Paenibacillus tuvanzhurensis for inhibiting plant pathogenic fungi and application thereof

Abstract

The present application relates to the field of biological control, and provides a bacillus toyonensis for inhibiting plant pathogenic bacteria, which has the ability of antagonizing sclerotinia sclerotiorum and has a certain application prospect. The bacillus toyonensis can inhibit sclerotinia sclerotiorum, and can also inhibit fusarium graminearum, alternaria solani, alternaria tenuis and valsa mali. The bacillus toyonensis can secrete protease, cellulase and chitinase. The bacillus toyonensis can be used for nitrogen fixation, and can secrete iron carrier and IAA.

Description

Technical Field

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

[0002] Biological control primarily utilizes beneficial microorganisms and their secreted secondary metabolites to control pathogens, thereby reducing plant disease incidence and achieving prevention and management of plant diseases. These microorganisms are called biocontrol strains or antagonistic strains.

[0003] Currently, the most researched biocontrol strains are actinomycetes, Trichoderma, and Bacillus. Numerous biocontrol strains have been developed for the control of plant diseases, but these applications suffer from limitations such as limited strain variety and poor adaptability, failing to adapt to different species, production areas, and ecological environments. Furthermore, the quality of these inoculants varies, with low and unstable strain survival rates. Therefore, developing biocontrol strains with strong adaptability and control capabilities is of paramount importance.

[0004] Numerous studies have identified a variety of potential biocontrol bacteria. Compared with agricultural and chemical control methods, biological control poses less harm to the environment and will become the main technical method for plant disease control in the future. Summary of the Invention

[0005] The purpose of this invention is to provide a Tuva Bacillus strain that inhibits plant pathogens, possesses the ability to antagonize Sclerotinia sclerotiorum var. sunflower, and is a biocontrol strain with certain application prospects.

[0006] This invention provides a strain of Bacillus toyonensis that inhibits plant pathogens. It is Bacillus toyonensis SJZ, which has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan City, on December 23, 2024, with accession number CCTCC NO: M 20242883.

[0007] The *Bacillus tuvanis* SJZ of this invention has white, round, opaque colonies that are easily picked up, raised, with smooth edges, no obvious luster, and emits an unpleasant volatile odor. The bacterial cells are rod-shaped, slender, and straight.

[0008] The Tuva Bacillus 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.

[0009] The 16S rDNA sequencing results of *Bacillus toyonensis* SJZ from this invention were submitted to the NCBI database, analyzed and compared using BLAST, and then a phylogenetic tree of *Bacillus toyonensis* SJZ was constructed using MEGA7 software. This strain is closely related to *Bacillus toyonensis*, with a sequence similarity of 100%. Based on the combined morphological observation results and physiological and biochemical results, *Bacillus toyonensis* SJZ was identified as *Bacillus toyonensis*.

[0010] The Tuva Bacillus SJZ of this invention is used to inhibit Sclerotinia sclerotiorum.

[0011] The Tuva Bacillus SJZ of this invention is used to inhibit Fusarium graminearum, Alternaria solanacea, Alternaria spp. and Alternaria spp.

[0012] The Tuva Bacillus SJZ of this invention can secrete protease, cellulase and chitinase.

[0013] The Tuva Bacillus SJZ of this invention is used for nitrogen fixation.

[0014] The Tuva Bacillus SJZ of this invention is used to secrete siderophores and IAA.

[0015] The beneficial effects of this invention are:

[0016] 1. The aseptic fermentation broth of Bacillus thuringiensis SJZ in Tuva has a strong inhibitory effect on the growth of Sclerotinia sclerotiorum, with an inhibition rate of 89.41%. It can significantly affect the hyphal morphology of Sclerotinia sclerotiorum, inhibit the growth of new hyphae, make the hyphae of Sclerotinia sclerotiorum thinner, more branched, and transparent, and "tumor-like" aggregates will also be formed in the middle and end of the hyphae.

[0017] 2. The aseptic fermentation broth of Bacillus thuringiensis SJZ can inhibit the secretion of oxalic acid by Sclerotinia sclerotiorum, with an inhibition rate of 75.22%.

[0018] 3. When Tuvan Bacillus SJZ was cultured in pairs with Sclerotinia sclerotiorum, it was found that it could secrete volatile substances with strong antibacterial properties, with an antibacterial rate of 79.02%.

[0019] 4. The clear zone experiment showed that Bacillus tuvanifolia SJZ has a strong ability to secrete protease and cellulase, and a certain ability to secrete chitinase, but no ability to secrete β-glucanase.

