A bacillus subtilis for preventing and treating poplar canker and a preparation method of a wettable powder thereof

Abstract

The present application relates to the technical field of forest disease prevention and microbial preparation, and particularly relates to a bacillus subtilis for preventing poplar canker disease and a preparation method of wettable powder thereof. The preservation number of the bacillus subtilis B13 is CGMCC No.39570. The present application constructs a complete technical system from pathogenic bacteria identification, endophytic antagonistic bacteria screening, fermentation condition optimization to wettable powder preparation and field application verification. The core of the system is to screen out the bacillus subtilis B13, which shows highly stable antagonistic activity in the primary screening and rescreening experiments, and the inhibition rate of the bacillus subtilis B13 to grape colletotrichum reaches 86.65%, which is much higher than that of most traditional strains, and the fluctuation of the inhibition rate in repeated experiments is less than 5%, which proves that the activity is highly reproducible, the effect is reliable, and the activity can be maintained for a long time in the laboratory and field environment.

Description

Technical Field

[0001] This invention relates to the field of forest tree disease control and microbial preparation technology, and in particular to a method for preparing Bacillus subtilis and its wettable powder for controlling poplar canker. Background Technology

[0002] Poplar ( Populus spp Due to its rapid growth, strong adaptability, and wide range of uses, forest trees are increasingly being applied in timber production, ecological restoration, carbon sequestration forestry, and bioenergy. However, with the expansion of planting scale and intensive management, forest diseases are becoming increasingly prominent, especially those caused by *Botrytis cinerea* (…). Botryosphaeria dothidea ) and rotten shell fungus ( Valsa spp Poplar canker, caused by diseases such as septicemia and malaria, has become a major bottleneck restricting the development of plantations. This disease is characterized by a long incubation period, high infectivity, and rapid spread. Young trees have a high incidence rate, with mortality rates reaching 30%–50%, and exceeding 70% in severely affected areas. In the early stages, lesions are brown to dark brown, gradually expanding and becoming sunken. In severe cases, they can encircle the branches and trunk, causing bark cracking, obstructing vascular tissue, and ultimately leading to branch and trunk dieback or the death of the entire tree. This severely damages the stand structure and reduces the trees' resistance to adverse conditions.

[0003] Existing control methods each have their limitations. Agricultural management and physical control (such as thinning, pruning, clearing diseased plant debris, and setting up isolation strips) are environmentally friendly and easy to implement, but their effectiveness is limited in high-density forest stands or severely diseased areas, and they cannot fundamentally block the spread of diseases. Chemical control (such as fungicides like carbendazim and tebuconazole) can quickly suppress the spread of pathogens in the short term, but long-term reliance on them can lead to pesticide residues, environmental pollution, and increased drug resistance in pathogens, making it difficult to meet the environmental protection requirements for sustainable forestry development.

[0004] In contrast, biological control has become a research hotspot for forest disease control due to its advantages such as being green and safe, highly targeted, and less prone to developing resistance. Existing research has confirmed that Bacillus spp. (…) Bacillus spp.), Pseudomonas ( Pseudomonas spp.) and Trichoderma ( Trichoderma Microorganisms such as *S. spp.* have shown good inhibitory effects on poplar canker and pine wilt disease. However, the research and development of biocontrol bacteria for poplar canker still has significant shortcomings: First, there is a lack of highly efficient, stable, and specific antagonistic strains, with most studies remaining at the in vitro antagonism stage and lacking a standardized activity evaluation system; second, the fermentation process conditions for biocontrol bacteria have not been systematically optimized, resulting in large fluctuations in antibacterial activity during large-scale production and field applications; third, the formulation and preparation process of microbial preparations are imperfect, and properties such as suspension, wettability, thermal stability, and UV protection are difficult to meet the actual spraying requirements; fourth, there is a lack of an integrated and complete chain from pathogen identification, strain screening, fermentation optimization to formulation development, which limits the large-scale promotion of biocontrol technology.

[0005] Meanwhile, the pathogen causing ulcer disease is highly adaptable to its environment and can spread through multiple pathways, including wind, rain, insects, wounds, and natural openings. It can also remain dormant in the tissues of branches and trunks for extended periods, greatly increasing the difficulty of control. Traditional single-method approaches are no longer sufficient to balance control effectiveness with environmental safety; therefore, there is an urgent need to develop a new green control solution that integrates systematic screening, efficient fermentation, and formulation.

[0006] Based on the above situation, this invention aims to provide a complete biological control technology system for poplar canker. By systematically screening highly efficient endophytic antagonistic strains from healthy poplars, optimizing their fermentation conditions, and further preparing stable wettable powders, this invention effectively solves key technical problems such as unstable strain activity and difficulty in formulation application, meeting the needs of large-scale and standardized forestry applications, and providing reliable technical support for the green control of poplar and other forest tree diseases.

[0007] In summary, the development of efficient and stable biocontrol agents and supporting application technologies has significant technical value and broad application prospects for alleviating the damage caused by poplar canker, reducing dependence on chemical pesticides, ensuring ecological security, and promoting the sustainable development of forestry. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing Bacillus subtilis and its wettable powder for controlling poplar canker. The core of this method lies in screening endophytic strains with antagonistic activity from healthy poplar branches, combined with fermentation process optimization and formulation design, to construct a complete technical system from pathogen identification, strain screening and evaluation, fermentation condition optimization to wettable powder preparation, achieving highly efficient inhibition of poplar branch diseases caused by pathogens such as *Botrytis cinerea*. This technology is not only applicable to poplar plantations and seedling cultivation, but can also be extended to the biological control of other forest trees or economic crops, possessing significant industrialization potential and application value.

