Bacillus velezensis for preventing and treating sugarcane leaf blight and application thereof

Abstract

The application discloses a bacillus velezensis for preventing and treating sugarcane leaf blight, which is bacillus velezensi NN02 with a preservation number of CGMCC No. 37628. The application also discloses application of the bacillus velezensi for preventing and treating sugarcane leaf blight, cabbage black spot and guava stem base rot. The bacillus velezensi NN02 for preventing and treating sugarcane leaf blight is separated from healthy mulberry variety Guiyou 12 branches, has antagonistic effect on sugarcane leaf blight and can effectively prevent and treat sugarcane leaf blight.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus bellis for the prevention and control of sugarcane leaf blight and its application. Background Technology

[0002] Sugarcane leaf blight is a serious fungal leaf disease of sugarcane, caused by the fungus *Stagonospora tainanensis*, belonging to the genus *Stagonospora* of the family Massarinaceae in the phylum Ascomycota, class Dothideomycetes, order Pleosporales. In the early stages, red or reddish-brown spots appear on young leaves, often surrounded by a yellow or red halo. These spots gradually enlarge and become elongated spindle-shaped. Later, the spots merge, causing the leaves to wither and die, resulting in a sharp reduction in sugarcane yield and causing significant economic losses to the sugar industry. It has now become one of the major obstacles to the development of my country's sugar industry.

[0003] Currently, the control of sugarcane leaf blight mainly relies on disease-resistant varieties and the application of chemical agents. However, the breeding of traditional disease-resistant varieties has significant limitations, such as the long time required. Furthermore, the overuse of chemical pesticides leads to environmental pollution, soil residues, and pesticide resistance, posing a potential threat to human health through bioaccumulation in the food chain. Therefore, developing environmentally friendly biological control technologies has become an urgent problem to be solved in the field of sugarcane leaf blight control. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a strain of Bacillus berberis NN02 isolated from branches of a healthy mulberry variety, Guiyou 12, which exhibits antagonistic activity against sugarcane leaf blight. This antagonistic activity is utilized for safe, effective, and environmentally friendly biological control of sugarcane leaf blight.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] A strain of Bacillus velezensi for controlling sugarcane leaf blight, namely Bacillus velezensi NN02, with accession number CGMCC No.37628, was deposited on February 2, 2026, at the China General Microbiological Culture Collection Center, located at No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0007] The application of Bacillus vesiculosus, as described above, in the control of sugarcane leaf blight.

[0008] The application of Bacillus berberis, which is used to control sugarcane leaf blight, in the control of black spot disease of cabbage, as described above.

[0009] The application of Bacillus vesiculosus, which is used to control sugarcane leaf blight as described above, in the control of passion fruit stem base rot.

[0010] Preferably, the application is performed after preparing a sterile fermentation broth of Bacillus vesiculosus for the prevention and control of sugarcane leaf blight.

[0011] Preferably, the amount of sterile fermentation broth added is 10% by volume.

[0012] Preferably, the sterile fermentation broth is prepared by inoculating 4 mL of antagonistic bacterial seed culture into 100 mL of commonly used Bacillus subtilis culture medium for fermentation, incubating at 28°C and 200 r / min for 72 h, centrifuging to collect the supernatant, and filtering it through a 0.22 µm bacterial filter to obtain the sterile fermentation broth.

[0013] Preferably, the seed culture is prepared by culturing Bacillus belye NN02 on LA medium for 48 hours, then inoculating a loopful of colonies into 5 mL of LB medium and culturing in a constant temperature shaker at 28°C and 200 rpm for 16-20 hours.

[0014] Preferably, the fermentation medium consists of 20.0g glucose, 15.0g peptone, 5.0g NaCl, 0.5g beef extract, and deionized water to a final volume of 1000mL, with a pH of 7.0-7.2; and is sterilized at 121℃ for 20 min.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The Bacillus velezensi NN02 strain used in this invention to control sugarcane leaf blight was isolated from branches of the healthy mulberry variety Guiyou 12. It has an antagonistic effect on sugarcane leaf blight and can effectively control the disease.

[0017] Preservation Information

[0018] Bacillus velezensi NN02 was deposited on February 2, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37628. Attached Figure Description

[0019] Figure 1This invention describes the inhibitory effect of Bacillus velezensi NN02 on plant pathogenic fungi. In the CK row, the pathogen was inoculated only in the center of the PDA medium. In the treatment row, the pathogen was inoculated in the center of the PDA medium and four patches of Bacillus velezensi NN02 were inoculated around the perimeter. The pathogens were: A: sugarcane leaf blight pathogen; B: cabbage black spot pathogen; C: passion fruit stem base rot pathogen; D: citrus / yellow peel anthracnose pathogen; E: gray mold; F: sugarcane ring spot pathogen; G: banana wilt pathogen; H: sugarcane top rot pathogen; I: lettuce sclerotium rot pathogen; J: peanut white mold pathogen; K: Rhizoctonia solani.

