Tobacco bacterial wilt antagonistic bacterium screening method and antibacterial mechanism method

Abstract

The invention relates to a screening method and an antibacterial mechanism method of antagonistic bacteria for tobacco bacterial wilt, and the screening method comprises the following steps: collection, separation and purification of bacterial strains, primary screening of bacterial wilt antagonistic bacteria activity results show that the bacterial strain y229-1 has the strongest bacteriostatic action on QK bacteria, and the bacterial strain y213 has the weakest bacteriostatic action on QK bacteria. The method comprises the following steps: determining a growth curve, determining the protein content, determining the AKP content, and processing data by adopting or i g i n software and Exc e l software. The method is based on a double-track research strategy of strain screening-mechanism verification, and mainly comprises the following steps: screening antagonistic bacteria; by means of analysis of an antibacterial mechanism, a tobacco bacterial wilt microorganism prevention and control technical framework is established, and a biological solution is provided for quality improvement and efficiency improvement of the tobacco industry in the karst landform area of Guizhou.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for screening tobacco bacterial wilt antagonistic bacteria and a method for antibacterial mechanism, and belongs to the technical field of biology. BACKGROUND

[0002] Tobacco (Nicotiana tabacum L.) is a plant of Solanaceae and Nicotiana, and is one of the main crops for promoting the economic development of China. The growth environment of tobacco is affected by factors such as soil, temperature, and altitude 0 . Tobacco is mostly planted in fixed areas, and the long-term continuous cropping and the use of various chemical fertilizers change the physicochemical properties of the soil and other factors, so that tobacco is easily infected with bacterial wilt during the growth period 0-0 . Bacterial wilt is one of the main diseases of tobacco, which is caused by Ralstonia solanacearum and has a disease effect on the roots, stems, and leaves of tobacco 00 . Tobacco bacterial wilt is a soil-borne disease under high temperature and high humidity, and the areas where tobacco is generally planted are mostly under high humidity and high temperature conditions. Before the maturation of tobacco, the tobacco is easily infected with bacterial wilt under the climate of high temperature and high humidity, which causes disaster and brings huge losses to the quality and yield of tobacco 0 . Studies have shown that the direct and indirect economic losses of Guizhou tobacco production due to tobacco bacterial wilt are as high as 3% to 8% per year, and the incidence rate in some areas reaches 100% 0 . It is of great significance to the sustainable development of Guizhou tobacco agriculture to carry out the screening of tobacco antagonistic bacteria.

Previous research progress

[0003] The present invention aims to address the technical problem of establishing a microbial control framework for tobacco bacterial wilt based on a dual-track research strategy of "strain screening-mechanism verification" (main: antagonistic bacteria screening; auxiliary: antibacterial mechanism analysis), providing a biological solution for improving the quality and efficiency of the tobacco industry in the karst landform area of ​​Guizhou.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0005] The screening method for antagonistic bacteria against tobacco bacterial wilt includes the following steps:

[0006] 1) Collection, isolation, and purification of bacterial strains:

[0007] Three endophytic bacteria obtained from the tissue culture plates of diseased tobacco stems were isolated and purified. After being streaked with LB medium on a sterile operating table, they were placed in a 30°C electric thermostatic incubator for 24 hours.

[0008] The target strain QK was streaked in a sterile operating table using bacterial wilt-specific medium and then incubated in a 30°C electric thermostatic incubator for 48 hours.

[0009] 2) Initial screening for bacterial wilt antagonistic bacteria activity:

[0010] In a sterile laminar flow hood, 1-2 loops of uncontaminated QK, b1029, y229-1, and y213 bacteria were picked and placed into 1.5 mL sterile centrifuge tubes. 1 mL of sterile water was added to each tube, and the mixture was vortexed. 500 μL of QK bacterial suspension was then pipetted onto LB agar. The suspension was spread evenly in the culture dish using a spreader. 6 mm filter paper was then placed in the culture dish coated with QK bacterial suspension using tweezers. 20 μL of b1029, y229-1, and y213 bacterial suspensions were pipetted onto the filter paper and labeled. The culture dishes were then incubated in a constant temperature incubator for 48 h. The results showed that strain y229-1 had the strongest inhibitory effect on QK pathogens, while strain y213 had the weakest inhibitory effect.

