Pseudo-ginseng pathogenic bacterium MA9 phage and application thereof

Abstract

The invention discloses a pseudo-ginseng pathogenic bacterium MA9 bacteriophage and application thereof, the bacteriophage is a myelocaudaceae bacteriophage, is classified and named as Chryseobacterium indologenes phage MA9V-1, has a preservation number of CCTCC NO: M2024592, is preserved in the China Center for Type Culture Collection (Wuhan University, Wuhan, China) on April 1, 2024, has a splitting action on indoleproducing Chryseobacterium MA9, and can be used for preparing the pseudoginseng pathogenic bacterium MA9. The bacteriophage MA9V-1 is used for preparing a biological preparation for preventing and treating panax notoginseng bacterial root rot, the biological preparation is composed of the bacteriophage MA9V-1 and a pharmaceutically acceptable carrier, the bacteriophage MA9V-1 reaches the highest infection efficiency when the MOI is 0.001-0.01, and a new way is provided for biological prevention and treatment of panax notoginseng root rot.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and relates to a Panax notoginseng pathogenic bacteria MA9 phage and application thereof. Background Art

[0002] Root rot is one of the most common diseases caused by continuous cropping of Panax notoginseng, and it is also the most serious disease of Panax notoginseng. It manifests as large-scale root rot of plants, which seriously affects the yield and quality of Panax notoginseng. Root rot occurs to varying degrees from March to October every year, causing a loss of 5% to 20% of Panax notoginseng, and in severe cases, more than 70%, or even a total crop failure. At present, researchers have conducted a lot of research and experiments on the problem of Panax notoginseng root rot, mainly focusing on fungal pathogens. For example, the isolation and identification of fungal pathogens, the screening of antagonistic fungi, the analysis of the structure and composition of fungal communities in the soil, and the PCR detection of fungi. With the in-depth understanding of the soil microecology of Panax notoginseng, people found that fungi only accounted for 2.10% of the total number of microorganisms in the soil of root rot plants, and bacteria still dominated. The occurrence of Panax notoginseng root rot is an extremely complex process, which is produced by the synergistic action of fungi, bacteria and nematodes. For example, Luo Wenfu et al. reported that Fusarium solani, Alternaria tenuis and Rhabditis elegans were isolated from the rotten roots of Panax notoginseng. The experiment found that when Pseudomonas, Fusarium solani and Alternaria tenuis were mixed and inoculated on Panax notoginseng plants, the incidence of Panax notoginseng was significantly higher than that of single pathogenic bacteria inoculation solution, especially the mixed solution with Rhabditis elegans, which had the highest incidence rate. In addition, the incidence rate of bacteria in the experimental group was much higher than that of bacteria-free. In other words, bacteria also play an important role in the occurrence and development of Panax notoginseng root rot. It is expected that the probability of root rot can be reduced by controlling the single pathogenic bacteria of root rot, thereby achieving the purpose of prevention and control. At present, people have also done some research on the bacterial pathogens of Panax notoginseng root rot. It is known that 6 different types of strains can cause Panax notoginseng root rot, such as Pseudomonas, Alcaligenes and Chryseobacterium indologenes MA9. In addition, compared with other root rot bacteria, the bacterial pathogen Chryseobacterium indologenes MA9 that can cause Panax notoginseng root rot was screened out by Ji Guanghai's team from the rotten roots of Panax notoginseng. This is the first bacterial pathogen reported in China that can infect Panax notoginseng alone, and its pathogenicity has been verified by Koch's postulates. However, research on the bacterial pathogenicity of Panax notoginseng is relatively lagging, and is still in the early stages of pathogen screening and identification, and there have been no reports on phage therapy. Summary of the invention

[0003] Based on the above research prospects, this application uses the bacterial pathogen MA9 of Panax notoginseng root rot as the host, screens phages that can inhibit indologenic Chryseobacterium MA9 by screening different samples, and uses them to prevent and treat Panax notoginseng root rot. The specific contents are as follows:

[0004] A phage MA9 of Chryseobacterium indologenes, a pathogenic bacterium of Panax notoginseng, the phage is classified and named Chryseobacterium indologenes phage MA9V-1, the deposit number is CCTCC NO: M2024592, and it was deposited in the China Center for Type Culture Collection on April 1, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0005] Furthermore, the bacteriophage Chryseobacterium indologenes phage MA9V-1 has a lytic effect on Chryseobacterium indologenes MA9.

