A bacillus strain and application thereof

Abstract

The application provides a bacillus strain and application thereof, the bacillus strain is preserved in Guangdong Microbial Culture Collection Center on July 24, 2025, and the preservation number is GDMCC No.66753; the application is preparation including the bacillus strain; the bacillus strain can effectively and durably inhibit the growth of plant pathogenic fungi, enhance the resistance of plants and promote the growth of plants.

Description

Technical Field

[0001] This invention relates to a Bacillus strain and its application, belonging to the field of microbial technology. Background Technology

[0002] Bacillus is a group of Gram-positive bacteria capable of forming spores. It can form spores under harsh environments to defend against attacks from adverse conditions. At the same time, Bacillus has a wide range of ecological functions, including promoting plant and animal growth, inhibiting the reproduction of harmful bacteria, decomposing organic matter, and improving the environment. It has applications in agriculture, aquaculture, environmental protection, and the food industry. Among these, biological control is environmentally friendly and has few side effects, making it a research hotspot in recent years and more in line with sustainable development. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the first objective of this invention is to provide a Bacillus strain that can effectively and persistently inhibit the growth of plant pathogenic fungi, enhance plant resistance, and promote plant growth.

[0004] The second objective of this invention is to provide an application of the above-mentioned Bacillus strain, which is to use the Bacillus strain to prepare a preparation and realize the function of the strain.

[0005] The first objective of this invention can be achieved by adopting the following technical solution: a Bacillus strain, classified and named... Neobacillus This Bacillus strain was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo. 66753.

[0006] Furthermore, the 16S rDNA of the Bacillus strain is shown in SEQ ID NO.1.

[0007] The second objective of this invention can be achieved by adopting the following technical solution:

[0008] The application of a Bacillus strain for the preparation of formulations containing the Bacillus strain.

[0009] Furthermore, the formulation is a plant growth promoter and / or disease resistant agent.

[0010] Furthermore, disease resistance refers to the inhibition of pathogenic bacteria in plants.

[0011] Furthermore, the pathogenic fungus is at least one of the following: rice blast fungus, *Chaetoceros globosum*, *Lacquer thunbergii*, and *Coccidioides praecox*.

[0012] Furthermore, the plant is at least one of the following: beans, rice, peanuts, and tomatoes.

[0013] Furthermore, the formulation is applied to the leaves or fruits of plants.

[0014] Furthermore, the formulation is prepared by inoculating a Bacillus strain into a fermentation substrate for fermentation, and then drying the obtained fermentation broth.

[0015] Furthermore, the concentration of the fermentation broth was 7 × 10⁻⁶. 6 -9×10 6 cfu / mL.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. The Bacillus of the present invention can effectively and persistently inhibit the growth of plant pathogens, and can be used as a broad-spectrum antibacterial biological fungicide to enhance plant resistance and promote plant growth;

[0018] 2. The Bacillus strain of the present invention can be used to prepare reagents that can be applied to plants. The application method is flexible, pollution-free, and in line with the sustainable development of the environment. Attached Figure Description

[0019] The Bacillus strain involved in this invention is classified and named as follows: Neobacillus This Bacillus strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No. 66753.

[0020] Figure 1 The colony morphology of strain HDS3-15;

[0021] Figure 2 Microscopic morphology of strain HDS3-15;

[0022] Figure 3 The growth curve for HDS3-15;

[0023] Figure 4 Photographs of the antibacterial test of rice blast fungus CK;

[0024] Figure 5 Photographs of the antibacterial test of rice blast fungus after 4 days;

[0025] Figure 6 Photographs of the antibacterial test of Chaetomium coccidioides CK;

[0026] Figure 7 Photographs of the antibacterial test of Chaetomium globulum on day 4;

[0027] Figure 8Photographs of the antibacterial test of *Russula dew-wet* CK;

[0028] Figure 9 These are photos of the antibacterial test of *Rust lacquer* after 4 days of exposure to dew.

[0029] Figure 10 Photographs of the antibacterial test of *Coccobacillus davidii* CK;

[0030] Figure 11 Photographs of the antibacterial test of *Coccidioidomyces beanus* on day 4;

[0031] Figure 12 The morphology of strain HDS3-15 on sodium selenite medium with a concentration of 0 μg / mL;

[0032] Figure 13 The morphology of strain HDS3-15 on sodium selenite medium with a concentration of 20 μg / mL;

[0033] Figure 14 The morphology of strain HDS3-15 on sodium selenite medium with a concentration of 40 μg / mL;

[0034] Figure 15 The morphology of strain HDS3-15 on sodium selenite medium with a concentration of 80 μg / mL;

[0035] Figure 16 The morphology of strain HDS3-15 on sodium selenite medium with a concentration of 160 μg / mL;

[0036] Figure 17 The morphology of strain HDS3-15 on sodium selenite medium at a concentration of 320 μg / mL is shown. Detailed Implementation

