Bacillus amyloliquefaciens and application thereof in prevention and treatment of willow rot disease

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens and application of the bacillus amyloliquefaciens to prevention and treatment of willow rot disease, the bacillus amyloliquefaciens is bacillus amyloliquefaciens 3JK1, the preservation number is GDMCC No 65533, and the bacillus amyloliquefaciens is preserved in Guangdong Microbial Culture Collection Center on November 25, 2024. The method comprises the following steps: collecting willow rhizosphere soil from Lazaka in the autonomous region of Tibet, and separating and purifying rhizosphere soil microorganisms to obtain the biocontrol bacterium bacillus amyloliquefaciens 3JK1 with a better antagonistic effect. The 3JK1 or a degerming solution or a sterilizing solution thereof is applied to willows suffering from rot diseases, and the 3JK1, the degerming solution and the sterilizing solution thereof can effectively prevent and treat the rot diseases of the willows and control the spreading of the diseases. The bacillus amyloliquefaciens mainly aims at willow rot diseases in the Tibet region, and a certain prevention and treatment method and theoretical basis are provided for forest tree rot diseases in the Tibet region.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Bacillus amyloliquefaciens and application thereof in the prevention and treatment of willow rot disease. Background Art

[0002] Due to its high altitude, Tibet has a scarce oxygen content. Forests play an important role in the global carbon cycle. Artificial afforestation is one of the simplest and most effective ways to increase oxygen content. Willows are suitable for planting and easy to survive, so they are widely selected in artificial afforestation and become the main tree species in Tibet's artificial forests. However, due to the single tree species and lack of diversity in artificial forests, the ecological environment is unbalanced and prone to pests and diseases. Rot, as one of the main diseases of artificial forests, seriously harms the growth of trees. Due to the special climate, geographical environment and high altitude in Tibet, the existing research and prevention technologies at home and abroad are not fully applicable in Tibet, and there are very few investigations, studies and prevention methods for willow rot in Tibet; and because the tree species in artificial forests are single and have low resistance to diseases, once they occur, they will spread over a large area, and in serious cases, large areas of willows will die.

[0003] The current prevention and control of willow rot in Tibet mainly relies on the use of chemical agents, but this method easily leads to drug resistance in pathogens, reducing the control effect; secondly, it will pollute the environment, and pesticide residues may pollute soil and water sources, causing poisoning to non-target organisms such as beneficial insects and birds; in addition, chemical control is complicated and requires precise control of the time and concentration of application. Over-reliance on chemical means may damage the tree or cause pesticide damage. Based on this, it is urgent to provide a new strategy for the prevention and control of willow rot. Summary of the invention

[0004] In order to effectively prevent and treat willow rot and control the spread of the disease, the present invention provides a Bacillus amyloliquefaciens for preventing and treating willow rot.

[0005] The technical solution adopted by the present invention is:

[0006] The invention provides a Bacillus amyloliquefaciens for preventing and treating willow rot disease. The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens 3JK1, with a preservation number of GDMCC No 65533, which was preserved in Guangdong Provincial Microbiological Culture Collection Center on November 25, 2024.

[0007] The second aspect of the present invention provides a Bacillus amyloliquefaciens bacterial liquid, wherein the Bacillus amyloliquefaciens bacterial liquid is obtained by culturing the Bacillus amyloliquefaciens in an LB liquid culture medium, and the culture conditions are: pH 3-10, 20°C-46°C for 3d-4d.

[0008] Preferably, the culture conditions are as follows: pH is 5, and culture is carried out at 24 °C for 3 days.

[0009] In the third aspect of the present invention, a filtered and sterilized solution is provided. The preparation method of the filtered and sterilized solution is as follows: the Bacillus amyloliquefaciens bacterial solution is centrifuged at 10,000 r / min for 5 min, the supernatant is collected, and then the supernatant is filtered. The obtained filtrate is the filtered and sterilized solution, and the diameter of the filter screen used during filtration is 0.22 μm.