[0006] Furthermore, the bacteriophage Chryseobacterium indologenes phage MA9V-1 is active in the pH range of 4 to 10 and at a temperature below 65°C.

[0007] The bacteriophage Chryseobacterium indologenes phage MA9V-1 is used in preparing a biological agent for treating Panax notoginseng bacterial root rot.

[0008] Furthermore, the bacteriophage MA9V-1 is used in the preparation of a biological agent for inhibiting Chryseobacterium indologenes MA9.

[0009] Furthermore, the bacteriophage MA9V-1 is used in the preparation of a preparation for preventing Panax notoginseng root rot.

[0010] Furthermore, the bacteriophage MA9V-1 is used in the preparation of a preparation for controlling Panax notoginseng root rot.

[0011] Furthermore, the biological preparation includes bacteriophage MA9V-1 and a pharmaceutically acceptable carrier.

[0012] Furthermore, the optimal multiplicity of infection MOI of the bacteriophage MA9V-1 in the biological preparation is 0.001.

[0013] Furthermore, the pH of the biological preparation is 4-10.

[0014] Compared with the prior art, the beneficial effects of the present invention are: using the bacterial pathogen of Panax notoginseng root rot C. indologenes MA9 as a host, the lytic phage MA9V-1 was screened out for the first time, and used to prepare a biological agent for preventing and controlling Panax notoginseng root rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the morphology of the bacteriophage MA9V-1 of the present application;

[0016] Among them, A is the plaque formed by bacteriophage MA9V-1 in a double-layer plate; B is the transmission electron microscopy image of bacteriophage MA9V-1;

[0017] Figure 2 This is a bar graph of the optimal infection multiplicity experiment of bacteriophage MA9V-1;

[0018] Figure 3 is the adsorption rate curve of bacteriophage MA9V-1;

[0019] Figure 4 is the one-step growth curve of bacteriophage MA9V-1;

[0020] Figure 5 Temperature (A) and pH stability (B) of bacteriophage MA9V-1

[0021] Figure 6 is the in vitro host lysis curve of bacteriophage MA9V-1;

[0022] Figure 7 The experimental results of bacteriophage MA9V-1 preventing Panax notoginseng root lesions, where "R" represents rotten Panax notoginseng roots and "H" represents healthy Panax notoginseng roots;

[0023] Figure 8 This is the result of the experiment of preventing Panax notoginseng root rot with bacteriophage MA9V-1.

[0024] A: spraying 20 mL of NA liquid culture medium as a blank control;

[0025] B: spraying 20 mL of MA9 as the positive control group;

[0026] C: At MOI = 0.01, 20 mL of the mixed solution of bacteriophage MA9V-1 and the host was sprayed as the prevention experimental group;

[0027] Fig. 9 This is the result of the experiment of controlling the root rot of Panax notoginseng with bacteriophage MA9V-1.

[0028] A: spraying 20 mL of NA liquid culture medium as a blank control;

[0029] B: First, spray the MA9 in the logarithmic phase to infect Panax notoginseng plants, and then spray the sterilized MA9 solution in the exponential phase as a positive control group;

[0030] C: First, the Panax notoginseng plants were infected by spraying MA9 in the logarithmic phase, and then 20 mL of a mixture of bacteriophage MA9V-1 and NA was sprayed as a control experimental group;

[0031] Fig.10 The incidence rate (A) and control rate (B) of the potted Panax notoginseng disease prevention group are statistically shown (P<0.05);

[0032] Fig.11 The incidence rate (A) and control rate (B) of the potted Panax notoginseng disease control group are statistically shown (P<0.05);

[0033] Fig.12 This is the complete genome map of MA9V-1;

[0034] Fig.13 A circular proteome tree constructed for bacteriophage MA9V-1 and related prokaryotic double-stranded DNA viruses;

[0035] Fig.14 The rectangular proteome tree of bacteriophage MA9V-1 and 11 myotail phage strains. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0037] The strain involved in the experiment of the present invention: Chryseobacterium indologenesMA9, was deposited in the China Center for Type Culture Collection on April 1, 2024, the depository address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2024594.

[0038] This bacteriophage Chryseobacterium indologenes phage MA9V-1 is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit number is: CCTCC NO:M2024592, and the deposit date is April 1, 2024.

