Bacterial strain for antagonizing pathogenic bacteria of tobacco bacterial wilt and application of bacterial strain in prevention and treatment of tobacco bacterial wilt

Abstract

The invention provides a bacterial strain for antagonizing pathogenic bacteria of tobacco bacterial wilt and application of the bacterial strain in prevention and treatment of the tobacco bacterial wilt, and relates to the technical field of agricultural biological prevention and treatment. According to the invention, rhizosphere soil of healthy tobacco plants is collected from a tobacco field suffering from bacterial wilt and is screened and separated to obtain antagonistic strains with an obvious inhibition effect on pathogenic bacteria of the tobacco bacterial wilt, and three strains with the effect of preventing and treating the bacterial wilt are obtained through secondary screening and antagonistic biomass metabolism promotion capability analysis. According to the present invention, the test research results show that the screened three strains, such as the pseudomonas strain ZL8, the bacillus velezensis strain XL104 and the bacillus amyloliquefaciens strain ZL4, have good control effects on bacterial wilt;

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural biological control, in particular to a strain antagonizing Ralstonia solanacearum and application thereof in preventing and treating Ralstonia solanacearum. BACKGROUND

[0002] Tobacco bacterial wilt is a typical bacterial soil-borne disease in tobacco production, which has a wide host range, and the main host plants are Solanaceae plants such as tobacco, pepper and tomato. It is difficult to prevent and control because of its strong destructive effect. Breeding of disease-resistant varieties is the most economical and effective method for preventing and treating tobacco bacterial wilt, but there are still key problems such as lack of disease-resistant variety resources, unknown resistance genetic mechanism and unclear disease resistance regulation mechanism. Although chemical agents have shown significant effect in inhibiting tobacco bacterial wilt, long-term use of chemical agents often causes a series of ecological problems: first, the pathogenic bacteria gradually develop drug resistance under the continuous drug selection pressure, resulting in decreasing control effect; second, chemical residues accumulate in the soil, which destroys the balance of microbial community and significantly reduces soil biodiversity; more seriously, these chemicals enter the surrounding environment through infiltration and runoff, causing persistent damage to the ecosystem, and finally forming a vicious cycle of "prevention-drug resistance-repeated prevention".

[0003] Microbial inoculants can reduce disease occurrence through competition, antagonism and parasitism with pathogenic bacteria, and can induce plant resistance, promote plant growth and regulate soil microecology, thereby effectively preventing and controlling soil-borne diseases. However, the prevention and treatment of microbial inoculants is a complex process, and factors such as inoculants, pathogenic bacteria and indigenous microorganisms and local climate conditions will affect the prevention and treatment.

[0004] Therefore, it is necessary and urgent to research and develop strains antagonizing Ralstonia solanacearum, and to provide new research resources for the discovery and exploration of high-efficiency antagonistic strains of tobacco bacterial wilt.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The present application aims to provide a group of strains antagonizing Ralstonia solanacearum, which have obvious prevention and treatment effect on tobacco bacterial wilt, and further provide new research resources for the discovery and exploration of high-efficiency antagonistic strains of tobacco bacterial wilt.

[0007] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0008] The present invention provides a strain that antagonizes the pathogen of tobacco bacterial wilt, wherein the strain is Pseudomonas sp. strain ZL8, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 20, 2025, with accession number GDMCC No: 66568.

[0009] Furthermore, the strain can be replaced with Bacillus velezensis strain XL104, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66567.

[0010] Furthermore, the strain can be replaced with Bacillus amyloliquefaciens strain ZL4, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66565.

[0011] The present invention provides a bacterial agent for antagonizing the pathogen of tobacco bacterial wilt, wherein the bacterial agent is a fermentation liquid, and the fermentation liquid includes Pseudomonas strain ZL8;

[0012] And / or, Bacillus belyssus strain XL104;

[0013] And / or, Bacillus amyloliquefaciens strain ZL4.

[0014] Furthermore, the concentration of the bacterial suspension in the fermentation broth is 10. 7 cfu / mL ~10 9 cfu / mL.

