Application of flagellin FliC 103211

Abstract

The present invention relates to an application of a flagellin FliC 103211, which is used for inhibiting at least one of Fusarium oxysporum, Fusarium solani and Ralstonia solanacearum. The flagellin FliC 103211 can inhibit at least one of Fusarium oxysporum, Fusarium solani and Ralstonia solanacearum, thereby preventing and controlling banana wilt, tomato root rot and bacterial wilt. The flagellin FliC 103211 has an ideal prevention and control effect, is non-polluting to the environment, and is non-toxic to humans and animals.

Description

Technical Field

[0001] The present invention relates to the technical field of plant control, and particularly relates to an application of flagellin FliC 103211, in particular to the application of flagellin FliC 103211 in inhibiting Fusarium oxysporum, Fusarium solani and Ralstonia solanacearum, thereby preventing and controlling tomato root rot, banana wilt and bacterial wilt. Background Art

[0002] With the intensive and modern development of agricultural planting, the use of pesticides and chemical fertilizers has continued to increase, resulting in the deterioration of the physical and chemical properties of the soil, the accumulation of pathogens, and the widespread spread of soil-borne diseases, which has led to frequent crop diseases and affected the yield and quality of agricultural products.

[0003] Common soil-borne pathogens include Fusarium oxysporum and Fusarium solani. Fusarium oxysporum can infect commercial crops such as bananas, tomatoes, cotton, potatoes, ginger, and strawberries, causing diseases such as wilt and root rot. When infected, plants begin to rot at the roots, with the stems and leaves gradually wilting upwards, severely damaging fruit and vegetable production. Fusarium oxysporum is the causative fungus of banana wilt, which has severely impacted banana cultivation and production. Current prevention and control measures include the use of pesticides and the development of resistant banana varieties, but these efforts have been minimal, and the problem of banana wilt has not been completely resolved. Fusarium oxysporum remains the primary pathogen to be conquered. Fusarium solani is also a common plant pathogen, commonly found in plants, soil, freshwater, and brackish water. Fusarium solani attacks similar plant parts to Fusarium oxysporum, damaging plant vascular bundles and causing the plants to wilt and die. Fusarium solani can infect a wide range of plants, including tomatoes, potatoes, ginger, apples, and ginseng, causing outbreaks of root rot and wilt, ultimately leading to severe economic losses.

[0004] Ralstonia solanacearum is a plant bacterial pathogen that infects cash crops such as tomatoes, peanuts, and bitter melon. This soil-borne vascular disease is highly pathogenic and has a high mortality rate in infected hosts. Severe cases can cause widespread crop death or even complete crop failure, resulting in reduced yield and quality, seriously impacting the healthy development of the crop industry. Summary of the Invention

[0005] Based on this, the object of the present invention is to provide an application of flagellin FliC 103211, wherein the flagellin FliC 103211 is used to inhibit at least one of Fusarium oxysporum, Fusarium solani and Ralstonia solanacearum, and then through the antibacterial effect of flagellin FliC 103211 on Fusarium oxysporum, Fusarium solani and Ralstonia solanacearum, to prevent and control tomato root rot, banana wilt and bacterial wilt, and the prevention and control effect is ideal, and the flagellin FliC 103211 is non-polluting to the environment and non-toxic to humans and animals.

[0006] Specifically, the flagellin FliC 103211 of the present invention is obtained by culturing, isolating, and purifying Bacillus B1032, a mangrove-derived strain. Bacillus B1032 was deposited with the China Center for Type Culture Collection on September 14, 2020, with the strain deposit number CCTCC NO: M 2020498 and the classification number Bacillus decolorationis B1032. The deposit address is Wuhan University, Wuhan, China.

[0007] Furthermore, it is used to prevent and control tomato root rot by inhibiting at least one of the Fusarium oxysporum and the Fusarium solani.

[0008] Furthermore, it is used to prevent and control banana wilt by inhibiting the Fusarium oxysporum.

[0009] Furthermore, it is used to prevent and control bacterial wilt by inhibiting the Ralstonia solanacearum.

