Bacillus velezensis YW91 and antibacterial application thereof

Abstract

The invention belongs to the field of microorganisms and biology, and provides a strain of Bacillus velezensis YW91 and bacteriostatic application thereof, the strain is preserved in China General Microbiological Culture Collection Center on July 23, 2025, the preservation number is CGMCC No.35342, the address of the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101. The bacillus velezensis YW91 disclosed by the invention can be used for simultaneously realizing strong inhibition on pathogenic fungi and pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and biotechnology, and particularly relates to a strain Bacillus velezensis YW91 and its antibacterial applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Tobacco bacterial wilt is caused by Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum Tobacco red spot disease is caused by Alternaria alternata, a bacterial disease that seriously threatens the tobacco industry and can ultimately lead to the complete death of plants. Alternaria alternata This disease is caused by *Anthracnose*, which primarily infects leaves, forming characteristic brown spots and causing leaf tissue necrosis, and can also lead to severe yield reduction. Tobacco anthracnose is caused by *Anthracnose spp.* (…). Colletotrichum nicotianae Grape gray mold is caused by *Botrytis cinerea*, primarily affecting leaves and stems. Lesions are circular, sunken, and exhibit concentric rings; severe cases can lead to leaf perforation and drop. Botrytis cinerea This disease is caused by a grayish-brown mold that primarily infects the fruit, leading to fruit rot and potentially severe losses under humid conditions. While traditional chemical control methods have some effectiveness, they increasingly face the dual challenges of growing pathogen resistance and escalating environmental pollution risks, necessitating safer and more effective control strategies.

[0004] Biocontrol strains, as microorganisms that control plant diseases through biological antagonism mechanisms, especially certain bacterial species, have shown great potential in tobacco disease control due to their diverse inhibitory mechanisms (such as producing antibiotics, competing for nutrients and space, and inducing plant resistance). However, there is still an urgent need to develop biocontrol strains that can simultaneously and effectively inhibit both pathogenic fungi and pathogenic bacteria.

[0005] A study has disclosed a strain of Bacillus belye isolated from tobacco rhizosphere soil. Bacillus velezensis EM-1, but its antibacterial effect still needs to be improved. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a strain of Bacillus belyssus YW91, which can simultaneously achieve potent inhibition against both pathogenic fungi and pathogenic bacteria.

[0007] The purpose of this invention is to develop novel microbial agents to meet the demand for highly efficient biological control products in the field of biological control. To achieve the above objective, this invention adopts the following technical solution: The first aspect of the present invention provides a strain of Bacillus belye. Bacillus velezensis YW91, this strain was deposited on July 23, 2025 at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 35342, address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postcode: 100101.

[0008] Studies have shown that the biocontrol strains of this invention can not only effectively inhibit the growth of pathogens of bacterial wilt, red spot disease, anthracnose, and grape gray mold, but also significantly reduce environmental pollution and prevent pathogens from developing drug resistance. This provides a sustainable and eco-friendly alternative for tobacco cultivation, achieving a win-win situation for disease control and environmental protection.

[0009] In a second aspect, the present invention provides the aforementioned Bacillus belye. Bacillus velezensis Application of YW91 in antibacterial activity.

[0010] A third aspect of the present invention provides the aforementioned Bacillus belye. Bacillus velezensis Application of YW91 in the control of tobacco bacterial wilt, tobacco red spot disease, tobacco anthracnose or grape gray mold.

[0011] A fourth aspect of the present invention provides an antibacterial agent, the antibacterial agent comprising: the above-mentioned Bacillus belyesii. Bacillus velezensis YW91 or Bacillus velezensis YW91 fermented crude extract.

[0012] A fifth aspect of the present invention provides a method for preparing an antibacterial agent, comprising: The Bacillus berberis Bacillus velezensis YW91 with 200-300 µL (OD) 600 Inoculate the culture medium with an inoculum of 0.3 g (=0.3 g), incubate at 28-30°C for 24 h or more, and collect the bacterial solution or crude fermentation extract to obtain the final product.

[0013] Beneficial effects of the present invention (1) The Bacillus vesiculus YW91 and its fermentation crude extract of the present invention can simultaneously achieve strong inhibition against pathogenic fungi and pathogenic bacteria.

[0014] (2) The fermentation method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The colony morphology of YW91 in Example 1 of this invention; Figure 2 This is the YW91 developmental tree in Embodiment 2 of the present invention; Figure 3 This illustrates the antagonistic effect of YW91 on RS10 in Embodiment 3 of the present invention. Figure 4 This illustrates the antagonistic effect of YW91 on CX06 (left), CN (middle), and 0201 (right) in Embodiment 4 of the present invention. Figure 5 The antagonistic effect of the crude extract of YW91 on RS10 in Example 5 of this invention; Figure 6 This describes the antagonistic effect of the crude extract of YW91 on CX06 in Example 6 of the present invention. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or product instructions. Similarly, unless otherwise specified, the test methods of this invention are performed in accordance with conventional methods in the art or industry-standard methods or practices. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0019] This invention provides a strain of Bacillus belye Bacillus velezensis YW91, this strain was deposited on July 23, 2025 at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 35342, address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postcode: 100101.

