Bacillus amyloliquefaciens MY-7 and application of bacillus amyloliquefaciens MY-7 in prevention and treatment of phytopathogen

Abstract

The bacillus albus MY-7 disclosed by the invention has a gene related to synthesis of bacteriocin, has obvious broad-spectrum antibacterial activity, and has an obvious inhibition effect on various plant pathogenic bacteria including cabbage alternaria alternata, fusarium tripteroides, alternaria alternata, geotrichum candidum, colletotrichum gloeosporioides, broad bean botrytis cinerea, penicillium digitatum and A. arundinis; and the bacillus subtilis has good environmental adaptability, is expected to be used for biological prevention and control and preparation of biological prevention and control agents, biological fertilizers, plant growth promoters and related microbial preparations, and is favorable for reducing the use of chemical pesticides and promoting the development of sustainable agriculture after being popularized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a white bacillus MY-7 and an application thereof in preventing and controlling plant pathogens. Background Art

[0002] The threat of microorganisms to human health and food safety is always of concern to the public and policymakers. Among them, plant diseases caused by outbreaks of plant pathogens are one of the most important threats to food safety. Plant diseases are the main constraint on crop production in all agricultural and horticultural systems. Almost all crops are susceptible to various plant pathogens. Plant diseases may cause property losses to individuals or countries, and in severe cases may even lead to famine.

[0003] Plant disease management is an important cost component in crop production. In traditional agricultural ecosystems, methods for controlling pathogens include breeding resistant crop varieties, preventing the spread of contaminated soil or seeds, and spraying pesticides. Although chemical pesticides are quite effective in controlling plant diseases, excessive use of chemical pesticides often leads to the following hazards: (1) resistance of plant pathogens and pests; (2) pollution of the atmosphere, soil, surface water and groundwater; (3) harm of pesticide residues in plants to non-target organisms, such as the step-by-step transmission and concentration of pesticides that are difficult to degrade and have strong bioaccumulation in different organisms. In recent years, the demand to replace these chemical pesticides has increased people's attention to biological control of plant diseases, and biological control has shown its appeal due to its unique advantages. Biological control is to inhibit diseases and prevent pathogens from infecting plants by applying biological control agents (BCAs) to plants or soil. BCAs are usually fungi, bacteria or viruses or a mixture of them. They have the characteristics of clear targets, low resistance, no pollution, no residue, and can regulate soil microecology. In fact, since 1990, the global sales of chemical pesticides have been on a continuous downward trend. The Action Plan for Reducing the Use of Chemical Pesticides by 2025 emphasizes further promoting the application of biopesticides and microbial fertilizers in a wider range and more scenarios. Internationally, the use of biopesticides has increased by nearly 10% each year, and it is expected that biopesticides will share the same market share with synthetic pesticides in the 2040s and 2050s.

[0004] Among bacteria, Bacillus has strong adaptability and survival ability, making it suitable as a biocontrol agent. Although it also has the stress resistance and broad-spectrum antibacterial activity of Bacillus, the antibacterial spectrum of different Bacillus species and the inhibitory effect on the same plant pathogen may still be significantly different. Therefore, it is still necessary to develop Bacillus with a broader antibacterial spectrum and stronger inhibitory effect on specific plant pathogens. Summary of the invention

[0005] In view of the defects of the prior art, the present invention has discovered a strain of Bacillus albus MY-7, which exhibits significant inhibitory effects on pathogens of several plant diseases.

[0006] In one aspect, the present invention provides a Bacillus albus MY-7, and the deposit number of the Bacillus albus MY-7 is CCTCC NO: M20241653.

[0007] In one or more embodiments, the Bacillus albicans MY-7 has a gene related to the synthesis of bacteriocin (Bacillibactin), and the gene related to the synthesis of bacteriocin is selected from one or more of DhbE, DhbB and DhbF.