[0015] Furthermore, the concentration of the bacterial suspension in the fermentation broth is 10. 8 cfu / mL ~10 9 cfu / mL.

[0016] Furthermore, the fermentation broth is a compound bacterial broth, which includes Pseudomonas strain ZL8, Bacillus belyssus strain XL104, and Bacillus amyloliquefaciens strain ZL4.

[0017] Furthermore, the mass ratio of Pseudomonas strain ZL8, Bacillus belyceae strain XL104, and Bacillus amyloliquefaciens strain ZL4 in the compound bacterial solution is 1:1:1.

[0018] The present invention provides strains of bacteria antagonizing the pathogen of tobacco bacterial wilt, or bacterial agents for antagonizing the pathogen of tobacco bacterial wilt, for the prevention and / or treatment of tobacco bacterial wilt caused by Ralstonia solanacearum.

[0019] This invention provides a method for preventing and / or treating tobacco bacterial wilt caused by Ralstonia solanacearum, the method comprising:

[0020] Apply the above-mentioned inoculants used to antagonize the pathogen of tobacco bacterial wilt to plants, whether they are infected or not.

[0021] Furthermore, the application method includes: applying the fermentation broth 1 to 3 times during the tobacco growing season, wherein the concentration of the bacterial suspension in the fermentation broth is 10. 7 cfu / mL ~10 9 The concentration of cfu / mL is 25–50 ml per application.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention involves collecting rhizosphere soil samples from healthy tobacco plants in tobacco fields affected by bacterial wilt and screening to isolate antagonistic strains that significantly inhibit the pathogen of bacterial wilt (bacterial wilt fungus). Through secondary screening and analysis of the antagonistic growth-promoting metabolic capacity, three strains with antibacterial activity against bacterial wilt were identified. The morphological characteristics and physicochemical properties of these three antagonistic strains were analyzed, and they were identified using molecular biology techniques. A tobacco pot experiment revealed that the aforementioned *Pseudomonas* strain ZL8, *Bacillus belyssus* strain XL104, and *Bacillus amyloliquefaciens* strain ZL4 all exhibit good control effects against bacterial wilt.

[0024] The strains of Ralstonia solanacearum antagonizing the pathogen of tobacco bacterial wilt provided by the present invention, or the bacterial agents used to antagonize the pathogen of tobacco bacterial wilt, can be widely used in the prevention and / or treatment of tobacco bacterial wilt caused by Ralstonia solanacearum. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is a colony characteristic diagram of single colony culture of Ralstonia solanacearum obtained by screening according to Example 1 of the present invention.

[0027] Figure 2The image shows the PCR identification results of the Ralstonia solanacearum strain obtained from screening in Example 1 of this invention.

[0028] Figure 3 This is an phylogenetic tree diagram of Ralstonia solanacearum obtained through screening, as provided in Example 1 of the present invention.

[0029] Figure 4 This is a diagram showing the inhibition zone effect of the seven antagonistic strains obtained through screening in Example 2 of the present invention.

[0030] Figure 5 The phylogenetic tree diagram of strains ZL8, ZL4, and XL104 provided in Example 3 of this invention;

[0031] Figure 6 Colony morphology diagrams of the antagonistic strains ZL8, ZL4, and XL104 provided in Example 3 of the present invention;

[0032] Figure 7 The electron micrographs of the antagonistic strains ZL8, ZL4, and XL104 provided in Example 3 of this invention;

[0033] Figure 8 This is a graph showing the metabolic capacity analysis of disease-resistant and growth-promoting substances of strains ZL8, ZL4, and XL104 provided in Example 3 of the present invention.

[0034] Figure 9 The diagram shows the control effect of antagonistic strains ZL8, ZL4, and XL104 provided in Example 4 of this invention on tobacco bacterial wilt. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] According to one aspect of the present invention, a strain antagonizing the pathogen of tobacco bacterial wilt, said strain being Pseudomonas sp. strain ZL8, which was deposited on June 20, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66568.

[0037] In a preferred embodiment of the present invention, the strain may be replaced with Bacillus velezensis strain XL104, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66567.

[0038] In a preferred embodiment of the present invention, the strain may be replaced with Bacillus amyloliquefaciens strain ZL4, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66565.

