Desert bacillus b-3 with broad-spectrum antibacterial activity and application thereof

Abstract

The application discloses a desert bacillus (Bacillus inaquosorum) B-3 with broad-spectrum antibacterial activity and an application thereof. The desert bacillus B-3 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is August 28, 2025, and the preservation number is CGMCC NO.35756. The B3 strain of the application is the first reported desert bacillus with antagonistic ability to plasmodiophoromycetes, and has antagonistic effects on a plurality of plant pathogenic fungi including botrytis cinerea, fusarium, sclerotinia sclerotiorum and oligoporus integrrins, and can be used as a biological pesticide, has a good effect on plasmodiophora diseases, and after the use of the B-3, the fresh weight of a plant infected by plasmodiophora brassicae is significantly increased, and the number of plant roots is significantly increased, indicating that the biocontrol fungus can effectively prevent and control the plasmodiophora diseases and has a bacteriostatic and growth-promoting effect.

Description

Technical Field

[0001] This invention belongs to the field of bio-agriculture and relates to a strain of Bacillus desertica B-3 with broad-spectrum antibacterial activity and its applications. Background Technology

[0002] Soil-borne plant diseases pose a persistent threat to agricultural production, with clubroot caused by *Cladophora brassicae* being particularly prominent, and its control is considered a global challenge. The dormant spores of this pathogen can survive in the soil for over ten years and spread through various routes, making it extremely difficult to eradicate once contaminated in the field. Currently, chemical pesticides have little effect on the dormant spores of this fungus and are ineffective against pathogens that have already invaded plant roots. While strong chemical methods such as soil fumigation have some effect, they are costly, dangerous to operate, and severely damage the soil microecology, running counter to sustainable agricultural development.

[0003] Faced with this severe challenge, microbial antagonists are considered a promising green control approach. However, existing biocontrol strains often exhibit significant limitations when dealing with clubroot fungi. Many strains have narrow antimicrobial spectra and cannot simultaneously combat secondary diseases caused by clubroot. More importantly, the acidic or infertile soil environments where clubroot often occurs severely restrict the survival, colonization, and activity of most biocontrol bacteria, leading to unstable field efficacy and difficulty in replicating laboratory results. Therefore, there is an urgent need in the field for a biocontrol microbial resource that can specifically adapt to and effectively act against this unique and stubborn pathogen, clubroot fungi. The ideal new strain should be able to effectively colonize in the harsh soil environments that clubroot fungi rely on for survival and possess broad-spectrum antagonistic activity to combat multiple infections, thus providing a completely new solution to overcome this intractable disease.

[0004] As an important member of the Bacillus genus, *Bacillus desertica* has seen its research and application in plant protection deepen due to its strong resistance and abundant metabolites. Publicly available research has shown that these strains have been proven to inhibit various plant pathogens, including *Fusarium cladosporium*, *Fusarium graminearum*, and *Fusarium wilt*, achieving disease control through mechanisms such as synthesizing antimicrobial compounds, competing for ecological niches, and inducing systemic resistance in plants. Some strains also possess multifunctional properties, such as promoting plant growth and degrading agricultural waste. Its culture conditions have been gradually standardized, and detection techniques have been continuously optimized, laying the foundation for large-scale application. However, to date, no publicly available reports have confirmed that *Bacillus desertica* has a function in controlling clubroot disease, and there is a lack of targeted strain screening and application research. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a strain of Bacillus desertica B-3 with broad-spectrum antibacterial activity and its applications.

[0006] Another object of the present invention is to provide a bacterial agent, supernatant and application of Bacillus desertica B-3.

[0007] Another objective of this invention is to provide a method for controlling clubroot disease in cruciferous crops.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A strain of Bacillus inaquosorum B-3 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 28, 2025, with accession number CGMCC NO.35756.

[0010] Bacillus inaquosorum B-3 was isolated from eggplant stems. It appears as opaque, yellowish-white, round colonies on LB agar plates. It can grow on LB, PDA, and YPDA media. The optimal growth temperature is 28°C. It can produce proteases and cellulases.

