Polaribacter pectinophilus bacteriophage S482 and applications thereof

Abstract

The present application relates to the field of microbial technology, and discloses a polar pectobacterium phage S482 and application thereof. The polar pectobacterium phage S482 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 46682, and the preservation date is October 13, 2025. The phage can lyse three kinds of pectobacterium, i.e. polar pectobacterium, brazilian pectobacterium and black pectobacterium, and can effectively inhibit the pathogenicity of the three kinds of pectobacterium, and reduce the disease index of potato soft rot and stem rot caused by the three kinds of pectobacterium. The phage of the present application enriches the broad-spectrum phage resources of potato soft rot and stem rot, widens the phage prevention and control spectrum, provides a new green prevention and control idea for potato bacterial diseases caused by pectobacterium, and lays a theoretical foundation for the development and application of subsequent phage preparations.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Polar Pectinobacterium phage S482 and its applications. Background Technology

[0002] By Pectinobacterium ( Pectobacterium Potato soft rot and stem rot caused by bacteria (spp.) are highly destructive bacterial diseases in potato production, causing significant economic losses to the potato industry. These diseases primarily affect the base of the potato stem, tubers, and roots. Under suitable environmental conditions, the pathogen invades the host tissue, secreting large amounts of pectinase, which degrades the pectin components of the cell walls, leading to cell disintegration, tissue softening, and resulting in blackening and rotting of the stem base and soft rot of the tubers. This severely impacts potato growth, yield, and quality. Compared to traditional chemical and physical control measures, bacteriophages, as biological control agents, offer advantages such as being green, highly efficient, and environmentally friendly, and have become a research hotspot in the field of plant bacterial disease control.

[0003] Currently, preliminary explorations have been made into phage control technologies for potato soft rot and stem rot, with multiple lytic phage strains isolated and their control potential confirmed. However, existing technologies still have the following shortcomings: most reported phages are limited to single-host specificity, resulting in a narrow host range that fails to cover the diversity of pathogen populations in the field. Furthermore, broad-spectrum phage resources capable of simultaneously lysing multiple pectinobacteria are still relatively scarce, limiting the widespread application of phages in the integrated management of potato soft rot and stem rot.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide polar pectinobacterium phage S482 and its application, aiming to solve the problems of potato soft rot and stem rot caused by pectinobacter spp. bacteria.

[0006] The technical solution of the present invention is as follows: Firstly, it provides polar pectinobacterium phage ( Pectobacterium polaris phage S482, the polar pectinobacterium phage S482, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.46682, and the deposit date is October 13, 2025.

[0007] In a second aspect, a biocontrol composition is provided, the biocontrol composition comprising: Polar Pectinobacterium phage S482 as described in the first aspect.

[0008] Optionally, the biocontrol composition further includes: biocontrol bacteria.

[0009] Further optionally, the biocontrol bacteria is Bacillus atrophus (Bacillus subtilis). Bacillus atrophaeus )4618, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34952 and deposit date of June 19, 2025.

[0010] Optionally, the biocontrol composition further includes: chemical agents.

[0011] Alternatively, the chemical agent is thiamethoxam.

[0012] Optionally, the biocontrol composition further includes agriculturally acceptable adjuvants.

[0013] Further optionally, the agriculturally acceptable adjuvant is selected from one or more of dispersants, stabilizers, fillers, and solvents.

[0014] Thirdly, the use of the Polar Pectinobacterium phage S482 as described in the first aspect or the biocontrol composition as described in the second aspect in the control of potato soft rot and / or stem rot.

[0015] Optionally, the soft rot and / or stem rot is caused by *Pectinobacterium polaris* (… Pectobacterium polaris ), Brazilian pectinobacterium ( Pectobacterium brasiliense ) and black shank pectinobacter ( Pectobacterium atrosepticum One or more of the following can cause this.

[0016] Beneficial Effects: This invention isolates a *Pectinobacter polaris* phage, S482, capable of lysing *Pectinobacter polaris* from soil. Its morphological characteristics, biological properties, and lysis spectrum are systematically characterized. Its control effect on potato soft rot is evaluated through potato chip experiments, and its control effect on potato stem rot is evaluated through pot experiments, both alone and in combination with chemical agents and / or biocontrol bacteria. Results show that the *Pectinobacter polaris* phage S482 of this invention can lyse *Pectinobacter polaris*, *Pectinobacter brasiliensis*, and *Pectinobacter blackshank*, and can effectively inhibit the pathogenicity of these three *Pectinobacter polaris*, reducing the disease index of potato soft rot and stem rot caused by them. This invention enriches the broad-spectrum phage resources for potato soft rot and stem rot, broadens the phage control spectrum, provides a new green control strategy for bacterial diseases of potato caused by *Pectinobacter polaris*, and lays a theoretical foundation for the subsequent development and application of phage preparations. Attached Figure Description

[0017] Figure 1 These are phage plaque images and transmission electron microscope images of Pectinobacter polaris phage S482 from Examples 1 and 2; where A is a phage plaque image and B is a transmission electron microscope image.