[0039] This invention involves collecting rhizosphere soil samples from healthy tobacco plants in tobacco fields affected by bacterial wilt and screening out antagonistic strains that significantly inhibit the pathogen of bacterial wilt (Rhizoctonia solani). Through secondary screening and analysis of the antagonistic growth-promoting metabolic capacity, three strains with antibacterial activity against bacterial wilt were identified. The morphological characteristics and physicochemical properties of these three antagonistic strains were analyzed, and they were identified using molecular biology techniques. A tobacco pot experiment revealed that the aforementioned Bacillus amyloliquefaciens, Bacillus belye, and Pseudomonas putida all exhibit good control effects against bacterial wilt.

[0040] According to one aspect of the present invention, a microbial agent for antagonizing the pathogen of tobacco bacterial wilt, said microbial agent is a fermentation broth, said fermentation broth comprising Pseudomonas strain ZL8;

[0041] And / or, Bacillus belyssus strain XL104;

[0042] And / or, Bacillus amyloliquefaciens strain ZL4.

[0043] The present invention provides a bacterial agent for antagonizing the pathogen of tobacco bacterial wilt, wherein the bacterial agent is a fermentation broth, and the fermentation broth includes at least one of Pseudomonas strain ZL8, Bacillus belye strain XL104 and Bacillus amyloliquefaciens strain ZL4.

[0044] In a preferred embodiment of the present invention, the concentration of the bacterial suspension in the fermentation broth is 10. 7 cfu / mL ~10 9 cfu / mL.

[0045] In the preferred embodiment described above, the concentration of the bacterial suspension in the fermentation broth is 10. 8 cfu / mL ~10 9 cfu / mL.

[0046] In a preferred embodiment of the present invention, the fermentation broth is a compound bacterial broth, which includes Pseudomonas strain ZL8, Bacillus belye strain XL104 and Bacillus amyloliquefaciens strain ZL4.

[0047] In the preferred embodiment described above, the mass ratio of Pseudomonas strain ZL8, Bacillus belyceae strain XL104, and Bacillus amyloliquefaciens strain ZL4 in the composite bacterial solution is 1:1:1.

[0048] According to one aspect of the present invention, the use of strains of Ralstonia solanacearum antagonizing the pathogen of tobacco bacterial wilt, or bacterial agents for antagonizing the pathogen of tobacco bacterial wilt, in the prevention and / or treatment of tobacco bacterial wilt caused by Ralstonia solanacearum.

[0049] The strains of Ralstonia solanacearum antagonizing the pathogen of tobacco bacterial wilt provided by the present invention, or the bacterial agents used to antagonize the pathogen of tobacco bacterial wilt, can be widely used in the prevention and / or treatment of tobacco bacterial wilt caused by Ralstonia solanacearum.

[0050] According to one aspect of the present invention, a method for preventing and / or treating tobacco bacterial wilt caused by Ralstonia solanacearum, the method comprising:

[0051] Apply the above-mentioned inoculants used to antagonize the pathogen of tobacco bacterial wilt to plants, whether they are infected or not.

[0052] In a preferred embodiment of the present invention, the application method includes: applying the fermentation broth 1 to 3 times during the tobacco growing season, wherein the concentration of the bacterial suspension in the fermentation broth is 10. 7 cfu / mL ~10 9 The concentration of cfu / mL is 25–50 ml per application.

[0053] The technical solution of the present invention will be further described below with reference to the embodiments.

[0054] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0056] Example 1: Isolation and Identification of Ralstonia solanacearum

[0057] Infected tobacco plants were collected from tobacco fields affected by bacterial wilt, pulverized, and extracted with sterile ultrapure water. 1 μL of the extract was then streaked onto TTC plates for separation. Single colonies of varying morphologies were observed. Colonies resembling *Ralstonia solanacearum* were selected, isolated, and purified. The selected test strains exhibited irregularly shaped, round colonies with a pink center and milky-white edges, and strong mobility. Figure 1 As shown.

[0058] Figure 1 This is a colony characteristic diagram of single colony culture of Ralstonia solanacearum obtained from Example 1 of this application.