[0011] The bacterial agent prepared from Bacillus inaquosorum B-3.

[0012] Preferably, the bacterial agent is prepared by inoculating Bacillus inaquosorum B-3 into fresh, sterile liquid LB and incubating at 28°C and 220 rpm for 3-4 days.

[0013] The supernatant prepared from Bacillus inaquosorum B-3.

[0014] Preferably, the supernatant is prepared by the following method: Bacillus inaquosorum B-3 is inoculated into fresh sterile liquid LB, and cultured at 28°C and 220 rpm for 3-4 days. The cultured bacterial solution is collected and centrifuged at 6000 rpm for 10 minutes. The supernatant after centrifugation is filtered through a bacterial filter and used immediately.

[0015] Any of the following applications of Bacillus inaquosorum B-3:

[0016] (1) Inhibits any one or more of clubroot pathogens, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0017] (2) Control of plant diseases caused by any one of clubroot fungus, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0018] (3) Prepare a biological pesticide, wherein the biological pesticide inhibits any one or more pathogens among clubroot fungus, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0019] (4) Promote plant growth.

[0020] Any of the following applications of the aforementioned microbial agent:

[0021] (1) Inhibits any one or more of clubroot pathogens, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0022] (2) Control of plant diseases caused by any one of clubroot fungus, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0023] (3) Prepare a biological pesticide, wherein the biological pesticide inhibits any one or more pathogens among clubroot fungus, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0024] (4) Promote plant growth.

[0025] Any of the following applications of the supernatant:

[0026] (1) Inhibits any one or more of clubroot pathogens, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0027] (2) Control of plant diseases caused by any one of clubroot fungus, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0028] (3) Prepare a biological pesticide, wherein the biological pesticide inhibits any one or more pathogens among clubroot fungus, Botrytis cinerea, Fusarium, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum;

[0029] (4) Promote plant growth.

[0030] A method for controlling clubroot disease in cruciferous crops includes applying the aforementioned Bacillus desertica B-3 or the aforementioned inoculant to the roots of cruciferous crops or to the planting soil.

[0031] Preferably, the application method is one or more of the following: root drenching, soil spraying, or soil mixing, with root drenching being preferred; the bacterial solution, after being cultured at 28°C and 220 rpm for 7 days, is evenly poured onto the roots of the plants, 5 ml per plant (final concentration approximately 1 x 10⁻⁶). 7 CFU / mL); Application time: 3-7 days after plant emergence.

[0032] Beneficial effects:

[0033] The B3 strain of this invention is the first reported desert Bacillus strain with antagonistic ability against clubroot bacteria. It can be used as a biological pesticide and has a good effect on clubroot disease. After using B-3, the fresh weight of plants infected with clubroot bacteria Pb4 increased significantly and the number of plant roots increased significantly, indicating that this biocontrol bacterium can effectively control clubroot disease and has antibacterial and growth-promoting effects.

[0034] B3 is simple to use for controlling plant diseases. A single root drenching after shaking culture is sufficient to effectively prevent the impact of clubroot infection on plant growth. It also has antagonistic effects on various plant pathogens, including *Botrytis cinerea*, *Fusarium*, *Sclerotinia sclerotiorum*, and *Sclerotium sclerotiorum*, significantly preventing the effects of clubroot infection on the host and promoting plant root growth. It produces various extracellular enzymes, and both the bacterial cells and the culture supernatant can antagonize multiple plant pathogens. Attached Figure Description

[0035] Figure 1 Colony morphology of Bacillus desertii B-3 on PDA

[0036] Figure 2 Developmental tree of 16S rRNA sequence alignment of Bacillus desertii B-3

[0037] Figure 3 Developmental tree of gryA sequence alignment of Bacillus desertii B-3

[0038] Figure 4 Whole genome sequencing and annotation comparison of Bacillus desertii B-3

[0039] Figure 5 Bacillus desertis B-3 exhibits antagonistic activity against various soil-borne pathogens; dual-cultural plate confrontation antagonism; VOCs antagonism; and CFS supernatant antagonism.