[0010] In the fourth aspect of the present invention, a high-temperature sterilized solution is provided. The preparation method of the high-temperature sterilized solution is as follows: the Bacillus amyloliquefaciens bacterial solution is sterilized at 119 °C to 123 °C for 25 min to 35 min, and then the high-temperature sterilized solution is obtained.

[0011] Preferably, the preparation conditions of the high-temperature sterilized solution are sterilization at 121 °C for 30 min.

[0012] In the fifth aspect of the present invention, an application of the Bacillus amyloliquefaciens, the Bacillus amyloliquefaciens bacterial solution, the filtered and sterilized solution or the high-temperature sterilized solution is provided. The application refers to preventing and / or inhibiting willow canker.

[0013] Preferably, the pathogen of the willow canker is Cytospora chrysosperma of the genus Cytospora.

[0014] In the sixth aspect of the present invention, a microbial pesticide is provided. The microbial pesticide is composed of a Bacillus amyloliquefaciens active ingredient and auxiliary materials;

[0015] The Bacillus amyloliquefaciens active ingredient includes at least one of the Bacillus amyloliquefaciens, the Bacillus amyloliquefaciens bacterial solution, the filtered and sterilized solution and the high-temperature sterilized solution.

[0016] Preferably, the auxiliary materials include at least one of a filler, a binder, a disintegrant, a lubricant and an antacid.

[0017] Preferably, the filler includes any one of microcrystalline cellulose, starch, lactose and mannitol.

[0018] Preferably, the binder includes any one of starch paste and hydroxypropyl methylcellulose.

[0019] Preferably, the disintegrant includes any one of starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

[0020] Preferably, the lubricant includes any one of magnesium stearate, talcum powder and polyethylene glycol.

[0021] Preferably, the antacid includes any one of calcium carbonate and aluminum hydroxide.

[0022] Bacillus amyloliquefaciens involved in the present invention:

[0023] Bacillus amyloliquefaciens 3JK1 was deposited in Guangdong Provincial Microbiological Culture Collection on November 25, 2024. The proposed taxonomic name of Bacillus amyloliquefaciens 3JK1 is Bacillusamyloliquefaciens, the deposit number is GDMCC No 65533, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0024] The pathogenic bacteria SDF-3 involved in the present invention is Cystosporum flavum, which is disclosed in the reference:

[0025] Zhang Xingyao, Chen Haiyan, Liang Jun, et al. Cultivation characteristics and vegetative compatibility of Cytospora chrysosperma[J]. Journal of Northwest Agriculture and Forestry University (Natural Science Edition), 2007, (03): 99-105. DOI: 10.13207 / j.cnki.jnwafu.2007.03.021.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a Bacillus amyloliquefaciens for preventing and treating willow rot, wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens 3JK1, with a deposit number of GDMCC No 65533, and is deposited in the Guangdong Provincial Microbial Culture Collection Center on November 25, 2024. The present invention collects willow rhizosphere soil from Lhasa, Tibet Autonomous Region, and separates and purifies rhizosphere soil microorganisms to obtain a biocontrol bacterium Bacillus amyloliquefaciens 3JK1 with good antagonistic effect. The present invention applies 3JK1 or its sterilizing solution or sterilizing solution to willows suffering from rot, and finds that 3JK1, its sterilizing solution and sterilizing solution can effectively prevent and treat willow rot diseases and control the spread of diseases. The Bacillus amyloliquefaciens of the present invention is mainly aimed at willow rot diseases in Tibet, and provides a certain prevention and control method and theoretical basis for forest rot diseases in Tibet. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the colony morphology of strain 3JK1.

[0029] Figure 2 This is the effect of strain 3JK1 on the growth of willow rot disease strains.

[0030] Figure 3 This is the Gram staining result of strain 3JK1.

[0031] Figure 4 Growth state of willow canker on PDA.

