Xenorhabdus bacteria for antagonizing ralstonia solanacearum, a biocontrol preparation and application thereof

Abstract

The present application relates to a kind of antagonistic serratia marcescens of ralstonia solanacearum, biocontrol preparation and its application, belong to the field of agricultural environmental microorganism technology.The serratia marcescens of the present application is named Serratia marcescens RS189, has been preserved in China General Microbiological Culture Collection Center on April 24, 2024, and the strain preservation number is CGMCC NO.30415.The Serratia marcescens RS189 described in the present application is isolated from the rhizosphere of tobacco infected with tobacco bacterial wilt, and the metabolic crude extract of the strain can effectively inhibit the growth of tobacco bacterial wilt pathogen;The metabolite of antagonistic bacteria Serratia marcescens RS189 can effectively prevent and treat tobacco bacterial wilt, and the relative control effect reaches 88.14%, and has good application prospect in the prevention and treatment of tobacco bacterial wilt.

Description

Technical Field

[0001] This invention relates to a Serratia marcescens antagonistic to Ralstonia solanacearum, a biocontrol agent and its application, belonging to the field of agricultural environmental microbiology technology. Background Technology

[0002] Bacterial wilt is one of the most destructive soil-borne bacterial diseases in the world. Its causative agent, *Ralstonia solanacearum* (commonly known as Bacterial Ralstonia), is highly diverse and has a wide host range (infecting over 450 plant species in more than 50 families). Massive proliferation of *Ralstonia solanacearum* can lead to soil microecological imbalance, seriously threatening global agricultural production. While chemical pesticides can control plant diseases to some extent, over-reliance on chemical pesticides and their unscientific use can damage the original ecological structure of the soil, causing soil acidification and other problems, resulting in latent degradation of soil organisms. With the rise of the global trend towards green agriculture and sustainable development, utilizing microorganisms or their metabolites to control plant diseases has become an important development trend.

[0003] Currently, the prevailing view is that the control effect of biocontrol bacteria is essentially an interaction between plants, antagonistic bacteria, and pathogens. On the one hand, antagonistic bacteria compete with pathogens for nutrients and living space, or directly inhibit pathogen growth by using their secondary metabolites (antibiotics, toxins, chitinases, etc.). On the other hand, antagonistic bacteria secrete certain metabolites to induce systemic resistance in plants or stimulate the plant's immune system, indirectly assisting plants in resisting pathogens. Besides resource competition with pathogens, other modes of action of antagonistic bacteria are achieved through secondary metabolites.

[0004] Chinese invention patent application CN117305184A, published on December 29, 2023, discloses *Serratia marcescens* YXNU08 and its application in controlling tobacco bacterial wilt. Specifically, it discloses that *Serratia marcescens* YXNU08 has a significant control effect on preventing tobacco plants from contracting bacterial wilt. However, the results of the plate antagonism method in its embodiments show that the inhibition zone of *Serratia marcescens* YXNU08 against *Ralstonia marcescens* is only 3 mm, indicating that its antagonistic ability against *Ralstonia marcescens* is limited. It mainly controls tobacco bacterial wilt through mechanisms such as niche competition and inducing plant resistance. This indicates that Serratia marcescens YXNU08 is necessary to treat tobacco plants when controlling bacterial wilt. However, the actual field conditions and greenhouse environments used in tobacco cultivation differ significantly from those in experiments. Therefore, the colonization effect of Serratia marcescens YXNU08 on plants is unpredictable, and its effectiveness in controlling bacterial wilt is also unknown. Summary of the Invention

[0005] The first objective of this invention is to provide a *Serratia marcescens* strain that antagonizes *Ralstonia solanacearum*, and to provide a biocontrol bacterium that inhibits *Ralstonia solanacearum* by secreting metabolites with good inhibitory effect.

[0006] The second objective of this invention is to provide a biocontrol agent antagonizing Ralstonia solanacearum, which provides a biocontrol agent with good control effect and high biosafety against Ralstonia solanacearum.

