Phage MA9V-2 and application thereof

Abstract

The invention discloses a bacteriophage MA9V-2 and application thereof, the bacteriophage MA9V-2 is classified and named as Chryseobacteria inogenes phage MA9V-2, the preservation number is CCTCC NO: M2024593, and the bacteriophage MA9V-2 is preserved in China Center for Type Culture Collection on April 1, 2024. Wuhan. Wuhan University), the bacteriophage can be used for cracking to produce the chrysobacter indole-3 MA9. Particularly, the bacteriophage MA9V-2 is widely applied to preparation of biological agents and can be used for preparing the biological agents for panax notoginseng bacterial root rot, preparations for producing chrysobacter indoleformans MA9 and preparations for preventing and controlling panax notoginseng root rot, and the biological agents contain the bacteriophage MA9V-2 and pharmaceutically acceptable carriers, so that the bacteriophage MA9V-2 can be applied to preparation of the biological agents for panax notoginseng bacterial root rot. The optimal infection complex number MOI of the bacteriophage MA9V-2 is 0.01, a new efficient biological prevention and treatment means is provided for prevention and treatment of panax notoginseng root rot, the use of chemical agents is expected to be reduced, and healthy development of the panax notoginseng planting industry is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to a pathogenic bacterium of Panax notoginseng, MA9 bacteriophage and its application, especially bacteriophage MA9V-2 and its application in the prevention and control of root rot of Panax notoginseng. Background Technology

[0002] Panax notoginseng, a unique economic crop in Yunnan, has roots and rhizomes containing various active pharmacological components, thus possessing extremely high medicinal value and bringing significant economic benefits to local farmers. However, the disease problems caused by continuous cropping obstacles have been hindering the healthy development of this industry, with root rot being the most prominent and causing the greatest losses.

[0003] The infection and pathogenesis of root rot in Panax notoginseng is an extremely complex process, with the combined incidence of fungal, bacterial, and nematode infections being far higher than that of a single pathogen. Current research primarily focuses on root rot caused by fungal pathogens, but the isolation and identification of the bacterial pathogen MA9 for Panax notoginseng root rot indicates that bacterial infection will also be a challenge that the Panax notoginseng industry must address for its healthy development. While traditional methods such as antibiotic treatment are relatively mature for bacterial infections, these methods are prone to problems such as excessive pesticide residues and increased drug resistance in pathogens, which have become a bottleneck in medical and agricultural antibacterial control. Phage therapy, on the other hand, is gaining increasing attention due to its environmental friendliness and low likelihood of inducing drug resistance. However, research on the bacterial pathogenicity of Panax notoginseng is relatively lagging, still in the early stages of pathogen screening and identification, and there have been no reports of phage therapy. Summary of the Invention

[0004] For the reasons mentioned above, this application uses MA9, a bacterial pathogen causing root rot in Panax notoginseng, as the host. A double-layer agar plate method was used to screen phages purified from different samples, identifying phages capable of inhibiting indole-producing bacillus MA9 for the prevention and control of root rot in Panax notoginseng. The specific details are as follows:

[0005] A bacteriophage MA9V-2 strain, classified as Chryseobacterium indologenesphage MA9V-2, with accession number CCTCC NO:M2024593, was deposited on April 1, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0006] Furthermore, the bacteriophage Chryseobacterium indologenes phage MA9V-2 exhibits lytic activity against indol-producing Chryseobacterium MA9.

[0007] Furthermore, the bacteriophage Chryseobacterium indologenes phage MA9V-1 exhibits lytic activity against indologen-producing Chryseobacterium MA9 at temperatures ranging from 4 to 40°C and at pH values ​​ranging from 4 to 10.

[0008] Application of bacteriophage MA9V-2 in the preparation of biological agents for bacterial root rot of Panax notoginseng.

[0009] Furthermore, the application of the bacteriophage MA9V-2 in the preparation of biological agents that inhibit indophora-producing bacillus MA9.

[0010] Furthermore, the application of the bacteriophage MA9V-2 in the preparation of a formulation for preventing root rot in Panax notoginseng.

[0011] Furthermore, the application of the bacteriophage MA9V-2 in the preparation of agents to control root rot in Panax notoginseng.

[0012] Furthermore, the biological agent comprises bacteriophage MA9V-2 and a pharmaceutically acceptable carrier.

[0013] Furthermore, the optimal multiplicity of infection (MOI) for bacteriophage MA9V-2 in the biological agent is 0.01.

[0014] Furthermore, the pH of the biological preparation is 4–10.

