheca agglomerata bacterial suspension A small piece of rotten tissue was taken from a walnut fruit infected with black spot and placed in sterile LB liquid medium. It was incubated at 37°C in a shaking incubator for 24 hours. Then, a sterile inoculating loop was used to streak the culture onto sterile LB solid medium. The solid plate was then incubated at 37°C for 16 hours. After colonies grew on the plate, a single colony was picked up with a sterile inoculating loop and streaked onto sterile LB solid medium, then incubated at 37°C for 16 hours. This streaking purification process was repeated three times to obtain clusters of pan-wine bacteria, PaPO3.

[0029] Pantothenia glutinosa PaP03 was streaked onto LB solid medium plates, and then single colonies were picked and transferred to LB liquid medium and incubated at 37°C in a shaking incubator until the bacterial OD reached the target value. 600 =0.6, store it in a 4℃ refrigerator for later use.

[0030] Example 2: Isolation and purification of bacteriophage vB_PanPg_DPHH-6 50 mL of wastewater was collected from a farmers' market in Yicheng District, Zhumadian City, Henan Province. The wastewater was aliquoted into centrifuge tubes and centrifuged at 12000 rpm for 10 min. The supernatant was then filtered through a 0.45 μm filter. 1 mL of the filtrate was added to 10 mL of cultured *Pantheraquinone* clumps and incubated at 37°C with shaking for 12 h. The mixture was then centrifuged and filtered again. The filtrate was serially diluted with sterile water. 100 μL of each dilution, 0.5 mL of the cultured bacterial suspension, and 5 mL of LB semi-solid medium were mixed and poured onto LB agar plates (double-layer plate method). After the plates solidified, they were inverted and incubated overnight at 37°C. A single plaque was picked from the double-layer plate and added to 5 mL of the cultured bacterial suspension. This process was repeated three times to purify and isolate the bacteriophage. Figure 1 As shown, the purified phage vB_PanPg_DPHH-6 formed uniformly shaped plaques on a double-layer plate.

[0031] Example 3 Electron microscopy of bacteriophage vB_PanPg_DPHH-6 A single purified phage plaque was added to 5 mL of cultured bacterial solution and incubated at 37°C with shaking for 20 h. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.45 μm filter to obtain a phage suspension. 10 μL of the phage suspension was added dropwise onto a copper grid for precipitation for 1 min. After standing at room temperature for 10 min, excess liquid was absorbed with filter paper. The phage was stained with 2% phosphotungstic acid for 2 min, excess stain was absorbed with filter paper, and the solution was dried at room temperature for 20 min. Phage morphology was observed using a JEM-1200EX transmission electron microscope at an accelerating voltage of 80 kV. Figure 2 As shown, bacteriophage vB_PanPg_DPHH-6 has an icosahedral head and a short tail, making it a typical short-tailed bacteriophage.

[0032] Example 4: Optimal Multiple of Infection for Bacteriophage vB_PanPg_DPHH-6 The phage suspension was serially diluted with sterile water. Then, 100 μL of each dilution of phage solution was mixed with 100 μL of bacterial suspension to achieve MOIs of 0.001, 0.01, 0.1, 1, 10, and 100. Each mixture was then added to 5 mL of LB broth and incubated at 37°C with shaking for 20 h. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.45 μm filter. The phage titer in each filtrate was determined using the double-layer plate method. Figure 3 It can be seen that bacteriophage vB_PanPg_DPHH-6 has the highest titer when the multiplicity of infection is 0.01, indicating that 0.01 is the optimal multiplicity of infection for bacteriophage.

[0033] Example 5: One-step growth curve of bacteriophage vB_PanPg_DPHH-6 To achieve the optimal multiplicity of infection (MOI), 1 mL of phage suspension and 1 mL of cultured bacterial solution were mixed and added to 8 mL of fresh LB broth. The mixture was then incubated at 37°C with shaking at 220 rpm. At 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min, 200 μL of culture was collected, centrifuged at 15000 rpm for 2 min, and 100 μL of the supernatant was serially diluted with sterile water. Finally, the phage titer at each time point was determined using the double-layer plate method. Figure 4 As shown, the incubation period of bacteriophage vB_PanPg_DPHH-6 is 20 min, the lysis period is 70 min, and it has a strong reproductive capacity.

[0034] Example 6: Thermal stability of bacteriophage vB_PanPg_DPHH-6 Six 1.5L centrifuge tubes were used, each containing 500 μL of phage suspension. The tubes were then incubated at 4℃, 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 6 hours. Before and after incubation, 100 μL of the phage suspension was serially diluted with sterile water. The phage titer in each group was then determined using the double-layer plate method. The thermostability of the phage was assessed by the ratio (percentage) of the titer after incubation to the titer before incubation. Figure 5 As shown, the titer of bacteriophage vB_PanPg_DPHH-6 remained essentially unchanged after incubation at 4-40℃ for 6 hours, demonstrating good thermal stability.

[0035] Example 7 pH tolerance of bacteriophage vB_PanPg_DPHH-6 Six 1.5 mL centrifuge tubes were used, each containing 100 μL of phage suspension. Then, 900 μL of LB broth at different pH values ​​(pH 2-12) were added, and the tubes were incubated at 30 °C for 6 h. Before and after incubation, 100 μL of the phage suspension was serially diluted with sterile water. The phage titer in each group was then determined using the double-layer plate method. The pH tolerance of the phage was assessed by the ratio (percentage) of the titer after incubation to the titer before incubation. Figure 6 As shown, the titer of bacteriophage vB_PanPg_DPHH-6 remained essentially unchanged after incubation at pH 6-9 for 6 hours, demonstrating good pH tolerance.

