Bacillus BFI-L3 with broad-spectrum antibacterial effect and application thereof
By using the bacterial agent and VOCs prepared by Bacillus vesiculosus BFI-L3, the problems of pathogen resistance and environmental pollution of anthracnose in tea trees have been solved, achieving green prevention and control in tea gardens and safe tea production.
Patent Information
- Application Number
- CN202511282130.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2026-02-03
AI Technical Summary
Existing chemical pesticides pose risks of pathogen resistance, environmental pollution, and pesticide residues in the control of anthracnose in tea trees. Biological control methods have not yet effectively solved the problem of compound diseases in tea gardens.
A strain of Bacillus belye BFI-L3 was used to prepare liquid or solid bacterial agents through cultivation and fermentation. Its broad-spectrum antibacterial activity and volatile organic compounds (VOCs) were used to combat pathogenic fungi and promote plant growth, thus replacing chemical fungicides.
It significantly inhibits anthracnose and other pathogenic fungi in tea trees, reduces the diseased area, promotes tea tree growth, reduces pesticide residue risk, and achieves green prevention and control.
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Figure CN121450489A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a Bacillus strain BFI-L3 with broad-spectrum antibacterial effects and its applications. Background Technology
[0002] Currently, the control of plant fungal diseases such as anthracnose in tea mainly relies on chemical control and preliminary exploration of biological control methods. Chemically synthesized fungicides are the most commonly used method for controlling anthracnose in tea gardens, with commonly used agents including carbendazim, chlorothalonil, and thiophanate-methyl. However, long-term use of chemical pesticides not only leads to drug resistance in pathogens but also causes soil compaction, environmental pollution, and pesticide residue risks, affecting tea varieties. With the improvement of people's living standards, the demand for safety, green practices, and environmental protection is increasing. Biological control, using microorganisms to prevent and treat fungal diseases, is green and safe, and contributes to the sustainable development of agricultural production, receiving increasing attention and expectation. The phyllosphere, as a key interface for plant-microorganism-pathogen interactions, has a microbial community structure, diversity, and balance that are closely related to plant health (including disease occurrence). Phyllosphere microorganisms can inhibit pathogens through nutrient competition, antagonism, or the production of antimicrobial substances (such as surfactants). Camellia anthracnose primarily affects mature leaves. Utilizing indigenous microorganisms (such as endophytic bacteria) from the foliage as biocontrol agents theoretically makes it easier for them to successfully colonize and exert their effects in the target ecological niche (leaf surface), providing a new approach to solving the problem of exogenous bacteria colonization. Previous studies have explored the use of Bacillus subtilis and Bacillus amyloliquefaciens for the biological control of plant fungal diseases. Summary of the Invention
[0003] This invention screened a novel Bacillus strain, BFI-L3, which exhibits broad-spectrum antibacterial activity and significant inhibitory effects against Anthracnose of Camellia sinensis, as well as pathogenic fungi such as Staphylococcus aureus and Alternaria. This allows for the simultaneous control of compound diseases in tea gardens. Furthermore, BFI-L3 also possesses plant growth-promoting capabilities; its VOCs (volatile organic compounds) significantly promote the growth of Arabidopsis thaliana.
[0004] The Bacillus BFI-L3 described in this invention is Bacillus velezensis. This strain was deposited on July 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Bacillus velezensis, with accession number CGMCC No. 35399.
[0005] The present invention also provides a culture of the Bacillus BFI-L3.
[0006] The present invention also provides a bacterial agent containing the Bacillus BFI-L3.
[0007] The present invention also provides secondary metabolites of the Bacillus BFI-L3.
[0008] The Bacillus spp. BFI-L3 bacterial agent described in this invention can be a liquid bacterial agent or a solid bacterial agent.
[0009] In this invention, the culture of Bacillus BFI-L3, the bacterial agent containing Bacillus BFI-L3, and the secondary metabolites of Bacillus BFI-L3 can be prepared using conventional techniques.
[0010] The present invention also provides the application of the Bacillus BFI-L3, the culture of the Bacillus BFI-L3, the bacterial agent containing the Bacillus BFI-L3, or the secondary metabolites of the Bacillus BFI-L3 in the prevention and control of Colletotrichum camelliae, Staphylococcus aureus, or Alternaria.