[0020] 5. Treatment of sunflower seeds with Tuvan Bacillus yunnanensis 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 sunflower plants treated with Sclerotinia sclerotiorum mycelial suspension showed slight wilting 24 hours later, wilting and lodging after 48 hours, and root rot and stem rot were observed at 54 hours. The use of Tuvan Bacillus yunnanensis SJZ reduced the incidence of sunflower strain infection to 38.33%.

[0021] 6. The broad-spectrum antibacterial activity and growth-promoting potential of *Bacillus yunnanensis* SJZ were investigated. It was found that its inhibitory effect on *Fusarium graminearum*, *Alternaria solanaceae*, and *Alternaria spp.* was weak, while its inhibitory effect on *Clostridium spp.* was strong, and it could cause discoloration of *Clostridium spp.* hyphae. *Bacillus yunnanensis* SJZ possesses nitrogen-fixing ability and can secrete small amounts of siderophores and IAA, indicating a certain growth-promoting potential. Attached Figure Description

[0022] Figure 1 A contrastive image of Tuva Bacillus yunnanensis SJZ secondary screening plates;

[0023] Figure 2 This is a colony morphology diagram of Bacillus thuringiensis SJZ from Tuva.

[0024] Figure 3 Microscopic morphology of Bacillus tuvaniformis SJZ;

[0025] Figure 4 The hydrolytic enzyme secretion capacity of Bacillus thuringiensis SJZ in Tuva;

[0026] Figure 5 Phylogenetic tree of Bacillus tumefaciens SJZ;

[0027] Figure 6 The effect of Tuvan Bacillus SJZ metabolites on the growth of Sclerotinia sclerotiorum;

[0028] Figure 7 The effect of Tuva Bacillus SJZ metabolites on oxalic acid secretion in Sclerotinia sclerotiorum;

[0029] Figure 8 The antibacterial effect of volatile substances in Bacillus thuringiensis SJZ from Tuva;

[0030] Figure 9 The effects of different treatments of Bacillus thuringiensis SJZ fermentation broth on sunflower seed pericarp infection;

[0031] Figure 10 The infection and control efficacy of Bacillus thuringiensis SJZ in sunflowers in a pot experiment;

[0032] Figure 11 Analysis of the broad-spectrum antibacterial activity of Bacillus yunnanensis SJZ in Tuva;

[0033] Figure 12 Analysis of the growth-promoting potential of Bacillus yunnanensis SJZ from Tuva. Detailed Implementation

[0034] 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.

[0035] Example 1:

[0036] The Tuvan Bacillus spp. used in this embodiment to inhibit plant pathogens is Bacillus spp. SJZ, which has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan City, on December 23, 2024, with accession number CCTCC NO: M 20242883.

[0037] The isolation and screening method of Bacillus thuringiensis SJZ in this embodiment:

[0038] Initial screening: Soil samples were collected from Beiji Village, Mohe City, Daxinganling Region, Heilongjiang Province. First, impurities and large particles were removed. The sterilized soil samples were sieved through a sieve to select fine soil particles. 5g of soil sample was added to a 45mL Erlenmeyer flask containing sterilized glass beads. The flask was incubated at 150rpm for approximately 20 hours to thoroughly mix and disperse bacterial cells. The suspension was then heated in an 80℃ water bath for 30 minutes to kill other bacteria and vegetative cells. Finally, the suspension was diluted 10-fold using a tenfold dilution method. -2 10 -3 10 -4 10 -5 10 -6 Five dilutions were performed. 100 μL of each soil dilution was then spread onto LB and NA media, with each dilution repeated three times. The media were then incubated at 28°C inverted mode to isolate several bacterial 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 in the center of PDA media, with the selected strains inoculated 1 cm from the edge of the plate. The plates were incubated inverted mode at 28°C, and the diameter of the *Sclerotinia sclerotiorum* was observed after 3 days. Strains forming a clear inhibition zone were selected for secondary screening.

[0039] 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 the primary screening strains inoculated 1 cm from the edge of the plate. The plates were incubated upside down at 28°C, and the width of the inhibition band was measured after 5 days. Antagonistic bacteria with an inhibition clear band width of 10 mm or more were screened, such as... Figure 1 As shown, strain SJZ was finally obtained through screening.

[0040] Example 2: Identification of strain SJZ

[0041] The colony morphology of Bacillus yunnanensis SJZ in Tuva is as follows: Figure 2 As shown, the microscopic morphology of strain SJZ is as follows: Figure 3 As shown. The colonies are white, round, opaque, easily picked up, raised, with smooth edges, no obvious luster, and emit an unpleasant volatile odor. The bacteria are rod-shaped, slender, and straight.