[0009] The technical problem this invention aims to solve is: how to efficiently and stably screen endophytic strains with specific antagonistic activity and establish a standardized evaluation system, while ensuring the maintenance of their activity during identification and formulation processing; based on this, it is necessary to systematically optimize fermentation process parameters (including carbon and nitrogen sources, pH value, temperature, time, inoculum amount, etc.) to ensure the large-scale and stable production of antibacterial active substances, and overcome the performance bottlenecks of wettable powders in terms of suspension, wettability, thermal stability and UV protection, and screen out the best combination of carriers and adjuvants; finally, it is necessary to conduct field efficacy tests under poplar seedling and plantation conditions to ensure the high efficacy, stability and environmental friendliness of the formulation in practical applications, and meet the urgent needs of green forestry disease control.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Bacillus subtilis strain for the prevention and control of poplar canker ( Bacillus subtilis B13, the Bacillus subtilis B13, was deposited on July 6, 2026 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 39570.

[0011] This invention provides the application of Bacillus subtilis B13 in the preparation of biocontrol agents for controlling poplar canker caused by Staphylococcus aureus.

[0012] This invention provides a fermentation method for Bacillus subtilis B13, comprising the following steps: The Bacillus subtilis B13 was inoculated into a fermentation medium and cultured with shaking at an initial pH of 6.0-8.0, a culture temperature of 30℃-40℃, and a shaking speed of 150-200 r / min for 12-48 h.

[0013] Preferably, the fermentation medium uses soluble starch as a carbon source and beef extract as a nitrogen source, and the inoculum amount of Bacillus subtilis B13 is 1% to 5%.

[0014] The present invention provides a wettable powder containing Bacillus subtilis B13, comprising Bacillus subtilis B13 fermentation product and adjuvants, wherein the adjuvants include a carrier, a dispersant, a wetting agent, a heat stabilizer and a UV protectant.

[0015] Preferably, the carrier is calcium carbonate, the dispersant and wetting agent is a combination of Tween 80 and sodium dodecyl sulfate, the heat stabilizer is potassium dihydrogen phosphate, and the ultraviolet protectant is ascorbic acid.

[0016] Preferably, the mass ratio of Tween 80 to sodium dodecyl sulfate is (4~8):(6~2), and the total amount of the Tween 80 and sodium dodecyl sulfate composition added is 2%~16% of the fermentation mass.

[0017] This invention provides a method for preparing the aforementioned wettable powder, comprising the following steps: (1) The Bacillus subtilis B13 was fermented and cultured to obtain a viable count of 2 × 10⁻⁶. 8 Fermentation broth with a CFU / mL or higher; (2) The fermentation liquid obtained in step (1) is mixed evenly with the carrier, and then dried, ground and sieved to obtain the microbial agent mother powder; (3) Add dispersant, wetting agent, heat stabilizer and ultraviolet protectant to the bacterial agent master powder and mix evenly to obtain the product.

[0018] Preferably, the fermentation broth and the carrier are mixed evenly at a ratio of (6~8):(4~2) (v / w).

[0019] This invention provides the application of the aforementioned wettable powder in the prevention and control of poplar canker.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention systematically constructs a complete technical system from pathogen identification, endophytic antagonistic bacteria screening, fermentation condition optimization to wettable powder preparation and field application verification. The core of this system lies in the screening of Bacillus subtilis B13, which exhibited highly stable antagonistic activity in both primary and secondary screening experiments, achieving an inhibition rate of 86.65% against *Botrytis cinerea*, far exceeding most traditional strains. Furthermore, the inhibition rate fluctuated by less than 5% in repeated experiments, demonstrating strong reproducibility and reliable efficacy. This highly efficient strain provides a solid microbial resource foundation for large-scale application and can maintain its activity for a long time in both laboratory and field environments.

[0021] Regarding fermentation process and formulation performance, this invention improves the antibacterial effect of the strain by approximately 40% through systematic optimization of parameters such as carbon source, nitrogen source, pH value, temperature, and rotation speed, while maintaining good batch-to-batch stability, allowing for direct integration with industrial fermentation production. In formulation preparation, through rational screening of carriers and adjuvants, and the addition of heat stabilizers and UV protectants, the resulting wettable powder maintains ≥85% activity under high temperature, strong light, and dry conditions, and the viable cell count remains ≥1×10⁻⁶ after three months of storage. 8 The formulation has a CFU / g concentration and an antibacterial rate of no less than 80%. At the same time, the formulation has excellent suspension and wettability, is easy to operate in the field, and effectively solves the problems of easy precipitation and decreased activity of traditional liquid formulations.

