Bacillus velezensis and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus velezensis F0b and an application of the bacillus velezensis F0b. The bacillus velezensis F0b is preserved in the Guangdong Microbial Culture Collection Center on April 26, 2024, the preservation date of the bacillus velezensis F0b is April 26, 2024, and the preservation number of the bacillus velezensis F0b is GDMCC (China General Microbiological Culture Collection Center): 64566. The bacillus velezensis has prevention and / or control effects on pyricularia oryzae, cabbage sclerotinia sclerotiorum, tomato fusarium wilt, banana fusarium wilt, rice leaf spot, peronophythora litchii, soybean carbon rot, colletotrichum capsici and corn leaf curvularia, and can be used for preparing broad-spectrum microbial source bactericides. The fermentation liquor of the bacillus velezensis can generate amylase, carboxymethyl cellulose and protease, and the fermentation liquor of the bacillus velezensis has prevention and / or control effects on vegetable pathogenic fungal diseases, namely cabbage sclerotiniose or pepper anthracnose.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of Bacillus velezensis F0b and an application thereof. Background Art

[0002] For a long time, the problem of plant disease prevention and control has been a difficult problem in the agricultural field. Plant disease types account for 80% of the total number of plant diseases, involving more than 30,000 plant species, which seriously affect the growth and development of plants, hinder the stable and increased production of crops, curb the world's food production and agricultural product safety, and make plant disease prevention and control difficult. It is urgent to explore microbial products that can replace chemical pesticides and reduce dependence on traditional pesticides. At present, chemical control is still a common means of plant disease prevention and control. Chemical control is popular among the majority of people because of its rapid effect, low prevention and control cost, convenient use, and wide range. However, the long-term use of chemical pesticides has caused many problems, such as environmental pollution, increased resistance, and excessive pesticide residues, which threaten human health and agricultural product safety. Therefore, it is necessary to implement eco-friendly alternatives in sustainable agriculture and implement green environmental protection needs, such as biological control to prevent and control plant diseases.

[0003] Biological control can inhibit the pathogens of various plant diseases, alleviate the problems caused by chemical pesticides, thereby improving the quality of agricultural products and increasing output. It is an effective and sustainable alternative or supplement to traditional pesticides in plant disease management. It mainly includes fungi, bacteria, actinomycetes, etc., among which bacterial antagonists are widely used in the biological control of plant diseases. Bacillus spp. plays a functional role in inhibiting plant diseases due to its environmental protection, good stability, rapid growth, broad antibacterial spectrum, strong stress resistance and high biosafety. It has become a hot topic for current scientific researchers to study, develop and apply, and is one of the most promising and commercially valuable biocontrol bacteria.

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

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

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a Bacillus velezensis F0b, which has been deposited in the Guangdong Microbiological Culture Collection Center, with a preservation date of April 26, 2024 and a preservation number of GDMCC No: 64566.

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

[0008] The present invention provides a Bacillus strain F0b isolated from healthy tomato plant leaves, and its bacterial body morphology is short rod-shaped. On LB culture medium, the colony is light yellow, smooth and opaque in the early stage, and the bacterial body is raised in the later stage, with wrinkled surface, opaque, dry, and irregular edges. The physiological and biochemical characteristics are Gram-positive, and physiological and biochemical identification shows that Bacillus F0b is positive for contact enzyme, methyl red, indigo matrix, nitrate reduction, starch hydrolysis, gelatin liquefaction, and VP reactions, and can utilize sucrose, glucose, and mannitol, and is negative for hydrogen sulfide and citrate reactions. Combined with the morphological characteristics of strain F0b, F0b is preliminarily identified as Bacillus, and its 16S rDNA sequence is shown in SEQ ID NO: 1, and it is identified as Bacillus Velezii.