[0039] The bacterial pathogens of Panax notoginseng root rot 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 the strain collection center. C. indologenes 01, 02, 03, and 04 were all screened from the soil of Panax notoginseng with root rot, and C. indologenes 05 and 06 were screened from sewage.

[0040] Example 1: Isolation and identification of bacteriophage

[0041] 1. Phage Screening

[0042] The samples for phage screening were collected from different locations as follows: healthy P. notoginseng rhizosphere soil and decayed P. notoginseng rhizosphere soil collected in Yanshan County, Wenshan Prefecture, Yunnan Province in November 2020; decayed roots of P. notoginseng and P. notoginseng root soil collected in Shilin Yi Autonomous Prefecture, Kunming City, Yunnan Province in January 2021; soils of different vegetables and fruit trees collected from farmlands near Kunming University of Science and Technology, Chenggong District, Kunming City, Yunnan Province in January 2021; and sewage collected from the First People's Hospital of Yunnan Province in February 2021.

[0043] (1) Solid sample: Grind the sample into powder, add it into a conical flask containing 100 mL of SM suspension, and let it stand at room temperature for 24 h;

[0044] (2) Liquid samples: Add the sewage sample and SM suspension in a ratio of 1:1 (50mL:50mL) into a 250mL conical flask, mix well and let stand at room temperature for 24 hours. Select a single MA9 colony in 5mL NA liquid culture medium and culture it overnight in a shaker at 28°C and 180rpm. The next day, take 2mL of overnight MA9 culture, 5mL SM suspension, and 10mL of treated sample static liquid, mix them, then add NA liquid culture medium to 50mL and culture it overnight under the same conditions. After the culture is completed, centrifuge the overnight culture at 15000×g and 4°C for 15min, filter the supernatant with a sterilized 0.22μm filter membrane, and store the filtrate at 4°C. Add the MA9 culture medium to 50mL NA liquid culture medium at a 2% inoculum and culture it under the same conditions until OD 600When the absorbance value is between 0.6 and 0.8, add 5 mL of the above filtrate, culture overnight at 28°C and 180 rpm in a shaker, treat the culture fluid under the same centrifugal conditions, and obtain the filtrate after the second enrichment. Similarly, continue to expand the culture volume and use 100 mL of NA liquid culture medium for the third phage enrichment culture. After filtration, obtain the pure phage culture fluid and store it at 4°C.

[0045] 2. Phage purification and optimization of infection conditions

[0046] Using the pathogenic bacteria MA9 as the host, a phage with strong lysis was isolated from sewage by the double-layer agar plate method. According to the scientific naming method of the International Committee on Taxonomy of Viruses (ICTV), it was named vB_CinP_MA9V-1, abbreviated as phage MA9V-1 in this article. The phage was deposited in the China Center for Type Culture Collection on April 1, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number: CCTCC NO: M2024592. In the determination of the optimal lysis conditions of the phage, the results were as follows: the incubation ratio of phage to host was 200:300 μL, the incubation time at 28°C was 15 min, and the ratio of the mixed solution to the semi-solid culture medium was 1:8.

[0047] The results showed that bacteriophage MA9V-1 could form clear circular plaques with a diameter of about 0.5 to 1.5 mm in a double-layer agar plate covered with MA9 biofilm ( Figure 1 A). The formation of plaques can clearly characterize the lytic ability of phages and also reflect the ability of phages to infect and replicate in the host.

[0048] 3. Phage Concentration, Gradient Centrifugation and Electron Microscope Morphology Observation

[0049] 1. Phage concentration and density gradient centrifugation

[0050] (1) Enrichment of phage lysate

[0051] A single colony of MA9 was picked from the plate and inoculated into a test tube containing 5 mL of NA liquid medium and cultured in a shaking incubator at 28°C for 12 h. The next day, the seed solution was transferred to 500 mL of NA medium at a 2% inoculum and cultured under the same culture conditions until the logarithmic growth phase. After adding the phage filtrate at the optimal multiplicity of infection, the culture was continued for 12 h to obtain a lysate containing phages.

[0052] (2) Polyethylene glycol (PEG) precipitation enrichment of phages

[0053] ① Let 500mL of phage lysis mixture stand, add DNase I and RNase A to the lysis solution to a final concentration of 1μg / mL, and incubate in a 37℃ incubator for 30min. The purpose is to digest the nucleic acid fragments in the lysis solution and prevent the phage from being adsorbed by the nucleic acid mucus.

