Bacillus composti DX-10 and application thereof

Abstract

The present application belongs to the field of microbial technology, and discloses a compost bacillus DX-10 and application thereof. The present application provides a compost bacillus, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251073. The strain has a good prevention and treatment effect on kiwi soft rot, and shows excellent inhibition on the pathogenic bacteria of kiwi soft rot, such as grape Coniothyrium and Diaporthe phaseolorum. The strain provides a strain resource for preparing a biocontrol agent, can alleviate the environmental hazards caused by excessive use of chemical agents, and is expected to become an important tool for developing green control drugs for kiwi soft rot.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and discloses a strain of Bacillus compostii DX-10 and its applications. Background Technology

[0002] Soft rot is a plant disease caused by bacteria or fungi, primarily affecting succulent vegetables, flowers, and tuber crops. Kiwi soft rot is a common post-harvest fungal rot disease in kiwifruit. The typical symptoms include brown or dark gray lesions on the fruit peel, mostly round or oval. The center of the lesion is milky white, surrounded by a yellowish-green ring, accompanied by a water-soaked ring around the edge, oozing a small amount of tissue fluid. In severe cases, it can lead to perforation and even complete rot of the kiwifruit. Many pathogens can cause soft rot in kiwifruit, but the most frequently reported and most virulent pathogens fall into two categories: fungi of the family Botryosphaeriaceae, such as *Botryosphaeria dothidea*; and fungi of the genus *Diaporthe*, such as *Diaporthe celeris*. Currently, chemical fungicides are generally used to control soft rot pathogens. However, the long-term use, improper abuse, and non-degradability of chemical fungicides can easily lead to environmental pollution, ecological damage, and pesticide residues on fruits and vegetables. Furthermore, the fixed mechanisms of action of chemical pesticides can easily lead to drug resistance in pathogens. Therefore, developing green, efficient, and non-toxic biological pesticides has become an urgent problem to be solved in the field of kiwifruit soft rot control.

[0003] Biological control, with its diverse antibacterial mechanisms and environmentally friendly characteristics, provides an effective approach to controlling kiwifruit soft rot. Bacillus, a type of bacteria widely found in nature, possesses advantages such as broad-spectrum antibacterial activity, rapid reproduction, strong resistance, and high safety, making it an ideal biocontrol agent. Bacillus can not only directly synthesize and secrete various bioactive and antibacterial substances, but also indirectly activate multiple defense response mechanisms, inducing systemic resistance in plants. Therefore, discovering more biocontrol Bacillus species has significant practical importance and application value for controlling kiwifruit soft rot. Summary of the Invention

[0004] To overcome the aforementioned problems in the prior art, this invention provides a strain of Bacillus compostii DX-10 and its applications. Compared with the prior art, the advantage of this invention is that it provides a strain of Bacillus compostii that has a good control effect on kiwifruit soft rot and exhibits excellent inhibitory effects on the pathogens of kiwifruit soft rot, such as Staphylococcus aureus and Staphylococcus mesenteroides.

[0005] In the technical solution provided by this invention, "DX-10" or "Bacillus stercoris DX-10" both refer to the Bacillus stercoris strain provided by this invention.

[0006] On one hand, this invention relates to a strain of Bacillus compostii, the preservation information of which is as follows:

[0007] Strain name: Bacillus compostii DX-10;

[0008] Classification and nomenclature: Bacillus stercoris strain;

[0009] Preservation period: May 15, 2025;

[0010] Depository: China Center for Type Culture Collection;

[0011] Accession number: CCTCC NO: M 20251073;

[0012] Address: Wuhan University, Wuhan, China.

[0013] On the other hand, the present invention relates to a microbial inoculant, the microbial inoculant comprising:

[0014] The aforementioned Bacillus compostii;

[0015] The volatile organic compounds produced by the composting Bacillus; and / or

[0016] The supernatant, fermentation product, filtrate, or extract of the culture of Bacillus compostii.

[0017] On the other hand, the present invention relates to the application of the aforementioned Bacillus compostii or the aforementioned microbial agent in the prevention and control of soft rot.

[0018] Furthermore, in the application provided by the present invention, the pathogenic plant of the soft rot is kiwifruit, and the pathogen of the soft rot is *Botryosphaeria dothidea* and / or *Diaporthe celeris*.

[0019] On the other hand, the present invention relates to a plant biocontrol agent, wherein the active ingredient of the plant biocontrol agent comprises:

[0020] The aforementioned Bacillus compostii;

[0021] The volatile organic compounds produced by the composting Bacillus; and / or

[0022] The supernatant, fermentation product, filtrate, or extract of the culture of Bacillus compostii.

