Method for improving the prevention and control of soil-borne bacterial wilt by using specific phage

Abstract

This invention discloses a biocontrol bacterium for bacterial wilt, YL-Ste-01evo (accession number CCTCC NO: M2022939); a biocontrol bacterium for bacterial wilt, YL-Ste-01 (accession number CCTCC NO: M2022324); and a lytic bacteriophage, YL-Ste-01P (accession number CCTCC NO: M2022460). This invention discloses the application of biocontrol bacterium YL-Ste-01evo, or biocontrol bacterium YL-Ste-01, or biocontrol bacterium YL-Ste-01evo in synergy with bacteriophage YL-Ste-01P in the control of soil-borne bacterial wilt in tomato. The biocontrol bacterium YL-Ste-01 can significantly inhibit the growth of Ralstonia solanacearum and reduce the incidence of bacterial wilt in tomato plants. Through phage stress, YL-Ste-01 evolved into a biocontrol bacterium YL-Ste-01evo with stronger phage resistance and better antibacterial effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to the application of using obligate bacteriophages to enhance the control of soil-borne bacterial wilt by biocontrol bacteria. Specifically, it involves a biocontrol bacterium YL-Ste-01, a lytic bacteriophage YL-Ste-01P that is obligate to target the host bacterium YL-Ste-01, an evolved biocontrol bacterium YL-Ste-01evo, and the application of biocontrol bacterium YL-Ste-01 or biocontrol bacterium YL-Ste-01evo or biocontrol bacterium YL-Ste-01evo synergistic with bacteriophage YL-Ste-01P in the control of soil-borne bacterial wilt in tomatoes. Background Technology

[0002] Soil-borne bacterial wilt caused by *Ralstonia solanacearum* (commonly known as bacterial wilt fungus) severely restricts sustainable agricultural development and has caused significant losses to farmers for many years. While physical and chemical control methods are fast-acting, they easily induce drug resistance in the pathogen, and their lack of targeted application disrupts the microbial community while killing *Ralstonia solanacearum*. Utilizing rhizosphere microorganisms to control soil-borne diseases has become a new and environmentally friendly biocontrol method. [1] Examples of pathogen-specific phages include beneficial bacteria, bacteriophages, and protozoa. Among these, bacteriophages are viruses that specifically infect bacteria, and studies have shown that specific bacteriophages of *Ralstonia solanacearum* play a crucial role in controlling soil-borne bacterial wilt. Besides pathogen-specific bacteriophages, the rhizosphere also contains a large number of bacteriophages belonging to biocontrol bacteria. However, research on the impact of specific bacteriophages of rhizosphere biocontrol bacteria on the reduction efficiency of biocontrol bacteria is relatively limited. As specific "killers" of bacteria, bacteriophages can effectively regulate the population density, physiology, and behavior of rhizosphere biocontrol bacteria; simultaneously, as an important driving force for bacterial evolution, bacteriophages, while inducing the evolution of resistance in host bacteria, also alter other life history characteristics of host bacteria, such as the growth, movement, and metabolism of biocontrol bacteria. [2] This can affect the rhizosphere colonization and competitive ability of biocontrol bacteria. Whether it is possible to use the specific bacteriophages of biocontrol bacteria to selectively acclimate them and enhance their efficiency in reducing the biological barrier caused by Ralstonia solanacearum in soil is still unclear and requires further research. Summary of the Invention

[0003] The purpose of this invention is to isolate a biocontrol bacterium YL-Ste-01 that can inhibit Ralstonia solanacearum from the rhizosphere of tomato plants, and to screen a bacteriophage YL-Ste-01P that specifically infects the biocontrol bacterium YL-Ste-01. The inventors discovered that under bacteriophage stress, the biocontrol bacterium YL-Ste-01 evolves, exhibiting enhanced inhibitory ability against Ralstonia solanacearum and increased resistance to the bacteriophage. This led to the further screening of a biocontrol bacterium YL-Ste-01evo that inhibits Ralstonia solanacearum. Greenhouse pot experiments showed that applying the evolved strain YL-Ste-01evo can significantly improve the control of bacterial wilt in tomatoes.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A bacterial wilt biocontrol bacterium, YL-Ste-01evo, classified as Stenotrophomonas maltophilia, was deposited at the China Center for Type Culture Collection on June 21, 2022, with accession number CCTCC NO:M2022939.

[0006] A bacterial wilt biocontrol bacterium, YL-Ste-01, classified as Stenotrophomonas maltophilia, was deposited at the China Center for Type Culture Collection on March 28, 2022, with accession number CCTCC NO:M2022324.

[0007] A lytic bacteriophage, YL-Ste-01P, belonging to the order Caudata, family Long-tailed Phages, and classified as Stenotrophomonas maltophilia phage, was deposited at the China Center for Type Culture Collection (CCTCC) on April 22, 2022, with accession number CCTCC NO:M 2022460.

