Bacillus subtilis p10-2 antagonizing multiple plant fungal disease pathogens and application thereof

Abstract

The application discloses a bacillus subtilis P10-2 antagonizing multiple plant fungal disease pathogens and an application thereof, the bacillus subtilis P10-2 is preserved in the China General Microbiological Culture Collection Center on November 27, 2025, the preservation number is CGMCC No. 36792, and the classification and naming is bacillus subtilis (Bacillus subtilis) Bacillus subtilis ). The bacillus subtilis P10-2 has good physiological characteristics and salt tolerance, can significantly inhibit the pathogens of six crops such as apples, corns and bananas, the highest inhibition rate reaches 89.32 %, can meet the comprehensive prevention and control demand of multiple crop diseases, and solves the problem that the existing biocontrol strains have a narrow inhibition range. In addition, the fermentation liquor of the strain can also significantly promote the growth of corn plants, so that the strain has the dual functions of growth promotion and biological control.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of microbiology, plant pathology and agricultural biotechnology, and specifically relates to a Bacillus subtilis P10-2 strain that antagonizes multiple plant fungal pathogens and its applications. Background Technology

[0002] Fungal diseases severely restrict the development of agricultural planting and cause huge economic losses. Fungal diseases not only reduce crop yield and quality but also affect food safety and pose a significant threat to the lives of humans and livestock. Currently, disease control mainly relies on three methods: agricultural control, chemical control, and biological control. Agricultural control mainly improves the crop growth environment and enhances plant resistance through agronomical measures such as rational crop rotation, formula fertilization, pruning and orchard sanitation, and water and temperature control. However, it has limitations such as slow effectiveness, high labor intensity, and weak ability to cope with large-scale outbreaks. Chemical control uses chemical fungicides (such as triazoles and methoxyacrylates) for spraying or application, which has the advantages of rapid antibacterial action and ease of operation. However, long-term use can easily cause soil pollution, excessive pesticide residues in agricultural products, and induce drug resistance in pathogens, disrupting the ecological balance. Biological control utilizes beneficial microorganisms (Bacillus, Trichoderma, Actinomycetes, etc.) and their metabolites to inhibit pathogen growth. It is environmentally friendly, ecologically safe, and has no risk of drug resistance, making it a core direction for green agricultural development.

[0003] Among existing biological control technologies, Bacillus has become one of the preferred resources for biocontrol microorganisms due to its rapid reproduction rate, strong resistance, and ease of industrial production, and has been widely used in crop disease control. For example, Bacillus belyssus F85 can effectively inhibit tobacco anthracnose (… C. fructicola The mycelial growth of *Bacillus ventriclis* is inhibited, while the germination of anthracnose conidia is also suppressed. *Bacillus belye* Apss-1 exhibits good inhibitory and control effects against potato scab, providing a new biocontrol resource for the biological control of potato scab. *Bacillus subtilis* A3-1 shows biocontrol potential against root rot of *Polygonatum sibiricum*. *Bacillus BC79 achieved an 89.9% control effect against rice blast under greenhouse conditions. *Bacillus shorthair* LS06, a biocontrol bacterium, shows significant biocontrol effects against *Fusarium oxysporum*, the pathogen causing tobacco root rot. However, most strains have problems such as a narrow spectrum of inhibition and insufficient stress resistance. Some biocontrol bacilli only inhibit single or a few pathogens, and their activity easily decreases under complex environments, making it difficult to meet the needs of integrated pest management for multiple crop diseases.

[0004] Therefore, it is urgent to find a biocontrol Bacillus strain with a broad antibacterial spectrum and strong stress resistance, which is of great significance for enriching the microbial strain bank and promoting the development of green prevention and control technologies in agriculture. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus subtilis P10-2 strain that antagonizes multiple plant fungal pathogens and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of Bacillus subtilis P10-2, which was deposited on November 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36792 and classified as Bacillus subtilis (…). Bacillus subtilis ).

