Bacillus velezensis strain EMM914 and application thereof in preparation of microbial pesticide for preventing and treating 19 fungal diseases
By screening and identifying Bacillus belyss EMM914, a microbial pesticide was prepared, which solved the problems of narrow control spectrum and environmental pollution in the existing technology, and achieved a highly efficient inhibitory effect on a variety of plant pathogens.
Patent Information
- Application Number
- CN202511558383.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-01-13
AI Technical Summary
Existing biocontrol agents have limited effectiveness against a variety of plant pathogens, and the long-term use of chemical pesticides has led to environmental pollution and pesticide resistance problems. There is a need to develop a broad-spectrum, environmentally friendly, and highly effective biocontrol agent.
Using Bacillus belyss strain EMM914, its 16S rRNA and gyrB nucleotide sequences were obtained through screening and identification, and used to prepare liquid or solid microbial pesticides, which are widely used to control 19 kinds of plant pathogenic fungi.
EMM914 significantly inhibits a variety of plant pathogens, with a control rate of over 65%, especially against black rot fungus with an inhibition rate as high as 84.92%, solving the problems of narrow control spectrum and environmental pollution in existing technologies.
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Figure CN121320167A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to Bacillus belye ( Bacillus velezensis ). Background Technology
[0002] Plant diseases are a key factor restricting the sustainable development of global agriculture. According to estimates by the Food and Agriculture Organization of the United Nations (FAO), crop losses due to plant diseases amount to hundreds of billions of US dollars annually. Soil-borne fungal diseases, due to their high degree of concealment and difficulty in control, have become the most prevalent type of disease, accounting for 70%–80% of all crop diseases. Soil-borne pathogens survive for extended periods in the soil as resilient structures such as conidia, chlamydospores, and sclerotia. They infect plant roots by secreting cell wall-degrading enzymes and toxins, damaging tissue structure, absorbing nutrients from the host, and causing crop wilting, root rot, and even death, resulting in severe economic losses.
[0003] Among the many soil-borne pathogens, Fusarium ( ) Fusarium Fungi are one of the most widespread and harmful groups of organisms, and the fungal threats to agricultural production extend far beyond this. Alternaria (… Alternaria The pathogen can cause early blight and black spot diseases in a variety of crops and vegetables, resulting not only in yield losses but also posing food safety risks due to its toxins; Anthracnose spp. Colletotrichum Anthracnose, caused by the genus *Hypertica*, affects almost all fruit trees and cash crops, leading to fruit rot and premature leaf drop. Valsa Fruit tree rot caused by pathogens such as spores and worms is often referred to as "the cancer of fruit trees," and in severe cases, it can destroy an entire orchard. These diverse pathogens collectively pose a persistent threat to agricultural production, making the development of a broad-spectrum biological control agent capable of simultaneously combating multiple pathogens particularly urgent.
[0004] Currently, the main control measures for these diseases in agricultural production include crop rotation, soil disinfection, and the application of chemical pesticides. Crop rotation is limited by arable land resources and planting systems, resulting in limited practical effectiveness. While traditional chemical pesticides are fast-acting, long-term and excessive use can lead to pesticide resistance in pathogens, and also poses problems such as pesticide residues, environmental pollution, and disruption of the ecological balance. In recent years, biological control has been considered the most promising alternative strategy due to its environmental friendliness and sustainability. Among them, Bacillus bacteria, due to their ability to produce various antibacterial substances and form resistant spores, have become a research hotspot in the development of biocontrol agents.
[0005] Existing biocontrol agents still generally suffer from two major bottlenecks: a relatively narrow spectrum of control, with many strains only effective against specific types of pathogens, failing to address the complex situation of multiple diseases occurring simultaneously in the field; and the antimicrobial activity of strains highly dependent on their secondary metabolites, but the metabolic profiles of different strains vary significantly. Common strains may only produce a few types of antimicrobial substances, making it difficult to guarantee their antimicrobial efficacy and stability.