[0008] The obligate lytic phage YL-Ste-01P can stress the biocontrol bacterium YL-Ste-01 to evolve into the biocontrol bacterium YL-Ste-01evo. Compared with the biocontrol bacterium YL-Ste-01, the biocontrol bacterium YL-Ste-01evo exhibits significantly enhanced resistance to pathogens and phage resistance. A method for phage YL-Ste-01P to stress the biocontrol bacterium YL-Ste-01 to evolve into the bacterial wilt biocontrol bacterium YL-Ste-01evo is as follows: YL-Ste-01 bacterial suspension in the logarithmic growth phase is obtained by culturing in NA liquid medium, and the OD of the bacterial suspension is adjusted with sterile water. 600 Adjust the value to 0.5; adjust the phage titer to 1×10⁻⁵ using sterile water. 8PFU / mL, placed at the optimal multiple of infection (MOI 1–10) in OD 600 YL-Ste-01 bacterial culture with a value of 0.5 was cultured at 30-33℃ and 160-180 rpm for 24 hours. The resulting biocontrol strain YL-Ste-01evo, which exhibited the strongest anti-phage ability and inhibition of Ralstonia solanacearum after evolution, was obtained by plating and screening.

[0009] Another object of the present invention is to provide a bacterial agent containing the biocontrol bacterium YL-Ste-01evo, wherein the concentration of the bacterial agent is 1×10⁻⁶. 8 ~ 5×10 8 CFU / mL.

[0010] A method for preparing a bacterial agent containing the biocontrol bacterium YL-Ste-01evo includes: picking a single bacterium of biocontrol bacterium YL-Ste-01evo, inoculating it into NA liquid medium, and culturing it at a temperature of 30-33℃ with shaking at a speed of 160-180 rpm to obtain a YL-Ste-01evo bacterial suspension in the logarithmic growth phase, and adjusting the OD of the bacterial suspension with sterile water. 600 A value of 0.45–0.55 indicates a concentration of 1 × 10⁻⁵. 8 ~5×10 8 CFU / mL bacterial agent.

[0011] Another object of the present invention is to provide a bacterial agent containing the biocontrol bacterium YL-Ste-01, wherein the concentration of the bacterial agent is 1×10⁻⁶. 8 ~ 5×10 8 CFU / mL.

[0012] A method for preparing a bacterial agent containing biocontrol bacteria YL-Ste-01 includes: inoculating biocontrol bacteria YL-Ste-01 into NA liquid medium, culturing at 30-33℃ with shaking at 160-180 rpm to obtain a YL-Ste-01 bacterial suspension in the logarithmic growth phase, and adjusting the OD of the bacterial suspension with sterile water. 600 A value of 0.45–0.55 indicates a concentration of 1 × 10⁻⁵. 8 ~5×10 8 CFU / mL bacterial agent.

[0013] A phage agent containing phage YL-Ste-01P is prepared by the following method: YL-Ste-01 bacterial suspension in the logarithmic growth phase is obtained by culturing in NA liquid medium; the OD of the bacterial suspension is then adjusted with sterile water. 600 Adjust the value to 0.5; adjust the phage titer to 1×10⁻⁵ using sterile water. 8 PFU / mL, inoculate phages into OD at the optimal multiple of infection (MOI 1–10). 600In a YL-Ste-01 bacterial culture with a titer of 0.5, the culture was incubated at 30–33 °C with shaking at 160–180 rpm for 12 h. The culture was then centrifuged at 10,000 rpm at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain a phage suspension. The titer of the phage suspension was adjusted to 1 × 10⁻⁶ with sterile water. 8 PFU / mL was used to obtain bacteriophage bacterial agents.

[0014] Another object of the present invention is to provide the application of the biocontrol bacterium YL-Ste-01 or the biocontrol bacterium YL-Ste-01evo or the biocontrol bacterium YL-Ste-01evo synergistic bacteriophage YL-Ste-01P in the control of soil-borne bacterial wilt of tomato.

[0015] Another objective of this invention is to provide a method for controlling soil-borne bacterial wilt of tomato, comprising: adding a biocontrol agent YL-Ste-01evo to the soil by root irrigation 6-7 days after transplanting tomato seedlings; or adding a biocontrol agent YL-Ste-01 to the soil by root irrigation 6-7 days after transplanting tomato seedlings; wherein the final concentration of the biocontrol agent YL-Ste-01evo is 1×10⁻⁶. 8 ~1×10 9 CFU / tree; final concentration of biocontrol bacteria YL-Ste-01 is 1×10⁻⁶. 8 ~1×10 9 CFU / tree.