[0022] Field efficacy verification results show that the formulation of this invention can significantly reduce the disease index of poplar canker, with a control efficacy of up to 87.50%, and it exhibits stability in both seedling and plantation environments, demonstrating good environmental adaptability. In summary, the beneficial effects of this invention are concentrated in the following aspects: first, it provides efficient and stable biocontrol bacteria resources; second, it establishes a systematic fermentation optimization process, ensuring industrial production; third, it prepares a high-performance wettable powder, adaptable to complex field environments; and fourth, it achieves a complete technical closed loop from strain to application, with strong operability. This invention not only effectively reduces the use of chemical pesticides and protects the ecological environment, but also provides a reliable technical model for the development of green forestry control products, possessing significant economic, ecological, and social benefits, and providing scientific support for sustainable forestry development. Attached Figure Description

[0023] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 Isolation and Identification of Pathogens of Poplar Canker

[0025] Note: (a) Colony characteristics of *Botrytis cinerea* after 7 days of culture; (b) Conidia of *Botrytis cinerea*; (c) Phylogenetic tree of *Botrytis cinerea* based on ITS sequence; (d) Morphology of poplar leaves after 3 days of inoculation with distilled water; (e) Morphology of poplar leaves after 3 days of inoculation with *Botrytis cinerea* spore suspension.

[0026] Figure 2 Schematic diagram of the antagonistic effects of endophytic bacteria and endophytic fungi on Staphylococcus aureus.

[0027] (a) Initial screening results of partial endophytic fungi antagonizing Staphylococcus aureus: the upper part shows the colonies of the tested antagonistic strains, and the lower part shows the colonies of the target pathogen Staphylococcus aureus; (b) Initial screening results of partial endophytic bacteria antagonizing Staphylococcus aureus: the left and right sides show the colonies of the tested antagonistic strains, and the center shows the colonies of the target pathogen Staphylococcus aureus; (c) Secondary screening results of partial endophytic fungi antagonizing Staphylococcus aureus: the four corners show the colonies of the tested antagonistic strains, and the center shows the colonies of the target pathogen Staphylococcus aureus; (d) Secondary screening results of partial endophytic bacteria antagonizing Staphylococcus aureus: the four sides show the colonies of the tested antagonistic strains, and the center shows the colonies of the target pathogen Staphylococcus aureus.

[0028] Figure 3 Screening and identification results of target biocontrol strains.

[0029] Figure 4 Optimization results of Bacillus subtilis B13 fermentation conditions

[0030] (a) Plate antagonistic effect of different carbon sources on the antibacterial activity of strain B13 fermentation filtrate, with CK as blank control; (b) Statistical analysis of the inhibition zone diameter of strain B13 fermentation broth against *Botrytis cinerea* under different carbon source conditions; (c) Plate antagonistic effect of different nitrogen sources on the antibacterial activity of strain B13 fermentation filtrate, with CK as blank control; (d) Statistical analysis of the inhibition zone diameter of strain B13 fermentation broth against *Botrytis cinerea* under different nitrogen source conditions; (e) Plate antagonistic effect of different initial pH on the antibacterial activity of strain B13 fermentation filtrate, with CK as blank control; (f) Plate antagonistic effect of different initial pH on strain B13 fermentation filtrate. 3. Statistical analysis of the inhibition zone diameter of fermentation broth against *Botrytis cinerea*; (g) Plate antagonistic effect of different culture temperatures on the antibacterial activity of fermentation filtrate of strain B13, with CK as blank control; (h) Statistical analysis of the inhibition zone diameter of fermentation broth of strain B13 against *Botrytis cinerea* under different culture temperatures; (i) Effect of different fermentation times on the antibacterial activity of fermentation broth of strain B13; (j) Effect of different inoculum amounts on the antibacterial activity of fermentation broth of strain B13; (k) Effect of different liquid volumes on the antibacterial activity of fermentation broth of strain B13; (l) Effect of different shaking speeds on the antibacterial activity of fermentation broth of strain B13.

[0031] Figure 5 Preparation and application effect test results of Bacillus subtilis B13 wettable powder

[0032] (a) Effect of different carrier materials on the adsorption capacity of strain B13 mother powder; (b) Effect of different carrier materials on the suspension rate of mother powder; (c) Effect of different carrier materials on the wetting time of mother powder; (d) Effect of different heat stabilizers on the spore yield of strain B13; (e) The prepared wettable powder of Bacillus subtilis B13; The control effect of wettable powder on poplar diseases caused by Botrytis cinerea ((f) is the treatment group sprayed with wettable powder after inoculation with pathogen, (g) is the control group inoculated only with pathogen).

[0033] Preservation Instructions

[0034] The Bacillus subtilis of the present invention ( Bacillus subtilis Strain B13 was deposited on July 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit registration number for this biological material is CGMCC No. 39570. Detailed Implementation

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] 1. This invention primarily targets the main pathogen of poplar canker—Botrytis cinerea (… Botryosphaeria dothidea Separation and identification were performed. Tissue samples were taken from branches infected with poplar canker on the campus of Northeast Forestry University in Harbin. A tissue block approximately 0.4 × 0.4 cm in size was taken from the junction of the lesion and healthy tissue. Surface disinfection was performed (immersion in 75% ethanol for 1 min, followed by treatment with 2% sodium hypochlorite for 2 min, and repeated rinsing with sterile water five times). 100 μL of the sterile water from the last rinse was spread onto a PDA plate and incubated at 25℃ for 48 h to verify the effectiveness of the surface disinfection.