[0040] Figure 6 Experiment on the antagonism of clubroot bacteria by Bacillus desertis B-3 inoculum.

[0041] Figure 7 Experiment on the antagonism of clubroot bacteria by supernatant of Bacillus desertis B-3

[0042] Information on the preservation of biological materials

[0043] B-3, classified as Bacillus inaquosorum, 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. The deposit date is August 28, 2025, and the accession number is CGMCC NO.35756. Detailed Implementation

[0044] Example 1 B-3 was obtained by isolating eggplant stems using the following method:

[0045] Cut the stems of eggplants with mild sclerotinia disease into small sections, soak them in 75% alcohol for disinfection, and rinse them twice with sterile water. Chop them in sterile water, let them stand for 2 minutes, dip an inoculation loop in the solution, streak the loop, and incubate at 28°C.

[0046] like Figure 1 As shown, B-3 forms opaque white, nearly circular colonies on LB plates; it can grow on LB, YPDA, and PDA plates; the optimal growth temperature is 28℃.

[0047] Based on morphological observation, 16S and gryA phylogenetic tree alignment, and whole-genome sequencing alignment, B-3 was identified as *Bacillus inaquosorum*. Figure 1-4 As shown.

[0048] Example 2 Preparation of bacterial agent

[0049] Inoculate a single colony of B-3 into fresh, sterile liquid LB and incubate at 28°C and 220 rpm for 72 hours. The resulting fresh bacterial solution is used as the inoculum.

[0050] Example 3: Preparation of Supernatant

[0051] Inoculate a single colony of B-3 into fresh, sterile liquid LB and incubate at 28°C and 220 rpm for 72 hours to obtain a fresh bacterial culture. Centrifuge the fresh bacterial culture from the previous step at 6000 rpm for 5 minutes. Transfer the supernatant from the centrifugation to a new 50 ml centrifuge tube and filter it through a 0.22 μm bacterial filter into a sterile container. The resulting fresh supernatant can be used immediately.

[0052] Example 4: Antagonism of B-3 inoculant, supernatant and volatile substances against Fusarium, Botrytis, Sclerotinia sclerotiorum and Sclerotinia sclerotiorum.

[0053] The inhibition rates of B-3 against Fusarium and Sclerotinia were determined using the plate confrontation method, VOCs antagonism, and supernatant antagonism experiment. Fusarium here specifically refers to *Fusarium oxysporum* and *Fusarium fjikuroi*, Botrytis cinerea is the gray mold, *Sclerotium rolfsii* is the regular sclerotium, and *Sclerotinia scleroriorum* is the sclerotinia. All pathogenic fungi used were isolated from susceptible plants, and their pathogenicity was determined using Koch's postulates.

[0054] The plate confrontation antagonism experiment method is as follows: Inoculate a 5 mm diameter fungal cake or a 1 mm diameter sclerotium in the center of a PDA plate, and simultaneously inoculate 5 μL of OD at a distance of 3.5 cm from the center of the plate. 600 The test strain with a value of 1, with the control strain not inoculated, was cultured at 28°C for 5 days. Each treatment was repeated 3 times.

[0055] The experimental method for antagonizing VOCs is as follows: Inoculate a 5 mm diameter fungal cake or a 1 mm diameter sclerotium in the center of a PDA plate; evenly spread 1 ml of B-3 bacterial solution (OD) on another plate. 600 =1.0), pipette to remove excess bacterial solution, and air dry; invert the two petri dishes with their edges aligned to seal, and incubate at 28 degrees Celsius for 4-7 days.

[0056] The supernatant antagonism experiment was conducted as follows: The supernatant obtained in Example 3 (B-3) was added to warm PDA solid medium at a volume ratio of 5%; before solidification, the medium was poured into sterile petri dishes; the pathogen to be tested was inoculated onto plates containing the supernatant. The plates were then incubated at 28 degrees Celsius for 4-7 days.