[0010] Furthermore, the amino acid sequence of the flagellin FliC 103211 is shown in the sequence listing SEQ ID NO.1.

[0011] Compared with the prior art, the present invention has the following advantages and effects:

[0012] (1) The present invention first discovered that the flagellin FliC 103211 has a significant inhibitory effect on Fusarium oxysporum, the pathogen that causes tomato root rot and banana wilt, and Fusarium solani, the pathogen that causes tomato root rot. Among them, the inhibition rates of 0.5 mg / ml flagellin FliC 103211 on Fusarium oxysporum and Fusarium solani were 83.7% and 84.3%, respectively. This provides a new biocontrol protein for the prevention and control of tomato root rot and banana wilt.

[0013] (2) The present invention also discovered for the first time that flagellin FliC 103211 also has a significant inhibitory effect on Ralstonia solanacearum, which causes plant wilt. Among them, the diameter of the inhibition zone of 0.5 mg / ml flagellin FliC 103211 against Ralstonia solanacearum is 23 mm, which also provides a new biocontrol protein for preventing and controlling plant wilt.

[0014] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The figure is an SDS-PAGE gel electrophoresis diagram of crude protein precipitated by 35% ammonium sulfate (separation gel concentration is 12%); wherein, M: marker, 1: flagellin FliC103211 fermentation supernatant; 2: 35% ammonium sulfate precipitation supernatant; 3: 35% ammonium sulfate precipitation crude protein;

[0016] Figure 2 The figure shows the SDS-PAGE gel electrophoresis of each component after the crude protein was purified by Sepharose Fast Flow ion exchange chromatography (separating gel concentration was 15%); wherein, M: Marker, 1: 35% ammonium sulfate precipitation of crude protein, 2: 0.02M Tris-HCl (pH = 7.6) buffer eluent, 3: 0.5M NaCl-Tris-HCl (pH = 7.6) buffer eluent, 4: 0.75M NaCl-Tris-HCl (pH = 7.6) buffer eluent, 5: 1M NaCl-Tris-HCl (pH = 7.6) buffer eluent;

[0017] Figure 3 This is the total ion current of the secondary mass spectrometry of flagellin FliC 103211;

[0018] Figure 4 The antibacterial activity experiments of flagellin FliC 103211 against Fusarium oxysporum and Fusarium solani, as well as the control experiments, were conducted. Figure 4 A: Fusarium oxysporum + sterile water (blank control); Figure 4 B: Fusarium oxysporum + 0.5 mg / mL purified protein; Figure 4 C: Fusarium oxysporum + 1 mg / mL carbendazim (positive control); Figure 4 D: Fusarium solani + sterile water (blank control); Figure 4 E: Fusarium solani + 0.5 mg / mL purified protein; Figure 4 F: Fusarium solani + 1 mg / mL carbendazim (positive control); the above purified protein is the flagellin FliC103211 eluted with 0.5 M NaCl-Tris-HCl (pH = 7.6) buffer;

[0019] Figure 5 This is an antibacterial activity experiment of flagellin FliC 103211 against Ralstonia solanacearum, as well as a control experiment. CK: negative control (H2O); P: positive control (10 mg / mL Amp, where Amp is ampicillin). The diameter of the inhibition zone is 24.2 mm.

[0020] S1: 0.5 mg / mL FliC103211, the inhibition zone diameter was 23 mm; S2: 1 mg / mL FliC103211, the inhibition zone diameter was 31.5 mm;

[0021] Figure 6 The results show the application effect of flagellin FliC 103211 in tomato cultivation; wherein, Figure 6 A: The effect of the blank control group, Figure 6 Panel B: Effect of the experimental group (flagellin FliC 103211-treated group). DETAILED DESCRIPTION

