Bacillus weizmannii for inhibiting plant pathogenic fungi and application thereof

Abstract

This invention relates to a strain of *Bacillus wiedmannii* that inhibits plant pathogens and its applications, within the field of biological control. It aims to address the negative impacts of chemical control methods on the ecological environment and human health. The *Bacillus wiedmannii* strain is identified as *Bacillus wiedmannii* SJZ, deposited at the China Center for Type Culture Collection (CCTCC) on December 23, 2024, with accession number CCTCC NO: M 20242884. This *Bacillus wiedmannii* strain inhibits *Sclerotinia sclerotiorum* and *Heterophyllaria rubra*. It secretes proteases, cellulases, and chitinases. This *Bacillus wiedmannii* strain is also used for nitrogen fixation, secreting siderophores and IAA (inorganic acid oxidase).

Description

Technical Field

[0001] This invention relates to the field of biological control, and more particularly to a strain of Bacillus weizmannii that inhibits plant pathogens and its application. Background Technology

[0002] Plant pathogenic microorganisms are key biological stressors affecting the stability and sustainability of global agricultural production systems, causing significant annual declines in global crop yields. Among the diverse types of plant pathogens, fungal diseases pose a major challenge to agricultural production due to their rapid spread, wide range of impacts, and difficulty in control. While traditional chemical control strategies are effective in the short term, long-term use leads to pathogen resistance, soil microbial imbalance, and pesticide residue pollution, negatively impacting the ecological environment and human health. Therefore, developing efficient and environmentally friendly disease control strategies has become an urgent need in the field of plant protection.

[0003] Biological control, as an environmentally friendly method of disease management, has received widespread attention in recent years. Its core principle is to utilize beneficial microorganisms or their metabolites to inhibit the growth and reproduction of pathogenic microorganisms. Its mechanisms of action mainly include nutrient and space competition, antimicrobial secretion, and induction of systemic resistance. Compared with chemical pesticides, biological control agents have multiple advantages: First, they have higher target specificity and better safety for non-target organisms and the environment; second, biocontrol microorganisms can work synergistically through multiple mechanisms to reduce the risk of pathogens developing resistance. Summary of the Invention

[0004] The present invention aims to address the negative impacts of chemical control methods on the ecological environment and human health caused by plant pathogens, and provides a strain of Bacillus weizmannii that inhibits plant pathogens and its applications.

[0005] The Bacillus wiedmannii strain used in this invention to inhibit plant pathogens is Bacillus wiedmannii SJZ, which has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, on December 23, 2024, with accession number CCTCC NO: M 20242884.

[0006] The colonies of *Bacillus weizmannii* SJZ of this invention are round or nearly round, opaque, with neat edges, a raised and dry surface, a relatively hard texture and a certain degree of stickiness, and a milky white or pale yellow color. The bacterial cells are rod-shaped, about 2-5 micrometers in length, and are usually arranged singly, in pairs or in short chains.

[0007] The *Bacillus weizmannii* SJZ of this invention is a Gram-positive bacterium that is positive for catalase reaction, positive for methyl red test, negative for VP test, negative for indole test, positive for gelatin liquefaction test, and positive for nitrate reduction test. It has the ability to secrete protease, cellulase, and chitinase.

[0008] The 16S rDNA sequencing results of *Bacillus wiedmannii* SJZ were submitted to the NCBI database, analyzed and compared using BLAST, and then a phylogenetic tree of *Bacillus wiedmannii* SJZ was constructed using MEGA 7 software. This strain is closely related to *Bacillus wiedmannii*, with a sequence similarity of 100%. Based on the combined morphological observation results and physiological and biochemical results, *Bacillus wiedmannii* SJZ was identified as *Bacillus wiedmannii*.

[0009] The Bacillus Weizmannii SJZ of this invention is used to inhibit Sclerotinia sclerotiorum.

[0010] The Bacillus Weizmannii SJZ of this invention is used to inhibit black rot bacteria.

[0011] The Bacillus Weizmannii SJZ of this invention can secrete proteases, cellulases, and chitinases.

