Bacillus velezensis sg27 and application thereof

Abstract

The present application relates to the technical field of plant disease control, and particularly relates to a bacillus velezensis SG27 and application thereof. Bacillus velezensis The present application provides a bacillus velezensis SG27, the Latin name of the bacillus velezensis SG27 is Bacillus velezensis , which is preserved in Guangdong Microbial Culture Collection Center, located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, and has a preservation date of January 28, 2026, and a preservation number of GDMCC No: 67754. The strain has obvious inhibitory effect on various plant pathogenic fungi, especially can effectively inhibit the pathogenic bacteria causing sugarcane leaf spot and sugarcane root rot, and the strain also has the effect of promoting the growth of sugarcane plants.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a Bacillus belye SG27 and its applications. Background Technology

[0002] A class of microbial communities that can coexist with the host plant for extended periods exists within plants. These microorganisms typically complete part or all of their life cycle within plant tissues without causing obvious disease symptoms. As a key component of the plant micro-ecosystem, they play a bridging role in the interaction between plants and the external environment. Studies have shown that these endophytic microorganisms can participate in plant nutrient metabolism, enhance the efficiency of plant absorption and utilization of mineral elements, and inhibit various plant pathogens. Therefore, they are considered important research targets for biological control and the development of agricultural microbial resources.

[0003] Currently, the long-term and large-scale application of chemical pesticides in plant disease control still faces challenges such as high costs, environmental pollution, and soil microecological imbalance. Furthermore, it may hinder the reproduction of beneficial microorganisms and affect the balance of the agricultural ecosystem. Therefore, cultivating endophytic Bacillus strains with clear origins, environmental compatibility, and stable efficacy for the biological control of plant diseases is of significant practical importance for reducing chemical pesticide use and improving crop health.

[0004] Plant diseases have always been a core factor restricting crop yield and quality. While traditional methods relying on chemical pesticides have limited the spread of pathogens to some extent, they have increasingly revealed problems such as environmental pollution, increased ecological threats, and growing pathogen resistance. Furthermore, chemical agents often destroy beneficial microbial communities in the soil and plants when effective against pathogens, leading to reduced stability of the agricultural ecosystem. Therefore, seeking safe, efficient, and sustainable plant disease control technologies has become a crucial task for modern agricultural development. Against the backdrop of the deepening concepts of ecological agriculture and green pest control, utilizing microbial resources for plant disease control is increasingly becoming an important trend in research and application. Compared to traditional control methods that rely on chemical pesticides or the breeding of disease-resistant varieties, endophytic bacteria exhibit significant advantages in disease control. These microorganisms can exert protective effects within plants without complex genetic modification, thus shortening research and development time, reducing application costs, and avoiding the safety controversies that may arise from genetically modified organisms (GMOs). Furthermore, endophytic bacteria are relatively safe for plants and the environment, have limited impact on non-target organisms, and maintain good colonization stability within the host, exerting a sustained disease-preventing effect even at low application doses. The biocontrol mechanisms of plant endophytic bacteria are diverse, mainly including secreting secondary metabolites with antibacterial activity, competing with pathogens for ecological niches and nutrients within the plant, inducing systemic resistance in the host, and secreting various extracellular enzymes to disrupt pathogen cell structure or weaken their pathogenicity. These mechanisms work synergistically, making endophytic bacteria a promising candidate for plant disease control.

[0005] Plant endophytic bacteria can inhibit the infection and spread of pathogenic microorganisms through various mechanisms, including secreting antimicrobial metabolites, competing with pathogens for survival resources and nutrients within plant tissues, inducing the host plant to initiate an immune response, and producing extracellular enzymes to degrade the cell structure of pathogens or reduce their toxicity. These mechanisms collectively establish the core position of endophytic bacteria in the biological control of plant diseases.

[0006] Bacillus belysinus ( Bacillus velezensis As a highly valued functional bacterium in agricultural biological control, it possesses advantages such as strong environmental adaptability, the ability to form stress-resistant spores, and ease of large-scale cultivation, and can generate a variety of bioactive secondary metabolites. However, since strains from different sources exhibit significant differences in biological characteristics and control efficacy, screening for specific strains with clear origins and stable characteristics remains of significant practical importance for specific crops.

[0007] sugar cane( Saccharum officinarumSugarcane roots are susceptible to various pathogenic microorganisms during their growth period, and their growth status directly affects the efficiency of agricultural production. As a key functional organ of sugarcane, the root system has a complex internal microecological environment, potentially harboring endophytic bacterial resources with biocontrol potential. However, current research on the isolation and identification of *Bacillus belyssae* endophytes in sugarcane roots and their application in plant disease control remains relatively limited.

