Application of Bacillus velezensis FG01 in the root system of perennial sugarcane

Abstract

This invention relates to the field of plant disease control technology, and in particular to a Bacillus belye species for the root system of perennial sugarcane (B. belye). Bacillus from Velez Application of FG01. This invention provides a *Bacillus belyceae* FG01, the Latin name of which is... Bacillus velezensis This strain 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: 67755. This strain exhibits significant inhibitory effects against sugarcane pathogenic fungi, particularly effectively inhibiting *Ulva globulus*, the pathogen causing sugarcane leaf spot disease. Simultaneously, this strain also promotes sugarcane plant growth.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a Bacillus belye species for the root system of perennial sugarcane (B. belye). Bacillus velezensis Application of FG01. Background Technology

[0002] Plant endophytes are a group of microorganisms that colonize plant tissues and establish long-term symbiotic relationships with the host plant. They typically complete part or all of their life cycle within the plant tissues and do not cause significant disease symptoms. As a key component of the plant micro-ecosystem, plant endophytes play a crucial role in the interaction between plants and their external environment. Existing research shows that plant endophytes can participate in plant nutrient metabolism processes, enhance the absorption and utilization efficiency of mineral elements, and inhibit various plant pathogenic microorganisms. Therefore, they are considered important research subjects for biocontrol and the development of agricultural microbial resources. However, the molecular mechanisms of interaction between endophytic microorganisms and host plants remain unclear, and their application potential in agricultural production has not yet been fully explored.

[0003] In the field of agricultural biotechnology, utilizing microbial resources for plant disease control has become a research hotspot. Compared to traditional control strategies relying on chemical pesticides or the breeding of disease-resistant varieties, endophytic bacteria exhibit unique advantages in disease control. These microorganisms can exert their defensive functions within plants without complex genetic modification methods, reducing research and development cycles and application costs, while avoiding the safety controversies associated with transgenic technology. Furthermore, endophytic bacteria are relatively safe for plants and the environment, have minimal impact on non-target organisms, and exhibit good stability within plants, enabling them to maintain their disease-preventing effects even at low application rates. The mechanisms of action of plant endophytic bacteria in biocontrol are diverse, mainly including secreting secondary metabolites with antibacterial activity, competing with pathogenic microorganisms for living space and nutrient resources within the plant, inducing systemic resistance responses in the host plant, and producing various extracellular enzymes to disrupt the cell structure of pathogens or reduce their pathogenicity. These mechanisms work synergistically, enabling endophytic bacteria to demonstrate excellent control effects in plant diseases. However, the endophytic bacteria currently discovered still have certain limitations in terms of the stability and broad-spectrum nature of their control effects and their compatibility with different plant varieties, making it difficult to meet the actual needs of large-scale agricultural production.

[0004] In terms of mechanisms of action, plant endophytic bacteria can limit the infection and spread of pathogenic microorganisms through multiple pathways. For example, they synthesize and secrete various secondary metabolites with broad-spectrum antibacterial activity, such as lipopeptides and polyketides. These substances can directly act on the cell membrane, cell wall, or key metabolic pathways of pathogens, thereby inhibiting their growth and reproduction. Within plant tissues, they compete with pathogens for living space and nutrient sources, inhibiting pathogen colonization and growth by seizing ecological niches and consuming key nutrients. They activate the host plant's defense response, inducing systemically acquired resistance (SAR) and induced systemic resistance (ISR), enhancing the plant's overall defense against pathogens. They also secrete extracellular enzymes, such as chitinase and glucanase, which disrupt the cell structure of pathogens or weaken their pathogenicity. These mechanisms collectively constitute the basis of endophytic bacteria in the biocontrol of plant diseases.

[0005] Bacillus belesiensis ( Bacillus velezensis *Pseudomonas aeruginosa* is a class of functional bacteria that has received widespread attention in the field of agricultural biological control. As a Gram-positive bacterium, it possesses extremely strong environmental adaptability, capable of surviving and colonizing plants under various extreme conditions. Its spore-forming structure endows it with excellent stress resistance and stability, facilitating large-scale industrial production and formulation processing. It can also synthesize a variety of biologically active secondary metabolites. However, strains from different isolation sources exhibit significant differences in biological characteristics and control efficacy. Most reported strains currently have drawbacks such as a narrow control spectrum, unstable control efficacy against specific pathogens, and significant differences in affinity with different plant varieties. Furthermore, the screening system for highly efficient strains targeting specific crops and diseases is still imperfect, severely restricting its widespread application in agricultural production. Therefore, screening for functional strains with clearly defined sources and stable performance for specific crops remains a practical necessity.