[0042] The physiological and biochemical results of Bacillus tuvaniformis SJZ are shown in Table 1.

[0043] Table 1. Detection results of physiological and biochemical indicators of Bacillus thuringiensis SJZ.

[0044]

[0045]

[0046]

[0047] Note: "+" indicates positive, and "-" indicates negative.

[0048] The hydrolytic enzyme secretion capacity of Bacillus thuringiensis SJZ in Tuva was determined, and the results are as follows: Figure 4 As shown, the details are as follows:

[0049] Determination of protease secretion capacity of Tuvan Bacillus SJZ: Tuvan Bacillus SJZ was inoculated onto protease selection medium and incubated upside down at 28°C for 3 days. Figure 4 As shown in figure a, a very clear zone was found to be formed on the plate, indicating the ability to secrete proteases.

[0050] Determination of cellulase secretion capacity of Tuvan Bacillus SJZ: Tuvan Bacillus 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, a distinct transparent zone was observed on the plate, indicating its strong ability to secrete cellulase.

[0051] Determination of chitinase secretion capacity of Tuvan Bacillus SJZ: Tuvan Bacillus SJZ 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 certain clear zone was observed in Bacillus tuvanifolia SJZ on the chitinase screening plate, indicating that it has a certain chitinase secretion activity.

[0052] Determination of β-glucanase secretion capacity of Bacillus yunnanensis SJZ from Tuvan: Bacillus yunnanensis 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 d, no clear zone appears on the outer edge of the colony, indicating that Bacillus tuvaniflorus SJZ does not have the ability to produce β-glucanase.

[0053] The 16S rDNA of strain SJZ was amplified by PCR, and the product was recovered and purified to obtain a specific band of 1404 bp in length. The band was sequenced, and the 16S rDNA sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0054] The 16S rDNA sequencing results of *Bacillus tuvanis* SJZ of this invention were submitted to the NCBI database, analyzed and compared using BLAST, and then a phylogenetic tree of *Bacillus tuvanis* SJZ was constructed using MEGA7 software, as follows: Figure 5 As shown, this strain is closely related to Bacillus toyonensis, with a sequence similarity of 100%. Based on the combined morphological and physiological / biochemical results, Bacillus toyonensis SJZ was identified as Bacillus toyonensis.

[0055] Example 3: Study on the antibacterial and control capabilities of Tuva Bacillus SJZ against Sclerotinia sclerotiorum.

[0056] 1. Effects of Tuva Bacillus SJZ metabolites on the growth of Sclerotinia sclerotiorum.

[0057] The seed culture of *Bacillus tuvanis* SJZ in the logarithmic growth phase was inoculated into BPY medium at a ratio of 1%, and cultured with shaking. The fermentation broth was collected, centrifuged, and the supernatant was filtered through a 0.22 nm aqueous microporous membrane to obtain sterile fermentation broth. The sterile fermentation broth was mixed into PDA medium at a ratio of 5%, and the plates were inoculated with *Sclerotinia sclerotiorum* mycelial cakes with a diameter of 8 mm. The plates were incubated upside down at 28°C for 5 days. Observation showed that the mycelial growth of *Sclerotinia sclerotiorum* was greatly inhibited, with an inhibition rate of 89.41%. The experimental results are as follows. Figure 6As shown, this indicates that the metabolites secreted by Bacillus tuvaniflorus SJZ have strong antibacterial activity and a good ability to secrete antibacterial substances that inhibit the growth of Sclerotinia sclerotiorum.

[0058] 2. Effects of Tuva Bacillus SJZ metabolites on the morphology of Sclerotinia sclerotiorum.

[0059] The agar blocks containing mycelia of *Sclerotinia sclerotiorum* treated with the above-mentioned aseptic fermentation broth of *Bacillus sclerotiorum* SJZ were cut off with a disposable scalpel and sliced ​​as thinly as possible. The mycelial morphology was observed using an optical microscope. It was found that it had a great influence on the mycelial growth and morphology of *Sclerotinia sclerotiorum*. The mycelia in the control group were thicker, with obvious branching and vigorous vitality. *Bacillus sclerotiorum* SJZ inhibited the formation of new mycelia of *Sclerotinia sclerotiorum*, making the mycelia thinner, more branched, and transparent. "Nodule-like" aggregates also appeared in the middle and at the ends of the mycelia.

[0060] 3. Effects of Tuva Bacillus SJZ metabolites on oxalic acid secretion in Sclerotinia sclerotiorum.

