Bacillus velezensis and application thereof

Abstract

The present application relates to the technical field of microorganisms, and particularly relates to a bacillus velezensis F0b and application thereof.The bacillus velezensis F0b has been preserved in the Guangdong Microbial Culture Collection Center on April 26, 2024, and the preservation number is GDMCC No: 64566.The bacillus velezensis has a preventive and / or control effect on rice blast fungus, plasmodiophora brassicae, fusarium solani, fusarium oxysporum f.sp.cubense, rice leaf spot fungus, pythium sylvaticum, diaporthe phaseolorum, colletotrichum gloeosporioides and curvularia inaequalis, and can be used to prepare a broad-spectrum microbial source bactericide.The bacillus velezensis can produce amylase, carboxymethyl cellulase and protease in fermentation liquor, and the fermentation liquor of the bacillus velezensis has a preventive and / or control effect on vegetable pathogenic fungal diseases, such as cabbage sclerotinia disease or pepper anthracnose disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Bacillus velezensis F0b and application thereof. BACKGROUND

[0002] For a long time, plant disease prevention and control has been a problem in the field of agriculture. Plant diseases account for 80% of the total number of plant diseases, involving more than 30,000 plant species. They seriously affect the growth and development of plants, hinder the stable and increased yield of crops, and curb the world's food production and agricultural product safety. Therefore, it is difficult to prevent and control plant diseases, and it is urgent to develop microbial products to replace chemical pesticides and reduce dependence on traditional pesticides. At present, chemical control is still a common means of plant disease control. Chemical control is favored by many people due to its rapid effect, low control cost, convenience and wide range of application. However, long-term use of chemical pesticides has caused many problems, such as environmental pollution, increased drug resistance, and pesticide residue exceeding the standard, thereby threatening human health and agricultural product safety. Therefore, in order to implement sustainable agriculture and meet the demand for green environmental protection, biological control is used to prevent and control plant diseases.

[0003] Biological control can inhibit various plant pathogenic organisms, alleviate the problems caused by chemical pesticides, and improve the quality and output of agricultural products. It is an effective and sustainable alternative or supplement to traditional pesticides in plant disease management. It mainly includes fungi, bacteria, actinomycetes, etc. Bacterial antagonists are widely used in biological control of plant diseases. Bacillus spp. has the advantages of environmental protection, good stability, rapid growth, broad-spectrum inhibition, strong stress resistance, and high biological safety, and plays a functional role in inhibiting plant diseases, thereby becoming a popular topic for researchers to study and develop and apply. It is one of the most potential and commercially valuable biocontrol agents.

[0004] Bacillus velezensis is a new species of Bacillus, which plays an important role in biological control, medicine, food industry and forestry. It is considered to be one of the most important biological antagonists for preventing plants from being harmed by pathogens. Early research reports are few, and it has gradually appeared in the research field since 2016. It can inhibit the growth of plant pathogens, including bacteria, fungi, viruses, and nematodes. Its metabolites are rich in broad-spectrum antibacterial activity and strong stress resistance. It has the characteristics of fast growth, good stability, rapid colonization, wide ecological niche, and environmental friendliness. It is the preferred biological agent for increasing crop yield, maintaining ecological environment and agricultural ecosystem. The present application provides a Bacillus velezensis that can inhibit a variety of plant pathogenic fungi, which has important biocontrol significance for plant fungal diseases. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a Bacillus velezensis F0b and its application. The Bacillus velezensis F0b is a green and environmentally friendly biocontrol bacterium, which has inhibitory effect on a variety of plant pathogenic fungi, which has important biocontrol significance for plant fungal diseases.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a Bacillus velezensis F0b, the strain has been preserved in Guangdong Microbial Culture Collection Center, the preservation date is April 26, 2024, and the preservation number is GDMCC No: 64566.

[0007] In some embodiments of the present application, the Bacillus velezensis F0b has an ITS1-5.8S-ITS4 rDNA gene sequence as shown in SEQ ID NO: 1.