[0054] ② Add 29.2 g of sodium chloride in a ratio of 5.84 g of solid sodium chloride per 100 mL of mixed solution (final concentration is 1 mol / L) and stir gently to dissolve it completely. Place it in an ice bath in ice water for 1 hour to use sodium chloride to promote the separation between phage particles and cells.

[0055] ③ Centrifuge the mixture at 4°C, 11,000×g for 10 min to remove cell debris, collect the supernatant, measure the volume of the supernatant with a measuring cylinder, and transfer it to a 2L conical flask.

[0056] ④ Add PEG 8000 at a ratio of 10% (m / v) and stir slowly with a magnetic stirrer to fully dissolve PEG 8000.

[0057] ⑤ Place the mixture in ice water again and leave it at 4°C overnight to allow PEG 8000 to adsorb the phage particles and precipitate at the bottom of the conical flask.

[0058] ⑥ The next day, centrifuge the above-mentioned treatment solution at 4°C, 11000×g for 15 minutes, discard the supernatant, invert the centrifuge tube to allow the supernatant to drain, and collect the phage precipitate after centrifugation.

[0059] (3) Chloroform extraction of bacteriophage

[0060] ① Gently resuspend the precipitate at the bottom of the centrifuge tube with SM buffer (add 3 mL SM buffer for every 100 mL of mixed solution) and leave it at room temperature for 1 hour to allow the phage to be completely suspended in the buffer.

[0061] ② Add the same volume of chloroform as the above suspension, slowly invert up and down 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 in the suspension.

[0062] ③ Extract the suspension with chloroform several times. The number of extractions is determined by the clarity of the aqueous phase, usually 3 to 5 times. Store at 4°C.

[0063] (4) Purification of phage by CsCl2 density gradient centrifugation

[0064] ① Using SM suspension as solvent, prepare three CsCl2 solutions with different concentrations (1.45, 1.50, 1.70 g / mL), and add 1.5 mL of CsCl2 solution into the ultracentrifuge tube in order from high to low.

[0065] ② Determine the volume of the hydrophilic phase after extraction, add CsCl2 at a ratio of 0.5g / mL, and gently stir with a pipette tip to dissolve the CsCl2, and then add 4.5mL of the solution to the top layer of the centrifuge tube.

[0066] ③After centrifugation at 4℃ and 150000×g for 10h, a layer of obvious white flocs will be observed. Use a 1mL syringe to poke from the outside of the centrifuge tube, and draw about 0.5-1mL of phage from the separation layer into a 2mL centrifuge tube. Wrap it with sealing film and store it at 4℃.

[0067] 2. Electron microscopic observation of bacteriophage morphology

[0068] The morphology of the purified phage particles was observed using a transmission electron microscope. 10 μL of the concentrated ultracentrifuged phage solution was added to a carbon-coated copper grid for adsorption for 10 to 15 minutes. After the sample was completely adsorbed, 2% phosphotungstic acid was added for staining for 5 to 10 minutes. After drying naturally, the phage was observed using a transmission electron microscope (Hitachi HT7820) at a biovoltage of 120 kV.

[0069] TEM morphological characteristics of lytic bacteriophage MA9V-1 ( Figure 1 B) Observations show that it has a protein "head" wrapped by a tetrahedron with a diameter of about 121.26nm. Studies have shown that the head of the myotail phage is an icosahedral structure, which not only gives the phage a unique appearance, but also enables the phage to have specific biological functions. Its main function is to protect the genetic material inside the phage and ensure that it remains stable during entry into the host cell and in the external environment. These genetic materials are essential for the survival and reproduction of the phage. In addition, the size of the contractile tail of the phage MA9V-1 is: about 170.34nm in length and about 44.62nm in width. This contractile tail undergoes a conformational conversion when the phage attaches to the host cell membrane, forming a channel that enables the phage genome to be injected into the host cytoplasm. According to the latest ICTV classification system, phage MA9V-1 belongs to the myotail phage of the Myoviridae family.

