Bacillus velezensis strain 3-25, culture thereof and application of bacillus velezensis strain 3-25 in prevention and treatment of early blight

Abstract

The invention provides a bacillus velezensis strain 3-25, a culture of the bacillus velezensis strain 3-25 and application of the bacillus velezensis strain 3-25 in prevention and treatment of early blight, and belongs to the technical scheme of functional microorganisms. The preservation number of the bacillus velezensis strain 3-25 is CGMCC (China General Microbiological Culture Collection Center) No. 31910. The strain 3-25 and a culture thereof have good bacteriostatic activity on pathogenic bacteria of early blight and have an efficient biocontrol effect on early blight, and meanwhile, the strain 3-25 also can secrete active ingredients for promoting growth and has a remarkable growth promoting effect. The bacillus velezensis strain 3-25 is used as a biological pesticide and / or bacterial fertilizer, and has a wide application prospect in prevention and treatment of crop early blight.

Description

Technical Field

[0001] The invention belongs to a functional microorganism technical solution, and specifically relates to a Bacillus velezensis strain 3-25 and a culture thereof, and an application of the strain 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, a variety of strategies are usually adopted, including breeding disease-resistant varieties, implementing agricultural management measures, using biological control technology and chemical control methods. Among them, chemical agents are prone to cause environmental and crop pollution, and are prone to dependence on chemical agents, and cannot achieve the purpose of high-efficiency control in a long-term and stable manner.

[0003] At present, the biological control microorganisms for potato early blight mainly include Bacillus subtilis, Bacillus subtilis, Pseudomonas fluorescens, Trichoderma harzianum, Streptomyces tenuifolius and Aspergillus niger. In addition, although the prior art discloses that the endophytic Bacillus Velezii SEB1 strain 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 object of the present invention is to provide a Velezac bacteria strain 3-25, which has good preventive effect on early blight and promotes crop growth.

[0005] The invention provides a Velezacacillus strain 3-25, whose preservation number is CGMCC No.31910.

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

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

[0008] The invention provides an antibacterial agent for early blight pathogenic bacteria, comprising the Velezella strain 3-25 or the culture.

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

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

[0011] When the antibacterial agent contains the metabolites of the Velezac bacteria strain 3-25, the volume percentage of the metabolites is 10% to 90%.

[0012] The invention provides a biological pesticide, comprising the Bacillus Velez strain 3-25 or the culture thereof and an auxiliary material acceptable in the pesticide field.

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

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

[0015] Preferably, the method for preventing and controlling early blight of crops comprises at least one of the following aspects:

[0016] Inhibit the hyphae growth of early blight pathogens;

[0017] The Bacillus Velez strain 3-25 is colonized in the rhizosphere soil of the crop;

[0018] and regulate the relative abundance of crop rhizosphere soil microbial flora.

[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 velez strain 3-25, with a deposit number of CGMCC No. 31910. The present invention uses potato early blight as a target disease, and through in vitro leaf bioassay experiments and field control experiments, it is proved that the strain 3-2 has a high-efficiency biocontrol effect on early blight, and the field control effect 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, experiments show that the strain 3-2 can stably colonize in the rhizosphere soil of crops and affect the microbial community of the rhizosphere soil of plants. In addition, the Bacillus velez strain 3-25 can produce growth-promoting substances and has a growth-promoting effect. Experiments have shown that the strain 3-25 improves the quality and per-acre yield of potato tubers, and also increases the commercial potato rate of potatoes and the starch content in 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 effect of preventing and treating early blight but also has the effect of promoting crop growth, and can be prepared into biological pesticides or biological fertilizers and applied to the soil, thus achieving multiple goals at one stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphological diagram of Bacillus velez strain 3-25 screened for the present invention;

[0022] Figure 2 is the phylogenetic tree of Bacillus velez strain 3-25;

[0023] Figure 3 The results of the determination of the properties of IAA, cellulase, siderophore, protease, amylase and phosphate solubilization of Bacillus velez strain 3-25;

[0024] Figure 4 This is the result of the inhibition of Bacillus Velez 3-25 against early blight pathogen;

[0025] Figure 5 The results of the control of early blight by Bacillus Velez 3-25 on detached leaves are shown;

[0026] Figure 6 The results of stability test of active substances in sterile supernatant of Bacillus velez 3-25 are shown;

[0027] Figure 7 The figure is a result of the determination of the inhibition of early blight pathogens by the culture solution, bacterial suspension and metabolic solution of Bacillus velez 3-25;

[0028] Figure 8 The results of the determination of the colonization of Bacillus Velez 3-25 in the root system of potato seedlings;

[0029] Fig. 9 Figure 1. Symptoms of potato early blight in the field;

[0030] Fig.10 This is the result diagram of the effect of Bacillus Velez 3-25 treatment on potato yield and quality;

[0031] Fig.11 The results are as follows: the effect of Bacillus Velez 3-25 treatment on the microbial flora in the potato rhizosphere soil;

[0032] Fig.12 Volcano and Manhattan plots for differential analysis of potato rhizosphere soil microorganisms after treatment with Bacillus Velez-Oehlers 3-25.

