Bacillus velezensis YP-5 and application thereof

Abstract

The invention discloses a bacillus velezensis YP-5 strain and an application thereof. Relates to the technical field of microorganisms. The invention provides a preservation number, a fungicide, a method for improving the planting capacity of bacillus velezensis in tea tree leaves and specific application. The bacillus velezensis YP-5 is high in control effect and wide in control spectrum. In addition, under the technological conditions of the invention, the quantity of colonized bacteria on tea tree leaves is increased by 1.9 times compared with that of a blank control group; after 7 days of treatment, the fixed planting bacteria quantity is 3.42 times of that of a blank control group. Meanwhile, when the microbial agent is sprayed to the tea trees in an indoor environment, the biological control effect on the tea anthracnose can be remarkably enhanced, and the biological control effect is improved by 10.29% compared with that of original YP-5 bacterial liquid. The problem that in the prior art, microbial thalli are weak in adhesion and prone to loss, and consequently the biocontrol capacity of the microbial thalli is weakened is effectively solved, and the method is suitable for industrial application of agricultural microbial preparations.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Bacillus belye YP-5 and its applications. Background Technology

[0003] Biological control technology, with its unique advantages such as environmental friendliness, high safety, long-lasting effect, no residue, and low likelihood of inducing drug resistance in pathogens, has become a key strategy to replace or reduce the use of chemical pesticides and is a core component of the current green pest control system in tea gardens. Among these, biocontrol microorganisms and plant-derived natural products, as core technologies of the biological control system, have shown significant application potential, exhibiting diverse mechanisms of action and good application prospects in suppressing tea anthracnose. However, in practical applications, biocontrol agents suffer from poor leaf adhesion and are easily washed away by rainwater, resulting in short colonization periods and unstable control effects. This is the core problem restricting the industrial application of biocontrol agents. Existing technologies often improve adhesion by adding polysaccharides, nonionic surfactants, or single surface modifiers, but this method relies solely on physical adsorption. Modifiers are easily shed with rainwater, and the issue of the microorganisms' own colonization ability on the leaf surface remains unresolved. Other studies have used chemical cross-linking agents to immobilize the microorganisms, but this may lead to microbial inactivation and poses environmental safety risks.

[0004] Therefore, whether it is possible to screen a new, highly specific and effective biocontrol strain for tea anthracnose and improve its leaf colonization ability is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Bacillus belyssus YP-5 and its applications. Beyond being limited to biocontrol bacteria, the present invention also proposes a synergistic strategy of "inducing biofilm formation - regulating leaf surface charge" from two dimensions: "enhancing the bacterial colonization ability" and "optimizing interfacial interactions." On the one hand, a specific carbon source induces the bacterial cells to form a biofilm, utilizing the "physical entanglement + chemical binding" of extracellular polysaccharides (EPS) and proteins in the biofilm to enhance the anchoring of the bacterial cells to the leaf surface. On the other hand, cationic chitosan neutralizes the negative charge repulsion between the leaf surface and the bacterial cells, while simultaneously forming an interpenetrating network with the EPS biofilm, improving erosion resistance. This synergistic mechanism enables the long-term colonization ability of biocontrol bacteria, overcoming the shortcomings of existing technologies.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Preservation information: Bacillus belesiensis ( Bacillus velezensisYP-5 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36088 and deposit date of September 26, 2025.

[0007] A strain of Bacillus belyssus, with accession number CGMCC No. 36088.

[0008] The present invention also provides a microbial inoculum containing the above-mentioned Bacillus belye.

[0009] This invention also provides a method for enhancing the colonization ability of Bacillus belye in tea leaves, comprising the following steps: (1) Preparation of YP-5 biofilm bacterial suspension: Bacillus belye, after activation, was inoculated and cultured to the logarithmic phase. The bacterial suspension was then inoculated into the biofilm induction medium and cultured. The cultured bacterial suspension was centrifuged, the supernatant was discarded, the bacterial film precipitate was resuspended, and the concentration of the bacterial suspension was adjusted to obtain the biofilm bacterial suspension. (2) YP-5 synergistic treatment: Take chitosan solution, place it evenly on the surface of tea leaves, and let it stand to form a positively charged film; add a biofilm suspension containing 1% mannitol to the same area; The Bacillus belyssus has the CGMCC No. 36088 preservation number.