[0059] The strains initially screened were identified by PCR using three primer pairs (759F / 760R, flic-F / R, pehA#3 / 6) previously reported for identifying Ralstonia solanacearum. The results are shown in [link to results]. Figure 2 .

[0060] Figure 2 The image shows the PCR identification results of the Ralstonia solanacearum strain obtained in Example 1 of this application. Figure 2 All lanes in the middle are covered with Ralstonia solanacearum.

[0061] Figure 3 This is an evolutionary tree diagram of Ralstonia solanacearum obtained from Example 1 of this application.

[0062] Depend on Figure 2 , Figure 3 It can be seen that the 759F / 760R primer pair can amplify a specific band in the tested strain, with a size of approximately 281 bp, consistent with the size of the characteristic amplified fragment of Ralstonia solanacearum previously reported. Its 16S rDNA phylogenetic tree is as follows: Figure 3 As shown. After the bacterial suspension samples were sent for testing and identification, the strain isolated and screened from the infected tobacco plants was determined to be the bacterial wilt pathogen.

[0063] Example 2 Screening of antagonistic strains

[0064] Rhizosphere soil from healthy tobacco plants was collected from tobacco fields affected by bacterial wilt. The microorganisms in the rhizosphere soil were cultured, isolated, and the strains with the best antagonistic effect against bacterial wilt bacteria were screened and analyzed.

[0065] 1. Isolation and purification of antagonistic strains

[0066] Weigh 10g of rhizosphere soil sample from healthy tobacco plants and add it to 90mL of sterile water. Place the sample in a shaker and shake at 180r / min for 30min. For rhizosphere soil samples, phosphate-buffered saline (PBS) should be added, and the sample should be treated in an ultrasonic cleaner for 30min to thoroughly wash away microorganisms. Serially dilute the soil suspension with sterile water to prepare 10... -3 10 -4 10 -5 10 -6 10 -7 Dilute the solution in multiples of 1:1. Each dilution was repeated three times to ensure experimental accuracy.

[0067] Bacterial isolation: Aspirate 10 -6 10 -7 0.1 mL of diluted bacterial solution was spread onto a nutrient agar (NA) medium plate.

[0068] Fungal isolation: Bengal red agar supplemented with streptomycin and chloramphenicol (final concentration 100 μg / mL) was used to spread the fungal culture medium into 10-layer plates. -6 10 -7 Diluted bacterial solution.

[0069] Actinomycete isolation: Using Gao's No. 1 medium, 10... -3 10 -4 10 -5 Diluted bacterial solution.

[0070] Invert the coated plates and incubate them in a 28°C incubator: bacteria and fungi for 3-5 days, actinomycetes for 7 days. Select dominant single colonies from each culture medium for multiple generations of purification culture.

[0071] 2. Preliminary screening of antagonistic strains

[0072] Bacterial plate inhibition method: Ralstonia solanacearum was prepared into a bacterial suspension of 1.0 × 10⁻⁶ CFU / mL and added at 1% to NA medium at approximately 50°C to prepare bacterial plates. After solidification, microorganisms were selected by spot inoculation and cultured at 28°C inverted position for 3–5 days. The presence or absence of a clear zone was observed, and the colony diameter (d) and clear zone diameter (D) were measured. The inhibition capacity D / d was calculated (Table 1). Antagonistic bacteria were inoculated into liquid medium and cultured at 28°C with shaking at 180 rpm until the logarithmic growth phase (OD600≈0.6). The bacterial suspension was mixed with 30% sterile glycerol at a 1:1 ratio, dispensed into 2.5 mL sterile cryovials, and stored at -80°C for long-term use.

[0073] Table 1. Determination of the plate inhibition ability of the initial screening antagonistic bacteria against Ralstonia solanacearum.

[0074]

[0075] As shown in Table 1, all strains exhibited a certain degree of plate inhibition. Strains XL104 and ZL50 showed particularly strong antagonistic activity, with D / d values ​​of 2.79 and 2.75, respectively. Other strains with good performance included ZL4 (2.69), ZL41 (2.52), ZL1011 (2.33), and ZL10822 (2.31), all of which had D / d values ​​exceeding 2, indicating strong inhibitory activity against Ralstonia solanacearum.

