Method for improving generation of beneficial bacterium antagonistic substance by using bacteriophage, antagonistic substance and application of antagonistic substance

Abstract

The invention discloses a method for improving generation of antagonistic substances of beneficial bacteria by utilizing bacteriophages. Under the continuous stress of the bacteriophage YL-Ste-01P, the yield of the bacteriostatic metabolic substances of the beneficial bacterium YL-Ste-01 is obviously increased. A bacterial wilt biocontrol bacterium YL-Ste-01 and a bacteriophage YL-Ste-01P are inoculated into an NA liquid culture medium for co-culture to obtain a culture solution, the culture solution is centrifuged to obtain supernate, and the supernate is filtered by a filter membrane to obtain fermentation liquor. According to the present invention, the co-culture fermentation broth of YL-Ste-01 and YL-Ste-01P, alpha-ketoglutaric acid, guanidinopropionic acid and fumagillin have significant inhibition effects on ralstonia solanacearum; the invention discloses application of an antagonistic substance in preparation of a reagent for inhibiting ralstonia solanacearum or prevention and control of bacterial wilt. The invention discloses application of alpha-ketoglutaric acid, guanidinopropionic acid or fumagillin to preparation of a reagent for inhibiting ralstonia solanacearum or prevention and control of bacterial wilt.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and relates to a method for enhancing the production of antagonistic substances by obligate bacteriophages in beneficial bacteria, and antagonistic substances and applications thereof, and specifically relates to antagonistic substances co-cultured with beneficial bacteria YL-Ste-01 and a lytic bacteriophage YL-Ste-01P that obligately targets host bacteria YL-Ste-01, and applications thereof in the prevention and control of bacterial wilt, and applications of α-ketoglutaric acid, guanidinopropionic acid or fumagillin with significantly increased production in the prevention and control of bacterial wilt. Background Art

[0002] Soil-borne bacterial wilt caused by Ralstonia solanacearum (Ralstonia solanacearum) has seriously restricted the sustainable development of agriculture and has caused serious losses to farmers over the years. Although physical and chemical control methods are effective quickly, they are easy to induce drug resistance in pathogens, and because of the lack of targeting during use, they kill Ralstonia and also destroy the microbial community. Using rhizosphere microorganisms to control soil-borne diseases has become a new environmentally friendly biological control method. [1] , such as beneficial bacteria, bacteriophages, and protozoa. Among them, bacteriophages are viruses that specifically infect bacteria. Studies have shown that the specific bacteriophages of Ralstonia solanacearum play an important role in the prevention and control of soil-borne bacterial wilt. In addition to the specific bacteriophages of pathogens, there are also a large number of bacteriophages of beneficial bacteria in the rhizosphere. However, there are relatively few studies on the effects of specific bacteriophages of beneficial bacteria in the rhizosphere on the beneficial bacteria's inhibition of pathogen growth. Summary of the invention

[0003] The inventors made creative discoveries through research: under the continuous stress of bacteriophage YL-Ste-01P and the NA liquid culture medium providing sufficient resources for the beneficial bacteria YL-Ste-01, the beneficial bacteria YL-Ste-01 was promoted to evolve rapidly, resulting in significant changes in the metabolites secreted by it, among which the production of antibacterial metabolites was significantly increased; the co-culture fermentation broth of beneficial bacteria YL-Ste-01 and bacteriophage YL-Ste-01P (i.e., antagonistic substances), α-ketoglutaric acid, guanidinopropionic acid, and fumagillin all had significant inhibitory effects on Ralstonia solanacearum, and can be used to prevent and control tomato bacterial wilt and other bacterial wilt diseases.

[0004] The purpose of the present invention is to provide a method for enhancing the production of antagonistic substances of beneficial bacteria by using bacteriophages, comprising: adjusting the OD of a suspension of bacterial wilt biocontrol bacteria YL-Ste-01 in a logarithmic growth phase; 600 is 0.4-0.6, and the titer of bacteriophage YL-Ste-01P suspension is 10 7 ~10 8PFU / mL, add YL-Ste-01 bacterial suspension to NA liquid culture medium at 1-2% of the total culture system volume, add phage YL-Ste-01P suspension to NA liquid culture medium at 1-2% of the total culture system volume, and culture at 28-30°C and 160-180rpm for 48-50h.

