Antagonistic bacterium and bacterial agent for tobacco bacterial wilt

Abstract

The present application relates to a strain of antagonistic Ralstonia solanacearum DW8 (Glutamicibacter soli), which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20232211. The antagonistic bacteria have good antagonistic effect on Ralstonia solanacearum strains with strong pathogenicity, not only show excellent prevention and control effect in indoor potting, but also confirm the excellent prevention and control effect in field test. The strain is a potential biological agent for prevention and control of Ralstonia solanacearum disease, has good development and application prospect, and greatly improves the prevention and control effect of chemical reagents for tobacco Ralstonia solanacearum disease, and is expected to be applied to the prevention and control of tobacco Ralstonia solanacearum disease to provide a new scheme for green and safe prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a tobacco bacterial wilt antagonist and its inoculant. Background Technology

[0002] Tobacco bacterial wilt is a soil-borne bacterial disease caused by *Ralstonia solanacearum*, a member of the Solanaceae family. It severely damages tobacco plant growth, causing enormous economic losses and is considered a devastating disease. It can infect hundreds of plant species from over 50 families. Bacterial wilt has been reported in my country, with tobacco bacterial wilt occurring extensively in 14 of the 22 major tobacco-producing regions, posing a significant threat to the tobacco industry. *Ralstonia solanacearum* primarily enters plant cells through wounds in the roots, root tips, or secondary roots. It develops in the intercellular spaces and subsequently enters the xylem, multiplying rapidly throughout the entire plant. It produces large amounts of extracellular polysaccharides that block the vascular bundles, causing wilting and ultimately leading to plant death. After infection, tobacco yield is significantly reduced, typically resulting in substantial economic losses for tobacco farmers.

[0003] Currently, the control of tobacco bacterial wilt mainly relies on chemical agents. Commonly used chemical agents on the market include inorganic copper, organochlorine, inorganic sulfur, and organocopper. Although chemical control has a good effect on tobacco bacterial wilt, the large-scale and continuous application of chemical agents easily leads to insurmountable problems such as pathogen resistance and environmental pollution, which are detrimental to ecological environment protection and sustainable agricultural development. It also increases pathogen resistance, reduces soil biodiversity in tobacco fields, and affects the quality of the tobacco field soil environment. Considering the problems of low tobacco leaf quality and deterioration of the soil ecological environment, green tobacco control technology is receiving increasing attention. Among them, biological control has become a more common method for controlling bacterial wilt in recent years, and research in this area is increasing, making it a key area for future development. Biological control of tobacco bacterial wilt utilizes the antagonistic, competitive, predatory, and parasitic relationships between microorganisms to inhibit the growth, development, and reproduction of pathogens, thereby reducing the occurrence of the disease. It is known that bacterial wilt in the Guangdong tobacco-growing area has been increasing year by year due to various factors such as long-term continuous cropping, increased chemical inputs, and abnormal climate change. The symptoms of bacterial wilt differ from those in the southwestern tobacco-growing area. In Guangdong, bacterial wilt outbreaks are concentrated after topping and can cause severe damage in a short period. Guangdong is one of the important tobacco-producing areas in my country, possessing typical characteristics and regional advantages. However, root and stem diseases such as bacterial wilt pose a serious challenge to the healthy and stable development of tobacco, causing severe losses in some areas and becoming a significant factor restricting tobacco production. In the process of healthy plant cultivation, microorganisms play a crucial role in the crop micro-ecosystem, and different rhizosphere micro-ecological community compositions determine the health of tobacco. Currently, significant research has been conducted on the microecology of bacterial wilt, but the antagonistic indigenous bacteria associated with the occurrence of bacterial wilt in the Guangdong tobacco-growing area remain unclear. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an antagonistic bacterium against tobacco bacterial wilt, and to provide a microbial agent containing the strain.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. A strain of *Glutamicibacter soli* resistant to bacterial wilt, wherein the *Glutamicibacter soli* is DW8, classified as *Glutamicibacter soli*, deposited at the China Center for Type Culture Collection, Wuhan, on November 13, 2023, with accession number CCTCC NO: M 20232211.

[0007] Furthermore, the colonies of the soil glutamate bacilli are milky yellow, round, with smooth, raised, and glossy edges, and are moist.

[0008] 2. A microbial agent is also provided, wherein the active ingredient of the microbial agent includes DW8 of *Soil Glutamicinus*.

[0009] Furthermore, the microbial agent comprises one or more of the following: bacterial cells, bacterial powder, and bacterial suspension selected from *Soil Glutamicinus DW8*.