[0008] In agricultural production, chemical agents remain one of the main means of controlling plant diseases. However, long-term or improper use can easily lead to environmental pollution, increased pathogen resistance, and soil microbial imbalance. Therefore, screening for endophytic Bacillus belyssus strains with clear origins, high safety, and excellent control efficacy, and applying them to the biological control of plant diseases, is of great significance for reducing dependence on chemical pesticides and promoting sustainable agricultural development. Isolating and screening Bacillus belyssus strains with significant antagonistic activity and application potential from the leaves of specific sugarcane varieties is of great significance for enriching plant endophytic biocontrol resources, reducing the use of chemical pesticides, and promoting green control of diseases in sugarcane and other crops. Summary of the Invention

[0009] The purpose of this invention is to provide a Bacillus vesiculosus SG27 that can prevent sugarcane diseases and promote sugarcane growth.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a *Bacillus belyceae* SG27, the Latin name of which is... Bacillus velezensis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 28, 2026, with accession number GDMCC No: 67754.

[0011] The present invention also provides a microbial preparation comprising the aforementioned Bacillus belyssus SG27.

[0012] Preferably, the dosage form of the microbial preparation is liquid or solid.

[0013] Preferably, the concentration of Bacillus vesiculus SG27 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

[0014] The present invention also provides the application of the aforementioned Bacillus vesiculosus SG27 or the aforementioned microbial preparation in the prevention and control of sugarcane diseases.

[0015] Preferably, the sugarcane diseases are fungal sugarcane diseases and sugarcane root rot.

[0016] Preferably, the pathogen of the sugarcane fungal leaf spot disease is *Ulva globosum*, and the pathogen of the sugarcane root rot disease is *Fusarium*.

[0017] The present invention also provides the application of the aforementioned Bacillus vesiculosus SG27 or the aforementioned microbial preparation in promoting sugarcane growth.

[0018] Preferably, the promotion of sugarcane growth is achieved through one or more of the following: the ability of Bacillus vesiculosus SG27 to solubilize inorganic phosphorus, potassium, produce iron carriers, produce indole-3-acetic acid, and produce 1-aminocyclopropane-1-carboxylic acid deaminase.

[0019] Beneficial effects: This invention provides a strain of *Bacillus belye* SG27, isolated from the internal tissue of healthy sugarcane underground seed stalks. This strain belongs to the sugarcane endophytic antagonistic bacteria category and possesses excellent plant endophytic colonization characteristics. Experimental results show that this strain has a significant inhibitory effect on various plant pathogenic fungi, especially effectively inhibiting pathogens causing sugarcane leaf spot and root rot. Simultaneously, this strain also promotes sugarcane plant growth, which can improve sugarcane disease resistance and growth performance while reducing the use of chemical pesticides, providing a safe, environmentally friendly, and effective technical approach for the green control of sugarcane diseases. Attached Figure Description

[0020] Figure 1 The colony morphology of Bacillus belyssus SG27; Figure 2 Microscopic structure of Bacillus belyssus SG27; Figure 3 Phylogenetic classification of Bacillus belyss SG27; Figure 4 Confrontation culture experiment between Bacillus vesiculosus SG27 and sugarcane root rot pathogen; Figure 5 Confrontation culture experiment between Bacillus vesiculosus SG27 and sugarcane leaf spot pathogen; Figure 6 It is the pathogen causing sugarcane root rot; Figure 7 It is caused by the fungus that causes sugarcane leaf spot disease; Figure 8 A schematic diagram for picking sugarcane root rot mycelium; Figure 9 A schematic diagram for picking out sugarcane leaf spot mycelia; Figure 10 Microstructure of the hyphae of Bacillus belyssus SG27-treated sugarcane root rot pathogen; Figure 11 The mycelial microstructure of the sugarcane root rot pathogen in the control group; Figure 12 Microstructure of hyphae of Bacillus belyssus SG27 after treatment with sugarcane leaf spot pathogen; Figure 13 The mycelial microstructure of the sugarcane leaf spot pathogen in the control group; Figure 14 Results of gene detection for growth-promoting genes in Bacillus belyssus SG27; Figure 15 The graph shows the phosphorus solubilization effect of Bacillus belyssus SG27; Figure 16 The potassium-solubilizing effect of Bacillus belyssus SG27 is shown in the figure. Figure 17 A diagram illustrating the siderogenic transport effect of Bacillus belyssus SG27; Figure 18 The results of the evaluation of the effects of Bacillus vesiculosus SG27 on preventing sugarcane root rot and promoting growth under sugarcane pot conditions; Figure 19 The results of the evaluation of the effects of Bacillus vesiculosus SG27 on the prevention of sugarcane leaf spot and the promotion of growth under sugarcane pot cultivation conditions.

[0021] Preservation Instructions

[0022] The Latin name of Bacillus belyssus SG27 is Bacillus velezensis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 28, 2026, with accession number GDMCC No: 67754. Detailed Implementation

[0023] This invention provides a *Bacillus belyceae* SG27, the Latin name of which is... Bacillus velezensis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 28, 2026, with accession number GDMCC No: 67754.

[0024] The present invention also provides a microbial preparation comprising the aforementioned Bacillus belyssus SG27.

[0025] In this invention, the dosage form of the microbial preparation is liquid or solid.

[0026] In this invention, the concentration of Bacillus vesiculus SG27 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL.