[0015] The working mechanism of the invention:

[0016] A lytic bacteriophage, vB_CinP_MA9V-2 (MA9V-2), was isolated using pathogenic bacterium MA9 as the host. The adsorption time for MA9V-2 to reach 75% was 25 min. Differences in adsorption rates resulted in a titer of 9.85 × 10⁻⁶ for MA9V-2 at the optimal multiple of infection (MOI) of 0.01. 9 With a latency of 30 PFU / mL, a lysis time of 100 min, and a lysis rate of ~6 PFU / cell, the short lifespan of the phage allows for rapid infection and lysis of MA9. It exhibits excellent acid-base and temperature stability, remaining stable within a pH range of 4–10 and maintaining lytic activity at temperatures ranging from 4 to 60°C. This characteristic not only closely matches the optimal pH of MA9 (7.2) and the optimal temperature of 28°C, ensuring optimal infection conditions for the phage, but also broadens its lytic spectrum. Furthermore, MA9V-2 can infect strains C. indologenes 02, 06, and 04, and this broad host spectrum allows the phage to maintain lytic activity even when facing evolving host resistance.

[0017] Methods such as root cuttings, root pathogenicity experiments, and CFU values ​​of pathogenic bacteria in experimental samples determined by dilution and spread assays showed that MA9 exhibits strong pathogenicity in in vitro root experiments, with a pathogenicity rate of approximately 75%. In potted root pathogenicity experiments, *Pseudomonas syringae* (PSS), a plant pathogen, and *Bacillus cereus* (BYM 41-22), a non-pathogenic bacterium, were used as control groups. The pathogenicity of these pathogens to *P. cereus* plants was further tested using spraying and root drenching methods, and the pathogenicity rate still reached 80%. This indicates that MA9 has a more prominent pathogenicity in potted experiments. Therefore, different treatments with bacteriophage MA9V-2 showed that it exhibited certain preventive and control effects in both the prevention and control groups, and can be used to prevent and treat bacterial root rot in *P. cereus*. Specifically, in the prevention experiment, the incidence rate after treatment with bacteriophage MA9V-2 was 16.7%, while the incidence rate in the control group was 72.1%, which was significantly lower than the 83.33% in the control group.

[0018] Compared with the prior art, the beneficial effects of the present invention are: using the bacterial pathogen C. indologenes MA9, which causes root rot of Panax notoginseng, as the host, the lytic bacteriophage MA9V-2 was screened for the first time and used to prepare a biological agent for the prevention and control of root rot of Panax notoginseng. Attached Figure Description

[0019] Figure 1 This is a morphological observation of bacteriophage MA9V-2;

[0020] A: Phage plaques formed by MA9V-2 and transmission electron microscopy image; B: Transmission electron microscopy observation of MA9V-2 adsorbed on the surface of host MA9 cell membrane.

[0021] Figure 2 The optimal infection multiple for bacteriophage MA9V-2.

[0022] Figure 3 The image shows the adsorption rate curve of bacteriophage MA9V-2.

[0023] Figure 4 This is the one-step growth curve of bacteriophage MA9V-2.

[0024] Figure 5 Temperature (A) and pH stability (B) of bacteriophage MA9V-2.

[0025] Figure 6 Experiment on the prevention of root rot of Panax notoginseng by bacteriophage MA9V-2.

[0026] A: Spray 20 mL of NA liquid culture medium as a blank control; B: Spray 20 mL of MA9 as a positive control group; C: Spray 20 mL of a mixture of bacteriophage MA9V-2 and the host as a prevention experimental group at MOI = 0.01.

[0027] Figure 7 Figure 1 shows the experimental results of bacteriophage MA9V-2 controlling root rot lesions in Panax notoginseng.

[0028] A: Spray 20 mL of NA liquid culture medium as a blank control; B: First, spray MA9 in the logarithmic stage to induce disease in Panax notoginseng plants, then spray sterilized MA9 bacterial solution in the exponential stage as a positive control group; C: First, spray MA9 in the logarithmic stage to induce disease in Panax notoginseng plants, then spray 20 mL of a mixture of bacteriophage MA9V-2 and NA as a control experimental group.

[0029] Figure 8 Results of pathogen and bacteriophage assays on leaves of Panax notoginseng plants (P<0.05);

[0030] A: Pathogenic bacteria on the leaves of Panax notoginseng plants in the prevention group; B: Detection of bacteriophage count in the prevention group (P<0.05)

[0031] C: Pathogenic bacteria in the leaves of Panax notoginseng plants in the control group; D: Bacteriophage count in the control group (P<0.05)

[0032] Note: NA: NA liquid blank group; PT: positive control group; MT2: MA9V-2 control experiment.

