A Bacillus velezensis strain 3-25, its culture, and its application in controlling early blight

Abstract

The present invention provides a Bacillus velezensis strain 3-25, its culture, and its application in preventing and treating early blight, belonging to the technical solution of functional microorganisms. A Bacillus velezensis strain 3-25, with the preservation number of CGMCC No. 31910. The strain 3-25 and its culture have good antibacterial activity against the pathogenic bacteria of early blight, and have a high biocontrol effect on early blight. At the same time, the strain 3-25 also has the ability to secrete growth-promoting active components, with a significant growth-promoting effect. The Bacillus velezensis strain 3-25 of the present invention is used as a biological pesticide and / or biofertilizer, and has broad application prospects in preventing and treating early blight of crops.

Description

Technical Field

[0001] The present invention belongs to the technical solution of functional microorganisms, and particularly relates to a Bacillus velezensis strain 3-25, its culture, and its application in preventing and controlling early blight. Background Art

[0002] The occurrence of early blight leads to a decline in crop quality and yield. In agricultural production activities, in order to effectively control this disease, various strategies are usually adopted, including cultivating disease-resistant varieties, implementing agricultural management measures, applying biological control technologies, and chemical control methods. Among them, chemical agents are prone to cause environmental and crop pollution, and are also prone to develop dependence on chemical agents, and cannot achieve the purpose of efficient prevention and control stably in the long term.

[0003] Currently, the microorganisms used for biological control of potato early blight mainly include Bacillus subtilis, Bacillus subtilis, Pseudomonas fluorescens, Trichoderma harzianum, Streptomyces microflavus, and Aspergillus niger. In addition, although the prior art discloses that the endophytic Bacillus velezensis strain SEB1 isolated and screened by Gorai et al. has a high inhibitory effect on Alternaria alternata, it does not disclose that it also has a growth-promoting effect. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a Bacillus velezensis strain 3-25, which has good control effect on early blight and can promote crop growth at the same time.

[0005] The present invention provides a Bacillus velezensis strain 3-25 with a preservation number of CGMCC No. 31910.

[0006] The present invention provides a culture of the Bacillus velezensis strain 3-25.

[0007] Preferably, it includes at least one of the culture solution, bacterial suspension, and metabolites of the Bacillus velezensis strain 3-25.

[0008] The present invention provides an antibacterial agent for early blight pathogenic bacteria, which includes the Bacillus velezensis strain 3-25 or the culture.

[0009] Preferably, when the antibacterial agent contains the Bacillus velezensis strain 3-25 or its bacterial suspension, the active concentration number of the Bacillus velezensis strain 3-25 is 10 8 ~10 10 CFU / mL;

[0010] When the antibacterial agent contains the culture solution of the Bacillus velezensis strain 3-25, the volume percentage of the culture solution is 10% - 90%;

[0011] When the bacteriostatic agent contains the metabolites of the Bacillus velezensis strain 3-25, the volume percentage of the metabolites is 10% to 90%.

[0012] The present invention provides a biological pesticide, comprising the Bacillus velezensis strain 3-25 or the culture and excipients acceptable in the pesticide field.

[0013] The present invention provides the application of the Bacillus velezensis strain 3-25, the culture, the bacteriostatic agent or the biological pesticide in preventing and controlling early blight of crops and / or promoting the growth of crops.

[0014] Preferably, the early blight of crops includes at least one of the following: early blight of potato, early blight of tomato and early blight of pepper.

[0015] Preferably, the prevention and control of early blight of crops includes at least one of the following aspects:

[0016] Inhibiting the mycelial growth of early blight pathogens;

[0017] The Bacillus velezensis strain 3-25 colonizes in the rhizosphere soil of crops;

[0018] And regulating the relative abundance of microbial flora in the rhizosphere soil of crops.

[0019] Preferably, the promotion of crop growth is manifested in at least one of the following aspects: promoting the growth of crop plants, increasing crop yield and improving crop quality.

[0020] The present invention provides a Bacillus velezensis strain 3-25 with a preservation number of CGMCC No. 31910. Taking early blight of potato as the target disease, through in vitro leaf bioassay experiments and field control efficacy experiments, the present invention proves that the strain 3-2 has a high biocontrol effect on early blight, and the field control efficacy is more than 79.9%. At the same time, the present invention also proves that the strain 3-2 and its culture have good antibacterial activity against early blight pathogens, and the antibacterial rate is more than 77.4%. At the same time, the experiments show that the strain 3-2 can stably colonize in the rhizosphere soil of crops and affects the microbial community in the plant rhizosphere soil. In addition, the Bacillus velezensis strain 3-25 can produce growth-promoting substances and has a growth-promoting effect. The experiments prove that the strain 3-25 improves the quality and mu yield of potato tubers, and also increases the commercial potato rate of potato and the starch content in the tubers. It can be seen that the strain 3-25 can not only promote the growth of crops but also improve the quality of crops. It can be seen that the strain 3-25 provided by the present invention not only has the function of preventing and controlling early blight but also has the function of promoting crop growth, and can be prepared into a biological pesticide or biological bacterial fertilizer and applied to the soil to achieve multiple purposes at one stroke. Description of the Drawings

[0021] Figure 1 Morphological diagram of Bacillus velezensis strain 3-25 screened for the present invention;

[0022] Figure 2 Phylogenetic tree of Bacillus velezensis strain 3-25;

[0023] Figure 3 Determination results of the production of indole-3-acetic acid (IAA), cellulase, siderophore, protease, amylase, and phosphate-solubilizing characteristics by Bacillus velezensis strain 3-25;

[0024] Figure 4 Inhibitory result diagram of Bacillus velezensis 3-25 against Alternaria solani;

[0025] Figure 5 Control results of early blight on detached leaves by Bacillus velezensis 3-25;

[0026] Figure 6 Determination results of the stability of active substances in the sterile supernatant of Bacillus velezensis 3-25;

[0027] Figure 7 Determination result diagram of the inhibition of Alternaria solani by the culture solution, bacterial suspension, and metabolite solution of Bacillus velezensis 3-25;

[0028] Figure 8 Determination results of the colonization of Bacillus velezensis 3-25 in the roots of potato seedlings;

[0029] Figure 9 Field disease symptom diagram of potato early blight;

[0030] Figure 10 Effect result diagram of Bacillus velezensis 3-25 treatment on potato yield and quality;

[0031] Figure 11 Effect results of Bacillus velezensis 3-25 treatment on the microbial flora in potato rhizosphere soil;

[0032] Figure 12 Volcano plot and Manhattan plot of differential analysis of soil microorganisms in potato rhizosphere after treatment with Bacillus velezensis 3-25.