[0027] The present invention also provides the application of the aforementioned Bacillus vesiculosus SG27 or the aforementioned microbial preparation in the prevention and control of sugarcane diseases.

[0028] In this invention, the sugarcane diseases are fungal sugarcane diseases and sugarcane root rot.

[0029] In this invention, the pathogen of the sugarcane fungal leaf spot disease is *Ulva globosum*; the pathogen of the sugarcane root rot disease is *Fusarium*.

[0030] The present invention also provides the application of the aforementioned Bacillus vesiculosus SG27 or the aforementioned microbial preparation in promoting sugarcane growth.

[0031] In this invention, the promotion of sugarcane growth is achieved through one or more of the following: the ability of Bacillus bereaves SG27 to solubilize inorganic phosphorus, potassium, produce siderophores, produce indole-3-acetic acid, and produce 1-aminocyclopropane-1-carboxylic acid deaminase.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] In an embodiment of the present invention, NA culture medium consisted of 5g peptone, 1g yeast extract, 3g beef extract, 10g glucose, and 15g agar, which were diluted with distilled water to a final volume of 1000mL. The pH was adjusted to 7.0, and the medium was sterilized at 121°C for 20 minutes. PDA medium: Weigh 200g of peeled potatoes, add 800mL of distilled water and boil for 30min, then filter. Take the filtrate, add 20g of glucose and 20g of agar, and distilled water to a final volume of 1000mL. Sterilize at 121℃ for 20min. NB liquid medium: Each 1000mL contains 5g peptone, 1g yeast extract, 3g beef extract, and 10g glucose, pH 7.0; Pikovskaya Inorganic Phosphorus-Solubilizing Solid Medium: (NH4)2SO4 0.5g / L, MgSO4·7H2O 0.3g / L, FeSO4·7H2O 0.03g / L, MnSO4·H2O 0.03g / L, Ca3(PO4)2 25g / L, glucose 10.0g / L, agar 118.0g, add deionized water to a final volume of 1L, and adjust the pH to 7.0–7.5; Silicate medium: 5.0g yeast extract, 0.1g calcium carbonate (CaCO3), 0.04g calcium chloride (CaCl2), 1.67g K2HPO4·3H2O, 0.87g KH2PO4, 0.004g FeCl3·6H2O, 0.1g MgSO4·7H2O, 21g agar, adjusted to pH 6.9±0.1 (25℃), dissolved in deionized water and brought to a final volume of 1L, sterilized at 121℃ for 20min; CAS agar plates: Dissolve 0.06 g of chromium azurite S (CAS) in 50 mL of double-distilled water and mix with 10 mL of iron(III) solution (1 mM FeCl3·6H2O, 10 mM HCl) to obtain solution A; dissolve 0.07 g of hexadecyltrimethylammonium bromide (HDTMA) in 40 mL of double-distilled water to obtain solution B; slowly add solution A to solution B with stirring and mix thoroughly to obtain a deep blue staining solution C; add 100 mL of 10x MM9 salt solution and 30.24 g of piperazine-1,4-diethanesulfonic acid (Pipes) to a conical flask containing 750 mL of double-distilled water, mix thoroughly, adjust the pH to 6.8 with 50% (w / w) NaOH solution, and add 15 g of agar to obtain medium D; combine staining solution C, medium D, 1 mM CaCl2 solution, and 1 mM... MgSO4·7H2O solution, 20% glucose solution, and 10% hydrolyzed casein solution were autoclaved at 120℃ for 20 min. When cooled to 50℃, 1 mL of CaCl2 solution, 20 mL of MgSO4·7H2O solution, 10 mL of glucose solution, and 30 mL of hydrolyzed casein solution were added to culture medium D. Then, dye solution C was slowly added along the bottle wall and mixed thoroughly to obtain blue CAS medium. The genes and primers used for detection were obtained from the following references: Joshi, R. and M. S. Gardener, Identification and Characterization of Novel Genetic Markers Associated with Biological Control Activities in Bacillus subtilis. Phytopathology, 2006.96(2):p.145. Mora I, Cabrefiga J, Montesinos E. Antimicrobial peptide genes in Bacillus strains from plant environments[J]. International Microbiology, 2011, 14(4):213-223. Li, B. The Antagonistic Mechanism of Bacillus amyloliquefaciens SQR9 against Soil-Borne Pathogens and In Situ Rhizosphere Antibiotic GeneExpression. Ph.D. Thesis, Nanjing Agriculture University, Nanjing, China, 2015.

[0034] Example 1: Screening of endophytic antagonistic bacteria in sugarcane

[0035] In May 2024, sugarcane underground seed tuber samples were collected using a five-point sampling method at the Second Experimental Base of the Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences in Kaiyuan City, Yunnan Province. The geographical coordinates of the sampling location were 103°04′E, 23°30′N. The average annual temperature of the sampling area was 19.8℃, the average annual precipitation was 800mm, and the soil type was typical sandy red soil of Yunnan Province. The collected sugarcane underground seed tuber samples were placed in sterile preservation bags and stored at 4℃ after being brought back to the laboratory for later use.