[0006] sugar cane( Saccharum officinarumSugarcane (Bacillus belye) is one of the world's most important sugar crops, widely cultivated in tropical and subtropical regions. Its yield and quality directly affect the economic benefits of the sugar industry and the sustainable development of agriculture. However, sugarcane is highly susceptible to various pathogenic microorganisms throughout its growth cycle, including fungi, bacteria, and viruses. These diseases can lead to leaf wilting, stem rot, and stunted growth, resulting in yield reductions of over 30% or even total crop failure in severe cases. Sugarcane roots, as vital organs for photosynthesis, possess a complex and diverse internal microecological environment, rich in endophytic bacterial resources. These endophytic bacteria form a close symbiotic relationship with sugarcane, not only promoting its growth and development but also potentially inhibiting pathogens through various mechanisms such as producing antibacterial substances, competing for ecological niches, and inducing plant disease resistance. They represent a biological control resource with significant development potential. Currently, research on the isolation and identification of Bacillus belye from sugarcane roots and its application in plant disease control remains limited, requiring further exploration and utilization.

[0007] In agricultural production, chemical pesticides remain a crucial means of plant disease control. However, long-term or improper use can easily lead to a series of serious problems. First, the extensive use of chemical pesticides causes environmental pollution, and pesticide residues can accumulate in soil, water bodies, and agricultural products, posing a potential threat to ecosystems and human health. Second, pathogens easily develop resistance to chemical pesticides, leading to a gradual decline in control effectiveness. This necessitates continuously increasing dosages or changing pesticide types, further exacerbating environmental pressure and production costs. Furthermore, while killing pathogens, chemical pesticides also disrupt beneficial microbial communities in the soil, causing soil microecological imbalance and affecting soil fertility and the long-term healthy growth of crops. Therefore, developing endophytic Bacillus belyssus strains with clear origins, high safety, and good disease control efficacy for the biological control of plant diseases is of significant practical importance and has broad application prospects for reducing the use of chemical pesticides, lowering pesticide residues, protecting the ecological environment, and promoting sustainable agricultural development. Summary of the Invention

[0008] The purpose of this invention is to provide a Bacillus berberis FG01 that can prevent sugarcane diseases and promote sugarcane growth.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Bacillus belyceae FG01, 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: 67755.

[0010] The present invention also provides a microbial preparation comprising the aforementioned Bacillus belyssus FG01.

[0011] Preferably, the dosage form of the microbial preparation is liquid or solid.

[0012] Preferably, the concentration of Bacillus berberis FG01 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

[0013] The present invention also provides the application of the aforementioned Bacillus berberis FG01 or the aforementioned microbial preparation in the prevention and control of sugarcane diseases.

[0014] Preferably, the sugarcane disease is a fungal sugarcane disease.

[0015] Preferably, the fungal sugarcane disease includes sugarcane fungal leaf spot; the pathogen of the sugarcane fungal leaf spot is *Ulva globosum*.

[0016] The present invention also provides the application of the aforementioned Bacillus berberis FG01 or the aforementioned microbial preparation in promoting sugarcane growth.

[0017] Preferably, the promotion of sugarcane growth is achieved through one or more of the nitrogen-fixing ability, potassium-solubilizing ability, siderophore-producing ability, indole-3-acetic acid-producing ability, and 1-aminocyclopropane-1-carboxylic acid deaminase-producing ability of Bacillus belye FG01.