[0016] Another objective of this invention is to provide a method for controlling soil-borne bacterial wilt using specific bacteriophages and enhanced biocontrol bacteria, comprising: 6-7 days after transplanting tomato seedlings, adding the biocontrol bacterium YL-Ste-01evo to the soil via root irrigation; and inoculating with bacteriophage YL-Ste-01P 2-3 days later; wherein the final concentration of the biocontrol bacterium YL-Ste-01evo is 1×10⁻⁶. 8 ~1×10 9 CFU / cell, the final titer of phage YL-Ste-01P was 1×10⁻⁶. 8 ~1×10 9 PFU / tree.

[0017] The beneficial effects of this invention are:

[0018] Indoor experiments showed that the biocontrol bacterium YL-Ste-01 could significantly inhibit the growth of Ralstonia solanacearum. Further evolution of YL-Ste-01 under phage stress resulted in the biocontrol bacterium YL-Ste-01evo, which exhibits stronger phage resistance but also better pathogen-inhibiting effects. This enhanced inhibition of Ralstonia solanacearum and resistance to phage infection and lysis further improved its biocontrol efficacy, demonstrating broad potential. In particular, the application of the biocontrol bacterium YL-Ste-01evo and phages to the soil via root irrigation to inhibit soil-borne Ralstonia solanacearum showed a synergistic effect between the evolved biocontrol strain and the phages, further enhancing the control efficacy against the disease. Attached Figure Description

[0019] Figure 1 The morphology of the biocontrol bacterium YL-Ste-01 is described.

[0020] Figure 2 Phylogenetic tree based on the 16S rRNA gene sequences of biocontrol bacterium YL-Ste-01 and related strains

[0021] Figure 3 The image shows a plaque formed by bacteriophage YL-Ste-01P on a host bacterial plate.

[0022] Figure 4 The morphology of bacteriophage YL-Ste-01P under a transmission electron microscope.

[0023] Figure 5 Phage YL-Ste-01P whole genome phylogenetic tree.

[0024] Figure 6 The growth curves of biocontrol bacteria YL-Ste-01 and biocontrol bacteria YL-Ste-01 under bacteriophage infection are shown.

[0025] Figure 7 The figure shows the resistance of biocontrol strain YL-Ste-01 and its evolved strains under bacteriophage infection; in the figure, the dashed line represents the characteristics of the original strain YL-Ste-01, and each dot represents an evolved strain.

[0026] Figure 8 The colonies formed by YL-Ste-01evo on NA solid plates.

[0027] Figure 9 Changes in the antibacterial ability of the biocontrol agent YL-Ste-01evo.

[0028] Figure 10The effects of the original strain and the evolved strain on the inhibition of bacterial wilt of tomato in greenhouse pots; in the figure, different letters represent significant differences at the P<0.05 level.

[0029] Biological Preservation Information:

[0030] YL-Ste-01, classified as Stenotrophomonas maltophilia, was deposited on March 28, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 2022324.

[0031] YL-Ste-01evo, classified as Stenotrophomonas maltophilia, was deposited on June 21, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 2022939.

[0032] YL-Ste-01P, classified as Stenotrophomonas maltophiliaphage, was deposited on April 22, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2022460. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0034] The culture medium formula is as follows:

[0035] NA liquid culture medium: 10g glucose, 5g peptone, 3g beef extract, 0.5g yeast powder, 1000mL deionized water, adjust pH to 7.2-7.4, autoclave at 115℃ for 30min.

[0036] NA solid medium: Add 25g of agar powder to 1L of NA liquid medium.

[0037] Gentamicin medium: 300 mL NA semi-solid medium, heated to 3 μL 1% gentamicin.

[0038] Ralstonia solanacearum (RFP) bacterial culture was prepared by the following method: Ralstonia solanacearum was activated with gentamicin solid medium, a single colony was picked and transferred to NA liquid medium, and cultured at 30℃ and 170 rpm for 24 h with shaking to obtain Ralstonia solanacearum bacterial culture in the logarithmic growth phase.