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

[0010] In some embodiments of the present invention, the plant pathogenic fungal diseases include rice blast disease, cabbage sclerotinia disease, tomato wilt disease, banana wilt disease, rice leaf spot disease, litchi downy mildew disease, soybean charcoal rot disease, pepper anthracnose disease or corn leaf spore disease.

[0011] Preferably, the plant pathogenic fungi include rice blast disease, cabbage sclerotinia disease or pepper anthracnose disease.

[0012] In another aspect, the present invention provides a broad-spectrum microbial-derived fungicide, wherein the broad-spectrum microbial-derived fungicide contains the fermentation broth of the Bacillus Velez subtilis F0b of the present invention.

[0013] In some embodiments of the present invention, the method for preparing the fermentation broth of the Bacillus Velez F0b is as follows: a single colony of Bacillus Velez F0b cultured on NA medium for 24 hours is selected and inoculated into LB medium for overnight culture to obtain a seed liquid, and then the seed liquid is inoculated into 100 / 250mL LB culture medium at a 1% inoculum amount for overnight culture to obtain a fermentation broth.

[0014] In some embodiments of the present invention, the fermentation broth of Bacillus Velez subtilis F0b can produce amylase, carboxymethyl cellulase, and protease.

[0015] In some embodiments of the present invention, the pH of the fermentation broth of Bacillus Velez F0b is 5-9.

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

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

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention provides a Bacillus strain F0b isolated from the leaves of healthy tomato plants and identified as Bacillus Velezii. The Bacillus Velezii has a preventive and / or control effect on rice blast pathogen, cabbage sclerotinia pathogen, tomato wilt pathogen, banana wilt pathogen, rice leaf spot pathogen, litchi downy mildew pathogen, soybean charcoal rot pathogen, pepper anthracnose pathogen and corn leaf curl spore pathogen. The Bacillus Velezii can be used to prepare a broad-spectrum microbial source fungicide.

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

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

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

[0023] Figure 3 This is the SrDNA phylogenetic tree of Bacillus velez F0b16.

[0024] Figure 4 This is the growth curve of Bacillus Velez F0b.

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

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

[0027] Figure 7 The extracellular enzyme characteristics of the fermentation broth of Bacillus Velez F0b, wherein a is carboxymethyl cellulase, b is β-glucanase, c is protease, and d is amylase.

[0028] Figure 8 The in vitro effect of Bacillus Velez F0b fermentation liquid on preventing and controlling cabbage sclerotinia disease, wherein a is treated with sterile water; b is treated with the original liquid of Bacillus Velez F0b fermentation liquid; c is 10 times of Bacillus Velez F0b fermentation liquid; d is 100 times of Bacillus Velez F0b fermentation liquid; e is 50 times of 7 billion CFU / g Bacillus Velez F0b wettable powder; f is 200 times of 7 billion CFU / g Bacillus Velez F0b wettable powder; g is 400 times of 7 billion CFU / g Bacillus Velez F0b wettable powder; h is 250 times of 40% Sclerotium Net WP.

[0029] Fig. 9 The in vitro effect of Bacillus Velez F0b fermentation liquid on preventing and controlling anthracnose fruit of pepper. DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific drawings and embodiments. In the embodiments, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0031] Example 1: This example is the separation, purification and screening of Bacillus velezensis F0b

[0032] 1. Test method:

[0033] (1) Surface disinfection of tomato leaves: Tomato leaves collected from Feng Village, Ningxi Town, Zengcheng District, Guangzhou City were washed with distilled water to remove large particles of impurities such as dust and soil attached to the surface, and the water was absorbed with filter paper before the surface disinfection of the tomato leaves was carried out. In the clean bench, the tomato leaves were soaked in a NaClO solution containing 1% effective chlorine for 5 minutes, and then slowly and thoroughly rinsed 5 times with sterile distilled water; soaked in a 75% ethanol solution for 3 minutes, and then slowly and thoroughly washed 3 times with sterile distilled water; soaked in 1% mercuric chloride for 1 minute, and then slowly and thoroughly rinsed 5 times with sterile distilled water, and the waste liquid from the last rinse was retained.