[0018] Figure 2 This is a graph showing the optimal multiplicity of infection (MLI) determination results for Pectinobacter polaris phage S482 in Example 4; where A is the test result in Pectinobacter brasiliensis SM19183, B is the test result in Pectinobacter nigra CD19130, and C is the test result in Pectinobacter polaris ZRIMU1222.

[0019] Figure 3 This is a graph showing the one-step growth curve of Pectinobacter polaris phage S482 in Example 5; where A is the test result of Pectinobacter brasiliensis SM19183, B is the test result of Pectinobacter nigra CD19130, and C is the test result of Pectinobacter polaris ZRIMU1222.

[0020] Figure 4 This is a graph showing the lysis kinetics curve of Pectinobacter polaris phage S482 in Example 6; where A is the test result of Pectinobacter brasiliensis SM19183, B is the test result of Pectinobacter nigra CD19130, and C is the test result of Pectinobacter polaris ZRIMU1222.

[0021] Figure 5 This is a phylogenetic tree diagram of Polar Pectinobacterium phage S482 in Example 7.

[0022] Figure 6 The graph shows the results of the potato chip experiment using Pectinobacter polaris phage S482 in Example 8; where A is the test result for Pectinobacter brasiliensis SM19183 as the pathogen, B is the test result for Pectinobacter nigra CD19130 as the pathogen, and C is the test result for Pectinobacter polaris ZRIMU1222 as the pathogen.

[0023] Figure 7 The figures shown are related results of the pot experiment of Pectinobacter polaris bacteriophage S482 in Example 8; where A is the test result of Pectinobacter brasiliensis SM19183 as the pathogen, B is the test result of Pectinobacter nigra as the pathogen, CD19130 as the pathogen, and C is the test result of Pectinobacter polaris ZRIMU1222 as the pathogen. Detailed Implementation

[0024] This invention provides Polar Pectinobacterium phage S482 and its applications. To make the objectives, technical solutions and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0025] Currently, the genus *Pectinobacter* is known to cause potato stem rot. Pectobacterium The main types of spp. include Pectobacterium atrosepticum, Pectobacterium brasiliense ,Pectobacterium carotovorum , Pectobacterium polaris , Pectobacterium parmentieri Different species exhibit significant differences in ecological adaptability, geographical distribution, and pathogenicity.

[0026] This invention provides Polar Pectinobacterium phage ( Pectobacterium polaris phage S482, the polar pectinobacterium phage S482, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46682, deposited on October 13, 2025, and classified as... Pectobacterium polaris phage .

[0027] Specifically, in this embodiment of the invention, *Pectobacterium polaris* phage S482 was isolated from soil samples from Xiertala, Hulunbuir City, Inner Mongolia Autonomous Region. Studies have found that *Pectobacterium polaris* phage S482, screened in this embodiment of the invention, is effective against various bacteria of the *Pectobacterium* genus (…). Pectobacterium brasiliense, Pectobacterium atrosepticum and Pectobacterium polaris Both showed good inhibitory effects, significantly reducing the disease index of potato soft rot and stem rot caused by Pectinobacterium, which is beneficial to improving the yield and quality of potato cultivation.

[0028] Based on the same inventive concept, embodiments of the present invention also provide a biocontrol composition comprising: Polar Pectinobacterium phage S482 as described in the first aspect.

[0029] In some embodiments, the biocontrol composition further includes, but is not limited to, biocontrol bacteria.

[0030] In some more specific embodiments, the biocontrol bacteria is Bacillus atrophus (Bacillus subtilis). Bacillus atrophaeus )4618, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34952 and deposit date of June 19, 2025.

[0031] In some embodiments, the biocontrol composition further includes, but is not limited to, chemical agents.

[0032] In some more specific embodiments, the chemical agent is thiamethoxam, but is not limited thereto.

[0033] In some embodiments, the biocontrol composition further includes, but is not limited to, agriculturally acceptable adjuvants.

[0034] In some more specific embodiments, the agriculturally acceptable adjuvant is selected from one or more of dispersants, stabilizers, fillers, and solvents, but is not limited thereto.

[0035] Based on the same inventive concept, embodiments of the present invention also provide the application of the above-described Polar Pectinobacterium phage S482 or the above-described biocontrol composition in the control of potato soft rot and / or stem rot.

[0036] In some embodiments, the soft rot and / or stem rot is caused by *Pectinobacillus polari* (… Pectobacterium polaris ), Brazilian pectinobacterium ( Pectobacterium brasiliense ) and black shank pectinobacter ( Pectobacterium atrosepticum One or more of the following can cause this, but are not limited to these:

[0037] The present invention will be further described below through specific embodiments.