[0057] Experimental results are as follows Figure 5 As shown, B-3 can significantly antagonize Fusarium, Sclerotinia sclerotiorum, Botrytis cinerea, and Sclerotium sclerotiorum.

[0058] Example 5: Antagonism of B-3 inoculant against clubroot bacteria

[0059] Experimental group (only biocontrol agent B-3 was applied): The B-3 bacterial fermentation liquid prepared in Example 2 was applied to the roots once, with 5 mL applied to each of the 6 plants. No additional bacterial liquid was applied during the treatment period, and the treatment lasted for 3 weeks.

[0060] Experimental group (treated with biocontrol agent B-3 and clubroot bacteria Pb4): The B-3 bacterial fermentation broth prepared in Example 3 was applied to the roots once, with 5 mL per plant, repeated for 6 plants. 24 hours after B-3 application, 1 mL of purified clubroot bacteria Pb4 spores (concentration 1 x 10⁻⁶) was applied to each plant. 8 mL / CFU. No additional B-3 bacterial suspension is required during the treatment period, which lasts for 3 weeks.

[0061] Control group (treated with clubroot bacteria Pb4): The clubroot tissue of the plant was ground, filtered, and collected to prepare a solution with a concentration of 1×10⁻⁶. 8 The spore suspension was prepared. The prepared clubroot spore suspension was used for root irrigation once, with 5 mL per plant. This was repeated for 6 plants. No additional spore suspension was needed during this period. The treatment lasted for 3 weeks.

[0062] Control group (treated with sterile PDA medium): The roots of each cruciferous plant were irrigated once with sterile PDA liquid medium, with a irrigation volume of 5 mL per plant, repeated for 6 plants. No additional medium was required during the treatment period, and the treatment lasted for 3 weeks.

[0063] Three weeks after all treatments, the roots were removed for inspection and photographic recording. The fresh weight of the plants was also recorded for analysis.

[0064] Depend on Figure 6 It can be seen that, compared with the treatment group that was only inoculated with clubroot bacteria, the fast-growing vegetables treated with biocontrol agent B-3 showed increased fresh weight, reduced clubroot size, and improved root growth. In conclusion, B-3 has significant antibacterial and growth-promoting effects and can significantly reduce the damage caused by clubroot disease.

[0065] Example 6: Antagonism of B-3 supernatant against clubroot bacteria

[0066] Experimental group (only supernatant of biocontrol bacteria B-3 was applied): The supernatant of bacteria B-3 prepared in Example 3 was applied to the roots once, with 5 mL applied to each plant. The treatment was repeated for 6 plants. No additional supernatant was applied during the treatment period. The treatment lasted for 3 weeks.

[0067] Experimental group (treated with biocontrol bacteria B-3 supernatant and clubroot bacteria Pb4): The supernatant of bacteria B-3 prepared in Example 3 was applied to the roots once, with 5 mL per plant, repeated for 6 plants. 24 hours after B-3 application, 1 mL of purified clubroot bacteria Pb4 spores (concentration 1 x 10⁻⁶) was applied to each plant. 8 mL / CFU. No additional B-3 bacterial suspension is required during the treatment period, which lasts for 3 weeks.

[0068] Control group (treated with clubroot bacteria Pb4): The clubroot tissue of the plant was ground, filtered, and collected to prepare a solution with a concentration of 1×10⁻⁶. 8 The spore suspension was prepared. The prepared clubroot spore suspension was used for root irrigation once, with 1 mL per plant. This was repeated for 6 plants. No additional spore suspension was needed during this period. The treatment lasted for 3 weeks.

[0069] Control group (applied with sterile PDA medium): roots were irrigated once with sterile PDA liquid medium, with 5 mL per plant, repeated for 6 plants. No additional medium was applied during the treatment period, and the treatment lasted for 3 weeks.

[0070] Three weeks after all treatments, the roots were removed for inspection and photographic recording. The fresh weight of the plants was also recorded for analysis.

