Bacillus siamensis BsiaSC07 and application thereof

Abstract

The invention provides a strain of bacillus siamensis Bsiamensis and application thereof, the strain is classified and named as bacillus siamensis Bsiamensis, the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO: 35785, and the preservation date is September 1, 2025. The bacterial suspension, the fermentation liquor and the ethyl acetate extract of the fermentation liquor of the bacterial strain have good bacteriostatic activity on fusarium graminearum, fusarium oxysporum, fusarium laminatum, alternaria longibrachiata, colletotrichum gloeosporioides and colletotrichum frutescens.

Description

Technical Field

[0001] This invention belongs to the field of biological control of plant diseases and management of pests and diseases in kiwifruit cultivation. More specifically, it relates to the discovery and application of highly effective antibacterial metabolites of Bacillus siamensis BsiaSC07. Background Technology

[0002] Kiwifruit is highly sought after by consumers for its unique flavor and rich nutritional components (rich in vitamin C, folic acid, potassium, etc.). In recent years, as an important economic crop, kiwifruit has seen a surge in pests and diseases due to the continuous expansion of its cultivation scale. Leaves, as crucial nutrient organs in the growth of kiwifruit, continuously supply nutrients to all parts of the plant through photosynthesis and transpiration. In kiwifruit cultivation, large-scale outbreaks of fungal leaf spot disease are often fatal to the industry. On the surface of diseased leaves, dark brown or grayish-brown spots of varying shapes and sizes appear in multiple areas, leading to leaf wilting and even large-scale leaf drop. It has been reported that there are many types of fungi that cause kiwifruit leaf spot, including *Fusarium spp.*, *Alternaria spp.*, and *Colletotrichum spp.*, all of which can infect leaves and cause leaf spot disease. Currently, the most common method for controlling this disease is still the use of chemical fungicides. However, the widespread overuse and misuse of chemical agents not only causes phytotoxicity and severely damages the natural environment, but also accelerates the increase in pathogen resistance. Therefore, there is an urgent need to explore new, efficient, and safe fungicides to enrich the means of treating kiwifruit leaf spot disease.

[0003] Bacillus species are exceptionally widespread in nature, inhabiting various ecological niches, including air, water, and soil, as well as existing on and inside plants. From a biotechnological perspective, a key characteristic of Bacillus species that has garnered significant attention is the rich diversity and structural variety of its secondary metabolites. These products can directly inhibit the growth of pathogenic microorganisms, induce plant resistance, and promote plant growth to control disease occurrence. Therefore, the discovery and development of secondary metabolites with antimicrobial activity has become a promising alternative to traditional chemical pesticides. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the discovery and application of highly effective antibacterial metabolites from a strain of Bacillus siamensis, BsiaSC07. This invention screened a biocontrol bacterium, BsiaSC07, from healthy kiwifruit leaves, demonstrating strong inhibitory activity against various pathogens causing kiwifruit leaf spot disease. Based on morphological characteristics and molecular biological methods, it was identified as Bacillus siamensis. Experiments verified that the bacterial suspension, fermentation broth, and ethyl acetate extract of the fermentation broth of BsiaSC07 all possess broad-spectrum antibacterial activity. Based on this, gas chromatography-mass spectrometry was used to identify and screen a compound with superior antibacterial effect, 3,5-di-tert-butylphenol (3,5-DTBP). The effects of this compound on the mycelial and spore growth and development of Fusarium graminearum were investigated, and its control effect on kiwifruit leaf spot disease was evaluated. Subsequently, by combining transcriptome data, visualization observation, and analysis of related substance content, it was clarified that the inhibition of Fusarium graminearum by 3,5-DTBP was mainly achieved by affecting its cell membrane integrity and energy metabolism.

[0005] The purpose and efficacy of this invention—the discovery and application of a *Bacillus amyloliquefaciens* strain for preserving kiwifruit—are achieved through the following specific technical means:

[0006] A strain of Bacillus sicca, BsiaSC07, was isolated from healthy kiwifruit leaves and screened using Fusarium graminearum, the pathogen causing kiwifruit leaf spot, as the target pathogen. Its accession number is CGMCC NO: 35785, and the accession date is September 1, 2025.

[0007] The strain can significantly inhibit the growth of various fungi that cause leaf spot disease in kiwifruit, including but not limited to Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Alternaria longipes, Colletotrichum gloeosporioides, and Colletotrichum gloeosporioides.

[0008] The fermentation method for a strain of Bacillus sicca, BsiaSC07, involved streaking BsiaSC07 onto nutrient agar (NA) medium for activation and culturing at 30°C for 24 h. A single colony was then picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of PYS fermentation medium (200 g / L potato, 10 g / L yeast extract, 18 g / L sucrose) and fermented for 5 days at 200 rpm and 30°C on a shaker. Finally, the supernatant was collected by centrifugation at 4000 rpm for 15 minutes and filtered three times through a 0.22 μm filter membrane to obtain the BsiaSC07 fermentation broth.

[0009] The discovery of highly effective antibacterial metabolites from Bacillus simonii BsiaSC07 was conducted by extracting antibacterial active compounds from the BsiaSC07 fermentation broth using ethyl acetate at a volume ratio of 2:1. Gas chromatography-mass spectrometry was then used to identify the compounds in the fermentation broth. Using the matching degree as a key indicator, the top ten compounds by relative abundance were selected to identify 3,5-DTBP, which showed the best inhibitory effect against Fusarium graminearum.

[0010] Applications of Bacillus sicca fermentation broth, ethyl acetate extract, and highly effective antibacterial metabolite 3,5-DTBP: The fermentation broth of BsiaSC07, its ethyl acetate extract, and the antibacterial metabolite 3,5-DTBP all exhibit good inhibitory activity against Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Alternaria longipes, Colletotrichum gloeosporioides, and Colletotrichum gloeosporioides.

[0011] The application effect of 3,5-DTBP, a highly effective antibacterial metabolite of Bacillus simonii BsiaSC07, shows that 3,5-DTBP has inhibitory activity on the growth of both vegetative and aerial mycelia of Fusarium graminearum, reduces the number of conidia of Fusarium graminearum, induces spore deformities, and leads to spore germination inhibition.