[0006] The core need of the current biopesticide industry lies in discovering novel, highly active bacterial strains with unique and rich antimicrobial metabolic profiles. These strains should be able to simultaneously inhibit multiple key pathogens such as Fusarium, Alternaria, Anthracnose, and Cordyceps, and their activity should remain stable and highly efficient under both laboratory and field conditions. Summary of the Invention
[0007] One technical problem to be solved by this invention is to provide a strain of Bacillus belyssus that has broad-spectrum, environmentally friendly, and highly effective inhibitory activity against a variety of plant pathogenic fungi. Bacillus velezensis ) strain EMM914.
[0008] Another technical problem to be solved by the present invention is to provide a use of Bacillus belyssus strain EMM914 in microbial pesticides.
[0009] The technical solutions to the above technical problems are as follows: A strain of Bacillus belye ( Bacillus velezensis Strain EMM914 was deposited on May 19, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251091, located at Wuhan University, Wuhan, Hubei Province, China. The nucleotide sequence of this strain consists of a 16S rRNA nucleotide sequence and a gyrB nucleotide sequence. The 16S rRNA nucleotide sequence is shown in SEQ ID NO:1, and the gyrB nucleotide sequence is shown in SEQ ID NO:2.
[0010] The 16S rRNA nucleotide sequence is as follows: gtgaaggcggcagctatacatgcaagtcgagcggacagatgggagcttgctccctgatgt tagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgg gaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggc ttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctc accaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagaca cggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctga cggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaa gaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggct aactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattggg cgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccgggg agggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtag cggtgaaatgcttacagatgtggagaaacaccacggtgcaaggcgactctctgggtctgt aactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtcca cgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaac gcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgg gggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttacca ggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgaca ggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgag cgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtga caaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctaca cacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccaca aatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctag taatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtc acaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgcc gaagtgacaggagt The gyrB nucleotide sequence is as follows: gcgggtgggatcggcggtctccgagtgtacggccgtctgtcgtaacgccttgtcgaccac tcttgacgttacggttcatcgtgacggaaaaatccactatcaggcgtacgagcgcggtgt acctgtggccgatcttgaagtgatcggtgatactgataagaccggaacgattacgcactt cgttccggatccggaaattttcaaagaaacaaccgaatacgactatgacctgctttcaaa ccgtgtccgggaattggccttcctgacaaaaggtgtaaacatcacgattgaagacaaacg tgaaggacaagaacggaaaaacgagtaccactacgaaggcggaatcaaaagctatgttga gtacttaaaccgttccaaagaagtcgttcatgaagagccgatttatatcgaaggcgagaa agacggcataacggttgaagttgcattgcaatacaacgacagctatacaagcaatattta ttctttcacaaataatatcaacacatacgaaggcggcacgcacgaagccggatttaaaac cggtctgacccgtgttataaacgactatgcaagaagaaaagggattttcaaagaaaatga tccgaatttaagcggggatgatgtgagggaagggctgactgccattatttcaattaagca ccctgatccgcaattcgaagggcagacgaaaacgaagctcggcaactccgaagcgagaac gatcactgatacgctgttttcttctgcgctggaaacattccttcttgaaaatccggactc agcccgcaaaatcgttgaaaaaggtttaatggccgcaagagcgcggatggcagcgaaaaa agcgcgggaattgacccgccgcaaaagtgcgcttgagatttccaatctgccgggcaaact ggcggactgttcttctaaagatccgagcatttccgagctgtatatcgtagagggtgactc tgcgggcggatcagcgaaacagggacgggaccgtcatttccaagccattctgccgctgcg cggtaagattctgaacgttgagaaagccagacttgataagattctctcaaacaatgaggt cagatcaatgatcacggccctcggaacagtactcatagcgacgacataaagacgcccgg
[0011] 1. Isolation and screening of Bacillus belyssus strain EMM914 Sampling: In October 2023, soil samples were collected from the rhizosphere of healthy Ephedra sinica in Yanchi County, Ningxia Autonomous Region, using a five-point sampling method. 10–15 g of soil sample was weighed and added to an Erlenmeyer flask containing 100 mL of sterile distilled water (containing sterile glass beads). The flask was incubated at 28℃ and 180 r / min with shaking for 40–60 min. The supernatant was collected as the stock solution for bacterial suspension and stored at 0±1℃ for later use.