[0020] The present invention also provides the above-mentioned Bacillus belesiensis. Bacillus velezensis Application of YW91 in antibacterial activity.

[0021] In some embodiments, the pathogens include: Ralstonia solanacearum and / or Alternaria alternata, anthrax fungi, and Botrytis cinerea. The Bacillus bellis YW91 and its crude fermentation extract of the present invention can simultaneously achieve potent inhibition against both pathogenic fungi and pathogenic bacteria.

[0022] The present invention does not impose any special limitation on the type of crop. In some embodiments, the crop is tobacco to obtain a better antibacterial effect.

[0023] The present invention also provides the above-mentioned Bacillus belesiensis. Bacillus velezensis Application of YW91 in the control of tobacco bacterial wilt, tobacco red spot disease, tobacco anthracnose or grape gray mold.

[0024] The present invention also provides an antibacterial agent, the antibacterial agent comprising: the above-mentioned Bacillus belye Bacillus velezensis YW91 or Bacillus velezensis YW91 fermented crude extract.

[0025] In some implementations, Bacillus belesiensis Bacillus velezensis The concentration of YW91 is OD 600 =0.3-0.4, to effectively prevent and control tobacco bacterial wilt, tobacco red spot disease, tobacco anthracnose, and grape gray mold, etc.

[0026] In some embodiments, the Bacillus velezensis The concentration of the crude extract from YW91 fermentation is 100-150 mg / mL (for RS10) or 10-20 mg / mL (for CX06), with different antimicrobial agent concentrations designed for different pathogens.

[0027] This invention also provides a method for preparing an antibacterial agent, comprising: The Bacillus berberis Bacillus velezensis YW91 with 200-300 µL (OD) 600 Inoculate the culture medium with an inoculum of 0.3 g (=0.3 g), incubate at 28-30°C for 24 h or more, and collect the bacterial solution or crude fermentation extract to obtain the final product.

[0028] In some implementations, the culture time is 24 hours, 3 days, or 5-7 days to allow the strain to grow sufficiently and accumulate enough metabolites.

[0029] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0030] In the following examples, all raw materials are commercially available products.

[0031] Example 1 Bacillus velezensis separation Add 10 g of soil from the humus layer of fallen leaves and branches in the tobacco forest to 90 mL of sterile ultrapure water. Shake at 28℃ and 180 rpm for 30 min to mix thoroughly, then let stand. Take 1 mL of the suspension and serially dilute it. Spread 100 μL of the diluted suspension onto nutrient agar (NA) medium and incubate at 28℃ for 5 days. Streaking the colonies on NA medium five times repeatedly purifies the bacteria. The purified single strain is cultured on NA medium, and the colony morphology on the medium is a smooth, raised, pale yellow, round colony (e.g., ...). Figure 1 As shown in the figure, the strain number is YW91.

[0032] Example 2 Bacillus velezensis Identification Genomic DNA was extracted from strain YW91. Following the instructions for using Novizan's 2×Taq Plus Master Mix II DNA polymerase, PCR amplification was performed using the extracted YW91 genomic DNA as a template and 16S rRNA universal primers. The PCR products were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0033] The sequences of the universal primers for 16S rRNA are as follows: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1), downstream primer (1492R): 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The PCR amplification system is shown in Table 1, and the PCR amplification program used is shown in Table 2.

[0034] Table 1 PCR amplification system

[0035] Table 2 PCR amplification program

[0036] The 16S rRNA amplification sequence of strain YW91 is as follows: SEQ ID NO.3:

[0037] Gene sequence alignment analysis using EzBioCloud (https: / / www.ezbiocloud.net / ) and NCBI Blast revealed that strain YW91 is similar to... Bacillus velezensis The CR-502(T) sequence showed a similarity of 99.93%, and phylogenetic trees were constructed in MEGA 11 software using amplified sequences of similar bacteria with high sequence similarity to strain YW91. Figure 2 As shown, strain YW91 belongs to Bacillus velezensis It was deposited on July 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 35342. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0038] Example 3 Bacillus velezensis YW91 antagonizes pathogenic bacteria The purified YW91 was activated and grown to the logarithmic growth phase in nutrient broth (NB) medium in shake flasks. The OD was then adjusted using sterile water. 600 The concentration was diluted to 0.3 and inoculated into the center of NA medium. After incubation in the dark at 28°C for 24 h, the OD value was... 600 The bacterial pathogen *Ralstonia solanacearum* (Ralstonia solanacearum) has a value of approximately 0.3. Ralstonia solanacearum RS10 bacterial suspension was evenly sprayed onto the culture medium surface to test the antagonistic effect of the strain on bacterial wilt. After 24 hours, the size of the inhibition zone was used to determine the strength of the inhibitory effect on bacterial wilt. Figure 3 As can be seen, a clear ring appeared around the YW91 colony in the center of the plate, indicating a significant antagonistic effect against RS10.