[0022] Currently, the commonly used methods for controlling soilborne crop diseases include physical control, chemical control, and biological control. Physical control typically involves adjusting ventilation and soil moisture during field operations based on crop characteristics to minimize the risk of disease. Chemical control typically involves pesticide use, which is often associated with residual pesticide ingredients, environmental pollution, biodiversity degradation, food safety issues, and even the development of antibiotic resistance in pathogens. Biological control utilizes the inhibitory effects of microorganisms and their metabolites on pathogens, thereby reducing pathogenic microorganisms and achieving effective control. Biological control offers the following advantages: 1. It reduces soil root pathogens, improves soil conditions, optimizes the microenvironment of cultivation facilities, and ensures crop quality; 2. It reduces pollution sources, fostering environmental friendliness; and 3. It reduces the overuse of pesticides and other chemical agents, reducing pollution at the source and ensuring healthy crops. To ensure healthy crop cultivation, the prevention and control of plant diseases, especially soilborne diseases, is crucial. Biological control using antagonistic effects on pathogens is a new approach to controlling plant diseases.

[0023] Fusarium oxysporum is the pathogenic fungus that causes banana wilt, and it can also infect tomatoes, causing outbreaks of root rot and wilt. Fusarium solani can infect tomatoes, damaging the plant's vascular bundles, causing the plants to wilt and die. Furthermore, Ralstonia solanacearum, a plant bacterial pathogen, can also infect tomatoes. The soil-borne vascular disease caused by bacterial wilt is highly pathogenic and has a high mortality rate in infected hosts. Therefore, it is highly desirable to find a more effective antibacterial protein for controlling Fusarium oxysporum, Fusarium solani, and Ralstonia solanacearum. The present inventors have isolated and purified the flagellin FliC 103211 from Bacillus B1032 for the first time. They have also discovered that the flagellin FliC 103211 has antibacterial effects against Fusarium oxysporum, Fusarium solani, and Ralstonia solanacearum, building on this finding to complete the present invention.

[0024] The present invention will be further described in detail below with reference to specific examples. The following examples are intended to illustrate the present invention only and are not intended to limit the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0025] Example 1

[0026] In this example, flagellin FliC 103211 was prepared by Bacillus B1032.

[0027] The preparation method of flagellin FliC 103211 comprises the following steps:

[0028] S1: crude protein preparation by Bacillus sp. B1032;

[0029] Specifically, the method includes the following steps:

[0030] S101: Inoculate the prepared Bacillus B1032 seed solution at a 5% inoculation volume and a concentration of 5.0×10 8 The culture medium was inoculated with 500 mL of LB liquid medium at 30°C and 220 rpm for 48 h to obtain a fermentation broth. Bacillus sp. B1032 was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2020498.

[0031] S102: The fermentation broth was centrifuged at 4°C and 8000 rpm for 5 minutes, and the supernatant was collected. Ammonium sulfate was added to the supernatant to a concentration of 35%, and the mixture was allowed to stand at 4°C for 4 hours to precipitate crude protein from the supernatant.

[0032] S103: The crude protein was centrifuged at 4°C and 10,000 rpm for 40 min, and the precipitate was collected. The precipitate was dissolved in Tris-HCl buffer (0.02 M, pH 7.6), placed in a dialysis bag, and dialyzed against Tris-HCl buffer (0.02 M, pH 7.6) of the same concentration at 4°C (dialysis bag pore size 3500 d). The dialysate was replaced every 4 h for a total of 5 times.

[0033] S104: freeze-drying the crude protein in the dialysis bag.

[0034] Furthermore, the method further comprises the following steps:

[0035] S105: Taking a portion of the crude protein for SDS-PAGE gel electrophoresis experiment, specifically comprising the following steps:

[0036] Preparation of electrophoresis gel: After preparing the separation gel, mix it thoroughly and inject it into two glass plates. Add deionized water as the sealant (use deionized water to seal the gel when the concentration is less than 10%, and use isopropyl alcohol to seal the gel when the concentration is greater than 10%). Let it stand at room temperature for 30 minutes. After the gel is coagulated, pour out the water (or isopropyl alcohol) and aspirate it. After preparing the concentration gel, inject it into the middle of the plate in the same way. Slowly insert the comb and let it stand at room temperature for 30 minutes. After the gel is coagulated, remove it and place the gel plate into the electrophoresis tank. Slowly add the electrode buffer.