[0007] A third objective of this invention is to provide the application of *Serratia marcescens* in the preparation of *Ralstonia solanacearum* inhibitors, thereby providing a new microbial resource for *Ralstonia solanacearum* inhibitors.

[0008] The fourth objective of this invention is to provide an application of a biocontrol agent in the control of bacterial wilt of plants, and to provide a method for controlling bacterial wilt of plants that is effective, biosafety-free, and environmentally friendly.

[0009] To achieve the above objectives, the technical solution of *Serratia marcescens* antagonizing *Ralstonia solanacearum* in this invention is as follows:

[0010] A *Serratia marcescens* species antagonistic to *Ralstonia solanacearum*, the taxonomic name of which is *Serratia marcescens* and the strain preservation number is CGMCC NO.30415.

[0011] The beneficial effects of the above technical solution are as follows: This invention, which describes a *Serratia marcescens* strain antagonistic to *Ralstonia solanacearum*, is a pioneering invention. This invention isolates an antagonistic bacterium, RS189, with strong inhibitory effects against *Ralstonia solanacearum* from the root surface soil of tobacco plants. Physiological, biochemical, and molecular biological identification confirms that this bacterium is *Serratia marcescens*, classified as *Serratia marcescens*. Furthermore, its colony characteristics indicate that the *Serratia marcescens* RS189 obtained by this invention does not secrete colored antibiotics such as undecylamine erythromycin.

[0012] To achieve the above objectives, the technical solution of the biocontrol agent antagonizing Ralstonia solanacearum in this invention is as follows:

[0013] A biocontrol agent antagonizing Ralstonia solanacearum, the biocontrol agent comprising the fermentation supernatant or metabolites of Serratia marcescens.

[0014] The beneficial effects of the above technical solution are as follows: This invention isolated a strain RS189 from the root surface soil of tobacco plants. After physiological, biochemical, and molecular biological identification, this bacterium was identified as *Serratia marcescens*, classified as *Serratia marcescens*. Screening experiments using the plate confrontation method demonstrated that *Serratia marcescens* RS189 exhibited an inhibition zone width of 12.20±0.15 mm and an inhibition zone diameter of 32.40±0.29 mm against *Ralstonia solanacearum*, demonstrating a good inhibitory effect on *Ralstonia solanacearum*.

[0015] Furthermore, antibacterial experiments on the metabolites of Serratia marcescens RS189 demonstrated that the metabolites of Serratia marcescens RS189 had a significant inhibitory effect on Ralstonia solanacearum. Moreover, the inhibitory effect of the metabolites on Ralstonia solanacearum was basically the same before and after high-temperature (105℃) treatment. This indicates that the metabolites of Serratia marcescens RS189 have good antibacterial ability and high resistance to high temperature. The high-temperature resistance of the metabolites is beneficial to the subsequent production and processing of the metabolites, and thus helps to expand their practical application range.

[0016] As a further improvement, the metabolite is an ethyl acetate extract of the fermentation supernatant of Serratia marcescens.

[0017] To achieve the above objectives, the technical solution of this invention for the application of *Serratia marcescens* in the preparation of *Ralstonia solanacearum* inhibitor is as follows:

[0018] Application of *Serratia marcescens* in the preparation of *Ralstonia solanacearum* inhibitor.

[0019] The beneficial effects of the above technical solution are as follows: Firstly, this invention demonstrates through plate confrontation experiments that *Serratia marcescens* RS189 possesses excellent antagonistic ability against *Ralstonia solanacearum*. Further analysis of its metabolites, high-temperature treated metabolites, and concentrated metabolites all show good inhibitory effects on *Ralstonia solanacearum*, indicating that the metabolites of this bacterium contain substances that effectively inhibit *Ralstonia solanacearum* (they still exhibit antibacterial activity after high-temperature treatment, indicating that this substance is not a conventional protein-based substance such as chitinase). This invention provides a new microbial resource for *Ralstonia solanacearum* inhibitors, and possesses good biosafety and environmental friendliness.