[0032] Figure 5 Research results on the antagonistic effects of 6 strains. A - F: 1JK52, 2JK11, 1JK53, 2JK9, 2JK8, and 3JK1 in sequence; G: Control inoculated with only the pathogen.

[0033] Figure 6 Phylogenetic tree results.

[0034] Figure 7 Inhibitory effect of filtered sterile solution on canker pathogen. A - E are in sequence: control, stock solution, 2 - fold dilution, 5 - fold dilution, and 10 - fold dilution.

[0035] Figure 8 Inhibitory effect of high - temperature sterilized solution on canker pathogen. A - E are in sequence: control, stock solution, 2 - fold dilution, 5 - fold dilution, and 10 - fold dilution.

[0036] Figure 9 Inhibitory effect of antagonistic strain 3JK1 on excised diseased willow branches. A: 3 - replicate parallel blank group; B: 3 - replicate parallel control group; C: 3 - replicate parallel treatment group. Detailed implementation manners

[0037] The present invention is further illustrated by the following specific embodiments, but does not limit the scope of the present invention. Modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and these modifications or substitutions all fall within the protection scope of the present invention.

[0038] The inventive concept of the present invention is as follows:

[0039] The existing prevention and control of willow canker in Tibet mainly rely on the use of chemical agents for prevention and control. However, this method is prone to cause the pathogen to develop drug resistance, reducing the prevention and control effect; secondly, it will also cause environmental pollution, and pesticide residues may pollute the soil and water sources, causing harm to non - target organisms such as beneficial insects and birds; in addition, chemical control is complex in operation, requiring precise control of the application time and concentration, and over - reliance on chemical means may damage the tree body or cause phytotoxicity.

[0040] Based on this, the present invention provides a Bacillus amyloliquefaciens for preventing and controlling willow canker. The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens 3JK1, with the preservation number GDMCC No65533, and was preserved in the Guangdong Provincial Microbial Culture Collection Center on November 25, 2024.

[0041] Most of the current research on Bacillus amyloliquefaciens focuses on diseases of crops and fruit and vegetable plants with higher economic value. There is little research on the control effect of Bacillus amyloliquefaciens on forest tree canker, and even less research on the diseases of artificial forests in Tibet. The present invention mainly studies the control effect of artificial forest canker in the Tibetan Plateau to effectively control willow canker disease and prevent the spread of the disease.

[0042] To enable those skilled in the art to better understand and implement the technical solution of the present invention, the following further illustrates the present invention with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0043] Example 1

[0044] A Bacillus amyloliquefaciens for controlling willow canker is as follows:

[0045] 1. Isolation of antagonistic bacteria.

[0046] The rhizosphere soil of poplar and willow collected from Lhasa City, Tibet Autonomous Region was placed in a triangular flask containing 100 mL of sterile water and shaken for 10 min, then the original solution of the bacterial suspension was collected, and the original solution of the bacterial suspension was diluted 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 , and the corresponding bacterial suspensions were collected. 50 μL of the original solution and the bacterial suspensions with different dilution multiples were respectively pipetted onto the LB solid medium for uniform coating, and incubated at a constant temperature of 25 °C. After single colonies grew, they were picked and streaked. After 48 hours, colonies grew, and single colonies with different characteristics such as morphology, color, and transparency were picked into a new LB medium for further cultivation and purification, and purified 5 times.

[0047] 2. Screening of antagonistic bacteria.

[0048] The pathogenic bacterium SDF-3 used in the present invention is a strain preserved by the 1st research group of the Plant Health Laboratory of the College of Plant Science, Tibet Agricultural and Animal Husbandry University. It was collected and isolated from the willow branches infected with canker disease in Sangda Village, Lhasa City, Tibet Autonomous Region in June 2023. After the strain was cultured well in the PDA medium, it was stored at 4 °C for standby. The growth of the strain in the medium is as Figure 4 shown.