[0037] Specifically, the separation gel and stacking gel used are configured as follows:

[0038] 12% separating gel (15 ml): 4.9 ml water; 6 ml 30% acrylamide solution; 3.8 ml separating gel buffer (1.5 M Tris-HCl pH = 8.8); 0.15 ml 10% SDS; 0.15 ml 10% ammonium persulfate; 0.006 ml TEMED.

[0039] 5% stacking gel (5 ml): 3.4 ml water; 0.83 ml 30% acrylamide solution; 0.63 ml stacking gel buffer (1.0 M Tris-HCl pH = 6.8); 0.05 ml 10% SDS; 0.05 ml 10% ammonium persulfate; 0.005 ml TEMED.

[0040] Sample treatment: Mix the sample with 5×SDS-PAGE Loading Buffer in a ratio of 4:1, boil in hot water for 3-5 minutes, load 20μL-50μL of sample, and load 5μL of protein marker.

[0041] Electrophoresis conditions: When the sample is in the stacking gel, use a constant voltage of 100V for electrophoresis; when the sample is in the separating gel, use a constant voltage of 150V for electrophoresis. Stop electrophoresis when bromophenol blue approaches the edge of the separating gel.

[0042] Staining: After electrophoresis, remove the gel sheet and carefully peel off the gel, place it in a staining dish, place it on a decolorizing shaker, add staining solution and stain for 1 hour.

[0043] Destaining: Place the stained gel in destaining solution and destain on a destaining shaker, changing the destaining solution several times until the protein bands are clear.

[0044] The solution used in the SDS-PAGE gel electrophoresis process was prepared as follows:

[0045] 30% acrylamide solution: Prepare with (purchased) deionized water, containing 29.2% (w / v) acrylamide and 0.8% (w / v) N,N'-methylene acrylamide. Filter through filter paper, place in a brown bottle, and store in the dark at 4°C until use.

[0046] Separation gel buffer (2M Tris-HCl pH=8.8): Dissolve 24.2 g of Tris base in 50 mL of water, adjust the pH to 8.8 with HCl (about 4 mL), make up to 100 mL, and keep at room temperature until use.

[0047] Stacking gel buffer (0.3 M Tris-HCl pH = 6.8): Dissolve 3.6 g of Tris base in 50 mL of water, adjust the pH to 6.8 with HCl (about 1.8 mL), make up to 100 mL, and keep at room temperature until use.

[0048] 10% ammonium persulfate (AP): Prepare to a concentration of 10% (w / v) with deionized water and store at 4°C until use.

[0049] 10× electrode buffer: 30g Tris, 144g glycine, 10g SDS, dissolved in water. Do not stir too vigorously to avoid bubbles. Make up to 1000mL. The pH of the solution should be 8.3 or above. Dilute (1×) before use and store at room temperature.

[0050] 50% (v / v) glycerol: Prepare 50% (w / v) glycerol with deionized water and keep at room temperature until ready to use.

[0051] 10% (w / v) SDS: Dissolve 10 g of SDS in 100 mL of deionized water and store at room temperature until use (prepare at 68°C for solubilization and use).

[0052] 1% (w / v) bromophenol blue: Dissolve 0.1 g of bromophenol blue in 10 mL of deionized water, filter, and store at room temperature until use.

[0053] 5×SDS-PAGE Loading Buffer: 8.5 mL of 0.3 M Tris-HCl (pH 6.8) stacking gel buffer, 8.0 mL of 50% glycerol, 8.0 mL of 10% SDS, 10 mL of 0.1% (w / v) bromophenol blue, 2.0 mL of β-mercaptoethanol, and distilled water to a total volume of 40 mL.

[0054] Staining solution: 45% methanol, 10% glacial acetic acid, 45% deionized water, 0.25% (w / v) Coomassie Brilliant Blue (Commassise blue R250), filtered (0.45 μm filter) and placed at room temperature for use.