[0033] Biological material preservation information

[0034] Bacillus velezensi 3-25 was deposited at the China General Microbiological Culture Collection Center on September 10, 2024. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 31910. Detailed implementation mode

[0035] The present invention provides a Bacillus velezensi strain 3-25, with the preservation number of CGMCC No. 31910.

[0036]

[0037] Phylogenetic tree construction showed that the Bacillus velezensis strain 3-25 clustered with Bacillus velezensis. Through morphological and molecular identification, it belongs to Bacillus velezensis.

[0038] In the present invention, the Bacillus velezensis strain 3-25 has the ability to produce auxin (IAA), cellulase, siderophore, protease, amylase and dissolve phosphorus. This enables the Bacillus velezensis strain 3-25 to have the ability to promote the growth of crops and even plants. At the same time, the Bacillus velezensis strain 3-25 has the characteristic of quickly colonizing the rhizosphere soil of crops when applied to the soil, which further ensures that the Bacillus velezensis strain 3-25 and its secretions can be quickly absorbed by plant roots, achieving the effects of promoting crop growth, increasing yield and improving quality.

[0039] The present invention provides a culture of the Bacillus velezensis strain 3-25.

[0040] In the present invention, the culture preferably includes at least one of the culture solution, bacterial suspension and metabolite of the Bacillus velezensis strain 3-25. The preparation method of the culture solution of the Bacillus velezensis strain 3-25 preferably inoculates the seed solution of the Bacillus velezensis strain 3-25 into a medium and performs shaking culture to obtain the culture solution. The medium is preferably LB liquid medium. The temperature of the shaking culture is preferably 36-38 °C, more preferably 37 °C. The rotation speed of the shaking culture is preferably 180 rpm-220 rpm, more preferably 200 rpm. The time of the shaking culture is preferably 45-50 h, more preferably 48 h. The inoculation amount of the seed solution is preferably 1%-5%, more preferably 2%-3%. The preparation method of the seed solution is preferably inoculating the activated Bacillus velezensis strain 3-25 into LB liquid medium and culturing, and performing shaking culture for 12-6 h to obtain. The metabolite exists in the form of culture supernatant. The preparation methods of the bacterial suspension and the metabolite preferably perform solid-liquid separation on the culture solution, collect the solid phase as the bacteria body, resuspend to obtain the bacterial suspension, and the separated liquid phase is the culture supernatant.

[0041] In the present invention, the pathogen of early blight is preferably Alternaria solani strain HCL-17, specifically referring to HCL-17 recorded in the prior art (Liu Lili. Study on sporulation induction, azoxystrobin sensitivity and varietal disease resistance of Alternaria solani in potatoes [D]. Hebei Agricultural University, 2013). The present invention does not impose special restrictions on the type of bacteria, and well-known strains in the art can be used. In the examples of the present invention, the early blight pathogen is taken as strain HCL-17 to illustrate the antibacterial effect of the plate confrontation experiment.

[0042] In the present invention, the culture solution, bacterial suspension and metabolites of the Bacillus velezensis strain 3-25 were respectively used to conduct a plate confrontation experiment on the pathogen of early blight. The results showed that the culture solution, bacterial suspension and metabolites of the Bacillus velezensis strain 3-25 could all effectively inhibit the mycelial growth of the pathogen of early blight, indicating that they had antibacterial activity against the pathogen of early blight. Among them, the antibacterial activity of the culture solution was the best, followed by the bacterial suspension, and the supernatant had relatively poor effects. In addition, the stability of the metabolites of the Bacillus velezensis strain 3-25 in inhibiting the pathogen of early blight under conditions such as high temperature, enzymatic hydrolysis, and ultraviolet irradiation was also investigated. The results showed that the metabolites showed stability under the conditions of proteinase K, high temperature, and ultraviolet light, and their antibacterial activity did not show significant changes.

[0043] In view of the antibacterial activity of the Bacillus velezensis strain 3-25 and its culture, the present invention provides an antibacterial agent for the pathogen of early blight, comprising the Bacillus velezensis strain 3-25 or the culture.

[0044] In the present invention, when the antibacterial agent contains the Bacillus velezensis strain 3-25 or its bacterial suspension, the active concentration number of the Bacillus velezensis strain 3-25 is preferably 10 8 ~10 10 CFU / mL, more preferably 10 8 ~10 9 CFU / mL, and most preferably 1.0×10 8 CFU / mL. When the antibacterial agent contains the culture solution of the Bacillus velezensis strain 3-25, the volume percentage of the culture solution is preferably 10% to 90%, more preferably 40% to 80% CFU / mL, and most preferably 50%. When the antibacterial agent contains the metabolites of the Bacillus velezensis strain 3-25, the volume percentage of the metabolites is preferably 10% to 90%, more preferably 40% to 80% CFU / mL, and most preferably 50%. The antibacterial agent also includes common excipients. The dosage form of the antibacterial agent includes an aqueous solution or a powder. The present invention has no special restrictions on the preparation method of the aqueous solution or the powder, and it can be prepared by using the well-known preparation methods of aqueous solutions or powders in the art. For example, for the preparation of an aqueous solution, the culture solution or bacterial suspension or metabolites can be mixed with glycerol. The volume percentage of the glycerol is preferably 30% to 80%. The powder is obtained by removing the moisture from the culture solution or bacterial suspension and then mixing it with an antifreeze protectant and freeze-drying. The present invention does not make special restrictions on the formula of the antifreeze protectant, and it can be prepared by using the well-known raw materials in the art, such as sugars (sucrose, lactose or trehalose), alcohols (isopropanol), proteins (milk powder), etc.