[0011] The identification methods for b1029, y229-1, and y213 are as follows:

[0012] 1) Extracting template DNA using the boiling template method: In a sterile operating table, scrape 2-3 inoculation loops of colonies numbered b1029, y229-1, and y213 and place them in sterile 1.5 mL centrifuge tubes. Add 1 mL of sterile water, vortex for 1 min, and then place them in a 100℃ boiling water bath for 10 min. Centrifuge at 12000 r / min for 10 min. Take the supernatant as the DNA template and store it at -20℃.

[0013] 2) PCR amplification and sequencing: Two primer sequences were used to amplify the DNA fragments of b1029, y229-1, and y213 by PCR. The PCR amplification reaction system was 20 μL of mixture (10 μL 2×Det PCR MasterMix, 0.5 μL each of forward and reverse primers, 1 μL bacterial DNA template, and 8 μL ddH2O). 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used as forward and reverse primers, respectively.

[0014] 3) The PCR amplification conditions for bacterial DNA were as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 50 s, 36 cycles, 72℃ extension for 5 min; after detection by 0.15 g / 15 mL agarose gel electrophoresis, the PCR products were sent to Shanghai Sangon Biotech for sequencing. The sequencing results were compared for homology in GenBank using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0015] 4) DNA Sequence Processing and Phylogenetic Tree Construction: BioEdit v7.2.6.1 software was used to select single-peak regions from the sequencing results. The NCBIBlast tool was used to search for and download the known sequence with the highest local sequence match: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. The downloaded sequences and sequencing results were arranged using MEGA X 64 software. The arranged result file was sent to the CIPRES Science Gateway to construct the phylogenetic tree: https: / / www.phylo.org / portal2 / login!input.action. The strains were identified as follows: b1029 was *Bacillus velezensis*, y229-1 was *Bacillus samyloliquefaciens*, and y213 was *Bacillus subtilis*.

[0016] The method for understanding the antibacterial mechanism of antagonistic bacteria against tobacco bacterial wilt includes the following steps:

[0017] 1) Determination of growth curve:

[0018] Strawberries y213, b1029, y229-1, and QK were streaked onto LB agar plates and incubated at 30°C for 24 h. Strawberry QK was incubated for 48 h. Single uncontaminated colonies were picked and transferred to LB liquid, shaken at 150 rpm, and incubated again at 30°C for 24 h. The QK suspension was diluted to 0.5 MCF (1.5 × 10⁸ CFU / mL) with MH broth, and the suspensions of y213, b1029, and y229-1 were diluted to 1.5 MCF. F (4.5×10⁸ cfu / mL); Take 200 μL of diluted QK bacterial suspension into an Erlenmeyer flask containing 30 mL of sterile nutrient broth, and then add 200 μL each of y213, b1029, and y229-1 bacterial suspensions. Use y213, b1029, y229-1, and QK bacterial suspensions as controls, respectively. Incubate at 37℃ and 120 r / min. Use an ELISA reader to monitor the growth of the test bacteria every 2 hours at OD600 and record the data.

[0019] 2) Determination of protein content:

[0020] Accurately weigh a certain amount of K₂HPO₄ and KH₂PO₄ to prepare a PBS buffer solution with a pH of 6.8, and sterilize it at 121℃ for 15 min. Inoculate the test bacteria QK (0.5 MCF), y213, b1029, and y229-1 (1.5 MCF) into 100 mL of MH broth and culture until the logarithmic growth phase. Take 10 mL of the bacterial suspension and transfer it to a 50 mL centrifuge tube. Centrifuge at 2700 rpm for 15 min, wash the bacterial cells three times with PBS buffer, and add 1 mL of PBS buffer. Bacterial suspensions of y213, b1029, and y229-1 were added to MH broth to bring the volume to 10 mL. The suspensions were incubated at 37°C for 4 hours, then centrifuged at 2700 rpm for 15 minutes to obtain the supernatant. Working solutions and BSA standards were prepared and diluted according to the instructions of the BCA (microplate method) protein assay kit, and a standard curve was plotted. The absorbance at 562 nm was measured using a spectrophotometer, with three parallel measurements performed. Each group used individual y213, b1029, y229-1, and QK as controls. Data were recorded, and protein concentrations were calculated based on the standard curve.

[0021] 3) Determination of AKP content:

[0022] After mixing, the absorbance at 510 nm was measured using a spectrophotometer. Each group was measured in triplicate with individual controls of y213, b1029, y229-1, and QK. The data were recorded and AKP activity was calculated using the following formula: (Definition of activity unit: 1 μmol of phenol produced per milligram of protein per minute at 37°C is one enzyme activity unit).