[0011] The Bacillus spp. BFI-L3 described in this invention can grow on R2A medium, and the culture temperature is generally 30-40℃.
[0012] The Bacillus spp. BFI-L3 described in this invention can be efficiently fermented in conventional LB medium, for example, at 37°C and 200 rpm for 48 hours. No complex nutrient optimization is required, significantly reducing production costs.
[0013] The Bacillus filamentosa BFI-L3, its culture, its inoculum, and its secondary metabolites described in this invention can be used to control anthracnose in tea trees caused by Colletotrichum camelliae, as well as plant diseases caused by pathogenic fungi such as Staphylococcus and Alternaria. Its core application is as an environmentally friendly biological control method to replace or reduce the use of chemical fungicides, thereby addressing issues such as excessive pesticide residues in tea, pathogen resistance, and environmental pollution, ensuring safe tea production and the construction of ecological tea gardens.
[0014] The Bacillus spp. BFI-L3, its inoculum, and secondary metabolites provided by this invention exhibit significant advantages in the control of plant diseases. Experiments have shown that this strain achieves a 63.84% inhibition rate against Colletotrichum camelliae in plate confrontation and a 95.06% inhibition rate in fermentation broth. Treatment of detached leaves with the inoculum reduced the lesion area by 77.78%.
[0015] The present invention also provides the Bacillus BFI-L3, a culture of the Bacillus BFI-L3, a bacterial agent containing the Bacillus BFI-L3, and the application of the secondary metabolites (VOCs) of the Bacillus BFI-L3 in promoting the growth of Arabidopsis thaliana.
[0016] Experiments have shown that the VOCs produced by Bacillus thaliana BFI-L3 provided by this invention have a significant growth-promoting effect on Arabidopsis thaliana.
[0017] This invention provides a green solution for multiple preventions of anthracnose and complex fungal diseases in tea trees, provides technical support for solving the bottleneck of pesticide residues in tea and promoting the construction of ecological tea gardens, and provides high-quality microbial resources for green prevention and control of tea gardens and biological control of plant diseases. Attached Figure Description
[0018] Figure 1 This image shows the colony morphology of strain BFI-L3 and its Gram staining microscopic observation.
[0019] Figure 2 This is a graph showing the inhibitory effect of strain BFI-L3 on *Anthracis chinensis* using the plate confrontation method.
[0020] Figure 3 This is a graph showing the inhibitory effect of strain BFI-L3 on various pathogenic fungi.
[0021] Figure 4 The graph shows the inhibitory effect of sterile fermentation broth of strain BFI-L3 on *Anthracis chinensis*.
[0022] Figure 5 The image shows the control efficacy of strain BFI-L3 against Camellia anthracnose in detached leaves.
[0023] Figure 6 This diagram illustrates the growth-promoting effect of VOCs produced by strain BFI-L3 on Arabidopsis thaliana. Detailed Implementation
[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0025] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0026] R2A medium: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15 g / L, pH 7.2 ± 0.2.
[0027] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.
[0028] PDA medium: 200 g / L potato extract, 20 g / L glucose, 14 g / L agar, pH 5.6 ± 0.2.
[0029] MS medium: Potassium nitrate 1.9 g / L, ammonium nitrate 1.65 g / L, potassium dihydrogen phosphate 0.17 g / L, magnesium sulfate 0.37 g / L, calcium chloride 0.44 g / L, potassium iodide 0.00083 g / L, boric acid 0.0062 g / L, manganese sulfate 0.0223 g / L, zinc sulfate 0.0086 g / L, sodium molybdate 0.00025 g / L, copper sulfate 0.000025 g / L. g / L, cobalt chloride 0.000025 g / L, disodium EDTA 0.0373 g / L, ferrous sulfate 0.0278 g / L, inositol 0.1 g / L, glycine 0.002 g / L, thiamine hydrochloride 0.0001 g / L, pyridoxine hydrochloride 0.0005 g / L, nicotinic acid 0.0005 g / L, sucrose 30 g / L, agar 7 g / L, pH 5.7 ± 0.1.