[0038] After sterilization, tissue blocks were placed on potato dextrose agar (PDA) and cultured for 10-15 days, with mycelial growth observed daily. Once mycelia emerged from the edges of the tissue blocks, purification was performed using the tip transfer method. Single colonies were then cultured on slant agar at 4°C for preservation. Morphological observation of the purified strains was performed, including colony color, texture, edge morphology, and conidia morphology, to preliminarily determine the genus. Pathogenicity was then verified using the Koch method, and a concentration of 1×10⁻⁶ was prepared. 6 A 20 μL suspension of pathogenic spores at spores / mL was inoculated onto healthy *Populus alba* leaves. The leaves were placed in 90 mm petri dishes lined with filter paper, with 1 mL of sterile water added to the filter paper to maintain humidity. Sterile water inoculation served as a control. The mixture was incubated at 25°C for 7 days to observe for the appearance of typical ulceration symptoms. To further ensure accurate identification, fungal DNA was extracted using a 2×T5 Direct PCR Kit. Amplification was performed using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3' as shown in SEQ ID NO: 1) / ITS4 (5'-TCCTCCGCTTATTGATATGC-3' as shown in SEQ ID NO: 2). The PCR amplification program was as follows: 94°C pre-denaturation for 5 min; followed by cycling: 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 1 min, for a total of 40 cycles; after cycling, a final extension at 72°C for 10 min was performed to ensure sufficient extension of the amplified fragment. After sequencing, the sequences were compared with those in the GenBank database, and phylogenetic analysis was performed using MEGA11 to support their species classification, providing a reliable target for subsequent antagonistic bacteria screening.

[0039] Results: Observation based on morphological characteristics ( Figure 1 a, 1b) and molecular biological identification ( Figure 1 c, Genbank accession number: KY788299.1) preliminarily identified the pathogen of poplar canker on the campus of Northeast Forestry University as Staphylococcus aureus (c). Botryosphaeria dothidea Based on this, through Koch's law ( Figure 1 (d, 1e) proved that the isolated strain was the pathogen of poplar canker.

[0040] 2. After identifying the pathogenic target, this invention isolates endophytic microorganisms (the surface disinfection part is the same as the diseased sample part) from healthy poplar branches using the method of separating pathogens. These microorganisms include endophytic bacteria and endophytic fungi. Hyphae or single colonies at the edge of the colony are picked and transferred to PDA medium for purification to obtain an endophytic microbial resource library.

[0041] During the initial screening, the plate confrontation method was used to assess antagonistic activity. For fungal strains, a two-point confrontation method was used, with pathogenic fungal discs collected using a 5 mm punch. The discs were inoculated onto 1 / 3 of a PDA plate, while endophytic fungal discs were inoculated onto 2 / 3. For bacterial strains, a streak confrontation method was used, with pathogenic fungal discs inoculated in the center of the PDA plate, and endophytic bacterial suspensions inoculated on both sides at a radius of 1 / 2 from the center. The control group was inoculated only with pathogenic bacteria in the center of the plate. The plates were incubated at 25°C in the dark for 3-5 days, and the formation of a clear inhibition zone or inhibition band was observed. All experiments were performed in triplicate.

[0042] To further verify the antagonistic activity of the antagonistic strains obtained in the initial screening against Staphylococcus aureus and to rule out false positives in the initial screening, a secondary screening was subsequently conducted. A 5 mm diameter pathogenic fungal disc was inoculated in the center of a PDA plate. For fungi, the four-point confrontation method was used, with the four corners (25-30 mm from the center) inoculated with the initially screened positive fungi; for bacteria, the cross-strike confrontation method was used, with four bacterial lines equidistantly streaked around the perimeter. The control group was inoculated only in the center of the plate. Each experiment was performed in triplicate. After the pathogenic colonies in the control group had fully colonized the plate, the diameter of the pathogenic colonies in the treatment group was measured using the cross-strike method, and the inhibition rate was calculated.

[0043] The formula for calculating the inhibition rate is: Inhibition rate (%) = (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - 5) × 100%.

[0044] Antagonistic bacterial strains with high inhibition rates were selected to determine the inhibitory effect of their sterile fermentation broth on the germination of pathogenic fungal spores. The strains that passed the second screening were inoculated into LB liquid medium and cultured at 37℃ and 180 r / min for 24 h. After centrifugation at 8000 r / min for 10 min, the broth was filtered through a 0.45 μm filter to obtain the sterile fermentation broth of the antagonistic strains. 100 μL of 1×10⁻⁶ slurry was then plated onto a PDA plate. 6 Spores / mL of pathogenic bacterial spore suspension were prepared by punching four wells in a plate using a 7 mm diameter punch. 80 μL of sterile fermentation filtrate containing different antagonistic bacteria was added to each well, with 80 μL of sterile water added as a control group. Each group was replicated three times. After incubation at 25°C for 3 days, the diameter of the inhibition zone was measured.

[0045] The diameter of the inhibition zone is calculated using the formula: Inhibition zone diameter (mm) = Measured inhibition zone diameter - 7.