[0045] The present invention provides a biological pesticide, comprising the Bacillus velezensis strain 3-25 or the culture and excipients acceptable in the pesticide field.

[0046] In the present invention, when the biological pesticide contains the Bacillus velezensis strain 3-25 or its bacterial suspension, the active concentration number of the Bacillus velezensis strain 3-25 is preferably 10 8 ~10 10 CFU / mL, more preferably 10 7 ~10 9 CFU / mL, and most preferably 1×10 8 CFU / mL. When the biological pesticide contains the culture solution of the Bacillus velezensis strain 3-25, the volume percentage of the culture solution is preferably 10% to 90%, more preferably 30% to 70%, and most preferably 50%. When the biological pesticide contains the metabolite of the Bacillus velezensis strain 3-25, the volume percentage of the metabolite is preferably 10% to 90%, more preferably 30% to 70%, and most preferably 50%.

[0047] The present invention provides the application of the Bacillus velezensis strain 3-25, the culture, the bacteriostatic agent or the biological pesticide in preventing and controlling early blight of crops and / or promoting the growth of crops.

[0048] In the present invention, the early blight of crops preferably includes at least one of the following: early blight of potato, early blight of tomato and early blight of pepper. The prevention and control of early blight of crops includes the following aspects: inhibiting the mycelial growth and spore germination of the early blight pathogen, the Bacillus velezensis strain 3-25 colonizing in the rhizosphere soil of crops and regulating the relative abundance of the microbial flora in the rhizosphere soil of crops.

[0049] In an embodiment of the present invention, in vitro leaf control efficacy experiments and field control experiments were carried out respectively. The Bacillus velezensis 3-25 showed good control efficacy against early blight on in vitro leaves, and the field control efficacy of the Bacillus velezensis 3-25 against early blight was 79.9%.

[0050] In another embodiment of the present invention, the effect of Bacillus velezensis strain 3-25 on the rhizosphere microbial community of potatoes was investigated. The results showed that the relative abundances of Proteobacteria and Firmicutes in the rhizosphere soil bacterial community increased, while those of Actinobacteria and Bacteroidetes decreased. The relative abundances of Pseudomonas and Citrobacter increased, while those of Paeniglutamicibacter, Arthrobacter, and Flavobacterium decreased. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil fungal community decreased. The relative abundances of Verticillium, Fusarium, Filobasidium, Naganishia, Sporobolomyces, and Colletotrichum decreased. By analyzing the compositions of the rhizosphere soil bacteria and fungi of potato plants, it was found that the rhizosphere bacteria and fungi communities of potatoes treated with strain 3-25 changed, altering the relative abundances of dominant species of bacteria and fungi at the phylum and genus levels, regulating the microbial community structure, and increasing the enrichment of beneficial microorganisms such as Bacillus and Pseudomonas.

[0051] In the present invention, the promotion of crop growth preferably manifests in at least one of the following aspects: promoting the growth of crop plants, increasing crop yields, and improving crop quality. Improving crop quality preferably includes increasing the nutrient content of the edible parts of crops and increasing the commercial qualification rate of the edible parts of crops. Increasing crop yields includes increasing the individual mass or per-acre yield of the edible parts of crops. Promoting the growth of crop plants preferably includes promoting an increase in the leaf area, stem thickening, and root elongation of crop plants.

[0052] In the embodiment of the present invention, compared with the blank control group and the control strain group, the potato tuber quality, the proportion of commercial potatoes, and the starch content increased when treated with Bacillus velezensis strain 3-25, showing an obvious growth-promoting effect.

[0053] The following is a detailed description of a Bacillus velezensis strain 3-25, its culture, and its application in the prevention and treatment of early blight provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] Isolation, purification and identification methods of Bacillus velezensis strain 3-25

[0056] First, pass the soil sample through a 20-mesh sieve. Take 0.2 g of the sieved soil and put it into a 50 mL centrifuge tube. Add 25 mL of sterile water and mix well by vortex oscillation. Add the suspension to 1000 mL of 1 / 10 TSA or NA liquid medium for gradient dilution. The dilution method is shown in Table 1.

[0057] Table 1 Dilution method of the suspension

[0058] Dilution factor Addition amount 2000-fold 500 μL 6000-fold 167 μL 18000-fold 56 μL 54000-fold 19 μL 162000-fold 6 μL

[0059] Note: The added amount indicates the amount of soil suspension added to 1 L of liquid medium.

[0060] Vortex and mix the dilution, pour it into a 13 cm × 13 cm square dish, and use a multi-channel pipette to aspirate 160 μL and add it to each well of a 96-well plate. Do 2-3 plates for each concentration as replicates, and set 3 blank control wells for each plate. Seal the 96-well plate with sealing film and incubate it at 30 °C. After 14 days, select the turbid wells and streak them on a TSA solid medium plate, and incubate at 30 °C. After two streaking purifications of the grown single colonies, pick a single colony and culture it in liquid TSA medium at 28 °C and 200 rpm for 24 h, mix it with 30% glycerol in a 1:1 ratio, and store it in a -80 °C refrigerator.