[0033] Biomaterial Deposit Information

[0034] Bacillus velezensi 3-25 was deposited on September 10, 2024 in the China General Microbiological Culture Collection, with the address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.31910. DETAILED DESCRIPTION

[0035] The invention provides a Bacillus velezensi strain 3-25, whose deposit number is CGMCC No.31910.

[0036]

[0037] The results of phylogenetic tree construction showed that the Bacillus velezensis strain 3-25 and Bacillus velezensis were clustered into one branch. Morphological and molecular identifications showed that the strains belonged to Bacillus velezensis.

[0038] In the present invention, the Velez Bacillus strain 3-25 has the ability to produce IAA, cellulase, siderophore, protease, amylase and phosphate dissolving. This enables Velez Bacillus strain 3-25 to promote the growth of crops and even plants. At the same time, Velez Bacillus strain 3-25 has the characteristic of rapid colonization in the rhizosphere soil of crops when applied to the soil, which further ensures that Velez Bacillus strain 3-25 and its secretions can be quickly absorbed by the plant root system, achieving the effect of promoting crop growth and improving yield and quality.

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

[0040] In the present invention, the culture preferably includes at least one of the culture solution, bacterial suspension and metabolites of the Velez Bacillus strain 3-25. The method for preparing the culture solution of the Velez Bacillus strain 3-25 is preferably to inoculate the Velez Bacillus strain 3-25 seed solution in the culture medium for shaking culture to obtain the culture solution. The culture medium is preferably an LB liquid culture medium. The temperature of the shaking culture is preferably 36 to 38°C, more preferably 37°C. The rotation speed of the shaking culture is preferably 180rpm to 220rpm, more preferably 200rpm. The time of the shaking culture is preferably 45 to 50h, more preferably 48h. The inoculation amount of the seed solution is preferably 1% to 5%, more preferably 2% to 3%. The method for preparing the seed solution is preferably to inoculate the activated Velez Bacillus strain 3-25 in the LB liquid culture medium for culture, and shake the culture for 12 to 6h. The metabolites exist in the form of culture supernatant. The method for preparing the bacterial suspension and metabolites preferably involves performing solid-liquid separation on the culture solution, collecting the solid phase as bacterial bodies, resuspending the solid phase to obtain a bacterial suspension, and the separated liquid phase as the culture supernatant.

[0041] In the present invention, the pathogenic bacteria of early blight is preferably Alternaria solani strain HCL-17, and specific reference is made to HCL-17 described in the prior art (Liu Lili. Study on spore induction, azoxystrobin sensitivity and disease resistance of potato early blight bacteria [D]. Hebei Agricultural University, 2013). The present invention does not impose any special restrictions on the type of bacteria, and strains well known in the art can be used. In the embodiments of the present invention, the pathogenic bacteria of early blight are 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 Velez Bacillus strain 3-25 were respectively subjected to a plate confrontation experiment of early blight pathogens. The results showed that the culture solution, bacterial suspension and metabolites of the Velez Bacillus strain 3-25 can effectively inhibit the mycelial growth of early blight pathogens, indicating that they have antibacterial activity against early blight pathogens, among which the culture solution has the best antibacterial activity, followed by the bacterial suspension, and the supernatant has a relatively poor effect. In addition, the stability of the metabolites of the Velez Bacillus strain 3-25 in inhibiting early blight pathogens under conditions such as high temperature, enzymolysis, and ultraviolet irradiation was also tested. The results showed that the metabolites showed stability under proteinase K, high temperature, and ultraviolet conditions, and their antibacterial activity did not change significantly.