[0012] Strain isolation: The original bacterial suspension was purified by sterile distilled water for 10 minutes. -1 -10 -6 Serial dilutions were performed, with 100 μL of each dilution spread onto PDA plates and incubated at 30°C for 24 h. Single colonies were then picked based on colony morphology and subjected to multiple streak purifications to obtain a pure culture strain, designated EMM914.
[0013] 2. Identification of Bacillus belyssus strain EMM914 (1) Morphological characteristics After culturing this strain on LB solid medium for 24–48 hours, the colonies are milky white, with neat edges, a dry and rough surface, flattened with slight wrinkles, and a granular texture. Figure 1 .exist Figure 1 In the image, A is a colony image, and B is a spore staining result image.
[0014] (2) Molecular biological identification Genomic DNA was extracted from strain EMM914 as a template, and universal primers for the bacterial 16S rRNA gene were selected: 16S-F: 5′-GGACTACHVGGGTWTCTATT-3′ 16S-R: 5′-GTGCCAGCMGCCGCGGTAA-3′ And gyrB gene primers: gyrB-F: 5′-GTCATAATGACRACTTTRTGRTAACGGGC-3′ gyrB-R: 5′-GCNGGNGGNAARTTYGACGGAAGCGG-3′ PCR amplification was performed. The reaction mixture (20 μL) consisted of: 10 μL of 2× Taq PCR MasterMix II, 1 μL each of 10 μM forward and reverse primers, 1 μL of template DNA, and water to a final volume of 20 μL. The reaction program was: 93℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for 30 cycles; and a final extension at 72℃ for 10 min. The PCR products were analyzed by 1% agarose gel electrophoresis and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.
[0015] The obtained 16S rRNA and gyrB sequences were BLAST aligned in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software. Figure 2 ,exist Figure 2 In the diagram, A represents a phylogenetic tree constructed using the 16S rRNA sequence, and B represents a phylogenetic tree constructed using gyrB. Combining morphological characteristics and molecular systematics analysis, this strain was identified as *Bacillus bereaves* (…). Bacillus velezensis It was named Bacillus belyssus EMM914.
[0016] The present invention relates to the use of Bacillus belysus strain EMM914 in the preparation of pesticides for controlling any one of the following fungal diseases: Fusarium moniliforme EMF994, Fusarium oxysporum EMF889, Colletotrichum gloeosporioides EMF991, Fusarium graminearum EMF897, Fusarium moniliforme EMF998, Fusarium solani EMF990, Fusarium solani EMF893, Fusarium oxysporum EMF996, Staphylococcus aureus EMF899, Fusarium solani EMF895, Fusarium trifidum EMF997, Fusarium pearis spp. EMFB1, Fusarium moniliforme EMF894, Fusarium trifidum EMF896, Alternaria alternata EMF993, Fusarium solani EMF1001, Fusarium moniliforme EMFB4, Trichoderma pinkis EMFB3, and Botrytis cinerea EMFHM.
[0017] The microbial pesticide of the present invention is: a liquid or solid formulation of Bacillus berleis strain EMM914 loaded on an agriculturally acceptable carrier, fermented, and prepared by conventional methods.
[0018] In the liquid-form microbial pesticide of this invention, the viable cell content of strain EMM914 is 1×10⁻⁶. 9 CFU / mL ~ 4 × 10 10 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM914 was 2 × 10⁻⁶. 8 CFU / g ~ 4×10 10 CFU / g.
[0019] In the liquid-form microbial pesticide of the present invention, the optimal viable cell content of strain EMM914 is 1.5 × 10⁻⁶. 9 CFU / mL; Among solid-form microbial pesticides, the optimal viable cell count for strain EMM914 is 3 × 10⁻⁶. 9 CFU / g.