[0010] Furthermore, in the microbial agent, soil glutamate bacillus DW8 is cultured to obtain a bacterial suspension, which is a liquid microbial agent.

[0011] Furthermore, the microbial agent also includes excipients acceptable for the preparation of the microbial agent.

[0012] Furthermore, the auxiliary material is a loaded solid, which includes one or more of rice bran, straw powder, diatomaceous earth, sodium alginate, zeolite, and biochar.

[0013] The beneficial effects of this invention are as follows: This invention isolates and screens functional microbial strains antagonistic to *Ralstonia solanacearum* from soil in tobacco-growing areas affected by bacterial wilt in Meizhou, Guangdong. These antagonistic bacteria exhibit good resistance to highly pathogenic *Ralstonia solanacearum* strains, demonstrating excellent control effects not only in indoor potted plants but also in field trials. They are potential biological agents for the control of bacterial wilt, with excellent development and application prospects. Compared to chemical agents used to control *Ralstonia solanacearum*, their effectiveness is significantly improved, and they are expected to provide a green and safe new control solution for *Ralstonia solanacearum*. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0015] Figure 1To investigate the incidence and antagonistic bacteria of bacterial wilt in tobacco plants in Guangdong tobacco-growing areas, rhizosphere soil was collected and antagonistic bacteria were isolated and purified.

[0016] Figure 2 Evaluation of the antagonistic activity of rhizosphere bacteria of tobacco in Guangdong tobacco-growing areas against Ralstonia solanacearum.

[0017] Figure 3 The antibacterial activity of antagonistic bacteria against Ralstonia solanacearum was isolated from the rhizosphere soil of healthy tobacco plants in Longwen Town.

[0018] Figure 4 The antibacterial activity of antagonistic bacteria against Ralstonia solanacearum was isolated from the rhizosphere soil of healthy tobacco plants in Wuxing Village, Songyuan Town.

[0019] Figure 5 The antibacterial activity of antagonistic bacteria against Ralstonia solanacearum was isolated from the rhizosphere soil of healthy tobacco plants in Xin'nan Village, Songyuan Town.

[0020] Figure 6 Phylogenetic trees were constructed for four strains based on their 16S rDNA sequences.

[0021] Figure 7 The activity (A), protease (B), cellulase (C), and clear zone radius (D) of antagonistic activity against Ralstonia solanacearum were measured in 30 bacterial strains.

[0022] Figure 8 The activity of four bacterial strains was antagonized by Ralstonia solanacearum (A), protease (B), cellulase (C), and hepatophilia (D).

[0023] Figure 9 To evaluate the control effect of antagonistic bacteria on tobacco bacterial wilt. The study included: (A) the effect of antagonistic bacteria on the disease index of tobacco bacterial wilt; (B) the control effect of antagonistic bacteria on tobacco bacterial wilt; and (C) potted plant photographs of tobacco bacterial wilt (20 days after inoculation).

[0024] Figure 10 The effects of four antagonistic bacteria on tobacco growth promotion were evaluated. The results included (A) plant height; (B) root length; (C) fresh weight; (D) dry weight; and (E) a photograph of the potted plant (20 days after inoculation).

[0025] Figure 11 To evaluate the outdoor control efficacy of four antagonistic bacteria against tobacco.

[0026] Figure 12 This is a colony morphology diagram of *Soil Glutamate Bacterium* MZ8-15.

[0027] Figure 13 The image shows field planting of strains MZ3-12, MZ8-15, MZ9-28, and a mixture of these three bacteria. Detailed Implementation

[0028] The preferred embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0029] Example 1

[0030] 1. Materials and Methods

[0031] 1.1 Materials

[0032] Soil samples for testing: In the Meizhou tobacco-growing area, rhizosphere soil samples were collected from tobacco plants that were not affected by bacterial wilt in tobacco fields that were frequently affected by bacterial wilt. These samples were then brought back to the Natural Products and Pesticides Research Laboratory of Southwest University for further processing.

[0033] The tested bacterial wilt strain: CQPS-1 was isolated from diseased tobacco plant samples collected in Pengshui, Chongqing by the Natural Products Pesticide Research Laboratory. After laboratory identification, it was found to be a bacterial wilt strain with strong pathogenicity. After isolation and purification, it was stored in the laboratory at -80℃.

[0034] Test instruments:

[0035]

[0036]

[0037] 1.2 Methods

[0038] 1.2.1 Soil Collection and Processing

[0039] The test soil was taken from the tobacco-growing area of ​​Meixian County, Meizhou City, Guangdong Province. Rhizosphere soil of diseased and normal tobacco plants in tobacco fields affected by bacterial wilt was collected 60 days and 80 days after transplanting. Six replicates were collected for both healthy and diseased plants, with one replicate per tobacco plant (six plants were collected for each sample).