[0018] Beneficial effects: This invention provides a strain of *Bacillus belye* FG01, isolated from the root tissue of healthy sugarcane in its fifth year of ratooning. This strain belongs to the endophytic antagonistic bacteria of sugarcane and possesses excellent endophytic colonization characteristics. Experimental results show that this strain has a significant inhibitory effect on sugarcane pathogenic fungi, especially effectively inhibiting *Ulva globosum*, the pathogen causing sugarcane leaf spot disease. Simultaneously, this strain also promotes sugarcane plant growth, which can improve plant 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

[0019] Figure 1 The colony morphology of Bacillus belyssus FG01; Figure 2 Microscopic structure diagram of Bacillus belyssus FG01; Figure 3 Phylogenetic classification of Bacillus belyss FG01; Figure 4The image shows a confrontation culture experiment between Bacillus belyssus FG01 and the pathogen of sugarcane leaf spot disease. The left image represents the treatment group, and the right image represents the control group. Figure 5 The results show the effect of Bacillus vesicles FG01 on the mycelial morphology of sugarcane leaf spot pathogen. The upper figure shows the mycelial morphology of sugarcane leaf spot pathogen after treatment with Bacillus vesicles FG01, and the lower figure shows the mycelial morphology of sugarcane leaf spot pathogen without treatment. Figure 6 The results of detection of genes related to biocontrol and growth promotion in Bacillus belyss FG01; Figure 7 The results of the plant growth-promoting function test of Bacillus belyssus FG01 are as follows: A is the result of nitrogen fixation capacity test, B is the result of potassium solubilization capacity test, and C is the result of iron carrier production capacity test. Figure 8 The results of the evaluation of the disease prevention and growth promotion effects of Bacillus belyssus FG01 under sugarcane pot cultivation conditions.

[0020] Preservation Instructions

[0021] The Latin name of Bacillus belyssus FG01 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: 67755. Detailed Implementation

[0022] This invention provides a Bacillus belyceae FG01, 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: 67755.

[0023] The present invention also provides a microbial preparation comprising the aforementioned Bacillus belyssus FG01.

[0024] In this invention, the dosage form of the microbial preparation is liquid or solid.

[0025] In this invention, the concentration of Bacillus berberis FG01 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL.

[0026] The present invention also provides the application of the aforementioned Bacillus berberis FG01 or the aforementioned microbial preparation in the prevention and control of sugarcane diseases.

[0027] In this invention, the sugarcane disease is a fungal sugarcane disease.

[0028] In this invention, the fungal sugarcane disease includes sugarcane fungal leaf spot; the pathogen of the sugarcane fungal leaf spot is *Ulva globosum*.

[0029] The present invention also provides the application of the aforementioned Bacillus berberis FG01 or the aforementioned microbial preparation in promoting sugarcane growth.

[0030] In this invention, the promotion of sugarcane growth is achieved through one or more of the nitrogen-fixing ability, potassium-solubilizing ability, siderophore-producing ability, indole-3-acetic acid (IAA)-producing ability, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing ability of Bacillus belye FG01.

[0031] 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.

[0032] In the embodiments of the present invention, the preparation method of the culture medium is as follows: NA medium: 5g peptone, 1g yeast extract, 3g beef extract, 10g glucose, 15g agar, add distilled water to a final volume of 1000mL, adjust pH to 7.0, and sterilize at 121℃ for 20min. 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; Ashby's nitrogen-free solid medium: mannitol 10.0g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4 0.1g, CaCO3 5.0g, agar 18.0g, add deionized water to a final volume of 1L, adjust pH to 6.8~7.0, and autoclave at 121℃ for 20min; Ashby's nitrogen-free medium: mannitol 10.0g, KH2PO4 0.2g, MgSO4 0.2g, NaCl 0.2g, CaSO4 0.1g, CaCO3 5g, agar 21g, pH 6.8~7.0, dissolved in deionized water and brought to a final volume of 1L; autoclave at 121℃ for 20min; 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.

[0033] Example 1: Screening of endophytic antagonistic bacteria in sugarcane

[0034] 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.

[0035] 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.

[0036] The disinfected underground seed tuber tissue was placed in a sterile mortar containing 9 mL of sterile water, sterile quartz sand was added, and the mixture was ground thoroughly. After standing for 15 minutes, 1 mL of the tissue suspension was taken and diluted to 10 mL. -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.

[0037] The obtained single strains were inoculated 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.

[0038] 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.