[0038] The materials involved in the following embodiments are specifically as follows: The potato variety is Hissen 6.

[0039] Polar pectinobacterium ( Pectobacterium polaris ZRIMU1222 was isolated from Xining City, Qinghai Province in September 2022; *Braziliana pectinata* ( Pectobacterium brasiliense SM19183 was isolated from Sanming City, Fujian Province in March 2019; *Pectinobacillus niger* ( Pectobacterium atrosepticum CD19130 was separated from Changde City, Hunan Province in April 2019.

[0040] Polar pectinobacterium phage ( Pectobacterium polaris phage S482 was isolated from a soil sample from Xiertala, Hulunbuir City, Inner Mongolia Autonomous Region in September 2024. It was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.46682. The classification name is... Pectobacterium polaris phage .

[0041] Bacillus atrophus ( Bacillus atrophaeus 4618 was isolated in September 2022 from the rhizosphere soil of maize at the Inner Mongolia University Farm in Hohhot, Inner Mongolia Autonomous Region. It was deposited on June 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.34952. The classification name is... Bacillus atrophaeus The relevant published patents are ZL202510868676.0 and ZL 202510868671.8.

[0042] Nutrient agar (NA) medium is prepared with water and includes: 10 g / L tryptone, 0.5 g / L sodium chloride (NaCl), 3 g / L beef extract, and 15 g / L agar, with a pH of 7.0-7.5.

[0043] Nutrient Broth (NB) was prepared with water and contained: 10 g / L tryptone, 0.5 g / L sodium chloride (NaCl), and 3 g / L beef extract, with a pH of 7.0–7.5.

[0044] Water agar is prepared with water and contains 7 g / L of agar.

[0045] Phage buffer is prepared with water and includes: sodium chloride (NaCl) 8.74 g / L, magnesium chloride hexahydrate (MgCl2·6H2O) 2.04 g / L, calcium chloride (CaCl2) 2.22 g / L, and trisbase 6.02 g / L, with a pH of 7.0~7.5.

[0046] The methods for resuscitation and purification of all strains include: The bacterial strains used in the examples were all derived from a bacterial strain bank stored in a laboratory freezer at -80°C. First, the frozen strains were removed from -80°C and then slowly thawed overnight at -20°C. After thawing, the bacterial solution was streaked onto NA medium, dried, and then incubated at 28°C for 1-2 days. Once colonies had grown, single colonies were picked and purified until uncontaminated pure single colonies were obtained. After purification, purified single colonies were inoculated into NB medium and incubated on a shaker at 200 rpm / min at 28°C for 1 day. The resulting bacterial solution was used for subsequent research.

[0047] Example 1 This embodiment describes the isolation, purification, and preservation of *Pectinobacter polaris* bacteriophage S482, as detailed below: Soil samples were collected from potato-growing areas in Xiertala, Hulunbuir City, Inner Mongolia Autonomous Region. 3.5 g of soil sample was weighed and placed in an Erlenmeyer flask. 500 μL of *Pectinobacter polaris* ZRIMU1222 bacterial suspension and 6 mL of phage buffer (pH=7.5) were added. The mixture was thoroughly mixed and placed in a shaker at 200 rpm / min, incubated at 28℃ for 2–3 days. After incubation, the sample was removed and placed in a laminar flow hood for 20 min. The supernatant was filtered through a 0.22 μm sterile filter to obtain the filtrate.

[0048] The presence of bacteriophages in the obtained filtrate was detected by plating and spotting. 400 μL of the filtrate and 400 μL of *Pectinobacter polaris* ZRIMU1222 bacterial suspension were added to a centrifuge tube, mixed, and allowed to stand for 5 min. Then, 7 mL of 0.7% (w / v) water agar (45-50℃) was added, mixed thoroughly, and poured into NA medium for plating. For spotting, another 400 μL of *Pectinobacter polaris* ZRIMU1222 bacterial suspension was added to 7 mL of 0.7% (w / v) water agar (45-50℃), mixed thoroughly, and poured into NA medium. After standing for 5 min until the water agar solidified, 10 μL of the filtrate was spotted. The spotted double-layer plates were incubated at 28℃ for 1-2 days to observe for plaque formation. If clear plaques appeared, it indicated the presence of bacteriophages capable of lysing *Pectinobacter polaris* ZRIMU1222 in the filtrate, allowing for further purification.

[0049] Select a single phage plaque with typical morphology and clear edges from a double-layer plate and immerse it in phage buffer (pH=7.5). Thoroughly break up the agar block containing the phage plaque and vortex to fully release the phage into the buffer. Collect the lysis buffer and filter it through a 0.22 μm sterile filter for sterilization. Perform serial dilutions of the obtained phage filtrate and re-plate it onto the plate, then incubate at 28°C for 1–2 days. Repeat the above purification steps 3–4 times until a genetically stable *Pectinobacter polaris* phage, designated S482, is obtained. The purified *Pectinobacter polaris* phage S482 plaques are shown below. Figure 1 As shown in Figure A. From Figure 1 As shown in Figure A, the plaques of Pectinobacter polaris phage S482 are uniformly sized, transparent, and round, with a diameter of approximately 1.62 ± 0.05 mm (n = 6).