[0036] The collected underground sugarcane stalks were thoroughly rinsed with sterile water and air-dried at room temperature. Under aseptic conditions, 10g of leaf samples were weighed, and the surface was first disinfected with 75% ethanol. Then, the leaves were cut into 2-3cm pieces with a sterile blade, soaked in 75% ethanol for 1 minute, and rinsed twice with sterile water. After that, the samples were treated with 0.1% sodium hypochlorite solution for 30 seconds and rinsed 6 times with sterile water to remove residual disinfectant.

[0037] The sterilized underground rhizome tissue was placed in a sterile mortar containing 9 mL of sterile water, sterile quartz sand was added, and the mixture was ground thoroughly and allowed to stand for 15 minutes. 1 mL of the tissue suspension was then diluted to a 10⁻¹⁰ solution. -4 10 -5 and 10 -6Gradients were used, with 0.1 mL taken from each dilution and evenly spread onto NA solid medium plates using a spreader method; sterile water from the last rinse of the underground rhizomes was used as a blank control; the petri dishes were incubated upside down at 30°C; after 2 days of incubation, when colonies appeared on the surface of the medium, colonies of different morphologies and sizes were selected and inoculated onto new NA solid medium plates for streak purification; after two consecutive purifications, several morphologically stable single strains of endophytic bacteria were obtained.

[0038] The obtained single strains were streaked onto NA solid slant medium and stored for a short period at 4°C; at the same time, the purified strains were added to sterile glycerol and stored for a long period at -80°C.

[0039] The pathogens used in the antagonism test were cultured on PDA solid medium; after the medium was cooled to 60°C, it was poured into 9cm diameter petri dishes at a rate of 20mL / dish to prepare plates.

[0040] Fusarium, the pathogen of sugarcane root rot ( Fusarium sp ) and the sugarcane leaf spot pathogen *Ulva globulus* ( Nigrospora sphaerica The pathogens (all pathogens were from the Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences) were inoculated onto PDA plates and cultured at 28℃ for 7 days. Then, mycelial cakes with a diameter of 5 mm were prepared using a sterile punch for later use.

[0041] The single strain was activated from NA slant medium stored at 4℃, streaked onto a new NA plate, and incubated upside down at 30℃ for 24 hours before being used for antagonism experiments.

[0042] Antagonistic activity was determined using the plate confrontation culture method: the pathogenic bacterial cake was placed in the center of a freshly prepared PDA plate and gently pressed to ensure full contact with the culture medium; then, the endophytic bacterial strain to be tested was inoculated 2 cm away from the bacterial cake; plates inoculated only with pathogenic bacteria and not with endophytic bacteria were used as controls; after sealing the petri dishes, they were incubated at 28°C for 24-72 h. By observing the growth of pathogen colonies, endophytic bacterial strains whose growth was significantly inhibited were identified as candidate strains with antagonistic activity. Further comparison of the inhibitory effects of different strains was conducted, and an endophytic bacterium that showed strong antagonistic activity against sugarcane leaf spot pathogen and sugarcane root rot pathogen was screened and named SG27.

[0043] Example 2: Morphological characteristics and species identification of strain SG27

[0044] (1) Colony morphology characteristics

[0045] The strain SG27 obtained in Example 1 was streaked into NA medium and cultured at 30°C for 24 hours. Morphological observation was then performed, and the colony morphology was as follows: Figure 1 As shown; The results showed that strain SG27 formed round, opaque colonies on the surface of NA solid medium. The colony surface was slightly dry, and the whole colony was slightly raised with slight wrinkles in the middle. The edges and the middle were slightly raised, irregular, and wavy.

[0046] (2) Morphological identification

[0047] The morphology of strain SG27 was observed using an optical microscope. The morphology under the optical microscope is as follows: Figure 2 As shown; The results showed that strain SG27 was arranged in a spherical pattern, with some cells being nearly rod-shaped.

[0048] The culture conditions for the strain were tested, and the initial pH and fermentation temperature of the culture medium were further optimized: the initial pH of the culture medium was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 using 0.1 mol / L HCl and 0.1 mol / L NaOH; the fermentation temperatures were set to 20, 24, 28, 32, and 36 °C. All other culture conditions for each treatment were consistent with those of the screening medium components, and the treatments were repeated three times. The results showed that the strain grew well at pH 7, and the optimal growth temperature was 37 °C.