[0055] Decolorizing solution: 5% methanol, 7.5% glacial acetic acid, 87.5% deionized water, set aside at room temperature.

[0056] TEMED (tetramethylethylenediamine): stock solution.

[0057] in, Figure 1 The electrophoresis results of SDS-PAGE gel electrophoresis of crude proteins are shown.

[0058] S2: Purification of flagellin FliC 103211 by DEAE Sepharose Fast Flow ion exchange chromatography;

[0059] Specifically, the method includes the following steps:

[0060] S201: Ion exchange chromatography packing pretreatment: DEAE is stored in 20% ethanol. A certain amount needs to be removed and placed in a small centrifuge tube. The ethanol is removed by filtration. The packing is washed with 1L of ultrapure water and then soaked in a small amount of ultrapure water.

[0061] S202: Equilibration and sample loading: Equilibrate the treated column with 0.02M Tris-HCl (pH=7.6) to compact the column packing, approximately 2-4 column volumes; load the treated active crude protein sample, the sample volume should not exceed the maximum adsorption capacity of the packing;

[0062] S203: Elution: First, use 0.02M Tris-HCl (pH=7.6) buffer to elute the unadsorbed components and impurities 3-5 times; then use 0.02M Tris-HCl (pH=7.6) buffer and 0.5M, 0.75M, and 1M NaCl-Tris-HCl (pH=7.6) buffer eluent linear gradient elution to elute all components adsorbed on the chromatography column packing;

[0063] S204: Collection: Pool the collected tubes corresponding to each elution peak and store at 4°C for future use;

[0064] S205: Dialysis: The collected component samples corresponding to the elution peak were dissolved in 0.02 M Tris-HCl (pH = 7.6) buffer, placed in a dialysis bag, and dialyzed at 4 ° C with 0.02 M Tris-HCl (pH = 7.6) buffer of the same concentration (dialysis bag pore size 3500d). The dialysate was replaced every 4 h for a total of 5 times to obtain the purified protein.

[0065] S206: Freeze drying: freeze drying the purified protein in the dialysis bag.

[0066] Furthermore, after the above step S204, the following steps are further performed:

[0067] Regeneration and storage of filler: After the ion exchange chromatography purification is completed, if the ion exchange column will not be used in the short term, it can be eluted online with a buffer containing 1M NaCl; if it is not used for a long time, the chromatography filler needs to be taken out and soaked in a buffer containing 0.5M NaOH and 1M NaCl, and slowly stirred with a magnetic stirrer for 30 minutes. Finally, the NaOH and NaCl are removed by filtration. The filler is washed with 1L ultrapure water, soaked in 20% ethanol, and stored at 4°C.

[0068] Example 2

[0069] In this example, SDS-PAGE gel electrophoresis experiment was performed on the purified flagellin FliC 103211.

[0070] The SDS-PAGE gel electrophoresis experiment in this embodiment differs from that in Example 1 only in that a different separating gel is used. This embodiment uses a 15% separating gel (15 ml). The specific composition of the 15% separating gel (15 ml) is: 3.4 ml of water; 7.5 ml of a 30% acrylamide solution; 3.8 ml of separating gel buffer (1.5 M Tris-HCl, pH = 8.8); 0.15 ml of 10% SDS; 0.15 ml of 10% ammonium persulfate; and 0.006 ml of TEMED. The solution configuration, specific steps of the gel electrophoresis experiment, and technical parameters in each step used in the SDS-PAGE gel electrophoresis experiment in this embodiment are the same as those in Example 1 and are therefore not described in detail here.

[0071] Figure 2 Shown are the SDS-PAGE gel electrophoresis results of each fraction after the crude protein was purified by Sepharose Fast Flow ion exchange chromatography.

[0072] Example 3

[0073] In this example, the purified protein was identified.

