Biocontrol bacterium, extracellular protein thereof, extraction method and application

Abstract

The invention relates to a biocontrol bacterium, an extracellular protein thereof, an extraction method and application, and belongs to the technical field of agricultural biological control. In order to solve the problems that an existing biocontrol bacterium agent is unstable in tomato fusarium wilt prevention and treatment effect and prone to inactivation, the invention provides a biocontrol bacterium which is classified and named as bacillus subtilis and has broad-spectrum antibacterial activity. The extracellular protein of the biocontrol bacteria disclosed by the invention has relatively strong bacteriostatic ability, can maintain relatively good bacteriostatic ability in a wide range of temperature, pH, ultraviolet irradiation and storage time, and has relatively good stability. The extracellular protein provided by the invention can be used for remarkably preventing and treating fusarium oxysporum of in-vitro leaves and in-vitro fruits of tomatoes; compared with a control group, the disease index is remarkably reduced; the compound has a remarkable prevention and treatment effect on tomato fusarium wilt in greenhouse cultivation, the activity of defensive enzymes PPO, POD and SOD of plants can be improved, the plant resistance is stimulated, a more comprehensive and efficient biological prevention and treatment effect is obtained, and the prevention and treatment range is wider.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and in particular relates to a biological control bacterium, an extracellular protein thereof, and an extraction method and application thereof. Background Art

[0002] Tomato wilt is a soil-borne vascular disease caused by Fusarium oxysporum. The pathogen exists in the soil and can also be spread over long distances through infected seeds. The pathogen often invades the vascular bundles through root wounds, natural cracks, and root hairs during transplanting and planting, reaches the vascular bundles and reproduces in them, blocks the ducts, and prevents the plant from absorbing water and fertilizer, causing the leaves to wilt and die, which has a serious impact on tomato yield and quality.

[0003] The prevention and control methods of tomato wilt include agricultural prevention, chemical prevention and biological prevention. Although agricultural prevention methods have the advantages of being environmentally friendly and sustainable, the prevention and control effects are often affected by a variety of environmental factors, such as weather conditions, soil texture, crop growth conditions, etc. These factors may lead to unstable prevention and control effects, so that the disease may still occur in some cases. The disadvantage of chemical prevention is that the abuse of chemical agents can easily lead to soil and water pollution and damage the ecological environment. Long-term use of chemical agents will cause pathogens to develop drug resistance, resulting in a decrease in prevention and control effects.

[0004] Biological control usually uses specific microbial preparations or plant extracts to control the pathogens of tomato wilt, so it is highly targeted. Microbial preparations can also improve the structure of soil microbial communities and enhance soil disease resistance, thereby reducing the recurrence of diseases.

[0005] However, the effect of biological control is greatly affected by environmental factors, and the control effect is not stable. For example, if the ambient temperature is too high or too low, it will affect the activity of microbial agents, thereby reducing the effect of biological control. Ultraviolet radiation can destroy the cell wall and cell membrane of microbial cells, resulting in inactivation of the microbial agents; it can also affect the metabolic activity of microbial cells, reducing the control effect of the microbial agents. The physical and chemical properties of the soil, such as pH value, will also affect the survival and reproduction of microorganisms, thereby affecting the effect of biological control. In addition, the health of crops will directly affect their resistance and adaptability to microbial agents. If the crops are already infected with diseases or are in a weak state, the effect of biological control will also be affected. Summary of the invention

[0006] In order to solve the problem that the existing biocontrol bacteria are unstable in effect and easy to inactivate in controlling tomato wilt, the present invention provides a biocontrol bacterium, an extracellular protein thereof, and an extraction method and application thereof.

[0007] The technical solution of the present invention:

[0008] A biocontrol bacterium, the classification name of the biocontrol bacterium is Bacillus subtilis, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on March 18, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30050.

[0009] An extracellular protein extracted from the fermentation liquid of the biocontrol bacteria of the invention.

[0010] A method for extracting extracellular protein according to the present invention comprises the following steps:

[0011] Step 1, inoculating the biocontrol bacteria of the present invention into a liquid culture medium for fermentation and culture;

[0012] Step 2: Centrifuge the fermentation liquid obtained in step 1 to collect the supernatant, sterilize the obtained centrifuged supernatant filter membrane, add solid ammonium sulfate to the obtained supernatant filtrate to a saturation of 80%, fully shake and mix, let stand at 4°C for 24 hours, then centrifuge to collect the precipitate, add PBS buffer to dissolve the precipitate, put the obtained dissolved solution into a dialysis bag with a molecular weight cutoff of 8000 to 14000D for dialysis desalination, collect the dialysate and filter the membrane for sterilization to obtain the extracellular extracted protein.

[0013] Furthermore, the liquid culture medium in step 1 is MSM basal salt medium, NB nutrient broth medium, Landy medium, LB bacterial basal medium, YSP yeast peptone medium, NA nutrient agar medium, PDA potato dextrose agar medium or CM complete medium; the liquid volume of the liquid culture medium is 60-100 mL / 250 mL; the fermentation culture conditions are 25-30 ° C fermentation temperature, 150-200 r / min shaking culture for 24-72 hours.

[0014] Furthermore, the liquid culture medium in step 1 is an MSM basal salt culture medium, and the liquid volume of the liquid culture medium is 80 mL / 250 mL; the fermentation culture conditions are 29 ° C fermentation temperature, 180 r / min shaking culture for 24 hours.

[0015] Furthermore, the centrifugation in step 2 is carried out at 4°C and 10,000 r / min for 20 min, and the dialysis desalination time is 24 h.

[0016] An application of the extracellular protein of the present invention in agricultural biological control, wherein the biological control comprises using the extracellular protein to control tomato wilt caused by Fusarium oxysporum.

[0017] An application of the extracellular protein of the present invention in agricultural biological control, wherein the biological control comprises using the extracellular protein to improve the disease resistance of crops.

[0018] Furthermore, the crop is tomato.

[0019] Beneficial effects of the present invention:

[0020] The present invention separates and obtains biocontrol bacteria XY-3 with good control effect on tomato wilt. Through physiological and biochemical identification, morphological observation and 16S rDNA gene sequence analysis, the strain is identified as Bacillus subtilis, which has broad-spectrum antibacterial activity, can secrete multiple hydrolases and siderophores, and does not produce HCN.

[0021] The extracellular protein of the biocontrol bacterium XY-3 has strong antibacterial ability, and can maintain good antibacterial ability in a wide range of temperature, pH, ultraviolet radiation and storage time, and has good stability.