[0070] Optimal Multiplicity of Infection

[0071] The optimal infection number (MOI) refers to the optimal ratio of phage particles to host cells during infection. MA9 was diluted to 10 by gradient dilution. -4 , 10 -5 , 10 -6The CFU value of the exponential growth phase was determined by the spread plate method. The phage filtrate of known titer was mixed with the host cells in the exponential phase at a multiplicity of infection of 0.001, 0.01, 0.1, 1, and 10, respectively, and cultured in a shaker at 180 rpm and 28°C for 12 hours. After enrichment, centrifuge at 15,000×g for 15 minutes, filter with a 0.22μm filter membrane, and determine the phage titer by the double-layer agar plate method. The experiment was repeated three times. The titer calculation formula 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 in Figure 2, when the infection multiplicity of phage MA9V-1 was 0.001, the titer of phage MA9V-1 was the highest, which was 1.25×10 10 PFU / mL, and the titer is about 10 at MOI = 0.01. 10 PFU / mL, so the optimal infection multiplicity of bacteriophage MA9V-1 is 0.001MOI, which is of great significance for understanding the infection efficiency of bacteriophages and formulating effective bacteriophage biological agents to control diseases and insect pests.

[0074] 5. Determination of adsorption rate

[0075] The adsorption rate is the ratio of phage particles adsorbed to host cells within a specific time. The adsorption rate curve of phage MA9V-1 is plotted with the adsorption rate percentage as the ordinate and time as the abscissa ( Figure 3 ). As can be seen from the figure, bacteriophage MA9V-1 can be quickly adsorbed to the surface of the host cell membrane. Within 8 minutes, >75% of the phage particles are adsorbed. After 12 minutes, about 90% of the phage particles are adsorbed, indicating that bacteriophage MA9V-1 has a high adsorption efficiency. A high adsorption rate allows the phage to effectively identify and infect host bacteria.

[0076] 6. The incubation period, rising period and lysis amount are three important parameters to characterize the one-step growth curve. The one-step growth curve results show that the three values ​​in the growth cycle of bacteriophage MA9V-1 are 20, 90min and ~10PFU / cell, respectively, indicating that the phage can infect the host MA9 and complete replication and lysis in a relatively short time (e.g. Figure 4 shown).

[0077] VII. Effects of Temperature and pH on Phage Stability

[0078] In the thermal stability experiment, after incubation at 30-50℃ for 1 hour, the infection ability of MA9V-1 did not change significantly. Even at 55℃ and 60℃, the phage MA9V-1 still had the infection ability, but the activity dropped to about 85% and 40%, respectively. Figure 5 A), completely lost infectivity at 65°C and subsequent temperatures. In addition, the infection ability of bacteriophage MA9V-1 did not change significantly when treated under different pH conditions of 5 to 8. However, under extreme pH conditions of 9, 10, and 11, it showed strong pH sensitivity and completely lost its activity ( Figure 5 B).

[0079] 8. In vitro lysis experiment of bacteriophage MA9V-1

[0080] A single colony of MA9 was picked from the plate and inoculated into a 5 mL test tube containing NA liquid medium. The tube was shaken overnight at 28°C for 12 hours. The next day, the seed solution was transferred to 50 mL NA medium at a 2% inoculum and cultured under the same conditions until the logarithmic growth phase. Bacteriophage MA9V-1 was added at different ratios of infection multiplicity MOI = 0.001, 0.01, 0.1, 1, and 10, and cultured under the above conditions. The OD measured after adding the phage was 600 The value was taken as the initial point 0h, and then 2mL of culture medium was taken into a centrifuge tube every 1h, and the absorbance value was measured by spectrophotometer, with 3 parallels in each group. The host culture medium without phage added MOI=0 was used as the control, and the experiment was repeated 3 times.

[0081] The entire growth process of MA9 was measured at MOI = 0. MA9 reached the stable phase after 8 hours of culture and presented a standard "S"-shaped growth curve. Figure 6 As shown in the figure, when the MOI was 0.001 and 0.01, the growth of MA9 was not significantly inhibited until 5 hours later, when the curve showed a downward trend and then quickly recovered. In addition, when MOI = 10, a phenomenon opposite to other MOIs was observed, that is, the host growth curve dropped sharply and slowly resumed growth after 9 hours. In general, after adding bacteriophage MA9V-1 with different MOI values, the growth of MA9 was inhibited to varying degrees, and the curve had a significant turning point at MOI = 0.001 and 0.01, indicating that MA9V-1 has a strong lysis ability against host bacteria.