[0023] On the other hand, the present invention relates to a plant biocontrol agent for the control of soft rot, wherein the active ingredient of the plant biocontrol agent for the control of soft rot comprises:

[0024] The aforementioned Bacillus compostii;

[0025] The volatile organic compounds produced by the composting Bacillus; and / or

[0026] The supernatant, fermentation product, filtrate, or extract of the culture of Bacillus compostii.

[0027] Spores or prespores, or hyphae, prespores, and / or combinations of spores of any of the Bacillus compostii or any active variants thereof provided herein, can be formulated into mycelial pastes, wettable powders, mycelial clumps, dust, granules, slurries, dry powders, aqueous or oil-based liquid products, etc. Such formulations will contain Bacillus compostii or its active variants and / or compositions derived therefrom provided herein, along with carriers and other reagents. The formulations can be used in a variety of methods disclosed elsewhere herein.

[0028] The various compositions and formulations disclosed herein may comprise a certain amount of hyphae, spores, or prespores, or a combination of hyphae, prespores, and / or spores of Bacillus compostii or any of its active variants; and / or may comprise a certain amount of the composition derived from any of Bacillus compostii or any of its active variants. Such amounts may include at least about 10 4 CFU / mL to approximately 10 11 CFU / mL, at least approximately 10 5 CFU / mL to approximately 10 11 CFU / mL, approximately 10 5 CFU / mL to approximately 10 10 CFU / mL, approximately 10 5 CFU / mL to approximately 10 12 CFU / mL, approximately 10 5 CFU / mL to approximately 10 6 CFU / mL, approximately 10 6 CFU / mL to approximately 10 7 CFU / mL, approximately 10 7 CFU / mL to approximately 10 8 CFU / mL, approximately 10 8 CFU / mL to approximately 10 9 CFU / mL, approximately 10 9 CFU / mL to approximately 10 10 CFU / mL, approximately 10 10 CFU / mL to approximately 10 11CFU / mL or approximately 10 11 CFU / mL to approximately 10 12 The strain concentration is CFU / mL. In other embodiments, the strain concentration includes at least approximately 10. 4 CFU / mL, at least approximately 10 5 CFU / mL, at least approximately 10 6 CFU / mL, at least approximately 10 7 CFU / mL, at least approximately 10 8 CFU / mL, at least approximately 10 9 CFU / mL, at least approximately 10 10 CFU / mL, at least approximately 10 11 CFU / mL, at least approximately 10 12 CFU / mL. The strain at the above concentration can be formed in any type of formulation for any purpose, including, for example, in liquid formulations, wettable powders, sprayed dry formulations, mycelial pastes, wettable granules, or freeze-dried formulations.

[0029] As used herein, "supernatant" refers to the liquid remaining after *Bacillus compostii* has been grown in a liquid culture medium or harvested from a solid culture medium into another liquid and removed by centrifugation, filtration, sedimentation, or other means known in the art. In some embodiments, the supernatant may be diluted with another substance, such as water, a buffer, fresh culture medium, and / or a formulation. The diluted supernatant is still considered to be the supernatant of this invention.

[0030] As used herein, "filtrate" refers to the liquid from a Bacillus composting fermentation culture in a liquid medium that passes through a membrane. The filtrate may contain a concentrated amount of the active compounds or metabolites compared to the concentration of the active compounds or metabolites in the fermentation culture or supernatant.

[0031] As used herein, "extract" refers to a substance removed from *Bacillus compostii* or its fermentation culture by means of a solvent (e.g., water, detergent, buffer, and / or organic solvent) or directly, and separated from *Bacillus compostii* by centrifugation, filtration, evaporation, or other methods known in the art. The extract may contain a concentrated or diluted amount of the active compound or metabolite compared to the concentration in the *Bacillus compostii* fermentation culture prior to extraction. Alternatively, the filtrate or extract may then be diluted with another composition, such as water, buffer, fresh culture medium, and / or formulation. Such diluted filtrate or extract is still considered the filtrate and extract of the present invention.

[0032] As used herein, “metabolite” or “metabolic product” refers to compounds, substances, or byproducts produced by Bacillus composting through fermentation. Effective compounds or metabolites are compounds present in the supernatant, fermentation culture containing Bacillus composting, or Bacillus composting itself, which, when applied in effective amounts to the target plant or the space in which the target plant is located, can control pathogens that cause plant soft rot.