[0061] Strain identification

[0062] 1 Bacterial genomic DNA extraction

[0063] (1) Use a sterile toothpick to pick the antagonistic strains 3-25, 2-5, 3-5-2, 2-1, 3-22 and 3-4 stored at -80 °C, streak and activate them on an LB medium plate, and culture them in a 37 °C constant temperature incubator for 12 h for standby;

[0064] (2) Use a sterile 200 μL pipette tip to pick a single colony with clear morphology of the test strain, inoculate it into a glass test tube containing 5 mL of LB liquid medium, and culture it at 37 °C and 200 rpm for 12 h;

[0065] (3) Aspirate 1.5 mL of the bacterial liquid and add it to a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, pour off the supernatant, and collect the bacterial cells;

[0066] (4) Add 1 mL of ddH2O to the centrifuge tube, use a vortex mixer for 30 s to mix the bacterial suspension until there are no obvious bacterial clumps suspended, centrifuge again, and pour off the supernatant;

[0067] (5) Add 60 μL of lysozyme with a concentration of 20 mg / mL into the tube, invert it up and down to mix well, incubate in a water bath at 37 °C for 40 min, and invert it again every 10 - 20 min during this period;

[0068] (6) Add 650 μL of nucleic acid lysis solution into the tube, invert it up and down to mix well;

[0069] (7) Add 250 μL of Protein precipitation solution into the tube, and shake it with a vortex mixer for about 1 min;

[0070] (8) Centrifuge at 12000 rpm for 5 min to obtain 900 μL of supernatant, and aspirate and transfer it to a new centrifuge tube with a pipette;

[0071] (9) Aspirate and add pre-cooled isopropanol at -20 °C into the tube, invert it slowly 10 times to mix well;

[0072] (10) Centrifuge at 12000 rpm for 5 min, and pour off the supernatant;

[0073] (11) Add 500 μL of 75% ethanol solution into the tube, invert it several times to mix well, centrifuge at 14000 rpm for 2 min, and pour off the supernatant;

[0074] (12) Repeat step (11), and then place the centrifuge tube in a laminar flow bench to dry the ethanol in the tube;

[0075] (13) Add 60 μL of ddH2O into the tube to resuspend the DNA, and store it in a -20 °C refrigerator for later use.

[0076] 2 PCR Amplification

[0077] (1) PCR reaction system: Use the DNA templates of strains 3 - 25, 2 - 5, 3 - 5 - 2, 2 - 1, 3 - 22, and 3 - 4 respectively, and amplify with the 16S rDNA gene fragment as the primer (27F / 1492R). The reaction system is as shown in Table 2 below:

[0078] Table 2 PCR reaction system

[0079] Reactant Dosage (μL) Template DNA 1.0 Forward primer 0.5 Reverse primer 0.5 Mix 12.5 <![CDATA[ddH2O]]> 10.5 Total 25.0

[0080] (2) PCR amplification conditions

[0081]

[0082] (3) Entrust the amplified PCR products to Beijing North Institute of Biological Technology for sequencing.

[0083] 3 Construction of phylogenetic tree

[0084] Use Mega7 software to construct a phylogenetic tree.

[0085] The morphological diagram of strain 3-25 is shown in Figure 1 . From Figure 1 it can be seen that on the LB solid medium, the colony surface is smooth, and later the colony surface has wrinkles and is rough. The whole is white. By Gram staining, the bacteria are purple and rod-shaped, and are Gram-positive bacteria.

[0086] The sequencing result of strain 3-25 is shown in SEQ ID NO:1. Using the Blast tool in the NCBI database for comparison, the sequence similarity with Bacillus velezensis is the highest, and the sequence similarity reaches 93%. Use Mega7 software to construct a phylogenetic tree with the sequences of strain 3-25 and its related species. The result is shown in Figure 2 . From Figure 2 it can be seen that strain 3-25 clusters with Bacillus velezensis. In summary, strain 3-25 is identified as Bacillus velezensis. This strain was sent to the China General Microbiological Culture Collection Center for preservation, and the preservation number is CGMCC No. 31910.

[0087] Example 2

[0088] Preparation method of the culture solution of Bacillus velezensis strain 3-25

[0089] (1) Take the test strain stored in an ultra-low temperature refrigerator at -80 °C. Use a sterile toothpick to dip a small amount of the bacterial solution of Bacillus velezensis strain 3-25 on an LB plate for activation. Seal the plate with a sealing film and invert it in a 37 °C constant temperature incubator for 12 h for standby;

[0090] (2) Use a sterile pipette tip to pick a single colony of the test strain and inoculate it into an LB liquid medium containing 5 mL. Shake culture at 37 °C and 200 rpm for 16 h as the seed liquid;

[0091] (3) According to a volume ratio of 1:50, inoculate the seed liquid into 200 mL of LB liquid medium and shake culture at 37 °C and 200 rpm for 48 h for standby. Adjust the viable bacteria concentration in the culture solution to 1.0×10 8 CFU / mL for standby.

[0092] Example 3

[0093] Detect the abilities of Bacillus velezensis 3-25 to produce indole-3-acetic acid (IAA), cellulase, siderophore, protease, amylase and phosphorus solubilization

[0094] (1) Detection of indole-3-acetic acid (IAA)

[0095] Use a sterile 200 μL pipette tip to pick a single colony of the biocontrol strain 3-25 that has been pre-activated, and inoculate it into an LB liquid medium containing 0.5 g / L L-try. Incubate at 37 °C with shaking at 200 rpm for 48 h, and then centrifuge at 12,000 g for 13 min. Pipette 2.0 mL of Salkowski reagent into a cell culture plate, and then add 1 mL of the supernatant of strain 3-25. Mix well, wrap the culture plate with aluminum foil to avoid light, and let it stand at room temperature in the dark for 30 min. If the mixture turns pink, it indicates that the biocontrol strain 3-25 has the ability to produce auxin.

[0096] (2) Detection of cellulase activity

[0097] Pick a single colony of the biocontrol strain 3-25 and inoculate it into 5 mL of LB liquid medium. After incubating at 37 °C with shaking at 200 rpm for 12 h, transfer it to 200 mL of LB liquid medium at a ratio of 1:50 and continue shaking for 12 h. Use a sterile borer to punch two holes at equal distances from the center on the left and right sides of the cellulose Congo red medium. Pipette 40 μL of the bacterial solution of strain 3-25 into the holes, using LB liquid medium as a control. Repeat each treatment 3 times and incubate in a 37 °C constant temperature incubator for 3 d. If a clear zone appears around the inoculation point, it indicates that the bacterium has the ability to produce cellulase.

