Bacillus subtilis m10 and application thereof

Abstract

The present application belongs to the field of microbiology, and relates to a bacillus subtilis M10 and application, the bacillus subtilis M10 has been preserved to China Microbial Culture Collection Committee General Microorganism Center on March 23, 2026, and the preservation number is CGMCC No.37999.The bacillus subtilis M10 provided in the present application can be used alone or in combination with artemisin to form a biocontrol preparation, inhibit tobacco phytophthora, especially effectively prevent and treat tobacco black shank caused by tobacco phytophthora, and has the advantages of significant synergistic effect, green, environmental protection and safety and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to a Bacillus species and its applications, particularly to a Bacillus subtilis M10 species and its applications. Background Technology

[0002] Tobacco is an important economic crop in my country, and its production faces various disease threats. Among them, tobacco black shank, caused by *Phytophthora nicotianae*, is one of the most destructive soil-borne diseases. This disease is prevalent in tobacco-growing areas worldwide, and in severe cases, it can lead to large-scale mortality in tobacco fields, causing significant economic losses (Yang Zhixiao et al., 2024). For a long time, the control of tobacco black shank has mainly relied on chemical pesticides and resistant varieties. However, the accompanying risks of pesticide resistance, environmental pollution, and the limited availability of resistance gene resources have forced tobacco production to continuously seek new, green, safe, and efficient control strategies (Peng Lijuan et al., 2014). Biological control, as one of the core strategies for sustainable agricultural development, is receiving increasing attention. Among these, the use of beneficial microorganisms for disease control has become a research hotspot (Luo Yuying et al., 2019). *Bacillus subtilis*, due to its strong adaptability, rapid reproduction, high safety, and ability to produce various antimicrobial substances, is considered a biocontrol resource with great application potential (Ao Jincheng et al., 2022). Multiple studies have confirmed that Bacillus subtilis strains from different sources have good control effects on tobacco black shank (Yan Fangfang et al., 2023). On the other hand, natural active ingredients derived from plants, due to their environmental friendliness and novel modes of action, have become an important source for the development of novel plant immune inducers or fungicides (Liu Xueqiu et al., 2024). Flavonoids, as a large class of plant secondary metabolites, are known to possess various biological activities such as antibacterial and antiviral activity (Liu Yijie et al., 2016). Geraniol, as a specific flavonoid, has been primarily studied for its medicinal value, such as anti-inflammatory and antitumor effects; its application in agricultural plant protection, especially its activity against plant pathogenic oomycetes, remains at the forefront of research. Scientifically combining highly effective biocontrol strains with active plant-derived compounds to achieve synergistic effects is an important direction in current biopesticide research and development. This "bacterium-drug" synergistic model is expected to reduce the dosage of single agents, delay the development of resistance, broaden the antibacterial spectrum, and enhance the stability of control effects (Song Ruimin et al., 2024). Therefore, exploring the combined application of superior Bacillus subtilis and geraniol to provide a novel technical approach for the green control of tobacco black shank is of great theoretical significance and application prospects. Summary of the Invention

[0003] In order to solve the above-mentioned technical problems in the background art, the present invention provides a Bacillus subtilis M10 with significant synergistic effects, green environmental protection and safety and broad application prospects and its applications.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A strain of Bacillus subtilis M10, characterized in that: the Bacillus subtilis M10 was submitted for preservation to the China General Microbiological Culture Collection Center on March 23, 2026, with the preservation number CGMCC No. 37999.

[0006] The application of Bacillus subtilis M10 in inhibiting Phytophthora intoxin, as mentioned above.

[0007] The application of Bacillus subtilis M10 in the prevention and control of tobacco black shank disease, as mentioned above.

[0008] The application of Bacillus subtilis M10 as described above in the prevention and control of tobacco black shank caused by Phytophthora infestans.

[0009] A biocontrol agent based on Bacillus subtilis M10 as described above, wherein the biocontrol agent is a seed liquid, culture medium, culture medium, bacterial liquid, fermentation broth and / or fermentation filtrate containing Bacillus subtilis M10.

[0010] A biocontrol agent, characterized in that: the biocontrol agent comprises flavonoids and Bacillus subtilis M10 as described above; preferably, the final concentration of Bacillus subtilis M10 is not less than 2 × 10⁻⁶. 7 CFU / mL; the final concentration of the flavonoid compound is not less than 0.1 mmol / L.

[0011] The flavonoid compound mentioned above is geraniol.

[0012] The application of biocontrol agents as described above in the inhibition of Phytophthora intoxin.

[0013] The application of biocontrol agents in tobacco black shank, as described above.

[0014] The application of the aforementioned biocontrol agents in tobacco black shank caused by Phytophthora indicum.