[0012] The mechanism of action of 3,5-DTBP, a highly effective antibacterial metabolite of Bacillus simonii BsiaSC07, is as follows: 3,5-DTBP affects the synthesis of lecithin, sphingolipids and ergosterol, which are components of the Fusarium graminearum cell membrane, reduces the content of intracellular pyruvate, and interferes with the normal process of energy metabolism.

[0013] The application method of highly effective antibacterial metabolites of Bacillus sicca BsiaSC07 is to prepare an appropriate concentration of 3,5-DTBP and spray it on the surface of kiwifruit leaves.

[0014] The discovery and application of highly effective antibacterial metabolites from Bacillus sicca SC07 have been applied in all or part of the following 1)-6):

[0015] 1) Application in suppressing the pathogen of kiwifruit leaf spot disease;

[0016] 2) Application in controlling the occurrence of kiwifruit leaf spot disease;

[0017] 3) Application in the preparation of pathogenic bacterial bioinhibitors;

[0018] 4) Application in the preparation of biological control agents for kiwifruit leaf spot disease;

[0019] 5) Application in the preparation of fungicides for Fusarium graminearum;

[0020] 6) Application in the preparation of pesticide formulations based on 3,5-DTBP.

[0021] The aforementioned pathogenic bioinhibitors have inhibitory effects on all or some of the following pathogens: Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Alternaria longipes, Colletotrichum gloeosporioides, and Colletotrichum gloeosporioides; the kiwifruit leaf spot biocontrol agent has inhibitory effects on all or some of the following diseases: kiwifruit brown spot, kiwifruit leaf black spot, and kiwifruit anthracnose; the Fusarium graminearum fungicide has all or some of the following functions: disrupting cell membrane integrity and inhibiting metabolic processes; the pesticide formulations of 3,5-DTBP have all or some of the following types: fumigants, suspension concentrates, microcapsule suspensions, and emulsifiable concentrates.

[0022] The present invention has at least the following beneficial effects:

[0023] The above-mentioned technical solution isolated and screened a biocontrol bacterium, *Bacillus sicca* BsiaSC07, with broad-spectrum antifungal activity from the surface of healthy kiwifruit leaves. The bacterial suspension, fermentation broth, and ethyl acetate extract of this strain showed significant inhibitory effects against six pathogens causing kiwifruit leaf spot disease. Furthermore, 3,5-DTBP, identified and screened from the fermentation products of BsiaSC07, also exhibited broad-spectrum antifungal activity, especially against *Fusarium graminearum*. This compound can inhibit the growth and development of mycelia and spores of the pathogen by disrupting the cell membrane integrity of *Fusarium graminearum* and interfering with energy metabolism, thereby controlling the occurrence of leaf spot disease. In conclusion, this strain and the discovered antifungal compound—3,5-DTBP—have broad application potential and value in the prevention and control of kiwifruit leaf spot disease. Attached Figure Description

[0024] Figure 1 This is the colony morphology of Bacillus sicca SC07 on NA plates according to the present invention.

[0025] Figure 2 The cell morphology of Bacillus sicca BsiaSC07 under a scanning electron microscope is shown in the present invention.

[0026] Figure 3 This is a multigene phylogenetic tree of Bacillus sicca SC07 of the present invention.

[0027] Figure 4 This invention describes the inhibitory effect of Bacillus sicca SC07 suspension on six pathogens causing leaf spot disease in kiwifruit.

[0028] Figure 5 This invention describes the inhibitory effect of Bacillus sicca SC07 fermentation broth on six pathogens causing leaf spot disease in kiwifruit.

[0029] Figure 6 This invention describes the inhibitory effect of the ethyl acetate extract of Bacillus sicca SC07 on six pathogens causing leaf spot disease in kiwifruit.

[0030] Figure 7 This is the total ion chromatogram of the ethyl acetate extract of Bacillus sicca SC07 under gas chromatography-mass spectrometry analysis according to the present invention.

[0031] Figure 8 This is the mass spectrum of the substance with a retention time of 25.34 minutes in the gas chromatography analysis of the ethyl acetate extract of Bacillus sicca SC07 in this invention.

[0032] Figure 9 This invention involves the screening of antibacterial compounds against Bacillus sicca (BsiaSC07).

[0033] Figure 10 This invention describes the inhibitory effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus sicca SC07 on six pathogens causing leaf spot disease in kiwifruit.

[0034] Figure 11 This invention demonstrates the inhibitory effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus simonii (BsiaSC07) on aerial mycelia of Fusarium graminearum.

[0035] Figure 12 The present invention relates to the effect of the highly effective antibacterial compound 3,5-DTBP of Bacillus simonii BsiaSC07 on the sporulation rate of Fusarium graminearum.

[0036] Figure 13 The present invention relates to the effect of the highly effective antibacterial compound 3,5-DTBP of Bacillus simonii BsiaSC07 on the germination of Fusarium graminearum spores.

[0037] Figure 14 The effect of the highly effective antibacterial compound 3,5-DTBP of Bacillus simonii BsiaSC07 on the morphology of Fusarium graminearum spores (optical microscope).

[0038] Figure 15 The effect of the highly effective antibacterial compound 3,5-DTBP of Bacillus simonii BsiaSC07 on the morphology of Fusarium graminearum spores (scanning electron microscopy).

[0039] Figure 16 This invention demonstrates the efficacy of the highly effective antibacterial compound 3,5-DTBP from Bacillus sicca SC07 against kiwifruit leaf spot disease.

[0040] Figure 17 This invention relates to the effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus simonii BsiaSC07 on the lecithin synthesis pathway of Fusarium graminearum.

[0041] Figure 18This invention relates to the effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus simonii BsiaSC07 on the sphingolipid synthesis pathway in Fusarium graminearum.

[0042] Figure 19 This invention investigates the effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus simonii BsiaSC07 on the expression levels of key genes in the ergosterol synthesis pathway of Fusarium graminearum.