[0039] Example 1

[0040] Isolation of Stenotrophomonas maltophilia

[0041] (1) Take 10g of rhizosphere soil from healthy tomato plants in Houcun Village, Qilin Town, Nanjing City, and place it in a 250mL Erlenmeyer flask containing 90mL of sterile water (with glass beads). Shake at 170rpm for 30min. After serially diluting the soil suspension 10 times, take 0.1mL of the suspension and spread it on NA medium plates. Incubate at 30℃ for 2-4 days. Pick single colonies and streak them on NA plates for purification. Pick single bacteria in NA liquid medium and incubate at 30℃ and 170rpm for 48h to obtain bacterial culture. Centrifuge at 6000r / min for 5min to obtain fermentation broth; (2) Use an inoculation loop to pick Ralstonia solanacearum RFP. [3] Bacterial cells were streaked onto gentamicin agar plates and incubated at 30°C for 36 h to obtain single bacteria. Single bacteria were then picked and cultured in NA liquid medium at 30°C with shaking at 170 rpm for 24 h to obtain a *Ralstonia solanacearum* suspension. The OD of the *Ralstonia solanacearum* suspension was adjusted with sterile water. 600 0.5. (3) Set up a control group (only use NA medium to culture Ralstonia solanacearum): Take NA liquid medium and add Ralstonia solanacearum at 1% of the system inoculation amount; Treatment group: Use NA liquid medium, add the fermentation broth obtained in step 1 to 10% of the total system to culture Ralstonia solanacearum, and add OD at 1% of the total volume. 600 The concentration of Ralstonia solanacearum was 0.5 μL; both the control and treatment groups were cultured at 30°C with shaking at 170 rpm, and the OD value was measured every 12 hours using a microplate reader. 600 The value below. Compared with the control group, if the OD value of the treatment group is lower... 600 If the OD level is significantly reduced, the bacteria corresponding to this treatment group are antagonistic to Ralstonia solanacearum. OD levels were then screened. 600 The lowest treatment group corresponding to the Ralstonia solanacearum antagonist bacterium was named YL-Ste-01 and stored at -80°C in 30% glycerol. YL-Ste-01 formed pale yellow, opaque, round colonies with a raised center, regular edges, smooth and moist surfaces, easily picked up, on NA agar plates. Figure 1 After DNA extraction, 16S rRNA gene sequencing was performed. The sequencing results were then analyzed using BLAST homology search in the GenBank database, and a phylogenetic tree was constructed using MEGA 4.1. Figure 2 Ultimately, the strain was identified as Stenotrophomonas maltophilia.

[0042] YL-Ste-01, classified as Stenotrophomonas maltophilia, was deposited on March 28, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 2022324.

[0043] Isolation of bacteriophages

[0044] Tomato rhizosphere soil samples were collected from the Qingjiankang area of ​​Houcun Village, Qilin Town, Nanjing City. A specific phage, YL-Ste-01, with highly efficient lytic ability, was screened using the double-layer agar plate method. The specific implementation plan is as follows:

[0045] The antagonistic bacteria YL-Ste-01 preserved in glycerol was activated by streaking on NA solid medium and cultured at 30℃ for 48 h. Single colonies from the plates were transferred to NA liquid medium and cultured at 30℃ and 170 rpm for 24 h to obtain YL-Ste-01 bacterial suspension in logarithmic growth phase for later use.

[0046] Weigh 1g of fresh soil sample from the rhizosphere of healthy tomato plants into a sterilized Erlenmeyer flask containing glass beads, add 9 mL of sterile water, mix well, and incubate at 30℃ and 170 rpm for 1 hour. Transfer the enriched soil suspension to a sterilized 2 mL centrifuge tube, centrifuge at 10000 rpm for 3 minutes, collect the supernatant, and filter through a 0.22 μm filter membrane (to remove bacteria). The filtrate is the phage stock solution. Mix 250 μL of YL-Ste-01 bacterial suspension in the logarithmic growth phase with 25 mL of NA semi-solid medium at a suitable temperature, and immediately pour the mixture onto solidified NA solid medium to prepare a double-layer plate containing YL-Ste-01. After the medium solidifies, dilute the phage suspension with sterile water, and spot-inoculate different areas of the plate with 10 μL of serially diluted (10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 The phage stock solution was incubated at 30℃ for 24 h to 48 h, and the presence or absence of phage plaques was observed.

[0047] Once plaques appear, a single plaque at its highest dilution is selected and inoculated into the YL-Ste-01 bacterial suspension. The suspension is incubated at 30°C with shaking at 170 rpm for 12 hours to allow phage proliferation. The resulting suspension is centrifuged at 10,000 rpm for 3 minutes, and the supernatant is collected. The supernatant is then filtered through a 0.22 μm filter membrane, and the resulting filtrate is passed through the double-layer agar plate method described above. [3] Perform the procedure; repeat 2-3 times to obtain a relatively pure phage, and designate the obtained phage as phage YL-Ste-01P. Take the purified phage suspension and perform serial dilutions (generally diluted to 10⁻⁶). -8Afterwards, drop (10-15 μL) onto the corresponding position on a semi-solid NA medium plate containing Ralstonia solanacearum (prepared as a double-layer plate according to the above method), and incubate at 30°C for 24 h. Phage YL-Ste-01P will then form phage plaques on the host bacteria plate. Figure 3 In the text, 4, 5, and 6 represent dilutions of 10⁻⁶. -4 10 -5 10 -6 ).

[0048] Example 2

[0049] Bacteriophage electron microscopy observation

[0050] 1. Culture the bacteriophage YL-Ste-01P to be observed and its corresponding host bacterium YL-Ste-01.