[0030] Example 1: Screening and Identification of Strains
[0031] Tea tree leaves collected from the Hunan Agricultural University Chang'an Base in Changsha County, Changsha City, Hunan Province were used as samples. The surface was washed with distilled water, transferred to a clean bench, and immersed in 75% alcohol for 30 seconds. The samples were then washed three to five times with sterile distilled water, immersed in sodium hypochlorite for 5 minutes, and washed five times with sterile distilled water. The samples were dried on clean petri dishes, transferred to a sterile mortar, and ground with liquid nitrogen. The samples were then resuspended in PBS buffer (0.4g sample: 2ml PBS buffer). The resulting suspension was serially diluted and used as an inoculum, spread on R2A medium, with three replicates for each treatment. The plates were then incubated at 37℃ for 5-10 days to obtain visible colonies. Colonies of different morphologies were streaked on LB medium to obtain single colonies, which were named strain BFI-L3. The purified single colonies of BFI-L3 were transferred to a final concentration of 25% glycerol and stored at -80℃ for later use.
[0032] The selected strain BFI-L3 was inoculated onto R2A medium for activation, morphological observation, and Gram staining. The results are as follows: Figure 1As shown. The colonies of this strain are round with neat edges, white and translucent; the cells are straight rod-shaped with round or square ends, and it is a Gram-positive bacterium.
[0033] The 16S rRNA gene of strain BFI-L3 was amplified and identified using primers 16S-PA (5'-AGAGTTTGATCCTGGCTCAG-3') and 16S-PB (5'-AAGGAGGTGATCCAGCCGCA-3'). The sequencing results were compared with BLAST homology in the NCBI database, and the results showed that strain BFI-L3 belongs to the genus Bacillus.
[0034] Further identification confirmed that strain BFI-L3 was Bacillus velezensis.
[0035] The strain BFI-L3 was deposited on July 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Bacillus velezensis, with accession number CGMCCNo.35399.
[0036] Example 2: Inhibitory effect of strain BFI-L3 on Bacillus anthracis
[0037] The *Colletotrichum camelliae* strain used in this invention is a known strain, screened from tea tree leaves. Using the PDA plate confrontation method, mycelial discs (5 mm in diameter, prepared according to the method in Example 1) of strain BFI-L3 were symmetrically inoculated at a distance of 2.5 cm from the center on 9 cm diameter PDA medium containing 10 mL of medium. A *Colletotrichum camelliae* mycelial disc (5 mm in diameter) was inoculated at the center of the plate. This was repeated three times. The plates were incubated at 28°C in the dark. After 5 days, the colony radius of *Colletotrichum camelliae* was measured, and the inhibition rate was calculated using the following formula:
[0038] Antibacterial rate (%) = (Growth diameter of control group - Growth diameter of treatment group) / Growth diameter of control group × 100%
[0039] See results Figure 2 The average inhibition rate of strain BFI-L3 against *Anthracis chinensis* reached 63.84%, indicating that strain BFI-L3 has a significant inhibitory effect on the growth of *Anthracis chinensis*.
[0040] Example 3: Inhibitory effect of strain BFI-L3 against various plant pathogens
[0041] Using the same method as in Experiment 2, the inhibitory effect of strain BFI-L3 on various plant pathogens was determined. All pathogens were screened from tea tree leaves. These included *Colletotrichum*, *Botryosphaeria*, and *Alternaria*, all known strains. The inhibitory effects of strain BFI-L3 on various plant pathogens are shown in Table 1 and... Figure 3 As shown.
[0042] Table 1
[0043] Target pathogens Antibacterial rate (%) Anthrax (Colletotrichum) 62.27 genus *Botryosphaeria* 65.20 genus *Alternaria* 69.40
[0044] From Table 1 and Figure 3 It can be seen that strain BFI-L3 has a good inhibitory effect on all tested pathogens, and the average inhibition rate of plate confrontation is higher than 60%, indicating that strain BFI-L3 has a broad-spectrum biocontrol effect.
[0045] Example 4: Inhibition of Anthracnose Bacillus thuringiensis by the biocontrol agent of strain BFI-L3.
[0046] The strain BFI-L3 was inoculated into LB medium and fermented at 37℃ and 200rpm for 48h. After centrifugation, the fermentation supernatant was filtered through a 0.22μm filter membrane to obtain the biocontrol agent.