[0022] The extracellular protein of the biocontrol bacterium XY-3 can significantly prevent and control Fusarium oxysporum in detached tomato leaves and fruits. Compared with the control group, the disease index was significantly reduced. It has a significant control effect on tomato wilt in greenhouse cultivation, and can increase the activity of the plant's defense enzymes PPO, POD and SOD, stimulate plant resistance, and obtain more comprehensive and efficient biological control effects with a wider range of control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 From left to right are the colony morphology photos, Gram staining photos and overall morphology photos of the XY-3 strain in Example 1;

[0024] Figure 2 The electrophoresis diagram of the 16S rDNA PCR amplification product of the XY-3 strain in Example 1;

[0025] Figure 3 This is a picture of the phylogenetic tree of the 16S rDNA sequence of the XY-3 strain in Example 1;

[0026] Figure 4 The comparison photos of the biofilm formation test results of the biocontrol bacteria XY-3 in Example 2;

[0027] Figure 5 This is a comparison photo of the test results of the HCN production capacity of the biocontrol bacteria XY-3 in Example 2;

[0028] Figure 6 The comparison photos of the results of the IAA secretion ability test of the biocontrol bacteria XY-3 in Example 2;

[0029] Figure 7The results of the enzyme production capacity and siderophore determination of the biocontrol bacteria XY-3 in Example 2 are shown in FIG. 1 , siderophore, 2 , cellulase, 3 , amylase, and 4 , protease;

[0030] Figure 8 The photos are the antibacterial activity test results of the supernatant, precipitate and ethyl acetate extract of the biocontrol bacteria XY-3 in Example 2, wherein 1 is the supernatant, 2 is the precipitate, and 3 is the ethyl acetate extract;

[0031] Fig. 9 The photo is a photo of the antibacterial activity test results of the extracellular protein and lipopeptide substances of the biocontrol bacteria XY-3 in Example 2, in which 1 is the extracellular protein and 2 is the lipopeptide substance;

[0032] Fig.10 The following are photos of the test results of volatile substances of the biocontrol bacteria XY-3 in Example 2, 1 is a blank control, 2 is the XY-3 strain;

[0033] Fig.11 This is a comparison chart of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 at different temperatures in Example 3;

[0034] Fig.12 This is a comparison chart of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 under different ultraviolet irradiation times in Example 3;

[0035] Fig.13 This is a comparison chart of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 under different pH conditions in Example 3;

[0036] Fig.14 This is a comparison chart of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 at different storage times in Example 3;

[0037] Fig.15 This is a comparison of the protective effects of different treatments on tomato detached leaves in Example 4, in which 1 is CK, 2 is PBS, 3 is extracellular protein, and 4 is pyrimethanil;

[0038] Fig.16 These are the trypan blue staining photos of detached tomato leaves with different treatments in Example 4;

[0039] Fig.17 These are photos of vein cells of tomato leaves stained with trypan blue under different treatments in Example 4;

[0040] Fig.18 These are photos of the protective effects of detached tomato fruits treated differently in Example 4 on days 1, 3, 5, and 7;

[0041] Fig.19 The photos are of greenhouse protection effect of different treatments on tomato plants in Example 5;

[0042] Fig. 20 This is a comparison of PPO activities in tomato leaves under different treatments in Example 6;

[0043] Fig.21 This is a comparison chart of SOD activity in tomato leaves under different treatments in Example 6;

[0044] Fig. 22 This is a comparison chart of POD activity in tomato leaves under different treatments in Example 6. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0046] Example 1

[0047] This example provides a method and result for identifying the species of biocontrol bacteria.

[0048] In this example, a bacterium with antagonistic effect on tomato wilt pathogen was isolated and purified from the rhizosphere soil of plants by using the plate dilution coating method and the plate streak method. The bacterium was numbered as XY-3, and the morphological characteristics, physiological and biochemical characteristics and molecular biology of the strain were identified.

[0049] (I) Morphological observation

[0050] Use an inoculating loop to dip into the purified XY-3 strain suspension, streak on LB solid culture medium, seal and mark, and culture in a 28°C constant temperature incubator for 48 hours. Observe the colony morphology, colony color, etc. according to the Bergey's Manual of Bacterial Identification (8th Edition).

[0051] Gram staining observation: Take a small amount of sterile water to rinse the slide, wait for it to dry, then use a pipette to draw an appropriate amount of XY-3 bacterial liquid and drop it on the slide, spread it evenly, bake it with an alcohol lamp to fix the shape, and cool it to room temperature.

[0052] Primary staining: Add an appropriate amount of crystal violet stain solution at a fixed position for 1 minute, rinse slowly with sterile water and dry.

[0053] Mordanting: Dye with appropriate amount of iodine solution for 2 minutes, rinse with sterile water and dry.

[0054] Elution: Repeatedly elute with 95% ethanol solution until the eluent is colorless, rinse with sterile water and dry.

[0055] Re-staining: add appropriate amount of safranin dye solution, let it stand for 1 minute, rinse the dye solution with sterile water until it is colorless, and dry the water with filter paper. Finally, place the stained slide under an oil microscope to observe its morphology, color, etc.

[0056] Electron microscopic observation: The XY-3 strain was inoculated into LB liquid culture medium and cultured in a shaking incubator at 28°C and 200 r / min for 48 h. The bacterial liquid was placed in a centrifuge tube and centrifuged at 4°C, 20 min, and 6000 r / min. The supernatant was discarded and the precipitate was rinsed evenly with PBS buffer, and repeated 2-3 times; the mixed solution was dehydrated with 70% ethanol for 15 min, and then dehydrated with 100% ethanol twice; it was replaced once with a 1:1 solution of 100% ethanol: tert-butanol, and then replaced twice with pure tert-butanol solution; the sample was placed in a -20°C refrigerator and frozen for 30 min, and then placed in a freeze dryer for freeze drying; the freeze-dried sample was attached to the electron microscope sample stage with conductive tape, and then a film (100-150 angstroms) was coated on the sample surface with an ion sputtering coating device, and the processed sample was observed under an electron microscope.

[0057] Figure 1 From left to right are the colony morphology photos, Gram staining photos and overall morphology photos of the XY-3 strain; the results show that the XY-3 colony is milky white at the beginning of culture, and turns light yellow after 7 days. It is dry, opaque, and wrinkled in the center. After Gram staining, it was found that the XY-3 bacteria are blue rods of varying lengths, distributed singly or in pairs, and are Gram-positive bacteria. Under an electron microscope, the overall morphology of the XY-3 strain is rod-shaped, with elliptical ends.

[0058] (II) Physiological and biochemical identification

[0059] (1) Glucose oxidation fermentation O / F test

[0060] Take two sterilized test tubes and add equal amounts of glucose oxidation fermentation medium. The closed tube is sealed with 0.5 cm liquid paraffin, and the open tube is not sealed. The XY-3 bacteria cultured at 28°C for 24 h are punctured and inoculated into the medium. The other two test tubes are controls and are not inoculated. The treatment is the same as the above group. After constant temperature culture at 28°C for 48 h, the color change in the test tube is observed. If both the open tube and the closed tube are yellow, it is a fermentation type. If the color of the open tube changes to yellow, it is oxidative. The experiment is repeated three times.

[0061] (2) Catalase test

[0062] Use an inoculation loop to scrape the bacteria cultured at 28°C for 24 hours and smear them evenly on a sterilized slide. Then add 10% H2O2 and observe whether small bubbles are generated. If it is positive, bubbles are generated, and if it is negative, no bubbles are generated. Repeat the test three times.