[0023] AKP enzyme activity (U / mg prot) = [C standard × (A test tube - A control tube) ÷ (A standard tube - A blank tube) × V sample] ÷ (Cpr × V sample) ÷ T = 0.167 × (A test tube - A control tube) / (A standard tube - A blank tube) ÷ Cpr

[0024] In the formula, Cstandard is the standard concentration, 2.5 μmol / mL; Vsample is the volume of supernatant added to the reaction system, 0.02 mL; T is the reaction time, 15 min; and Cpr is the sample protein concentration, mg / mL.

[0025] 4) Use Origin and Excel software to process the data.

[0026] The beneficial effects of adopting the above technical solution are:

[0027] This invention screened three bacterial strains with antagonistic effects against the pathogen of bacterial wilt: *Bacillus amyloliquefaciens*, *Bacillus belycera*, and *Bacillus subtilis*. The inhibition zones of these three antagonistic bacteria ranged from 12.0 to 18.0 mm. The growth curves of the antagonistic bacteria were significantly lower than those of the bacterial wilt pathogen after their addition, and both protein and AKP contents were significantly increased. They exerted excellent antibacterial effects by disrupting the cell wall and cell membrane permeability of the bacterial wilt pathogen. This expands the biocontrol resources for tobacco bacterial wilt and provides a reliable prevention solution for tobacco cultivation in Guizhou, promoting the stable development of the tobacco industry. Attached Figure Description

[0028] Figure 1 A phylogenetic tree for strain b1029 based on its 16S rDNA sequence.

[0029] Figure 2 A phylogenetic tree for strain y213 based on its 16S rDNA sequence.

[0030] Figure 3 A phylogenetic tree for strain y229-1 based on its 16S rDNA sequence.

[0031] Figure 4 The growth curves of antagonistic bacteria y229-1, b1029, and y213 and pathogenic bacteria QK are shown.

[0032] Figure 5 This is the BSA standard curve.

[0033] Figure 6 The effect of three antagonistic bacteria on the dissolution of QK protein.

[0034] Figure 7 The effect of three antagonistic bacteria on the AKP dissolution of QK. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0036] 1. Materials and Methods

[0037] 1.1 Test Materials

[0038] 1.1.1 The tested strain QK Ralstonia solanacearum was obtained from Zhejiang University of Science and Technology; Bacillus amyloliquefaciens y229-1, Bacillus subtilis y213, and Bacillus velezensis b1029 were all isolated and purified from the stem tissue of diseased tobacco plants in Guizhou.

[0039] 1.1.2 Reagents: Peptone, Beijing Aoboxing Biotechnology Co., Ltd.; Glucose, Tryptone, Yeast Extract, Shanghai Maclean Biochemical Technology Co., Ltd.; Acid-hydrolyzed Casein (Casein Amino Acids), Shanghai Yuanye Biotechnology Co., Ltd.; Agar Powder, Hangzhou Kangbeis Biotechnology Co., Ltd.; Sodium Chloride, Chengdu Kelong Chemical Reagent Factory; Dipotassium Hydrogen Phosphate, Chongqing Wansheng Chuandong Chemical Co., Ltd.; Potassium Dihydrogen Phosphate, Shanghai Chemical Reagent Factory; Biowest 111860 Regular AGAROSE G-10, Shanghai Yubo Biotechnology Co., Ltd.; BCA Protein Concentration Assay Kit, Tissue and Blood Alkaline Phosphatase (AKP / ALP) Activity Assay Kit, Beijing Solarbio Science & Technology Co., Ltd.; Matrix, Jiangsu Xingnong Matrix Technology Co., Ltd.

[0040] 1.1.3 Culture medium for bacterial wilt: 10.0g peptone, 10.0g glucose, 1.0g acid-hydrolyzed casein, 20.0g agar powder, 1000ml water, pH approximately 7.0; Luria-Bertani medium (LB): 2.0g tryptone, 1.0g yeast extract, 2.0g NaCl, 4.0g agar powder, 200ml water.