[0049] The plate confrontation method was adopted: a pathogen agar disc with a diameter of 5 mm was inoculated at the center of the PDA medium, and bacteria were streaked and inoculated at a position 2.5 cm away from the center pathogen position. The plate inoculated only with the pathogen was used as a control and incubated at a constant temperature of 25°C. When the mycelium of the control pathogen covered the plate, the diameter of the pathogen inoculated with bacteria was observed and measured, and the inhibition rate was calculated. Each treatment was set with 3 replicates, and the formula for calculating the inhibition rate was as follows:

[0050] where the units of the control diameter, treatment diameter, and agar disc diameter are all mm.

[0051] The strains with the best antagonistic effect against canker were selected for the following research.

[0052] 3. Gram staining of the strains.

[0053] Reagents: Hecker's crystal violet solution, Lugol's iodine solution, 95% ethanol by volume fraction, and 0.5% safranin solution by mass percentage.

[0054] Staining operation steps:

[0055] 1) Smear fixation: Drop a drop of distilled water on a glass slide, pick a single colony grown for 20 h and place it in the distilled water, spread it evenly, and dry it on alcohol.

[0056] 2) Drop 2 drops of crystal violet stain on the glass slide, let it stand for about 1 min, and then rinse it slowly with water.

[0057] 3) Wash away the excess water with iodine solution, let it stand for 1 min, and blot it dry after washing with water.

[0058] 4) Decolorize with 95% ethanol for 30 s, and immediately rinse it slowly with water after it becomes colorless.

[0059] 5) Counterstain with 0.5% safranin aqueous solution for 2 min, then wash with water, dry it with alcohol, and finally examine it under a microscope.

[0060] 6) Observe the individually dispersed bacterial cells with an oil immersion lens. Bacterial cells that are blue-violet are Gram-positive G + bacteria, and those that are red are Gram-negative G - bacteria.

[0061] 4. Study on the physiological and biochemical characteristics of the strains.

[0062] (1) Catalase: For each liter of nutrient agar medium NA: peptone 10.0 g, beef powder 3.0 g, NaCl 5.0 g, agar 16 g, pH 7.3, made up to 1000 mL with distilled water. Reagent: 3% hydrogen peroxide. Inoculate the strains on the medium and culture at 25°C for 24 h. Drop 3% hydrogen peroxide on the colonies and let it stand. If bubbles are produced, it is positive.

[0063] (2) Citrate: Per liter of medium: 5.0 g of sodium citrate, 0.2 g of magnesium sulfate MgSO4·7H2O, 1.0 g of ammonium dihydrogen phosphate NH4H2PO4, 5.0 g of sodium chloride NaCl, 1.0 g of yeast extract, 16 g of agar, 3 mL of 0.5% phenol red solution, and make up to 1000 mL with distilled water. Spot inoculate the strain on the medium and incubate at an appropriate temperature for 4 days. If the medium changes from the original pink color to rose red, it is positive.

[0064] (3) Acetylmethylcarbinol: Per liter of medium: 5.0 g of peptone, 5.0 g of dipotassium hydrogen phosphate K2HPO4, 5.0 g of glucose, 1000 mL of distilled water, pH 7.0, and make up to 1000 mL with distilled water. Reagents: Solution A: 5% α-naphthol, prepare and use immediately; Solution B: 40% NaOH solution. Inoculate the strain into the above culture solution and incubate at 25°C for 1 day. Each time, take 1 mL of the culture solution, add an equal amount of 40% NaOH and 5% α-naphthol, shake vigorously, and observe the color change after 30 minutes. Red is positive and yellow is negative.

[0065] (4) Ammonia production: Medium: 0.5% peptone solution, pH 7.2. Aliquot into test tubes and sterilize at 121°C for 20 minutes. Reagent: Nessler's reagent: Solution A: 10.0 g of potassium iodide, 20.0 g of mercuric iodide, 100 mL of distilled water; Solution B: 20 g of potassium hydroxide. After Solutions A and B are cooled, mix them, and store the supernatant in a brown bottle for later use. Inoculate the strain into the test tube and incubate at an appropriate temperature for 1 day, using the test tube without inoculating the strain as a control. Each time, take a small amount of the culture solution and add 1 drop of Nessler's reagent. The appearance of a yellow or brown precipitate is positive.