[0063] (3) Aerobic and anaerobic test

[0064] Wait for the LB solid culture medium to cool to about 45°C in the clean bench, inoculate 1% of the XY-3 bacterial solution that has been cultured at 28°C for 24 hours into the 45°C culture medium, stir and mix with a sterilized glass rod, seal and culture at 28°C for 3-7 days, observe the growth of the colonies during this period, the aerobic type only grows on the surface of the culture medium, the anaerobic type only grows at the bottom of the culture medium, and the facultative anaerobic type can grow on both the surface and the bottom of the culture medium. Repeat the experiment three times.

[0065] (4) Methyl red test

[0066] Take two sterile test tubes, add glucose peptone water culture medium, use an inoculation loop to scrape a small amount of XY-3 bacteria cultured at 28℃ for 24h and inoculate it into one test tube. The other test tube is used as a control and is not inoculated. After constant temperature culture at 28℃ for 48h, slowly add an appropriate amount of methyl red indicator along the wall of the tube. If the tube turns red, it is positive, and if it turns yellow, it is negative. Repeat the test three times.

[0067] (5) Gelatin liquefaction test

[0068] Take two sterilized test tubes, add appropriate amount of gelatin liquefied culture medium, tilt them until solidified, use an inoculation loop to scrape a small amount of XY-3 bacteria cultured at 28℃ for 24h and inoculate it into one test tube. The other test tube is not inoculated with bacteria as a control and cultured at 28℃ for 3-7d. If the gelatin liquefied culture medium in the test tube is liquid, it is positive. If it is solid, it is negative. Repeat the test three times.

[0069] (6) VP test

[0070] Take two sterilized test tubes, add glucose peptone water culture medium, use an inoculation loop to scrape a small amount of XY-3 bacteria cultured at 28℃ for 24h and inoculate it into one test tube. The other test tube is used as a control and is not inoculated. After constant temperature culture at 28℃ for 48h, mix the XY-3 bacterial suspension with equal volumes of 40% sodium hydroxide, add appropriate amount of creatine powder to the mixture, if the mixture turns red after ten minutes, or turns red after waiting longer, it is positive, and repeat the test three times.

[0071] Table 1 shows the results of identification of physiological and biochemical characteristics of strain XY-3.

[0072] Table 1

[0073] Physiology and Biochemistry Strain XY-3 Aerobic or anaerobic Aerobic Glucose oxidative fermentation Fermentation Gelatin liquefaction + Catalase test + Methyl red reaction - VP -

[0074] Note: “+” means positive; “-” means negative

[0075] The results in Table 1 show that strain XY-3 is an aerobic bacterium, showing a fermentation type in the glucose oxidation and fermentation test, being positive in the gelatin liquefaction test and the catalase test, and being negative in the methyl red MR test and the VP test.

[0076] (III) 16S rDNA sequence analysis

[0077] (1) The genomic DNA of strain XY-3 was extracted by column method. The specific operation is as follows:

[0078] 1) Take a 1.5 mL centrifuge tube, add 200 μL of pretreatment solution and a small amount of glass beads, then add an appropriate amount of bacterial sample, and grind it in a grinder until it is fully ground.

[0079] 2) Add 20 μL of Proteinase K solution and 200 μL of lysis buffer in sequence, mix thoroughly and let stand at 70°C for 10 min.

[0080] 3) Add 200 μL of anhydrous ethanol and mix well. Perform short centrifugation to remove droplets on the inner wall and top cover of the centrifuge tube.

[0081] 4) Pass the adsorption column, wash once with washing solution and wash twice with rinsing solution.

[0082] 5) The adsorption column is placed at room temperature for 3 to 5 minutes to allow the residual rinsing liquid in the adsorption material to evaporate completely.

[0083] 6) Transfer the adsorption column into a new centrifuge tube, take 50-100 μL ddH2O and drop it in the middle of the adsorption membrane, leave it at room temperature for 3-5 minutes, centrifuge it at 12000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

[0084] (2) The extracted XY-3 genomic DNA was used as a template and PCR amplification was performed using universal primers. The sequences of amplification primers and sequencing primers are shown in Table 2. The PCR amplification reaction system and reaction conditions are shown in Table 3.

[0085] Table 2

[0086]

[0087] Table 3

[0088]

[0089] Prepare gel and place it in the electrophoresis tank. Use a pipette to take 3 μL of the PCR amplified product and spot it. Figure 2 This is the electrophoresis diagram of the 16S rDNA PCR amplification product of the XY-3 strain. By observing the electrophoresis results, a PCR amplification product of about 1400 bp was obtained, and the obtained PCR amplification fragment was consistent with the conventional 16S rDNA sequence length.

[0090] (3) The PCR amplification product was recovered and purified. The purification process strictly followed the standard operation procedure of magnetic bead purification. The purified product was sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing to obtain the nucleotide sequence of 16S rDNA as shown in SEQ ID No: 1.

[0091] The 16S rDNA sequence of XY-3 strain was compared with NCBI-BLAST and compared with the reported model strains. The phylogenetic tree of 16S rDNA sequences of XY-3 strain and 15 strains was constructed by the neighbor joining method using MEGA6.06 software, and the similarity was calculated repeatedly 1000 times. Figure 3 This is a picture of the phylogenetic tree of the 16S rDNA sequence of the XY-3 strain.

[0092] Combined with the morphological identification, physiological and biochemical identification and 16SrDNA sequence analysis of the above XY-3 strain, it was shown that the XY-3 strain was Bacillus subtilis. It was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 18, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30050.

[0093] Example 2

[0094] This example provides a fermentation culture method of the biocontrol bacterium XY-3, a method for extracting extracellular proteins, and an investigation of the characteristics of the strain.

[0095] (I) Biofilm formation ability

[0096] The purified XY-3 strain was inoculated into LB liquid culture medium and cultured in a shaker at 28°C and 200r / min for 24h. 5mL of bacterial solution was taken with a pipette and injected into a sterilized test tube. After culturing at 28°C for 24h, the bacterial solution was discarded and the inner wall was slowly washed 2-3 times with clean water. Subsequently, 5mL of crystal violet dye solution was taken with a pipette and added to the test tube. The test tube was allowed to stand for about 20min, the crystal violet was discarded, and the inner wall was slowly washed 3 times with clean water. The blank CK control was an empty test tube with 5mL of crystal violet dye solution added and allowed to stand for 20min, the crystal violet was discarded, and the test tube wall was observed to see if it was bluish purple. If so, it indicated that the strain produced biofilm.

[0097] Figure 4 Comparison photos of the test results of biofilm formation of biocontrol bacteria XY-3; the photos show that after the test tubes in which XY-3 bacterial liquid was cultured were rinsed with clean water, obvious purple substance was formed, indicating that the XY-3 strain can produce biofilm.