[0046] The selected high-efficiency strains will be further identified by morphological and physiological biochemical characteristics and 16S rDNA sequencing to clarify their taxonomic status. In this invention, the selected high-efficiency strains are identified by Gram staining. Single colonies grown on LB medium for 2-3 days are picked, sterile distilled water is added and evenly spread on a glass slide, and after drying, they are heat-fixed by passing them over an alcohol lamp flame twice. After fixation, ammonium oxalate crystal violet staining solution is added for 1 min, followed by washing with water; Lugol's iodine solution is added for mordanting for 1 min, followed by washing with water; 95% ethanol is added for decolorization for 30-45 s, followed by washing with water; finally, safranin counterstaining solution is added for counterstaining for 1 min, followed by washing with water and drying. The color is observed under a microscope: those with purple cells are Gram-positive bacteria, and those with red cells are Gram-negative bacteria. Physiological and biochemical characteristics are determined with reference to the "Common Bacterial System Identification Manual". The following indicators are determined by micro-biochemical reaction tubes and cultured at 37℃: (1) VP test: inoculated into glucose peptone water medium and cultured for 24-48 h. Add an equal amount of VP reagent, shake for 2 min, and incubate in a 30℃ water bath for 30 min. A red color indicates a positive result, while a colorless color indicates a negative result; (2) Starch hydrolysis test: Inoculate into starch medium and culture for 24-48 h. Add iodine solution and observe. A clear zone around the colony indicates a positive result, while the absence of a clear zone indicates a negative result; (3) Methyl red test: Inoculate into glucose peptone water medium and culture for 48 h. Add methyl red indicator. A red color indicates a positive result, while a yellow color indicates a negative result; (4) Gelatin liquefaction test: Inoculate into gelatin high-layer medium and culture for 7 d. If the gelatin liquefies but cannot solidify, it is a positive result, while if it remains solidified, it is a negative result; (5) Citrate utilization test: Inoculate into Simon's citrate medium and culture for 24-48 h. If the medium turns blue, it is a positive result, while if it does not change color, it is a negative result; (6) Glycolysis test: Inoculate into fermentation medium containing glucose, sucrose, xylose, mannose, galactose, fructose, and maltose and culture for 24-48 h. (7) Nitrate reduction test: Inoculate into nitrate medium containing Durham tubes and incubate for 24-48 h. A red color or bubbles in Gaglis' reagent indicate a positive result; no color change indicates a negative result. (8) Salt tolerance test: Inoculate into medium containing 7% NaCl and incubate for 48-168 h. Growth of the strain indicates a positive result; no growth indicates a negative result.

[0047] This invention uses a bacterial genomic DNA extraction kit to extract total DNA from bacterial strains and performs multi-gene sequence (16S rDNA, GyrB, AtpD) analysis and identification. The primers used for amplification were: 27F (5'-AGAGTTTGATCCTGGCTCAG-3' as shown in SEQ ID NO: 3) / 1492R (5'-GGTTACCTTGTTACGACTT-3' as shown in SEQ ID NO: 4), U-gyrB (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3' as shown in SEQ ID NO: 5) / D-gyrB (5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3' as shown in SEQ ID NO: 6), and AtpD-F (5'-ACAAGTTTGTCTTCCTCGCC-3' as shown in SEQ ID NO: 7') / AtpD-R (5'TGAGGTCGCTCTTCATTTAGG-3' as shown in SEQ ID NO: 8). The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The PCR amplification system and procedure were the same as above. Sequencing was performed and compared with sequences in the GenBank database. Phylogenetic analysis was then conducted using MEGA11 to support the species classification.

[0048] Results: Sixty endophytic fungi and 51 endophytic bacteria were isolated from healthy poplar branches. Figure 2 The antagonistic activity was assessed through primary and secondary screening. Primary screening results showed that some endophytic fungi and bacteria could form inhibition zones against Staphylococcus aureus on agar plates, but the antibacterial effect of most fungi was unstable. Bacterial strain B13, however, exhibited significant antibacterial activity (inhibition rate 86.65%), forming a clear and stable inhibition zone. Secondary screening further confirmed that the antibacterial ability of strain B13 was reproducible and stable, and the diameter of its inhibition zone was significantly larger than that of other strains (Table 1).

[0049] Table 1. Inhibitory effects of eight antagonistic bacteria on Staphylococcus aureus

[0050] Note: Data are mean ± standard deviation. Differences between groups were assessed using Student's t-test. Different lowercase letters in the same column indicate significant differences. P <0.05).

[0051] The sterile fermentation broth antibacterial experiment showed that the B13 fermentation broth formed a significant inhibition zone against Staphylococcus aureus, and the antibacterial effect was significantly better than that of the control group. Figure 3 a). Statistical analysis showed that the diameter of the inhibition zone did not differ significantly between different batches of fermentation broth (P>0.05), indicating that the antibacterial activity of the fermentation broth was stable. Figure 3 b). Analysis shows that the excellent antibacterial properties of B13 may be mainly attributed to its efficient production of antibacterial metabolites, which exhibit stable activity under different culture conditions, laying the foundation for subsequent fermentation process optimization.

[0052] The selected high-efficiency strain B13 was subjected to morphological observation and physiological and biochemical characterization analysis. B13 was Gram-positive ( Figure 3 c) Spore formation is obvious, and it can utilize carbon sources such as soluble starch, glucose, and sucrose, as well as nitrogen sources such as beef extract and yeast extract (Table 2). Multi-gene (16S rDNA, GyrB, AtpD) PCR amplification and sequencing were performed, and the obtained sequences have been uploaded to the NCBI database (accession numbers: 16S rDNA, PZ600699; GyrB, PZ613085; AtpD, PZ613086). Multiple sequence alignment results showed that B13 is related to Bacillus subtilis (…). Bacillus subtilis The homology is higher than 99%, and phylogenetic analysis further supports its classification. Figure 3 d).