[0005] Another object of the present invention is to provide an antagonistic substance obtained by using bacteriophage to enhance the production of antagonistic substances of beneficial bacteria.

[0006] Another object of the present invention is to provide an antagonistic substance, wherein the antagonistic substance is obtained by inoculating the bacterial wilt biocontrol bacteria YL-Ste-01 and the bacteriophage YL-Ste-01P into a NA liquid culture medium for co-culturing to obtain a culture solution, centrifuging the culture solution to obtain a supernatant, and filtering the supernatant through a filter membrane to obtain a fermentation solution.

[0007] The antagonistic substance contains alpha-ketoglutaric acid, guanidinopropionic acid and fumagillin.

[0008] The antagonistic substance is prepared by the following method: adjusting the OD value of the bacterial suspension of bacterial wilt biocontrol bacteria YL-Ste-01 in the logarithmic growth phase 600 is 0.4-0.6, and the titer of bacteriophage YL-Ste-01P suspension is 10 7 ~10 8 PFU / mL, add YL-Ste-01 bacterial suspension to NA liquid culture medium at 1-2% of the total volume of the culture system, add phage YL-Ste-01P suspension to NA liquid culture medium at 1-2% of the total volume of the culture system, culture at 28-30°C, 160-180rpm shaking for 48-50h, centrifuge the culture solution, take the supernatant, filter the supernatant through a filter membrane to obtain a fermentation broth.

[0009] The centrifugal speed is 10000-12000 rpm, and the centrifugal time is 3-5 minutes.

[0010] The filter membrane is a 0.22 μm filter membrane.

[0011] The bacterial wilt biocontrol bacterium YL-Ste-01 suspension is prepared by the following method: inoculating the bacterial wilt biocontrol bacterium YL-Ste-01 into a NA liquid culture medium, culturing at a temperature of 28 to 30°C and a rotation speed of 160 to 180 rpm to obtain a YL-Ste-01 bacterial suspension in a logarithmic growth phase, and adjusting the OD of the bacterial suspension with sterile water. 600 The value is between 0.4 and 0.6.

[0012] The bacteriophage YL-Ste-01P suspension is prepared by the following method: using NA liquid medium to culture the bacterial wilt biocontrol bacteria YL-Ste-01 in the logarithmic growth phase, and adjusting the OD of the bacterial suspension with sterile water. 600 The value was 0.4-0.6, and the titer of bacteriophage YL-Ste-01P was 10 7 ~10 8 PFU / mL, inoculate the bacteriophage YL-Ste-01P into the bacterial suspension of YL-Ste-01, a biocontrol bacterium for bacterial wilt, at an optimal multiplicity of infection (MOI) of 0.1-1, and culture at 28-30°C with shaking at 160-180 rpm for 12 h. Centrifuge the culture solution, take the supernatant, and filter it with a 0.22 μm filter membrane to obtain a suspension of bacteriophage YL-Ste-01. Adjust the titer of the suspension of bacteriophage YL-Ste-01 to 10 with sterile water. 7 ~10 8 PFU / mL; wherein the centrifugal temperature is room temperature, the centrifugal speed is 10000-12000 rpm, and the centrifugal time is 3-5 min.

[0013] Another object of the present invention is to provide the use of the antagonistic substance in the preparation of an agent for inhibiting Ralstonia solanacearum.

[0014] Another object of the present invention is to provide the use of the antagonistic substance in preventing and controlling bacterial wilt.

[0015] The bacterial wilt biocontrol bacteria YL-Ste-01 and bacteriophage YL-Ste-01P are both known microorganisms, recorded in the invention patent application filed by the applicant on November 1, 2022, with the invention name "Method for using obligate bacteriophages to enhance the biocontrol bacteria for controlling soil-borne bacterial wilt", application number "2022113553017", and publication number: CN 115651867 A.