[0043] Figure 20 This invention demonstrates the disruptive effect of the highly effective antibacterial compound 3,5-DTBP (BsiaSC07) on the cell membrane structure of Fusarium graminearum hyphae.

[0044] Figure 21 The effect of the highly effective antibacterial compound 3,5-DTBP of Bacillus simonii BsiaSC07 on the leakage of intracellular substances from Fusarium graminearum mycelia.

[0045] Figure 22 This invention relates to the effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus sicca SC07 on the content of cell membrane components in Fusarium graminearum hyphae.

[0046] Figure 23 This invention relates to the effects of the highly effective antibacterial compound 3,5-DTBP from Bacillus simonii (BsiaSC07) on energy metabolism-related pathways in Fusarium graminearum.

[0047] Figure 24 This invention relates to the effect of the highly effective antibacterial compound 3,5-DTBP from Bacillus simonii BsiaSC07 on the content of related substances in the energy metabolism of Fusarium graminearum. Detailed Implementation

[0048] The embodiments of the present invention will be described in further detail below through examples. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0049] In the description of this invention, unless otherwise stated, "a plurality of" means two or more; the terms "coaxial," "bottom," "one end," "top," "middle," "other end," "upper," "side," "top," "inner," "front," "center," "both ends," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0050] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "setting," "connection," "fixing," "screw connection," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Unless otherwise explicitly limited, those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0051] Example:

[0052] This invention provides the discovery and application of highly effective antibacterial metabolites from Bacillus sicca SC07, as shown in the attached document. Figure 1-23 As shown, the Bacillus sicca strain BsiaSC07 described in this invention was deposited on September 1, 2025, at the China General Microbiological Culture Collection Center (Address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Postcode: 100101, Tel: 010-64807596), accession number: CGMCC NO: 35785.

[0053] The plants involved in this invention are as follows:

[0054] (1) Kiwi leaves

[0055] The plant pathogenic fungi involved in this invention are as follows:

[0056] (1) Fusarium graminearum;

[0057] (2) Fusarium oxysporum;

[0058] (3) Fusarium proliferatum;

[0059] (4) Alternaria longipes;

[0060] (5) Colletotrichum gloeosporioides;

[0061] (6) Colletotrichum fructicola.

[0062] The culture media and solutions involved in this invention are as follows:

[0063] NA medium: 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 20 g / L agar powder, 1000 mL sterile water.

[0064] NB medium: 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 1000 mL sterile water.

[0065] PYS medium: 200 g / L potato, 10 g / L yeast extract, 18 g / L sucrose, 1000 mL sterile water.

[0066] PDA medium: 200 g potato, 20 g glucose, 17 g agar powder, 1000 mL sterile water.

[0067] PDB medium: 200 g potato, 20 g glucose, 1000 mL sterile water.

[0068] Mung bean culture medium: 30 g mung beans, 1000 mL sterile water.

[0069] YEPD medium: 200 g potato, 20 g sucrose, 1000 mL sterile water.

[0070] 1. Isolation and screening of biocontrol bacteria

[0071] Healthy kiwifruit leaves were collected in July 2022 from Miluo Town, Shuicheng County, Liupanshui City, Guizhou Province, China (104.9669°E, 26.3983°N). Fresh, healthy kiwifruit leaves were disinfected, and then 5 g of leaves were thoroughly ground and transferred to a 250 mL Erlenmeyer flask containing 50 mL of sterile water. The flask was incubated at 30°C and 150 rpm for 2 h with shaking. 80 μL of the supernatant was spread onto NA medium and incubated upside down at 28°C for 48 h. Single colonies were then picked, purified, and preserved. Finally, using *Fusarium graminearum* as the target pathogen, the biocontrol bacterium with the best inhibitory activity was screened and named BsiaSC07.

[0072] 2. Morphological observation of biocontrol bacteria

[0073] Using an inoculation loop, pick up a suspension of the biocontrol bacterium BsiaSC07 and quickly streak it onto NA medium. On the medium, BsiaSC appears pale yellow, with raised wrinkles around the colony. Figure 1 On the other hand, the biocontrol bacteria were centrifuged at 10,000 rpm for 1 min to collect the bacterial cells, washed three times with PBS, and then fixed with 2.5% glutaraldehyde at 4°C for 12 h. Scanning electron microscopy (SEM) revealed that the bacterial cells were long rods, approximately 9 μm in length. Figure 2 ).

[0074] 3. Molecular biological identification

[0075] The 16S rDNA, gyrA gene, and gyrB gene of BsiaSC07 were amplified using 27F (5′-AGAGTTTGATCCTGGCTCAG–3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), gyrA-F (5′-CAGTCAGGAAATGCGTACGTCTT-3′) and gyrA-R (5′-CAAGGTAATGCTCCAGGCATTGCT-3′), and gyrB-F (5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′) and gyrB-R (5′-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′). The amplified products were sent to Shanghai Sangon Biotech for sequencing. The sequencing results were then submitted to GenBank, and strains with high homology were selected. A phylogenetic tree was constructed using MEGA 11 with the maximum likelihood method. The experimental results show that BsiaSC07 and Bacillus siamensis GUWM35 exhibit extremely high homology, reaching 100% ( Figure 3 Therefore, based on the above experimental results, the taxonomic status of BsiaSC07 is clearly defined as B. siamensis.

[0076] 4. Preparation of BsiaSC07 fermentation broth and extraction of antibacterial substances

[0077] A BsiaSC07 bacterial suspension was streaked onto NA medium. A single colony was picked and inoculated into a 50 mL NB Erlenmeyer flask (250 mL volume), and incubated with shaking at 180 rpm and 28°C for 18 h. Then, 0.5 mL of the bacterial suspension was inoculated into an Erlenmeyer flask containing 50 mL PYS fermentation medium (250 mL), and fermented on a shaker at 200 rpm and 30°C for 5 days. Afterward, the precipitate was discarded by centrifugation at 10,000 rpm, and the broth was filtered three times through a 0.22 μm sterile membrane to obtain a sterile fermentation broth.