[0008] Preferably, the white Bacillus MY-7 has genes related to the synthesis of bacteriocin, and the genes related to the synthesis of bacteriocin are DhbE, DhbB and DhbF. In another aspect, the present invention provides the use of the white Bacillus MY-7 as described in any embodiment of the present invention in the production of bacteriocin.

[0009] In another aspect, the present invention provides a bacterial agent, comprising the white Bacillus MY-7 or its culture solution as described in any embodiment herein.

[0010] In one or more embodiments, the bacterial agent is bacterial powder obtained after drying.

[0011] Preferably, the bacterial powder is freeze-dried bacterial powder or spray-dried bacterial powder.

[0012] In another aspect, the present invention provides the use of Bacillus albus MY-7 as described in any embodiment herein or the bacterial agent as described in any embodiment herein in controlling plant pathogens, wherein the plant pathogens are selected from one or more of Alternaria brassicicola, Fusarium tricinctum, Alternaria alternat, Geotrichum candidum, Colletotrichum gloeosporioides, Botrytis fabae, Penicillium digitatum, and Apiospora arundinis (A.arundinis).

[0013] Preferably, the plant pathogen is selected from one or more of Alternaria brassicicola, Fusarium tricinctum, Geotrichum candidum, Colletotrichum gloeosporioides, Botrytis fabae, Penicillium digitatum and Apiospora arundinis (A.arundinis).

[0014] More preferably, the plant pathogen is selected from one or more of Alternaria brassicicola, Geotrichum candidum, Colletotrichum gloeosporioides, Botrytis fabae, and Apiospora arundinis (A.arundinis).

[0015] In another aspect, the present invention provides a method for preparing the bacterial agent as described in any embodiment of the present invention, the method comprising inoculating Bacillus albus MY-7 into a liquid culture medium for culturing, and diluting the culture solution by an appropriate multiple to obtain the bacterial agent.

[0016] In one or more embodiments, the method comprises inoculating Bacillus albus MY-7 into a liquid culture medium for activation culture; after the activation culture, taking an appropriate amount of the activation culture solution and inoculating it into a fermentation culture medium for fermentation culture; after the fermentation culture, taking the fermentation culture solution and diluting it by an appropriate multiple to obtain the bacterial agent.

[0017] In one or more embodiments, the volume fraction of the Bacillus albus MY-7 inoculated in the liquid culture medium is 1 to 10%, and the activation culture time is 8 to 24 hours.

[0018] Preferably, the volume fraction of the Bacillus albicans MY-7 inoculated in the liquid culture medium is 2-5%. More preferably, the volume fraction of the Bacillus albicans MY-7 inoculated in the liquid culture medium is 2%.

[0019] Preferably, the activation culture time is 10 to 18 hours. More preferably, the activation culture time is 12 hours.

[0020] In one or more embodiments, the volume fraction of the activated culture solution inoculated into the fermentation medium is 1 to 10%, and the fermentation culture time is 18 to 48 hours.

[0021] Preferably, the volume fraction of the activated culture solution inoculated into the fermentation medium is 2-5%. More preferably, the volume fraction of the activated culture solution inoculated into the fermentation medium is 2%.

[0022] Preferably, the fermentation culture time is 24 to 36 hours. More preferably, the fermentation culture time is 24 hours or 36 hours.

[0023] In one or more embodiments, the dilution factor is preferably 0 to 22 times.

[0024] Preferably, the suitable dilution multiple is 2 to 20 times. More preferably, the suitable dilution multiple is 2 times, 5 times, 10 times, 15 times or 20 times.

[0025] In one or more embodiments, the method further comprises freeze-drying or spray-drying the bacterial agent to prepare freeze-dried bacterial powder or spray-dried bacterial powder.

[0026] In one or more embodiments, the inlet temperature of the spray drying is 100-150°C, the feed rate is 400-1000 mL / h, and the air inlet rate is 10-60 M 3 / h, the spray pressure is 0.1~1.0MPa.