[0098] (II) HCN (hydrogen cyanide) production detection

[0099] Take a sterile test tube, add 10mL of XY-3 strain suspension, then add a drop of sulfuric acid and mix slowly, take 1mL of the mixture to evenly soak the HCN test paper, observe whether the test paper changes color after 15 minutes, and wait overnight when the concentration is low. The HCN test paper changes from light blue to dark blue. If there is no color change, it proves that HCN is not produced. Repeat the test three times.

[0100] Figure 5 Comparison photos of the test results of the HCN production ability of the biocontrol bacteria XY-3; the picture shows that the XY-3 bacterial solution was mixed with sulfuric acid and soaked in HCN test paper, and then left to stand overnight, and no color change was found, indicating that the strain does not produce HCN.

[0101] (III) IAA (indole-3-acetic acid) secretion detection

[0102] Prepare 50mL LB liquid culture medium, add 1% L-tryptophan, inoculate XY-3 strain into the liquid culture medium, use the liquid culture medium without inoculation of XY-3 strain as control, and culture in a shaker at 28°C and 200r / min for 48h; put the bacterial solution into a 2mL centrifuge tube, centrifuge at 10000rpm for 20min, take 1mL supernatant and add it into a test tube, add 2mL Salkowaski reagent and two drops of orthophosphoric acid in turn and mix evenly, place the mixed solution in a 28°C water bath for 2h, observe whether the mixed solution turns red, if it turns red, it means that the biocontrol bacteria can secrete IAA, and repeat the test 3 times.

[0103] Figure 6 These are comparison photos of the results of the IAA secretion ability test of the biocontrol bacteria XY-3; as shown in the figure, the test tube on the right is the test group, and the test tube on the left is the control group. After adding Salkowaski reagent and orthophosphoric acid to the supernatant test tube after centrifugation, it turns red significantly, indicating that the XY-3 strain produces IAA.

[0104] (IV) Enzyme production capacity and siderophore

[0105] Extracellular enzyme detection: Prepare protease identification medium, amylase identification medium, and cellulase identification medium, pour the plate on the clean bench, cool and solidify, put a sterile round filter paper (Φ5mm) in the middle of the culture dish, use a pipette to take an appropriate amount of bacterial liquid and drop it on the filter paper on the culture medium, and culture at 28℃ for 3-5 days. Observe whether a transparent circle is formed around the colony in the protease identification medium every day; add an appropriate amount of iodine solution to the amylase identification medium for staining, and observe whether a transparent circle is formed around the colony; add 0.1g / L Congo red to the cellulase identification medium for staining, and then wash it with 1mol / L sodium chloride solution to observe whether a transparent circle is formed around the colony. Repeat the test three times.

[0106] Siderophore detection: prepare chrome azuro blue CAS culture medium, pour it into a plate, let it dry and solidify, then add sterile circular filter paper (Φ5mm), use a pipette to take an appropriate amount of bacterial solution and drop it onto the circular filter paper in the center of the culture medium, culture at 28℃ for 3-5 days, observe whether orange circles are formed around the colonies, and repeat the test three times.

[0107] Figure 7 These are photos of the enzyme production capacity and siderophore determination results of the biocontrol bacteria XY-3. In the figure, 1 is siderophore, 2 is cellulase, 3 is amylase, and 4 is protease. As shown in the figure, after the XY-3 strain has grown in the amylase medium for 3-5 days, iodine solution is added for staining, and an obvious transparent circle appears, indicating that the strain produces amylase; after the XY-3 strain has grown in the protease medium for 3-5 days, a white transparent circle is produced, indicating that the strain produces protease; after the XY-3 strain has grown in the cellulase medium for 3-5 days, 0.1g / L Congo red dye is added and then washed with 1mol / L sodium chloride solution, a white transparent circle appears, indicating that the strain produces cellulase; after the XY-3 strain has been cultured in the chrome azure CAS medium for 3-5 days, orange-yellow circles are found, indicating that the strain produces siderophore.

[0108] (V) Antibacterial activity of supernatant and precipitate

[0109] The biocontrol bacteria XY-3 was inoculated into LB liquid culture medium, placed in a shaking incubator at 28°C and 200 r / min for 48 hours to obtain a fermentation liquid, the fermentation liquid was added to a 50 mL large centrifuge tube, and centrifuged at 4°C and 10000 r / min for 15 minutes, the supernatant and the precipitate were collected in 50 mL centrifuge tubes respectively, the supernatant was filtered through a 0.22 μm filter membrane, and the precipitate was shaken and dissolved with sterile water; the Oxford cup confrontation culture method was adopted, a tomato wilt pathogen block was placed on one side of a PDA plate, and an Oxford cup with 100 μL of supernatant, precipitate or ethyl acetate extract was placed on the other side, and an Oxford cup without liquid was used as a control, and the plate was cultured at a constant temperature of 28°C for 5-7 days, and the antibacterial effect was observed, and the experiment was repeated three times.

[0110] Figure 8 The following are photos of the antibacterial activity test results of the supernatant, precipitate, and ethyl acetate extract of biocontrol bacteria. In the figure, 1 is the supernatant, 2 is the precipitate, and 3 is the ethyl acetate extract. The results are as follows: Figure 8 As shown, the width of the antibacterial zone of the supernatant is 8.36 mm, the width of the antibacterial zone of the precipitate is 5.38 mm, and the width of the antibacterial zone of the ethyl acetate extract is 5.89 mm. The width of the antibacterial zone of the supernatant is significantly greater than that of the precipitate and the ethyl acetate extract, and the width of the antibacterial zone of the ethyl acetate extract is slightly greater than that of the precipitate. This indicates that the content of antibacterial substances in the supernatant of the fermentation broth is higher than that in the precipitate, the content of antibacterial substances in the ethyl acetate extract is not as high as that in the supernatant, and the content of antibacterial substances in the precipitate is not as high as that in the ethyl acetate extract.

[0111] (VI) Antibacterial activity of extracellular proteins and lipopeptides

[0112] The biocontrol bacteria XY-3 was inoculated into LB liquid culture medium at an inoculum rate of 2%, and cultured in a shaking incubator at 28°C and 200 r / min for 3 days. Then, the culture medium was added into a 50 mL large centrifuge tube and centrifuged at 4°C and 10000 r / min for 20 minutes. The supernatant after centrifugation was collected and sterilized by a 0.22 μm filter membrane to obtain the metabolite solution. The extracellular antibacterial substances were crudely extracted by ammonium sulfate graded precipitation method. Solid ammonium sulfate was added to the supernatant filtrate to a saturation of 80%, and the mixture was fully shaken and mixed. The mixture was allowed to stand at 4°C for 24 hours. The mixture was then added into a 50 mL large centrifuge tube and centrifuged at 4°C for 24 hours. Centrifuge in a centrifuge tube for 20 minutes at 4°C and 10,000 r / min. Discard the supernatant, add PBS buffer to dissolve the precipitate, select a dialysis bag with a molecular weight cutoff of 8,000-14,000 D, and place the dissolved solution in a dialysis bag (8,000-14,000 D) after pretreatment. Clamp both ends with clamps, place the dialysis bag in a beaker, add PBS buffer to cover the dialysis bag, and perform 24-hour dialysis desalination. Pass the dialysate through a 0.22 μm filter membrane and adjust the volume to 1 / 20 of the metabolite solution to obtain the extracellular extracted protein.