[0053] Table 2 Physiological and biochemical characteristics of strain B13

[0054] Note: "+" indicates positive, and "-" indicates negative.

[0055] 3. To ensure the maintenance of antibacterial activity of the selected high-efficiency strains during production and application, this invention systematically optimized the fermentation conditions of Bacillus subtilis B13. Using the diameter of the inhibition zone as an evaluation index, the effects of carbon source, nitrogen source, initial pH, temperature, fermentation time, inoculum size, liquid volume, and rotation speed on antibacterial activity were investigated. Based on a basal fermentation medium (10 g glucose, 5.0 g peptone, 1.0 g KH2PO4, 0.5 g MgSO4·7H2O, 1000 mL distilled water), fructose, maltose, sucrose, galactose, and soluble starch were used to replace glucose as a single carbon source, and the medium was cultured at 37℃ and 160 r / min for 24 h with shaking. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to screen for the optimal carbon source. Based on the selected optimal carbon source, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, tryptone, and beef extract, each containing an equimolar amount of nitrogen, were used as single nitrogen sources and cultured at 37℃ and 160 r / min for 24 h with shaking. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to screen for the optimal nitrogen source.

[0056] Based on the optimal carbon and nitrogen sources, the initial pH of the culture medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, and cultured at 37℃ with shaking at 160 r / min for 24 h. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to screen for the optimal pH. Based on the optimal carbon and nitrogen sources and pH, the inoculated culture medium was placed at 25℃, 28℃, 30℃, 34℃, 37℃, and 40℃ with shaking for 24 h. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to screen for the optimal temperature. Under the above optimal conditions, the inoculated culture medium was cultured with shaking at 160 r / min for 3, 6, 12, 24, 48, and 72 h, respectively. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to determine the optimal fermentation time. Under the above optimal conditions, the seed culture of the strain was inoculated at volume ratios of 1%, 3%, 5%, 7%, 9%, and 10%, and fermented at the optimal fermentation temperature and time. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to identify the optimal inoculum size. Under the above optimal conditions, 30, 50, 80, 100, 150, and 200 mL of optimized culture medium were added to 250 mL Erlenmeyer flasks, respectively. The strain was inoculated at the optimal inoculum size, and fermented at the optimal fermentation temperature and time. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to identify the optimal liquid volume. Under the above optimal conditions, the culture was placed in shakers at 110, 130, 150, 180, and 200 r / min, respectively. The sterile fermentation filtrate was collected, and its inhibitory effect on pathogen spore germination was determined to identify the optimal rotation speed. During the optimization process, all experiments were performed in triplicate to ensure data reproducibility, and statistical analysis was used to verify significance. P <0.05). By optimizing fermentation parameters, this invention solves the technical problem of unstable activity of high-efficiency strains in large-scale production, and provides highly active fermentation raw materials for the preparation of wettable powders.

[0057] Results: Systematic optimization of B13 fermentation conditions was carried out, with the diameter of the inhibition zone as the evaluation index, and the carbon source was investigated ( Figure 4 Group A: a. Maltose, b. Soluble starch, c. Glucose, d. Sucrose, e. Fructose, f. Galactose, ck. Distilled water), Nitrogen source ( Figure 4 Group c: a. tryptone, b. ammonium sulfate, c. beef extract, d. ammonium chloride, e. ammonium nitrate, f. urea, ck. distilled water), initial pH ( Figure 4 Group e: 4-9 represent pH 4-9, ck distilled water, and temperature, respectively. Figure 4Group g: a 25℃, b 28℃, c 30℃, d 34℃, e 37℃, f 40℃, ck distilled water), effects of fermentation time, inoculum size, liquid volume, and rotation speed. Results showed: (1) Carbon source: Soluble starch has the best effect and the largest inhibition zone diameter. Figure 4 a, 4b); (2) Nitrogen source: Beef extract is the best. Figure 4 c, 4d); (3) pH value: pH 7.0 is the most suitable. Figure 4 e, 4f); (4) Temperature: The antibacterial activity was highest at 37℃. Figure 4 g, 4h); (5) Other factors: When the fermentation time was 24 h, the inoculum size was 3%, the liquid volume was 50 mL / 250 mL, and the rotation speed was 180 r / min, the diameter of the inhibition zone reached 14 mm. Figure 4 il).

[0058] 4. This invention further prepares the highly efficient antagonistic strain obtained through fermentation into a wettable powder for easy field application. First, the highly efficient strain is inoculated into LB liquid medium and cultured at 37°C and 180 r / min for 12 h with shaking to obtain a seed culture. The culture is then shaken and cultured under optimized conditions. When the viable cell count in the fermentation broth reaches 2 × 10⁻⁶... 8 The culture was terminated when the concentration of CFU / mL was reached, yielding the fermentation broth. Suitable carriers were then screened, including calcium carbonate, bentonite, diatomaceous earth, kaolin, and talc. 5 g of each carrier was weighed and sterilized at 121℃ for 20 min. In a clean bench, the fermentation broth was slowly added while stirring until the mixture was fluid and free of condensation. The volume of fermentation broth used (v) was recorded. The maximum adsorption capacity of the carrier (H, L / kg) was calculated using the following formula: H = v / m × 1000 In the formula, H is the maximum adsorption capacity (L / kg), v is the amount of fermentation broth used (L), and m is the carrier mass (kg).