[0040] In this tobacco-growing area, three plots of land where bacterial wilt has occurred for many years were randomly selected. Rhizosphere soil samples were collected from diseased and healthy tobacco plants in the diseased plots. The sampling and subsequent processing methods are as follows: Approximately 60 and 80 days after tobacco transplanting (harvest period), 4-6 diseased and 4-6 healthy tobacco plants were selected. The topsoil was dug up with a hoe, the roots were dug out, and large soil clumps adhering to the roots were removed. 50g of soil adhering to the surface of the tobacco plant roots (0-4mm) was collected, mixed well, packed in a self-sealing bag and sealed. The soil was then brought back to the laboratory, sieved through a 2mm standard sieve, and stored at 4℃ for subsequent experiments. Figure 1 To investigate the incidence and antagonistic bacteria of bacterial wilt in tobacco plants in Guangdong tobacco-growing areas, rhizosphere soil was collected and antagonistic bacteria were isolated and purified.

[0041] 1.2.2 Isolation and Purification of Soil Bacteria

[0042] 1.2.2.1 Isolation of bacteria: Plate dilution method was used.

[0043] Weigh 5g of fresh soil and put it into a 100mL Erlenmeyer flask. Add 45mL of sterile water and then shake it in a constant temperature shaker at 180r / min for 20min to disperse the soil particles evenly in the water and make a soil suspension.

[0044] 1. Using a pipette, transfer 1 mL of soil suspension into a centrifuge tube containing 9 mL of sterile water. Mix thoroughly by aspiration several times. Then, dilute the soil suspension sequentially using the 10x method until a final concentration of 10 is reached. -6 ;

[0045] 2. Dilute to a factor of 10 -4 -10 -6 100 μL of soil suspension was taken onto TSB medium and immediately spread with sterile glass beads until the surface of the medium was dry and no more scratches from the rolling of the glass beads appeared. Two plates were made for each sample.

[0046] 3. Seal the plate and invert it in a 30℃ constant temperature incubator for 2-3 days until colonies grow.

[0047] 1.2.2.2 Bacterial Purification

[0048] 1. Observe the growth of single colonies and number them according to their morphology and color.

[0049] 2. Select single colonies according to their numbers and streak them onto TSA plates, repeating each colony three times. After streaking, seal the plates with sealing film and incubate them upside down in a 30°C incubator for 2-3 days.

[0050] 3. Remove the plate, pick a single colony again, and repeat the above steps to purify 2-3 times;

[0051] 4. After the purification step is completed, pick one single colony from each sample and put it into an Erlenmeyer flask containing 20 mL of sterile TSB medium. Place the flask in a constant temperature shaker and incubate overnight at 30°C and 180 r / min until the bacterial OD600 = 0.8-1.0.

[0052] 5. Take 1 mL of bacterial culture into a sterile centrifuge tube and centrifuge at 8000 r / min for 5 min; discard the supernatant, add 1 mL of sterile water, shake up and down to form a uniform suspension, repeat three times, and store at room temperature for later use.

[0053] 6. Use a pipette to draw 100 μL of bacterial suspension into a 96-well plate, then add an equal volume of 50% glycerol, and mix thoroughly by repeated pipetting. Seal the 96-well plate with sealing film and store it in a -80°C freezer for later use.

[0054] 1.2.3 Antagonistic test of Ralstonia solanacearum for preliminary screening of bacteria

[0055] 1. Take out the preserved rhizosphere bacteria, streak 1 ml of bacterial solution onto TSA medium (small), and incubate at 30℃ for 2 days. At the same time, streak CQPS-1 onto B medium and incubate at 30℃ for 2 days.

[0056] 2. Select a single colony from the cultured rhizosphere bacteria and place it in the center of 1 / 16 of a 14cm square petri dish. Repeat this process twice and incubate for 1 day. Simultaneously, select a single colony of cultured Ralstonia solanacearum and mix it with 15ml of LB medium. Place the Erlenmeyer flask containing the bacterial solution in a shaker and incubate at 170 rpm and 30°C for 12 hours (until the OD value reaches 0.8-1.0).

[0057] 3. Transfer the bacterial wilt bacteria to a sterilized spray bottle and spray it evenly onto the square dish;

[0058] 4. After one day of incubation, observe the inhibition zone to determine whether there is an antibacterial effect. Use the cross-sectional method to measure the diameter of the inhibition zone to evaluate the antibacterial effect and take a picture.