[0020] Figure 2 This invention describes the inhibitory effect of Bacillus velezensi NN02 fermentation broth on plant pathogenic fungi. In the control row, pathogens were inoculated only in the center of PDA medium, while in the treatment row, pathogens were inoculated in the center of PDA medium containing 10% (v / v) of NN02 fermentation broth. The pathogens included: A: Sclerotinia sclerotiorum var. lettuce; B: Black spot fungus of cabbage; C: Rhizoctonia solani; D: Leaf blight fungus of sugarcane; E: Anthracnose fungus of citrus / yellow peel; F: Gray mold; G: Sugarcane ring spot fungus; H: Peanut white spot fungus; I: Banana wilt fungus; J: Passion fruit stem base rot fungus; K: Sugarcane top rot fungus.

[0021] Figure 3 This invention relates to the effect of Bacillus velezensi NN02 on the mycelium of sugarcane leaf blight pathogen. Row A represents the control (CK). The leftmost image shows the colony of sugarcane leaf blight pathogen on PDA medium, the middle image shows a 2000x magnified view of the mycelium at the edge of the colony on PDA medium, and the rightmost image shows a 5000x magnified view of the mycelium at the edge of the colony on PDA medium.

[0022] Line B shows the mycelium of *Bacillus velezensi* NN02 fermentation broth treated with this invention. The leftmost image shows *Bacillus velezensi* colonies on PDA medium containing 10% sterile fermentation filtrate; the middle image shows a 2000x magnified view of the mycelium at the edge of the colonies on PDA medium containing 10% sterile fermentation filtrate; and the rightmost image shows a 5000x magnified view of the mycelium at the edge of the colonies on PDA medium containing 10% sterile fermentation filtrate.

[0023] Figure 4 This invention describes the effects of different treatments on the severity of sugarcane leaf blight; wherein, A is treatment 2 of Example 5; B is treatment 1 of Example 5; C is treatment 3 of Example 5; D is treatment 4 of Example 5; and E is treatment 6 of Example 5. Detailed Implementation

[0024] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the embodiments are commercially available.

[0025] The tested pathogens are: *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, *Brachystomata var. chinensis*, and *Brachystomata var. chinensis*. All of the above pathogens were isolated, identified, and preserved at -80℃ by the State Key Laboratory of Subtropical Agricultural Biological Resources Conservation and Utilization. The above pathogens isolated from the diseased parts of relevant crops in the natural environment and verified to be correct can be used in the following experiments.

[0026] The culture medium used in the examples is as follows:

[0027] Potato glucose agar (PDA) medium: 6.0 g potato starch, 20.0 g glucose, 20.0 g agar, and deionized water to a final volume of 1.0 L.

[0028] Potato glucose water culture medium (PDW): 6.0 g potato flour, 20.0 g glucose, and deionized water to a final volume of 1.0 L.

[0029] LB medium: 10.0 g tryptone, 5.0 g yeast extracts, 10.0 g NaCl, and deionized water to a final volume of 1.0 L, pH 7.0-7.2.

[0030] LA medium: yeast extract 5.0g, tryptone 10.0g, NaCl 10.0g, agar 20.0g, deionized water to a final volume of 1.0L, pH 7.0~7.2.

[0031] Common culture medium for Bacillus subtilis: 20.0g glucose, 15.0g peptone, 5.0g NaCl, 0.5g beef extract, and deionized water to a final volume of 1.0L, pH 7.0~7.2.

[0032] Corn flour agar medium (CMA): 30 g corn flour, 20 g agar, add water to a final volume of 1.0 L.

[0033] All the above culture media were sterilized at 121℃ for 20 min.

[0034] Example 1

[0035] Screening, isolation, and purification of Bacillus belyssus NN02:

[0036] Mulberry branches of the Guiyou 12 variety, collected from the mulberry orchard of the Guangxi Sericulture Technology Extension Station, were washed with running water and dried. The roots were then cut into 2-3 cm sections and disinfected with 75% alcohol for 3 minutes and 1% NaClO for 3 minutes. The disinfected branches were placed in sterilized petri dishes, and 10 mL of sterile water was added. The tissue was then minced using sterile scissors, and the suspension was diluted to 10-10. 3 100 μL of each culture was plated onto LA medium plates and incubated at 28°C for 48 h. Single colonies with significant differences in colony morphology were selected based on colony size, color, and surface morphology and purified by streak plating. The purified colonies were then transferred to fresh LA medium and named NN02.