[0007] Furthermore, the Bacillus subtilis P10-2 is effective against Alternaria alternata, which causes apple leaf spot disease. Alternaria alternate ), the fungus that causes apple rot ( ), Cytospora mali Fusarium solani, which causes apple replant disease ( Fusarium solani Fusarium graminearum, which causes corn stalk rot, is a fungus that causes corn stalk rot. F. graminearum) And Fusarium oxysporum race Cuban specific type 1, which causes banana wilt disease (… Fusarium oxysporum f.sp. cubense race 1 Foc1) and Fusarium oxysporum Cuban specialized race 4 ( Fusarium oxysporum f.sp. Cuban tropical race 4 FocTR4 has an inhibitory effect.

[0008] Secondly, the present invention provides a microbial preparation containing the above-mentioned Bacillus subtilis P10-2 or its fermentation broth.

[0009] Furthermore, the fermentation broth is prepared by inoculating a single colony of Bacillus subtilis P10-2 into LB liquid medium, shaking at 180 rpm, and incubating at 37°C for 24-48 h to obtain the fermentation broth.

[0010] Thirdly, the present invention provides the application of the above-mentioned Bacillus subtilis P10-2 or the above-mentioned microbial preparation in the prevention and control of plant diseases.

[0011] Furthermore, the plant diseases include apple rot, apple leaf spot, apple replant disease, banana wilt, and corn stem rot.

[0012] Fourthly, the present invention provides the application of the above-mentioned Bacillus subtilis P10-2 or the above-mentioned microbial preparation in promoting plant growth.

[0013] Furthermore, the plant in question is corn.

[0014] Furthermore, when applying this method, corn seeds should be soaked in a microbial preparation for 0.5 to 2 hours before sowing.

[0015] Furthermore, the promotion of plant growth includes increasing plant height, biomass, total root length of seedlings, root surface area of ​​seedlings, and number of root tips of seedlings.

[0016] The beneficial effects of this invention are as follows: This invention isolated a strain of Bacillus subtilis with good physiological characteristics and salt tolerance from earthworm castings. Bacillus subtilis P10-2, this strain has a significant inhibitory effect on pathogens of six crops, including apple, corn, and banana, with an inhibition rate of up to 89.32%. It can meet the needs of integrated pest management for multiple crop diseases and solves the problem of narrow inhibition range of existing biocontrol strains. In addition, the fermentation broth of this strain can also significantly promote the growth of corn plants, giving this strain the dual function of growth promotion and biological control. Attached Figure Description

[0017] Figure 1 This is the single colony morphology of strain P10-2.

[0018] Figure 2 Phylogenetic tree of strain P10-2.

[0019] Figure 3 Salt tolerance test for strain P10-2.

[0020] Figure 4 The inhibitory effect of strain P10-2 on six pathogenic fungi was studied.

[0021] Figure 5 The biocontrol effect of strain P10-2 against the pathogen of apple rot was investigated. Detailed Implementation

[0022] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0023] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0025] The PDA medium used in the following examples comprises: 200 g / L potato, 20 g / L glucose, 20 g / L agar, and autoclaved at 121 °C for 20 min.

[0026] Example 1: Isolation, purification and identification of strains 1. Isolation of strains Collect fresh earthworm castings, remove impurities, and grind them in a mortar. Take 1 g of the ground earthworm castings, add 90 mL of sterile water, and shake at 37 ℃ for 30 min until the sample is evenly dispersed to obtain a suspension. Take 10 mL of the suspension and serially dilute it to prepare 1×10⁻⁶ ppm. -3 1×10 -4 1×10 -5 Sample dilution. 100 μL of the dilution was spread onto LB agar plates and incubated at 37 °C. After the strain grew, it was purified by repeated streaking and culturing on LB agar plates to obtain the purified strain P10-2, which was then stored in glycerol.

[0027] 2. Strain identification (1) Morphological observation: The strain was streaked and incubated at 37 ℃ for 24 h. Single-cell colonies were observed and colony characteristics were recorded. Results are shown in […]. Figure 1 The colonies of strain P10-2 are light yellow, with a rough surface, irregular edges, and are opaque.

[0028] (2) Physiological and biochemical tests: The physiological and biochemical characteristics of the strain were tested with reference to the "Handbook of Systematic Identification of Common Bacteria" and "Microbiological Experiment". The results of the physiological and biochemical characteristics test of strain P10-2 are shown in Table 1. Strain P10-2 has the ability to dissolve organic and inorganic phosphorus, potassium, and produce iron carriers. It can produce protease, β-glucanase, amylase and catalase. At the same time, the methyl red, VP and nitrate reduction tests were positive.