[0078] The sterile fermentation broth was treated at 121°C for 2 h to remove unstable antibacterial active substances. It was then mixed with ethyl acetate at a 1:2 ratio and transferred to 50 mL centrifuge tubes, with each tube brought to a final volume of 30 mL. Extraction was carried out by shaking at 150 rpm for 24 h. The supernatant ethyl acetate solution was collected, concentrated by rotary evaporation, and the product was dissolved in DMSO.

[0079] 5. Antibacterial activity of BsiaSC07 bacterial suspension

[0080] Six mycelial discs (6 mm) of six pathogens causing kiwifruit leaf spot were inoculated into the center of PDA medium. Then, 6 mm sterile filter paper discs were immersed in a BsiaSC07 bacterial suspension for 1 min and air-dried. The filter paper discs were then inoculated at four points 25 mm away from the pathogenic mycelial discs. The control group used NB medium instead of the BsiaSC07 bacterial suspension. The treated petri dishes were incubated at 28°C for 72 h.

[0081] Inhibition rate (%) = (Diameter of pathogen in control group - Diameter of pathogen in treatment group) / Diameter of pathogen in control group × 100

[0082] Experimental results showed that the biocontrol bacterium BsiaSC07 exhibited good inhibitory activity against six postharvest pathogens of kiwifruit. Figure 4 Compared with the control group, the growth of pathogens was significantly inhibited, with the best inhibitory effect observed against Fusarium graminearum, with an inhibition rate of 76.40%.

[0083] 6. Aseptic fermentation broth and ethyl acetate extract (EAE)

[0084] Six pathogenic bacteria mycelial cakes (6 mm) were inoculated onto PDA medium containing 2.5% and 5% sterile fermentation broth and 50 mg / L and 100 mg / L ethyl acetate extract, respectively, and incubated at 28℃ for 72 h. PYS medium and DMSO were used as controls instead of sterile fermentation broth and EAE, respectively, and the inhibition rate was calculated according to formula in section 5. The results showed that both concentrations of sterile fermentation broth also exhibited good antibacterial activity against the six pathogenic bacteria, with the best inhibition rates against *Fusarium graminearum* being 55.88% and 67.59%, respectively. Figure 5 On the other hand, compared to sterile fermentation broth, the two lower concentrations of EAE showed better inhibitory effects against six pathogens, but the best inhibitory effect was still against Fusarium graminearum, with inhibition rates of 67.18% and 77.89%, respectively. Figure 6 ).

[0085] 7. Identification of BisaSC07 metabolites

[0086] To screen for stable antibacterial substances in the fermentation products of BisaSC07, stable components were determined by gas chromatography-mass spectrometry (GC-MS). EAE (extraction emulsion) was transferred to an extraction flask, preheated for 20 min, and then the extraction needle was inserted into the flask at a distance of 1 cm from the sample surface. Headspace extraction was performed for 40 min followed by GC-MS analysis. Chromatographic conditions were as follows: HP-5MS (30.0 m × 250 μm, 0.25 μm) column, vaporization chamber temperature of 250 °C, transfer line temperature of 260 °C, helium as carrier gas, and flow rate of 1.0 mL / min. Mass spectrometry conditions were as follows: ion source temperature of 230 °C, electron energy of 70 eV, quadrupole temperature of 150 °C, full scan mode, and scan mass range of 50–450 u. Qualitative analysis of the raw data was performed using the NIST 11 standard mass spectrometry library (https: / / webbook.nist.gov / chemistry / ) and retention time, and quantitative analysis was performed using peak area normalization. From the analytical results, the top ten compounds with a matching degree ≥70% and the highest relative abundance were selected, namely: 3,4-dimethylbenzaldehyde, 3,5-di-tert-butylphenol (3,5-DTBP), 2,4-dimethoxybenzaldehyde, di-n-butyl phthalate, diisobutyl phthalate, 2,3,6-trimethylphenol, 2,6-dimethylanisole, benzaldehyde, n-octadecane, and n-heptadecane (Figure 7). The 3,5-DTBP mass spectrum analysis at a retention time of 25.34 minutes corresponded to its structure, verifying the presence of this substance (Figure 8).

[0087] 8. Screening of antibacterial substances

[0088] Based on GC-MS analysis, using *Fusarium graminearum* as the target fungus, 6 mm fungal discs were inoculated into the center of PDA medium containing ten compounds. The effective concentration of the ten compounds in the medium was uniformly prepared to 100 mg / L. DSMO was used as a control group instead of the compounds. After incubation at 28℃ for 72 h, the inhibition rate of each compound was calculated according to the formula in section 5. The experimental results showed that only three compounds had an inhibition rate higher than 50% against *Fusarium graminearum*: 2,3,6-trimethylphenol, diisobutyl phthalate, and 3,5-DTBP. Among them, 3,5-DTBP showed the strongest antibacterial activity, with an inhibition rate of 92.54%. Figure 9 ).

[0089] 9. In vitro antifungal activity of 3,5-DTBP

[0090] Six pathogenic fungi causing kiwifruit leaf spot were selected, and the mycelial growth rate method was used to evaluate the inhibitory activity of 3,5-DTBP on these pathogens. Five different concentration gradients were set up for each pathogen, and the treatments were carried out according to the experimental method described in section 8. DSMO was used as a control instead of 3,5-DTBP. Finally, the colony diameter of each fungus was recorded, and the inhibition rate of each treatment was calculated according to the formula in section 5. The concentration-based regression equation was determined using DPS software, and EC was calculated. 50 The experimental results showed that 3,5-DTBP had good inhibitory activity against six pathogenic fungi: *Fusarium graminearum*, *Fusarium oxysporum*, *Fusarium solani*, *Alternaria stalkata*, *Colletotrichum gloeosporioides*, and *Colletotrichum candida*, with EC50 values ​​of 5.3217, 9.0824, 14.1649, 6.7282, 9.7504, and 7.9539 μg / mL, respectively. Figure 10 ).