[0066] (5) Indole production: Medium: 1% tryptone aqueous solution, pH 7., aliquot 5 mL into each tube and sterilize at 112°C for 30 minutes. Reagent: 2.0 g of p-dimethylaminobenzaldehyde, 190 mL of 95% ethanol, 40 mL of concentrated hydrochloric acid. After inoculating the strain into the culture solution, incubate at an appropriate temperature. Take the culture solution after 1 day of incubation, slowly add 5 mm high indole reagent along the tube wall. A red color at the liquid layer interface indicates the production of indole, which is positive.

[0067] (6) Starch hydrolysis: Medium: Common nutrient agar medium plus 0.2% soluble starch; Reagent: Lugol's iodine solution. Use an inoculation loop to take a small amount of the strain and spot inoculate it on the plate medium, and incubate at an appropriate temperature for 5 days. Open the petri dish lid, drop iodine solution on the colony. If the area around the colony is blue, it indicates that there is no hydrolysis; if there is a colorless transparent circle around the colony, or it is purple-red, it proves that the starch has been hydrolyzed. The size of the transparent circle indicates the strength of the enzyme activity.

[0068] (7) Methyl Red: Reagent: 0.1 g of methyl red, 300 mL of 95% ethanol, and 200 mL of distilled water. Inoculate the strain into the above culture medium and incubate at an appropriate temperature for 1 day. Each time, take 1 mL of the culture medium, add 1 - 2 drops of methyl red reagent, shake vigorously, and observe the color change after 30 minutes. Red is positive and yellow is negative.

[0069] (8) Utilization of carbon sources: Per liter of culture medium: 2.0 g of ammonium sulfate (NH4)2SO4, 0.5 g of dipotassium hydrogen phosphate K2HPO4, 0.1 g of calcium chloride CaCl2·2H2O, 0.5 g of sodium dihydrogen phosphate NaH2PO4·H2O, 0.2 g of magnesium sulfate MgSO4·7H2O, make up to 1000 mL with distilled water, pH 6.5; use sucrose, maltose, glucose, and lactose as carbon sources, and no carbon as the control. Take 1 ml of the bacterial solution into the culture medium and incubate at an appropriate temperature for 24 h. Use the basal medium without added carbon source as the control. When observing, if the growth of the test strain in the basal medium containing carbon compounds is significantly more than that in the basal medium, it is positive, otherwise it is negative.

[0070] (9) Utilization of nitrogen sources: Per liter of culture medium: 2.0 g of glucose, 0.5 g of dipotassium hydrogen phosphate K2HPO4, 0.1 g of calcium chloride CaCl2·2H2O, 0.5 g of sodium dihydrogen phosphate NaH2PO4·H2O, 0.2 g of magnesium sulfate MgSO4·7H2O, make up to 1000 mL with distilled water, pH 6.5; use peptone, tryptone, malt extract, and urea as nitrogen sources, and no nitrogen as the control. Take 1 ml of the bacterial solution into the culture medium and incubate at an appropriate temperature for 24 h. Use the basal medium without added nitrogen source as the control. When observing, if the growth of the test strain in the basal medium containing nitrogen compounds is significantly more than that in the basal medium, it is positive, otherwise it is negative.

[0071] 5. Sequencing and analysis of bacterial 16S rDNA.

[0072] Send the selected bacteria to Shanghai Jiao Tong University for testing. Use the 16S rDNA universal primers 27F and 1492 to amplify the target fragment, and compare the test results on the NCBI website. Select the strains with higher credibility and use the MEGA11 software to construct a phylogenetic tree. The sequences of 27F and 1492R are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0073] SEQ ID NO.1: 5′-AGAGTTTGATCMTGGCTCAG-3′.