[0020] As a further improvement, the active ingredient of the inhibitor is the fermentation supernatant or metabolites of Serratia marcescens.

[0021] To achieve the above objectives, the technical solution for the application of a biocontrol agent in the control of bacterial wilt of plants according to the present invention is as follows:

[0022] Application of a biocontrol agent in the control of bacterial wilt in plants.

[0023] The beneficial effects of the above technical solution are as follows: Experiments have demonstrated that the metabolites of *Serratia marcescens* RS189, the metabolites treated at high temperatures, and the concentrated metabolites all exhibit good inhibitory effects against *Ralstonia solanacearum*. Simultaneously, experiments on the control of tobacco bacterial wilt by *Serratia marcescens* RS189 metabolites have shown that the metabolites of this bacterium can effectively prevent tobacco bacterial wilt. These conclusions fully demonstrate that the metabolites of *Serratia marcescens* RS189 can be used as a biocontrol agent for the control of plant bacterial wilt. Using these metabolites to control plant bacterial wilt overcomes the influence of environmental factors present when traditional bacterial solutions are applied directly, thus enhancing their control effect.

[0024] As a further improvement, biocontrol agents are used to treat the plants by root irrigation.

[0025] As a further improvement, the plant is tobacco. Attached Figure Description

[0026] Figure 1 This refers to the plate confrontation experiment of the antagonistic bacteria RS189 in Example 1 of the present invention;

[0027] Figure 2 This is a colony morphology diagram of the antagonistic bacterium RS189 in Example 2 of the present invention;

[0028] Figure 3 This is the phylogenetic tree constructed based on the 16S rRNA gene sequence of strain RS189 in Example 2 of the present invention;

[0029] Figure 4 The crude extract of RS189 metabolism, high-temperature treatment of the crude extract, and the inhibition of Ralstonia solanacearum by the crude extract concentrate in Example 3 of this invention;

[0030] Figure 5 This is a metabolic profile of the crude ethyl acetate extract of strain RS189 in Example 3 of the present invention;

[0031] Figure 6 This is a predicted metabolite diagram of strain RS189 in Example 3 of the present invention;

[0032] Figure 7 This invention demonstrates the control effect of the RS189 strain metabolite on tobacco bacterial wilt in Example 4 of this invention.

[0033] Figure 8 This invention illustrates the effect of RS189 strain metabolites on tobacco growth in Example 4 of this invention. Detailed Implementation

[0034] Existing technologies using *Serratia marcescens* YXNU08 to control tobacco bacterial wilt employ mechanisms such as niche competition and induction of plant resistance. However, the actual field conditions differ significantly from the experimental greenhouse environments, making it difficult to predict the colonization effectiveness of *Serratia marcescens* YXNU08 on plant plants, and its effectiveness in controlling bacterial wilt remains unknown. This invention provides *Serratia marcescens* RS189, which, through experiments, demonstrates the production of metabolites with strong antibacterial properties and heat resistance.

[0035] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, all reagents, instruments and other items used in the embodiments are commercially available products.

[0036] The culture medium and reagent components involved in the following embodiments of the present invention:

[0037] NA liquid culture medium: glucose 2.5 g / L, beef extract 3.0 g / L, peptone 5.0 g / L, add 1000 mL of distilled water, natural pH, autoclave at 115℃ for 30 min.

[0038] NA solid medium: glucose 2.5 g / L, beef extract 3.0 g / L, peptone 5.0 g / L, agar 18.0 g / L, add 1000 mL of distilled water, natural pH, autoclave at 115℃ for 30 min.

[0039] TSB liquid medium: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L NaCl, add 1000 mL distilled water, pH 7.0, autoclave at 115℃ for 30 min.