[0113] Using tomato Fusarium wilt as the indicator bacteria, the Oxford cup confrontation culture method was used to observe the antibacterial activity of extracellular proteins. The wilt pathogen blocks were placed on one side of the PDA plate, and an Oxford cup with 100μL of extracellular protein was placed on the other side. The Oxford cup without liquid was used as the control. The plates were cultured at a constant temperature of 28℃ for 5-7 days, and the antibacterial effect was observed. The experiment was repeated three times.

[0114] The biocontrol bacteria XY-3 was inoculated into LB liquid culture medium, cultured in a shaker at 28°C and 200r / min for 3 days, the bacterial solution was centrifuged for 30 minutes at 4°C and 10000r / min, the supernatant was collected in a test tube, the pH was adjusted to 2.0 with 6mol / L HCl, and the culture was placed in a constant temperature refrigerator at 4°C for 24h, and centrifuged for 30 minutes under the same conditions. The precipitate was dried and extracted twice with ultrapure water. The extract obtained was lipopeptide substances. Similarly, tomato wilt pathogen was used as an indicator bacteria, and the Oxford cup confrontation culture method was used to observe the antibacterial activity of extracellular proteins. The wilt pathogen block was placed on one side of the PDA plate, and an Oxford cup with 100μL lipopeptide substances was placed on the other side. The Oxford cup without liquid was used as a control. The plate was cultured at a constant temperature of 28°C for 5-7d, and the antibacterial situation was observed. The experiment was repeated three times.

[0115] Fig. 9 The photos are the results of the antibacterial activity test of extracellular proteins and lipopeptides of biocontrol bacteria. 1 is the extracellular protein, 2 is the lipopeptide; Fig. 9As shown, the width of the antibacterial zone of extracellular protein is 8.91 mm, and the width of the antibacterial zone of lipopeptides is 5.41 mm. The width of the antibacterial zone of extracellular protein is significantly larger than that of lipopeptides, indicating that the antibacterial ability of the extracellular protein of the XY-3 strain is stronger than that of lipopeptides, and is also significantly higher than the width of the antibacterial zone of the supernatant, precipitate and ethyl acetate extract. Therefore, extracellular protein can be used as the main substance for studying the prevention effect of Fusarium wilt.

[0116] (VII) Volatile substance detection

[0117] Prepare 200 mL of LB medium and PDA medium, pour them into both sides of a two-grid culture dish, inoculate one side of the experimental group with the purified XY-3 strain, and the other side with the tomato wilt pathogen blocks, and the control group with only the tomato wilt pathogen blocks. Culture at 28°C for 3-5 days. After the control group is full of pathogens, observe the growth of pathogens in the experimental group. Repeat the experiment three times.

[0118] Fig.10 The following are photos of the test results of volatile substances of biocontrol bacteria. 1 is the blank control, and 2 is the XY-3 strain. Fig.10 As shown in the figure, after the tomato wilt pathogen grew on one side of the two-grid plate in the control group, it began to spread to the other grid. After inoculation with the biocontrol bacteria XY-3, the tomato wilt pathogen could not spread to the other grid, indicating that the XY-3 strain produced volatile substances during its growth process to inhibit the growth of pathogenic fungi.

[0119] Example 3

[0120] This example provides an investigation on the stability of the antibacterial ability of the extracellular protein of the biocontrol bacterium XY-3.

[0121] (I) Effect of temperature on the antibacterial ability of extracellular proteins

[0122] The extracellular protein of the biocontrol bacteria XY-3 obtained by extraction in Example 2 was added to a 2.0 mL centrifuge tube and placed at 0°C, 20°C, 40°C, 60°C, 80°C, and 100°C for 30 min, respectively. After cooling to room temperature, the tube was cultured in confrontation with the tomato wilt pathogen using an Oxford cup. A wilt pathogen cake was placed in the center of the PDA plate. Three Oxford cups were placed equidistantly 2 cm away from the cake, and 100 μL of extracellular protein was added to each tube. After constant temperature culture at 28°C, the width of the inhibition zone was determined. The test was repeated three times.

[0123] Fig.11 The figure is a comparison of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 at different temperatures; Fig.11As shown in the figure, from 0℃ to 100℃, the antibacterial ability of its extracellular protein shows a trend of first increasing and then decreasing. After being treated in a water bath at 40℃ for 30min, the antibacterial ability is the strongest, with an antibacterial diameter of 7.35mm, and the antibacterial ability is the weakest after being treated in a water bath at 100℃ for 30min, with an antibacterial diameter of 5.09mm. The difference between the two antibacterial diameters is 2.26mm. After being treated in a water bath at 0℃-40℃ for 30min, the antibacterial diameter becomes larger and larger, indicating that its antibacterial ability shows an increasing trend at 0℃-40℃, and the difference in its antibacterial diameter is 1.15mm. It is speculated that the extracellular enzyme has an optimal temperature at this time; after being treated in a water bath at 40℃-100℃ for 30min, its antibacterial diameter decreases significantly, and the antibacterial ability is greatly weakened. After exceeding 40℃, the extracted extracellular substances are increasingly affected by temperature, indicating that the extracellular protein is thermally unstable above 40℃ and relatively stable below 40℃.

[0124] (II) Effect of UV irradiation on the antibacterial ability of extracellular proteins

[0125] The extracellular protein of the biocontrol bacteria XY-3 obtained in Example 2 was added to a 2.0 mL centrifuge tube, and placed under ultraviolet light for 0.5 h, 1 h, 3 h, 6 h, 9 h, and 12 h. The tomato wilt pathogen was cultured in an Oxford cup. The wilt pathogen cake was placed in the center of the PDA plate. Three Oxford cups were placed equidistantly 2 cm away from the cake. 100 μL of extracellular protein was added to each of the cups. After constant temperature culture at 28° C., the width of the inhibition zone was determined. The extracellular protein without ultraviolet light irradiation was used as a control, and the experiment was repeated three times.

[0126] Fig.12 The figure is a comparison of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 under different ultraviolet irradiation conditions; Fig.12 As shown in the figure, the antibacterial activity of extracellular protein weakened with the extension of ultraviolet irradiation time. The CK group had the strongest antibacterial ability with an antibacterial diameter of 7.27 mm. The antibacterial diameter after 12 hours of ultraviolet irradiation was 1.77 mm lower than that of the CK group, indicating that ultraviolet irradiation would destroy the effective antibacterial components of extracellular protein. After 12 hours of ultraviolet irradiation, the extracellular protein still had a strong antibacterial ability, indicating that the extracellular protein had good stability under ultraviolet irradiation.