[0049] (3) 16S rDNA gene sequence analysis

[0050] Genomic DNA of the strain was extracted and purified using the TSINGKE Plant DNA Extraction Kit (Universal); using the extracted genomic DNA as a template, the 16S rDNA fragment was amplified by PCR using universal primers 27F and 1492R; after amplification, the PCR product was sent to TSINGKE Biotechnology Co., Ltd. for sequencing. The sequence of upstream primer 27F is: 5′-AGTTTGATCMTGGCTCAG-3′ (SEQ ID No. 1). The sequence of the downstream primer 1492R is: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID No. 2); The PCR amplification system and amplification conditions are shown in Table 1 and Table 2, respectively. Table 1 16S rDNA amplification system

[0051] Table 2 16S rDNA Amplification Conditions

[0052] The obtained 16S rDNA sequences were submitted to the NCBI database for BLAST alignment analysis, and a phylogenetic tree was constructed using MEGA 11 software based on the Neighbor-Joining (NJ) method (e.g., ...). Figure 3 (As shown); the classification and identification process refers to reference materials such as "Handbook of Systematic Identification of Common Bacteria" and "Experimental Techniques in Agricultural Microbiology"; Sequence alignment results showed that the 16S rDNA sequence of strain SG27 was similar to that of previously reported Bacillus belye (…). Bacillus velezensis The type strain sequences are highly similar; in the phylogenetic tree, strain SG27 is similar to... Bacillus velezensis Those that cluster together in the same branch are the most closely related.

[0053] Based on the culture characteristics, morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis results of strain SG27, this strain was identified as *Bacillus belyesense*. Bacillus velezensis ).

[0054] Example 3: Verification of the genetic stability of the strain's antagonistic activity against fungi

[0055] The Bacillus belyss SG27 obtained in Example 1 was streaked into NA slant medium, cultured at 30°C for 24 h, and then stored at 4°C. Every 15 days, the strain was re-inoculated into a new NA slant medium for subculture. The subculture was carried out for 20 generations according to the above subculture method. The strains from the 5th, 10th, 15th and 20th generations of subculture were selected for activation treatment. The activated strains were then subjected to antagonistic activity tests against the fungal leaf spot pathogen and the root rot pathogen of sugarcane using the plate confrontation culture method described in Example 1. The experimental results showed that each generation of strains after continuous subculture maintained an inhibitory effect on the tested pathogens comparable to that of the initial strain, and the antagonistic activity of each generation of strains did not change significantly, indicating that Bacillus belye SG27 has good genetic stability in its antagonistic activity against the fungal leaf spot pathogen and the root rot pathogen of sugarcane.

[0056] Example 4: Determination of the antagonistic activity of the strain against fungi

[0057] Antagonistic activity was determined using the plate confrontation culture method: The fungal leaf spot pathogen of sugarcane, *Ichthyophthirius multifiliis* (Saccharomyces cerevisiae), was separately... Nigrospora sphaerica ), Fusarium, the pathogen of sugarcane fungal root rot ( Fusarium spInoculate the bacteria onto PDA plates and incubate at 28°C for 7 days. Then, use a sterile punch to prepare bacterial cakes with a diameter of 5 mm for later use. Inoculate Bacillus belye SG27 onto NB liquid medium and incubate at 28°C on a shaker for 24 hours to prepare a bacterial suspension for later use. On freshly prepared PDA plates (8.4 cm in diameter), the center position was marked using a cross-marking method. Pathogen bacterial pellets were inoculated into the center of the plate. Then, approximately 2 cm from the pellets, *Bacillus belyssioides* SG27 strain was dotted along the cross-marking lines. Plates inoculated only with the pathogen and without antagonistic bacteria served as controls. Each treatment was repeated in triplicate and cultured at 28°C for 7 days. After culture, the diameter of the pathogen colonies was measured, and the inhibition rate was calculated. Figures 4-7 ); The formula for calculating the inhibition rate is as follows: Inhibition rate (%) = [(Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / (Coronavirus colony diameter in control group - Mycelium cake diameter)] × 100%; The results showed that the growth of *Fusarium oxysporum* and *Ulva canis* leaf spot pathogens in the treatment group was significantly inhibited, and their colony sizes were significantly smaller than those in the control group, indicating that strain SG27 had a strong inhibitory effect on these pathogens. *Bacillus belyss* SG27 showed an inhibition rate of 63.86% against *Fusarium oxysporum* and 71.27% against *Ulva canis*, the pathogen of sugarcane fungal leaf spot. Therefore, *Bacillus belyss* SG27 has a significant inhibitory effect on both *Fusarium oxysporum* and *Ulva canis*, the pathogen of sugarcane fungal leaf spot.