[0016] The bacterial wilt biocontrol bacterium YL-Ste-01, classified and named Stenotrophomonas maltophilia, was deposited in the China Center for Type Culture Collection on March 28, 2022, with the deposit number CCTCCNO: M2022324.

[0017] Bacteriophage YL-Ste-01P belongs to the order Caudovirales, family Stenotrophomonas, and is classified as Stenotrophomonas maltophilia phage. It was deposited in the China Center for Type Culture Collection on April 22, 2022, with the deposit number CCTCC NO:M 2022460.

[0018] Another object of the present invention is to provide the use of α-ketoglutaric acid, guanidinopropionic acid or fumagillin in the preparation of an agent for inhibiting Ralstonia solanacearum.

[0019] Another object of the present invention is to provide the use of α-ketoglutaric acid, guanidinopropionic acid or fumagillin in preventing and controlling bacterial wilt.

[0020] Another object of the present invention is to provide an agent for preventing and controlling bacterial wilt, wherein the agent contains α-ketoglutaric acid, guanidinopropionic acid or fumagillin.

[0021] The bacterial wilt bacteria described are tomato bacterial wilt, tobacco bacterial wilt, peanut bacterial wilt, eggplant bacterial wilt and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Relative inhibition rate of beneficial bacteria products on pathogenic Ralstonia solanacearum after different treatments.

[0023] Figure 2 To explore the effect of phage infection on the metabolites of beneficial bacteria based on the metabolome; A is the overall impact map, and B is the metabolites with significant changes.

[0024] Figure 3 It is the change multiple of three types of metabolites (α-ketoglutarate, guanidinopropionic acid, and fumagillin) that are significantly upregulated after phage infection of beneficial bacteria.

[0025] Figure 4 It is the effect of three types of metabolites (α-ketoglutaric acid, guanidinopropionic acid, and fumagillin) on the growth of pathogenic Ralstonia solanacearum. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0027] The culture medium formula is as follows:

[0028] NA liquid culture medium: 10 g glucose, 5 g peptone, 3 g beef extract, 0.5 g yeast powder, 1000 mL deionized water, adjust pH to 7.2-7.4, and sterilize at 115°C for 30 min.

[0029] NA solid medium: Add 25 g agar powder to 1 L NA liquid medium.

[0030] Gentamicin solid medium: Heat 300 mL of NA semi-solid medium and add 3 μL of 1% gentamicin.

[0031] The bacterial suspension of Ralstonia solanacearum was prepared by the following method: Ralstonia solanacearum QL-Rs1115 was activated with gentamicin solid culture medium, a single colony was picked and transferred to NA liquid culture medium, and cultured with shaking at 30°C and 170rpm for 24h to obtain the bacterial suspension of Ralstonia solanacearum in the logarithmic growth phase.

[0032] Preparation of YL-Ste-01 bacterial suspension: The biocontrol bacteria YL-Ste-01 was inoculated into NA liquid culture medium, and cultured at 30°C and 180 rpm to obtain YL-Ste-01 bacterial suspension in the logarithmic growth phase. The OD of the bacterial suspension was adjusted with sterile water. 600 The value is 0.5, which means the concentration is 10 8 CFU / mL of bacterial agent.

[0033] Preparation of bacteriophage YL-Ste-01P suspension: Use NA liquid medium to obtain YL-Ste-01 bacterial suspension in the logarithmic growth phase, and use sterile water to adjust the OD of the bacterial suspension to 600 The value was adjusted to 0.5 and the phage titer was 10 7 ~10 8 PFU / mL, and the phage was inoculated at OD according to the optimal infection multiplicity (MOI 0.1). 600 The YL-Ste-01 bacterial suspension with a titer of 0.5 was cultured at 30°C and 180 rpm for 12 h. The culture solution was centrifuged at 10000 rpm for 5 min at room temperature. The supernatant was filtered with a 0.22 μm filter membrane to obtain a phage YL-Ste-01P suspension. The titer of the phage YL-Ste-01P suspension was adjusted to 10 with sterile water. 8 PFU / mL.