[0074] The purified protein bands from the SDS-PAGE gel electrophoresis in Example 2 were cut with a sterile scalpel, placed in a 1.5 ml centrifuge tube, submerged in sterile water, and sent to Wuhan Jinkairui Bioengineering Co., Ltd. for LC-MS / MS mass spectrometry identification. Figure 3The total ion current of the secondary mass spectrometry of flagellin FliC 103211 is shown, and the amino acid sequence shown in the sequence listing SEQ ID NO.1 is also obtained; the amino acid sequence is then aligned through NCBI, and the alignment result is shown as flagellin (Flagellin Hag Protein), Sequence ID (sequence number): WP_003228021.1.

[0075] Example 4

[0076] This example tests the antibacterial activity of purified flagellin FliC 103211 against Fusarium oxysporum and Fusarium solani, comprising the following steps:

[0077] The purified flagellin FliC 103211 was dissolved in 0.02 M Tris-HCl (pH = 7.6) buffer to prepare a flagellin FliC 103211 solution with a concentration of 0.5 mg / ml, and sterilized by filtration through a 0.22 μm filter membrane;

[0078] Using the plate confrontation method, take the Fusarium oxysporum and Fusarium solani cakes in the center of the PDA culture medium, punch 3 holes (aperture 6 mm) every 120° at a distance of 15 mm from the cake, add 50 μL of flagellin FliC 103211 solution to each well, and culture in a 30℃ incubator for 5 days.

[0079] Among them, the formula of PDA solid culture medium is: 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 20% potato extract (200g potatoes), 1.5% agar, a total of 1L.

[0080] Figure 4 A-4C shows the antibacterial activity of flagellin FliC 103211 against Fusarium oxysporum and the results of a control experiment. Figure 4 As shown in A-4C, flagellin FliC 103211 exhibits an antibacterial effect against Fusarium oxysporum. Since Fusarium oxysporum is the primary pathogen causing tomato root rot and banana wilt, the results of the above antibacterial activity experiments against Fusarium oxysporum suggest that flagellin FliC 103211 can be used to inhibit Fusarium oxysporum and thereby control tomato root rot and banana wilt.

[0081] and Figure 4 D-4F shows the antibacterial activity of flagellin FliC 103211 against Fusarium solani and the results of the control experiment. Figure 4As shown in D-4F, flagellin FliC 103211 also has an antibacterial effect against Fusarium solani. Since Fusarium solani is also the main pathogen of tomato root rot, the results of the above antibacterial activity experiments against Fusarium solani indicate that flagellin FliC 103211 can be used to inhibit Fusarium solani and prevent tomato root rot.

[0082] Example 5

[0083] This example tests the antibacterial activity of purified flagellin FliC 103211 against Ralstonia solanacearum, comprising the following steps:

[0084] The purified flagellin FliC 103211 was dissolved in 0.02 M Tris-HCl (pH = 7.6) buffer to prepare flagellin FliC 103211 solutions with concentrations of 0.5 mg / ml and 1 mg / ml, and sterilized by filtration through a 0.22 μm filter membrane;

[0085] Culture Ralstonia solanacearum to OD 600 =1.5, the Ralstonia solanacearum solution was spread on LB glucose medium, four filter paper pieces (6 mm in diameter) were placed equidistantly in the middle of the medium to form four spotting areas, 10 μL of sterile water, 10 mg / mL Amp (ampicillin) solution, 0.5 mg / mL FliC103211 solution, and 1 mg / mL FliC103211 solution were added respectively; and the mixture was cultured in a 30°C incubator for 1 day.

[0086] The formula of LB glucose liquid medium is as follows: 10 g of tryptone, 5 g of yeast powder, 7 g of sodium chloride, and 5 g of glucose, and distilled water is added to make up 1 L; solid LB glucose medium is further added with 1.5% agar.

[0087] Figure 5 The results of the antibacterial activity experiment of flagellin FliC 103211 against Ralstonia solanacearum and the control experiment are shown. Figure 5 It can be seen that flagellin FliC 103211 has an antibacterial effect on Ralstonia solanacearum. Figure 5 As shown by the sizes of the inhibition zones, the antibacterial effect of flagellin FliC103211 at a concentration of 1 mg / mL was significantly superior to that of flagellin FliC103211 at a concentration of 0.5 mg / mL, and also superior to that of traditional Amp. Since Ralstonia solanacearum is the primary pathogen causing plant bacterial wilt, the results of the above antibacterial activity experiments against Ralstonia solanacearum indicate that flagellin FliC 103211 can inhibit Ralstonia solanacearum and thus control plant bacterial wilt.