[0008] The Bacillus strain F0b provided by the present application is isolated from the leaves of a healthy tomato plant. The cell morphology is short rod-shaped. On the LB medium, the colony is light yellow, smooth and opaque in the early stage, and the cell is raised, the surface is wrinkled, opaque, dry, and the edge is irregular in the later stage. The physiological and biochemical characteristics are gram-positive. Physiological and biochemical identification shows that the Bacillus F0b is positive for contact enzyme, methyl red, indigo substrate, nitrate reduction, starch hydrolysis, gelatin liquefaction, and V-P reaction. It can utilize sucrose, glucose, mannitol, hydrogen sulfide, and citrate. The reaction is negative. Combined with the morphological characteristics of the strain F0b, the F0b is initially identified as Bacillus, and its 16S rDNA sequence is shown in SEQ ID NO: 1. It is identified as Bacillus velezensis.

[0009] On the other hand, the present application provides the application of the Bacillus velezensis F0b in preventing and / or controlling plant pathogenic fungal diseases.

[0010] In some embodiments of the present application, the plant pathogenic fungal diseases include rice Magnaporthe oryzae disease, cabbage Sclerotinia sclerotiorum disease, tomato Fusarium oxysporum disease, banana Fusarium oxysporum disease, rice Cochliobolus miyabeanus disease, litchi Phytophthora citri disease, soybean Diaporthe phaseolorum disease, pepper Colletotrichum gloeosporioides disease, or corn Curvularia inaequalis disease.

[0011] Preferably, the plant pathogenic fungi include rice Magnaporthe oryzae disease, cabbage Sclerotinia sclerotiorum disease, or pepper Colletotrichum gloeosporioides disease.

[0012] In another aspect, the present application provides a broad-spectrum microbial fungicide containing the fermentation liquor of Bacillus velezensis F0b.

[0013] In some embodiments of the present application, the fermentation liquor of Bacillus velezensis F0b is prepared by inoculating a single colony of Bacillus velezensis F0b cultured on NA medium for 24 hours into LB medium for overnight culture to obtain a seed liquor, and then inoculating the seed liquor into 100 / 250 mL of LB culture medium at a 1% inoculation amount for overnight culture.

[0014] In some embodiments of the present application, the fermentation liquor of Bacillus velezensis F0b can produce amylase, carboxymethyl cellulase and protease.

[0015] In some embodiments of the present application, the pH of the fermentation liquor of Bacillus velezensis F0b is 5-9.

[0016] In another aspect, the present application provides the use of the broad-spectrum microbial fungicide in preventing and treating vegetable pathogenic fungal diseases.

[0017] In some embodiments of the present application, the vegetable pathogenic fungal disease is cabbage sclerotinia disease or pepper anthracnose disease.

[0018] Compared with the prior art, the present application has the following advantages:

[0019] (1) The present application provides a Bacillus strain F0b isolated from the leaves of a healthy tomato plant, which is identified as Bacillus velezensis. The Bacillus velezensis has a preventive and / or control effect on rice blast fungus, cabbage sclerotinia fungus, tomato wilt fungus, banana wilt fungus, rice leaf spot fungus, litchi blight fungus, soybean charcoal rot fungus, pepper anthracnose fungus and corn flexuose spot fungus, and can be used to prepare a broad-spectrum microbial fungicide.

[0020] (2) The fermentation liquor of the Bacillus velezensis provided by the present application can produce amylase, carboxymethyl cellulase and protease, and the fermentation liquor of the Bacillus velezensis has a preventive and / or control effect on vegetable pathogenic fungal diseases such as cabbage sclerotinia disease or pepper anthracnose disease. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1The figure shows the inhibitory effect of Bacillus belyssus F0b on several plant pathogenic fungi. In the figure, A, B, C, D, E, F, G, H, I, J, and K are blank control groups, while a, b, c, d, e, f, g, h, i, j, and k are treatment groups of strain F0b. The pathogens corresponding to a, b, c, d, e, f, g, h, i, j, and k are: rice blast fungus, peanut white mold fungus, tomato wilt fungus, banana wilt fungus, rice leaf spot fungus, litchi downy mildew fungus, rice sheath blight fungus, soybean anthracnose fungus, pepper anthracnose fungus, corn leaf blight fungus, and cabbage sclerotinia rot fungus.

[0022] Figure 2 The images show the colony morphology and Gram staining results of Bacillus belyssus 1. In the images, a and b are colony morphology images of strain F0b on NA medium; c and d are Gram staining results of strain F0b (×100).

[0023] Figure 3 Phylogenetic tree of Bacillus belyss F0b16 SrDNA.