[0034] Example 1

[0035] Effects of bacteriophages on the ability of host bacteria to antagonize pathogens

[0036] By setting up three treatments of single culture, co-culture and cell disruption, and keeping other conditions the same, the OD was measured using an enzyme reader. 600 Used to characterize the growth of pathogens.

[0037] (1) Specific operations of the three processes:

[0038] Single culture treatment group: Sterile water was used to adjust the OD of the YL-Ste-01 bacterial suspension in the logarithmic growth phase. 600 The inoculum size was 0.5, and the YL-Ste-01 bacterial suspension was added to the NA liquid culture medium at a 1% inoculation rate. The culture was shaken at 30°C and 170 rpm in a shaker for 48 h. The culture solution was centrifuged at 10000 rpm for 3 min at room temperature. The supernatant was taken and filtered with a 0.22 μm filter membrane to obtain a single culture fermentation broth.

[0039] Co-culture treatment group: Sterile water was used to adjust the OD of the YL-Ste-01 bacterial suspension in the logarithmic growth phase. 600 is 0.5, and the titer of the bacteriophage YL-Ste-01P suspension is 10 8 PFU / mL, add YL-Ste-01 to NA liquid culture medium at an inoculation amount of 1% of the total culture system volume, add phage YL-Ste-01P suspension to NA liquid culture medium at an inoculation amount of 1% of the total culture system volume, shake and culture in a shaker at 30°C and 170rpm for 48h, centrifuge the culture solution at 10000rpm at room temperature for 3min, take the supernatant, filter it with a 0.22μm filter membrane, and obtain the co-culture fermentation broth.

[0040] Cell disruption treatment group: Sterile water was used to adjust the OD of the YL-Ste-01 bacterial suspension in the logarithmic growth phase. 600 The inoculum volume of YL-Ste-01 bacterial suspension was 0.5, and the inoculation amount of 1% of the total volume of the culture system was added to the NA liquid culture medium. The culture was shaken in a shaker at 30°C and 170rpm for 48h. The culture medium was disrupted by an ultrasonic cell disruptor. The program was set to a power of 500W, a total disruption time of 5min, and a disruption interval of 5s for 5s. The disrupted liquid was then centrifuged at 10000rpm for 3min at room temperature. The supernatant was taken and filtered with a 0.22μm filter membrane to obtain a cell lysis product.

[0041] (2) Dip the inoculation loop into the bacterial cells of Ralstonia solanacearum QL-Rs1115 and streak them on a gentamicin solid culture medium plate, and culture them at 30°C for 36 hours to obtain a single bacterium; pick a single bacterium and culture it in NA liquid culture medium, and shake and culture it at 30°C and 170 rpm for 24 hours to obtain a Ralstonia solanacearum suspension, and adjust the OD of the Ralstonia solanacearum suspension with sterile water. 600 In a 96-well plate 200 μL system, add Ralstonia solanacearum suspension at 1% inoculation volume of the total culture system volume, add single culture fermentation broth, co-culture single culture or cell disruption lysis product at 10% inoculation volume (blank control group added equal volume of NA liquid culture medium instead of fermentation broth), set 8 replicates; shake culture at 30°C, 170 rpm in a shaker for 24 h, and measure the absorbance at 600 nm wavelength.

[0042] Inhibition rate = (blank group OD 600 - Treatment group OD 600 ) / blank group OD 6000 ×100%

[0043] Results Figure 1 The cell disruption treatment group had the lowest inhibition rate, and the co-culture treatment group (i.e. Figure 1The inhibition rate of the group "+ phage" was the highest, indicating that the enhanced ability of the host's beneficial bacteria to antagonize Ralstonia solanacearum under phage stress is not caused by phage lysis of host cells and release of intracellular substances, but rather by phage stress causing changes in the metabolic substances secreted by the bacterial host.

