A Bacillus species and its applications

Abstract

This invention provides a Bacillus and its applications. The Bacillus was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 6, 2025, with accession number GDMCC No. 66483. One application of the Bacillus is to prepare preparations that promote plant growth and / or disease resistance. Another application is to use the Bacillus to inhibit microorganisms. This Bacillus can effectively and persistently inhibit the growth of plant pathogens and can be used as a broad-spectrum antibacterial biological control agent to enhance plant resistance and promote plant growth.

Description

Technical Field

[0001] This invention relates to a Bacillus species and its applications, 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 is applied in agriculture, animal husbandry, environmental protection, and the food industry. Among these, soil improvement, promoting plant growth, and biological control are among the hot research topics in recent years. 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 that 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.

[0004] A second objective of the present invention is to provide an application of the above-mentioned Bacillus, wherein the application is to use Bacillus to prepare preparations that promote plant growth and / or resist disease or inhibit microorganisms.

[0005] The first objective of this invention can be achieved by adopting the following technical solution: a Bacillus species, classified and named... Neobacillus The sp. strain was deposited on June 6, 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 GDMCC No. 66483.

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

[0007] Furthermore, the primers 27F and 1492R used to identify Bacillus are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0008] The second objective of this invention can be achieved by adopting the following technical solution: one of the technical solutions:

[0009] An application of Bacillus, used for the preparation of formulations containing Bacillus.

[0010] Furthermore, the formulation is a plant growth promoter and / or disease resistant agent.

[0011] Furthermore, the formulation is prepared by the following method: inoculating the bacterial solutions of Bacillus and Lactobacillus plantarum into a fermentation substrate for fermentation, and then drying the obtained bacterial solution.

[0012] Furthermore, the mass ratio of Lactobacillus plantarum to Bacillus spp. is 1:3-5.

[0013] The second technical solution mentioned above:

[0014] An application of Bacillus, specifically the use of Bacillus to inhibit microorganisms.

[0015] Furthermore, the microorganism is at least one of rice blast fungus, dew-wet lacquer fungus, and bean spore-forming fungus.

[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 of the present invention can be used in combination with other bacterial strains to prepare reagents for promoting plant growth and / or resisting disease. Attached Figure Description

[0019] The Bacillus involved in this invention is classified as... Neobacillus The sp. was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2025, with accession number GDMCC No. 66483.

[0020] Figure 1 The colony morphology of strain BL5-Ba4;

[0021] Figure 2 Microscopic morphology of strain BL5-Ba4;

[0022] Figure 3 The growth curve of BL5-Ba4;

[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 *Russula dew-wet* CK;

[0026] Figure 7 Photographs of the antibacterial test of *Russula pulveratula* after 4 days of exposure to de

[0027] Figure 8 Photographs of the antibacterial test of *Coccobacillus davidii* CK;

[0028] Figure 9 Photographs of the antibacterial test of *Coccidioidomyces beanus* on day 4;

[0029] Figure 10 Photographs of the antibacterial test of Chaetomium coccidioides CK;

[0030] Figure 11 Photographs of the antibacterial test of Chaetomium globulum on day 4;

[0031] Figure 12 Leaves inoculated only with Fusarium oxysporum mycelium blocks of tomato;

[0032] Figure 13 Image showing the inoculation of *Fusarium oxysporum* var. *tomatoides* after soaking fermentation broth containing BL5-Ba4. Detailed Implementation

[0033] The present invention will now be further described with reference to the accompanying drawings and specific embodiments:

[0034] Example 1:

[0035] A type of Bacillus, obtained by the following method:

[0036] 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 -5 The 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.

[0037] The isolated and purified single colonies were identified using the following primers:

[0038] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2)

[0039] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3);

[0040] The 16S rDNA sequence of Bacillus was obtained, as shown in SEQ ID NO.1, and the strain was named Bacillus BL5-Ba4.

[0041] 2) The sequence was compared and analyzed using the EZBioCloud database. The results showed that the BL5-Ba4 strain was closely related to known type strains in the database. Neobacillus pocheonensis The homology was 98.14%, suggesting it is a novel species of Bacillus.

[0042] 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.

[0043] Table 1 Calculation of ANI and dDDH in the BL5-BA4 genome

[0044]

[0045] The genome of BL5-BA4 and closely related type strains Neobacillus pocheonensis Compared to KCTC 13943, the ANI value is 71.07%, which is less than the threshold of 95%, and the dDDH value is 23.10%, which is less than the threshold of 70%, so it is a novel species of Bacillus. Figure 1 This refers to the colony morphology of the bacterial strain. Figure 2 It is in microscopic form.

[0046] 4) Growth curve determination of BL5-Ba4: 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 17 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD 600 nm 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 BL5-Ba4 was measured every 10 minutes to obtain the growth curve (3 replicates).

[0047] like Figure 3 As shown, BL5-Ba4 grew to OD600nm of 1.0 in 10 hours and reached the stable phase in 17 hours.

[0048] Detection:

[0049] 1) Detection of the antibacterial effect of BL5-Ba4 strain:

[0050] 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 17 h. The resulting fermentation broth was then used for further processing.

[0051] Add 100 μL of BL5-Ba4 strain fermentation broth 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 1–12 days, observing whether the strain grows.