[0037] Example 2

[0038] Inhibitory effect of Bacillus belyssus strain NN02 on pathogens

[0039] The *Bacillus bellsii* NN02 obtained after purification in Example 1 was activated on LA plates, and the above-mentioned test pathogens were activated on PDA plates for later use.

[0040] Using a sterilized pipette tip, collect 6mm diameter mycelial discs from the edge of activated pathogen colonies and inoculate them into the center of a PDA plate. At four symmetrical locations approximately 2.2cm from the mycelial disc, inoculate pre-activated *Bacillus belyssiensis* NN02 single colonies using sterilized toothpicks. A control group was prepared by inoculating only with pathogen mycelial discs (without *Bacillus belyssiensis* NN02). Each treatment was repeated three times, and incubated at 28℃. Mycelial growth was observed daily. After the control group's pathogen colonies had fully colonized the plate, the diameter of the pathogen colonies in each treatment group was measured using the cross-sectional method to calculate the antibacterial effect.

[0041] Antibacterial inhibition rate calculation: Antibacterial inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / (Coronary diameter of control group) × 100

[0042] The results of the plate confrontation test (Table 1) showed that Bacillus belye NN02 strain exhibited varying degrees of antagonism against all 11 tested plant pathogenic fungi. Figure 1 It did not show a significant inhibitory effect on Rhizoctonia solani, and its antibacterial spectrum had a certain degree of specificity.

[0043] Bacillus belycei NN02 showed the best inhibitory effect on sugarcane leaf blight pathogens, with a mycelial growth inhibition rate of (87.01±2.02)%. It also exhibited inhibition rates exceeding 70% against cabbage black spot pathogens and passion fruit stem rot pathogens, at (78.40±0.60)%, (74.15±2.30)%, and (72.11±2.16)%, respectively. Inhibition rates against citrus / yellow peel anthracnose pathogens, gray mold, and sugarcane ring spot pathogens ranged from 65% to 70%. The inhibitory effects on peanut white mold pathogens, banana wilt pathogens, sugarcane top rot pathogens, and lettuce sclerotinia pathogens were relatively weak, with inhibition rates ranging from 53.62% to 59.46%.

[0044] Table 1. Inhibition rate of strain NN02 against plant pathogenic fungi

[0045]

[0046] Example 3

[0047] Inhibitory effect of aseptic fermentation broth of Bacillus vesiculosus NN02 on pathogens

[0048] Bacillus belyssus NN02 was cultured on LA medium for 48 h. A loopful of colony was picked and inoculated into 5 mL of LB medium. The culture was incubated at 28 °C and 200 rpm for 16-20 h to obtain the antagonistic bacterial seed culture. 4 mL of this seed culture was inoculated into 100 mL of Bacillus subtilis medium and incubated at 28 °C and 200 rpm for 72 h to obtain the antagonistic bacterial fermentation broth. The fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain the sterile fermentation supernatant of Bacillus belyssus NN02. 1.5 mL of the sterile fermentation broth was thoroughly mixed with 13.5 mL of PDA medium melted and cooled to approximately 50 °C and poured onto a plate. A blank control was prepared by mixing 1.5 mL of sterile Bacillus subtilis medium with 13.5 mL of PDA medium. A cultured pathogenic fungal disc (6 mm in diameter) was inoculated in the center of each plate, with three replicates per group, and incubated at 28°C. After the control group colonies had fully grown on the plates, the colony diameter was measured and the mycelial growth inhibition rate was calculated according to the method in Example 2. The results are as follows. Figure 2 As shown.

[0049] The results of the antibacterial test of the sterile fermentation broth (Table 2) showed that the sterile fermentation broth of Bacillus bellis NN02 had a significantly better inhibitory effect on the 13 plant pathogenic fungi tested than the plate confrontation method, with only a very weak inhibitory effect on Phytophthora nicotineis. It had a broader antibacterial spectrum and stronger antibacterial activity.