[0044] Example 2

[0045] Effects of bacteriophages on host secretion of metabolites

[0046] Metabolome sequencing analysis was performed on the fermentation broth obtained by culturing YL-Ste-01 alone and co-culturing YL-Ste-01 and YL-Ste-01P for 24 hours. The specific operations were as follows:

[0047] Prepare YL-Ste-01 bacterial suspension and bacteriophage YL-Ste-01P suspension, adjust the concentration of YL-Ste-01 bacterial suspension to 10 with sterile water. 8 CFU / mL, the concentration of bacteriophage YL-Ste-01P suspension was adjusted to 10 with sterile water. 8 PFU / mL.

[0048] Co-culture of YL-Ste-01 and YL-Ste-01P: Add NA liquid medium to a 24-well plate, inoculate YL-Ste-01 bacterial suspension at 1% of the total volume of the culture system, and inoculate phage YL-Ste-01P suspension at 1% of the total volume of the culture system; YL-Ste-01 culture alone: ​​Add NA liquid medium to a 24-well plate, inoculate YL-Ste-01 bacterial suspension at 1% of the total volume of the culture system. Each treatment was repeated four times, shaken at 30°C and 170rpm for 24h, transferred to a centrifuge tube, centrifuged at 4°C and 3000rpm for 10min, and quickly filtered with a 0.22μm pore size filter membrane to obtain a filtrate, which was quickly frozen in liquid nitrogen. The sample was sequenced by Jiaxing Maiwei Metabolic Biotechnology Co., Ltd. for non-targeted metabolome sequencing. Thaw the sample until there is no ice in the sample, vortex for 10 seconds to mix, and transfer 50 μL of the sample to the corresponding numbered centrifuge tube; add 150 μL of 20% acetonitrile methanol internal standard extract, vortex for 3 minutes, centrifuge at 4°C and 12000 rpm for 10 minutes, transfer 150 μL of the supernatant to another corresponding numbered centrifuge tube, let it stand in a -20°C refrigerator for 30 minutes, centrifuge at 4°C and 12000 rpm for 3 minutes, and transfer 120 μL of the supernatant to the inner liner of the corresponding injection bottle for analysis on the machine. Liquid chromatography conditions: chromatographic column: Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm×100 mm); mobile phase A: ultrapure water (0.1% formic acid); mobile phase B: acetonitrile (0.1% formic acid); volume ratio of mobile phase A and mobile phase B: 0 min A: B is 95:5 (v / v), 11 min A: B is 10:90 (v / v), 14 min A: B is 95:5 (v / v); instrument column temperature: 40 °C; flow rate: 0.40 mL / min; injection volume: 2 μL.

[0049] Results Figure 2 After bacteriophage YL-Ste-01P infected bacterial host YL-Ste-01, 384 substances in the host fermentation liquid were down-regulated and 99 substances were up-regulated. Figure 3 As shown, the production of α-ketoglutaric acid (α-Ketoglutaric Acid), guanidinopropionic acid (Guanidinopropionic acid) and fumagillin (Fumagillin) in the fermentation broth of YL-Ste-01 infected by bacteriophage YL-Ste-01P was significantly increased, with α-ketoglutaric acid increased by 928.2%, guanidinopropionic acid increased by 132.2%, and fumagillin increased by 80.9 times.

[0050] Example 3

[0051] Effects of host-secreted metabolites on Ralstonia solanacearum

[0052] The effects of α-ketoglutaric acid, guanidinopropionic acid and fumagillin on Ralstonia solanacearum were further verified. The specific operations were as follows:

[0053] 1335 μL NA liquid medium was added to a 24-well plate (Costar), and 15 μL Ralstonia solanacearum suspension (OD 600 The concentration of α-ketoglutaric acid, guanidinopropionic acid and fumagillin solution (all prepared with sterile water, with a final concentration of 0.1 mM in the system) was 0.5, the same as in Example 1), 150 μL of α-ketoglutaric acid, guanidinopropionic acid and fumagillin solution (all prepared with sterile water, with a final concentration of 0.1 mM in the system) were added to the three experimental groups respectively, and an equal volume of sterile water was added to the control group. Six replicates were used for each treatment, and the culture was shaken in a shaker at 30°C and 170 rpm for 24 h, and the absorbance at a wavelength of 600 nm was measured.