[0047] 600 μL of the biocontrol agent was evenly spread on a PDA culture dish, dried, and then a 5 mm diameter *Colletotrichum camelliae* mycelial cake was inoculated in the center of the dish. In the control group, an equal volume of sterile water was spread, and a 5 mm diameter *Colletotrichum camelliae* mycelial cake was inoculated in the center. The experiment was repeated three times. The plates were incubated at 28°C in the dark, and the colony area of *Colletotrichum camelliae* was measured after 5 days. The inhibition rate was calculated using the following formula:
[0048] Antibacterial rate (%) = (Growth area of control group - Growth area of treatment group) / Growth area of control group × 100%
[0049] See results Figure 4 The biocontrol agent of strain BFI-L3 showed an average inhibition rate of over 95.06% against *Anthracnose cambogia*, indicating that the biocontrol agent of strain BFI-L3 has an effective inhibitory effect on *Anthracnose cambogia*.
[0050] Example 5: Determination of the control efficacy of strain BFI-L3 on detached leaves
[0051] Single colonies of strain BFI-L3 obtained in Experiment 1 were placed in 2 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 8 h. Then, 1% (v / v) of the culture was added to 100 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 8 h, yielding a bacterial suspension of strain BFI-L3 (OD≈0.6). Mycelia of *Colletotrichum camelliae* were inoculated into PDB medium and cultured at 28°C with shaking at 200 rpm for 4 days. After filtration through sterile gauze, a *Colletotrichum camelliae* spore suspension (1×10⁻⁶) was obtained. 5 -1×10 7 (spores / mL).
[0052] After surface disinfection by soaking in sodium hypochlorite, tea leaves were needled. The experimental group was inoculated with a suspension of *Anthracis chinensis* spores at the needle-puncture sites, along with a bacterial suspension of strain BFI-L3. The control group was inoculated with a suspension of *Anthracis chinensis* spores and an equal volume of sterile water. The blank group received only an equal volume of sterile water. After incubation at 28℃ under high humidity for 2 days, the affected area was calculated, and the inhibition rate was calculated using the following formula:
[0053] Inhibition rate (%) = (Affected area in control group - Affected area in treatment group) / Affected area in control group × 100%
[0054] The results are as follows Figure 5 As shown in the figure, the anthrax lesion area decreased by 77.78%.
[0055] Example 6: VOCs-promoting effect of strain BFI-L3 on Arabidopsis thaliana
[0056] Half of the double-layered dish was used to prepare LB solid medium, and the other half was used to prepare MS solid medium. Treatment group: 10 μL of bacterial culture of strain BFI-L3 (same as in Example 5) was added to the LB solid medium, and Arabidopsis thaliana with uniform growth was inoculated into the MS solid medium after 4 days. Control group: No bacterial culture was inoculated. After 14 days of co-cultivation, growth was photographed and fresh weight was measured. The growth-promoting effect was calculated according to the following formula:
[0057] Fresh weight growth rate (%) = [(Weight of Arabidopsis thaliana in the treatment group - Weight of Arabidopsis thaliana in the control group) / Colony weight of the control group] × 100%
[0058] The results are as follows Figure 6 As shown in the figure. The results showed that VOCs produced by strain BFI-L3 promoted the fresh weight growth rate of Arabidopsis thaliana by 164.44%.
[0059] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. Bacillus BFI-L3, which is Bacillus velezensis, with accession number CGMCC No.35399.
2. The culture of Bacillus BFI-L3 as described in claim 1.
3. An inoculum containing Bacillus BFI-L3 as described in claim 1.
4. The secondary metabolites of Bacillus BFI-L3 as described in claim 1.
5. The use of Bacillus BFI-L3 of claim 1, the culture of Bacillus BFI-L3 of claim 2, the bacterial agent containing Bacillus BFI-L3 of claim 3, or the secondary metabolite of Bacillus BFI-L3 of claim 4 in the prevention and control of Colletotrichum camelliae, Staphylococcus aureus or Alternaria.
6. The use of Bacillus BFI-L3 of claim 1, the culture of Bacillus BFI-L3 of claim 2, the bacterial agent containing Bacillus BFI-L3 of claim 3, or the secondary metabolite of Bacillus BFI-L3 of claim 4 in promoting Arabidopsis thaliana growth.