[0091] Effects of 10,3,5-DTBP on aerial mycelia of Fusarium graminearum

[0092] Each is configured with 0.5 EC 50 EC 50 and 2 EC 50 PDA medium was prepared. 20 mL of the prepared PDA mixed medium was poured into a glass test tube. After the medium solidified, a 6 mm Fusarium graminearum mycelium was inoculated into the center. The tube was then incubated at 28℃ for 72 h. An equal volume of DSMO was added to the PDA as a control group. The length of the aerial mycelium of the pathogen was measured every 24 h during this period. The growth inhibition rate of the aerial mycelium was calculated using the following formula:

[0093] Inhibition rate (%) = (Hyphae length of control group - Hyphae length of treatment group) / Hyphae length of control group × 100

[0094] The experimental results showed that 3,5-DTBP not only effectively inhibited the growth of vegetative mycelia of Fusarium graminearum, but also significantly reduced the growth of its aerial mycelia. At 72 h, the inhibition rates of the three concentrations of 3,5-DTBP on the aerial mycelia of Fusarium graminearum were 53.39%, 71.99%, and 88.21%, respectively. Figure 11 ).

[0095] 11. Effects of 3,5-DTBP on sporulation of Fusarium graminearum

[0096] Five 6 mm Fusarium graminearum mycelial discs were inoculated separately with 0.5 EC. 50 EC 50 and 2 EC 50In a mung bean culture medium, the samples were incubated for 5 days with shaking at 150 rpm and 25°C. The filtrate was then collected after filtering three times using sterile gauze. The absorbance of the filtrate at 600 nm was measured using a microplate reader to assess the number of sporozoites produced by *Fusarium graminearum*. The results showed that 3,5-DTBP significantly reduced the number of *Fusarium graminearum* sporozoites, which directly affects the spread and reproduction of the pathogen. Figure 12 ).

[0097] Effects of 12,3,5-DTBP on Fusarium graminearum spore germination

[0098] Add 10 mL of Fusarium graminearum spores (1 × 10⁻⁶) 6 (CFU / mL) was inoculated into 90 mL containing 0.5 EC 50 EC 50 and 2EC 50 In YEPD medium, spores were incubated for 12 h in a shaker at 180 rpm and 28°C. Spore germination was observed every 4 h starting from h 0, and the number of germinating spores was recorded. The experiment was repeated three times, with three randomly selected fields of view for each repeat, recording 25 ± 3 spores per field. The results showed that in the first 4 h after treatment, spores treated with all three concentrations showed almost no germination, with 0.5 EC... 50 The spore germination rate in the treatment group was only 2.15%, EC 50 and 2EC 50 No germination occurred in any of the treatment groups. However, after 4 hours, 0.5 EC... 50 In the treatment group, the number of spores germinating significantly increased, and at 12 h, the germination rate was not significantly different from that of the control group. However, EC... 50 and 2 EC 50 Both concentrations significantly inhibited spore germination throughout the entire treatment period. 2 EC 50 The concentration showed the best inhibitory effect on spore germination, with spore germination rates of 0% and 2.14% at 8 h and 12 h, respectively. Figure 13 )

[0099] 13. Effects of 3,5-DTBP on the spore morphology of Fusarium graminearum

[0100] Following the experimental steps in section 12, single spores were selected using an optical microscope at each time point to observe changes in spore morphology. Furthermore, spores were collected at 12 h of treatment, washed three times with PBS, fixed with 2.5% glutaraldehyde at 4℃ for 12 h, and then dehydrated and sputter-coated with gold according to the method in section 2.2 before SEM observation and analysis. The results showed that, according to SEM observation, although *Fusarium graminearum* spores could still germinate normally after 12 h of treatment with 0.5% EC50, the spores were swollen compared to the control group. Higher concentrations of EC50... 50 and 2 EC 50 After treatment, obvious lumps appeared on the surface of the spores, and the higher the concentration, the denser the distribution of the lumps. Figure 14 Furthermore, observations using a higher-magnification optical microscope also confirmed that 3,5-DTBP has an irreversible effect on the morphology of Fusarium graminearum spores during the spore germination stage. Figure 15 )

[0101] 14. Prevention and control effects

[0102] Select healthy kiwifruit leaves with uniform growth, rinse them with 75% alcohol for 30 seconds, then rinse three times with sterile water and air dry in a sterile environment. Next, divide the leaves into groups of nine and place them in a preservation box. Spray 5 mL of Fusarium graminearum spore suspension evenly at a distance of 20 cm from the leaf surface. After 12 hours, spray 0.5 EC of the suspension into three different boxes. 50 EC 50 and 2EC 50 Leaves treated with 3,5-DTBP were used, while the control group was sprayed with only DMSO. The treated leaves were placed in a climate incubator at 25°C and 75% relative humidity, with each of the four treatments repeated three times. After 7 days, changes in leaf wet weight and lesion area were recorded, and the disease symptoms were graded as follows: 0 = no obvious lesions; 1 = lesion area <5%; 3 = 5%-25%; 5 = 26%-50%; 7 = 51%-75%; 9 = lesion area >75%. Three leaves were grouped together, and the disease index and control effect of leaves treated with different concentrations of 3,5-DTBP were calculated using the following two formulas.

[0103] Disease index = (Number of leaves per grade × Disease grade) / (Total number of leaves × Highest disease grade) × 100

[0104] Prevention efficacy (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100

[0105] The experimental results show that the control group exhibited numerous brown, withered lesions on the leaves. However, after applying 3,5-DTBP, the area of ​​leaf lesions was significantly controlled, and the lesion area decreased with increasing 3,5-DTBP concentration. Furthermore, compared to the control, the reduction in leaf fresh weight due to disease was significantly controlled under all three concentrations of 3,5-DTBP. However, within the same component, the difference between treatments before and after applying 2 EC... 50 Except for the kiwifruit leaves, the fresh weight of other components of the kiwifruit leaves decreased significantly. Figure 16 ).