[0027] Preferably, the inlet temperature of the spray drying is 110°C, 120°C or 130°C; preferably, the feed rate is 500mL / h, 600mL / h or 700mL / h; preferably, the air inlet rate is 20M 3 / h,25M 3 / h or 30M 3 / h; preferably, the spray pressure is 0.2MPa, 0.25MPa or 0.3MPa.

[0028] Bacillus albus is a new type of Bacillus species discovered in recent years. It has the functions of degrading aniline and ammonia nitrogen organic matter, degrading aflatoxin, degrading microplastics, degrading glyphosate, treating tobacco wastewater, preparing microbial inoculants for bio-organic fertilizer, preparing γ-polyglutamic acid and promoting plant growth. At the same time, it can also inhibit plant pathogens such as Xanthomonas oryzae, Fusarium oxysporum, Curvularia pathogens, Agrobacterium tumefaciens and Rhizoctonia solani. The Bacillus albus MY-7 discovered by the present invention shows a significant inhibitory effect on the pathogens of several plant diseases. Through whole genome sequencing, it was found that the strain has a complete bacteriocin synthesis pathway and has good application prospects. MY-7 is expected to be used for biological control, preparation of biological control agents, biological fertilizers, plant growth promoters and related microbial preparations. Its promotion will help reduce the use of chemical pesticides and promote the development of sustainable agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the colony morphology of MY-7.

[0030] Figure 2 This is the cell morphology of MY-7 under a microscope.

[0031] Figure 3 This is the cell morphology of MY-7 under a scanning electron microscope.

[0032] Figure 4 This is the phylogenetic tree of MY-7 based on 16S rRNA sequences.

[0033] Figure 5 This is the whole genome circle map of MY-7.

[0034] Figure 6 This is the genome circle map of MY-7 plasmid A.

[0035] Figure 7 It is a gene related to the synthesis of MY-7 bacteriocin.

[0036] Figure 8 This is a 1% agarose gel electrophoresis diagram of the qPCR products of the MY-7 bacteriocin synthesis-related genes.

[0037] Fig. 9 This is a graph showing the inhibitory effect of MY-7 fermentation liquid on 9 plant pathogenic fungi. Fig. 9 Figure A is a graph showing the inhibitory effect of 12h+24h fermentation liquid of MY-7 on 9 kinds of plant pathogenic fungi; Figure B is a graph showing the inhibitory effect of 12h+36h fermentation liquid of MY-7 on 9 kinds of plant pathogenic fungi.

[0038] Preservation matters

[0039] The Bacillus albus MY-7 obtained in the present invention was submitted to the China Center for Type Culture Collection (CCTCC) located at Wuhan University, Wuhan, China for preservation on July 22, 2024, with the preservation number being CCTCC NO: M20241653. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] The inventors of this application isolated a strain from stinky tofu in Pudong, Shanghai, and identified it as Bacillus albus, named MY-7, based on colony morphology observation, 16S rRNA and whole genome sequencing. Further research found that the Bacillus albus MY-7 has broad-spectrum antibacterial activity against pathogenic bacteria and has broad application prospects. The Bacillus albus MY-7 was submitted to the China Center for Type Culture Collection (CCTCC) for preservation on July 22, 2024, with the preservation number CCTCC NO: M20241653.

[0042] Part of the culture medium of the present invention is prepared according to the following formula:

[0043] LB medium (LB liquid medium): 5 g yeast extract powder, 10 g tryptone, 10 g sodium chloride, dilute to 1 L with distilled water, adjust the pH to 7±0.05, sterilize at 115°C for 20 min, and set aside.

[0044] LB solid medium (LB plate): 5 g yeast extract powder, 10 g tryptone, 10 g sodium chloride, 20 g agar, dilute to 1 L with distilled water, adjust the pH to 7 ± 0.05, sterilize at 115 ° C for 20 minutes, and set aside.

