Bacillus Velez HF01 and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a Velez bacillus HF01 and its application; Velez bacillus HF01 has been deposited as a patented strain in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC NO: 33137. The strain has a significant effect in preventing and treating wax gourd damping-off disease caused by plant pathogens; it has a good inhibitory effect on wax gourd damping-off disease caused by Rhizoctonia solani, and can also promote the growth of wax gourd.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to Bacillus Velezii and applications thereof. Background Art

[0002] Winter melon damping-off is a winter melon disease caused by the hyphae fusion group of Rhizoctonia solani Kühn AG-4, which can occur in both the seedling and adult stages. In the seedling stage, the cotyledons turn yellow and dry, the young stems, leaves and growing points wilt, and the base of the rhizomes turn brown and constrict; in the adult stage, some leaves wilt at noon, recover in the morning and evening, the leaf color becomes lighter, and then all wilt. Transmission route and disease conditions Mycelium or thick-walled spores stay in the soil with the diseased residues to overwinter, becoming the primary source of infection. Thick-walled spores can survive in the soil for 5-10 years. After germination, they first grow bud tubes and invade from the root wound or the root cap cell gap. Winter melon damping-off is a common winter melon disease, which plays a vital role in its yield. Chemical agents are the main means of preventing and controlling winter melon damping-off. Commonly used chemical agents include Jinggangmycin, Methacin and Thiram. However, long-term use will produce drug resistance and will also have a certain impact on the soil.

[0003] Bacillus, a family of bacteria, is a rod or coccus that can form spores (endospores). It includes Bacillus, Sporolactobacillus, Clostridium, Desulfur Enterobacter, and Sporosarcina. They are highly resistant to harmful factors from the outside world and are widely distributed in soil, water, air, and animal intestines. Among them, Bacillus Velezii, as a new type of Bacillus, has antibacterial activity against some pathogenic fungi and even some bacteria, and is harmless to humans and animals and does not pollute the environment.

[0004] If a new type of Bacillus Velezii can be obtained and used for biological control of wax gourd damping-off disease, the yield of wax gourd will be greatly increased and is worthy of promotion. Summary of the invention

[0005] In view of the above problems, the present invention provides a new strain HF01 of Bacillus Velezii, which has a significant effect in preventing and treating wax gourd damping-off disease caused by plant pathogens.

[0006] In a first aspect, the present invention provides a Bacillus velezensis HF01, which is classified and named Bacillus velezensis. The strain has been deposited as a patented strain in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with a deposit number of CGMCC NO: 33137.

[0007] The 16S rRNA nucleotide sequence of the Bacillus Velezii HF01 is shown in SEQ ID NO: 1.

[0008] The gyrB nucleotide sequence of the Bacillus Velezii HF01 is shown in SEQ ID NO:2.

[0009] In a second aspect, the present invention provides the use of Bacillus Velezii HF01 in preventing and controlling wax gourd damping-off disease caused by plant pathogens.

[0010] As a preferred embodiment, before the wax gourd buds and blooms, the agent containing Bacillus Velez subtilis HF01 is sprayed on the wax gourd; the agent is sprayed once every 7-10 days.

[0011] As a preferred embodiment, the plant pathogen is Rhizoctonia solani.

[0012] In a third aspect, the present invention provides a method for culturing Bacillus Velezii HF01, comprising the following steps: (1) Inoculate Bacillus Velezii HF01 into a slant culture medium prepared from a marine habitat Bacillus culture medium to prepare a primary slant seed, and culture at a constant temperature of 25-30°C for 70-80 hours; (2) Inoculate the slant seeds in step (1) into a flask or fermentation tank containing Bacillus liquid culture medium and culture at a constant temperature for 17-24 hours to obtain liquid seeds; (3) Inoculate the liquid seeds into liquid fermentation medium and culture for 20-48 hours to obtain the product.

[0013] As a preferred embodiment, in step (1), the culture is carried out at a constant temperature of 28°C.