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

[0044] In the present invention, when the antibacterial agent comprises the Velezac bacteria strain 3-25 or its bacterial suspension, the active concentration of the Velezac bacteria strain 3-25 is preferably 10 8 ~10 10 CFU / mL, more preferably 10 8 ~10 9 CFU / mL, the most preferred is 1.0×10 8 CFU / mL. When the bacteriostatic agent contains the culture solution of the Velez Bacillus 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 bacteriostatic agent contains the metabolite of the Velez Bacillus strain 3-25, the volume percentage of the metabolite is preferably 10% to 90%, more preferably 40% to 80% CFU / mL, and most preferably 50%. The bacteriostatic agent also includes common excipients. The dosage form of the bacteriostatic 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 the aqueous solution or the powder preparation method known in the art can be used for preparation. For example, the preparation of the aqueous solution can be obtained by mixing the culture solution or the bacterial suspension or the metabolite with glycerol. The volume percentage of the glycerol is preferably 30% to 80%. The powder comprises removing the water from the culture solution or the bacterial suspension and mixing it with an antifreeze protective agent, and freeze-drying it. The present invention does not impose any special restrictions on the formula of the antifreeze protectant, and it can be prepared using raw materials well known in the art, such as sugars (sucrose, lactose or trehalose), alcohols (isopropanol), proteins (milk powder), etc.

[0045] The invention provides a biological pesticide, comprising the Bacillus Velez strain 3-25 or the culture thereof and an auxiliary material acceptable in the pesticide field.

[0046] In the present invention, when the biopesticide comprises the Velezac bacteria strain 3-25 or its bacterial suspension, the active concentration of the Velezac bacteria strain 3-25 is preferably 10 8 ~10 10 CFU / mL, more preferably 10 7 ~10 9 CFU / mL, the most preferred is 1×10 8 CFU / mL. When the biopesticide contains the culture solution of the Velezac 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 biopesticide contains the metabolite of the Velezac 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 use of the Bacillus velez strain 3-25, the culture, the antibacterial agent or the biological pesticide in preventing and controlling early blight of crops and / or promoting crop growth.

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

[0049] In one embodiment of the present invention, an in vitro leaf control experiment and a field control experiment were carried out respectively. Bacillus Velez 3-25 showed good control effect on early blight on detached leaves, and the field control effect of Bacillus Velez 3-25 on early blight was 79.9%.

[0050] In another embodiment of the present invention, the effect of Bacillus Velez strain 3-25 on the rhizosphere microbial community of potato was studied, and the results showed that the relative abundance of Proteobacteria and Firmicutes in the rhizosphere soil bacterial community increased, and the relative abundance of Actinobacteria and Bacteroidetes decreased; the relative abundance of Pseudomonas and Citrobacter increased, and the relative abundance of Paeniglutamicibacter, Arthrobacter and Flavobacterium decreased. The relative abundance of Ascomycota and Basidiomycota in rhizosphere soil fungal community decreased; the relative abundance of Verticillium, Fusarium, Filobasidium, Naganishia, Sporobolomyces and Colletotrichum decreased. By analyzing the composition of 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, the relative abundance of dominant species of bacteria and fungi at the phylum and genus levels, the structure of microbial communities was adjusted, and the enrichment of beneficial microorganisms such as Bacillus and Pseudomonas was increased.

[0051] In the present invention, the promotion of crop growth is preferably manifested 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 nutritional content of the edible part of the crop and increasing the commercial qualification rate of the edible part of the crop. Increasing crop yield includes increasing the single mass or per mu yield of the edible part of the crop. The promotion of crop plant growth preferably includes promoting the increase of leaf area, stem thickening and root elongation of crop plants.

[0052] In the embodiment of the present invention, the Velezac bacteria strain 3-25 has increased tuber quality, commercial potato ratio and starch content compared with the blank control group and the control strain group, and has a significant growth-promoting effect.

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

[0054] Example 1

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

[0056] First, the soil sample was passed through a 20-mesh sieve, 0.2 g of the sieved soil was placed in a 50 mL centrifuge tube, 25 mL of sterile water was added, and vortexed to mix. The suspension was added to 1000 mL of 1 / 10 TSA or NA liquid culture medium for gradient dilution. The dilution method is shown in Table 1.

[0057] Table 1 Suspension dilution method

[0058] Dilution multiple Addition amount 2000 times 500μL 6000 times 167μL 18000 times 56μL 54,000 times 19μL 162000 times 6μL

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

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

[0061] Strain identification

[0062] 1. Bacterial genomic DNA extraction

[0063] (1) Using a sterile toothpick, pick the bacterial suspensions of antagonistic strains 3-25, 2-5, 3-5-2, 2-1, 3-22, and 3-4 stored at -80°C, streak them on LB medium plates for activation, and culture them in a constant temperature incubator at 37°C for 12 h for later use;

[0064] (2) Pick a single colony of the test strain with a sterile 200 μL pipette tip and inoculate it into a glass test tube containing 5 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 12 h.