[0041] 1.1.4 Clean bench, Suzhou Antai Air Technology Co., Ltd. (Suzhou Jingjing Group); PCR instrument, Bio-Rad Laboratories, Inc.; Electric thermostatic water bath, Shanghai Shangdao Instrument Co., Ltd.; Electric thermostatic incubator, Shanghai Qixin Scientific Instrument Co., Ltd.; WGZ-2XJ turbidimeter (bacteria), Shanghai Xinrui Instrument Co., Ltd.; Vortex mixer, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; Micro-volume benchtop high-speed centrifuge, Hunan Xiangxin Instrument Co., Ltd.; XFH-75CA electric pressure steam sterilizer, Zhejiang Xinfeng Medical Instrument Co., Ltd.; Multifunctional electric furnace, Zhejiang Yongqian Electric Co., Ltd.; Blue light gel cutting instrument D1008E, Sangon Biotech (Shanghai) Co., Ltd.; Microplate reader, Thermo Fisher Scientific Oy; Ultraviolet spectrophotometer, Shanghai Jinghua Instrument Co., Ltd.; Shaker, Shanghai Bilang Instrument Manufacturing Co., Ltd.; Benchtop low-speed centrifuge, Changsha Dongwang Experimental Instrument Co., Ltd.; Electronic balance, Kunshan Youke Weite Electronic Technology Co., Ltd.

[0042] 1.2 Test Methods

[0043] 1.2.1 Collection, Isolation, and Purification of Strains: Three endophytic bacteria obtained from the tissue culture plates of diseased tobacco stems were isolated and purified. After streaking with LB medium on a sterile operating table, the culture plates were incubated at 30°C for 24 hours. The target strain, designated QK, was streaked with bacterial wilt-specific medium on a sterile operating table and incubated at 30°C for 48 hours.

[0044] 1.2.2 Initial Screening of Antibacterial Activity Against Bacterial Wilt: In a sterile laminar flow hood, 1-2 inoculation loops of uncontaminated QK, b1029, y229-1, and y213 bacteria were picked and placed in 1.5 mL sterile centrifuge tubes. 1 mL of sterile water was added to each tube, and the mixture was vortexed. 500 μL of QK bacterial solution was pipetted onto LB medium and spread evenly in the culture dish using a spreader. 6 mm filter paper was then placed in the culture dish coated with QK bacterial solution using tweezers. 20 μL of b1029, y229-1, and y213 bacterial solutions were pipetted onto the filter paper and labeled. The antibacterial culture dishes were then incubated in a constant temperature incubator for 48 h. The antibacterial results were observed and recorded.

[0045] 1.2.3 Identification of strains b1029, y229-1, and y213

[0046] 1.2.3.1 Extraction of template DNA by boiling method: In a sterile operating table, scrape 2-3 inoculation loops of colonies numbered b1029, y229-1, and y213 and place them in sterile 1.5 mL centrifuge tubes. Add 1 mL of sterile water, vortex for 1 min, and then place them in a 100℃ boiling water bath for 10 min. Centrifuge at 12000 r / min for 10 min. Take the supernatant as the DNA template and store it at -20℃.

[0047] 1.2.3.2 PCR amplification and sequencing were performed according to the method described by Pu Yongyu et al. 0 The method used two primer sequences to amplify DNA fragments of b1029, y229-1, and y213 by PCR. The PCR amplification reaction system was 20 μL of mixture (10 μL 2×Det PCRMasterMix, 0.5 μL each of forward and reverse primers, 1 μL bacterial DNA template, and 8 μL ddH2O). 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used as forward and reverse primers, respectively.

[0048] The PCR amplification conditions for bacterial DNA were as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 50 s, 36 cycles, followed by a final extension at 72℃ for 5 min. After detection by 0.15 g / 15 mL agarose gel electrophoresis, the PCR products were sent to Shanghai Sangon Biotech for sequencing. The sequencing results were compared for homology using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) in GenBank.

[0049] 1.2.3.3 DNA Sequence Processing and Phylogenetic Tree Construction

[0050] BioEdit v7.2.6.1 software was used to select single-peak regions in the sequencing results. The NCBI Blast tool was used to search for and download the known sequence with the highest local sequence match (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). The downloaded sequences and sequencing results were arranged using MEGAX 64 software. The arranged result file was then sent to CIPRES ScienceGateway to construct a phylogenetic tree. https: / / www.phylo.org / portal2 / login!input.action ), and identify the strains.