[0010] Preferred method for preparing biofilm induction medium: Add any carbon source of sucrose, mannitol, or glucose to NB liquid medium until the w / v concentration is 1.0%.

[0011] Preferred carbon source: mannitol.

[0012] Preferred method: Step (1) specifically involves: Bacillus belyssus YP-5, activated with NA solid medium, inoculated into NB liquid medium, cultured at 32℃ and 180 r / min with shaking until the logarithmic phase, then 1 mL of bacterial suspension is inoculated into biofilm induction medium, cultured at 32℃ and 180 rpm with shaking for 24 h; the cultured bacterial suspension is centrifuged at 3000 rpm for 10 min, the supernatant is discarded, the bacterial film precipitate is resuspended with sterile physiological saline, and the bacterial suspension concentration is adjusted to 1×10⁻⁶. 8 CFU / mL was used to obtain a biofilm suspension.

[0013] Preferred step (2) specifically involves: accurately pipetting 100 μL of 0.1% chitosan solution and uniformly adding it to a 2 cm × 2 cm area on the surface of tea leaf, allowing it to stand for 10–15 min to form a positively charged film; then adding 100 μL of 1 × 10⁻⁶ solution containing 1% mannitol to the same area. 8 CFU / mL biofilm bacterial suspension.

[0014] The present invention also provides the application of the above-mentioned Bacillus berberis, the above-mentioned microbial inoculant, and any of the above-mentioned methods in the preparation of microbial preparations and in tea tree cultivation.

[0015] Preferred: Microbial preparations: pesticides, antibacterial agents.

[0016] Preferred: inhibits anthracnose of tea, tea leaf spot fungus, tea ring spot fungus, and fruit anthracnose fungus.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a strain of Bacillus belyssus YP-5 and its application, and the technical effects achieved are as follows: The strain Bacillus belye YP-5 of this invention is not only highly effective but also has a broad spectrum of control. It has good control effects on a variety of major tea tree diseases, such as tea anthracnose, tea leaf spot, and tea leaf spot. Among them, the highest inhibitory activity against tea anthracnose bacteria reaches 84.61%.

[0018] This invention discloses a method for enhancing the colonization ability of *Bacillus belye* on tea leaves. Under the conditions of this process, the colonization amount on tea leaves is 1.9 times higher than that of the control group; after 7 days of treatment, the colonization amount is 3.42 times higher than that of the control group. Simultaneously, spraying this agent on tea trees in an indoor environment significantly enhances the biocontrol effect against tea anthracnose, improving the biocontrol effect by 10.29% compared to the original YP-5 bacterial solution. This effectively solves the problem of weak microbial cell adhesion and easy loss leading to weakened biocontrol ability in existing technologies, and is suitable for the industrial application of agricultural microbial preparations. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The attached figure shows the inhibitory effects of different biocontrol bacteria provided by this invention on tea anthrax bacteria.

[0021] Figure 2 The attached figure shows the colony morphology of YP-5 provided by this invention.

[0022] Figure 3 The attached figure shows an electrophoresis diagram of the 16S rDNA and gyrA gene amplification of the YP-5 strain provided by this invention.

[0023] Figure 4The attached figure is a phylogenetic tree of the YP-5 strain constructed based on the 16S rDNA sequence provided by the present invention.

[0024] Figure 5 The attached figure is a phylogenetic tree of the YP-5 strain constructed based on the gyrA gene sequence provided by this invention.

[0025] Figure 6 The attached figure shows the antibacterial effect of YP-5 provided by the present invention against tea tree pathogens.

[0026] Figure 7 The attached figure shows the indoor efficacy of the YP-5 biocontrol agent provided by this invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention discloses a strain of Bacillus belyssus YP-5 and its applications.

[0029] All raw materials not mentioned in the examples are commercially available, and all methods not mentioned are conventional methods, which will not be described in detail here.