[0033] Figure 9 The statistical results of the incidence and control rates of diseases in the potted Panax notoginseng disease prevention group and control group (P<0.05);

[0034] A: Incidence rate in the prevention group; B: Control rate in the prevention group; C: Incidence rate in the control group; D: Control rate in the prevention group;

[0035] Note: NA: NA liquid blank group; PT: positive control group; MT2: MA9V-2 control experiment.

[0036] Figure 10 This is the complete genome map of MA9V-2. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, in the following descriptions, well-known structures and techniques have been omitted to avoid unnecessarily obscuring the concept of the invention.

[0038] The strain involved in this invention experiment, Chryseobacterium indologenes MA9, was deposited at the China Center for Type Culture Collection (CCTCC) on April 1, 2024. The depository address is Wuhan University, Wuhan, China, and the accession number is CCTCC NO:M 2024594.

[0039] This bacteriophage, Chryseobacterium indologenes phage MA9V-2, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M2024593, on April 1, 2024.

[0040] The bacterial pathogens causing root rot in Panax notoginseng, namely MA9, Chryseobacterium indologenes ATCC 29897, Bacillus cereus MYB41-22, Pseudomonas syringae CGMCC 1.3070, and Escherichia coli ATCC 11303, were all standard strains purchased from a bacterial culture center. C. indologenes 01, 02, 03, and 04 were screened from soil containing Panax notoginseng with root rot, while C. indologenes 05 and 06 were screened from sewage.

[0041] Example 1: Isolation and Identification of Bacteriophage MA9V-2

[0042] Phage screening was performed using glucose-free NA medium, and subsequent experiments also used glucose-free NA medium. The specific experimental procedures are as follows:

[0043] Solid samples: After grinding the sample into powder, add it to an Erlenmeyer flask containing 100 mL of SM suspension and let it stand at room temperature for 24 h;

[0044] Liquid Samples: Wastewater samples and SM suspension were added to a 250 mL Erlenmeyer flask at a 1:1 ratio (50 mL:50 mL), mixed thoroughly, and allowed to stand at room temperature for 24 h. Single colonies of MA9 were selected and cultured overnight in 5 mL of NA liquid medium at 28 °C and 180 rpm on a shaker. The next day, 2 mL of the overnight MA9 culture, 5 mL of SM suspension, and 10 mL of the treated sample solution were mixed, and the volume was increased to 50 mL with NA liquid medium. The mixture was then incubated overnight under the same conditions. After incubation, the overnight culture was centrifuged at 15000 × g at 4 °C for 15 min. The supernatant was filtered through a sterile 0.22 μm filter membrane, and the filtrate was stored at 4 °C. MA9 culture medium was added to 50 mL of NA liquid medium at a 2% inoculum level. The culture was incubated under the same conditions until the OD600 absorbance value reached between 0.6 and 0.8. Then, 5 mL of the above filtrate was added, and the culture was incubated overnight at 28°C and 180 rpm in a shaker. The culture medium was then treated under the same centrifugation conditions to obtain the second enriched filtrate. Similarly, the culture volume was further increased, and a third phage enrichment culture was performed using 100 mL of NA liquid medium. After filtration, pure phage culture was obtained and stored at 4°C.

[0045] Phage purification and optimization of infection conditions

[0046] Using pathogenic bacterium MA9 as the host, a highly lytic bacteriophage was isolated from sewage using the double-layer agar plate method. Following the scientific nomenclature of the International Committee on Taxonomy of Viruses (ICTV), it was named vB_CinP_MA9V-2, which is abbreviated as bacteriophage MA9V-2 in this paper. The optimal lysis conditions for the bacteriophage were determined as follows: a bacteriophage-to-host incubation ratio of 200:300 μL, an incubation time of 15 min at 28°C, and a mixture-to-semi-solid culture medium ratio of 1:8.

[0047] The results are as follows Figure 1 As shown in Figure A, the "head" of bacteriophage MA9V-2 has a diameter of approximately 112.62 nm, and its retractable tail measures approximately 165.39 nm in length and 40.72 nm in width. This retractable tail undergoes a conformational change upon attachment to the host cell membrane, forming a channel that allows the bacteriophage genome to be injected into the host cytoplasm. According to the latest ICTV classification system, bacteriophage MA9V-2 belongs to the Myoviridae family, specifically the Myotail phage. Furthermore, TEM clearly shows bacteriophage MA9V-2 adsorbing onto the surface of the host MA9 cell membrane. Figure 1 B) indicates that the phage adsorption receptor is located on the surface of the host cell membrane, while the thick capsule on the host surface hinders the phage selection, resulting in the intermittent appearance of the phage during the phage selection process.