[0029] Table 1. Physiological and biochemical test results of strain P10-2

[0030] (3) Molecular biological identification: Genomic DNA was extracted according to the instructions in the bacterial DNA extraction kit (Sangon Biotech, Shanghai). The 16S rRNA gene was amplified under the following conditions: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 1 min, 55 ℃ annealing for 1 min, 72 ℃ extension for 1 min, 35 cycles; 72 ℃ extension for 10 min. The PCR products were detected by 1% agarose gel electrophoresis and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing analysis. The 16S rDNA sequence of strain P1-3 is shown in SEQ ID NO.1. The sequence results were compared on the NCBI website, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11 software. After comparison, strain P10-2 was found to be similar to Bacillus subtilis (…). Bacillus subtilis The homology between the two species reached 99.93%. This was confirmed by a phylogenetic tree (...). Figure 2 ), and strain P10-2 was found to be related to Bacillus subtilis HZ9 Based on the close phylogenetic relationship, strain P10-2 was identified as Bacillus subtilis (… Bacillus subtilis This strain P10-2 was deposited on November 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36792, and classified as Bacillus subtilis (…). Bacillus subtilis ).

[0031] (3) Salt tolerance analysis of the strain Strain strain P10-2 was inoculated into solid LB medium containing 5%, 10%, 15%, and 20% NaCl, and cultured at 37 °C for 24 h. The growth of the strain was observed to determine its salt tolerance. Results are as follows: Figure 3 As shown, strain P10-2 grew on both 5% and 10% NaCl media, indicating that strain P10-2 has a certain degree of salt tolerance.

[0032] Example 2: Detection of antibacterial activity of strain P10-2 The plate confrontation culture method was used to determine the interaction between strain P10-2 and various pathogenic fungi. The specific method is as follows: Using an inoculation loop, Bacillus subtilis P10-2 glycerol-preserved bacterial suspension was streaked onto LB solid medium for activation. The activated bacteria were then subjected to a plate confrontation test. In a 9 cm PDA petri dish, purified test bacteria were aseptically streaked on both sides, 2 cm from the center, and incubated for 1 day. A 0.8 cm diameter mycelial cake of the plant pathogen was then placed in the center of the petri dish using a sterile punch. A control group was set up, inoculated only with pathogenic mycelial cakes. Both groups were incubated at 28 ℃, with each treatment repeated three times. After 7 days of incubation, the colony diameters of the control and treatment groups were measured, and the inhibition rate was calculated. Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control plate × 100%; values ​​in the table are mean ± standard deviation. Figure 4 It is known that strain P10-2 has a significant growth-inhibiting effect on a variety of plant pathogenic fungi, including Alternaria, which causes apple leaf spot disease. Alternaria alternata ), the fungus that causes apple rot ( ), Cytospora mali Fusarium solani, which causes apple replant disease ( Fusarium solani Fusarium graminearum, which causes corn stalk rot, is a fungus that causes corn stalk rot. F. grasses) And Fusarium oxysporum race Cuban specific type 1, which causes banana wilt disease (… Fusarium oxysporum f.sp. cubense race 1 Foc1) and Fusarium oxysporum Cuban specialized tropical race 4 ( Fusarium oxysporum f.sp. Cuban tropical race 4 , FocTR4).

[0033] The inhibition rate of strain P10-2 against the pathogen is shown in Table 2. The results indicate that strain P10-2 has a broad spectrum of inhibition.