[0061] A 1% seed culture of *Bacillus tuvanis* SJZ in the logarithmic growth phase was inoculated into 100 mL of BPY medium and cultured at a constant temperature with shaking to obtain an 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 a sterile fermentation broth. *Sclerotinia sclerotiorum* mycelial suspension was inoculated into 100 mL of PD medium and cultured at 28°C with shaking at 120 rpm for 3 days to obtain an activated *Sclerotinia sclerotiorum* mycelial suspension. The sterile fermentation broth was added at a 5% ratio to 20 mL of PD medium, followed by 1 mL of the activated *Sclerotinia sclerotiorum* mycelial suspension. The mixture was cultured at a constant temperature with shaking for 5 days, and mycelial growth was observed. The secretion of oxalic acid by *Sclerotinia sclerotiorum* was detected using a colorimetric method.

[0062] The effect of Tuva Bacillus SJZ on oxalate secretion by Sclerotinia sclerotiorum was determined by colorimetric method. Figure 7 As shown in the figure. Observations revealed that *Bacillus thuringiensis* SJZ can inhibit the growth of *Sclerotinia sclerotiorum* and suppress oxalic acid production, with an oxalic acid content inhibition rate of 75.22%. Oxalic acid is one of the main pathogenic factors of *Sclerotinia sclerotiorum*, which can reduce plant resistance and provide a suitable environment for the pathogen's hydrolytic enzymes. The *Bacillus thuringiensis* SJZ of this invention has a good ability to inhibit oxalic acid secretion and can inhibit the pathogenicity of *Sclerotinia sclerotiorum* to a certain extent.

[0063] 4. Analysis of the inhibitory effect of volatile substances from Tuvan Bacillus Flourishing SJZ on Sclerotinia sclerotiorum.

[0064] Tuvan *Bacillus yunnanensis* SJZ was inoculated into LB broth and cultured with shaking to obtain activated seed culture. The seed culture was then spread onto LB agar plates. The LB plates were then inverted onto PDA agar plates inoculated with *Sclerotinia sclerotiorum* mycelial cakes and cultured for 5 days. It was observed that *Bacillus yunnanensis* SJZ produced volatile antibacterial substances that inhibited the growth of *Sclerotinia sclerotiorum*. The antibacterial activity of the volatile substances is as follows: Figure 8 As shown, the antibacterial rate reached 79.02%, indicating that Bacillus tuvaniflorus SJZ has a strong ability to secrete antibacterial volatile substances.

[0065] 5. Shell Infection Experiment

[0066] Preparation of Tuvan Bacillus Flourishing SJZ bacterial culture: The Tuvan Bacillus Flourishing SJZ seed culture in the logarithmic growth phase was inoculated into BPY medium at an inoculum of 1%, and cultured at 28℃ and 180r / min for 3 days to obtain the original bacterial culture.

[0067] Disinfection and washing of sunflower seeds: Soak sunflower seeds in sterile water for 2 minutes; then soak sunflower seeds in 75% alcohol for 1 minute, repeat twice; soak sunflower seeds in 2% sodium hypochlorite solution for 2 minutes; then rinse sunflower seeds twice with 2% Na2S2O3 solution to remove residual chlorine; then rinse sunflower seeds three times with sterile water; finally, use sterile filter paper to absorb the moisture from the seed surface.

[0068] 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.

[0069] 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.

[0070] 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 observed 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:

[0071]

[0072] In the formula, M represents the incidence rate (%), I represents the number of infected seeds (seeds), and T represents the total number of seeds (seeds).

[0073]

[0074] In the formula, 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 level representative value.

[0075] 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 In the table, CK1 and CK2 represent untreated blank controls, SJZ represents the original bacterial solution, and 1 / 20 represents 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 and the 20-fold diluted bacterial solution. Detailed analysis is shown in Table 2.

[0076] Table 2. Effects of different treatments of Bacillus yunnanensis SJZ bacterial suspension on sunflower seed coat infection.

[0077]

[0078]

[0079] 6. Sunflower pot experiment

[0080] 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.

[0081] Pot infection experiment: *Sclerotinia sclerotiorum* mycelial suspension was inoculated into PD medium and cultured at 28℃ with shaking at 120 rpm for 5 days to obtain activated *Sclerotinia sclerotiorum* mycelial suspension. Experimental group: 30 mL of *Sclerotinia sclerotiorum* mycelial suspension was used for root irrigation, followed by 1 cm of soil coverage; 20 pots were treated in total. Control group 1: An equal volume of sterile water was used for root irrigation, followed by 1 cm of soil coverage; 20 pots were treated in total. Control group 2: An equal volume of PD medium was used for root irrigation, followed by 1 cm of soil coverage; 20 pots were treated in total. The susceptibility and symptoms of sunflower plants were observed and recorded every 12 hours.