[0030] Example 1 Screening and identification of biocontrol strains Isolation of biocontrol strains The bacterial strain was isolated using the dilution plating method. Fresh, disease-free tea leaves were ground and a suspension of 1 mL was inoculated into a test tube containing 9 mL of NB liquid medium. After mixing, the suspension was incubated in an 80℃ water bath for 10 min, then cooled to room temperature. The test tube was then transferred to a 32℃ incubator for enrichment culture for 24 h. The enriched bacterial suspension was then serially diluted with sterile water to obtain 10-1... - ¹ to 10 -6 Bacterial suspensions were prepared. 0.1 mL of bacterial suspensions at different dilutions were plated onto manganese salt nutrient agar plates, with three replicates for each dilution. All plates were incubated upside down in a 32 ℃ incubator for 48 h. Single colonies were then picked for purification and preservation.

[0031] Beef extract peptone liquid medium (g / L, NB): peptone 10, beef extract 3, sodium chloride 5, pH 7.2-7.4.

[0032] Manganese salt nutrient agar medium (g / L): peptone 10, beef extract 3, yeast extract 3, manganese sulfate 0.01, agar 15, pH 7.2-7.4.

[0033] Screening of biocontrol bacteria The isolated strains were screened using the streak plate contrast method. A 5 mm *C. fructicola* mycelial disc was inoculated into the center of a PDA plate, followed by inoculation of the isolated strains 2.0 cm to either side of the disc. A control group was prepared by inoculating only *C. fructicola* mycelial discs. Each experiment was performed in triplicate and incubated at 26°C for 9 days. The colony radius of the pathogens in the control and treatment groups was measured using the cross-hatching method to calculate the inhibition rate.

[0034] Potato dextrose agar medium (g / L, PDA): potato flour 6, glucose 20, agar 20, pH 5.4-5.8, Solarbio Beijing Technology Co., Ltd.

[0035] Inhibition rate (%) = (Coronary radius of control group - Colony radius of treatment group) / Colony radius of control group × 100 Experimental results: A total of 19 bacterial strains were isolated and purified from healthy tea leaves. Four strains showing good antagonistic activity against the pathogen causing tea anthracnose were screened using a plate confrontation test. Figure 1 The strains were named YP-4, YP-5, YP-6, and YP-9, respectively. The antibacterial effects of different biocontrol strains on *C. fructicola* were measured (Table 1). The results showed that strain YP-5 exhibited the strongest antibacterial activity, with the smallest pathogen growth radius (8.42±1.85 mm) and the highest inhibition rate of 79.34%. In summary, YP-5 showed the best overall performance in inhibiting the growth of *C. fructicola*, demonstrating potential as a biocontrol agent. Therefore, it was selected as the target strain for further research.

[0036] Identification of biocontrol strains Morphological characteristics of YP-5 strain like Figure 2 As shown, after culturing YP-5 on NA medium at 32℃ for 48 hours, it forms typical colony characteristics: colonies are round or nearly round, with a dry, rough, and opaque surface, and irregularly wavy or lobed edges. The colonies are distinctly raised, initially milky white, gradually turning pale yellow with prolonged incubation. Dense wrinkles or radial striations are commonly seen in the central area, exhibiting a typical waxy luster. The bacterial cells are dense and easy to pick up, and red pigment is observed at the bottom of the culture medium. According to the *Handbook of Systematic Identification of Common Bacteria*, the morphological characteristics of YP-5 colonies conform to those of Bacillus, and can be used as a morphological basis for the preliminary identification of this strain.