[0076] 3. Secondary screening of antagonistic strains

[0077] Based on the initial screening results, seven antagonistic strains with good antagonistic ability were selected for secondary screening. First, NA medium containing Ralstonia solanacearum was prepared (method as above). A 9mm diameter hole was punched in the center of the solidified medium using a sterile punch, ensuring the hole walls were smooth. 100μL of antagonistic bacterial suspension in the logarithmic growth phase (OD600≈0.8) was inoculated. After incubation at 30℃ for 48h, the inhibition zone was observed and its diameter was measured using the cross-sectional method. Each treatment was repeated three times. Finally, the inhibition rate was calculated based on the measured colony diameter to evaluate the inhibitory effect of the antagonistic strains on the growth of Ralstonia solanacearum (see [link to relevant documentation]). Figure 5 (Table 2).

[0078] The formula for calculating the antibacterial rate is as follows:

[0079]

[0080] Figure 4 This is a diagram showing the inhibition zone effect of the 7 antagonistic strains obtained in Example 2 of the present invention.

[0081] Table 2. Determination of plate inhibition rate of antagonistic bacteria against Ralstonia solanacearum during secondary screening.

[0082]

[0083] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate that there is a significant difference at the P<0.05 level using the Duncan test. n=3.

[0084] Table 2 shows that there are significant differences in the antibacterial effects among different antagonistic strains. The antagonistic ability, from strongest to weakest, is ZL4 > ZL50 > XL104 > ZL41 > ZL8 > ZL108 > ZL3. Strains ZL50 and ZL4 showed better inhibition rates of 77.39% and 78.57%, respectively. Other strains, such as XL104, had inhibition rates between 72.93% and 75.75%, indicating some disease prevention effects.

[0085] Based on the results of the secondary screening, three groups of antagonistic strains, namely ZL8, ZL4, and XL104, were selected for subsequent efficacy verification.

[0086] Example 3: Analysis, Identification and Detection of Antagonistic Strains

[0087] 1. Molecular identification

[0088] The 16S rDNA sequences of three antagonistic strains were amplified and sequenced, yielding target gene fragments of approximately 1500 bp each, with NCBI accession numbers ZL8 (MN889396.1), ZL4 (MT613661.1), and XL104 (MT626060.1), respectively. BLAST searches were performed and sequences were compared with those in the NCBI database. Strains with detailed information were selected as reference strains, and a phylogenetic tree was constructed using the MEGA7 neighbor-joining (NJ) method.

[0089] Figure 5 This is an phylogenetic tree diagram of strains ZL8, ZL4, and XL104 provided in Example 3 of the present invention.

[0090] The results showed that strain ZL4 (MT613661.1) was Bacillus amyloliquefaciens, and the 16S rRNA of ZL4 (MT613661.1) was shown in SEQ ID NO:1.

[0091] The strain XL104 (MT626060.1) is Bacillus belye, and the 16S rRNA of XL104 (MT626060.1) is shown in SEQ ID NO:2.

[0092] Strain ZL8 (MN889396.1) is a *Pseudomonas putida*, and the 16S rRNA of ZL8 (MN889396.1) is shown in SEQ ID NO:3.

[0093] 2. Morphological characteristics

[0094] Figure 6 Colony morphology diagrams of the antagonistic strains ZL8, ZL4, and XL104 provided in Example 3 of the present invention;

[0095] Figure 7 Electron micrographs of the antagonistic strains ZL8, ZL4, and XL104 provided in Example 3 of this invention.

[0096] like Figure 6 , Figure 7As shown, the colonies of *Pseudomonas putida* strain ZL8 are milky white, round, about 0.5–2.5 cm in diameter, moist, smooth, and translucent, with coccobacillus cells; the colonies of *Bacillus belyssus* strain XL104 are milky white with wrinkled, hill-like surfaces, smooth edges, and a viscous texture, with rod-shaped, large oval cells and a colony diameter of about 2.0–3.0 cm; the colonies of *Bacillus amyloliquefaciens* strain ZL4 are pale yellow, with a rough, opaque, moist surface and wavy edges, a colony diameter of about 0.5–1.0 cm, and appear as short rods under electron microscopy.