[0098] (3) Detection of phosphate-solubilizing ability

[0099] Refer to the method for detecting cellulase activity to complete the inoculation of the bacterial solution in the calcium phosphate medium, setting LB liquid medium as a control. Repeat each treatment 3 times and incubate in a 37 °C incubator for 3 d. If a clear zone appears, it indicates that the strain has the ability to solubilize phosphate.

[0100] (4) Detection of siderophore ability

[0101] Refer to the method for detecting cellulase activity to complete the inoculation of the bacterial solution of strain 3-25 in the CAS solid medium, setting LB liquid medium as a control. Repeat each treatment 3 times and incubate in a 37 °C incubator for 3 d. If a clear zone appears, it indicates that the strain has the ability to produce siderophores.

[0102] (5) Detection of protease activity

[0103] Refer to the method for detecting cellulase activity to complete the inoculation of the bacterial solution of strain 3-25 in the skim milk solid medium, setting LB liquid medium as a control. Repeat each treatment 3 times and incubate in a 37 °C incubator for 24 h, 48 h, and 72 h, then observe around the inoculation holes. If a clear zone appears, it indicates that the strain produces protease, and the diameter of the clear zone represents the amount of protease produced by the strain.

[0104] (6) Detection of amylase activity

[0105] The inoculation of bacterial liquid in amylase detection medium was completed by referring to the cellulase activity detection method, and LB liquid medium was set as the control. Each treatment was repeated 3 times and cultured in a 37°C constant temperature incubator for 48 hours. The culture was stained with diluted Lugol's electrophoresis solution for 15 seconds, and the surface floating color was cleaned with 70% ethanol to observe whether a transparent circle was produced. If so, it means that the bacteria have the ability to produce amylase.

[0106] Results Figure 3 The strains 3-25 of Bacillus velez were all capable of producing IAA, cellulase, siderophore, protease, amylase and phosphate solubilizing enzymes.

[0107] Example 4

[0108] Plate confrontation experiment between Bacillus velez strain 3-25 and pathogenic bacteria of early blight

[0109] (1) Use a sterilized toothpick to pick up the stored test strain liquid, activate it on an LB plate, seal it with a sealing film, place it upside down in a 37°C constant temperature incubator and culture it overnight for later use;

[0110] (2) Activate and culture the Alternaria solani strain HCL-17, the pathogen of early blight, on a PDA plate with a diameter of 90 mm. Take a bacterial cake and inoculate it in the center of a new PDA plate. Use a sterilized toothpick to pick up a single colony of the activated test strain and inoculate it at equal distances around the bacterial cake of the early blight pathogen. The PDA plate inoculated with only the pathogenic bacteria cake is used as the control group (CK). Place it upside down in a 25°C constant temperature incubator for dark culture. Repeat 3 plates for each treatment. When CK grows all over the PDA plate, observe and record the antagonistic effect on the early blight pathogen HCL-17.

[0111] Results Figure 4 The inhibition rate of Bacillus Velezii strain 3-25 against early blight pathogen reached 77.4%.

[0112] Example 5

[0113] Detection of the protective effect of Bacillus velez strain 3-25 against early blight on detached leaves

[0114] (1) Select healthy potato plants, collect leaves with good growth status and uniform size, cut them with scissors, and temporarily store them in a refrigerator at 4°C;

[0115] (2) First, disinfect the leaf surface with 2% sodium hypochlorite solution for 30 seconds, then soak the leaf in 75% alcohol for 5 seconds, and finally rinse the leaf three times with sterile water and place it on sterile gauze to absorb the moisture on the leaf surface;

[0116] (3) Using LB liquid medium and sterile water as controls respectively, immerse the leaves in 200 mL of the biocontrol strain culture solution, LB liquid medium, and sterile water for 10 min. Each treatment has 3 replicates, and each replicate has 10 leaves.

[0117] (4) Place a sterile filter paper on a petri dish, add 1 mL of sterile water to moisten the filter paper, place the leaves on the filter paper with the abaxial side facing up. After drying in a laminar flow hood, place them in a sealed box lined with four layers of moistened gauze, seal the box with plastic wrap, and incubate in a light incubator with a 12 h light cycle at 25 °C for 24 h. After 24 h, inoculate the leaf abaxial surface with an Alternaria solani spore cake of consistent activity, put it back into the sealed box, seal it with plastic wrap, and continue to incubate in a light incubator with a 12 h light cycle at 25 °C for 4 - 5 d. During this period, observe the disease incidence carefully and pay attention to replenishing water and maintaining humidity. 4 - 5 d after inoculating the pathogen, count the disease grades of the potato leaves, calculate the control effect, and take pictures for record.

[0118] The results are shown in Table 3.

[0119] Table 3 Control results of Alternaria solani on detached leaves

[0120] Treatment Disease index Control effect (%) Clear water 68.9±1.4d — LB 82.2±1.1b — 3-25 54.3±0.4e 33.9

[0121] Note: The letter abcd marking method is used to represent the significant differences between different groups.

[0122] From Table 1 and Figure 5 it can be seen that the field control effect of Bacillus velezensis strain 3 - 25 against Alternaria solani is 79.9%. At the same time, there are also increases in the tuber quality, the proportion of marketable tubers, and the starch content compared with the control group, showing an obvious growth - promoting effect.

[0123] Example 6

[0124] Industrial fermentation method of Bacillus velezensis strain 3 - 25

[0125] Fermentation process: Pick the colonies of the biocontrol strain 3 - 25 and inoculate them into a 2 L Erlenmeyer flask containing 300 mL of liquid LB medium, shake - culture at 37 °C and 200 rpm for 16 h to prepare the seed liquid; inoculate the seed liquid into an industrial fermenter for industrial fermentation. After 36 h, take samples for microscopic examination. When the spore - forming rate of bacillus in the field of view reaches 80%, the fermentation can be stopped; filter the fermentation broth of the biocontrol strain, and detect the viable bacteria content of the biocontrol strain before and after filtration by the viable bacteria counting method.

[0126] The measured viable bacteria content of Bacillus velezensis strain 3 - 25 in the fermentation broth is about 8×10 10 CFU / mL.