[0015] The advantages of this invention are:

[0016] 1. Significant Synergistic Effect: This invention is the first to combine Bacillus subtilis M10 with geraniol, exhibiting a significant synergistic effect. While geraniol cannot directly inhibit the mycelial growth and spore germination of Phytophthora nicotianae, its combination with Bacillus subtilis M10 significantly enhances its antagonistic effect against Phytophthora nicotianae. Bacillus subtilis M10, as a dominant rhizosphere microorganism, can rapidly colonize the tobacco rhizosphere and secrete various antibacterial substances. Geraniol, as a signaling molecule or cofactor, may indirectly enhance the biocontrol activity of Bacillus subtilis M10 by regulating microbial metabolism or influencing the physiological state of pathogens. The combined use of Bacillus subtilis M10 and geraniol shows a significantly higher inhibitory effect against Phytophthora nicotianae than either alone, effectively blocking the infection cycle of tobacco black shank and reducing the incidence of the disease, providing a novel and highly efficient combination solution for the green control of tobacco black shank.

[0017] 2. Green, Environmentally Friendly, and Safe: The Bacillus subtilis M10 strain used in this invention is isolated from tobacco rhizosphere soil and is a dominant indigenous strain. It exhibits high adaptability and colonization ability in the tobacco root environment, and its use will not disrupt the original rhizosphere microecological balance, avoiding the ecological risks that may arise from the introduction of exogenous microorganisms. Simultaneously, the geraniol used in this invention is a natural plant-derived secondary metabolite widely found in various plants. It is biodegradable, easily decomposes in soil and the environment, leaves no residual toxicity, and is safe for humans, animals, and non-target organisms. This combination fully complies with the concept of green pest control in modern agriculture, effectively controlling tobacco black shank disease while being environmentally friendly and conducive to sustainable agricultural development.

[0018] 3. Broad application prospects: The biocontrol agent formed by Bacillus subtilis M10 and geraniol provided by this invention is simple to prepare and has low cost. It can be processed into various formulations (such as wettable powder, aqueous solution, granules, etc.) and is suitable for different application methods during tobacco seedling raising, transplanting and field growth. It has good promotion and application value. Attached Figure Description

[0019] Figure 1 This is a phylogenetic tree of five Bacillus strains constructed using the neighbor-joining method based on the 16S rRNA gene sequence;

[0020] Figure 2 This refers to the colony morphology of Bacillus subtilis M10 used in this invention;

[0021] Figure 3 This is the electron micrograph of Bacillus subtilis M10 used in this invention;

[0022] Figure 4It is the antagonistic effect of different Bacillus strains on Phytophthora tobaccoii.

[0023] Figure 5 The inhibition zone width and inhibition rate of different Bacillus strains against Phytophthora tobaccois;

[0024] Figure 6 This invention demonstrates the antibacterial effect of the Bacillus subtilis M10 aseptic fermentation filtrate on Phytophthora intolis.

[0025] Figure 7 The aseptic fermentation filtrate of Bacillus subtilis M10 used in this invention inhibits the growth of Phytophthora intoxin.

[0026] Figure 8 This invention demonstrates the biocontrol effect of Bacillus subtilis M10 against Phytophthora in tobacco.

[0027] Figure 9 This is a statistical analysis of the incidence and control efficacy of tobacco black shank after inoculation with Bacillus subtilis M10 used in this invention;

[0028] Figure 10 It describes the types and composition of tobacco rhizosphere soil metabolites;

[0029] Figure 11 This is a validation of the OPLS-DA model of rhizosphere soil metabolites;

[0030] Figure 12 This is a metabolic volcano diagram showing the difference between crop rotation and continuous cropping;

[0031] Figure 13 The effect of geraniol on the growth of Bacillus subtilis M10;

[0032] Figure 14 The effect of geraniol on the growth of Phytophthora intoxin;

[0033] Figure 15 It is the antagonistic effect of geraniol combined with Bacillus subtilis M10 against Phytophthora intoxin;

[0034] Figure 16 This study verifies the potted plant control efficacy of the combination of geraniol and Bacillus subtilis M10 against Phytophthora in tobacco. Detailed Implementation

[0035] This invention was completed or verified through the following experiments.

[0036] 1. Dominant microorganisms in tobacco rhizosphere soil can be cultured and isolated.

[0037] 1.1 Overview of the test site and sample collection

[0038] Soil samples used in the experiments of this invention were collected from the Chengjiang Experimental Base of the Yunnan Provincial Tobacco Agricultural Science Research Institute (24.65°N, 102.87°E). This base has long-term experimental fields for tobacco continuous cropping (CC) and tobacco-rice rotation (CR) for 16 years, with the main variety being Yunyan 87, covering an area of ​​approximately 100 mu. Except for the tillage system, the soil fertility level, fertilization management, and pest and disease control are consistent. Healthy tobacco plants under the two different tillage systems were selected as test materials. Surface vegetation residues and debris were cleared from the sampling points, and rhizosphere soil was collected according to an X-shaped sampling pattern. Five subsamples were taken from both CC and CR treatments and mixed together. After noting the collection time, location, and personnel, the samples were properly transported back to the laboratory and stored at 4°C for microbial isolation.