[0050] The purified *Pectinobacter polaris* phage S482 was co-cultured with *Pectinobacter polaris* ZRIMU1222 using the double-layer agar plate method described above. The upper agar layer was harvested, homogenized, and mixed with an appropriate amount of phage buffer (pH=7.5). The supernatant was centrifuged (10000g, 15 minutes, 4℃) and filtered through a 0.22μm sterile filter to obtain a suspension of *Pectinobacter polaris* phage S482, which was stored at 4℃ for short-term use. Separately, an appropriate amount of the *Pectinobacter polaris* phage S482 suspension was mixed with 40% (v / v) glycerol, transferred to cryovials, and stored at -80℃ for long-term preservation.

[0051] Example 2 This embodiment describes the morphological observation of Pectinobacter polaris phage S482, as follows: Morphological observation of *Pectinobacter polaris* phage S482 was performed using transmission electron microscopy (TEM). The sample preparation process is as follows: After amplifying the purified *Pectinobacter polaris* phage S482 using the double-layer plate method described in Example 1, DNase I and RNase A were added to the *Pectinobacter polaris* phage S482 suspension to adjust their final concentrations to 1 μg / mL. After thorough mixing, the mixture was incubated in a 37°C water bath for 30 min. Subsequently, the *Pectinobacter polaris* phage S482 suspension underwent salting-out precipitation: pre-weighed sodium chloride (29.2 g / 500 mL) was slowly added to the above system. After the sodium chloride was completely dissolved, the mixture was placed in an ice bath for 1 h. To remove cell debris, the mixture was centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was carefully transferred to a new centrifuge tube, and PEG8000 was slowly added to the supernatant to adjust its final concentration to 10% (m / v). After thorough mixing, the liquid was placed in an ice bath overnight for precipitation. The following day, the precipitated liquid was centrifuged at low temperature at 4℃ and 4000 rpm for 30 min. After centrifugation, the supernatant was discarded, and the bottom precipitate was retained. The precipitate was eluted with an appropriate amount of phage buffer and fully resuspended to obtain a suspension of *Pectinobacter polaris* phage S482. Subsequently, the suspension was extracted with chloroform. After extraction, the treated suspension was stored at 4℃ for later use.

[0052] A suspension of *Pectinobacter polaris* phage S482 was dropped onto a copper mesh and soaked for 5-8 minutes to allow the phage to fully adsorb onto the mesh surface. After soaking, excess unadsorbed liquid was blotted off with filter paper. The copper mesh containing the adsorbed *Pectinobacter polaris* phage S482 was then immersed in a 2% (w / v) phosphotungstic acid solution for negative staining for 1 minute. Excess staining solution was gently blotted off from the edges of the mesh with filter paper. After the mesh was allowed to air dry, it was placed under a transmission electron microscope to observe the morphological characteristics of *Pectinobacter polaris* phage S482 and typical morphologies were photographed and recorded. Specific results are shown below. Figure 1 As shown in B. From Figure 1 As shown in section B, the head of *Pectinobacter polaris* phage S482 has a hexagonal outline with a diameter of approximately 59.24 ± 0.14 nm (n=3); it also has a short tail with a length of approximately 12.8 ± 0.84 nm (n=3). According to the latest morphological classification standards released by the International Committee on Taxonomy of Viruses (ICTV) in 2024, *Pectinobacter polaris* phage S482 belongs to... Autographivirales head,Autonotataviridae division.

[0053] Example 3 This embodiment determines the host range of Polar Pectinobacterium phage S482, as detailed below: Three random bacterial strains collected from soils in different regions of China were used to determine the host range of *Pectinobacter polaris* phage S482 using a spotting method. 400 μL of the bacterial suspension was thoroughly mixed with 7 mL of 0.7% (w / v) water agar (45–50 °C), then slowly poured into NA medium and plated. After standing for 10 min until the upper layer of water agar was completely solidified, 10 μL of the purified *Pectinobacter polaris* phage S482 suspension was spotted onto the water agar surface. After the plate surface was allowed to air dry, it was placed in a 28 °C incubator for 1–2 days. The formation of phage plaques was observed, with three replicates for each strain. The appearance of phage plaques at the spotting site was recorded as "+", indicating that *Pectinobacter polaris* phage S482 could effectively lyse the test strain. Specific results are shown in Table 1.