[0048] Phage concentration, gradient centrifugation and electron microscopic morphological observation

[0049] Phage Concentration and Density Gradient Centrifugation

[0050] Enrichment of phage lysate

[0051] A single MA9 colony was picked from a petri dish and inoculated into a test tube containing 5 mL of NA liquid medium. The culture was then incubated at 28°C in a shaker for 12 h. The next day, the seed culture was transferred to 500 mL of NA medium at an inoculation rate of 2%. The culture was then incubated under the same conditions until the logarithmic growth phase. Phage filtrate was added at the optimal multiplicity of infection, and the culture was continued for 12 h to obtain a lysate containing phages.

[0052] Polyethylene glycol (PEG) precipitation enrichment of bacteriophages

[0053] 500 mL of phage lysis buffer was allowed to stand, then DNase I and RNase A were added to the lysis buffer to a final concentration of 1 μg / mL, and incubated at 37°C for 30 min. The purpose was to digest the nucleic acid fragments in the lysis buffer and prevent the phages from being adsorbed by nucleic acid mucus.

[0054] Add 29.2g of sodium chloride at a ratio of 5.84g of solid sodium chloride (final concentration of 1mol / L) per 100mL of mixed solution and stir gently until completely dissolved. Then place in an ice bath for 1 hour to utilize sodium chloride to promote the separation of bacteriophage particles from cells.

[0055] Centrifuge the above mixture at 4°C and 11000×g for 10 min to remove cell debris, collect the supernatant, measure the volume of the supernatant using a graduated cylinder, and transfer it to a 2L conical flask.

[0056] Add PEG 8000 at a ratio of 10% (m / v) and stir slowly with a magnetic stirrer to fully dissolve PEG 8000.

[0057] The mixture was placed in ice water again and left to stand overnight at 4°C, allowing PEG 8000 to adsorb the phage particles and precipitate at the bottom of the conical flask.

[0058] The next day, the above-treated solution was centrifuged at 11000×g for 15 minutes at 4°C. The supernatant was discarded, the centrifuge tube was inverted to allow the supernatant to drain completely, and the centrifuged phage precipitate was collected.

[0059] Chloroform extraction of bacteriophages

[0060] Gently resuspend the precipitate at the bottom of the centrifuge tube with SM buffer (add 3 mL of SM buffer per 100 mL of mixture) and incubate at room temperature for 1 hour to allow the phages to be completely suspended in the buffer.

[0061] Add the same volume of chloroform as the suspension, slowly invert for 30 seconds, and centrifuge at 3000×g for 10 minutes at 4°C to recover the hydrophilic phase containing phage particles and remove PEG and cell debris from the suspension.

[0062] The suspension is extracted multiple times with chloroform. The number of extractions depends on the clarity of the aqueous phase, generally 3 to 5 times. The suspension is then stored at 4°C.

[0063] CsCl2 density gradient centrifugation to purify bacteriophages

[0064] Using SM suspension as a solvent, three different concentrations of CsCl2 solution (1.45, 1.50, and 1.70 g / mL) were prepared. 1.5 mL of CsCl2 solution was added to an ultracentrifuge tube in descending order of concentration.

[0065] Measure the volume of the hydrophilic phase after extraction, add CsCl2 at a ratio of 0.5 g / mL, and gently stir with a pipette tip to dissolve CsCl2. Then add 4.5 mL of the solution to the top layer of the centrifuge tube.

[0066] After centrifuging at 150,000×g for 10 hours at 4℃, a very obvious white flocculent layer will be observed. Use a 1mL syringe to puncture the outside of the centrifuge tube and aspirate about 0.5 to 1mL of the phage from the separated layer into a 2mL centrifuge tube. Wrap the tube with sealing film and store at 4℃.

[0067] morphological observation of bacteriophages under electron microscopy

[0068] The morphology of the purified phage particles was observed using a transmission electron microscope. 10 μL of the concentrated, ultrapure phage solution was added to a carbon-coated copper grid for adsorption for 10–15 min. After complete adsorption, 2% phosphotungstic acid was added for staining for 5–10 min. After air drying, the phages were observed using a transmission electron microscope (Hitachi HT7820) at 120 kV biovoltage.

[0069] TEM morphological characteristics of lysed bacteriophage MA9V-2 Figure 1A) Observations show that it has a protein "head" enclosed by an icosahedron with a diameter of approximately 121.26 nm. Studies have shown that the head of myotail bacteriophage has an icosahedral structure, which not only gives the bacteriophage its unique appearance but also endows it with specific biological functions. Its main function is to protect the genetic material inside the bacteriophage, ensuring its stability during entry into the host cell and in the external environment. This genetic material is crucial for the survival and reproduction of the bacteriophage. In addition, the retractable tail of bacteriophage MA9V-2 has dimensions of approximately 170.34 nm in length and approximately 44.62 nm in width. This retractable tail undergoes a conformational change when the bacteriophage attaches to the host cell membrane, forming a channel that allows the bacteriophage genome to be injected into the host cytoplasm. According to the latest ICTV classification system, bacteriophage MA9V-2 belongs to the Myotail bacteriophage family.