[0039] 1.2 Preparation of culture media and soil suspensions and isolation of culturable microorganisms

[0040] Bacteria in the soil were isolated and cultured using nutrient broth (NB) medium. 3g of beef extract, 10g of peptone, 5g of NaCl, and 20g of agar were weighed and added to 1000mL of distilled water, stirring until completely dissolved. The pH was adjusted to 7.0-7.2 with NaOH or HCl solution. After aliquoting, the medium was autoclaved at 121℃ for 30min. Once cooled to approximately 50℃, the medium was poured into plates in a clean bench for later use. The soil sample collected in section 1.1 was removed from the sterile self-sealing bag, and 5g was weighed and placed in an Erlenmeyer flask. 50mL of 0.01mol / L sterile PBS was added, and the flask was incubated at 25℃ and 180rpm for 30min on a shaker to obtain a soil suspension for dilution and spreading. The soil suspension was diluted with sterile water to a final concentration of 10. -5 ~10 -7 Take 100 μL of the sample and spread it evenly on NB medium. Set up 3 replicates to culture and isolate the bacterial community in the soil sample.

[0041] 1.3 Purification and Molecular Biological Identification of Dominant Rhizosphere Microorganisms

[0042] Based on the morphological characteristics of the strains, counts were performed on three replicate plates from both CC and CR treatments. Strains accounting for more than 10% of the total bacterial count were selected and further purified into single clones in freshly prepared NB solid medium. These single clones were then inoculated into NB liquid medium and cultured at 30°C and 170 rpm for 48 hours until the culture medium became turbid. Nucleic acid extraction was performed using the freeze-thaw method to obtain the DNA template (Feng Guangda 2012). Molecular biological identification was performed using the universal primers 341F / 806R for the V3-V4 region of bacterial 16S rDNA. The upstream primer 341F sequence was CCTAYGGGRBGCASCAG (e.g., SEQ ID NO.2), and the downstream primer 806R sequence was GGACTACNNGGTATCTAAT (e.g., SEQ ID NO.3). The amplified products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained bacterial 16S rDNA splice sequences were compared online using BLAST in the NCBI nucleic acid database to determine the phylogenetic relationships between the strains.

[0043] 1.4 Data Processing

[0044] The data obtained from the experiment were organized and calculated in Microsoft Excel 2019. BLAST comparisons were performed with the type species using the NCBI website (https: / / www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using the maximum likelihood method in MEGA 11 software.

[0045] 1.5 Results and Analysis

[0046] Seventeen bacterial species were cultured and isolated from the rhizosphere soil of tobacco continuous cropping (CC) and tobacco-rice rotation (CR). Based on phylogenetic analysis of 16S rRNA gene sequences and morphological observation, the names, classifications and quantities of the dominant microorganisms were statistically analyzed, as shown in Table 1. Bacteria isolated from tobacco continuous cropping (CC) soil were mainly classified as Stenotrophomonas (1 strain), Ensifer (1 strain), Brevibacillus (1 strain), Terrabacter (1 strain), and Bosea (1 strain); bacteria isolated from tobacco-rice rotation (CR) soil were mainly classified as Bacillus (5 strains), Microbacterium (2 strains), Arthrobacter (1 strain), Paenarthrobacter (1 strain), Variovorax (1 strain), Delftia (1 strain), and Devossia (1 strain).

[0047] Table 1. Statistics on dominant bacteria and their quantities in rhizosphere soil of tobacco continuous cropping and tobacco-rice rotation.

[0048]

[0049] Note: The increase rate of each indicator of crop rotation compared to continuous cropping is expressed as "ΔH", and the increase rate ΔH = (crop rotation - continuous cropping) / continuous cropping × 100%.

[0050] Based on the above results, the genus *Bacillus* was found to be the most diverse among culturable bacteria, with a total of 5 strains: Z12, Y1, M10, L45, and K22. Their phylogenetic relationships are as follows: Figure 1 As shown. Compared with tobacco continuous cropping (CC), the number of these five morphologically different Bacillus strains in the rhizosphere soil of the rotation crops was significantly increased (1.8 to 16.5 times), with Bacillus subtilis M10 being the most abundant, reaching 0.35 × 10⁻⁶. 6 CFU / g. 1.6 Determination of 16S rDNA sequence of Bacillus subtilis M10.

[0051] The 16S rDNA sequence of Bacillus subtilis M10, which was the most abundant obtained in step 1.5, was amplified by PCR, and the 16S rDNA sequence is as follows:

[0052]

[0053] 2. Purification and morphological identification of Bacillus subtilis M10

[0054] 2.1 Streak purification

[0055] Single colonies of Bacillus subtilis M10 obtained from the initial screening in section 1.5 were picked and purified by streaking continuously on NB solid plates 3-5 times using the three-zone streak method until a pure culture was obtained. Referring to materials such as the "Handbook of Systematic Identification of Common Bacteria", the neatness, size, color, shape, and transparency of the colony edges were observed and recorded by the naked eye. Gram staining was then performed for observation and photography.