[0082] Example 2: Experiment on the prevention of Panax notoginseng root lesions by a single bacteriophage (MA9V-1)

[0083] ① Inoculate the host strain MA9 at a 2% inoculum into 50 mL NA liquid culture medium, culture at 28°C and 180 rpm until the exponential growth phase, and then dispense 2 mL into each centrifuge tube.

[0084] ②Add the corresponding phage filtrate (10 10 PFU / mL) and incubated in a 28°C incubator for 15 min.

[0085] ③ Add 200 μL of the mixed solution to each sample (for the Panax notoginseng root experiment, the mixed solution should be dripped onto the wound), and place the sample in a 28°C incubator, adding samples every 24 hours. After 7 days, observe whether the Panax notoginseng roots have rotted.

[0086] ④The blank control group was NA liquid culture medium.

[0087] The results are shown below: The preventive effect of MA9V-1 on the pathogenic bacteria MA9 provides a basis for the subsequent phage joint prevention and control experiments. Among them, the enlarged images of the second sample in the MOI = 0.01 experimental group and the fifth sample in the MOI = 10 experimental group (circled in red) more intuitively reflect the difference between the rotten and non-rotten samples ( Figure 7 B, C). According to Figure 7 A The results showed that compared with the negative control group, more than 85% of the Panax notoginseng root samples in the positive control had rotted and smelled bad, indicating that the pathogenic bacteria MA9 does have a strong pathogenicity. However, in the experimental groups treated with different MOI (MA9V-1), only some samples showed mild rot symptoms, and some were even asymptomatic. Especially when the MOI was 0.01 and 0.1, both could reduce the incidence of Panax notoginseng roots by more than 50%, and had a strong preventive effect on Panax notoginseng root rot. This experiment preliminarily showed that the bacteriophage MA9V-1 has an inhibitory effect on the formation of disease symptoms caused by the pathogenic MA9, revealing its potential in the prevention and treatment of root rot.

[0088] Example 3: Pathological changes in potted Panax notoginseng plants

[0089] (1) Experiment on the prevention of disease in potted Panax notoginseng plants by using bacteriophage

[0090] The prevention experiment was divided into three groups: ① a blank control group with an equal volume of NA liquid culture medium added; ② a positive control group with MA9 in the exponential growth phase added; ③ a mixture of bacteriophage MA9V-1 and host bacteria at an MOI of 0.01, and then the experimental operation was carried out according to the following steps (see Table 1 for detailed experimental design):

[0091] Step 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 conditions as the experimental group.

[0092] Step 2: NA liquid culture medium was used as the blank control group; MA9 in the exponential growth phase was used as the positive control group, and samples were added every 24 hours.

[0093] Step 3: Mix the bacteriophage MA9V-1 and the host at the optimal MOI condition, incubate at 28°C for 15 minutes, and then spray it on the surface of plant leaves.

[0094] Step 4: Add samples every 24 hours, and observe the growth status of the Panax notoginseng plants after 7 days.

[0095] Table 1 Design of the experimental study on the prevention of phages in potted Panax notoginseng

[0096]

[0097] (2) Experiment on controlling lesions of potted Panax notoginseng plants with bacteriophage

[0098] The control experiment was divided into 3 groups, namely: ① a blank control group with an equal volume of NA liquid culture medium added; ② a positive control group in which MA9 cultured to the logarithmic growth phase was first used to cause disease, and then sterilized MA9 in the logarithmic growth phase was added; ③ MA9 cultured to cause disease, and then treated with bacteriophage MA9V-1 (MOI = 0.01). The detailed operation is as follows (see Table 2 for detailed experimental design):

[0099] Step 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 conditions as the experimental group.

[0100] Step 2: NA liquid culture medium was used as the blank control group; MA9 cultured to the exponential growth phase was first used to cause disease, and then sterilized MA9 in the exponential growth phase was added as the positive control group, and samples were added every 24 hours.

[0101] Step 3: When the Panax notoginseng in the experimental group shows mild pathological symptoms, such as leaf tips drying up, turning black, and falling over, this time is selected as the starting point.

[0102] Step 4: In this experiment, plants treated with pathogenic bacteria MA9 for 3 days were selected as the starting point, and the treatment solution of bacteriophage MA9V-1 was prepared according to the optimal infection conditions (MOI=0.01) and sprayed onto the surface of plant leaves.