[0040] TSB solid medium: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L NaCl, 20 g / L agar, 1000 mL distilled water added, pH 7.0, autoclaved at 115°C for 30 min.

[0041] Specific embodiments of the present invention regarding an antagonistic Serratia marcescens against Ralstonia solanacearum, a biocontrol agent, and its application:

[0042] The *Serratia marcescens* strain of this invention is named RS189 and classified as *Serratia marcescens*. It was deposited on April 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCCNO.30415. Plate confrontation experiments demonstrated that *Serratia marcescens* RS189 exhibited an inhibition zone width of 12.20±0.15 mm and an inhibition zone diameter of 32.40±0.29 mm against *Ralstonia solanacearum*, demonstrating a good inhibitory effect on *Ralstonia solanacearum*. Furthermore, metabolic assays revealed a significant activity peak in the crude metabolic extract of *Serratia marcescens* RS189 at a retention time of 11.05 min, and the substances contained in this peak are highly likely to be the key antibacterial substances of RS189. Simultaneously, experiments on the control of tobacco bacterial wilt demonstrated that *Serratia marcescens* RS189 effectively prevents tobacco bacterial wilt. The specific implementation procedures are as follows:

[0043] Example 1: Isolation and Screening of Antagonistic Bacteria

[0044] In this embodiment, an antagonistic bacterium with good inhibitory ability against Ralstonia solanacearum was isolated from the root topsoil of tobacco. The specific implementation operation is as follows:

[0045] 1. Strains Isolation

[0046] Six rhizosphere samples each from healthy and diseased tobacco plants were collected from Tianzhu County, Guizhou Province. 10g of tobacco rhizosphere soil was weighed and added to an Erlenmeyer flask at a ratio of 90mL sterile water per 10g sample. The flask was then placed on a 28℃ constant-temperature shaker and shaken at 180rpm for 20-30min. A gradient dilution method was used to dilute the soil suspension to 10... -2 10 -3 10 -4 10 -5 10 -6 Different gradients were used. 100 μL of appropriately diluted soil solution was placed in TSB solid medium and spread evenly (in this experiment, 10 μL was selected). -4 10 -5 10 -6 (Three gradients were applied). The culture was incubated at 28℃ for 24 hours, and single colonies were picked. The obtained strains were purified using the parallel streak method until single colonies were obtained. The strains were then numbered and stored at 4℃ for later use.

[0047] 2. Screening of antagonistic bacteria

[0048] Antagonistic bacteria were initially screened using the plate confrontation method. The preserved strains were inoculated into TSB liquid medium and cultured at 28°C and 200 rpm for 24 h on a shaker. Ralstonia solanacearum, stored at -80°C, was inoculated into NA liquid medium and cultured overnight at 30°C and 200 rpm on a shaker. The activated Ralstonia solanacearum was then cultured according to the Ralstonia solanacearum suspension (2.0 × 10⁻⁶ viable cells). 8 CFU / mL): Culture medium ratio = 1:600 ​​(30 mL culture medium + 50 μL bacterial suspension) added to NA solid culture medium at approximately 45°C (temperature should not be too high, otherwise the bacteria will be easily killed; nor should it be too low, to prevent the generation of a large number of air bubbles during mixing). Mix well and pour into plates. After the plates solidify, use an 8 mm diameter punch to make a hole in the center of the plate. Use sterile forceps or a toothpick to remove any remaining culture medium from the hole. Add the bacterial suspension of the test strain (viable count 1.0 × 10⁻⁶) to the culture medium. 8 Add 100-120 μL of cfu / mL to the central well (ensuring no overflow of sterile solution), incubate at 28°C, observe for the formation of inhibition zones, and measure the diameter of the inhibition zone.

[0049] 3. Antibacterial effect of antagonistic bacteria

[0050] The strain with the best antibacterial effect was selected, strain number RS189, and its plate confrontation results are as follows: Figure 1 As shown in Table 1, the width and diameter of the inhibition zone of RS189 were statistically analyzed, indicating that the width of the inhibition zone reached 12.20±0.15mm and the diameter of the inhibition zone reached 32.40±0.29mm.