[0012] The Bacillus Weizmannii SJZ of this invention is used for nitrogen fixation.

[0013] The Bacillus Weizmannii SJZ of this invention is used to secrete siderophores and IAA.

[0014] The beneficial effects of this invention are:

[0015] 1. The *Bacillus weizmannii* SJZ of this invention has a strong inhibitory effect on the growth of *Sclerotinia sclerotiorum*, preventing it from producing almost no white, cottony mycelium, with an inhibition rate of 89.41%. It can secrete metabolites with strong antibacterial activity, effectively inhibiting the growth of *Sclerotinia sclerotiorum*, significantly reducing the diameter of *Sclerotinia sclerotiorum* hyphae, thinning the cell wall and causing local permeability, significantly increasing the frequency of hyphal branching, and resulting in irregular branching angles, often accompanied by local swelling at the branching points. The growth of the hyphal apex is significantly inhibited, with the apical extension area becoming blunt or exhibiting irregular swelling.

[0016] 2. Bacillus Weizmannii SJZ can inhibit the secretion of oxalic acid by Sclerotinia sclerotiorum, with an inhibition rate of 18.38%.

[0017] 3. When Bacillus Weizmannii SJZ was cultured in pairs with Sclerotinia sclerotiorum, it was found that it could secrete volatile substances with strong antibacterial properties, and the antibacterial rate reached 90.00%.

[0018] 4. The clear zone experiment showed that Bacillus Weizmannii SJZ has a strong ability to secrete protease and cellulase, and a certain ability to secrete chitinase, but no ability to secrete β-glucanase.

[0019] 5. Treatment of sunflower seeds with Bacillus Weizmannii SJZ bacterial suspension and a 20-fold dilution of the bacterial suspension showed that it could inhibit the infection of sunflower seeds by Sclerotinia sclerotiorum. Pot experiments showed that 24 hours after applying the Sclerotinia sclerotiorum mycelial suspension, the plants showed mild wilting symptoms and the leaves drooped due to water loss; after 48 hours, the wilting intensified and some plants collapsed; after 54 hours, the root and stem showed rot symptoms, accompanied by tissue browning and softening. The use of Bacillus Weizmannii SJZ bacterial suspension for control reduced the incidence of sunflower strain infection to 43.33%.

[0020] 6. The broad-spectrum antibacterial activity and growth-promoting potential of *Bacillus weizmannii* SJZ were investigated. It was found that its inhibitory effect on *Fusarium graminearum*, *Alternaria solanaceae*, and *Alternaria spp.* was relatively limited, while its inhibitory ability on *Clostridium pachycarba* was strong, and it could cause discoloration of the *Clostridium pachycarba* hyphae. *Bacillus weizmannii* SJZ has nitrogen-fixing ability and can secrete small amounts of siderophores and IAA, thus possessing certain growth-promoting potential. Attached Figure Description

[0021] Figure 1 A contrastive image of Bacillus Weizmannii SJZ rescreening plates;

[0022] Figure 2 This is a colony morphology diagram of Bacillus Weizmannii SJZ.

[0023] Figure 3 This is a cell morphology diagram of Bacillus Weizmannii SJZ.

[0024] Figure 4 The hydrolytic enzyme secretion capacity of Bacillus weizmannii SJZ;

[0025] Figure 5 Phylogenetic tree of Bacillus Weizmannii SJZ;

[0026] Figure 6 The effect of Bacillus Weizmannian SJZ metabolites on the growth of Sclerotinia sclerotiorum;

[0027] Figure 7 The effect of Bacillus Weizmannii SJZ metabolites on oxalic acid secretion in Sclerotinia sclerotiorum;

[0028] Figure 8 The antibacterial effect of volatile substances in Bacillus Weizmannii SJZ;

[0029] Figure 9 The effects of different treatments of Bacillus Weizmann SJZ fermentation broth on sunflower seed pericarp infection;

[0030] Figure 10 The infection and control efficacy of Bacillus Weizmannii SJZ in sunflower pot experiments;

[0031] Figure 11 Analysis of the broad-spectrum antibacterial activity of Bacillus weizmannii SJZ;

[0032] Figure 12 Analysis of the growth-promoting potential of Bacillus Weizmannii SJZ. Detailed Implementation

[0033] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0034] Example 1:

[0035] This embodiment is Bacillus wiedmannii SJZ, which has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, on December 23, 2024, with accession number CCTCCNO: M 20242884.