[0020] Its usage method is as follows: When using EMM914 liquid microbial pesticide, dilute it with water at a volume ratio of 1:300-400 and spray it evenly at the roots of crops to control the 19 plant pathogens mentioned above. Alternatively, the solid biological pesticide can be diluted with water at a mass ratio of 1:100-400 and applied to the root zone. The dosage is 2-10 kg per acre, applied every 7-14 days. The specific dosage and frequency should be determined according to the degree of damage caused to the plants by the plant pathogens. It can control the 19 plant pathogens mentioned above.
[0021] Compared with the prior art, the present invention has the following beneficial effects: Solid or liquid microbial pesticides prepared using the strain EMM914 of this invention have been demonstrated in laboratory tests to have a significant control effect on root rot disease of Astragalus membranaceus caused by Fusarium trifidum, with a comprehensive control rate of 72.98%. For the other 18 pathogenic fungi, except for Fusarium tumefaciens EMF894, the control rate is above 65%, demonstrating a significant control effect. This pesticide can be used in the preparation of microbial pesticides for the control of any one of the aforementioned 19 fungal diseases. Attached Figure Description
[0022] Figure 1 The image shows the growth morphology and Gram staining results of Bacillus belyss strain EMM914.
[0023] Figure 2 The tree construction results for Bacillus belyss strain EMM914 are shown in the figure.
[0024] Figure 3 The inhibition diagram of EMM914 and 19 pathogenic fungi on PDA plates. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to these embodiments. Example 1
[0026] A strain of Bacillus belye ( Bacillus velezensisStrain EMM914 was deposited on May 19, 2025, at the China Center for Type Culture Collection, Wuhan University, Hubei Province, China, with accession number CCTCC NO: M 20251091. Its nucleotide sequence consists of the 16S rRNA nucleotide sequence, gyrB The nucleotide sequence composition is as follows: the 16S rRNA nucleotide sequence is shown in SEQ ID NO:1, and the gyrB nucleotide sequence is shown in SEQ ID NO:2.
[0027] The 16S rRNA nucleotide sequence is as follows: gtgaaggcggcagctatacatgcaagtcgagcggacagatgggagcttgctccctgatgt tagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgg gaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggc ttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctc accaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagaca cggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctga cggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaa gaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggct aactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattggg cgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccgggg agggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtag cggtgaaatgcttacagatgtggagaaacaccacggtgcaaggcgactctctgggtctgt aactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtcca cgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaac gcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgg gggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttacca ggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgaca ggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgag cgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtga caaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctaca cacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccaca aatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctag taatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtc acaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgcc gaagtgacaggagt The gyrB nucleotide sequence is as follows: gcgggtgggatcggcggtctccgagtgtacggccgtctgtcgtaacgccttgtcgaccac tcttgacgttacggttcatcgtgacggaaaaatccactatcaggcgtacgagcgcggtgt acctgtggccgatcttgaagtgatcggtgatactgataagaccggaacgattacgcactt cgttccggatccggaaattttcaaagaaacaaccgaatacgactatgacctgctttcaaa ccgtgtccgggaattggccttcctgacaaaaggtgtaaacatcacgattgaagacaaacg tgaaggacaagaacggaaaaacgagtaccactacgaaggcggaatcaaaagctatgttga gtacttaaaccgttccaaagaagtcgttcatgaagagccgatttatatcgaaggcgagaa