[0037] Molecular biological identification of YP-5 strain Genomic DNA was extracted from strain YP-5, and its 16S rDNA and gyrA gene sequences were amplified using conventional PCR technology. Gel electrophoresis of the PCR products showed two distinct bands at approximately 1500 bp and 1000 bp (Figure 3). These bands were excised from the gel and sent to Shanghai Sangon Biotech Sequencing Co., Ltd. for sequencing. The 16S rDNA gene sequencing results were compared with BLAST results on the NCBI website, revealing over 99% homology with several Bacillus velezensis species. A phylogenetic tree based on the maximum likelihood (MLLM) method was constructed using the 16S rDNA gene sequence of strain YP-5. The results are as follows: Figure 4 As shown, strain YP-5 clustered with five published Bacillus velezensis isolates into a single independent branch, with a 100% self-expansion support rate, indicating a high confidence level. Therefore, strain YP-5 was preliminarily identified as Bacillus velezensis. To further determine the species-level taxonomic position of strain YP-5, the gyrA gene sequence of strain YP-5 was sequenced, and the phylogenetic tree constructed based on this sequence is shown below. Figure 5 As shown, the comparison results support the above identification conclusion, indicating that strain YP-5 is Bacillus velezensis.

[0038] Example 2 Investigating the antibacterial spectrum of YP-5 Using a 5 mm punch, pathogens of tea anthracnose, tea ring spot, and tea leaf spot were inoculated into the center of PDA medium. YP-5 strain was then streaked 2 cm to either side of each pathogen using an inoculation loop. A control group was prepared by inoculating the pathogens individually. All culture dishes were incubated upside down in a 26°C incubator for 9 days. The mycelial radius of the pathogens was measured using the cross-cross method, and the inhibition rate of YP-5 against each pathogen was calculated.

[0039] The results are as follows Figure 6 As shown, Bacillus vesiculosus YP-5 exhibits good inhibitory effects against various pathogens. The inhibition rates of YP-5 against different pathogens were determined, and the results are shown in Table 2. YP-5 showed the best inhibitory effect against the two main dominant pathogens of tea anthracnose. The inhibition rate of YP-5 against *Anthracnose spp.* was significantly higher than that against other pathogens, reaching a maximum of 84.61%, followed by *Anthracnose spp.* (75.7%). The control efficacy of YP-5 against *Tea leaf spot* was 70.87%. In comparison, although the inhibitory ability of YP-5 against *Tea leaf spot* was relatively weaker than that against other pathogens, with an inhibition rate of 64.87%, it still falls within the range of good control efficacy.

[0040] The results showed that Bacillus belye YP-5 had a good inhibitory effect on a variety of major pathogens of tea trees, especially a significant antagonistic effect on anthracnose pathogens of tea trees, demonstrating good biocontrol potential for controlling anthracnose of tea trees.

[0041]

[0042] Example 3 Indoor efficacy test of YP-5 biocontrol agent Two-year-old tea cultivar Longjing 43, susceptible to anthracnose, was selected as the experimental material. Healthy, disease-free tea leaves were punctured with sterile insect stingers, and the bacterial solution was diluted to 1×10⁻⁶. 8 A CFU / mL suspension of Bacillus vesiculosus YP-5 was sprayed onto tea leaves until liquid dripped from the leaves. After drying for 1 hour, a 5 mm diameter anthracnose fungal cake was inoculated into the wound. The leaves were kept moist with cotton. NB medium spraying was used as a control. Each treatment was repeated 3 times, with 3 tea seedlings in each group. The seedlings were placed in an artificial climate incubator with the following conditions: 26℃, 90% humidity, 15000 lux, 12L / 12D. After 14 days, the diameter of the lesions was investigated and measured to determine the control efficacy.

[0043] like Figure 7 As shown in Table 3 (top image is the control group, bottom image is the treatment group), YP-5 fermentation broth has a significant control effect on tea anthracnose. The experimental results are shown in Table 3. After spraying tea trees with YP-5 fermentation broth and then inoculating them with the pathogen, the average diameter of tea anthracnose lesions was 2.34 mm, while the diameter of lesions in the control group reached 9.46 mm, showing a significant difference between the two. The calculated control efficacy of YP-5 fermentation broth was 75.26%, indicating its good application potential in controlling tea anthracnose.