[0050] The aseptic fermentation broth of Bacillus belliferae NN02 showed 100% inhibition against Sclerotinia sclerotiorum var. rubrum in lettuce, with no growth of pathogen colonies in the treated group. The inhibition rates against Black Spot Bacterium tumefaciens and Rhizoctonia solani were (96.75±0.77)% and (94.60±0.34)%, respectively, demonstrating extremely significant inhibition. The inhibition rates against Sugarcane Leaf Blight Bacterium, Citrus / Humidorum Anthracnose Bacterium, and Gray Mold ranged from 84% to 93%. The inhibition rates against Sugarcane Ring Spot Bacterium, Peanut White Spot Bacterium, and Banana Fusarium wilt ranged from 70% to 78%. The inhibition rates against Passion Fruit Stem Base Rot Bacterium and Sugarcane Top Rot Bacterium decreased, to (62.50±1.18)% and (44.66±2.48)%, respectively. The inhibition rate against Pseudomonas aeruginosa was only (19.13±3.78)%, and the inhibition rate against Phytophthora tobaccois was the lowest, only (2.10±8.45)%.

[0051] Table 2. Inhibition rate of NN02 aseptic fermentation broth against plant pathogenic fungi

[0052]

[0053] Example 4

[0054] Bacillus bellis strain NN02 was cultured on LA medium for 48 h. A loopful of colony was picked and inoculated into 5 mL of LB medium and cultured at 28 °C and 200 rpm for 16–20 h to obtain the antagonistic bacterial seed culture. 4 mL of the obtained antagonistic bacterial seed culture was inoculated into 100 mL of Bacillus subtilis culture medium and cultured at 28 °C and 200 rpm for 72 h to obtain the antagonistic bacterial fermentation broth. The obtained antagonistic bacterial fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain a sterile fermentation supernatant. Take 1.5 mL of NN02 sterile fermentation broth and mix thoroughly with 13.5 mL of PDA medium melted and cooled to about 50℃, then pour the mixture onto a plate. Use 1.5 mL of sterile Bacillus subtilis culture medium mixed with 13.5 mL of PDA as a blank control. Inoculate the center of each plate with cultured pathogenic bacterial cakes (6 mm in diameter). Perform three replicates per group. After incubating at 28℃ for 9 days, take 2-3 bacterial cakes from the edge of each colony, add electron microscopy fixative, and send the samples to Chengdu Lilai Biotechnology Co., Ltd. for electron microscopy observation.

[0055] Electron microscopy results as follows Figure 3 As shown, the surface of the mycelium of *Bacillus thuringiensis* cultured without sterile fermentation filtrate was smooth, while the mycelium cultured with sterile fermentation filtrate showed obvious wrinkling, shriveling, and curling deformities, indicating that the sterile fermentation broth of *Bacillus belyssiensis* NN02 can inhibit the growth of *Bacillus thuringiensis* mycelium.

[0056] Example 5

[0057] Pot experiment on the control of sugarcane leaf blight by Bacillus beryl NN02

[0058] 1. Preparation of sugarcane leaf blight spore suspension

[0059] Use a blue torch to scrape the activated sugarcane leaf blight mycelium, add it to 20 mL of PDW medium (containing 50 ug / ml streptomycin), shake on a shaker at 28°C for 24 h, then remove it; use a handheld electric grinder to break up the mycelium (3 times, more than 5 seconds each time), place it on a shaker at 28°C for 24 h again, then remove it and use a handheld electric grinder to break up the mycelium (3 times, more than 5 seconds each time), filter through two layers of gauze to collect the broken mycelium, and finally resuspend the mycelium in sterile water to obtain a suspension.

[0060] Add 150 μL of streptomycin (50 μg / mL) to 150 mL of CMA medium, shake well, pour into a plate, use a 1000 μL pipette to take 100 μL of mycelial suspension, spread it on the CMA medium plate, air dry in a clean bench for about 10 minutes, seal the petri dish with sealing film, and place it in a 25°C black light incubator.

[0061] On day 3 of incubation, remove the plates. At this time, a layer of white mycelium can be observed on the surface of the plates. Add 200 μL of sterile water to each plate, briefly burn the mycelium with a coverslip heated with an alcohol lamp, spread the scraped mycelium evenly on the plate, let it dry for 10 minutes, then reseal it with sealing film and put it back into the black light incubator.

[0062] On day 7 of incubation, numerous mature conidiophores can be observed on the plates. Add 2 ml of sterile water to each plate, and briefly scorch the hyphae with a coverslip heated by an alcohol lamp. Conidia will be released into the sterile water. Filter the sterile water through two layers of gauze, then add another 1 ml of sterile water to the plate and filter to collect the remaining conidia. After collecting all plates, use a blue pipette tip to squeeze the gauze to wash off the remaining conidia. Add another 2 ml of sterile water to the gauze and squeeze to collect the spores, obtaining a spore suspension. Dilute or concentrate the spore concentration as needed (each square on a hemocytometer contains 4 spores, which equals 10⁶ spore suspension) to obtain a 1×10⁶ spore suspension. 6 A spore suspension of CFU / mL.