[0024] Figure 4 The growth curve of Bacillus belyssus F0b is shown.

[0025] Figure 5 The effect of different pH values ​​on the growth of Bacillus belye F0b.

[0026] Figure 6 The effect of different sodium chloride contents on the growth of Bacillus berleis F0b.

[0027] Figure 7 The extracellular enzyme characteristics of Bacillus belyssus F0b fermentation broth are shown, where a is carboxymethyl cellulase; b is β-glucanase; c is protease; and d is amylase.

[0028] Figure 8 The in vitro efficacy of Bacillus berberis F0b fermentation broth in controlling sclerotinia stem rot in cabbage was determined by the following parameters: a) sterile water treatment; b) treatment with the original Bacillus berberis F0b fermentation broth; c) 10-fold dilution of Bacillus berberis F0b fermentation broth; d) 100-fold dilution of Bacillus berberis F0b fermentation broth; e) 50-fold dilution of 7 billion CFU / g Bacillus berberis F0b wettable powder; f) 200-fold dilution of 7 billion CFU / g Bacillus berberis F0b wettable powder; g) 400-fold dilution of 7 billion CFU / g Bacillus berberis F0b wettable powder; and h) 250-fold dilution of 40% sclerotinia WP.

[0029] Figure 9 The in vitro effect of Bacillus vesicle F0b fermentation broth on the prevention and control of anthracnose in pepper fruits. Detailed Implementation

[0030] For better illustrating the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0031] Example 1: This example is the isolation, purification and screening of Bacillus velezensis FOb

[0032] 1. Test method:

[0033] (1) Tomato leaf surface disinfection: Tomato leaves collected from Fengcun, Ningxi Town, Zengcheng District, Guangzhou City were washed with distilled water to remove large particle impurities such as sand dust and soil, and then the water was absorbed with filter paper. The tomato leaves were then surface-disinfected by immersing them in a 1% NaCIO solution containing effective chlorine for 5 min, and then slowly and thoroughly rinsing them with sterile distilled water for 5 times. The tomato leaves were then immersed in a 75% ethanol solution for 3 min, and then slowly and thoroughly washed with sterile distilled water for 3 times. The tomato leaves were then immersed in a 1% mercury chloride solution for 1 min, and then slowly and thoroughly rinsed with sterile distilled water for 5 times, and the waste liquid from the last rinse was reserved.

[0034] (2) Isolation and purification of Bacillus FOb: In a clean bench, the scalpel and tweezers were surface-disinfected, and then the testa was cut into squares of about 1 cm x 1 cm using surgical scissors, and then attached to NA medium, with 5 replicates per medium. The medium was then incubated at 30°C in the dark, and continuously observed for 7 days. When colonies were observed, they were immediately picked using a sterile inoculation needle, and then streaked on NA medium until a single colony was isolated and purified. The purified bacteria were stored at -80°C in a low-temperature freezer for later use. The culture conditions were as follows: temperature (25 ± 1) °C, relative humidity 60% to 80%, and light cycle L:D = 16:8 in an artificial climate chamber.

[0035] (3) Screening: rice blast, cabbage sclerotinia, tomato wilt, soybean root rot, cucumber wilt, banana wilt, soybean charcoal rot, maize leaf spot, litchi blight, citrus brown spot were inoculated on PDA medium, and cultured at 28°C until the colony diameter was about 6 cm for standby. Sclerotinia scleroriorum (Lib.) de Bary, Fusarium oxysporum f. sp. Lycopersici, Colletotrichum capsici, Pyricularia oryzae Cav., Helminthosporium oryzae Breda de Haan, Peronophythora litchii, Fusarium oxysporum f. sp. cubense, Macrophomina phaseolina, Curvularia lunata, Sclerotium rolfsii, Rhizoctonia solani were used as target bacteria, and the target bacteria were punched (d = 9 mm) on the plate, 9 mm fungus cake and 50 μL of the test endophytic bacteria were inoculated into the prepared confrontation plate, and the treatment of inoculating sterile LB liquid medium was used as control, and cultured at 22°C for 7d, each treatment was repeated 3 times, the growth changes of the colonies were observed, including size, color, and finally one strain with the best antagonistic effect was selected. When the control group of pathogenic fungi grew full plate, the cross method was used to measure the colony radius, and the inhibition rate effect was calculated, and the target bacteria with good effect were selected for subsequent experiments.