[0034] (2) Isolation and purification of Bacillus F0b: After disinfecting the surface of the scalpel and tweezers in a clean bench, use surgical scissors to cut the outer seed coat into a square of about 1 cm × 1 cm, and attach it to NA medium. Set up 5 replicates for each medium, and then place it in the dark at 30°C for continuous observation for 7 days. When colonies grow, immediately use an inoculation needle to pick them up, and streak them in NA medium until a single colony is isolated and purified. The purified bacteria are stored in a -80°C ultra-low temperature refrigerator for future use. The culture conditions are: temperature (25±1)°C, relative humidity 60% to 80%, and light cycle L:D=16:8 in an artificial climate box.

[0035] (3) Screening: Take rice blast, cabbage sclerotinia, tomato wilt, soybean root rot, cucumber wilt, banana wilt, soybean charcoal rot, corn leaf spot, litchi downy mildew, and citrus brown spot on PDA medium and culture at 28°C until the colony diameter is about 6 cm for later use. The pathogens included Sclerotinia scleroriorum (Lib.) deBary, Fusarium oxysporum f.sp. Lycopersici, Colletortrichum capsici, Pyricularia oryzae Cav., Helminthosporium oryzae Breda de Haan, Peronophythoralitchii, Fusarium oxysporum f.sp. cubense, Macrophominaphaseolina, Curvularia lunata, Sclerotium rolfsii, Rhizoctonia solani) as the target bacteria, punch holes (d = 9mm) on the target bacteria plate, inoculate 9mm bacterial cakes of pathogenic bacteria and 50μL of the endophytic bacteria to be tested into the prepared confrontation plates, respectively, and take the treatment inoculated with sterile LB liquid culture medium as the control, dark culture at 22℃ for 7d, and repeat each treatment 3 times to observe the growth changes of the colonies, including size and color, and finally select a strain with the best antagonistic effect. When the pathogenic fungi in the control group overgrow the plate, the colony radius is measured by the cross method, and the antibacterial rate effect is calculated, and the target bacteria with good effect are selected for subsequent experiments.

[0036] (4) Inhibition rate (%) = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%.

[0037] 2. Test results and analysis

[0038] Table 1 Antibacterial activity of strain F0b

[0039]

[0040] Note: The data in Table 1 are the mean ± standard error of 3 replicates. Different lowercase letters after the data in the same column indicate significant differences (p<0.05) tested by DMRT method.

[0041] Through the plate confrontation test method, an antagonistic bacterium with obvious antibacterial effect on pathogenic fungi was obtained from the obtained tomato leaves, and it was named strain F0b. The above-mentioned strain F0b had weak antibacterial effects on rice sheath blight and peanut white rot, with inhibition rates of 8.27% and 8.54% respectively. It had obvious antibacterial effects on rice blast, cabbage sclerotinia, tomato wilt, banana wilt, rice leaf spot, litchi downy mildew, soybean charcoal rot, pepper anthracnose, and corn curved spore fungus, with inhibition rates of more than 50%, indicating that strain F0b has a broad-spectrum antibacterial activity. The antibacterial effect diagram is shown in the figure. Figure 1 shown.

[0042] Example 2: This example identifies the aforementioned strain F0b

[0043] 1. Test method:

[0044] Identification of morphological characteristics: The pure culture of strain F0b obtained above was streaked on a NA medium plate, and after culturing at 28°C for 48 hours, the color, transparency, presence of wrinkles and other characteristics of the colony were observed, and the Gram staining changes were observed under an optical microscope.

[0045] Physiological and biochemical identification was carried out according to the experimental methods specified in the Manual of Identification of Common Bacterial Systems. The physiological and biochemical indicators tested included hydrogen sulfide reaction, nitrate reduction reaction, methyl red reaction, VP reaction, starch hydrolysis, citric acid utilization, sugar oxidation and fermentation reaction, catalase, gelatin liquefaction reaction, etc. Each treatment was repeated three times.