[0065] (3) Pipette 1.5 mL of bacterial solution into a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, and collect the bacteria;

[0066] (4) Add 1 mL of ddH 2 O, use a vortexer for 30 seconds to mix the bacterial suspension until there are no obvious bacterial clumps suspended, centrifuge again, and discard the supernatant;

[0067] (5) Add 60 μL of 20 mg / mL lysozyme to the tube, invert it to mix thoroughly, and place it in a water bath at 37°C for 40 min, inverting it again every 10-20 min to mix thoroughly;

[0068] (6) Add 650 μL of nucleic acid lysis buffer to the tube and invert it to mix thoroughly;

[0069] (7) Add 250 μL of protein precipitation solution to the tube and vortex it for about 1 min;

[0070] (8) Centrifuge at 12000 rpm for 5 min to obtain 900 μL of supernatant, which was transferred to a new centrifuge tube using a pipette;

[0071] (9) Pipette isopropanol pre-cooled at -20°C into the tube and slowly invert 10 times to mix thoroughly;

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

[0073] (11) Add 500 μL of 75% ethanol solution to the tube, invert it several times to mix thoroughly, centrifuge it at 14,000 rpm for 2 min, and discard the supernatant;

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

[0075] (13) Add 60 μL ddHO to the tube. 2 Resuspend the DNA in 4% CO and store it in a -20°C refrigerator for later use.

[0076] 2PCR amplification

[0077] (1) PCR reaction system: DNA templates of strains 3-25, 2-5, 3-5-2, 2-1, 3-22 and 3-4 were used for amplification using 16SrDNA gene fragments as primers (27F / 1492R). The reaction system is shown in Table 2 below:

[0078] Table 2 PCR reaction system

[0079] Reactants Dosage (μL) Template DNA 1.0 Forward primer 0.5 Reverse primer 0.5 Mix 12.5 <![CDATA[ddH 2 The]]> 10.5 total 25.0

[0080] (2) PCR amplification conditions

[0081]

[0082] (3) The amplified PCR product was entrusted to Beijing Nosai Genome Research Center Co., Ltd. for sequencing.

[0083] 3 Construction of phylogenetic tree

[0084] The phylogenetic tree was constructed using Mega7 software.

[0085] The morphology of strain 3-25 is shown in Figure 1 .Depend on Figure 1 It can be seen that the surface of the colony on the LB solid culture medium is smooth, and in the later stage the surface of the colony is wrinkled and rough, and the whole is white. Gram staining observes that the bacteria are purple and rod-shaped, which are Gram-positive bacteria.

[0086] The sequencing results of strain 3-25 are shown in SEQ ID NO: 1. Using the Blast tool in the NCBI database, the sequence similarity with Bacillus velez is the highest, with a sequence similarity of 93%. The phylogenetic tree of strain 3-25 and its closely related species was constructed using Mega7 software. Figure 2 .Depend on Figure 2 It can be seen that strain 3-25 and Bacillus velez belong to one branch. In summary, strain 3-25 was identified as Bacillus velez. The strain was sent to the China General Microbiological Culture Collection Administration Center for preservation, and the preservation number is CGMCC No.31910.

[0087] Example 2

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

[0089] (1) Take the test strain stored in a -80°C ultra-low temperature refrigerator, use a sterile toothpick to dip a small amount of the bacterial solution of Bacillus Velezii strain 3-25 on an LB plate for activation in an ultra-clean workbench, seal it with a sealing film and invert the plate in a 37°C constant temperature incubator for 12 h for later use;

[0090] (2) Pick a single colony of the test strain with a sterile pipette tip and inoculate it into 5 mL of LB liquid medium. Shake and incubate at 37°C and 200 rpm for 16 h as seed solution.

[0091] (3) Inoculate the seed solution into 200 mL LB liquid culture medium at a volume ratio of 1:50 and shake at 37°C and 200 rpm for 48 h as the culture medium for later use. Adjust the viable bacterial concentration in the culture medium to 1.0 × 10 8 CFU / mL is reserved.

[0092] Example 3

[0093] Testing the ability of Bacillus velez 3-25 to produce IAA, cellulase, siderophore, protease, amylase and phosphate solubilization

[0094] (1) Auxin (IAA) detection

[0095] Use a sterile 200μL pipette tip to pick up a single colony of the pre-activated biocontrol strain 3-25, inoculate it into LB liquid culture medium containing 0.5g / L L-try, shake at 37℃, 200rpm, incubate for 48h, and centrifuge at 12000g for 13min. Pipette 2.0mL of Salkowski reagent and add it to the cell culture plate, then add 1mL of the supernatant of strain 3-25, mix well, wrap the culture plate with tin foil to avoid light, and let it stand at room temperature in the dark for 30min. If the mixture turns pink, it means that the biocontrol strain 3-25 has the ability to produce auxin.