[0039] Example 4 Bacillus velezensis YW91 antagonistic pathogenic fungi Red star pathogen (Alternaria alternata, Alternaria alternate CX06) was activated using oat solid medium (OA), and the anthrax pathogen ( Colletotrichum nicotianae Averna CN) and grape gray mold pathogen ( Botrytis cinerea 0201) The mycelium was activated using potato dextrose agar (PDA), and the mycelial cake was taken using a 5 mm punch and inoculated into the center of the culture medium. The purified YW91 was activated and grown to the logarithmic growth phase in NB medium in shake flasks, and the OD was... 600 Dilute to 0.3 and inoculate on both sides of the pathogenic fungus. Incubate in the dark at 25℃ for 5-7 days, observing the antibacterial effect daily (the above pathogenic fungal strains are preserved in the National Agricultural Environmental Microbial Germplasm Resource Bank). Figure 4It is evident that on day 7, strain YW91 significantly inhibited the growth of CX06, CN, and 0201, indicating that it has an antagonistic effect on the three pathogenic fungi.

[0040] Example 5 Bacillus velezensis YW91 fermentation crude extract antagonism Ralstonia solanacearum RS10 To further investigate the antagonistic effect of YW91 metabolites on pathogens, its metabolites were extracted. First, YW91 underwent large-scale fermentation: OD... 600 200 μL of bacterial culture with a pH of 0.3 was inoculated into 500 mL of NB medium and fermented for 3 days. The resulting fermentation broth was centrifuged, and the supernatant was lyophilized into powder to obtain the crude extract. The crude extract was used for antagonistic testing at a concentration of 100 mg / mL. The crude extract was weighed and extracted with 500 μL of methanol to extract the active ingredients. This extraction was repeated three times. The methanol extracts obtained from the three extractions were dried under nitrogen and then dissolved in a 30% methanol solution. The solutions were then filtered through a 0.22 μm filter membrane for sterilization before analysis.

[0041] OD 600 A bacterial suspension of *Ralstonia solanacearum* with a pH of approximately 0.3 was evenly sprayed onto the surface of NA medium. After the bacterial suspension air-dried, holes were punched using a puncher, and the center medium was removed with an inoculation needle. 50 μL of crude extract solution was added to the holes. The medium was incubated in the dark at 28°C for 24 h, and the size of the inhibition zone was observed. Figure 5 It is evident that, compared to the control (CK), the plate containing crude YW91 extract exhibited antagonistic effects against RS10, showing a distinct transparent zone, indicating that the metabolites of YW91 have an inhibitory effect on RS10.

[0042] Example 6 Bacillus velezensis YW91 fermentation crude extract antagonism Alternaria alternate CX06 The crude extract was obtained according to the procedure in Example 5 and tested at a concentration of 10 mg / mL. *Aureobasidium adenophorum* was activated and cultured in oat (OA) medium for approximately 7 days. The crude extract solution was first dissolved in OA medium, and then the plates were poured. After the plates solidified and cooled, *Aureobasidium adenophorum* pellets were placed in the center of the medium using a 5 mm punch. The plates were then incubated in the dark at 25°C for 7-10 days, and the antibacterial effect was observed. Figure 6 It is evident that the growth diameter of CX06 in the plate with added crude extract of YW91 was significantly smaller than that in the CK treatment, indicating that the metabolites of YW91 have an inhibitory effect on CX06.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belye Bacillus velezensis YW91, characterized in that, This strain was deposited on July 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35342. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

2. The Bacillus belesiensis as described in claim 1 Bacillus velezensis Application of YW91 in antibacterial activity.

3. The application as described in claim 2, characterized in that, Pathogens include: Ralstonia solanacearum, Alternaria alternata, anthrax bacteria, and Botrytis cinerea.

4. The application as described in claim 2, characterized in that, The crop is tobacco.

5. The Bacillus belesiensis as described in claim 1 Bacillus velezensis Application of YW91 in the control of tobacco bacterial wilt, tobacco red spot disease, tobacco anthracnose or grape gray mold.

6. An antibacterial agent, characterized in that, The bacterial agent comprises: Bacillus belesiensis as described in claim 1. Bacillus velezensis YW91 or Bacillus velezensis YW91 fermented crude extract.

7. The antibacterial agent as described in claim 6, characterized in that, Bacillus belesiensis Bacillus velezensis The concentration of YW91 is OD 600 =0.3-0.

5.

8. The antibacterial agent as described in claim 6, characterized in that, The Bacillus velezensis The concentration of the crude extract from YW91 fermentation is 100-150 mg / mL or 10-20 mg / mL.

9. A method for preparing an antibacterial agent, characterized in that, include: The Bacillus berberis Bacillus velezensis YW91 with 200-300 µL (OD) 600 Inoculate the culture medium with an inoculum of 0.3 g (=0.3 g), incubate at 28-30°C for 24 h or more, and collect the bacterial solution or crude fermentation extract to obtain the final product.

10. The method for preparing the antibacterial agent as described in claim 9, characterized in that, The incubation period is 24 hours, 3 days, or 5-7 days.

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