[0036] (4) Inhibition rate (%) = [(control pathogenic colony diameter - treatment colony diameter) / (control colony diameter - fungus cake diameter)] x 100%.

[0037] 2. Test results and analysis

[0038] Table 1 Inhibition activity of strain F0b

[0039]

[0040] Note: The data in Table 1 is the average value ± standard error of 3 repeated tests, and the same column data after different lowercase letters represent significant difference (p < 0.05) by DMRT method.

[0041] A strain of antagonistic bacteria with obvious bacteriostatic effect on pathogenic fungi was obtained from the tomato leaves, and was named as strain F0b. The strain F0b had weak bacteriostatic effect on the rice sheath blight fungus and the peanut white thread fungus, with the bacteriostatic rates of 8.27% and 8.54%, respectively. The strain F0b had obvious bacteriostatic effect on the rice blast fungus, the cabbage sclerotium fungus, the tomato fusarium wilt fungus, the banana fusarium wilt fungus, the rice leaf spot fungus, the litchi downy mildew fungus, the soybean charcoal rot fungus, the pepper anthracnose fungus, and the corn curved leaf spot fungus, with the bacteriostatic rates all reaching more than 50%, indicating that the strain F0b had broad-spectrum bacteriostatic activity. The bacteriostatic effect diagram is shown in Figure 1 .

[0042] Example 2: In this example, the strain F0b was identified

[0043] 1. Test method:

[0044] Morphological characteristic identification: The pure culture of the strain F0b was streaked on the NA medium plate, and was cultured at 28°C for 48h. The color, transparency, wrinkle, and other characteristics of the colony were observed, and the gram staining change was observed through an optical microscope.

[0045] Physiological and biochemical identification was performed according to the experimental method specified in the Common Bacteria System Identification Manual. The tested physiological and biochemical indexes included hydrogen sulfide reaction, nitrate reduction reaction, methyl red reaction, V-P reaction, starch hydrolysis, citric acid utilization, sugar oxidation fermentation reaction, contact enzyme, gelatin liquefaction reaction, and the like. Each treatment was repeated three times.

[0046] Molecular biology identification: The strain F0b was sent to Beijing Qianke Biological Technology Co., Ltd. (Guangzhou) for sequencing. After sequencing, homology analysis was performed. BLAST software was used for comparison, and MEGA7.0 software was used to construct a system evolution tree.

[0047] The method of TSINGKE plant DNA extraction kit was used to extract the strain F0b, and the DNA of the strain sample was extracted to obtain the genomic DNA. The 16S rDNA of the target strain was amplified and sequenced using bacterial 16S rDNA universal primers 27F (SEQ ID NO: 2, 5'-AGTTTGATCMTGGCTCAG-3') and 1492 (SEQ ID NO: 3, 5'-GGTTACCTTGTTACGACTT-3'). The components of the PCR reaction system were as follows: 1xTSE101Mix 45 uL, 2 uL of each primer, and 1 uL of DNA template. The reaction conditions were as follows: pre-denaturation at 98°C for 2 min; cycle stage at 98°C for 10 s, 56°C for 10 s, and 72°C for 10 s for 35 cycles; extension at 72°C for 5 min. After the PCR product was detected by agarose gel electrophoresis, the PCR amplified sample was sent to Beijing Qikong Biotechnology Co., Ltd. (Guangzhou Branch) for sequencing analysis. The sequencing results were used for BLAST comparison in the NCBI database, so as to preliminarily identify the sample, and the MEGA7.0 software was used to construct a phylogenetic tree.

[0048] 2. Experimental results and analysis

[0049] The morphological characteristics showed that the strain F0b was light yellow, raised, wrinkled surface, opaque, dry, and irregular edge. Under a microscope, the bacterial morphology was short rod-shaped, and Figure 2 The physiological and biochemical identification showed that the bacillus F0b was positive in contact enzyme, methyl red, indole substrate, nitrate, starch hydrolysis, gelatin liquefaction, and V-P reaction, and could utilize sucrose, glucose, mannitol, hydrogen sulfide, oxidase, and citrate reaction. The morphological characteristics of the strain F0b were preliminarily identified, and the F0b was preliminarily identified as Bacillus. The DNA genome of the strain F0b was extracted for sequencing, and the BLAST sequence comparison was performed in the NCBI database. The MEGA7.0 software was used to construct a phylogenetic tree, and the results were shown in Figure 3 . The strain F0b belonged to the same branch as Bacillus velezensis (MG705703.1, LC191186.1, CP054714.1). Based on the morphological characteristics and the results of physiological and biochemical experiments, the strain F0b was identified as Bacillus velezensis.