[0074] SEQ ID NO.2: 5′-GGTTACCTTGTTACGACTT-3′.

[0075] 6. Mycelial growth inhibition test.

[0076] The antagonistic bacteria were inoculated into LB liquid medium and cultured at 28 °C and 180 r / min for 3 days for standby to obtain Bacillus amyloliquefaciens bacterial liquid. A part of the Bacillus amyloliquefaciens bacterial liquid was centrifuged at 10000 r / min for 5 min, and the supernatant was filtered through a 0.22-μm bacterial filter to obtain a filtered and sterilized solution; another part of the Bacillus amyloliquefaciens bacterial liquid was sterilized at 121 °C for 30 min to obtain a high-temperature sterilized solution.

[0077] The high-temperature sterilized solution and the filtered and sterilized solution were respectively diluted with sterile water to 2-fold, 5-fold, and 10-fold solutions of the original solution. Then, different concentrations of the high-temperature sterilized culture solution and the filtered and sterilized culture solution were added to the PDA sterilized and cooled to 60 °C at a ratio of 1:1 and mixed evenly, and poured into plates. The plates prepared with ordinary PDA were used as controls. The test pathogens were inoculated at the center of each plate, with 3 replicates for each concentration, and incubated in the dark at a constant temperature of 25 °C. When the colonies in the control group were fully grown, the diameter of each colony was measured by the cross method, and the calculation method of the mycelial inhibition rate was as follows:

[0078]

[0079] 7. In vitro shoot control efficacy test.

[0080] The collected healthy willow branches were cut into 15 cm, first soaked in 1% sodium hypochlorite for 2 min, then soaked in 75% alcohol for 5 min, and finally rinsed 3 times with sterile water. After drying, one end was sealed with paraffin, and the other end was wrapped with sterile absorbent cotton moistened with sterile water. The bark was scalded with a 5-mm puncher, and the antagonistic bacteria suspension was applied to the branches with sterile absorbent cotton. After culturing at 25 °C for 24 h, a pathogen cake was inoculated at the scalded site, which was the treatment group; in another group, a pathogen cake was directly inoculated at the scalded site, which was the control group; and in another group, a PDA medium cake was inoculated at the scalded site, which was the blank group. A sterile absorbent cotton moistened with sterile water was placed 1 cm above the inoculated cake, and the absorbent cotton and the inoculation site were wrapped together with plastic wrap, with three replicates for each treatment. The branches were put into a tray and sealed with plastic wrap for moisture retention. After culturing at 25 °C for 7 d, the disease occurrence of the branches was observed, and the calculation method of the inhibition effect was as follows:

[0081]

[0082] 8. Study on the optimal culture conditions.

[0083] 8.1 Effect of different pH values on the growth of antagonistic bacteria.

[0084] Place the LB liquid medium in a 250 ml Erlenmeyer flask and make up the volume to 100 ml. Adjust the pH of the made-up LB liquid medium to ten values ranging from 3 to 12 with sterilized 1 mol / L NaOH and 1 mol / L HCl respectively, and then inoculate 1000 μL of the Bacillus amyloliquefaciens 3JK1 seed liquid. Each treatment has 3 replicates. Place it on a shaker at 180 r / min and incubate at a constant temperature of 28 °C. Measure the OD600 after 24 h.

[0085] 8.2 Effects of different temperatures on the growth of antagonistic bacteria

[0086] Inoculate 1000 μL of the Bacillus amyloliquefaciens 3JK1 seed liquid into 100 mL of the LB liquid medium with a pH of 5 and a fixed volume. Place the Erlenmeyer flasks containing the bacterial suspension in shakers at different temperatures ranging from 20 °C to 50 °C and culture at 180 r / min. Measure the OD600 after 24 h. Each temperature has 3 replicates.