[0036] The method for obtaining Bacillus wiedmannii SJZ in this embodiment is as follows:

[0037] Initial screening: Soil samples were collected from Beiji Village, Mohe City, Daxinganling Region, Heilongjiang Province. Using a five-point sampling method, six soil samples were collected from different sampling points at depths of 1-3m in Mohe. First, impurities and large particles were removed. The sterilized soil samples were sieved to remove fine soil particles. Then, 5g of each sample was added to a 45mL Erlenmeyer flask containing sterilized glass beads and incubated on a shaker at 150rpm for approximately 20 hours to thoroughly mix and disperse bacterial cells. The flask was then heated in an 80℃ water bath for 30 minutes to kill other bacteria and vegetative cells. Finally, using a tenfold dilution method, 1mL of the supernatant was transferred to a test tube filled with 9mL of distilled water and diluted to 100%. -2 10 -3 10 -4 10 -5 10 -6Five dilutions were performed. 100 μL of each soil dilution was then spread onto LB agar plates. Each dilution was repeated three times. The plates were then incubated at 28°C inverted to isolate several strains. The strains were activated by streaking on LB agar plates to obtain single colonies. Using the plate confrontation method, 8.0 mm diameter *Sclerotinia sclerotiorum* mycelial discs were inoculated into the center of PDA agar plates, with *Bacillus* inoculated 1 cm away from the plate. The plates were incubated inverted at 28°C for 3 days, and the diameter of the *Sclerotinia sclerotiorum* was observed. Strains forming a clear inhibition zone were selected for further screening.

[0038] Secondary screening: Using the plate confrontation method, 8.0 mm diameter *Sclerotinia sclerotiorum* mycelial discs were inoculated in the center of a PDA medium plate, with two *Bacillus* points inoculated 1 cm away from the plate. The plates were incubated upside down at 28°C, and the width of the inhibition zone was measured after 5 days. Antagonistic bacteria with an inhibition zone width of 10 mm or more were screened, such as... Figure 1 As shown, Figure 1 The left side of the image shows the control group, and the right side shows the results of the rescreening of Bacillus Weizmann SJZ. Bacillus Weizmann SJZ was finally selected.

[0039] Example 2: Identification of Bacillus Weizmannii SJZ

[0040] 1. Morphological observation of Bacillus Weizmann SJZ

[0041] The colonies of *Bacillus weizmannii* SJZ are round or nearly round, opaque, with neat edges, a raised and dry surface, a relatively hard and somewhat sticky texture, and a milky white or pale yellow color. The bacteria are rod-shaped, approximately 2-5 micrometers in length, and usually arranged singly, in pairs, or in short chains. They are Gram-positive bacteria. A colony photograph of *Bacillus weizmannii* SJZ is shown below. Figure 2 As shown in the image, the bacterial cell photograph is as follows: Figure 3 As shown.

[0042] 2. Physiological and biochemical characteristics of Bacillus Weizmannii SJZ

[0043] The physiological and biochemical results of Bacillus Weizmannii SJZ are shown in Table 1.

[0044] Table 1. Results of physiological and biochemical indicators of Bacillus Weizmann.

[0045]

[0046]

[0047]

[0048] Note: "+" indicates positive, and "-" indicates negative.

[0049] The results of the hydrolytic enzyme secretion capacity test of Bacillus Weizmannii SJZ are as follows: Figure 4 As shown.

[0050] Determination of protease secretion capacity of Bacillus Weizmannii SJZ: Bacillus Weizmannii SJZ was inoculated onto a protease selection plate and incubated upside down at 28°C for 3 days. Figure 4 As shown in Figure a, the opaque, milky solid culture medium is hydrolyzed into a transparent state under the action of proteases, indicating that it has the ability to secrete proteases.