[0070] Optimal infection multiple

[0071] The optimal multiplicity of infection (MOI) refers to the optimal ratio of phage particles to host cells at the time of infection. MA9 was diluted to 10^6 using a serial dilution method. -4 10 -5 10 -6 The CFU value of phages during the exponential growth phase was determined using the spread plate method. Phage filtrate with known titers was mixed with host cells in the exponential phase at multiples of infection of 0.001, 0.01, 0.1, 1, and 10, respectively, and incubated for 12 h at 180 rpm and 28 °C in a shaker. After enrichment, the phages were centrifuged at 15,000 × g for 15 min, filtered through a 0.22 μm filter, and the phage titer was determined using the double-layer agar plate method. The experiment was repeated three times. The formula for calculating the titer is as follows:

[0072] Phage titer (PFU / mL) = 1000 / volume of phage added (μL) × average number of plaques × dilution factor (PFU: plaque forming unit).

[0073] The results are as follows Figure 2 As shown, when the MOI is 0.01, the highest phage titer of MA9V-2 is approximately 9.84 × 10⁻⁶. 9 The MOI value for bacteriophage MA9V-2 is 0.01, which is significantly lower than other MOI values. Therefore, the optimal MOI for MA9V-2 is 0.01, which is of great significance for understanding the infection efficiency of bacteriophages and developing effective bacteriophage biological agents to control pests and diseases.

[0074] Adsorption rate determination

[0075] Adsorption rate, which is the ratio of phage particles adsorbed onto host cells within a specific time period, is plotted as an adsorption rate curve for phage MA9V-2, with the percentage of adsorption rate as the ordinate and time as the abscissa. Figure 3 As shown in the figure, bacteriophage MA9V-2 can rapidly adsorb onto the surface of the host cell membrane. Within 8 minutes, >75% of the bacteriophage particles are adsorbed, and after 12 minutes, about 90% of the bacteriophage particles are adsorbed. This indicates that bacteriophage MA9V-2 has a high adsorption efficiency, and the high adsorption rate allows the bacteriophage to effectively recognize and infect the host bacteria.

[0076] Further analysis of the growth curve revealed that the latency, ascent phase, and lysis rate of bacteriophage MA9V-2 were 30 min, 100 min, and ~6 PFU / cell, respectively, indicating that MA9V-2 possesses a strong lytic ability against the host. Figure 4 Furthermore, among 11 different host strains, MA9V-2 could infect not only *C. indologenes* MA9, *C. indologenes* 02, and *C. indologenes* 06, but also *C. indologenes* 04, demonstrating a broad host spectrum for this bacteriophage. *C. indologenes* 05 and 06 were both screened from hospital sewage, and both bacteriophages could infect *C. indologenes* 06. These results provide a data basis for the clinical application of bacteriophages (Table 3.2). In addition, neither bacteriophage infects other bacteria across genera, indicating the high specificity of the bacteriophages and ensuring they do not affect other microorganisms in their application for disease control. The significance of determining the host spectrum range of bacteriophages lies in confirming their infectivity and providing important evidence for subsequent bacteriophage-based disease control research.

[0077] Table 3.2 phage host spectrum range

[0078]

[0079]

[0080] In vitro lysis experiment of bacteriophage MA9V-2

[0081] Plot the temperature and pH stability curves of MA9V-2 with the phage infection rate as the ordinate. Figure 5 The results show that the infectivity of bacteriophage MA9V-2 is only affected under extreme temperature and pH conditions, specifically between 4 and 40°C. Figure 5 A) and pH 4–10 ( Figure 5B) It remains active within a certain range, has a wide pH and temperature adaptability, and closely matches the optimal pH=7.2 and optimal temperature 28℃ of the host MA9, ensuring the best infection conditions for the phage and maximizing its lysis activity.

[0082] A single MA9 colony was picked from a Petri dish and inoculated into a test tube containing 5 mL of NA liquid medium. The culture was incubated overnight at 28°C with a shaker for 12 h. The next day, the seed culture was transferred to 50 mL of NA medium at a 2% inoculation rate and cultured under the same conditions until the logarithmic growth phase. Then, bacteriophage MA9V-2 was added at different ratios (MOI = 0.001, 0.01, 0.1, 1, 10), and cultured under the same conditions. The OD value was measured after the addition of the bacteriophage. 600 The initial value was 0h. Subsequently, 2mL of culture medium was collected into centrifuge tubes every 1h, and the absorbance value was measured using a spectrophotometer. Each group was tested in triplicate. The host culture medium without phages had an MOI of 0 as a control. The experiment was repeated 3 times.