[0037] The present invention will now be further described with reference to the accompanying drawings and specific embodiments:

[0038] Example 1:

[0039] A strain of Bacillus was obtained by the following method:

[0040] 1) Isolation and purification of the strain: After sampling, fermentation broth was prepared. 100 μL of the sample solution was added to 900 μL of sterile water, and then the sample was diluted to 10 μL. -4 10 -5The concentration was determined by incubating the medium in a 70°C water bath for 20 minutes. 100 μL of the resulting liquid was then spread onto NA plates and incubated at 28°C for 72 hours. Bacterial growth was observed on the plates. Once colonies appeared, typical single colonies were streaked onto NA solid medium for isolation and purification. Single colonies were inoculated into 5 mL of sterilized NB broth and incubated at 28°C for 72 hours. The culture was then stored at -80°C with 50% v / v glycerol for later use.

[0041] The isolated and purified single colonies were identified using the following primers:

[0042] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2)

[0043] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3);

[0044] The 16S rDNA sequence of Bacillus HDS3-15 strain was obtained, as shown in SEQ ID NO.1.

[0045] 2) The sequence was compared and analyzed using the EZBioCloud database. The results showed that strain HDS3-15 was closely related to known type strains in the database. Neobacillus soli NBRC 102451 T The homology was 97.43%, suggesting it is a new species of *Bacillus* genus from soil.

[0046] 3) Upload the genome of the target strain to the Korean EzBioCloud website. Download the sequence of a type strain with high similarity and use the EzBioCloud online tool ANICalculator to calculate the ANI value. Use 95-96% as the cutoff value to determine whether the strains are different species. Upload the genomes of the target strain and related type strains to the online tool Genome-to-Genome Distance Calculator 3.0 on the German Culture Collection website. Use 70% as the cutoff value and calculate the dDDH value to determine whether the strains are different species.

[0047] Table 1 Calculation of ANI and dDDH in HDS3-15 genome

[0048]

[0049] The genome of HDS3-15 and closely related type strains Neobacillus soli NBRC 102451 TIn comparison, the ANI value is 75.18%, which is less than the threshold of 95%, and the dDDH value is 20.70%, which is less than the threshold of 70%. Therefore, HDS3-15 is a novel species of Bacillus and is named Bacillus Novelans. Neobacillus sp . HDS3-15. Figure 1 This refers to the colony morphology of the bacterial strain. Figure 2 It is in microscopic form.

[0050] 4) Growth curve determination of HDS3-15: The bacterial culture in the cryopreservation tube was inoculated into NB medium at an inoculation rate of 2% v / v and cultured in an incubator at 30℃ for 72 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD600nm of the strain was adjusted to 1.0 with PBS buffer. The strain was then inoculated into NB medium at an inoculation rate of 2% v / v and cultured in a growth curve analyzer at 30℃. The growth curve of HDS3-15 was measured every 10 minutes to obtain the growth curve (3 replicates).

[0051] like Figure 3 As shown, HDS3-15 took 6.5 hours to grow to an OD of 1.0 at 600 nm and 50 hours to reach the stable phase.

[0052] Detection:

[0053] 1) Antibacterial effect test of HDS3-15 strain:

[0054] The bacterial culture in the cryopreservation tube was inoculated into NB medium at an inoculation rate of 2% v / v and cultured at 30°C for 72 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD600nm of the strain was adjusted to 1.0 with PBS buffer. The strain was then inoculated into NB medium at an inoculation rate of 2% v / v and cultured at 30°C for 50 h. The resulting fermentation broth was then used for further processing.

[0055] Add 100 μL of fermentation broth of strain HDS3-15 to each of the four wells. Finally, place a uniformly sized bacterial block in the center of the solidified plate and incubate at 30°C for 4-10 days, observing whether the strain grows.

[0056] Prepare mycelial blocks of pathogenic fungi such as rice blast fungus, Chaetomium globosum, Lacquer thunbergii, and Coccidioides praecox, and repeat the experiment according to the above method.

[0057] Table 2. Antibacterial test results of HDS3-15: Antibacterial rate (%)

[0058]

[0059] Figures 4-11The images show the control (CK) for each pathogen and the antibacterial test results of *Orychophragmus oryzae*, *Chaetoceros glomeratus*, *Eucalyptus glomeratus*, and *Coccidioidomyces coccidioides* over 4 days.

[0060] 2) The control effect of HDS3-15 strain on pathogenic bacteria infecting detached leaves of common bean:

[0061] Prepare the HDS3-15 strain suspension for later use. Inoculate the strain into NB medium at an inoculation rate of 2% v / v and incubate at 30℃ for 50 h. Prepare the fermentation broth for later use.