[0051] Determination of cellulase secretion capacity of Bacillus Weizmannii SJZ: Bacillus Weizmannii SJZ was inoculated onto cellulase selection medium using sterile toothpicks and incubated upside down at 28°C for 3 days. The medium was then stained with Congo red for 10 min and destained with 1 mol / L NaCl for 10 min. Figure 4 As shown in b, Congo red can form a stable red complex with the macromolecular polysaccharide cellulose. When cellulase secreted by the bacteria degrades cellulose in the plate into smaller sugar molecules, the structure of cellulose is destroyed, preventing it from binding with Congo red and thus forming a transparent hydrolysis zone on the plate. This phenomenon indicates that this strain has a strong cellulase secretion capacity and can effectively degrade cellulose substrates.

[0052] Determination of chitinase secretion capacity of Bacillus Weizmannii SJZ: Bacillus Weizmannii was inoculated onto chitinase selection medium using sterile toothpicks and incubated upside down at 28°C for 5 days. Figure 4 As shown in Figure c, a distinct transparent hydrolysis zone was observed around *Bacillus weizmannii* SJZ on the chitinase selection plate, indicating that this strain can secrete chitinase, thereby degrading chitin substrates in the culture medium. This result confirms that *Bacillus weizmannii* SJZ possesses certain chitinase secretion activity.

[0053] Determination of β-glucanase secretion capacity of Bacillus Weizmannii SJZ: Bacillus Weizmannii SJZ was inoculated onto β-glucanase selection medium using sterile toothpicks and incubated upside down at 28°C for 3 days. The medium was then stained with Congo red for 30 min and destained with 1 mol / L NaCl for 10 min. Figure 4 As shown in Figure d, no transparent hydrolysis zone was observed around the colony, indicating that this strain failed to degrade the β-glucan substrate in the culture medium. This result confirms that Bacillus Weizmannii SJZ does not possess the ability to secrete β-glucanase.

[0054] 3. Molecular identification of Bacillus Weizmannii SJZ

[0055] Genomic DNA was extracted from *Bacillus weizmannii* SJZ, and the 16S rDNA sequence was amplified by PCR. The PCR amplification product was subjected to agarose gel electrophoresis, and sequencing yielded a 1425 bp 16S rDNA sequence, as shown in SEQ ID NO: 1 in the sequence listing. The sequencing results were uploaded to the NCBI database for BLAS alignment, and a phylogenetic tree was constructed as follows: Figure 5 As shown. Based on 16S rDNA sequence alignment and phylogenetic analysis, this strain is closely related to *Bacillus wiedmannii*. Combining morphological observations and physiological and biochemical results, this strain was identified as *Bacillus wiedmannii*.

[0056] Example 3: Study on the antibacterial and preventive capabilities of Bacillus weizmannii SJZ

[0057] 1. Effects of Bacillus Weizmannii SJZ metabolites on the growth of Sclerotinia sclerotiorum.

[0058] Bacillus Weizmannii SJZ seed culture in the logarithmic growth phase was inoculated into BPY medium at a ratio of 1%, and cultured with shaking. The fermentation broth was aspirated, centrifuged, and the supernatant was filtered through a 0.22 nm aqueous microporous membrane to obtain sterile fermentation broth. The sterile fermentation broth of Bacillus Weizmannii SJZ was mixed into PDA medium at a ratio of 5%, and the plates were inoculated with 8 mm diameter Sclerotinia sclerotiorum mycelium. The plates were incubated upside down at 28°C for 5 days, and the results were observed. The control group consisted of 8 mm diameter Sclerotinia sclerotiorum mycelium inoculated onto PDA plates without the addition of sterile fermentation broth.

[0059] Experimental results showed that the growth of *Sclerotinia sclerotiorum* mycelium was significantly inhibited compared to the control group, with almost no white cottony mycelium produced. The inhibition rate against *Sclerotinia sclerotiorum* reached 89.41%. Figure 6 As shown in the figure. This result confirms that Bacillus Weizmannii SJZ can secrete metabolites with strong antibacterial activity, effectively inhibiting the growth of Sclerotinia sclerotiorum, indicating that it has a good ability to secrete antibacterial substances.