[0127] (III) Effect of pH on the antibacterial ability of extracellular proteins

[0128] The extracellular protein of the biocontrol bacteria XY-3 extracted in Example 2 was adjusted to pH 3, 5, 7, 9, 11, and 13 with 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solution, placed in a 4°C constant temperature refrigerator for 24 hours, adjusted to neutrality, and cultured in confrontation with the tomato wilt pathogen using an Oxford cup, the wilt pathogen cake was placed in the center of the PDA plate, and three Oxford cups were placed equidistantly 2 cm away from the cake, 100 μL of extracellular protein was added to each, and the width of the inhibition zone was measured after constant temperature culture at 28°C. The extracellular protein without pH adjustment was used as a control, and the experiment was repeated three times.

[0129] Fig.13 The figure is a comparison of the antibacterial activity of the extracellular protein of the biocontrol bacterium XY-3 under different pH conditions; Fig.13 As shown in the figure, the antibacterial diameter of the extracellular protein increased first and then decreased as the pH value increased from 3 to 13. When the pH value was 7 and 5, the antibacterial diameter was not significantly different from that of the CK group, which were 7.16 mm and 6.79 mm, respectively. When the pH value was 3, 9, 11, and 13, the antibacterial diameter was significantly lower than that of the control group, which indicates that the antibacterial effect of the extracellular protein is relatively stable when the pH value is 5-7.

[0130] (IV) Effect of storage time on the antibacterial ability of extracellular proteins

[0131] The extracellular protein of the biological control bacteria XY-3 extracted in Example 2 was stored in a 4°C constant temperature refrigerator. Starting from the 30th day, the Oxford cup was used for confrontation culture with tomato wilt pathogens every 10 days. The wilt pathogen cake was placed in the center of the PDA plate, and three Oxford cups were placed equidistantly 2 cm away from the cake. 100 μL of extracellular protein was added to each of them. After constant temperature culture at 28°C, the width of the inhibition zone was measured, and the duration was 60 days. The newly extracted extracellular protein was used as a control, and the experiment was repeated three times.

[0132] Fig.14 This is a comparison chart of the antibacterial activity of the extracellular protein of the biocontrol bacteria XY-3 at different storage times; Fig.14 As shown in the figure, with the increase of storage time, the antibacterial effect of extracellular protein gradually weakened compared with the CK group, but the antibacterial diameter could still reach 6.22 mm after 80 days of storage, indicating that the antibacterial effect of extracellular protein was relatively stable within 80 days of storage.

[0133] Example 4

[0134] This example provides an investigation on the in vitro control effect of the extracellular protein of the biocontrol bacterium XY-3 on tomato Fusarium wilt.

[0135] (I) The control effect of extracellular protein of biocontrol bacteria XY-3 on detached leaves

[0136] Take tomato leaves of uniform size and vigorous growth, spray the surface with 3% sodium hypochlorite for 5 minutes, then rinse with sterile water 2-3 times and dry, spread two layers of soaked sterile filter paper on the bottom of the sterilized plate, place the leaves in the center of the filter paper, and wrap the petiole with moistened absorbent cotton. The experiment is divided into four groups of treatments, each group treats 10 leaves, sterile water is used as a blank control, and absorbent cotton is dipped in 800-fold dilution of 40% pyrimethanil, PBS buffer, and the extracellular protein of the biocontrol bacteria XY-3 extracted in Example 2 and gently smeared on the leaf surface. After drying, inoculate wilt bacteria blocks, and culture at 28°C for 5-7 days. Calculate the incidence rate, disease index and control effect, use SPSS19.0 software for significance analysis, and repeat the experiment three times.

[0137] Effects of different treatments on the control of tomato detached leaves Fig.15 And as shown in Table 4.

[0138] Table 4

[0139] deal with Disease index Control effect (%) Extracellular proteins 25.93±3.40b 58.19±5.84a Pyrimethanil 20.00±2.22b 67.66±4.50a PBS 61.48±7.81a 0.72±10.35b CK 61.85±2.79a -

[0140] The disease index of each treatment was CK (61.85) > PBS (61.48) > extracellular protein (25.93) > pyrimethanil (20.00). There was no significant difference in the disease index between the CK group and the PBS buffer treatment group, and there was no significant difference in the disease index between the extracellular protein treatment and the pyrimethanil treatment, and both disease indexes were significantly lower than the CK group.

[0141] The control effects of different treatments were as follows: pyrimethanil (67.66) > extracellular protein (58.19) > PBS (0.72), among which pyrimethanil had the best control effect. There was no significant difference between the control effects of the extracellular protein treatment and the pyrimethanil treatment, and the PBS buffer treatment was significantly lower than the pyrimethanil and extracellular protein treatments.

[0142] This indicates that the extracellular protein of the biocontrol bacterium XY-3 has a good control effect on tomato Fusarium wilt and can significantly reduce the disease index of tomato Fusarium wilt.

[0143] (II) Trypan blue staining of detached leaves by extracellular proteins of biocontrol bacteria XY-3

[0144] Select green tomato leaves of similar size and shape, soak them in a petri dish filled with 75% alcohol, let stand for 1 min, rinse with sterile water, then soak them in 3% sodium hypochlorite, let stand for 3 min, rinse with sterile water, lay two layers of soaked sterile filter paper on the bottom of the sterilized plate, place the leaves in the center of the sterilized petri dish, and wrap the petiole with soaked absorbent cotton. The extracellular protein of the biocontrol bacteria XY-3 extracted in Example 2 was gently applied to the leaf surface, the CK group was sprayed with sterile water, and then 1×10 6cfu / mL spore suspension of Fusarium oxysporum was stained 3-5 days after treatment.

[0145] Trypan blue staining solution is often used as a staining tool to identify the life and death of cells in cells and tissues. During staining, living cells with complete cell structure and function will not be stained, while cells with damaged structure and no normal cell function are often stained blue by the staining solution.

[0146] Soak the leaves in a 50mL large centrifuge tube of trypan blue dye solution, place the large centrifuge tube in an 80℃ water bath for 3 minutes, dye at room temperature for 24 hours after the water bath, rinse with distilled water, and soak the leaves in 2.5g / mL chloral hydrate until completely decolorized. After complete decolorization, soak the leaves in 75% alcohol for storage, observe the leaf staining, and use a microscope to photograph the changes in leaf vein cell staining. Healthy cells appear green, damaged or dead cells appear blue, and the experiment is repeated three times.

[0147] Fig.16 These are the epigenetic photos of detached tomato leaves stained with trypan blue under different treatments; Fig.17 These are photos of vein cells of tomato leaves stained with trypan blue under different treatments; Fig.16 and Fig.17 As shown, most of the leaves in the CK group were stained blue, while the stained area of ​​the leaves treated with the XY-3 extracellular protein solution was significantly smaller than that of the CK group, with only a small part being stained. Under a microscope, the vein cells and vein tissues in the CK group were stained blue, while the vein cells and vein tissues treated with the XY-3 extracellular protein solution were not stained, indicating that the extracellular protein of the biocontrol bacterium XY-3 can inhibit the invasion of pathogens on vein cells and tissues and maintain the normal function of the leaves.