[0045] Fermentation medium: 5 g yeast extract powder, 10 g tryptone, 10 g sodium chloride, 20 g glucose, dilute to 1 L with distilled water, adjust pH to 7±0.05, sterilize at 115°C for 20 min, and set aside.

[0046] YPD solid culture medium (YPD plate): 20 g of peptone, 10 g of yeast extract powder, 20 g of glucose, 20 g of agar, dilute to 1 L with distilled water, sterilize at 115°C for 20 minutes, and set aside.

[0047] Example 1: Isolation, screening and identification of Bacillus albus MY-7

[0048] 1.1 Isolation and purification of bacteria

[0049] The stinky tofu samples were collected from the vegetable market in Pudong New District, Shanghai, and the samples were placed in clean bags. 10 g of the collected samples were mixed evenly with 90 mL of sterile water. 1 mL of the mixture was added to a test tube containing 9 mL of sterile water to obtain 1×10 -1 Diluent, gradient dilution, successively diluted to 1×10 -3 , 1×10 -5 The concentration ranged from 1×10 -1 , 1×10 -3 , 1×10 -5Take 100 μL of the dilution of the concentration and spread it on the LB plate, spread it evenly, and place it in a 30℃ constant temperature incubator for 1 to 2 days, and observe the growth of the colony regularly. After a single colony grows, use a sterile pipette tip to dip colonies of different morphologies and streak them on the LB plate for culture. Repeat several times until the colony morphology is stable. The obtained strains are numbered and made into glycerol strains, which are stored in a -80℃ refrigerator.

[0050] 1.2 Morphological characteristics

[0051] Take out MY-7 from the -80℃ refrigerator, inoculate it into LB medium at a 2% (v / v) inoculation amount, and activate and culture it for 10 hours; streak the activated cultured bacterial liquid on the LB plate, and culture it in a 30℃ incubator for 16.5 hours. After it grows into a single colony, observe the colony morphology and take photos to record it. The observation results are as follows: Figure 1 The observation contents include colony shape, color, surface, edge, transparency, ridge shape, etc.

[0052] Depend on Figure 1 It can be seen that the colonies are round, milky white, smooth, with regular edges, opaque, and slightly raised.

[0053] 1.3 Microscopic morphological characteristics

[0054] Select the above-mentioned cultured bacterial strains for Gram staining microscopy, according to the method of "Common Bacterial System Identification Manual", and the microscopic examination results are as follows: Figure 2 As shown, the bacteria are short rod-shaped and Gram-stained positive (blue-purple).

[0055] 1.4 Scanning electron microscopy morphological characteristics

[0056] MY-7 was inoculated in LB liquid medium at an inoculum of 2% (v / v), and cultured at 30°C shaking for 10 hours to activate the first generation. The activated MY-7 bacterial solution was then inoculated in a fermentation medium at an inoculum of 2% (v / v), and cultured at 30°C shaking for 11 hours to the logarithmic growth phase; the bacterial solution in the logarithmic growth phase was taken, centrifuged at 5000rpm for 3 minutes, the supernatant was discarded, and the bacteria were washed 3 times with PBS; after discarding the PBS, 2.5% glutaraldehyde was added to resuspend and fix the bacteria for 16 hours; after fixation, the solution was centrifuged at 4°C, 5000rpm for 3 minutes, the fixative was discarded, 1 mL PBS solution was added to resuspend and wash the bacteria, and after standing for 10 minutes, the solution was centrifuged at 4°C, 5000rpm for 3 minutes, and the supernatant was discarded. The washing process of resuspending, standing, centrifuging, and discarding the supernatant was repeated 2 more times.