[0097] 3. Physiological and biochemical tests

[0098] 3.1 Siderophore Capacity Detection Method: Antagonistic strains were spot-inoculated onto CAS plates using sterilized toothpicks and incubated at 30℃ for 3 days. The formation of a yellow halo around the colonies was observed. Since siderophores compete for iron ions chelated by EDTA in the culture medium, the CAS medium can change from blue to yellow. This phenomenon can be used to determine whether the strain produces siderophores.

[0099] 3.2 Biofilm Formation Ability Detection Method: 100 μL of overnight cultured Bacillus amyloliquefaciens was added to a 2 mL centrifuge tube containing 1 mL of LB medium and incubated at 30℃ for 24 hours. After incubation, the bacterial culture in the tube was discarded, and the centrifuge tube was rinsed several times with sterile water. 1 mL of pre-prepared 1% crystal violet was added and stained for 15 min. The stain was then rinsed off with sterile water. If a purple ring was observed on the tube wall at the location of the bacterial culture volume, it indicated that the strain could form a biofilm. Each treatment was performed in triplicate.

[0100] 3.3 Method for identifying IAA production capacity: The Salkowski colorimetric method was used to detect whether bacteria could produce indole-3-acetic acid (IAA) and to determine its concentration.

[0101] 3.4 Protease degradation ability detection method: After activating the antagonistic bacteria, a single colony was picked up with a sterile toothpick and inoculated onto the protease detection medium. The medium was placed in a constant temperature incubator at 30℃ and cultured in the dark for 48 hours. The presence or absence of a clear zone around the colony was observed to determine the protease production ability of the strain.

[0102] 3.5 Method for detecting cellulase production capacity: The activated antagonistic strain was spotted onto a sodium carboxymethyl cellulose agar plate using a sterile toothpick. After incubation at 30℃ for 2 days, Congo red solution (1 g / L) was added to the petri dish for staining for 30 min, followed by fixation with 1 mol / L sodium chloride solution for 30 min. Finally, the plate was rinsed with water, and the presence of a clear zone around the colony was observed. Cellulase can decompose cellulose into cellobiose and glucose. Congo red can react with cellulose in the culture medium to form a red complex, but it does not react with cellobiose and glucose, resulting in a clear zone around the cellulose-producing colony. This method can be used to determine the cellulase production of the strain.

[0103] 3.6 Method for identifying nitrogen fixation and phosphorus solubility: After activating the antagonistic bacteria by streaking on solid LB medium, a single colony was picked up with a sterile toothpick and lightly dotted onto prepared nitrogen-free medium and insoluble inorganic phosphorus medium. The colonies were incubated at 30°C for 3 days. The ability of the colonies to fix nitrogen was determined by observing whether they could grow on the nitrogen-free medium. The presence of a transparent ring around the edge of the colony on the insoluble inorganic phosphorus medium was also observed to determine whether the strain had phosphorus solubility.

[0104] The test results showed that the isolated antagonistic bacteria had the ability to produce siderophores, form biofilms, and produce auxins, proteases, and cellulases, but lacked nitrogen fixation and phosphorus solubilization capabilities. Figure 8 (Table 3).

[0105] Figure 8 This is a graph showing the metabolic capacity analysis of disease-resistant and growth-promoting substances of strains ZL8, ZL4, and XL104 provided in Example 3 of the present invention.

[0106] Table 3. Analysis of the metabolic capacity of antagonistic bacteria for disease resistance and promoting growth.

[0107]

[0108] Example 4: Antagonistic effect of strains

[0109] 1. Test Methods

[0110] (I) The three antagonistic strains (Bacillus vesiculus XL104, Bacillus amyloliquefaciens ZL4, and Pseudomonas ZL8) obtained by screening were fermented in a shaker at 30℃ and 180r / min for 48h, and the OD600 was measured to be 0.8-1, thus obtaining single-strain antagonistic bacterial solutions.