[0088] Example 6

[0089] This example studies the application effect of flagellin FliC 103211 in tomato cultivation.

[0090] The experiment was conducted in a tomato greenhouse at the Experimental Teaching Base of South China Agricultural University in Zengcheng District, Guangzhou, Guangdong Province. The greenhouse experienced severe outbreaks of tomato root rot caused by Fusarium oxysporum and Fusarium solani, as well as bacterial wilt caused by Ralstonia solanacearum, resulting in a tomato plant mortality rate exceeding 30% last season.

[0091] Experimental group: 6 rows, 50 plants per row. The flagellin FliC 103211 solution obtained in Example 2 was diluted to a final concentration of 0.5 mg / ml. At planting, the roots of the tomato seedlings were dipped in the diluted flagellin FliC 103211 solution for half a minute before being planted in soil. After planting, the roots were drip-irrigated with a 0.5 mg / ml flagellin FliC 103211 solution, with 50 ml of the protein solution applied to each row of tomato seedlings. Eighteen days after planting, the roots were drip-irrigated with a 0.2 mg / ml flagellin FliC 103211 solution, with 50 ml of the protein solution applied to each row of tomato seedlings, for a total of three applications. The number of surviving plants was counted at peak fruiting stage, and the survival rate was calculated.

[0092] Blank control group: 6 rows, 50 trees in each row. The experimental process was carried out in parallel with the experimental group, except that water was used instead of the flagellin FliC103211 solution.

[0093] Among them, the mortality rate = the number of dead plants / the total number of plants × 100%, and the survival rate = the number of surviving plants / the total number of plants × 100%.

[0094] Figure 6 shows the results of tomato cultivation, by Figure 6 As can be seen, the final survival rate of the 300 tomatoes in the experimental group was 96%, and the survival rate of the blank control group was 70%. The use of the flagellin FliC 103211 of the present invention greatly improved the survival rate of tomatoes and reduced the mortality rate of tomatoes.

[0095] Compared with the prior art, the present invention has the following advantages and effects:

[0096] (1) The present invention first discovered that the flagellin FliC 103211 has a significant inhibitory effect on Fusarium oxysporum, the pathogen that causes tomato root rot and banana wilt, and Fusarium solani, the pathogen that causes tomato root rot. Among them, the inhibition rates of 0.5 mg / ml flagellin FliC 103211 on Fusarium oxysporum and Fusarium solani were 83.7% and 84.3%, respectively. This provides a new biocontrol protein for the prevention and control of tomato root rot and banana wilt.

[0097] (2) The present invention also discovered for the first time that flagellin FliC 103211 also has a significant inhibitory effect on Ralstonia solanacearum, which causes plant wilt. Among them, the diameter of the inhibition zone of 0.5 mg / ml flagellin FliC 103211 against Ralstonia solanacearum is 23 mm, which also provides a new biocontrol protein for preventing and controlling plant wilt.

[0098] The above-described embodiments merely represent several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, and the present invention is intended to encompass such modifications and variations.

Claims

1. An application of flagellin FliC 103211, characterized in that: Used to inhibit at least one of Fusarium oxysporum, Fusarium solani and Ralstonia solanacearum, wherein the amino acid sequence of the flagellin FliC 103211 is shown in the sequence listing SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Tomato root rot is controlled by inhibiting at least one of the Fusarium oxysporum and the Fusarium solani.

3. The use according to claim 1, characterized in that: Banana wilt disease is prevented and controlled by inhibiting the Fusarium oxysporum fungus.

4. The use according to claim 1, characterized in that: The bacterial wilt disease is prevented and controlled by inhibiting the Ralstonia solanacearum.

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