[0046] Molecular biological identification: strain F0b was sent to Beijing Qingke Biotechnology Co., Ltd. (Guangzhou) for sequencing, and homology analysis was performed after sequencing. BLAST software was used for comparison, and MEGA7.0 software was used to construct a phylogenetic tree.

[0047] The strain F0b was extracted by TSINGKE plant DNA extraction kit, and DNA was extracted from the strain sample to obtain genomic DNA. The genomic DNA was used as a template and the bacterial 16SrDNA universal primers 27F (sequence number: SEQ ID NO: 2, 5'-AGTTTGATCMTGGCTCAG-3') and 1492 (sequence number: SEQ ID NO: 3, 5'-GGTTACCTTGTTACGACTT-3') were used to amplify and sequence the target strain 16SrDNA. The components of the PCR reaction system are as follows: 1×TSE101Mix45 uL, 2uL of each upper and lower primer, and 1uL of DNA template. The reaction conditions are: pre-denaturation at 98℃ for 2min; cycle stage at 98℃ for 10s, 56℃ for 10s, and 72℃ for 10s, 35 cycles; extension at 72℃ for 5min. After the PCR product was detected by agarose gel electrophoresis, the PCR amplified sample was sent to Beijing Qingke Biotechnology Co., Ltd. (Guangzhou Branch) for sequencing analysis. The sequencing results were used to perform BLAST comparison in the NCBI database to preliminarily identify the samples, and the phylogenetic tree was constructed using MEGA7.0 software.

[0048] 2. Experimental results and analysis

[0049] The morphological characteristics showed that strain F0b was light yellow, raised, wrinkled, opaque, dry, and had irregular edges. Under a microscope, its bacterial body was short rod-shaped. Figure 2 Physiological and biochemical identification showed that Bacillus F0b was positive for contact enzyme, methyl red, indigo matrix, nitrate, starch hydrolysis, gelatin liquefaction, and VP reactions, and was negative for sucrose, glucose, mannitol, hydrogen sulfide, oxidase, and citrate reactions. The morphological characteristics of strain F0b were preliminarily identified, and F0b was preliminarily identified as Bacillus. The DNA genome of strain F0b was extracted and sequenced, and the BLAST sequence comparison was performed using the NCBI database. The phylogenetic tree was constructed using the MEGA7.0 software. The results are shown in Figure 3 The strain F0b belongs to the same branch as Bacillus Velezii (MG705703.1, LC191186.1, CP054714.1). Based on its morphological characteristics and physiological and biochemical experimental results, the strain F0b was identified as Bacillus Velezii.

[0050] Example 3 Biological characteristics of Bacillus Velez F0b

[0051] Preparation of fermentation broth: A single colony cultured on NA medium for 24 h was selected and inoculated into LB medium for overnight culture to obtain seed solution. The seed solution was then inoculated into 100 / 250 mL LB culture medium at a 1% inoculum amount and cultured overnight (16 h) to obtain an OD 600 =1 fermentation liquid and set it aside.

[0052] Determination of F0b growth curve: Use a pipette to take 100 μL of seed solution and add it to a 250 mL conical flask containing 100 mL LB liquid medium, and place it in a constant temperature oscillator for cultivation at 30°C and 180 r / min. Take samples every 2 hours and measure their absorbance OD 600 , the growth curve of Bacillus Velez F0b was drawn, and each treatment was repeated 3 times.

[0053] The results showed that 0h~6h was the delayed growth period of the strain, 6h~16h was the logarithmic growth period of the strain, the strain grew slowly after 16h, 16h~38h was the stable period of the strain, and the strain began to enter the decline period after 38h. The strain grew vigorously and multiplied quickly during 6h~16h, so the culture medium cultivated for 16h was selected as the fermentation liquid of Bacillus Velez F0b. The growth curve of Bacillus Velez F0b is shown in Figure 4 .