[0148] (III) Effect of extracellular protein of biocontrol bacterium XY-3 on detached tomato fruit

[0149] Take the horn tomatoes of uniform size, similar maturity, no disease and no wound on the surface, apply 3% sodium hypochlorite on the surface for disinfection for 5 minutes, use a sterile puncher to punch 1cm×1cm circular holes at equal distances on the surface of the fruit. The experiment is divided into four groups of treatments, each treatment has 10 fruits, and they are sprayed with 800-fold dilution of 40% pyrimethanil, PBS buffer, and the extracellular protein of the biocontrol bacteria XY-3 extracted in Example 2, respectively. Sterile water is used as a blank control, and the fruits are dried and left to stand for 12 hours, and then sprayed with 1×10 6 The bacterial suspension of 1.5 cfu / mL of Fusarium wilt was placed in a sterile culture bottle and cultured at 28℃ for 7 days. Samples were taken on the 1st, 3rd, 5th and 7th days to observe the disease. The experiment was repeated three times.

[0150] Effects of different treatments on the control of tomato detached fruits Fig.18 And as shown in Table 5.

[0151] Table 5

[0152] deal with Disease index Control effect (%) Extracellular proteins 30.00±1.11b 59.24±2.59a Pyrimethanil 33.33±2.33b 54.81±1.33a PBS 71.85±3.40a 2.35±7.48b CK 73.70±2.80a -

[0153] As shown in Table 5, the disease index CK>PBS>pyrimethanil>extracellular protein. There was no significant difference in the disease index of PBS compared with the control group. The fruit protection effect of PBS was only 2.35, and it had no inhibitory effect on fruit disease. The control effect of extracellular protein was slightly higher than that of the biological agent pyrimethanil, which was 59.24%.

[0154] Fig.18 These are photos of the protective effects of different treatments on tomato detached fruits on the 1st, 3rd, 5th and 7th days; Fig.18 As shown in the figure, pyrimethanil and extracellular protein treatments had a significant inhibitory effect on tomato wilt. Three days before the treatment, there was no significant change in the fruits of each treatment, no lesions appeared, and the fruits felt hard; from the 4th to the 7th day, small lesions began to appear in the CK group and the PBS group, and gradually expanded, and corruption appeared, and the fruits felt significantly soft. Most of the pyrimethanil and extracellular protein treatment groups had no obvious lesions, and a small part had lesions and became soft.

[0155] The results showed that the extracellular protein of the biocontrol bacterium XY-3 had a significant inhibitory effect on the occurrence of tomato fruit wilt, and the protective effect of pyrimethanil on the fruit was lower than that on the seedling stage.

[0156] Example 5

[0157] This example provides an investigation on the efficacy of the extracellular protein of the biocontrol bacterium XY-3 against tomato wilt in greenhouse cultivation.

[0158] (I) Preparation of suspension of pathogenic bacteria of Fusarium wilt

[0159] Inoculate Fusarium oxysporum with a culture dish, culture at 28℃ for 7 days, observe the mycelial growth, select a culture dish with full mycelium and no foreign bacteria, scrape the mycelium with an inoculation loop, add an appropriate amount of sterile water to rinse it, filter the scraped liquid with sterile gauze, which is the Fusarium oxysporum suspension, and adjust the OD value of the suspension to 600 Adjust the value to 0.5 (concentration is about 1x10 6 cfu / mL), the color is light purple, and it is placed in a 4℃ refrigerator for later use.

[0160] (II) Potted Plant Experiment

[0161] The pot experiment was conducted at the Horticulture Station of Northeast Agricultural University and the artificial climate room of our research group. Tomato seeds (AC) were first disinfected and germinated, then sown in 12×6 hole trays and watered thoroughly. After germination, they were cultured for 15 days and then moved to nutrient pots. When they grew to 4-5 true leaves in the nutrient pots, they were treated. The four treatments are as follows:

[0162] (1) Blank control group: spray only the suspension of Fusarium wilt (concentration of about 1x10 6cfu / mL).

[0163] (2) Treatment group 1: Spray PBS buffer first, and then spray Fusarium wilt fungus suspension 12 hours later.

[0164] (3) Treatment group 2: The extracellular protein of the biocontrol bacterium XY-3 extracted in Example 2 was first sprayed, and then the suspension of Fusarium wilt was sprayed 12 hours later.

[0165] (4) Positive control group: first spray 40% pyrimethanil 800 times diluted solution, and then spray Fusarium wilt fungus suspension 12 hours later.

[0166] After treatment, the plants were placed in a 25℃ artificial climate chamber for moisturizing culture. After 15 days, the disease conditions of each treatment were observed. The leaves of the plants were investigated according to the disease grading standard (Table 6), and the disease index and control effect were calculated according to the formula. SPSS19.0 software was used for significance analysis.

[0167] (III) Classification standards for wilt diseases

[0168] When the wilt pathogen infects tomato leaves, the leaves will lose their green color and wilt, forming dead spots. The leaves are graded according to the area of ​​leaf spots, the area of ​​chlorosis, or the number of fruit diseases (Table 6), and the disease index and control effect are calculated according to the following formula.

[0169] Table 6

[0170] Disease level Leaf spot area / % Leaf chlorosis area / % Number of diseased fruits 0 0 0 No fruit disease 1 0-5 0-15 S(onset)≤5% 3 6-10 15-25 6% < S (incidence) ≤ 15% 5 11-25 25-50 16% < S (incidence) ≤ 25% 7 26-50 50-75 26% < S (incidence) ≤ 50% 9 50-100 75-100 S(onset)>50%

[0171] Calculation formula:

[0172]

[0173] (IV) Greenhouse protection results

[0174] Table 7

[0175] deal with Disease index Control effect (%) Extracellular proteins 31.85±1.28b 51.65±1.67b Pyrimethanil 25.92±3.57c 60.73±4.28a PBS 63.70±1.28a 3.19±5.85c CK 65.93±3.40a -

[0176] The greenhouse protection results are shown in Table 7 and Fig.19 As shown, the disease index of each treatment was CK (65.93) > PBS (63.70) > pyrimethanil (25.92) > extracellular protein (31.85). There was no significant difference in the disease index between the PBS group and the CK group. PBS had no significant inhibitory effect on tomato plant wilt. The disease index of the pyrimethanil treatment group was the smallest, which was 25.92, and the control effect was also the largest, which was 60.73. The disease index of the extracellular protein treatment was slightly higher than that of the pyrimethanil treatment, and its control effect was close to that of the pesticide preparation pyrimethanil.

[0177] Example 6

[0178] This example provides an investigation on the effect of the extracellular proteins of the biocontrol bacterium XY-3 on the induction of related enzyme activities in tomato leaves.

[0179] After the tomato seedlings were grown in nutrient pots to 4-5 true leaves, different treatments were performed. Four treatments were set:

[0180] (1) Sterile water treatment group;

[0181] (2) Tomato wilt pathogen (concentration is about 1x10 6 cfu / mL) spray treatment;

[0182] (3) Spray treatment of the extracellular protein of the biocontrol bacterium XY-3 extracted in Example 2;

[0183] (4) Spray the extracellular protein in advance and then spray the tomato wilt pathogen suspension.