[0082] Potted plant control experiment: *Bacillus tuvanis* 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 tuvanis* SJZ bacterial solution. Experimental group: 50 mL of *Bacillus tuvanis* SJZ bacterial solution 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.

[0083]

[0084] In the formula, 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.

[0085] The disease susceptibility of sunflowers in the experimental group during the potted plant control experiment is shown in the figure. Figure 10 Compared with the water-based blank control group and the PD medium experimental control group, the *Sclerotinia sclerotiorum* infection group showed significantly increased susceptibility to the disease. The sunflower pot infection experiment demonstrated that *Sclerotinia sclerotiorum* can cause disease in sunflower seedlings within a short period. Observations showed that sunflower seedlings exhibited slight wilting 24 hours after mycelial drenching, followed by wilting and lodging 48 hours later, and root and stem rot at 54 hours. Compared with the water and PD medium control groups, the disease index and incidence rate of the sunflower strain treated with *Bacillus tuvani* SJZ were 38.33%.

[0086] Example 4: Broad-spectrum antibacterial activity analysis of Bacillus thuringiensis SJZ from Tuva

[0087] Plant pathogens *Fusarium graminearum*, *Alternaria dauci*, *Alternaria nees*, and *V. sordida* (purchased from Mingzhou Biotechnology Co., Ltd.) were selected to prepare mycelial cakes. Using the plate confrontation method, mycelial cakes with a diameter of 8.0 mm were inoculated in the center of PDA medium, with *Bacillus* inoculated 1 cm from the edge of the plate. The plates were incubated upside down at 28℃, and the diameter of the pathogens was observed after 5 days. The experimental results are as follows: Figure 11 As shown, Figure 11A represents *Fusarium graminearum*, B represents *Alternaria alternata*, C represents *Alternaria solanacea*, and D represents *Alternaria spp.* The results show that *Bacillus tuvani* SJZ can inhibit the growth of *Fusarium graminearum*, *Alternaria spp.*, and *Alternaria solanacea* to a small extent. *Bacillus tuvani* SJZ can also inhibit the growth of *Alternaria alternata*; the hyphae of *Alternaria alternata* near *Bacillus tuvani* SJZ turn yellow to brown. This may be because the antibacterial substance secreted by *Bacillus tuvani* SJZ causes the pathogenic hyphae to break down, leading to leakage and oxidation of the hyphal contents. This indicates that *Bacillus tuvani* SJZ has certain application potential in the field of agricultural and forestry disease control.

[0088] Example 5: Growth-promoting potential analysis of Bacillus thuringiensis SJZ from Tuva

[0089] The growth-promoting abilities of *Bacillus tuvani* SJZ strains mainly include five aspects: nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore secretion, and IAA secretion. Single colonies of *Bacillus tuvani* SJZ were inoculated onto Assumption nitrogen fixation selection medium, Monkina organic phosphorus phosphorus solubilization selection medium, inorganic phosphorus phosphorus solubilization selection medium, potassium solubilization selection medium, and CAS medium, and incubated upside down at 28°C for 3 days. If *Bacillus tuvani* 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 the strain produced an orange-yellow clear zone on CAS medium, the strain possessed the ability to secrete siderophores. Inoculate Tuva Bacillus SJZ into LB medium containing 0.1% L-Try and incubate at 37°C and 180 rpm for 2 days. Take out the bacterial culture, centrifuge at 8000 rpm for 5 min, take out 100 μL of the supernatant, add 200 μL of Salkowski reagent, and develop color in the dark for 30 min. If it turns pink, it is positive. If it is difficult to distinguish, measure its OD530 nm.

[0090] The 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. It can be seen that *Bacillus tuvani* SJZ has a certain nitrogen-fixing ability, but lacks phosphorus and potassium solubilization capabilities. It possesses the ability to secrete siderophores and produce IAA, indicating its potential for growth promotion and suggesting promising applications in agricultural disease control and the development of biocontrol and growth-promoting bacteria.

Claims

1. The application of Tuva Bacillus subtilis, characterized in that, The *Bacillus tuvani* is used to inhibit *Sclerotinia sclerotiorum*, *Fusarium graminearum*, *Alternaria alternata*, and *Alternaria spp.* var. *tuvani*. The *Bacillus tuvani* is *Bacillus tuvani* (…). Bacillus toyonensis SJZ has been deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan City, on December 23, 2024, with accession number CCTCC NO: M 20242883.

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

3. The Tuva Bacillus as described in claim 1 is used for nitrogen fixation.

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