[0051] Table 1 Results of inhibition zone determination

[0052]

[0053] Example 2: Morphological observation and molecular biological identification of antagonistic bacteria

[0054] This embodiment utilizes morphological and molecular biological methods to identify the antagonistic bacterium RS189 screened in Example 1. The specific implementation steps are as follows:

[0055] 1. Colony characteristics

[0056] The colony characteristics of strain RS189 are as follows: after culturing on TSB medium for 12 hours, single colonies can be formed. The colonies are relatively small, round, milky white in color, moist, with a raised surface, irregular edges, and are opaque. No pigment is produced (e.g., Figure 2 (As shown).

[0057] 2. Determination and analysis of 16S rDNA of antagonistic bacterium RS189

[0058] Antagonistic bacteria RS189 were streaked onto TSB plates. Single colonies were then picked from the plates and activated in TSB liquid medium. The colonies were incubated at 28°C and 200 rpm for 24 h until the logarithmic growth phase, and the cells were collected. Bacterial DNA was extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 1) and 1492R (5'-TACGGCTACCTTGTTACCACTT-3', SEQ ID No. 2).

[0059] The amplification reaction system was 50 μL: 2 μL each of primers 27F and 1492R, 2 μL of DNA template, 25 μL of 2×TransTaq HiFiPCR SuperMixⅡ, and 19 μL of ddH2O.

[0060] The reaction program was as follows: 95℃ for 2 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, 30 cycles; 72℃ for 10 min.

[0061] PCR amplification products were detected by 1% agarose gel electrophoresis and then sent to Beijing Liuhe BGI Genomics Co., Ltd. for 16S rDNA gene sequence analysis. The sequencing results are shown in SEQ ID NO.3 of the nucleotide sequence listing. The sequencing results were compared with sequences in the GeneBank database, and a phylogenetic tree was constructed using MEGA 6.0 software (e.g., ...). Figure 3 (As shown).

[0062] Based on the above identification results, the antagonistic bacterium RS189 strain belongs to Serratia marcescens and was deposited on April 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the accession number CGMCC NO.30415.

[0063] Example 3: Inhibition of Ralstonia solanacearum metabolite crude extract on Ralstonia solanacearum

[0064] This embodiment extracts metabolites from Serratia marcescens RS189 to investigate the inhibitory effect of these metabolites on Ralstonia solanacearum. The specific procedures are as follows:

[0065] 1. Preparation and processing of crude metabolic extracts

[0066] The RS189 strain was inoculated into a wide-mouth Erlenmeyer flask containing 500 mL of TSB liquid medium and cultured for 5 days at 28 °C and 200 rpm in a constant temperature shaker. The fermentation broth was centrifuged at 4000 rpm for 20 min after 5 days, and the supernatant was collected and extracted three times with an equal volume of ethyl acetate to obtain a crude metabolic extract. To test the heat tolerance of the metabolites, the crude metabolic extract was subjected to high-temperature treatment at 105 °C. To test the inhibitory effect of the metabolite concentration on the pathogen, the crude metabolic extract was concentrated using a vacuum rotary evaporator.