[0054] Effect of different pH on the growth of Bacillus Velez F0b: The fermentation broth OD 600 = 1 for standby use, adjust the pH of LB liquid culture medium to 3.0, 5.0, 7.0, 9.0, 11.0, and the volume of LB liquid culture medium is 100mL / 250mL. Determine OD 600 .

[0055] The results showed that after culturing in LB liquid medium with different pH for 24 hours, the growth of strain F0b was significantly different. When the pH was 5-9, Bacillus F0b grew well, and the growth was best at pH 7. When the pH was 1, 3, and 11, the growth of Bacillus F0b was significantly inhibited and almost did not grow. The effect of different pH on the growth of Bacillus Velez F0b is shown in Figure 5 .

[0056] Effect of different sodium chloride contents on the growth of Bacillus Velez F0b: 50 mL of LB liquid culture medium was placed in a 150 mL conical flask, and the sodium chloride content in the culture 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 F0b cultured overnight was fermented at a 1% inoculum (OD 600 =1) Add to different treatments, repeat 3 times for each treatment, place at 30°C, shake at 180r / min for 24h, take out, and measure OD 600 value, and determine the optimal sodium chloride content of strain F0b.

[0057] When the sodium chloride content was 0g / L, 5g / L, 10g / L, 25g / L, 50g / L, and 100g / L, the growth of Bacillus Velez F0b was good, among which the growth was the best at 10g / L, with significant differences. When the sodium chloride content was 200g / L, the growth of Bacillus Velez F0b was significantly inhibited and almost did not grow, indicating that Bacillus Velez F0b had good salt tolerance. The effects of different sodium chloride contents on the growth of Bacillus Velez F0b are shown in Figure 6 .

[0058] Example 4 Determination of extracellular enzymes of Bacillus Velez F0b

[0059] Place Φ = 5 mm sterile filter paper in the center of the test medium containing carboxymethyl cellulase, β-glucanase, amylase and protease, and add 2 μL F0b fermentation liquid (OD 600 =1.0) on the filter paper, detect the extracellular enzyme activity of Bacillus Velez F0b, under the same conditions, add 2 μL of sterile water to the filter paper as a control, each treatment is repeated 3 times, all plates are cultured in a 30°C constant temperature incubator for 3d to 5d, and observe whether there is a transparent circle. If there is a transparent circle, it means that the strain can produce the extracellular enzyme. Among them, the carboxymethyl cellulase detection adopts 0.2% Congo red staining for 30min, and then the dye is thoroughly washed away with distilled water and 1mol / L NaCl in turn, and then the color is fixed with 5% acetic acid solution, and whether there is a transparent hydrolysis circle around the colony; for the amylase detection, a small amount of Lugol's iodine solution is added to the plate, gently rotated, so that the iodine solution is evenly spread over the entire plate, and observe whether a transparent circle is formed around the colony. The size of the colony and the diameter of the transparent circle were measured with a vernier caliper, and the enzyme activity was determined according to the following formula. The enzyme activity was expressed as the ratio of the transparent circle to the colony diameter. The larger the ratio, the stronger the ability of the strain to produce extracellular enzymes.

[0060] Enzyme activity = diameter of transparent zone / diameter of colony

[0061] The results showed that the colonies of strain Fob produced transparent circles on carboxymethyl cellulase, protease and amylase plates, but no transparent circle on β-glucanase. Therefore, F0b has the ability to produce carboxymethyl cellulase, protease and amylase, but not β-glucanase (extracellular enzyme characteristics of Bacillus Velez F0b fermentation broth can be found in Figure 7 ), the HC values ​​of protease, amylase and carboxymethyl cellulase in the extracellular enzymes were 2.71, 2.79 and 1.52 respectively.