[0057] After washing three times, 1 mL of 50% (v / v) ethanol aqueous solution was added to resuspend the bacteria, and the cells were allowed to stand for dehydration for 15 min. The cells were centrifuged at 5000 rpm for 3 min, and the supernatant was discarded. The same operation was then performed in sequence, with 60% (v / v), 70% (v / v), 80% (v / v), 90% (v / v), and 100% (v / v) ethanol aqueous solutions for gradient dehydration, wherein the 100% ethanol dehydration was repeated twice, and finally the bacteria were resuspended with 1 mL of 100% ethanol to obtain ethanol-resuspended bacterial solution A.

[0058] Take 20μL of ethanol-resuspended bacterial solution A and drop it on the coverslip, pre-freeze it in a -80℃ refrigerator for 15 minutes, and then freeze-dry it in a freeze dryer at -55℃ for 2 hours. The freeze-dried cells are dehydrated in 30% (v / v), 50% (v / v), 60% (v / v), 70% (v / v), 80% (v / v), 90% (v / v) and 100% (v / v) ethanol in a gradient manner (i.e., resuspend, stand and centrifuge) for 10 minutes. The 100% ethanol dehydration is repeated twice, and finally the bacteria are resuspended with an appropriate amount of 100% ethanol to obtain ethanol-resuspended bacterial solution B. Pipette ethanol-resuspended bacterial solution B and drop it on the coverslip, pre-freeze it in a -80℃ refrigerator for 1 hour, and then freeze-dry it for 15 hours to obtain the observation sample. Spray gold on the surface of the observation sample, and then use an S-3400N scanning electron microscope to observe and take pictures. The observation results are as follows Figure 3 shown.

[0059] Depend on Figure 3 It can be seen that MY-7 has a rod-shaped morphology observed under a scanning electron microscope, which is consistent with the typical characteristics of Bacillus.

[0060] 1.5 Identification of physiological and biochemical characteristics

[0061] With reference to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems, the physiological and biochemical performance indicators of MY-7, including D-glucose utilization ability, sucrose utilization ability, D-galactose utilization ability, fructose utilization ability, starch hydrolysis ability, gelatin liquefaction ability, casein hydrolysis ability, indole test, H2S production test, VP test, and methyl red test were observed. The results are shown in Table 1 below; "+" represents the ability, and "-" represents the lack of or almost no ability.

[0062] Table 1: Physiological and biochemical test results

[0063] Physiological and biochemical performance indicators result D-Glucose + sucrose + D-Galactose + fructose + Starch hydrolysis + Gelatin liquefaction + Casein Hydrolyzate + Indole test - <![CDATA[H2S production test]]> - VP test - Methyl red test +

[0064] 1.6 Molecular Biological Identification

[0065] The DNA of strain MY-7 was extracted using a bacterial DNA extraction kit, and the 16S primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were selected to perform PCR amplification on the 16S rRNA sequence of strain MY-7. The resulting PCR product was sequenced by Qingke Biotechnology (Shanghai) Co., Ltd.

[0066] The 16S rRNA sequence of strain MY-7 was compared with the NCBI database, and the sequencing results were analyzed using MEGA 11 software. The Neighbour-Joining method (NJ) was used to construct a phylogenetic tree. The bootstrap test was used to estimate the reliability of the constructed phylogenetic tree. The multi-gene phylogenetic tree was constructed with 1000 repetitions to clarify the classification status of strain MY-7. The results of the phylogenetic tree construction are shown in the figure. Figure 4 As shown, it can be seen that according to the multi-gene phylogenetic tree of strain MY-7 16S rRNA, the strain is clustered on the same branch with Bacillus albus, with the highest similarity.

[0067] According to the above analysis of the results, combined with the strain morphological characteristics and phylogenetic tree construction and identification, strain MY-7 belongs to Bacillus albus.

[0068] 1.7 Analysis of the whole genome structure of MY-7

[0069] Bacillus albus MY-7 was cultured to the exponential growth phase. After centrifugation to obtain bacterial pellets, the bacterial cells were cryopreserved and sent to Meiji Biotechnology Co., Ltd. (Shanghai) for DNA extraction. The whole genome was sequenced and assembled using a combination of second-generation Illumina and third-generation PacBio sequencing technologies.