[0111] The concentration of the bacterial suspension in the single-strain antagonistic bacterial culture is 10. 8 cfu / mL ~10 9 cfu / mL.

[0112] The compound bacterial solution is formed by compounding strains ZL4, ZL8, and XL104. The compounding method is to ferment the strains separately and then mix them: each strain is inoculated into LB liquid medium at an inoculation amount of 1%, shaken at 180 rpm and 28°C for 24 h, and then mixed in equal proportion.

[0113] The concentration of the bacterial suspension in the compound bacterial solution is 10. 8 cfu / mL ~10 9 cfu / mL.

[0114] Single colonies of Ralstonia solanacearum obtained in Example 1 were picked and fermented on LB medium at 30°C and 180 r / min for 48 h on a shaker. The OD600 was measured to be approximately 0.8-1.0, thus obtaining the Ralstonia solanacearum fermentation broth.

[0115] (II) Four treatments were set up for potted plants:

[0116] Treatment 1 (T1): Sterile water (CK);

[0117] Process 2 (T2): Add 1×10 7 cfu / mL Ralstonia solanacearum inoculum (RS);

[0118] Treatment 3 (T3): Simultaneous addition of Ralstonia solanacearum inoculum and ZL8 inoculum (Pseudomonas putida) inoculum.

[0119] Treatment 4 (T4): Simultaneous addition of Ralstonia solanacearum inoculum and XL104 inoculum (Bacillus velezensis) inoculum.

[0120] Treatment 5 (T5): Simultaneous addition of Ralstonia solanacearum inoculum and ZL4 inoculum (Bacillus amyloliquefaciens) inoculum.

[0121] Treatment 6 (T6): Simultaneously add Ralstonia solanacearum inoculum solution and compound inoculum solution;

[0122] The specific potted plant experiment is as follows:

[0123] (1) Each treatment consisted of 10 pots, with three replicates. The specific steps are as follows: After transplanting the tobacco seedlings, cultivate them for 30 days. Then, make a 10cm deep and 2cm wide longitudinal cut in the soil 2cm away from the base of the stem on both sides.

[0124] Treatments 1 and 2 were irrigated with 25 mL of distilled water; treatments 3, 4, and 5 were irrigated with 25 mL of the corresponding single-strain fermentation broth (OD600 = 0.8-1); treatment 6 was irrigated with 25 mL of compound microbial fermentation broth.

[0125] (2) Two days later, during the incubation process, Ralstonia solanacearum inoculum was inoculated. Treatment 1 was watered with 25 mL of distilled water; treatments 2 to 6 were watered with 25 mL of Ralstonia solanacearum inoculum. Subsequently, 14 days after the inoculation with pathogen (Ralstonia solanacearum inoculum), the incidence rate and disease index were measured.

[0126] Note: The concentration of Ralstonia solanacearum inoculum added in each of the above treatment groups was 1×10⁻⁶. 7 CFU / mL; the concentration of the bacterial suspension in the bacterial culture of 3-5 single antagonistic bacteria was 10. 8 cfu / mL ~10 9 CFU / mL; The concentration of the bacterial suspension in the compound bacterial solution of treatment 6 was 10. 8 cfu / mL ~10 9 cfu / mL.

[0127] Disease classification: Refer to "YC / T39—1996 Tobacco Disease Classification and Investigation Methods" to investigate the disease severity level of each tobacco plant, record the number of diseased plants at each level, and calculate the disease severity index and control efficacy index according to the following formula.

[0128] Table 4. Criteria for Classification of Bacterial Wilt Disease Levels

[0129]

[0130] Disease index (%) = ∑(Ni×Vi) / (N×V)×100%

[0131] In the formula: Ni is the number of diseased plants at each level, Vi is the disease level value, N is the total number of plants surveyed, and V is the highest level value.

[0132] Efficacy index (%) - [(Ick-Itr) / Ick] × 100%

[0133] In the formula: Ick is the disease index (%) of the control group, and Itr is the disease index (%) of the treatment group.