[0103] Step 5: Add samples every 24 hours, and observe the growth status of the Panax notoginseng plants after 7 days.

[0104] Table 2 Experimental design of phage control in potted Panax notoginseng

[0105]

[0106] from Figure 8A It can be seen that after spraying 20mL of sterilized NA liquid culture medium onto the surface of the leaves of Panax notoginseng plants in a spraying manner and continuing the treatment for 7 days, there was no change in the leaves of the plants. This experimental result ruled out the possibility that the NA culture medium had an impact on the plants. In addition, after spraying the Panax notoginseng leaves with MA9 bacterial liquid in the logarithmic growth period and continuing the treatment for 7 days, it can be clearly observed that the Panax notoginseng leaves dried up, turned black, and the roots and stems fell over, which once again proved that the pathogenic bacteria MA9 has a strong pathogenicity to Panax notoginseng (4.4B). In summary, both the root in vitro experiment and the Panax notoginseng potted experiment show that MA9 has a strong pathogenicity to Panax notoginseng.

[0107] In the phage prevention and control experiment, there are two treatment methods: one is to pre-mix the phage filtrate with the MA9 bacterial solution in the logarithmic growth period according to the optimal MOI conditions, and incubate it at 28°C for 15 minutes before spraying it on the surface of the leaves of Panax notoginseng plants. The purpose of this treatment method is to study the role of phage in preventing root rot or slowing down the infection rate of pathogenic bacteria under the condition of MA9 pathogenic bacteria infection; the second is to first use the pathogenic bacteria MA9 to infect Panax notoginseng plants, and when the strain shows mild symptoms, such as scorched and dried leaves, and slight lodging of rhizomes, etc., then use a mixture of phage and NA liquid culture for prevention and control treatment to observe whether the Panax notoginseng plants can delay or stop the symptoms of the plants, or even the possibility of resuming growth. Figure 8 The results of the C prevention experiment showed that the Panax notoginseng plants had less severe lesions than the positive control group, while the symptoms of the Panax notoginseng in the positive control group were very obvious, and even necrosis occurred. In the prevention experimental group, the MA9V-1 prevention group only showed mild rhizome lodging symptoms, indicating that the prevention effect of the bacteriophage was significant.

[0108] In the experiment of controlling the root rot of Panax notoginseng ( Fig. 9 C), the positive control group was the Panax notoginseng plants after infection and was used as the starting point of the experiment, and the sterilized MA9 bacterial solution in the growth exponential stage was added later. Compared with the lodging, yellowing, and dryness of the Panax notoginseng plants in the positive control group, the experimental group (treatment experimental group) after 7 days of treatment, the plants treated with MA9V-1 also all showed a state of lodging, but compared with the positive control group, no yellowing of the leaves was observed.

[0109] From the above experiments, we can see that: after Panax notoginseng plants show mild symptoms, even if they are treated with bacteriophage MA9V-1 in time, the plants cannot be restored to a healthy growth state, but the rate of disease can be effectively slowed down; compared with the prevention experimental group, plants that have already become diseased are difficult to control with bacteriophages because the plants themselves have lost the ability to absorb water and nutrients, so once the disease occurs, it will be uncontrollable, and bacteriophages can only delay the disease.

[0110] The results of the potted Panax notoginseng plant lesion experiment showed that the incidence of Panax notoginseng root rot prevention group MP1 after phage treatment was about 44.3% ( Fig.10 A), which was significantly lower than the 83.3% in the control group and lower than the 63.9% in the prevention and control group ( Fig.11 A). There was no significant difference in the incidence rate within the same group in the control experiment, indicating that the addition of bacteriophages after the Panax notoginseng plants became diseased had a very limited control effect on Panax notoginseng root rot. In terms of control rate, there was a significant difference in the bacteriophage prevention experiment. In addition, although the control rate of the control group was not significant, only about 25% ( Fig.11 B), but compared with the control group, they all played a controlling role on root rot.