[0096] (2) Cellulase activity detection

[0097] A single colony of the biocontrol strain 3-25 was picked and inoculated into 5 mL of LB liquid culture medium. After shaking at 37°C and 200 rpm for 12 h, it was transferred to 200 mL of LB liquid culture medium at a ratio of 1:50 and continued to shake for 12 h. Two holes were punched on the left and right sides of the cellulose Congo red culture medium at an equidistant distance from the center using a sterile cork borer, 40 μL of the biocontrol strain 3-25 bacterial solution was drawn into the holes, and LB liquid culture medium was used as a control. Each treatment was repeated 3 times and the cells were placed in a constant temperature incubator at 37°C for 3 days. If a transparent circle was produced around the inoculation point, it indicated that the bacteria had the ability to produce cellulase.

[0098] (3) Phosphorus dissolving capacity test

[0099] Refer to the cellulase activity detection method to complete the inoculation of the bacterial liquid in the calcium phosphate-dissolving medium, set LB liquid medium as the control, repeat each treatment 3 times, and culture in a 37°C incubator for 3 days. If a transparent circle is produced, it means that the strain has the ability to dissolve phosphate.

[0100] (4) Siderophore capacity test

[0101] The inoculation of strain 3-25 bacterial liquid on CAS solid culture medium was completed by referring to the cellulase activity detection method, and LB liquid culture medium was set as the control. Each treatment was repeated 3 times and cultured in a 37°C incubator for 3 days. If a transparent circle was produced, it indicated that the strain had the ability to favor siderophore.

[0102] (5) Protease activity detection

[0103] The strain 3-25 bacterial liquid was inoculated into skim milk solid culture medium according to the cellulase activity detection method. LB liquid culture medium was set as the control. Each treatment was repeated 3 times. The culture was placed in a 37°C incubator for 24h, 48h, and 72h, and then the area around the inoculation hole was observed. If a transparent circle was produced, it indicated that the strain produced protease, and the diameter of the transparent circle represented the amount of protease produced by the strain.

[0104] (6) Amylase activity detection

[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, the leaves were placed in 200 mL of biocontrol strain culture medium, LB liquid medium and sterile water and soaked for 10 min, respectively. Each treatment was repeated 3 times, with 10 leaves in each group.

[0117] (4) Place a piece of sterile filter paper on the culture dish, add 1 mL of sterile water to moisten the filter paper, place the leaf on the filter paper with the back of the leaf facing up, blow dry on the clean bench, place in a sealed box covered with four layers of moist gauze, seal the sealed box with plastic wrap, and place in a 12-h photoperiod, 25°C light incubator for moisturizing for 24 hours; after 24 hours, inoculate the early blight fungus cake with consistent activity on the back of the leaf, put it back into the sealed box, seal it with plastic wrap, and continue to culture in a 12-h photoperiod, 25°C light incubator for 4-5 days. During this period, pay attention to observe the disease situation and pay attention to hydration and moisturizing; 4-5 days after inoculation of the pathogen, count the disease level of the potato leaves, calculate the prevention and control effect, and take photos for record.

[0118] The results are shown in Table 3.

[0119] Table 3 Early blight control results on detached leaves

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

[0121] Note: abcd notation is used to indicate significant differences between different groups.

[0122] From Table 1 and Figure 5 It can be seen that the field control effect of Bacillus Velez strain 3-25 on early blight is 79.9%. At the same time, the tuber quality, commercial potato proportion and starch content are also increased compared with the control group, which has a significant growth-promoting effect.

[0123] Example 6

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

[0125] Fermentation process: pick colonies of 3-25 biocontrol strains respectively and inoculate them into a triangular flask containing 300mL of liquid LB culture medium and 2L of medium, shake and culture at 37°C and 200rpm for 16h to prepare seed solution; connect the seed solution to an industrial fermentation tank for industrial fermentation, take samples for microscopic examination after 36h, and stop fermentation when the bacillus production in the field of view reaches 80%; filter the fermentation liquid of the biocontrol strain, and detect the bacterial content of the biocontrol strain before and after filtration by the live bacteria counting method.

[0126] The test results showed that the bacterial content of biocontrol strain 3-25 in the fermentation broth was about 8×10 10 CFU / mL.