[0056] The purified M10 strain was inoculated into NB liquid medium and cultured at 30℃ and 180 rpm for 18-24 h with shaking. The bacterial culture was then mixed with 15% sterile skim milk at a volume ratio of 1:1 and stored at -80℃ for a long time. At the same time, slant culture medium was prepared and stored at 4℃ for a short time.

[0057] 2.2 Scanning electron microscopy (SEM) observation of bacterial cell morphology

[0058] Single colonies of Bacillus subtilis M10 obtained in step 2.1 were inoculated into NB liquid medium and cultured at 30°C with shaking at 180 rpm until OD reached. 600 =0.5~0.8, centrifuge the bacterial suspension at 7000rpm for 5min, collect the bacterial precipitate the size of mung bean, discard the supernatant, add 4% glutaraldehyde fixative at room temperature, gently pipette to mix, pre-fix at room temperature for 30min, then transfer to 4℃ refrigerator for overnight storage. The sample is then sealed in a low-temperature package and sent to the Scientific Compass platform. After pretreatment by the Scientific Compass platform, including CO2 critical point drying and sputtering a 5~10nm platinum / palladium alloy film, observation and imaging are performed using a ZEISS Sigma 360 field emission scanning electron microscope at 20000x magnification with an accelerating voltage of 3.00kV, a working distance of 5.4mm, an aperture of 20.00m, and an SE2 secondary electron detector.

[0059] 2.3 Results and Analysis

[0060] like Figure 2 As shown, Bacillus subtilis M10 colonies grown on NB medium are large, appearing as grayish-white, turbid, round or irregular shapes, approximately 3 mm in diameter. The edges are smooth, but the surface is rough and irregular, with raised areas and wrinkles. The colony texture is relatively fine and viscous. Bacillus subtilis M10 is Gram-positive, purple rod-shaped, arranged singly or in short chains, with slender cells, blunt ends, and exhibiting active motility. Scanning electron microscopy observation (results as shown) Figure 3As shown in the image, the strain is approximately 2.3–2.8 μm long and 0.6–0.8 μm wide. The cell surface is smooth and intact, without obvious wrinkles or depressions, and no obvious damage or autolysis was observed. Some cells in the stable phase show oval spores located in the center or near the end of the cell, which do not cause cell swelling, consistent with the typical morphological characteristics of Bacillus subtilis.

[0061] 2.4 Preservation of bacterial strains

[0062] Subsequently, the Bacillus subtilis M10 obtained in section 2.3 was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Bacillus subtilis M10, with accession number CGMCC No. 37999.

[0063] 3. Antagonistic effect of Bacillus subtilis M10 and its metabolites on Phytophthora indicum

[0064] 3.1 Bacterial antagonism assay

[0065] The antibacterial activity of Bacillus subtilis M10 against Phytophthora tobaccois, the pathogen of tobacco black shank, was determined using the plate confrontation method. The pathogen was inoculated onto oat (OA) agar plates (prepared by sterilizing the oat kernels at 121℃ for 30 min, then boiling and filtering through four layers of gauze; the other reagents were heated and mixed with the filtrate, and finally poured into Erlenmeyer flasks and autoclaved at 121℃ for 30 min; the plates were then poured out while still hot). After incubation at 28℃ in the dark for 7 days, pathogenic bacterial cakes were punched from the edge of the colonies using a 5 mm diameter punch and inoculated into the center of new oat agar plates. Three Bacillus subtilis M10 bacterial cakes (5 mm in diameter) were symmetrically inoculated 2 cm away from the pathogenic bacterial cakes. Each treatment was replicated in triplicate, and a control plate inoculated only with pathogenic bacterial cakes was also included. After incubating the plates in a 28℃ incubator for 5 days, the antibacterial effect was observed. The width of the inhibition zone was measured using the cross-sectional method, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = (r1-r2) / r1×100%, where: r1 is the colony radius (mm) of the pathogen in the control group; r2 is the colony radius (mm) of the pathogen in the treatment group.

[0066] 3.2 Antagonistic assay of aseptic fermentation filtrate

[0067] Bacillus subtilis M10 was inoculated into NB liquid medium and incubated in a shaker at 30°C and 180 rpm for 24 hours. The culture was then removed to allow the bacterial culture to reach its OD value. 600>1.5, yielding the fermentation broth of Bacillus subtilis M10. The fermentation broth was centrifuged at 10,000 rpm for 10 min at 4℃, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane to obtain the sterile filtrate of Bacillus subtilis M10. The antibacterial activity of the sterile fermentation filtrate (Sff) of Bacillus subtilis M10 against Phytophthora indica was determined using the agar mixing method. The sterile filtrate was mixed with unconsolidated OA medium at a ratio of 1:4 (V:V) and shaken well. An equal volume of sterile distilled water and unconsolidated OA medium was mixed as a control. The mixture was poured into plates, and after the medium solidified, an activated pathogenic bacterial cake (5 mm in diameter) was inoculated in the center of the plate. The plates were incubated in the dark at 28℃ for 5 days. The diameter of the pathogenic colony was measured using the cross-cross method, and the inhibition rate of the Bacillus subtilis M10 metabolites was calculated.