[0033] On the other hand, the present invention relates to a method for preventing and controlling soft rot, comprising: causing the Bacillus compostii or the plant biocontrol agent to act on the pathogens of soft rot and / or their habitats;

[0034] The pathogenic bacteria are *Botryosphaeria dothidea* and / or *Diaportheceleris*.

[0035] Furthermore, in the method provided by the present invention, their habitat is a kiwifruit, a kiwifruit growing area, or a kiwifruit storage area.

[0036] Furthermore, in the method provided by the present invention, the Bacillus compostii or the plant biocontrol agent is applied to pathogens causing soft rot or to plants at risk of developing soft rot.

[0037] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages.

[0038] This invention provides a strain of Bacillus compostii, or a modified strain, its active variants, and / or compositions derived therefrom, which can be used with any plant species or its habitat to control plant soft rot. The control of plant soft rot can be, but is not limited to: preventing plants from being infected by the pathogens causing plant soft rot; treating plants already infected by the pathogens causing plant soft rot; controlling the formation of inoculum in plants or the habitats of the pathogens causing plant soft rot; and controlling the formation of inoculum in risk areas where the pathogens may cause plant soft rot.

[0039] The Bacillus composting strain provided by this invention is a biocontrol bacterium isolated from kiwifruit infected with soft rot in a cold storage facility in Zhouzhi County, Xi'an City, Shaanxi Province. This Bacillus composting strain, as a plant endophyte, is an environmentally friendly raw material for biological agents. Compared to rhizosphere and soil microorganisms, endophytes have a unique growth environment within the plant, do not compete with soil microorganisms for survival, are less affected by field operations and climate change, and can enhance the plant's endogenous resistance, showing promising application prospects. The Bacillus composting strain provided here, for the control of kiwifruit soft rot, features rapid growth, strong establishment ability, simple cultivation, good stability, and environmental friendliness, demonstrating good development and application prospects for the biological control of soft rot. Attached Figure Description

[0040] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This image shows the antibacterial effect of Bacillus compostii DX-10 on the pathogen causing soft rot in kiwifruit.

[0042] Figure 2 This is a colony morphology diagram of Bacillus compostii DX-10.

[0043] Figure 3 The diagram shows the construction results of the phylogenetic tree of Bacillus compostii DX-10.

[0044] Figure 4 This is an example of the control effect of Bacillus compostii DX-10 on soft rot in kiwifruit.

[0045] Figure 5 The diagram shows the inhibitory effect of VOCs produced by Bacillus composting DX-10 on the pathogen of soft rot.

[0046] Figure 6 The graph shows the inhibitory effect of the fermentation supernatant of Bacillus composting DX-10 on the pathogen of soft rot.

[0047] Figure labeling: Control or CK represents the blank control group; DX-10 represents the results of the experiment using Bacillus compostii DX-10; B. dothidea represents the results of the experiment using Botryosphaeria dothidea as an antagonistic pathogen; Diaporthe sp. represents the results of the experiment using Diaporthe celeris as an antagonistic pathogen. Detailed Implementation

[0048] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, % and ‰ in the following embodiments refer to mass percentage and mass thousandths, respectively.

[0049] In the following examples, the *Botryosphaeria dothidea* and *Diaporthe celeris*, which cause soft rot in kiwifruit, were preserved and provided by the Shaanxi Institute of Microbiology.

[0050] Example 1

[0051] This example provides the isolation and screening of bacterial strains.

[0052] In March 2025, kiwifruit infected with soft rot was selected from a cold storage facility in Zhouzhi County, Xi'an City, Shaanxi Province. After cleaning and disinfection, pulp tissue from the junction of the infected and diseased areas was collected and placed on coarse-sieved LB solid medium. After incubation at 37°C for 3 days, single colonies were picked for purification culture, yielding a total of 13 strains. The plate confrontation method was used to screen for biocontrol strains with significant antagonistic effects against the kiwifruit soft rot pathogen: *Botrytis cinerea* and *Mesochrysis fusiformis* (preserved and provided by the Shaanxi Provincial Institute of Microbiology) mycelial discs causing kiwifruit soft rot (9 mm in diameter) were inoculated at the center of PDA plates. The purified biocontrol strains were then inoculated at 3 cm from the center of the pathogen (four equidistant inoculations). Plates without biocontrol strains served as a control group. The plates were incubated at 28°C until the control pathogen nearly covered the entire plate. The presence of inhibition zones was observed, and the diameter of the pathogen colonies was measured using the cross-crossing method. The pathogen growth inhibition rate was calculated using the following formula:

[0053] Pathogen growth inhibition rate % = (control pathogen diameter mm - experimental pathogen diameter mm) / (control pathogen diameter mm - inoculated bacterial cake diameter 9 mm) × 100%.