[0063] 2. Pot-based control experiment design

[0064] The antagonistic bacterial fermentation broth was obtained according to the method in "Example 3". Healthy sugarcane seedlings of the Zhongzhe No. 1 variety with consistent growth were selected as experimental materials, and six treatment groups were set up, with 30 sugarcane seedlings in each treatment:

[0065] Treatment 1: First spray with Bacillus vesiculosus NN02 fermentation broth, and then spray with sugarcane leaf blight spore suspension 3 days later;

[0066] Treatment 2: First spray with a suspension of sugarcane leaf blight spores, and then spray with fermentation liquid of strain NN02 3 days later;

[0067] Treatment 3: First spray with a suspension of sugarcane leaf blight spores, and then spray with 500g / L thiophanate-methyl suspension 3 days later;

[0068] Treatment 4: Spray only a suspension of sugarcane leaf blight spores;

[0069] Treatment 5: Spray only with 500g / L thiophanate-methyl suspension;

[0070] Treatment 6: Spray only with sterile water.

[0071] All spore suspensions for each treatment were sprayed at the same time. All reagents were sprayed evenly on both sides of the sugarcane leaves using a spraying method until the leaves were dripping wet.

[0072] 3. Vaccination Trials and Surveys

[0073] After sugarcane was inoculated with spore suspension, the seedlings were kept moist in a greenhouse with 80% humidity for 3 days, and then transferred to a conventional greenhouse for management. Fifteen days later, 30 leaves were randomly sampled from each group to investigate the severity of sugarcane leaf blight. The disease was graded according to the sugarcane leaf blight grading standard (Table 3), and the disease index and control effect were calculated. Disease index (%) = ∑(number of diseased plants at each level × disease grade value) / (total number of leaves investigated × highest grade value) × 100; Control effect (%) = (disease rate in control group - disease rate in treatment group) / disease rate in control group × 100%.

[0074] Table 3 Grading Standards for Severity of Sugarcane Leaf Blight

[0075]

[0076] The results of the pot experiment are as follows Figure 4 As shown in Table 4, the control efficacy was better when the pathogen spore suspension was inoculated first, followed by spraying with Bacillus vesiculosus fermentation broth 3 days later (treatment 2), reaching 46.84%, which is close to the efficacy of the 500 g / L thiophanate-methyl suspension treatment (treatment 3, control efficacy 48.85%). The control efficacy was relatively weaker when spraying NN02 fermentation broth first and then inoculating with pathogen spore suspension (treatment 1), with a control efficacy of 25.57%.

[0077] Table 4. Control effect of Bacillus berberis NN02 on sugarcane leaf blight in potted plants.

[0078]

[0079] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain of Bacillus velezensi for controlling sugarcane leaf blight, characterized by: It is Bacillus velezensi NN02, with accession number CGMCC No. 37628.

2. The application of Bacillus berberis, as described in claim 1, in the control of sugarcane leaf blight.

3. The application of Bacillus berberis, as described in claim 1, in the control of sugarcane leaf blight and black spot disease of cabbage.

4. The application of Bacillus vesiculosus, as described in claim 1, for controlling sugarcane leaf blight in the control of passion fruit stem base rot.

5. The application according to claims 2-3, characterized in that: This was prepared to obtain a sterile fermentation broth of Bacillus vesiculosus for the prevention and control of sugarcane leaf blight.

6. The application according to claim 5, characterized in that: The amount of sterile fermentation broth added is 10% by volume.

7. The application according to claim 6, characterized in that: The sterile fermentation broth was prepared by inoculating 4 mL of antagonistic bacterial seed culture into 100 mL of commonly used Bacillus subtilis culture medium and fermenting it at 28°C and 200 r / min for 72 h. After centrifugation, the supernatant was collected and filtered through a 0.22 µm bacterial filter to obtain the sterile fermentation broth.

8. The application according to claim 7, characterized in that: The seed culture was prepared by culturing Bacillus belye NN02 on LA medium for 48 hours, then inoculating a loopful of colonies into 5 mL of LB medium and culturing in a constant temperature shaker at 28°C and 200 rpm for 16-20 hours.

9. The application according to claim 7, characterized in that: The fermentation medium consisted of 20.0g glucose, 15.0g peptone, 5.0g NaCl, 0.5g beef extract, and deionized water to a final volume of 1000mL, with a pH of 7.0-7.2; it was sterilized at 121℃ for 20 min.

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