[0014] As a preferred embodiment, in step (1), the marine habitat Bacillus culture medium is composed of the following components: 7g peptone, 2g yeast powder, 2g glucose, 6g sodium chloride, 0.06g potassium chloride, 0.5g magnesium chloride heptahydrate, 18g agar per liter of culture medium, and water is added to make up to 1 liter.

[0015] As a preferred embodiment, in step (2), the Bacillus liquid culture medium comprises the following components: 10-13 g / L glucose, 20-28 g / L peanut powder, 0.02-0.05 g / L iron phosphate, 0.05-0.08 g / L potassium chloride, 6-8 g / L sodium chloride, 0.5-1.0 g / L magnesium chloride, and a pH value of 7.5.

[0016] As another preferred embodiment, in step (2), the Bacillus liquid culture medium is composed of the following components: 7 g peptone, 2 g yeast powder, 2 g glucose, 6 g sodium chloride, 0.06 g potassium chloride, and 0.5 g magnesium chloride heptahydrate per liter of culture medium, and water is added to make up to 1 liter.

[0017] As a preferred embodiment, in step (3), the number of Bacillus spores in the product reaches 1.0×10 9 cfu / mL or above.

[0018] As a preferred embodiment, in step (3), the culture medium composition is: 40 g / L glucose, 30 g / L starch, 30 g / L peanut cake, 10 g / L yeast extract, 0.02 g / L iron phosphate, 0.1 g / L magnesium sulfate, 0.06 g / L potassium chloride, 6 g / L sodium chloride, 0.4 g / L magnesium chloride, 0.1 g / L calcium chloride, 1.5 g / L potassium dihydrogen phosphate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L light calcium carbonate, 0.05 g / L manganese sulfate, and 1 ml / L folate.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention discloses Bacillus velezensis HF01, which is a new strain of Bacillus velezensis and has a good control effect on wax gourd damping-off disease, can reduce the application dosage of chemical pesticides, and reduce pesticide residues and environmental pollution.

[0020] 2. The method for culturing Bacillus Velez subtilis provided by the present invention adopts liquid culture medium to prepare Bacillus Velez subtilis subtilis subtilis subtilis subtilis subtilis, which not only has mild culture conditions and simple culture steps, but also can make Bacillus Velez subtilis subtilis HF01 proliferate in large quantities, and the number of Bacillus spores in the product reaches 1.0×10 9 cfu / mL or above. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a growth curve diagram of Bacillus Velez HF01 of the present invention; Figure 2 This is an electron microscope image of the growth process of Bacillus Velez HF01 of the present invention; Figure 3 The figure is a comparison diagram of the plate growth antagonism test between Bacillus velez and Rhizoctonia solani of the present invention. DETAILED DESCRIPTION

[0022] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described in detail below with reference to specific embodiments. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0023] The present application provides a Bacillus velezensis, which is named Bacillus velezensis HF01 (Bacillus velezensis HF01). The strain has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on December 20, 2024, with the deposit number CGMCC: No. 33137.

[0024] Example 1 Identification of Bacillus velezensis HF01 Bacillus velezensis HF01 was isolated from the rhizosphere soil of a wax gourd plant in Quzhou, Zhejiang Province, and morphological and molecular identification was performed.

[0025] The dominant strain was activated on the plate and cultured in a constant temperature incubator for 72 hours. The colony color, growth rate, shape, edge, degree of protrusion, surface condition, etc. were observed and recorded. The culture morphology of Bacillus Velezii HF01: On LB solid medium, it presents white opaque colonies with a rough surface and a rod shape in Gram staining. The details are shown in Table 1.

[0026] Table 1 Cell morphology of Bacillus velez HF01 strain Test Project result Test Project result Test Project result Test Project result Cell morphology Rod Gram stain Positive Catalase + Oxidase + Biological identification results The Bacillus Velez subtilis HF01 of the present invention has a 16S rRNA gene sequence as shown in SEQ ID NO:1 and a gyrB gene sequence as shown in SEQ ID NO:2.

[0027] The results of the growth experiment and the chemical sensitivity experiment are shown in Table 2 and Table 3, respectively.