[0060] 2. Effects of Bacillus Weizmannii SJZ metabolites on the morphology of Sclerotinia sclerotiorum.

[0061] The agar blocks containing hyphae of *Bacillus wesmannii* treated with the aseptic fermentation broth of *Bacillus wesmannii* SJZ were cut into thin sections using a disposable scalpel, and the hyphal morphology was observed using an optical microscope. The results showed that the *Bacillus wesmannii* SJZ treatment group exhibited a significant phenotype of inhibited hyphal development. In the control group, the hyphae were uniform in diameter, with intact cell wall structure, high cytoplasmic density, and vigorous metabolic activity. There was no obvious swelling or abnormal structure at the branching points. The hyphae were milky white to light gray overall, with moderate translucency and no obvious transparency. Normal hyphal fusion was observed, forming a complex hyphal network structure. The *Bacillus wesmannii* SJZ treatment group showed a typical fungal growth inhibition phenotype: significantly reduced hyphal diameter, thinner cell walls with localized translucency, significantly increased hyphal branching frequency, irregular branching angles, and frequent localized swelling at the branching points. Tip growth was significantly inhibited, with the tip extension area becoming blunt or irregularly swollen.

[0062] 3. Effects of Bacillus Weizmannii SJZ metabolites on oxalic acid secretion in Sclerotinia sclerotiorum.

[0063] Bacillus Weizmannii SJZ seed culture in logarithmic growth phase was inoculated at a ratio of 1% into 100 mL of BPY medium and cultured at a constant temperature with shaking to obtain active fermentation broth. The active fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was carefully aspirated and filtered through a disposable aqueous microporous membrane with a pore size of 0.22 nm to obtain sterile fermentation broth. Sclerotinia sclerotiorum mycelial suspension was inoculated into 100 mL of PD medium and cultured at 28℃ with shaking at 120 rpm for 3 days to obtain activated Sclerotinia sclerotiorum mycelial suspension. The sterile fermentation broth was added at a ratio of 5% to 20 mL of PD medium, followed by 1 mL of activated Sclerotinia sclerotiorum mycelial suspension. The culture was then cultured at a constant temperature with shaking for 5 days. Mycelial growth was observed, and the oxalic acid secretion of Sclerotinia sclerotiorum was detected using a colorimetric method. 1 mL of Sclerotinia sclerotiorum mycelial suspension was added to 20 mL of PD medium as a blank control.

[0064] Experimental results showed that *Bacillus weizmannii* SJZ significantly inhibited the growth of *Sclerotinia sclerotiorum* and its oxalic acid synthesis, with an inhibition rate of 18.38%. The effect of *Bacillus weizmannii* SJZ on oxalic acid secretion by *Sclerotinia sclerotiorum* was determined by colorimetric method as follows: Figure 7 As shown, oxalic acid, a key pathogenic factor of *Sclerotinia sclerotiorum*, can disrupt plant cell wall structure, inhibit host defense responses, and provide a suitable acidic environment for cell wall-degrading enzymes secreted by the pathogen. *Bacillus weizmannii* SJZ can, to some extent, inhibit the pathogenicity of *Sclerotinia sclerotiorum* by suppressing oxalic acid synthesis.

[0065] 4. Analysis of the inhibitory effect of volatile substances from Bacillus Weizmannii SJZ on Sclerotinia sclerotiorum.

[0066] Bacillus Weizmannii SJZ was inoculated into LB liquid medium and cultured with shaking to obtain activated seed culture. The seed culture was then spread onto LB agar plates. The LB agar plates containing the seed culture were then inverted onto PDA agar plates inoculated with Sclerotinia sclerotiorum mycelium and cultured for 5 days. It was observed that Bacillus Weizmannii SJZ produced volatile antibacterial substances that inhibited the growth of Sclerotinia sclerotiorum. The antibacterial activity of the volatile substances from Bacillus Weizmannii SJZ is as follows: Figure 8 As shown, the antibacterial rate reached 90.00%, indicating that this strain has a strong ability to synthesize and secrete volatile antibacterial substances.