[0059] 1.2.4 Antagonistic test of Ralstonia solanacearum

[0060] 1. Activation of Ralstonia solanacearum and its antagonistic bacteria: Add the preserved bacterial culture to TSB medium at a ratio of 1:200 (use B medium for Ralstonia solanacearum) and incubate overnight until OD. 600 =1.0, take 1 mL of bacterial culture into a sterile centrifuge tube, centrifuge at 8000 r / min for 5 min; discard the supernatant, add 1 mL of sterile water, shake up and down to mix evenly to form a suspension, repeat three times, and set aside for later use;

[0061] 2. Select three single colonies from the cultured rhizosphere bacteria and place them in an equilateral triangle on a 90mm round petri dish, with appropriate spacing. Repeat each colony twice and incubate for 1 day. At the same time, select one cultured Ralstonia solanacearum single colony and mix it with 15ml of LB liquid medium. Place the Erlenmeyer flask containing the bacterial solution in a shaker and incubate at 170 rpm and 30℃ for 12 hours (until the OD value reaches 0.8-1.0).

[0062] 3. Transfer the Ralstonia solanacearum solution to a sterilized spray bottle, spray it evenly onto the petri dishes, and incubate for 1 day;

[0063] 4. Cross-multiplication method for measuring the inhibition zone: Take the average of the three radii of the measured transparent zone as the radius of the inhibition zone on the plate. The radii of the two replicate inhibition zones are taken as the radius of the sample. Note: The cross-multiplication method is used for all subsequent measurements of the transparent zone.

[0064] 1.2.5 Determination of bacterial hydrolytic enzyme activity

[0065] 1.2.5.1 Protease activity assay

[0066] Three single colonies were inoculated on a protease plate (A: 8g skim milk powder dissolved in 300mL water, sterilized at 115℃ for 10min; B: 8g agar, diluted to 300mL, sterilized at 121℃ for 20min; A and B were sterilized separately and then mixed) to form an equilateral triangle with appropriate spacing. Each treatment was performed twice. After incubation at 30℃ for 3 days, the presence and size of the clear zone were observed and recorded.

[0067] 1.2.5.2 Cellulose Activity Assay

[0068] Following Ghose's method, the prepared bacterial strains were inoculated onto cellulase activity assay plates (10g peptone, 10g yeast extract, 10g sodium carboxymethyl cellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, 18g agar, pH=7.0). Three single colonies were inoculated into an equilateral triangle with appropriate spacing. Each colony was treated twice. After incubation at 30℃ for 3 days, the plates were stained with 1g / L Congo red for 1 hour, the stain was discarded, and the plates were then soaked in 1mol / L NaCl solution for 1 hour. The presence and size of the clear zone were observed and recorded.

[0069] 1.2.6 Assay for Ferrophilic Activity

[0070] The prepared strains were inoculated into the pre-purchased and prepared CAS solid medium. Three single colonies were inoculated into an equilateral triangle with appropriate spacing. Each colony was treated twice. After incubation at 30°C for 1 day, the presence and size of the clear zone were observed and recorded.

[0071] 1.2.7 Value Assignment Evaluation for Bacteria

[0072] According to the method of Faltin et al., the biocontrol potential of antagonistic bacteria was evaluated by assigning values ​​as follows: 3 points each for protease production, heparin production, and inhibition of Ralstonia solanacearum, of which 1 point is given for inhibition zone or clear zone radius of 0-3mm, 2 points for 3-6mm, and 3 points for >6mm; 1 point is given for cellulase production, and the total score is 10 points.

[0073] 1.2.8 Molecular identification of antagonistic bacteria

[0074] DNA from antagonistic bacteria was extracted using a kit, and then the extracted DNA was amplified for 16S-specific variable region using PCR primers 27F / 1492R. The primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGCTACCTTGTTACGAC-3'). The total volume of the PCR reaction system was 25 μL.

[0075]

[0076] 1. The PCR amplification conditions were: 94℃ pre-denaturation for 5 min, 30 cycles of 94℃ denaturation for 1 min, 51℃ annealing for 1 min, 72℃ extension for 3 min, and a final extension at 72℃ for 10 min.