[0059] The fermentation broth and sterile carrier were mixed evenly at a ratio of 7:3 (v / w) (based on H) and dried in a 50°C forced-air drying oven. The dried mixture was ground into a fine powder and sieved to obtain the microbial agent mother powder. The suspension rate, wetting time, and adsorption capacity of the mother powder prepared with different carriers were measured and compared. Based on the maximum adsorption capacity and cost, the optimal carrier was screened. Subsequently, the optimal adjuvants and their dosages were screened.

[0060] 1) Screening of dispersants and wetting agents: The masterbatch was mixed with different wetting agents and dispersants. The optimal adjuvants were screened based on short wetting time, high suspension rate, and minimal spore content loss. The optimal wetting agent and dispersant were mixed in a series of ratios from 1:9 to 9:1, and then compounded with the masterbatch at a 10% addition level to determine the best ratio for short wetting time and high suspension rate. This optimal ratio adjuvant was then mixed with the masterbatch in gradients from 2% to 16% to determine the optimal addition amount.

[0061] 2) Screening of heat stabilizers: 2% of the tested stabilizers (sodium citrate, CMC-Na, K3PO4, KH2PO4, EDTA) were added to the mother powder to prepare the formulation. After heat storage at 54±2℃ for 14 days, the viable cell count was determined using the plate count method. The stabilizer with the highest spore survival rate after heat storage was selected as the optimal heat stabilizer.

[0062] 3) Screening of UV protectants: 2% of the tested UV protectants (dextrin, ascorbic acid, lignin) were added to the mother powder. Control groups were set up: a UV-treated group without protectant and a no-treatment group without protectant. Each treatment was irradiated at a distance of 40cm from the UV lamp for 30 minutes, and the spore survival rate was counted to screen the optimal UV protectant. Finally, the wettable powder was prepared and its quality was tested. The fermentation product was pulverized through a 300-mesh sieve and mixed evenly with the selected optimal carrier, dispersant, wetting agent, heat stabilizer, and UV protectant in a mixer according to the optimized ratio to obtain the wettable powder. Testing was performed according to GB 20287-2006 standard. 10 g of sample was weighed, 100 mL of sterile water was added, and the mixture was shaken for 10 minutes to prepare 10 g of wettable powder. -1 Diluent. After serial dilution, 100 μL was spread onto LB plates and incubated at 28°C for 18–24 h, then counted.

[0063] Results: Based on the fermentation broth activity and strain characteristics, B13 was prepared as a wettable powder. The optimal ratio of carrier, dispersant, wetting agent, heat stabilizer, and UV protectant was determined through screening. (1) Calcium carbonate was chosen as the carrier, as it had the highest adsorption rate. Figure 5 a, P <0.05); (2) The combination of dispersant and wetting agent is Tween 80 and sodium dodecyl sulfate in a ratio of 7:3, with a total addition of 10%, which results in the best suspension rate and wetting time. Figure 5 bc, Table 3-4); (3) The heat stabilizer KH2PO4 and the ultraviolet protectant ascorbic acid can effectively maintain spore activity. Figure 5 d. Table 5).

[0064] The prepared wettable powder is uniform and fine, with a spore content of 7.85 × 10⁻⁶. 11CFU / g, survival rate after three months of storage was 81.78%. It exhibits good suspension properties and moderate wetting time, meeting the requirements for field application. Figure 5 e).

[0065] Table 3 Screening results of optimal wetting agents and dispersants

[0066] Note: Data are mean ± standard deviation. Differences between groups were assessed using Student's t-test. Different lowercase letters in the same column indicate significant differences. P <0.05). Spore count was determined by plate counting.

[0067] Table 4. Screening Results of Optimal Ratio of Wetting Agent and Dispersant

[0068] Note: Data are mean ± standard deviation. Differences between groups were assessed using Student's t-test. Different lowercase letters in the same column indicate significant differences. P <0.05).

[0069] Table 5. Effects of different UV protectants on the UV tolerance of strain B13 spores.

[0070] Note: Data are mean ± standard deviation. Differences between groups were assessed using Student's t-test. Different lowercase letters in the same column indicate significant differences. P <0.05).

[0071] 5. The efficacy of the wettable powder was verified under poplar seedling and plantation conditions. Three-month-old *Populus alba* seedlings were used as experimental material. An epidermal incision was made at the base of the seedling stem, 2-3 cm above the soil surface. A 6 mm diameter *Botrytis cinerea* mycelial block was placed at the incision site, with a sterile PDA agar block used as a control. The seedlings were incubated under moist conditions after inoculation. The wettable powder was applied by spraying. The *Botrytis cinerea* mycelial block was inoculated onto the seedling stem using a scab inoculation method. 24 hours later, a 100-fold diluted wettable powder was evenly sprayed onto the inoculated site (until the solution began to drip). A blank control group treated with water was used as a control. Each treatment was applied three times, with an interval of 7 days between each application. The spread of lesions was observed regularly, and the disease index and control effect were recorded. Three to five seedlings were randomly selected from each group for replication to ensure the representativeness and reliability of the experimental data. The dosage per seedling was controlled at 0.5-1 mL. Three to five seedlings were randomly selected from each group for replication to ensure the representativeness and reliability of the experimental data. The infection status around the wound after inoculation with the pathogen was observed, and the incidence rates in the treatment and control groups were statistically analyzed.