[0127] Example 7

[0128] Determination of the Stability of Active Substances in the Fermentation Supernatant of Bacillus velezensis Strain 3-25

[0129] Using a sterile 200 μL pipette tip, pick single colonies with clear morphology of the above strains respectively, inoculate them into 5 mL LB test tubes, and culture them at 37 °C and 200 rpm for 12 h. Then, transfer them to 500 mL Erlenmeyer flasks containing 200 mL of liquid LB medium at a ratio of 1:50 and continue shaking culture for 36 h. Respectively, pipette 1.5 mL of the culture broth of each above strain into sterilized 2.0 mL centrifuge tubes, centrifuge at 12000 rpm for 5 min to obtain the supernatant and the bacterial cells. Transfer the supernatant to a new sterilized 2.0 mL centrifuge tube and filter it using a 0.22 μm bacterial filter to obtain a sterile supernatant. Wash the obtained bacterial cells with 1.5 mL of sterile water, vortex and centrifuge again, repeat 3 times, add 1.5 mL of sterile water to the finally obtained bacterial cells, and vortex to prepare a bacterial suspension.

[0130] Mix the sterile supernatant of the biocontrol strain 3-25 and the proteinase K solution with a concentration of 10 mg / mL evenly at a volume ratio of 20:1, and react at 37 °C for 60 min. In addition, irradiate two portions of the sterile supernatant of strain 3-25 under a 20 W ultraviolet lamp for 0.5 h and 2 h respectively, with the supernatant 10 cm away from the ultraviolet lamp. Heat the three portions of sterile supernatant in a water bath at 60 °C, 80 °C, and 100 °C for 30 min respectively.

[0131] Use a sterile punch to punch 3 evenly distributed holes at an equal distance from the center of the PDA plate; use a sterile punch to punch a mycelial cake from the edge of the Alternaria solani HCL-17 mycelial cake colony on the 7th day of culture and inoculate it to the center position of the PDA medium. Respectively, pipette 40 μL of the sterile supernatant of the biocontrol bacterium 3-25 and the sterile supernatant under the above different treatments into the holes, and repeat each treatment on 3 plates; place the plates in a 25 °C climate incubator and culture them in the dark for 10 d, measure the radius of the pathogenic bacteria in the control group, the radius of the pathogenic bacteria in the treatment group, and the width of the inhibition zone, and take pictures for recording.

[0132] The results are shown in Figures 6 - 7 and Table 4.

[0133] Table 4 Results of Stability Test

[0134]

[0135] Note: The letters abcd are used to indicate significant differences between different groups.

[0136] There was no significant difference in the effect of the sterile supernatant of protease K-treated strain 3-25 and the supernatant of untreated strain 3-25 on Alternaria solani; after heat treatment of the sterile supernatant of strain 3-5 at 60 °C, 80 °C, and 100 °C, there was also no significant difference in the inhibitory effect on Alternaria solani; similarly, the antibacterial activity of the supernatant of strain 3-25 remained stable when irradiated with ultraviolet light for 0.5 h and 2 h. The results showed that the antibacterial active substance in the sterile supernatant of strain 3-25 showed stability under the conditions of protease K, high temperature, and ultraviolet light, and its antibacterial activity did not change significantly. The culture solution, bacterial suspension, and metabolite of Bacillus velezensis 3-25 could all inhibit the growth of Alternaria solani hyphae, among which the inhibitory activity of the culture solution was the strongest, followed by the bacterial suspension, and the metabolite was relatively poor.

[0137] Example 8

[0138] Treatment of the root system of potato seedlings with the labeled strain of Bacillus velezensis strain 3-25

[0139] The labeled strain of Bacillus velezensis strain 3-25 was obtained by transforming the Escherichia coli pGFP-78 plasmid containing the GFP protein coding gene into Bacillus velezensis strain 3-25 and verifying it. The specific steps are as follows.

[0140] 1. Extraction of Escherichia coli pGFP-78 plasmid DNA containing the GFP protein coding gene

[0141] Using the LQ-3GFP-labeled detection strain (see the prior art: Li Rong, Hong He, Jiang Wenxiao, et al. Effect of Bacillus velezensis LQ-3 on controlling wheat sharp eyespot and its rhizosphere colonization ability [J]. Journal of Triticeae Crops, 2024, 44(07): 947-954.) as the material, the Escherichia coli pGFP-78 plasmid containing the GFP protein coding gene was extracted. The plasmid DNA extraction was carried out according to the operation manual of the plasmid DNA mini extraction kit of Tiangen Biochemical Technology Co., Ltd.

[0142] 2. Preparation of competent cells of Bacillus velezensis strain 3-25 and electroporation transformation

[0143] (1) Use a sterile inoculation loop to pick a fresh 3-25 single colony and inoculate it into 5 mL of LB liquid medium, and shake culture overnight;

[0144] (2) Inoculate the seed liquid into the LB growth medium at a ratio of 1:100 and shake culture at 37 °C and 200 rpm (until OD600 is 0.35-0.55);

[0145] (3) Add weakening agents (2% glycine, 1% DL-threonine), continue shaking culture at 37 °C and 200 rpm until the OD600 is just less than the OD value before adding the weakening agents (20 - 40 min);

[0146] (4) Collect the culture medium, place it on ice bath for 20 min, centrifuge at 5000×g and 4 °C for 10 min, and discard the supernatant;

[0147] (5) Take out the electroporation buffer from the refrigerator, wash the bacterial cells 4 - 5 times with it, each time using 1 mL, centrifuge for 5 min, collect the bacterial cells, and do not pour out completely for the last time;

[0148] (6) Add the corresponding electroporation buffer according to the number of electroporations, pre-cool a 1.5 mL centrifuge tube in advance on ice, and aliquot into it;

[0149] (7) Take out the plasmid to be electroporated from the refrigerator, take out the recovery medium preheated in a 37 °C heat block, turn on the electroporator, add 7 - 8 μL of the plasmid to be electroporated into a centrifuge tube containing 100 μL of competent cells, pipette several times to mix evenly, then transfer to an electroporation cuvette, and place it in the slot for electroporation;

[0150] (8) Take out, quickly add 900 μL of recovery medium, mix evenly and transfer to a 15 mL centrifuge tube, shake culture at 160 rpm for 3 - 5 h, and spread on the corresponding resistant LB plate.