[0051] 2. Prepare a double-layer plate containing the host bacterium YL-Ste-01 according to Example 1 "Isolation of bacteriophage". Add four drops of 50 μL bacteriophage suspension to the plate and incubate at 30°C for 12-24 h until phage plaques appear.

[0052] 3. Pick out the semi-solid plaques and transfer them one by one into a 15mL centrifuge tube containing 4mL of SM solution. Place the tube flat on a shaker and shake at 30℃ and 170rpm for 2 hours.

[0053] 4. Centrifuge and filter three times to obtain the supernatant: First, centrifuge at 3000 rpm for 2 min, collect the supernatant, and filter; centrifuge the filtrate a second time (6000 rpm), collect the supernatant, and filter a second time; centrifuge the filtrate a third time (6000 rpm), collect the supernatant, and filter a third time. The resulting filtrate (phage suspension) is the phage electron microscopy specimen.

[0054] 5. Take an appropriate amount of phage suspension and perform negative staining with 2% phosphotungstic acid solution. After staining for about 90 seconds, remove it from the solution and let it air dry at room temperature.

[0055] 6. Observe the morphology of the bacteriophage using a transmission electron microscope. Bacteriophage YL-Ste-01P has a typical icosahedral head and a non-retractable tail. Figure 4 After comparison with the classification standards of the International Committee on Taxonomy of Viruses. [4] After extracting phage DNA, 16S rRNA gene sequencing was performed. The sequencing results were then used for BLAST homology searching in the GenBank database, and a phylogenetic tree was constructed using MEGA 4.1 analysis. Figure 5 Ultimately, it was determined that the bacteriophage belonged to the order Caudophages and the family Longtailophages.

[0056] Phage YL-Ste-01P, classified as Stenotrophomonas maltophilia phage, was deposited on April 22, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2020460.

[0057] Example 3

[0058] Optimal Multiple of Infection Determination

[0059] The multiplicity of infection (MOI) is the ratio of the number of bacteriophages to the number of host bacteria before the bacteriophage adsorbs and infects the host bacteria. The optimal MOI is the MOI that yields the highest progeny bacteriophage production.

[0060] Following the method in Example 1, a single bacterium of biocontrol bacteria YL-Ste-01 was picked and cultured in NA liquid medium at 30°C and 170 rpm with shaking to obtain a YL-Ste-01 bacterial suspension in the logarithmic growth phase. The OD was then diluted with sterile water. 600 Adjusting the value to 0.5 corresponds to a concentration of 1×10⁵. 8 YL-Ste-01 bacterial agent with CFU / mL.

[0061] The phage suspension purified in Example 1 was added dropwise to NA liquid medium containing YL-Ste-01 bacterial culture and cultured at 30°C and 170 rpm for 12 h with shaking. The culture medium was centrifuged at 10000 rpm / min for 3 min at room temperature, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the phage suspension. The titer of the phage suspension was adjusted to 1×10⁻⁶ with sterile water. 8 PFU / mL was used to obtain bacteriophage bacterial agents.

[0062] Take YL-Ste-01 bacterial agent (OD) 600 Add 0.5 g of phage agent to each well of a 48-well plate, 10 μL per well. Then, add 10 μL of phage agent at MOIs of 0.01, 0.1, 1, 10, and 100 (each MOI is tested in triplicate). Shake the mixture thoroughly and incubate at 30°C and 170 rpm for 12 h. Transfer the co-culture suspension from the 48-well plate to sterilized 2 mL centrifuge tubes and centrifuge at 10,000 rpm for 3 min. Collect the supernatant and filter it through a 0.22 μm filter membrane to obtain the phage suspension. After serial dilution of the phage suspension, use the double-layer agar plate method. [3] The optimal multiplicity of infection can be determined by measuring and recording the titer.

[0063] Table 1. Optimal Multiplicity of Infection for Bacteriophage YL-Ste-01P

[0064]

[0065] As shown in Table 1, serially diluted co-culture medium with a multiplicity of infection (MOI) of 0.01 was added to double-layer plates containing Ralstonia solanacearum and incubated for 24 h, and no plaques were observed. The highest phage titers were observed at MOIs of 1 and 10, suggesting that the optimal MOI for phage YL-Ste-01P to the host bacterium YL-Ste-01 is between 1 and 10.