[0106] 15. Transcriptome detection and analysis

[0107] Collect 2 g of Fusarium graminearum mycelium and transfer it to a medium containing EC. 50 In PDB medium, the mycelia were cultured at 180 rpm and 28℃ for 12 h with shaking. Afterwards, 1 g of mycelia from each treatment were collected and washed three times with PBS at 4℃. Total RNA was then extracted from the mycelia, and the purity, content, and integrity of the obtained RNA were assessed. The samples were then sent to Shanghai Sangon Biotech for testing and analysis. In short, libraries were constructed for the samples, and qualified libraries were sequenced on an Illumina Novaseq 6000 platform. The gene sequence of the type strain *Fusarium graminearum* H-1 was selected as a reference for analysis (accession number: GCA_000240135.3). Differential gene expression analysis was performed using edgeR, with FDR ≤ 0.05 and |log2FC| ≥ 1 set as thresholds for significant differential gene expression. The results showed that multiple coding genes related to lecithin synthesis in *Fusarium graminearum* were significantly downregulated after 3,5-DTBP treatment. Figure 17 Five genes encoding the synthesis of ceramides and sphingosine in the sphingolipid metabolism pathway were significantly upregulated, while the sphingosine kinase gene, which directly encodes the conversion of sphingosine to sphingokinase 1-phosphate, was significantly downregulated. Figure 18 Furthermore, significant upregulation and downregulation of gene expression were observed in genes encoding ergosterol synthesis, severely impacting the normal synthesis of ergosterol. Figure 19 On the other hand, during energy metabolism, the expression levels of 13 genes in the three pathways related to pyruvate synthesis were significantly downregulated. When pyruvate is converted to acetyl-CoA and enters the tricarboxylic acid cycle, the genes encoding isocitrate dehydrogenase and succinate dehydrogenase are significantly downregulated, while the three genes encoding succinyl-CoA synthase are significantly upregulated. Furthermore, genes encoding acetyl-CoA converted via the propionate metabolic pathway also showed significant downregulation. Figure 20 ).

[0108] 16. Damage to the cell membrane by 3,5-DTBP

[0109] Use 0.5 EC respectively 50 EC 50 and 2 EC 50 Fusarium graminearum hyphae were treated with 3,5-DTBP for 12 h, then washed three times with PBS at 4°C and stored at 4°C for later use. On one hand, small amounts of hyphae treated with different methods were stained with 5 μL propidium iodide dye in the dark for 10 min, and the fluorescence intensity and area of ​​the hyphae were observed using a fluorescence microscope. On the other hand, some hyphae were fixed with 2.5% glutaraldehyde at 4°C for 12 h, then dehydrated, resin-embedded, and sectioned, and the hyphae were observed using SEM and transmission electron microscopy (TEM). The results showed that, in the propidium iodide fluorescence staining experiment, the red fluorescence intensity and area gradually increased with increasing 3,5-DTBP concentration, while the control group showed almost no red fluorescence. SEM observation revealed that, compared with the control group, although 3,5-DTBP caused the hyphae to become rough, wrinkled, and concave, no obvious mycelial rupture was observed. TEM also showed that when the concentration of 3,5-DTBP increased, the internal structure of the pathogenic fungus became disordered, and plasmolysis occurred due to cell membrane damage, especially at a concentration of 2 EC. 50 At that time, the cell membrane was completely destroyed. Figure 21 ).

[0110] 17. Detection of intracellular substances and cell membrane components

[0111] Using EC 50 Two g of *Fusarium graminearum* mycelia were treated with 3,5-DTBP for 12 h, followed by filtration to collect the mycelia and filtrate separately. 200 μL of the filtrate was used to measure the change in relative conductivity, and the absorbance at 260 nm and 280 nm was measured to evaluate the leakage of nuclei and proteins. 0.5 g (fresh weight) of the treated mycelia was used to determine the ergosterol content in the mycelia by high-performance liquid chromatography (HPLC). Additionally, 1 g (fresh weight) of mycelia was used to determine the contents of lecithin, sphingosine-1-phosphate, sphingosine, and ceramide in the mycelia using a kit. The results showed that using 2 EC... 50 After 12 h of treatment with 3,5-DTBP, the contents of lecithin, sphingosine monophosphate, and ergosterol in Fusarium graminearum mycelium decreased significantly, while the contents of ceramide and sphingosine increased significantly. Figure 22 Furthermore, compared with the control, the relative conductivity and OD... 260 OD 280 All three indicators also rose significantly. Figure 23 ).

[0112] 18. Effects on energy metabolism processes

[0113] Follow the kit instructions at 2 EC50 After treating mycelia with 3,5-DTBP for 12 h, the contents of ATP, ATPase, pyruvate, succinate dehydrogenase, acetyl-CoA, and oxaloacetate in the mycelia were measured. The results showed that after 12 h of treatment with 3,5-DTBP, the contents of pyruvate, acetyl-CoA, oxaloacetate, and succinate dehydrogenase in Fusarium graminearum mycelia all decreased significantly. In addition, the contents of ATP (an energy storage substance in energy metabolism) and ATPase (an energy-converting enzyme) also decreased simultaneously. Figure 24 ).

[0114] 19. Sequence List:

[0115] Seq No. 1 16S rDNA sequence, gyrB sequence and gyrA sequence of Bacillus sicca SC07

[0116] SEQUENCE LISTING

[0117] <110> Guizhou University

[0118] <120> Application of Bacillus sicca strain BsiaSC07 and its highly effective antibacterial metabolites

[0119] <130> 2025

[0120] <160> 3

[0121] <170> PatentIn version 3.5

[0122] <210> 1

[0123] <211> 1545

[0124] <212> DNA

[0125] <213> Bacillussiamensis

[0126] <400> 1 (16S rDNA)