[0051] 1.2.4 Preliminary study on antibacterial mechanism

[0052] 1.2.4.1 Determination of Growth Curves: Strains y213, b1029, y229-1, and QK were streaked onto LB solid medium and incubated at 30°C for 24 h. Strain QK was incubated for 48 h. Single uncontaminated colonies were picked and transferred to LB liquid, shaken at 150 rpm, and incubated again at 30°C for 24 h. The QK bacterial suspension was diluted with MH broth to a concentration of 0.5 MCF (1.5 × 10⁻⁶). 8 CFU / mL), the concentration of bacterial suspensions of Y213, B1029, and Y229-1 was adjusted to 1.5 MCF (4.5 × 10⁻⁶ CFU / mL). 8 (cfu / mL); Take 200 μL of diluted QK bacterial suspension into an Erlenmeyer flask containing 30 mL of sterile nutrient broth, then add 200 μL each of y213, b1029, and y229-1 bacterial suspensions, using y213, b1029, y229-1, and QK bacterial suspensions as controls, respectively. Incubate at 37℃ and 120 r / min, and use a microplate reader to measure the OD value. 600 The growth of the tested bacteria was monitored and data were recorded every 2 hours.

[0053] 1.2.4.2 Determination of Protein Content

[0054] Accurately weigh a certain amount of K2HPO4 and KH2PO4 to prepare a PBS buffer solution with a pH of 6.8, and sterilize it at 121℃ for 15 min. Inoculate the test bacteria QK (concentration of 0.5 MCF), y213, b1029, and y229-1 (concentration of 1.5 MCF) into 100 mL of MH broth and culture them to the logarithmic phase. Take 10 mL of the bacterial suspension into a 50 mL centrifuge tube, centrifuge at 2700 r / min for 15 min, wash the bacterial cells three times with PBS buffer, add 1 mL of y213, b1029, and y229-1 bacterial suspension, and then add MH broth to make up to 10 mL. The sample was incubated at 37℃ for 4 hours, centrifuged at 2700 rpm for 15 minutes, and the supernatant was obtained. The working solution and BSA standard were prepared and diluted according to the instructions of the BCA (microplate method) protein content assay kit, and a standard curve was plotted. The absorbance at 562 nm was measured with a spectrophotometer, and the measurement was repeated in triplicate. Each group was controlled by y213, b1029, y229-1, and QK. The data were recorded, and the protein concentration was calculated based on the standard curve.

[0055] 1.2.4.3 Determination of AKP content

[0056] Follow the instructions for the AKP activity assay kit and refer to the protein content determination method to obtain the supernatant and perform the assay according to Table 1.

[0057] Table 1 AKP Testing Operation Table

[0058] Table 1 AKP Detection Operation Table

[0059]

[0060] After mixing, the absorbance at 510 nm was measured using a spectrophotometer. Each group was measured in triplicate with individual controls of y213, b1029, y229-1, and QK. The data were recorded and AKP activity was calculated using the following formula: (Definition of activity unit: 1 μmol of phenol produced per milligram of protein per minute at 37°C is one unit of enzyme activity)

[0061] AKP enzyme activity (U / mg prot) = [C standard × (A test tube - A control tube) ÷ (A standard tube - A blank tube) × V sample] ÷ (Cpr × V sample) ÷ T = 0.167 × (A test tube - A control tube) / (A standard tube - A blank tube) ÷ Cpr

[0062] In the formula, Cstandard is the standard concentration, 2.5 μmol / mL; Vsample is the volume of supernatant added to the reaction system, 0.02 mL; T is the reaction time, 15 min; and Cpr is the sample protein concentration, mg / mL.

[0063] 1.2.5 Data Processing

[0064] The data was processed using Origin and Excel software.

[0065] 2 Results and Analysis

[0066] 2.1 Initial screening of bacterial wilt antagonistic bacteria activity

[0067] This experiment used the filter paper disc method to screen strains b1029, y229-1, and y213 as the target pathogen. The diameter of the inhibition zone was measured multiple times and the average value was taken. All three strains showed stable inhibitory effects against QK. The strain numbers and the size of their inhibition zones are shown in Table 2. Among all tested strains, strain y229-1 showed the strongest inhibitory effect against QK, while strain y213 showed the weakest inhibitory effect.

[0068] Table 2. Antibacterial effects of different antagonistic bacteria against bacterial wilt pathogen.

[0069] Table 2 Antibacterial effects of different antagonistic bacteriaagainst bacterial wilt

[0070]

[0071] 2.2 Identification of strains b1029, y229-1, and y213: Genomic DNA was extracted from strains b1029, y229-1, and y213 using the template boiling method. 16S rDNA was amplified by PCR, and agarose gel electrophoresis yielded the target bands in all strains, with band sizes around 1200 bp. The obtained sequences were BLAST-aligned against the NCBI database, and phylogenetic trees were constructed for homology analysis of each strain. Figures 1-3 As shown, the antagonistic strains were preliminarily identified as Bacillus velezensis, Bacillus samyloliquefaciens, and Bacillus subtilis.