[0067] 5. Shell Infection Experiment

[0068] Preparation of Bacillus Weizmann SJZ bacterial culture: The bacterial seed culture was inoculated into BPY medium at an inoculum of 1% and cultured at 28℃ and 180r / min for 3 days.

[0069] Disinfection and washing of sunflower seeds: Soak sunflower seeds in sterile water for 2 minutes; then soak the seeds in 75% alcohol for 1 minute, repeat twice; soak the seeds in 2% sodium hypochlorite solution for 2 minutes; then wash the sunflower seeds twice with 2% Na2S2O3 solution to remove residual chlorine; then wash the sunflower seeds three times with sterile water; finally, use sterile filter paper to dry the surface of the seeds.

[0070] Sunflower seed retreatment: Take the seeds that have undergone the above treatment and divide them into four portions of 15 seeds each. Add the original bacterial solution that covers the seeds to one portion, add an equal volume of bacterial solution diluted 20 times to one portion, add an equal volume of distilled water to one portion, and add an equal volume of BPY culture medium to one portion. Soak each portion of seeds for 30 minutes, and set up 3 replicates for each group.

[0071] Shell infection experiment: The four groups of sunflower seeds treated above were evenly placed on plates covered with Sclerotinia sclerotiorum mycelium after being cultured at 28℃ for 5 days. Each plate contained 15 treated sunflower seeds, and each group was replicated 3 times.

[0072] Two days later, the disease status of sunflower seeds was observed, and the seeds were turned over with tweezers. Observation was repeated two days later. The disease status was categorized into three levels: Level 0: No mycelium visible on the seed surface; Level 1: A small amount of mycelium visible on the seed surface; Level 2: The seed surface is clearly covered with mycelium. A disease index was used to describe the disease status of the sunflower seeds, and the results for the experimental and control groups were recorded separately. The formulas for the incidence rate and disease index are shown below:

[0073]

[0074] Where M represents the incidence rate (%), I represents the number of infected seeds (seeds), and T represents the total number of seeds (seeds).

[0075]

[0076] Where D represents the disease index, N represents the number of diseased seeds at each level, R represents the representative value at each level, T represents the total number of seeds surveyed, and H represents the highest representative value.

[0077] See the following for the disease index, incidence rate, and seed coat infection status of sunflower seeds in each treatment group: Figure 9 . Figure 9 CK1 represents treatment with sterile water, CK2 represents treatment with BPY culture medium, SJZ represents treatment with the original bacterial solution, and 1 / 20 represents treatment with a 20-fold dilution of the bacterial solution. Observations and calculations after 4 days revealed that the incidence of seed coat infection was reduced in both the original bacterial solution treatment and the 20-fold dilution treatment. Detailed analysis is shown in Table 2.

[0078] Table 2. Effects of different treatments of Bacillus Weizmann bacterial suspension on sunflower seed coat infection.

[0079]

[0080] 6. Sunflower pot experiment

[0081] This experiment used the potted root irrigation method, using commercially available nutrient soil mixed with vermiculite in a ratio of 4:1. The mixture was placed in disposable seedling pots (12cm×10.5cm), and 1-2 sunflower seeds were sown in each pot. 100mL of water was added, and the seeds were covered with 1cm of soil. The seeds were germinated indoors and then moved outdoors. 100mL of water was added every 2 days. Once the sunflowers had grown four true leaves, one sunflower plant was kept in each pot to conduct the potted infection and potted control experiment.

[0082] Pot infection experiment: *Sclerotinia sclerotiorum* mycelial suspension was inoculated into PD medium and cultured at 28℃ and 120 rpm for 5 days to obtain the *Sclerotinia sclerotiorum* mycelial suspension. Experimental group: 30 mL of the prepared *Sclerotinia sclerotiorum* mycelial suspension was used for root irrigation, and the soil was covered with 1 cm of soil. A total of 20 pots were treated. Control group 1: An equal volume of sterile water was used for root irrigation, and the soil was covered with 1 cm of soil. A total of 20 pots were treated. Control group 2: An equal volume of PD medium was used for root irrigation, and the soil was covered with 1 cm of soil. A total of 20 pots were treated. The susceptibility and symptoms of the sunflower plants were observed and recorded every 12 hours.