[0077] 2. The PCR products obtained from the reaction were detected by 1% agarose gel electrophoresis, and finally sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0078] 3. Homology comparisons were performed between the sequencing results and the 16S rRNA gene sequences in the Genbank database. The strain names with the highest QueryCover value (greater than 99%) and Per.Ident value were selected as the genus names for identifying unknown strains. Phylogenetic trees were constructed using the MEGAX software based on the sequencing results. The MUSCLE method was used to align the sequences, the optimal model was calculated, and the phylogenetic tree was constructed using the ML method with a bootstrap value of 1000.

[0079] 1.2.9 Verification of Potted Plant Effect

[0080] 1.2.9.1 Verification of disease resistance effect

[0081] Based on the assigned score evaluation results and the molecular identification results of the antagonistic bacteria, four distinct antagonistic bacteria with assigned scores greater than 6 were selected for pot experiment verification. Four-leaf, one-heart tobacco seedlings of the varieties Yunyan 87 and K326 were prepared for use. The isolated antagonistic bacteria were activated, and the antagonistic bacterial culture was cultured to the logarithmic growth phase, then diluted to 10⁻⁶. 8 The plants were treated with a concentration of cfu / mL by root drenching, with 10 mL per plant. After 3 days of antagonistic bacterial treatment, the plants were inoculated with the pathogenic bacterium *Ralstonia solanacearum*, and the bacterial culture, which had reached the logarithmic growth phase, was diluted to 10⁻⁶.8 The concentration of cfu / mL was used to treat the roots of the plants by drenching, with 10 mL per plant. After inoculation, the plants were placed in a greenhouse under the following conditions: 14h light / 10h dark, 30±1℃, and 75% relative humidity. The plants were monitored daily for 20 days after inoculation. The severity of tobacco bacterial wilt in the indoor environment was graded from 0 to 4, as follows (per plant):

[0082] Grade 0: All healthy leaves on the plant;

[0083] Grade 1: 1 to 2 leaves are semi-wilted;

[0084] Grade 2: 2 to 3 leaves are wilted;

[0085] Grade 3: Only 1 to 2 healthy leaves remain;

[0086] Level 4: No healthy leaves.

[0087] Disease severity was statistically analyzed according to the disease severity criteria proposed by Kempe et al.

[15] . The formulas for disease severity and control efficacy are as follows:

[0088]

[0089] 1.2.9.2 Verification of the growth-promoting effect

[0090] The treatment method for antagonistic strains on tobacco seedlings was as described in section 1.2.9.1. The control group was treated with an equal volume of sterile water. Each treatment was replicated 10 times. The treated seedlings were placed in a greenhouse with 14 hours of light and 10 hours of darkness, at 30±1℃ and 75% relative humidity. Their growth was observed daily. After 21 days, fresh weight, plant height, root length, and dry weight were statistically analyzed. Each treatment was measured three times.

[0091] 1.2.10 Field effect verification

[0092] Based on the results of indoor disease resistance and growth promotion experiments, antagonistic bacteria that can significantly resist bacterial wilt and promote tobacco plant growth were screened and their field effects were verified.

[0093] (1) Test site

[0094] The experimental site was selected from areas with a high incidence of bacterial wilt due to continuous cropping. The experimental site was located in the tobacco-growing area of ​​Meizhou, Guangdong Province.

[0095] (2) Test methods

[0096] The experiment consisted of 5 treatments, 3 replicates, and 15 plots, with each plot covering approximately 0.1 acres. Protective rows were included, resulting in an experimental plot area of ​​approximately 1.8 acres. Root drenching treatments were administered 25 days after transplanting and at the initial stage of disease development.

[0097] Process 1: MZ3-12 10 6cfu / mL 100mL;

[0098] Process 2: MZ8-15 10 6 cfu / mL 100mL;

[0099] Process 3: MZ9-28 10 6 cfu / mL 100mL;

[0100] Treatment 4: Mix equal volumes of the three bacterial agents;

[0101] Process 5: Blank control

[0102] (3) Disease investigation

[0103] The occurrence of tobacco diseases was investigated according to the national standard GB / 23222-2008 "Classification and Investigation Methods for Tobacco Diseases and Pests". Based on local disease characteristics, a systematic investigation was conducted on various key diseases. The number of diseased plants and the severity level in each plot were investigated to calculate the incidence rate. Disease investigations could be conducted simultaneously with the determination of tobacco agronomic traits. Depending on the occurrence of different diseases, investigations began at the initial stage of disease, with investigations conducted every 5 days for at least 4 consecutive times.

[0104]

[0105] Results and Analysis of Example 2

[0106] 2.1 Isolation of bacteria from the rhizosphere soil of healthy tobacco plants

[0107] Nine samples of rhizosphere soil from healthy tobacco plants in tobacco fields chronically affected by bacterial wilt were collected from Songyuan Town and Longwen Town, Meixian County, Meizhou City, Guangdong Province. Antagonistic bacteria were isolated and purified from these samples. The bacteria were isolated and purified on TSA medium, ultimately yielding 274 strains of tobacco rhizosphere bacteria.