[0068] 3.3 Verification of potted plant efficacy

[0069] 3.3.1 Preparation and use of Phytophthora tobacco spore suspension

[0070] After culturing Phytophthora tobaccoii on OA plates for 21 days, surface hyphae were scraped and soaked in 0.1% KNO3 solution for 72 hours, followed by the addition of sterile water and mixing. The mixture was then incubated at 4°C for 40 minutes and then transferred to a 25°C incubator for 20 minutes. Afterward, a zoospore suspension was prepared, and its concentration was determined to be 1.2 × 10⁻⁶ using the dilution-spreading method. 7 CFU / mL. Add 1% glucose solution to the suspension for later use. Finally, drench each plant with 50 mL of the prepared Phytophthora tobacco spore suspension.

[0071] 3.3.2 Preparation and use of Bacillus subtilis M10 bacterial suspension

[0072] Bacillus subtilis M10 was pre-activated to obtain a seed culture. For each preparation, 1% of the seed culture was inoculated into a new liquid culture medium and incubated at 30°C and 160 rpm for 1.5 days. After incubation, the culture was removed, and the concentration was determined by dilution and spread, and then adjusted to 10⁻⁶. 7 For each plant, apply 100 mL of the prepared biocontrol solution to the roots.

[0073] 3.3.3 Potted plant treatment and index statistics

[0074] Seedlings were raised in 32-cell trays for 4 weeks. When the seedlings reached the large cross stage (the fifth true leaf appeared), they were transplanted. Three days before transplanting, the seedlings should be hardened off. A mixture of substrate and vermiculite in a 1:3 (V:V) ratio was placed in a 2L culture pot. After transplanting, 200mL of water was added to settle the seedlings. The pots were then placed in a glass greenhouse to ensure sufficient light and proper ventilation.

[0075] Seven days after transplanting, tobacco seedlings were treated. The experiment included four treatments: a water control (Ctrl), a positive control (Ctrl+), a pathogen treatment (Pn), and an antagonistic treatment (M10). The efficacy of each treatment was verified by drenching the roots with a suspension of Phytophthora tobacco spores and a Bacillus subtilis M10 bacterial solution or a chemical agent (0.67 mL / L metalaxyl-mancozeb aqueous solution). Ten tobacco plants were used for each treatment. In each round, equal amounts of the liquid from different treatments were added first, followed by equal amounts of pathogens 2 days later. Three rounds of treatment were conducted, with a 5-day interval between each round. During this period, normal quantitative fertilization and routine pest and disease management were maintained.

[0076] Three days after the third round of root irrigation treatment, a survey of the tobacco black shank disease index was conducted. The tobacco black shank disease grading was carried out according to the national industry standard GB / T 23222-2008 Classification and Survey Methods of Tobacco Diseases and Pests. The incidence rate, disease index, control effect and relative control efficacy were statistically analyzed. The calculation formulas are as follows: (1) Incidence rate (%) = Number of diseased plants / Total number of plants × 100%; (2) Disease index = {∑(Disease level × Disease grade index)} × 100 / Total index of survey. (Total index of survey = Total number of plants or total number of leaves × Highest grade index); (3) Control effect (%) = (Incidence rate of control group − Incidence rate of treatment group) / Incidence rate of control group × 100%; (4) Relative control efficacy = (F CK -F t ) / F CK ×100%. (F) CK F represents the disease index of the blank group. t (This indicates the disease severity index of the treatment group).

[0077] 3.4 Data Processing

[0078] The data obtained from the experiment were organized and calculated in Microsoft Excel 2019, and statistical and difference analysis (Waller-Duncan) was performed on the data using SPSS 20.0. Origin Pro 2023b was used to plot the data.

[0079] 3.5 Results and Analysis

[0080] To investigate the antagonistic potential of dominant Bacillus strains (Bacillus spp.) in the rhizosphere soil of crop rotation against Phytophthora tobacco, the inhibitory effect was determined using the plate confrontation method. The results showed significant differences in the antagonistic abilities of the five Bacillus strains (e.g., ...). Figure 4 (As shown). Among them, Bacillus subtilis M10 had the strongest antibacterial effect, with an inhibition rate of 67.87%; Bacillus thuringiensis Z12 was second, with an inhibition rate of 57.94%; Bacillus geysersis L45 showed a weaker antagonistic effect, with an inhibition rate of 12.25% (as shown). Figure 5(as shown in the figure). Under these conditions, no antagonistic effect against Phytophthora nicotineis was detected in Bacillus paramicularis K22 and Bacillus dwarfusus Y1.

[0081] To explore the potential mechanism of antagonism, the effects of sterile fermentation filtrates from various Bacillus species on the mycelial growth of *Phytophthora tobaccotae* were further investigated. The results are as follows: Figure 6 As shown, only the sterile fermentation filtrate of Bacillus subtilis M10 exhibited significant inhibitory activity, with an inhibition rate of 34.16%, indicating that it can secrete extracellular metabolites with antagonistic activity (such as...). Figure 7 (As shown). The fermentation filtrates of the other four strains did not show significant inhibitory effects under the experimental conditions.