[0070] The sequencing results showed that the whole genome of Bacillus albus MY-7 was a 5,269,880 bp circular chromosome and a natural circular plasmid, with a GC content of 35.56% and 5288 coding genes. The visualized genome of strain MY-7 is shown as follows: Figure 5 and Figure 6 Circle diagram of the genome shown.

[0071] Through antiSMASH online prediction and NCBI BLAST comparison analysis, the secondary metabolites encoded by Bacillus albus MY-7 include petrobactin, bacillibactin, fengycin and molybdenum cofactor. The similarities between the gene clusters encoding these secondary metabolites and the existing gene clusters are 100%, 85%, 40% and 23%, respectively. The results are shown in Table 2.

[0072] The above whole genome structure analysis reflects the unique characteristics of the Bacillus albus MY-7 genome.

[0073] Table 2: Identification results of MY-7 secondary metabolite gene cluster

[0074] Coding gene cluster Similarity (%) Petroleobactin 100 Bacteriocin 85 Fengyuansu 40 Molybdenum cofactor 23

[0075] Example 2: Genes related to bacteriocin synthesis in MY-7

[0076] The KEGG database was used to retrieve genes related to bacteriocin synthesis, including DhbE, DhbB and DhbF. The three genes are involved in the complete pathway of bacteriocin synthesis, and their corresponding relationships with the bacteriocin structural formula are shown in the following figure. Figure 7 As shown; DhbF is involved in the synthesis of the mother nucleus (in the dotted box) of bacteriocin, and DhbE and DhbB enzymes are involved in the synthesis of the complete structure of bacteriocin. Since the DhbF gene is 7149 bp long, it is divided into three segments (DhbF1, DhbF2 and DhbF3). The target gene sequence was imported into NCBI, and the length of the PCR target gene was selected to be 150-250 bp. NCBI was further used to design specific primers for the target gene. The primers and their sequences are shown in Table 3. Total RNA was extracted according to the instructions of the Bacteria RNA Extraction Kit R403 kit, and the RNA was purified according to the method of the PCR kit. III RT SuperMix for qPCR (+gDNA wiper) R323 kit instructions to remove genomic DNA, perform reverse transcription reaction, PCR, and finally obtain PCR products containing the target gene. Preliminary confirmation was performed by 1% agarose gel electrophoresis. The results are as follows Figure 8As shown in the figure (M represents Marker DL2503; 1 to 5 represent the PCR products of genes DhbE, DhbB, DhbF1, DhbF2 and DhbF3 in strain MY-7 sample 1; 6 to 10 represent the PCR products of genes DhbE, DhbB, DhbF1, DhbF2 and DhbF3 in strain MY-7 sample 2). The PCR products were simultaneously sent to Qingke Biotechnology (Shanghai) Co., Ltd. for sequencing verification, and the sequence matching degree of all products and genes was 100%, proving that MY-7 has all genes in the bacteriocin synthesis pathway and can synthesize bacteriocin.

[0077] Table 3: RT-PCR primer sequences

[0078]

[0079]

[0080] Example 3: Broad-spectrum antibacterial experiment of MY-7 against plant pathogens