agacggcataacggttgaagttgcattgcaatacaacgacagctatacaagcaatattta ttctttcacaaataatatcaacacatacgaaggcggcacgcacgaagccggatttaaaac cggtctgacccgtgttataaacgactatgcaagaagaaaagggattttcaaagaaaatga tccgaatttaagcggggatgatgtgagggaagggctgactgccattatttcaattaagca ccctgatccgcaattcgaagggcagacgaaaacgaagctcggcaactccgaagcgagaac gatcactgatacgctgttttcttctgcgctggaaacattccttcttgaaaatccggactc agcccgcaaaatcgttgaaaaaggtttaatggccgcaagagcgcggatggcagcgaaaaa agcgcgggaattgacccgccgcaaaagtgcgcttgagatttccaatctgccgggcaaact ggcggactgttcttctaaagatccgagcatttccgagctgtatatcgtagagggtgactc tgcgggcggatcagcgaaacagggacgggaccgtcatttccaagccattctgccgctgcg cggtaagattctgaacgttgagaaagccagacttgataagattctctcaaacaatgaggt cagatcaatgatcacggccctcggaacagtactcatagcgacgacataaagacgcccgg
[0028] The above-mentioned EMM914 strain is used in the preparation of pesticides for controlling any one of the following fungal diseases: Fusarium moniliforme EMF994, Fusarium oxysporum EMF889, Colletotrichum gloeosporioides EMF991, Fusarium graminearum EMF897, Fusarium moniliforme EMF998, Fusarium solani EMF990, Fusarium solani EMF893, Fusarium oxysporum EMF996, Staphylococcus aureus EMF899, Fusarium solani EMF895, Fusarium trifleum EMF997, Fusarium pearis spp. EMFB1, Fusarium moniliforme EMF894, Fusarium trifleum EMF896, Alternaria alternata EMF993, Fusarium solani EMF1001, Fusarium moniliforme EMFB4, Trichoderma pinkis EMFB3, and Botrytis cinerea EMFHM.
[0029] The aforementioned microbial pesticide is: EMM914 fermented and prepared into liquid or solid formulations using conventional methods. The solid formulation uses an agriculturally acceptable carrier to adsorb Bacillus berleis strain EMM914.
[0030] In liquid formulations of microbial pesticides, the viable cell count of strain EMM914 is 1×10⁻⁶. 9 CFU / mL ~ 4 × 10 10CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM914 was 2 × 10⁻⁶. 8 CFU / g ~ 4×10 10 CFU / g. In this example, the viable cell count of strain EMM914 in the liquid formulation of microbial pesticide was 1.5 × 10⁻⁶ CFU / g. 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM914 was 3 × 10⁻⁶. 9 CFU / g.
[0031] Its usage method is as follows: When using EMM914 liquid microbial pesticide, dilute it to water at a volume ratio of 1:300-400 and spray it evenly on the roots of crops to inhibit or kill the 19 plant pathogens mentioned above. Alternatively, the solid biological pesticide can be diluted to water at a mass ratio of 1:100-400 and applied with the water to the root zone. The dosage is 2-10 kg per acre, applied every 7-14 days. The specific dosage and frequency should be determined according to the degree of damage caused to the plants by the plant pathogens. This method can control the 19 plant pathogens mentioned above. Example 2
[0032] This embodiment of Bacillus berberis (Bacillus velezensis) The nucleotide sequence of strain EMM914 is the same as that in Example 1, and the 19 plant pathogens are the same as those in Example 1.
[0033] The microbial pesticides used to control 19 plant pathogens are: Bacillus beleilliformis strain EMM914 loaded on an agriculturally acceptable carrier, and prepared into liquid or solid formulations by conventional methods after fermentation.
[0034] In liquid formulations of microbial pesticides, the viable cell count of strain EMM914 is 1×10⁻⁶. 9 CFU / mL ~ 4 × 10 10 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM914 was 2 × 10⁻⁶. 8 CFU / g ~ 4×10 10 CFU / g. The microbial pesticide in this embodiment is a liquid formulation microbial pesticide with a viable count of 1×10⁻⁶ CFU / g for strain EMM914. 9 In solid-form microbial pesticides, the viable cell count of strain EMM914 is 2 × 10⁻⁶ CFU / mL. 8 CFU / g.
[0035] The method of use is the same as in Example 1. Example 3
[0036] This embodiment of Bacillus belesi ( Bacillus velezensis The nucleotide sequence of strain EMM914 is the same as that in Example 1, and the 19 plant pathogens are the same as those in Example 1.
[0037] The microbial pesticides used to control 19 plant pathogens are: Bacillus beleilliformis strain EMM914 loaded on an agriculturally acceptable carrier, and prepared into liquid or solid formulations by conventional methods after fermentation.