[0127] ttcggagagtttgatcctggctcaggacgaacgctggcggcgtgcctaatacatgcaagt 60

[0128] cgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtg 120

[0129] ggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttg 180

[0130] tttgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccg 240

[0131] cggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacct 300

[0132] gagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagca 360

[0133] gtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaag 420

[0134] gttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggc 480

[0135] accttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaata 540

[0136] cgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttctta 600

[0137] agtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttga 660

[0138] gtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggagga 720

[0139] acaccagtggcgaaggcgactctctgggctgtaactgacgctgaggagcgaaagcgtggg 780

[0140] gagcgaacaggattagaaaccctggtagtccacgccgtaaacgatgagtgctaagtgtta 840

[0141] gggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacg 900

[0142] gtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtgg 960

[0143] tttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctaga 1020

[0144] gataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtg 1080

[0145] tcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcat 1140

[0146] tcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtc 1200

[0147] aaatcatcatgccccttttgacctgggctacacacgtgctacaatgggcagaacaaaggg 1260

[0148] cagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtct 1320

[0149] gcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaa 1380

[0150] tacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaag 1440

[0151] tcggtgaggtaacctttatggagggagccgccgaaggtgggacagatgattggggtgaag 1500

[0152] tcgtaacaaggtagccgtatcggaaggtgcggctggatcacctcc 1545

[0153] <210>2

[0154] <211> 2454

[0155] <212> DNA

[0156] <213>Bacillussiamensis

[0157] <400>2(gyrA)

[0158] gagtgaacaaaacacaccacaagtacgtgaagtcaatatcagtcaggaaatgcggacatc 60

[0159] cttcctggactatgcaatgagcgttatcgtatcccgggcgcttccggatgtgcgtgacgg 120

[0160] tctgaagccggttcacagacggattttgtacgcaatgaatgatttaggcatgaccagtga 180

[0161] caaaccatataaaaaatctgcccgtatcgtcggcgaagttatcggtaagtaccacccgca 240

[0162] cggtgattcagcggtttacgaatcaatggtcagaatggcgcaggattttaactaccgcta 300

[0163] catgcttgttgacggacacggcaacttcggttcagttgacggcgactctgcggctgcgat 360

[0164] gcgttacacagaagcgagaatgtcaaaaatcgcaatggaaattctgcgtgacattacgaa 420

[0165] agacacgattgactatcaagacaactatgacggttcagaaagagaacctgccgtcatgcc 480

[0166] ttcgagatttccgaatctgctcgtaaacggagctgccggtattgcggtcggaatggcgac 540

[0167] aaacattcccccgcatcagcttggagaagtcattgaaggcgtgcttgccgtaagtgagaa 600

[0168] tcctgagatcacaatccaagagctgatggaatatattccgggcccggattttccgacggc 660

[0169] cggtcagattttaggccggagcggcatccgcaaggcatatgaatccggacggggatcaat 720

[0170] cacgatccgggctaaggctgaaatcgaagagacatcctcgggaaaagaaagaattattgt 780

[0171] cacggaacttccttatcaggtcaacaaagcgagattaattgaaaaaatcgcagatctcgt 840

[0172] ccgggacaaaaaaatcgaaggaatcaccgatctgcgtgacgaatccgaccgtaacggaat 900

[0173] gagagtcgtcatcgagatccgccgtgacgccaatgctcacgtcattctgaataacctgta 960

[0174] caaacaaacggccctgcagacgtcttttggaatcaacctgctggcgctcgttgacggaca 1020

[0175] gccgaaggtattaaacctgaaagaatgtctggagcattatcttgatcaccaaaaggtcgt 1080

[0176] gatccgacgcagaacggcttacgaacttcgcaaagcggaggcgagagctcacattctgga 1140

[0177] agggttaagaatcgctctcgatcatttagacgcggtgatctcttatccgtagttctca 1200

[0178] gacggctgaaattgcgagaacgggtttaatcgaacaatttcactgactgaaaaacaggc 1260

[0179] gcaagccattctggacatgcgtcttcagcgattaaccggtggcgcgaaaagatcga 1320

[0180] agaggaatatcaatcacttgcgcttatcgccgagctgaaagatattttagccaatga 1380

[0181] agaaagagtgctcgagatcatccgtgaagaactcaatcaatcaaagagcggttcaatga 1440

[0182] tgagacgcactgaaatcgttacgtccggtcttgaaacaattgaggatgaagatctcat1500

[0183] cgagagagaaacatcgtcatcacgctgactcataacggctatgcgtctcccagc 1560

[0184] gtccacataccgcagccaaaaacggggcggaaaggcgtgcagggaatgggaacgaatga 1620

[0185] agatgactttgtggaacacttaatttctacatcgacgcacgatacgattctattcttctc1680

[0186] tataaagggaaggtatatcgttcaaaaggatatgaaattcctgaatacggccgtaccgc 1740

[0187] aaaaggcattccgattattaacctcctggaggttgaaaaaggcgaatggattaatgccat 1800

[0188] tattccggtcagcacatttgatgaggagctttacctcttcttcacgaccaaacaaggggt1860

[0189] gtcgaaacgtacggcactgtcccagtttgcgaatatacggaataacggactgatcgcttt 1920

[0190] aggtcttcgggaagacgatgaactgatggccgtccgtctgactgacggcaagaaacaaat 1980

[0191] catcatcggaacgaagaacggcttgctgatccgtttccctgaagaagatgtaagacaaat 2040

[0192] ggggcgcaccgctgcaggtgtcaaaggcatcacgctgacagatgatgatgtcgtggtagg 2100

[0193] catggagattcttgaagaagactcccatgttctcatagtgacagaaaacggctacggcaa 2160

[0194] acgtacgccggcctcagaatacagagtccaaagccggggcggaaaaggccttaaaacatg 2220

[0195] taaaatcactgacagcaacggtccgctagtaacagtaaaagctacaagaggcgaagaaga 2280

[0196] cttaatgattattacggcaagcggcgtgttaatcagaatggatattaacgatatctccac 2340

[0197] tacgggacgtgtaacacaaggcgtccgtctgatccgcatgtccgatgaagagcacgtggc 2400

[0198] cactgtggcactagtggaaaagaatgaagaagagccggaagaaacggaagaaga 2454

[0199] <210>3

[0200] <211> 1077

[0201] <212> DNA

[0202] <213>Bacillussiamensis

[0203] <400>3(gyrB)