[0083] At MOI values ​​of 0.001 and 0.01, the growth capacity of MA9 was not significantly inhibited until 2 hours later, when the MOI=0.01 curve dropped rapidly and then rebounded at a relatively fast rate. Similarly, at MOI=0.001, the same phenomenon occurred after 3 hours. However, under other MOI conditions, the opposite was observed: after phage addition, OD... 600 The absorbance value decreased sharply, but the decrease was relatively gradual, which is suspected to be caused by the addition of bacteriophage leading to a decrease in absorbance value; then growth slowly resumed over 2 hours until the end of the growth cycle. Overall, the addition of bacteriophage at different MOI values ​​inhibited the growth of MA9 to some extent, and this inhibition continued until the end of the lysis curve determination, with the strongest inhibitory effect observed at MOI = 0.01.

[0084] In summary, (1) using C. indologenes MA9 as the host, vB_CinP_MA9V-2 (MA9V-2) was isolated from sewage samples and formed clear circular patches with a diameter of about 0.4 to 1.5 mm in double-layer agar plates. TEM observation showed that the phage had a "head" wrapped with icosahedral protein and a contractile "tail". The diameter was about 112.62 nm, the tail length was about 165.39 nm, and the width was about 40.72 nm. According to the latest ICTV classification system, it was identified as Myoviridae phage. The adsorption time of MA9V-2 to 75% was 25 min. The difference in adsorption rate led to the titer of MA9V-2 being 9.85 × 10⁻⁶ under the condition of optimal multiple of infection (MOI) of 0.01. 9The latency, lysis time, and lysis rate of bacteriophage MA9V-2 are 30, 100 min, and ~6 PFU / cell, respectively. Its short lifespan allows for rapid infection and lysis of MA9 cells. Furthermore, it exhibits excellent acid-base and temperature stability, remaining stable within a pH range of 4–10 and maintaining activity at temperatures between 4 and 60°C. This characteristic not only closely matches the optimal pH of the host MA9 (7.2) and the optimal temperature of 28°C, ensuring optimal infection conditions for the phage, but also expands its lysis spectrum. Additionally, MA9V-2 can infect strains C. indologenes 02, 06, and 04, and this broad host spectrum allows the phage to maintain lysis activity even when facing evolving host resistance.

[0085] Example 2: Experiment on bacteriophage prevention of disease in potted Panax notoginseng plants

[0086] bacteriophage experiment to prevent disease in potted Panax notoginseng plants

[0087] The prevention experiment was divided into three groups: ① a blank control group with only an equal volume of NA liquid culture medium; ② a positive control group with MA9 in the exponential growth phase; and ③ a mixture of bacteriophage MA9V-2 and the host bacteria at MOI = 0.01. The detailed operation is as follows (see Table 4.1 for the experimental design).

[0088] ① Continue to cultivate the transplanted Panax notoginseng plants, water them daily and check the humidity. After 1-2 weeks of cultivation, select plants with good growth as the experimental group.

[0089] ②NA liquid culture medium was used as a blank control group; MA9, which was in the exponential growth phase, was used as a positive control group, and samples were added every 24 hours.

[0090] ③ Mix bacteriophage MA9V-2 with the host under optimal MOI conditions, incubate at 28°C for 15 min, and then spray onto the surface of plant leaves.

[0091] ④ Add samples every 24 hours and observe the growth status of Panax notoginseng plants after 7 days.

[0092] Table 4.1 Experimental Design for Phage Prevention of Panax notoginseng in Pots

[0093]

[0094] bacteriophage control of disease in potted Panax notoginseng plants

[0095] The control experiment was divided into three groups: ① a blank control group with only an equal volume of NA liquid culture medium; ② a positive control group, which was first infected with MA9 bacteria cultured to the logarithmic growth phase, and then sterilized MA9 bacteria in the logarithmic growth phase were added; ③ a positive control group, which was first infected with MA9 bacteria culture and then treated with bacteriophage MA9V-2 (MOI = 0.01). Detailed procedures are as follows (experimental design is shown in Table 4.2):

[0096] The transplanted Panax notoginseng plants were further cultivated, and watered and humidity was monitored daily. After 1-2 weeks of cultivation, plants with good growth were selected as the experimental group.

[0097] NA liquid culture medium was used as a blank control group; after inducing disease with MA9 bacterial suspension cultured to the exponential growth stage, sterilized MA9 in the exponential growth stage was added as a positive control group, and samples were added every 24 hours.