[0039] The pathogen of sugarcane leaf spot disease, *Ichthyophthirius multifiliis* (… Nigrospora sphaerica (From the Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences) The pathogen was inoculated onto PDA plates and cultured at 28°C for 7 days. Then, 5 mm diameter mycelial cakes were prepared using a sterile punch for later use.

[0040] 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.

[0041] 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 the pathogen of sugarcane leaf spot was obtained and named FG01.

[0042] Example 2: Morphological characteristics and species identification of strain FG01

[0043] (1) Colony morphology characteristics

[0044] The strain FG01 obtained in Example 1 was inoculated 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 FG01 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.

[0045] (2) Morphological identification

[0046] The morphology of strain FG01 was observed using an optical microscope. The morphology under the optical microscope is as follows: Figure 2 As shown; The results showed that strain FG01 was nearly spherical, with some cells arranged in short rod-like formations.

[0047] The culture conditions for the strain were tested, and the initial pH of the culture medium and fermentation temperature 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; and the fermentation speed was set to 90, 120, 150, 180, and 210 rpm. 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.

[0048] (3) 16S rDNA gene sequence analysis

[0049] 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

[0050] Table 2 16S rDNA Amplification Conditions

[0051] 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 FG01 was similar to that of previously reported Bacillus belye (…). Bacillus velezensis The type strain sequences are highly similar; in the phylogenetic tree, strain FG01 is similar to... Bacillus velezensis Those that cluster together in the same branch are the most closely related.

[0052] Based on the comprehensive analysis of the culture characteristics, morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence of strain FG01, this strain was identified as *Bacillus belyesense*. Bacillus velezensis ).

[0053] Example 3: Validation of the genetic stability of the strain's antagonistic activity against the fungal leaf spot pathogen of sugarcane.

[0054] The Bacillus belyssus FG01 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 new NA slant medium for subculture in the same manner. The subculture was carried out for 20 generations in accordance with the above subculture method. Strains from the 5th, 10th, 15th and 20th generations were selected for activation treatment. The activated strains were then subjected to antagonistic activity against the fungal leaf spot 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 pathogen comparable to that of the initial strain, and the antagonistic activity of each generation of strains did not change significantly, indicating that Bacillus belyssus FG01 has good genetic stability in its antagonistic activity against the fungal leaf spot pathogen of sugarcane.

[0055] Example 4: Determination of the antagonistic activity of the strain against the fungal leaf spot pathogen of sugarcane.

[0056] Antagonistic activity was determined using the plate confrontation culture method: The fungal leaf spot disease of sugarcane is caused by *Ichthyophthirius multifiliis* (Sugarcane spores). Nigrospora sphaerica The bacteria were inoculated onto PDA plates and cultured at 28°C for 7 days. Then, 5 mm diameter bacterial cakes were prepared using a sterile punch for later use. Bacillus berberis FG01 was inoculated onto NB liquid medium and cultured 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* strain FG01 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. Figure 4 ); 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 *Ichthyophthirius multifiliis* leaf spot pathogen in the treatment group was significantly inhibited, and its colony size was significantly smaller than that in the control group, indicating that strain FG01 had a strong inhibitory effect on *Ichthyophthirius multifiliis* leaf spot pathogen. Calculations showed that *Bacillus belyssioides* FG01 had an inhibition rate of 69.77% against *Ichthyophthirius multifiliis*, the pathogen of sugarcane fungal leaf spot; all of these showed significant inhibitory effects.

[0057] Example 5: Effects of the strain on the hyphal morphology of the fungal leaf spot pathogen.