[0054] One biocontrol bacterium with the best antagonistic effect against the pathogen of soft rot was selected, and its plate confrontation results were as follows: Figure 1 As shown, the DX-10 specimen showed an inhibition rate of 78.62% against Staphylococcus aureus and an inhibition rate of 89.47% against Metasporum tobira.

[0055] Example 2

[0056] In this embodiment, the selected DX-10 strain was identified by morphological, physiological and biochemical characteristics and 16S rDNA gene.

[0057] like Figure 2 As shown, the colonies of strain DX-10 are large, milky white, opaque, raised, and have irregular edges.

[0058] The physiological and biochemical characteristics of strain DX-10 were determined according to Bergey's Manual of Systematic Bacteriology. The physiological and biochemical characteristics are shown in the table below.

[0059] Table 1: Physiological and Biochemical Characteristics of DX-10

[0060]

[0061]

[0062] Single colonies of the biocontrol bacterium DX-10 were picked and inoculated into LB liquid medium and cultured overnight (37℃, 180 rpm). The bacterial cells were then collected by centrifugation (8000 rpm, 5 min). Bacterial DNA was extracted according to the procedures outlined in the bacterial genomic DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.). The extracted DNA was amplified by PCR using universal primers for bacterial 16S rDNA (27F, 1492R). The PCR products were verified by agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing sequences were compared using BLAST in NCBI data, and phylogenetic analysis was performed using MEGA 7.0 software. Based on morphological and physiological / biochemical characteristics, strain DX-10 was ultimately identified as *Bacillus stercoris* strain. This strain was deposited at the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China), with accession number CCTCCNO: M 20251073, and deposit date: May 15, 2025.

[0063] Example 3

[0064] This embodiment provides an evaluation of the in vivo control effect of Bacillus compostii DX-10 on soft rot of kiwifruit.

[0065] Clean the surface of the kiwifruit to be tested, and use a sterile punch to make a 9mm diameter wound on the surface of the kiwifruit. Incubate Bacillus compostii DX-10 for 48 hours (37℃, 180rpm) to obtain OD. 600The bacterial suspension had a pH of 1.4. Each fruit wound was inoculated with 50 μL of *Bacillus compostii* DX-10 bacterial suspension, and a 9 mm diameter pathogenic fungal cake was placed over the wound. The fruit was then placed in a tray containing moistened sterile gauze, sealed with plastic wrap, sprayed with sterile water to maintain humidity, and incubated at 28°C. After 48 h, the pathogenic cake was removed, and the disease incidence of the kiwifruit was observed after 96 h. The blank control consisted of healthy kiwifruit, the negative control consisted of fruit inoculated only with 50 μL of *Bacillus compostii* DX-10 bacterial suspension, the positive control consisted of fruit inoculated only with soft rot pathogenic fungal cake, and the experimental group consisted of fruit inoculated with both DX-10 bacterial suspension and pathogenic fungal cake. Each treatment was repeated 20 times.

[0066] Depend on Figure 4 The results showed that after inoculation with DX-10 bacterial solution, the diameter of lesions on the fruit in the experimental group of kiwifruit was significantly reduced compared to the positive control groups of both pathogens. This indicates that DX-10 effectively reduced the infectivity of the pathogens to kiwifruit, thereby delaying fruit rot. Furthermore, the results of the negative control group (inoculated only with DX-10 bacterial solution) showed that DX-10 was not pathogenic to kiwifruit. These results indicate that DX-10 has a good control effect on kiwifruit soft rot caused by *Botryosphaeria dothidea* and *Diaportheceleris*, and it could be developed into a biocontrol agent for the green control of kiwifruit soft rot in the future.

[0067] Example 4

[0068] This embodiment provides an evaluation of the antagonistic effect of volatile organic compounds produced by Bacillus composting DX-10 on soft rot pathogens.

[0069] The effect of volatile organic compounds (VOCs) produced by strain DX-10 on the growth of *Botryosphaeria dothidea* and *Diaporthe celeris* was determined using the plate-on-plate method. Holes were punched in the pathogen colonies using a sterile puncher to obtain mycelial cakes with a diameter of 9 mm. These mycelial cakes were then inoculated into the center of a PDA solid plate using a sterile toothpick. Separately, 50 μL of DX-10 bacterial suspension activated overnight on a LB solid plate was spread onto the LB plate. The two plates were then inverted and sealed with sealing film. The control group's LB plate was coated only with 50 μL of distilled water. The inverted plates were incubated at 28°C. When the pathogens in the control group were about to completely cover the plate, the inverted plates were opened, and the radial growth of the pathogen colonies and the accumulation of mycelial biomass in the experimental groups were observed. The inhibition rate was calculated as in Example 3.