[0058] Example 5: Effects of strain on fungal hyphal morphology

[0059] Bacillus vesiculosus SG27 was selected and compared with *Ichthyophthirius multifiliis*, the pathogen of sugarcane fungal leaf spot disease. Nigrospora sphaerica ), Fusarium, the pathogen of sugarcane fungal root rot ( Fusarium sp The culture plates after 7 days of plate confrontation culture were used as the treatment group; under aseptic conditions, the newly formed hyphae of the pathogen near the Bacillus belyss SG27 colony were picked up with sterile toothpicks. Figure 8 and Figure 9 Temporary slides were prepared; newly formed hyphae at the edge of pathogen colonies cultured alone on PDA medium for 7 days were used as a control group; the morphological characteristics of pathogen hyphae in the treatment group and the control group were observed and compared using an optical microscope at 20× magnification. The results are as follows: Figures 10-13 As shown; The results showed that after treatment with Bacillus belye SG27, Fusarium ( FusariumThe hyphae of the control group (Fusarium sp.) showed obvious swelling and deformity, with the hyphal tips swollen into a round shape and the interseptal spacing shortened; the hyphae of the control group (Fusarium sp.) were smooth, uniform in thickness, and grew naturally in a straight direction. After treatment with Bacillus vesiculosus SG27, *Ichthyophthirius multifiliis* (a type of mold) was found in sugarcane. Nigrospora sphaerica The control group showed abnormal phenomena such as swelling, deformation, and end enlargement of the hyphae, and obvious aggregation of protoplasm inside the hyphae; the control group showed intact hyphae structure, regular morphology, and no obvious abnormalities were observed.

[0060] Therefore, Bacillus belye SG27 can significantly affect the normal hyphal growth of Fusarium spp., the pathogen of sugarcane root rot, and Mycospora sugarcane, the pathogen of sugarcane fungal leaf spot. It is speculated that it inhibits the growth of pathogens by disrupting their hyphal structure.

[0061] Example 6: Detection of genes related to biocontrol and growth promotion of the strain

[0062] To further analyze the biocontrol and plant growth-promoting potential of Bacillus belyssus SG27, genes related to antagonistic activity and growth-promoting function in this strain were detected. Twelve genes related to secondary metabolite synthesis and biocontrol function were amplified and analyzed using PCR. The detected genes included: SrfAA , yndJ , fenD , ituD , yngG , bamD , dhb , dfn , bae , mln , bac and SboA The specific steps are as follows: (1) Preparation of genomic DNA Strains SG27 were inoculated onto NA solid medium using the streak method and activated by culturing at 37°C for 24 h. Single colonies were picked up with sterile toothpicks and placed in centrifuge tubes containing 50 μL of lysis buffer. The mixture was thoroughly mixed and lysed in a water bath at 80°C for 15 min. The mixture was then centrifuged at 8000 r / min for 1 min, and the supernatant was used as the genomic DNA template for PCR amplification.

[0063] (2) PCR amplification

[0064] The primer sequences for each gene used for detection are shown in Table 3, the PCR amplification system is shown in Table 4, the total reaction volume is 25 μL, and the amplification program is shown in Table 5.

[0065] Table 3. Genes and primer sequences related to biocontrol and growth promotion.

[0066] Table 4. Biocontrol / Growth-Promoting Gene Amplification System

[0067] Table 5. Biocontrol / Growth-Promoting Gene Amplification Procedure

[0068] (3) Analysis of test results

[0069] The PCR amplification products were detected by agarose gel electrophoresis, and the results are as follows: Figure 14 As shown.

[0070] The results showed that, except SrfAA、bamD Besides genes, the remaining 10 genes ( bae、yndJ , fenD , ituD , yngG , dhb , dfn , mln , bac and SboA All of these genes were successfully amplified in the genomic DNA of Bacillus belyssus SG27. Therefore, strain SG27 contains a variety of functional genes related to antagonistic activity and biocontrol, providing a molecular-level basis for its application in the biological control of sugarcane diseases and plant growth promotion.

[0071] Example 7: Detection of plant growth-promoting function of the strain

[0072] To evaluate the plant growth-promoting potential of Bacillus belyss SG27, its ability to solubilize inorganic phosphorus, potassium, produce siderophores, produce IAA, and produce ACC deaminase was tested. The specific methods are as follows: (1) Phosphorus solubilization capacity test Bacillus belye SG27 was inoculated onto Pikovskaya inorganic phosphate-solubilizing solid medium and cultured at 30°C for 7 days. If a clear halo formed around the colony, the strain was considered to have the ability to solubilize inorganic phosphate. The results were as follows: Figure 15 As shown.

[0073] (2) Potassium solubilization capacity test

[0074] The potassium-solubilizing ability of the strain was tested using silicate medium (with a poorly soluble potassium source as the sole potassium source): Strain SG27 was inoculated onto medium plates and cultured at 30°C for 7 days; if a clear or erosion zone formed around the colony, the strain was considered to have potassium-solubilizing ability. The results are as follows: Figure 16 As shown.

[0075] (3) Testing of iron production capacity

[0076] The siderophore-producing ability of the strain was detected using the CAS (Chrome Azurol S) plate method. In the CAS plate, Chrome Azurol S, ferric ions, and hexadecyltrimethylammonium bromide form a blue complex. When the strain secretes siderophores and chelates Fe... 3+ When the complex is destroyed, the color of the plate changes from blue to yellow; Strain strain SG27 was inoculated onto CAS medium plates and cultured at 30°C for 7 days. If a distinct yellow halo appeared around the colony, the strain was considered to have siderophore-producing ability. The results are as follows: Figure 17 As shown.