[0038] In liquid formulations of microbial pesticides, the viable cell count of strain EMM914 is 1×10⁻⁶. 9 CFU / mL ~ 4 × 10 10 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM914 was 2 × 10⁻⁶. 8 CFU / g ~ 4×10 10 CFU / g. In this example, the viable cell count of strain EMM914 in the liquid formulation of microbial pesticide was 4 × 10⁻⁶. 10 In solid-form microbial pesticides, the viable cell count of strain EMM914 is 4 × 10⁻⁶ CFU / mL. 10 CFU / g.
[0039] The method of use is the same as in Example 1.
[0040] To determine the control efficacy of strain EMM914 against plant pathogens including *Fusarium moniliforme* EMF994, *Fusarium oxysporum* EMF889, *Colletotrichum gloeosporioides* EMF991, *Fusarium graminearum* EMF897, *Fusarium moniliforme* EMF998, *Fusarium solani* EMF990, *Fusarium solani* EMF893, *Fusarium oxysporum* EMF996, *Botrytis cinerea* EMF899, *Fusarium solani* EMF895, *Fusarium trifidum* EMF997, *Fusarium pearii* EMFFB1, *Fusarium oxysporum* EMF894, *Fusarium trifidum* EMF896, *Alternaria alternata* EMF993, *Fusarium solani* EMF1001, *Fusarium moniliforme* EMFFB4, *Trichoderma pinkii* EMFFB3, and *Botrytis cinerea* EMFHM, the inventors conducted extensive laboratory research experiments using strain EMM914 from Example 1 of this invention. The experimental results are as follows: 1. Inhibitory effect of strain EMM914 on the above 19 plant pathogenic fungi. The inhibitory effect of Bacillus belycei EMM914 on the mycelial growth of the aforementioned 19 plant pathogenic fungi was determined using the plate confrontation method. The experimental results after 7 days of plate confrontation are as follows: This experiment only uses the beneficial effect of Bacillus belyss strain EMM914 in controlling Fusarium trifidum EMF896 as an example. The environment, materials, reagents, dosage, and conditions of various experiments with the other 18 pathogens were the same as those used in controlling Fusarium trifidum EMF896. Under the same conditions, 19 experimental groups of pathogens and their respective control groups were set up simultaneously. The specific experiments are as follows: (1) Inhibitory effect of strain EMM914 on Fusarium trifidum EMF896 In the experimental group, *Bacillus belyssioides* strain EMM914 and *Fusarium trifidum* strain EMF896 were inoculated onto potato agar solid medium on opposite sides. The control group was a blank. *Fusarium trifidum* strain EMF896 was stored in the Industrial Microbiology Laboratory of the School of Life Sciences, Shaanxi Normal University. Both the experimental and control cultures were incubated at 28℃ for 5 days, and the growth of pathogens was recorded daily. At the end of the incubation, the inhibition rate was calculated based on the colony diameters R1 and R2 of the pathogens in the experimental and control groups, and the inhibition rate η was determined using the following formula:
[0041] Each treatment was performed in triplicate. The inhibition rate (η) of Bacillus belyss strain EMM914 against Fusarium trifidum EMF896 was 74.49%. The experimental results are shown in Table 1.
[0042] (2) In vitro experiments Pretreatment of Astragalus membranaceus: Take healthy 2-year-old Astragalus membranaceus without disease spots on the surface, wash it, soak it in 1% sodium hypochlorite solution for 1 minute, rinse off the sodium hypochlorite on the surface with sterile water, soak it in 75% alcohol for 30 seconds, rinse it 3 times with sterile water, and air dry it. Use a sterile knife to cut the Astragalus membranaceus into 6 cm long sections, and make a 0.5 cm long and 0.3 cm deep wound in the center of the Astragalus membranaceus and 2 cm away from the center.
[0043] Experimental group 1: The inoculation concentration for each wound was 5 × 10⁻⁶. 7 Dilute 50 μL of EMM914 liquid formulation with CUF / mL, and inoculate with 50 μL of 8×10⁻⁶ concentration 2 hours later. 5 CFU / mL Fusarium trifidum EMF896 spore suspension.