[0082] Given the excellent performance of Bacillus subtilis strain M10 in in vitro antagonism and metabolite activity detection, this invention further verified its control effect on Phytophthora tobaccoii after inoculation into tobacco plants through pot experiments. The experiment included four groups: a water control (Ctrl), a positive control (Ctrl+) (30% metalaxyl-mancozeb aqueous solution), a pathogen treatment (Pn, Phytophthora tobaccoii spore suspension), and an antagonistic treatment (M10, fermentation broth). The results are as follows: Figure 8 As shown, compared with the Pn treatment group, the treatment group pre-treated with Bacillus subtilis strain M10 showed significantly reduced symptoms of Phytophthora blight in tobacco plants, with a 50% decrease in incidence and a significant drop in the disease index from 80.0 to 32.2. Based on formula (3.3.3), the control rate of Bacillus subtilis strain M10 against Phytophthora blight in tobacco reached 50%, with a relative control efficacy as high as 59.8%, demonstrating good potential for biocontrol applications compared to traditional chemical control (e.g., ...). Figure 9 (As shown).

[0083] The above results indicate that under the tobacco-rice rotation (CR) system, the dominant Bacillus strains isolated from the rhizosphere soil, especially Bacillus subtilis M10, have strong antagonistic effects and biocontrol potential against Phytophthora indicum, suggesting that they may play an important role in building a healthy rhizosphere microecology and resisting pathogen invasion.

[0084] 4. Metabolomics analysis of tobacco rhizosphere soil on the effects of tobacco-rice rotation

[0085] 4.1 Overview of the test site

[0086] Same as 1.1

[0087] 4.2 Sample Collection and Preparation of Rhizosphere Soil Extract

[0088] Rhizosphere soil samples were collected using a sterilized shovel, removing plant and animal debris and other impurities to ensure sample purity. Three biological replicates were randomly selected for both treatments (CC and CR) to eliminate experimental errors caused by individual differences. Samples were taken approximately 20 cm deep around the plant roots, collected at clockwise locations, and placed in the same sealed bag. The soil was sieved through a 0.85 mm sieve, placed in centrifuge tubes, and stored in the laboratory with ice packs. 100 g of fresh rhizosphere soil was added to 500 ml of deionized water, shaken and extracted for 3 hours, then centrifuged at low temperature for 5 minutes (20℃, 8000 rpm). Approximately 50 ml of the supernatant was retained, filtered, and the water was evaporated. Methanol was added to dissolve the residue, and the mixture was allowed to evaporate. The mixture was washed 2-3 times, dissolved in 5 ml of methanol, stored at -80℃, and sent to Wuhan Metawell Biotechnology Co., Ltd. for testing.

[0089] 4.3 Plant broad-targeted metabolomics assays

[0090] Metabolomics analysis was performed using a broad-targeted metabolomics approach based on UPLC-ESI-MS / MS. Lyophilized samples were extracted with 70% methanol containing an internal standard. After vortex mixing, sonication, and centrifugation, the supernatant was filtered through a 0.22 μm filter membrane for subsequent analysis.

[0091] Metabolite profiling was performed using an ExionLC™ AD ultra-high performance liquid chromatography system coupled with a QTRAP mass spectrometer (Sciex, USA). Chromatographic separations were performed using an Agilent SB-C18 column (1.8 μm, 2.1 × 100 mm) with a gradient elution program of water and acetonitrile containing 0.1% formic acid. Mass spectrometry data were acquired in multiple reaction monitoring (MRM) mode under electrospray ionization (ESI) conditions.

[0092] 4.4 Data Processing

[0093] Peak detection, alignment, and normalization were performed on the raw data. Multivariate statistical analysis was conducted using R software. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to assess metabolic differences between treatment groups. Differential metabolites were identified based on variable importance in the projection (VIP>1), fold change (FC>2 or <0.5), and Student's t-test (p<0.05).

[0094] 4.5 Results and Analysis

[0095] To identify potential signaling molecules or key metabolites involved in alleviating continuous cropping obstacles, untargeted metabolomics (LC-MS) analysis was performed on soil samples from continuous cropping (CC) and crop rotation (CR) systems. A total of 2,347 metabolites were detected in the soil samples, belonging to 12 categories. Results are as follows:Figure 10 As shown. Partial least squares discriminant analysis (OPLS-DA) revealed significant differences in metabolic profiles between the CC and CR groups (e.g., Figure 11 R²X and R²Y measure the goodness of fit of the model, and Q² tests its predictive ability, indicating that different farming regimes lead to significant metabolic reprogramming. To screen for the most critical differentially expressed metabolites (DEMs), stringent criteria were used: projected variable importance VIP > 1, p < 0.05, and |Log²FoldChange| > 2. Based on these thresholds, a total of 79 DEMs were identified, including 56 upregulated and 23 downregulated metabolites (compared to the CR and CC groups). The results are shown below. Figure 12 As shown in the figure. Notably, among the significantly upregulated metabolites, diosmetin exhibited the highest fold change (Log2FC = 2.89, p < 0.05). As a natural flavonoid known for its diverse biological activities, diosmetin is presumed to play a key role in inhibiting soil-borne pathogens.