[0058] Bacillus berleis FG01 and the fungal leaf spot pathogen of sugarcane, *Ichthyophthirius multifiliis* (Saccharomyces cerevisiae), were selected. Nigrospora spherical The culture plates that underwent a 7-day plate confrontation culture served as the treatment group. Under aseptic conditions, newly formed hyphae of the pathogen near the *Bacillus belyssae* FG01 colony were picked up with sterile toothpicks to prepare temporary slides. Newly formed hyphae at the edge of the pathogen colony, cultured alone on PDA medium for 7 days, served as the control group. The morphological characteristics of the pathogen hyphae in the treatment and control groups were observed and compared using an optical microscope at 40× magnification. The results are as follows: Figure 5 As shown; As shown in the figure, after treatment with Bacillus vesicularis FG01, the mycelia of *Ulva canis* exhibited abnormal phenomena such as swelling, deformation, and end enlargement, with obvious aggregation of protoplasm inside the mycelia. In contrast, the mycelia of *Ulva canis* in the control group had intact structure and regular morphology, with no obvious abnormalities observed. Therefore, Bacillus vesicularis FG01 can significantly affect the normal growth of the mycelia of *Ulva canis*, the pathogen of sugarcane fungal leaf spot disease, and it is speculated that it inhibits the growth of the pathogen by destroying the mycelial structure.

[0059] Example 6: Detection of genes related to biocontrol and growth promotion of the strain

[0060] To further analyze the biocontrol and plant growth-promoting potential of Bacillus belyssus FG01, 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 , indJ , science , ituD , new , bamD , dhb , burial , UAE , million , tax and SboA The specific steps are as follows: (1) Preparation of genomic DNA Strains of strain FG01 were inoculated onto NA solid medium using the streak method and cultured at 37°C for 24 h to activate them. 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.

[0061] (2) PCR amplification

[0062] 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.

[0063] Table 3. Genes and primer sequences related to biocontrol and growth promotion.

[0064]

[0065] Table 4. Biocontrol / Growth-Promoting Gene Amplification System

[0066] Table 5. Biocontrol / Growth-Promoting Gene Amplification Procedure

[0067] (3) Analysis of test results

[0068] The PCR amplification products were detected by agarose gel electrophoresis, and the results are as follows: Figure 6 As shown.

[0069] The results showed that all genes ( UAE , SrfAA , indJ , science , ituD , new , bamD , dhb , burial , million , tax and SboA All of these genes were successfully amplified in the genomic DNA of Bacillus belyssus FG01; therefore, strain FG01 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 plant diseases and the promotion of plant growth.

[0070] Example 7: Detection of plant growth-promoting function of the strain

[0071] To evaluate the plant growth-promoting potential of Bacillus belyssus FG01, its nitrogen-fixing capacity, potassium-solubilizing capacity, siderophore production capacity, IAA production capacity, and ACC deaminase production capacity were tested. The specific steps are as follows: (1) Nitrogen fixation capacity Strawberry strain FG01 was streaked onto Ashby nitrogen-free medium plates and cultured at 30°C for 7 days. Strains that could grow on nitrogen-free medium and form a clear zone or growth ring around the colony were deemed to have nitrogen-fixing ability. The results are as follows: Figure 7 As shown in A in the diagram.

[0072] (2) Potassium solubilization capacity test

[0073] The potassium-solubilizing ability of the strain was tested using silicate medium (with a poorly soluble potassium source as the sole potassium source): Strain FG01 was inoculated onto agar 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 7 As shown in B in the diagram.

[0074] (3) Testing of iron production capacity

[0075] 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 FG01 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 7 As shown in C.

[0076] (4) Quantitative determination of indole-3-acetic acid (IAA)

[0077] 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.

[0078] (5) ACC deaminase activity assay

[0079] 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)).

[0080] (6) Results Analysis

[0081] The results showed that *Bacillus belye* FG01 could grow normally on nitrogen-free and potassium-solubilizing media, and formed a transparent halo on CAS medium. The IAA content was 2.082 μg / mL. The α-butanone content in FG01 without ACC was 0.958 μmol, and the α-butanone content in FG01 with ACC was 4.89 μmol. The protein content in FG01 was 0.682 mg / mL, and the enzyme activity of FG01 was 0.240 μmol / (mg·h). This indicates that the strain has the ability to fix nitrogen, solubilize potassium, produce siderophores, produce IAA, and produce ACC deaminase. Therefore, *Bacillus belye* FG01 has multiple functional characteristics related to sugarcane nutrient acquisition and growth promotion, providing experimental evidence for its application in sugarcane growth promotion and agricultural production.

[0082] Example 8 Evaluation of the disease prevention and growth promotion effects of the strain under sugarcane pot cultivation conditions

[0083] (1) Preparation of materials for sugarcane potted plants

[0084] 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.