[0098] When the Panax notoginseng in the experimental group showed mild pathological symptoms, such as dry and blackened leaf tips and lodging, this point was selected as the starting point.

[0099] In this experiment, plants treated with pathogenic bacterium MA9 for 3 days were selected as the initial site. A treatment solution containing bacteriophage MA9V-2 was prepared under optimal infection conditions (MOI = 0.01) and sprayed onto the leaf surface of the plants. Samples were added every 24 hours for 7 days, after which the growth status of the Panax notoginseng plants was observed.

[0100] Table 4.2 Experimental Design of Bacteriophage Control in Potted Panax notoginseng

[0101]

[0102] from Figure 6 As shown in Figure A, spraying 20 mL of sterilized NA liquid culture medium onto the leaf surface of Panax notoginseng plants for 7 consecutive days did not result in any changes to the leaves, thus ruling out the possibility that the NA culture medium had any effect on the plants. Furthermore, spraying Panax notoginseng leaves with MA9 bacterial solution in the logarithmic growth phase for 7 consecutive days clearly showed signs of leaf drying, blackening, and rootstock lodging, further demonstrating the strong pathogenicity of the pathogen MA9 to Panax notoginseng. Figure 6 B). In conclusion, both the in vitro root experiment and the Panax notoginseng pot experiment show that MA9 has a strong pathogenicity to Panax notoginseng.

[0103] from Figure 6 The results of the C-prevention experiment showed that the Panax notoginseng plants exhibited milder disease symptoms than the positive control group, where the disease symptoms were very pronounced, even leading to necrosis. In the prevention experiment group (MA9V-2 prevention experiment group), only mild yellowing and drying of leaves were observed, indicating that the preventive effect of the bacteriophage was significant.

[0104] In the experiment on the control of root rot disease in Panax notoginseng ( Figure 7 C) The positive control group consisted of diseased Panax notoginseng plants, serving as the initial point of the experiment. Sterilized MA9 bacterial suspension in the growth index stage was subsequently added. Compared to the positive control group, which exhibited lodging, yellowing, and wilting symptoms, the MA9V-2 treatment group, after 7 days of treatment, did not show lodging, but exhibited yellowing and wilting leaves. In summary, the experiments indicate that even with timely bacteriophage treatment after mild symptoms appear, Panax notoginseng plants cannot be restored to a healthy growth state, but the rate of disease progression can be effectively slowed. Therefore, it is difficult to control diseased plants using bacteriophages, as the plants have already lost their ability to absorb water and nutrients, leading to uncontrollable disease progression once it occurs. Bacteriophages can only play a role in delaying the progression of the disease.

[0105] Leaf pathogen count determination

[0106] Rotten and dried leaves from Panax notoginseng plants in each experimental group were randomly picked, crushed in a sterilized mortar, and then added to 50 mL of NA liquid medium. The mixture was incubated at 28°C and 180 rpm for 2 h. The turbidity solution was then diluted and spread on NA solid medium. After 12 h of incubation, the number of pathogens in the plates was counted and the pathogen count was calculated.

[0107] The results are as follows Figure 8 As shown, the results are as follows Figure 8 As shown, the results of the Panax notoginseng pot experiment indicated that the number of pathogens in the PT positive control group was approximately 10. 8 CFU / mL, after different treatment methods, the number of pathogens in other experimental groups differed significantly from that in the control group. Specifically, the number of pathogens in the prevention experimental group (MP2) was higher than that in the control group, while the number of pathogens in the control group was lower than that in the prevention group because only bacteriophages were subsequently added. Figure 8 (A and 8C)

[0108] Leaf phage titer determination

[0109] Rotten and dried leaves from Panax notoginseng plants in the experimental group were randomly selected, crushed in a sterilized mortar, and then added to 10 mL of SM buffer. The mixture was shaken at 180 rpm for 2 hours, then allowed to stand at room temperature for 2 hours. The supernatant was transferred to a 2 mL sterilized centrifuge tube, centrifuged at 15000 × g at 4°C for 15 minutes, and then filtered through a filter membrane. Different concentrations of phage solution were obtained through serial dilution, and the titer was determined using the double-layer agar plate method. The mixture was incubated overnight at 28°C. The number of phage plaques on each plate was counted, and the phage titer was calculated.

[0110] In the phage titer assay experiment ( Figure 8 In the control group (B and 8D), MA9 phage was undetectable due to the absence of phage. In the prevention group, the presence of pathogenic bacteria enabled phages to infect and replicate in the host. In the treatment group, the reduced number of host bacteria led to a corresponding decrease in phage numbers, but the daily addition replenished these numbers, resulting in no significant difference in phage numbers between the different treatments. This indicates that, in the presence of phage concentrations, the prevention group has a stronger disease prevention ability than the control group. After early-stage disease development, the amount of phage does not "save" already infected plants; it only slows down the rate of disease progression.