[0087] The above results are as follows:

[0088] (1) Using the plate confrontation method, a total of 6 strains with antagonistic effects were screened out. It was found that the 3JK1 strain had the best antagonistic effect, as shown in Figure 1 , Figure 2 , Figure 5 and Table 1. Among them, the inhibition rate of 3JK1 on hyphae was as high as 74.61%. The subsequent experiments selected 3JK1 with the best antagonistic effect.

[0089] Table 1 6 strains with antagonistic effects

[0090] Bacterial number Mycelial inhibition rate 1JK52 53.01％±0.92％b 2JK11 52.57％±0.09％b 1JK53 51.10％±1.54％b 2JK9 47.43％±0.76％c 2JK8 53.50％±0.55％b 3JK1 74.61％±4.02％a

[0091] Note: Different letters in the column of hypha inhibition rate indicate significant differences, p < 0.05.

[0092] (2) The results of Gram staining and physiological and biochemical tests are shown in the following table:

[0093] On the LB solid medium, 3JK1 formed an opaque milky white nearly circular colony. At the initial stage, the surface was smooth, and it was viscous when picked up. At the later stage, the surface was rough and shrank, and the middle of the colony was sunken and the surrounding was raised, as shown in Figure 1 .

[0094] Gram staining showed purple, as shown in Figure 3 , and it was a Gram-positive bacterium. The results of physiological and biochemical characteristics were: catalase, citrate, V-P, indole, starch hydrolysis, etc. were positive, as shown in Table 2. The above manifestations and results indicate that 3JK1 all conform to the morphological characteristics and physiological and biochemical characteristics of the genus Bacillus.

[0095] Table 2 Gram staining and physiological and biochemical characteristics

[0096] Item Result Gram staining + Catalase + Citrate + Acetylmethylcarbinol + Ammonia production + Indole production + Starch hydrolysis + Methyl red － Sucrose + Maltose + Glucose + Lactose + Peptone + Malt extract + Tryptone + Urea －

[0097] Note: In Table 2, "+" indicates positive and "-" indicates negative.

[0098] (3) 16S rDNA sequence and phylogenetic tree results of the antagonistic bacterium 3JK1.

[0099] The genomic sequence of strain 3JK1 was obtained by third-generation sequencing technology, and the 16S rDNA sequence was analyzed. The submitted test results were compared on the NCBI website. The sequence information of the 16S rDNA of 3JK1 after sequencing is shown in SEQ ID NO.3. Strains with relatively high credibility were selected to construct a phylogenetic tree using MEGA11 software. The phylogenetic tree is as Figure 6 shown. The results show that the similarity between the 16S rDNA and Bacillus amyloliquefaciens is the highest, reaching 99.87%. Therefore, through the results of physiological and biochemical characteristics, Gram staining results and homology analysis, combined with phylogenetic tree alignment analysis, strain 3JK1 was finally identified as Bacillus amyloliquefaciens and named Bacillus amyloliquefaciens 3JK1.

[0100] SEQ ID NO.3:

[0101]

[0102] Example 2

[0103] Application of Bacillus amyloliquefaciens for preventing and controlling willow canker, specifically as follows:

[0104] (1) Inhibitory effects of the filtered and sterilized solution of 3JK1 and the high-temperature sterilized solution on willow canker pathogen.

[0105] The inhibitory effects of the filtered and sterilized solution and the high-temperature sterilized solution on willow canker pathogen are shown in Table 3, Table 4, Figure 7 and Figure 8 .

[0106] Table 3 Inhibitory effect of the filtered and sterilized solution on willow canker pathogen

[0107] Filtered sterile solution Mycelial inhibition rate Stock solution 100.00％ 2-fold solution 100.00％ 5-fold solution 63.19％±13.76％ 10-fold solution 45.70％±6.57％

[0108] Table 4 Inhibitory effect of the high-temperature sterilized solution on willow canker pathogen

[0109] High-temperature sterilized solution Mycelial inhibition rate Stock solution 79.01％±3.64％a 2-fold solution 41.00％±4.50％b 5-fold solution 29.50％±2.20％c 10-fold solution 19.43％±3.83d

[0110] (2) Antibacterial effect of antagonistic strain 3JK1 on diseased willow detached branches, and the results are shown in Table 5 and Figure 9 .