[0085] (2) Preparation of Bacillus belye fermentation broth

[0086] Bacillus belye FG01, stored in glycerol at -80℃, was activated and inoculated onto NA solid medium using the streak method. The medium was incubated at 30℃ for 24 h. Single colonies were then 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 (OD) 600 =1), used as a treatment solution for Bacillus vesiculosus in pot experiments.

[0087] (3) Preparation of pathogen spore suspension

[0088] The fungal leaf spot pathogen of sugarcane, *Ichthyophthirius multifiliis* (… Nigrospora sphaerica The fungal discs 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 (OD) 600 =0.01), for backup.

[0089] (4) Grouping and treatment of potted plants

[0090] Sugarcane potted seedlings were randomly divided into 4 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) The pathogen treatment group was irrigated only with a suspension of sugarcane fungal leaf spot pathogen spores; 3) Growth-promoting group: Bacillus beleibacillus FG01 fermentation broth was applied to the roots only on the 7th day; 4) Biocontrol treatment group: First, irrigate with a suspension of sugarcane fungal leaf spot pathogen spores, and then irrigate with Bacillus beleibasis FG01 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.

[0091] (5) Disease survey and evaluation of prevention efficacy

[0092] Thirty 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 Table 6, and the control effect of Bacillus berreatus FG01 on sugarcane fungal leaf spot was calculated accordingly.

[0093] (6) Disease grading standards and evaluation of control effects

[0094] The following standards were used to classify sugarcane root rot and sugarcane fungal leaf spot diseases.

[0095] Table 6 Grading Standards for Sugarcane Leaf Spot Disease

[0096] 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%.

[0097] (7) Experimental Results and Analysis

[0098] On day 30 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 in the three treatment groups is as follows: Figure 8 As shown in Table 7, the incidence of diseases in the root system and leaves of each group was statistically analyzed. Table 7. Statistics on the Grading of Sugarcane Leaf Spot Disease

[0099] Note: In Table 7, (1) group was irrigated with NA liquid culture medium alone; (2) group was inoculated with pathogens alone; (3) group was inoculated with Bacillus belysinus FG01 fermentation broth alone; and (4) group was inoculated with pathogens and irrigated with Bacillus belysinus FG01 fermentation broth.

[0100] Based on the above statistical data and formula calculations, we can conclude that: The disease index of the pathogen control group (inoculated only with pathogen) was 83.33; the disease index of the biocontrol group (inoculated with Bacillus belyssus FG01) was significantly reduced to 26.67; the disease index of the blank control group and the group inoculated only with FG01 was close to 0. Calculations showed that Bacillus belyss FG01 achieved a comprehensive control effect of 56.66% against sugarcane fungal leaf spot disease; Therefore, Bacillus belyssus FG01 ( Bacillus velezensis FG01 has strong biocontrol capabilities and can effectively control the infection and damage of sugarcane black spore leaf spot pathogen, significantly reducing disease losses. It is a biocontrol strain with important application potential.

[0101] As can be seen from the above embodiments, the present invention provides a *Bacillus belyssioides* species for the root system of perennial sugarcane (…). Bacillus velezensis Application of FG01. This strain has a significant inhibitory effect on sugarcane pathogenic fungi, especially effectively inhibiting *Ulva globulus*, the pathogen that causes sugarcane leaf spot disease. At the same time, this strain also promotes the growth of sugarcane plants.

[0102] 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 FG01, characterized in that, The Latin name of the Bacillus belysium FG01 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: 67755.

2. A microbial preparation, characterized in that, Includes Bacillus belyssus FG01 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 FG01 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

5. The use of Bacillus berberis FG01 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; the pathogen of the sugarcane fungal leaf spot is *Ulva globosum*.

8. The use of Bacillus berberis FG01 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 4 in promoting sugarcane growth.

9. The application according to claim 8, characterized in that, The promotion of sugarcane growth is achieved through one or more of the nitrogen-fixing ability, potassium-solubilizing ability, siderophore-producing ability, indole-3-acetic acid-producing ability, and 1-aminocyclopropane-1-carboxylic acid deaminase-producing ability of Bacillus belye FG01.

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