[0028] Table 2 Growth experiment results Negative control - α-D-Glucose + gelatin - p-Hydroxyphenylacetic acid - dextrin + D-Mannose + L-Alanine + Methyl pyruvate + D-Maltose + D-Fructose + L-Aspartic Acid + D-Methyl lactate - D-Trehalose + D-Galactose - L-Glutamic Acid + L-Lactic Acid + D-Cellobiose + 3-Methyl-D-glucose - L-Histidine + Citric Acid + Gentianobiose + D-fucose - L-Pyroglutamic acid - α-Ketoglutarate - sucrose + L-fucose - L-Serine - D-Malic Acid - turanose + L-Rhamnose - Pectin + L-Malic Acid + Stachyose + Inosine + D-galacturonic acid + Bromosuccinic acid - D-Raffinose + D-Sorbitol + L-Galactone + Twain 40 - α-D-Lactose + D-Mannitol + D-Gluconic acid + GABA - D-Mi Ertang + D-arabitol - D-Glucuronic acid + Alpha-Hydroxybutyrate - β-Methyl-D-glucose + Inositol + Glucuronamide - β-Hydroxy-D-,L-butyric acid - D-Salicin + glycerin + mucic acid - α-Ketobutyric Acid - N-Acetyl-D-Glucosamine + D-Glucose-6-phosphate - Quinic acid + Acetoacetic acid + N-Acetyl-β-D-mannosamine - D-fructose-6-phosphate + Glucose acid - Propionic acid - N-Acetyl-D-Galactosamine - D-Aspartic Acid - L-Arginine + Acetic acid - N-acetylneuraminic acid - D-serine - Glycine-L-Proline - Formic acid - Table 3 Chemical sensitivity test results (+ insensitive, - sensitive) Positive Control + 1% Sodium Lactate + Lincomycin - Nalidixic acid - pH 6.0 + Fusidic acid - Guanidine hydrochloride + Lithium chloride + pH5.0 + D-serine - Sodium Tetradecane Sulfate - Potassium tellurite + 1%NaCl + Troleandomycin - Vancomycin - Chlortrexone + 4%NaCl + Rifamycin SV - Tetrazolium violet + Sodium Butyrate + 8%NaCl + Minocycline - Tetrazolium blue + Sodium bromate + Based on the comprehensive analysis of experimental data such as cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, gyrB gene sequence, and reference to the Berges Manual of Systematic Bacteriology and relevant research papers in the International Journal of Systematic and Evolutionary Microbiology, the strain was identified as Bacillus velezensis. The selected strain was named Bacillus velezensis HF01.

[0029] Example 2 Cultivation method of Bacillus velez HF01 (1) The isolated Bacillus Velez subtilis HF01 strain was inoculated into a slant culture medium prepared from marine habitat Bacillus culture medium (each liter of culture medium contained 7 g peptone, 2 g yeast powder, 2 g glucose, 6 g sodium chloride, 0.06 g potassium chloride, 0.5 g magnesium chloride heptahydrate, 18 g agar, and made up to 1 liter with water) to prepare a primary slant seed, which was cultured in a constant temperature incubator at 28°C for 72 h.

[0030] (2) Inoculate the slant seeds into a flask or fermenter filled with a marine habitat Bacillus liquid culture medium, wherein the concentration of the slant seeds in the liquid culture medium is 10%. Incubate the slant seeds at a constant temperature of 35°C and 200 rpm for 17-24 hours to obtain liquid seeds.

[0031] The liquid culture medium does not contain agar, and the other ingredients are the same as the slant culture medium. The liquid culture step can be replaced with this culture medium: 12.5 g / L glucose, 20 g / L peanut powder, 0.02 g / L iron phosphate, 0.06 g / L potassium chloride, 6 g / L sodium chloride, 0.5 g / L magnesium chloride, pH 7.5.