[0111] Table 5 Antibacterial effect of antagonistic strain 3JK1 on diseased willow detached branches

[0112] Strain number Inhibitory effect 3JK1 79.7％±11.39％

[0113] From the above data, it can be seen that the antibacterial effect of antagonistic strain 3JK1 on diseased willow detached branches is 79.7%.

[0114] (3) The optimal culture conditions of antagonistic strain 3JK1 are shown in Table 6 and Table 7.

[0115] Table 6 Growth status at different pH values

[0116] pH OD600, 24h 3 0.0032±0.0015d 4 0.0177±0.0213d 5 1.0448±0.0588a 6 0.9431±0.0725b 7 0.9088±0.0591b 8 0.6847±0.0822c 9 0.0055±0.0038d 10 0.0047±0.0037d 11 0 12 0

[0117] Table 7 Growth status at different temperatures

[0118]

[0119]

[0120] It can be seen from Table 6 and Table 7 that when the pH is 5 and the temperature is 24 °C, it is most suitable for the growth of strain 3JK1.

[0121] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0122] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens for preventing and controlling willow canker, characterized in that, The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens 3JK1, with the preservation number of GDMCC No 65533, and it was preserved in the Guangdong Microbial Culture Collection Center on November 25, 2024.

2. A Bacillus amyloliquefaciens bacterial liquid, characterized in that, The Bacillus amyloliquefaciens bacterial liquid is obtained by culturing the Bacillus amyloliquefaciens described in claim 1 in LB liquid medium, and the culture conditions are: pH 3 - 10, culturing at 20°C - 46°C for 3d - 4d.

3. The Bacillus amyloliquefaciens bacterial liquid according to claim 2, characterized in that, The culture conditions are: pH is 5, culturing at 24°C for 3d.

4. A filtering and sterilizing liquid, characterized in that, The preparation method of the filtered and sterilized liquid is as follows: Centrifuge the Bacillus amyloliquefaciens bacterial liquid described in claim 2 at 10000r / min for 5min, collect the supernatant, and then filter the supernatant. The obtained filtrate is the filtered and sterilized liquid, and the diameter of the filter mesh used during filtration is 0.22μm.

5. A high-temperature sterilizing liquid, characterized in that, The preparation method of the high-temperature sterilized liquid is as follows: Sterilize the Bacillus amyloliquefaciens bacterial liquid described in claim 2 at 119°C - 123°C for 25min - 35min to obtain the high-temperature sterilized liquid.

6. The high-temperature sterilizing liquid according to claim 5, wherein, The preparation condition of the high-temperature sterilized liquid is sterilization at 121°C for 30min.

7. Use of the Bacillus amyloliquefaciens according to claim 1, the Bacillus amyloliquefaciens bacterial liquid according to claim 2, the filtered and sterilized liquid according to claim 4, or the high-temperature sterilized liquid according to claim 5, characterized in that, The application refers to preventing and / or inhibiting willow canker.

8. The application according to claim 7, wherein the pathogen of the willow canker is Cytospora chrysosperma.

9. A microbial pesticide, characterized in that, The microbial pesticide is composed of an active ingredient of Bacillus amyloliquefaciens and auxiliary materials; The active ingredient of Bacillus amyloliquefaciens includes at least one of the Bacillus amyloliquefaciens described in claim 1, the Bacillus amyloliquefaciens bacterial liquid described in claim 2, the filtered and sterilized liquid described in claim 4, and the high-temperature sterilized liquid described in claim 5.

10. The microbial pesticide according to claim 9, wherein The auxiliary materials include at least one of a filler, a binder, a disintegrant, a lubricant, and an antacid.

Images