[0083] Potted plant control experiment: *Bacillus weizmannii* SJZ seed culture was inoculated at a ratio of 1% into BPY medium and cultured at 28℃ with shaking at 180 rpm for 3 days to obtain *Bacillus weizmannii* bacterial suspension. Treatment group: 50 mL of *Bacillus weizmannii* SJZ bacterial suspension was used for root irrigation. After 3 days, 30 mL of *Sclerotinia sclerotiorum* mycelial suspension was used for root irrigation, and the soil was covered with 1 cm of soil. A total of 20 pots were treated. Control group 1: 50 mL of sterile water was used for root irrigation. After 3 days, 30 mL of *Sclerotinia sclerotiorum* mycelial suspension was used for root irrigation, and the soil was covered with 1 cm of soil. A total of 20 pots were treated. Control group 2: 50 mL of BPY medium was used for root irrigation. After 3 days, 30 mL of *Sclerotinia sclerotiorum* mycelial suspension was used for root irrigation, and the soil was covered with 1 cm of soil. A total of 20 pots were treated. Each experiment was repeated three times. The susceptibility and symptoms of sunflower plants were observed and recorded every 12 hours. The disease susceptibility of sunflowers was described using a disease index, with level 0 indicating no disease, level 1 indicating slight disease, and level 2 indicating disease. The results for the experimental group and the control group were recorded separately.

[0084]

[0085] Where D represents the disease index, N represents the number of diseased plants at each level, R represents the representative value at each level, T represents the total number of plants surveyed, and H represents the highest representative value.

[0086] The pathogenicity of *Sclerotinia sclerotiorum* and the biocontrol effect of *Bacillus weizmannii* SJZ were evaluated through a sunflower pot infection experiment. The susceptibility of sunflowers in the treatment groups in the pot control experiment is shown in [link to experiment]. Figure 10 The results showed that, compared with the water-based blank control group and the PD medium control group, the *Sclerotinia sclerotiorum*-infected group exhibited obvious disease symptoms. After inoculation with *Sclerotinia sclerotiorum*, sunflower seedlings exhibited a typical disease progression within a short period: 24 hours after mycelial drenching, the plants showed mild wilting symptoms, with leaves drooping due to water loss; 48 hours later, the wilting intensified, and some plants collapsed; 54 hours later, rot symptoms appeared in the rootstock, accompanied by tissue browning and softening. In contrast, the disease index and incidence rate of sunflower plants treated with *Bacillus weizmannii* SJZ were 43.33%.

[0087] Example 4: Analysis of the broad-spectrum antibacterial activity of Bacillus Weizmannii SJZ

[0088] Plant pathogens *Fusarium graminearum*, *Alternaria dauci*, *Alternaria nees*, and *V. sordida* (purchased from Mingzhou Biotechnology Co., Ltd.) were selected and mycelial cakes were prepared. Using the plate confrontation method, mycelial cakes with a diameter of 8.0 mm were inoculated in the center of PDA medium, with *Bacillus weizmannii* SJZ inoculated 1 cm away from the plate. The plates were incubated upside down at 28℃. After 5 days, the diameter of the pathogens was observed. A pure culture of pathogens without *Bacillus weizmannii* SJZ was used as a control. The experimental results are as follows: Figure 11 As shown, Figure 11 A represents *Fusarium graminearum*, B represents *Clostridium spp.*, C represents *Alternaria alternata*, and D represents *Alternaria spp.* The results show that *Bacillus weizmannii* SJZ exhibits differentiated antifungal activity against various plant pathogenic fungi. Its growth-inhibiting effect on *Fusarium graminearum*, *Alternaria alternata*, and *Alternaria alternata* is relatively limited, with only a slight slowdown in colony expansion. However, this bacterium shows a significant inhibitory effect on *Clostridium spp.*, with obvious phenotypic changes observed in the hyphae near *Bacillus weizmannii* SJZ: the hyphae gradually change from normal white to yellowish-brown, and the apical extension zone of the hyphae becomes blunted. This color change may be related to the leakage and oxidation of intracellular substances after the integrity of the hyphal cell wall is damaged, indicating that *Bacillus weizmannii* SJZ may interfere with the normal growth and development of pathogens by secreting secondary metabolites. These phenomena suggest that *Bacillus weizmannii* SJZ has certain application potential in the biological control of agricultural and forestry diseases.