[0111] Example 4: Whole genome analysis

[0112] The sequencing results showed that the phage MA9V-1 genome sequence was a linear double-stranded DNA (dsDNA) with a length of 213,507 bp and a GC content of 35.99% ( Fig.12 ). The genome of bacteriophage MA9V-1 contains 263 putative open reading frames (ORFs); among them, there are 257 and 6 ORFs in the forward and reverse strands, respectively. The longest and shortest ORF gene sequence lengths are 7,122 bp and 90 bp, respectively, encoding a hypothetical protein containing 2374 amino acids and a hypothetical protein containing 30 amino acids, respectively. According to the BLAST results, 39 ORFs have homology with genes encoding known functional proteins, of which 14 functional proteins are grouped into three modules of phage structure, host lysis and DNA replication, and the remaining 25 are other types of functional proteins. In addition, the absence of integrase in the genome indicates that bacteriophage MA9V-1 is a lytic phage targeting C. indologenes MA9, rather than a lysogenic phage. In addition, no related tRNA genes, lysogen genes, resistance genes and virulence genes were found in the bacteriophage MA9V-1 genome, which indicates that bacteriophage MA9V-1 depends on the translation of host proteins and meets the recommended standards for safe use when preventing and controlling MA9. During the application of bacteriophage, horizontal transfer of resistance genes and virulence genes will not occur, preventing the impact on subsequent bacteriophage prevention and control experiments, and will not cause "environmental pollution" when used on a large scale.

[0113] The proteome evolutionary tree of bacteriophage MA9V-1 was constructed by integrating the ViPtree database and related phage genomes, which included 2201 viruses and 482 host bacteria. Fig.13As shown in the figure, the circular proteome tree of phage MA9V-1 shows that its genome is located in the gray module of "other" regardless of the "virus type" or "host bacteria" type. The gray module represents that there is no phage with high similarity in the database. This result shows that phage MA9V-1 is a potential new group of phages with pathogenic bacteria MA9 as host. In addition, this paper also constructed a rectangular proteome evolutionary tree, which contains 11 myotail phages related to the target phage. The results show that phage MA9V-1 is in different branches with Sphingomonas phage PAU (accession number: NC_019521), Tenacibaculum phage PTm1 (accession number: NC_049340) and Tenacibaculum phage PT24 (accession number: NC_049383), and has the closest relationship with Sphingomonas phage PAU and the highest similarity. The specific homology relationship needs further analysis ( Fig.14 Subsequent multiple linear alignment analysis and genetic evolution relationship analysis of the phage MA9V-1 genome showed that MA9V-1 is a potential new group of myotail phages targeting the pathogenic bacterium MA9.

[0114] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A phage of the pathogenic bacteria of Panax notoginseng, Chryseobacterium indologenes MA9, characterized in that: The bacteriophage is classified and named Chryseobacteriumindologenes phage MA9V-1, with a preservation number of CCTCCNO: M2024592. It was deposited in the China Center for Type Culture Collection on April 1, 2024, and the preservation address is Wuhan University, Wuhan, China.

2. The Panax notoginseng pathogenic bacteria Chryseobacterium indologenes MA9 bacteriophage according to claim 1, characterized in that: The bacteriophage Chryseobacteriumindologenes phage MA9V-1 has a lytic effect on Chryseobacterium indologenes MA9.

3. The Panax notoginseng pathogenic bacteria Chryseobacterium indologenes MA9 bacteriophage according to claim 1, characterized in that: The bacteriophage Chryseobacteriumindologenes phage MA9V-1 is active when the pH value is in the range of 5 to 8 and the temperature is lower than 65°C.

4. The Panax notoginseng pathogenic bacteria Chryseobacterium indologenes MA9 bacteriophage according to claim 1, characterized in that: The bacteriophage Chryseobacteriumindologenes phage MA9V-1 is used in preparing biological preparations for treating Panax notoginseng bacterial root rot.

5. The use according to claim 3, characterized in that: The bacteriophage MA9V-1 is used in preparing a biological preparation for inhibiting indologenic Chryseobacterium MA9.

6. The use according to claim 4, characterized in that: The bacteriophage MA9V-1 is used in preparing a preparation for preventing Panax notoginseng root rot.

7. The use according to claim 4, characterized in that: The bacteriophage MA9V-1 is used in preparing a preparation for controlling Panax notoginseng root rot.

8. The use according to claim 3, characterized in that: The biological preparation comprises bacteriophage MA9V-1 and a pharmaceutically acceptable carrier.

9. The use according to claim 7, characterized in that: The optimal infection multiplicity MOI of bacteriophage MA9V-1 in the biological preparation is 0.

001.

10. The use according to claim 7, characterized in that: The pH value of the biological preparation is 5-8.

Images