[0066] Example 4

[0067] Changes in host bacterial growth rate under bacteriophage stress

[0068] By setting the presence or absence of bacteriophages as the sole variable and keeping all other conditions constant, the OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 This was used to characterize the growth of the host bacterium YL-Ste-01. The specific procedure was as follows: Co-culture treatment group: The OD value of the YL-Ste-01 bacterial culture in the logarithmic growth phase was adjusted. 600 The titer was 0.5, and the titer of phage YL-Ste-01P was 10. 8 PFU / mL (same as the phage agent in Example 3), YL-Ste-01 was added to NA liquid medium at a 1% inoculum volume, and phage was added to NA liquid medium at a 1% inoculum volume of the total system. A 200 μL microplate culture system was used, and the culture was carried out at 30℃ and 170 r / min in a shaker for 48 h. Six replicates were set up, and the absorbance at 600 nm was measured every 8 h. Single culture treatment group: The OD of YL-Ste-01 bacterial culture in the logarithmic growth phase was adjusted. 600 YL-Ste-01 was added to NA liquid medium at an inoculum size of 0.5% and cultured in 200 μL microplates at 30 °C and 170 r / min for 48 h. Six replicates were set up and the absorbance at 600 nm was measured every 8 h.

[0069] The results are as follows Figure 6 The results indicate that in the single-culture treatment group, YL-Ste-01 grew rapidly in the first 24 hours, and then its growth stabilized. In the co-culture treatment group, the host bacterium YL-Ste-01 experienced very low growth rates in the early stages due to infection and lysis by bacteriophage YL-Ste-01P, especially in the first 24 hours, with significantly lower growth compared to the single-culture group. However, as time progressed, the number of biocontrol bacteria YL-Ste-01 increased rapidly after 24 hours, and the OD value at 48 hours was lower than that of the single-culture group. This suggests that bacteriophage YL-Ste-01P has an inhibitory effect on the biocontrol bacteria YL-Ste-01, especially with significant inhibition in the initial 24 hours.

[0070] Example 5

[0071] Changes in the resistance of biocontrol bacterium YL-Ste-01 after bacteriophage stress

[0072] Bacteriophages and bacteria have evolved continuously for survival through long-term interaction, with bacterial hosts needing to change their own characteristics to evade infection. [5] Therefore, after co-culturing the host bacterium YL-Ste-01 and bacteriophage YL-Ste-01P for 24 h, the bacteria were plated and cultured at 30℃ for 24 h. 188 strains were randomly selected from the plates and cultured at 30℃ and 170 rpm for 24 h. Single colonies were obtained by streaking the plates and these single bacteria were regarded as evolutionary strains. Their bacteriophage resistance and antibacterial ability were compared with those of the original strain YL-Ste-01. Specific operation: (1) The evolutionary strains in the logarithmic growth phase and the original strain YL-Ste-01 (control) were cultured in NA liquid medium with shaking for 24 h and then adjusted to OD with sterile water. 600 The value was 0.5, and the phage titer was 10. 8 PFU / mL (same as the phage agent in Example 3), the bacterial suspension and phage were added to NA liquid medium at 1% of the total system inoculation volume. Six replicates were set up for each strain. The culture was carried out at 30°C and 170 rpm for 24 h with shaking. The OD was measured by an enzyme-linked immunosorbent assay (ELISA) reader. 600 Value; OD 600 The larger the value, the stronger the resistance of the strain to bacteriophage. (2) Adjust the evolved strain and the original strain YL-Ste-01 (control) in the logarithmic growth phase to OD. 600 The OD value was adjusted to 0.5, and 1% was added to NA liquid medium. The culture was incubated at 30°C and 170 rpm for 48 h using a shaker. The resulting fermentation broths of the evolved strain and the original strain YL-Ste-01 were obtained by filtration. Ralstonia solanacearum cells were streaked onto gentamicin agar plates and incubated at 30°C for 36 h to obtain single cells. Single cells were then picked and incubated in NA liquid medium at 30°C and 170 rpm for 24 h to obtain a Ralstonia solanacearum suspension. The OD values ​​of the evolved strain fermentation broth, the original strain YL-Ste-01 fermentation broth, and the Ralstonia solanacearum suspension were adjusted with sterile water, respectively. 600 All values ​​were 0.5. In a 200 μL inoculation system of a 96-well plate, Ralstonia solanacearum suspension was added at 1% of the total inoculation volume, and fermentation broth was added at 10% of the total inoculation volume. Six replicates were set up. The plates were incubated at 30°C and 170 rpm for 24 h with shaking. RFUs were then measured. (587-610) (Excitation light: 587nm, emission light: fluorescence value at 610nm), the higher the value, the worse the inhibitory ability of the strain against Ralstonia solanacearum.

[0073] Antiphage ability = (OD of evolved strain) 600 -Original strain OD 600 ) / Original strain OD 600×100%

[0074] Inhibition ability of Ralstonia solanacearum = (original strain RFUs) 587-610 - Evolutionary strains RFUs 587-610 ) / Original strain RFUs 587-610 ×100%

[0075] The results are as follows Figure 7 As shown in the figure, the dashed lines represent the phage resistance and Ralstonia solanacearum inhibition ability of the original strain YL-Ste-01, and each dot represents an evolved strain. The results show that most of the evolved strains are in the upper right quadrant, indicating that the original strain YL-Ste-01 evolved under phage infection stress. The evolved strains have better resistance to phages and better inhibition of Ralstonia solanacearum than the original strain.