[0071] Depend on Figure 7It can be seen that, compared with the treatment group inoculated only with clubroot bacteria, the *Gnaphalium affine* treated with the supernatant of biocontrol bacterium B-3 showed increased fresh weight, significantly reduced clubroot, and improved root growth. In conclusion, the supernatant of B-3 has a significant antibacterial and growth-promoting effect, and can significantly reduce the occurrence of clubroot disease. This also indicates that its supernatant contains biocontrol substances and has extremely high biocontrol potential.

Claims

1. A strain of desert Bacillus ( Bacillus inaquosorum B-3, characterized in that, It is deposited at the China General Microbiological Culture Collection Center; deposit date: August 28, 2025, accession number: CGMCC NO.35756.

2. The desert Bacillus (as described in claim 1) Bacillus inaquosorum B-3 is a microbial agent prepared by the microbial agent.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is prepared by the following method: The desert Bacillus (…) Bacillus inaquosorum B-3 was inoculated into liquid LB and incubated at 28°C and 220 rpm for 3-4 days.

4. The desert Bacillus (as described in claim 1) Bacillus inaquosorum The supernatant prepared by B-3.

5. The supernatant according to claim 4, characterized in that, The supernatant was prepared by the following method: Bacillus desertis B-3 was inoculated into fresh sterile liquid LB, and cultured at 28°C and 220 rpm for 3-4 days. The cultured bacterial solution was then centrifuged at 6000 rpm for 10 min. The supernatant was then filtered through a bacterial filter and used immediately.

6. The desert Bacillus (as described in claim 1) Bacillus inaquosorum B-3 can be applied in any of the following ways: (1) Inhibits any one or more of the following pathogens: clubroot, botrytis cinerea, fusarium oxysporum, fusarium tumefaciens, sclerotium sclerotiorum, and sclerotium sclerotiorum; (2) Control of plant diseases caused by any one of the following fungi: clubroot, Botrytis cinerea, Fusarium oxysporum, Fusarium tumefaciens, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum; (3) Prepare a biological pesticide, wherein the biological pesticide inhibits any one or more pathogens among clubroot fungus, Botrytis cinerea, Fusarium oxysporum, Fusarium tumefaciens, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum; (4) Promote plant growth.

7. Any one of the following applications of the microbial agent according to claim 2 or 3: (1) Inhibits any one or more of the following pathogens: clubroot, botrytis cinerea, fusarium oxysporum, fusarium tumefaciens, sclerotium sclerotiorum, and sclerotium sclerotiorum; (2) Control of plant diseases caused by any one of the following fungi: clubroot, Botrytis cinerea, Fusarium oxysporum, Fusarium tumefaciens, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum; (3) Prepare a biological pesticide, wherein the biological pesticide inhibits any one or more pathogens among clubroot fungus, Botrytis cinerea, Fusarium oxysporum, Fusarium tumefaciens, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum; (4) Promote plant growth.

8. Any of the following applications of the supernatant as described in claim 4 or 5: (1) Inhibits any one or more of the following pathogens: clubroot, botrytis cinerea, fusarium oxysporum, fusarium tumefaciens, sclerotium sclerotiorum, and sclerotium sclerotiorum; (2) Control of plant diseases caused by any one of the following fungi: clubroot, Botrytis cinerea, Fusarium oxysporum, Fusarium tumefaciens, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum; (3) Prepare a biological pesticide, wherein the biological pesticide inhibits any one or more pathogens among clubroot fungus, Botrytis cinerea, Fusarium oxysporum, Fusarium tumefaciens, Sclerotinia sclerotiorum, and Sclerotium sclerotiorum; (4) Promote plant growth.

9. A method for controlling clubroot disease in cruciferous crops, characterized in that, This includes applying the Bacillus desertis B-3 of claim 1, the inoculum of claim 2, or the supernatant of claim 4 to the roots of cruciferous crops or to the planting soil.

10. The prevention and control method according to claim 9, characterized in that, The application method is one or more of the following: root irrigation, soil spraying, or soil mixing; apply 5 ml of the described microbial agent or the described supernatant to each plant; application time: within 3-7 days after the plant emerges.

11. The prevention and control method according to claim 9, characterized in that, The application method is root irrigation.

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