[0111] Based on this incidence rate calculation method, plant disease status was divided into five levels, and incidence rate and control rate were statistically analyzed (results are shown in the figure). Figure 9 The incidence rate in the prevention group (MP2) after phage treatment was approximately 16.7%. Figure 9 A), significantly lower than the control group's 83.3%, and also lower than the prevention and control group's 72.1% incidence rate. Figure 9 C). There was no significant difference in disease incidence within the same control group, indicating that the addition of bacteriophages after the disease on Panax notoginseng plants had a very limited effect on controlling root rot. However, the bacteriophage prevention experiments showed more significant differences in control rates.

[0112] In the aforementioned potted Panax notoginseng control experiment, it was found that the control rate of the prevention experimental group was higher than that of the control group. The prevention experimental group was treated by adding a mixture of incubated bacteriophage and host. This method resulted in a lower number of pathogens added to the prevention group each time compared to the control group. This is because after 15 minutes of incubation, the bacteriophage can quickly adsorb onto the surface of the pathogen and complete subsequent replication and release steps in a short time, which greatly reduces the number of pathogens and thus effectively slows down the rate of Panax notoginseng leaf wilting.

[0113] Example 3: Whole Genome Analysis

[0114] Genome Map

[0115] Phage genome analysis is crucial for identifying specific functional proteins in the genome and the safety of phage applications. The phage MA9V-2 described in this application has been uploaded to the NCBI database, accession number NCBI accession ID: OR513085. Sequencing results show that the phage MA9V-2 genome is linear double-stranded DNA (dsDNA), with a full length of 218,539 bp and a GC content of 36.23% (…). Figure 10The MA9V-2 genome contains 269 ORFs. Of these, 263 are in the forward strand and 6 in the reverse strand. The longest and shortest ORF protein gene sequences are 7104 bp and 102 bp, respectively, encoding a putative protein of 2368 amino acids and a putative protein of 34 amino acids. According to BLAST results, 50 ORF proteins are homologous to genes encoding proteins with known functions. Of these, 19 can be clustered into three modules, involving aspects such as phage structure, host lysis, and DNA replication. The remaining 31 genes encode other biologically functional proteins. Furthermore, the MA9V-2 genome lacks integrase, indicating that it is not a lysogenic phage. In addition, no related tRNA genes, lysogenic genes, drug resistance genes, or virulence genes were found in the MA9V-2 phage genome, suggesting that phage MA9V-2 can be used for control applications and holds promise as a biocontrol agent for the pathogenic *C. indologenes* MA9.

[0116] It should be understood that the specific embodiments described above are merely illustrative of the invention or for explaining the principles of the invention, and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. Bacteriophage MA9V-2, characterized in that, The bacteriophage is classified as Chryseobacterium indologenes phage MA9V-2, with accession number CCTCC NO:M2024593, and was deposited on April 1, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. The bacteriophage MA9V-2 according to claim 1, characterized in that, The bacteriophage Chryseobacterium indologenes phage MA9V-2 has a lytic effect on indole-producing Chrysobacterium MA9.

3. The indole-producing chrysogena MA9 bacteriophage of Panax notoginseng pathogens according to claim 2, characterized in that, The bacteriophage Chryseobacterium indologenes phage MA9V-1 exhibits lytic activity against indole-producing Chryseobacterium MA9 at temperatures ranging from 4 to 40°C and at pH values ​​ranging from 4 to 10.

4. The application of bacteriophage MA9V-2 according to claim 1 in the preparation of a biological agent for bacterial root rot of Panax notoginseng.

5. The application according to claim 4, characterized in that, The application of bacteriophage MA9V-2 in the preparation of biological agents that inhibit indole-producing Aureobacterium MA9.

6. The application according to claim 5, characterized in that, The application of bacteriophage MA9V-2 in the preparation of a formulation to prevent root rot in Panax notoginseng.

7. The application according to claim 5, characterized in that, The application of bacteriophage MA9V-2 in the preparation of agents to control root rot in Panax notoginseng.

8. The application according to claim 4, characterized in that, The biological agent comprises bacteriophage MA9V-2 and a pharmaceutically acceptable carrier.

9. The application according to claim 5, characterized in that, The optimal multiplicity of infection (MOI) for bacteriophage MA9V-2 in the biological agent is 0.

01.

10. The application according to claim 5, characterized in that, The pH value of the biological agent is 4 to 10.

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