[0062] *C. pratensis* was inoculated onto PDA medium and cultured at 30°C for 3 days. Mycelial blocks from the colony edges were collected using an inoculation spatula as inoculum. Isolated bean leaves were immersed in the fermentation broth for half an hour, then removed, and the leaf roots were wrapped with moistened defatted cotton balls. The leaves were placed in disposable petri dishes lined with moistened defatted cotton and allowed to stand for 24 hours before inoculation with the pathogen. A three-needle puncture treatment was used to inoculate the *C. pratensis* mycelial blocks onto the puncture sites of the isolated leaves. The leaves were cultured at 30°C for 11 days to observe disease progression. HDS3-15 showed a significant control effect of 93.67% against *C. pratensis* infection in isolated bean leaves.

[0063] 3) Determination of the available phosphorus capacity of HDS3-15 strain:

[0064] The strain (adjusted to OD=1.0) was inoculated at a rate of 2% v / v into 50 mL centrifuge tubes containing 30 mL of organic and inorganic phosphorus liquid culture medium. A blank control (CK) of uninoculated organic and inorganic phosphorus liquid culture medium was used. The tubes were cultured at 28℃ with shaking at 200 rpm for 5 days. Samples were taken at 24h, 48h, 72h, 96h, and 120h, with 5 mL samples taken each time. The samples were centrifuged at 10000 rpm at 4℃ for 5 min, and the supernatant was stored at 4℃ (3 replicates). The soluble phosphorus content in the supernatant at 24h, 48h, 72h, 96h, and 120h was determined using the molybdenum antimony colorimetric method.

[0065] Table 3. Determination of available phosphorus capacity: soluble phosphorus concentration (μg / mL)

[0066]

[0067] 4) Determination of IAA production capacity of HDS3-15 strain:

[0068] A bacterial suspension with OD600=1 was inoculated into 5 mL of NB liquid medium (containing 100 mg / L L-tryptophan) at an inoculation rate of 2% v / v (using 10 mL tubes) and cultured at 28℃ with shaking at 200 rpm for 5 days (3 replicates). 500 μL of the bacterial suspension at different time points (24 h, 48 h, 72 h, 96 h, and 120 h) were collected in 1.5 mL EP tubes, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. 50 μL of the supernatant was transferred to a 96-well plate, and 50 μL of Salkowski colorimetric solution (a mixture of solution A and solution B) was added simultaneously. After incubating the 96-well plate at room temperature in the dark for 30 min, the absorbance at OD530 nm was measured using a microplate reader.

[0069] Table 4 IAA Concentration (mg / L)

[0070]

[0071] 5) New selenium-enriched species (sodium selenite converted to monoselenium, qualitative analysis):

[0072] Prepare NB solid culture medium and autoclave at 121℃ for 30 minutes. Cool to 60-80℃ and add sodium selenite solution (stock solution concentration of 50mg / mL) to make the sodium selenite concentrations in the culture medium 0μg / mL, 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL, and 320μg / mL, respectively. After inverting and cooling the plates, inoculate HDS3-15 strain on the culture medium and incubate at 37℃ for 24h. If the strain can grow, absorb and convert sodium selenite on the sodium selenite plate, coffee red monomeric selenium will be formed on the plate surface and stored in the bacterial cells.

[0073] Figures 12-17 The morphology of HDS3-15 strain on sodium selenite plates at concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL, respectively.

[0074] Example 2:

[0075] Application of HDS3-15 strain preparations:

[0076] The HDS3-15 strain was inoculated into the fermentation substrate and fermented at 28-32℃ for 60 days. After 84 hours, the concentration of the fermentation broth obtained was 7 × 10⁻⁶. 6 -9×10 6 The concentration of cfu / mL is obtained by drying the sample.

[0077] When using, dilute at 0.5-4wt% to obtain a plant antibacterial agent.

[0078] This plant-based antibacterial agent has a rapid onset of action and a stable and sustained antibacterial effect. It can be applied to seeds, seedlings, leaves, and fruits through spraying, dipping, coating, encapsulation, and / or powdering.

[0079] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A Bacillus strain, characterized in that, The Bacillus strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No. 66753.

2. The Bacillus strain according to claim 1, characterized in that, The 16S rDNA of the Bacillus strain is shown in SEQ ID NO.

1.

3. The application of a Bacillus strain as described in claim 1, characterized in that, The application is for preparing formulations containing Bacillus strains.

4. The application as described in claim 3, characterized in that, The preparation is a plant disease-resistant preparation; the disease resistance is the inhibition of plant pathogens; the pathogens are at least one of rice blast fungus, Chaetomium globosum, Lacquer thunbergii, and Coccidioides praecox.

5. The application as described in claim 4, characterized in that, The plant is at least one of green beans, rice, peanuts, and tomatoes.

6. The application as described in claim 4, characterized in that, The preparation is applied to the leaves or fruits of plants.

7. The application as described in claim 3, characterized in that, The preparation is obtained by the following method: Bacillus strains are inoculated into a fermentation substrate for fermentation, and the obtained fermentation broth is dried.

8. The application as described in claim 7, characterized in that, The concentration of the fermentation broth is 7×10⁻⁶. 6 -9×10 6 cfu / mL.

Images