[0127] Example 7

[0128] Stability determination of active substances in the fermentation supernatant of Bacillus velezinsis strain 3-25

[0129] Use a sterile 200μL pipette tip to pick up single colonies with clear morphology of the above strains, inoculate them into 5mL LB test tubes, culture them at 37°C and 200rpm for 12h, transfer them to a 500mL Erlenmeyer flask containing 200mL liquid LB culture medium at a ratio of 1:50, and continue to shake and culture for 36h; respectively take 1.5mL of the culture solution of each of the above strains and add it to a sterilized 2.0mL centrifuge tube, centrifuge at 12000rpm for 5min to obtain supernatant and bacteria; transfer the supernatant to a new sterilized 2.0mL centrifuge tube, filter it with a 0.22μm bacterial filter to obtain a sterile supernatant; wash the obtained bacteria with 1.5mL of sterile water, vortex and centrifuge again, repeat 3 times, add 1.5mL of sterile water to the finally obtained bacteria, vortex and oscillate, and then make a bacterial suspension.

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

[0131] Use a sterile cork hole puncher to punch three evenly distributed holes at equal distances from the center of the circle on the PDA plate; use a sterile cork hole puncher to punch a bacterial cake at the edge of the colony of the early blight pathogen HCL-17 on the 7th day of culture, and inoculate it into the center of the PDA culture medium; respectively, aspirate 40 μL of the sterile supernatant of biocontrol bacteria 3-25 and the sterile supernatant under the above different treatments and add them to the holes, and repeat 3 plates for each treatment; place the plate in a 25℃ climate incubator and culture in the dark for 10 days, measure the radius of the pathogen in the control group, the radius of the pathogen in the treatment group and the width of the inhibition zone, and take photos for record.

[0132] Results Figure 6-7 and Table 4.

[0133] Table 4 Stability test results

[0134]

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

[0136] There was no significant difference between the sterile supernatant of strain 3-25 treated with proteinase K and the untreated supernatant of strain 3-25 on early blight. After the sterile supernatant of strain 3-5 was treated at 60℃, 80℃ and 100℃, the inhibitory effect on early blight was also significantly different. Similarly, the antibacterial activity of the supernatant of strain 3-25 remained stable when irradiated with ultraviolet light for 0.5h and 2h. The results showed that the antibacterial active substances in the sterile supernatant of strain 3-25 showed stability under the conditions of proteinase K, high temperature and ultraviolet light, and their antibacterial activity did not change significantly. The culture solution, bacterial suspension and metabolic solution of Bacillus velez 3-25 can inhibit the growth of early blight hyphae, among which the culture solution had the strongest inhibitory activity, followed by the bacterial suspension, and the metabolic solution was relatively poor.

[0137] Example 8

[0138] Treatment of potato seedling roots with Bacillus velezensis strain 3-25

[0139] The Velez Bacillus strain 3-25 marker strain is obtained by transforming the Escherichia coli pGFP-78 plasmid containing the GFP protein encoding gene into the Velez Bacillus strain 3-25, and it is verified that the Velez Bacillus strain 3-25 marker strain containing the GFP protein encoding gene is obtained. The specific steps are as follows.

[0140] 1. Extraction of E. coli pGFP-78 plasmid DNA containing GFP protein encoding gene

[0141] The LQ-3GFP-marked detection strain (see the prior art: Li Rong, Hong He, Jiang Wenxiao, et al. Effect of Velezella LQ-3 on the prevention and treatment of wheat sheath blight and its rhizosphere colonization ability [J]. Journal of Wheat Crops, 2024, 44(07): 947-954.) was used as the material to extract the Escherichia coli pGFP-78 plasmid containing the GFP protein encoding gene. 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 and electroporation of competent cells of Bacillus velezine strain 3-25

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

[0144] (2) The seed solution was inoculated into LB growth medium at a dilution of 1:100 and cultured at 37°C with shaking at 200 rpm (until OD600 was 0.35-0.55);

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

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

[0147] (5) Take out the electroporation buffer from the refrigerator and use it to wash the cells 4-5 times, using 1 mL each time. Centrifuge for 5 min and collect the cells. Do not pour out all the cells for the last time.

[0148] (6) Add the appropriate electroporation buffer according to the number of electroporations, pre-cool the 1.5 mL centrifuge tubes on ice, and dispense the contents into the tubes;

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

[0150] (8) Take out the tube and quickly add 900 μL of recovery medium. Mix well and transfer to a 15 mL centrifuge tube. Shake at 160 rpm for 3 to 5 h and spread on the corresponding resistance LB plate.