[0072] 2.3 Study on antibacterial mechanism

[0073] 2.3.1 Growth curve analysis of strains: Growth curves of strains at concentrations of 1.5 MCF (4.5 × 10⁻⁶) were determined. 8 The effects of antagonistic bacteria Y229-1, Y213, and B1029 (CFU) on the growth of bacterial wilt pathogen QK were investigated, and the results are as follows: Figure 4 As shown, it was found that both the pathogenic bacteria and the antagonistic bacteria entered the logarithmic growth phase after 6 hours of culture under normal conditions, with OD... 600 The growth exhibited an exponential pattern. The pathogenic bacterium QK plateaued after 20 hours, while the antagonistic bacteria y229-1 and b1029 plateaued after 16 hours, and y213 plateaued after 18 hours. Furthermore, when antagonistic bacteria were added to the pathogenic bacterium QK for co-culture, the OD values ​​compared to the growth curves of the pathogenic bacterium alone were significantly different. 600 All three strains showed a significant reduction; among them, the inhibitory effect of antagonistic b1029 was the most significant, indicating that all three antagonistic bacteria could effectively inhibit the growth of pathogenic bacterium QK.

[0074] 2.3.2 Protein content determination

[0075] 2.3.1 Construction of the BSA Standard Curve A standard curve of BSA was plotted with concentrations of 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, and 0.05 mg / mL as the x-axis and the corresponding absorbance (A) values ​​as the y-axis. The results are as follows: Figure 5 As shown, there is a linear correlation between concentration and absorbance, with a linear regression equation of y = 13.99x + 0.190, R0. 2 =0.993.

[0076] 2.3.2 Protein Concentration Analysis: 1 mL of bacterial suspensions (y229-1, b1029, and y213) cultured to the logarithmic growth phase were added to each suspension. The absorbance values ​​of each mixed bacterial suspension at 562 nm were higher than those of the single pathogenic bacteria and antagonistic bacteria. The protein concentration was calculated based on the standard curve. The results are as follows: Figure 6 As shown, the protein concentrations increased to varying degrees after the addition of bacterial suspensions y229-1, b1029, and y213, indicating that the antagonistic bacteria y229-1, b1029, and y213 disrupted the permeability of the bacterial cell membrane, leading to protein leakage and an increase in their content.

[0077] 2.3.3 The determination of AKP content is shown in Table 3. 1 mL of bacterial suspension cultured to the logarithmic growth phase, y229-1, b1029, and y213 were added respectively. The absorbance values ​​of each mixed bacterial suspension at 510 nm were higher than those of the single pathogenic bacteria and antagonistic bacteria. The corresponding concentrations were calculated using the formula. The results are as follows: Figure 7 As shown, the AKP content after adding bacterial suspensions y229-1, b1029, and y213 was higher than that of the pathogenic bacteria and antagonistic bacteria alone, indicating that the antagonistic bacteria y229-1, b1029, and y213 may have interacted to disrupt the bacterial cell wall, leading to the dissolution of bacterial AKP and an increase in its content.

[0078] Table 3. Effect of antagonistic bacteria on absorbance value A of *Ralstonia solanacearum* at a wavelength of 510 nm.

[0079] Table 3 Effect of antagonistic bacteria on the absorbance value A of bacterial wilt at wavelength 510nm

[0080]

[0081]