[0081] Sterilize at 115℃ and cool to about 55℃, pour 15mL YPD medium that has not yet solidified into a sterile culture dish, and make a YPD plate after it solidifies. Frozen cakes of 9 pathogenic fungi, including Alternaria brassicicola, Fusarium tricinctum, Alternaria alternat, Geotrichum candidum, Colletotrichum gloeosporioides, Botrytis fabae, Penicillium digitatum, and Apiospora arundinis (A.arundinis), which were screened from the soil of the vegetable greenhouse in Qingpu, Shanghai, were used to pick a small amount of mycelium with a sterile inoculation needle and inoculated in the middle of the PDA plate, and cultured in a constant temperature incubator at 28±1℃ until the mycelium covered the entire plate, and then set aside. MY-7 was inoculated into LB liquid medium at a 2% (v / v) inoculum and activated and cultured at 30°C for 12 h. The activated MY-7 bacterial solution was diluted with fermentation medium to an OD of 600The concentration of MY-7 fermentation culture medium was about 0.2, and the diluted bacterial solution was fermented and cultured in a shaking incubator at 30°C for 24h and 36h respectively; the MY-7 fermentation culture solution was first diluted 1-fold with the fermentation medium, and the diluted fermentation solution was diluted with the YPD medium cooled to 55±1°C and not yet solidified at a ratio of 1:9. After fully mixing, 10mL was pipetted into a sterile culture dish with a sterile pipette, and the culture dish was gently shaken to make it evenly flattened. After solidification, a detection plate was made, and a plate prepared by mixing the corresponding sterile culture medium of equal volume with the YPD medium was used as a blank control plate for later use.

[0082] Use a sterile borer (inner diameter 5.5mm) to punch a hole in the center of the activated indicator bacteria plate with the same radius edge, and place the mycelium face down in the middle of the prepared test plate. Perform three parallel tests for each pathogenic fungus. Place the plate with the mycelium upright in a constant temperature incubator at 28±1℃, and culture until the mycelium of the blank control plate covers 2 / 3 or less of the entire plate (not completely covered). Take out the incubated plate, and use the cross method to measure the diameter of the pathogen colony on each control plate and the test plate. The average value of the two measurements is the diameter of the pathogen colony. The growth of the pathogen colonies on each control plate and the test plate is shown in the figure. Fig. 9 shown.

[0083] The inhibition rate of MY-7 on the growth of each pathogen colony is shown in Table 4, where the inhibition rate is calculated by the following formula:

[0084] Inhibition rate (%) = [(pathogen colony diameter on control plate - 5.5) (mm) - (pathogen colony diameter on test plate - 5.5) (mm)] / (colony diameter on control plate - 5.5) (mm) × 100%.

[0085] Table 4: Inhibition rate of MY-7 on the growth of plant pathogens

[0086]

[0087]

[0088] Depend on Fig. 9 The results showed that the fermentation broth of MY-7 had obvious inhibitory effects on the above-mentioned plant pathogens, among which the inhibitory effects on Apiospora arundinis, Botrytis cinerea, Geotrichum candidum, Alternaria cruzi and Colletotrichum gloeosporioides were particularly significant.

[0089] Example 4: Preparation of bacterial powder by spray drying of MY-7 fermentation broth

[0090] MY-7 was fermented in a 5L fermenter (liquid volume 3L) for 36 hours to obtain a fermentation solution, which could be directly placed in the feed port of a spray dryer for drying.

[0091] Since spray drying is a high-temperature stress process, the number of viable bacteria in the product after spray drying is affected by many factors. After preliminary screening, four factors, namely, inlet temperature (A), feed rate (B), air inlet rate (C) and spray pressure (D), were selected for orthogonal experiments at three levels to determine the optimal spray drying conditions. The parameters are shown in Table 5.

[0092] Table 5: Orthogonal experimental parameters for spray drying conditions

[0093]

[0094] 1L of MY-7 fermented bacterial liquid was placed in the feed port of the spray dryer for spray drying. The number of viable bacteria in the obtained bacterial powder was used as the evaluation standard. The results are shown in Table 6. The spray temperature corresponding to No. 3 was 110°C, the feed rate was 600mL / h, and the air inlet rate was 25M 3 / h and a spray pressure of 0.25MPa were the optimal spray conditions. After testing, the number of live bacteria in the spray-dried bacterial powder reached 6.77×10 9 CFU / g, the number of live bacteria in the other serial numbered powders can also be maintained at the same order of magnitude. It can be seen that after MY-7 is made into spray-dried powder, it can still maintain most of the activity of the strain, which is conducive to further improving its stability in storage and transportation, and has significant application potential.