[0067] 2. Inhibitory effect of crude metabolic extract on Ralstonia solanacearum

[0068] The inhibitory effect of crude metabolic extracts on *Ralstonia solanacearum* was detected using the plate confrontation method. *Ralstonia solanacearum* was prepared according to the following conditions: *Ralstonia solanacearum* suspension (2.0 × 10⁻⁶ viable cells). 8 CFU / mL: Culture medium ratio = 1:600 ​​(30 mL culture medium + 50 μL bacterial suspension) was added to NA solid culture medium at approximately 45℃, mixed well, and poured into plates. After the plates solidified, a hole was punched in the center of the plate using an 8 mm diameter punch. The remaining culture medium in the hole was removed using sterile forceps or a toothpick. 100 μL of crude metabolic extract, high-temperature treated crude metabolic extract, and concentrated crude metabolic extract were added to the central wells, respectively. The plates were incubated at 28℃, and the presence of inhibition zones was observed. The results showed that the crude metabolic extract, high-temperature treated crude metabolic extract, and concentrated crude metabolic extract all had significant inhibitory effects on *Ralstonia solanacearum*, with the concentrated crude extract showing a stronger antibacterial effect (e.g., CFU / mL). Figure 4 As shown in the figure, this indicates that the antibacterial substance of RS189 is heat-resistant and concentration-dependent.

[0069] 3. Metabolic detection of crude extract

[0070] The crude extract of RS189 metabolites obtained by ethyl acetate extraction was dried to constant weight in a vacuum rotary evaporator. An internal standard extractant of 80% methanol-water was added, and the mixture was vortexed. The process of rapid freezing, thawing, and vortexing in liquid nitrogen was repeated three times. The mixture was then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected for non-targeted chromatographic detection. Reverse high-performance liquid chromatography (RP-HPLC) was performed on a C18 column using an Agilent 1290 micrometer. The mobile phase was 100% acetonitrile and water, the flow rate was 1 mL / min, the injection volume was 20 μL, and the detection wavelength was 326 nm. Chromatographic analysis showed that the crude extract of RS189 metabolites exhibited a significant activity peak at a retention time of 11.05 min. The substances contained in this active peak are highly likely to be the key antibacterial substances of RS189 (such as...). Figure 5 (As shown).

[0071] 4. Prediction of secondary metabolites

[0072] Secondary metabolites of RS189 were simultaneously predicted using the online anti-SMASH software (https: / / antismash.secondarymetabolites.org / ). To compare with common *Serratia marcescens* strains, metabolite predictions were also performed on RS101 (*Serratia marcescens*), another isolate with *Ralstonia solanacearum* antibacterial activity, producing styraxin, but with significantly weaker antibacterial effects than RS189, and the differences between the two strains were compared.

[0073] The predicted results of secondary metabolites for the two strains are as follows: Figure 6 As shown, both RS101 and RS189 possess 11 gene clusters for secondary metabolites. Consistent with previously observed strain phenotypes, RS101 has a gene cluster for synthesizing prodigiosin (100% similarity), but this was not detected in the genome of RS189. Because RS189 produces substances with stronger antibacterial effects, analysis of RS189-specific gene clusters revealed five gene clusters (Clusters 1, 6, 8, 9, and 11) that are completely different from those in RS101. No gene clusters homologous to Cluster 1 (hserlactone gene cluster) and Cluster 11 (NRPS gene cluster) were detected. Cluster 6 (Rev response element gene cluster), Cluster 8 (NRPS gene cluster), and Cluster 9 (arylpolyene gene cluster) showed similarity to gene clusters that synthesize synechobactin (9% similarity), 5-dimethylallylindole-3-acetonitrile (33% similarity), and andrimid (5% similarity), respectively, but the similarity was very low. Therefore, this invention suggests that there are new antibacterial substances in the metabolites of RS189.

[0074] Example 4: Control effect of crude metabolic extract of Serratia marcescens RS189 on tobacco bacterial wilt.

[0075] 1. Preparation of metabolites

[0076] The RS189 strain was inoculated into a wide-mouth Erlenmeyer flask containing 500 mL of TSB liquid medium and cultured for 5 days at 28°C and 200 rpm in a constant temperature shaker. The fermentation broth after 5 days of culture was centrifuged at 4000 rpm for 20 min to remove the bacterial cells, and the supernatant was collected, which is the metabolite of strain RS189.