[0096] 5. Effects of geranium lignin on the growth of Bacillus subtilis M10 and its antagonistic effect on Phytophthora indicum

[0097] 5.1 Experimental Materials and Methods

[0098] To verify the effects of key metabolites obtained from metabolomics analysis on the growth of Bacillus subtilis M10 and their antagonistic activity against Phytophthora nicotine, shake flask culture and in-plate experiments were conducted. The metabolite Diosmetin (>98%) used in this invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and was dissolved in 1% methanol. All control treatments were treated with an equal volume of 1% methanol to eliminate solvent effects (Eloff 1998).

[0099] In shake-flask culture, the effect of metabolites on the growth of Bacillus subtilis M10 was first evaluated. Diosmetin was added to NB liquid medium to final concentrations of 1 mmol / L and 0.1 mmol / L (see international patent WO2009 / 049747), with a medium without added metabolites serving as a control. Subsequently, Bacillus subtilis M10 (B. subtilis) culture was inoculated at 1% of the total volume and cultured at 28°C with shaking at 180 rpm. The OD of the culture was measured periodically during the culture process. 600 The absorbance values ​​were calculated and the growth curve was plotted.

[0100] In the in-plate experiment, 7 mm diameter filter paper discs soaked in Diosmetin (three concentration gradients: 0 (Crtl), 0.1 mmol / L and 1 mmol / L) for 20 min were added to OA medium and inoculated with P. nicotianae to assess the direct effect of metabolites on the growth of the pathogen.

[0101] 5.2 Data Processing

[0102] The data obtained from the experiment were organized and calculated in Microsoft Excel 2019, and statistical and difference analysis (Waller-Duncan) was performed on the data using SPSS 20.0. The ChiPlot platform was used to plot the data.

[0103] 5.3 Results and Analysis

[0104] To verify whether geraniol, a metabolite significantly enriched in crop rotation soils, could affect the growth of Bacillus subtilis M10, 1 mmol / L and 0.1 mmol / L geraniol were added to the liquid culture system, with 1% methanol treatment as a control. Growth curve results are shown (e.g., Figure 13 As shown), OD values ​​between treatments during the initial incubation period (0-5h) 600 The differences in values ​​were small; with the extension of culture time, the growth trends of each treatment group were basically the same. After 10 h, the geraniol treatment group showed no significant difference compared with the control group. In the later stage of culture (15–20 h), the cell density of different concentrations of geraniol treatment was similar to that of the control group, with no significant overall change, indicating that exogenous addition of geraniol had no significant effect on the growth and reproduction of Bacillus subtilis M10.

[0105] To further verify the antagonistic effect of geraniol on pathogen growth, pathogenic fungal discs with a diameter of 7 mm were inoculated in three gradients on the left and right sides, respectively, and then filter paper discs with a diameter of 7 mm soaked in the corresponding concentration were inoculated on the right side. The results are as follows. Figure 14 As shown, no significant difference in the growth of Phytophthora tobaccois colonies was observed between the three gradients and the control treatment, indicating that geraniol itself does not have a direct inhibitory effect on Phytophthora tobaccois, and its antibacterial effect is mainly achieved by regulating the activity of antagonistic bacteria (Bacillus subtilis M10).

[0106] 6. The control effect of Bacillus subtilis M10 combined with geraniol on Phytophthora intoxin

[0107] 6.1 Experimental Materials and Methods

[0108] The antagonistic effect of the combination of Bacillus subtilis M10 and geraniol against Phytophthora nicotianae was evaluated using an in-vessel antagonistic culture method. Five-mm diameter mycelial discs were created at the edge of P. nicotianae colonies cultured for 5 days. These discs were then inoculated into the center of OA medium. Three 5mm diameter Bacillus subtilis M10 discs were inoculated 2cm around the pathogen discs. Geraniol was pre-mixed with Bacillus subtilis M10 discs at final concentrations of 1 mmol / L and 0.1 mmol / L. The media were incubated at 28℃. When the control pathogen colonies were about to cover the entire culture dish, the colony diameter of each treatment was measured, and the inhibition rate was calculated. The calculation method was the same as in section 3.1.