[0044] Control group: Inoculation concentration at wound site was 4×10 5 100 μL of Fusarium trifidum EMF896 spore suspension at CFU / mL.
[0045] Placed in a 25℃ constant temperature and humidity incubator, cultured for 7 days, and the changes in lesion area in each treatment group were recorded.
[0046] The experimental results are shown in Table 1.
[0047] Table 1. Results of in vitro assays of Bacillus belye EMM914 killing Fusarium trilobum EMF896.
[0048] Note: Different letters indicate significant differences between samples. P <0.05).
[0049] (3) Pot experiment The experimental groups and methods were the same as in the in vitro experiment. After the experiment, the samples were placed in flowerpots measuring 90 cm long, 25 cm wide, and 30 cm high. Each pot was filled with soil to a height of 25 cm, with a soil mass of 8 kg. Twenty biological replicates were set up for each experimental group. Statistical analysis of the experimental results was performed, and the control effect is shown in Table 2.
[0050] Table 2. Results of pot experiment on the killing effect of strain EMF914 on Fusarium trifidum EMF896
[0051] Note: Different letters indicate significant differences between samples. P <0.05).
[0052] The overall incidence rate in the experimental group was 72.98% lower than that in the control group. P <0.05, the difference is significant.
[0053] The experimental methods for the antibacterial effects against 18 other plant pathogens, including Fusarium moniliforme EMF994, Fusarium oxysporum EMF889, Colletotrichum gloeosporioides EMF991, Fusarium graminearum EMF897, Fusarium moniliforme EMF998, Fusarium solani EMF990, Fusarium solani EMF893, Fusarium oxysporum EMF996, Staphylococcus aureus EMF899, Fusarium solani EMF895, Fusarium trifidum EMF997, Staphylococcus pearis spp. EMF1, Fusarium moniliforme EMF894, Alternaria alternata EMF993, Fusarium solani EMF1001, Fusarium moniliforme EMF4, Trichoderma pinkis EMF3, and Botrytis cinerea EMFHM, were the same as those for Fusarium trifidum EMF896.
[0054] The inhibition rate η of strain EMM914 against 19 pathogenic fungi is shown in the figure. Figure 3 And Table 3.
[0055] exist Figure 3In the study, AS were: Fusarium moniliforme EMF994, Fusarium oxysporum EMF889, Colletotrichum gloeosporioides EMF991, Fusarium graminearum EMF897, Fusarium moniliforme EMF998, Fusarium solani EMF990, Fusarium solani EMF893, Fusarium oxysporum EMF996, Staphylococcus aureus EMF899, Fusarium solani EMF895, Fusarium trifidum EMF997, Fusarium pearis spp. EMFFB1, Fusarium moniliforme EMF894, Fusarium trifidum EMF896, Alternaria alternata EMF993, Fusarium solani EMF1001, Fusarium moniliforme EMFFB4, Trichoderma pinkis EMFFB3, and Botrytis cinerea EMFHM; 1 was the control group, and 2 was the experimental group.
[0056] As shown in Table 3, the inhibition rate statistics indicate that compared with the uninoculated strain EMM914 and the normally growing control group, the mycelial growth of pathogens in the confrontation culture experimental group was inhibited to varying degrees. Strain EMM914 showed good inhibitory effects on all 19 plant pathogen strains, with the strongest inhibitory effect on EMF990 of black rot fungus, with an inhibition rate as high as 84.92%. Except for Fusarium oxysporum EMF894, it showed good inhibitory effects on other pathogens, with inhibition rates above 65%.
[0057] 2. Control efficacy of strain EMM914 against 19 pathogenic fungi. The experimental method was the same as that used in Experiment 1 to determine the inhibitory effect of strain EMM914 on 19 plant pathogenic fungi.
[0058] The experimental results are shown in Table 3. The control efficacy of strain EMM914 against 19 pathogenic fungi of common crops or economic crops was determined using the conventional plate confrontation method.