[0076] Screening for the antibacterial activity of an evolved biocontrol bacterium YL-Ste-01evo

[0077] The strain with the strongest resistance to bacteriophages and inhibition of Ralstonia solanacearum was selected from 188 evolutionary strains and denoted as YL-Ste-01evo. YL-Ste-01evo colonies on NA solid plates are pale yellow, opaque, round, raised in the center, with a smooth, moist surface, and are easily picked up. Figure 8 ).

[0078] YL-Ste-01evo, classified as Stenotrophomonas maltophilia, was deposited on June 21, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 2022939.

[0079] Referring to Example 1, a single bacterium of biocontrol bacteria YL-Ste-01 was picked and cultured in NA liquid medium at 30°C and 170 rpm with shaking to obtain the original strain YL-Ste-01 in the logarithmic growth phase. The original strain YL-Ste-01 bacterial suspension (control) was adjusted to OD using sterile water. 600 A value of 0.5 corresponds to a concentration of 1 × 10⁻⁵. 8 YL-Ste-01 bacterial agent at CFU / mL; single bacteria of biocontrol bacterium YL-Ste-01evo were picked and cultured in NA liquid medium at 30℃ and 170 rpm with shaking to obtain a bacterial suspension of biocontrol bacterium YL-Ste-01evo in the logarithmic growth phase. The YL-Ste-01evo bacterial suspension was adjusted to OD value with sterile water. 600 A value of 0.5 corresponds to a concentration of 1 × 10⁻⁵. 8 YL-Ste-01evo bacterial agent at CFU / mL.

[0080] YL-Ste-01 and YL-Ste-01evo bacterial inoculants were added to NA liquid medium at 1% of the total inoculum, respectively. The mixtures were cultured at 30℃ and 170 rpm for 48 h on a shaker, centrifuged at 6000 rpm for 5 min, and filtered through a 0.22 μm filter to obtain the fermentation broth of the evolved strain YL-Ste-01evo and the original strain YL-Ste-01. In a 200 μL system of 96-well plates, Ralstonia solanacearum suspension (OD200) was added at 1% of the total inoculum. 600 (For a concentration of 1.0), fermentation broth of either the evolved strain YL-Ste-01evo or the original strain YL-Ste-01 was added as an inoculum of 10% of the total system. Six replicates were set for both the evolved strain YL-Ste-01evo and the original strain YL-Ste-01. The mixture was incubated at 30℃ and 170 rpm for 24 h using a shaker, and the fluorescence value (RFUs) was measured. (587，610) The results are shown below. Figure 9 Compared with the original strain, the antibacterial rate of the evolved strain was significantly increased, indicating that the biocontrol strain evolved after adding bacteriophage stress has a stronger antibacterial effect.

[0081] Example 6

[0082] Greenhouse pot experiment of primitive biocontrol bacteria and evolved biocontrol bacteria

[0083] The effects of original and evolved strains on the control of soil-borne bacterial wilt in tomatoes were investigated in a greenhouse at Nanjing Agricultural University.

[0084] The tomatoes were grown using a substrate and were of the Red Dwarf variety.

[0085] The experiment was set up with four treatments, as follows:

[0086] Control group (CK): Inoculated only with pathogens;

[0087] Treatment 1, YL-Ste-01: Inoculate with pathogenic bacteria and biocontrol bacteria YL-Ste-01.

[0088] Treatment 2, YL-Ste-01evo: Inoculate with pathogenic bacteria and biocontrol bacteria YL-Ste-01evo.

[0089] Treatment 3, YL-Ste-01+YL-Ste-01P: Inoculated with pathogen, biocontrol bacteria YL-Ste-01, and bacteriophage YL-Ste-01P.

[0090] Treatment 4, YL-Ste-01evo+YL-Ste-01P: Inoculated with pathogen, biocontrol bacteria YL-Ste-01evo, and bacteriophage YL-Ste-01P.

[0091] YL-Ste-01 bacterial agent and YL-Ste-01evo bacterial agent were prepared according to Example 5; bacteriophage bacterial agent was prepared according to Example 3.

[0092] Transplant the tomato seedlings into six-cell trays, filling the trays with the prepared substrate. Water thoroughly after transplanting. One week after transplanting, follow the 10-day planting schedule. 8 CFU / seedling inoculated with biocontrol bacteria, two days later according to 10 8 PFU / seedling inoculated with phage, transplanted two weeks later, according to 10 8 CFU / seedling was inoculated with the pathogen Ralstonia solanacearum. The incidence rate was recorded daily after the first plant showed symptoms, until the disease situation in the tomato plants in the greenhouse stabilized.