[0036] Example 8: Genomic analysis of bacteriophage vB_PanPg_DPHH-6 The genome of phage vB_PanPg_DPHH-6 was extracted using phenol-chloroform-isoamyl alcohol and resuspended in sterile water. Genome sequencing was performed using the Illumina NovaSeq 6000 platform. The quality of the raw sequencing data was analyzed using FastQCv0.11.5, and low-quality reads and sequencing adapter regions were filtered using the default parameters Trimmomatic 0.36. The selected high-quality reads were assembled using SPAdes v3.13.0 to obtain the complete phage genome sequence. Homology comparison of the phage vB_PanPg_DPHH-6 genome with phage genomes in the NCBI database showed that it was most closely related to *Pantoea phage* LIMElight (NCBI accession number: NC_019454.1), with a genome sequence similarity of 86.48%, significantly lower than the 95% threshold commonly used for defining new species. Therefore, bacteriophage vB_PanPg_DPHH-6 exhibits significant genetic diversity and is a novel bacteriophage species.

[0037] The open reading frames (ORFs) of the vB_PanPg_DPHH-6 genome were predicted using the RAST online service, and then the functions of the proteins encoded in the phage genome were annotated using the Basic Local Protein Alignment Search (BLASTp) tool from the NCBI website. The genome map was then constructed using the online visualization tool Proksee.

[0038] Sequencing analysis revealed that the genome of bacteriophage vB_PanPg_DPHH-6 is a double-stranded linear DNA molecule, 45593 bp in length, with a G+C content of 54%. For example... Figure 7 The genome of bacteriophage vB_PanPg_DPHH-6 contains 55 ORFs, of which 18 are known functional coding sequences, and the remaining genes are annotated as protein-coding sequences with unknown functions. The annotated proteins in this genome mainly involve four functional modules, including: phage replication, regulation, assembly, and structure.

[0039] Example 9 Host spectrum of bacteriophage vB_PanPg_DPHH-6 Following the bacterial isolation and purification method described in Example 1, clusters of Pantotheca acuminata PaP01, PaP02, PaP03, PaP04, PaP05, PaP06, PaP07, and PaP08 were isolated from diseased corn, Chinese cabbage, peach, and apple as test bacteria. Each strain was streaked onto LB agar, and then single colonies were picked and inoculated into 5 mL of fresh LB broth, and cultured at 37°C with shaking until the bacterial culture reached OD. 600 =0.6. Then, 0.5 mL of bacterial suspension was added to 5 mL of LB semi-solid medium, mixed thoroughly, and poured onto LB solid plates. After the plates solidified, 2 μL of bacteriophage vB_PanPg_DPHH-6 suspension was added to the center of each plate. Finally, the plates were incubated at 37℃ for 12 h. After incubation, plaques were observed on each plate. If plaques were formed, it indicated that bacteriophage vB_PanPg_DPHH-6 could lyse the corresponding test bacteria. The test results are shown in Table 1.

[0040] Table 1. Results of host spectrum determination for bacteriophage vB_PanPg_DPHH-6

[0041] "-" indicates no cleavage; "+" indicates cleavage.

[0042] Example 10: Bactericidal ability of bacteriophage vB_PanPg_DPHH-6 Take fresh Chinese cabbage leaves and divide them into 6 groups. Spray each group with a cultured Pantotheca acuminata PaPO3 solution (1×10⁻⁶). 8CFU / mL of bacteriophage was applied to the surface of Chinese cabbage. The first group was then sprayed with sterile water (control group), while groups 2 through 6 were sprayed with different concentrations of bacteriophage vB_PanPg_DPHH-6 suspension (MOIs of 0.001, 0.01, 0.1, 1, and 10, respectively), and allowed to stand at room temperature for 12 hours. Then, a portion of cabbage leaves from each group was soaked in sterile water, followed by serial dilutions. The number of clumps of pantothenic bacteria in each group was detected using the plate count method. Figure 8 As shown, compared with the control group that did not undergo phage treatment, even with 1×10 5 Phage treatment at PFU / mL reduced the number of bacteria by 5 orders of magnitude, indicating that phage vB_PanPg_DPHH-6 has extremely strong bactericidal ability.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lytic, clustering pan-based bacteriophage, characterized in that, It was deposited on October 30, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20252393; classified and named as follows: Pantoea agglomerans phage vB_PanPg_DPHH-6.

2. The lytic, clustering pan-based bacteriophage according to claim 1, characterized in that, The complete genome sequence of the bacteriophage is shown in SEQ ID NO.

1.

3. The use of the lytic clump-forming pantothenic phage as described in claim 1 or 2 in the preparation of products that inhibit and / or kill clump-forming pantothenic bacteria.

4. The application of the lytic clustering pantothenic phage as described in claim 1 or 2 in the prevention and control of pantothenic infection in economic crops.

5. An environmental biological disinfectant, characterized in that, Includes the lytic, clustered pan-based bacteriophage as described in claim 1 or 2.

6. The environmental biological disinfectant according to claim 5, characterized in that, The environment includes agricultural product planting and storage environments, agricultural product and feed processing spaces, and hospital environments.

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