[0082] 3 Discussion

[0083] In the antagonistic bacteria screening experiment, three strains were found to have inhibitory effects on bacterial wilt: *Bacillus amyloliquefaciens*, *Bacillus belyceae*, and *Bacillus subtilis*. These three antagonistic strains belong to the genus *Bacillus*. *Bacillus amyloliquefaciens* and *Bacillus subtilis* are facultative anaerobes and are very similar in morphology, culture characteristics, and physiological and biochemical properties. *Bacillus amyloliquefaciens* colonies appear as pale yellow, opaque colonies on LB agar. *Bacillus amyloliquefaciens* can produce antibacterial proteins, lipopeptide antibiotics, and other antibacterial substances, and can inhibit various plant pathogens, such as those causing wilt, root rot, canker, and brown spot. 00In addition to its antagonistic effect on Fusarium wilt pathogens, it also has a significant growth-promoting effect on cucumbers. 0 Bacillus subtilis forms milky-white colonies on LB agar. Its main antibacterial substances include lipopeptides, proteins, phenols, and polyenes, which inhibit various pathogens causing plant diseases. In plant disease control, it shows good efficacy against tobacco anthracnose, red spot disease, black shank, root rot, and wilt, and also promotes plant growth to some extent, making it widely used. 00 Bacillus belye appears as milky-white colonies on LB agar. It is an aerobic bacterium that effectively inhibits various plant pathogens, such as Phytophthora capsici, Fusarium solani, Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. It exerts its antibacterial effect by altering the structure and permeability of the cell membrane through the production of antimicrobial proteins, lipopeptide antibiotics, and polyketide antibiotics, causing leakage of intracellular substances and ultimately leading to cell death. 00 The *Bacillus amyloliquefaciens* Y299-1, *Bacillus subtilis* Y213, and *Bacillus belyss* B1029 screened in this experiment all effectively inhibited the growth of pathogenic bacterium QK. The protein concentration after adding the bacterial suspension was significantly increased compared to the control group, and the AKP content was higher than that of the pathogenic bacterium alone and the antagonistic bacteria. This indicates that the screened antagonistic bacteria have good control potential against tobacco bacterial wilt pathogens. However, whether the antagonistic bacteria have other effects on tobacco plants requires further research and verification.

[0084] 4. Conclusion

[0085] This invention screened three bacterial strains with antagonistic effects against the pathogen of bacterial wilt: *Bacillus amyloliquefaciens*, *Bacillus belycera*, and *Bacillus subtilis*. The inhibition zones of these three antagonistic bacteria ranged from 12.0 to 18.0 mm. The growth curves of the antagonistic bacteria were significantly lower than those of the bacterial wilt pathogen after their addition, and both protein and AKP contents were significantly increased. They exerted excellent antibacterial effects by disrupting the cell wall and cell membrane permeability of the bacterial wilt pathogen. This expands the biocontrol resources for tobacco bacterial wilt and provides a reliable prevention solution for tobacco cultivation in Guizhou, promoting the stable development of the tobacco industry.

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Claims

1. A method for screening antagonistic bacteria against tobacco bacterial wilt, characterized in that: Includes the following steps: 1) Collection, isolation, and purification of bacterial strains: Three endophytic bacteria obtained from the tissue culture plates of diseased tobacco stems were isolated and purified. After being streaked with LB medium on a sterile operating table, they were placed in a 30°C electric thermostatic incubator for 24 hours. The target strain QK was streaked in a sterile operating table using bacterial wilt-specific medium and then incubated in a 30°C electric thermostatic incubator for 48 hours. 2) Initial screening for bacterial wilt antagonistic bacteria activity: In a sterile laminar flow hood, 1-2 loops of uncontaminated QK, b 1029, y229-1, and y213 bacteria were picked and placed into 1.5 mL sterile centrifuge tubes. 1 mL of sterile water was added to each tube, and the mixture was vortexed. 500 μL of QK bacterial suspension was then pipetted onto LB agar. The suspension was spread evenly in the culture dish using a spreader. A 6 mm filter paper was placed in the culture dish coated with QK bacterial suspension using tweezers. 20 μL of b 1029, y229-1, and y213 bacterial suspensions were then pipetted onto the filter paper and labeled. The culture dishes were then incubated in a constant temperature incubator for 48 hours. The results showed that strain y229-1 exhibited the strongest inhibitory effect against QK bacteria, while strain y213 showed the weakest inhibitory effect.