[0077] 2. Greenhouse pest control effect

[0078] Tobacco (Red Flower Dajinyuan) seeds were sown in seedling trays filled with a mixture of nutrient soil and vermiculite. Once the seedlings reached the small cross stage (two true leaves and two cotyledons), they were transplanted into 10×9 seedling trays filled with the same nutrient soil and vermiculite mixture. One seedling was transplanted per tray, and the seedlings were divided into four groups: CK (no inoculation), RS189 (applied only with RS189 metabolites), Rs (inoculated only with Ralstonia solanacearum Rs), and RS189+Rs (applied with RS189 metabolites followed by inoculation with Ralstonia solanacearum Rs). Each treatment had three replicates, with 15 seedlings per replicate. After 10 days of cultivation in a greenhouse, the RS189 and RS189+Rs treatment groups were treated with the metabolites by drenching each plant with 20 mL of RS189 metabolites. Cultivation continued for another 10 days, and the same drenching treatment was repeated for the RS189 and RS189+Rs groups. Four days later, plants from the Rs and RS189+Rs groups were inoculated with Ralstonia solanacearum. 5 mL of OD200 solution was used to inoculate the plants with Ralstonia solanacearum. 600 A 0.5% solution of Ralstonia solanacearum suspension was used to drench the roots of each plant. After the plants in the Rs group showed disease characteristics, photos were taken and samples were collected. The incidence of bacterial wilt was investigated daily, and the incidence rate of the plants was calculated after 15 days.

[0079] Incidence rate (%) = Number of infected plants / Total number of plants surveyed × 100%;

[0080] Prevention and control effect = (control incidence rate - treatment incidence rate) / control incidence rate × 100%.

[0081] Table 2. Control effects of Serratia marcescens RS189 metabolites on tobacco bacterial wilt.

[0082]

[0083] Fifteen days after inoculation with Ralstonia solanacearum, the disease incidence rate in tobacco plants in the Rs treatment group was 88.52%, while the disease incidence rate in the RS189+Rs treatment group was 10.50%, significantly reducing the disease incidence rate (Table 2). No plants in the uninoculated treatment group showed disease incidence. The growth status of plants in each treatment group is as follows: Figure 7 As shown, metabolites 189 significantly enhanced the resistance of tobacco to bacterial wilt. Statistical analysis was performed on tobacco plant height, fresh weight, root length, and root weight (e.g., ...). Figure 8 As shown in the figure, in the uninoculated Ralstonia solanacearum treatments (CK and RS189), compared with the blank control, RS189 metabolites had no significant effect on tobacco plant height, fresh weight, root length, and root weight. In the Ralstonia solanacearum treatments (Rs and RS189+Rs), RS189 metabolites significantly alleviated the effects of Ralstonia solanacearum on tobacco growth.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of *Serratia marcescens* antagonistic to *Ralstonia solanacearum*, characterized in that: The strain of *Serratia marcescens* is classified as *Serratia marcescens* and its preservation number is CGMCC NO.30415.

2. A biocontrol agent antagonizing Ralstonia solanacearum, characterized in that: The biocontrol agent includes the fermentation supernatant or metabolites of Serratia marcescens as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that: The metabolite is an ethyl acetate extract of the fermentation supernatant of Serratia marcescens.

4. The use of Serratia marcescens as described in claim 1 in the preparation of Ralstonia solanacearum inhibitor.

5. The application of *Serratia marcescens* according to claim 4 in the preparation of *Ralstonia solanacearum* inhibitor, characterized in that: The active ingredient of the inhibitor is the fermentation supernatant or metabolites of Serratia marcescens.

6. The application of a biocontrol agent as described in claim 2 or 3 in the control of bacterial wilt in plants.

7. The application of the biocontrol agent according to claim 6 in the control of bacterial wilt of plants, characterized in that: Plants are treated with root irrigation using biocontrol agents.

8. The application of the biocontrol agent according to claim 6 or 7 in the control of bacterial wilt in plants, characterized in that: The plant in question is tobacco.

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