[0151] After the above operations, strain 3-25 was labeled with a plasmid fused with GFP protein to obtain strain 3-25(pGFP78). Single colonies were picked and inoculated into ordinary LB medium, and the seed liquid was cultured overnight to obtain the culture solution of strain 3-25(pGFP78) labeled strain. Potato plant seedlings with the same growth vigor were selected, and the roots were irrigated with the culture solution of strain 3-25 labeled strain. Each potato seedling was irrigated with 10 mL of the culture solution, and each treatment was repeated 4 times. At 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d after the roots of potato plants were irrigated with the labeled strain, the potato root tissues were collected, rinsed thoroughly with PBS buffer, weighed and recorded, placed in a sterilized 2.0 mL centrifuge tube, ground into a homogenate with a sterilized mortar rod, added 1 mL of sterile water and mixed well, and then diluted by gradient dilution method. 100 μL of the diluted suspension was pipetted and spread on the LB medium plate containing 20 mg / mL tetracycline. 4 plants were collected at each time point, and each treatment was repeated on 3 plates, and placed in a constant temperature incubator at 37 °C for culture. When the colonies grew evenly, the count was carried out. The roots of the collected potato plants were gently shaken to remove the floating soil, and the soil on the rhizosphere surface 1-2 mm was collected, weighed, and then diluted by gradient dilution. 100 μL of the diluted suspension was pipetted and spread on the LB medium plate containing 20 mg / mL tetracycline. Each treatment was repeated on 3 plates, and placed in a constant temperature incubator at 37 °C for culture. When the colonies grew evenly, the count was carried out.

[0152] The results are shown in Figure 8 . It can colonize in both the rhizosphere soil and roots of potato seedlings. At 1 d, the colonization amount of strain 3-25(pGFP78) in the rhizosphere soil of potato plant seedlings was 1.5×10 5 CFU / g·w, and the colonization amount in the seedling roots was 5.2×10 4 CFU / g·w. Subsequently, the colonization amount of strain 3-25(pGFP78) in the rhizosphere soil showed a decreasing trend. At 10 d, the colonization amount in the rhizosphere soil of potatoes decreased to 9.8×10 4 CFU / g·w, while the colonization amount of strain 3-25(pGFP78) in the potato roots first showed an increasing trend, reached the highest value at 7 d, 5.8×10 4 CFU / g·w, and then decreased slightly after 10 d.

[0153] Example 9

[0154] Field control efficacy test against early blight

[0155] 1. Test site

[0156] Dongbahao, Siziwang Banner, Ulanqab City, Inner Mongolia Autonomous Region; Longitude: 111°42′39.13″; Latitude: 41°25′36.64″; Altitude: 1556m.

[0157] 2. The potato variety planted is Zhongjia No. 7.

[0158] 3. Planting method: Each ridge is 40 cm wide and 120 m long. The interval between each potato tuber is 20 cm, and the planting depth is 10 cm. Design every 2 ridges as a group of treatments, and design 2 ridges of protective rows between different treatments. Among them, the length of the ridge for the field control efficacy experiment is 30 m, and the length of the ridge for the field growth promotion experiment is 70 m. The interval between the control efficacy experiment and the growth promotion experiment is 20 m as the isolation row. Use clear water as the blank control.

[0159] 4. Application method of biocontrol strains

[0160] Spray the first time after the potato plants emerge. 24 hours after the first spraying of the biocontrol strain fermentation broth, inoculate a spore suspension of Alternaria solani with a concentration of 1.0×10 4 CFU / mL in the field to ensure the occurrence of early blight in the field and simulate a field with early blight of potatoes. Subsequently, spray the antagonistic bacteria liquid once every 25 days during the growth period of potatoes, for a total of 3 sprays. Use an electric sprayer to spray the biocontrol strain fermentation broth, with the fermentation broth moistening the potato leaves but not flowing away. The water consumption is about 675 L·hm -2 . The fermentation broth should be sprayed in weather without strong wind, rain and excessive temperature. Start investigating the disease situation 7 days after the last spraying of the fermentation broth (see Table 5 and Figure 9 ).

[0161] Table 5 Grading standard for early blight of potatoes

[0162] Level Disease incidence Grade 0 No lesions Grade 1 Lesion area less than 5% of the entire leaf area Grade 3 Lesion area 6% - 10% of the entire leaf area Grade 5 Lesion area 11% - 20% of the entire leaf area Grade 7 Lesion area 21% - 50% of the entire leaf area Grade 9 Lesion area more than 51% of the entire leaf area

[0163] Divide each treatment into three plots. Randomly take five points in each plot, and investigate 3 plants at each point. Investigate 15 leaves on the upper, middle and lower layers of each potato plant respectively. Grade according to the proportion of the early blight lesion area on the leaves to the total leaf area, and calculate the diseased leaf rate, disease index and relative control effect.

[0164] The results are shown in Table 6 and Figure 10 .

[0165] Table 6 Field control and growth promotion results of biocontrol strain 3-25

[0166]

[0167] Note: The letters abcd are used to represent significant differences between different groups.

[0168] The field control effect of Bacillus velezensis strain 3-25 against early blight was 79.9%. At the same time, there were also increases in the quality of potato tubers, the proportion of marketable potatoes, and starch content compared to the control group, showing an obvious growth-promoting effect.