[0077] (4) Quantitative determination of indole-3-acetic acid (IAA)

[0078] The quantitative determination of IAA followed the Glickmann method. Activated test strains were inoculated into liquid Landy medium (purchased from Guangdong Huankai Biotechnology Co., Ltd., batch number 250811A50) containing L-tryptophan and without tryptophan. After continuous culture for 5 days at 28℃ and 160 rpm in a shaker, 1 mL of the bacterial culture was centrifuged at 10000 rpm for 5 min, and the supernatant was used as the test sample. The Salkowski colorimetric method was used to determine the IAA content in the test sample. An IAA secretion amount was obtained by preparing an IAA standard curve. The Salkowski colorimetric method was used to accurately determine the IAA secretion amount of the test strain under tryptophan-containing / non-tryptophan conditions, exploring the strain's ability to synthesize IAA and the effect of tryptophan on its synthesis.

[0079] (5) ACC deaminase activity assay

[0080] The α-butanolic acid content was obtained by culturing in ADF liquid medium. The Bradford method is based on the characteristic that Coomassie Brilliant Blue G-250 changes color from brownish-red to blue after binding with protein. Within the concentration range of up to 25 mg / mL, the protein concentration and the absorbance value at 595 nm showed a linear relationship. A standard curve was plotted by measuring the absorbance of a known concentration of bovine serum albumin (BSA) standard solution, and then the protein concentration of unknown samples was calculated based on the sample absorbance. ACC deaminase activity (μmol / (mg·h)) = (α-butanolic acid content generated in the reaction system (μmol) ÷ bacterial protein amount (mg) ÷ reaction time (h)).

[0081] (6) Results Analysis

[0082] As shown in the figure, Bacillus belye SG27 formed distinct clear zones on inorganic phosphorus-solubilizing and potassium-solubilizing media, and a clear halo on CAS medium. The IAA content was 10.2963 μg / mL. The α-butanone content in SG27 without ACC was 0.313 μmol, while the α-butanone content with ACC was 2.90 μmol. The protein content in SG27 was 0.406 mg / mL, and the enzyme activity of SG27 was 0.265 μmol / (mg·h). This indicates that the strain has the ability to solubilize inorganic phosphorus, potassium, produce siderophores, produce IAA, and produce ACC deaminase.

[0083] Example 8 Evaluation of the disease prevention and growth promotion effects of the strain under sugarcane pot cultivation conditions

[0084] (1) Preparation of materials for sugarcane potted plants

[0085] Healthy sugarcane seedlings obtained from tissue culture were selected, and after washing away the residual culture medium from the roots, they were transplanted into seedling bags for acclimatization. When the sugarcane seedlings grew to 3-4 true leaves (about 1 month), they were transplanted into plastic pots containing sterilized substrate. The plastic pots were 11 cm in diameter and 12 cm in height. After transplanting, water regularly to keep the substrate moist, and fertilize once a week. The fertilization method is to apply compound fertilizer at a rate of 2g per sugarcane seedling, wherein the mass fraction of nitrogen (N), phosphorus pentoxide (P2O5) and potassium oxide (K2O) in the compound fertilizer is 15%. When the sugarcane seedlings grow to 5-6 true leaves, they will be used for subsequent potted disease prevention and growth promotion experiments.

[0086] (2) Preparation of Bacillus belye fermentation broth

[0087] Bacillus vesiculus SG27 stored at -80℃ in glycerol was activated and inoculated onto NA solid medium using the streak method, and cultured at 30℃ for 24 h. A single colony was picked and inoculated into 200 mL of NA liquid medium, and cultured at 37℃ and 220 rpm for 48 h with shaking to obtain the fermentation broth. The fermentation broth was diluted with sterile water to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL was used as the treatment solution for Bacillus belyssus in pot experiments.

[0088] (3) Preparation of pathogen spore suspension

[0089] Fusarium, the pathogen of sugarcane root rot ( Fusarium sp.) and the fungal leaf spot pathogen of sugarcane, *Ichthyophthirius multifiliis* (sp.) Nigrospora sphaericaThe mycelial cakes were inoculated into liquid PDA medium and cultured with shaking at 28℃ and 220 r / min for 3 days. After the culture was completed, the culture medium was filtered through four layers of sterile gauze to collect the spore suspension. The pathogenic fungal spore suspension was diluted to 1×10⁻⁶ with sterile water. 6 CFU / mL, for later use.

[0090] (4) Grouping and treatment of potted plants

[0091] Sugarcane potted seedlings were randomly divided into 6 groups, with 5 plants in each group, and the experiment was repeated 3 times. The treatments are as follows: 1) Control group: Only the roots were irrigated with NA liquid culture medium; 2) Pathogen treatment group I: Irrigation only with sugarcane root rot pathogen spore suspension; 3) Pathogen Treatment Group II: Irrigation only with a suspension of sugarcane fungal leaf spot pathogen spores; 4) Growth-promoting group: Bacillus beleibasis SG27 fermentation broth was applied to the roots only on the 7th day; 5) Biocontrol treatment group I: First, irrigate with a suspension of sugarcane root rot pathogen spores, and then irrigate with Bacillus belye SG27 fermentation liquid on the 7th day; 6) Biocontrol treatment group II: First, irrigate with a suspension of sugarcane fungal leaf spot pathogen spores, and then irrigate with Bacillus belye SG27 fermentation liquid on the 7th day; Before irrigation, the sugarcane roots were slightly damaged, and each plant was irrigated with 200 mL each time. After the treatment, the plants in each group were placed under the same environmental conditions for cultivation.