[0095] Table 6: Results of orthogonal test for spray drying parameter optimization

[0096]

[0097] Note: n=3, SPSS19.0 software was used for Duncan multiple range test and one-way analysis of variance. Different letters indicate significant differences among treatments (p<0.05)

[0098] According to the above examples, the Bacillus albus MY-7 of the present invention has obvious broad-spectrum antibacterial activity, and has obvious inhibitory effects on various plant pathogens including Alternaria brassicae, Fusarium trilineata, Alternaria alternata, Geotrichum candidum, Colletotrichum gloeosporioides, Botrytis cinerea, Penicillium digitatum, and A.arundinis, and also has good environmental adaptability. Therefore, MY-7 is expected to be used for biological control, preparation of biological control agents, biological fertilizers, plant growth promoters and related microbial preparations, and its promotion will help reduce the use of chemical pesticides and promote the development of sustainable agriculture.

[0099] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, but they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification based on the spirit of the present invention, such as using the Bacillus albus MY-7 of the present invention to control other plant pathogens, or making the Bacillus albus MY-7 of the present invention into freeze-dried bacterial powder or other bacterial agents, etc., should be covered within the protection scope of the present invention.

Claims

1. A Bacillus albus MY-7, characterized in that: The deposit number of the white Bacillus MY-7 is CCTCC NO: M20241653.

2. The white Bacillus MY-7 according to claim 1, characterized in that The albicans MY-7 has a bacteriocin synthesis-related gene, and the bacteriocin synthesis-related gene is selected from one or more of DhbE, DhbB and DhbF.

3. Use of the white Bacillus MY-7 as claimed in claim 1 or 2 in the production of bacteriocins.

4. A bacterial agent, characterized in that The bacterial agent comprises the white Bacillus MY-7 or its culture solution as claimed in claim 1 or 2.

5. The bacterial agent according to claim 4, characterized in that The bacterial agent is bacterial powder obtained after drying.

6. Use of the white Bacillus MY-7 according to claim 1 or 2 or the bacterial agent according to claim 4 in controlling plant pathogens, characterized in that: The plant pathogenic bacteria is selected from one or more of Alternaria brassicicola, Fusarium tricinctum, Alternaria alternat, Geotrichum candidum, Colletotrichum gloeosporioides, Botrytis fabae, Penicillium digitatum and Apiospora arundinis (A.arundinis).

7. A method for preparing the bacterial agent according to claim 4, characterized in that: The method comprises the steps of inoculating white bacillus MY-7 into a liquid culture medium for culturing, and diluting the culture medium to a suitable multiple to obtain the bacterial agent.

8. The method according to claim 7, characterized in that The method comprises inoculating white bacillus MY-7 into a liquid culture medium for activation culture; after the activation culture, taking an appropriate amount of the activation culture liquid and inoculating it into a fermentation culture medium for fermentation culture; after the fermentation culture, taking the fermentation culture liquid and diluting it by an appropriate multiple to obtain the bacterial agent.

9. The method according to claim 8, characterized in that The volume fraction of the white bacillus MY-7 inoculated in the liquid culture medium is 1-10%, and the activation culture time is 8-24 hours.

10. The method according to claim 8, characterized in that The volume fraction of the activated culture solution inoculated into the fermentation medium is 1-10%, and the fermentation culture time is 18-48 hours.

11. The method according to claim 8, characterized in that The suitable dilution multiple is 0 to 22 times.

12. The method according to any one of claims 7 to 11, characterized in that The method further comprises freeze-drying or spray-drying the bacterial agent to prepare freeze-dried bacterial powder or spray-dried bacterial powder.

13. The method according to claim 12, characterized in that The inlet temperature of the spray drying is 100-150°C, the feed rate is 400-1000 mL / h, and the air inlet rate is 10-60 M 3 / h, the spray pressure is 0.1~1.0MPa.

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