[0062] Table 2 The ability of Bacillus Velez F0b to produce extracellular enzymes

[0063]

[0064] Example 5 In vitro control effect of Bacillus Velez F0b fermentation liquid on the control of cabbage sclerotinia

[0065] The in vitro leaf method was used. Healthy cabbage leaves with the same leaf age were selected for the experiment. The surface was disinfected with 75% alcohol, rinsed with sterile water, and dried for later use. The 7 billion CFU / g Velez Bacillus WP F0b 200 times was prepared in the laboratory. F0b fermentation liquid was prepared and the concentration of the fermentation liquid was adjusted to 1×10 8 CFU / mL, a total of 8 treatments were set up in the experiment: (a) sterile water treatment, (b) F0b fermentation broth, (c) F0b fermentation broth 10 times, (d) F0b fermentation broth 100 times, (e) 7 billion CFU / g Velez subtilis wettable powder F0b 50 times, (f) 7 billion CFU / g Velez subtilis wettable powder F0b 200 times, (g) 7 billion CFU / g Velez subtilis wettable powder F0b 400 times, (h) 40% Sclerotium net WP 250 times. The cabbage leaves were soaked in the drug solution of each treatment dose for 30 minutes, dried naturally, and placed in a culture dish with moist filter paper spread inside. The wet cotton was placed on the petiole. A Φ=6mm sclerotinia fungus disk was inoculated on each cabbage leaf, the lid was closed, and the leaves were placed in a light incubator for 5 days (16h light / 8h dark, 20°C, relative humidity above 70%). The diameter of the lesions was measured, and each treatment was repeated 3 times.

[0066] The results showed that in the cabbage detached leaf method disease prevention experiment, 5 days after treatment, the diameter of the lesions on the leaves of the control group treated with sterile water was 5.13 cm. The diameter of the lesions on the cabbage leaves treated with the original solution of Bacillus Velez F0b fermentation liquid and the prepared 7 billion CFU / g Bacillus Velez F0b wettable powder was significantly reduced (see Table 3), and the treatment effect was significantly decreased. The 50 times solution of 7 billion CFU / g Bacillus Velez F0b wettable powder was the most obvious. The original solution, 10-fold dilution, 100-fold dilution and the prepared wettable powder of Bacillus Velez F0b fermentation liquid all had obvious antibacterial effect on cabbage sclerotinia rot, and the diameters of the lesions were 0 cm, 1.28 cm and 2.38 cm, respectively. The original solution of Bacillus Velez F0b fermentation liquid did not cause the disease, and the control effect was equivalent to that of the chemical agent 40% Sclerotium Cleaner WP 250 times treatment in the same period (the in vitro effect of Bacillus Velez F0b on the prevention and control of cabbage sclerotinia rot is shown in Figure 8 ), indicating that F0b fermentation broth has a good inhibitory effect on cabbage sclerotinia rot.

[0067] Table 3 The efficacy of Velez Bacillus F0b against Sclerotinia sclerotiorum of Cabbage

[0068]

[0069] Note: The data in Table 3 are the mean ± standard error of 3 replicates. Different lowercase letters after the data in the same column indicate significant differences (p<0.05) tested by DMRT method.

[0070] Example 6 In vitro effect of Bacillus Velez F0b fermentation liquid on preventing and treating pepper anthracnose fruit

[0071] Select pepper fruits with uniform appearance and size and no symptoms of pests and diseases, wipe the surface of the pepper fruits with 75% alcohol, rinse with sterile water for 2 to 3 times, and spray different treatment solutions after drying. Set up 5 treatments: (1) 1×108 CFU / mL of Bacillus Velez F0b fermentation liquid, (2) 50 times of 7 billion CFU / g Bacillus Velez F0b wettable powder, (3) 200 times of 60% difenoconazole, (4) positive control-sterile water treatment, (5) negative control-no inoculation of pathogens. After the fruits are dried, inoculate 6 mm pepper anthracnose bacteria cakes in treatments (1) to (5). Use the puncture inoculation method to pierce the middle waist of the fruit with a sterile toothpick, and repeat 3 times for each treatment. Place the plates at 28°C and L:D of 16:8 and culture them under alternating moisturizing conditions. Seal the fresh-keeping boxes containing pepper fruits with plastic wrap until the positive control shows significant anthracnose symptoms. Measure the diameter of the lesions using the cross method and calculate the control effect using the following formula.