[0054] Table 1. Host spectrum of Polar Pectinobacterium phage S482

[0055] Example 4 This example demonstrates the determination of the optimal multiple of infection (MOI) for Pectinobacter polaris phage S482, as detailed below: The host bacteria used include: *Braziliana brasiliensis* (… Pectobacterium brasiliense SM19183, Blackleg Pectinobacterium ( Pectobacterium atrosepticum CD19130 and Polar Pectin Bacillus ( Pectobacterium polaris ZRIMU1222. Before the experiment, the phage titer (PFU) of the *Pectinobacter polaris* phage S482 suspension and the total colony count (CFU) of the host bacterial culture were determined and serially diluted for later use. 100 μL of *Pectinobacter polaris* phage S482 suspension was mixed thoroughly with an equal volume of host bacterial culture at eight different MOI ratios: 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001. The mixture was then inoculated into 1.8 mL of NB medium and cultured with shaking at 28°C and 200 rpm / min for 4 h. After culture, the culture was filtered through a 0.22 μm sterile filter, and the filtrate was serially diluted, plated, and incubated at 28°C. The PFU values ​​were then counted, and the phage titer values ​​at each MOI gradient were calculated. Ultimately, the MOI parameter corresponding to the treatment group with the highest phage titer is the optimal multiplicity of infection between the phage and the host bacteria.

[0056] The results are as followsFigure 2 As shown. Figure 2 In the table, A represents the test results for *Pectinobacter brasiliensis* SM19183, B represents the test results for *Pectinobacter nigra* CD19130, and C represents the test results for *Pectinobacter polaris* ZRIMU1222; data are expressed as x±SE (n=6), and data points marked with different letters indicate significant differences. p <0.05). By Figure 2 As shown in section A, the MOI for the highest titer of *Pectinobacter polaris* phage S482 in *Pectinobacter brasiliensis* SM19183 was 0.01, corresponding to a titer of 1.62 × 10⁻⁶. 10 PFU / mL; by Figure 2 According to B, the MOI for the highest titer of *Pectinobacter polaris* phage S482 in *Pectinobacter niger* CD19130 was 0.01, corresponding to a titer of 4.67 × 10⁻⁶. 8 PFU / mL; by Figure 2 According to C, the MOI for the highest titer of *Pectinobacter polaris* phage S482 in *Pectinobacter polaris* ZRIMU1222 was 10, corresponding to a titer of 1.75 × 10⁻⁶. 6 PFU / mL.

[0057] Example 5 This embodiment measures the one-step growth curve of *Pectinobacter polaris* phage S482, as detailed below: The host bacteria used were the same as in Example 4. Before the experiment, the phage titer (PFU) of the *Pectinobacter polaris* phage S482 suspension and the total colony count (CFU) of the host bacteria suspension were determined to determine the optimal multiple of infection (MOU). 1 mL of the host bacteria suspension was taken, centrifuged, and resuspended. The resuspended suspension was then thoroughly mixed with 1 mL of *Pectinobacter polaris* phage S482 suspension according to the optimal MOU, and incubated at 28°C for 30 min to allow for complete adsorption of the host bacteria by *Pectinobacter polaris* phage S482. After incubation, the bacterial precipitate was collected by centrifugation and washed twice with NB medium to remove any unadsorbed free *Pectinobacter polaris* phage S482. 2 mL of the washed resuspension was mixed with 48 mL of NB medium and transferred to an Erlenmeyer flask. Immediately, 500 μL of the mixture was filtered through a 0.22 μm sterile filter as the starting sample. The remaining mixture was incubated in a shaker at 28℃ and 200 rpm / min. 500 μL samples were taken every 20 min and filtered promptly to serve as samples for the corresponding time points. The samples at each time point were serially diluted and the phage titer was determined using the double-layer agar plate method to plot a one-step growth curve, and the phage latency, lysis period, and outbreak rate were calculated accordingly.

[0058] The results are as follows Figure 3 As shown. Figure 3In the figures, A represents the test results for *Pectinobacter brasiliensis* SM19183, B represents the test results for *Pectinobacter nigra* CD19130, and C represents the test results for *Pectinobacter polaris* ZRIMU1222; data are expressed as x ± SE (n=6). Figure 3 As shown in section A, the latent period of polar pectinobacterium phage S482 in *Pectinobacter brasiliensis* SM19183 is approximately 20 minutes, followed by a rapid lysis phase. The titer plateaus at 100 minutes, with a final titer of approximately 10. 8 PFU / mL, calculated to have a burst rate of 286 PFU / infected cell; Figure 3 According to B, the latent period of polar pectinobacterium phage S482 in *Pectinobacter nigra* CD19130 is approximately 20 min, the lysis period lasts until 80 min before entering the plateau phase, and the final titer is approximately 10. 7 PFU / mL, calculated to have a burst dose of 33 PFU / infected cell; Figure 3 As shown in C, the latent period of Pectinobacter polaris phage S482 in Pectinobacter polaris ZRIMU1222 is approximately 40 min, the lysis period lasts until 100 min before entering the plateau phase, and the final titer is approximately 10. 7 The PFU / mL concentration yielded a calculated burst dose of 12 PFU / infected cell. In summary, the proliferation kinetics of *Pectinobacter polaris* phage S482 differed significantly across different host bacteria, indicating its distinct host specificity.