[0034] Table 2. Inhibition rate of strain P10-2 against six pathogenic fungi

[0035] Example 3: Detection of the effect of strain P10-2 on the growth of maize seedlings The effect of bacterial strain P10-2 on the growth of maize (Xinruipu 826) seedlings was investigated using the soaking method. Single colonies of strain P10-2 were inoculated into sterile LB broth and incubated at 37 °C for 24 h with shaking at 180 rpm in a constant temperature shaking incubator to obtain the fermentation broth (OD). 600 Approximately 1). Maize seeds were divided into a control (CK) group and a P10-2 group, with 50 seeds in each group. The CK group seeds were soaked in sterile LB liquid for 1 h, while the P10-2 group seeds were soaked in fermentation broth for 1 h. The maize seeds were then sown in seed trays (8 cm in diameter, 10 cm in height) filled with substrate (organic matter content ≥35%, pH neutral) and grown under natural light at 25 °C. Two weeks after sowing, plant height and maximum root length were measured using a ruler; stem diameter was measured using calipers; aboveground fresh weight, underground fresh weight, and plant biomass were measured using an electronic balance; root structure was analyzed using a root phenotypic analysis system to obtain total root length, average root diameter, root volume, root surface area, root projected area, and number of root tips. Table 3 shows the effects of strain P10-2 on maize seedlings. Before inoculation, maize seedlings soaked in fermentation broth were 3.95% taller and 4.94% more biomass than the control (CK) group, but their stem diameter, aboveground fresh weight, and maximum root length were similar to those of the CK group. Further analysis of root structure revealed that the total root length, root surface area, and number of root tips in the P10-2 group were all higher than those in the CK group.

[0036] Table 3 Effects of strain P10-2 on maize seedlings

[0037] Example 4: Detection of the biocontrol effect of the strain Healthy apple leaves of uniform size were selected, surface-sterilized with 75% alcohol for 30 seconds, and then rinsed thoroughly with sterile water. The leaves were then immersed in fermentation broth of P10-2 (prepared using the same method as in Example 3, P10-2 group) and LB liquid medium for 30 seconds (CK group), respectively, to ensure uniform wetting of the leaf surface. The leaves were placed in petri dishes lined with sterile filter paper and moistened with sterile water. After 12 hours, 8 mm diameter apple leaf spot pathogen cakes were inoculated onto the surfaces of the different treated leaves, and the leaves were incubated in a 28 ℃ light incubator. Results were observed at 3 and 6 days. Figure 5To assess the biocontrol effect of strain P10-2 on detached leaves of apple leaf spot disease, compared with the control group (CK), the lesions in the treatment group were significantly smaller, indicating a significant biocontrol effect.

[0038] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this utility model are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A strain of Bacillus subtilis P10-2, characterized in that: The Bacillus subtilis P10-2 strain was deposited on November 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36792, and classified as Bacillus subtilis (…). Bacillus subtilis ).

2. The Bacillus subtilis P10-2 according to claim 1, characterized in that: The Bacillus subtilis P10-2 strain is effective against Alternaria alternata, which causes apple leaf spot disease. Alternaria alternata ), the fungus that causes apple rot ( ), Cytospora mali Fusarium solani, which causes apple replant disease ( Fusarium solani Fusarium graminearum, which causes corn stalk rot, is a fungus that causes corn stalk rot. F. graminearum) And Fusarium oxysporum race Cuban specific type 1, which causes banana wilt disease (… Fusarium oxysporum f.sp. cubense race 1 ) and Fusarium oxysporum Cuban specialized tropical race 4 ( Fusarium oxysporum f.sp. cubense tropical race 4 It has an inhibitory effect.

3. A microbial preparation, characterized in that: Contains Bacillus subtilis P10-2 or its fermentation broth as described in claim 1.

4. The microbial preparation according to claim 3, characterized in that: The fermentation broth is prepared by inoculating a single colony of Bacillus subtilis P10-2 into LB liquid medium, shaking at 180 rpm, and incubating at 37°C for 24-48 h to obtain the fermentation broth.

5. The application of Bacillus subtilis P10-2 as described in claim 1 or the microbial preparation as described in claim 3 in the prevention and control of plant diseases.

6. The application according to claim 5, characterized in that: The plant diseases mentioned include apple rot, apple leaf spot, apple replant disease, banana wilt, and corn stem base rot.

7. The application of Bacillus subtilis P10-2 as described in claim 1 or the microbial preparation as described in claim 3 in promoting plant growth.

8. The application according to claim 7, characterized in that: The plant in question is corn.

9. The application according to claim 7, characterized in that: When applying the treatment, soak the corn seeds in the microbial preparation for 0.5 to 2 hours before sowing.

10. The application according to claim 7, characterized in that: The promotion of plant growth includes increasing plant height, biomass, total root length of seedlings, root surface area of ​​seedlings, and number of root tips of seedlings.

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