[0108] 2.2 Preliminary screening of rhizosphere bacteria with antagonistic effects against Ralstonia solanacearum

[0109] The antagonistic activity of rhizosphere bacteria against Rhizosphere bacteria was evaluated using a plate culture method in square petri dishes. Antagonistic bacteria were cultured on TSA medium 24 hours in advance. Then, a suspension of Rhizosphere bacteria containing different antagonistic bacteria was sprayed onto the square petri dishes. The antagonistic activity of the rhizosphere bacteria was evaluated based on the size of the inhibition zone. The antibacterial activity of 274 rhizosphere bacteria against Rhizosphere bacteria was evaluated in this study. Figure 2 This image shows an experimental evaluation of the antagonistic activity of some rhizosphere bacteria from tobacco-growing areas in Guangdong against *Ralstonia solanacearum*. The results showed that all rhizosphere bacteria isolated from Guangdong tobacco-growing areas exhibited certain antibacterial activity against *Ralstonia solanacearum*, with a total of 84 strains showing antagonistic activity. 25 antagonistic bacteria were isolated from Longwen Town. Figure 3 To investigate the antibacterial activity of these 25 antagonistic bacteria against Ralstonia solanacearum, and to isolate 30 antagonistic bacteria from Wuxing Village, Songyuan Town (…). Figure 4 To investigate the antibacterial activity of these 30 antagonistic bacteria against Ralstonia solanacearum, and to isolate 29 antagonistic bacteria from Xin'nan Village, Songyuan Town (…). Figure 5 (This study investigated the antibacterial activity of 29 antagonistic bacteria against Ralstonia solanacearum. 30 strains showed inhibition zones exceeding 10 mm in diameter, including MZ2-3, MZ2-14, MZ2-30, MZ3-12, MZ3-30, MZ4-2, MZ4-8, MZ4-9, MZ4-13, MZ4-25, MZ4-26, MZ5-11, MZ5-26, MZ5-28, MZ6-8, MZ6-15, MZ6-19, MZ6-21, MZ7-4, MZ7-8, MZ7-11, and MZ7-13.) MZ7-27, MZ7-30, MZ8-13, MZ8-15, MZ8-21, MZ8-27, MZ9-22 and MZ9-28 showed good antagonistic activity against Ralstonia solanacearum. Further tests were conducted on these 30 bacterial strains to determine their individual anti-Ralstonia solanacearum activity, hydrolytic enzyme activity, and ferrophilic activity.

[0110] 2.3 Molecular identification of antagonistic bacteria isolated from tobacco-growing areas in Guangdong

[0111] 16S rRNA gene sequence analysis was used to molecularly identify these 30 antagonistic bacteria. The target gene sequences of the antagonistic bacteria were analyzed in the GenBank database, compared with BLAST, and a phylogenetic tree was constructed. Some results are shown in Table 1. Figure 6 Phylogenetic trees were constructed for the four strains based on their 16S rDNA sequences. The results showed that strain MZ3-12 was *Bacillus cereus*, strain MZ4-13 was *Bacillus subtilis*, strain MZ8-15 was *Glutamicibacter soli*, and strain MZ9-28 was *Bacillus amyloliquefaciens*. Figure 12 The image shows the colony morphology of strain MZ8-15. The colonies are milky yellow, round, with smooth, raised, glossy edges, and moist. Soil glutamate bacterium MZ8-15 is classified and named Glutamicibacter soli DW8, and is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan, on November 13, 2023, with accession number CCTCCNO: M 20232211.

[0112] Table 1. Partial results of molecular identification of antagonistic bacteria isolated from tobacco-growing areas in Guangdong.

[0113]

[0114] 2.4 Evaluation Results of Value Assignment

[0115] Experiments were conducted on these 30 antagonistic bacteria to determine their antagonistic activity, hydrolytic enzyme activity, and heparin production capacity. The score for each antagonistic bacterium was assigned based on the size of its clear zone: 3 points each for protease production, heparin production, and inhibition of *Ralstonia solanacearum*, with 1 point for a clear zone radius of 0–3 mm, 2 points for 3–6 mm, and 3 points for >6 mm; 1 point was awarded for cellulase production, for a total score of 10 points. Results Figure 7 The results are shown in Table 1. Among the strains with a total score greater than 6, strains with different molecular identification results were selected for indoor pot experiments, namely MZ3-12, MZ4-13, MZ8-15, and MZ9-28. Figure 8 The graph shows the activity test results of these four bacteria against Ralstonia solanacearum (A), protease (B), cellulase (C), and hepatophilia (D).