2. The method for screening antagonistic bacteria against tobacco bacterial wilt according to claim 1, characterized in that: The identification methods for b 1029, y229-1, and y213 are as follows: 1) Extracting template DNA using the boiling template method: In a sterile operating table, scrape 2-3 inoculation loops of colonies numbered b1029, y229-1, and y213 and place them in sterile 1.5 mL centrifuge tubes. Add 1 mL of sterile water, vortex for 1 min, and then place them in a 100℃ boiling water bath for 10 min. Centrifuge at 12000 r / min for 10 min. Take the supernatant as the DNA template and store it at -20℃. 2) PCR amplification and sequencing: Two primer sequences were used to amplify the DNA fragments of b1029, y229-1, and y213. The PCR amplification reaction system was 20 μL of mixture (10 μL of 2×Det PCR Master Mix, 0.5 μL each of forward and reverse primers, 1 μL of bacterial DNA template, and 8 μL of ddH2O). 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used as forward and reverse primers, respectively. 3) The PCR amplification conditions for bacterial DNA were as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 50 s, 36 cycles, 72℃ extension for 5 min; after detection by 0.15 g / 15 mL agarose gel electrophoresis, the PCR products were sent to Shanghai Sangon Biotech for sequencing. The sequencing results were compared for homology in GenBank using BLAST (http: / / blast.ncbi.nlm.nih.gov / Blost.cgi). 4) DNA Sequence Processing and Phylogenetic Tree Construction: BioEdit v7.2.6.1 software was used to select single-peak regions from the sequencing results. The NCBIBlast tool was used to search for and download the known sequence with the highest local sequence match: https: / / blast.ncbi.nlm.nih.gov / blast.cgi?PROGRAM=blastn&PAGE_TYPE=blastSearch&LINK_LOC=blasthome. The downloaded sequences and sequencing results were arranged using MEGA X 64 software. The arranged result file was then sent to the CIPRES Science Gateway to construct the phylogenetic tree: https: / / www.phylo.org / portal2 / login! The input.action method was used to identify the antagonistic strains, and b1029 was identified as Bacillus svelezensis, y229-1 as Bacillus samyloliquefaciens, and y213 as Bacillus subtilis.

3. The method for detecting the antibacterial mechanism of tobacco bacterial wilt antagonistic bacteria as described in claim 1, comprising the following steps: 1) Determination of growth curve: Strawberries y213, b 1029, y229-1, and QK were streaked onto LB agar plates and incubated at 30°C for 24 h. Strawberry QK was incubated for 48 h. Single uncontaminated colonies were picked and transferred to LB liquid, shaken at 150 rpm, and incubated again at 30°C for 24 h. The QK bacterial suspension was diluted to 0.5 MCF (1.5 × 10⁸ CFU / mL) with MH broth, and the y213, b 1029, and y229-1 bacterial suspensions were diluted to 1.5 MCF (4.5 × 10⁸ CFU / mL). 200 μL of the diluted QK bacterial suspension was transferred to a flask containing 30 mL of sterile nutrient broth, and then 200 μL each of the y213, b 1029, and y229-1 bacterial suspensions were added. Strawberries y213, b 1029, and y229-1 were cultured separately. 1029, y229-1, and QK bacterial suspensions were used as controls. They were incubated at 37℃ and 120 r / min. The growth of the test bacteria was monitored and recorded every 2 hours using an ELISA reader at OD600. 2) Determination of protein content: Accurately weigh a certain amount of K2HPO4 and KH2PO4 to prepare a PBS buffer solution with a pH of 6.8, and sterilize it at 121℃ for 15 min for later use. The test bacteria QK (0.5 MCF), y213, b 1029, and y229-1 (1.5 MCF) were inoculated into 100 mL of MH broth and cultured to the logarithmic phase. 10 mL of the bacterial suspension was transferred to a 50 mL centrifuge tube and centrifuged at 2700 rpm for 15 min. The cells were washed three times with PBS buffer, and 1 mL of y213, b 1029, and y229-1 bacterial suspension was added, followed by MH broth to a final volume of 10 mL. The mixture was incubated at 37°C for 4 h, centrifuged at 2700 rpm for 15 min, and the supernatant was collected. The working solution and BSA standard were prepared according to the BCA (microplate method) protein assay kit instructions, and a standard curve was plotted. The absorbance at 562 nm was measured using a spectrophotometer, with three replicates. Each group was prepared using y213, b 1029, and y229-1 alone. 1029, y229-1, and QK were used as controls. Data were recorded, and protein concentration was calculated based on the standard curve. 3) Determination of AKP content: After mixing, the absorbance at 510 nm was measured using a spectrophotometer. Each group was measured in triplicate with individual y213, b1029, y229-1, and QK as controls. The data were recorded and AKP activity was calculated according to the following formula: (Definition of activity unit: 1 μmol of phenol produced per minute per milligram of protein at 37℃ is one enzyme activity unit). AKP enzyme activity (U / mg prot) = [C standard × (A test tube - A control tube) ÷ (A standard tube - A blank tube) × V sample] ÷ (Cpr × V sample) ÷ T = 0.167 × (A test tube - A control tube) / (A standard tube - A blank tube) ÷ Cpr In the formula, Cstandard is the standard concentration, 2.5 μmol / mL; Vsample is the volume of supernatant added to the reaction system, 0.02 mL; T is the reaction time, 15 min; and Cpr is the sample protein concentration, mg / mL. 4) Use Origin and Excel software to process the data.

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