[0072] The severity of lesions is classified into five levels: Level 0 (healthy / no lesions), Level 1 (lesion width < 1 / 3 of stem circumference), Level 2 (lesion width 1 / 3-1 / 2 of stem circumference), Level 3 (lesion width 1 / 2-3 / 4 of stem circumference), and Level 4 (lesion width > 3 / 4 of stem circumference or plant death). The disease index and control efficacy are calculated using the following formula: Disease index = ∑(Number of diseased plants at each level × Corresponding disease level) / (Total number of plants surveyed × Highest disease level) × 100% Disease control efficacy = (Control disease index - Treatment disease index) / Control disease index × 100% All data were performed in triplicate, and results are expressed as mean ± standard deviation. Significance was indicated by lowercase letters: lowercase letters indicate statistically significant differences ( ). P <0.05, phylogenetic analysis was performed using MEGA11 software, and a phylogenetic tree was constructed based on the Neighbor-Joining method.

[0073] Results: A pot experiment was conducted on Populus tomentosa seedlings to show efficacy (…). Figure 5 f, g). For example... Figure 5 As shown in treatment group f, after spraying with 100-fold diluted B13 wettable powder, the expansion of lesions on the stems of seedlings in the treatment group was significantly inhibited, and the leaves maintained good greenness and health, with a disease index of 8.33%; while the control group ( Figure 5 g) The leaves showed obvious yellowing and leaf drop, and the overall plant growth was weak. The disease index was as high as 66.67%, which was about 58.34% lower than that of the blank control, and the control effect reached 87.50%. The difference between the treatment group and the control group was significant (P<0.05). Repeated experiments under different temperature and humidity conditions showed stability, indicating that the preparation has good environmental adaptability.

[0074] In summary, this invention establishes a complete technical system from pathogen identification, endophytic antagonistic bacteria screening, fermentation condition optimization to wettable powder preparation and application verification, achieving stable activity of highly effective antagonistic strains and practicality of the formulation. The screened Bacillus subtilis B13 exhibits high inhibition rate and reproducibility, and can stably exert control effects in both laboratory and field environments. Optimized fermentation conditions significantly increased the yield of antibacterial substances, providing highly active raw materials for formulation preparation. The formulation preparation method solves practical problems such as suspension, wettability, thermal stability, and UV stability, enabling the wettable powder to demonstrate high efficacy, ease of operation, and environmental friendliness in field applications. This invention not only provides a green and scalable control solution for poplar canker but also offers a referable technical model and application reference for the biological control of other forest tree diseases and the development of microbial agents, possessing significant application value and industrialization potential.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis B13 strain for controlling poplar canker, characterized in that, The Bacillus subtilis B13 was deposited at the China General Microbiological Culture Collection Center on July 6, 2026, with accession number CGMCC No. 39570.

2. The use of Bacillus subtilis B13 as described in claim 1 in the preparation of a biocontrol agent for controlling poplar canker caused by Staphylococcus aureus.

3. A fermentation method for Bacillus subtilis B13 according to claim 1, characterized in that, Includes the following steps: The Bacillus subtilis B13 was inoculated into a fermentation medium and cultured with shaking at an initial pH of 6.0-8.0, a culture temperature of 30℃-40℃, and a shaking speed of 150-200 r / min for 12-48 h.

4. The fermentation method according to claim 3, characterized in that, The fermentation medium uses soluble starch as the carbon source and beef extract as the nitrogen source, and the inoculum amount of Bacillus subtilis B13 is 1%~5%.

5. A wettable powder containing Bacillus subtilis B13 as described in claim 1, characterized in that, It contains Bacillus subtilis B13 fermentation product and adjuvants, said adjuvants including carriers, dispersants, wetting agents, heat stabilizers and ultraviolet protectants.

6. The wettable powder according to claim 5, characterized in that, The carrier is calcium carbonate, the dispersant and wetting agent is a combination of Tween 80 and sodium dodecyl sulfate, the heat stabilizer is potassium dihydrogen phosphate, and the ultraviolet protectant is ascorbic acid.

7. The wettable powder according to claim 6, characterized in that, The mass ratio of Tween 80 to sodium dodecyl sulfate is (4~8):(6~2), and the total amount of the Tween 80 and sodium dodecyl sulfate composition added is 2%~16% of the fermentation material mass.

8. The method for preparing the wettable powder according to any one of claims 5-7, characterized in that, Includes the following steps: (1) Fermenting and culturing the Bacillus subtilis B13 according to claim 1 yields a viable count of 2 × 10⁻⁶. 8 Fermentation broth with a CFU / mL or higher; (2) The fermentation liquid obtained in step (1) is mixed evenly with the carrier, and then dried, ground and sieved to obtain the microbial agent mother powder; (3) Add dispersant, wetting agent, heat stabilizer and ultraviolet protectant to the bacterial agent master powder and mix evenly to obtain the product.

9. The method for preparing the wettable powder according to claim 8, characterized in that, The fermentation broth and carrier are mixed evenly at a ratio of (6~8):(4~2) (v / w).

10. The use of the wettable powder according to any one of claims 5-7 in the prevention and control of poplar canker.

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