[0050] Example 3 Biological characteristics of Bacillus velezensis F0b

[0051] Preparation of fermentation broth: a single colony cultured on NA medium for 24 h was inoculated in LB medium to obtain a seed liquid after overnight culture. Then, the seed liquid was inoculated in 100 / 250 mL of LB culture medium at a 1% inoculation amount to obtain an OD 600 = 1 fermentation broth after overnight culture (16 h), which was prepared for use.

[0052] Determination of Fob growth curve: 100 μL seed liquid was taken by a pipette and added into a 250 mL conical flask containing 100 mL LB liquid medium, which was placed in a constant temperature shaker and cultured under the condition of 30°C and 180 r / min. The sample was taken every 2 h, and the absorbance OD 600 was determined to draw the growth curve of Bacillus velezensis Fob, with 3 repeats for each treatment.

[0053] The results showed that 0 h-6 h was the lag phase of the strain, 6 h-16 h was the logarithmic growth phase of the strain, 16 h later the strain grew slowly, 16 h-38 h was the stationary phase of the strain, and 38 h later the strain began to enter the decline phase. The strain grew vigorously and reproduced quickly in 6 h-16 h, so the culture solution cultured for 16 h was selected as the fermentation broth of Bacillus velezensis Fob, and the growth curve of Bacillus velezensis Fob is shown in Figure 4 .

[0054] Effect of different pH on the growth of Bacillus velezensis Fob: the fermentation broth OD 600 = 1 was prepared, the pH of LB liquid medium was adjusted to 3.0, 5.0, 7.0, 9.0, 11.0, and the liquid volume of LB liquid medium was 100 mL / 250 mL, and the OD 600 was determined.

[0055] The results showed that after 24 h of culture in LB liquid medium with different pH, the growth of strain Fob was significantly different. Bacillus velezensis Fob grew well at pH 5-9, with the best growth at pH 7. Bacillus velezensis Fob was obviously inhibited and almost did not grow at pH 1, 3 and 11. The effect of different pH on the growth of Bacillus velezensis Fob is shown in Figure 5 .

[0056] Effect of different sodium chloride content on the growth of Bacillus velezensis Fob: 50 mL of LB liquid medium was added into a 150 mL conical flask, and the sodium chloride content in the medium was 0 g / L, 5 g / L, 10 g / L, 25 g / L, 50 g / L, 100 g / L and 200 g / L, respectively. After sterilization and cooling, the strain Fob cultured overnight (OD 600 = 1) was added to different treatments with 3 repeats for each treatment, and was placed in a 30°C, 180 r / min shaker for 24 h. Then the OD 600 value was determined to determine the optimal sodium chloride content of strain Fob.

[0057] The Bacillus velezensis F0b grew well when the sodium chloride content was 0 g / L, 5 g / L, 10 g / L, 25 g / L, 50 g / L, and 100 g / L, and the growth was best when the sodium chloride content was 10 g / L. The growth of the Bacillus velezensis F0b was significantly inhibited when the sodium chloride content was 200 g / L, and the Bacillus velezensis F0b hardly grew, indicating that the Bacillus velezensis F0b had good salt tolerance. The effects of different sodium chloride contents on the growth of the Bacillus velezensis F0b are shown in Figure 6 .

[0058] Example 4: Determination of the extracellular enzymes of the Bacillus velezensis F0b