[0116] Table 2. Evaluation Results of Antagonistic Bacteria Value Assignment

[0117]

[0118] 2.5 Study on the indoor control effect of four antagonistic bacteria against tobacco bacterial wilt

[0119] To verify the control effect of the screened antagonistic bacteria on tobacco bacterial wilt, we selected four rhizosphere bacteria with different molecular identification results (MZ3-12, MZ4-13, MZ8-15, and MZ9-28) from those with a score greater than 6 for pot experiments. The control effects of these four antagonistic bacteria on tobacco bacterial wilt were evaluated as follows: Figure 9 The results show that (A) the effect of antagonistic bacteria on the disease index of tobacco bacterial wilt; (B) the control effect of antagonistic bacteria on tobacco bacterial wilt; and (C) potted plants affected by antagonistic bacteria (20 days after inoculation). The incidence and disease index of each strain on tobacco bacterial wilt are shown in Tables 3 and 4. The results showed that, compared with the control, the application of antagonistic bacteria significantly reduced the incidence of tobacco bacterial wilt. Antagonistic bacteria MZ3-12, MZ8-15, and MZ9-28 all had significant control effects on tobacco bacterial wilt, with strain MZ8-15 showing the best control effect. The relative control efficacies at 10 and 15 days after inoculation were 85.61% and 67.00%, respectively. These results preliminarily demonstrate that the indigenous microorganisms of Guangzhou screened in this study have a relatively good control effect on tobacco bacterial wilt.

[0120] Table 3. Incidence of Tobacco Bacterial Wilt by Various Strains

[0121]

[0122] Table 4 shows the disease index of each strain on the occurrence of tobacco bacterial wilt.

[0123]

[0124]

[0125] 2.6 Study on the growth-promoting effects of four antagonistic bacteria on tobacco in indoor and field environments.

[0126] While verifying the control efficacy of the four selected antagonistic bacteria against tobacco bacterial wilt, we also conducted a study on whether the antagonistic bacteria had a growth-promoting effect on tobacco plants in both indoor and field environments. The results showed that all four bacteria had varying degrees of growth-promoting effects on tobacco plants. Figure 10 The effects of four antagonistic bacteria on tobacco growth promotion were evaluated. (A) Tobacco plant height; (B) Tobacco plant root length; (C) Tobacco plant fresh weight; (D) Tobacco plant dry weight; (E) Photograph of potted plants with increased growth (20 days after inoculation); Specific data are shown in Table 5. As shown in the figure, all four antagonistic bacteria significantly increased tobacco plant height, root length, fresh weight, and dry weight. Figure 10 (AD). Furthermore, in terms of tobacco plant growth, the size and quantity of tobacco leaves in the treatment group were significantly better than those in the control group (AD). Figure 10 Table 6 shows the effects of different strains on the agronomical morphology of tobacco in the field. Figure 13 The image shows field planting of strains MZ3-12, MZ8-15, MZ9-28, and a mixture of these three bacteria.

[0127] Table 5. Effects of different strains on tobacco growth and development

[0128]

[0129] Table 6. Effects of different strains on tobacco agronomic morphology (field study)

[0130]

[0131]

[0132] 2.7 Study on the outdoor control effect of antagonistic bacteria on tobacco bacterial wilt

[0133] The occurrence of bacterial wilt in tobacco is accompanied by a complex natural micro-ecological environment. The control efficacy of a single antagonistic bacteria in pot experiments is insufficient to fully demonstrate its control value. To further explore the field control efficacy of native antagonistic bacteria screened indoors in the Meizhou tobacco-growing area of ​​Guangdong, we conducted an outdoor control effect study of the antagonistic bacteria in tobacco fields where bacterial wilt occurred in Meizhou, Guangdong. Based on the results in section 2.5, the treatment groups we selected were strains MZ3-12, MZ8-15, MZ9-28, and a mixture of the three bacteria (Mix). The results are as follows: Figure 11As shown in Tables 7 and 8, the incidence and disease index of each strain against tobacco bacterial wilt in the field trial are presented. The results indicate that all four treatments exhibited varying degrees of relative control efficacy, with MZ8-15 showing the best effect, achieving relative control efficiencies of 86.72% and 60.19% at 25 and 40 days post-inoculation, respectively.