[0044]

[0045] Example 4 A method to enhance the colonization ability of Bacillus belye in tea leaves based on "induced biofilm formation-leaf surface charge regulation". Principle: Under specific carbon source conditions such as sucrose, glucose, and mannitol, Bacillus can activate extracellular polysaccharide (EPS) and protein (such as TasA) synthesis genes to form a biofilm structure. The EPS in the biofilm anchors the bacteria firmly to the leaf surface through a dual mechanism of physical entanglement (forming hydrogen bonds and hydrophobic interactions with leaf waxes and cuticles) and chemical bonding (covalent / ionic bonding). Plant leaf waxes and cuticles contain carboxyl / phenolic groups and are negatively charged; the bacterial cell wall is also negatively charged, generating electrostatic repulsion and hindering initial attachment. Chitosan, as a positively charged polysaccharide, has been proven to improve the adhesion and retention of pesticides on leaves. Chitosan protonates (-NH3) under acidic conditions. + Carrying a positive charge, chitosan can electrostatically attract the negative charges on the leaf and bacterial surfaces, reducing the repulsion barrier and promoting the bacteria's approach to the leaf surface. Furthermore, chitosan molecular chains can interpenetrate and cross-link with the polysaccharide chains in the EPS biofilm, forming a more robust composite membrane structure, further enhancing adhesion strength and resistance to rain erosion.

[0046] Preparation of biofilm induction medium: Add any one of the following carbon sources, such as sucrose, mannitol, or glucose, to NB liquid medium to a concentration of 1.0% (w / v); The optimal carbon source is mannitol.

[0047] Preparation of chitosan solution: Dissolve chitosan with a degree of deacetylation ≥90% in 0.1 mol / L acetic acid to prepare a concentration of 0.1% (w / v), and then adjust the pH to 6.0 with 1 mol / L NaOH to make it consistent with the pH of the tea leaf surface; Two-year-old Longjing 43 seedlings were selected as experimental subjects. Mature leaves of uniform size and free from pests and diseases were selected, rinsed with sterile water and dried, and a 2cm×2cm detection area was marked.

[0048] Preparation of YP-5 biofilm suspension: Bacillus belyeis YP-5 (CGMCC No. 36088), after activation on NA solid medium, was inoculated onto NB liquid medium and cultured at 32℃ with shaking at 180 rpm until the logarithmic growth phase. 1 mL of the bacterial suspension was then inoculated onto biofilm induction medium and cultured at 32℃ with shaking at 180 rpm for 24 h. The cultured bacterial suspension was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the bacterial film precipitate was resuspended in sterile physiological saline. The concentration of the bacterial suspension was adjusted to 1×10⁻⁶. 8 CFU / mL was used to obtain a biofilm suspension.

[0049] YP-5 synergistic treatment: First, accurately pipette 100 μL of 0.1% chitosan solution and evenly drop it onto the marked 2cm × 2cm area on the surface of tea leaves. Let it stand at room temperature for 10–15 min to form a positively charged film. Then, drop 100 μL of a biofilm suspension containing 1% mannitol (1 × 10⁻⁶) onto the same area. 8 (CFU / mL), ensuring the liquid covers the marked area without spilling.

[0050] The control group was operated in the same way: the blank control group was given a bacterial suspension without any treatment, the single biofilm group was given a bacterial suspension without any treatment, and the single chitosan group was given chitosan and then a bacterial suspension without any treatment. All the tea seedlings were placed in an artificial climate incubator and the culture conditions were set at 26℃ and 70% humidity for 2 hours to complete the initial adhesion and colonization.

[0051] Initial colonization effect test: After 24 hours, the marked area of ​​the treated leaf was cut off with sterile scissors and placed in an Erlenmeyer flask containing 10 mL of 0.1% Tween-80 eluent. The mixture was shaken at 150 rpm for 30 min. The eluent was then serially diluted 10-fold (10... - ¹~10 -5 ), 100 μL of each dilution was spread on NB plates, incubated at 32℃ for 48 h, and the colonization amount of the leaves was counted (CFU / cm²).

[0052] As shown in Table 4, the initial colonization of tea leaves in the synergistic treatment group was significantly higher than that in the control groups, increasing by 1.9 times compared to the blank control. This demonstrates that the synergistic effect of "inducing biofilm formation - regulating leaf surface charge" can effectively enhance the initial binding ability of bacteria to tea leaves.