[0032] (3) Inoculate the liquid seeds into the liquid fermentation medium. The medium composition is: glucose 40g / L, starch 30g / L, peanut cake 30g / L, yeast extract 10g / L, iron phosphate 0.02g / L, magnesium sulfate 0.1g / L, potassium chloride 0.06g / L, sodium chloride 6g / L, magnesium chloride 0.4g / L, calcium chloride 0.1g / L, potassium dihydrogen phosphate 1.5g / L, dipotassium hydrogen phosphate 3g / L, light calcium carbonate 0.5g / L, manganese sulfate 0.05g / L, 1ml / L of chloranil, pH 7.5, 180r / min, 35℃ temperature, culture for 20-48h. The spore count of the product reaches 1.0×10 9 cfu / mL or more. Measure the OD600 value during the process, measure 3 times at each time, take the average value and draw a growth curve, and observe by electron microscope test ( Figure 2 ). Some growth curves obtained from specific experiments are shown in Figure 1 As shown. Figure 1 The growth curve of Figure 2 The experimental results of (20 hours, 25 hours, 35 hours, 40 hours and 45 hours) show that when cultured for 20 hours, the OD600 value can reach a maximum value of about 57, after which the growth of Bacillus Velez HF01 begins to gradually stabilize. The OD600 value stabilizes between 55 and 57 between 48 hours. Therefore, according to the experimental results, the optimal culture time of Bacillus Velez HF01 is preferably controlled at 20 to 48 hours using the culture method of the present application.

[0033] Example 3 Antagonism experiment of Bacillus velez HF01 First, the Velez subsp. HF01 and the Bacillus (control bacteria) cultured in Example 2 were inoculated on PDA plates. Each plate was inoculated with the Velez subsp. HF01 and the control bacteria at the same time, and the same Bacillus were inoculated oppositely and placed in a 28° C. incubator for culture.

[0034] Observe the growth of Bacillus, and inoculate Rhizoctonia solani after about one day of growth. Take a freshly grown Rhizoctonia solani plate, take a 5mm diameter bacterial disc from the periphery of the colony, with the mycelium facing down, and inoculate it in the center of the PDA plate; in addition to inoculating the plate antagonistic with Bacillus, inoculate the Rhizoctonia solani blank control, and culture them in a 28℃ incubator together. After 5-7 days of inoculation, observe the plate antagonistic effect, and the plate is completed when the Rhizoctonia solani blank control grows all over the plate.

[0035] The inhibition rate was calculated according to the inhibition rate (%) = (control pathogen colony diameter - treated pathogen colony diameter) / control diameter × 100%.

[0036] The antibacterial rate = (31.73-8.92) / 31.73×100% = 71.9%, that is, the antibacterial rate is 71.9%.

[0037] When Bacillus Velezii HF01 was antagonized against the pathogen causing winter melon damping-off, the inhibition rate reached 71.9%. Figure 3 As shown (A is the control plate, B is the antagonistic plate), it is shown that the Bacillus Velezii of the present application has a good antibacterial effect on Rhizoctonia solani, the pathogen of wax gourd blight.

[0038] Example 4 Field Test Field trials were conducted using Example 2 and commercially available oxadipamide as examples to test their control effects on damping-off disease of wax gourd. The trial was conducted at a wax gourd planting base in Quzhou, Zhejiang Province, with 4 repetitions per treatment, each repetition area of ​​4 square meters, 5 points were investigated in double diagonal lines in each plot, all leaves of all wax gourd plants at each point were investigated, and the occurrence of the disease was counted.

[0039] The severity of the disease is recorded using a grading method, and the grading standards are as follows: Level 0: no disease; Level 1: The area of ​​lesions is less than 5% of the entire leaf area; Level 3: The area of ​​lesions accounts for less than 6%-10% of the entire leaf area; Level 5: The area of ​​lesions accounts for less than 11%-20% of the entire leaf area; Level 7: The area of ​​lesions accounts for less than 21%-50% of the entire leaf area; Level 9: The area of ​​lesions accounts for more than 50% of the entire leaf area.