[0134] Table 7. Incidence of Tobacco Bacterial Wilt by Various Strains

[0135]

[0136] Table 8 shows the disease index of each strain on the occurrence of tobacco bacterial wilt.

[0137]

[0138] 3. Discussion and Conclusion

[0139] Numerous factors influence the yield and quality of cash crops and the rhizosphere microecology, with soil-borne diseases being a major biological factor that significantly restricts agricultural production. A significant portion of the occurrence of soil-borne diseases is due to the enrichment of certain fungi, bacteria, and other microorganisms in the soil, leading to an imbalance in the soil microbial ecosystem. Furthermore, there are many types of soil-borne pathogens, mainly fungi, bacteria, and viruses, among which bacterial soil-borne diseases are most severe, with bacterial wilt caused by *Ralstonia solanacearum*. Bacterial wilt primarily affects solanaceous crops, such as tobacco and tomatoes, often causing significant losses in yield and quality. We isolated 274 bacterial strains from healthy rhizosphere soil in diseased tobacco fields in Meizhou City, Guangdong Province. 84 strains exhibited antagonistic activity, and 30 strains had inhibition zones exceeding 10 mm in diameter. Subsequent independent plate antagonistic activity assays against *Ralstonia solanacearum*, hydrolytic enzyme activity assays, ferroptophilic production capacity assays, and molecular identification were performed on these 30 bacterial strains. Based on the above experimental results, we screened out four antagonistic bacteria: MZ3-12, MZ4-13, MZ8-15, and MZ9-28. These bacteria showed different molecular identification results and exhibited good antagonistic activity, enzyme activity, and heparin production. To further confirm and explore the indoor disease resistance or growth-promoting effects of these four antagonistic bacteria, we conducted an indoor pot experiment. The results showed that MZ3-12, MZ8-15, and MZ9-28 had good control effects against *Ralstonia solanacearum*, with MZ8-15 showing the best control effect. The relative control efficacies at 10 and 15 days after inoculation were 85.61% and 67.00%, respectively. Compared with the control, the treatments of MZ3-12, MZ4-13, MZ8-15, and MZ9-28 all showed significant growth-promoting effects on tobacco plants, as reflected in plant height, root length, fresh weight, and dry weight, respectively. Besides MZ4-13, MZ3-12, MZ8-15, and MZ9-28 all showed significant control efficacy. Therefore, we conducted a field efficacy experiment using these three antagonistic bacteria in tobacco-growing areas affected by bacterial wilt in Meizhou, Guangdong. The results showed that MZ3-12, MZ8-15, MZ9-28, and the combination of the three antagonistic bacteria all had certain control efficacy against tobacco bacterial wilt, with MZ8-15 showing the best effect. The relative control efficacy at 25 days and 40 days after inoculation was 86.72% and 60.19%, respectively. Existing literature (Hydroxycoumarins: New, effective plant-derived compounds reduce Ralstonia pseudosolanacearumpopulations and control tobacco bacterial wilt) reported that, through indoor pot experiments, the control effect of the conventional chemical control agent, thiabendazole copper, on tobacco bacterial wilt was approximately 38.36%.The three native antagonistic bacteria strains from Guangdong that were finally screened in this experiment showed good disease control against tobacco bacterial wilt in both indoor pot experiments and outdoor field experiments, and their biocontrol mechanism is worthy of further in-depth research.

[0140] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A soil-borne glutamate bacterium antagonizing bacterial wilt, characterized in that, The soil glutamate bacillus is DW8, and the classification name is: Glutamicibacter soli , and is preserved in the China Center for Type Culture Collection, Wuhan, on November 13, 2023, with the preservation number CCTCC NO: M 20232211.

2. A microbial preparation, characterized in that, The active ingredient of the microbial preparation comprises one or more of the following: bacterial cells, bacterial powder, and bacterial suspension of *Soil Glutamate Bacillus DW8* as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, Soil glutamate bacillus DW8 was cultured to obtain a bacterial suspension, which is a liquid microbial inoculant.

4. The microbial preparation according to claim 2, characterized in that, The microbial agent also includes excipients acceptable for the preparation of the microbial agent.

5. The microbial preparation according to claim 4, characterized in that, The auxiliary material is a loaded solid, which includes one or more of rice bran, straw powder, diatomaceous earth, sodium alginate, zeolite, and biochar.

6. A method for the preparation of the microbial preparation according to claim 2 or 3, characterized in that The soil bacillus DW8 strain was activated and inoculated into LB liquid medium and cultured at 28-30°C until the OD 600 nm reached 0.6-1.0.

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