[0059] Example 6 This embodiment measures the lysis kinetics curve of Pectinobacter polaris phage S482, as detailed below: The host bacteria used were the same as in Example 4. Before the experiment, the host bacteria were brought to the logarithmic phase and their optical density (OD) values ​​were measured. The control group consisted of a single culture of the host bacteria, with the host bacterial suspension mixed 1:1 with NB medium. The treatment group consisted of co-cultured *Pectinobacter polaris* phage S482 and the host bacteria, with the host bacterial suspension mixed 1:1 with *Pectinobacter polaris* phage S482 suspension at the optimal multiple of infection. The mixtures were cultured in a shaker at 28°C and 200 rpm / min, and OD values ​​were measured every 3 hours. 600 The value was continuously monitored for 24 hours, and the pyrolysis kinetic curve was plotted.

[0060] The results are as follows Figure 4 As shown. Figure 4In the table, A represents the test results in *Pectinobacter brasiliensis* SM19183, where SM19183 is the control group cultured alone, and S482+SM19183 is the co-culture treatment group; B represents the test results in *Pectinobacter nigra* CD19130, where CD19130 is the control group cultured alone, and S482+CD19130 is the co-culture treatment group; C represents the test results in *Pectinobacter polaris* ZRIMU1222, where ZRIMU1222 is the control group cultured alone, and S482+ZRIMU1222 is the co-culture treatment group; data are expressed as x±SE (n=6). Figure 4 It can be seen that all three host bacteria can grow normally when cultured alone. The OD values ​​of *Pectinobacter brasiliensis* SM19183, *Pectinobacter nigra* CD19130, and *Pectinobacter polaris* ZRIMU1222 after 24 hours of individual culture were [not specified]. 600 The values ​​increased from the initial 0.28, 0.23, and 0.13 to 0.72, 0.61, and 0.55, respectively; while in the treatment groups with the addition of *Pectinobacter polaris* phage S482, the growth of all host bacteria was significantly inhibited, with the OD value of the S482+SM19183 treatment group being the highest. 600 The value rapidly decreased to 0.05 within 6 hours and remained at a low level. The OD values ​​of the S482+CD19130 and S482+ZRIMU1222 treatment groups... 600 The values ​​were consistently significantly lower than those of the corresponding control group, indicating that the polar pectinobacterium phage S482 had good lytic activity against all three host bacteria, and the inhibitory effect on Brazilian pectinobacterium SM19183 was the most significant.

[0061] Example 7 This embodiment performs a whole genome analysis of Pectinobacter polaris phage S482, as detailed below: I. Phage Genome Assembly Next-generation whole-genome sequencing of *Pectinobacter polaris* phage S482 was performed using the Illumina MiSeq platform. Soapnuke (v2.0.5) was used for quality control of the raw sequencing data to obtain high-quality clean reads. Subsequently, BWA (v0.7.17, default parameter: mem–k 30) software was used to align the clean reads to the host genome and remove host-derived sequences, ultimately obtaining 5358712 bp of phage sequencing data. Megahit software (v1.1.2, default parameters: --presets meta-large --min-contig-len 300) was used to assemble the data to obtain the complete sequence. The whole genome length of *Pectinobacter polaris* phage S482 is 41700 bp, and it was identified as a double-stranded DNA phage. Specific results are shown in Table 2.

[0062] Table 2. Genome assembly results of Pectinobacter polaris phage S482

[0063] II. Phage Phylogenetic Analysis Based on the genome sequence of Pectinobacter polaris phage S482, 14 strains of the genus Pectinobacter were screened. Pectobacterium The relevant phage sequence was obtained, and an Enterobacter phage strain was selected. Enterobacteria PhageBA14 was used as an outgroup, and a phylogenetic tree was constructed using the online tool GGDC, as follows: Figure 5 As shown. Figure 5 The annotation bars on the right, from left to right, represent the virus's classification at the family, genus, and species levels, its genomic GC content (46-52%), and its sequence length, respectively. Color differences distinguish their classification at the family, genus, and species levels, as well as differences in GC content; the same color indicates belonging to the same category. The length of the rightmost bar represents the genome length; a longer bar indicates a higher number of base pairs in the genome. Figure 5 It can be seen that S482 and Pectobacterium phagePP2 is located in the same evolutionary branch and is the most closely related.