[0059] A Φ = 5 mm sterilized filter paper piece was placed in the center of a detection medium containing carboxymethyl cellulase, β-glucanase, amylase, and protease, respectively, 2 μL of the F0b fermentation liquor (OD 600 = 1.0) was added dropwise on the filter paper piece, the extracellular enzyme activity of the Bacillus velezensis F0b was detected, 2 μL of sterile water was added dropwise on the filter paper piece under the same conditions as the control, 3 repetitions were set for each treatment, all plates were cultured in a 30°C constant temperature incubator for 3-5 days, and whether a transparent circle appeared was observed. If a transparent circle appeared, it indicated that the strain could produce the extracellular enzyme. For the carboxymethyl cellulase detection, 0.2% congo red was dyed for 30 min, and then the dye was washed away with distilled water and 1 mol / L NaCl in sequence, and then 5% acetic acid solution was used to fix the color, and whether a transparent hydrolysis circle appeared around the colony was observed; for the amylase detection, a small amount of Lugol's iodine solution was added to the plate and rotated gently to make the iodine solution evenly cover the entire plate, and whether a transparent circle was formed around the colony was observed. The size of the colony and the transparent circle diameter was measured by using a vernier caliper, the enzyme activity was determined according to the following formula, and the enzyme activity was expressed by the ratio of the transparent circle diameter to the colony diameter. The larger the ratio, the stronger the ability of the strain to produce extracellular enzymes.

[0060] Enzyme activity = transparent circle diameter size / colony diameter size

[0061] The results showed that the colonies of the strain Fob grew on the carboxymethyl cellulase, protease, and amylase plates and transparent circles appeared, and no transparent circle appeared on the β-glucanase plate, indicating that the F0b had the ability to produce carboxymethyl cellulase, protease, and amylase, but had no ability to produce β-glucanase (see the extracellular enzyme properties of the Bacillus velezensis F0b fermentation liquor in Figure 7 ), and the HC values of the protease, amylase, and carboxymethyl cellulase in the extracellular enzymes were 2.71, 2.79, and 1.52, respectively.

[0062] Table 2: Ability of the Bacillus velezensis F0b to produce extracellular enzymes

[0063]

[0064] Example 5 In vitro control effect of Bacillus velezensis F0b fermentation broth on cabbage Sclerotinia rot

[0065] The in vitro leaf method was used. Healthy and uniform leaf age cabbage leaves were selected for the test, and the leaves were surface sterilized with 75% alcohol, washed with sterile water and dried for standby. The 70 billion CFU / g Bacillus velezensis wettable powder F0b 200 times used in the experiment was prepared in the laboratory. The F0b fermentation broth was prepared, and the concentration of the fermentation broth was adjusted to 1 x 10 8 CFU / mL. A total of 8 treatments were set up for the experiment: (a) sterile water treatment, (b) F0b fermentation broth stock solution, (c) F0b fermentation broth 10 times dilution, (d) F0b fermentation broth 100 times dilution, (e) 70 billion CFU / g Bacillus velezensis wettable powder F0b 50 times, (f) 70 billion CFU / g Bacillus velezensis wettable powder F0b 200 times, (g) 70 billion CFU / g Bacillus velezensis wettable powder F0b 400 times, (h) 40% Sclerotinia rot WP 250 times. The cabbage leaves were soaked in the treatment liquid for 30 min, naturally dried, placed in a culture dish with internal wet filter paper, and cotton was wet and placed on the petiole. Each cabbage leaf was inoculated with a Φ = 6 mm Sclerotinia disc, covered with a lid, and placed in a light incubator for 5 days (16 h light / 8 h dark, 20°C, relative humidity above 70%). The lesion diameter was measured, and each treatment was repeated 3 times.

[0066] The results showed that the cabbage in vitro leaf method for disease prevention experiment 5d after treatment, the lesion diameter of the control group of sterile water treated leaves was 5.13 cm, the lesion diameter of the cabbage leaves treated with Bacillus velezensis F0b fermentation broth stock solution and the prepared 70 billion CFU / g Bacillus velezensis wettable powder F0b was significantly reduced (see Table 3), and the lesion diameter was significantly reduced. The treatment effect of 70 billion CFU / g Bacillus velezensis wettable powder F0b 50 times was the most obvious. Bacillus velezensis F0b fermentation broth stock solution, 10 times dilution, 100 times dilution and the prepared Bacillus velezensis F0b wettable powder had obvious bacteriostatic effect on cabbage Sclerotinia rot, and the lesion diameter was 0 cm, 1.28 cm and 2.38 cm respectively. The cabbage leaves treated with Bacillus velezensis F0b fermentation broth stock solution did not develop disease, and the control effect was equivalent to that of the chemical agent 40% Sclerotinia rot WP 250 times (see Figure 8 ), indicating that the F0b fermentation broth had good inhibitory effect on cabbage Sclerotinia rot.