[0169] Example 10

[0170] Determination of the microbial flora in the rhizosphere soil of potatoes

[0171] 1. Collection of potato rhizosphere soil

[0172] For each treatment group, the random sampling method was used. Three points were randomly selected, and the roots of three potato plants at each point were randomly dug out with a spade. The loose non-rhizosphere soil on the outer layer of the roots was gently shaken off, the roots of the plants were cut off with scissors, and the soil attached to the roots within 1-2 mm was collected as rhizosphere soil and placed in a sterile sampling bag. The sterile bag containing the soil and many ice bags frozen in a -20°C refrigerator were placed in a foam box and taken back to the laboratory as soon as possible. After removing large impurities such as stones and plant roots from the soil, the soil sample was sieved through a 100-mesh sieve disinfected with 75% alcohol. After thoroughly mixing the sieved soil, it was aliquoted into 2 mL centrifuge tubes sterilized by high-temperature and high-pressure and stored in a -80°C ultra-low temperature refrigerator for subsequent extraction of soil genomic DNA.

[0173] 2. Extraction and sequencing of potato rhizosphere soil DNA

[0174] It was extracted according to the FastDNA SPIN Kit for soil soil extraction kit, and the extracted rhizosphere soil DNA was entrusted to Majorbio Co., Ltd. for high-throughput sequencing.

[0175] The results are shown in Figure 11 and Figure 12After treatment with strain 3-25, the relative abundances of Proteobacteria and Firmicutes in the rhizosphere soil bacterial community increased, while those of Actinobacteria and Bacteroidetes decreased; the relative abundances of Pseudomonas and Citrobacter increased, while those of Paeniglutamicibacter, Arthrobacter, and Flavobacterium decreased. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil fungal community decreased; the relative abundances of Verticillium, Fusarium, Filobasidium, Naganishia, Sporobolomyces, and Colletotrichum decreased, while the relative abundance of Alternaria increased. In the rhizosphere soil bacterial community of potato treated with strain 3-25, 63 ASVs were up-regulated and 75 ASVs were down-regulated; among them, the levels of Gammaproteobacteria and Bacilli in the rhizosphere soil of potato were up-regulated, while the levels of Alphaproteobacteria, Betaproteobacteria, Actinobacteria, Flavobacteriia, and Sphingobacteriia were down-regulated. In the rhizosphere soil fungal community of potato, 24 ASVs were up-regulated and 23 ASVs were down-regulated; among them, the level of Dothideomycetes was up-regulated, while the levels of Tremellomycetes, Microbotryomycetes, Sordariomycetes, Pezizomycetes, and Laboulbeniomycetes were down-regulated.

[0176] Comparative Example 1

[0177] Comparative experiment on control efficacy and growth promotion effect

[0178] Multiple strains of bacteria were isolated from healthy potato soil according to the method of Example 1. Using the same morphological and molecular identification methods, it was found that strains 2-5, 3-5-2, 3-22, 2-1, and 3-4 all belong to Bacillus velezensis.

[0179] Using Bacillus velezensis strain 3-25, Bacillus velezensis strain 2-5, Bacillus velezensis strain 3-5-2, Bacillus velezensis strain 3-22, Bacillus subtilis strain 2-1, and Bacillus subtilis strain 3-4 as experimental strains, a field control efficacy experiment was carried out, and the method was the same as that in Example 9. At the same time, a blank control group was set up.

[0180] The results are shown in Table 7.

[0181] Table 7 Field disease control and growth promotion results of strain 3-25 and its control strains

[0182]

[0183] Note: The letter abcd marks are used to represent significant differences between different groups.

[0184] The field control efficacy experiment showed that the fermentation broths of the six antagonistic bacteria all had a certain control effect on potato early blight. Among them, the field control effects of strains 3-25, 2-5, 3-5-2, and 2-1 all reached more than 70%, which were 79.9%, 73.9%, 72.9%, and 71.3% respectively. The field control effects of strains 3-22 and 3-4 reached 67.2% and 64.9%. Among them, the field control effect of strain 3-25 was the best. The results of the field growth promotion experiment showed that the six strains also increased in terms of potato tuber quality, the proportion of marketable potatoes, and starch content compared with the control group, and had an obvious growth promotion effect. However, strain 3-25 simultaneously had an efficient field control effect and the effect of promoting potato yield and quality.

[0185] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Bacillus velezensis ( Bacillus velezensis ) strain 3-25, characterized in that The preservation number is CGMCC No. 31910.

2. A culture of the Bacillus velezensis strain 3-25 according to claim 1.

3. The culture according to claim 2, wherein, It includes at least one of the culture solution, bacterial suspension and metabolites of the Bacillus velezensis strain 3-25.

4. An antibacterial agent for the pathogen causing early blight, characterized in that, It includes the Bacillus velezensis strain 3-25 according to claim 1 or the culture according to claim 2 or 3.

5. The bacteriostatic agent according to claim 4, wherein When the bacteriostatic agent contains the Bacillus velezensis strain 3-25 or its bacterial suspension, the active concentration number of the Bacillus velezensis strain 3-25 is 10 8 ~10 10 CFU / mL; When the culture solution of the Bacillus velezensis strain 3-25 is included in the bacteriostatic agent, the volume percentage of the culture solution is 10% - 90%; When the metabolite of the Bacillus velezensis strain 3-25 is included in the bacteriostatic agent, the volume percentage of the metabolite is 10% - 90%.

6. A biological pesticide, characterized in that, It includes the Bacillus velezensis strain 3-25 according to claim 1 or the culture according to claim 2 or 3 and excipients acceptable in the pesticide field.

7. The application of the Bacillus velezensis strain 3-25 according to claim 1, the culture according to claim 2 or 3, the bacteriostatic agent according to claim 4 or 5, or the biological pesticide according to claim 6 in preventing and controlling early blight of crops and / or promoting the growth of crops, wherein the early blight of crops is early blight of potato, early blight of tomato and early blight of pepper, and the crop for promoting the growth of crops is potato.

8. The application according to claim 7, wherein The prevention and control of early blight of crops includes at least one of the following aspects: Inhibiting the mycelial growth of the early blight pathogen; The Bacillus velezensis strain 3-25 colonizes in the rhizosphere soil of crops; And regulating the relative abundance of the microbial flora in the rhizosphere soil of crops.

9. The application according to claim 7, wherein The promotion of crop growth is manifested in at least one of the following aspects: promoting the growth of crop plants, increasing crop yield and improving crop quality.

Images