[0064] Example 8 This embodiment evaluates the biocontrol efficacy of Pectinobacter polaris phage S482, as detailed below: I. Preparation of bacterial culture The pathogens used include: *Braziliana brasiliensis* (… Pectobacterium brasiliense SM19183, Blackleg Pectinobacterium ( Pectobacterium atrosepticum CD19130 and Polar Pectin Bacillus ( Pectobacterium polaris ZRIMU1222. Single colonies of the resuscitated and purified pathogen were cultured in NB medium. The concentration of the bacterial suspension was then determined using the dilution plate method. After centrifugation, the supernatant was discarded, and the suspension was resuspended in sterile water. The final concentration was adjusted to 10. 8 CFU / mL.

[0065] The biocontrol bacteria is Bacillus atrophus ( Bacillus atrophaeus 4618. Single colonies of the revived and purified biocontrol bacteria were cultured in NB medium. The concentration of the bacterial suspension was then determined using the dilution plate method. After centrifugation, the supernatant was discarded, and the suspension was resuspended in sterile water. The final concentration was adjusted to 10. 8 CFU / mL.

[0066] II. Preparation of bacteriophage suspension The titer of Pectinobacter polaris phage S482 was determined. After centrifugation, the supernatant was removed, and the phage was resuspended in phage buffer. The phage concentration was adjusted according to the optimal MOI for each bacterial culture.

[0067] III. Preparation of Chemical Reagents Commercially available thiamethoxam fungicide was used as the chemical agent. Its active ingredient is 3% (w / v) thiamethoxam, and the formulation is water-dispersible granules. The thiamethoxam fungicide was diluted 500 times before use in the experiment.

[0068] IV. Potato Chip Experiment The effects of Polar Pectinobacterium phage S482 on Pectinobacter spp. were evaluated using a potato chip experiment. Pectobacterium The control effects of spp. on bacterial soft rot are as follows: Select healthy potato tubers of uniform size. First, wash off the soil adhering to the surface, dry them, and then spray them with 75% (v / v) alcohol for disinfection. Place the treated potatoes in a sterile workbench and irradiate them under ultraviolet light along with a fruit knife and work board for further sterilization. Cut the sterilized potatoes into slices about 1 cm thick and place them in a petri dish lined with moist sterile filter paper.

[0069] The experimental grouping included four conditions: (1) Single pathogen treatment group, abbreviated as H; (2) The pathogen and the polar pectin bacteriophage S482 were mixed in equal volumes and treated as H+P; (3) The group treated with only Polar Pectinobacterium phage S482 was abbreviated as P; (4) Sterile water blank control group, abbreviated as blank control.

[0070] 15mm diameter circular filter paper discs were pre-soaked in the liquids used in each group. Then, using sterile forceps, the soaked filter paper discs were placed in the center of potato slices and placed in an incubator set at 28℃ for 48 hours. Disease development was observed, the area of ​​lesions on the potato slices was measured, and statistical graphs were plotted. Each group was set up with three replicates, and the entire experiment was repeated twice.

[0071] The results are as follows Figure 6 As shown. Figure 6 In the table, A represents the test results for *Pectinobacter brasiliensis* SM19183, B represents the test results for *Pectinobacter nigra* CD19130, and C represents the test results for *Pectinobacter polaris* ZRIMU1222. Data are expressed as x±SE (n=6), and data points marked with different letters indicate significant differences. p <0.05).

[0072] Depend on Figure 6It can be seen that all groups treated with individual pathogens exhibited obvious soft rot symptoms. The average lesion area in the groups treated with *Pectinobacter brasiliensis* SM19183, *Pectinobacter nigra* CD19130, and *Pectinobacter polaris* ZRIMU1222 was approximately 10 cm². 2 7cm 2 and 5cm 2 However, after treatment with *Pectinobacter polaris* phage S482, the lesion area in each treatment group was significantly reduced, decreasing to approximately 3 cm², 3 cm², and 4 cm², respectively. p <0.05); no symptoms of disease were observed in either the phage-only treatment group or the blank control group. In conclusion, *Pectinobacter polaris* phage S482 itself is non-pathogenic to potato tissue and can effectively inhibit the pathogenicity of the three *Pectinobacter* strains.

[0073] V. Potted Plant Experiment Pot experiments were conducted to evaluate the effects of Polar Pectinobacter phage S482 alone and in combination with biocontrol agents and chemical agents on Pectinobacter spp. ( Pectobacterium The control effects of spp. on potato aerial stem rot are as follows: After sprouting the potato seed tubers, they were planted in flowerpots with a diameter of 21cm. Healthy potato plants with a height of about 30cm were selected for phage control experiments.