[0070] Depend on Figure 5It was found that VOCs produced by DX-10 significantly reduced the radial growth and mycelial biomass of *Diaporthe celeris*, with a radial growth inhibition rate of 82.60%. While the radial growth of *Botryosphaeria dothidea* was not significantly inhibited by VOCs produced by DX-10, the mycelial biomass of this fungus was significantly reduced. These results indicate that VOCs produced by DX-10 have a strong inhibitory effect on the growth of soft rot pathogens, especially *Diaporthe celeris*.

[0071] Example 5

[0072] This embodiment provides an evaluation of the antagonistic effect of Bacillus composting DX-10 fermentation supernatant on soft rot pathogens.

[0073] Take the DX-10 strain preserved in glycerol tubes, streak it on an LB agar plate, and incubate it upside down at 37°C overnight. Pick a single colony from the plate and inoculate it into 50 mL of LB liquid medium. Incubate on a shaker for 72 h (37°C, 180 rpm). Centrifuge the bacterial culture (8000 rpm, 10 min), and filter the supernatant through a 0.22 μm sterile filter membrane to obtain the DX-10 fermentation broth. The mycelial cakes (9 mm in diameter) of the soft rot pathogens (Botryosphaeria dothidea and Diaporthe celeris) were inoculated into the center of new PDA solid plates. Four holes (9 mm in diameter) were then punched at equal intervals 3 cm from the center of the pathogen using a sterile punch. 50 μL of DX-10 fermentation broth was added to each hole. The control group was inoculated with the pathogen only, without the addition of fermentation broth. The plates were incubated at 28°C. When the mycelium in the control group was about to cover the entire plate, the colony diameter was measured using a cross-sectional method. The inhibition rate of the DX-10 fermentation broth on the growth of the pathogen was calculated using the same formula as in Example 3.

[0074] Depend on Figure 6 It can be seen that the fermentation broth of DX-10 significantly inhibited the mycelial growth of Botryosphaeria dothidea and Diaporthe celeris, with inhibition rates of 88.43% and 50.19%, respectively, indicating that the fermentation supernatant of DX-10 has a significant antagonistic effect on the growth of soft rot pathogens.

[0075] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A strain of Bacillus compostii ( Bacillus stercoris strain ), characterized in that, The Bacillus compostii is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251073.

2. The metabolites of Bacillus compostii according to claim 1, characterized in that, The metabolites are volatile organic compounds produced by the Bacillus compostii and / or the supernatant of the Bacillus compostii culture.

3. The application of the Bacillus compostingus of claim 1 or the metabolite of claim 2 in the prevention and control of soft rot, characterized in that, The pathogen causing the soft rot is Staphylococcus aureus. Botryosphaeria dothidea and / or metasomatitis Diaporthe celeris .

4. The application according to claim 3, characterized in that, The plant that causes soft rot is kiwifruit.

5. A plant biocontrol composition for kiwifruit, characterized in that, The active ingredients of the plant biocontrol composition include: The composting Bacillus as described in claim 1; The volatile organic compounds produced by the composting Bacillus as described in claim 1; and / or The supernatant of the culture of Bacillus compostii as described in claim 1.

6. A plant biocontrol composition for the control of soft rot in kiwifruit, characterized in that the active ingredients of the plant biocontrol composition for the control of soft rot in kiwifruit include: The composting Bacillus as described in claim 1; The volatile organic compounds produced by the composting Bacillus as described in claim 1; and / or The supernatant of the culture of Bacillus compostii as described in claim 1.

7. The plant biocontrol composition for controlling soft rot of kiwifruit according to claim 6, characterized in that, The pathogen causing the soft rot is Staphylococcus aureus. Botryosphaeria dothidea and / or metasomatitis Diaporthe celeris .

8. A method for preventing and controlling soft rot in kiwifruit, characterized in that, include: This enables the Bacillus compostii of claim 1 or the plant biocontrol composition of claim 5 to act on the pathogens of kiwifruit soft rot and / or their habitats; The pathogenic bacterium is Staphylococcus aureus. Botryosphaeria dothidea and / or metasomatitis Diaporthe celeris .

9. The method according to claim 8, characterized in that, Their habitats are kiwi fruit growing areas or kiwi fruit storage areas.

10. The method according to claim 8, characterized in that, Apply the Bacillus compostii or the plant biocontrol composition to the pathogen causing soft rot or to plants at risk of developing soft rot.

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