[0151] After the above operation, strain 3-25 was marked with a plasmid fused with GFP protein to obtain strain 3-25 (pGFP78). A single colony was picked and inoculated into ordinary LB medium, and the seed solution was cultured overnight to obtain the culture solution of strain 3-25 (pGFP78). Potato seedlings with the same growth potential were selected and root-irrigated with the culture solution of 3-25 marked strain. Each potato seedling was root-irrigated with 10 mL of culture solution, and each treatment was repeated 4 times. The potato root tissues were collected at 1d, 3d, 5d, 7d, 10d and 14d after the roots of potato plants with marked strains were irrigated. The collected roots were rinsed with PBS buffer, and their weights were weighed and recorded. The roots were placed in a sterilized 2.0mL centrifuge tube, and the root tissues were ground into a homogenate with a sterilized mortar and pestle. 1mL of sterile water was added to mix thoroughly, and then diluted by gradient dilution method. 100μL of the diluted suspension was applied to an LB medium plate containing 20mg / mL tetracycline. Four strains were collected at each time point, and three plates were repeated for each treatment. The plates were placed in a 37℃ constant temperature incubator for culture, and counted when the colonies grew evenly. Gently shake off the loose soil from the roots of the collected potato plants, collect 1-2 mm of soil from the rhizosphere surface, weigh it, and then perform gradient dilutions on it. Pipette 100 μL of the diluted suspension and spread it on an LB medium plate containing 20 mg / mL tetracycline. Repeat 3 plates for each treatment and culture them in a constant temperature incubator at 37°C. Count the colonies when they grow evenly.

[0152] Results Figure 8 The strain 3-25 (pGFP78) could colonize in the rhizosphere soil and root system of potato seedlings. On the first day, the colonization rate of strain 3-25 (pGFP78) in the rhizosphere soil of potato seedlings was 1.5×10 5 CFU / g·w, the colonization amount in the seedling root system was 5.2×10 4 CFU / g·w, and then the colonization of strain 3-25(pGFP78) in the rhizosphere soil showed a decreasing trend. On the 10th day, the colonization of strain 3-25(pGFP78) in the rhizosphere soil of potato decreased to 9.8×10 4 CFU / g·w, while the colonization of strain 3-25(pGFP78) in potato roots first showed an increasing trend and reached the highest value of 5.8×10 4 CFU / g·w, and then the colonization amount decreased slightly after 10 days

[0153] Example 9

[0154] Field early blight control effectiveness testing

[0155] 1. Trial location

[0156] No. 8, East 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 40cm wide and 120m long in total. Each potato tuber is 20cm apart and planted at a depth of 10cm. Two ridges are designed as a group of treatments. Two ridge protection rows are designed between different treatments. The ridge length for the field control experiment is 30m, and the ridge length for the field growth promotion experiment is 70m. There is a 20m interval between the control experiment and the growth promotion experiment as an isolation row. Clear water is used as a blank control.

[0159] 4. Application methods of biocontrol strains

[0160] The first spraying was carried out after the potato plants emerged, and 24 hours after the first spraying of the fermented liquid of the biocontrol strain, the inoculation concentration in the field was 1.0×10 4 The spore suspension of early blight fungus was prepared at a concentration of 100 / mL to ensure the occurrence of potato early blight in the field, simulating the field where potato early blight occurred. The antagonistic bacteria solution was then sprayed every 25 days during the potato growth period, for a total of 3 times. The fermentation liquid of the biocontrol strain was sprayed with an electric sprayer until the fermentation liquid wetted the potato leaves but did not leak. The water consumption was about 675 L·hm -2 The fermentation liquid should be sprayed in weather without strong winds, rain and high temperatures. The disease situation was investigated 7 days after the last fermentation liquid spraying (see Table 5 and Fig. 9 ).

[0161] Table 5 Potato early blight classification standards

[0162] level Disease Level 0 No disease spots Level 1 The lesion area accounts for less than 5% of the entire leaf area Level 3 The lesion area accounts for 6%-10% of the entire leaf area Level 5 The lesion area accounts for 11%-20% of the entire leaf area Level 7 The lesion area accounts for 21%-50% of the total leaf area Level 9 The lesion area accounts for more than 51% of the entire leaf area

[0163] Each treatment was divided into three plots, and five random sampling points were taken in each plot. Three plants were investigated at each point. Fifteen leaves on the upper, middle and lower layers of each potato plant were investigated respectively. The leaves were graded according to the proportion of the early blight spot area on the leaves to the total leaf area, and the diseased leaf rate, disease index and relative control effect were calculated.