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

[0073] The experimental results are as follows Fig. 9 5 days after inoculation, pepper anthracnose can infect normally. The disease in the control group is more serious. The color of the fruit inoculation site deepens, the lesions are sunken, and the diameter is 8.37mm. The pepper fruit treated with the original solution of Velez F0b fermentation liquid and 50 times solution of 7 billion CFU / g Velez F0b wettable powder has a significantly lighter disease. The inoculation site is slightly sunken, the color is normal, and the diameter of the lesions is significantly reduced. The diameter of the lesions is 0.91mm and 2.12mm respectively. The control effect of 7 billion CFU / g Velez F0b wettable powder on anthracnose of detached fruit is 74.68%, and the control effect of Velez F0b fermentation liquid on anthracnose of detached pepper fruit is 89.11%, which is significantly better than chemical agents.

[0074] Table 4 Control effect of Bacillus Velez F0b fermentation liquid on tomato wilt

[0075]

[0076] Note: The data in Table 4 are the mean ± standard error of 3 replicates. Different lowercase letters after the data in the same column indicate significant differences (p<0.05) tested by DMRT method.

Claims

1. A Bacillus velezensis F0b, characterized in that The strain has been deposited in the Guangdong Microbiological Culture Collection Center, with the deposit date being April 26, 2024, and the deposit number being GDMCCNo: 64566.

2. The Bacillus velezensis F0b according to claim 1, wherein The Bacillus Velez F0b has the ITS1-5.8S-ITS4 rDNA gene sequence shown in SEQ ID NO:

1.

3. Use of Bacillus velezensis F0b according to claim 1 or 2 in preventing and / or controlling plant pathogenic fungal diseases.

4. The use according to claim 3, characterized in that The plant pathogenic fungal diseases include rice blast disease, cabbage sclerotinia disease, tomato wilt disease, banana wilt disease, rice leaf spot disease, litchi downy mildew disease, soybean charcoal rot disease, pepper anthracnose disease or corn leaf spore disease.

5. The use according to claim 4, characterized in that The plant pathogenic fungi include rice blast disease, cabbage sclerotinia disease or pepper anthracnose disease.

6. A broad-spectrum microbial-derived bactericide, characterized in that: The broad-spectrum microbial-derived fungicide contains the fermentation broth of Bacillus Velez F0b according to claim 1 or 2.

7. The broad-spectrum microbial-derived bactericide according to claim 6, characterized in that: The preparation method of the fermentation liquid of the Velez subtilis F0b is as follows: a single colony of Velez subtilis F0b cultured on a NA medium for 24 hours is selected and inoculated into an LB medium for overnight culture to obtain a seed liquid, and then the seed liquid is inoculated into a 100 / 250 mL LB culture medium at a 1% inoculum amount for overnight culture to obtain a fermentation liquid.

8. The broad-spectrum microbial-derived bactericide according to claim 6, characterized in that: The fermentation liquid of the Bacillus Velez subtilis F0b can produce amylase, carboxymethyl cellulase and protease; the pH value of the fermentation liquid of the Bacillus Velez subtilis F0b is 5-9.

9. Use of the broad-spectrum microbial fungicide according to any one of claims 6 to 8 in preventing and controlling vegetable pathogenic fungal diseases.

10. The use according to claim 9, characterized in that The vegetable pathogenic fungal disease is cabbage sclerotinia disease or pepper anthracnose disease.

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