[0040] Calculation method of protection effect Diseased leaf rate (%) = number of diseased leaves / total number of leaves surveyed × 100% Disease index = ∑ (number of diseased leaves at each level × relative level value) / (total number of leaves investigated × 9) × 100 The control effect (%) = [1-(the disease index of the blank control area before the drug × the disease index of the treated area after the drug) / (the disease index of the blank control area after the drug × the disease index of the treated area before the drug)] × 100%.

[0041] The pesticide was applied twice at the seedling stage of wax gourd (the experimental group used the pesticide prepared in Example 2 at a concentration of 1.0×10 9 cfu / mL, the control group was sprayed with 500 times diluted carbendazim), each application was 7 days apart, and the prevention effect was counted 7 days after the second application. After the test was completed, the prevention effect of winter melon damping-off disease was focused on and the efficacy was calculated. The results are shown in Table 4.

[0042] Table 4 Statistics of field efficacy test results pesticide Diseased leaf rate (%) Disease index Damping-off disease control effect (%) Experimental Group 23±4.32 0.08±0.072 80.64±5.62 Control group 26±6.34 0.100±0.091 76.91±4.52 Statistical analysis showed that the isolated Bacillus Velez HF01 had a good protective effect against wax gourd damping-off, which was comparable to or even superior to the commercially available chemical pesticides such as oxazolidinone. Bacillus Velez HF01 can completely reduce the use of chemical pesticides, and in addition to inhibiting the growth and reproduction of harmful microorganisms such as harmful bacteria and pathogens, it can also provide mineral nutrients such as N, P, K and Fe for plant growth through metabolism, synthesize growth hormones to promote the growth of wax gourd, induce it to produce systemic disease resistance, and compete with pathogens when coexisting, achieving the effect of prevention and treatment.

[0043] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. A person skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features may be replaced by equivalents. These modifications or replacements should also be regarded as within the scope of protection of the present invention.

Claims

1. A Bacillus velezensis strain HF01, with a deposit number of CGMCC No.33137.

2. The Bacillus Velez subtilis HF01 according to claim 1, characterized in that The 16S rRNA nucleotide sequence of the Bacillus Velezii HF01 is shown in SEQ ID NO:

1.

3. The Bacillus Velez subtilis HF01 according to claim 1, characterized in that The gyrB nucleotide sequence of the Bacillus Velezii HF01 is shown in SEQ ID NO:

2.

4. Use of the Bacillus Velezii HF01 according to claim 1 in preventing and treating damping-off disease of wax gourd caused by plant pathogens.

5. The use according to claim 4, characterized in that: Before the wax gourd buds and blooms, apply a pesticide containing Bacillus Velez HF01 to the wax gourd; spray once every 7-10 days.

6. The use according to claim 4, characterized in that: The plant pathogen is Rhizoctonia solani.

7. A method for culturing Bacillus Velez HF01, characterized in that: The following steps are involved: (1) Inoculate Bacillus Velezii HF01 into a slant culture medium prepared from a marine habitat Bacillus culture medium to prepare a primary slant seed, and culture at a constant temperature of 25-30°C for 70-80 hours; (2) Inoculate the slant seeds in step (1) into a flask or fermentation tank containing Bacillus liquid culture medium and culture at a constant temperature for 17-24 hours to obtain liquid seeds; (3) Inoculate the liquid seeds into liquid fermentation medium and culture for 20-48 hours to obtain the product.

8. The method for culturing Bacillus Velez subtilis HF01 according to claim 7, characterized in that: In the step (1), the culture is carried out at a constant temperature of 28°C.

9. The method for culturing Bacillus Velez subtilis HF01 according to claim 7, characterized in that: In the step (2), the Bacillus liquid culture medium includes the following components: 10-13 g / L glucose, 20-28 g / L peanut powder, 0.02-0.05 g / L iron phosphate, 0.05-0.08 g / L potassium chloride, 6-8 g / L sodium chloride, 0.5-1.0 g / L magnesium chloride, and the pH value is 7.

5.

10. The method for culturing Bacillus Velez subtilis HF01 according to claim 7, characterized in that: In step (3), the number of Bacillus spores in the product reaches 1.0×10 9 cfu / mL or above.

Images