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

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

[0166]

[0167] Note: abcd are used to indicate significant differences between different groups.

[0168] The field control effect of Bacillus Velez strain 3-25 on early blight was 79.9%. At the same time, the tuber quality, commercial potato ratio and starch content also increased compared with the control group, showing a significant growth-promoting effect.

[0169] Example 10

[0170] Determination of Microbial Community in Potato Rhizosphere Soil

[0171] 1. Collection of potato rhizosphere soil

[0172] Each treatment group adopted the random sampling method. Three points were randomly selected, and the roots of three potato plants in each point were randomly dug with a shovel. The loose non-rhizosphere soil on the outer layer of the roots was gently shaken off. The roots of the plants were cut with scissors, and the 1-2mm soil attached to the roots was collected as the rhizosphere soil and put into a sterile sampling bag. The sterile bags containing soil and more ice packs frozen in a -20℃ refrigerator were placed in a foam box and brought back to the laboratory as soon as possible; after removing larger impurities such as stones and plant roots in the soil, the soil samples were sieved with a 100-mesh sieve disinfected with 75% alcohol. The sieved soil was fully mixed and then divided into 2mL centrifuge tubes sterilized with high temperature and high pressure, and stored in a -80℃ ultra-low temperature refrigerator for subsequent soil genome extraction.

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

[0174] The DNA from the rhizosphere soil was extracted using the Fast DNAS SPIN Kit for soil, and the extracted DNA was entrusted to Meiji Biotech for high-throughput sequencing.

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

[0176] Comparative Example 1

[0177] Comparative experiment on prevention and growth promotion effects

[0178] According to the method of Example 1, multiple strains of bacteria were isolated from healthy potato soil, and the same morphological and molecular identification methods were used to obtain strains 2-5, 3-5-2, 3-22, 2-1 and 3-4, all of which belonged to Bacillus velezensis.

[0179] Bacillus velez strain 3-25, Bacillus velez strain 2-5, Bacillus velez strain 3-5-2, Bacillus velez strain 3-22, Bacillus subtilis strain 2-1, and Bacillus subtilis strain 3-4 were used as experimental strains to carry out field control efficacy experiments, and the method was the same as that of Example 9. A blank control group was also set up.

[0180] The results are shown in Table 7.

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

[0182]

[0183] Note: abcd are used to indicate significant differences among different groups.

[0184] Field control experiments showed that the fermented liquid of the six antagonistic bacteria 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%, 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 which strain 3-25 had the best field control effect. The results of the field growth promotion experiment showed that the six strains also increased the tuber quality, commercial potato proportion and starch content compared with the control group, and had a significant growth promotion effect, but strain 3-25 also had a high-efficiency field control effect and the effect of promoting potato yield and quality.

[0185] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

2. A culture of the Bacillus Velezii strain 3-25 of claim 1.

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

4. A bacteriostatic agent for early blight pathogenic bacteria, characterized in that: It comprises the Bacillus Velez strain 3-25 of claim 1 or the culture of claim 2 or 3.

5. The antibacterial agent according to claim 4, characterized in that When the antibacterial agent comprises the Velez Bacillus strain 3-25 or its bacterial suspension, the active concentration of the Velez Bacillus strain 3-25 is 10 8 ~10 10 CFU / mL; When the antibacterial agent contains the culture solution of the Velezac strain 3-25, the volume percentage of the culture solution is 10% to 90%; When the antibacterial agent contains the metabolites of the Velezac bacteria strain 3-25, the volume percentage of the metabolites is 10% to 90%.

6. A biological pesticide, characterized in that: The method comprises the Bacillus Velez strains 3-25 according to claim 1 or the culture according to claim 2 or 3 and auxiliary materials acceptable in the pesticide field.

7. Use of the Bacillus velezensis strain 3-25 of claim 1, the culture of claim 2 or 3, the antibacterial agent of claim 4 or 5, or the biological pesticide of claim 6 in preventing and controlling early blight of crops and / or promoting crop growth.

8. The use according to claim 7, characterized in that: The crop early blight includes at least one of the following: potato early blight, tomato early blight and pepper early blight.

9. The use according to claim 7 or 8, characterized in that: The method for preventing and controlling early blight of crops comprises at least one of the following aspects: Inhibit the hyphae growth of early blight pathogens; The Bacillus Velez strain 3-25 is colonized in the rhizosphere soil of the crop; and regulate the relative abundance of crop rhizosphere soil microbial flora.

10. The use according to claim 7 or 8, characterized in that: 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.

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