[0089] Example 5: Analysis of the growth-promoting potential of Bacillus Weizmannii SJZ

[0090] The growth-promoting abilities of *Bacillus weizmannii* SJZ strains mainly include five aspects: nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore secretion, and IAA secretion. Single colonies of *Bacillus weizmannii* SJZ were inoculated onto Assumption nitrogen fixation selection medium, Monkina organic phosphorus solubilization selection medium, inorganic phosphorus solubilization selection medium, potassium solubilization selection medium, and CAS medium, and incubated upside down at 28°C for 3 days. If *Bacillus weizmannii* SJZ produced a clear zone on Assumption nitrogen fixation selection medium, Monkina phosphorus solubilization selection medium, and potassium solubilization selection medium, the strain possessed nitrogen fixation, phosphorus solubilization, and potassium solubilization abilities, respectively. If *Bacillus weizmannii* SJZ produced an orange-yellow clear zone on CAS medium, the strain possessed the ability to secrete siderophores. Bacillus Weizmannii SJZ was inoculated into LB medium containing 0.1% L-Try and cultured at 37°C and 180 rpm for 2 days. The bacterial suspension was then centrifuged at 8000 rpm for 5 min, and 100 μL of the supernatant was collected. 200 μL of Salkowski's reagent was added, and the mixture was incubated in the dark for 30 min. A pink color change indicates a positive result. If the color change is difficult to distinguish, the OD value was measured. 530nm.

[0091] Experimental results are as follows Figure 12 As shown, Figure 12 In the diagram, a represents nitrogen fixation, b represents siderophore production, and c represents IAA production. *Bacillus weizmannii* SJZ exhibits certain plant growth-promoting characteristics. This strain possesses nitrogen-fixing ability and can grow normally on nitrogen-free media, indicating that it may provide nitrogen nutrition to plants through nitrogen fixation. However, in phosphorus and potassium solubilization tests, no obvious phosphate or potassium solubilization zones were observed, indicating that it does not possess significant phosphate or potassium solubilization capabilities. Further testing revealed that this strain can secrete siderophores, forming a significant color change on CAS test plates, indicating its ability to chelate iron ions. Furthermore, colorimetric detection confirmed that *Bacillus weizmannii* SJZ can produce the auxin indole-3-acetic acid (IAA), showing certain growth-promoting potential. These characteristics suggest that *Bacillus weizmannii* SJZ has certain application value in the biological control of agricultural diseases and plant growth promotion, and can be further studied as a potential biocontrol bacterium or rhizosphere growth-promoting bacterium.

Claims

1. A strain of Bacillus Weizmannii that inhibits plant pathogens, characterized in that, The Bacillus Weizmannii is Bacillus Weizmannii ( Bacillus wiedmannii SJZ has been deposited at the China Center for Type Culture Collection on December 23, 2024, with accession number CCTCC NO: M 20242884.

2. The Bacillus Weizmannii as described in claim 1 is used to inhibit Sclerotinia sclerotiorum.

3. The Bacillus Weizmannii as described in claim 1 is used to inhibit the black rot of the shell fungus.

4. The Bacillus Weizmannii as described in claim 1 is capable of secreting proteases, cellulases, and chitinases.

5. The Bacillus Weizmannii as described in claim 1 is used for nitrogen fixation.

6. The Bacillus Weizmannii strain as described in claim 1 is used to secrete siderophores and IAA.

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