[0204] gccttgtcgaccactcttgatgttacggttcatcgtgatgggaaaatccattatcaggcg 60

[0205] tatgaacgcggtgttccggcggctgatcttgaagtgatcggtgacactgacaagaccggg 120

[0206] acgattacgcacttcgttcccgacccggaaattttcaaagaaacaactgtatatgactat 180

[0207] gatcttctgtcaaaccgtgtccgcgaattggccttcctgacaaaaggcgtaaacatcacg 240

[0208] attgaagacaaacgtgaaggacaagaacggaaaaacgagtaccactacgaaggcggaatc 300

[0209] aaaagctacgttgagtacttaaaccgttccaaagaagtcgttcacgaagagccgatttat 360

[0210] atcgaaggcgagaaagacggcatcacggttgaagttgcattgcaatacaacgacagctat 420

[0211] acaagcaatatttattctttcgcgaataatatcaacacatacgaaggcggcacgcacgag 480

[0212] gccggatttaaaaccggtctgacccgtgtcataaacgactatgcaagaagaaaagggatt 540

[0213] ttcaaagaaaatgatccgaatttaagcggggatgatgtgagagaagggctgactgccatt 600

[0214] atttcaattaagcaccctgatccgcaatttgaagggcagacgaaaacgaagctcggcaac 660

[0215] tctgaagcaagaacgatcactgatacgctgttttctgctgcgctggaaacattccttctt 720

[0216] gaaaatccggattcagcccgcaaaatcgttgaaaaaggcttaatggccgcaagagcgcgg 780

[0217] atggcggcgaaaaaagcgcgggaattgacccgccgcaaaagcgcgcttgagatttccaat 840

[0218] ctgccgggcaaactggcggactgttcttctaaagatccgagcatttccgagctctatatc 900

[0219] gtagagggtgactctgcgggcggatcagcgaaacagggacgggaccgccatttccaagcc 960

[0220] attctgccgctgcgcggtaagattctgaacgttgagaaagccagacttgataagattctc 1020

[0221] tcaaataatgaggtcagatcaatgattacggctctcggcacgggtatcggagaagat 1077

[0222] The biocontrol bacterium BsiaSC07, isolated and screened from the surface of healthy kiwifruit, and its metabolite, 3,5-di-tert-butylphenol (3,5-DTBP), demonstrated its efficacy in controlling kiwifruit leaf spot disease. In experiments, the superior inhibitory effect of the biocontrol bacterium BsiaSC07 and its fermentation products on the pathogenic fungus causing kiwifruit leaf spot was verified, and 3,5-DTBP was identified from its fermentation products for the first time. This compound exhibited excellent inhibitory effects against six pathogenic fungi, especially *Fusarium graminearum*. Under the action of 3,5-DTBP, the normal growth of both vegetative and aerial mycelia of *Fusarium graminearum* was significantly inhibited, the number of conidia decreased significantly, and the spores became swollen and even deformed, leading to inhibited spore germination. In the leaf spot disease control efficacy test, 3,5-DTBP also effectively controlled leaf damage caused by *Fusarium graminearum*, significantly reducing the area of ​​leaf lesions. Transcriptomic analysis revealed that 3,5-DTBP inhibits the growth and development of *Fusarium graminearum*, likely by affecting the synthesis of cell membrane components and interfering with energy metabolism. Subsequent visualization analysis further validated the effects of 3,5-DTBP on the cell membrane structure and permeability of *Fusarium graminearum*, and confirmed that 3,5-DTBP indeed led to a decrease in the content of cell membrane components and key substances in energy metabolism pathways. Based on these findings, the biocontrol agent *Bacillus siamensis* BsiaSC07 and 3,5-DTBP hold promise as broad-spectrum biological control agents, enriching the management methods for kiwifruit leaf spot disease at the planting stage.

[0223] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0224] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A strain of Bacillus sicca, characterized in that: The strain was classified and named Bacillus siamensis BsiaSC07, with accession number CGMCC NO: 35785 and accession date of September 1, 2025.

2. The *Bacillus sicca* strain according to claim 1, characterized in that: The strain was isolated from healthy kiwifruit leaves and screened using Fusarium graminearum, the pathogen causing kiwifruit leaf spot disease, as the target pathogen.

3. The application of a strain of Bacillus sicca as described in claim 1, characterized in that: The strains inhibited the growth of Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Alternaria longipes, Colletotrichum gloeosporioides, and Colletotrichum gloeosporioides.

4. The method for applying the Bacillus sicca strain according to claim 3, characterized in that: The method is as follows: BsiaSC07 is activated by streaking on nutrient agar (NA) medium and cultured at 30℃ for 24 h. Then, a single colony is picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of PYS fermentation medium (200 g / L potato, 10 g / L yeast extract, 18 g / L sucrose). Fermentation is carried out in a shaker at 200 rpm and 30℃ for 5 days. Finally, the supernatant is collected by centrifugation at 4000 rpm for 15 minutes and then filtered three times through a 0.22 μm filter membrane to obtain the fermentation broth of BsiaSC07.

5. The method of applying the Bacillus sicca strain according to claim 4, characterized in that: The method is as follows: antibacterial active compounds in the BsiaSC07 fermentation broth are extracted with ethyl acetate at a volume ratio of 2:

1. Then, the types of compounds in the fermentation broth are identified by gas chromatography-mass spectrometry. Using the matching degree as the key indicator, the top ten compounds with the highest relative abundance are selected to screen out the compound with the best inhibitory effect on Fusarium graminearum—3,5-di-tert-butylphenol (3,5-DTBP). 10~50 μg / mL of 3,5-DTBP is prepared and sprayed on the surface of kiwifruit leaves.

6. The method of applying the Bacillus sicca strain according to claim 4 or 5, characterized in that: The method involves the following: the fermentation broth of BsiaSC07, the ethyl acetate extract of the fermentation broth, and the antibacterial metabolite 3,5-DTBP exhibit inhibitory activity against Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Alternaria longipes, Colletotrichum gloeosporioides, and Colletotrichum spp.

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