[0067] Table 3 Control effect of Bacillus velezensis F0b on cabbage Sclerotinia rot

[0068]

[0069] Note: The data in Table 3 are the average values ± standard errors of 3 replicates, and the same lowercase letters after the data in the same column indicate significant differences (p < 0.05) by DMRT.

[0070] Example 6 In vitro effect of Bacillus velezensis F0b fermentation broth on controlling pepper anthracnose fruit

[0071] Select pepper fruits with uniform appearance and no disease and pest symptoms, clean the surface of the pepper fruits with 75% alcohol, rinse with sterile water for 2-3 times, and then spray different treatment liquids after drying. Set up 5 treatments: (1) Bacillus velezensis F0b fermentation broth 1 x 108 CFU / mL, (2) Bacillus velezensis wettable powder F0b 50 times of 7 billion CFU / g, (3) Difenoconazole 200 times of 60%, (4) positive control-sterile water treatment, (5) negative control-no inoculation of pathogen. After the fruits are dried, inoculate 6mm pepper anthracnose fungus discs on (1)-(5) treatments, use sterile toothpicks to make wounds on the middle waist of the fruits, and repeat 3 times for each treatment. Place in 28℃, L:D is 16:8, alternate moisture culture, seal the preservation box with preserved fruits with preservation film, until the positive control appears significant anthracnose symptoms, measure the lesion diameter by cross method, and calculate the control effect according to the following formula.

[0072] Control effect (%) = ((blank control lesion diameter - preparation treated lesion diameter) / blank control diameter) x 100

[0073] The experimental results are as follows Figure 9 After 5 days of inoculation, pepper anthracnose fungus can normally invade, and the control group has more serious disease, the color of the inoculation site of the fruits deepens, the lesion is sunken, and the diameter is 8.37mm. The disease of pepper fruits treated with Bacillus velezensis F0b fermentation broth and 7 billion CFU / g Bacillus velezensis F0b wettable powder 50 times is significantly lighter, the inoculation site is slightly sunken, the color is normal, and the lesion diameter is significantly smaller, with lesion diameters of 0.91mm and 2.12mm respectively. The control effect of 7 billion CFU / g Bacillus velezensis F0b wettable powder on in vitro fruit anthracnose is 74.68%, and the control effect of Bacillus velezensis F0b fermentation broth on in vitro pepper fruit anthracnose is 89.11%, which is significantly better than chemical agents.

[0074] Table 4 Control effect of Bacillus velezensis F0b fermentation broth on tomato fusarium wilt

[0075]

[0076] Note: The data in Table 4 are the average values ± standard errors of 3 replicates, and the same lowercase letters after the data in the same column indicate significant differences (p < 0.05) by DMRT.

Claims

1. A Bacillus velezensis F0b, characterized in that, The Bacillus belyssus F0b has been deposited at the Guangdong Microbial Culture Collection Center on April 26, 2024, with accession number GDMCCNo: 64566; the Bacillus belyssus F0b has the ITS1-5.8S-ITS4 rDNA gene sequence as shown in SEQ ID NO:

1.

2. The application of Bacillus velezensis F0b as described in claim 1 in the prevention and control of plant pathogenic fungal diseases, wherein the plant pathogenic fungal diseases are rice blast fungus, cabbage sclerotinia rot fungus, tomato wilt fungus, or pepper anthracnose fungus.

3. A broad-spectrum microbial bactericide, characterized in that, The broad-spectrum microbial bactericide contains the fermentation broth of Bacillus berberis F0b as described in claim 1; The fermentation broth of Bacillus belyssus F0b is prepared as follows: a single colony of Bacillus belyssus F0b cultured on NA medium for 24 h is inoculated into LB medium and cultured overnight to obtain a seed culture. The seed culture is then inoculated into LB medium at a 1% inoculum amount and cultured overnight to obtain the fermentation broth. The fermentation broth of *Bacillus beryl* F0b can produce amylase, carboxymethyl cellulase, and protease; the pH of the fermentation broth of *Bacillus beryl* F0b is 5–9.

4. The application of the broad-spectrum microbial fungicide as described in claim 3 in the control of pathogenic fungal diseases in vegetables, characterized in that, The pathogenic fungal diseases of vegetables mentioned are sclerotinia rot of cabbage, anthracnose of pepper, or wilt of tomato.

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