[0134] 2. Test Results

[0135] From Table 5 and Figure 9 The results showed that the incidence rate of bacterial wilt was 94.64% and the disease index was 88.84% when Ralstonia solanacearum inoculum was added alone; the control effects of single-strain treatments T3, T5, and T6 were 77.19%, 50.2%, and 61.7%, respectively; and the incidence rate was 53.57% and the control effect was 52.20% when Ralstonia solanacearum inoculum and combined microbial inoculum were added simultaneously. This indicates that the combined microbial inoculum treatment not only significantly improved the control effect of individual single strains, but also significantly reduced the incidence rate of bacterial wilt in tobacco.

[0136] Figure 9The diagram shows the control effect of antagonistic strains ZL8, ZL4, and XL104 provided in Example 4 of this invention on tobacco bacterial wilt. See Table 5 below for specific effects.

[0137] Table 5. Control efficacy of antagonistic strains against bacterial wilt.

[0138]

[0139] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate that there is a significant difference at the P<0.05 level using the Duncan test. n=3.

[0140] In summary, the *Bacillus amyloliquefaciens*, *Bacillus belyceae*, and *Pseudomonas putida* strains of this invention all exhibit good control effects against bacterial wilt. The composite bacterial flora formed by combining these three strains can significantly delay the onset of tobacco bacterial wilt, providing a green solution for the prevention and control of this disease.

[0141] 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; and these 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 strain antagonistic to the pathogen of tobacco bacterial wilt, characterized in that, The strain is Pseudomonas sp. strain ZL8, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66568.

2. The strain antagonizing the pathogen of tobacco bacterial wilt according to claim 1, characterized in that, The strain can be replaced by Bacillus velezensis strain XL104, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66567. And / or, the strain may be replaced with Bacillus amyloliquefaciens strain ZL4, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 20, 2025, with accession number GDMCC No: 66565.

3. A fungal agent for antagonizing the pathogen of tobacco bacterial wilt, characterized in that, The bacterial agent is a fermentation broth, which includes Pseudomonas strain ZL8. And / or, Bacillus belyssus strain XL104; And / or, Bacillus amyloliquefaciens strain ZL4.

4. The inoculant for antagonizing the pathogen of tobacco bacterial wilt according to claim 3, characterized in that, The concentration of the bacterial suspension in the fermentation broth is 10. 7 cfu / mL ~10 9 cfu / mL.

5. The inoculant for antagonizing the pathogen of tobacco bacterial wilt according to claim 4, characterized in that, The concentration of the bacterial suspension in the fermentation broth is 10. 8 cfu / mL ~10 9 cfu / mL.

6. The inoculant for antagonizing the pathogen of tobacco bacterial wilt according to claim 3, characterized in that, The fermentation broth is a compound bacterial broth, which includes Pseudomonas strain ZL8, Bacillus belyssus strain XL104, and Bacillus amyloliquefaciens strain ZL4.

7. The inoculant for antagonizing the pathogen of tobacco bacterial wilt according to claim 6, characterized in that, The mass ratio of Pseudomonas strain ZL8, Bacillus belyssus strain XL104, and Bacillus amyloliquefaciens strain ZL4 in the compound bacterial solution is 1:1:

1.

8. The strain of Ralstonia solanacearum antagonizing the pathogen of tobacco bacterial wilt as described in claim 1 or 2, or the bacterial agent of Ralstonia solanacearum antagonizing the pathogen of tobacco bacterial wilt as described in claims 3 to 7, in the prevention and / or treatment of tobacco bacterial wilt caused by Ralstonia solanacearum.

9. A method for preventing and / or treating tobacco bacterial wilt caused by Ralstonia solanacearum, characterized in that, The method includes: Apply the fungal agents of claims 3 to 7, which are used to antagonize the pathogen of tobacco bacterial wilt, to plants, whether infected or not.

10. The method for preventing and / or treating tobacco bacterial wilt caused by Ralstonia solanacearum according to claim 9, characterized in that, The method of application includes: The fermentation broth was applied 1-3 times during the tobacco growing season, with the concentration of the bacterial suspension in the fermentation broth being 10%. 7 cfu / mL ~10 9 The concentration of cfu / mL is 25–50 ml per application.

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