[0184] After different treatments, starting from the second day, leaves with consistent growth conditions were taken from the growth position at the same time for seven consecutive days, placed in 1.5 mL centrifuge tubes, preserved in liquid nitrogen, and stored in a -80°C refrigerator for later use.

[0185] When determining the activity of related enzymes in tomato leaves, the kit method was used for detection. The kits used were provided by Suzhou Keming Biotechnology Co., Ltd., and orign 2019b software was used for drawing and analysis.

[0186] (I) Determination of polyphenol oxidase (PPO) activity

[0187] Weigh 0.1g of leaves accurately, cut them into pieces and put them in a mortar with ice bath, grind them into homogenate with ultra-low temperature grinder, centrifuge for 10min, centrifuge at 4℃, 10000r / min, put the supernatant into centrifuge tube, store on ice for later use, operate strictly according to the instructions of the kit provided by the company, set up control tube and test tube, preheat the microplate reader for more than 30min, add the required reagents to the 96-well microplate in turn and mix, measure the absorbance value A of the control tube and test tube at 525nm, and record the data, ΔA=A 测定 -A 对照 , PPO enzyme activity was calculated according to the calculation formula, and the experiment was repeated three times.

[0188] Calculation formula:

[0189] Fig. 20 The comparison chart of PPO activity in tomato leaves under different treatments; Fig. 20As shown in the figure, treatments 2, 3, and 4 can significantly increase the PPO enzyme activity. The PPO enzyme activity of treatments 2 and 3 reached the maximum value on the 4th day, which was 3664.33U / gFW and 8826.33U / g FW, respectively. The maximum value of treatment 4 was 11295.67U / g FW on the 5th day. Compared with CK, treatment 4 had the strongest ability to induce plant resistance, followed by treatment 3.

[0190] (II) Determination of superoxide dismutase (SOD) activity

[0191] Accurately weigh 0.1g of leaves, cut them into pieces and put them in a mortar with an ice bath, grind them into a homogenate with an ultra-low temperature grinder, centrifuge for 10 minutes at 4°C and 10,000 r / min, put the supernatant into a centrifuge tube, and store it on ice for later use. Strictly follow the instructions of the kit provided by the company, preheat the ELISA instrument for more than 30 minutes, add the required reagents to the 96-well ELISA plate in turn and mix well, let it stand at room temperature for 30 minutes, measure the absorbance A of each well at 560nm, record the data, calculate the SOD enzyme activity according to the calculation formula, and repeat the experiment three times.

[0192] Calculation formula:

[0193]

[0194] Fig.21 The comparison chart of SOD activity in tomato leaves under different treatments; Fig.21 As shown in the figure, treatments 2, 3, and 4 can significantly increase the PPO enzyme activity. The PPO enzyme activity of treatments 2 and 3 reached the maximum value on the 4th day, which was 3664.33U / gFW and 8826.33U / g FW, respectively. The maximum value of treatment 4 was 11295.67U / g FW on the 5th day. Compared with CK, treatment 4 had the strongest ability to induce plant resistance, followed by treatment 3.

[0195] (III) Determination of peroxidase (POD) activity

[0196] Accurately weigh 0.1g of leaves, cut them into pieces and put them in a mortar with an ice bath, grind them into a homogenate with an ultra-low temperature grinder, centrifuge for 10min at 4℃ and 10000r / min, put the supernatant into a centrifuge tube, and store it on ice for later use. Strictly follow the instructions of the kit provided by the company, preheat the ELISA instrument for more than 30min, add the required reagents to the 96-well ELISA plate in turn and mix well, quickly measure the absorbance A1 after 1min and the absorbance A2 after 2min at 470nm, record the data, calculate the POD enzyme activity according to the calculation formula, and repeat the experiment three times.

[0197] Calculation formula:

[0198]

[0199] Fig. 22 The comparison chart of POD activity in tomato leaves under different treatments; Fig. 22 As shown in the figure, treatments 2, 3, and 4 can significantly affect the POD activity of plant leaves, and the overall trend is to increase first and then decrease; the changes in POD enzyme activity in treatments 3 and 4 are greater than that in treatment 2, indicating that both treatments 3 and 4 can induce plants to produce certain disease resistance. The POD enzyme activity of treatment 2 reached a maximum of 7805U / g FW on the 4th day, treatment 3 reached a maximum of 11170.7U / gFW on the 5th day, and treatment 4 reached a maximum of 13438.7U / g FW on the 3rd day. Therefore, treatment 4 can accelerate the reaction time of POD enzyme and increase the change range of enzyme activity.

Claims

1. A biocontrol bacterium, characterized in that: The biocontrol bacteria are classified and named Bacillus subtilis, and were deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 18, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30050.

2. An extracellular protein extracted from the fermentation broth of the biocontrol bacteria according to claim 1.

3. A method for extracting extracellular protein as claimed in claim 2, characterized in that: The steps include: Step 1, inoculating the biocontrol bacteria according to claim 1 into a liquid culture medium for fermentation culture; Step 2: Centrifuge the fermentation liquid obtained in step 1 to collect the supernatant, sterilize the obtained centrifuged supernatant filter membrane, add solid ammonium sulfate to the obtained supernatant filtrate to a saturation of 80%, fully shake and mix, let stand at 4°C for 24 hours, then centrifuge to collect the precipitate, add PBS buffer to dissolve the precipitate, put the obtained dissolved solution into a dialysis bag with a molecular weight cutoff of 8000 to 14000D for dialysis desalination, collect the dialysate and filter the membrane for sterilization to obtain the extracellular extracted protein.

4. The method for extracting extracellular protein according to claim 3, characterized in that: The liquid culture medium in step 1 is MSM basal salt culture medium, NB nutrient broth culture medium, Landy culture medium, LB bacterial basal culture medium, YSP yeast peptone culture medium, NA nutrient agar culture medium, PDA potato dextrose agar culture medium or CM complete culture medium; the liquid volume of the liquid culture medium is 60-100mL / 250mL; the fermentation culture conditions are 25-30°C fermentation temperature, 150-200r / min shaking culture for 24-72h.

5. The method for extracting extracellular protein according to claim 4, characterized in that: The liquid culture medium in step 1 is MSM basal salt culture medium, and the liquid volume of the liquid culture medium is 80mL / 250mL; the fermentation culture condition is 29°C fermentation temperature and 180r / min shaking culture for 24h.

6. The method for extracting extracellular protein according to claim 5, characterized in that: The centrifugation in step 2 is carried out at 4°C and 10,000 r / min for 20 min, and the dialysis desalination time is 24 h.

7. A use of the extracellular protein as claimed in claim 2 in agricultural biological control, characterized in that: The biological control comprises using the extracellular protein to control tomato wilt caused by Fusarium oxysporum.

8. A use of the extracellular protein as claimed in claim 2 in agricultural biological control, characterized in that: The biological control comprises using the extracellular protein to improve the disease resistance of crops.

9. The use of the extracellular protein in agricultural biological control according to claim 8, characterized in that: The crop is tomato.

Images