[0059] Table 3. Inhibition rate η of strain EMM914 against 19 pathogenic fungi
[0060] Table 3 shows the inhibition rate (η) of strain EMM914 against 19 pathogenic fungi. In Table 3, strain EMM914 showed the following inhibition rates: 77.38% against *Fusarium moniliforme* EMF994, 73.91% against *Fusarium oxysporum* EMF889, 69.23% against *Colletotrichum gloeosporioides* EMF991, 77.96% against *Fusarium graminearum* EMF897, 66.10% against *Fusarium solani* EMF998, 84.92% against *Heterophyllum heliotropium* EMF990, 68.29% against *Fusarium solani* EMF893, 65.26% against *Fusarium oxysporum* EMF996, and [the inhibition rate was not specified in the original text]. The inhibition rates were as follows: 74.68% against Fusarium solani EMF895, 72.73% against Fusarium trifurcation EMF997, 78.02% against *Fusarium solani* EMFFB1, 70.01% against *Fusarium solani* EMF894, 48.89% against *Fusarium trifurcation* EMF896, 74.49% against *Alternaria alternata* EMF993, 69.01% against *Fusarium solani* EMF1001, 70.73% against *Fusarium moniliforme* EMFFB4, 84.90% against *Trichoderma pinkis* EMFFB3, and 81.11% against *Botrytis cinerea* EMFHM.
[0061] As shown in Table 3, strain EMM914 has good control effects against 19 plant pathogen strains. It has the strongest inhibitory effect on EMF990 of black rot fungus, with a control rate η as high as 84.92%. It also has good control effects against other pathogens. Except for Fusarium moniliforme EMF894, the inhibition rate η is above 65%.
Claims
1. A strain of Bacillus velezensis, EMM914, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251091, on May 19, 2025, at Wuhan University, Wuhan, Hubei Province, China. The nucleotide sequence of this strain consists of a 16S rRNA nucleotide sequence and a gyrB nucleotide sequence. The 16S rRNA nucleotide sequence is shown in SEQ ID NO:1, and the gyrB nucleotide sequence is shown in SEQ ID NO:
2.
2. The use of the Bacillus berberis strain EMM914 of claim 1 in the preparation of a pesticide for controlling any one of the following fungal pathogens: Fusarium moniliforme EMF994, Fusarium oxysporum EMF889, Colletotrichum gloeosporioides EMF991, Fusarium graminearum EMF897, Fusarium moniliforme EMF998, Fusarium solani EMF990, Fusarium solani EMF893, Fusarium oxysporum EMF996, Staphylococcus aureus EMF899, Fusarium solani EMF895, Fusarium trifleum EMF997, Fusarium pearis spp. EMFB1, Fusarium moniliforme EMF894, Fusarium trifleum EMF896, Alternaria alternata EMF993, Fusarium solani EMF1001, Fusarium moniliforme EMFB4, Trichoderma pinkis EMFB3, and Botrytis cinerea EMFHM.
3. The use of Bacillus belyssus strain EMM914 according to claim 2 in the preparation of pesticides for controlling any one of 19 fungal diseases, characterized in that... The microbial pesticide mentioned is: EMM914 fermented and prepared into liquid or solid formulations using conventional methods. The solid formulation uses an agriculturally acceptable carrier to adsorb Bacillus berleis strain EMM914.
4. The use of Bacillus berberis strain EMM914 according to claim 2 in the preparation of a pesticide for controlling any one of 19 fungal diseases, characterized in that: In the liquid-form microbial pesticide, the viable cell count of strain EMM914 is 1×10⁻⁶. 9 CFU / mL ~ 4.0 × 10 10 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM914 was 2 × 10⁻⁶. 8 CFU / g ~ 4.0 × 10 10 CFU / g.
5. The use of Bacillus belyssus strain EMM914 according to claim 3 or 4 in the preparation of a pesticide for controlling any one of 19 fungal diseases, characterized in that: In the liquid-form microbial pesticide, the viable cell count of strain EMM914 is 1.5 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticide formulations, the viable cell count of strain EMM914 was 3 × 10⁻⁶. 9 CFU / g.