[0074] The experimental grouping included ten conditions: (1) Single pathogen treatment group, abbreviated as H; (2) The pathogen and the polar pectin bacteriophage S482 were mixed in equal volumes and treated as H+P; (3) The pathogen and biocontrol bacteria are mixed in equal volumes and the treatment group is abbreviated as H+B; (4) The pathogen and chemical agent are mixed in equal volumes for treatment, abbreviated as H+C; (5) The treatment group consisting of equal volumes of pathogens, polar pectin bacteriophage S482 and chemical agents is abbreviated as H+P+C; (6) The treatment group consisting of equal volumes of pathogens, polar pectin bacteriophage S482 and biocontrol bacteria is abbreviated as H+P+B; (7) The group treated with only Polar Pectinobacterium phage S482 was abbreviated as P; (8) The group treated with chemical agents alone is abbreviated as C; (9) The single biocontrol bacteria treatment group is abbreviated as B; (10) Sterile water blank control group, abbreviated as blank control.

[0075] In all single-treatment groups and the control group, 100 μL of liquid was injected into the base of the plant stem using a 1 mL syringe. For the mixed-treatment group, 100 μL of pathogenic bacterial solution was first injected into the stem base, and 4 hours later, 100 μL of control agent (Polar Pectinobacterium phage S482 suspension, biocontrol bacterial solution, and chemical agent) was injected along the previously injected site. After all injections were completed, the upper part of the plant was covered with a bag to maintain humidity. The disease status of the plants was observed, and data were recorded and photographed after 48 hours to calculate the disease index. The experimental plants in the mixed-treatment group numbered 30, while those in the single-treatment group and the control group numbered 10 each. The entire experiment was repeated twice.

[0076] Wherein, the disease index = [∑(number of diseased plants at each level × representative value of that level) / (total number of plants surveyed × 9)] × 100; The disease grading method is as follows: Grade 0: The entire plant is disease-free; Grade 1: Stem lesions do not exceed one-third of the stem circumference, and / or individual leaves wilt; Grade 3: Stem lesions do not exceed half of the stem circumference, and / or less than half of the leaves are slightly wilted, or / and a few lower leaves have lesions; Level 5: Stem lesions cover more than half of the stem circumference, and / or more than half of the leaves show mild wilting; Grade 7: Stem lesions surround the stem circumference, and / or more than two-thirds of the leaves wither; Level 9: All leaves of the diseased plant wither or die.

[0077] The results are as follows Figure 7 As shown. Figure 7 In the table, A represents the test results for *Pectinobacter brasiliensis* SM19183, B represents the test results for *Pectinobacter nigra* CD19130, and C represents the test results for *Pectinobacter polaris* ZRIMU1222. Data are expressed as x±SE, and data points marked with different letters indicate significant differences. p <0.05).

[0078] Depend on Figure 7 It was found that the disease indices of *Pectinobacter brasiliensis* SM19183, *Pectinobacter niger* CD19130, and *Pectinobacter polaris* ZRIMU1222 treated individually were 66.67, 65.74, and 45.99, respectively. The disease indices of all mixed treatment groups were significantly reduced, with the mixed treatment group (H+P) of the pathogen and *Pectinobacter polaris* bacteriophage S482 showing the best effect, with disease indices decreasing to 19.26, 23.15, and 21.48, respectively, all significantly lower than their respective pathogen control groups. p<0.05); no symptoms were observed in the treatment groups treated with only *Pectinobacter polaris* phage S482, biocontrol bacteria, chemical agents, or the blank control group. In conclusion, *Pectinobacter polaris* phage S482 can effectively inhibit the pathogenicity of the three pathogens, and its control effect when used alone is superior to combined treatment with biocontrol bacteria or chemical agents.

[0079] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Polar pectinobacterium phage ( Pectobacterium polaris phage S482, characterized in that, The polar pectinobacterium phage S482 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46682 and deposit date of October 13, 2025.

2. A biological control composition, characterized in that, The biocontrol composition comprises: Polar Pectinobacterium phage S482 as described in claim 1.

3. The biological control composition according to claim 2, characterized in that, The biocontrol composition also includes: biocontrol bacteria.

4. The biological control composition according to claim 3, characterized in that, The biocontrol bacteria is Bacillus atrophus ( Bacillus atrophaeus )4618, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34952 and deposit date of June 19, 2025.

5. The biological control composition according to claim 2, characterized in that, The biological control composition also includes chemical agents.

6. The biological control composition according to claim 5, characterized in that, The chemical agent is thiamethoxam.

7. The biological control composition according to claim 2, characterized in that, The biological control composition also includes agriculturally acceptable adjuvants.

8. The biological control composition according to claim 7, characterized in that, The agriculturally acceptable adjuvants are selected from one or more of dispersants, stabilizers, fillers, and solvents.

9. The application of the *Pectinobacterium polaris* bacteriophage S482 as described in claim 1 or the biocontrol composition as described in any one of claims 2 to 8 in the control of potato soft rot and / or stem rot, characterized in that, The soft rot and / or stem rot are caused by Polar pectinobacter ( Pectobacterium polaris ), Brazilian pectinobacterium ( Pectobacterium brasiliense ) and black shank pectinobacter ( Pectobacterium atrosepticum One or more of the following can cause this.

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