[0093] The formula for calculating the incidence rate is as follows:

[0094] Incidence rate (%) = Number of diseased plants in the plot / Total number of plants in the plot × 100%

[0095] The results are as follows Figure 10 As shown, inoculating the rhizosphere of tomatoes with only the biocontrol bacterium YL-Ste-01 significantly reduced the incidence of bacterial wilt. However, when both the biocontrol bacterium YL-Ste-01 and the bacteriophage YL-Ste-01P were inoculated simultaneously, the incidence rate was not significantly different from the control, indicating that the bacteriophage YL-Ste-01P in the soil could reduce the biocontrol effect of the biocontrol bacterium YL-Ste-01 by lysing it. In contrast, inoculation with the evolved strain YL-Ste-01evo, which evolved under bacteriophage stress, significantly reduced the incidence of disease regardless of the presence of the bacteriophage, demonstrating a good biocontrol effect unaffected by the bacteriophage (P<0.05).

[0096] In summary, bacteriophages reduce the sensitivity of biocontrol bacteria to bacteriophages while enhancing their own antibacterial properties through stress-induced evolution, providing a new approach for controlling soil-borne bacterial wilt using biological methods.

[0097] References:

[0098] [1].Keswani C,Singh HB,García-Estrada C,et al.Antimicrobialsecondary metabolites from agriculturally important bacteria as next-generation pesticides[J].Applied Microbiology and Biotechnology,2020,104:1013-1034.

[0099] [2].Hsu Bryan B.,Gibson Travis E.,YeliseyevVladimir,LiuQing,LyonLorena,BryLynn,Silver Pamela A.,Gerber Georg K..Dynamic Modulation of theGut Microbiota and Metabolome by Bacteriophages in a Mouse Model[J].CellHost&Microbe,2018,25(6).

[0100] [3]. Wei Zhong. Study on the effect and mechanism of bio-organic fertilizer in controlling soil-borne bacterial wilt of tomato [D]. Nanjing Agricultural University, 2012.

[0101] [4].Breitbart M, Rohwer F. Here a virus, there a virus, everywhere the same virus? Trends Microbiol2005;13:278-84.

[0102] [5].Ofir G,Sorek R.Contemporary Phage Biology:From Classic Models toNew Insights.Cell 2018;172:1260-70.

[0103] [6]. Aa, Haeruman, Azam, et al. Bacteriophage-host arm race: an update on the mechanism of phage resistance in bacteria and revenge of the phage with the perspective for phage therapy. [J]. Applied Microbiology&Biotechnology, 2019, 103: 2121-2131.

Claims

1. A bacterial wilt biocontrol bacterium, YL-Ste-01evo, was classified and named Stenotrophomonas maltophilia (…). Stenotrophomonas maltophilia It was deposited at the China Center for Type Culture Collection on June 21, 2022, with accession number CCTCC NO: M 2022939.

2. A bacterial agent containing the biocontrol bacterium YL-Ste-01evo as described in claim 1, wherein the concentration of the bacterial agent is 1×10⁻⁶. 8 ~5×10 8 CFU / mL.

3. A lytic bacteriophage, YL-Ste-01P, was classified and named Stenotrophomonas maltophilia bacteriophage (…). Stenotrophomonas maltophilia The phage was deposited at the China Center for Type Culture Collection on April 22, 2022, with accession number CCTCC NO: M2022460.

4. The application of the biocontrol bacterium YL-Ste-01evo as described in claim 1, or the biocontrol bacterium YL-Ste-01evo as described in claim 1 in combination with the bacteriophage YL-Ste-01P as described in claim 3, in the control of soil-borne bacterial wilt of tomato.

5. A method for controlling soil-borne bacterial wilt of tomato, characterized in that: include: Six to seven days after transplanting tomato seedlings, the biocontrol agent YL-Ste-01evo (as claimed in claim 2) was added to the soil by root irrigation.

6. The method for controlling soil-borne bacterial wilt of tomato according to claim 5, characterized in that: The final concentration of the biocontrol bacterium YL-Ste-01evo was 1×10⁻⁶. 8 ~1×10 9 CFU / tree.

7. A method for controlling soil-borne bacterial wilt by utilizing obligate bacteriophages to enhance biocontrol bacteria, characterized in that: Six to seven days after transplanting tomato seedlings, the biocontrol agent YL-Ste-01evo (as claimed in claim 2) was added to the soil by root irrigation. Two to three days later, bacteriophage YL-Ste-01P was inoculated.

8. The method for controlling soil-borne bacterial wilt using obligate bacteriophages to enhance biocontrol bacteria according to claim 7, characterized in that: The final concentration of the biocontrol bacterium YL-Ste-01evo was 1×10⁻⁶. 8 ~1×10 9 CFU / cell, the final titer of phage YL-Ste-01P was 1×10⁻⁶. 8 ~1×10 9 PFU / tree.

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