[0109] A pot experiment was conducted to verify the regulatory effect of geraniol on the control of tobacco black shank disease by Bacillus subtilis M10. The experiment was carried out in a research greenhouse, and the tested tobacco variety was Yunyan 87. Four treatments were set up: natural growth control (Ctrl), inoculated with Phytophthora indica (Pn), inoculated with Phytophthora indica and inoculated with 2×10 7 CFU / mL (OD) 600 The treatments included Bacillus subtilis bacterial suspension (Pn+M10) with a concentration approximately 1.0 (≈1.0) and inoculation with Phytophthora indicum followed by simultaneous application of equal concentrations of M10 and 0.1 mmol / L geraniol (Pn+M10+0.1 mMDiosmetin), with 10 replicates for each treatment. Seven days after transplanting, tobacco seedlings were inoculated with Bacillus subtilis M10 bacterial suspension and geraniol via root irrigation. Two days later, Phytophthora indicum spore suspension was inoculated. This process was repeated three times, with a one-week interval between each round. All treatments were cultured under the same irrigation volume and light conditions. Seven days after the third round of inoculation, a disease survey was conducted. Disease severity grading and disease index calculation methods were performed according to the national industry standard GB / T 23222-2008. The control effects of different treatments on tobacco black shank were evaluated by statistically analyzing the incidence rate and disease index, using the same method as in section 3.3.3.

[0110] 6.2 Data Processing

[0111] The data obtained from the experiment were organized and calculated in Microsoft Excel 2019, and statistical and differential analysis (Waller-Duncan) was performed on the data using SPSS 20.0.

[0112] 6.3 Results and Analysis

[0113] This invention evaluates the effect of geraniol on the antagonistic effect of Bacillus subtilis M10 against Phytophthora tobaccora using a plate confrontation experiment. The results are as follows: Figure 15As shown, in the treatment with only Bacillus subtilis M10 inoculation, the strain exhibited a significant inhibitory effect on Phytophthora indicum, with an inhibition zone width of 0.95±0.20 cm and an inhibition rate of 62.11%. However, after adding two concentrations of geraniol to the culture system, the inhibitory area of ​​Bacillus subtilis M10 against Phytophthora indicum further expanded, and the inhibitory effect was significantly enhanced. Among these, the 0.1 mmol / L geraniol treatment showed the strongest antagonistic effect, with an inhibition zone width of 1.61±0.15 cm and an inhibition rate of 74.66%, which was significantly different from the treatment with only Bacillus subtilis M10 inoculation (p<0.001).

[0114] To further verify the above results, the results of the pot experiment showed (e.g.) Figure 16 As shown, among the following treatments: ①Ctrl; ②Pn; ③Pn+M10; ④Pn+M10+0.1 mM Diosmetin), no disease was observed in the Ctrl treatment; in the treatment with only Phytophthora infestans, 7 pots showed obvious disease symptoms, with a disease index as high as 54.4; after inoculation with Bacillus subtilis M10, the number of diseased pots decreased to 5, the disease index decreased to 30.0, and the relative control efficacy was 44.9%, indicating that Bacillus subtilis M10 has a certain disease-suppressing effect in the potted system; while inoculation with Bacillus subtilis M10 treated with 0.1 mmol / L geraniol (2×10) 7 After treatment with CFU / mL, the number of diseased pots further decreased to 2, and the disease index also dropped significantly to 17.8, indicating a significant reduction in disease occurrence. Compared with the single M10 treatment, the relative control efficacy increased by 22.4%. These results indicate that geraniol can significantly enhance the disease-suppressing effect of Bacillus subtilis M10 in the plant system.

[0115] The combined results of antagonistic experiments and pot experiment verification show that geraniol can promote the growth of Bacillus subtilis M10 and further enhance its antagonistic ability against Phytophthora intoxin, thereby improving its disease-suppressing effect in tobacco plants.

Claims

1. A strain of Bacillus subtilis M10, characterized in that: The Bacillus subtilis M10 was submitted for preservation to the China General Microbiological Culture Collection Center on March 23, 2026, with the preservation number CGMCC No. 37999.

2. The application of Bacillus subtilis M10 according to claim 1 in inhibiting Phytophthora intoxin.

3. The application of Bacillus subtilis M10 according to claim 1 in the prevention and control of tobacco black shank disease.

4. The application of Bacillus subtilis M10 according to claim 1 in the prevention and control of tobacco black shank caused by Phytophthora infestans.

5. A biocontrol agent based on Bacillus subtilis M10 according to claim 1, wherein the biocontrol agent is a seed liquid, culture medium, culture medium, bacterial liquid, fermentation broth and / or fermentation filtrate containing Bacillus subtilis M10.

6. A biocontrol agent, characterized in that: The biocontrol agent comprises flavonoids and Bacillus subtilis M10 as described in claim 1; preferably, the final concentration of Bacillus subtilis M10 is not less than 2 × 10⁻⁶. 7 CFU / mL; the final concentration of the flavonoid compound is not less than 0.1 mmol / L.

7. The biocontrol agent according to claim 6, characterized in that: The flavonoid compound is geraniol.

8. The application of the biocontrol agent according to claim 6 or 7 in inhibiting Phytophthora indicans.

9. The application of the biocontrol agent according to claim 6 or 7 in tobacco black shank disease.

10. The application of the biocontrol agent according to claim 6 or 7 in tobacco black shank caused by Phytophthora indicum.

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