[0054] Results Figure 4 All three substances have significant inhibitory effects on Ralstonia solanacearum. The inhibition rates of α-ketoglutaric acid, guanidinopropionic acid and fumagillin on Ralstonia solanacearum are 66.04%, 16.22% and 16.33% respectively. This indicates that after the bacteriophage YL-Ste-01P infects the bacterial host YL-Ste-01, it promotes the production of α-ketoglutaric acid, guanidinopropionic acid and fumagillin, which are antagonists of beneficial bacteria in the host. Moreover, the above three antagonistic substances can significantly inhibit Ralstonia solanacearum and can be used to prevent and control tomato bacterial wilt.

Claims

1. A method for enhancing the production of antagonistic substances of beneficial bacteria by using bacteriophage, characterized in that: include: Adjust the OD of bacterial suspension of bacterial wilt biocontrol bacterium YL-Ste-01 in logarithmic growth phase 600 is 0.4-0.6, and the titer of bacteriophage YL-Ste-01P suspension is 10 7 ~10 8 PFU / mL, adding YL-Ste-01 bacterial suspension to NA liquid culture medium according to 1-2% of the total volume of the culture system, adding bacteriophage YL-Ste-01P suspension to NA liquid culture medium according to 1-2% of the total volume of the culture system, and culturing at a temperature of 28-30°C and shaking at 160-180rpm for 48-50h; wherein the preservation number of the bacterial wilt biocontrol bacteria YL-Ste-01 is CCTCC NO: M 2022324, and the preservation number of the bacteriophage YL-Ste-01P is CCTCC NO: M 2022460.

2. An antagonistic substance obtained by the method of enhancing the production of antagonistic substances of beneficial bacteria by using bacteriophage as claimed in claim 1.

3. An antagonist substance, characterized in that: The antagonistic substance is prepared by inoculating bacterial wilt biocontrol bacteria YL-Ste-01 and bacteriophage YL-Ste-01P into NA liquid culture medium for co-culturing to obtain culture solution, centrifuging the culture solution to obtain supernatant, and filtering the supernatant through a filter membrane to obtain fermentation liquid; wherein the preservation number of the bacterial wilt biocontrol bacteria YL-Ste-01 is CCTCC NO: M2022324, and the preservation number of the bacteriophage YL-Ste-01P is CCTCC NO: M 2022460.

4. The antagonist substance according to claim 1, characterized in that: The antagonistic substance is prepared by the following method: adjusting the OD value of the bacterial suspension of bacterial wilt biocontrol bacteria YL-Ste-01 in the logarithmic growth phase 600 is 0.4-0.6, and the titer of bacteriophage YL-Ste-01P suspension is 10 7 ~10 8 PFU / mL, add YL-Ste-01 bacterial suspension to NA liquid culture medium at 1-2% of the total volume of the culture system, add phage YL-Ste-01P suspension to NA liquid culture medium at 1-2% of the total volume of the culture system, culture at 28-30°C, 160-180rpm shaking for 48-50h, centrifuge the culture solution, take the supernatant, filter the supernatant through a filter membrane to obtain a fermentation broth.

5. Use of the antagonistic substance according to claim 2 or 3 in the preparation of an agent for inhibiting Ralstonia solanacearum.

6. Use of the antagonistic substance according to claim 2 or 3 in preventing and controlling bacterial wilt.

7. Use of α-ketoglutaric acid, guanidinopropionic acid or fumagillin in the preparation of reagents for inhibiting Ralstonia solanacearum.

8. Application of α-ketoglutaric acid, guanidinopropionic acid or fumagillin in the prevention and control of bacterial wilt.

9. An agent for preventing and controlling bacterial wilt, characterized in that: The reagent contains alpha-ketoglutaric acid, guanidinopropionic acid or fumagillin.

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