[0092] (5) Disease survey and evaluation of prevention efficacy

[0093] Forty-five days after treatment, the disease incidence of sugarcane plants in each treatment group was investigated. The disease index was calculated according to the disease grading standards shown in Tables 6 and 7, and the control effect of Bacillus vesiculosus SG27 on sugarcane fungal leaf spot and sugarcane root rot was calculated accordingly.

[0094] (6) Disease grading standards and evaluation of control effects

[0095] The following standards were used to classify sugarcane root rot and sugarcane fungal leaf spot diseases.

[0096] Table 6 Grading Standards for Sugarcane Root Rot Disease

[0097] Table 7 Grading Standards for Sugarcane Leaf Spot Disease

[0098] Disease index and prevention and control effectiveness are calculated using the following formula: , Prevention and control effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.

[0099] (7) Experimental Results and Analysis

[0100] On day 45 after sugarcane seedlings were transplanted into pots, the disease incidence of sugarcane plants in each treatment group was investigated; the growth status of the plants after the four treatments is as follows: Figure 18 and Figure 19 As shown, the occurrence of diseases in the leaves of each group was statistically analyzed, and the results are shown in Tables 8 and 9. Table 8. Statistics on the classification of sugarcane root rot

[0101] Table 9. Statistics on the Grading of Sugarcane Leaf Spot Diseases

[0102] Note: In Tables 8 and 9, (1) group was irrigated with NA liquid culture medium alone; (2) group was inoculated with sugarcane root rot pathogen alone; (3) group was inoculated with sugarcane leaf spot pathogen alone; (4) group was inoculated with Bacillus belye SG27 fermentation broth alone; (5) group was inoculated with sugarcane root rot pathogen and applied with Bacillus belye SG27 fermentation broth; (6) group was inoculated with sugarcane leaf spot pathogen and applied with Bacillus belye SG27 fermentation broth.

[0103] Based on the above statistical data and formula calculations, we can conclude that: The average disease index of the pathogen control group (inoculated only with the pathogen) was 88.33; the average disease index of the sugarcane leaf spot pathogen control group (inoculated only with the leaf spot pathogen) was 76.67; the disease index of the biocontrol group (inoculated with root rot pathogen + Bacillus belyss SG27) was significantly reduced to 20; the disease index of the biocontrol group (inoculated with leaf spot pathogen + Bacillus belyss SG27) was significantly reduced to 26.67; the disease index of the blank control group and the growth promotion group was close to 0. Calculations showed that *Bacillus belye* SG27 achieved integrated control efficacy of 68.33% against sugarcane root rot and 50% against sugarcane fungal leaf spot. Therefore, *Bacillus belye* SG27 possesses strong biocontrol capabilities, effectively controlling the infection and damage caused by the pathogens of sugarcane root rot and sugarcane black spore leaf spot, significantly reducing disease losses. It is a biocontrol strain with significant application potential.

[0104] As can be seen from the above embodiments, the present invention provides a strain of Bacillus belye G27 and its applications. This strain has a significant inhibitory effect on a variety of plant pathogenic fungi, especially effectively inhibiting pathogens that cause sugarcane leaf spot and sugarcane root rot. At the same time, this strain also promotes the growth of sugarcane plants.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Bacillus belye SG27, characterized in that, The Latin name of the Bacillus belyssus SG27 is Bacillus velezensis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 28, 2026, with accession number GDMCC No: 67754.

2. A microbial preparation, characterized in that, Includes Bacillus vesiculosus SG27 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The dosage form of the microbial preparation is liquid or solid.

4. The microbial preparation according to claim 3, characterized in that, The concentration of Bacillus vesiculus SG27 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

5. The use of Bacillus vesiculosus SG27 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 4 in the prevention and control of sugarcane diseases.

6. The application according to claim 5, characterized in that, The sugarcane disease mentioned is a fungal sugarcane disease.

7. The application according to claim 6, characterized in that, The fungal sugarcane diseases mentioned include sugarcane fungal leaf spot and sugarcane root rot.

8. The application according to claim 7, characterized in that, The pathogen of the sugarcane fungal leaf spot disease is *Ulva globosum*; the pathogen of the sugarcane root rot disease is *Fusarium*.

9. The use of Bacillus vesiculosus SG27 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 4 in promoting sugarcane growth.

10. The application according to claim 9, characterized in that, The promotion of sugarcane growth is achieved through one or more of the following: the ability of Bacillus vesiculosus SG27 to solubilize inorganic phosphorus, potassium, produce siderophores, produce indole-3-acetic acid, and produce 1-aminocyclopropane-1-carboxylic acid deaminase.

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