[0052] Prepare mycelial blocks of pathogenic fungi such as rice blast fungus, dew-wet lacquer spot fungus, bean shell coccidioidomyces, and spherical scab fungus, and repeat the experiment according to the above method.

[0053] Table 2. Antibacterial rate (%) of Bacillus BL5-Ba4 in antibacterial experiment.

[0054]

[0055] Figure 4-11 The images show the control (CK) for each pathogen and the antibacterial test results for *Orychophragmus oryzae*, *Epipremnum aureum*, *Coccidioidomyces comatus*, and *Chaetoceros globosum*, taken after 4 days.

[0056] 2) The control effect of strain BL5-Ba4 on pathogenic bacteria infecting detached peanut leaves:

[0057] Prepare the BL5-Ba4 strain suspension for later use. Inoculate the strain into NB medium at an inoculation rate of 2% v / v and incubate at 30°C for 17 hours. The resulting fermentation broth is then prepared for later use.

[0058] Fusarium oxysporum tomatoense was inoculated onto PDA medium and cultured at 30°C for 3 days. Mycelial blocks from the edge of colonies were collected using an inoculation spatula as inoculum. Peanut leaves were immersed in 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 balls and allowed to stand for 24 hours before inoculation with pathogenic fungi. The leaves were then inoculated with Fusarium oxysporum tomatoense mycelial blocks at the puncture sites using a three-needle puncture treatment. The leaves were then cultured at 30°C for 11 days to observe disease development. Figure 12 Leaves inoculated only with Fusarium oxysporum mycelium blocks of tomato, Figure 13 The leaves of peanuts inoculated with Fusarium oxysporum mycelium blocks after being soaked in BL5-Ba4 fermentation broth showed that BL5-Ba4 had a significant control effect on Fusarium oxysporum infection of peanut detached leaves, with a control effect of 90.67%.

[0059] Table 2 shows that strain BL5-Ba4 exhibits good antibacterial effects, with a noticeable effect observed after 4 days and sustained antibacterial activity after 10 days. Combined with the in vitro peanut leaf infection experiment, it is believed that strain BL5-Ba4 can be used to prepare antibacterial agents for crops such as corn, wheat, rice, soybeans, peanuts, cucumbers, peppers, tomatoes, eggplants, green beans, and melons. Furthermore, strain BL5-Ba4 does not show significant inhibition against *Chaetoceros globosum*, which has a significant growth-promoting effect on plants; therefore, a synergistic effect on plant growth promotion is possible.

[0060] 3) Determination of the available phosphorus capacity of strain BL5-Ba4:

[0061] 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℃ and 200 rpm with shaking for 5 days. Samples were taken at 24h, 48h, 72h, and 96h, 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, and 96h was determined using the molybdenum antimony colorimetric method.

[0062] Table 3. Soluble phosphorus concentration (μg / mL) for determination of available phosphorus capacity

[0063]

[0064] 4) Determination of IAA production capacity of BL5-Ba4 strain:

[0065] 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.

[0066] Table 4 IAA Concentration (mg / L)

[0067]

[0068] Example 2:

[0069] Application of BL5-Ba4 strain preparations:

[0070] The bacterial solutions of BL5-Ba4 strain and Lactobacillus plantarum were inoculated into the fermentation substrate at a ratio of 4:1 and fermented at 28-32℃ for 60-84 hours. After centrifugation and washing, the bacterial solution was dried to obtain the final product.

[0071] When using, dilute at 0.5-4wt% to obtain a plant antibacterial agent.

[0072] Testing the antibacterial efficacy of plant antibacterial agents:

[0073] Prepare mycelial blocks of rice blast fungus, dew-wet lacquer spot fungus, and bean scab fungus, and repeat the test according to the method for detecting the antibacterial effect of BL5-Ba4 strain in Example 1.

[0074] Table 5. Antibacterial rate (%) of plant antibacterial agents in antibacterial experiments

[0075]

[0076] 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, and plants through spraying, dipping, coating, encapsulation, and / or powdering.

[0077] 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 species, characterized in that, The Bacillus (Latin scientific name: Neobacillus The sp. was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2025, with accession number GDMCC No. 66483.

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

1.

3. The Bacillus strain as described in claim 1, characterized in that, Primers 27F and 1492R used to identify Bacillus are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

4. A formulation, characterized in that, The formulation includes Bacillus as described in claim 1; the formulation is an inhibitor of microorganisms on plants; the microorganism is at least one of Rice Blast Fungus, Dewdrop Mold Fungus, and Common Bean Coccidioides.

5. The formulation as described in claim 4, characterized in that, The preparation is a plant growth promoter and / or disease resistant preparation.

6. The formulation according to claim 4, characterized in that, The preparation is obtained by the following method: inoculating the bacterial solutions of Bacillus and Lactobacillus plantarum into a fermentation substrate for fermentation, and then drying the obtained bacterial solution.

7. The formulation according to claim 6, characterized in that, The mass ratio of added Lactobacillus plantarum and Bacillus spp. is 1:3-5.

8. An application characterized in that, The application is to use the Bacillus as described in claim 1 to inhibit microorganisms on plants; the microorganisms are at least one of rice blast fungus, dew-wet lacquer fungus, and bean scab.

Images