[0053]

[0054] Long-term colonization bacteria count detection: After initial colonization, tea seedlings were placed in an artificial climate chamber (26℃, 16h light / 8h darkness, 80% humidity). Samples were taken at 1, 3, 5 and 7 days after treatment to detect the colonization bacteria count in the marked areas of the leaves.

[0055] As shown in Table 5, the bacterial count in all treatment groups gradually decreased over time. The highest bacterial count was observed in the synergistic treatment group YP-5 7 days after colonization of tea leaves, with a leaf colonization count of 1.06 × 10⁻⁶. 6 The CFU / cm² concentration was 3.42 times that of the blank control group, demonstrating that this synergistic method can effectively prolong the colonization time of Bacillus belye on tea leaves.

[0056]

[0057] YP-5 synergistic treatment efficacy determination: Following the method described in the example for enhancing the colonization ability of *Bacillus belye* on tea leaves, conventional YP-5 fermentation broth was sprayed onto tea leaves in stages, with a sterile fermentation medium as a control. The specific method was the same as in Example 3. The experimental results are shown in Table 6. Spraying tea trees with this synergistic treatment agent under indoor conditions significantly enhanced the biocontrol effect against tea anthracnose, achieving a control efficacy of 83.45%, which was 10.29% higher than the initial fermentation broth efficacy. This effectively solves the problem of weak microbial cell adhesion and easy loss, leading to weakened biocontrol ability in existing technologies.

[0058]

[0059] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0060] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus belesii, characterized in that, The accession number is CGMCC: NO.36088.

2. A microbial inoculum containing Bacillus belye as described in claim 1.

3. A method for enhancing the colonization ability of Bacillus belye in tea leaves, characterized in that, Includes the following steps: (1) Preparation of YP-5 biofilm bacterial suspension: Bacillus belye, after activation, was inoculated and cultured to the logarithmic phase. The bacterial suspension was then inoculated into the biofilm induction medium and cultured. The cultured bacterial suspension was centrifuged, the supernatant was discarded, the bacterial film precipitate was resuspended, and the concentration of the bacterial suspension was adjusted to obtain the biofilm bacterial suspension. (2) YP-5 synergistic treatment: Take chitosan solution, place it evenly on the surface of tea leaves, and let it stand to form a positively charged film; add a biofilm suspension containing 1% mannitol to the same area; The Bacillus belyssus has the accession number CGMCC: NO.36088.

4. The method as described in claim 3, characterized in that, The preparation method of the biofilm induction medium is as follows: add any carbon source of sucrose, mannitol, or glucose to NB liquid medium until the w / v concentration is 1.0%.

5. The method as described in claim 4, characterized in that, The carbon source is mannitol.

6. The method as described in claim 5, characterized in that, Step (1) is as follows: Bacillus belye YP-5, after activation on NA solid medium, is inoculated into NB liquid medium and cultured at 32℃ and 180 rpm until the logarithmic phase. 1 mL of the bacterial suspension is then inoculated into biofilm induction medium and cultured at 32℃ and 180 rpm for 24 h. The cultured bacterial suspension is centrifuged at 3000 rpm for 10 min, the supernatant is discarded, and the bacterial film precipitate is resuspended in sterile physiological saline. The bacterial suspension concentration is adjusted to 1×10⁻⁶. 8 CFU / mL was used to obtain a biofilm suspension.

7. The method as described in claim 6, characterized in that, Step (2) specifically involves: accurately pipetting 100 μL of 0.1% chitosan solution and uniformly adding it to a 2 cm × 2 cm area on the surface of tea leaf, allowing it to stand for 10–15 min to form a positively charged film; then adding 100 μL of 1 × 10⁻⁶ solution containing 1% mannitol to the same area. 8 CFU / mL biofilm bacterial suspension.

8. The application of Bacillus belye as described in claim 1, the microbial agent as described in claim 2, and the method described in any one of claims 3-7 in the preparation of microbial preparations and in tea tree cultivation.

9. The application as described in claim 8, characterized in that, The microbial preparations mentioned include pesticides and antibacterial agents.

10. The application as described in claim 9, characterized in that, It inhibits anthracnose fungus, tea spot fungus, tea ring spot fungus, and fruit anthracnose fungus.

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